Parasites and Pathogens of Insects. Parasites

Parasites and Pathogens of Insects

Volume 1: Parasites

E d i t e d by

Ν. E. Beckage S. N. Thompson B. A. Federici Department of Entomology University of California Riverside, California

A c a d e m i c P r e s s , I n c . A Division of H a r c o u r t Brace & Company

San Dieao New York Boston London Svdnev Tokvo Toronto

T h i s b o o k is p r i n t e d o n ac id - f r ee p a p e r . ©

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United Kingdom Edition published by Academic Press Limited 2 4 - 2 8 O v a l R o a d , L o n d o n N W 1 7 D X

L i b r a r y o f C o n g r e s s C a t a l o g i n g - i n - P u b l i c a t i o n D a t a

P a r a s i t e s a n d p a t h o g e n s o f i n s e c t s / e d i t e d b y Ν . E . B e c k a g e , S . N .

T h o m p s o n , B . A . F e d e r i c i .

p . c m .

I n c l u d e s b i b l i o g r a p h i c a l r e f e r e n c e s a n d i n d e x .

I S B N 0 - 1 2 - 0 8 4 4 4 1 - 9 (v . 1) . - I S B N 0 - 1 2 - 0 8 4 4 4 2 - 7 (v . 2 )

1. I n s e c t s — P a r a s i t e s . 2 . I n s e c t s — P a t h o g e n s . 3 . H o s t — p a r a s i t e s

r e l a t i o n s h i p s . I. B e c k a g e , Ν . E . ( N a n c y Ε . ) Π. T h o m p s o n , S . N .

I I I . F e d e r i c i , B r i a n A .

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Contents o f Vo lume 2

Chapter 1 Tripartite Interactions between Symbiotically Associated Entomopathogenic Bacteria, Nematodes, and Their Insect Hosts

Ray J. Akhurst and Gary B. Dunphy

Chapter 2 The Insect Immune Proteins and the Regulation of Their Genes

Ingrid Faye and Dan Hultmark

Chapter 3 Interactions of Bacillus thuringiensis Endotoxins with the Insect Midgut Epithelium

Patricia V. Pietrantonio, Brian A. Federici, and Sarjeet S. Gil l

Chapter 4 Viral Pathobiology in Relation to Insect Control

Brian A. Federici

Chapter 5 Baculoviruses, Vertebrate Viruses, and Cytoskeletons

Carol A. Charlton and Loy E. Volkman

Chapter 6 Baculovirus Enhancing Proteins as Determinants of Viral Pathogenesis

Bartholomew G. Corsaro, Mark Gijzen, Ping Wang, and Robert R. Granados

Chapter 7 Invertebrate Transposable Elements in the Baculovirus Genome: Characterization and Significance

Paul D. Friesen

xi

Contents of Volume 2

Genetic Manipulation of the Baculovirus Genome for Insect Pest Control

R. E. Hawtin and R. D. Possee

Insect Resistance to Viruses

James R. Fuxa

Biology and Mechanisms of Insect-Cuticle Invasion by Deuteromycete Fungal Pathogens

Raymond St. Leger

Host—Parasitoid—Pathogen Interactions

Wayne M. Brooks

xii

Chapter 8

Chapter 9

Chapter 10

Chapter 11

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Nancy E. Beckage (25) , D e p a r t m e n t of E n t o m o l o g y , U n i v e r s i t y of Cal iforn ia , R ive r s ide , Ca l i fo rn ia 92521

Bruce M. Christensen (245) , D e p a r t m e n t of A n i m a l H e a l t h a n d B i o m e d i c a l Sc iences , U n i v e r s i t y of W i s c o n s i n , M a d i s o n , W i s c o n s i n 53706

Thomas Coudron (227) , Biological C o n t r o l of In sec t s L a b , U S D A - A R S , C o l u m b i a , M i s s o u r i 65205

Douglas L Dahlman (145) , D e p a r t m e n t of En tomology , U n i v e r s i t y of K e n tucky, L e x i n g t o n , K e n t u c k y 40546

Jo-Ann G. W. Fleming (189) , D e p a r t m e n t of En tomology , T e x a s A & M U n i versity, Co l l ege S t a t i on , T e x a s 77843

David R. Horton (107) , Y a k i m a A g r i c u l t u r a l L a b , U S D A - A R S , Y a k i m a , W a s h i n g t o n 98902

Hilary Hurd (87) , D e p a r t m e n t of Biological Sc iences , U n i v e r s i t y of Kee le , S taf fordshi re , S T 5 5 B G U n i t e d K i n g d o m

Davy Jones (227) , G r a d u a t e C e n t e r for Toxicology, U n i v e r s i t y of K e n t u c k y , L e x i n g t o n , K e n t u c k y 40506

Michael R. Kanost (317) , D e p a r t m e n t of B iochemis t ry , K a n s a s S t a t e U n i v e r sity, M a n h a t t a n , K a n s a s 66506

Richard D. Karp (305) , D e p a r t m e n t of Biological Sc iences , U n i v e r s i t y of C i n c i n n a t i , C i n c i n n a t i , O h i o 45221

Peter J. Krell (189) , D e p a r t m e n t of Microb io logy , U n i v e r s i t y of G u e l p h , G u e l p h , O n t a r i o N I T 2 W 1 , C a n a d a

Beatrice Lanzrein (59) , Div i s ion of D e v e l o p m e n t a l Biology, Zoo log ica l I n s t i t u t e , U n i v e r s i t y of B e r n e , 3012 B e r n e , S w i t z e r l a n d

xiii

xiv Contributors

Pauline O. Lawrence (59), D e p a r t m e n t of Zoology, U n i v e r s i t y of F l o r i d a , Ga inesv i l l e , F l o r i d a 32611

M. Mackauer (1), C e n t r e for Pest M a n a g e m e n t , D e p a r t m e n t of Bio logica l Sc iences , S i m o n F r a s e r Univers i ty , B u r n a b y , Br i t i sh C o l u m b i a , C a n a d a V 5 A 1S6

Janice Moore (107) , D e p a r t m e n t of Biology, C o l o r a d o S t a t e Univers i ty , Fo r t Col l ins , C o l o r a d o 80523

Norman A. Ratcliffe (267) , B i o m e d i c a l a n d Phys io log ica l R e s e a r c h G r o u p , School of Biological Sc iences , U n i v e r s i t y Col lege of S w a n s e a , S ing le ton Pa rk , S w a n s e a , SA2 8 P P Wales , U n i t e d K i n g d o m

R. Sequeira (1), School of Biological Sc iences , U n i v e r s i t y of E a s t A n g l i a , N o r w i c h N R 4 7 T G , E n g l a n d

David W. Severson (245), D e p a r t m e n t of A n i m a l H e a l t h a n d B i o m e d i c a l Sc iences , U n i v e r s i t y of W i s c o n s i n , M a d i s o n , W i s c o n s i n 53706

Donald B. Stoltz (167) , D e p a r t m e n t of Microb io logy , D a l h o u s i e Univers i ty , Ha l i fax , N o v a Scot ia , B 3 H 4 H 7 C a n a d a

Manickam Sugumaran (317) , D e p a r t m e n t of Biology, U n i v e r s i t y of M a s s a chuse t t s a t Bos ton , Bos ton , M a s s a c h u s e t t s 02125

S. N. Thompson (125) , D e p a r t m e n t of En tomology , U n i v e r s i t y of Ca l i fo rn ia , R ive r s ide , Ca l i fo rn ia 92521

S. Bradleigh Vinson (145) , D e p a r t m e n t of En tomology , T e x a s A & M U n i v e r sity, Col lege S t a t i on , Texas 77843

Preface

T h e focus of this t w o - v o l u m e set is t h e interface b e t w e e n insec ts a n d the i r a s soc i a t ed p a r a s i t e s a n d p a t h o g e n s , w i t h p a r t i c u l a r e m p h a s i s p l aced o n t h e bas i c biology, b iochemis t ry , a n d m o l e c u l a r b io logy of t hese i n t i m a t e a n d i n t r i g u i n g r e l a t i o n s h i p s . T h e in i t ia l i n s p i r a t i o n for th is book w a s t h e r ecogn i t ion t h a t a l t h o u g h t h e p o t e n t i a l u se as b io logica l con t ro l a g e n t s of m a n y p a r a s i t e s a n d p a t h o g e n s of insec ts h a s b e e n in tens ive ly inves t iga t ed , few r ecen t works have a t t e m p t e d to a d d r e s s t h e b i o c h e m i c a l a n d m o l e c u l a r in te r ac t ions o c c u r r i n g b e t w e e n insec t hos t s a n d the i r i n v a d e r s . Yet t hese i n t e r ac t ions c lear ly a r e cr i t ica l to t h e u l t i m a t e o u t c o m e of t he con f ron ta t i on b e t w e e n a n y i n v a d e r a n d its hos t . T h e p a r a s i t e o r p a t h o g e n m u s t e v a d e o r s u p p r e s s t h e hos t i m m u n e r e s p o n s e a n d , wh i l e u l t i m a t e l y c o m p l e t i n g i ts d e v e l o p m e n t in t h e hos t , m u s t n o t s t ress it p r e m a t u r e l y s u c h t h a t t h e su rv iva l of b o t h p a r t n e r s is c o m p r o m i s e d . I n a d d i t i o n , t he hos t e n v i r o n m e n t m u s t satisfy t h e p a r a s i t e s ' n u t r i t i o n a l a n d m e t a b o l i c n e e d s .

O u r i n t e n t is to s u m m a r i z e d e v e l o p m e n t s a n d t echno log ica l a p p r o a c h e s c u r r e n t l y b e i n g exp lo i t ed to m o n i t o r t h e b i o c h e m i c a l , i m m u n o l o g i c a l , m e t a bol ic , a n d b e h a v i o r a l a l t e r a t i o n s in insec ts infected w i t h p a r a s i t e s a n d p a t h o gens , w i t h p a r t i c u l a r e m p h a s i s on i n t e r ac t i ons o c c u r r i n g a t t h e m o l e c u l a r level. Add i t iona l ly , o u r a u t h o r s p r e s e n t severa l novel ideas for exp lo i t a t i on of this i n f o r m a t i o n in t h e m a n i p u l a t i o n of insec t pes t s .

P r e s s u r e to m i n i m i z e s y n t h e t i c c h e m i c a l insec t ic ides in insec t c o n t r o l p r o g r a m s h a s led to i n c r e a s e d in t e re s t in t h e use a n d s t u d y of p a r a s i t e s a n d p a t h o g e n s c a p a b l e of l imi t ing insec t p o p u l a t i o n s . T h i s in te res t , in c o m b i n a t ion w i t h r a p i d a d v a n c e s in t h e t e c h n i q u e s n o w ava i l ab le to s t u d y b io logica l s y s t e m s , h a s r e su l t ed in a n e n o r m o u s i nc rea se in k n o w l e d g e a b o u t t h e b io c h e m i s t r y a n d phys io logy of t h e p a r a s i t e s a n d p a t h o g e n s t h a t a t t a c k insec t s , as well as the i r i n t e r ac t i ons w i t h t h e hos t . T h i s k n o w l e d g e is so d iverse a n d ex tens ive t h a t m o s t j o u r n a l rev iews d e a l on ly w i t h h igh ly spec ia l i zed a s p e c t s of t h e overa l l field. W h i l e s u c h rev iews a r e of g r e a t v a l u e , o u r i n t e n t for t he se two v o l u m e s w a s to a s s e m b l e a m o r e ex tens ive su rvey of th is r a p i d l y d e v e l o p ing field b y p u b l i s h i n g rev iews on se lec ted topics d e a l i n g w i t h i n t e r ac t i ons w i t h p a r a s i t e s ( V o l u m e 1) a n d p a t h o g e n s ( V o l u m e 2) of insec t s . I t w o u l d be

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xvi Preface

imposs ib l e , even in two v o l u m e s , to rev iew all of t h e r ecen t findings cons id e red m a j o r a d v a n c e s . T h e r e f o r e , in se lec t ing o u r top ics , we chose to focus o n sub jec t a r e a s of l o n g - s t a n d i n g in te res t (e.g. , insec t a n t i b a c t e r i a l p r o t e i n s a n d p a r a s i t e - h o s t d e v e l o p m e n t a l in t e rac t ions ) a n d those d e a l i n g w i t h r e c e n t b r e a k t h r o u g h s t h a t a p p e a r s ignif icant a n d likely to b e of v a l u e in con t ro l l i ng insec ts (e.g. , d e v e l o p m e n t of r e c o m b i n a n t bacu lov i ruses a n d f o r m u l a t i o n of " s u p e r " p a t h o g e n s ) .

T h e i n t e n d e d a u d i e n c e for these v o l u m e s i nc ludes upper - l eve l u n d e r g r a d u a t e s w i t h spec ia l ty in te res t s in pa r a s i t o logy a n d en tomology , g r a d u a t e s t u d e n t s , a n d p o s t - g r a d u a t e r e s ea r che r s w h o m a y use th is i n f o r m a t i o n to dev ise n e w technolog ies for m a n i p u l a t i o n of insec ts of i m p o r t a n c e to ag r i cu l t u r e a n d h u m a n h e a l t h . We h o p e these v o l u m e s will find a n i c h e o n bookshelves in m a n y p e r s o n a l a n d profess ional l ib ra r ies focusing o n paras i to logy , en tomology , i m m u n o l o g y , ep idemiology , phys io log ica l ecology, e v o l u t i o n a r y biology, a n d o t h e r a r e a s d e a l i n g w i th va r ious a spec t s of h o s t - p a r a s i t e re la t i onsh ips .

I n sec t p a r a s i t o l o g y a n d p a t h o l o g y have c lear ly b e c o m e m u l t i d i s c i p l i n a r y fields. For e x a m p l e , t he d e v e l o p m e n t of n e w technolog ies for g e n e t rans fe r a n d a r t h r o p o d t r a n s f o r m a t i o n m a y benefi t f rom t h e exp lo i t a t i on of n a t u r a l l y o c c u r r i n g t r a n s p o s a b l e e l e m e n t s in insec t v i rus g e n o m e s . Useful t a r g e t genes for m a n i p u l a t i o n m i g h t i n c l u d e those t h a t a r e cr i t ica l to t h e n o r m a l func t ioning of t he i m m u n e sys t em as well as those pa r a s i t e - a s s o c i a t ed factors invoking d e v e l o p m e n t a l d i s r u p t i o n o r s ter i l i ty of insec t pes t s ( i .e. , e n d o c r i n e r e g u la to r s ) . G e n e s a s soc ia t ed w i t h ref ractor iness in vec to r a r t h r o p o d s a r e a lso i m p o r t a n t a n d a r e u n d e r s c ru t iny as poss ib le m e a n s for g e n e r a t i n g eng i n e e r e d vec tors w i t h r e d u c e d capac i ty to t r a n s m i t p a r a s i t e s .

I n c o n t r a s t to t h e re la t ively l imi ted i n fo rma t ion ava i l ab le o n m o l e c u l a r h o s t - p a r a s i t e a n d h o s t - p a t h o g e n in t e r ac t ions in insec t hos t s , a w e a l t h of r ecen t ly p u b l i s h e d m a t e r i a l desc r ibes t he r e l a t i onsh ips b e t w e e n p a r a s i t e s a n d p a t h o g e n s of m a m m a l s w i t h the i r r espec t ive hos t s . I n p a r t , th i s difference reflects i n t ense r e s e a r c h efforts d i r ec t ed t o w a r d d e v e l o p i n g n e w t h e r a p e u t i c t r e a t m e n t s for d i sease b a s e d on these i n t e r ac t i ons in t h e h o p e of ident i fy ing cr i t ica l po in t s of vu lne rab i l i t y t h a t m a y b e m a n i p u l a t e d b y d r u g s o r o t h e r a g e n t s . A s imi l a r ex tens ive l i t e r a tu r e d e s c r i b i n g t h e m o l e c u l a r in te r ac t ions of p l a n t s w i t h p a t h o g e n s a n d p a r a s i t e s a l so n o w exis ts . O u r knowl edge of t h e m o l e c u l a r m e c h a n i s m s of i n t e r ac t ions b e t w e e n p l a n t s a n d p l a n t p a t h o g e n s is t h u s m u c h m o r e soph i s t i ca t ed t h a n o u r u n d e r s t a n d i n g of t h e m o l e c u l a r m e c h a n i s m s o p e r a t i n g in insec ts as they confront i n v a d e r s . W h i l e we have yet to identify v i ru l ence genes o r av i ru l en t m u t a n t s of t hose genes in species t h a t a t t a c k insec ts , for e x a m p l e , s u c h genes a l r e a d y h a v e b e e n isol a t ed in species t h a t a r e p a t h o g e n i c to p l a n t s a n d m a m m a l s . M o r e o v e r ,

Preface xvii

t h o u g h m e c h a n i s m s of v i rus r e s i s t ance have b e e n p i n p o i n t e d in p l a n t s a n d m a m m a l s , o u r i n f o r m a t i o n a b o u t insect an t i v i r a l defenses r e m a i n s r u d i m e n tary. I n d u c t i o n of r e s i s t ance h a s b e e n d o c u m e n t e d , b u t its m e c h a n i s m s re m a i n to be ident i f ied.

N e v e r t h e l e s s , we d o have s ignif icant i n fo rma t ion a b o u t t h e c o m p l e x i t y of s o m e p rocesses , s u c h as t h e a n t i b a c t e r i a l defenses of insec t s , a n d h o w they pa ra l l e l s imi l a r p rocesses in m a m m a l i a n hos t s . H o r m o n a l h o s t - p a r a s i t e re la t i onsh ips have b e e n in tens ive ly s c ru t in i zed in severa l i n v e r t e b r a t e species , i n c l u d i n g m a n y insec ts , w h i c h a p p e a r to be p a r t i c u l a r l y a p p r o p r i a t e m o d e l s for s t u d y i n g e n d o c r i n e i n t e r ac t i ons . M o r e o v e r , t he a s soc ia t ion of t h i r d - p a r t y e l e m e n t s w i t h p a r a s i t e s is p e r h a p s bes t i l l u s t r a t ed by species t h a t a t t a c k insec ts ; for i n s t a n c e , b o t h w a s p a n d n e m a t o d e p a r a s i t e s s h o w i n t i m a t e re lat i onsh ips w i t h v i ruses a n d bac t e r i a , respect ively, d u r i n g p a r a s i t i s m of insec t hos t s .

I n t h e s h o r t t e r m , these v o l u m e s a r e d i r ec t ed a t filling a void in t h e l i t e r a t u r e by e m p h a s i z i n g bas i c i n t e r ac t i ons a t t he b i o c h e m i c a l a n d m o l e c u l a r levels. I n t h e long t e r m , we expec t t h a t m a n y of these i n t e r ac t i ons will p r o v i d e a v e n u e s for exp lo i t a t ion to e i the r e n h a n c e t he r a t e s of "bene f i c i a l " p a r a s i t i s m (in b io logica l con t ro l , for e x a m p l e ) o r r e d u c e t h e r a t e of d i sease t r a n s m i s s i o n a n d the r a t e of infect ion of v e r t e b r a t e hos t s . O u r h o p e is t h a t t h e i n f o r m a t i o n a s s e m b l e d h e r e will have s ignif icant i m p a c t o n a g r i c u l t u r e a n d h u m a n h e a l t h a n d t h a t these v o l u m e s will s t i m u l a t e fresh a p p r o a c h e s to t h e inves t iga t ion of these fasc ina t ing a n d in t r i ca t e i n t e r ac t i ons .

We expres s t h a n k s to t h e a u t h o r s for t he i r t ime ly c o n t r i b u t i o n s . I n a d d i t ion , t h e scope a n d d e p t h of th is w o r k d e m a n d e d t h a t we enl is t t h e a s s i s t ance of m a n y sc ient i s t s to rev iew the c h a p t e r s . Fo r the i r t i m e a n d o u t s t a n d i n g efforts in th is endeavor , we s incere ly t h a n k the following i n d i v i d u a l s : D r s . T h e o d o r e A n d r e a d i s , C h r i s t o p h e r B a y n e , G a r y Bl i s sa rd , D r i o n B o u c i a s , J o h n B r o w n , Pe te r B r y a n t , J o h n B u r a n d , T h o m a s C o u d r o n , S a m u e l D a l e s , R o b i n D e n e l l , D o u g l a s D a h l m a n , G o r d o n G o r d h , R o b e r t G r a n a d o s , Pa t r i ck G r e a n y , L e a h H a i m o , Alfred H a n d l e r , K i y o s h i H i r u m a , H i l a r y H u r d , D a v y J o n e s , M i c h a e l K a n o s t , J a m e s K e r w i n , K a r l K r a m e r , Lesl ie Lewis , M i c h a e l Locke , R o b e r t L u c k , Lois Mi l le r , Lou i s Mi l le r , E d P la tze r , J o h n Pos t l e th -wa i t , L y n n Ridd i fo rd , J u s t i n S c h m i d t , D o n Stol tz , M i c h a e l S t r a n d , J u s t V l a k , B r u c e W e b b , J o h n Webs te r , A l a n W o o d , T i m o t h y Yosh ino , Rolf Z ieg-ler, a n d M a r l e n e Z u k .

I n a d d i t i o n , we a c k n o w l e d g e t h e excel lent sec re ta r i a l staff of t h e E n t o m o l o g y D e p a r t m e n t of t h e U n i v e r s i t y of Ca l i fo rn ia , R ive r s ide . We exp re s s spec ia l t h a n k s to M s , D i a n a H a n s o n a n d M s . P a m H o a t s o n for the i r efficient efforts in p roces s ing m a n u s c r i p t s a n d c o r r e s p o n d i n g w i t h o u r a u t h o r s .

xviii Preface

Last ly, b u t m o s t i m p o r t a n t l y , t he ed i to r s express d e e p a p p r e c i a t i o n to Dr . Phyl l is B . M o s e s of A c a d e m i c Press for h e r u n w a v e r i n g e n t h u s i a s m for this pro jec t . H e r scientific i n p u t a n d ed i to r ia l adv ice w e r e i n v a l u a b l e to us first in deve lop ing the idea of a s s e m b l i n g these v o l u m e s a n d s u b s e q u e n t l y in oversee ing the pro jec t to frui t ion. We a lso acknowledge A c a d e m i c Press for i ts s p o n s o r s h i p .

Ν. E. Beckage S. N. Thompson

B. A. Federici

Chapter 1

Patterns of Development in Insect Parasites M. Mackauer Centre for Pest Management Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada

R. Sequeira1

Centre for Pest Management Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada

I. Introduction

II. The Idiobiont-Koinobiont Dichotomy

III. Patterns of Parasite Development and Growth A. Idiobionts B. Koinobionts

IV. Parasite Development and Host Ecology A. Seasonal Adaptations B. Influence of Host Nutrition

V. Developmental Strategies

VI. Conclusions Acknowledgment References

I. Introduction

Successful p a r a s i t i s m by insec t p a r a s i t o i d s 2 r equ i r e s t he so lu t ion of t h r e e k i n d s of g e n e r a l p r o b l e m s : (1) w h i c h hos t to select; (2) h o w to i n t e g r a t e d e v e l o p m e n t a n d g r o w t h w i t h those of t he hos t ; a n d (3) w h a t l i fe-history tac t ics to a d o p t . T h e first p r o b l e m c o n c e r n s b e h a v i o r a l a d a p t a t i o n s r e l a t i n g to hos t se lec t ion a n d p r o g e n y a l loca t ion b y the a d u l t female ( C h a r n o v a n d Sk inner , 1985; W a a g e a n d Godfray, 1985; v a n A l p h e n a n d Vet , 1986; W a a g e , 1986) a n d will no t b e c o n s i d e r e d h e r e for th is r e a s o n . I n s t e a d we will focus m a i n l y o n t h e t h i r d a n d , to a lesser ex ten t , t he second p r o b l e m , b o t h of w h i c h c o n c e r n p a r a s i t e g r o w t h a n d d e v e l o p m e n t .

M o s t ea r ly s tud ie s have e m p h a s i z e d m e c h a n i s m s of p a r a s i t e g r o w t h a n d d e v e l o p m e n t , w i t h less a t t e n t i o n p a i d to t h e a d a p t i v e s ignif icance of t hese m e c h -

^urrent address: School of Biological Sciences, University of East Anglia, Norwich NR4 7TG, England.

2Henceforth we use the term parasite to mean parasitoid. These protelean parasites include a large group of mostly hymenopteran and dipteran species that, as immatures, are obligate parasites of insects and other arthropods and are free-living as adults. Hymenopterous parasites often are called parasitic wasps as opposed to the nonparasitic, aculeate wasps and bees.

Parasites and Pathogens of Insects Volume 1: Parasites 1

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

2 Μ. Mackauer and R. Sequeira

a n i s m s . I n a c a u s a l a p p r o a c h , p a r a s i t e d e v e l o p m e n t a n d g r o w t h m a y b e c h a r a c t e r i z e d in t e r m s e i the r of t he effects of p a r a s i t i s m o n t h e hos t (e.g. , D o u t t , 1963; Sal t , 1964; Smi lowi tz a n d I w a n t s c h , 1973; C l o u t i e r a n d M a c k aue r , 1979, 1980; L a w r e n c e , 1982; T h o m p s o n , 1982, 1983; Beckage a n d T e m p l e t o n , 1985, 1986; S t r a n d , 1986; G u n a s e n a et al., 1989; V i n s o n , 1990; S t r a n d a n d Dover , 1991) or of t he pa ra s i t e ' s r e s p o n s e to v a r i a t i o n s in t h e n u t r i t iona l s t a t e a n d phys io logy of t he hos t (e.g. , C o r b e t , 1968; Wese loh , 1984; H e b e r t a n d C lou t i e r , 1990; L a w r e n c e , 1990; K o u a m e a n d M a c k a u e r , 1991 ; S t r a n d etal., 1991; S e q u e i r a a n d M a c k a u e r , 1992a ,b) . I n th is r e g a r d , L a w r e n c e (1986, 1990) cons ide r ed hos t r egu l a t i on ( V i n s o n , 1975; V i n s o n a n d I w a n t s c h , 1980a) a n d flexibility of p a r a s i t e d e v e l o p m e n t ( C o r b e t , 1968; Wese loh , 1984) as a l t e r n a t e d e v e l o p m e n t a l s t ra teg ies . T h i s v i e w p o i n t s t resses p r o x i m a t e m e c h a n i s m s of p a r a s i t e surv iva l b a s e d on qua l i t a t i ve d e s c r i p t i o n s of t h e phys io log ica l a n d b i o c h e m i c a l i n t e r ac t ions b e t w e e n the p a r a s i t e s a n d the i r hos t s .

Howeve r , func t iona l c o n s t r a i n t s o n t he pa r a s i t e ' s g r o w t h a n d d e v e l o p m e n t on o r in different hos t s c a n a lso b e cons ide red as va r i ab l e s w i t h i n a b r o a d e r evo lu t i ona ry f r amework . T h i s a p p r o a c h e m p h a s i z e s q u e s t i o n s a b o u t t he fitness va lue of d e v e l o p m e n t a l cha rac te r i s t i c s a n d p a t t e r n s of hos t u t i l i za t ion . B e c a u s e p r o t e l e a n p a r a s i t e s d e p e n d exclusively o n hos t -de r ived n u t r i e n t s for the i r l a rva l d e v e l o p m e n t a n d g r o w t h , n a t u r a l se lect ion w o u l d b e e x p e c t e d to favor m e c h a n i s m s t h a t m a x i m i z e t h e efficient u t i l i za t ion of t hese r e sou rce s . O p t i m a l r e sou rce a l loca t ion to a d u l t b o d y size, cons ide r ed t h e p e r h a p s m o s t i m p o r t a n t c o m p o n e n t of D a r w i n i a n fitness in p a r a s i t i c w a s p s ( K i n g , 1987), d e p e n d s on the insec t ' s g r o w t h r a t e a n d d e v e l o p m e n t t i m e . T h e a l loca t ion of l imi ted (hos t ) r e sources to c o m p e t i n g fitness funct ions m a y resu l t in t r a d e offs t h a t d e t e r m i n e a n " o p t i m a l c h a r a c t e r s e t " a n d , in d o i n g so , m a y s h a p e the evo lu t ion of a species ' l ife-history s t r a t egy (Sibly a n d Calow, 1986).

We use t he t e r m strategy in a c c o r d a n c e w i t h D o m i n e y (1984) to refer to a set of g e n e r a l ru les t h a t specify w h i c h a l t e r n a t i v e p a t t e r n of r e sponses will b e a d o p t e d in a p a r t i c u l a r s i tua t ion ; these ru les a r e typ ica l for each species a n d d e t e r m i n e its a d a p t e d n e s s to t he e n v i r o n m e n t , h e r e t h e hos t . Tac t i cs , b y c o n t r a s t , refer to severa l a l t e rna t i ve o p t i o n s o r m e c h a n i s m s b y w h i c h these e v o l u t i o n a r y object ives a r e ach ieved ; these o p t i o n sets m a y v a r y a m o n g different i n d i v i d u a l s o r p h e n o t y p e s . Also , we d i s t i ngu i sh b e t w e e n h o s t su i t ab i l i ty (Sal t , 1938; V i n s o n a n d I w a n t s c h , 1980b) a n d h o s t qua l i ty , two t e r m s often u sed synonymous ly . A hos t species is su i t ab l e if it n o r m a l l y s u p p o r t s t h e successful d e v e l o p m e n t of p a r a s i t e offspring; consequen t ly , su i t ab i l i ty is a cha rac t e r i s t i c of t he hos t species a n d is gene t ica l ly d e t e r m i n e d , o r l a rge ly so . I n c o m p a r i s o n , we use t he t e r m q u a l i t y to de sc r ibe v a r i a t i o n s in t h e s t a t e o r c o n d i t i o n of t h e hos t t h a t affect p rocess d y n a m i c s , s u c h as t h e r a t e s of p a r a si te g r o w t h a n d d e v e l o p m e n t . S u c h s t a t e va r i ab l e s i n c l u d e , o r m a y b e co r r e l a t ed w i t h , hos t age , s t age of d e v e l o p m e n t , size, sex, a n d n u t r i t i o n a l s t a t u s .

1. Patterns of Development in Insect Parasites 3

T h e c h a p t e r is o r g a n i z e d as follows. F i r s t , we i n t r o d u c e t h e i d i o b i o n t -k o i n o b i o n t d i c h o t o m y , w h i c h we use as a ( m a c r o e v o l u t i o n a r y ) o r g a n i z i n g s c h e m e . S e c o n d , we d e s c r i b e severa l d e v e l o p m e n t a l p a t t e r n s t h a t c h a r a c t e r ize b r o a d differences b e t w e e n i d i o b i o n t a n d k o i n o b i o n t p a r a s i t e s . N e x t , we d i scuss s ea sona l a d a p t a t i o n s a n d t h e inf luence of hos t n u t r i t i o n o n p a r a s i t e d e v e l o p m e n t , i n c l u d i n g s u p e r p a r a s i t i s m a n d s t a r v a t i o n . Final ly , we p r o p o s e t h r e e m o d e l s of p a r a s i t e d e v e l o p m e n t in r e s p o n s e to hos t c o n s t r a i n t s . We sugges t t h a t t h e essen t ia l c o m p o n e n t s of a n y d e v e l o p m e n t a l s t r a t e g y a r e t h e p a r a s i t e ' s g r o w t h r a t e , d e v e l o p m e n t t ime , a n d a d u l t b i o m a s s , w h i c h a r e c o n s t r a i n e d by h o s t q u a l i t y in a n associa t ion-speci f ic m a n n e r .

II. The Idiobiont—Koinobiont Dichotomy

H a e s e l b a r t h (1979) first d r e w a t t e n t i o n to a n i m p o r t a n t m a c r o e v o l u t i o n a r y d iv i s ion b e t w e e n p a r a s i t e s d e v e l o p i n g in hos t s t h a t c o n t i n u e to g r o w a n d m e t a m o r p h o s e d u r i n g t h e in i t ia l s t ages of p a r a s i t i s m (cal led k o i n o p h y t e s ) a n d those t h a t deve lop in n o n g r o w i n g a n d p a r a l y z e d hos t s (ca l led id-i ophy t e s ) . A s k e w a n d S h a w (1986) i n t r o d u c e d t h e t e r m s k o i n o b i o n t a n d i d i o b i o n t to d e s c r i b e these a l t e r n a t i v e hos t - exp lo i t a t i on s t r a t eg ie s .

B e c a u s e t h e h o s t of a n i d i o b i o n t does n o t feed, grow, o r m e t a m o r p h o s e d u r i n g t h e c o u r s e of t h e i n t e r ac t i on , it c o n t a i n s a fixed a m o u n t of r e sou rces for t h e p a r a s i t e l a rva , w i t h l a rge hos t s b e i n g a s s u m e d of h i g h e r q u a l i t y t h a n s m a l l h o s t s . H o w e v e r , un less t h e hos t is ki l led by t h e female a t ov ipos i t ion , age - r e l a t ed v a r i a t i o n s in q u a l i t y m a y resu l t f rom d e v e l o p m e n t a l c h a n g e s w i t h i n a p a r t i c u l a r hos t s t age , s u c h as eggs a n d p u p a e ( S t r a n d , 1986; K i n g , 1990a) . By c o n t r a s t , hos t s p a r a s i t i z e d b y a k o i n o b i o n t c o n t i n u e to feed, grow, a n d d e v e l o p d u r i n g m u c h of t h e i n t e r ac t i on . C o n s e q u e n t l y , hos t q u a l i t y a s a r e s o u r c e for t h e p a r a s i t e l a r v a is in f luenced by fu ture h o s t g r o w t h , w h i c h d e p e n d s o n t h e hos t ' s a g e a n d s t a g e of d e v e l o p m e n t , r a t h e r t h a n o n its s ize, a t t h e t i m e of p a r a s i t i z a t i o n ( M a c k a u e r , 1986; K i n g , 1989; K o u a m e a n d M a c k -aue r , 1991; S e q u e i r a a n d M a c k a u e r , 1992a) .

B l a c k b u r n (1991) n o t e d t h a t k o i n o b i o n t s h a v e longe r p u p a l p e r i o d s a n d p r e a d u l t life s p a n s t h a n id iob ion t s , sugges t i ng t h a t these t w o g r o u p s h a v e p r o b a b l y evolved u n d e r different c o n s t r a i n t s w i t h r e g a r d to r e s o u r c e u s a g e . A s k e w a n d S h a w (1986) c o m p a r e d i d i o b i o n t a n d k o i n o b i o n t s t r a t eg ies as a co r r e l a t e of h o s t r a n g e . T h e y p r o p o s e d t h a t k o i n o b i o n t s a r e m o r e likely to s h o w a n a r r o w spec ia l i za t ion b e c a u s e of t he i r g r e a t e r d e p e n d e n c e o n hos t phys io logy a n d d e v e l o p m e n t . G a u l d a n d Bo l ton (1988) n o t e d t h a t i d iob ion t s typ ica l ly a r e synovigen ic (i .e. , females m a t u r e eggs c o n t i n u o u s l y t h r o u g h o u t life), p r o d u c e re la t ive ly l a rge , l ec i tha l (or a n h y d r o p i c ) eggs c o n t a i n i n g suffic ien t r e sou rces for ea r ly e m b r y o n i c d e v e l o p m e n t , a n d d e v e l o p as ec to -


p a r a s i t e s on concea l ed hos t s . K o i n o b i o n t s , b y c o n t r a s t , a r e typ ica l ly e n d o -p a r a s i t e s , p r o d u c e sma l l n u t r i e n t - p o o r , a l ec i tha l (or h y d r o p i c ) eggs , a n d a t t a ck f ree-moving hos t s .

I d i o b i o n t a n d k o i n o b i o n t s t ra teg ies show p o o r co r r e l a t i on w i t h so l i t a ry a n d g r e g a r i o u s d e v e l o p m e n t (Askew a n d Shaw, 1986). T h e o r e t i c a l r esu l t s (Godfray, 1987), a n d s o m e e x p e r i m e n t a l ev idence ( C r u z , 1981), sugges t t h a t so l i ta ry a n d g r e g a r i o u s d e v e l o p m e n t r e p r e s e n t d i s t inc t r e p r o d u c t i v e a n d / o r d e v e l o p m e n t a l s t ra teg ies (Waage , 1986). However , m a n y g r o u p s of p a r a s i t e s , i n c l u d i n g severa l g e n e r a such as Cotesia, c o n t a i n b o t h so l i t a ry a n d g r e g a r i o u s species . I n m o s t cases it is n o t c lear w h e t h e r s u c h d e v e l o p m e n t a l v a r i a t i o n s a r e d u e to hos t - r e l a t ed cond i t i ons (le M a s u r i e r , 1987, 1991) o r reflect phy -logene t ic c o n s t r a i n t s ( G a u l d , 1988; G a u l d a n d Bo l ton , 1988), o r b o t h .

III. Patterns of Parasite Development and Growth

A species ' l ife-history s t r a t egy r e p r e s e n t s a u n i q u e c o m b i n a t i o n of r e sponses ( tact ics) t h a t a r e s h a p e d by n a t u r a l se lect ion from a n o p t i o n se t a s soc i a t ed w i t h each p h e n o t y p i c cha rac t e r . O p t i o n sets m a y b e empi r i ca l l y def ined as t he q u a n t i t a t i v e r e sponses of the i r c o r r e s p o n d i n g l ife-history c h a r a c t e r s . W i t h r e g a r d to h o s t - p a r a s i t e i n t e r ac t ions , it is c o n v e n i e n t to d i s t i n g u i s h b e t w e e n two r e s p o n s e ca tegor ies : t h e inf luence of t he hos t ' s p h e n o t y p e o n t h e p a r a s i t e ' s p h e n o t y p e , a n d the fitness c o n s e q u e n c e s of v a r i a t i o n s in t h e p a r a si te 's p h e n o t y p i c a t t r i b u t e s . T h e first c a t ego ry inc ludes t h e inf luence of t h e species of hos t , of its s t age of d e v e l o p m e n t , a n d of i ts n u t r i t i o n a l s t a t u s o n t h e p a r a s i t e ' s g r o w t h r a t e , d e v e l o p m e n t , a n d size a t m a t u r i t y .

I n this a n d the following sec t ion , we use se lec ted host—paras i te assoc ia t ions to identify va r i ab l e s t h a t a r e likely to r e p r e s e n t m a j o r c o m p o n e n t s of m i c r o e v o l u t i o n a r y s t r a t eg ies .

A. Idiobionts

1. E g g Paras i t e s (Trichogramma)

T h e genera l i s t egg p a r a s i t e s of t h e g e n u s Trichogramma a r e a m o n g t h e m o s t t h o r o u g h l y s t u d i e d id iob ion t s . T h e s e species a r e easi ly r e a r e d in t h e l a b o r a to ry o n the i r h a b i t u a l as well as on va r ious fact i t ious hos t s (Bigler et al.} 1987; S c h m i d t a n d S m i t h , 1987). I n his s e m i n a l s tud ie s , Sa l t (1940) d e m o n s t r a t e d a close co r re l a t ion b e t w e e n the size of hos t eggs a n d t h e a d u l t size of t h e p a r a s i t e . Eggs t h a t were too sma l l to satisfy all t he n u t r i t i o n a l r e q u i r e m e n t s of t he deve lop ing p a r a s i t e l a rva p r o d u c e d " r u n t s " w i t h s t r u c t u r a l p e c u -


l iar i t ies cha rac t e r i s t i c of s t a r v a t i o n . Also , d e v e l o p m e n t t i m e va r i ed w i t h t h e egg 's size a n d t h e th ickness of i ts cho r ion .

A l t h o u g h species of Trichogramma exh ib i t d i s t inc t p a t t e r n s of hos t - s ize a n d species u s a g e ( H i n t z a n d Andow, 1990), a c c u r a t e a s s e s s m e n t of a d a p t i v e life-h i s to ry v a r i a t i o n a n d d e v e l o p m e n t a l s t r a t eg ies is m a d e difficult b y facul ta t ive g r e g a r i o u s d e v e l o p m e n t . Pak (1986) r ecogn ized six d i s t inc t p a t t e r n s in t h e r e l a t i o n s h i p b e t w e e n hos t age a t p a r a s i t i z a t i o n a n d d e v e l o p m e n t a l success in Trichogramma. F ive of t hese p a t t e r n s w e r e n o n l i n e a r , a fact i n d i c a t i n g t h a t h o s t q u a l i t y for p a r a s i t e d e v e l o p m e n t is a n o n l i n e a r func t ion of hos t age a n d , m o r e i m p o r t a n t p e r h a p s , t h a t these q u a n t i t a t i v e r e l a t i o n s h i p s a r e associa t ion-speci f ic . W h i l e p a r a s i t e size a n d fecundi ty i n c r e a s e d w i t h h o s t size (Rezn ik a n d U m a r o v a , 1990), d e v e l o p m e n t t i m e va r i ed n o n l i n e a r l y w i t h hos t s ize, poss ib ly ref lect ing differences in re la t ive hos t q u a l i t y r a t h e r t h a n in a b s o l u t e hos t size o r age . B a r r e t t a n d S c h m i d t (1991) sugges t ed t h a t , in s m a l l hos t eggs , t h e p a r a s i t o i d ' s size is r e s t r i c t ed b y the n u t r i e n t s a n d space avai l a b l e . H o w e v e r , in hos t s above a t h r e s h o l d v o l u m e , t h e size of t h e r e su l t i ng w a s p s d e p e n d s o n t h e n u m b e r of eggs a l loca t ed b y t h e female a t ov ipos i t ion .

E g g s m a y be cons ide r ed as t r a n s i t o r y hos t s t ages in t h a t r a p i d e m b r y -ogenes is c a n qu ick ly d e p l e t e t h e a m o u n t a n d the ava i lab i l i ty of s t o r ed re sources ( A n d e r s o n , 1972). T h u s , egg p a r a s i t e s a r e u n d e r se lec t ion to m i n i m i z e d e v e l o p m e n t t i m e a n d to m a x i m i z e g r o w t h r a t e ( S t r a n d , 1986). S t r a n d (1986) sugges t ed t h a t , in Trichogramma spec ies , p o l y p h a g y m a y b e a conse q u e n c e of t h e phys io log ica l un i fo rmi ty of insec t eggs as a food r e s o u r c e , especia l ly b e c a u s e eggs lack s ignif icant ce l lu la r defenses a n d a r e often u n p r o t e c t e d . I f th is h y p o t h e s i s is in fact cor rec t , we w o u l d expec t l i t t le v a r i a t i o n in t h e g r o w t h r a t e s of gene ra l i s t (egg) p a r a s i t e s . H o w e v e r , m o r e d e t a i l e d s tud i e s of t h e g r o w t h , d e v e l o p m e n t , a n d b i o n o m i c s of egg p a r a s i t e s m a y well revea l s u b s t a n t i a l hos t - r e l a t ed v a r i a t i o n in p a r a s i t e p e r f o r m a n c e ( M a r s t o n a n d E r t l e , 1973; Bigler et al, 1987; Rezn ik a n d U m a r o v a , 1990; B a r r e t t a n d S c h m i d t , 1991).

2 . P u p a l Paras i t e s (Pimpla, Coccygomimus)

T h e g r o w t h a n d d e v e l o p m e n t of p u p a l e n d o p a r a s i t e s a n d of t hose t h a t kill t he h o s t a t o r sho r t ly after ov ipos i t ion is less well s t u d i e d . A r t h u r a n d Wyl ie (1959) s h o w e d t h a t , in t h e i c h n e u m o n i d Pimpla turionellae, b o d y size i n c r e a s e d w i t h t h e p u p a l size of different hos t spec ies , whi le d e v e l o p m e n t t i m e w a s gene ra l l y l onge r in l a rge t h a n in sma l l p u p a e . S a n d l a n (1982) found t h a t t h e a d u l t size of Coccygomimus turionellae d e c r e a s e d w i t h a n i nc r ea se in hos t a g e w i t h i n a h o s t species b u t w a s n o t co r r e l a t ed w i t h size differences b e t w e e n h o s t species ; however , p a r a s i t e d e v e l o p m e n t t i m e i n c r e a s e d w i t h p u p a l a g e .

H o s t size-, age- , a n d spec ies -usage p a t t e r n s in p u p a l p a r a s i t e s , i n d e x e d b y the p e r c e n t a g e of p a r a s i t i s m , m a y v a r y w i t h i n a n d b e t w e e n hos t species


( H a l l a n d Fischer , 1988; K i n g , 1990b) . F u r t h e r m o r e , L e g n e r (1969) r e p o r t e d t h a t p a r a s i t e d e v e l o p m e n t t i m e was co r r e l a t ed , in a n associa t ion-speci f ic m a n n e r , w i t h hos t size a t p a r a s i t i z a t i o n in p u p a l e n d o - b u t n o t e c t o p a r a s i t e s .

B. Koinobionts

M o s t of t he l i t e r a tu r e o n h o s t - p a r a s i t e d e v e l o p m e n t a l i n t e r ac t i ons is conc e r n e d w i th ko inob ion t s , especia l ly so l i ta ry e n d o p a r a s i t e s . I n these spec ies , t he d y n a m i c s of p a r a s i t i s m is d e t e r m i n e d by the m e c h a n i s m s t h a t con t ro l t h e g r o w t h a n d d e v e l o p m e n t of t he p a r a s i t e i m m a t u r e s in r e s p o n s e to hos t c o n d i t ions , w h i c h in t u r n m a y be a l t e red in r e sponse to p a r a s i t i s m . S u c h a " c o u n t e r b a l a n c e d s t a t e of o p p o s i n g c o n d i t i o n s " was t e r m e d enantiostasis by M a n -g u m a n d Towle (1977) .

1. A p h i d i i d W a s p s (Aphidius, Ephedras)

T h e p e a a p h i d , Acyrthosiphon pisum, s u p p o r t s a b r o a d r a n g e of so l i t a ry e n d o p a r a s i t e s be long ing to two h y m e n o p t e r a n famil ies , A p h i d i i d a e a n d A p h -e l in idae . A l t h o u g h m a n y of these p a r a s i t e s c a n successfully d e v e l o p in all t h e n y m p h a l s tages a n d in a d u l t a p h i d s , p a r a s i t i s m of different hos t s t ages a n d m o r p h s h a s v a r i a b l e c o n s e q u e n c e s for p a r a s i t e l a rva l on togeny .

S e q u e i r a a n d M a c k a u e r (1992a) c o m p a r e d the g r o w t h t ra jec tor ies of Aphidius ervi d eve lop ing in different n y m p h a l i n s t a r s of a p t e r o u s v i r g i n o p a r a e . P a r a s i t e g r o w t h r a t e s va r i ed n o n l i n e a r l y w i t h t he hos t ins ta r , w i t h t h e g r o w t h of ea r ly l a rvae b e i n g h ighes t in second in s t a r s a n d lowest in t h i r d i n s t a r s of t h e p e a a p h i d . U s i n g p a r a s i t e g r o w t h as a m e a s u r e of hos t qua l i ty , S e q u e i r a a n d M a c k a u e r (1992a) r a n k e d the four n y m p h a l i n s t a r s in t h e o r d e r L 2 > L 4

> L j > L 3 . T h i s r a n k i n g o r d e r w a s cons i s t en t w i t h differences in r e p r o d u c t ive a c h i e v e m e n t (as i n d e x e d by the in t r ins ic r a t e of i nc rease , r m ) b e t w e e n A. ervi females r e a r e d on these i n s t a r s ( S e q u e i r a a n d M a c k a u e r , 1992b) . C h a n g e s in p a r a s i t e fitness a t t r i b u t e s in r e spon s e to v a r i a t i o n in hos t re sources a r e ind ica t ive of t he pa ra s i t e ' s l ife-history s t ra tegy. F i r s t - a n d four th -i n s t a r p e a a p h i d s differ in b i o m a s s by o n e o r d e r of m a g n i t u d e o r m o r e . I n A. ervi, a d u l t d r y m a s s w a s co r re l a t ed w i t h m a x i m u m la rva l m a s s ach ieved j u s t p r i o r to p u p a t i o n (Seque i r a a n d M a c k a u e r , 1992a) . P a r a s i t e m a s s a n d deve l o p m e n t t i m e were co r r e l a t ed w i t h hos t size ( S e q u e i r a a n d M a c k a u e r , 1992b) . D r y m a s s a n d d e v e l o p m e n t t i m e were posi t ively co r r e l a t ed in L j - L ^ a p h i d s b u t n o t co r r e l a t ed in l a rge L 4 a p h i d s . T h e pe r iod f rom ovipos i t ion to p u p a t ion w a s i n d e p e n d e n t of hos t size a t p a r a s i t i z a t i o n . T h e s e resu l t s c lear ly show t h a t t h e p a r a s i t e r e s p o n d s to a d y n a m i c n u t r i t i o n a l a n d phys io log ica l h o s t e n v i r o n m e n t t h r o u g h va r i a t i ons in t he l a rva l g r o w t h t ra jec tor ies . T h u s , in A. ervi d eve lop ing in hos t s be low a ce r t a in size t h r e s h o l d ( L j - L ^ ) , t he o p t i m i z a t ion of b o d y size took p r e c e d e n c e over t h e m i n i m i z a t i o n of d e v e l o p m e n t t i m e . However , p a r a s i t e s deve lop ing in hos t s above a t h r e s h o l d size u t i l ized t h e


a d d i t i o n a l r e sou rces b o t h to i nc rea se b o d y m a s s a n d to d e c r e a s e d e v e l o p m e n t t i m e .

Similar ly , in Ephedras californicus, a d u l t b o d y size i n c r e a s e d w i t h p e a a p h i d size u p to a t h r e s h o l d v a l u e . O n c e t h e l a rva h a d ach ieved th is t h r e s h o l d in d r y m a s s , a d d i t i o n a l hos t r e sou rces w e r e a l loca ted t o w a r d t h e s i m u l t a n e o u s m i n i m i z a t i o n of d e v e l o p m e n t t i m e a n d m a x i m i z a t i o n of b o d y m a s s . I n cont r a s t to A. ervi, t h e t i m e - t o - p u p a t i o n w a s l onge r in L 2 t h a n in L j a n d L 4 hos t s , a difference i n d i c a t i n g t h a t E. californicus d i d n o t ad jus t t h e l a rva l g r o w t h t ra jec to ry to hos t c o n d i t i o n s ( S e q u e i r a a n d M a c k a u e r , 1993).

R e p r o d u c t i v e success in a p h i d p a r a s i t e s is f r equen t ly a s s u m e d to b e co r r e l a t ed w i t h a d u l t size (C lou t i e r et al., 1981; L iu , 1985; I r v i n e , 1991). A l t h o u g h b o d y size c a n b e a useful i n d e x of a w a s p ' s p o t e n t i a l fecundi ty , s u c h r e l a t ion sh ips m a y b e n o n l i n e a r a n d h e n c e m u s t b e i n t e r p r e t e d w i t h c a u t i o n , poss ib ly b e i n g a n ar t i fact of t h e s tep-s ize se lec ted for t h e va r i ab l e s ( L e a t h e r , 1988). Fo r e x a m p l e , in A. ervi, t he in t r in s i c r a t e of i nc rease for c o h o r t s of females r e a r e d o n different a p h i d i n s t a r s w a s a q u a d r a t i c func t ion of b o d y d r y m a s s (R. S e q u e i r a a n d M . M a c k a u e r , u n p u b l i s h e d ) . I t is w o r t h n o t i n g t h a t a d u l t size is h igh ly p l a s t i c in a p h i d i i d w a s p s . I n Aphidius smithi, ve ry s m a l l b u t v i ab l e a n d fertile offspring d e v e l o p e d from eggs d e p o s i t e d in a p h i d e m b r y o s wh i l e still i n s ide the i r m o t h e r ( M a c k a u e r a n d K a m b h a m p a t i , 1988). T h i s f inding sugges t s t h a t t h e l a rva l d e v e l o p m e n t of this p a r a s i t e m a y n o t b e c o n s t r a i n e d b y t h e n e e d to ach ieve a cr i t ica l size for m o l t i n g o r p u p a t i o n .

Different ia l d e v e l o p m e n t r a t e s m a y have a p r o f o u n d effect o n c o m p e t i t i v e i n t e r ac t i ons a m o n g different species of p a r a s i t e s t h a t explo i t t h e s a m e h o s t r e sou rce , i n d e p e n d e n t of e n v i r o n m e n t a l c o n d i t i o n s . Fo r e x a m p l e , d e v e l o p i n g in t h i r d n y m p h a l i n s t a r s of t h e p e a a p h i d , Praon pequodorum r e q u i r e d m o r e t i m e to c o m p l e t e t he e m b r y o n i c s t age , a n d showed g r e a t e r d e v e l o p m e n t a l v a r i a t i o n , t h a n A. smithi; however , b o t h p a r a s i t e s m o l t e d to t h e s e c o n d l a rva l ins t a r a t a p p r o x i m a t e l y t h e s a m e t i m e ( C h o w a n d M a c k a u e r , 1984). B e c a u s e P. pequodorum phys ica l ly a t t a cks a n d kills p o t e n t i a l c o m p e t i t o r s d u r i n g t h e ea r ly first l a rva l s t age , a slow e m b r y o n i c d e v e l o p m e n t i n c r e a s e d t h e p r o b a bi l i ty of i ts su rv iva l in hos t s a l r e a d y p a r a s i t i z e d by A. smithi over a b r o a d r a n g e of a g e differences.

2 . Hyposoter exiguae

T h e i c h n e u m o n i d H. exiguae deve lops as a so l i ta ry e n d o p a r a s i t e in l a rvae of v a r i o u s l e p i d o p t e r a n spec ies . T h e ov ipos i t iona l a n d d e v e l o p m e n t a l success of t h e p a r a s i t e a p p e a r s to v a r y w i t h t he species a n d t h e s t age of h o s t se lec ted . Fo r e x a m p l e , P u t t i e r (1961) r e p o r t e d t h a t w a s p s d e p o s i t e d the i r eggs in t h e first t h r e e i n s t a r s of Peridroma saucia, a l t h o u g h l a rva l d e v e l o p m e n t w a s often i n h i b i t e d in t h i rd - in s t a r hos t s . By c o m p a r i s o n , Heliothis zea w a s successful ly p a r a s i t i z e d in i n s t a r s 1-4, b u t t h e p a r a s i t o i d w a s gene ra l ly u n a b l e to ovipos i t in fifth i n s t a r s b e c a u s e of the i r aggress iveness ( C a m p b e l l a n d Duffey, 1979).


T h e in t e r ac t ions b e t w e e n H. exiguae a n d its va r i ous hos t species a r e cha r ac te r ized by hos t d e v e l o p m e n t a l a r r e s t m e n t , w h i c h m a y v a r y w i t h t h e species of hos t ( T h o m p s o n , 1982, 1983, 1985a ,b ; Beckage a n d T e m p l e t o n , 1985). I n Trichoplusia ni, t he p a r a s i t e l a rva e m e r g e d from the four th o r t h e fifth hos t ins ta r , r ega rd le s s of t h e hos t s t age a t p a r a s i t i z a t i o n . I n c o n t r a s t , p a r a s i t e e m e r g e n c e from Manduca sexta l a rvae va r i ed w i th hos t age a t p a r a s i t i z a t i o n (Beckage a n d T e m p l e t o n , 1985). T h e s e differences in hos t exp lo i t a t i on a p p e a r to b e r e l a t ed to hos t species-specif ic n u t r i t i o n a l a n d phys io log ica l in te r ac t ions (Beckage a n d T e m p l e t o n , 1985), i n c l u d i n g effects m e d i a t e d by p o l y d -n a v i r u s a n d o t h e r pa r a s i t e -de r i ved p r o d u c t s injected a t ov ipos i t ion ( V i n s o n , 1990; F l e m i n g , 1992).

I n c o m p a r i s o n w i t h phys io log ica l a n d b i o c h e m i c a l i n t e r ac t i ons , t h e re lat i o n s h i p b e t w e e n a d u l t l ife-history a t t r i b u t e s a n d the l a rva l o n t o g e n y of H. exiguae, especia l ly as inf luenced b y v a r i a t i o n in hos t size (or q u a l i t y ) , h a s rece ived less a t t e n t i o n . To ta l d e v e l o p m e n t t i m e from egg to a d u l t ec los ion a p p e a r s to d e c r e a s e ( a n d b o d y size a p p e a r s to inc rease ) w i t h hos t size o r age a t p a r a s i t i z a t i o n (Pu t t i e r , 1961; Smi lowi tz a n d I w a n t s c h , 1973; C a m p b e l l a n d Duffey, 1979). W h a t ev idence is ava i lab le sugges t s t h a t H. exiguae r e qu i r e s a cr i t ica l hos t b i o m a s s to m a t u r e o r m o l t to t h e n e x t l a rva l s t age (Beckage a n d T e m p l e t o n , 1985; B l o e m a n d Duffey, 1990), a l t h o u g h l a rva l o n togeny a p p e a r s n o t to b e d i rec t ly inf luenced by the hos t ' s e n d o c r i n e s y s t e m .

3· Microplitis S p e c i e s

T h e in t e r ac t ions b e t w e e n sol i ta ry b r a c o n i d w a s p s a n d the i r l e p i d o p t e r a n hos t s a r e well exempl i f ied by species of Microplitis ( J o n e s a n d Lewis , 1971; W e b b a n d D a h l m a n , 1985, 1986; S t r a n d a n d Dover , 1991). S t r a n d et al (1988) c o m p a r e d the r e sponses oiHeliothis virescens l a rvae to p a r a s i t i s m b y M. croceipes a n d M. demolitor. U n l i k e H. exiguae, these p a r a s i t e s feed exclus ively o n hos t h e m o l y m p h t h r o u g h o u t the i r l a rva l d e v e l o p m e n t . T h e g r o w t h t ra jec to ry of u n p a r a s i t i z e d H. virescens is s igmoid ; however , in l a rvae p a r a s i t i z e d by M. demolitor a n d M. croceipes, t he g r o w t h r a t e is l inea r a n d severely d e p r e s s e d . I n c o m p a r i s o n , p a r a s i t i s m of H. zea l a rvae by M. croceipes r e su l t s in a g r o w t h t ra jec tory s imi la r in s h a p e , b u t r e d u c e d in m a g n i t u d e , to t h a t of u n p a r as i t ized l a rvae ( J o n e s a n d Lewis , 1971). S t r a n d et al. (1988) c o n c l u d e d t h a t t he pa ra s i t e ' s g r o w t h r a t e is d e t e r m i n e d by the hos t i n s t a r a t p a r a s i t i z a t i o n .

Different Microplitis species exh ib i t d i s t inc t p a t t e r n s of hos t -s ize p re fe rence ( R a j a p a k s e a n d Ashley, 1985; T i l l m a n a n d Powell, 1989). Fo r e x a m p l e , M. croceipes p re fe r red t h i r d - i n s t a r l a rvae of Heliothis species , w i t h t he a c c e p t a n c e r a t e of o t h e r i n s t a r s dec l i n ing s y m m e t r i c a l l y a b o u t t he t h i rd i n s t a r (Lewis , 1970; H o p p e r a n d K i n g , 1984). D e v e l o p m e n t t i m e w a s co r r e l a t ed w i t h i n s t a r p re fe rence ; p a r a s i t e s deve lop ing in t h i r d - i n s t a r hos t s deve loped fastest ( H o p p e r a n d K i n g , 1984) a n d ach ieved the l a rges t b o d y size ( H o p p e r , 1986).


IV. Parasite Development and Host Ecology

A. Seasonal Adaptations

M a n y insec ts e n t e r a s t a t e of a r r e s t e d d e v e l o p m e n t a n d r e p r o d u c t i o n to su rv ive d u r i n g pe r iods of un favorab le c o n d i t i o n s , s u c h as low a n d h i g h t e m p e r a t u r e s . T a u b e r et al. (1984) d i s t i n g u i s h e d b e t w e e n two k i n d s of a d a p t a t ions to h a z a r d o u s s ea sona l cond i t i ons : qu i e scence , a p e r i o d d u r i n g w h i c h g r o w t h a n d d e v e l o p m e n t a r e on ly de l ayed un t i l c o n d i t i o n s a r e favorab le a g a i n , a n d d o r m a n c y , d u r i n g w h i c h g r o w t h a n d r e p r o d u c t i o n a r e s u p p r e s s e d b y d i a p a u s e .

1. T e m p e r a t u r e R e q u i r e m e n t s for D e v e l o p m e n t

O b l i g a t e p a r a s i t e s c a n n o t su rv ive in t he a b s e n c e of the i r hos t s a n d , t h u s , m u s t s y n c h r o n i z e the i r d e v e l o p m e n t a n d r e p r o d u c t i o n w i t h hos t a b u n d a n c e . S y n c h r o n y r e q u i r e s t h a t t w o c o n d i t i o n s b e satisfied: (1) t h e p a r a s i t e m u s t in i t i a t e d e v e l o p m e n t in r e s p o n s e to t he s a m e , o r to a closely c o r r e l a t e d , set of c o n d i t i o n s as t he hos t ; a n d (2) t h e p a r a s i t e m u s t cease d e v e l o p m e n t w h e n h o s t a b u n d a n c e dec l ines or, a l te rna t ive ly , shift to a different h o s t spec ies . I n v a r i a b l e c l ima t e s , ea r ly -season s y n c h r o n y is p a r t i c u l a r l y i m p o r t a n t . C a m p bell et al. (1974) c o m p a r e d t h e t e m p e r a t u r e r e q u i r e m e n t s of severa l species of a p h i d p a r a s i t e s w i t h those of the i r hos t s . Pa ra s i t e s h a d a h i g h e r t e m p e r a t u r e t h r e s h o l d t h a n the a p h i d s , a difference t h a t e n s u r e s the i r e m e r g e n c e f rom d i a p a u s e after hos t s b e c o m e ava i l ab le in sp r i ng . In te res t ing ly , t h e p r i m a r y p a r a s i t e s h a d a c o n s i d e r a b l y lower t h r e s h o l d t h a n t h e h y p e r p a r a s i t e Asaphes lucens, cons i s t en t w i th this species ' re la t ive l a te a p p e a r a n c e in t h e s ea son . V a r i a t i o n s in t he t e m p e r a t u r e r e q u i r e m e n t s b e t w e e n different p o p u l a t i o n s i n d i c a t e t h a t p o p u l a t i o n s a r e local ly a d a p t e d ( C a m p b e l l et al., 1974; K a -m b h a m p a t i a n d M a c k a u e r , 1989). A p p a r e n t l y hos t size m a y inf luence a p a r a s i t e ' s t e m p e r a t u r e t h r e s h o l d for d e v e l o p m e n t . Al len a n d Ke l l e r (1991) found t h a t Cotesia urabae, w h i c h is a so l i ta ry k o i n o b i o n t e n d o p a r a s i t e , h a d a h i g h e r t h r e s h o l d for egg- la rva l d e v e l o p m e n t w h e n d e v e l o p i n g in s m a l l r a t h e r t h a n in mid - s i ze l a rvae of its hos t , t he n o c t u i d Uraba lugens.

2 . P a r a s i t e D i a p a u s e

I n a d d i t i o n to e x t e r n a l ab io t i c cues , w h i c h m a y be m e d i a t e d by c h a n g e s in t h e hos t ' s e n d o c r i n e sy s t em, t h e in i t i a t ion a n d m a i n t e n a n c e of d i a p a u s e in p a r a s i t e s often is inf luenced b y hos t cues . Severa l hos t c o n d i t i o n s h a v e b e e n i m p l i c a t e d in d i a p a u s e i n d u c t i o n , especia l ly in e n d o p a r a s i t e s , for e x a m p l e , hos t a g e (in Cotesia glomerata; S c h o o n h o v e n , 1962), hos t size (in Aphidius nigripes; B r o d e u r a n d M c N e i l , 1989), a n d hos t species (in Nasonia vitripennis;


S a u n d e r s et al., 1970). B r o w n et al. (1990) showed t h a t , in l a rvae of t h e cod l ing m o t h , Cydia pomonella, e x p o s u r e to sho r t d a y l e n g t h r e su l t ed in dor m a n c y c a u s e d by fai lure of t he e n d o c r i n e sys t em. T h e c h a n g e d e n d o c r i n e mi l ieu , in t u r n , i n d u c e d d i a p a u s e in first-instar l a rvae of t h e b r a c o n i d e n d o -p a r a s i t e , Ascogaster quadridentata. A d i rec t effect of t he hos t o n d i a p a u s e in i t ia t ion w a s d e m o n s t r a t e d recen t ly in Aphidius matricariae a n d Praon volucre, t w o so l i ta ry e n d o p a r a s i t e s of t he g r e e n p e a c h a p h i d , Myzus persicae (Po lga r et al., 1991). U n d e r iden t i ca l l o n g - d a y cond i t i ons , p a r a s i t e l a rvae d e v e l o p i n g in n y m p h s of ov ipa r ae e n t e r e d a p r e p u p a l d i a p a u s e , w h e r e a s those d e v e l o p i n g in p a r t h e n o g e n e t i c a l l y r e p r o d u c i n g m o r p h s ( funda t r i ces , f u n d a t r i g e n i a e , a n d v i r g i n o p a r a e ) d id no t . T h e a u t h o r s sugges t ed t h a t , in t h e fall, t h e p r o d u c t i o n of o v i p a r a e in holocycl ic a p h i d s , w h i c h is usua l ly a s soc ia t ed w i t h s h o r t - d a y c o n d i t i o n s , c a n serve as a n u n a m b i g u o u s s igna l of d e c l i n i n g h o s t n u m b e r s to these p a r a s i t e s .

3 . D e v e l o p m e n t a l A r r e s t m e n t

H a g e n (1964) d i scussed va r ious e x a m p l e s of d e l a y e d d e v e l o p m e n t in insect p a r a s i t e s . Severa l species of T r i g o n a l i d a e ( H y m e n o p t e r a ) a n d T a c h -in idae ( D i p t e r a ) lay so-cal led m i c r o t y p e eggs d i rec t ly o n foliage. Fo r t he egg to h a t c h , it m u s t be inges ted by a p o t e n t i a l hos t ; t he l a r v a t h e n p e n e t r a t e s t h e g u t wal l a n d deve lops e n d o p a r a s i t i c a l l y ( W i s h a r t , 1956; C o p p e l , 1958). D e layed la rva l d e v e l o p m e n t h a s a lso b e e n found in s o m e a p h i d h y p e r p a r a s i t e s , for e x a m p l e , Alloxysta vitrix a n d A. megourae. Fema le s depos i t t he i r eggs in t h e l a rva , o r occas iona l ly t h e e m b r y o n a t e d egg, of a p r i m a r y a p h i d p a r a s i t e s u c h as Aphidius. Howeve r , t h e egg does n o t h a t c h un t i l t h e p r i m a r y p a r a s i t e h a s comple t e ly c o n s u m e d a n d m u m m i f i e d t he hos t a p h i d ; t h e h y p e r p a r a s i t e larv a t h e n feeds o n a n d even tua l ly kills t h e p r i m a r y l a r v a ( M a t e j k o a n d Su l l ivan , 1979; Sul l ivan , 1987).

D e v e l o p m e n t a l a r r e s t m e n t m a y a lso o c c u r a t a l a t e r s t age in p a r a s i t e d e v e l o p m e n t , for e x a m p l e , d u r i n g t h e first ins ta r . T h e p a r a s i t e l a r v a con t i n ues to deve lop a n d g row only after t he h o s t h a s r e a c h e d a specific size o r i n s t a r ( H a g e n , 1964; Al len a n d Kel ler , 1991). I t is n o t a l w a y s c lea r w h e t h e r s u c h e x a m p l e s r e p r e s e n t n o r m a l va r i a t i ons in t h e pa r a s i t e ' s g r o w t h r a t e , p e r h a p s co r r e l a t ed w i t h differences in hos t qual i ty , o r a n a r r e s t m e n t of g r o w t h in t he a b s e n c e of a specific ( h o r m o n a l ) s t i m u l u s ( L a w r e n c e , 1990; S t r a n d et al., 1991).

B. Influence of Host Nutrition

A n y func t iona l c o n s t r a i n t s o n o p t i o n sets a n d trade-offs b e t w e e n l ife-history c h a r a c t e r s m a y b e revea led w h e n t h e h o s t - p a r a s i t e s y s t e m is sub jec t ed to a d d i t i o n a l s t ress . T w o a p p r o a c h e s wou ld s e e m p a r t i c u l a r l y p r o m i s i n g for


s u c h s tud i e s , especia l ly in e n d o p a r a s i t e s : t h e use of s u p e r p a r a s i t i s m a n d hos t s t a r v a t i o n . I n s u p e r p a r a s i t i s m , n o t all p a r a s i t e offspring c a n c o m p l e t e deve l o p m e n t b e c a u s e e i the r t h e hos t r e sou rces a r e insufficient to s u p p o r t all l a rvae ( typica l ly g r e g a r i o u s species) o r t h e l a rvae c o m p e t e w i t h each o t h e r w i t h on ly o n e s u r v i v i n g ( typica l ly so l i t a ry species) (Sal t , 1961; M a c k a u e r , 1990). I n c o n t r a s t , s t a r v a t i o n causes a g e n e r a l r e d u c t i o n in insec t g r o w t h a n d d e v e l o p m e n t (S lansky a n d Scr iber , 1985), a c h a n g e t h a t p r e s u m a b l y lowers h o s t qua l i ty .

1. S u p e r p a r a s i t i s m

P a r a s i t i s m by m a n y e n d o p a r a s i t e s resu l t s in d i m i n i s h e d hos t g r o w t h a n d c o n s u m p t i o n r a t e s ( T h o m p s o n , 1985a) . P a r a s i t i s m of t he p e a a p h i d b y A. smithi t h u s m a y b e a typ i ca l in t h a t p a r a s i t i z e d a p h i d s i nges t ed m o r e food ( b u t a s s i m i l a t e d it less efficiently) t h a n the i r u n p a r a s i t i z e d c o u n t e r p a r t s ; al l a p h i d s w e r e m a i n t a i n e d o n a n art if icial d i e t (C lou t i e r a n d M a c k a u e r , 1979). B e c a u s e s u p e r p a r a s i t i z e d a p h i d s i n c o r p o r a t e d food m o r e efficiently a n d g r e w faster t h a n s ingly p a r a s i t i z e d a p h i d s (C lou t i e r a n d M a c k a u e r , 1980), t h e g r o w t h p o t e n t i a l of t h e su rv iv ing p a r a s i t e l a rva m a y b e h igher . T h i s s u p p o s i t ion w a s conf i rmed recent ly . M a l e s of A. ervi t h a t , as l a rvae , d e v e l o p e d in s u p e r p a r a s i t i z e d p e a a p h i d s h a d g r e a t e r a d u l t m a s s t h a n t hose f rom s ingly p a r a s i t i z e d hos t s ; however , d e v e l o p m e n t t i m e d i d n o t differ b e t w e e n t h e two g r o u p s (Bai a n d M a c k a u e r , 1992). T h e s e resu l t s sugges t t h a t , in s o m e h o s t -p a r a s i t e a s soc ia t ions a t leas t , t he hos t m a y r e s p o n d to t h e a d d e d d e m a n d s of t h e d e v e l o p i n g p a r a s i t e l a rva (e ) b y i n c r e a s e d food u p t a k e .

I n c o n t r a s t , in g r e g a r i o u s species , t h e su rv iv ing w a s p s f rom supe r p a r a s i t i z e d hos t s gene ra l ly a r e s m a l l e r t h a n the i r c o u n t e r p a r t s f rom n o n -s u p e r p a r a s i t i z e d hos t s ; however , d e v e l o p m e n t t i m e is less c o m m o n l y affected. Wyl ie (1965) showed t h a t , in t h e e c t o p a r a s i t e N. vitripennis, t h e m e a n p e r c e n t a g e of l a rvae t h a t su rv ived in s ame-s i zed h o u s e fly p u p a e d e c l i n e d w i t h t h e p a r a s i t e l oad . S u p e r n u m e r a r y l a rvae w e r e e l i m i n a t e d b y s t a r v a t i o n , u sua l l y in t h e las t ins ta r . I n cases of h e a v y s u p e r p a r a s i t i s m ( ^ 5 0 l a rvae p e r p u p a in i t ia l ly) , t h e hos t w a s comple t e ly c o n s u m e d before t h e p a r a s i t e l a rvae r e a c h e d t h e las t ins ta r , a n d h e n c e all p a r a s i t e s d i ed . T h e s t a r v e d l a rvae of g r e g a r i o u s species a r e u sua l ly n o t e a t e n b y t h e su rv ivor s , a b e h a v i o r t h a t c a n resu l t in t h e w a s t a g e of hos t r e sou rces (Sal t , 1961). B e c a u s e m o r t a l i t y w a s h i g h e r a m o n g female t h a n m a l e l a rvae in Eupteromalus dubius, Wyl ie (1976) sugges t ed t h a t s u p e r p a r a s i t i s m m a y b ias t h e offspring sex r a t i o . H o w e v e r , T a y l o r (1988) found n o ev idence of differential offspring m o r t a l i t y in Bracon hebetor, c o n c l u d i n g t h a t a n y b i a s r e p o r t e d for o t h e r species ( W a a g e , 1986; K i n g , 1987) m a y have b e e n d u e to m a t e r n a l sex r a t i o m a n i p u l a t i o n .

B e c k a g e a n d Ridd i fo rd (1978 , 1983) e x a m i n e d t h e g r o w t h a n d d e v e l o p m e n t of t h e g r e g a r i o u s e n d o p a r a s i t e C. congregata in r e l a t i on to t h e p a r a s i t e


load a n d the n u t r i t i o n a l s t a t u s of its hos t , M. sexta l a rvae . I n f i r s t - ins tar h o s t s , t h e n u m b e r of p a r a s i t e l a rvae d e t e r m i n e d the t i m i n g of the i r e m e r g e n c e f rom t h e hos t . E m e r g e n c e usua l ly o c c u r r e d d u r i n g the four th o r fifth hos t i n s t a r ; however , p a r a s i t e s e m e r g e d in the t h i rd o r a s u p e r n u m e r a r y s ix th i n s t a r in cases of a n ex t r eme ly l ight o r heavy p a r a s i t e load , respect ively. T h e first l a rva l m o l t of t he p a r a s i t e o c c u r r e d after hos t ecdysis to t h e t e r m i n a l s t age , r ega rd l e s s of w h e t h e r t h a t s t age w a s t he four th , fifth, o r s ix th ins ta r . W h e r e a s t he to ta l we t m a s s of t he h o s t - p a r a s i t e c o m p l e x w a s posi t ively c o r r e l a t e d w i t h t he p a r a s i t e load , the m a s s of i n d i v i d u a l l a rvae or of a d u l t w a s p s w a s less in heavi ly p a r a s i t i z e d hos t s . I n c o n t r a s t , d e v e l o p m e n t t i m e a p p e a r e d to be i n d e p e n d e n t of t he p a r a s i t e load or t he hos t i n s t a r p a r a s i t i z e d in C. glomerata, a n e n d o p a r a s i t e of Pieris rapae; t he g r e g a r i o u s l a rvae e m e r g e d a l ways from fifth-instar ca t e rp i l l a r s ( F u h r e r a n d Keja , 1976).

2 . H o s t S tarvat ion

T h e p a r a s i t e C. congregata failed to e m e r g e if newly ecdysed , t e r m i n a l - s t a g e hos t l a rvae , M. sexta, we re s t a rved . By p o s t p o n i n g t h e s t a r v a t i o n t r e a t m e n t to a l a te r t ime , Beckage a n d Ridd i fo rd (1983) were ab l e to i nc r ea se t he p e r c e n t age of p a r a s i t e s r e a c h i n g the a d u l t s t age ; they sugges t ed t h a t hos t l a rvae m u s t feed d u r i n g a specific pe r iod to satisfy t he d e v e l o p m e n t a l r e q u i r e m e n t s of t he p a r a s i t e . Similar ly, t he d e v e l o p m e n t t i m e of t he t ach in id Compsilura concinnata w a s unaffected by s t a r v a t i o n if t h e p a r a s i t e deve loped in a l a rge gypsy m o t h la rva ; however , p a r a s i t e s e m e r g e d ear l i e r f rom severely s t ressed hos t s (Weseloh, 1984). I n the a p h i d p a r a s i t e E. californicus, w a s p s e m e r g e d ea r l i e r f rom s t a rved a p h i d s , a n d they were smal le r , t h a n the i r c o u n t e r p a r t s from n o n s t a r v e d hos t s ( K o u a m e a n d M a c k a u e r , 1991). By c o n t r a s t , t h e d e v e l o p m e n t t i m e of t he so l i ta ry i c h n e u m o n i d Campoletis sonorensis i n c r e a s e d if f ou r th - in s t a r hos t l a rvae , Heliothis virescens, were s t a rved (Dover a n d V i n s o n , 1990).

C r o w d i n g m a y have a s imi la r effect as hos t s t a r v a t i o n in t h a t b o t h ac t as g e n e r a l s t ress factors . Fo r e x a m p l e , in Leptopilina boulardi, a cyn ip id e n d o p a r a s i t e of Drosophila l a rvae , a d u l t size a n d d e v e l o p m e n t t i m e w e r e r e d u c e d in hos t s m a i n t a i n e d u n d e r c r o w d e d cond i t i ons ( W a j n b e r g et al., 1990).

V Developmental Strategies

Diver se hos t exp lo i t a t ion a n d d e v e l o p m e n t a l s t ra teg ies have evolved in insec t p a r a s i t e s t h a t a d a p t t he i m m a t u r e s ' m e t a b o l i c r e q u i r e m e n t s to t h e ava i l ab le b i o m a s s , g r o w t h po t en t i a l , a n d surv iva l of t he p a r a s i t i z e d hos t . A h i g h d e g r e e of n u t r i t i o n a l a n d phys io log ica l i n t e g r a t i o n b e t w e e n the hos t a n d t h e p a r a s i t e sy s t em is cha rac t e r i s t i c of s u c h assoc ia t ions ( T h o m p s o n , 1985b, 1990; Bar -


re t t a n d S c h m i d t , 1991). We have s h o w n in Sec t ions I I I a n d I V t h a t t h e p a r a s i t e ' s g r o w t h r a t e , d e v e l o p m e n t t i m e , a n d a d u l t b i o m a s s a r e t h e essen t ia l c o m p o n e n t s of a n y d e v e l o p m e n t a l s t r a t egy ; they a r e c o n s t r a i n e d by h o s t q u a l i t y in a n associa t ion-speci f ic m a n n e r .

M e t a b o l i c p rocesses t h a t govern g r o w t h , d e v e l o p m e n t , a n d u l t i m a t e l y fitness a r e u sua l ly s i z e - d e p e n d e n t (Pe te rs , 1983; S c h m i d t - N i e l s e n , 1984). I n insec t p a r a s i t e s , b o d y size is gene ra l l y co r r e l a t ed w i t h l ife-history a n d d e m o g r a p h i c c h a r a c t e r s ( K i n g , 1987), a fact sugges t ing t h a t b o d y size is t he m a i n t a r g e t of se lec t ion . However , a d u l t size a n d d e v e l o p m e n t r a t e m a y have i n d e p e n d e n t , a n d even o p p o s i t e , effects on fitness (Roff, 1981; Sibly a n d Calow, 1986). C o n s e q u e n t l y , t he p a r t i c u l a r va lues a d o p t e d by a p a r a s i t e species for these a t t r i b u t e s a r e condi t ion-spec i f ic a n d d e t e r m i n e d , for e x a m ple , by a n y fitness t rade-off b e t w e e n size a n d d e v e l o p m e n t t i m e . Sibly a n d C a l o w (1986) n o t e d t h a t se lect ion e i t he r m a y p u s h p h e n o t y p e s a n d the i r u n d e r l y i n g phys io logies to the i r l imi t i m p o s e d by s ize- re la ted c o n s t r a i n t s , o r m a y o p t i m i z e p h e n o t y p e s be low size c o n s t r a i n t s in r e s p o n s e to fitness t r a d e offs b e t w e e n i n d i v i d u a l c h a r a c t e r s . T h u s , a pa ra s i t e ' s " d e c i s i o n " to g row bigger , o r to m a t u r e ear l ier , o r to c o m p r o m i s e b o t h , will d e p e n d o n a d a p t i v e va r i ab i l i ty in t he g r o w t h r a t e . W h e n the g r o w t h r a t e is a t its phys io log ica l m a x i m u m , a n i nc rea se in b o d y size c a n on ly be ach ieved a t t he cost of a n i nc rea se in d e v e l o p m e n t t ime . Al te rna t ive ly , a l a rge r b o d y size c o m b i n e d w i t h a d e c r e a s e in d e v e l o p m e n t t i m e r e q u i r e s a n i n c r e a s e d g r o w t h r a t e .

E m p i r i c a l d a t a (see Sec t ions I I I a n d I V ) sugges t t h a t t h r e e b r o a d l y differen t d e v e l o p m e n t a l p a t t e r n s have evolved a m o n g insec t p a r a s i t e s in r e s p o n s e to hos t c o n s t r a i n t s . T h e first m o d e l (Fig . 1) a s s u m e s t h a t t h e h o s t is a c losed r e sou rce e n v i r o n m e n t a n d t h a t p a r a s i t e g r o w t h a n d d e v e l o p m e n t a r e cons t r a i n e d solely by hos t qual i ty . T h e hos t c o n t a i n s a finite a m o u n t of r e sou rces t h a t is d e t e r m i n e d a t t h e t i m e of p a r a s i t i z a t i o n . H o s t q u a l i t y does n o t vary, o r var ies very l i t t le , d u r i n g the cou r se of t h e in t e rac t ion ; however , q u a l i t y m a y va ry b e t w e e n different species a n d ages of hos t s . T h e p a r a s i t e ' s s t r a t e g y is to m a x i m i z e a d u l t b i o m a s s ( a s s u m e d to b e t h e chief d e t e r m i n a n t of fi tness) p e r un i t of hos t r e sou rces . T h e g r o w t h t ra jec tory is re la t ively i n v a r i a n t a n d m a x i m i z e d for a g iven set of hos t a n d e n v i r o n m e n t a l cond i t i ons . T h e r e f o r e , p a r a site a d u l t size is posi t ively co r r e l a t ed w i t h hos t qua l i t y ; l a rge hos t s give r ise to l a rge ( a n d p r e s u m a b l y m o r e fecund) p a r a s i t e s t h a t , however , m a y r e q u i r e m o r e t i m e to c o m p l e t e d e v e l o p m e n t t h a n s m a l l i n d i v i d u a l s .

T h e second m o d e l (Fig . 2) a s s u m e s t h a t t he hos t r e p r e s e n t s a n o p e n r e sou rce e n v i r o n m e n t a n d , as for M o d e l 1, t h a t p a r a s i t e g r o w t h a n d d e v e l o p m e n t a r e c o n s t r a i n e d solely b y hos t qual i ty . H o s t q u a l i t y a n d r e s o u r c e avai l abi l i ty v a r y over t i m e a n d w i t h hos t age a t p a r a s i t i z a t i o n . H o s t feeding a n d g r o w t h a r e e x p e c t e d to c o n t i n u e d u r i n g t h e in i t ia l p h a s e s of p a r a s i t i s m , a lbe i t a t modi f ied r a t e s . I n low-qua l i ty hos t s , p a r a s i t e d e v e l o p m e n t m a y i n c l u d e a


min max Parasite development time

Figure 1 Parasite growth and development in a closed resource environment: bio-mass per unit of host resources is maximized (Model 1). The parasite grows at a constant rate that is independent of host quality. Parasite biomass is directly proportional to host quality (and development time).

lag min max Parasite development time

Figure 2 Parasite growth and development in an open resource environment: biomass per unit of host resources is maximized (Model 2). The parasite must reach a critical mass within a variable development time. By delaying the beginning of exponential growth (= lag phase) until the host has reached a larger size, the parasite may increase its growth potential in low-quality hosts but cannot offset any initial delay.


l ag p h a s e d u r i n g w h i c h t i m e t h e h o s t c o n t i n u e s to grow. As in M o d e l 1, t h e p a r a s i t e ' s s t r a t e g y is to m a x i m i z e b i o m a s s p e r u n i t of hos t r e s o u r c e s . A p p a r ently, p a r a s i t e s g r o w a t the i r phys io log ica l l imi t . W h e r e a s fi tness g a i n s f rom i n c r e a s e d b i o m a s s m u s t b e b a l a n c e d a g a i n s t fi tness losses f rom i n c r e a s e d d e v e l o p m e n t t i m e , t h e fo rmer a r e a s s u m e d to b e of re la t ive ly g r e a t e r i m p o r t a n c e for fitness. T h i s s t r a t e g y favors t h e evo lu t ion of a m i n i m u m o r cr i t ica l b o d y size for successful d e v e l o p m e n t to t h e a d u l t s t age .

T h e t h i r d m o d e l (Fig . 3) a s s u m e s t h a t t h e hos t r e p r e s e n t s a n o p e n re sou rce e n v i r o n m e n t a n d t h a t p a r a s i t e g r o w t h a n d d e v e l o p m e n t a r e r e s o u r c e -a n d t ime- l imi t ed . T h e d y n a m i c s of hos t q u a l i t y a r e t h e s a m e as in t h e s e c o n d m o d e l ; however , th is m o d e l a s s u m e s a d e v e l o p m e n t a l s t r a t e g y t h a t o p t i m i z e s a set of c h a r a c t e r s t h a t j o i n t l y d e t e r m i n e fitness, for e x a m p l e , b o d y size a n d d e v e l o p m e n t t ime . T i m e c o n s t r a i n t s c a n t ake severa l fo rms , s u c h a s a fixed p e r i o d of h o s t su rv iva l after p a r a s i t i z a t i o n o r a s h a r p dec l ine in a d u l t p a r a s i t e su rv iva l w i t h i n c r e a s e d age . I n low-qua l i ty o r sma l l hos t s , p a r a s i t e g r o w t h m a y b e r e sou rce - l imi t ed d u r i n g e i the r ea r ly d e v e l o p m e n t ( lag p h a s e ) o r l a t e d e v e l o p m e n t (p recoc ious d e a t h ) . T h e p a r a s i t e ' s g r o w t h t ra jec to ry is v a r i a b l e , ref lect ing h o s t age - o r s tage-specif ic c o n s t r a i n t s o n n u t r i t i o n a l r e s o u r c e s .

lag min max Parasite development time

Figure 3 Parasite growth and development in an open resource environment: the optimal phenotype is determined by a trade-off between adult biomass and development time (Model 3). As in Model 2, a parasite developing in a low-quality host may delay the beginning of exponential growth ( = lag phase) until the host has reached a larger size. This initial delay is offset by compensatory feeding (and increased growth) during late larval development.


T h u s , a n ini t ia l d e l a y in the onse t of e x p o n e n t i a l p a r a s i t e g r o w t h m a k e s a c o m p e n s a t o r y inc rease in t he g r o w t h r a t e d u r i n g l a te r s t ages neces sa ry in o r d e r to b a l a n c e a n y fitness ga ins (from inc reased b i o m a s s ) a n d losses (from inc rea sed d e v e l o p m e n t t ime) .

W h e r e a s M o d e l 1 conforms to t he d e v e l o p m e n t a l p a t t e r n of m o s t id iob ion t s , M o d e l s 2 a n d 3 a r e r e p r e s e n t a t i v e of k o i n o b i o n t life h i s to r ies . H o s t b i o m a s s a t t he t i m e of p a r a s i t i z a t i o n r e p r e s e n t s t he m a i n c o n s t r a i n t o n id io-b i o n t a d u l t b i o m a s s a n d d e v e l o p m e n t t ime . E g g p a r a s i t e s typica l ly c a u s e t h e a r r e s t m e n t of hos t e m b r y o g e n e s i s ( S t r a n d , 1986), t h u s e n s u r i n g t h a t h o s t q u a l i t y r e m a i n s la rge ly u n c h a n g e d d u r i n g the cour se of i n t e r ac t i on . T h e s t r a t e g y a d o p t e d by the p a r a s i t e is to g row un t i l hos t r e sou rces a r e e x p e n d e d (Fig . 1). I n s o m e species , d e v e l o p m e n t m a y be c o m p r o m i s e d by t h e p a r a s i t e ' s inab i l i ty to ut i l ize all t he r e sources c o n t a i n e d in l a rge hos t s (Sal t , 1964; Wyl ie , 1965). Severa l ko inob ion t s deve lop ing in l e p i d o p t e r a n hos t s r e q u i r e a cr i t ica l hos t s i z e / b i o m a s s for c o m p l e t i o n of l a rva l d e v e l o p m e n t (Beckage a n d T e m p l e t o n , 1985; B l o e m a n d Duffey, 1990) o r m o l t i n g (Slansky, 1986). H o w ever, s o m e c h a n g e s in p a r a s i t e g r o w t h a n d d e v e l o p m e n t m a y b e m e d i a t e d b y the hos t ' s e n d o c r i n e sys t em ( C o r b e t , 1968; Beckage , 1985; L a w r e n c e , 1990; S t r a n d et al., 1991). T h e d e v e l o p m e n t a l cha rac te r i s t i c s of H. exiguae a n d Microplitis species a p p e a r to be cons i s t en t w i th t he p a t t e r n s h o w n in Fig . 2.

I n c o m p a r i s o n , t he d e v e l o p m e n t a l s t r a t egy of A. ervi a p p e a r s to b e b a s e d on trade-offs t h a t resu l t in t he o p t i m i z a t i o n of b o t h a d u l t b i o m a s s a n d devel o p m e n t t i m e (Fig . 3) . T h e su rv ivo r sh ip of p a r a s i t i z e d a p h i d s is i n d e p e n d e n t of age a t p a r a s i t i z a t i o n ( C a m p b e l l a n d M a c k a u e r , 1975; L iu a n d H u g h e s , 1984), a cond i t i on l imi t ing r e sou rce avai lab i l i ty to a fixed p e r i o d before t he hos t d ies . I n low-qual i ty hos t s , p a r a s i t e l a rvae e x p e r i e n c e d slow in i t ia l g r o w t h b u t c o m p l e t e d d e v e l o p m e n t a t a c o m p e n s a t o r y r a t e . U n d e r different o p t i o n sets , p a r a s i t e b o d y size w a s o p t i m i z e d before d e v e l o p m e n t t i m e (Seq u e i r a a n d M a c k a u e r , 1992a,b) .

VI. Conclusions

F r o m the p a r a s i t e ' s pe r spec t ive , a hos t is obv ious ly m o r e t h a n its n u t r i t i o n a l qual i ty , i n c l u d i n g its avai labi l i ty, behav ior , a n d as soc ia t ed c o m p e t i t o r s a n d n a t u r a l e n e m i e s . However , to u n d e r s t a n d the evo lu t ion of different life-h i s to ry s t ra teg ies in insect p a r a s i t e s , we n e e d to k n o w the func t iona l cons t r a i n t s o n the i r d e v e l o p m e n t a n d r e p r o d u c t i o n in va r ious hos t spec ies . I n this r e g a r d , a d u l t b i o m a s s m a y i n d e e d serve as a useful i n d e x of p a r a s i t e fitness. As we have shown , t he bes t s t r a t egy for t he m a x i m i z a t i o n of b i o m a s s will b e d e t e r m i n e d by the b a l a n c e b e t w e e n t i m e a n d r e sou rce l im i t a t i ons o n p a r a s i t e g r o w t h . O p t i m a l i t y t heo ry ( M a y n a r d S m i t h , 1982; P a r k e r a n d M a y -


n a r d S m i t h , 1990) a s s u m e s t h a t , for different species , on ly c e r t a i n cons t r a i n e d c o m b i n a t i o n s of f i tness- re la ted c h a r a c t e r s c a n be rea l ized in p r a c t ice. T h e s e o p t i o n sets differ b e t w e e n i d iob ion t a n d k o i n o b i o n t a n d , poss ib ly as well , b e t w e e n so l i ta ry a n d g r e g a r i o u s species of insec t p a r a s i t e s . For e x a m ple , a s t r a t e g y b a s e d on a flexible g r o w t h t ra jec tory m a y e n a b l e a k o i n o b i o n t to r e s p o n d to v a r i a b l e hos t q u a l i t y b u t w o u l d have l i t t le u t i l i ty in i d iob ion t s .

A d i s t i nc t i on b e t w e e n " c o n f o r m e r s " a n d " r e g u l a t o r s , " as p r o p o s e d b y L a w r e n c e (1986, 1990) a n d V i n s o n (1990) , focuses o n the p r o x i m a t e m e c h a n i s m s by w h i c h p a r a s i t e s explo i t hos t r e sou rces . By def in i t ion , al l fo rms of p a r a s i t i s m a r e e x p e c t e d to a l t e r hos t phys io logy a n d on togeny , a lbe i t in w a y s t h a t m a y b e obv ious a n d p a t h o l o g i c a l in s o m e assoc ia t ions ( " r e g u l a t o r s " ) a n d s u b t l e in o t h e r s ( " c o n f o r m e r s " ) . Similar ly , the i n v o l v e m e n t in hos t a l ter a t i on of p a r a s i t e - d e r i v e d p r o d u c t s , s u c h as p o l y d n a v i r u s a n d v e n o m s (Sto l tz , 1986; S t r a n d a n d Dover , 1991; F l e m i n g , 1992), does n o t in p r i n c i p l e c h a n g e t h e n a t u r e of t he i n t e r ac t ions , a l t h o u g h these m e c h a n i s m s e n l a r g e t he o p t i o n set for hos t exp lo i t a t ion .

D e t a i l e d s tud ie s of t he d y n a m i c s of p a r a s i t e d e v e l o p m e n t u n d e r different c o n s t r a i n t s have b e e n m a d e on only few species , n o t a b l y so l i ta ry k o i n o b i o n t s . T h u s , t h e t h r e e m o d e l s p r e s e n t e d h e r e a r e b a s e d o n f r a g m e n t a r y i n f o r m a t i o n a n d s h o u l d be seen as p r e l i m i n a r y only. I n p a r t i c u l a r , t h e m o d e l s a n d the i r u n d e r l y i n g a s s u m p t i o n s m u s t be t es ted empir ica l ly . O f in t e re s t w o u l d b e a c o m p a r i s o n of t h e g r o w t h t ra jec tor ies of i n d i v i d u a l l a rvae b e t w e e n so l i ta ry a n d g r e g a r i o u s species , especia l ly as r e l a t ed to s u p e r p a r a s i t i s m a n d n u t r i t iona l s t ress r e su l t i ng from, for e x a m p l e , s t a r v a t i o n a n d c r o w d i n g . A n o t h e r a r e a t h a t is la rge ly u n e x p l o r e d a n d n e e d s a t t e n t i o n is t h e t i m i n g of t h e e n d of l a rva l feeding a n d of hos t d e a t h . S u c h c o m p a r a t i v e s tud ies of a s soc ia t ion -specific func t iona l c o n s t r a i n t s o n p a r a s i t e o n t o g e n y m a y h e l p e x p l a i n t h e su rv iva l v a l u e of different l ife-history s t ra teg ies in insec t p a r a s i t e s .

Acknowledgment

We thank the Natural Sciences and Engineering Research Council of Canada for financial support.

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cha t r 2 ^ a m e s P a r a s ^ e s Play: The a p e r Dynamic Roles of Proteins

and Peptides in the Relationship between Parasite and Host Nancy E. Beckage Department of Entomology University of California Riverside, California

I. Parasitism-Induced Changes: Host Responses or Parasite-Directed Host Manipulation?

II. Parasitism- and Polydnavirus-lnduced Peptides and Proteins in Insect Hosts

III. Parasitic Effects on Endogenous Host Proteins, Enzymes, and Peptides

IV. Comparisons with Parasitism- and Virus-Induced Changes in Other Systems

Acknowledgments References

I. Parasitism-Induced Changes: Host Responses or Parasite-Directed Host Manipulation?

P a r a s i t i s m inflicts m u l t i p l e c h a n g e s in t h e phys io log ica l a n d b i o c h e m i c a l func t ions of h o s t insec ts a s ide from a l t e r i ng the i r behav io r , h o r m o n e t i t e r s , a n d d e v e l o p m e n t as e m p h a s i z e d e l sewhere in th is v o l u m e . G i v e n t h e s o p h i s t i ca t ed b i o c h e m i c a l a n d m o l e c u l a r t e c h n i q u e s n o w ava i l ab le , m o n i t o r i n g t h e effects of p a r a s i t i s m on hos t p r o t e i n syn thes i s , a n d con f i rma t ion t h a t p a r a s i t i s m s o m e t i m e s i n d u c e s syn thes i s of novel p r o t e i n s in t he hos t , n o w c o n s t i t u t e s o m e of t h e m o s t r ead i ly d o c u m e n t a b l e c h a n g e s o c c u r r i n g d u r i n g insec t e n d o p a r a s i t i s m . T h e goals of this r ev iew a r e to s u m m a r i z e t h e m a j o r l ines of ev idence d e m o n s t r a t i n g t h a t p a r a s i t i z e d insec ts exh ib i t m a j o r c h a n g e s invo lv ing p r o t e i n s a n d p e p t i d e s , a n d pose q u e s t i o n s a b o u t t h e n a t u r e of the i r p o t e n t i a l b io logica l s ignif icance a n d i m p a c t u p o n t h e h o s t - p a r a s i t e r e l a t ion sh ip .

I m p o r t a n t l y , a b r o a d t a x o n o m i c su rvey reveals t h a t p a r a s i t i s m i n d u c e s a l t e r a t i o n s in t h e p r o t e i n s a n d p e p t i d e s p r e s e n t in a w i d e va r i e ty of o r g a n -

Parasites and Pathogens of Insects Volume 1: Parasites 2 5


2 6 Nancy Ε. Beckage

i sms a s ide from insec ts , i n d i c a t i n g t hey likely r e p r e s e n t m a j o r p l aye r s in t he r e l a t i o n s h i p b e t w e e n p a r a s i t e a n d hos t in m a n y sys t ems . M a n y m a m m a l s , in a d d i t i o n to severa l c lasses of i n v e r t e b r a t e s , i n c l u d i n g mol luscs a n d c r u s t a c e a n s , exh ib i t m a j o r c h a n g e s in the i r b lood c o m p o n e n t s o r h e m o l y m p h d u r ing p a r a s i t i s m , a r g u i n g t h a t s imi l a r b i o c h e m i c a l c h a n g e s o c c u r in wide ly r a n g i n g t a x a as a resu l t of p a r a s i t i s m . I n s o m e sys t ems , t h e p a r a s i t e s t h e m selves d i rec t ly i n t e rvene in c a u s i n g these c h a n g e s , w h e r e a s in o t h e r s t he i r inf luence a p p e a r s to b e m o r e ind i rec t . Severa l p a r a s i t e s d e v e l o p i n g in m a m m a l i a n hos t s (or even p l an t s ) have b e e n s h o w n to secre te p r o t e i n s a n d enz y m e s t h a t faci l i tate, o r a r e abso lu t e ly r e q u i r e d for, i nvas ion of hos t t i ssues o r m a i n t e n a n c e of a n a p p r o p r i a t e b i o c h e m i c a l e n v i r o n m e n t t h a t m a r k e d l y enh a n c e s p a r a s i t e success .

O n e of t he m o s t cr i t ica l fea tures of p a r a s i t i c i nvas ion obv ious ly is t h a t t h e p a r a s i t e s successfully e scape or s u p p r e s s t he h o s t i m m u n e r e s p o n s e , a n d s o m e of t h e molecu le s p r o d u c e d or o the rwi se a s soc i a t ed w i t h p a r a s i t e s a r e t h o u g h t to serve i m p o r t a n t roles in act ively d i s e n g a g i n g t h e h o s t i m m u n e r e sponse , t h e r e b y p r e v e n t i n g i m m u n o d e t e c t i o n . Yet o t h e r species of p a r a s i t e s a r e t h o u g h t to e m p l o y hos t " m i m i c r y " to c i r c u m v e n t hos t defenses b y incorp o r a t i n g hos t l ike a n t i g e n s (e i ther p a r a s i t e - o r hos t -de r ived molecu les ) o n t o the i r surface, t h e r e b y a l lowing the p a r a s i t e s to e s c a p e r ecogn i t i on as foreign d u r i n g d e v e l o p m e n t in hos t t i ssues . P ro t e in s t h u s p l a y i m p o r t a n t roles in " m a s k i n g " t he surface of p a r a s i t e s o r o the rwi se p r o v i d i n g p r o t e c t i o n .

Unfo r tuna t e ly , insec ts have b e e n c o n s i d e r a b l y less in tens ive ly s t u d i e d c o m p a r e d to m a m m a l s w i t h r e spec t to t h e p rec i se m e c h a n i s m s w h e r e b y the i r p a r a s i t e s c a u s e hos t i m m u n o s u p p r e s s i o n or e scape i m m u n o r e c o g n i t i o n a n d re jec t ion. C o n s e q u e n t l y , o u r b i o c h e m i c a l ins igh t s i n to t h e n a t u r e of t h e cr i t i cal molecu les a n d m e c h a n i s m s func t ion ing a t t h e h o s t - p a r a s i t e i m m u n o l o g i cal in terface in insec t sy s t ems r e m a i n very l imi ted . I n o n e i n s t a n c e , it a p p e a r s t h a t t h e v i rus l ike pa r t i c l es injected b y a w a s p p a r a s i t o i d (Venturia canescens) express " h o s t l i k e " ep i topes on the surface of t he v i r ions . Fol lowing oviposi t ion , l a rge n u m b e r s of v i r ions a d h e r e to t h e surface of t h e d e v e l o p i n g p a r a s i t e eggs , t h u s p r o t e c t i n g t h e a c c o m p a n y i n g p a r a s i t e s b y a p rocess ak in to m i m i cry of hos t a n t i g e n s ( S c h m i d t a n d S c h u c h m a n n - F e d d e r s e n , 1989). H o w e v e r , these v i ru s pa r t i c l e s a r e u n i q u e a m o n g t h e po lydnav i ru s l i ke e l e m e n t s c a r r i e d by p a r a s i t o i d s in t h a t t hey a p p e a r to lack a n y nuc le ic ac ids ye t have evolved p o t e n t p ro t ec t ive s t ra teg ies b a s e d u p o n h o s t p r o t e i n r ecogn i t i on p rocesses ( S c h m i d t et al, 1990).

T h e m e c h a n i s m s o p e r a t i n g in o t h e r species of p a r a s i t i z e d insec t s a r e less clear . Fo r e x a m p l e , a l t h o u g h it is k n o w n t h a t t h e h e m o c y t e s of p a r a s i t i z e d insec ts show ex tens ive behav io ra l t r a n s f o r m a t i o n s t h a t p r e c l u d e t he i r inv o l v e m e n t in e n c a p s u l a t i o n (Stol tz , V o l u m e 1, C h a p t e r 8) , t h e t r a n s d u c i n g p a t h w a y s w h e r e b y t h e pa r a s i t o id - a s soc i a t ed p o l y d n a v i r u s e s c a u s e these eel-

2. Games Parasites Play 2 7

l u l a r c h a n g e s r e m a i n whol ly specu la t i ve . T h e m o l e c u l a r m e c h a n i s m s o p e r a t ing to s u p p r e s s h o s t h u m o r a l a n d ce l lu la r defenses d u r i n g p a r a s i t i s m t h u s r e m a i n to b e def ined. T h i s s i t u a t i o n hopeful ly soon will b e d r a m a t i c a l l y c h a n g e d as we explo i t t h e r e c e n t r a p i d pro l i fe ra t ion of p r o b e s a n d t e c h n i q u e s ava i l ab le for us to d issec t t h e d y n a m i c n a t u r e a n d va r ious levels of m o l e c u l a r i n t e r ac t i ons o p e r a t i n g b e t w e e n p a r a s i t e s a n d the i r r espec t ive h o s t s . O n t h e bas i s of o u r k n o w l e d g e of m a m m a l i a n s y s t e m s , we m i g h t s u s p e c t t h a t p r o te ins , p e p t i d e s , g lycopro t e in s , lec t ins , l i p o p h o s p h o g l y c a n s , a n d o t h e r cell-su r face-assoc ia ted molecu le s m i g h t p l a y m a j o r roles .

I n m a m m a l s , r e cen t a d v a n c e s in d e v e l o p m e n t a l b io logy h a v e p r o v e n insightful in t h a t m a n y of t h e s a m e molecu le s here tofore r ecogn ized a s m a j o r p l aye r s in ce l l - ce l l r ecogn i t i on p rocesses in i m m u n i t y a lso a p p e a r to p l a y cr i t ica l ro les in cell p ro l i fe ra t ion , t i ssue o r g a n i z a t i o n , a n d o t h e r d e v e l o p m e n ta l p rocesses . C y t o k i n e s a n d severa l g r o w t h factors p l u s t he i r r e c e p t o r s u l t i m a t e l y m a y b e s h o w n to h a v e s u c h d u a l roles in m a m m a l s . Fo r e x a m p l e , m e m b e r s of t r a n s f o r m i n g g r o w t h factor β-family p l a y a role in defense to p a t h o g e n s , for e x a m p l e , in d e t e r m i n i n g m a c r o p h a g e suscep t ib i l i ty to l e i s h m a n i a p a r a s i t e s ( B a r r a l - N e t t o et al., 1992), whi le a l so func t ion ing d u r i n g e m b r y o n i c d e v e l o p m e n t (Fe rguson a n d A n d e r s o n , 1992; J e s se l l a n d M e l t o n , 1992). I n insec t s , a s imi l a r d u a l func t iona l i ty of s o m e molecu le s a p p e a r s likely ( N a t o r i , 1990). I n Drosophila, a n e u r o g l i a n b e l o n g i n g to t h e i m m u n o g l o b u l i n supe r fami ly p l ays a n i m p o r t a n t ro le d u r i n g gl ial a n d n e u r a l cell a d h e s i o n d u r i n g d e v e l o p m e n t of t h e n e r v o u s s y s t e m (Biebe r et al., 1989) a n d a n o t h e r insec t i m m u n o g l o b u l i n l i k e m o l e c u l e ca l led h e m o l i n a p p e a r s to b i n d to t h e surface of b a c t e r i a a n d t a r g e t t h e m for d e s t r u c t i o n ( S u n et al., 1990). H e n c e , s o m e of t h e m e c h a n i s m s involved in d i s t i n g u i s h i n g "self" v e r s u s "non-se l f " d u r i n g a n i m a l d e v e l o p m e n t ve r sus infect ion m a y involve s o m e of t h e s a m e m o l e c u l a r s igna ls a n d r ecogn i t i on p rocesses d i r e c t e d a t ident i fy ing different deg ree s of " fo re ignnes s " in t h e a n i m a l .

Severa l a u t h o r s s t u d y i n g insec t sy s t ems have ident i f ied " p a r a s i t i s m -specif ic" o r " p a r a s i t i s m - i n d u c e d " p r o t e i n s in insec t h o s t s , w h i c h b e c o m e d e t e c t a b l e in t h e h o s t a t v a r y i n g t imes p o s t p a r a s i t i z a t i o n (see Sec t ion I I ) . T h e i r de novo syn thes i s m a y beg in as ea r ly as a few h o u r s after t h e h o s t is p a r a s i t i z e d , o r t h e y m a y n o t a p p e a r in t h e h e m o l y m p h u n t i l t h e p a r a s i t e s h a v e n e a r l y m a t u r e d , w i t h t h e p rec i se t e m p o r a l p a t t e r n of t he i r a p p e a r a n c e v a r y i n g a c c o r d i n g to t h e species of h o s t a n d t h e p a r a s i t e s involved . Severa l species of l e p i d o p t e r a n s (Cook et al., 1984; B e c k a g e et al., 1987, 1989; Sol-dev i l a a n d J o n e s , 1991 , 1993) a n d d i p t e r a n s ( L a w r e n c e , 1990) p a r a s i t i z e d b y e n d o p a r a s i t i c w a s p s h a v e b e e n s h o w n to syn thes i ze pa ras i t i sm-spec i f i c p r o te ins . I n s o m e species (e .g. , Manduca sexta, see Sec t ion I I ) t h e h o s t h e m o l y m p h shows ev idence of t h e p r e s e n c e of different sets of " e a r l y " a n d " l a t e " pa ras i t i sm-spec i f i c p r o t e i n s , w i t h t h e t e m p o r a l p a t t e r n of t he i r exp res s ion


b e i n g prec ise ly r e g u l a t e d . I m p o r t a n t l y , t he p r o t e i n s a p p e a r u n i q u e to p a r a si t ized l a rvae , b e i n g n o n i n d u c i b l e by o t h e r forms of "phys io log ica l s t r e s s " such as h e a t o r cold shock (Fi t t inghoff a n d Ridd i fo rd , 1990; J o p l i n et al., 1990), p a t h o g e n i c infect ion (Beckage et al., 1989; see t he following d i scuss ion ) , o r phys i ca l t r a u m a a n d in jury (i .e. , s h a m inject ions) (Ferkovich et al., 1983). W h i l e n e w p ro t e in s a r e p r o d u c e d in r e sponse to t h e l a t t e r cha l l enges , careful ana lys i s reveals t h a t they differ in m o l e c u l a r we igh t a n d o t h e r cha rac t e r i s t i c s f rom those i n d u c e d by p a r a s i t i z a t i o n . For e x a m p l e , a l t h o u g h w o u n d i n g o c c u r s following p u n c t u r e of t h e h e m o c o e l d u r i n g ovipos i t ion , t h e molecu le s t h a t h a v e t h u s far b e e n c h a r a c t e r i z e d as b e i n g syn thes i zed in r e s p o n s e to phys i ca l in jury (e.g. , s a r c o t o x i n s — s e e K a n a i a n d N a t o r i , 1989) a p p e a r n o t a b l y differen t from those i n d u c i b l e by p a r a s i t i z a t i o n . T h u s , t hey d o n o t r e p r e s e n t gen era l ized s t ress p ro t e in s b u t i n s t ead c o n s t i t u t e a specific r e s p o n s e .

A survey of r ecen t inves t iga t ions sugges t s t h a t t he a b s e n c e of a n y q u a n t i ta t ive o r qua l i t a t i ve effect of p a r a s i t i s m on hos t h e m o l y m p h e n z y m e s o r cons t i tu t ive p ro t e in s likely r ep re sen t s the excep t ion r a t h e r t h a n t h e ru le . T h e i n d u c t i o n of h o r m o n e - r e l a t e d c h a n g e s a lso is now recogn ized as b e i n g c o m m o n l y assoc ia ted w i th insect e n d o p a r a s i t i s m . Hypo the t i ca l l y , t he a n t i b a c ter ia l p ro t e in s i n d u c e d by bac te r i a l cha l l enge (see references in Faye a n d H u l t m a r k , V o l u m e 2, C h a p t e r 2; K a n o s t et al., 1990) m i g h t have close p a r a l lels in insects a t t a cked by mu l t i ce l l u l a r o r g a n i s m s , s u c h t h a t hos t s r e s p o n d by p r o d u c i n g novel p ro t e in s o r p e p t i d e s , w h i c h a r e specifically ( a n d u n i q u e l y ) a s soc ia ted w i t h t he pa r a s i t i z ed s t a t e . Howeve r , t h e s i t u a t i o n is m o r e c o m p l i c a t e d w i t h respec t to p a r a s i t i s m in t h a t in s o m e cases t he p a r a sites have " s y m b i o t i c " o r t h i r d - p a r t y e l e m e n t s s u c h as v i ruses o r b a c t e r i a a s soc ia t ed w i th t h e m , t h u s c o n f o u n d i n g i n t e r p r e t a t i o n of e x p e r i m e n t s d e s igned to p i n p o i n t t he or ig in(s ) of t he c o g n i z a n t molecu les (i .e. , genes of t he hos t or its p a r a s i t e s o r t he e l e m e n t involved) . Moreove r , these p r o t e i n s often a p p e a r co r r e l a t ed wi th successful p a r a s i t i s m , r a t h e r t h a n a lack thereof. D u r i n g n o r m a l d e v e l o p m e n t of t he p a r a s i t e s , t h e p r o t e i n s a r e p r o d u c e d , a n d the i r syn thes i s h e n c e is co r r e l a t ed w i t h p a r a s i t e p r o t e c t i o n a n d a b s e n c e of e n c a p s u l a t i o n . T h u s , t he p a t t e r n of the i r o c c u r r e n c e differs f rom t h a t of t he a n t i b a c t e r i a l p r o t e i n s , w h i c h cons t i t u t e a n efficacious " p r o t e c t i v e " hos t r e s p o n s e t h a t checks p a t h o g e n invas ion .

A n ana lys i s of th is c o m p l e x scena r io w o u l d no t b e c o m p l e t e w i t h o u t reference to p a r a s i t i s m - i n d u c e d modi f ica t ions in the hos t ' s e n d o g e n o u s p r o t e i n s . T i t e r s of m a n y of those molecu les a r e s ignif icant ly a l t e r ed d u r i n g p a r a s i t i s m . Fac to r s a s soc ia ted w i t h p a r a s i t i s m m a y affect t h e r a t e s of t r a n s c r i p t i o n o r t r a n s l a t i o n of hos t genes a n d be r e spons ib l e for i n d u c i n g these c h a n g e s . I n t he leas t c o m p l i c a t e d s i t ua t ion , t he s i m p l e p r e s e n c e of t he p a r a s i t e s w i t h i n t he b o d y cavi ty of t he hos t m a y b e sufficient to affect t h e r a t e s of t r a n s c r i p t ion or t r a n s l a t i o n , t h e r e b y h a v i n g a n i m p a c t on hos t p r o t e i n syn thes i s . T i m e


cou r se s tud i e s sugges t c h a n g e s o c c u r early, before t he w a s p s h a t c h . L a t e r , d e p l e t i o n of a m i n o ac ids a n d o t h e r n u t r i e n t s d u e to p a r a s i t e c o n s u m p t i o n of hos t h e m o l y m p h c o n s t i t u e n t s conce ivab ly cou ld modify t he r a t e of hos t p r o tein syn thes i s , s ince t he pools of p r e c u r s o r s r e q u i r e d for o p t i m a l t r a n s c r i p t i o n a n d t r a n s l a t i o n of hos t m R N A s m i g h t c o n s e q u e n t l y b e p r e s e n t in s u b o p t i m a l c o n c e n t r a t i o n s o r r a t ios d u e to d ive rs ion to " c o m p e t i n g " t i ssues , t h a t is, t h e p a r a s i t e s . T h u s , s o m e m e t a b o l i c effects ( T h o m p s o n , V o l u m e 1, C h a p t e r 6) likely a r e t igh t ly i n t e rwoven w i t h p a r a s i t i s m - i n d u c e d modi f i ca t ions in hos t p r o t e i n p r o d u c t i o n a n d t r a n s p o r t .

Add i t iona l ly , factors sec re ted o r o t h e r w i s e a s soc ia t ed w i t h t h e p a r a s i t e s , s u c h as t e r a tocy te s ( D a h l m a n a n d V i n s o n , V o l u m e 1, C h a p t e r 7) a n d p o l y d -nav i ru se s (Stol tz , V o l u m e 1, C h a p t e r 8; F l e m i n g a n d K r e l l , V o l u m e 1, C h a p te r 9) , m a y a lso have a r e g u l a t o r y inf luence . E x a m p l e s of t h e e n d o g e n o u s p r o t e i n s n o w k n o w n to b e affected b y insec t e n d o p a r a s i t i s m i n c l u d e t hose i n t i m a t e l y a s soc ia t ed w i t h l a rva l d e v e l o p m e n t a n d m e t a m o r p h o s i s (e .g. , a r -y l p h o r i n s ) , r e p r o d u c t i o n (e.g. , v i t e l logen in) , a n d t h e hos t i m m u n e r e s p o n s e (e.g. , p h e n o l o x i d a s e ) . As s o m e of these c h a n g e s a r e d e s c r i b e d in de t a i l in o t h e r c h a p t e r s , on ly a br ief s u m m a r y is p r e s e n t e d h e r e to i l l u s t r a t e t h e n a t u r e a n d ex t en t to w h i c h the hos t ' s e n d o g e n o u s p r o t e i n s a r e modif ied d u r i n g e n d o p a r a s i t i s m .

A n un re so lved , b u t none the l e s s i n t r i gu ing , q u e s t i o n c o n c e r n s t h e d e g r e e to w h i c h these c h a n g e s reflect ac t iv i ty of p a r a s i t e (or p a r a s i t e - a s s o c i a t e d elem e n t ) " d e s i g n e r g e n e s " w h o s e expres s ion d i rec t ly m a n i p u l a t e s t h e phys io logy of t he hos t for t he p a r a s i t e s ' benefi t . T h e c o n c e p t of " h o s t r e g u l a t i o n " is f requent ly invoked by phys io log is t s a n d pa ras i to log i s t s to exp l a in t he a d a p t i v e s ignif icance of m a n y of t he i m m u n o l o g i c a l , m e t a b o l i c , a n d b e h a v i o r a l c h a n g e s o b s e r v e d in hos t o r g a n i s m s d u r i n g p a r a s i t i s m ; yet t he s u p p o r t i n g ev idence for s u c h r e g u l a t o r y p h e n o m e n a is d i s a p p o i n t i n g l y s p a r s e , especia l ly w i t h r e spec t to insec t s y s t e m s . Unfo r tuna t e ly , t he t ru ly i n t i m a t e coevo lu t i ona ry n a t u r e of t h e h o s t - p a r a s i t e r e l a t i o n s h i p r e n d e r s i n t e r p r e t a t i o n of m a n y of t h e b i o c h e m i cal c h a n g e s seen in hos t insec ts exceed ing ly p r o b l e m a t i c , d u e to t h e c o m p l e x i ties involved in unravel ing the web of interact ions. S imul taneously one m u s t cons ider t h e i n t r i g u i n g i n t e r p l a y of t he b i o c h e m i c a l " a t t a c k " s t r a teg ies evolved by p a r a s i t e s a n d t h e begu i l i ng c o u n t e r s t r a t e g i e s evolved b y hos t insec ts for the i r defense , no t to m e n t i o n the roles of p a r a s i t e - a s s o c i a t e d e l e m e n t s .

A m o n g p l a n t pa tho log i s t s , t he i dea of gene-for-gene coevo lu t ion b e t w e e n p a r a s i t e s a n d hos t s h a s n o w b e e n wide ly accep t ed s ince th is c o n c e p t w a s first p r o p o s e d by F lo r (1942) . S ince its i ncep t i on , th is w o r k i n g h y p o t h e s i s h a s g e n e r a t e d m u c h d e b a t e a n d e x p e r i m e n t a l r e s e a r c h , a n d for tu i tous ly h a s led to t he ident i f ica t ion of severa l " v i r u l e n c e " a n d " a v i r u l e n c e " genes in p l a n t p a t h o g e n s , h e n c e verifying its usefulness as a n e x p e r i m e n t a l p a r a d i g m (for review, see T h o m p s o n a n d B u r d o n , 1992). V i r u l e n t g e n o t y p e s r ecen t ly have


also b e e n ident if ied in o t h e r p a r a s i t e s , for e x a m p l e , p r o t o z o a n p a r a s i t e s of m a m m a l s (Sibley a n d B o o t h r o y d , 1992). T h i s c o n c e p t u a l f r a m e w o r k s e e m ingly h a s b e e n less w a r m l y e m b r a c e d by insec t pa ra s i to log i s t s a n d p a r a s i t o i d spec ia l i s t s , w h o h a v e i n t e r p r e t e d t h e p a n o r a m a of h o s t c h a n g e s a s s y m p t o m a t i c of hos t " s t r e s s , " " r e d i r e c t i o n , " o r " r e g u l a t i o n " w i t h o u t a n y d i r ec t reference to the i r i m p o r t a n c e as i n d i c a t o r s of evo lu t ion of p a r a s i t e v i ru l ence cha rac te r i s t i c s r e q u i r e d for hos t i nvas ion or l o n g - t e r m surv iva l in w h a t m i g h t o t h e r w i s e b e a n o n c o m p a t i b l e hos t e n v i r o n m e n t .

However , as r ecen t r e s e a r c h o n m a l a r i a h a s e m p h a s i z e d , a de l i ca t e in te r p l a y of hos t genes e n c o d i n g factors r e l a t ed to suscep t ib i l i ty a n d r e s i s t ance cha rac te r i s t i c s , i n t e r a c t i n g w i t h t h e p r o d u c t s of p a r a s i t e v i ru l ence a n d avir-u l ence genes , likely d e t e r m i n e s t h e o u t c o m e of m a n y h o s t - p a r a s i t e e n c o u n ters (Sayles a n d W a s s o m , 1988). T h e de l i ca t e c o u n t e r b a l a n c e of p a r a s i t e offense ve r sus hos t defense p r e s u m a b l y r equ i r e s a s imi l a r s t r a t e g y of g e n e -for-gene coevo lu t ion t h a t u l t i m a t e l y r e n d e r s t he r e l a t i o n s h i p a "success fu l" o n e w h e n the p a r a s i t e s b e c o m e c a p a b l e of d e v e l o p m e n t to m a t u r i t y w i t h i n a p rev ious ly " h o s t i l e " o r n o n h a b i t u a l hos t species (or s t r a i n the reo f ) . T h e p a r a s i t e t h u s m a y b e cons ide r ed t h e p a r t n e r w i t h t h e " d r i v e r " genes d i r ec t ing t h e hos t t o w a r d evolu t ion of r e s i s t ance - re l a t ed c h a r a c t e r s .

T h e ident i f ica t ion of pa r a s i t e - a s soc i a t ed " v i r u l e n c e " o r "k i l l e r " genes , a n d the i r c o r r e s p o n d i n g a v i r u l a n t m u t a n t al leles , u n f o r t u n a t e l y still awa i t s us w i t h r e spec t to sy s t ems invo lv ing p a r a s i t e s of insec t hos t s . T h e successful express ion of g e n e p r o d u c t s by the i r a s soc ia t ed " t h i r d - p a r t y " e l e m e n t s (e.g. , p o l y d n a v i r u s e s a s soc ia t ed w i t h w a s p s , o r bac t e r i a a s soc i a t ed w i t h n e m a todes) a lso likely is cr i t ica l to d e t e r m i n i n g the o u t c o m e of p a r a s i t i c i n t e r a c t ions invo lv ing a va r i e ty of insec t hos t s . T h u s , in these c o m p l e x s y s t e m s invo lv ing severa l " p a r t n e r s " it a p p e a r s likely t h a t b o t h t h e e l e m e n t ( such as a p o l y d n a v i r u s ) a n d t h e p a r a s i t e m u s t be c a p a b l e of c o m p l e t i n g i ts life cycle to e n s u r e successful p r o p a g a t i o n in t he hos t sys t em, w h i c h c u l m i n a t e s in m a t u r a t i o n of t h e p a r a s i t e s followed by d i s s e m i n a t i o n a n d infect ion of n e w h o s t s .

II. Parasitism- and Polydnavirus-lnduced Peptides and Proteins in Insect Hosts

M a n y s tud ie s have s h o w n t h a t insec t h e m o l y m p h p r o t e i n s a r e a l t e r ed d u r i n g p a r a s i t i s m (see references in following d i scuss ion , p lu s ea r l i e r s t ud i e s b y F i s h e r a n d G a n e s a l i n g a m , 1970; K i n g a n d Rafa i , 1970; V i n s o n a n d B a r r a s , 1970; B a r r a s et al., 1972; B r e w e r et al., 1973; Smi lowi tz , 1973; Smi lowi tz a n d S m i t h , 1977; V i n s o n a n d I w a n t s c h , 1980). I n a few cases of p a r a s i t i s m of l e p i d o p t e r a n l a rvae b y b r a c o n i d a n d i c h n e u m o n i d w a s p s , novel " p a r a s i t i s m -

2. Games Parasites Play 31

specif ic" h e m o l y m p h p r o t e i n s h a v e b e e n s h o w n to b e syn thes i zed de novo fol lowing p a r a s i t i z a t i o n , as i n d i c a t e d by in vivo a m i n o ac id r a d i o l a b e l i n g of t h e p r o t e i n ( s ) p r o d u c e d in newly p a r a s i t i z e d hos t s (Cook et al., 1984; Becka g e et al., 1987). T h i s l ine of ev idence sugges t s t h a t t hey a r e n o t mo lecu l e s s h u t t l e d f rom t h e a d u l t p a r a s i t o i d to t he hos t d u r i n g p a r a s i t i z a t i o n , b u t i n s t e a d r e p r e s e n t newly syn thes i zed molecu le s . M o s t w o r k h a s focused o n assess ing c h a n g e s in h e m o l y m p h - b o r n e molecu le s , w h i c h a r e therefore e m p h a s i z e d in th is c h a p t e r ; a va r i e ty of i n t r ace l lu l a r o r t i s sue -as soc ia t ed c h a n g e s likely a lso o c c u r b u t h a v e b e e n c o n s i d e r a b l y less well s t u d i e d .

T h e p o l y d n a v i r u s e s in jec ted b y t h e w a s p p a r a s i t o i d Campoletis sonorensis t o g e t h e r w i t h t h e eggs in i t i a t e v i ra l g e n e t r a n s c r i p t i o n fol lowing t rans fe r of t h e v i rus from t h e w a s p " v e c t o r " to t he l e p i d o p t e r a n " h o s t . " Different s u b sets of v i ra l genes a r e exp res sed in each species ; a t h i r d set of " c o n s t i t u t i v e " genes a p p e a r s to b e expres sed ini t ia l ly in t h e w a s p ovary a n d expres s ion c o n t i n u e s following t ransfe r to t h e l e p i d o p t e r a n hos t ( T h e i l m a n n a n d S u m m e r s , 1988; F l e m i n g a n d K r e l l , V o l u m e 1, C h a p t e r 9) . I n th i s s y s t e m , m R N A t r a n s c r i p t s have b e e n i so la ted b y h y b r i d i z a t i o n of e x t r a c t e d R N A s to v i ra l p r o b e s , a l t h o u g h t h e c o r r e s p o n d i n g p r o t e i n s awa i t fu ture c h a r a c t e r i z a t i o n (Bl i ssard et al., 1986; T h e i l m a n n a n d S u m m e r s , 1988). T h e s e m a y r e p r e s e n t t h e novel p r o t e i n s d e s c r i b e d in ea r l i e r s tud ie s of p a r a s i t i z e d Heliothis l a rvae ( V i n s o n a n d B a r r a s , 1970; B a r r a s et al., 1972; B r e w e r et al., 1973). I n t e r estingly, s o m e p a r t i c u l a r l y a b u n d a n t "pa ra s i t i sm-spec i f i c " p r o t e i n s de t ec t a b l e in h o s t insec ts a r e i n d e e d p o l y d n a v i r u s - i n d u c e d t r a n s c r i p t s , w h i c h a r e n o t p r o d u c e d w h e n t h e v i rus is i nac t iva t ed (Cook et al., 1984; B e c k a g e et al., 1987; H a r w o o d , 1993; H a r w o o d et al., 1993a) . O t h e r mo lecu l e s a p p a r e n t l y a r e syn thes i zed by t h e d e v e l o p i n g p a r a s i t e l a rvae o r the i r a s soc i a t ed ter-a tocyes , a n d obv ious ly a r e g e n e r a t e d in sufficient a m o u n t s to b e d e t e c t a b l e in t h e hos t ' s b lood ( L a w r e n c e , 1990; Soldevi la a n d J o n e s , 1993). T h u s , t h e o r ig in a n d s i te(s) of p r o d u c t i o n of these p r o t e i n s a r e v a r i a b l e a n d likely species-specif ic , w i t h s o m e p r o t e i n s p r o b a b l y h a v i n g m u l t i p l e exp res s ion loci w i t h i n t h e hos t (wasp , p o l y d n a v i r u s , hos t ) .

T h e m o l e c u l a r we igh t s of t h e p a r a s i t i z a t i o n - i n d u c e d molecu le s d e s c r i b e d t h u s far v a r y f rom < 10,000 d a l t o n s to > 150,000 d a l t o n s , a n d w i t h i n a s ingle hos t , m u l t i p l e p r o t e i n s o r p e p t i d e s m a y b e p r o d u c e d a c c o r d i n g to a t igh t ly r e g u l a t e d t e m p o r a l p a t t e r n v a r y i n g a c c o r d i n g to t h e s t age of p a r a s i t i s m . For e x a m p l e , different se ts of pa ras i t i sm-spec i f i c p r o t e i n s a r e p r o d u c e d in " e a r l y " ve r sus " l a t e " s t age hos t l a rvae of t h e t obacco h o r n w o r m , Manduca sexta, following p a r a s i t i z a t i o n by t h e b r a c o n i d w a s p Cotesia congregata ( D a h l m a n a n d G r e e n e , 1981; B e c k a g e et al., 1987, 1989; B e c k a g e a n d T e m p l e t o n , 1986; B e c k a g e a n d K a n o s t , 1993; H a r w o o d , 1993; H a r w o o d a n d B e c k a g e , 1993). Fo l lowing t h e d i s a p p e a r a n c e of t h e " e a r l y " p o l y p e p t i d e s severa l d a y s pos t -p a r a s i t i z a t i o n , o t h e r " l a t e " a p p e a r i n g p r o t e i n s s u b s e q u e n t l y a p p e a r in las t -


i n s t a r hos t s , w h i c h c o n t i n u e to c i r cu la te in t he hos t ' s h e m o l y m p h un t i l it even tua l ly d ies severa l d a y s following the w a s p s ' e m e r g e n c e (Beckage a n d T e m p l e t o n , 1986) d u e to n e u r e n d o c r i n e d i s r u p t i o n ( Z i t n a n et aL, 1993).

T h e t e m p o r a l p a t t e r n of a p p e a r a n c e of the " l a t e " p r o t e i n s m i r r o r s t h a t seen in o t h e r sys t ems . I n c a b b a g e looper l a rvae p a r a s i t i z e d b y Chelonus n e a r curvimaculatus, a novel h i g h - m o l e c u l a r - w e i g h t p r o t e i n (ca. 160 k D a o n S D S -P A G E gels) a p p e a r s in t he h e m o l y m p h of newly m o l t e d m a t u r e h o s t l a rvae , w h i c h t h e n , as resu l t of p a r a s i t i s m , a r e i n d u c e d to u n d e r g o p recoc ious m e t a m o r p h o s i s a n d w a n d e r following w h a t w o u l d n o r m a l l y b e t he p e n u l t i m a t e i n s t a r ( J o n e s et aL, 1985, 1986; J o n e s , 1989; Soldevi la a n d J o n e s , 1991). T h i s p r o t e i n is of low a b u n d a n c e re la t ive to o t h e r h e m o l y m p h p r o t e i n s , a n d beg ins to a p p e a r a t h e a d capsu l e s l ippage p r e c e d i n g the las t l a rva l ecdysis ; it is u n d e t e c t a b l e in n o n p a r a s i t i z e d la rvae of t h e s a m e species , r ega rd l e s s of the i r s t age of d e v e l o p m e n t . Moreove r , the p r o t e i n pers i s t s in t he h e m o l y m p h of t he hos t un t i l t he w a s p e m e r g e s (Soldevi la a n d J o n e s , 1991, 1993), s imi l a r to t h e " l a t e " p ro t e in s cha rac t e r i s t i c of p a r a s i t i z e d M. sexta.

T h e p r o t e i n fails to a p p e a r in p s e u d o p a r a s i t i z e d T. ni l a rvae , def ined as l a rvae in w h i c h n o p a r a s i t e is p r e s e n t b u t i n t o w h i c h v e n o m a n d p o l y d -n a v i r u s have b e e n injected, t he p a r a s i t e h a v i n g b e e n kil led by chi l l ing or o t h e r e x p e r i m e n t a l m a n i p u l a t i o n s ( J o n e s et aL, 1986), a r g u i n g t h a t t h e p r e s ence of a deve lop ing p a r a s i t e is r equ i s i t e for its syn thes i s (Soldevi la a n d J o n e s , 1991, 1993). Recen t d a t a show the p r o t e i n ac tua l ly o r ig ina t e s f rom the p a r a s i t e s a n d the w a s p s show a posi t ive r eac t ion w i t h a n t i b o d i e s d i r e c t e d a g a i n s t t he p r o t e i n . E v i d e n c e a lso exists for its de novo b iosyn thes i s by t he w a s p s , w h i c h cou ld a lso a c q u i r e t h e p r o t e i n b y inges t ion of t he i r hos t ' s h e m o l y m p h . H e n c e , Soldevi la a n d J o n e s (1991 , 1993) c o n c l u d e d t h a t t h e m o s t likely s cena r io is t h a t the p r o t e i n is p a r a s i t e - d e r i v e d . Ear l i e r , it was t h o u g h t t h a t t he p r e s e n c e of t he m a t u r e w a s p s (or the i r a s soc ia t ed factors) s o m e h o w i n d u c e s t h e hos t ' s t issues to syn thes ize t h e p r o t e i n b e g i n n i n g w h e n the hos t in i t ia tes its final l a rva l mo l t .

T h e p r e d o m i n a n t " e a r l y " expressed paras i t i sm-spec i f ic p o l y p e p t i d e s seen in M. sexta a r e in t he size r a n g e of 3 3 - 3 8 k D a a n d a r e very a b u n d a n t p r o te ins , r e p r e s e n t i n g u p to 8 - 1 0 % of t he to ta l c i r cu l a t i ng h e m o l y m p h p r o t e i n a n a l y z e d a t 24 h r p o s t p a r a s i t i z a t i o n . O n e of t he t h r e e m a j o r b a n d s d e t e c t a b l e in th is size r a n g e h a s b e e n conf i rmed to be a p o l y d n a v i r a l g e n e p r o d u c t ( H a r w o o d , 1993; H a r w o o d et aL, 1993a) . Severa l l ower -molecu la r -we igh t p o l y p e p t i d e s in t he 17- to 2 0 - k D a r a n g e a lso a r e i n d u c e d d u r i n g t h e s a m e t e m p o r a l window, a n d l ikewise d i s a p p e a r s y n c h r o n o u s l y w i t h t h e h igher -m o l e c u l a r - w e i g h t p ro t e in s several d a y s following ovipos i t ion (Beckage et aL, 1989). T h e s i m u l t a n e o u s u p - a n d d o w n - r e g u l a t i o n of severa l of th is a r r a y of " e a r l y " p o l y p e p t i d e s is s t r ik ing a n d sugges t s they m a y b e c o o r d i n a t e l y r egu l a t ed (Beckage et aL, 1987, 1989). Moreove r , s ince all of t h e m a p p e a r i n d u e -


ib le b y in ject ion of pur i f ied p o l y d n a v i r u s i n t o n o n p a r a s i t i z e d M. sexta l a rvae , o u r o b s e r v a t i o n s sugges t t hey m i g h t have a c o m m o n or ig in , t h a t is, coord i n a t e ^ r e g u l a t e d v i ra l genes .

I n p a r a s i t i z e d t o b a c c o h o r n w o r m s a very l a rge p o l y p e p t i d e ( > 1 2 0 k D a ) a p p e a r s as a m a j o r d e n s e b a n d on S D S - P A G E gels b e g i n n i n g a few d a y s fol lowing t h e hos t ' s ecdysis to its final ins ta r , j u s t a few d a y s p r i o r to t h e w a s p s ' e m e r g e n c e (Beckage a n d T e m p l e t o n , 1986). In te res t ing ly , th i s p r o te ins a p p e a r s s y n c h r o n o u s l y w i t h a m u c h less a b u n d a n t ca . 100-kDa polyp e p t i d e a few d a y s p r i o r to w a s p e m e r g e n c e , j u s t a t t h e t i m e t h e w a s p s u n d e r g o the i r first l a rva l ecdysis to t he s econd i n s t a r (Beckage a n d T e m p l e t o n , 1986). T h e onse t of syn thes i s of these p r o t e i n s s eems r e m a r k a b l y well-s y n c h r o n i z e d w i t h th is L j - L 2 t r a n s i t i o n in t h e w a s p s , sugges t ing t hey m a y b e of p a r a s i t e o r ig in o r syn thes i zed b y h o s t t i ssues in r e s p o n s e to t h e p r e s e n c e of th is specific p a r a s i t e s t age . M o r e o v e r , t he i r a p p e a r a n c e is s u p p r e s s e d in hos t s t r e a t e d w i t h t h e insec t g r o w t h r e g u l a t o r a z a d i r a c h t i n , w h i c h ac t s a s a n ecdys is i n h i b i t o r so t he p a r a s i t e s p r o d u c e a s e c o n d - i n s t a r cu t ic le b u t d ie as p h a r a t e L 2 l a rvae t r a p p e d w i t h i n t h e hos t (Beckage et al., 1988). F a i l u r e of t h e p a r a s i t e s to ecdyse p r e v e n t s a p p e a r a n c e of t he " l a t e " p r o t e i n s , fu r the r c o r r o b o r a t i n g the i r co r r e l a t i on w i t h onse t of t he p a r a s i t e s ' L 2 s t age . Yet a n o t h e r pa ras i t i sm-spec i f i c p o l y p e p t i d e of 56 k D a m o l e c u l a r w e i g h t is p r e s e n t in hos t M. sexta h e m o l y m p h b e g i n n i n g a t its ecdysis to t h e las t i n s t a r (Beckage a n d T e m p l e t o n , 1986). I t s t i m i n g of a p p e a r a n c e is r e m i n i s c e n t of t h e p r o t e i n d e t e c t e d in p a r a s i t i z e d c a b b a g e loopers by Soldevi la a n d J o n e s ( 1 9 9 1 , 1993); however , t h e re la t ive a b u n d a n c e a n d m o l e c u l a r we igh t s of t h e p r o t e i n s i so la ted in t he two species a r e s ignif icant ly different, sugges t i ng t hey m a y b e a n a l o g o u s b u t no t i den t i ca l p a r a s i t i s m - a s s o c i a t e d p r o t e i n s .

I n M. sexta w e h y p o t h e s i z e d t h a t t h e p a r a s i t e s m i g h t b e t h e sou rce of t hese " l a t e " p r o t e i n s ; however , c u l t u r i n g t h e p a r a s i t e s in vitro w i t h [ 3 5 S ] m e t h i o n i n e followed b y f l u o r o g r a m ana lys i s of p r o t e i n s sec re ted i n t o t h e m e d i u m over a 24 -h r p e r i o d p r o d u c e d n o ev idence of the i r de novo p r o d u c t i o n by t h e w a s p s , a l t h o u g h severa l p r o t e i n s w e r e r e l eased by t h e w a s p s (Fig . 1). T h e ter-a tocy te s d e v e l o p i n g in t he hos t h e m o c o e l a lso d o n o t a p p e a r to b e t h e synthe t i c s o u r c e (de B u r o n et al., 1993). O u r c u r r e n t w o r k i n g h y p o t h e s i s is t h a t t hey a r e l ikely hos t - r a t h e r t h a n p a r a s i t e - d e r i v e d . Add i t iona l ly , in vivo s t ud i e s h a v e s h o w n t h a t t h e " l a t e " p r o t e i n s pers i s t in t h e h e m o l y m p h of t h e hos t for severa l d a y s fol lowing p a r a s i t e e m e r g e n c e f rom t h e hos t , f u r the r sugges t i ng t hey m a y b e of h o s t or ig in . I n c o n t r a s t to t h e " e a r l y " i n d u c e d p r o t e i n s , t hey c lear ly a r e n o t i n d u c i b l e b y t he in ject ion of p o l y d n a v i r u s i n to n o n p a r a s i t i z e d l a rvae (Beckage et al., 1993), sugges t i ng t h a t t hey d o n o t r e q u i r e ac t iv i ty of v i ra l genes for the i r express ion . I n s u m m a r y , a l t h o u g h t h e " e a r l y " p r o t e i n s r e p r e s e n t p r o t e i n p r o d u c t s of p o l y d n a v i r u s genes , t h e o r ig in of t h e " l a t e " p r o t e i n s d e t e c t e d in t he hos t r e m a i n s ( f rus t ra t ingly) unc l ea r .

3 4

A

Nancy Ε. Beckage

- 9 4 -

- 6 7 -

- 4 3 »

- 3 0

- 2 0 -

Figure Ί (A) Production of radiolabeled polypeptides by Cotesia congregata larvae dissected from host larvae on day 2 (D2) or 3 (D3) of the fifth instar, and cultured for 24 hr with sterile Grace's medium (formulated without methionine added) that was supplemented with [ 3 5 S ] methionine to label proteins synthesized de novo. For culture procedures see de Buron et al. (1993) and Riddiford et al. (1979). Proteins recovered from the media (M), whole-body homogenates of the parasites (P), and parasite

{continues)

I m p o r t a n t l y , n o n e of these p r o t e i n s c o r r e s p o n d s to a n y of t h e a n t i b a c t e r i a l p r o t e i n s cha rac te r i s t i ca l ly p r o d u c e d in n o n p a r a s i t i z e d t o b a c c o h o r n w o r m la rvae following inject ion of n o n p a t h o g e n i c bac t e r i a ( B e c k a g e et al., 1989; K a n o s t et al., 1990; Faye a n d H u l t m a r k , V o l u m e 2, C h a p t e r 2) . N o r does in ject ion of o t h e r D N A v i ruses i n d u c e the i r syn thes i s . Fo r e x a m p l e , in jec t ion of l e tha l dosages of Autographa californica n u c l e a r po lyhed ros i s v i ru s ( N P V ) does n o t i n d u c e t h e s a m e p r o t e i n s (Fig . 2) , i n d i c a t i n g they a r e u n i q u e l y i n d u c e d by p a r a s i t i s m . T h e t obacco h o r n w o r m r e p r e s e n t s a s e m i p e r m i s s i v e h o s t of t h e N P V v i rus a n d lives for a n e x t e n d e d p e r i o d fol lowing in jec t ion of

P a r a s i t e L a r v a e

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M D 2 M D 3 M D 3 P H D 2 P D 2 P D 3 P D 3 P D 3


Figure 1 (continued) hemolymph (PH) were analyzed by SDS-PAGE and fluorography (for techniques, see Beckage et al., 1987, 1989). Note that the media samples contain many radiolabeled polypeptides synthesized and secreted by the wasps; however, the major "parasitism-specific" bands detected in last-instar hosts (Beckage and Templeton, 1986) do not appear to be of wasp origin as determined by comparison to previously published gels (Beckage and Templeton, 1986; Beckage et al., 1989). (B) Appearance of second-instar parasites similar to those used for the in vitro culture experiments described in Fig. l a legend. Note the bulbous anal vesicle at the posterior end of each larva; these were punctured and hemolymph samples from several parasites were pooled and analyzed as described in Beckage et al. (1989). At this stage the mature second-instar parasites are ca. 3 m m in length and weigh 3 mg (Beckage and Riddiford, 1982). Photograph by Frances F. Tan.

th is v i ru s ; in jec ted l a rvae p r o d u c e severa l novel p r o t e i n s in r e s p o n s e to N P V (Fig . 2) a n d u l t i m a t e l y d ie f rom the v i ra l infect ion ca . 1 0 - 1 2 d a y s l a t e r after m o l t i n g to t h e fifth i n s t a r ( G r e t c h et al., 1991). A s ide-by-s ide c o m p a r i s o n shows t h a t t h e p r o t e i n s d e t e c t e d in l a rvae following N P V in jec t ion c lear ly differ in M W from those i n d u c e d following in ject ion of t h e C. congregata P D V (Fig . 2) (Beckage et al., 1987, 1989; H a r w o o d , 1993; H a r w o o d et al., 1993a) . T h u s , these t w o classes of insec t D N A v i ruses i n d u c e different sets of p r o t e i n s to b e exp re s sed in t h e s a m e " h o s t . " T h e s e differences a r e n o t s u r p r i s i n g g iven t h a t a t l eas t s o m e of t he novel p r o t e i n s d e t e c t e d r e p r e s e n t p r o d u c t s of v i ra l

- « 9 4

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AcNPV Control PDV


genes ( H a r w o o d , 1993; H a r w o o d et al., 1993a, b) w h o s e s e q u e n c e s m a y n o t b e h igh ly conse rved a m o n g insec t D N A v i ruses .

U n f o r t u n a t e l y , w h a t is c u r r e n t l y l ack ing is a d i r ec t d e m o n s t r a t i o n of t h e b io logica l s ignif icance of "pa ra s i t i sm-spec i f i c " p r o t e i n s a n d ev idence t h a t t hey p l a y a cr i t ica l role in t he r e l a t i o n s h i p b e t w e e n p a r a s i t e a n d hos t . S ince t h e " e a r l y " p r o t e i n s a p p e a r p r i o r to h a t c h i n g of t h e first-instar p a r a s i t e s , t h e v i ruses a n d the i r g e n e p r o d u c t s m a y serve as p ro tec t ive a g e n t s d u r i n g th is ea r ly v u l n e r a b l e p h a s e of t he w a s p s ' d e v e l o p m e n t , wh i l e d u r i n g l a t e r s t ages t h e p a r a s i t e s t hemse lves b e c o m e c a p a b l e of func t ion ing in th is capac i ty . T h o u g h th is i dea is a t t r ac t i ve b a s e d on the re la t ive t i m e cou r se of a p p e a r a n c e of t h e p r o t e i n s , s t r o n g e x p e r i m e n t a l ev idence for th is h y p o t h e s i s is l ack ing .

C o n c e i v a b l y a t leas t s o m e of t h e p a r a s i t i s m - i n d u c e d p r o t e i n s have a funct iona l ro le in r e g u l a t i n g s o m e of t h e f u n d a m e n t a l i n t e r ac t i ons cha rac t e r i s t i c of i n t i m a t e o r " h a b i t u a l " h o s t - p a r a s i t e r e l a t i onsh ip s d u r i n g ea r ly p h a s e s of t h e r e l a t i onsh ip . I n d e e d , m a n y of t h e paras i t i sm-spec i f i c p r o t e i n s c h a r a c te r ized t h u s far a r e extens ively g lycosy la ted , s imi l a r to o t h e r surface-i n t e r a c t i n g molecu le s . S ince g lycos ida ted r e s idues a r e n o w k n o w n to p l a y m a j o r roles in ce l l - ce l l r ecogn i t i on a n d defense p rocesses in o t h e r a n i m a l sy s t ems as well as p l a n t s , t hey m a y r e p r e s e n t p a r t i c u l a r l y cr i t ica l m o l e c u l a r d e t e r m i n a n t s of p a r a s i t e - h o s t i n t e r ac t i on . For e x a m p l e , t hey cou ld b i n d to p a r a s i t e surfaces a n d p r e v e n t s t i m u l a t i o n of h e m o c y t e e n c a p s u l a t i o n ; a l ter nat ively, t hey cou ld i n t e r ac t w i t h t he cells t hemse lves to elicit t r a n s f o r m a t i o n to t h e n o n e n c a p s u l a t i v e s t a t e (Stol tz , V o l u m e 1, C h a p t e r 8) .

A r e a s o n a b l e coro l la ry m i g h t b e t h a t r e q u i r e d pa ras i t i sm-spec i f i c p r o t e i n s m a y n o t b e p r o d u c e d a t t h e n o r m a l r a t e o r the i r funct ion m a y b e i m p a i r e d in n o n h a b i t u a l i n t e r ac t ions c u l m i n a t i n g in t h e e v e n t u a l d e a t h o r d e m i s e of p a r a s i t e s in " a l i e n " hos t s . C o o k et al. (1984) n o t e d t h a t p r o d u c t i o n of a 5 5 -

Figure 2 SDS-PAGE analysis of hemolymph samples collected from fourth-instar nonparasitized tobacco hornworm larvae 24 hr after injection of 10 6 pfu of the budded form of the Autographa californica nuclear polyhedrosis virus (AcNPV) diluted in sterile Grace's medium, control larvae injected with Grace's only, and larvae injected with ca. 2 wasp equivalents of filter-purified Cotesia congregata polydnavirus (Beckage et aL, 1993). T h e dose of AcNPV used is lethal for the larvae though they live for several days following virus injection (Gretch et al., 1991). Hemolymph sampling and S D S -PAGE analysis were carried out as described previously (Beckage et aL, 1987). Arrows (left) denote novel proteins present only in samples collected from AcNPV-injected larvae; stars (right) denote the "parasitism-specific" proteins induced by injection of PDV (Beckage etaL, 1987, 1989; Harwood, 1993; Harwood etal., 1993a). Note that the majority of the proteins induced by the two agents appear to have different mobilities; a few of the induced proteins in the AcNPV and PDV lanes appear to have roughly similar, though not identical, molecular weights. At least one of the novel proteins produced in the PDV-injected insects represents a viral transcript (see text).


k D a g lycopro te in in hos t l a rvae pa r a s i t i z ed by the i c h n e u m o n Hyposoter exiguae w a s co r r e l a t ed w i t h t he pa ra s i t e ' s e v e n t u a l successful d e v e l o p m e n t ; moreover , t he p r o t e i n was n o t expressed in n o n h a b i t u a l hos t s in w h i c h t h e p a r a s i t e s w e r e e n c a p s u l a t e d . T h e i r s t u d y fur ther showed t h a t t h e p r o t e i n w a s i n d u c i b l e by inject ion of t h e w a s p ' s p o l y d n a v i r u s i n to n o n p a r a s i t i z e d insec t s , w h e r e a s w h e n the v i rus w a s inac t iva ted th is p r o t e i n failed to b e i n d u c e d .

Similar ly, in s p h i n g i d s t h a t a r e n o n p e r m i s s i v e or pa r t i a l l y pe rmi s s ive for t he d e v e l o p m e n t of C. congregata, syn thes i s of t he m a j o r ear ly " p o l y d n a v i r u s -i n d u c e d " p r o t e i n a p p e a r s a l t e r ed (or m a r k e d l y r e d u c e d ) in cases w h e r e p a r a s i t i sm is no t whol ly effective a n d the p a r a s i t e s u l t i m a t e l y b e c o m e e n c a p s u l a t ed ( H a r w o o d , 1993; H a r w o o d et al., 1993b) . Howeve r , t h e co r r e l a t i on of p r o t e i n p r o d u c t i o n w i t h pe rmiss iv i ty is n o t en t i re ly c lea r -cu t (as in a p l u s / m i n u s co r re l a t ion ) , as s o m e p r o d u c t i o n of t h e p r o t e i n occu r s in n o n p e r miss ive hos t s , a lbe i t a t s ignif icant ly r e d u c e d levels; moreover , W e s t e r n b lo t s show t h a t the i r syn thes i s s o m e t i m e s ceases p r e m a t u r e l y in cases w h e r e t h e p a r a s i t e s a r e d e s t i n e d for e n c a p s u l a t i o n ( H a r w o o d , 1993; H a r w o o d et al., 1993b) . W h e t h e r t he r e d u c e d p r o d u c t i o n of these pa ras i t i sm-spec i f i c p r o t e i n s o r a l t e red t i m i n g of the i r syn thes i s c o n t r i b u t e s to t h e p a r a s i t e s ' e n c a p s u l a t i o n a n d fai lure to e m e r g e from the hos t l a rva r e m a i n s to b e e s t ab l i shed ; conceivably, t he lack of p r o t e i n p r o d u c t i o n cou ld r e p r e s e n t a n a s soc ia t ed , b u t n o t c ausa l , p h e n o m e n o n . However , in species t h a t have b e e n d e t e r m i n e d to b e fully pe rmis s ive a l t e rna t i ve hos t species , s u c h as Hyles lineata, t h e full spec t r u m of b o t h " e a r l y " a n d " l a t e " p ro t e in s a r e p r o d u c e d ( H a r w o o d , 1993; H a r w o o d et al., 1993b) . T h e s e o b s e r v a t i o n s s u p p o r t t he h y p o t h e s i s t h a t t he i r syn thes i s m a y be l inked w i th successful p a r a s i t i s m of s p h i n g i d hos t s b y C. congregata b u t a d d i t i o n a l e x p e r i m e n t a l ev idence is r e q u i r e d to yield def ini t ive conc lus ions a b o u t the i r func t iona l ro le(s ) . A fu ture a p p r o a c h will b e to inject a n t i s e n s e nuc leo t ides i n to newly pa ra s i t i z ed hos t s , w h i c h w o u l d b e e x p e c t e d to d e c r e a s e p a r a s i t e success if t h e c o m p l e m e n t a r y g e n e p r o d u c t s a r e a b s o lu te ly essent ia l for successful p a r a s i t i s m .

T h u s , o u r c u r r e n t w o r k i n g hypo thes i s in t h e M. sexta s y s t e m is t h a t t h e " e a r l y " p r o t e i n s s o m e h o w act a t t h e in i t ia l i m m u n o l o g i c a l in terface b e t w e e n p a r a s i t e a n d hos t . W h e r e a r e they p r o d u c e d ? N o r t h e r n b lo t s show t h e p r e d o m i n a n t " e a r l y " p ro t e in s a r e p r o d u c e d p r i m a r i l y by t h e fat b o d y a n d h e m o -cytes , a l t h o u g h o t h e r t i ssues a lso syn thes ize t he m R N A s to a lesser d e g r e e ( H a r w o o d , 1993; H a r w o o d a n d Beckage , 1993). E x p e r i m e n t s u s i n g in vitro c u l t u r e of hos t fat b o d y (Fig . 3) a n d h e m o c y t e s ( d a t a n o t s h o w n ) h a v e verified t h a t t he " e a r l y " p ro t e in s a r e syn thes i zed by fat b o d y cells a n d sec re ted i n t o t he m e d i u m . Similar ly, fat b o d y a n d h e m o c y t e s r e p r e s e n t t h e p r i m a r y t i ssue sources of a n t i b a c t e r i a l p r o t e i n s , wh i l e severa l o t h e r s p l a y a lesser role ( D u n n , 1986; Faye a n d H u l t m a r k , V o l u m e 2, C h a p t e r 2) .

T h e nex t cr i t ica l s t ep will b e to a s say effects of pur i f ied pa ras i t i sm-spec i f i c p r o t e i n s on h e m o c y t e behav io r a n d o t h e r hos t phys io log ica l func t ion to m o r e

Figure 3 Results of SDS—PAGE and fluorograph analysis of proteins synthesized and secreted into Grace's medium by the fat bodies of unparasitized fourth-instar tobacco hornworm larvae (U) and newly parasitized (P) larvae 24 hr postparasitiza-tion. Culture techniques and fluorography were carried out as described for ter-atocyte cultures (de Buron et al., 1993) and cultures of M. sexta tissues (Riddiford et al., 1979). Stars (right) indicate bands present in the cultures of parasitized fat body that were undetected in the control media containing fat body from unparasitized larvae. Note multiple novel bands appearing in the media of the parasitized sample, in the < 2 0 and 30—40 kDa M W ranges; these likely represent the early "parasitism-specific" proteins described in the text (Beckage et al., 1987, 1989; Harwood, 1993).

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22

14


di rec t ly a d d r e s s the i r b iological role . M u l t i c e l l u l a r p a r a s i t e s obv ious ly m u s t ut i l ize different m e c h a n i s m s a s ide from d i rec t ce l lu la r i nvas ion to d i r ec t hos t b i o c h e m i c a l c h a n g e s , a n d these ex t race l lu la r sec re ted molecu le s w o u l d a p p e a r to be o n e p l aus ib l e m e c h a n i s m of phys io log ica l i n t e r ac t i on w i t h t he hos t . As m e n t i o n e d earl ier , s o m e of the bes t c lues a b o u t t h e n a t u r e of t h e r equ i s i t e h o s t - p a r a s i t e m o l e c u l a r i n t e rac t ions m a y ar i se f rom e x a m i n a t i o n of s i t ua t i ons in w h i c h the hos t is comple t e ly n o n p e r m i s s i v e , o r exh ib i t s on ly p a r t i a l suscep t ib i l i ty to p a r a s i t i s m , so t h a t t he p a r a s i t e s beg in d e v e l o p i n g b u t t h e n b e c o m e e n c a p s u l a t e d before e m e r g i n g . Severa l s cena r ios a r e p l a u s i b l e , a n d s ide-by-s ide c o m p a r i s o n s of t he " s u i t e " of b i o c h e m i c a l c h a n g e s o c c u r r i n g in suscep t ib l e , n o n p e r m i s s i v e , a n d semipe rmis s ive hos t s m a y facil i tate d i s c r i m i n a t i o n of t he m o s t cr i t ical genes w h o s e express ion i n v a r i a b l y is l inked to "successfu l" p a r a s i t i s m (or, a l te rna t ive ly , cons t i t u t e a n effective " a n t i p a r a s i t e p r o t e c t i o n " ) . A n o t h e r poss ibi l i ty is t h a t t he a b s e n c e (or a t leas t r e d u c e d syn thes i s ) of " i n c o m p a t i b l e " hos t p r o t e i n s m a y r e p r e s e n t w h a t is r e q u i r e d . I n t he fo rmer case , p r e s u m a b l y t he n o r m a l express ion of a n y cr i t ica l genes w o u l d b e h a m p e r e d (poss ibly to ta l ly i nh ib i t ed ) in a n o n p e r m i s s i v e hos t env i r o n m e n t , w h e r e a s express ion of nonc r i t i ca l genes w o u l d n o t s h o w a n y m o d ifying effect of e n d o p a r a s i t i s m . N o n p e r m i s s i v e hos t s t h u s w o u l d a p p e a r s imila r to n o n p a r a s i t i z e d a n i m a l s , w h e r e a s "successful ly" p a r a s i t i z e d i n d i v i d u a l s shou ld show a n a t t e n u a t i o n o r s i lenc ing of express ion of a n y t ru ly n o n c o m -p a t i b l e p r o t e i n w h o s e p r e s e n c e m i g h t t a rge t t he p a r a s i t e for e n c a p s u l a t i o n . T h e s e m i g h t r e p r e s e n t " a s s a s s i n " genes , t he express ion of w h i c h m u s t b e k n o c k e d o u t by t he p a r a s i t e ( s ) to p r e v e n t a po t en t i a l l y l e tha l s i t ua t i on f rom deve lop ing .

I n h o r n w o r m la rvae injected w i th l a rge a m o u n t s of P D V , t he " e a r l y " p ro t e in s a r e i n d u c e d , b u t o t h e r u n i q u e p ro t e in s even tua l ly a p p e a r , w h i c h a r e neve r seen in n a t u r a l l y p a r a s i t i z e d hos t s o r n o n p a r a s i t i z e d l a rvae . Specifically, a d m i n i s t r a t i o n of two or m o r e w a s p equ iva l en t s of pur i f ied C. congregata p o l y d n a v i r u s i n d u c e s t he " e a r l y " p ro t e in s d e s c r i b e d ear l ier , b u t a lso s o m e p r o t e i n s n o t n o r m a l l y d e t e c t a b l e following n a t u r a l e x p o s u r e to w a s p p a r a s i to ids ; a t 6 - 1 0 d a y s pos t in jec t ion large a m o u n t s of heavi ly g lycosy la ted 18-to 2 2 - k D a M W p o l y p e p t i d e s a r e d e t e c t a b l e in these P D V - t r e a t e d " h o s t s " (Beckage et aL, 1993). E i t h e r these p a r t i c u l a r p r o t e i n s a r e n o t i n d u c i b l e d u r i n g n a t u r a l p a r a s i t i s m , o r for s o m e r e a s o n the i r syn thes i s is s u p p r e s s e d in hos t s w h o s e p a r a s i t e s deve lop normal ly . Al te rna t ive ly , t hey m a y be i n d u c i b l e on ly by inject ion of m e g a d o s e s of p o l y d n a v i r u s no t typ ica l ly de l ive red b y female w a s p s to hos t s d u r i n g p a r a s i t i z a t i o n . In t r igu ing ly , these e n i g m a t i c p r o t e i n s beg in to a p p e a r w h e n the l a rvae beg in to show m a j o r p i g m e n t a t i o n a n o m a l i e s d u e to P D V inject ion, w i th t he i n t e g u m e n t a c q u i r i n g a d i s t inc t ive rosy co lo ra t ion (Beckage et aL, 1990), b u t w h e t h e r t he two p h e n o m e n a , t h a t is, t h e a p p e a r a n c e of t he p ro t e in s a n d p i g m e n t s , a r e phys io log ica l ly l inked is


u n k n o w n . S imi l a r p i g m e n t a t i o n c h a n g e s o c c u r in n a t u r a l l y p a r a s i t i z e d larvae , b u t t hey a r e often e x a g g e r a t e d in l a rvae in jected w i t h l a rge a m o u n t s of P D V . A d d i t i o n a l s tud ie s a r e u n d e r w a y to c h a r a c t e r i z e these u n u s u a l p r o te ins a n d assess the i r poss ib le r e l evance to o t h e r phys io log ica l c h a n g e s in d u c e d b y P D V inject ion.

O t h e r poss ib le roles of t h e " e a r l y " i n d u c e d p r o t e i n s i n c l u d e i n d u c t i o n of hos t m e t a b o l i c o r b e h a v i o r a l effects, o r m o d u l a t i o n of t h e hos t ' s e n d o c r i n e o r n e u r o e n d o c r i n e s y s t e m c u l m i n a t i n g in i n d u c t i o n of hos t a r r e s t . S u c h ac t ion a p p e a r s p a r t i c u l a r l y p l a u s i b l e for t h e " l a t e " p ro t e in s p r o d u c e d d u r i n g t h e p e r i o d w h e n t h e hos t shows m u l t i p l e m e t a b o l i c a n d h o r m o n a l d e v i a t i o n s f rom t h e n o r m a l n o n p a r a s i t i z e d s t a t e . Howeve r , e x p e r i m e n t a l ev idence for a role of p e p t i d e s o r p r o t e i n s in a l t e r ing t h e hos t ' s e n d o c r i n e s t a t u s , behav io r , a n d d e v e l o p m e n t a l p r o g r a m m i n g r e m a i n s a " m i s s i n g l ink" (Beckage , 1990) a n d the i r p o t e n t i a l r e g u l a t o r y role in gove rn ing s u c h c h a n g e s r e m a i n s p u r e l y specu l a t i ve a t th is po in t .

Amaz ing ly , t he d e g r e e to w h i c h insec t p a r a s i t e s t hemse lves invoke c h a n g e s in t h e h e m o l y m p h p r o t e i n c o n t e n t of the i r hos t h a s rece ived surp r i s ing ly l i t t le a t t e n t i o n . T h e p a u c i t y of e x p e r i m e n t a t i o n in th is a r e a s h a r p l y c o n t r a s t s w i t h r a p i d r ecen t p r o g r e s s in ident i f ica t ion of t h e s p e c t r u m of mo lecu le s sec re t ed by p a r a s i t e s of v e r t e b r a t e hos t s ; l i tera l ly t h o u s a n d s of r e cen t p a p e r s h a v e d e s c r i b e d m e m b r a n e - a s s o c i a t e d p r o t e i n s a n d secret o r y / e x c r e t o r y a n t i g e n s in m a n y spec ies , u sua l ly w i t h a focus o n exp lo i t ing t h a t i n f o r m a t i o n for t he p u r p o s e s of d i sease d i agnos i s a n d vacc ine d e v e l o p m e n t (see t h e fol lowing). I n d e e d , g iven t h e lack of r e s e a r c h d i r e c t e d t o w a r d specific c h a r a c t e r i z a t i o n of p a r a s i t e - o r p a r a s i t o i d - s e c r e t e d molecu le s in insect hos t s , o n e m i g h t logical ly a s s u m e t h a t e n d o p a r a s i t i c w a s p s r e p r e s e n t a " s i l en t p a r t n e r " o r m i n o r c o m p o n e n t in t he phys io log ica l i n t e r a c t i o n b e t w e e n h o s t a n d p a r a s i t e . T h e c o m p r e h e n s i v e p i c t u r e likely i nc ludes c o n t r i b u t i o n s from severa l i n t e r a c t i n g c o m p o n e n t s , i n c l u d i n g t h e p a r a s i t e s as well as the i r a s soc i a t ed t e r a tocy t e s a n d p o l y d n a v i r u s e s , p lus t h e t issues of t h e hos t w h o s e phys io log ica l func t ions a r e va r ious ly modif ied d u r i n g p a r a s i t i s m .

For e x a m p l e , s tage-specif ic p r o t e i n s have b e e n s h o w n to b e p r o d u c e d b y e m b r y o s of Microplitis croceipes (Ferkovich a n d D i l l a rd , 1986; T i l d e n a n d Fer-kovich , 1987) a n d Copidosoma floridanum (Baeh recke et al., 1992), b u t w h e t h e r a n y e m b r y o - d e r i v e d p r o t e i n s a r e sec re ted in to t he s u r r o u n d i n g h o s t t i ssues w a s n o t d e t e r m i n e d in t h e s tud ies c i ted . I n t he t e p h r i t i d fruitfly Anastrepha suspensa, t h e se rosa l cells (i .e. , t e r a tocy te s ) of t h e b r a c o n i d w a s p Biosteres longicaudatus p r o d u c e a 2 4 - k D a m o l e c u l a r we igh t pa ras i t i sm-spec i f i c p o l y p e p t ide d e t e c t e d in t h e p h a r a t e p u p a l hos t ; t h e p a r a s i t e s a lso secre te t h e s a m e p r o t e i n , sugges t i ng it cou ld b e p r o d u c e d b y all p a r a s i t e - d e r i v e d cell l ineages ( L a w r e n c e , 1990). O t h e r t e r a tocy t e - sec re t ed molecu les a r e d e s c r i b e d b y D a h l m a n (1991) , D a h l m a n a n d V i n s o n ( V o l u m e 1, C h a p t e r 7), a n d d e Bur -


on et al. (1993) . I n t he l a t t e r p a p e r , m u l t i p l e ( > 3 0 ) p r o t e i n s were o b s e r v e d to b e syn thes ized a n d sec re ted by t e r a tocy te s of C. congregata; d u e to t h e l a rge n u m b e r s ( l i teral ly h u n d r e d s of t h o u s a n d s ) of cells p r e s e n t in each hos t , t he i r c o n t r i b u t i o n to t he hos t h e m o l y m p h mi l ieu w o u l d be s ignif icant .

I n o n e case , a p e p t i d e p rev ious ly c h a r a c t e r i z e d as "pa ra s i t i sm-spec i f i c " was r ecen t ly d e t e c t e d in n o n p a r a s i t i z e d i nd iv idua l s of t h e s a m e species ( H a y -a k a w a , 1992). T h u s , t he p r ev ious d e s i g n a t i o n of this p e p t i d e as " p a r a s i t i s m -specif ic" w a s a m i s n o m e r ( H a y a k a w a , 1990, 1992) w i t h t he a u t h o r n o w c o n c l u d i n g t h a t p a r a s i t i s m causes a n o m a l o u s mi sexp re s s ion of th is e n d o g e n o u s mo lecu l e a t a s t age l a te r t h a n it n o r m a l l y w o u l d b e d e t e c t e d ( H a y a k a w a , 1992) (see t he following). I n c o n t r a s t , t he p r o t e i n s c i ted ear l i e r a p p e a r to be t ru ly "pa ras i t i sm-spec i f i c " a n d c a n n o t b e " t u r n e d o n " in n o n p a r a s i t i z e d insects sub jec ted to v a r i o u s forms of phys io log ica l s t ress o r m a n i p u l a t i o n , a s ide f rom p a r a s i t i s m .

B e c a u s e of the i r u n i q u e assoc ia t ion w i t h p a r a s i t i z e d insec t s , a fu ture aven u e for exp lo i t a t ion of paras i t i sm-spec i f ic hos t h e m o l y m p h p r o t e i n c h a n g e s m a y be to ut i l ize prote in-speci f ic p r o b e s to conf i rm p a r a s i t i z a t i o n in cases w h e r e t h e hos t s a r e s imp ly too sma l l to b e easi ly d i s sec ted . W e s t e r n slot b lo t s o r E L I S A tests t h e n cou ld b e u sed to deve lop insec t " s q u a s h b l o t s " to est i m a t e t he p e r c e n t a g e of pa r a s i t i z ed insects in t h e hos t insec t p o p u l a t i o n . I n a g r i c u l t u r a l en tomology , a n a c c u r a t e a s s e s s m e n t of t h e p e r c e n t a g e of insec ts p a r a s i t i z e d following re lease of b iological con t ro l a g e n t s is cr i t ica l to m o n i t o r ing t he success of b iocon t ro l p r o g r a m s , ye t t e s t ing is difficult to c a r r y o u t , p a r t i c u l a r l y w i t h l a rge p o p u l a t i o n s of sma l l insec t s . J u s t as p a r a s i t e -a s soc ia t ed a n t i g e n s a r e now be ing ut i l ized for de t ec t i on of p a r a s i t i c infect ion in m a m m a l i a n hos t s , a n d for de t ec t i on of a r t h r o p o d - b o r n e p a r a s i t e s (e .g . , m a l a r i a , l e i s h m a n i a ) in vec to rs , so too m i g h t t he pa ras i t i sm-spec i f i c a n t i g e n s d e s c r i b e d h e r e be used for d i a g n o s t i c p u r p o s e s in s c r een ing insec ts for evid e n c e of p a r a s i t i s m .

A n o t h e r s t r a t egy for " t e c h n o l o g y t r ans fe r " m a y b e to ut i l ize g e n e t rans fe r to explo i t genes e n c o d i n g paras i t i sm-spec i f ic p ro t e in s for gene t i c e n g i n e e r i n g of p a r a s i t e s , poss ib ly u s i n g p o l y d n a v i r u s p r o m o t e r s d i r e c t i n g " e a r l y " exp re s s ion of c ruc ia l genes n e e d e d for successful i n t e rac t ion . S i t e -d i rec ted m u t a genes is first c a n b e u s e d to identify t h e m o s t cr i t ica l d o m a i n s n e e d e d for a p p r o p r i a t e i n t e rac t ion , w h i c h t h e n w o u l d b e t r ans fe r red to o t h e r p a r a s i t e s o r p o l y d n a v i r u s e s ; o n e goa l m i g h t b e to r e n d e r a species h a v i n g a n e x t r e m e l y n a r r o w hos t r a n g e m o r e p r o m i s c u o u s , for e x a m p l e . M o l e c u l a r " a s s a s i n " molecu le s m i g h t be f o r m u l a t e d for t a r g e t i n g to hos t h e m o c y t e s , t h e r e b y r en d e r i n g t h e m i n c a p a b l e of m o u n t i n g a successful defense . A l t h o u g h the se s t r a teg ies m a y b e cons ide red futur is t ic a t th is j u n c t u r e , r e cen t successes in g e n e t ransfer a n d t r a n s f o r m a t i o n in o t h e r b iological con t ro l a g e n t s s u c h as beneficial p r e d a t o r y mi t e s (P resna i l a n d Hoy, 1992) a r e e n c o u r a g i n g a n d


l ead to t h e o p t i m i s t i c p r e d i c t i o n t h a t th is t ype of b io t echno log ica l a p p r o a c h m i g h t b e m a d e feasible for p a r a s i t i c species in t h e n e x t d e c a d e .

III. Parasitic Effects on Endogenous Proteins, Enzymes, and Peptides

A s i d e f rom s t i m u l a t i n g de novo syn thes i s of n e w p r o t e i n s , p a r a s i t i s m freq u e n t l y a lso d r a m a t i c a l l y affects t h e t i te rs of e n d o g e n o u s h o s t h e m o l y m p h p r o t e i n s . T h e s e c h a n g e s m a y a r i se v ia a l t e r a t i ons in t h e r a t e ( s ) of t r a n s c r i p t ion of h o s t genes o r t r a n s l a t i o n a l even t s . N o t surpr i s ingly , t h e levels of to ta l h e m o l y m p h p r o t e i n often a r e u l t i m a t e l y affected, w i t h s o m e spec ies of h o s t insec ts s h o w i n g a d e c r e a s e in to ta l h e m o l y m p h p r o t e i n w h e r e a s o t h e r s d i s p l a y a s ignif icant i nc rease . As ide f rom h a v i n g d i r ec t effects o n h o s t p r o t e i n express ion , p a r a s i t i s m m a y a lso a l t e r h o s t b lood v o l u m e , t h e r e b y ind i r ec t ly affecting p r o t e i n c o n c e n t r a t i o n ; h e m o l y m p h v o l u m e m a y in t u r n b e a l t e r e d v ia effects o n d iu re s i s , food a n d fluid i n t a k e , o r o t h e r p rocesses affecting t h e d i l u t i o n of e n d o g e n o u s p r o t e i n s in t h e b lood . T h u s , h o s t h e m o l y m p h p r o t e i n c o n c e n t r a t i o n reflects t he s u m m a t i o n of ac t ion of m u l t i p l e phys io log ica l facto r s .

H o w m i g h t t h e express ion of hos t genes b e affected b y p a r a s i t i s m ? Severa l h y p o t h e t i c a l r o u t e s a r e p l aus ib l e . S o m e a l t e r a t i ons m a y ar i se v ia m e c h a n i s m s m e d i a t e d b y effects o n t r a n s c r i p t i o n factors , he l icases , R N A p roces s ing e n z y m e s , o r o t h e r modif iers t h a t u l t i m a t e l y i m p a c t t h e r a t e of hos t g e n e express ion . T r a n s l a t i o n a n d p r o t e i n p roce s s ing m a y a lso b e modif ied (see t h e fol lowing). Pa ra s i t i c effects on hos t n u t r i t i o n a n d t h e re la t ive a b u n d a n c e of a m i n o ac ids a n d o t h e r mo lecu le s r e q u i r e d for syn thes i s of specific e n z y m e s , p e p t i d e s , a n d cons t i t u t ive o r t r a n s p o r t p r o t e i n s m i g h t a l so b e e x p e c t e d to h a v e a d a m p i n g effect o n t he r a t e of h o s t p r o t e i n syn thes i s . O b v i o u s l y o n e c o m p l i c a t i n g factor is t h a t t h e feeding b e h a v i o r of t he h o s t f r equen t ly is a l t e r e d d u r i n g p a r a s i t i s m . T h i s inf luences t h e r a t e of n u t r i e n t i n t a k e re la t ive to t h a t o c c u r r i n g in n o n p a r a s i t i z e d insec t s , a n d m a n y species of p a r a s i t i z e d insec ts s h o w a dec l ine o r c o m p l e t e cessa t ion of food c o n s u m p t i o n d u r i n g t h e final s t ages of p a r a s i t i s m . Levels of a m i n o ac ids a r e often r e d u c e d w h e n t h e h o s t insec t is e x p e r i e n c i n g s e m i s t a r v a t i o n a n d is feeding a t a s ignif icant ly s lower r a t e , t h u s d e c r e a s i n g n u t r i e n t t ransfe r f rom t h e g u t to t h e h e m o l y m p h . A n a c r o s s - t h e - b o a r d d e c r e a s e in all h e m o l y m p h p r o t e i n s logical ly m i g h t e n s u e ; however , in m a n y cases t h e effects of p a r a s i t i s m a r e pro te in-spec i f ic , w i t h s o m e p r o t e i n s s h o w i n g n o a l t e r a t i o n s o r q u a n t i t a t i v e effects, w h e r e a s o t h e r s s h o w signif icant i nc reases o r d e c r e a s e s d u r i n g p a r a s i t i s m . T h i s select ivi ty s t rong ly a r g u e s t h a t a nonspeci f ic lower ing o r s h u t d o w n in t h e r a t e of h o s t p r o t e i n syn thes i s is n o t r e spons ib l e . I n s t e a d , t h e effects a p p e a r h igh ly


specific a n d t a r g e t e d to ce r t a in genes w h o s e levels of express ion a r e va r ious ly modif ied d u r i n g p a r a s i t i s m .

O n e class of p ro t e in s c o m m o n l y affected d u r i n g p a r a s i t i s m of l a rva l s t age hos t s is t he a r y l p h o r i n s . T h e s e h i g h - m o l e c u l a r - w e i g h t s t o r a g e p ro t e in s a r e syn thes ized d u r i n g the l a rva l feeding s t age a n d funct ion as a n a m i n o acid reservoi r for use d u r i n g d e v e l o p m e n t a l r e s t r u c t u r i n g ( W e b b a n d R idd i fo rd , 1988a ,b) . L a b e l i n g s tud ies have s h o w n t h a t t he a m i n o acid c o m p o n e n t s of a r y l p h o r i n a r e pe r iod ica l ly b r o k e n d o w n a n d t h e n i n c o r p o r a t e d in to t h e cut ic le a n d o t h e r t issues d u r i n g la rva l m o l t i n g a n d m e t a m o r p h o s i s , p r o v i d i n g ev idence for the i r role in a m i n o acid " r e p r o c e s s i n g " ( W e b b a n d R idd i fo rd , 1988a) . D u r i n g s t a r v a t i o n a n d o t h e r nonfeed ing pe r iods s u c h as m o l t i n g , t he syn thes i s of a r y l p h o r i n ceases t rans ien t ly , b u t is qu ick ly r e s u m e d u p o n re s u m p t i o n of feeding ( W e b b a n d Ridd i fo rd , 1988a ,b) .

T w o d i s t inc t t r e n d s a r e ev iden t in p a r a s i t i z e d insec ts . I n a few i n s t a n c e s p a r a s i t i s m causes p recoc ious express ion of h igh levels of a r y l p h o r i n s d u r i n g w h a t w o u l d n o r m a l l y be the p e n u l t i m a t e ins ta r , as exempl i f ied b y w a s p s b e l o n g i n g to t he g e n u s Chelonus, w h i c h i nva r i ab ly i n d u c e th is p h e n o m e n o n in hos t l e p i d o p t e r a n l a rvae ( J o n e s et al., 1985, 1986; J o n e s , 1989; K u n k e l et aL, 1990). I n o t h e r s , h e m o l y m p h a r y l p h o r i n levels a r e m a r k e d l y d e p r e s s e d d u r ing p a r a s i t i s m , as seen in l e p i d o p t e r a n la rvae p a r a s i t i z e d by severa l species of Cotesia. For e x a m p l e , in fifth-instar l a rvae of M. sexta p a r a s i t i z e d b y C. congre-gata, t he hos t ' s h e m o l y m p h shows a t e m p o r a l dec l ine in t h e c o n c e n t r a t i o n of a r y l p h o r i n following hos t ecdysis to t he fifth ins ta r , w h e r e a s t h a t of s imi la r ly aged n o n p a r a s i t i z e d a n i m a l s exh ib i t s a d r a m a t i c fourfold i nc rea se in ary l p h o r i n c o n t e n t (Beckage a n d K a n o s t , 1993). In te res t ing ly , d e s p i t e t h e lowered level of h e m o l y m p h a r y l p h o r i n , t he fat b o d y of p a r a s i t i z e d l a rvae cont a ins r o u g h l y s imi l a r a r y l p h o r i n m R N A levels (pe r m g t issue) as t hose d e t e c t a b l e in t i ssues excised from u n p a r a s i t i z e d p r e w a n d e r i n g l a rvae , as d e t e r m i n e d by N o r t h e r n h y b r i d i z a t i o n s ca r r i ed o u t u s ing r a d i o l a b e l e d M. sexta a r y l p h o r i n p r o b e s to a n a l y z e fat b o d y m R N A s (Beckage a n d W e b b , u n p u b l i s h e d d a t a ) . T h e l a t t e r s t u d y a lso showed t h a t these m R N A s a r e t r a n s l a t ab l e in vitro u s ing the t e c h n i q u e s of W e b b a n d Ridd i fo rd (1988b) . I n s u c h s i t ua t ions w h e r e n o r m a l m R N A levels c o n t i n u e to be p r o d u c e d a l t h o u g h h e m o l y m p h t i ters of the p r o t e i n a r e m a r k e d l y d e p r e s s e d , o n e logical conc lu sion is t h a t a r y l p h o r i n express ion m a y be r e g u l a t e d pos t t r ansc r ip t i ona l ly . T h u s , s imi la r to h e a t shock ( a n d o t h e r s t ress - re la ted) p r o t e i n s , v e r t e b r a t e t r a n s p o r t p r o t e i n s , a n d n u m e r o u s v i ra l gene p r o d u c t s ( I l a n , 1987; T h a c h , 1992; Her shey , 1992), a r y l p h o r i n p r o t e i n express ion d u r i n g p a r a s i t i s m m a y b e r e g u l a t e d pos t - t r ansc r ip t iona l ly , r a t h e r t h a n a t ear l ie r s t eps in f luenc ing the r a t e of a c c u m u l a t i o n of hos t m R N A s . P e r h a p s t r a n s l a t i o n a l con t ro l is logical for t he insec t s t o r age p r o t e i n s , w h o s e syn thes i s m a y be qu ick ly u p - o r d o w n - r e g u l a t e d s y n c h r o n o u s w i t h r a p i d c h a n g e s in n u t r i t i o n a l s t a t e ; d u r i n g


nonfeed ing p e r i o d s , i ts a c c u m u l a t i o n s tops ( W e b b a n d R idd i fo rd , 1988a) . P l aus ib l e m e c h a n i s m s m i g h t involve fas t -ac t ing t r a n s l a t i o n b lock ing a g e n t s or o t h e r factors . As o t h e r a u t h o r s have n o t e d a d e c r e a s e in hos t h e m o l y m p h s t o r a g e p r o t e i n t i te rs following p a r a s i t i z a t i o n by r e l a t ed species of Cotesia ( J u n n i k k a l a , 1966), t he m e c h a n i s m s effecting a s u p p r e s s i o n of a r y l p h o r i n a c c u m u l a t i o n m a y b e w i d e - r a n g i n g a m o n g species .

B e c a u s e s t o r a g e p r o t e i n s r e p r e s e n t a l a rge p r o p o r t i o n of t h e to ta l h e m o l y m p h p r o t e i n , p a r t i c u l a r l y d u r i n g the p r e w a n d e r i n g feeding p e r i o d i m m e d ia te ly p r e c e d i n g the onse t of m e t a m o r p h o s i s , it is no t s u r p r i s i n g to find t h a t p a r a s i t i s m f requen t ly causes a lower ing of t he to ta l p r o t e i n c o n t e n t of t h e b lood d u r i n g this pe r iod . H y p o p r o t e i n e m i a h a s b e e n r e p o r t e d in a w i d e r a n g e of p a r a s i t i z e d insec ts , i n d i c a t i n g t h a t it likely is a c o m m o n effect of p a r a s i t i s m ( V i n s o n a n d I w a n t s c h , 1980). Effects a r e often c o m p l e x , t h a t is, in f o u r t h - i n s t a r newly p a r a s i t i z e d M. sexta l a rvae , t he to ta l p r o t e i n c o n c e n t r a t ion in i t ia l ly is e leva ted following ovipos i t ion ( p r e s u m a b l y d u e to i n d u c t i o n of t he pa ras i t i sm-spec i f i c p ro t e ins ) t h e n is s ignif icant ly d e c r e a s e d re la t ive to t he p r o t e i n c o n c e n t r a t i o n m e a s u r e d in n o n p a r a s i t i z e d l a rvae , following m o l t ing to t he fifth i n s t a r (Beckage et αι., 1989). T h i s l a t t e r dec l i n ing t r e n d likely reflects t he re la t ive d e c r e a s e in a r y l p h o r i n t i ters (Beckage a n d K a n o s t , 1993). T h u s , b o t h t r a n s i t o r y hype r - a n d h y p o - r e g u l a t o r y effects c a n b e o b s e r v e d in t he s a m e hos t .

M i g h t a n o t h e r factor c o n t r i b u t i n g to hos t h y p o p r o t e i n u r i a b e p a r a s i t e u p t a k e of hos t h e m o l y m p h p ro te ins? Pa ra s i t e eggs d o n o t a p p e a r to a b s o r b o r ut i l ize hos t a p o p r o t e i n s o r the i r d e g r a d a t i o n p r o d u c t s (Ferkovich a n d Di l -l a r d , 1986) t h o u g h t h e s i t u a t i o n m i g h t be different w i t h l a rvae . P rev ious ly we h a d s u s p e c t e d t h a t t h e r a p i d l y d e v e l o p i n g s e c o n d - i n s t a r C. congregata l a rvae s e q u e s t e r e d a r y l p h o r i n following u p t a k e from the hos t h e m o l y m p h . H o w ever, W e s t e r n b lo ts u s ing a n t i b o d i e s to M. sexta a r y l p h o r i n s h o w e d t h a t t h e l a t t e r p r o t e i n w a s u n d e t e c t a b l e in h e m o l y m p h or w h o l e - b o d y h o m o g e n a t e s of t h e w a s p s ; a l t h o u g h t h e w a s p s a p p e a r e d to have e n d o g e n o u s s t o r a g e p r o te in l ike mo lecu l e s w i t h m o l e c u l a r we igh t s s imi la r to M. sexta a r y l p h o r i n s u b u n i t s , t h e w a s p p r o t e i n s d id n o t c ross - reac t w i t h t h e M. sexta a r y l p h o r i n a n t i b o d i e s (Beckage et al., 1989). Howeve r , t h e poss ib i l i ty still exists t h a t h o s t p r o t e i n s i n c l u d i n g a r y l p h o r i n a r e c o n s u m e d , r a p i d l y d e g r a d e d , t h e n re p roces sed b y t h e w a s p s . Moreove r , p a r a s i t o i d s t h a t a r e c u l t u r e d in vitro freq u e n t l y r e q u i r e t h e p r e s e n c e of p r o t e i n - o r l i pop ro t e in - r i ch s u p p l e m e n t s (in t h e form of fetal bov ine s e r u m , bov ine s e r u m a l b u m i n , etc .) in t h e m e d i u m to faci l i tate n o r m a l d e v e l o p m e n t (Greany , 1986; Ferkovich et al., 1991; Pen-n a c h i o et al., 1992). I t h a s t h u s b e e n inferred t h a t acqu i s i t i on of h o s t p r o t e i n s m i g h t b e cr i t ica l in fulfilling t he p a r a s i t e s ' n o r m a l n u t r i t i o n a l r e q u i r e m e n t s , a l t h o u g h the specific roles of hos t p r o t e i n s o r l i pop ro t e in s h a v e yet to b e e s t ab l i shed .


A r y l p h o r i n is n o t t h e on ly hos t h e m o l y m p h p r o t e i n affected b y p a r a s i t i s m . H e m o l y m p h se rp in levels a l so a r e d r a m a t i c a l l y lowered d u r i n g t h e final s t ages of p a r a s i t i s m by C. congregata (Beckage a n d K a n o s t , 1993). Se r ine p r o t e a s e s a r e i n t i m a t e l y involved in m o d u l a t i n g act iv i ty of p h e n o l o x i d a s e s a n d p a r t i c i p a t e as well in o t h e r e n z y m e ca scades ; t h u s , i n d u c t i o n of s o m e modi f ica t ions in hos t b i o c h e m i c a l r e g u l a t o r y p a t h w a y s m i g h t b e e x p e c t e d to e n s u e c o n c o m i t a n t w i t h a r e d u c t i o n in s e rp in t i te rs . A n t i b a c t e r i a l p r o t e i n s a lso f requen t ly a r e lowered d u r i n g p a r a s i t i s m , t h u s e n h a n c i n g sens i t iv i ty to p a t h o g e n i c infect ion in m a n y species (Brooks , V o l u m e 2, C h a p t e r 11). P a r a si t ized M. sexta l a rvae a r e m o r e suscep t ib l e to bac t e r i a l infect ion as c o m p a r e d to n o n p a r a s i t i z e d l a rvae , a n d b o t h cell- a n d h u m o r a l - m e d i a t e d i m m u n e m e c h a n i s m s likely a r e s u p p r e s s e d d u r i n g p a r a s i t i s m (Ross a n d D u n n , 1989).

Howeve r , t he vas t ma jo r i ty of hos t h e m o l y m p h p r o t e i n s in M. sexta r e m a i n unaffected b y p a r a s i t i s m a n d effects of p a r a s i t i s m a p p e a r h igh ly select ive. I n c o n t r a s t to t h e d e p r e s s i o n in levels of a r y l p h o r i n a n d se rp in s , i n sec t i cyan in a n d l i p o p h o r i n a r e p r e s e n t a t n e a r - n o r m a l c o n c e n t r a t i o n s in t h e b lood (Beckage a n d K a n o s t , 1993). T h e a b s e n c e of a n y effect o n l i p o p h o r i n is s u r p r i s i n g for two r e a s o n s . F i r s t , p r ev ious s tud ies have n o t e d a r e d u c t i o n in l ipid a n d e leva t ion in g lycogen c o n t e n t of t h e fat b o d y of p a r a s i t i z e d l a rvae c o m p a r e d to n o n p a r a s i t i z e d l a rvae ( T h o m p s o n et aL, 1990); th i s effect o n s to red l ip ids m i g h t logical ly be expec t ed to a l t e r t he t i te rs of l ip id t r a n s p o r t p r o t e i n s c i r cu l a t i ng in t h e b lood , yet t h e levels r e m a i n u n a l t e r e d . T h e lack of effect o n l i p o p h o r i n w a s a l so u n e x p e c t e d g iven the r e d u c e d we igh t s of p a r a s i t i z e d l a rvae . T h u s , p a r a s i t i z e d l a rvae p rov ide s o m e clues as to its m o d e of r egu l a t ion in t h a t t h e l i p o p h o r i n t i ter of 3 - to 4-g hos t l a rvae w i t h newly e m e r g e d w a s p s is t h e s a m e as t h a t r e p o r t e d for 8- to 10-g n o n p a r a s i t i z e d p r e w a n d e r -ing l a rvae , sugges t ing t h a t l a rva l we igh t p e r se h a s n o m a j o r r e g u l a t o r y inf luence (Beckage a n d K a n o s t , 1993). L i p o p h o r i n h a s r ecen t ly b e e n s h o w n to s ignif icant ly r e d u c e h e m o c y t e a d h e s i o n b e h a v i o r ( C o o d i n a n d Caveney , 1992), l e a d i n g th is a u t h o r to specu la t e : M i g h t t h e re la t ive a b u n d a n c e of th is p r o t e i n be r e l a t ed to t h e cha rac t e r i s t i c " t r a n s f o r m a t i o n s " in hos t h e m o c y t e b e h a v i o r o c c u r r i n g d u r i n g p a r a s i t i s m ?

A n o t h e r hos t p r o t e i n f requent ly affected by p a r a s i t i s m is t h e e n z y m e p h e -no lox idase (a lso see C h r i s t e n s e n a n d Severson , V o l u m e 1, C h a p t e r 11; D a h l -m a n a n d V i n s o n , V o l u m e 1, C h a p t e r 7). I n insec ts a t t a c k e d b y i c h n e u m o n i d (e.g. , Hyposoter exiguae) a n d b r a c o n i d {Cotesia congregata) w a s p s , p o l y d n a v i r a l i nvas ion of hos t h e m o c y t e s elicits a r e d u c t i o n of p h e n o l o x i d a s e ac t iv i ty (Stol tz a n d C o o k , 1983; Beckage et aL, 1990), poss ib ly b y affecting t r a n s c r i p t ion of t h e hos t p h e n o l o x i d a s e gene ( s ) , o r s u b s e q u e n t t r a n s l a t i o n of t h e m R N A s following v i rus u p t a k e by the cells. A n o t h e r i n t e r p r e t a t i o n w a s p r e sen t ed b y S roka a n d V i n s o n (1978) , w h o a t t r i b u t e d p h e n o l o x i d a s e i n h i b i t i o n in p a r a s i t i z e d Heliothis l a rvae to ty ros ine deficiency; in o t h e r spec ies , p h e -


no lox ida se i n h i b i t o r s m a y a lso b e p r e s e n t in p a r a s i t i z e d l a rvae ( F u h r e r a n d Wi l l e r s , 1986; K i t a n o et al, 1990).

Bacu lov i ruses , w h i c h i n v a d e h o s t h e m o c y t e s s imi l a r to p o l y d n a v i r u s e s , a l so c a u s e a s u p p r e s s i o n of h e m o l y m p h p h e n o l o x i d a s e ac t iv i ty a n d m e l a n i z a -t ion ( A n d e r s o n s et al, 1990), sugges t ing t h a t a w i d e r a n g e of v i ruses m a y e m p l o y a c o m m o n m e c h a n i s m to d o w n - r e g u l a t e p h e n o l o x i d a s e p r o d u c t i o n in infected insec t s . Poss ibly a n y t ype of v i ra l o r bac te r i a l infect ion t h a t h a s a d v e r s e effects o n h e m o c y t e s , i n c l u d i n g p a r a s i t i s m b y e n t o m o p h a g o u s n e m a todes a n d the i r a s soc ia t ed b a c t e r i a (Yokoo et al., 1992), m a y l ikewise i n h i b i t p h e n o l o x i d a s e activity, s ince t h e e n z y m e n o r m a l l y is p r o d u c e d b y these cells a n d re leased i n t o t h e p l a s m a . H e m o c y t e d e p l e t i o n o r p a t h o l o g y m i g h t a l so c o n t r i b u t e to a lowered capac i ty of t h e hos t to p r o d u c e a n t i b a c t e r i a l p r o t e i n s , as well a s o t h e r s of h e m o c y t e o r ig in .

E n z y m e s cr i t ica l to e n d o c r i n e r e g u l a t i o n a r e a lso i m p a c t e d by p a r a s i t i s m , t h e r e b y affecting t he hos t ' s d e v e l o p m e n t a l p r o g r a m . T h e e n z y m e s k n o w n to b e affected b y p a r a s i t i s m i n c l u d e j u v e n i l e h o r m o n e es t e rase , w h i c h is e i t he r d e p r e s s e d to n e a r n o n d e t e c t a b l e levels (Beckage a n d Ridd i fo rd , 1982; D a h l m a n et al, 1990; H a y a k a w a , 1990; S t r a n d et al, 1990; Z h a n g et al, 1992) o r p recoc ious ly i n c r e a s e d ( J o n e s et al, 1986), a n d e c d y s o n e 2 0 - m o n o o x y g e n a s e m a y a lso b e affected (Beckage a n d T e m p l e t o n , 1986). B o t h of t hese e n z y m e s a r e n o r m a l l y p r o d u c e d by t h e fat body, sugges t ing o n c e a g a i n t h a t th is t i s sue r e p r e s e n t s a m a j o r s i te w h e r e effects of p a r a s i t i s m a r e exp re s sed o r " t r a n s d u c e d , " t h e r e b y modi fy ing the hos t ' s phys io logy a n d d e v e l o p m e n t . I n m a n y species , t h e fat b o d y of p a r a s i t i z e d l a rvae often shows m a j o r deb i l i t a t ive effects of p a r a s i t i s m in t h a t it fails to pro l i fe ra te a t t h e n o r m a l r a t e , p e r h a p s t h u s a c c o u n t i n g for s o m e of t h e i n h i b i t o r y effects of p a r a s i t i s m o n e n z y m e p r o d u c t i o n . A g a i n , p a r a s i t i c effects a p p e a r t a r g e t e d to specific e n z y m e s , as exempl i f ied b y t h e fact t h a t in M. sexta h e m o l y m p h k e t o r e d u c t a s e ac t iv i ty a p p e a r s unaffected by p a r a s i t i s m ( D . B . G e l m a n a n d Ν . E . B e c k a g e , u n p u b l i s h e d ) . T h i s e n z y m e conver t s t h e 3 - d e h y d r o e c d y s o n e p r o d u c e d b y t h e p r o t h o r a c i c g l a n d s to e c d y s o n e (Ki r i i sh i et al, 1990) a n d a p p e a r s to b e a loof to a n y modi fy ing effect of p a r a s i t i s m , in c o n t r a s t to J H es t e ra se t i te rs in t h e s a m e l a rvae , w h i c h a r e d r a m a t i c a l l y r e d u c e d . T h u s , hos t h o r m o n a l c h a n g e s m a y a r i se f rom p a r a s i t i c effects o n t h e ac t iv i ty of r e g u l a t o r y e n z y m e s in t h e p a t h w a y s l e a d i n g from p r e c u r s o r s to h o r m o n e s to m e t a b o l i t e s , b u t n o t all e n z y m e s a r e affected equal ly .

H a y a k a w a (1990, 1991) r e p o r t e d t h e p r e s e n c e of a g r o w t h - b l o c k i n g j u v e ni le h o r m o n e e s t e ra se supp re s s ive factor in t h e h e m o l y m p h of l a s t - i n s t a r l a rvae of t h e a r m y w o r m Pseudaletia separata p a r a s i t i z e d b y t h e w a s p Apanteles ( = Cotesia) kariyai. A l t h o u g h ini t ia l ly h e h y p o t h e s i z e d t h a t th is factor of 4500 k D a M W w a s u n i q u e l y p r e s e n t in p a r a s i t i z e d l a rvae ( H a y a k a w a , 1990, 1991) a n d ac t ed to p r e v e n t the i r m e t a m o r p h o s i s b y r ep re s s ing J H e s t e r a se activity,


a m o r e r ecen t s t u d y ( H a y a k a w a , 1992) i n d i c a t e d t h a t t he factor is n o r m a l l y p r e s e n t in p e n u l t i m a t e - s t a g e a n d y o u n g e r n o n p a r a s i t i z e d l a rvae b u t d i s a p p e a r s in the las t ins ta r . The re fo r e , p a r a s i t i s m e x t e n d s t h e p e r i o d w h e n the p e p t i d e is p r o d u c e d , p r e s u m a b l y by s u p p r e s s i n g m e t a m o r p h o s i s .

T h i s p e p t i d e is h y p o t h e s i z e d to be r e q u i r e d for m a i n t e n a n c e of l a rva l cha rac te r i s t i c s a n d s p e c u l a t e d to be a h o r m o n e l i k e mo lecu l e ( H a y a k a w a , 1992). However , a n o t h e r i n t e r p r e t a t i o n is p l aus ib l e . In jec t ion of pur i f ied p e p t i d e i n to n o n p a r a s i t i z e d la rvae causes i nh ib i t i on of food c o n s u m p t i o n a n d g r o w t h ( H a y a k a w a , 1991), a n d lack of n o r m a l feeding b e h a v i o r m i g h t b e expec t ed to d e p r e s s levels of h e m o l y m p h j u v e n i l e h o r m o n e es t e rase . P r ev ious s tud ies have s h o w n t h a t s t a r v a t i o n of n o n p a r a s i t i z e d l a rvae e levates t he J H t i te r a n d dep re s se s J H es te rase , t h e r e b y d e l a y i n g m e t a m o r p h o s i s ( C y m -borowsk i et al., 1982). T h u s , effects of the p e p t i d e o n J H es t e rase cou ld b e ind i rec t , m e d i a t e d via o t h e r m e c h a n i s m s such as r e d u c t i o n of t h e hos t ' s feeding activity.

IV. Comparisons with Parasitism- and Virus-Induced Changes in Other Systems

Simi l a r i n t e rac t ions involv ing p r o t e i n s a n d p e p t i d e s occu r in a w i d e r a n g e of p a r a s i t i z e d a n i m a l s . I n m a m m a l s , p a r a s i t i s m i n d u c e s m a j o r a l t e r a t i o n s in t he p r o t e i n s a n d o t h e r c o m p o n e n t s of t he hos t ' s b lood , f r equen t ly affecting levels of the hos t ' s e n d o g e n o u s p ro t e in s in con junc t ion w i t h i n d u c i n g n e w p ro t e in s ( some of w h i c h a r e pa r a s i t e -de r ived ) to a p p e a r in t h e c i r cu l a t i on . A var ie ty of pa r a s i t e - s ec r e t ed molecu les a r e re leased in to t h e hos t mi l i eu , a n d s o m e a r e n o w ut i l ized as d i agnos t i c tools to d o c u m e n t t h e o c c u r r e n c e of infect ion. Severa l of these so-cal led m e m b r a n e - a s s o c i a t e d a n t i g e n s , e x o a n -t igens , o r e x c r e t o r y / s e c r e t o r y p r o d u c t s have b e e n p roven to have a s ignif icant i m p a c t on the i r hos t ' s phys io logy (Turco , 1990; B lax t e r et al., 1992; L o k e r et al., 1992; N o d a , 1992). I n a d d i t i o n to b e i n g ex t r eme ly useful for d i agnos i s of a c u t e a n d c h r o n i c p a r a s i t e m i a s , s o m e of these molecu le s a r e a lso b e i n g exp lo i ted in d e v e l o p m e n t of n e w m e t h o d s of vacc ine f o r m u l a t i o n b a s e d u p o n these pa r a s i t e -de r i ved a n t i g e n s (see references in C o o m b s a n d N o r t h , 1991).

A n excep t iona l ly w e l l - d o c u m e n t e d e x a m p l e of t h e i n d u c t i o n of syn thes i s of novel p e p t i d e s by p a r a s i t i s m t h a t s u b s e q u e n t l y in ter fere w i t h n o r m a l hos t funct ion is i l l u s t r a t ed by s tud ies of sch is tosome- infec ted sna i l s ( s u m m a r i z e d in d e J o n g - B r i n k , 1992). T h e hos t exper iences p a r a s i t i c c a s t r a t i o n , a n d the m e c h a n i s m s c a u s i n g cessa t ion of r e p r o d u c t i o n in t h e f resh-wate r sna i l Lym-naea stagnalis h ave now b e e n ident i f ied. T h e p r i m a r y m e d i a t o r of c a s t r a t i o n is a novel p e p t i d e t e r m e d sch i s tosomin , w h i c h h a s a p r o n o u n c e d i n h i b i t o r y


effect o n r e p r o d u c t i o n of t h e hos t sna i l (Schal l ig et al., 1991; d e J o n g - B r i n k , 1992), a n d h a s b o t h p e r i p h e r a l a n d c e n t r a l effects o n t h e phys io logy of t h e hos t sna i l . T h i s p e p t i d e is i n d u c e d by t h e p r e s e n c e of s c h i s t o s o m e ce rca r i ae in t h e hos t , conce ivab ly by h u m o r a l factors sec re ted by t h e p a r a s i t e s . I t h a s a n i n h i b i t o r y effect o n r e p r o d u c t i o n by a n t a g o n i z i n g t h e ac t ion of t h e sna i l s ' female g o n a d o t r o p i c h o r m o n e s ( c a u d o d o r s a l cell h o r m o n e a n d ca l f luxin) , s u p p r e s s i n g the i r s t i m u l a t o r y ac t ion o n , respect ively, t h e g o n a d a n d t h e a l b u m e n g l a n d , t h e female accessory sex g l a n d t h a t p r o d u c e s per iv i te l l in fluid to n o u r i s h t h e d e v e l o p i n g e m b r y o (de J o n g - B r i n k et al., 1988). S c h i s t o s o m i n a l so h a s c e n t r a l effects o n t he n e u r o e n d o c r i n e cen te r s r e g u l a t i n g r e p r o d u c t ion a n d g r o w t h , specifically by ac t ing o n the c a u d o d o r s a l cells a n d l ight g r e e n cells (de J o n g - B r i n k , 1992; d e J o n g - B r i n k et al., 1992). A l t h o u g h it p r ev ious ly w a s s p e c u l a t e d t h a t e cdys t e ro id s sec re ted by t h e p a r a s i t e s i n d u c e t he syn thes i s of s ch i s to somin by hos t t i ssues , e x p e r i m e n t s sugges t th is is n o t t h e case (Schal l ig , 1991). T h u s , t h e p r o x i m a t e i n d u c i n g fac tor(s) , as well a s t h e t i ssues p r o d u c i n g t h e m , awa i t fu ture i so la t ion a n d c h a r a c t e r i z a t i o n . Conce ivab ly , t he ident i f ica t ion of t hese r e g u l a t o r y mo lecu le s , p l u s t h e m o d u l a t o r y factors in f luenc ing s ch i s to somin p r o d u c t i o n , u l t i m a t e l y m a y yie ld i n f o r m a t i o n r e l evan t to d e v e l o p m e n t of n e w s t ra teg ies to c o n t r o l p ro l i fe ra t ion of un in fec ted sna i l s .

I n p a r a s i t i z e d c r a b s , p r o t e i n s t h a t a p p e a r "pa ra s i t i sm-spec i f i c " ( H e r b e r t s , 1982) have b e e n i so la ted ; s o m e of these a p p e a r to i n h i b i t hos t sper m a t o g e n e s i s a n d r e p r o d u c t i o n ( R u b i l i a n i , 1983), b u t c o n s i d e r a b l y less is k n o w n a b o u t t hose molecu le s c o m p a r e d to s ch i s to somin . I n insec t s , severa l species of insec t p a r a s i t o i d s c a u s e hos t c a s t r a t i o n by i n h i b i t i n g n o r m a l deve l o p m e n t of t h e g o n a d s d u r i n g the l a rva l s t age ( J u n n i k k a l a , 1985; R e e d -L a r s e n a n d B r o w n , 1990). T h o u g h it is c lea r t h a t t h e a g e n t s c a u s i n g th i s effect a r e t r ans fe r r ed to t he hos t d u r i n g a def ined t e m p o r a l w i n d o w a t p a r a s i t i za t ion , v i r tua l ly n o t h i n g is k n o w n a b o u t t he n a t u r e of t h e m o l e c u l a r m e d i a t o r s s u p p r e s s i n g d i f ferent ia t ion of t he tes tes ( R e e d - L a r s e n a n d B r o w n , 1990; B r o w n a n d K a i n o h , 1992). P r o t e i n s (or pep t i de s ) m a y m e d i a t e c a s t r a t ion a s o c c u r s d u r i n g s u p p r e s s i o n of s p e r m a t o g e n e s i s in c r a b s infected w i t h r h i z o c e p h a l a n b a r n a c l e p a r a s i t e s ; t h e l a t t e r p a r a s i t e s secre te mo lecu l e s (est i m a t e d to be 25 to 30 k D a m o l e c u l a r we igh t ) t h a t ac t o n t h e hos t g o n a d direct ly , a s well as h a v i n g ind i r ec t effects m e d i a t e d v ia t h e s inus g l a n d a n d t h e h o s t n e u r o s e c r e t o r y s y s t e m ( R u b i l i a n i , 1983).

S tud i e s of o t h e r p a r a s i t e t a x a have conf i rmed t h a t t hey sec re te a n a s t o u n d i n g n u m b e r of p o t e n t mo lecu le s t h a t i n c l u d e , a s ide f rom h o r m o n e s , a b a t t e r y of p r o t e a s e s a n d o t h e r p r o t e i n s ( C o o m b s a n d N o r t h , 1991 ; Beie r et al., 1992), ch i t i nases (Schle in et al., 1992), cyc looxygenases ( K a i s e r et al., 1992), g r o w t h - p r o m o t i n g factors ( P h a r e s a n d W a t t s , 1988), a n d o t h e r p e p t ides ( T h o r n d y k e , 1990). T h e s e p a r a s i t e - g e n e r a t e d " c o c k t a i l s " likely influ-


ence t h e hos t ' s phys io logy to v a r y i n g deg rees , m o s t often for t h e p a r a s i t e ' s u l t i m a t e benefi t . S o m e in t eg ra l m e m b r a n e p ro t e in s a p p e a r to b e p a r t i c u l a r l y cr i t ica l in t h e h o s t - p a r a s i t e interface; these a r e i m p r e g n a t e d in t h e p a r a s i t e s ' surface a n d ac t p ro tec t ive ly to p r e v e n t i m m u n o r e c o g n i t i o n p rocesses f rom b e i n g mob i l i zed to des t roy t h e p a r a s i t e s (B lax te r et al., 1992) o r m e d i a t e c e l l -cell r ecogn i t i on d u r i n g invas ion of hos t cells. Fo r e x a m p l e , surface lec t ins m a y m e d i a t e con t ac t a n d c y t o a d h e r e n c e w i t h specific s u b s e t s of h o s t cells ( T h e a et al., 1992). A n t i g e n s h e d d i n g a lso occu r s a n d a l lows t h e p a r a s i t e to c o n t i n u o u s l y r e n e w the molecu les exposed on its surface, a l lowing n e w g e n e p r o d u c t s to be expres sed d u r i n g different s tages of p a r a s i t i s m . R e c e p t o r s for hos t h o r m o n e s a n d g r o w t h factors m a y a lso be exp res sed o n t h e p a r a s i t e ' s surface , t h u s faci l i ta t ing a r e s p o n s e to hos t h o r m o n e s o r i m m u n e m o d u l a t o r s s u c h as t u m o r necros is factor re leased d u r i n g a n i m m u n e r e s p o n s e to p a r a s i t ic infect ion ( A m i r i et al., 1992). Unfo r tuna t e ly , o u r k n o w l e d g e of t h e a n t i g e n s a s soc i a t ed w i t h , a n d secre ted by, p a r a s i t e s deve lop ing in insec t hos t s a r e c o n s i d e r a b l y less well c h a r a c t e r i z e d in c o m p a r i s o n to those p r o d u c e d b y p r o t o z o a n , h e l m i n t h , a n d o t h e r p a r a s i t e s t h a t i n v a d e a n d pro l i fe ra te in m a m m a l i a n hos t s .

S imi l a r to p a r a s i t e s , m a n y v i ruses l ikewise affect t r a n s c r i p t i o n a n d / o r t r a n s l a t i o n of h o s t g e n e p r o d u c t s , a n d m a y d o so w i t h o u t p e r t u r b i n g v i t a l cell func t ions . For e x a m p l e , s tud ies of l y m p h o c y t i c c h o r i o m e n i n g i t i s v i ru s ac t ion in p i t u i t a r y cells sugges t t he v i rus interferes w i t h t r a n s c r i p t i o n of g r o w t h h o r m o n e genes , v ia a n effect o n g r o w t h h o r m o n e t r a n s a c t i v a t o r factor, b u t expres s ion of p ro l ac t i n a n d o t h e r r e q u i r e d genes r e m a i n s unaffec ted (de la T o r r e a n d O l d s t o n e , 1992). T h e l a t t e r a u t h o r s c o n c l u d e d t h a t a va r i e ty of non ly t i c v i ruses t h a t pe r s i s t en t ly infect cells of t h e e n d o c r i n e , i m m u n e , o r n e r v o u s t issues m a y c a u s e select ive effects on h o r m o n e p r o d u c t i o n , i m m u n e funct ion , a n d n e u r o t r a n s m i t t e r re lease , w h i c h here tofore w e r e n o t r ecogn ized as h a v i n g a v i ra l etiology.

H o w m i g h t these findings r e l a t e to t h e p h e n o m e n a o c c u r r i n g in p a r a si t ized insects? M a n y of t he phys io log ica l c h a n g e s o b s e r v e d in insec t hos t s m a y reflect ac t ion of these a g e n t s a s soc ia t ed w i t h insec t p a r a s i t e s . C o n c e i v ably, t h e p o l y d n a v i r u s e s h a v e s imi l a r non ly t i c , b u t none the l e s s h igh ly m o d ula tory , roles in t h e t a rge t l e p i d o p t e r a n insec ts in jec ted w i t h these v i ruses . M o r e o v e r , severa l r e cen t s tud ies have verified t h a t p o l y d n a v i r u s D N A "pe r s i s t s " in hos t insec t cells for t he d u r a t i o n of t he a s soc ia t ion b e t w e e n p a r a s i t e a n d hos t , as d e m o n s t r a t e d u s ing d o t o r slot b lo ts for de t ec t i on of " l i n g e r i n g " v i ra l D N A s (Stol tz et al., 1988; F . F . T a n a n d Ν . E . Beckage , u n p u b l i s h e d ; S t r a n d et al., 1992). H e n c e , " l a t e n t " infect ion of cells of t h e fat b o d y a n d o t h e r t i ssues cou ld inf luence hos t gene express ion from the m o m e n t of t h e in i t ia l con t ac t b e t w e e n p a r a s i t e a n d hos t un t i l t he w a s p s successfully m a t u r e , so t h a t s m a l l dosages of P D V m a y affect hos t e n d o c r i n e r e g u l a t i o n as well as i m m u n i t y ( D u s h a y a n d Beckage , 1993).


W h a t r e m a i n s to be u n r a v e l e d a r e t h e p o t e n t i a l m e c h a n i s m s involved in modi fy ing hos t g e n e expres s ion . Clear ly , t h e fact t h a t s o m e p r o t e i n s a r e select ively d e p r e s s e d whi l e o t h e r s a r e m a i n t a i n e d a t t i te rs a p p r o a c h i n g nor m a l levels a r g u e s s t rong ly t h a t p a r a s i t i s m h a s c o m p l e x effects u p o n t h e h o s t g e n o m e . V i r a l t r a n s c r i p t i o n factors a n d t r a n s l a t i o n a l con t ro l m e c h a n i s m s affect hos t g e n e express ion in a w i d e r a n g e of a n i m a l s (no t to m e n t i o n p l a n t s ) , sugges t i ng t h a t p o l y d n a v i r u s e s , a c t i ng i n d e p e n d e n t l y o r c o o r d i n a t e l y w i t h t h e p a r a s i t e s c a r r y i n g t h e m , m a y h a v e factors a s soc i a t ed w i t h t h e m c a p a b l e of in f luenc ing hos t g e n e express ion for t he d u r a t i o n of t h e p a r a s i t i c r e l a t i onsh ip .

T h u s , h o s t genes m a y be r e g u l a t e d e i the r a t t h e t r a n s c r i p t i o n a l level ( P t a s h n e , 1988) o r a t s u b s e q u e n t p o i n t s in t h e p r o t e i n s y n t h e t i c p a t h w a y t h r o u g h a l t e r a t i o n s in t h e r a t e of t r a n s l a t i o n of hos t m R N As invo lv ing in i t i a t ion , e l onga t i on , a n d p roces s ing of t h e n a s c e n t p r o t e i n s . T h e l a t t e r m a y o c c u r v ia d i r ec t i n t e r r u p t i o n of t h e n o r m a l t r a n s l a t i o n p rocess , o r ind i rec t ly v ia s u p p r e s s i o n of p r o t e i n syn thes i s d u e to c o m p e t i t i o n of t he hos t a n d i ts p a r a si tes for ava i l ab le n u t r i e n t s a n d t h e a m i n o ac ids r e q u i r e d for p r o t e i n s y n t h e sis. S ince t h e effects of p a r a s i t i s m often a r e h igh ly specific, t h e l a t t e r p h e n o m e n o n a p p e a r s un l ike ly to b e t he m a j o r factor a c c o u n t i n g for m a n y of t h e o b s e r v e d c h a n g e s in t h e h e m o l y m p h p r o t e i n profile in hos t insec t s . A l t h o u g h t h e ident i f ica t ion of t he m e c h a n i s m s of t r a n s c r i p t i o n a l / t r a n s l a t i o n a l c o n t r o l a n d i so la t ion of t r a n s c r i p t i o n factors , t r a n s l a t i o n a l r ep re s so r p r o t e i n s , a n d o t h e r modi fy ing e l e m e n t s h a s p r o g r e s s e d r a p i d l y in m a m m a l i a n a n d o t h e r s y s t e m s , we u n f o r t u n a t e l y have on ly r u d i m e n t a r y i n fo rma t ion a b o u t t hese p rocesses in insec t s . O b v i o u s l y g r e a t cha l l enges awa i t us w i t h r e g a r d to d e c i p h e r i n g t h e m e c h a n i s m s w h e r e b y p a r a s i t i s m a l t e r s t h e h e m o l y m p h p r o te ins of insec t hos t s , a n d a t p r e s e n t we c a n on ly s p e c u l a t e a b o u t t h e f r equency of t he i r o c c u r r e n c e a n d p o t e n t i a l ro le(s) a t t h e in terface b e t w e e n p a r a s i t e a n d hos t . W e c a n on ly a s s u m e t h a t m a n y of t he o b s e r v e d hos t c h a n g e s l ikely h a v e s ignif icant select ive a d v a n t a g e s for t h e p a r a s i t e s involved (as e x e m plified b y t h e a d v a n t a g e o u s i n d u c t i o n of insec t hos t b e h a v i o r a l c h a n g e s re p o r t e d h e r e i n by H o r t o n a n d M o o r e ) , a n d work f rom t h a t p o i n t f o rw a rd in assess ing t he i r ro les . A l t h o u g h m a n y p o i n t s in th is r ev iew h a v e b e e n obv i ous ly a n d de l i be ra t e ly h igh ly specu la t ive , hopeful ly t hey will s t i m u l a t e n e w d i r ec t i ons in r e s e a r c h a n d p rov ide a s t r o n g i m p e t u s for fu tu re m o l e c u l a r s t ud i e s of these c o m p l e x , t h o u g h inc red ib ly fasc ina t ing , i n t e r ac t i ons in insec t hos t s .

Acknowledgments

The author sincerely acknowledges Drs. Bruce Webb, Davy Jones, Steve Harwood, Mitch Dushay, and several other reviewers for their critical comments; Ms. Frances Tan, Ms. Therese Reignier, and Ms. Dorothy Nesbit for technical assistance. Many undergrade assisted with


various aspects of this work; Anthony Nguyen, Lisa Cherubin, Roni Lane, and Trang Vo deserve special mention. Studies of the Manduca sexta system cited in this review were funded by grants from the NSF, USDA, and University of California-Riverside, all of which are gratefully acknowledged by the author.

The author dedicates this review, as well as her efforts in assembling these volumes, to her son Ross, in joyful celebration of his tenth birthday and his infectious enthusiasm for nature of all sorts; and acknowledges family members, especially her mother Pauline and brother Paul, and many friends for their enduring and much-appreciated support.

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Chapter 3 • • ι • . , ·

Hormonal Interactions between Insect Endoparasites and Their Host Insects Pauline O. Lawrence Beatrice Lanzrein Department of Zoology Division of Developmental Biology University of Florida Zoological Institute Gainesville, Florida University of Berne

Berne, Switzerland

I. Introduction and Terminology

II. Hormones Controlling Insect Development: An Overview

III. Parasite Hormones Relative to Those of Their Hosts

IV. Direct or Indirect Host Endocrine Effects on Endoparasites

V. Direct or Indirect Effects of Endoparasites on Host Endocrine System

A. Substances Secreted by Serosal Membranes/Teratocytes

B. Calyx Fluid, Viruses, and Venom Gland Secretions

VI. Limitations

VII. Possible Solutions

VIII. Summary Acknowledgments References

I. Introduction and Terminology

Para s i t i c o r g a n i s m s l iv ing w i t h i n the i r hos t s necessar i ly e n c o u n t e r a r a n g e of phys io log ica l a n d b i o c h e m i c a l c o n d i t i o n s t h a t reflect t h e d e v e l o p m e n t a l , m a t u r a t i o n a l , a n d s t r e s s - re la t ed c h a n g e s t h e hos t s u n d e r g o . T h e i m m u nolog ica l , n u t r i t i o n a l , a n d h o r m o n a l c o n d i t i o n s of t h e h o s t inf luence a n d a r e t hemse lve s in f luenced by s u c h c h a n g e s . E n d o p a r a s i t e s therefore r eac t to t h e p r e v a i l i n g h o s t e n v i r o n m e n t to m i n i m i z e d a m a g e to t hemse lves a n d to c a p i ta l ize o n those n u t r i t i o n a l a n d phys io log ica l c o n d i t i o n s t h a t will u l t i m a t e l y e n h a n c e t he i r o w n fi tness. I n s e c t p a r a s i t e s h a v e essent ia l ly t h e s a m e fami ly of h o r m o n e s a s the i r insec t hos t s . C o n s e q u e n t l y , s o m e a r e likely to ut i l ize t he se h o r m o n e s d i rec t ly to r e g u l a t e the i r o w n d e v e l o p m e n t a l a n d m e t a b o l i c p r o cesses w h e r e a s o t h e r s m a y ut i l ize t h e fluctuations in hos t h o r m o n e t i te rs a n d / o r h o r m o n e - m e d i a t e d m e t a b o l i c a n d n u t r i t i o n a l s t a t u s as cues to in i t i -

ParasiUs and Pathogens of Insects Volume 1: Parasites 5 9


6 0 Pauline Ο. Lawrence and Beatrice Lanzrein

a t e the i r o w n e n d o c r i n e a n d m e t a b o l i c mach ine ry . S o m e e n d o p a r a s i t e s m a y r e q u i r e cond i t i ons of h o m e o s t a s i s t h a t a r e s imi l a r to the i r hos t s ' a n d m a y read i ly ut i l ize these cond i t i ons w i t h m i n i m a l d i s r u p t i o n of t h e hos t ' s e n d o c r ine o r m e t a b o l i c p a t h w a y s . C o n s e q u e n t l y , a t p a r a s i t e l oads o p t i m a l for the i r d e v e l o p m e n t , these conformers d o n o t s ignif icant ly d i s r u p t the i r hos t ' s b e h a v i o r o r d e v e l o p m e n t o r c a u s e macroeco log ica l n i che a l t e r a t i o n s of the i r hos t s ( L a w r e n c e , 1986a, 1990b) . Still o t h e r p a r a s i t e s ( r egu la to r s ) d i s r u p t t h e n o r m a l m e t a b o l i c a n d e n d o c r i n e p rocesses of the i r hos t s w h o s e m i c r o e n -v i r o n m e n t a l cond i t i ons a r e s u b o p t i m a l for p a r a s i t e g r o w t h a n d d e v e l o p m e n t ( L a w r e n c e , 1986a, 1990b) . C o n s e q u e n t l y , these r e g u l a t o r s a l t e r t he deve lop m e n t , behav ior , a n d u l t imate ly , t he n i che of the i r hos t s . A l a rge c a d r e of e n d o p a r a s i t i c insec ts m a y ut i l ize a c o m b i n e d c o n f o r m e r - r e g u l a t o r s t r a t egy d e p e n d i n g on the s tage(s ) of t he hos t they a t t ack a n d in w h i c h they deve lop , on t he c h a n g e s in the i r n u t r i t i o n a l r e q u i r e m e n t s ( L a w r e n c e , 1990b) , a n d p r o b a b l y o n the ex ten t of the coevo lu t iona ry r e l a t i o n s h i p b e t w e e n t h e p a r t i c u l a r hos t a n d p a r a s i t e species (see L a w r e n c e , 1988c).

T h e p rocess of p a r a s i t i s m s t i m u l a t e s r ec ip roca l r e sponses f rom the hos t . For e x a m p l e , feeding of p a r a s i t e l a rvae dep le t e s t he hos t ' s n u t r i e n t s a n d these n u t r i t i o n a l c h a n g e s likely a l t e r t he e n d o c r i n e mi l ieu of t h e hos t , t h e r e b y i m p a c t i n g t h e p a r a s i t e ( L a w r e n c e , 1990b) . T h u s , a neces sa ry u n d e r p i n n i n g of d i scuss ions of e n d o c r i n e in t e rac t ions b e t w e e n insec t hos t s a n d the i r e n d o p a r a s i t e s is t he a p p r e c i a t i o n of t h e i n t r i ca t e i n t e r p l a y a m o n g m e t a b o l i s m , n u t r i t i o n a l s t a t u s , a n d e n d o c r i n e feedback m e c h a n i s m s t h a t o c c u r in insec ts (Ni jhou t , 1975; C y m b o r o w s k y et al, 1982; D o w n e r a n d Laufer , 1983; K e r k u t a n d G i lbe r t , 1985).

A p p r o x i m a t e l y 1 0 % of all species of a n i m a l s a r e p a r a s i t i c insec ts (Askew, 1971). A l a rge n u m b e r of these insec ts a r e p a r a s i t i c a s a d u l t s b u t a n even g r e a t e r n u m b e r a r e p a r a s i t i c d u r i n g the i r i m m a t u r e s tages a n d a r e therefore t e r m e d p r o t e l e a n p a r a s i t e s (Askew, 1971). P r o t e l e a n insec t p a r a s i t e s t h a t u l t i m a t e l y kill the i r insect hos t s a n d l a t e r e m e r g e as free-l iving a d u l t s a r e t e r m e d p a r a s i t o i d s ( D o u t t , 1964). (However , s ince p a r a s i t o i d s b e h a v e essential ly like o t h e r mu l t i ce l lu l a r p a r a s i t e s d u r i n g the i r d e v e l o p m e n t w i t h i n the i r hos t s , t he t e r m p a r a s i t e s will be u t i l ized t h r o u g h o u t th is c h a p t e r . )

I n s e c t p a r a s i t e s a r e ca tegor i zed on the bas i s of t he hos t s t age t hey a t t a c k a n d from w h i c h they exit as m a t u r e l a rvae or a d u l t s , as well as o n t h e o p t i m a l n u m b e r of p r o g e n y t h a t c a n deve lop p e r hos t . T h u s , egg- la rva l p a r a s i t e s a t t a c k t he hos t egg a n d e m e r g e from the l a rva l s t age , a n d so on . So l i t a ry a n d g r e g a r i o u s p a r a s i t e s deve lop o n e a n d m o r e t h a n o n e p r o g e n y p e r hos t , re spect ively ( D o u t t , 1964). I n p o l y e m b r y o n i c species , severa l offspring d e v e l o p from a s ingle egg. A hos t is sa id to be s u p e r p a r a s i t i z e d if it h a s m o r e p r o g e n y of a s ingle species t h a n c a n deve lop . I t is m u l t i p l y p a r a s i t i z e d w i t h p r o g e n y from two or m o r e p a r a s i t e species ( D o u t t , 1964).

3. Hormonal Interactions of Endoparasites and Hosts 61

T h e ma jo r i ty of p a r a s i t i c species o c c u r in t h e o r d e r s H y m e n o p t e r a a n d D i p t e r a , a n d exh ib i t a va r i e ty of d e v e l o p m e n t a l a n d phys io log ica l i n t e r a c t ions w i t h the i r hos t s . T h e s e i n t e r ac t i ons p r e s u m a b l y o c c u r a l o n g a c o n t i n u u m , w i t h confo rmers a n d r e g u l a t o r s a t t h e e x t r e m e s ( L a w r e n c e , 1986a) . H o w e v e r , t h e p a u c i t y of e n d o c r i n e s tud ie s h a s res t r i c ted o u r ab i l i ty to b e t t e r u n d e r s t a n d these i n t e r ac t i ons . T h e ava i lab i l i ty of sensi t ive b i o c h e m i c a l a n d m o l e c u l a r t e c h n i q u e s h a s faci l i ta ted s e p a r a t e a n d d i r ec t e v a l u a t i o n s of t h e e n d o g e n o u s h o r m o n e fluctuations a n d r e l a t ed b i o c h e m i c a l even t s in insec t hos t s a n d the i r e n d o p a r a s i t e s . H o w e v e r , m a n y h o s t - p a r a s i t e t a x a r e m a i n to b e s t u d i e d in o r d e r to facil i tate a hol is t ic a p p r e c i a t i o n of t he i r e n d o c r i n e i n t e r ac t i ons . T h e p u r p o s e of this p a p e r is to (1) rev iew o u r c u r r e n t k n o w l e d g e on t h e h o r m o n a l i n t e r ac t ions b e t w e e n h y m e n o p t e r a n e n d o p a r a s i t e s ( the m o s t s t u d i e d g r o u p of p a r a s i t i c insects) a n d the i r hos t s a n d (2) identify p r o b l e m s t h a t res t r ic t o u r u n d e r s t a n d i n g of these i n t e r ac t i ons a n d sugges t poss ib le so lu t ions to t h e m .

II. Hormones Controlling Insect Development: An Overview

T h r e e types of h o r m o n e s p l ay m a j o r roles in insec t d e v e l o p m e n t : p e p t i d e s p r o d u c e d by the b r a i n o r o t h e r n e u r a l t i ssues , ecdys t e ro ids (Fig . 1) p r o d u c e d b y t h e p r o t h o r a c i c g l a n d (or r i ng g l a n d in D i p t e r a ) , a n d j u v e n i l e h o r m o n e s ( J H s ) (F ig . 2) , w h i c h a r e s e s q u i t e r p e n o i d s p r o d u c e d by t h e c o r p o r a a l l a t a ( C A ) . T h e r e a d e r is referred to rev iews by Lafon t a n d K o o l m a n (1984) o n ecdys t e ro id s a n d B a k e r (1990) on J H s . T h e m o s t i m p o r t a n t n e u r o p e p t i d e s a r e t h e p r o t h o r a c i c o t r o p i c h o r m o n e s ( P T T H s ) , w h i c h s t i m u l a t e t h e s y n t h e sis a n d re lease of e c d y s o n e b y t h e p r o t h o r a c i c g l a n d ; a l l a t o t r o p i n s a n d a l -l a t o s t a t i n s , w h i c h s t i m u l a t e a n d inh ib i t , respect ively, t h e p r o d u c t i o n of J H b y the C A ; a n d eclosion h o r m o n e , w h i c h elicits eclosion behav io r .

D a t a f rom Manduca sexta i n d i c a t e t h a t t h e ecdys t e ro ids 2- a n d 3 - d e h y d r o -e c d y s o n e a r e p r o d u c e d by the p r o t h o r a c i c g l a n d s a n d a r e i m m e d i a t e l y conve r t ed to e c d y s o n e ( rev iewed b y G i lbe r t , 1989). I t a p p e a r s t h a t , d e p e n d i n g o n t h e d e v e l o p m e n t a l s t a g e of t h e insec t , e c d y s o n e ac ts a s a h o r m o n e b y i tself o r as a p r e c u r s o r (or p r o h o r m o n e ) of 2 0 - h y d r o x y e c d y s o n e , t h e m o s t c o m m o n l y o c c u r r i n g m o l t i n g h o r m o n e . 2 0 , 2 6 - D i h y d r o x y e c d y s o n e is gene ra l ly c o n s i d e r e d a m e t a b o l i t e of 2 0 - h y d r o x y e c d y s o n e b u t m a y exer t its o w n m o r p h o g e n e t i c effects. I t h a s b e e n sugges t ed t h a t in e m b r y o s of M. sexta, 26-h y d r o x y e c d y s o n e a n d / o r 2 0 , 2 6 - d i h y d r o x y e c d y s o n e m a y ac t m o r p h o g e n e t -ically ( rev iewed in G i lbe r t , 1989). M a k i s t e r o n e A is u n i q u e a m o n g t h e ecdys t e ro id s in t h a t it c o n t a i n s a m e t h y l s u b s t i t u e n t a t C - 2 4 of t h e s ide c h a i n


C H 2 - R 3

OH

HO

HO

(I) R 1 = R 2 = R 3 = H

(II) R 1 = O H , R 2 = R 3 = H

(III ) R 1 = O H , R 2 = C H 3 , R 3 = H Η ι

Ο

(IV) R 1 = R 3 = H , R 2 = C H 3

(V) R 1 = R 3 = O H , R 2 = C H 3

Figure 1 Structures of ecdysteroids of insects. (I) ecdysone; (II) 20-hydroxyecdysone; ( I I I ) makisterone A; (IV) 20-deoxymakisterone A; (V) 26-hydroxymakisterone A.

a n d is n o t syn thes i zed from choles te ro l as a r e all t he o t h e r ecdys t e ro ids . I t h a s b e e n ident if ied in severa l H e m i p t e r a , as well as in t h e h o n e y b e e ( H y m e n o p te ra ) a n d Drosophila ( D i p t e r a ) (Kel ly et al, 1981 , 1984; Feldlaufer , 1989).

A m a j o r funct ion of J H s in l a rva l insec ts is to d i c t a t e t h e c h a r a c t e r of t h e mol t : t he p r e s e n c e of J H d u r i n g a ce r t a in pe r iod in t h e m o l t i n g cycle l eads to

a R R' R"

COOR M M

JH III JH II JHI J H O

R = R' = R" = Me R = Et, R' = R" = Me R = R' = Et, R" = Me R = R' = R" = Et

R"' = R m = R'" = R'" = R , M =

Η Η Η Η Me 4 - M e J H I R = R' = Et, R" = Me

R' for JHs = Me for JH acids = Η

b

J Η bisepoxide

Figure 2 Structures of juvenile hormones in insects, (a) J H s found in vivo, (b) J H bisepoxide synthesized by cyclorraphous dipteran ring glands in vitro.

3. Hormonal Interactions of Endoparasites and Hosts 6 3

l a rva l m o l t s , a n d its a b s e n c e l eads to m e t a m o r p h i c mo l t s (for r ev iews , see D o w n e r a n d Laufer , 1983; K e r k u t a n d G i l b e r t , 1985; S e h n a l , 1989; G u p t a , 1990). J H I I I a p p e a r s to b e t h e m o s t u b i q u i t o u s J H as it w a s found in all o r d e r s i nves t iga t ed , i n c l u d i n g L e p i d o p t e r a , H y m e n o p t e r a , a n d D i p t e r a , w h e r e a s t h e h i g h e r h o m o l o g s J H I I , J H I , J H 0, a n d 4 - m e t h y l - J H I h a v e so far on ly b e e n found in l a rva l a n d / o r e m b r y o n i c L e p i d o p t e r a ( rev iewed in Baker , 1990). I n p r e p u p a l s t ages of M. sexta a n d poss ib ly o t h e r L e p i d o p t e r a , t h e C A swi tch f rom sec re t ing J H to sec re t ing J H acid , w h i c h c a n b e m e t h y l a t ed to J H by i m a g i n a l d isks ( B h a s k a r a n et al., 1986). J H I I I b i s epox ide h a s so far on ly b e e n obse rved in vitro u p o n i n c u b a t i o n of r i ng g l a n d s of l a rva l Drosophila melanogaster a n d r e l a t ed D i p t e r a a n d m a y r e p r e s e n t a b iological ly ac t ive fo rm of J H in c y c l o r r a p h o u s D i p t e r a ( R i c h a r d et al., 1989). A l t h o u g h insec t d e v e l o p m e n t is p u n c t u a t e d b y m o l t s , it is a c o n t i n u o u s p r o c e s s in w h i c h b o t h ecdys t e ro ids a n d J H s have funct ions in a d d i t i o n to t hose g iven h e r e (see G i lbe r t , 1989; S e h n a l , 1989).

T h e fluctuations in t h e t i te r of J H s a n d ecdys t e ro ids a r e b r o u g h t a b o u t b y v a r i a t i o n s in b o t h the i r r a t e s of b iosyn thes i s a n d d e g r a d a t i o n . I n t h e h e m o l y m p h J H is m e t a b o l i z e d by es te r hydro lys i s to J H acid ( rev iewed by H a m m o c k , 1985), b u t in t h e w h o l e insec t epox ide hydro lys i s l e a d i n g to J H diol a n d J H ac id-d io l a p p e a r s to b e equa l ly i m p o r t a n t ( K l a g e s a n d E m m e r i c h , 1979; H a l a r n k a r a n d Schooley, 1990). T h e m a j o r r o u t e s of d e g r a d a t i o n of 20-h y d r o x y e c d y s o n e a r e 2 6 - h y d r o x y l a t i o n l e a d i n g to 2 0 , 2 6 - d i h y d r o x y e c d y s o n e a n d t h e s u b s e q u e n t f o rma t ion of 20 -hydroxy-ecdysono ic ac id . I t is , however , i m p o r t a n t to n o t e t h a t e c d y s o n e m e t a b o l i s m p a t h w a y s v a r y w i t h spec ies , d e v e l o p m e n t a l s t age , a n d t i ssue ( rev iewed b y Lafon t a n d C o n n a t , 1989).

D u r i n g l a rva l m o l t s a n d m e t a m o r p h o s i s , c o m p l e x i n t e r e n d o c r i n e i n t e r a c t ions o c c u r a n d a r e essent ia l ly s imi l a r in d i p t e r a n s a n d l e p i d o p t e r a n s (see R i c h a r d s , 1981a ,b ; Redfe rn , 1984; G i lbe r t , 1989; Baker , 1990; R a u s c h e n -b a c h , 1991), b u t l e p i d o p t e r a n sy s t ems have b e e n s t u d i e d m o r e extensively. C u r r e n t m o d e l s for l e p i d o p t e r a n m e t a m o r p h o s i s a r e t h a t a cr i t ica l size m u s t first b e ach ieved to a l low t h e J H t i t e r to fall a n d P T T H to b e r e l eased . T h e a b s e n c e of J H a p p e a r s to b e neces sa ry for b o t h P T T H re lease to o c c u r a n d for p r o t h o r a c i c g l a n d s to g a i n s t e ro idogen ic c o m p e t e n c e , t h a t is , to r e s p o n d to P T T H b y p r o d u c i n g e c d y s o n e (see rev iews b y Bo l l enbache r , 1988; W a t s o n a n d Bo l l enbache r , 1988; W a t s o n et al., 1989). A s l ight i nc r ea se in e c d y s o n e a n d / o r 2 0 - h y d r o x y e c d y s o n e in t h e a b s e n c e o f J H i n d u c e s p u p a l c o m m i t m e n t (Ridd i fo rd , 1980). T h e r e a f t e r a s econd re lease of P T T H i n d u c e s a s econd , m u c h h i g h e r i nc rea se in e c d y s o n e a n d 2 0 - h y d r o x y e c d y s o n e , a n d J H inc reases a g a i n a l o n g w i t h J H ac ids (see rev iew by Bo l l enbache r , 1988). P r e s u m a b l y th i s i nc r ea se in J H is n e c e s s a r y for fu r the r ac t iva t ion of t h e p r o tho rac i c g l a n d ( G r u e t z m a c h e r et al., 1984; W a t s o n et al., 1989) a s wel l as for t h e p r o p e r f o r m a t i o n of t h e p u p a ( H i r u m a , 1980, 1986). I n s o m e species th is p r e p u p a l i nc r ea se in J H is a l so p r e s u m e d to i n d u c e j u v e n i l e h o r m o n e es-


t e ra se ( J H E ) ( J o n e s a n d H a m m o c k , 1983) b u t in o t h e r species th is s e e m s no t to b e t he case (Baker et al., 1987). I n still o t h e r species , a l l a t e c t o m i z e d l a rvae u n d e r g o n o r m a l p u p a t i o n (Sehna l a n d G r a n g e r , 1975), i n d i c a t i n g species-specif ic i m p o r t a n c e of t he p r e p u p a l J H peak . T h e h o r m o n a l c h a n g e s a s soc ia t ed w i th t he in i t i a t ion of insect m e t a m o r p h o s i s a l so d ra s t i ca l ly influence t he syn thes i s a n d s to rage of p r o t e i n s , l ip ids , a n d g lycogen in t h e fat b o d y (Col l ins , 1974; Locke , 1980).

III. Parasite Hormones Relative to Those of Their Hosts

T h e q u a l i t a t i v e a n d q u a n t i t a t i v e c h a n g e s of J H s a n d ecdys t e ro ids in t h e cour se of d e v e l o p m e n t of l e p i d o p t e r a n a n d d i p t e r a n hos t s c r ea t e a va r i e ty of b i o c h e m i c a l cond i t i ons t h a t e n d o p a r a s i t e s e n c o u n t e r . T h e hos t mi l i eu c lear ly var ies w i t h t he species , t he e n v i r o n m e n t a l a n d n u t r i t i o n a l c o n d i t i o n s , t h e ins ta r , a n d the t i m e p o i n t w i th in a n ins ta r . E n d o p a r a s i t e s h a v e different n u t r i t i o n a l a n d phys io log ica l r e q u i r e m e n t s (see rev iew by L a w r e n c e , 1990b) . C o n s e q u e n t l y , p a r t i c u l a r p a r a s i t e s will select p a r t i c u l a r hos t spec ies , w h e t h e r eggs , ear ly i n s t a r s , o r l a te i n s t a r s ( L a w r e n c e , 1990b) . T h e r a n g e of hos t species (n iche b r e a d t h ) se lected d e p e n d s on w h e t h e r p a r a s i t e s a r e m o n o - o r p o l y p h a g o u s . The re fo r e , a p a r a s i t e , especia l ly a p o l y p h a g o u s spec ies , will l ikely e n c o u n t e r hos t s of va r ious e n d o c r i n e a n d phys io log ica l s t a t e s . T h u s , e i the r r egu l a t i on of o r con fo rma t ion to t he ex is t ing hos t c o n d i t i o n s o r a c o m b i n e d c o n f o r m e r - r e g u l a t o r coevo lu t iona ry r e l a t i o n s h i p w i t h t he hos t will en sue .

J H a n d ecdys te ro id t i ters have b e e n m e a s u r e d in on ly very few p a r a s i t i c h y m e n o p t e r a n s a n d the i r hos t s . F i r s t - ins t a r l a rvae of t h e b r a c o n i d Di-achasmimorpha (Biosteres) longicaudata, a l a r v a l - p u p a l p a r a s i t e of Anastrepha sus-pensa ( D i p t e r a , T e p h r i t i d a e ) , c o n t a i n e d h i g h q u a n t i t i e s of J H I I I ( L a w r e n c e et al., 1990). T h e c o r r e s p o n d i n g la te t h i r d - i n s t a r a n d p h a r a t e p u p a l hos t s h a d a n a b n o r m a l l y h igh level of J H I I I c o m p a r e d w i t h t h e u n p a r a s i t i z e d con t ro l s ( L a w r e n c e et al., 1990). I t is no t k n o w n w h e t h e r this e leva ted J H t i te r is a c o n s e q u e n c e of i nc reased J H p r o d u c t i o n by the hos t o r w h e t h e r it r e p r e s e n t s J H t h a t w a s re leased from the p a r a s i t e o r its se rosa ( e x t r a e m b r y o n i c m e m b r a n e ) . In te res t ing ly , t h e h igh J H t i te r in p h a r a t e p u p a l hos t s c o - o c c u r r e d w i t h a d e c r e a s e in J H E , whi le J H a n d J H E levels in u n p a r a s i t i z e d i n d i v i d u als w e r e low a n d h igh , respect ively ( L a w r e n c e et al., 1990), as e x p e c t e d in h e a l t h y d i p t e r a n s ( R a u s c h e n b a c h , 1991). E c d y s t e r o i d s in D. longicaudata first i n s t a r s w i t h i n p h a r a t e p u p a e of A. suspensa i nc reased w i t h each dec l ine of t he p h a r a t e p u p a l ecdys te ro id peaks of t he hos t ( L a w r e n c e , 1988b, 1991). T h i s r e l a t i o n s h i p is fu r the r i n d i c a t e d in in vitro e x p e r i m e n t s ; p a r a s i t e s


p icked u p [ 3 H ] e c d y s o n e from c u l t u r e m e d i u m a n d c o n v e r t e d it to [ 3 H ] - 2 0 -h y d r o x y e c d y s o n e ( L a w r e n c e a n d H a g e d o r n , 1986), w h i c h t h e y r e q u i r e to syn thes i ze a s econd l a rva l cut ic le ( L a w r e n c e , 1988d) . S tud i e s in vitro i nd i c a t e d t h a t t h e a d d i t i o n of 2 0 - h y d r o x y e c d y s o n e a t va r i ous in t e rva l s to c u l t u r e m e d i a r e su l t ed in ecdys t e ro id inc reases in first- a n d s e c o n d - i n s t a r D. long-icaudata a n d s t i m u l a t e d m o l t i n g to s u b s e q u e n t i n s t a r s , a l t h o u g h a s m a l l per c e n t a g e of these l a t e r i n s t a r s have b e e n o b s e r v e d to m o l t w i t h o u t c o n t i n u e d h o r m o n e s u p p l e m e n t ( L a w r e n c e , 1991 , a n d u n p u b l i s h e d ) . T h e ecdys t e ro id r e q u i r e m e n t by D. longicaudata in vitro sugges t s a poss ib le e n d o c r i n e d e p e n d e n c e on t h e hos t .

T h i s a p p e a r s n o t to be t h e case in t h e b r a c o n i d Chelonus sp . , a n egg- la rva l p a r a s i t e of Trichoplusia ni ( L e p i d o p t e r a , N o c t u i d a e ) . I n th is p a r a s i t e J H I I I w a s ident i f ied w i t h p h y s i c o c h e m i c a l m e t h o d s in l a te s e c o n d - i n s t a r l a rvae w h e r e a s J H I I w a s ident i f ied in t h e hos t ( J o n e s et al., 1990). T h i s is t h e on ly p a r a s i t e / h o s t s y s t e m w h e r e fluctuations in J H I I , J H I I I , a n d v a r i o u s ecd y s t e r o i d s w e r e m e a s u r e d in p a r a s i t e s a n d c o r r e s p o n d i n g h o s t h e m o l y m p h (Grossn ik l aus -B i i rg in a n d L a n z r e i n , 1990b) . I n l a te first-instar p a r a s i t e s a very h i g h t i te r (60 n g / g ) of J H I I I w a s seen followed by a s m a l l e r p e a k (30 n g / g ) in t he m i d d l e of t h e s econd i n s t a r whi le t he t i te r of J H I I in h o s t h e m o l y m p h fluctuated b e t w e e n 0 a n d 3 n g / m l (Grossn ik l aus -B i i rg in a n d L a n z r e i n , 1990b) . E c d y s t e r o i d s in t h e p a r a s i t e i n c r e a s e d before t h e m o l t to t h e s econd i n s t a r a n d fluctuated a t h i g h levels in s econd- a n d t h i r d - i n s t a r l a rvae wh i l e e c d y s o n e a n d 2 0 - h y d r o x y e c d y s o n e w e r e low in c o r r e s p o n d i n g hos t h e m o l y m p h ; in t h i r d - i n s t a r p a r a s i t e s ecdys t e ro ids w e r e t en ta t ive ly ident i f ied b y the use of h i g h - p e r f o r m a n c e l iqu id c h r o m a t o g r a p h y ( H P L C ) c o m b i n e d w i t h r a d i o i m m u n o a s s a y ( R I A ) ( two different a n t i b o d i e s ) a s e c d y s o n e , 2 0 - h y d r o x y e c d y s o n e , 2 0 , 2 6 - d i h y d r o x y e c d y s o n e , a n d u n k n o w n s , a n d gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y ( G C - M S ) a n a l y s e s con f i rmed t h e p r e s e n c e of 2 0 - h y d r o x y e c d y s o n e , 2 0 , 2 6 - d i h y d r o x y e c d y s o n e , e c d y s o n e , a n d poss ib ly 2 6 - h y d r o x y e c d y s o n e in a p r o p o r t i o n of 1 0 0 : 1 4 . 8 : 1 1 . 3 : 2 . 0 (Grossn ik l aus -B i i rg in a n d L a n z r e i n , 1990b) . T o o u r k n o w l e d g e th is r e p r e sen t s t h e on ly ident i f ica t ion of ecdys t e ro id s in a n e n d o p a r a s i t i c insec t . A s n e i t h e r J H s n o r ecdys t e ro ids fluctuated in a cons i s t en t p a t t e r n in t h e p a r a s i t e re la t ive to t hose in t he hos t a n d w e r e in g e n e r a l h i g h e r in t h e former , a n d as t h e p a r a s i t e c o n t a i n s J H I I I whi le t h e hos t c o n t a i n s J H I I , th i s p a r a s i t e c lear ly m a k e s its o w n h o r m o n e s a t s t ages after t h e l a te first i n s t a r (Gros sn ik l aus -B i i rg in a n d L a n z r e i n , 1990b) .

T a k e n t o g e t h e r t he few d a t a ava i l ab le so far sugges t t h a t in e n d o p a r a s i t i c as in free-l iving h y m e n o p t e r a n s t he m a j o r J H is J H I I I . I t s c o n c e n t r a t i o n in p a r a s i t e s is very h igh , p a r t i c u l a r l y in t h e first a n d second i n s t a r s . N o d a t a o n m e t a b o l i s m of J H by p a r a s i t e s h a v e b e e n r e p o r t e d to d a t e b u t p r e l i m i n a r y in vitro d a t a o b t a i n e d w i t h h o m o g e n a t e s of first- a n d s e c o n d - i n s t a r l a rvae of t h e


p a r a s i t e Chelonus inanitus i nd i ca t e t h a t l abe led J H I I I is d e g r a d e d on ly a t a very low r a t e a n d t h a t t he m a j o r m e t a b o l i t e is J H I I I d iol (B . L a n z r e i n a n d B . D . H a m m o c k , u n p u b l i s h e d ) . T h u s t h e h igh J H t i te rs seen in p a r a s i t e s m i g h t reflect low ra t e s of J H d e g r a d a t i o n r a t h e r t h a n h i g h r a t e s of J H b io syn thes i s . So far t he r e exists n o ev idence t h a t p a r a s i t e s re lease h o r m o n e s i n to t h e hos t . T h i s poss ib i l i ty a p p e a r s to b e a t t r ac t i ve a s it w o u l d offer to t h e p a r a s i t e m u l t i p l e possibi l i t ies of in f luenc ing t h e hos t ' s e n d o c r i n e s y s t e m , p a r t i c u l a r l y in v iew of t h e c o m p l e x i n t e r e n d o c r i n e r e g u l a t o r y m e c h a n i s m s . However , two a spec t s have to b e cons ide red . (1) I t is n o t c lea r b y w h i c h m e c h a n i s m h o r m o n e s p r o d u c e d ins ide t he p a r a s i t e cou ld r e a c h t h e hos t ' s h e m o l y m p h w i t h o u t b e i n g d e g r a d e d . (2) A l t h o u g h t h e c o n c e n t r a t i o n of J H a n d ecdys t e ro ids w a s found to b e h i g h in t he p a r a s i t e s inves t iga t ed so far, t h e q u a n t i t y in a b s o l u t e t e r m s a n d in c o m p a r i s o n to t h e hos t h e m o l y m p h v o l u m e is very sma l l . C o n s e q u e n t l y , p a r a s i t e s w o u l d have to p r o d u c e very l a rge a m o u n t s of h o r m o n e s in o r d e r to subs t an t i a l l y a l t e r t h e t i te r in t h e hos t . T h e poss ib i l i ty of re lease of h o r m o n e m e t a b o l i t e s s eems m o r e likely.

E x p e r i m e n t s w i t h Chelonus sp./T. ni, w h e r e l abe led e c d y s o n e w a s in jec ted in to t he hos t , followed by s u b s e q u e n t ana lys i s of l abe led ecdys t e ro id s in p a r a s i t e a n d hos t , sugges ted t h a t this p a r a s i t e m a k e s a p o l a r ecdys t e ro id s t h a t a r e re leased in to t he hos t ( G r o s s n i k l a u s - B u r g i n et al., 1989). Also t h e p r e s ence of J H I I I acid in la te s e c o n d - i n s t a r Chelonus sp . a n d c o r r e s p o n d i n g ca rcasses of T. ni ( J o n e s et al., 1990) cou ld b e i n t e r p r e t e d a s re lease of J H I I I acid from t h e p a r a s i t e i n to t he hos t . T h e r e f o r e t h e r e is i n d i c a t i o n t h a t s o m e p a r a s i t e s use a n d m e t a b o l i z e hos t h o r m o n e s a n d / o r re lease h o r m o n e m e t a b oli tes i n t o t h e hos t b u t n o c o h e r e n t p i c t u r e e m e r g e s ye t as to t h e e n d o c r i n e e x c h a n g e s b e t w e e n p a r a s i t e s a n d hos t s .

IV. Direct or Indirect Host Endocrine Effects on Endoparasites

T h e i m p a c t of hos t q u a l i t y o n p a r a s i t e d e v e l o p m e n t h a s b e e n t e r m e d hos t su i t ab i l i ty ( D o u t t , 1964), w h i c h is inf luenced b y t h e n u t r i t i o n a l , e n d o c r i n e , b i o c h e m i c a l , a n d i m m u n o l o g i c a l factors w i t h i n t h e h o s t ( V i n s o n a n d I w a n t s c h , 1980a) . T h e s e factors in t u r n m a y i m p a c t each o t h e r t h r o u g h feedback loops . E n v i r o n m e n t a l l y i n d u c e d s t ress c o n d i t i o n s m a y a lso a l t e r t h e d y n a m i c i n t e r p l a y of t he v a r i o u s phys io log ica l factors t h a t m o d u l a t e h o s t su i t ab i l i ty ( L a w r e n c e , 1988c).

T h e r e h a v e b e e n n u m e r o u s s tud ies o n p a r a s i t i s m - i n d u c e d e n d o c r i n e c h a n g e s in t h e insec t hos t (see Sec t ion V ) b u t very few o n t h e hos t ' s i m p a c t o n t h e p a r a s i t e (see rev iews by L a w r e n c e , 1986a, 1990b) . T h i s p a u c i t y p r o b -


a b l y reflects t h e i n h e r e n t difficulty in s e p a r a t i n g h o s t - r e l a t e d ve r sus p a r a s i t e -r e l a t ed factors a n d ident i fy ing t he i r r e spec t ive causes a n d effects in vivo.

H o s t e n d o c r i n e effects o n e n d o p a r a s i t e s w e r e d e m o n s t r a t e d ind i rec t ly in t h e Optus concolor ( H y m e n o p t e r a , Braconidae)-C*rafr7w capitata ( D i p t e r a , T e -p h r i t i d a e ) l a r v a l - p u p a l p a r a s i t e - h o s t sy s t em. T h e pa r a s i t e ' s first l a rva l m o l t w a s i n d u c e d by t h e in ject ion of e c d y s o n e in to t h e hos t after t he i r d e v e l o p m e n t a l a r r e s t b y y i r r a d i a t i o n ( C a l s - U s c i a t i , 1969, 1975). S ince p u p a r i a -t i o n / p u p a t i o n of t he in jec ted h o s t p r e c e d e d t h e p a r a s i t e ' s l a rva l m o l t , it is n o t c lea r w h e t h e r t h e h o r m o n e effects d i rec t ly o n t h e p a r a s i t e o r i nd i r ec t ly b y m e d i a t i n g i m p o r t a n t b i o c h e m i c a l c h a n g e s a s soc ia t ed w i t h h o s t p u p a r i a -t ion a n d p u p a t i o n . I n t h e r e l a t e d H y m e n o p t e r a - D i p t e r a p a r a s i t e - h o s t syst e m s Opius (Diachasmimorpha) tryoni-C. capitata a n d Optus melleus-Rhagoletis pomonella, t h e p a r a s i t e s ' first l a rva l m o l t a p p e a r s to b e in f luenced b y a n d o c c u r s in r e l a t ion to t h e p u p a r i a t i o n / p u p a t i o n of the i r hos t s ( P e m b e r t o n a n d W i l l a r d , 1918; L a t h r o p a n d N e w t o n , 1933). T h o u g h s ignif icant e n d o c r i n e c h a n g e s a r e k n o w n to b e neces sa ry for m o l t i n g , p u p a r i a t i o n , p u p a t i o n , a n d d i a p a u s e , t he i r ro le in in f luenc ing e n d o p a r a s i t e s n e e d s c lar i f ica t ion. O n l y co r r e l a t i ons b e t w e e n p a r a s i t e b e h a v i o r a n d d e v e l o p m e n t a n d hos t e n d o c r i n e -m e d i a t e d even t s (see Me l l i n i , 1975; L a w r e n c e , 1986a, 1990b) a n d h o s t ho r m o n e t i ters h a v e so far b e e n e s t ab l i shed ( P l a n t e v i n et al., 1986; G r o s s n i k l a u s -Bi i rg in a n d L a n z r e i n , 1990b; S t r a n d et al., 1991) in t h e ma jo r i t y of s t ud i e s to d a t e . In vitro c u l t u r e a n d h o r m o n e t h e r a p y of a few p a r a s i t e species h a v e d e m o n s t r a t e d a d i r ec t effect of hos t h o r m o n e s o n p a r a s i t e d e v e l o p m e n t . Fo r e x a m p l e , N e n o n (1972) first d e m o n s t r a t e d t h e necess i ty of J H a n d e c d y s o n e for g r o w t h a n d d e v e l o p m e n t of t h e h y m e n o p t e r a n p a r a s i t e Ageniaspis fitsciollis in vitro. M o r e recent ly, L a w r e n c e (1988d) d e m o n s t r a t e d t h a t 20 -hydroxye c d y s o n e s u p p l e m e n t a t i o n of c u l t u r e m e d i u m w a s n e c e s s a r y for cu t i cu lo -genes is of D. longicaudata s e c o n d i n s t a r s p r e c e d i n g the first l a rva l m o l t . F u r t h e r m o r e , 2 0 - h y d r o x y e c d y s o n e cell-free c u l t u r e m e d i u m c o n t a i n i n g D. longicaudata l a rvae effected inc reases in p a r a s i t e e n d o g e n o u s ecdys t e ro id t i t e rs a n d s u b s e q u e n t l a rva l mo l t s ( L a w r e n c e , 1991). T h e d e v e l o p m e n t of n e w in vitro c u l t u r e m e d i a m u s t necessar i ly p r e c e d e s tud ies o n a l a r g e r n u m b e r of e n d o p a r a s i t e s to d e t e r m i n e hos t e n d o c r i n e effects. I t s h o u l d b e n o t e d t h a t t h e effects of 2 0 - h y d r o x y e c d y s o n e , J H , a n d o t h e r insec t h o r m o n e s o n e n d o p a r a s i t e s in vitro a r e n o t s u r p r i s i n g b u t t h e a b s e n c e of p a r a s i t e d e v e l o p m e n t in h o r m o n e - f r e e m e d i u m prov ides s t r o n g ev idence for a d i r ec t h o s t h o r m o n a l r o l e — p r o v i d i n g t h e m e d i u m is o p t i m a l for p a r a s i t e g r o w t h .

I n t h e a b s e n c e of e m p i r i c a l ev idence of v a r i o u s hos t e n d o c r i n e effects o n e n d o p a r a s i t e s , we will briefly a d d r e s s se lec ted hos t factors a n d t h e poss ib le w a y s in w h i c h they likely i m p a c t t h e d e v e l o p i n g p a r a s i t e d i rec t ly o r v ia t h e e n d o c r i n e s y s t e m . T h e e n d o c r i n e a n d n u t r i t i o n a l s t a t u s of h o s t insec t s is k n o w n to i m p a c t t h e d e v e l o p m e n t a n d v iabi l i ty of the i r e n d o p a r a s i t e s . For


e x a m p l e , Sa l t (1937) d e m o n s t r a t e d t h a t Trichogramma evanescens r e a r e d o n a n e u r o p t e r a n hos t (Sialis) e m e r g e d as a p t e r o u s a d u l t s , w h e r e a s those r e a r e d on the u s u a l l e p i d o p t e r a n hos t eggs were n o r m a l (w inged ) . H e a r g u e d t h a t t he n u t r i e n t s in Sialis we re s u b o p t i m a l for a l a t e a d u l t d e v e l o p m e n t . T h e size a n d q u a l i t y of t he hos t in inf luencing p a r a s i t e d e v e l o p m e n t w e r e fu r the r d e m o n s t r a t e d by the r e a r i n g of Trichogramma o n eggs of different-s ized l ep i d o p t e r a n hos t s . Sal t (1941) o b t a i n e d p a r a s i t e a d u l t s t h a t w e r e s t u n t e d o r h a d m a l f o r m e d wings from sma l l hos t eggs w h e r e a s n o r m a l - s i z e d i n d i v i d u a l s e m e r g e d from t h e u s u a l l e p i d o p t e r a n hos t eggs . S o m e p a r a s i t e s c o n t a i n t h e s a m e types of a m i n o ac ids as the i r hos t s ( T h o m p s o n , 1986) a n d p r o b a b l y s e q u e s t e r all o r s o m e of these from the i r hos t s . C o n s e q u e n t l y , a def ic iency in s u c h n u t r i e n t s w i t h i n t he hos t cou ld b e de le t e r ious to t h e d e v e l o p i n g p a r a s i t e . C a l s - U s c i a t i (1969, 1975) a n d severa l o t h e r s have s h o w n t h a t l a rva l e n d o p a r a s i t e s feed on hos t h e m o l y m p h d u r i n g t h e first l a rva l s ta d i u m a n d m a y even p u n c t u r e hos t cells w i t h the i r m a n d i b l e s to ut i l ize cell c o n t e n t s .

T h e n u t r i e n t s ava i l ab le in a hos t a r e inf luenced b y the food e a t e n b y t h e hos t itself as well as by t he e n d o c r i n e a n d d e v e l o p m e n t a l s t a t u s of t h e hos t (see L a w r e n c e , 1990b, for rev iew) . B a r b o s a (1988) h a s rev iewed t h e effects of s u b s t a n c e s such as n ico t ine , r u t i n , a n d o t h e r a l l e lochemica l s w i t h i n t h e hos t ' s food on e n d o p a r a s i t e s . T h e y inh ib i t p a r a s i t e g r o w t h , p r o l o n g l a rva l deve lop m e n t , a n d even c a u s e m o r t a l i t y in a l a rge p e r c e n t a g e of e n d o p a r a s i t e s . S o m e p l a n t - d e r i v e d s u b s t a n c e s like c o u m a r i n s c a n cross t h e insec t g u t wal l a n d b i n d to s t o r age p r o t e i n s in t he h e m o l y m p h ( S h a p i r o et al., 1988) a n d a r e therefore e n c o u n t e r e d by the p a r a s i t e l a rvae . P h y t o h o r m o n e s , s u c h as j u v a d e c e n e ( N i s h i d a et al., 1983) a n d p h y t o e c d y s t e r o i d s , p l u s a n t i h o r m o n e s such as p r e c o c e n e , c a n d i rec t ly o r ind i rec t ly i m p a c t t h e d e v e l o p i n g p a r a s i t e . A z a d i r a c t i n , a n e e m t ree de r iva t ive , w a s found to d i s r u p t v i te l logenes is as well as J H a n d ecdys te ro id levels in t he exposed insec ts ( R e m b o l d a n d S c h m u t t e r e r , 1981). T h u s , e n d o p a r a s i t e s t h a t a t t ack act ively feeding l a rvae of p h y t o p h a g o u s insec ts a r e t he m o s t likely to be affected b y p l a n t - d e r i v e d s u b s t a n c e s f rom the hos t ' s food. S u c h effects m a y o c c u r w i t h i n a s h o r t t i m e after t he hos t feeds o r a t a l a te r d a t e in cases w h e r e s u b s t a n c e s like c o u m a r i n s m a y be b o u n d to a r y l p h o r i n s ( S h a p i r o et al., 1988), l i pop ro t e in s , o r o t h e r s t o r a g e molecu le s . S e c o n d - o r d e r effects of a l l e lochemica l s o n benefic ia ls h a v e b e e n s t u d i e d in a few cases ( B a r b o s a , 1988). However , t he i m p a c t of t hese a l l e lochemica l s on hos t a n d p a r a s i t e e n d o c r i n e sy s t ems , t h o u g h likely, h a s no t b e e n e v a l u a t e d .

T h e level of h e m o l y m p h n u t r i e n t s in insects is k n o w n to be in f luenced by the i r e n d o c r i n e s t a t u s (Locke , 1980; W y a t t , 1980). N o s tud ies have b e e n d o n e on the i m m e d i a t e a n d u l t i m a t e effects of e n d o c r i n e - i n d u c e d n u t r i t i o n a l c h a n g e s in hos t s on the i r deve lop ing e n d o p a r a s i t e s . O n e c a n on ly s p e c u l a t e


o n t h e poss ib le effects. For e x a m p l e , act ively feeding l e p i d o p t e r a n l a rvae m a y h a v e a h i g h e r level of free h e m o l y m p h t r eha lose t h a n nonfeed ing l a rvae . C o n s e q u e n t l y , h o r m o n e s t h a t s t i m u l a t e t he m e t a b o l i s m of g lycogen a n d t h e p r o d u c t i o n of t r eha lose (Col l ins , 1974) m a y differ in t i te r b e t w e e n m a t u r e l a rvae a n d y o u n g e r s t age feeding l a rvae w i t h c o r r e s p o n d i n g l y different effects on the i r r espec t ive e n d o p a r a s i t e s . Severa l of these h o r m o n e s t h a t r e g u l a t e insec t m e t a b o l i s m have b e e n rev iewed ( G o l d s w o r t h y a n d G a d e , 1983; Ker -k u t a n d G i l b e r t , 1985). T h e i r poss ib le d i r ec t a n d ind i r ec t effects o n d e v e l o p ing e n d o p a r a s i t e s have b e e n d i scussed ( L a w r e n c e , 1990b) b u t e m p i r i c a l s t ud i e s a r e n e e d e d to d e t e r m i n e the i r i m p a c t o n e n d o p a r a s i t e s . Resu l t s f rom s u c h s tud i e s cou ld b e p a r t i c u l a r l y v a l u a b l e in deve lop ing art if icial m e d i a for r e a r i n g e n d o p a r a s i t e s .

Severa l h o r m o n e l i k e s u b s t a n c e s m a y a lso work v ia t he h o s t to adve r se ly i m p a c t t h e d e v e l o p i n g p a r a s i t e . T h e s e i n c l u d e h o r m o n e a n a l o g s ( W r i g h t a n d S p a t e s , 1972; M c N e i l , 1975; Beckage , 1985), a n t i h o r m o n e s (Bowers , 1983), h o r m o n e agon i s t s ( W i n g , 1988), a n d ch i t in syn thes i s i n h i b i t o r s ( L a w r e n c e , 1981). A l t h o u g h m u c h effort is b e i n g m a d e to deve lop pes t i c ides w i t h m i n i m a l de l e t e r ious e n v i r o n m e n t a l effects, l i t t le effort is b e i n g m a d e to a d d r e s s t h e i m p a c t of these s u b s t a n c e s in c o m p r e h e n s i v e pes t con t ro l p r o g r a m s in w h i c h beneficial insec t s , p r i m a r i l y e n d o p a r a s i t e s , a r e e m p l o y e d . For e x a m ple , m o r t a l i t y of t he e n d o p a r a s i t e D. longicaudata, t r e a t e d w i t h t h e ch i t in syn thes i s i n h i b i t o r d i f l u b e n z u r o n , as first i n s t a r s w i t h i n p h a r a t e p u p a e of t h e d i p t e r a n hos t , A. suspensa, w a s lower t h a n t h a t of p a r a s i t e s t h a t w e r e e i the r four th i n s t a r o r p u p a e w h e n t h e hos t w a s t r e a t e d ( L a w r e n c e , 1981). H o w e v e r , g r e a t e r t h a n 7 0 % of those p a r a s i t e s ( t r e a t e d as first i n s t a r s ) t h a t e m e r g e d as a d u l t s w e r e m a l f o r m e d d u e to a b n o r m a l sc ler i te d e v e l o p m e n t ( L a w r e n c e , 1981 , 1983). P r e s u m a b l y , t h e i n h i b i t o r y effect of d i f l u b e n z u r o n o n iV-ace ty lg lucosamine u p t a k e d u r i n g ch i t in syn thes i s ( M a y e r et aL, 1980) m a y h a v e i m p a c t e d t he p a r a s i t e s a n d p r e v e n t e d t he n o r m a l f o r m a t i o n of p u p a l a n d a d u l t cu t ic les .

T h e newly deve loped e c d y s o n e agon i s t R H 5849 is a p o t e n t i a l insec t i c ide t h a t b i n d s e c d y s o n e r ecep to r s a n d h a s s imi l a r effects o n m o l t i n g as 2 0 - O H E ( W i n g , 1988). T h i s agon i s t is m o r e effective in vivo t h a n n a t u r a l e c d y s t e r o i d s b e c a u s e it is n o t as r ead i ly m e t a b o l i z e d by e n d o g e n o u s e n z y m e s as a r e t h e ecdys t e ro id s . S ince s o m e p a r a s i t e s s u c h as C. congregata a p p a r e n t l y ut i l ize i n c r e a s e d e c d y s o n e t i te rs as a s igna l to exit the i r t r e a t e d hos t s (Beckage , 1985), to w h a t ex t en t cou ld this pes t i c ide inf luence p r e m a t u r e e m e r g e n c e of e n d o p a r a s i t e l a rvae f rom the i r t r e a t e d hos ts? I n t h e case of t hose p a r a s i t e s r e q u i r i n g hos t ecdys t e ro ids to in i t i a t e cut ic le syn thes i s a n d s u b s e q u e n t deve l o p m e n t , w h a t i m p a c t will s u b s t a n c e s like R H 5849 have o n p a r a s i t e d e v e l o p m e n t w h e n a p p l i e d a t s u b l e t h a l d o s a g e s to d i s r u p t t he pes t hos t ' s deve lop m e n t o r r e p r o d u c t i o n ?


V. Direct or Indirect Effects of Endoparasites on Host Endocrine System

T h e effects of e n d o p a r a s i t e s on hos t d e v e l o p m e n t a r e n u m e r o u s a n d d ive r se ( rev iewed in V i n s o n a n d I w a n t s c h , 1980b; Beckage , 1985, 1990a ,b ; Lawrence , 1986a, 1988a) . I n m o s t cases it is n o t k n o w n w h e t h e r p a r a s i t e s d i rec t ly in terfere w i t h t he hos t ' s e n d o c r i n e s y s t e m o r w h e t h e r p a r a s i t e effects a r e ind i rec t a n d c a u s e d by s t ress a n d / o r inf luences o n the n u t r i t i o n a l s t a t u s of t h e hos t . I n t h e following we shal l d i scuss va r ious types of d e v e l o p m e n t a l a n d e n d o c r i n e effects of p a r a s i t e s on the i r hos t s by focusing o n se lec ted a n d well-inves t iga ted p a r a s i t e / h o s t sy s t ems . T a b l e 1 s u m m a r i z e s t h e effects of severa l h y m e n o p t e r o u s p a r a s i t e s on the i r l e p i d o p t e r u s hos t s . I t shows t h e d e v e l o p m e n t a l effects, t he e n d o c r i n e man i f e s t a t i ons , a n d w h e r e ava i l ab le , t h e p u t a tive factors involved . I t i nc ludes p a r a s i t e s t h a t p r o l o n g l a rva l life of t h e hos t a n d / o r c a u s e its d e v e l o p m e n t a l a r r e s t in t h e las t ins ta r , o r t h a t i n d u c e p r e c o c ious onse t of m e t a m o r p h o s i s , o r t h a t p r o l o n g t h e feeding p h a s e in t h e las t ins ta r .

T h e b r a c o n i d s C. congregata, Apanteles kariyai, Cardiochiles nigriceps, a n d Microplitis croceipes a r e all l a rva l p a r a s i t e s t h a t c a u s e d e v e l o p m e n t a l a r r e s t of t h e hos t in t h e las t ins ta r . T h i s is reflected in lowered J H E a n d ecdys t e ro id levels as well as a l t e r ed sensi t ivi ty of p r o t h o r a c i c g l a n d s to P T T H a n d / o r a l t e r ed sens i t iv i ty of t a r g e t t i ssues to ecdys t e ro ids , d e p e n d i n g o n t h e s y s t e m . C a l y x (wh ich c o n t a i n s v i rus ) , v e n o m , a n d t e r a tocy te s a p p e a r to b e involved in c a u s i n g s o m e of t he e n d o c r i n e c h a n g e s in t he hos t b u t the i r p rec i se func t ion h a s n o t ye t b e e n e luc ida t ed (see be low) . T h e i c h n e u m o n i d Campoletis sonorensis a l so causes d e v e l o p m e n t a l a r r e s t of t h e hos t a n d p o l y d n a v i r u s e s i n d u c e d e g e n e r a t i o n of t he p r o t h o r a c i c g l a n d a n d s u p p r e s s e c d y s o n e p r o d u c t i o n b u t on ly if l a s t - i n s t a r l a rvae a r e a t t a c k e d (Dover a n d V i n s o n , 1990). T h i s a p p e a r s to b e excep t iona l u n d e r n a t u r a l cond i t i ons , a n d this effect m i g h t t h u s r a t h e r r e p r e s e n t a safety m e a s u r e t h a t w o u l d al low s o m e p a r a s i t e s to su rv ive w h e n n o y o u n g e r l a rvae a r e ava i lab le . C. sonorensis is a h a b i t u a l p a r a s i t e of t h i r d -i n s t a r H. virescens (Davies et al., 1987).

W h i l e t he l a rva l p a r a s i t e s d i scussed in t h e foregoing p r o l o n g l a rva l life of t h e hos t a n d / o r p r e v e n t hos t p u p a t i o n , egg- la rva l p a r a s i t e s of t h e g e n u s Chelonus (Bracon idae ) i n d u c e p recoc ious cocoon s p i n n i n g of t h e hos t in t h e p e n u l t i m a t e i n s t a r followed by p a r a s i t e e m e r g e n c e ( J o n e s et al., 1981). T h e s e so l i t a ry p a r a s i t e s oviposi t i n t o eggs of va r i ous l e p i d o p t e r a n spec ies . T h e first i n s t a r h a t c h e s w h e n the hos t is a la te e m b r y o o r a newly h a t c h e d f i rs t - ins tar l a rva . T h e p a r a s i t e s r e m a i n in the i r first s t a d i u m for severa l d a y s a n d m o l t


i n to t h e s econd i n s t a r on ly after t h e hos t ' s ecdysis i n to its p r ecoc ious las t i n s t a r ( G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990b) .

V a r i o u s e n d o c r i n e p a r a m e t e r s have b e e n m e a s u r e d in t he cou r se of deve l o p m e n t in n o n p a r a s i t i z e d a n d p a r a s i t i z e d T. ni l a rvae . T h e p a t t e r n of J H E ( B u h l e r et aL, 1985), t he v a r i a t i o n s in J H I I a n d v a r i o u s ecdys t e ro id s ( G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990b) , a n d the m e t a b o l i s m of e c d y s o n e ( G r o s s n i k l a u s - B u r g i n et aL, 1989) w e r e found to be s imi l a r in p a r a s i t i z e d p r ecoc ious l a s t - i n s t a r l a rvae a n d in n o n p a r a s i t i z e d l a s t - i n s t a r l a rvae (Tab le 1). A l so t h e s t o r a g e p r o t e i n a r y l p h o r i n w a s seen to a p p e a r p recoc ious ly ( K u n k e l et aL, 1990). All of these d a t a i n d i c a t e t h a t th is p a r a s i t e i n d u c e s a t r u e p r ecoc ious in i t i a t ion of m e t a m o r p h o s i s . E x p e r i m e n t s c a r r i e d o u t w i t h p s e u d o p a r a s i t i z e d l a rvae ( la rvae d e v e l o p i n g from s t u n g eggs b u t c o n t a i n i n g n o live p a r a s i t e ) t h a t b e c o m e d e v e l o p m e n t a l l y a r r e s t e d in t h e p r e p u p a l s t age i n d i c a t e d s u p p r e s s i o n of e c d y s o n e p r o d u c t i o n a n d m e t a b o l i s m ( J o n e s , 1986) b u t n o i n d i c a t i o n of e i the r s u p p r e s s i o n of e c d y s o n e p r o d u c t i o n o r conve r s ion to 2 0 - h y d r o x y e c d y s o n e w a s seen in p a r a s i t i z e d T. ni u p to t h e s t age w h e n t h e p a r a s i t e s exi t t h e hos t ( G r o s s n i k l a u s - B u r g i n et aL, 1989; G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990b) . I t is n o t c lea r w h i c h factors a r e r e spons ib l e for i n d u c t ion of t h e hos t ' s p r ecoc ious m e t a m o r p h o s i s .

T h e o b s e r v a t i o n t h a t p s e u d o p a r a s i t i z e d l a rvae go in to p r e c o c i o u s m e t a m o r p h o s i s a n d t h e fact t h a t r e m o v a l of s e c o n d - i n s t a r p a r a s i t e s f rom h o s t l a rvae does n o t p r e v e n t in i t i a t ion of p r ecoc ious m e t a m o r p h o s i s ( B u h l e r et aL, 1985) i n d i c a t e t h a t t he s e c o n d - i n s t a r p a r a s i t e a l one c a n n o t i n d u c e p r e c o c i o u s m e t a m o r p h o s i s . F r o m ind i r ec t a p p r o a c h e s it h a s b e e n c l a i m e d t h a t m a t e r i a l f rom t h e female w a s p a lone is r e spons ib l e for i n d u c i n g p r e c o c i o u s m e t a m o r p h o s i s ( J o n e s , 1987) b u t so far d i r ec t ev idence is l ack ing a n d a n involvem e n t of e m b r y o n i c o r f i rs t - ins tar p a r a s i t e s o r t e r a tocy te s c a n n o t ye t b e exc l u d e d . In jec t ion of ca lyx fluid p lu s v e n o m from Chelonus insularis i n to Spodoptera ornithogalli w a s seen to i m i t a t e t he g r o w t h - r e d u c i n g effect of p a r a s i t i sm b u t n o t t h e p recoc ious in i t i a t ion of m e t a m o r p h o s i s (Abies a n d V i n s o n , 1981). In jec t ion of ca lyx fluid, pur i f ied v i rus , o r v e n o m from Chelonus inanitus also d i d n o t i n d u c e p recoc ious m e t a m o r p h o s i s in e i the r S. littoralis o r S. exigua ( C . G r o s s n i k l a u s - B u r g i n a n d B . L a n z r e i n , u n p u b l i s h e d ) . T h i s sugges t s t h a t factors in a d d i t i o n to v i rus a n d v e n o m a r e neces sa ry to i n d u c e p r e c o c i o u s m e t a m o r p h o s i s .

A c o m p a r i s o n of J H s in n o n p a r a s i t i z e d eggs of T. ni a n d eggs p a r a s i t i z e d b y Chelonus sp . r evea led t h a t before h a t c h i n g p a r a s i t i z e d eggs c o n t a i n e d h i g h q u a n t i t i e s of J H ( J H I , J H I I , a n d J H I I I ) w h e r e a s n o n p a r a s i t i z e d ones c o n t a i n e d on ly l i t t le J H ( J H I a n d J H I I ) . A t th is s t age t h e p a r a s i t e s h a t c h a n d re lease t h e se rosa l m e m b r a n e , a n d it w a s sugges t ed t h a t t h e l a t t e r p r o d u c e s J H I I I ( G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990b) . T h e difference in

Tabl

e 1

Effe

cts

of P

aras

ites

on H

ost

Deve

lopm

ent a

nd E

ndoc

rinol

ogy,

and

Put

ativ

e Pa

rasit

e Fa

ctor

s In

volv

ed in

The

se E

ffect

s

Para

site

H

ost

Cat

egor

y D

evel

opm

enta

l ef

fect

s E

ndoc

rine

m

anif

esta

tion

in

host

Fa

ctor

s R

efer

ence

Cote

sia c

ongr

egat

a B

raco

nida

e

Apan

tele

s ka

riyai

B

raco

nida

e

Card

ioch

iles

nigr

icep

s B

ra

coni

dae

Man

duca

sex

ta

Sphi

ngid

ae

Gre

gari

ous

larv

al

Pseu

dale

tia s

epar

ata

Gre

gari

ous

Noc

tuid

ae

larv

al

Hel

ioth

is vi

resc

ens

Noc

tuid

ae

Gre

gari

ous

larv

al

Dev

elop

men

tal

arre

st

in l

ast

inst

ar,

occa

si

onal

ly s

uper

nu

mer

ary

larv

al

inst

ar

Prol

onga

tion

of

lar

val

life,

dev

elop

m

enta

l ar

rest

in

last

sta

dium

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onga

tion

of

lar

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life,

dev

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rest

in

last

sta

dium

JH

titer

ele

vate

d, J

H

este

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low

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, ec

dyso

ne 2

0-m

onoo

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su

ppre

ssed

, se

nsi

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o ec

-dy

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biti

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f P

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synt

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, lo

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to

20-

hydr

oxye

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one,

pr

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raci

c gl

and

sens

itiv

ity

to

PT

TH

su

ppre

ssed

; JH

est

eras

e su

ppr

essi

ng f

acto

r

Func

tion

of

pro-

thor

acic

gla

nd a

nd

its

sens

itiv

ity

to

PT

TH

de

pres

sed

Cal

yx +

ve

nom

+

tera

tocy

tes

Cal

yx -

I- ve

nom

Rev

iew

ed i

n B

ecka

ge

and

Tem

plet

on

(198

6);

Bec

kage

(1

990b

)

Tan

aka

et a

l. (1

987)

; T

anak

a (1

987)

; W

ani

etal

. (1

990)

; H

ayak

awa

(199

0)

Tan

aka

and

Vin

son

(199

1)

72

Mic

ropl

itis

croc

eipe

s B

raco

nida

e H

elio

this

vire

scen

s N

octu

idae

So

litar

y la

rval

D

evel

opm

enta

l ar

rest

in

las

t st

adiu

m

Cam

pole

tis so

nore

n-sis

Ich

-ne

umon

idae

Hel

ioth

is vi

resc

ens

Noc

tuid

ae

Solit

ary

larv

al

Dev

elop

men

tal

arre

st

whe

n la

st i

nsta

r la

rvae

are

att

acke

d

Chel

onus

sp.

Bra

co

nida

e Tr

icho

plus

ia n

i, N

octu

idae

So

litar

y eg

g-la

rval

Pr

ecoc

ious

ons

et o

f m

etam

orph

osis

, em

erge

nce

of p

ara

site

fro

m p

reco

ci

ous

prep

upa

Copi

doso

ma

flori

- Tr

icho

plus

ia n

i, Po

lyem

bryo

nic

Prol

onga

tion

of

feed

-da

num

Enc

yr-

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tuid

ae

egg-

larv

al

ing

in l

ast

stad

ium

ti

dae

Ecd

yste

roid

s lo

wer

ed,

J Η

est

eras

e lo

w

ered

Deg

ener

atio

n of

pro

-th

orac

ic g

land

s,

ecdy

ster

oids

lo

w

ered

Prec

ocio

us a

ppea

ran

ce o

f pr

emet

-am

orph

ic c

hang

es

in J

H,

ec

dyst

eroi

ds,

and

JH

este

rase

; el

evat

ed

JH t

iter

in p

ara

siti

zed

eggs

JH t

iter

elev

ated

, JH

es

tera

se a

nd e

c-dy

ster

oid

incr

ease

de

laye

d

Ter

atoc

ytes

, ca

ly

x

Cal

yx o

r po

lyd-

Poly

dnav

irus

?

Ter

atoc

ytes

?

Web

b an

d D

ahlm

an

(198

6);

Zha

ng a

nd

Dah

lman

(1

989)

; D

ahlm

an e

t al

. (1

990a

,b)

Dov

er e

t al.

(198

7,

1988

a,b,

19

89);

D

over

and

V

inso

n (1

990)

Jone

s et

al

(198

1);

Buh

ler

et a

l. (1

985)

G

ross

nikl

aus-

Biir

gin

and

Lan

zrei

n (1

990b

)

Stra

nd e

t al.

(199

0,

1991

)

73


J H t i te r a p p e a r s n o t to be d u e to different r a t e s of m e t a b o l i s m of J H as l abe led J H I I I was seen to b e d e g r a d e d to t he s a m e e x t e n t b y h o m o g e n a t e s of p a r a s i t i z e d a n d n o n p a r a s i t i z e d eggs in t he closely r e l a t ed s y s t e m C. inan-itus/S. exigua (B . L a n z r e i n , u n p u b l i s h e d ) .

T h e p o l y e m b r y o n i c egg- la rva l p a r a s i t e Copidosoma floridanum ( E n c y r t i d a e ) pa ra s i t i ze s T. ni a n d o t h e r p lus i ine L e p i d o p t e r a . C. floridanum ov ipos i t s i n t o t h e eggs a n d d u r i n g t h e first t h r o u g h four th s t a d i a t h e p a r a s i t e e m b r y o prol i fera tes to form a n ave rage of 1200 m o r u l a s t age e m b r y o s . A p p r o x i m a t e l y 40 of these e m b r y o s in i t i a te m o r p h o g e n e s i s d u r i n g th is p e r i o d to form a m o r p h referred to as t he p recoc ious l a rva ( S t r a n d , 1989). T h e s e l a rvae cease to be fo rmed in t he four th s t a d i u m of T. ni, d o n o t mo l t , a n d d ie in t h e fifth s t a d i u m . T h e r e m a i n i n g e m b r y o s deve lop in to a s econd m o r p h , refer red to as t he r e p r o d u c t i v e l a rva . Dif ferent ia t ion of m o r u l a e beg ins w i t h t h e T. ni m o l t to t h e fifth s t a d i u m (Baehrecke a n d S t r a n d , 1990) a n d t h e r e p r o d u c t i v e l a rvae eclose o n d a y 3 of t h e hos t ' s fifth s t a d i u m . Pa ra s i t i z ed l a rvae a t t a i n a l a rge r final we igh t a n d in i t i a te w a n d e r i n g o n e d a y l a t e r t h a n n o n p a r a s i t i z e d l a rvae . J H t i ter m e a s u r e m e n t s showed t h a t p a r a s i t i z e d T. ni c o n t a i n e d t h r e e t imes m o r e J H t h a n n o n p a r a s i t i z e d ones d u r i n g the m o l t to t h e four th s t a d i u m ( S t r a n d et al., 1991). As the iden t i ty of t he J H w a s n o t d e t e r m i n e d , it is n o t c l ea r w h e t h e r it is of p a r a s i t e o r hos t o r ig in . I n t h e fifth s t a d i u m t h e J H t i ter of p a r a s i t i z e d la rvae fell o n e d a y l a te r c o m p a r e d to t h a t of n o n p a r a s i t i z e d l a rvae ( S t r a n d et al., 1991) a n d c o r r e s p o n d i n g l y ecdys t e ro id a n d J H E inc reases w e r e a lso de l ayed ( S t r a n d et al., 1990). I t is n o t k n o w n w h e t h er t he d e l a y in t h e in i t i a t ion of m e t a m o r p h o s i s a n d t h u s t h e i nc rea se in l e n g t h of t h e hos t ' s feeding p h a s e a r e c a u s e d d i rec t ly b y h o r m o n a l s igna l s f rom t h e p a r a s i t e o r w h e t h e r it is a c o n s e q u e n c e of a l t e r ed hos t physiology.

I n t he following we d i scuss t h e p u t a t i v e e n d o c r i n e effects of t h e v a r i o u s p a r a s i t e - a s s o c i a t e d factors .

A. Substances Secreted by Serosal Membranes/Teratocytes

Tera tocy t e s a r e cells de r ived from the serosal m e m b r a n e of B r a c o n i d a e , T r i -c h o g r a m m a t i d a e , a n d Sce l ion idae ; these cells a p p a r e n t l y a s s u m e different roles ( t roph ic , i m m u n o s u p p r e s s i o n , secre tory) in different species ( rev iewed by D a h l m a n , 1990). Endocr ino log ica l ly , t h e l ink b e t w e e n t h e se rosa l m e m b r a n e / t e r a tocy t e s a n d t h e J H s y s t e m is m o s t i n t r i g u i n g . S o m e o b s e r v a t i o n s sugges t t h a t se rosa l m e m b r a n e s a n d y o u n g t e r a tocy te s of s o m e species cont a in a n d / o r re lease J H . I n t he sys t em D. longicaudata-A. suspensa, c o m p a r a tive J H I I I m e a s u r e m e n t s in n o n p a r a s i t i z e d a n d p a r a s i t i z e d A. suspensa a n d d i s sec ted p a r a s i t e s sugges ted t h a t se rosa l m e m b r a n e s p r o b a b l y c o n t a i n J H I I I ( L a w r e n c e et al., 1990). T h e s a m e hypo thes i s w a s p u t f o r w a r d in t h e sys t em Chelonus sp.-T. ni, w h e r e p a r a s i t i z e d eggs were seen to c o n t a i n m u c h


h i g h e r q u a n t i t i e s of J H t h a n n o n p a r a s i t i z e d ones a t t he s t a g e of re lease of se rosa l m e m b r a n e s i n to t he hos t ( G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990b) . I n t h e sys t em C. nigriceps-H. virescens, in ject ion of t e r a tocy t e s w a s seen to h a v e J H - l i k e effects, w i t h y o u n g e r t e r a tocy te s b e i n g m o r e effective ( V i n s o n , 1970), a n d J H ana ly se s m a d e by Galleria b ioa s say revea led h i g h c o n c e n t r a t i o n s of J H in 7-day-old t e r a tocy t e s a n d m u c h lower c o n c e n t r a t i o n s in t e r a tocy t e s of m i x e d ages ( J o i n e r et aL, 1973). In te res t ing ly , se rosa l e p i t h e -lia of t h e locus t Locusta migratoria h a v e b e e n s h o w n to p r o d u c e J H I I I ( H a r t -m a n n et aL, 1987), w h i c h d e m o n s t r a t e s t h e capab i l i t y of th is t i ssue to s y n t h e size J H .

O l d e r t e r a tocy te s of s o m e species a p p e a r to in ter fere w i t h t h e J H s y s t e m a n d to i nc r ea se t h e J H t i te r in t h e h o s t b y ac t ing o n t h e J H E . I n t h e s y s t e m M. croceipes-H. viresecens, in ject ion of t e r a tocy te s w a s seen to i n h i b i t l a r v a l -p u p a l t r a n s f o r m a t i o n in t h e hos t in a dose - a n d a g e - d e p e n d e n t m a n n e r , w i t h y o u n g e r t e r a tocy t e s b e i n g m o r e act ive a n d y o u n g e r hos t s b e i n g m o r e sens i t ive ( Z h a n g a n d D a h l m a n , 1989). I t is i n t e r e s t i ng to n o t e t h a t C. congregata t e r a tocy t e s col lec ted from M. sexta h a d n o effect w h e n in jec ted i n t o H. virescens; m e a s u r e m e n t of J H E in h e m o l y m p h of / / , virescens l a rvae in jec ted w i t h t e r a tocy t e s f rom M. croceipes r evea led t h a t J H E w a s d r a m a t i c a l l y d e p r e s s e d w h e n t h e n o r m a l n u m b e r (750) of t e r a tocy t e s f rom a s ingle p a r a s i t e w a s in jec ted , b u t n o t w h e n t e r a tocy te s f rom C. congregata w e r e in jec ted ( Z h a n g a n d D a h l m a n , 1989). I n t h e s y s t e m Apanteles karyiai-Pseudaletia separata, in ject ion of t e r a tocy t e s d e l a y e d l a r v a l - l a r v a l a n d l a r v a l - p u p a l ecdys is ( W a n i et aL, 1990). I n th is s y s t e m h e m o l y m p h of l a rvae col lec ted 3 - 6 d a y s after p a r a s i t i z a t i o n w a s seen to c o n t a i n a factor t h a t s u p p r e s s e s J H E ; th is factor w a s pur i f ied , found to b e a p r o t e i n , a n d pa r t i a l l y s e q u e n c e d ( H a y a k a w a , 1990), b u t i ts o r ig in (hos t , t e r a tocy t e s , p o l y d n a v i r u s ) h a s n o t b e e n inves t i g a t e d . I n al l of these s y s t e m s , p a r a s i t i z a t i o n causes d e v e l o p m e n t a l a r r e s t before p u p a t i o n (Tab le 1). I n t h e e n d o c r i n e c h a n g e s a s soc i a t ed w i t h m e t a m o r p h o s i s a n a b s e n c e of J H is neces sa ry n o t on ly for p u p a l c o m m i t m e n t b u t a l so for t h e r e g u l a t i o n of P T T H a n d s t e ro idogen ic c o m p e t e n c e of t h e p r o -tho rac i c g l a n d s (see Sec t ion V.B) , a n d c o n s e q u e n t l y a n in te r fe rence w i t h t h e r e m o v a l of J H b y es te rases h a s m u l t i p l e effects.

B. Calyx Fluid, Viruses, and Venom Gland Secretions

V i r u s e s h a v e b e e n i m p l i c a t e d in i m m u n o s u p p r e s s i o n a n d in a l t e r i n g t h e n u t r i t i o n a l a n d h o r m o n a l b a l a n c e of t h e hos t (Stol tz a n d V i n s o n , 1979). I n t h e s y s t e m C. congregata-M. sexta, p o l y d n a v i r u s h a s b e e n s h o w n to i n d u c e t h e a p p e a r a n c e of pa ras i t i sm-spec i f i c p r o t e i n s in t h e h o s t (Beckage , 1990a) . I n t h e s y s t e m C. sonorensis/H. virescens, in ject ion of ca lyx fluid o r p o l y d n a v i r u s i n t o f if th-instar l a rvae led to d e g e n e r a t i o n of t he p r o t h o r a c i c g l a n d a n d r e -


d u c e d ecdys te ro id t i ters (Dover et al., 1987, 1988a ,b , 1989). H o s t s s t u n g in t he four th s t a d i u m showed n o s igns of p r o t h o r a c i c g l a n d d e g e n e r a t i o n a n d inject ion of ca lyx in to ear ly f o u r t h - s t a d i u m la rvae d i d n o t c a u s e p r o t h o r a c i c g l a n d d e g e n e r a t i o n , sugges t ing t h a t i n d u c t i o n of p r o t h o r a c i c g l a n d d e g e n e r a t ion by p o l y d n a v i r u s is specific to t he las t s t a d i u m of t he hos t (Dover a n d V i n s o n , 1990). I n t he sys t em M. croceipes-H. virescens, in ject ion of ca lyx fluid i n h i b i t e d g r o w t h a n d d e v e l o p m e n t as well as J H E activity, effects a l so seen u p o n the inject ion of t e r a tocy tes (see T a b l e 1 a n d ear l i e r d i scuss ion ) , a n d it was sugges t ed t h a t t e r a tocy tes c o n t a i n p o l y d n a v i r u s o r s o m e of i ts g e n o m i c i n f o r m a t i o n ( D a h l m a n et al., 1990b) .

V e n o m g l a n d secre t ions exh ib i t a w ide r a n g e of act ivi t ies . T h o s e p r o d u c e d by e c t o p a r a s i t e s a r e often p a r a l y t i c , t hose of s o m e e n d o p a r a s i t e s have b e e n s h o w n to c o n t r i b u t e to t he a b r o g a t i o n of t he hos t ' s i m m u n e sy s t em, a n d o t h e r s have b e e n i m p l i c a t e d in t he p e r t u r b a t i o n of t h e e n d o c r i n e s y s t e m of t he hos t (Stol tz a n d V i n s o n , 1979; Stol tz , 1986). A n i m p o r t a n t func t ion of v e n o m s a p p e a r s to be the i r role as synerg is t s of v i ruses in t h a t t hey faci l i tate p e n e t r a t i o n a n d surv iva l of v i rus in hos t cells (Stol tz et al., 1988). I n t h e sys t em A. karyiai-P. separata (see T a b l e 1), ca lyx fluid p lus v e n o m have b e e n s h o w n to s u p p r e s s t he sensi t iv i ty of p r o t h o r a c i c g l a n d s to P T T H , a n d in t h e sys t em C. nigriceps-H. virescens (see T a b l e 1), t he two c o m p o n e n t s t o g e t h e r have b e e n s h o w n to d e p r e s s t he funct ion of t he p r o t h o r a c i c g l a n d a n d its c o m p e t e n c e to r e s p o n d to P T T H ( T a n a k a a n d V i n s o n , 1991).

F r o m these de sc r ip t i ons it is obv ious t h a t t he m e c h a n i s m s a n d factors t h r o u g h w h i c h p a r a s i t e s a l t e r the i r hos t ' s e n d o c r i n e s y s t e m v a r y w i t h t he p a r a s i t e species involved a n d w i th t he hos t species a n d s t age a t t a c k e d . S o m e effects m a y be d u e to pa r a s i t e -de r i ved s u b s t a n c e s ac t ing o n e n d o c r i n e g l a n d s , o n h o r m o n e m e t a b o l i s m , o r o n t h e sens i t iv i ty of t a r g e t g l a n d s a n d t i s sues , o r in te r fe r ing w i t h i n t e r e n d o c r i n e axes in t he P T T H / e c d y s o n e a n d J H s y s t e m a n d feedback loops . I n t e r a c t i o n s w i t h m e t a b o l i c h o r m o n e s have n o t ye t b e e n inves t iga t ed b u t a r e a lso a likely t a rge t for p a r a s i t e ac t ion . A t leas t in o n e sys t em it h a s b e e n s h o w n t h a t t he t h r e e factors ca lyx, v e n o m , a n d t e r a tocy t e s in jected in a s e q u e n c e t h a t s i m u l a t e d t he in vivo s i t ua t i on s i m u l a t e d t h e d e v e l o p m e n t a l effect of p a r a s i t i s m ( W a n i et al., 1990). T h i s i n d i c a t e s t h a t t h e va r ious pa r a s i t e - a s soc i a t ed factors act in conce r t a n d m i g h t exp la in w h y a p p l i c a t i o n of on ly o n e factor usua l ly does no t i n d u c e t h e full s p e c t r u m of p a r a s i t i s m l i k e a l t e r a t i o n s .

VI. Limitations

T h e foregoing review clear ly d e m o n s t r a t e s t h a t o u r s t u d y of p a r a s i t e - h o s t e n d o c r i n e i n t e r ac t ions is in its infancy a n d m u c h n e e d s to b e d o n e before we


c a n a t t e m p t to u n d e r s t a n d th is very c o m p l i c a t e d a s soc ia t ion . A m o n g t h e factors t h a t c o n t r i b u t e to o u r l imi ted p rog re s s a r e t h e following: (1) o u r inab i l i ty to s e p a r a t e (successfully) p a r a s i t e f rom hos t over a p r o l o n g e d p e r i o d in o r d e r to s t u d y the physiology, n u t r i t i o n a l b iochemis t ry , a n d e n d o c r i n o l o g y s e p a r a t e l y ; (2) t h e lack of a p p r o p r i a t e t echno logy t h a t a l lows t h e s t u d y of very sma l l o r g a n i s m s such as insec t p a r a s i t e s , p a r t i c u l a r l y t h e H y m e n o p t e r a , a n d t h e m i n u t e v o l u m e s of h e m o l y m p h ava i l ab le for a c c u r a t e h o r m o n e ana ly sis, b o t h q u a l i t a t i v e a n d q u a n t i t a t i v e ; (3) o u r i n t e r p r e t a t i o n s of t h e effects of h o r m o n e s o n hos t s a n d p a r a s i t e s , for e x a m p l e , i nc reases in h o r m o n e t i te r m a y faci l i tate c e r t a i n ce l lu la r even t s n o t r ead i ly d e t e c t a b l e b u t a dec l ine in t h e s a m e h o r m o n e m a y t h e n resu l t in s o m e m e a s u r a b l e c h a n g e ; a n d (4) n u m e r o u s p e p t i d e h o r m o n e s o c c u r in insec ts b u t m o s t paras i te—host s t ud i e s have b e e n conf ined to J H a n d ecdys t e ro ids . W h a t roles d o p e p t i d e h o r m o n e s p l a y in h o s t - p a r a s i t e in t e rac t ions?

T h e m a j o r l imi t a t i ons in d o i n g endoc r ino log ica l s tud ie s w i t h insec t p a r a sites a r i se f rom t h e sma l l size of p a r a s i t e eggs a n d l a rvae (fract ions of a m i c r o g r a m to a few mi l l i g r ams ) . Fo r m e a s u r e m e n t of J H s for i n s t a n c e , t h e m o s t r e l i ab le m e t h o d is G C - M S , w h i c h al lows prec i se quan t i f i c a t i on of i nd i v i d u a l J H s (for de ta i l s , see Baker , 1990), b u t th is m e t h o d r e q u i r e s q u a n t i t i e s of b iological m a t e r i a l t h a t a r e often n o t ava i l ab le a n d on ly a few l a b o r a t o r i e s have t h e neces sa ry cost ly e q u i p m e n t a n d the exper t i se .

R a d i o i m m u n o a s s a y s of b io logica l ex t rac t s d o n o t p r o v i d e a b s o l u t e inform a t i o n a b o u t t he c o n c e n t r a t i o n of i n d i v i d u a l ecdys t e ro ids . T h i s is p r i m a r i l y b e c a u s e t h e a n t i b o d i e s c o m m o n l y u s e d c ross - reac t w i t h severa l e cdys t e ro id s to v a r y i n g deg rees (for de ta i l s , see W a r r e n a n d G i lbe r t , 1986; R e u m a n d K o o l m a n , 1989). C o n s e q u e n t l y , a c o m b i n a t i o n of R I A w i t h c h r o m a t o g r a p h i c m e t h o d s s u c h as t h in - l aye r c h r o m a t o g r a p h y or p re fe rab ly H P L C al lows quan t i f i c a t i on of i n d i v i d u a l ecdys t e ro ids , p r o v i d e d t h e c ross - reac t iv i ty of t h e v a r i o u s ecdys t e ro ids w i t h t he a n t i b o d y is k n o w n . For m e a s u r i n g J H s , severa l a p p r o a c h e s a r e b e i n g used : p h y s i c o c h e m i c a l m e t h o d s ( G C - M S ) , b i o a s s ay s , R I A s , a n d c o m b i n a t i o n s of H P L C w i t h b ioas say o r R I A . I n f o r m a t i o n a b o u t t h e fluctuation of i n d i v i d u a l J H s c a n be bes t o b t a i n e d by G C - M S . T h e c o m b i n a t i o n of H P L C w i t h b ioas say or R I A c a n a lso fulfill th i s goa l , p r o v i d e d s t a n d a r d cu rves for t h e different J H s a r e ava i l ab le (b ioassays ) o r c ross -react iv i t ies of t h e a n t i b o d y w i t h t h e i n d i v i d u a l J H s a r e k n o w n ( R I A ) .

I n cases w h e r e specific m e t h o d s were used a n d m e a s u r e m e n t s t a k e n a t s h o r t i n t e rva l s ( 6 - 8 h r ) , h o r m o n e t i te rs w e r e seen to fluctuate rap id ly . For e x a m p l e , P T T H w a s seen to b e re l eased in b u r s t s ( rev iewed in G i l b e r t , 1989), i n d i v i d u a l J H s d i s p l a y e d s h o r t a n d s h a r p p e a k s ( B a k e r et aL, 1987; G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990a) , a n d a l so e c d y s o n e a n d 20-h y d r o x y e c d y s o n e were seen to fluctuate r a p i d l y (Lafon t et aL, 1975; G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n , 1990a) . I n a d d i t i o n , s ince very m i n o r


h o r m o n e inc reases a n d dec reases c a n b e biological ly s ignif icant , it w o u l d b e m o s t d e s i r a b l e to a n a l y z e prec ise ly s t aged insec ts a t s h o r t i n t e rva l s .

T h e sma l l size of p a r a s i t e s m a k e s it imposs ib l e to m e a s u r e h o r m o n e t i t e rs in t h e h e m o l y m p h or to rou t ine ly m e a s u r e r a t e s of h o r m o n e syn thes i s by i n c u b a t i n g g l a n d s (GA, p r o t h o r a c i c g l a n d s , o r b r a i n s ) in vitro. C o n s e q u e n t l y , m a n y a p p r o a c h e s u sed to inves t iga te t he e n d o c r i n e s t a t u s of p a r a s i t e s a r e ind i rec t . I n t he case of h o r m o n e a p p l i c a t i o n to t h e hos t , it is n o t poss ib le to k n o w if a pa r a s i t e ' s r eac t ion is c a u s e d by t h e h o r m o n e itself o r b y h o r m o n e -i n d u c e d c h a n g e s in t he hos t . L ikewise , ana lys i s of h o r m o n e m e t a b o l i s m b y p a r a s i t e s in vivo is very difficult as t he l abe led h o r m o n e h a s to b e in jected i n t o t h e hos t , w h i c h a lso me tabo l i ze s it. I f t he s a m e h o r m o n e m e t a b o l i t e s a r e ident if ied in p a r a s i t e a n d hos t it is no t poss ib le to k n o w w h e t h e r t hey w e r e m a d e b y t h e p a r a s i t e o r s imp ly t a k e n u p a s m e t a b o l i t e s . I f p a r a s i t e s u se as yet un iden t i f i ed , paras i te -spec i f ic h o r m o n e s , t he i r ana lys i s w o u l d b e ext r eme ly difficult b e c a u s e of t he lack of specific b ioas says .

VII. Possible Solutions

T o d a t e , t he p h y s i o l o g y / e n d o c r i n o l o g y of on ly very few h o s t - p a r a s i t e syst e m s have b e e n s t ud i ed . C o n s e q u e n t l y , fu r the r ana lyses of p a r a s i t e h o r m o n e s in r e l a t ion to those of the i r hos t s s h o u l d b e ca r r i ed o u t . E m p h a s i s o n hos t s t h a t have a l r e a d y b e e n endocr ino log ica l ly a n a l y z e d w o u l d faci l i tate t h e task . However , t h e ma jo r i t y of these hos t species a r e l e p i d o p t e r a n s . T h e r e f o r e , a d d i t i o n a l efforts s h o u l d be m a d e to i n c l u d e hos t g r o u p s s u c h as t h e h e m i p -t e r a n s a n d d i p t e r a n s s ince r e a s o n a b l e endoc r ino log ica l s tud ie s h a v e b e e n d o n e o n se lected m e m b e r s of these g r o u p s . F u r t h e r m o r e , severa l species of h e m i p t e r a n s a r e m a j o r a g r i c u l t u r a l pes t s a n d n u m e r o u s d i p t e r a n species a r e e i the r themse lves p a r a s i t e s o r vec tors of h u m a n a n d a n i m a l d i seases . C o n s e quen t ly , s tud ie s on t he h o r m o n a l i n t e r ac t ions of p a r a s i t e s of t hese hos t s w o u l d b e v a l u a b l e in pes t con t ro l efforts.

W h e n ana lyses of J H s a r e be ing u n d e r t a k e n , they s h o u l d b e m e a s u r e d ind iv idua l ly . I n t he case of severa l l e p i d o p t e r o u s hos t s , th is w o u l d a l low d i s c r i m i n a t i o n b e t w e e n the h y m e n o p t e r a n p a r a s i t e - p r o d u c e d J H I I I a n d hos t J H I I a n d J H I , o r in t he case of l e p i d o p t e r a n hos t eggs , J H 0. H o w e v e r , t h e p r o b l e m s will b e m o r e difficult in h y m e n o p t e r a n — d i p t e r a n p a r a s i t e - h o s t sy s t ems in w h i c h J H I I I is c o m m o n to b o t h g r o u p s . O n e a p p r o a c h t h a t cou ld a l lev ia te this p r o b l e m a n d s i m u l t a n e o u s l y e luc ida t e t h e effect of p a r a s i t e s o n t h e e n d o c r i n e g l a n d s of t he hos t a n d vice ve rsa is t h e ana lys i s of t h e p r o d u c t ion in vitro of h o r m o n e s o r o t h e r s u b s t a n c e s by in t ac t p a r a s i t e s , o r by g l a n d s of hos t s a n d the i r n o n p a r a s i t i z e d c o u n t e r p a r t s . T h i s a p p r o a c h h a s b e e n suc cessfully u sed to d e m o n s t r a t e p a r a s i t e u p t a k e of l abe led p r o t e i n s (Beckage et


al., 1989) a n d a m i n o ac ids (Ferkovich a n d D i l l a rd , 1986), l abe l ed h o r m o n e s ( L a w r e n c e a n d H a g e d o r n , 1986), t h e re lease of p r o t e i n s b y p a r a s i t e s (Lawr e n c e , 1990a) , a n d hos t p r o t h o r a c i c g l a n d act iv i ty ( T a n a k a et al., 1987). O t h e r in vitro s t ud ie s on p a r a s i t e s h a v e i n c l u d e d t h e successful r e a r i n g of p u p a l p a r a s i t e s a n d a s t u d y of t h e n u t r i t i o n a l r e q u i r e m e n t s of o t h e r s (for r ev iews , see G r e a n y , 1986; Ne t t l e s , 1990) a n d t h e r e q u i r e m e n t s of s o m e p a r a s i t e s for e x o g e n o u s h o r m o n e s ( N e o n , 1972; L a w r e n c e , 1986b, 1988d, 1991).

O t h e r a s ye t un re so lved q u e s t i o n s in host—paras i te i n t e r ac t i ons r e l a t e to t hose p a r a s i t e - d e r i v e d factors t h a t d i s r u p t t h e hos t ' s e n d o c r i n o l o g y (see Sect ion V ) . T h e i r inf luence on s u s p e c t e d t a r g e t t i ssues a n d g l a n d s of t h e h o s t cou ld b e e v a l u a t e d in vitro.

VIII. Summary

I n t h e p r e c e d i n g d i scuss ion , we have a t t e m p t e d to focus p r i m a r i l y o n hy-m e n o p t e r o u s p r o t e l e a n e n d o p a r a s i t e s t h a t a t t a c k eggs a n d l a rvae of l e p i d o p -t e r a n s a n d l a rvae of d i p t e r a n s . We have u t i l ized ex is t ing i n f o r m a t i o n o n t h e g e n e r a l e n d o c r i n e sys t ems a n d i n t e r e n d o c r i n e i n t e r ac t ions k n o w n in insec t s as t h e bas i s of o u r d i scuss ion a n d h a v e c o m p l e m e n t e d th is w i t h t h e l imi t ed d a t a ava i l ab le f rom a few h o s t - p a r a s i t e sy s t ems .

T h e i n f o r m a t i o n ava i l ab le so far i nd i ca t e s t h a t t h e d e p e n d e n c e of p a r a s i t e s on t h e hos t ' s e n d o c r i n e sys t em var ies f rom s t r o n g to m a r g i n a l a n d is c h a r a c ter is t ic of each p a r a s i t e - h o s t sy s t em. I t a p p e a r s t h a t t he e n d o c r i n e i n t e r a c t ions b e t w e e n p a r a s i t e s a n d the i r hos t s va ry f rom those t h a t r e q u i r e t h e hos t ' s h o r m o n e s (especia l ly those g o v e r n i n g g r o w t h , d e v e l o p m e n t , a n d m e t a m o r p h o s i s ) for t he i r o w n d e v e l o p m e n t (conformers ) to t hose t h a t signific a n t l y modify the i r hos t ' s e n d o c r i n e s y s t e m in o r d e r to r e t a i n t h e h o s t for s o m e p e r i o d of t i m e in a p a r t i c u l a r phys io log ica l a n d likely, n u t r i t i o n a l s t a t e ( r egu l a to r s ) . A t h i r d c a t e g o r y of p a r a s i t e s a p p e a r s to u t i l ize a c o m b i n e d s t ra tegy. I n th is l a t t e r g r o u p , ea r ly s t ages ( e m b r y o s to first-instar l a rvae) a p p e a r to d i s p l a y a h i g h e r d e g r e e of d e p e n d e n c e t h a n l a t e r s t ages . P a r a s i t e effects b e c o m e man i fes t in l a t e r s t ages of hos t d e v e l o p m e n t a n d often involve a d i s t u r b a n c e of t h e P T T H / e c d y s o n e axis a n d / o r t h e J H ax i s . I n c a u s i n g these effects, all factors , name ly , v i ru s , v e n o m , a n d t e r a tocy t e s (if p r e s e n t ) , a p p e a r to b e involved b u t the i r i n t e r r e l a t i o n s h i p a n d i m p a c t o n t h e hos t ' s e n d o c r i n e s y s t e m a r e n o t ye t u n d e r s t o o d . S o m e p a r a s i t e s have b e e n s h o w n to p r o d u c e the i r o w n h o r m o n e s (a t l eas t after t h e l a te first i n s t a r ) a n d th is m i g h t b e gene ra l l y t r u e , b u t it is n o t c lea r w h e t h e r p a r a s i t e h o r m o n e s o r m e t a b o l i t e s t he reo f a r e ever r e l eased i n t o t h e hos t a n d u s e d to inf luence i ts d e v e l o p m e n t .


Acknowledgments

Financial support from the National Science Foundation, Grant DCB 9005514, and the USDA, CRGO Grant 9001300, to P.O.L., and the Swiss National Science Foundation, Grant 31-27723.89, to B. L. is gratefully acknowledged.

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Vinson, S. B. (1970). Development and possible function of teratocytes in the host-parasite association. J. Invertebr. Pathol. 16:93-101.

Vinson, S. B., and Iwantsch, G. F. (1980a). Host suitability for insect parasitoids. Anna. Rev. Entomol. 25 , 397-419.

Vinson, S. B., and Iwantsch, G. F. (1980b). Host regulation by insect parasitoids. Q. Rev. Biol. 55: 143-165.

Wani, M., Yagi, S., and Tanaka, T. (1990). Synergistic effect of venom, calyx and teratocytes of Apanteles kariyai on the inhibition of larval-pupal ecdysis of the host, Pseudaletia separata. Entomol. Exp. Appl. 57:101-104.

Warren, J. T , and Gilbert, L. I. (1986). Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta. Insect Biochem. 16:65—82.

Watson, R. D., and Bollenbacher, W. E. (1988). Juvenile hormone regulates the steroidogenic competence of Manduca sexta prothoracic glands. Mol. Cell. Endocrinol. 57:251-259.

Watson, R. D., Spaziani, E., and Bollenbacher, W. E. (1989). Regulation of ecdysone biosynthesis in insects and crustaceans: A comparison. In "Ecdysone" (J. Koolman, ed.), pp. 188-203. Thieme, Stuttgart.

Webb, Β. Α., and Dahlman, D. L. (1986). Ecdysteroid influence on the development of the host Heliothis virescens and its endoparasite Microplitis croceipes. J. Insect Physiol. 32:339—345.


Wing, S. (1988). RH 5849, a nonsteroidal ecdysone agonist: Effects on a Drosophila cell line. Science 241:467-469.

Wright, J. E., and Spates, G. E. (1972). A new approach in integrated control: Insect juvenile hormone plus a hymenopteran parasite against the stable fly. Science 178:1292-1293.

Wyatt, G. R. (1980). The fat body as a protein factory. In "Insect Biology in the Future—VBW 80" (M. Locke and D. S. Smith, eds.), pp. 201-225. Academic Press, New York.

Zhang, D. , and Dahlman, D. L. (1989). Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae. Arch. Insect Biochem. Physiol. 12:51-61 .

Chapter 4

ΗReproductive Disturbances Induced by Parasites and Pathogens of Insects Hilary Hurd Department of Biological Sciences University of Keele Staffordshire, United Kingdom

I. Introduction

II. Microparasites A. Microsporidia

III. Nematodes

IV. Cestodes

V. Endoparasitic Insects

VI. Conclusions References

I. Introduction

Paras i t e s a n d p a t h o g e n s p l a y a m a j o r ro le as r e g u l a t o r s of h o s t life h i s to r ies a n d p o p u l a t i o n d y n a m i c s (see, e.g., D o b s o n , 1988; H o l m e s a n d Z o h a r , 1990; M a y a n d A n d e r s o n , 1990) a n d the i r inf luence u p o n t h e evo lu t ion a n d m a i n t e n a n c e of h o s t s exua l b e h a v i o r h a s b e e n rev iewed recen t ly ( R e a d , 1990). I t is m y i n t e n t i o n to confine th is r ev iew to o r g a n i s m s t h a t affect insec t p o p u l a t ions , n o t as l e tha l a g e n t s , b u t b y r e d u c i n g r e p r o d u c t i v e o u t p u t o r c a u s i n g h o s t steri l i ty. Pa ra s i t i zed i n v e r t e b r a t e s c o m m o n l y exh ib i t s o m e form of r e p r o d u c t i v e c u r t a i l m e n t ( H u r d , 1990a) a n d d iverse a spec t s of phys io logy a n d b e h a v i o r c a n b e affected, r a n g i n g f rom i r revers ib le d e s t r u c t i o n of r e p r o d u c tive t i ssue ( t r u e c a s t r a t i o n ) to c h a n g e s in s exua l b e h a v i o r o r j u v e n i l i z a t i o n . A l t h o u g h e x a m p l e s of r e p r o d u c t i v e dys func t ion in infected insec ts a r e a b u n d a n t , a t t e m p t s to e l u c i d a t e t h e u n d e r l y i n g m e c h a n i s m s h a v e r a re ly b e e n m a d e . W h e r e v e r poss ib le I will focus u p o n these s tud ie s b u t m a t e r i a l is a r r a n g e d to p r e s e n t a t a x o n o m i c r a t h e r t h a n m e c h a n i s t i c survey. T h u s , t h e effects of m i c r o p a r a s i t e s [def ined b y A n d e r s o n a n d M a y (1981) as s m a l l p a t h o g e n s w i t h a h i g h r a t e of r e p r o d u c t i o n w i t h i n t h e hos t a n d a s h o r t g e n e r a t i o n t i m e ] , n e m a t o d e s , ces todes , a n d e n d o p a r a s i t i c insec t s u p o n insec t r e p r o d u c t i o n will b e e x a m i n e d .

Parasites and Pathogens of Insects Copyright © 1993 by Academic Press, Inc. Volume 1: Parasites 8 7 All rights of reproduction in any form reserved.

8 8 Hilary Hurd

II. Microparasites

T h e i m p a c t of n o n l e t h a l v i ra l , fungal , a n d bac te r i a l infect ions u p o n insec t p o p u l a t i o n d y n a m i c s is very g r e a t , b o t h hos t r e p r o d u c t i v e o u t p u t a n d surv ivo r sh ip of offspring b e i n g affected. However , a su rvey of th is field w o u l d r e q u i r e m o r e space t h a n is ava i l ab le a n d I shal l therefore c o n c e n t r a t e u p o n those m i c r o p a r a s i t e s genera l ly r e g a r d e d as b e i n g w i t h i n t h e r e m i t of p a r a sitology, t he P ro t i s t a . A l t h o u g h m a n y insec ts ac t as hos t s for p r o t o z o a w i t h s ing le-s tage life cycles , s o m e a r e vec tors o r i n t e r m e d i a t e hos t s for p a r a s i t e s w i t h v e r t e b r a t e definit ive hos t s , often c a u s i n g d i seases of m e d i c a l o r e c o n o m ic i m p o r t a n c e . O u r k n o w l e d g e of t he in t e rac t ion b e t w e e n t h e P ro t i s t a a n d insec t -hos t r e p r o d u c t i v e o u t p u t is su rp r i s ing ly scanty , especia l ly in v iew of t he i m p o r t a n c e of, for e x a m p l e , t he D i p t e r a in t he ep idemio logy of p a r a s i t i c d i sease . T h e ma jo r i ty of s tud ies have c o n c e n t r a t e d u p o n m i c r o s p o r i d i a l infections a n d these will be dea l t w i th separa te ly .

A c o m p a r i s o n of r e p r o d u c t i v e o u t p u t in six s t r a ins of Aedes aegypti (Hacke r , 1971) showed a m a r k e d difference in t he r e d u c t i o n in fecund i ty c a u s e d b y Plasmodium gallinaceum, w i t h t he R O C K s t r a in b e i n g the m o s t affected. T h e poss ib le i m p a c t of t he p a r a s i t e u p o n t h e gene t i c s t r u c t u r e of a p o l y m o r p h i c hos t p o p u l a t i o n , a n d its role in t he m a i n t e n a n c e of a b a l a n c e d p o p u l a t i o n s t r u c t u r e , w a s d i scussed . F u r t h e r s tud ies ( H a c k e r a n d K i l a m a , 1974) showed t h a t a r e d u c t i o n in m o s q u i t o fecundi ty o c c u r r e d a t very low levels of p a r a s i t emia , m e a s u r e d in t e r m s of b o t h n u m b e r of p a r a s i t e s p e r 100 ch icken e ry th rocy t e s in the b lood m e a l a n d oocys t dens i t y in t h e m o s q u i t o gu t . H o s t fecundi ty w a s no t r e l a t ed to p a r a s i t e b u r d e n in this s tudy.

F re i e r a n d F r i e d m a n (1976) conf i rmed t h a t P. gallinaceum-'mfected A. aegypti la id fewer eggs ( an ave rage of 3 5 % less) t h a n those fed o n un in fec ted ch ickens a n d a lso d e m o n s t r a t e d t h a t egg v iabi l i ty w a s unaffected b y infect ion . T h e size of t h e b lood m e a l t a k e n by A. aegypti is k n o w n to inf luence t h e n u m b e r of eggs la id (see d i scuss ion in F re i e r a n d F r i e d m a n , 1976). F e m a l e m o s q u i t o e s feeding on pa ra s i t i z ed ch ickens were s h o w n to t ake 4 3 % less b lood t h a n those feeding on un infec ted b lood a n d d e c r e a s e in b l o o d m e a l size a n d fecundi ty w e r e b o t h a s soc ia t ed w i t h a r ise in b lood p a r a s i t e m i a (F re ie r a n d F r i e d m a n , 1976), a l t h o u g h c o n s i d e r a b l e va r iab i l i ty exis ted b e t w e e n i nd i v i d u a l insec ts . Infec ted m o s q u i t o e s a r e k n o w n to exh ib i t a l t e r ed p r o b i n g behav io r , p r o b a b l y assoc ia ted w i t h t he p r e s e n c e of sporozo i tes in t h e sa l iva ry g l a n d s (see e.g., M o l y n e u x a n d Jeffer ies , 1976). F r e i e r a n d F r i e d m a n (1976) found t h a t p r o b i n g b e h a v i o r of un infec ted females d i d n o t differ w h e n feeding on infected ch ickens b u t b lood inges t ion r a t e s were r e d u c e d from 1.23 to 0.66 m g / m i n . F u r t h e r m o r e , feeding choice e x p e r i m e n t s i n d i c a t e d t h a t females p re fe r red un infec ted ch ickens a n d could d i s t i ngu i sh b e t w e e n b i r d s sus -

4. Reproductive Disturbances Induced by Parasites and Pathogens 8 9

p e n d e d 1 c m from t h e cage . T h i s r e p o r t i nd i ca t e s t h a t factors o t h e r t h a n , o r in a d d i t i o n to , q u a l i t y of b lood m e a l a r e involved in feeding cho ice a n d the a u t h o r s c o n c l u d e t h a t olfact ion a n d g u s t a t i o n m a y b e n o n i n t e r a c t i n g factors affecting feeding behav io r .

T h e r e is c lear ly m u c h w o r k still to b e d o n e to clarify t h e r e l a t i o n s h i p b e t w e e n b lood i n t a k e a n d fecundity. I nves t i ga t i ons n e e d to b e p e r f o r m e d to d e t e r m i n e t he effect of m a l a r i a on egg p r o d u c t i o n in t h e s econd a n d s u b s e q u e n t g o n o t r o p h i c cycles, w h e n t h e p a r a s i t e is e s t ab l i shed in t h e m o s q u i t o . I n a d d i t i o n t h e r e h a v e b e e n n o de t a i l ed s tud ies of t he effect of Plasmodium s p p . u p o n egg l ay ing in Anopheles m o s q u i t o e s , t h e vec tors of h u m a n m a l a r i a . Sh i eh a n d Ross igno l (1992) d i scussed t h e inf luence of t he a n a e m i a a s soc i a t ed w i t h b l o o d - b o r n e p a r a s i t e s u p o n m o s q u i t o feeding a n d s u b s e q u e n t fecundi ty . T h e y conf i rmed t h e D a n i e l a n d K i n g s o l v e r m o d e l p r e d i c t i o n s b y d e m o n s t r a t i n g t h a t Aedes aegypti feed faster o n a n a e m i c b lood a n d a lso d e m o n s t r a t e d t h e n e g a t i v e inf luence of a n a e m i c b lood on egg l ay ing . T h e s ignif icance of these findings to t he field s i t u a t i o n , w h e r e fast feeding m a y b e i m p o r t a n t w h e n feeds a r e f requen t ly i n t e r r u p t e d d u e to hos t defences , w a s d i scussed .

T h e a m o e b a Malamoeba locustae infects t he M a l p i g h i a n t u b u l e s of t h e d e s e r t locus t a n d causes a p rogress ive a t t r i t i o n of t h e hos t fat body. C h a n g e s in fat b o d y p r o t e i n m e t a b o l i s m resu l t in a r e d u c t i o n in h e m o l y m p h p r o t e i n t i ter , w h i c h m a y a c c o u n t for t h e s ignif icant d e l a y in oocy te d e v e l o p m e n t o b s e r v e d in infected females (Pap i l lon a n d Cass ie r , 1978). F a t b o d y a t t r i t i o n is a l so a s soc i a t ed w i t h M. locustae infect ion of g r a s s h o p p e r s .

U s i n g t h e M. locustaelgrasshopper m o d e l J a c k s o n et al. (1968) m a d e a n e x a m i n a t i o n of egg l ip ids . A l t h o u g h n o c h a n g e in to ta l l ip id c o n t e n t o r l ip id c o m p o s i t i o n o c c u r r e d in infected g r a s s h o p p e r s , t h e re la t ive a b u n d a n c e of fat ty ac ids w a s a l t e r ed b y infect ion. T h e a u t h o r s were u n a b l e to d e t e r m i n e w h e t h e r th is difference a c c o u n t e d for t h e d e c r e a s e in egg v iab i l i ty a s soc i a t ed w i t h infect ion.

A. Microsporidia

T h e ma jo r i t y of m i c r o s p o r i d i a n - i n s e c t a s soc ia t ions p r o d u c e c h r o n i c , n o n -l e tha l infect ions (Weiser, 1974) of w h i c h a c o m m o n fea tu re is a r e d u c t i o n in h o s t r e p r o d u c t i v e o u t p u t . T h i s r e p r o d u c t i v e c u r t a i l m e n t is e n g e n d e r e d in a n u m b e r of w a y s a n d g e n e r a l i z a t i o n is h a m p e r e d b y t h e v a r i a t i o n in assess m e n t p a r a m e t e r s a d o p t e d b y inves t iga to r s . Howeve r , a r e d u c t i o n in m a t i n g success , fecundi ty , a n d egg h a t c h h a v e b e e n d e s c r i b e d b y m a n y a u t h o r s as s h o w n in T a b l e 1. M e r c e r a n d W i g l e y (1987) m e a s u r e d m a t i n g success in Nosema sp . (NGS)- in fec ted p o r o p o r o s t e m b o r e r s , Sceliodes cordalis, a s t h e ab i l i ty to lay v i ab l e eggs . I n a d d i t i o n , G a u g l e r a n d Brooks (1975) o b s e r v e d a h i g h e r i n c i d e n c e of locked m a t i n g s (fai lure to s e p a r a t e after c o p u l a t i o n ) in

Tabl

e 1

The

Effe

ct o

f Mic

rosp

orid

ial

Infe

ctio

ns u

pon

the

Repr

oduc

tive

Succ

ess

of T

heir

Inse

ct H

osts

Mat

ing

Egg

V

erti

cal

Ven

erea

l Pa

rasi

te

Hos

t su

cces

s*

Fecu

ndit

y ha

tch

tran

smis

sion

tr

ansm

issi

on

Sour

ce

Nos

ema

pyra

ustra

O

strin

ia n

ubila

lis

? -v

e -v

e ρ

? W

inde

ls e

t al.

(197

6)

ρ -v

e -v

e +

Sieg

el e

t al.

(198

6)

ρ ρ

? +

+ (h

o)

Saju

p an

d L

ewis

(19

88)

Nos

ema

sp.

Scel

iode

s cor

dalis

-v

e -v

e -v

e +

+ (s

s)

Mer

cer

and

Wig

ley

(198

7)

(NSC

)

Nos

ema

white

i Tr

ibol

uim

spp

. ρ

-ve

0 N

o M

ilne

r (1

972)

T.

cas

tane

um

-ve

-ve*

?

ρ A

rmst

rong

and

Bas

s (1

986)

N

osem

a he

lioth

idis

Hel

ioth

us z

ea

-ve

-vec

0

+ G

augl

er a

nd B

rook

s (1

975)

N

osem

a al

gera

e Ae

des

aegy

pti

ρ -v

e -v

e +

+ (h

o)

Nna

kum

usan

a (1

986)

Cu

lex

Jatig

ans

? -v

e -v

e +

+ (h

o)

Nos

ema

carp

ocap

sae

Cydi

a po

mon

ella

-v

e -v

e -v

e +

+ (h

o)

(ss)

N

o M

alon

e an

d W

igle

y (1

981)

N

osem

a ki

ngi

Dro

soph

ila w

illist

oni

ρ ρ

ρ +

Yes

A

rmst

rong

(1

977)

N

osem

a pl

odia

e Pl

odia

inte

rpun

ctel

la

ρ -v

e -v

e +

Yes

K

elle

n an

d L

inde

gren

(1

971)

N

osem

a bo

mby

sis

Bom

byx

mor

i ρ

? -v

e +

+ (s

s)

No

Han

and

Wat

anab

e (1

988)

N

osem

a sp

. M

il

ρ ρ

-ve

+ +

(ss)

N

o N

osem

a ste

gom

yiae

Cu

lex

Jatig

ans

ρ -v

e -v

e N

o Po

ssib

le

Rey

nold

s (1

971)

N

osem

a Jum

ifera

na

Chon

stone

ura

fitm

ifera

na

0 -v

e 0

+ ?

Bau

er a

nd N

ordi

n (1

989)

M

icro

spor

idiu

m

Listr

onot

us b

onar

iens

is ρ

-ve

0 ρ

ρ M

alon

e (1

987)

iti

iti

a-v

e, p

aras

ite

adve

rsel

y af

fect

s th

e ho

st; 0

, no

chan

ge o

ccur

s w

ith

infe

ctio

n; ?

, fea

ture

not

inv

esti

gate

d; +

, tr

ansm

issi

on o

ccur

s; +

+ ,

tran

sova

rial

tra

nsm

issi

on

dem

onst

rate

d by

sur

face

ste

riliz

atio

n; o

f eg

gs (

ss)

or b

y hi

stol

ogic

al o

bser

vati

on (

ho).

bF

ecun

dity

mea

sure

d as

off

spri

ng e

mer

genc

e.

'Onl

y oc

curs

whe

n in

sect

s ar

e in

fect

ed p

er o

s.

4. Reproductive Disturbances Induced by Parasites and Pathogens 91

Nosema heliothidis infect ions of Heliothis zea, a n d A r m s t r o n g a n d Bass (1986) m e a s u r e d m a t i n g f r equency in N. whitei-'mfected Tnbolium castaneum. F e c u n d i ty is u s u a l l y m e a s u r e d as t h e n u m b e r of eggs la id (e.g. , Ke l l en a n d L i n -d e g r e n , 1971 ; M a l o n e a n d Wigley, 1981; M a l o n e , 1987) a n d egg v iab i l i ty is d e t e r m i n e d b y egg h a t c h r a t e . H o w e v e r , M e r c e r a n d W i g l e y (1987) d i s t in g u i s h e d infert i le eggs f rom those u n d e r g o i n g i n c o m p l e t e e m b r y o g e n e s i s ( a n d therefore n o t h a t c h i n g ) , t h o u g h they d i d n o t d e t e r m i n e w h e t h e r t h e signific a n t d e c r e a s e in successful e m b r y o d e v e l o p m e n t w a s d u e to d e p l e t i o n of food reserves o r in te r fe rence w i t h e m b r y o g e n e s i s .

T h e m e c h a n i s m s u n d e r l y i n g th is r e d u c e d fecund i ty a r e d ive r se . Severa l a u t h o r s h a v e sugges t ed t h a t p a r a s i t e - i n d u c e d n u t r i t i o n a l d e p r i v a t i o n is t h e c a u s e of r e d u c e d egg p r o d u c t i o n ( M a l o n e , 1987). D e p l e t i o n of fat a n d p r o t e i n reserves is a s soc i a t ed w i t h m i c r o s p o r i d i a l infect ion of insec t fat b o d i e s (e .g . , T h o m p s o n a n d Sikorowski , 1979) a n d m a y resu l t in r e d u c e d v i te l logen in p r o d u c t i o n ( G a u g l e r a n d Brooks , 1975).

T h e i n v o l v e m e n t of t h e hos t e n d o c r i n e s y s t e m in t h e i n t e r a c t i o n b e t w e e n m i c r o s p o r i d i a a n d l a rva l insec t hos t s w a s p r o p o s e d b y F i s h e r a n d S a n b o r n (1962 , 1964) to exp l a in t h e d i s t u r b a n c e of l a rva l m o l t s a n d o c c u r r e n c e of a d u l t o i d s t h a t t hey o b s e r v e d in these a s soc i a t ions . T h e y d e m o n s t r a t e d t h a t Nosema s p p . s ec re to ry p r o d u c t s r e s to red t h e n y m p h a l o r l a rva l m o l t s to a l -l a t e c t o m i z e d insec ts w h e n t h e p r o t o z o a w e r e a p p l i e d ex t e rna l l y a n d s e p a r a t e d f rom t h e h e m o c o e l b y a 0 .45 -μη ι mi l l i po re filter (F i she r a n d S a n b o r n , 1964). J u v e n i l e h o r m o n e - l i k e ac t iv i ty w a s d e m o n s t r a t e d in s p o r e ex t r ac t s f rom Nosema (F i she r a n d S a n b o r n , 1964), Farinocystis tribolii ( R a b i n d r a et al, 1981), a n d N. bombycis ( K a r a v a e v a , 1987). H o w e v e r , M i l n e r (1972) w a s u n a b l e to d e m o n s t r a t e t h e d i s t u r b a n c e of l a rva l m o l t s d e s c r i b e d b y F i s h e r a n d S a n b o r n (1962 , 1964) a n d severa l a u t h o r s have a t t r i b u t e d t h e p a r a s i t e a s so c i a t ed d i s t u r b a n c e of insec t d e v e l o p m e n t to fat b o d y invas ion (e .g . , Listov, 1977). I t h a s b e e n sugges t ed t h a t d e s t r u c t i o n of fat b o d y t i s sue w o u l d r e d u c e t h e p r o d u c t i o n of j u v e n i l e h o r m o n e d e g r a d a t i o n e n z y m e s a n d l ead to a n a c c u m u l a t i o n of t h e h o r m o n e , t h u s r e su l t i ng in a b n o r m a l l a rva l d e v e l o p m e n t ( R a b i n d r a et al, 1981).

U l t r a s t r u c t u r a l s t ud i e s of c o r p o r a a l l a t a f rom Nosema ajfrw-infected w o r k e r bees d e m o n s t r a t e d t h a t n e u r o s e c r e t o r y a x o n g r a n u l e s w e r e r a r e ly p r e s e n t in infected bees (L iu , 1986). T h e a u t h o r sugges t ed t h a t g l a n d ac t iv i ty m a y b e affected b u t n o his to lys is o r d e g r a d a t i o n w a s o b s e r v e d . L a t e r s t ud i e s of t h e effect of j u v e n i l e h o r m o n e I I I on t h e h o n e y b e e h y p o p h a r a n g e a l g l a n d s h o w e d t h a t t h e h o r m o n e c a u s e d a d e p l e t i o n of r i b o s o m e s in t h e c y t o p l a s m of t h e cells , r e su l t i ng in i nh ib i t i on of p r o t e i n syn thes i s . T h e o b s e r v a t i o n t h a t Nosema infect ion d id n o t c a u s e r i b o s o m e d e p l e t i o n led to t h e conc lus ion t h a t t h e ac t ion of N. apis m a y n o t b e s y n o n y m o u s w i t h J H ac t ion (L iu , 1989, 1990). Clear ly , fu r the r s tud i e s a r e r e q u i r e d to e l u c i d a t e t h e ro le of e n d o c r i n e

92 Hilary Hurd

factors in m i c r o s p o r i d i a l - i n s e c t a s soc ia t ions a n d in p a r t i c u l a r the i r p a r t i c i p a t i o n , if any, in t he r e d u c t i o n of hos t r e p r o d u c t i v e o u t p u t .

His to log ica l s tud ie s of t he ovar ies of N. pyrausta-mfected Ostrinia nubilalis (Sa jup a n d Lewis , 1988) d e m o n s t r a t e d mass ive g e r m cell d e s t r u c t i o n , w h i c h w o u l d a c c o u n t for r e d u c e d fecundity. However , o v a r i a n invas ion b y m i c r o -spo r id i a does no t a lways d e c r e a s e egg v iabi l i ty as infected l a rvae a r e p r o d u c e d in m a n y assoc ia t ions . Sa jup a n d Lewis (1988) p o s t u l a t e d t h a t t h e c o n c e n t r a t i o n of m i c r o s p o r i d i a in the cen t e r of oocytes m a y p r e v e n t in te r ference w i th deve lop ing e m b r y o s .

A l t h o u g h m a n y m i c r o s p o r i d i a a r e conf ined to hos t t issues s u c h as fat b o d y or gu t , those t h a t i n v a d e the r e p r o d u c t i v e s y s t e m a r e p r o v i d e d w i t h a n a v e n u e for t r a n s m i s s i o n . T h u s , b o t h h o r i z o n t a l t r a n s m i s s i o n of p a r a s i t e s b e t w e e n hos t s a n d ver t ica l t r a n s m i s s i o n to t he nex t g e n e r a t i o n c a n o c c u r w i th in t he s a m e assoc ia t ions . S o m e a u t h o r s have d i s t i n g u i s h e d b e t w e e n t r ansova r i a l a n d t r a n s o v u m t r a n s m i s s i o n (see T a b l e 1). I n t h e former , infect ion occu r s whi le t he egg is deve lop ing w i th in t he ovary, a n d in t he l a t t e r t h e p a t h o g e n is a d s o r b e d o n t o the egg surface a n d infect ion of t h e l a rvae o c c u r s d u r i n g or after h a t c h i n g . T h u s , S a j u p a n d Lewis (1988) o b s e r v e d t h a t N. pyrausta m a y pas s in to deve lop ing oocytes w i t h n u t r i e n t s , f rom the t ro -p h o c y t e s of 0. nubilalis a n d , in a d d i t i o n , infect ion of t h e ep i the l i a l cells of t he ov iduc t m a y resu l t in t r a n s o v u m t r a n s m i s s i o n . If h i s to logica l s tud ie s have n o t b e e n p e r f o r m e d , surface s te r i l iza t ion of eggs is a n essen t ia l s t ep in t h e d e t e r m i n a t i o n of w h e t h e r t r ue t r ansova r i a l t r a n s m i s s i o n h a s o c c u r r e d w h e n infected la rvae e m e r g e . Vene rea l t r a n s m i s s i o n , r e su l t i ng in infected l a rvae b e i n g p r o d u c e d by non infec ted females m a t e d to infected m a l e s h a s a lso b e e n de sc r i bed . T h i s m a y be d u e to c o n t a m i n a t i o n of t he egg surface (Ke l l en a n d L i n d e g r e n , 1971) o r t r u e infect ion of t he female r e p r o d u c t i v e t i ssues ( A r m s t rong , 1977).

T h e hos t r e p r o d u c t i v e sys t em t h u s a p p e a r s to p l ay a m a j o r ro le in m i c r o -spor id i a l t r a n s m i s s i o n (see T a b l e 1) a l t h o u g h the exac t m o d e ( t r ansova r i a l o r t r a n s o v u m ) h a s no t a lways been d e t e r m i n e d . N n a k u m u s a n a (1986) sugges ted t h a t th is is t he sole m e t h o d of t r a n s m i s s i o n for m o r e t h a n 30 species of m i c r o s p o r i d i a l l a rva l p a t h o g e n s of d i p t e r a n vec tors . S m i t h a n d D u n n (1991) recen t ly d i scussed c y t o p l a s m i c i n h e r i t a n c e of i n t r ace l lu l a r s y m b i o n t s a n d s t ressed the se lect ion p r e s s u r e s faced by p a r a s i t e s c a u s i n g p a t h o l o g y in h o s t offspring. I n v iew of th i s , it is s u r p r i s i n g t h a t this is the sole r o u t e of t r a n s m i s sion for so m a n y m i c r o s p o r i d i a .

Few ecological s tud ies of the effects of m i c r o s p o r i d i a l infect ions h a v e b e e n p e r f o r m e d ( b u t see A n d r e a d i s , 1986). However , it is c lear t h a t a k n o w l e d g e of t he d e g r e e of i m p o r t a n c e of h o r i z o n t a l a n d ver t ica l t r a n s m i s s i o n is a n essential factor in t he c o n s t r u c t i o n of m o d e l s of t he inf luence of m i c r o s p o r i d i a u p o n hos t p o p u l a t i o n d y n a m i c s ( A n d e r s o n a n d M a y , 1981; O n s t a d a n d


M a d d o x , 1989). T h e p o t e n t i a l for t h e use of insec t p a t h o g e n s as m i c r o b i a l con t ro l a g e n t s of pes t species h a s long b e e n recogn ized . A l t h o u g h m i c r o spo r id i a l infect ions a r e usua l ly s u b l e t h a l , t hey resu l t in severe r e d u c t i o n in f ecund i ty a n d , in t he case of N. pyrausta-O. nubilalis infect ions , have b e e n s h o w n to d o w n - r e g u l a t e t he hos t p o p u l a t i o n be low t h e c a r r y i n g capac i t y of t h e e n v i r o n m e n t ( O n s t a d a n d M a d d o x , 1989). For e x a m p l e , N. locusta spo re s have b e e n s h o w n to be effective p o p u l a t i o n r e g u l a t o r s b o t h a l o n e ( R a i n a et aL, 1987) a n d in c o m b i n a t i o n w i t h insec t ic ides ( G e r m i d a et aL, 1987; J o h n son a n d H e n r y , 1987). M o r e o v e r , t he p o t e n t i a l for m i c r o s p o r i d i a as con t ro l a g e n t s of s to red p r o d u c t C o l e o p t e r a h a s r ecen t ly b e e n e m p h a s i z e d b y K h a n a n d S e l m a n (1989) .

III. Nematodes

T h e r a t i o of p a r a s i t e to hos t t i ssue u sua l ly a t t a i n s far g r e a t e r p r o p o r t i o n s in e n t o m o g e n o u s n e m a t o d e infect ions t h a n in t he i n s e c t - m i c r o p a r a s i t e assoc ia t ions d i scussed in t he foregoing. T h u s , t he p o t e n t i a l d r a i n o n hos t r e sou rces is likely to b e g rea t e r . A l t h o u g h l i t t le d i r ec t g o n a d a l t i ssue invas ion h a s b e e n obse rved , s o m e a u t h o r s r e p o r t e x a m p l e s of n e m a t o d e - i n d u c e d r e p r o d u c t i v e c u r t a i l m e n t r a n g i n g from s l ight r e d u c t i o n s in egg l ay ing to m a l e a n d / o r female s te r i l i za t ion . I n m a n y cases p a r a s i t e b u r d e n h a s b e e n c o r r e l a t e d w i t h loss of r e p r o d u c t i v e o u t p u t a n d ev idence sugges t ing t h a t n u t r i t i o n a l d e p r i v a t ion is l inked to hos t - r e sou rce r ea l loca t ion is d i scussed in t h e fol lowing.

A l l a n t o n e m a t i d s infect a va r i e ty of b a r k bee t les (Scoly t idae) a n d a l t e r severa l a s p e c t s of the i r r e p r o d u c t i o n . Behav io ra l p a t t e r n s s u c h as s w a r m i n g w e r e s h o w n to b e affected (Lieu t ie r , 1981) a n d m a n y of t h e infected b a r k bee t les t h a t still la id eggs w e r e found to excava te a b e r r a n t ga l le r ies , w h i c h h a d n o egg n i ches . E x a m p l e s of t h e effect of a l l a n t o n e m a t i d s o n b a r k bee t l e r e p r o d u c t i o n a r e g iven in T a b l e 2 a n d r a n g e from p a r a s i t e - i n d u c e d c a s t r a t ion of m a l e a n d female hos t s to a s soc ia t ions w i th n o o b s e r v a b l e effects u p o n hos t g a m e t o g e n e s i s . I n t he ma jo r i ty of cases female insec ts a r e m o r e severely affected t h a n m a l e s .

H i s to log ica l e x a m i n a t i o n of hos t g o n a d s in severa l h o s t - p a r a s i t e assoc ia t ions led T o m a l a k et al. (1990) to obse rve t h a t t he scale of d a m a g e infl icted w a s r e l a t ed to t h e t i m i n g of infect ion, re lease of j u v e n i l e n e m a t o d e s i n t o t h e h e m o c o e l , a n d the i r re la t ive a b u n d a n c e . For e x a m p l e , in Sulphuretylenchus pseudoundulatus-'mfected Polygraphus rufipennis, l a rge n u m b e r s of j u v e n i l e s w e r e r e l eased s y n c h r o n o u s l y w h e n the m a t e r n a l n e m a t o d e cut ic le w a s r u p t u r e d . D e v e l o p m e n t of g e r m cells in t he tes tes was i n h i b i t ed , t r o p h a r i a in t h e ovaries w e r e r e d u c e d , a n d n o foll icular d e v e l o p m e n t o c c u r r e d in infected fem a l e s . H o w e v e r , Contortylenchus reversus females d e p o s i t e d j u v e n i l e s i n t o t h e

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h o s t h e m o c o e l over a l ong t i m e p e r i o d a n d in this a s soc ia t ion t h e a u t h o r s found t h a t oogenes i s a n d vi te l logenes is p r o c e e d e d in h o s t ovar ies , a l t h o u g h a t a r e d u c e d r a t e , a n d egg l ay ing o c c u r r e d (see T a b l e 2) .

T h e lack of t i ssue d a m a g e a s soc i a t ed w i t h a l l a n t o n e m a t i d infect ions h a s led to t h e sugges t ion t h a t h o s t r e p r o d u c t i v e c u r t a i l m e n t m a y b e d u e to p a r a s i t e -i n d u c e d n u t r i e n t d e p r i v a t i o n r e su l t i ng in i n c r e a s e d fat b o d y c a t a b o l i s m a n d d e c r e a s e d a n a b o l i s m ( T o m a l a k etal.3 1990). L i t t l e ev idence h a s b e e n p r o d u c e d to s u p p o r t th is h y p o t h e s i s , a l t h o u g h L ieu t i e r (1984a ,b ) d e m o n s t r a t e d a r e d u c t ion in t h e p r o t e i n c o n t e n t of fat bod ie s t a k e n from n e m a t o d e - i n f e c t e d Ips sexdentatus. T h e a u t h o r sugges t ed t h a t t h e d e l a y in o v a r i a n m a t u r a t i o n w a s a r e su l t of r e d u c e d p r o t e i n s e q u e s t r a t i o n , a l t h o u g h r a t e of fat b o d y syn thes i s a n d h e m o l y m p h p r o t e i n c o n t e n t w e r e n o t e x a m i n e d . U s i n g p o l y a c r y l a m i d e gel e l ec t rophores i s ( P A G E ) , T h o n g a n d W e b s t e r (1975a) d e m o n s t r a t e d t h a t Con-tortylenchus reversus a l so d e p l e t e d t he h e m o l y m p h p r o t e i n s of i ts hos t , Dendroc-tonus pseudotsugae. I m p o r t a n t l y , a l ink has b e e n e s t ab l i shed b e t w e e n fat b o d y p r o t e i n syn thes i s , h e m o l y m p h v i te l logenin t i te rs , a n d o v a r i a n s e q u e s t r a t i o n in a c e s t o d e - i n s e c t a s soc ia t ion (see t h e following).

M e r m i t h i d infect ions a lso resu l t in hos t r e p r o d u c t i v e d i s t u r b a n c e s (see rev iew b y G o r d o n , 1981). I n v e s t i g a t i o n s i n t o t h e u n d e r l y i n g m e c h a n i s m s have focused u p o n locus t a n d blackfly infect ions . G o r d o n a n d his a ssoc ia tes (1973) i nves t iga t ed t h e phys io log ica l m e c h a n i s m s involved in t h e cessa t ion of egg p r o d u c t i o n in Mermis nigrescens-'mfected Schistocerca gregaria. O v a r i e s f rom female locus t s infected 3 d a y s after t h e i m a g i n a l m o l t exh ib i t ed i m p a i r e d s e q u e s t r a t i o n of v i te l logenic p r o t e i n s in t h e s econd week of infect ion a n d , b y week 3 , t e r m i n a l a n d p e n u l t i m a t e oocytes w e r e r e s o r b e d . D e t e r m i n a t i o n of q u a n t i t a t i v e a n d q u a l i t a t i v e c h a n g e s in fat b o d y a n d h e m o l y m p h p r o t e i n s r evea led t h a t t e r m i n a t i o n of v i te l logenes is w a s n o t d u e to d e p l e t i o n of h e m o l y m p h v i te l logen ins (wh ich w a s n o t s ignif icant un t i l 1 week l a te r ) a n d w a s t h o u g h t to r e su l t f rom a n i m p a i r m e n t of t h e ab i l i ty of t h e fol l icular e p i t h e l i u m to s e q u e s t e r p r o t e i n s . A b s o r p t i o n of oocy tes m a y h a v e r e su l t ed in t h e a c c u m u l a t i o n of v i te l logenic p r o t e i n s in t h e hos t h e m o c o e l , o b s e r v e d in t h e four th week of infect ion, however , t h e a u t h o r s d i d n o t m a k e th is a s soc ia t ion . T h e effect of t h e n e m a t o d e s o n fat b o d y p r o t e i n t u r n o v e r w a s a s c r i b e d to t h e p rov i s ion of a d i e t a r y sou rce of a m i n o ac ids for t h e n e m a t o d e s w i t h i n t h e h e m o l y m p h . I n a d d i t i o n , t h e a u t h o r s sugges t ed t h a t p a r a s i t e - i n d u c e d c h a n g e s in hos t n e u r o s e c r e t o r y ac t iv i ty m a y affect fat b o d y m e t a b o l i s m a n d t h a t a r e d u c t i o n in c i r cu l a to ry J H m a y i n h i b i t o v a r i a n s e q u e s t r a t i o n of yolk p r o t e i n s . A l t h o u g h t h e r e is l i t t le ev idence to s u p p o r t o r refute th is view, Neomesomermis flumenalis w a s s h o w n to i nc r ea se t h e n u c l e a r D N A / R N A activi ty ( m e a s u r e d in t e r m s of n u c l e a r / c y t o p l a s m i c ra t ios ) , t h e v o l u m e of t h e h o s t c o r p o r a a l l a t a ( C A ) , a n d t h e a m o u n t of n e u r o s e c r e t o r y m a t e r i a l a c c u m u l a t e d in t h e c e r e b r a l n e u r o s e c r e t o r y cells of Simulium venustum ( C o n d o n a n d G o r d o n , 1977).

9 6 Hilary Hurd

A r e d u c t i o n in C A v o l u m e was a lso assoc ia ted w i t h Sphaerularia bombis infect ions of Bombus terrestris. N o o v a r i a n d e v e l o p m e n t o c c u r r e d in infected q u e e n bees , a n d t h e h e m o l y m p h v i te l logenin t i te r w a s r e d u c e d (Rose ler a n d Roseler , 1973). As b o t h biological ly act ive j u v e n i l e h o r m o n e a n d j u v e n i l e h o r m o n e a n a l o g u e s (Rogers , 1973) a n d ecdys t e ro ids (see B a r k e r a n d Rees , 1990) o c c u r in n e m a t o d e s , t he r e is c lear ly a need to reassess t h e p o t e n t i a l for i n t e r ac t ion b e t w e e n insect a n d n e m a t o d e e n d o c r i n e sy s t ems .

R e d u c e d egg p r o d u c t i o n in D i p t e r a infected w i t h filarial n e m a t o d e s h a s b e e n co r r e l a t ed w i t h p a r a s i t e b u r d e n . U s i n g refractory a n d suscep t ib l e s tocks of Aedes aegypti, J a v a d i a n a n d M a c d o n a l d (1974) d e m o n s t r a t e d a n e g a tive l inea r co r re l a t ion b e t w e e n p a r a s i t e l oad a n d egg p r o d u c t i o n in infect ions w i th o lde r l a rvae of Brugia pahangi a n d in A. aegypti infected w i t h Dirofilaria repens, in b o t h ear ly a n d la te infect ions. A nega t ive l i nea r co r r e l a t i on b e t w e e n p a r a s i t e b u r d e n a n d m e a n n u m b e r of b a s a l follicles deve loped p e r female w a s also d e m o n s t r a t e d in t he B. pahangi/A. aegypti a s soc ia t ion ( G a a b o u b , 1976). I n D. immitis-infected A. trivittatus, r e d u c e d egg p r o d u c t i o n h a s a lso b e e n r e l a t ed to p a r a s i t e b u r d e n , w i th those exceed ing 15 j u v e n i l e s p e r m o s q u i t o severely l imi t ing r e p r o d u c t i o n ( C h r i s t e n s e n , 1981). Dirofilaria immitis a n d B. pahangi a r e M a l p i g h i a n t u b u l e a n d flight m u s c l e dwel le r s , respect ively, a n d n o ev idence of d i r ec t d a m a g e to hos t ovar ies h a s b e e n d e s c r i b e d .

T h e fecundi ty of w i l d - c a u g h t Simulium damnosum w a s found to b e r e d u c e d by Onchocerca sp . infect ions ( C h e k e et al., 1982) a n d two species of U n i t e d K i n g d o m blackflies exh ib i t ed r e d u c t i o n s in fecundi ty r e l a t ed to 0. lienalis b u r d e n w h e n infected by b lood feeding o r by i n t r a t h o r a c i c in jec t ions ( H a m a n d B a n y a , 1984). F u r t h e r e x p e r i m e n t s d e m o n s t r a t e d a n a p p r o x i m a t e l y 5 0 % r e d u c t i o n in egg l ay ing in S. ornatum in jected w i t h 30 microf i la rae ( H a m a n d G a l e , 1984). I n t r a t h o r a c i c in ject ions ru l ed o u t t he poss ib le effect of p a t h o l o g y c a u s e d by g u t p e n e t r a t i o n on fecundity. Howeve r , b o t h t h e q u a l i t y a n d q u a n t i t y of b lood inges ted by infected vec tors m a y a l t e r n u t r i e n t avai l abi l i ty for t he s y m b i o n t s . U s i n g r ad io i so tope e s t i m a t i o n s of b lood m e a l size, C o u r t n e y et al. (1985) d e m o n s t r a t e d a r e d u c e d i n t a k e w h e n A. aegypti w e r e feeding on D. immitis-infected b lood a n d sugges ted t h a t n u t r i e n t d e p r i v a t i o n w a s t he c a u s e of r e d u c e d fecundi ty in infected m o s q u i t o e s . H a m a n d B a n y a (1984) found a r e d u c t i o n in feeding r a t e in blackflies fed o n infected b lood .

IV. Cestodes

A l t h o u g h ces tode p a r a s i t i z a t i o n of insec ts does no t resu l t in hos t c a s t r a t i o n , r e p r o d u c t i v e o u t p u t is r e d u c e d in h y m e n o l e p i d infect ions of T e n e b r i o n i d a e ( C o l e o p t e r a ) . E m b r y o n a t e d " e g g s " of t he r a t t a p e w o r m Hymenolepis diminuta a r e pass ive ly t r a n s m i t t e d to i n t e r m e d i a t e hos t s w h e n these s to red g r a i n pests


feed o n feces f rom infected defini t ive hos t s . T h e o n c o s p h e r e is r e l eased in t h e gu t , p e n e t r a t e s t h e m i d g u t wal l , a n d deve lops i n to a m e t a c e s t o d e in 8 - 1 2 d a y s a t 26°C. T h e p a r a s i t e s a b s o r b n u t r i e n t s from t h e h e m o l y m p h a n d n o t i ssue i nvas ion o c c u r s .

A m a r k e d l o n g - t e r m d e p r e s s i o n of hos t p o p u l a t i o n s w a s o b s e r v e d in Hy-menolepis diminuta infect ions of Tribolium castaneum ( K e y m e r , 1981) a n d r e d u c t ion of h o s t fecundi ty w a s r e l a t ed to t o t a l p a r a s i t e b i o m a s s ( K e y m e r , 1980). M a e m a (1986) a s soc ia t ed hos t egg o u t p u t w i t h age of infect ion, finding a s ignif icant r e d u c t i o n 14 or m o r e d a y s pos t infec t ion . H u r d a n d A r m e (1986a) a lso d e m o n s t r a t e d r e d u c e d ovipos i t ion in t he l a rge r Tenebrio molitor 1 3 - 1 6 d a y s pos t in fec t ion w i t h H. diminuta, a l t h o u g h on ly w h e n t h e hos t s w e r e m a i n t a i n e d in very c r o w d e d c o n d i t i o n s w a s th is r e d u c t i o n still e v i d e n t 30 d a y s pos t in fec t ion . E g g v iabi l i ty w a s d e p r e s s e d by 9 . 4 % in p a r a s i t i z e d Tenebrio molitor k e p t as s ingle p a i r s a n d by 1 2 % in bee t les k e p t in c r o w d e d c o n d i t i o n s ( H u r d a n d A r m e , 1986b) . I n a d d i t i o n , a s ignif icant r e d u c t i o n in t h e n u m b e r of l a rvae p r o d u c e d d u r i n g a 6-week pe r iod w a s r e c o r d e d ( H u r d , 1990b) .

T h e r e p r o d u c t i v e phys io logy of Tenebrio molitor-'mfected w i t h Hymenolepis diminuta h a s b e e n extens ive ly inves t iga t ed by the a u t h o r . T h e s e findings h a v e b e e n rev iewed recen t ly ( H u r d , 1990a ,b) a n d t h u s will n o t b e d e a l t w i t h in g r e a t de t a i l in th is c h a p t e r . Howeve r , p e r t u r b a n c e of hos t egg p r o d u c t i o n in th is s y s t e m a p p e a r s to have s o m e pa ra l l e l s w i t h t h e m e r m i t h i d infect ions of locus t s , a d d r e s s e d ear l ier , a n d t h u s these a spec t s will b e d i s cus sed . H o s t r e p r o d u c t i v e c u r t a i l m e n t p r o b a b l y resu l t s f rom a r e t a r d a t i o n of oocy te m a t u r a t i o n a n d is u n d o u b t e d l y t he resu l t of i m p a i r e d v i te l logenes is . D e s p i t e a s ignif icant r e d u c t i o n in yolk v o l u m e in eggs f rom bee t les infected w i t h H. diminuta ( H u r d a n d A r m e , 1986b) , h e m o l y m p h p r o t e i n t i te rs w e r e n o t d e p l e t ed , b u t i n c r e a s e d by 4 6 . 7 % , in females h a r b o r i n g m a t u r e p a r a s i t e s . D e n -s i t o m e t r i c ana lys i s of h e m o l y m p h s e p a r a t e d on S D S - P A G E gels r evea led t h a t th is i nc r ea se w a s en t i re ly d u e to i n c r e a s e d t i te rs of t h e v i te l logenin s u b u n i t s ( H u r d a n d A r m e , 1984). T h u s , in c o m m o n w i t h t h e m e r m i t h i d / l o c u s t a s so c ia t ion , v i t e l logen in is p r e s e n t in a b u n d a n c e a n d r e d u c e d egg o u t p u t is n o t a d i r ec t r esu l t of n u t r i e n t d e p r i v a t i o n . O v a r i a n s e q u e s t r a t i o n of yolk w a s inves t i ga t ed in vivo b y m o n i t o r i n g t h e i n c o r p o r a t i o n of 1 4 C - l a b e l e d v i te l logen in a n d s h o w n to b e d e c r e a s e d b y 5 1 . 5 % in p a r a s i t i z e d females ( H u r d a n d A r m e , 1986a) . T h e t e r m i n a l follicles of these t e lo t rop ic ovar ies exh ib i t a s ignif icant r e d u c t i o n in p a t e n c y ( H u r d a n d A r m e , 1987) a n d it is p r o p o s e d t h a t th is r e d u c t i o n resu l t s in a n i m p a i r m e n t of yolk u p t a k e a n d h e n c e a r e d u c t i o n in fecundi ty.

T h e m e c h a n i s m s u n d e r l y i n g th is p a r a s i t e - i n d u c e d p e r t u r b a n c e of vi te l logenesis a r e n o t k n o w n . I t is t h o u g h t t h a t j u v e n i l e h o r m o n e I I I r egu l a t e s v i te l logenes is in Tenebrio molitor ( L a v e r d u r e , 1970). H o w e v e r , C A secre t ion , c i r c u l a t i n g t i t e r s , a n d r a t e of d e g r a d a t i o n of j u v e n i l e h o r m o n e a r e n o t af-

9 8 Hilary Hurd

fected b y infect ion ( H u r d a n d Weaver , 1987; H u r d et aL, 1990). A t t e m p t s to d e m o n s t r a t e c h a n g e s in ecdys t e ro id t i ters in infected bee t les h a v e a l so failed ( H u r d et aL, 1987) a n d , a l t h o u g h ecdys te ro ids h a v e b e e n d e m o n s t r a t e d in a d u l t H. diminuta ( M e r c e r et aL, 1987), n o free ecdys te ro id ac t iv i ty w a s found in t h e m e t a c e s t o d e s u s i n g R I A m e t h o d s ( M e r c e r et aL, 1987) o r i m m u -n o c h e m i s t r y ( H . D . F . H . Schal l ig a n d H . H u r d , p e r s o n a l o b s e r v a t i o n s ) . D e sp i te t h e a p p a r e n t n o r m a l i t y of h o r m o n e t i te rs in infected females , a j u v e n i l e h o r m o n e a n a l o g u e , m e t h o p r e n e , a p p l i e d to bee t les a t t h e t i m e of infect ion, p r o d u c e d a r e scue effect b y p r e v e n t i n g t h e e leva t ion of h e m o l y m p h p r o t e i n t i ters a s soc ia t ed w i t h infect ion ( H u r d et aL, 1990). I n a d d i t i o n , o t h e r j u v e n i l e h o r m o n e - c o n t r o l l e d even t s s u c h as fat b o d y v i te l logenin syn thes i s ( H u r d a n d A r m e , 1986a) a n d sex p h e r o m o n e p r o d u c t i o n ( H u r d a n d Par ry , 1991) a r e a lso d o w n - r e g u l a t e d by H. diminuta m e t a c e s t o d e s . I s it a p p a r e n t t h a t a l t h o u g h n o n e of t h e inves t iga t ions p e r f o r m e d so far d i rec t ly s u p p o r t s t h e p r o p o s i t i o n t h a t m e t a c e s t o d e s m a n i p u l a t e hos t e n d o c r i n e c o n t r o l of r e p r o d u c t i o n , n e w avenues n e e d to b e exp lo red . Pa ras i t e s m a y n o t c h a n g e hor m o n e t i te r b u t cou ld , for e x a m p l e , i n d u c e a c h a n g e in hos t r e s p o n s e to m e d i a t o r s u b s t a n c e s (i .e. , J H ) in t h e t a r g e t t i ssues .

V. Endoparasitic Insects

I n s e c t p a r a s i t o i d s u sua l ly a t t a c k i m m a t u r e insec ts ( K u r i s , 1974) a n d kill t he i r hos t s before t hey r each sexua l m a t u r i t y . H o s t c a s t r a t i o n is t h u s r a re ly obse rved in i n s e c t - i n s e c t a s soc ia t ions . Howeve r , J u n n i k k a l a (1985) n o t e d a r e d u c t i o n in t h e v o l u m e a n d wal l th i ckness of d e v e l o p i n g tes tes in Pieris brassicae l a rvae p a r a s i t i z e d b y Apanteles glomeratus, w i t h t h e d e g r e e of r e t a r d a t ion n o t b e i n g r e l a t ed to p a r a s i t e b u r d e n . B u n d l e s of s p e r m w e r e , however , p r o d u c e d before t he p a r a s i t o i d kil led its hos t a t t h e p r e p u p a l s t age , i n d i c a t ing t h a t t h e g o n a d w a s still func t iona l a t th is s t age . T h e t i m i n g of a t t a c k d e t e r m i n e d t h e d e g r e e of g o n a d a l d a m a g e in t h e c o d l i n g m o t h , Cydia pomonella, p a r a s i t i z e d b y Ascogaster quadridentata ( R e e d - L a r s o n a n d B r o w n , 1990). C a s t r a t i o n o c c u r r e d in l a rvae deve lop ing f rom eggs exposed to a d u l t w a s p s d u r i n g t h e first 36 h ; however , s o m e la rvae d e v e l o p e d s m a l l tes tes if a t t a c k e d b e t w e e n 36 a n d 48 h a n d those p a r a s i t i z e d a t 6 4 h h a d tes tes t h a t , a t t h e four th s t a d i u m , exh ib i t ed p recoc ious d e v e l o p m e n t . R e e d - L a r s o n a n d B r o w n (1990) d i scussed the p o t e n t i a l benefi t to p a r a s i t o i d s of h o s t c a s t r a t i o n a n d sugges t ed t h a t t h e e l im ina t i on of hos t t a r g e t o r g a n s m a y m a k e m o r e e c d y s o n e ava i l ab le to ac t as a cue for A. quadridentata d e v e l o p m e n t , a n d a l so r e m o v e p o t e n t i a l c o m p e t i t i o n for g lycogen a n d l ip id reserves .

T w o w e l l - d o c u m e n t e d cases in w h i c h t h e infected female hos t r e a c h e s


a d u l t h o o d a r e o u t l i n e d in t h e following. I n b o t h e x a m p l e s , i n t e r ac t i on b e t w e e n t h e p a r a s i t e a n d h o s t e n d o c r i n e s y s t e m h a s b e e n inves t iga t ed .

T r i u n g u l i n i d l a rvae of t h e p a r a s i t i c w a s p Xenos vesparum i n v a d e Polistis s pp . ( H y m e n o p t e r a ) l a rvae in t h e s p r i n g . P a r a s i t e g r o w t h p r o c e e d s slowly in h o s t l a rvae a n d t h e las t l a rva l i n s t a r s a n d n y m p h a l a n d i m a g i n a l m o l t s o c c u r in y o u n g a d u l t w a s p hos t s . M a l e p a r a s i t e s e m e r g e from the i r h o s t s , m a t e , a n d d i e , w h e r e a s females , infect ing fu tu re q u e e n w a s p s , ove rwin t e r in the i r hos t s (Nassonov, 1910; H o f f m a n n , 1914). Pa ra s i t i c c a s t r a t i o n ( s ty lop iza t ion synd r o m e ) is i n d u c e d b y s u p p r e s s i o n of oocy te m a t u r a t i o n a n d t h e o c c u r r e n c e of in te r sexes in s ty lop ized bees is well k n o w n (Wulker , 1975).

S t r a m b i et al. (1982) d e m o n s t r a t e d t h a t , a t t h e t i m e of h o s t e m e r g e n c e from d i a p a u s e , t e r m i n a l oocy tes w e r e s ignif icant ly r e d u c e d in infected fem a l e s . C o n t r o l X. vesparum females h a d h i g h e r b lood p r o t e i n t i te rs a t th i s t i m e ( S t r a m b i et al., 1982) a n d , a l t h o u g h p r e s e n t , v i te l logenin t i te rs w e r e r e d u c e d in p a r a s i t i z e d females ( R o u x , 1973). In fec ted a d u l t s w e r e found to h a v e r e d u c e d fat bod i e s , ovar ies , a n d C A v o l u m e . After ove rwin t e r i ng , syn thes i s of j u v e n i l e h o r m o n e by t h e C A r e a c h e d 4 p m o l e s p e r p a i r of g l a n d s p e r h o u r in c o n t r o l w a s p s b u t on ly 0.2 p m o l e s w e r e p r o d u c e d b y C A from infected w a s p s (Roseler et al., 1980). I n a d d i t i o n , h e m o l y m p h j u v e n i l e h o r m o n e t i te rs w e r e s ignif icant ly r e d u c e d in infected females (A. S t r a m b i , p e r s o n a l o b s e r v a t i o n s ) a n d C A i m p l a n t a t i o n in to p a r a s i t i z e d females i n d u c e d v i te l logenes is a n d o v a r i a n d e v e l o p m e n t ( S t r a m b i a n d G i r a r d i e , 1973). T h u s , S t r a m b i et al. (1982) m a i n t a i n e d t h a t it w a s a j u v e n i l e h o r m o n e deficiency t h a t i n h i b i t e d egg m a t u r a t i o n in infected insec t s . Cy to log ica l ev idence sugges t t h a t , un l ike in un in fec ted c o u n t e r p a r t s , l a rge a m o u n t s of n e u r o s e c r e t o r y m a t e r i a l a r e r e l eased f rom infected w a s p s a n d t h a t th is m a y con t ro l C A func t ion ing (e .g. , S t r a m b i , 1966; S t r a m b i a n d S t r a m b i , 1973). T h e m e t h o d b y w h i c h t h e p a r a si te exer t s i ts inf luence o n t h e n e u r o s e c r e t o r y cells r e m a i n s to b e e l u c i d a t e d .

I n Metacemyia calloti ( D i p t e r a , T a c h i n i d a e ) infect ions of t h e g r a s s h o p p e r Anacridium aegyptium, i m p a i r m e n t of h o s t oocy te d e v e l o p m e n t h a s b e e n assoc i a t e d w i t h a d e p l e t i o n of h e m o l y m p h p r o t e i n s . Syn thes i s of v i te l logenic p r o te ins w a s r e d u c e d a n d v i te l logenins were s h o w n to b e invo lved in t h e n u t r i t iona l r e q u i r e m e n t s of t h e p a r a s i t e s ( G i r a r d i e , 1977). U n l i k e t h e s t r e p s i p t e r a n infect ion o u t l i n e d ear l ier , h o s t j u v e n i l e h o r m o n e p r o d u c t i o n w a s n o t r e d u c e d , b u t e leva ted in infected females ( G i r a r d i e a n d Jo ly , 1975). U l t r a s t r u c t u r a l s tud ie s conf i rmed t h e s t a t u s of C A from infected insec t s a n d C A i m p l a n t a t i o n failed to in i t i a t e oocy te d e v e l o p m e n t in p a r a s i t i z e d females ( G i r a r d i e a n d G r a n i e r , 1974). Howeve r , a c o m b i n a t i o n of r ad ioac t ive t r a ce r s tud i e s a n d l ight a n d e lec t ron m i c r o s c o p y d e m o n s t r a t e d t h e lack of ac t iv i ty in t h e m e d i a n n e u r o s e c r e t o r y cells of infected g r a s s h o p p e r s . E lec t r i ca l s t i m u l a t ion of t h e p a r s i n t e r ce r eb ra l i s e n h a n c e d o v a r i a n d e v e l o p m e n t ( G i r a r d i e a n d

100 Hilary Hurd

G i r a r d i e , 1977). G i r a r d i e (1977) p o s t u l a t e d t h a t h e m o l y m p h p r o t e i n defic iency m a y be t he resu l t of b o t h p a r a s i t e n u t r i t i o n a l r e q u i r e m e n t s a n d h y p o -act iv i ty of t he m e d i a n n e u r o s e c r e t o r y cells r e su l t i ng in d e c r e a s e d p r o t e i n syn thes i s , poss ib ly v ia J H con t ro l .

VI. Conclusions

T h e assoc ia t ions se lected for d i scuss ion in this c h a p t e r a p t l y i l l u s t r a t e t h e w i d e s p r e a d o c c u r r e n c e of p a r a s i t e - i n d u c e d r e d u c t i o n in hos t fecundi ty . D a w -kins (e.g. , 1982, 1990) r e g a r d e d p a r a s i t i c c a s t r a t i o n as a n e x a m p l e of p a r a s i t e m a n i p u l a t i o n of hos t s v ia a n ex tens ion of t he inf luence of p a r a s i t e genes . T h e p o t e n t i a l t h a t th is s t r a t egy p rov ides for e n h a n c i n g s y m b i o n t su rv iva l b y d ive rs ion of m e t a b o l i c r e sources a w a y from hos t r e p r o d u c t i o n h a s b e e n d i s cussed b y m a n y a u t h o r s (e.g. , B a u d o i n , 1975; H u r d , 1990a) .

T h e ma jo r i ty of m e t h o d s w h e r e b y hos t r e p r o d u c t i o n is s u p p r e s s e d fall i n t o o n e of t h r e e ca tegor ies : t he i nh ib i t i on of hos t m a t i n g d u e to m o r p h o l o g i c a l o r b e h a v i o r a l c h a n g e s , t h e d e s t r u c t i o n of hos t r e p r o d u c t i v e t i ssue , a n d t h e re d u c t i o n of egg o u t p u t d u e to ma l func t ion of o v a r i a n yolk s e q u e s t r a t i o n . I n all t h r ee cases , n u t r i e n t s , w h i c h in con t ro l insec ts w o u l d b e u t i l ized b y deve lop ing oocytes , a r e m a d e ava i l ab le to s u s t a i n t h e hos t a n d d e v e l o p i n g p a r a s i t e s . O u r u n d e r s t a n d i n g of t h e m o d e of p a r a s i t e ac t ion is r u d i m e n t a r y b u t in m o s t a s soc ia t ions it a p p e a r s to involve a m o r e sub t l e i n t e r p l a y t h a n t h e s i m p l e s cena r io of p a r a s i t e u t i l i za t ion of n u t r i e n t s d e s t i n e d for egg p r o d u c t i o n . I n d i rec t d ive rs ion of n u t r i e n t s v ia a l t e r a t i o n of fat b o d y m e t a b o l i s m a n d / o r p e r t u r b a n c e of n e u r o e n d o c r i n e con t ro l m e c h a n i s m s a r e u n d o u b t e d l y involved in s o m e assoc ia t ions a n d p u t a t i v e p a r a s i t e sec re t ion of m e d i a t o r s u b s t ances , w h i c h m a y a l t e r g o n a d a l sens i t iv i ty o r affect h o r m o n e m e t a b o l i s m , h a s b e e n d i scussed recen t ly ( H o l m e s a n d Z o h a r , 1990). H o w e v e r , a t t h e m o m e n t t h e r e is n o e x a m p l e of p a r a s i t e - i n d u c e d d i s t u r b a n c e of insec t r e p r o d u c t i o n in w h i c h t h e u n d e r l y i n g m e c h a n i s m s have b e e n fully e l u c i d a t e d a n d th is r e m a i n s a cha l l enge for t he fu ture .

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Raina, S. K., Rai, Μ. M., and Khurad, A. M. (1987). Grasshopper and locust control using microsporidian insecticides. In "Biotechnology of Invertebrate Pathology and Cell Culture" (K. Maramorosch ed.), pp. 345-366. Academic Press, Orlando, FL.

Read, A. F. (1990). Parasites and the evolution of host sexual behaviour. In "Parasitism and Host Behaviour" (C. B. Barnard and J. M. Behnke, eds.), pp. 117-158. Taylor & Francis, London.

Reed-Larson, D. Α., and Brown, J. J. (1990). Embryonic castration of the codling moth, Cydia pomonella, by an endoparasitoid, Ascogaster quadridentata. J. Insect Physiol. 36:111-118.

Reynolds, D. G. (1971). Parasitization of Culex fatigans by Nosema stegomyiae. J. Invertebr. Pathol. 18:429.

Rogers, W. P. (1973). Juvenile and moulting hormones from nematodes. Parasitology 67:105—113. Roseler, I., and Roseler, P. F. (1973). Anderungen im Muster der Haemolymphproteine von

Adulten Koniginen der Hummelart Bombus terrestris. J. Insect Physiol. 19:1741-1752. Roseler, P. F., Roseler, I., and Strambi, A. (1980). The activity of corpora allata in dominant and

subordinated females of the wasp Polistes gallicus. Insect Soc. 27:97-107. Roux, C. (1973). Etude comparee de la proteinemie chez les imagos de Polistes (Hymenoptores,

Vespides) sains ou parasites par le Strepsiptore Xenos vesparum Rossi. C.R. Hebd. Seances Acad. Sci. 276:3159-3162.

Sajup, A. S., and Lewis, L. C. (1988). Histopathology of transovarial transmission οϊ Nosema pyrausta in the European corn borer, Ostrinia nubilalis. J. Invertebr. Pathol. 52:147-153.

Schvester, D. (1957). Contribution de l'etude de Coleopteres scolytids. Ann. Inst. Natl. Rech. Agron., Ser. C 8:1-162.

Shieh, J.-N., and Rossignol, P. A. (1992). Opposite effects of host anaemia on blood feeding rate and fecundity of mosquitos. Parasitology 105:159-163.

Siegel, J. P., Maddox, J. V., and Ruesink, W. G. (1986). Lethal and sublethal effects οϊ Nosema pyrausta on the European corn borer (Ostrinia nubilalis) in central Illinois. J. Invertebr. Pathol. 48:167-173.

Smith, J. E., and Dunn, A. M. (1991). Transovarial transmission. Parasitol. Today 7:146-148.


Strambi, A. (1966). Action de Xenos vesparum Rossi (Strepsiptere) sur la neurosecretion des fondatrices filles de Polistes gallicus L. (Hymenoptere, Vespide) en diapause. C.R. Hebd. Seances Acad. Sci. 263:533-535.

Strambi, Α., and Girardie, A. (1973). Effect de l'implanation de corpora allata actifs de Locusta migratoria (Orthoptere) dans des femelles de L. (Hymenoptere) saines et parasitees par Xenos vesparum Rossi (Insecte, Strepsiptere). C.R. Hebd. Seances Acad. Sci. 276:3319-3322.

Strambi, Α., and Strambi, C. (1973). Influence du developpement du parasite Xenos vesparum Rossi (Insecte, Strepsiptere) sur le systeme neuroendocrinien des femelles de Polistes (Hymenoptere, Vespide) au debut de leur vie imaginale. Arch. Anat. Microsc. Morphol. Exp. 6 2 : 3 9 -54.

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Thong, C.H.S., and Webster, J. M. (1975a). Effects of Contortylenchus reversus (Nematoda: Sphae-rulariidae) on haemolymph composition and oocyte development in the beetle Dendroctonus pseudopsugae (Coleoptera: Scolytidae). J. Invertebr. Pathol. 26:91-98 .

Thong, G.H.S., and Webster, J. M. (1975b). Effects of the bark beetle nematode, Contortylenchus reversus, on gallery construction, fecundity, and egg viability of the Douglas fir beetle, Dendroctonus pseudotsugae (Coleoptera: Scolytidae). J. Invertebr. Pathol. 26:235-238.

Tomalak, M., Michalski, J., and Grocholski, J. (1984). The influence of nematodes on the structure and genitalia of Tomicus piniperda (Coleoptera: Scolytidae). J. Invertebr. Pathol. 43:358-362 .

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Chapter 5

ΗBehavioral Effects of Parasites and Pathogens in Insect Hosts David R. Horton Janice Moore Yakima Agricultural Laboratory Department of Biology USDA-ARS Colorado State University Yakima, Washington Fort Collins, Colorado

I. Introduction

II. Review of Altered Behavior in Parasitized Insects A. Changes in Microhabitat Preference B. Changes in Level of Activity C. Reproductive Behavior by Castrated

Insects

III. Potential Benefits to Parasite

A. Increased Parasite Dissemination B. Increased Parasite Survival

IV. Potential Benefits to Host A. Increased Survival of Kin B. Host Defense: Behavioral Fever

V. Considerations for Future Research Acknowledgments References

I. Introduction

R e s p o n s e s of insec t hos t s to p a r a s i t i s m i n c l u d e phys io log ica l , m o r p h o l o g i c a l , a n d b e h a v i o r a l effects, m a n y of w h i c h affect p a r a s i t e o r h o s t f i tness. C h a n g e s in h o s t b io logy a s soc ia t ed w i t h p a r a s i t i s m a r e often infer red to b e u n d e r p a r a s i t e con t ro l , p a r t i c u l a r l y w h e n t h e c h a n g e s benefi t t h e p a r a s i t e (e .g . , V i n s o n a n d I w a n t s c h , 1980). T h e e n t o m o l o g i c a l l i t e r a t u r e e m p h a s i z e s t h e effects of p a r a s i t i s m o n h o s t phys io logy ( V i n s o n a n d I w a n t s c h , 1980; Beckage , 1985; J o n e s , 1985); effects o n hos t b e h a v i o r a r e less well d e s c r i b e d ( b u t see M c A l l i s t e r a n d R o i t b e r g , 1987; B r o d e u r a n d M c N e i l , 1989). N o n e t h e l e s s , b e h a v i o r a l c h a n g e s c a u s e d b y p a r a s i t i s m a r e c o m m o n , a n d o n e m a y h y p o t h esize t h a t t h e c h a n g e s a r e p r o d u c t s of n a t u r a l se lec t ion ac t i ng on t h e h o s t o r t h e p a r a s i t e .

H e r e we s u m m a r i z e a diffuse l i t e r a t u r e d e s c r i b i n g p a r a s i t e - i n d u c e d b e h a v i o r a l c h a n g e s , cover ing a va r i e ty of m i c r o - (e.g. , fungi a n d v i ruses ) a n d m a c r o p a r a s i t i c (e.g. , p a r a s i t o i d s a n d n e m a t o d e s ) o r g a n i s m s (sensu A n d e r s o n a n d M a y , 1979). T h r e e b r o a d ca tegor ies of b e h a v i o r a l a l t e r a t i o n s i n c l u d e : (1)

Parasites and Pathogens of Insects Copyright © 1993 by Academic Press, Inc. Volume 1: Parasites 107 All rights of reproduction in any form reserved.

108 David R. Horton and Janice Moore

c h a n g e s in m i c r o h a b i t a t p references , i n c l u d i n g d i u r n a l ac t iv i ty b y n o r m a l l y n o c t u r n a l hos t s ; (2) c h a n g e s in act ivi ty level; a n d (3) r e p r o d u c t i v e ac t iv i ty expres sed u n d e r i n a p p r o p r i a t e cond i t i ons , t h a t is, by c a s t r a t e d hos t s . M o d ifications of feeding b e h a v i o r in insec t vec tors a r e exc luded , as th is l i t e r a t u r e h a s b e e n rev iewed ( M o l y n e u x a n d Jeffries, 1986).

Behav io ra l modi f i ca t ions c a u s e d by p a r a s i t i s m c a n be i n t e r p r e t e d to h a v e a r i sen (see a lso M i n c h e l l a , 1985): (1) t h r o u g h n a t u r a l se lec t ion a n d beneficial to t h e p a r a s i t e ; (2) t h r o u g h n a t u r a l se lect ion a n d beneficial to t h e hos t ; a n d (3) as a c o n s e q u e n c e of p a t h o l o g y o r s t ress , w h i c h m a y o r m a y n o t benefi t e i the r par ty . M o o r e (1984) , J o n e s (1985) , M o o r e a n d Gote l l i (1990) , a n d C o m b e s (1991) e m p h a s i z e d the i m p o r t a n c e of d e c i d i n g a m o n g these h y p o t h e s e s if o n e is to u n d e r s t a n d the evo lu t i ona ry s ignif icance of b e h a v i o r a l a l t e r a t i ons . We will first cons ide r how b e h a v i o r a l c h a n g e s m i g h t benefi t t h e p a r a s i t e , e m p h a s i z i n g p a r a s i t e d i s s e m i n a t i o n a n d surv iva l . Second , behaviora l c h a n g e s t h a t m a y benefi t t he hos t t h r o u g h e i the r k in se lec t ion ( "hos t su i c ide" ; S h a p i r o , 1976) o r i nc r ea sed hos t b o d y t e m p e r a t u r e ( " b e h a v i o r a l fever") a r e d i scussed . Finally, r e s e a r c h a r e a s t h a t s h o u l d e n h a n c e o u r u n d e r s t a n d i n g of b e h a v i o r a l modi f ica t ions will be p r o p o s e d .

II. Review of Altered Behavior in Parasitized Insects

A. Changes in Microhabitat Preference

Paras i t i zed insec ts often show c h a n g e s in m i c r o h a b i t a t p re fe rence (Tab le 1), i n c l u d i n g m o v e m e n t to e leva ted loca t ions , m o v e m e n t to concea l ed o r exposed loca t ions , c h a n g e s in t e m p e r a t u r e pre fe rences , a n d a l t e r e d r e s p o n s e to l ight ; add i t iona l ly , n o c t u r n a l insec ts m a y b e c o m e act ive d u r i n g t h e day. E leva t ion - seek ing b e h a v i o r a p p e a r s c o m m o n l y in hos t s infected b y fungal a n d v i ra l p a t h o g e n s [e.g. , fungal " s u m m i t d i s e a s e " (Mar ikovsky , 1962) a n d v i ra l " t r e e - t o p d i s e a s e " ( S t e i n h a u s , 1949) ] , a l t h o u g h m a c r o p a r a s i t e s s u c h as p a r a s i t o i d s a n d the t r e m a t o d e " b r a i n - w o r m " ( A n o k h i n , 1966) a lso i n d u c e t he behav ior . In sec t s no t typica l ly found o n p l a n t s m a y c l i m b to t h e tops of vege t a t i on ( T h a x t e r , 1888; Biinzli a n d Bii t t iker , 1959); s u b t e r r a n e a n insec ts often move to t he soil surface u p o n b e i n g p a r a s i t i z e d (Tab le 1).

M o v e m e n t to exposed loca t ions is i n d u c e d by a va r i e ty of p a r a s i t e s , inc l u d i n g fungi, v i ruses , p a r a s i t o i d s , t r e m a t o d e s , a n d ces todes (Tab le 1). M o v e m e n t to concea led loca t ions by a p h i d s p a r a s i t i z e d by a b r a c o n i d w a s p h a s b e e n d o c u m e n t e d ( B r o d e u r a n d M c N e i l , 1989, 1990). B o t h of these effects poss ib ly reflect c h a n g e s in l ight reac t ions of p a r a s i t i z e d hos t s (Bro-

5. Behavioral Effects of Parasites and Pathogens in Insect Hosts 109

A. Elevation-seeking behavior Fungi Lepidoptera: several species 24, 43, 48, 49

of caterpillars Orthoptera: grasshoppers 24, 32, 38, 43

cricket 43 Hymenoptera: ants 7, 19, 24 Coleoptera 28*, 43 Diptera 3 Homoptera: aphids 14*, 31

Viruses Lepidoptera: several species 1, 9, 11*, 16*, 17, of caterpillars 21, 22, 29, 41, <

Rickettsiella Coleoptera 27* Parasitoids Lepidoptera 37, 42

Hymenoptera: ant 12 Trematodes Hymenoptera: ants 2, 33

Diptera: mosquito larva 45 '

. Behavior seeking exposed locations Fungi Diptera 23, 25 Viruses Lepidoptera 15, 36, 47

Hymenoptera: sawfly 39 Parasitoid Homoptera: aphid 5 Trematode Hymenoptera: ant 8 Cestode Coleoptera 13 '

(continues)

"References: (1) Allen (1921); (2) Anokhin (1966); (3) Baird (1957); (4) Boorstein and Ewald (1987); (5) Brodeur and McNeil (1989); (6) Brodeur and McNeil (1990); (7) Bunzli and Buttiker (1959); (8) Carney (1969); (9) Edland (1965); (10) Eilenberg (1987); (11) Fowler and Robertson (1972); (12) Gosswald (cited in Wickler, 1976); (13) Graham (1966); (14) Harper (1958); (15) Hostetter and Biever (1970); (16) Kalmakoff and Moore (1975); (17) King and Atkinson (1928); (18) Lewis (1960); (19) Loos-Frank and Zimmerman (cited in Romig et al., 1980); (20) Louis et al. (1986); (21) Lounsbury (1913); (22) Lower (1954); (23) MacLeod et al., 1973; (24) Marikovsky (1962); (25) Miller and McClanahan (1959); (26) Moore (1983a); (27) Niklas (cited in Martignoni, 1964); (28) Nirula (1957); (29) Paschke and Hamm (1961); (30) Poinar and Gyrisco (1964); (31) Rockwood (1950); (32) Roffey (1968); (33) Romig et al. (1980); (34) Sato et al. (1983); (35) Schmid-Hempel and Schmid-Hempel (1990); (36) Semel (1956); (37) Shapiro (1976); (38) Skaife (1925); (39) Smirnoff (1960); (40) SmirnofT(1965); (41) Stairs (1965); (42) Stamp (1981); (43) Thaxter (1888); (44) Watson et al. (1993); (45) Webber et al. (1987a); (46) Whitlock (1974); (47) Wygant (1941); (48) Y e n (1962); (49) Y e n d o l and Paschke (1967). * Soil-dwelling insects to surface. 'Parasitized higher in water column. 'Decreased photophobia.

Table 1 Change in Microhabitat Preference by Parasitized Insects

Parasite or pathogen Host References 0


Table 1 (continued)

Parasite or pathogen Host References'1

C. Behavior seeking concealed locations Parasitoid Homoptera: aphid 5, 6

D. Change in reaction to light Parasitoid Lepidoptera 18' Acanthocephala Dictyoptera: cockroach 26*

E. Diurnal behavior by nocturnal insects Virus Lepidoptera 40 Parasitoid Lepidoptera 34 Nematode Coleoptera 30

F. Change in temperature preference Rickettsiella Orthoptera: cricket 20 Microsporidium Orthoptera: grasshopper 4 Fungus Diptera 44

G. Change in oviposition or foraging sites Fungus Diptera 10 Diptera Hymenoptera: bumblebee 35

'Increased photophobia.

d e u r a n d M c N e i l , 1989), as n o t e d a lso in o t h e r hos t s (Lewis , 1960; G r a h a m , 1966; M o o r e , 1983a) . D i u r n a l act ivi ty by n o r m a l l y n o c t u r n a l hos t s w a s o b served in p a r a s i t i z e d ca te rp i l l a r s a n d beet les (Tab le 1). Final ly , p a r a s i t i z e d cr ickets , g r a s s h o p p e r s , a n d flies select t e m p e r a t u r e s h i g h e r t h a n those selec ted by h e a l t h y conspecif ics (Tab le 1).

B. Changes in Level of Activity

V i r u s e s c a u s e g r e g a r i o u s sawflies to e n g a g e in so l i t a ry w a n d e r i n g b e h a v i o r (Smirnoff, 1960, 1961 , 1965); v i rus- infec ted l e p i d o p t e r a n l a rvae m a y l ikewise exh ib i t u n d i r e c t e d w a n d e r i n g (Lower , 1954; S m i t h et al., 1956; T a m a s h i r o a n d H u a n g , 1963). C o c k r o a c h e s pa r a s i t i z ed b y Moniliformis moniliformis s p e n d m o r e t i m e in act iv i ty ( M o o r e , 1983a) a n d m o v e a g r e a t e r d i s t a n c e in a n a r e n a (Wi l son a n d E d w a r d s , 1986) t h a n d o paras i te - f ree insec t s . F o r a g i n g w o r k e r b u m b l e b e e s (Bombus) p a r a s i t i z ed by c o n o p i d flies s p e n d m o r e t i m e a w a y from t h e nes t t h a n d o h e a l t h y bees ( S c h m i d - H e m p e l a n d M u l l e r , 1991). Q u e e n b u m b l e b e e s p a r a s i t i z e d by t h e n e m a t o d e Sphaerularia bombi fly close to t he g r o u n d , occas iona l ly a l i gh t ing a n d d igg ing a d e p r e s s i o n in t h e soil (Po ina r a n d v a n d e r L a a n , 1972; L u n d b e r g a n d Svensson , 1975). J u v e ni le n e m a t o d e s a r e expel led in to t h e ho le , a n d t h e p rocess is r e p e a t e d . F l i gh t is c o n t i n u e d long after h e a l t h y bees h a v e in i t i a t ed a b r o o d , h e n c e p a r a s i t i z e d bees have b e e n ca l led " t h e e t e r n a l s eeke r s " (Po ina r a n d v a n d e r L a a n , 1972).


O t h e r p a r a s i t i z e d insec ts show r e d u c e d activity. A l t h o u g h th i s effect m a y ar i se la rge ly from pa tho logy , t h e d e c r e a s e in act iv i ty c a n affect p a r a s i t e o r hos t fi tness (see t h e fol lowing). S o m e p a t h o g e n s c a u s e hos t s to b e c o m e s luggish ( H e n r y a n d O m a , 1981; J o h n s o n , 1989); o t h e r s p r o d u c e pa ra ly s i s ( K u l i n c e v i c et al., 1970; R e i n g a n u m et al., 1970). A n t s p a r a s i t i z e d b y ces todes ( P l a t e a u x , 1972) o r t r e m a t o d e s (Carney , 1969) m o v e slowly c o m p a r e d to h e a l t h y conspecif ics . T r e m a t o d e - p a r a s i t i z e d a n t s (Leptothorax) m a y s t ay in t h e nes t , un l ike h e a l t h y a n t s ( P l a t e a u x , 1972). L a r v a l Aedes aegypti p a r a s i t i z e d w i t h a t r e m a t o d e (Plagiorchis noblei) s how r e d u c e d ac t iv i ty ( W e b b e r et al., 1987a) .

C. Reproductive Behavior by Castrated Insects

H o s t s t h a t h a v e b e e n c a s t r a t e d b y n e m a t o d e s m a y none the l e s s r e t a i n n o r m a l o r s l ight ly modif ied r e p r o d u c t i v e behav io r . Blackflies (S imul i idae ) c a s t r a t e d b y m e r m i t h i d n e m a t o d e s exh ib i t b e h a v i o r s imi l a r to t h a t s h o w n b y h e a l t h y flies. B o t h h e a l t h y a n d p a r a s i t i z e d females fly u p s t r e a m to ovipos i t , b u t p a r a s i t i z e d females re lease n e m a t o d e s i n s t e a d of eggs ( G r u n i n , 1949; H u n t e r a n d M o o r h o u s e , 1976; C o l b o a n d Por te r , 1980; Molloy, 1981). M u s c i d a n d sc ia r id flies s h o w " m o c k ov ipos i t i on" ( N a p p i , 1973), d e p o s i t i n g n e m a t o d e s i n s t e a d of fly eggs (Poinar , 1965; Stoffolano, 1970, 1973). P a r a s i t i s m b y n e m a t o d e s p r o d u c e s in te r sexes in s o m e c h i r o n o m i d s ; m i d g e s r e t a i n female r e p r o d u c t i v e b e h a v i o r s , b u t depos i t n e m a t o d e s i n s t e a d of eggs ( R e m p e l , 1940).

F u n g i in t h e g e n u s Massospora infect c i c a d a s ( H o m o p t e r a ) . I n t h e a d u l t insec t , t hey a r e res t r i c t ed to t he a b d o m i n a l t i ssues ( T h a x t e r , 1888). Succes sive a b d o m i n a l s e g m e n t s b e c o m e infected a n d s lough off, even tua l l y p r o d u c ing a c i c a d a c o m p o s e d p r i m a r i l y of h e a d a n d t h o r a x (Go lds t e in , 1929). M. cicadina c o n s u m e s t he eggs of i ts hos t (Go lds t e in , 1929); even a t ea r ly s t ages of infect ion, females fail to oviposi t (L loyd et al., 1982). D e s p i t e t h e loss of b o d y s e g m e n t s , infected c i cadas c i r cu la t e a m o n g the m a t i n g p o p u l a t i o n ( S p e a r e , 1921; Soper , 1963). Infec ted m a l e s c o n t i n u e to s ing (Sope r et al., 1976) a n d a t t r a c t h e a l t h y females w i t h w h i c h they a t t e m p t to c o p u l a t e (Soper , 1963). H e a l t h y m a l e s m a y a t t e m p t to c o p u l a t e w i t h infected females (Sope r et al., 1976).

III. Potential Benefits to Parasite

A. Increased Parasite Dissemination

Severa l b e h a v i o r a l a l t e r a t i o n s s h o u l d e n h a n c e p a r a s i t e d i s s e m i n a t i o n . For e x a m p l e , a fungus - o r v i r u s - l o a d e d c a d a v e r o n t he g r o u n d s h o u l d b e a p o o r e r sou rce of i n o c u l u m t h a n o n e a t t r e e t o p level. V i r u s e s t h a t c a u s e e leva t ion-


seeking b e h a v i o r in ca te rp i l l a r s w e a k e n t h e hos t ' s e p i d e r m i s ; flaccid c a d a v e r s ("wil t d i s ease" ) even tua l ly b r e a k a n d spill infect ious v i rus o n t o t h e foliage be low (e.g. , T a n a d a , 1963). E leva t ion - seek ing b e h a v i o r by hos t s infected b y fungal p a t h o g e n s exposes n e i g h b o r s to a n infective r a i n of spo res ( M a c L e o d et al., 1966). Fungus - in fec t ed so ld ier beet les (Chauliognathus) g r i p t h e p l a n t w i t h m a n d i b l e s a n d s p r e a d e ly t rae a n d h i n d w ings . T h e b e h a v i o r exposes t h e surface of t he a b d o m e n , from w h i c h fungal spores a r e p r o d u c e d ( C a r n e r , 1980).

D i s s e m i n a t i o n m a y a lso be e n h a n c e d by inc reases in hos t activity. P a t h o gens t h a t a t t a ck t he hos t ' s gu t often a r e re leased in t he hos t ' s feces o r r egu rg i t a t i ons (e.g. , Smirnoff, 1961), c o n t a m i n a t i n g foliage e a t e n by h e a l t h y insec t s . T h e s e p a t h o g e n s t e n d to kill the i r hos t s slowly, p r o l o n g i n g d i s s e m i n a t i o n . For e x a m p l e , a l a rva l sod w e b w o r m (Crambus trisectus) m a y p a s s u p to 100 mi l l ion Nosema spores in its feces p r io r to d e a t h , a n d c o n t a i n a n a d d i t i o n a l 200 mi l l ion spores a t d e a t h ( M a d d o x , 1973). G r a s s h o p p e r s (Melanoplus) m a y p r o d u c e u p w a r d s of 1 0 9 spores of Nosema locustae (Henry , 1972). P a t h o g e n s t h a t c a u s e s lugg i shness m a k e the hos t v u l n e r a b l e to c a n n i b a l i s m by h e a l t h y conspecif ics ( H e n r y a n d O m a , 1981), o r to p r e d a t o r s t h a t in t u r n d i s s e m i n a t e t he p a t h o g e n (Young a n d Year ian , 1989).

For p a r a s i t e s r e q u i r i n g a v e r t e b r a t e hos t , e l eva t ion-seek ing behav io r , c h a n g e s in activity, a n d inc reased e x p o s u r e all e n h a n c e t r a n s m i s s i o n ( M o o r e , 1984). For i n s t a n c e , a n t s p a r a s i t i z e d by the " b r a i n - w o r m " t r e m a t o d e a t t a c h themse lves to t he t ips of g rass s t e m s w h e r e they a r e inges t ed by g r a z i n g sheep , t he defini t ive hos t of t he p a r a s i t e ( A n o k h i n , 1966). T h e i n c r e a s e d act ivi ty a n d d e c r e a s e d p h o t o p h o b i a s h o w n by cockroaches p a r a s i t i z e d by M. moniliformis cou ld m a k e cockroaches c o n s p i c u o u s to t he p a r a s i t e ' s def ini te hos t , Rattus ( M o o r e , 1983a) . S imi l a r benefi ts resu l t f rom r e d u c e d hos t activity. T r e m a t o d e s c a u s e r e d u c t i o n s in act iv i ty of l a rva l Aedes aegypti, l e a d i n g to i nc r ea sed p r e d a t i o n by the vole defini t ive hos t ( W e b b e r et al., 1987a ,b) .

Behav io r of c a s t r a t e d hos t s d i s s e m i n a t e s t he p a r a s i t e . T h e n e m a t o d e -d e p o s i t i n g act ivi t ies of b u m b l e b e e s (Sect ion I I . B ) likely s p r e a d n e m a t o d e s over t he w i n t e r i n g h a b i t a t of t he bee , p r o d u c i n g infect ions t he fol lowing s p r i n g (Po ina r a n d v a n d e r L a a n , 1971). U p s t r e a m "ov ipos i t i on" b e h a v i o r b y c a s t r a t e d blackflies he lps m a i n t a i n t he m e r m i t h i d p a r a s i t e in t h e s t r e a m (Golbo a n d Por ter , 1980). " M o c k ov ipos i t ion" by m u s c i d s s p r e a d s n e m a t o d e s over a r e a s f r equen ted by h e a l t h y flies ( N a p p i , 1973). M a t i n g a t t e m p t s by Atomr/wra- infec ted c i cadas m a y t r a n s m i t fungal spores to h e a l t h y insec ts (Sope r et al., 1976).

B. Increased Parasite Survival

M o v e m e n t to n e w m i c r o h a b i t a t s by hos t s c a n favor p a r a s i t e s by r e d u c i n g p r e d a t i o n or h y p e r p a r a s i t i s m . S t a m p (1981) n o t e d t h a t Euphydryas phaeton


ca t e rp i l l a r s p a r a s i t i z e d by a b r a c o n i d w a s p m o v e d to e leva ted loca t ions , lower ing r a t e s of h y p e r p a r a s i t i s m . A p h i d s c o n t a i n i n g p a r a s i t o i d s m o v e d to concea l ed loca t ions , r e su l t i ng in d e c r e a s e d r a t e s of h y p e r p a r a s i t i s m ( B r o d e u r a n d M c N e i l , 1989) a n d , potent ia l ly , i nc r ea sed w i n t e r su rv iva l for t he p a r a s i toid ( B r o d e u r a n d M c N e i l , 1990). A l i t e r a t u r e rev iew (Fr i t z , 1982) c o n c l u d ed t h a t p r e d a t i o n r a t e s often w e r e lower on p a r a s i t i z e d hos t s t h a n o n h e a l t h y hos t s , poss ib ly b e c a u s e of c h a n g e s in hos t behav ior . H o s t c a s t r a t i o n c a n be v iewed as a p a r a s i t e surv iva l s t r a t e g y if it r ed i rec t s r e sou rces u s e d by the h o s t for r e p r o d u c t i o n t o w a r d p rocesses beneficial to t h e p a r a s i t e ( B a u d o i n , 1975).

IV. Potential Benefits to Host

A. Increased Survival of Kin

S h a p i r o (1976) h y p o t h e s i z e d t h a t b e h a v i o r a l c h a n g e s cou ld i n c r e a s e t h e hos t ' s inc lus ive fitness. L a r v a l Chlosyne harrissii p a r a s i t i z e d by a b r a c o n i d w a s p p e r c h a t t he tops of vege ta t ion w h e r e they a r e h igh ly c o n s p i c u o u s ( S h a p i r o , 1976). S h a p i r o s p e c u l a t e d t h a t if n e a r b y ca t e rp i l l a r s w e r e r e l a t e d to t h e p a r a s i t i z e d hos t , t he hos t cou ld ga in in inc lus ive fitness by expos ing itself to p r e d a t o r s ( "hos t su i c ide" ) . P r e d a t i o n w o u l d kill t h e p a r a s i t o i d a n d e l i m i n a t e d a n g e r to re la t ives from t h e act ivi t ies of t h a t p a r t i c u l a r w a s p .

T h e hos t su ic ide h y p o t h e s i s w o u l d s e e m to r e q u i r e severa l fairly res t r ic t ive c o n d i t i o n s ( S h a p i r o , 1976; S m i t h T r a i l , 1980; S t a m p , 1981; M o o r e , 1984; M c A l l i s t e r a n d Ro i tbe rg , 1987): p a r a s i t i z e d hos t s s h o u l d b e r e p r o d u c t i v e l y d e a d ; k in s h o u l d b e a g g r e g a t e d [e.g. , as in social insec ts (see M c A l l i s t e r a n d R o i t b e r g , 1987) ] ; hos t a n d p a r a s i t e p h e n o l o g y s h o u l d al low t h e d e v e l o p i n g p a r a s i t e to even tua l ly p a r a s i t i z e k in of t he infected hos t ; a n d b e h a v i o r a l c h a n g e s s h o u l d inc rease m o r t a l i t y of t he hos t . A n u m b e r of these c o n d i t i o n s qua l i t a t i ve ly a p p e a r to b e m e t in insec t h o s t / p a r a s i t e a s soc ia t ions . P a r a s i t ized insec ts suffer f rom p r e d a t i o n by a va r i e ty of a n i m a l s ( W h i t e a n d Du tky , 1940; Smirnoff, 1959; H o s t e t t e r a n d Biever, 1970; Tos towaryk , 1971). Social insec ts show p a r a s i t i s m - i n d u c e d b e h a v i o r a l c h a n g e s t h a t m a y i nc r ea se mor ta l i ty . For e x a m p l e , t he e leva t ion-seek ing r e s p o n s e of p a r a s i t i z e d a n t s i nc reases the i r risk of p r e d a t i o n (Sec t ion I I I . A ) . Final ly , p a r a s i t i c c a s t r a t i o n is c o m m o n , a n d s h o u l d m i n i m i z e t h e cost of su ic ide to t h e hos t ; t h a t is, benefi ts n e e d n o t b e too g r e a t for su ic ide to b e a d v a n t a g e o u s ( M o o r e , 1984), s ince t h e hos t is evo lu t ionar i ly d e a d ( K u r i s , 1974).

M c A l l i s t e r a n d R o i t b e r g (1987) showed t h a t p e a a p h i d s (Acyrthosiphon pisum) p a r a s i t i z e d by a b r a c o n i d w a s p e n g a g e d in b e h a v i o r t h a t t h e a u t h o r s i n t e r p r e t e d as b e i n g su ic ida l . Pa ra s i t i zed a p h i d s f rom h o t d r y h a b i t a t s d r o p p e d from t h e hos t p l a n t , r e su l t i ng in des i cca t ion , in r e s p o n s e to t he a p p r o a c h of a p r e d a t o r . I n we t t e r a r e a s , w h e r e t he b e h a v i o r w a s unl ike ly to


resu l t in des icca t ion , a p h i d s were less likely to d r o p f rom t h e hos t (see a lso L a t t a , 1987; T o m l i n s o n , 1987; McAl l i s t e r a n d R o i t b e r g , 1988; M c A l l i s t e r et al, 1990).

Final ly, kin m a y benefi t from hos t b e h a v i o r even if t h e b e h a v i o r a l c h a n g e is n o t " s u i c i d a l . " Pa thogen- in fec ted hos t s t h a t m o v e to n e w m i c r o h a b i t a t s m a y avoid b e i n g c a n n i b a l i z e d b y h e a l t h y k in . Also , a l t h o u g h s o m e behav iora l c h a n g e s (e.g. , e leva t ion-seeking) m a y d i s s e m i n a t e infective p r o p a g u l e s over l a rge a r e a s , t he b e h a v i o r cou ld r e d u c e t r a n s m i s s i o n to n e a r b y hos t s ; t h a t is, un less t he p a t h o g e n is h igh ly infective a n d r equ i r e s on ly a few p r o p a g u l e s to beg in a n e w infect ion, w i d e s p r e a d d i s s e m i n a t i o n of p r o p a g u l e s cou ld r e d u c e i n o c u l u m dens i t y to t he ex t en t t h a t n e a r b y k in a r e unl ike ly to c o n t a c t a n infective dose ( C . M a c V e a n , p e r s o n a l c o m m u n i c a t i o n ) .

B. Host Defense: Behavioral Fever

H o s t s w o u l d benefi t if a l t e r ed b e h a v i o r ass i s ted in c o m b a t i n g t h e p a r a s i t e . M a n y e c t o t h e r m s r e s p o n d to p a r a s i t i s m b y ra i s ing b o d y t e m p e r a t u r e s ; t h a t is, p a r a s i t i z e d i n d i v i d u a l s select w a r m e r m i c r o h a b i t a t s t h a n d o h e a l t h y a n i m a l s ( "behav io ra l fever"; see K l u g e r , 1979, 1990). T h i s o p t i o n m a y b e avai l ab l e to insec ts , s ince i nc reased t e m p e r a t u r e s a r e often d e t r i m e n t a l to p a r a s i t e success (Tab le 2; see a lso H e i n r i c h , 1981). Severa l b e h a v i o r a l c h a n g e s a r e cons i s t en t w i t h a fever e x p l a n a t i o n . M o v e m e n t to exposed loca t ions m a y inc rease b o d y t e m p e r a t u r e s . M a r i k o v s k y (1962) sugges t ed t h a t fungus -infected insec ts benef i ted from " so l a r i r r a d i a t i o n " a n d n o t e d t h a t b a s k i n g g r a s s h o p p e r s occas iona l ly recovered f rom the p a t h o g e n . D i u r n a l ac t iv i ty b y n o r m a l l y n o c t u r n a l hos t s m a y ra i se b o d y t e m p e r a t u r e s , a s m i g h t m o v e m e n t to t h e surface b y s u b t e r r a n e a n hos t s . I n c r e a s e d activity, p a r t i c u l a r l y flight, a lso ra ises b o d y t e m p e r a t u r e ( H e i n r i c h , 1981).

Five s tud ies have s h o w n t h a t insec ts cha l l enged w i t h tox ins o r p a t h o g e n s e leva ted the i r b o d y t e m p e r a t u r e s (Brons te in a n d C o n n e r , 1984; Lou i s et al., 1986; Boors t e in a n d E w a l d , 1987; M c C l a i n et al., 1988; W a t s o n et al., 1993); in two cases this was s h o w n to i nc rease hos t su rv iva l (Boors te in a n d E w a l d , 1987; W a t s o n et al., 1993; see a lso Lou i s et al., 1986; C a r r u t h e r s et al., 1992).

V. Considerations for Future Research

W i t h s o m e excep t ions (e.g. , S t a m p , 1981; Boors t e in a n d E w a l d , 1987; McAl l i s t e r a n d R o i t b e r g , 1987; B r o d e u r a n d M c N e i l , 1990), ev idence ty ing b e h a v i o r a l a l t e r a t i ons to hos t o r p a r a s i t e success h a s b e e n a n e c d o t a l . Fu l l u n d e r s t a n d i n g will r e q u i r e e x p e r i m e n t a t i o n , specifically: (1) q u a n t i t a t i v e d e sc r ip t ions of b e h a v i o r a l c h a n g e s f rom e x p e r i m e n t s t h a t i n c o r p o r a t e s u i t a b l e


Table 2 Selected Examples of Effects of High Temperature on Survival of Parasitized Hosts or Mortality of Parasites

Parasite or Effects of temperature pathogen Host on host or parasite Reference*

Viruses Caterpillars Host survival at 32.2°C > 26.7°C, 21.1°C, 15.6°C

Host survival at 35°C > 25°C Host survival at 35°C, 30°C > 25°C Host survival at 40°C > 37°C > 33°C

> 30°C

2

7 11 10

Fungi Aphids Fungal growth on agar ceased at 27— 36°C

5

Caterpillar Host survival at 31°C > 26°C, 21°C, 16°C

Host survival at 30°C > 25°C, 20°C, 15°C

3

8

Microsporidium Caterpillar Loss of parasite at culture temperatures of 35°C

12

Bacterium Grasshopper Host survival at 28°C > 20°C 9

Parasitoids Aphid Upper lethal temperature lower for parasitoid than for host

4

Caterpillar Upper lethal temperature lower for parasitoid than for host

1, 6

"References: (1) Allen and Smith (1958); (2) Boucias et al. (1980); (3) Boucias et al. (1984); (4) Force and Messenger (1964); (5) Hall and Bell (1960); (6) Kaya and Tanada (1969); (7) Kobayashi et al. (1981); (8) Mohamed et al. (1977); (9) Pospelov (1926); (10) Steinhaus and Dineen (1960); (11) Tanada and Tanabe (1965); (12) Wilson and Sohi (1977).

con t ro l s ; (2) q u a n t i t a t i v e tests of w h e t h e r e i the r p a r t y benefi ts ; a n d (3) tes ts s e p a r a t i n g b e h a v i o r a l c h a n g e s t h a t a r e p r e s e n t b e c a u s e of n a t u r a l se lec t ion o p e r a t i n g o n t h e c o n t e m p o r a r y a s soc ia t ion f rom those t h a t a r e p r e s e n t o n l y for tu i tous ly [i .e. , as ca r ryovers f rom s o m e h i s to r ica l a s soc ia t ion ( M o o r e a n d Gote l l i , 1990) o r as c o n s e q u e n c e s of p a t h o l o g y ] . T h e l a t t e r t w o a p p r o a c h e s a r e i m p o r t a n t in t h a t t r e a t m e n t s of a l t e r e d hos t b io logy m a y o v e r e m p h a s i z e t h e i m p o r t a n c e of t h e p a r a s i t e in t h e i n t e r ac t i on (see a lso M i n c h e l l a , 1985) ; e x p e r i m e n t a t i o n s h o u l d r e d u c e th is t e m p t a t i o n . We c o n c l u d e b y d i s cus s ing these t h r e e a p p r o a c h e s .

A l t h o u g h it is t e m p t i n g to infer b e h a v i o r a l a l t e r a t i ons f rom differences in t h e d i s t r i b u t i o n s of h e a l t h y a n d p a r a s i t i z e d i n d i v i d u a l s , th i s c a n b e m i s l e a d i n g ( S t a m p , 1981). J a y n e s (1954) n o t e d t h a t s p r u c e b u d w o r m l a rvae col lec ted f rom t h e u p p e r t ree c r o w n w e r e m o r e likely to b e p a r a s i t i z e d b y Apanteles w a s p s t h a n w e r e ca t e rp i l l a r s lower in t he t ree , w h e r e a s t h e r e w e r e n o d i s t r i -


b u t i o n differences for ca te rp i l l a r s pa r a s i t i z ed by Glypta w a s p s . H e n c e , o n e m a y h y p o t h e s i z e e i the r t h a t Apanteles i n d u c e d b e h a v i o r a l c h a n g e s in t h e h o s t whi le Glypta d id no t , o r t h a t t he two p a r a s i t o i d s differed in fo rag ing behav io r . D o a n e (1970) i n d i c a t e d t h a t the t r e e top a c c u m u l a t i o n of v i rus-ki l led gypsy m o t h s (Lymantria dispar) was d u e to t he act ivi t ies of b o t h h e a l t h y a n d infected ca te rp i l l a r s ; infected ca te rp i l l a r s d i ed a t t r e e t o p level, w h e r e a s h e a l t h y insects m o v e d d o w n the t ree as they a g e d . I n s u m , d e m o n s t r a t i n g t r u e behaviora l modi f ica t ions r equ i r e s e x p e r i m e n t a t i o n w i t h su i t ab l e con t ro l s .

Second , from s tud ies r e p o r t e d h e r e , it is n o t poss ib le to d e t e r m i n e t he benef ic iary of a g iven behav io ra l a l t e r a t i on ; i n d e e d , s o m e a l t e r a t i o n s , d e p e n d i n g u p o n the p a r a s i t e , cou ld be cons i s t en t w i t h all four h y p o t h e s e s p r e s e n t e d in this p a p e r (Tab le 3). C o s t s a n d benefi ts m a y a c c r u e to b o t h p a r a s i t e a n d hos t . For e x a m p l e , d e v e l o p m e n t r a t e s of M. moniliformis in cockroaches inc rease as t e m p e r a t u r e inc reases from 20 to 28°C; a t t e m p e r a t u r e s h i g h e r t h a n 28°C, p a r a s i t e s show d e v e l o p m e n t a l a b n o r m a l i t i e s (Lack ie , 1972). T h a t is, a s l ight i nc rease in hos t t e m p e r a t u r e benefi ts t h e p a r a s i t e , w h e r e a s a l a rge inc rease does no t . Moreove r , t r a n s m i s s i o n r e q u i r e s inges t ion by a v e r t e b r a t e hos t . H e n c e , even if a b e h a v i o r a l c h a n g e w e r e , say, a fever r e s p o n s e , if t he act ivi ty a lso i nc reased hos t c o n s p i c u o u s n e s s , t h e p a r a s i t e m i g h t benefi t t h r o u g h e n h a n c e d d i s s e m i n a t i o n .

Table 3

Potential Benefits of Host Behavioral Changes to Parasite or Host*

Parasite adaptive Host adaptive

Increased Increased Increased Behavioral Host behavioral modification dissemination survival survival of kin fever

Elevation-seeking Movement to exposed locations Movement to concealed locations Preference for higher tempera

ture Altered light reaction Increase in activity Decrease in activity Diurnal activity by nocturnal in

sects Reproductive activity by cas

trated hosts or castration itself

a*-Modification potentially consistent with hypothesis for at least a subset of parasites that induce the behavioral change; representative examples discussed in text. O-Modification not obviously consistent with hypothesis or not explored in text.

* * * * 0 * 0 0

* 0 0 *

* 0 * *

* ο * * * 0 * 0

* ο * *

* * 0 0


T h u s , e x p e r i m e n t a t i o n is cr i t ica l for d e t e r m i n i n g t h e benef ic iary of a c h a n g e in behav io r . G i v e n the n u m b e r of poss ib le o u t c o m e s (Tab le 3), th i s a p p r o a c h cou ld b e c o m e q u i t e c o m p l e x . Fami l i a r i t y w i t h t h e s y s t e m m a y e l i m i n a t e s o m e h y p o t h e s e s . For e x a m p l e , s o m e sys t ems m a y b e less sub jec t to k in se lec t ion t h a n a r e o t h e r s ( S m i t h T r a i l , 1980; S t a m p , 1981; M o o r e , 1984; M c A l l i s t e r a n d R o i t b e r g , 1987). I n o n e c a t e r p i l l a r - p a r a s i t o i d s y s t e m , S t a m p (1981) n o t e d t h a t t h e g r e g a r i o u s n a t u r e of t h e hos t m i g h t in i t ia l ly l ead o n e to look for hos t su ic ide . However , s h e a lso n o t e d t h a t su ic ide w a s un l ike ly to evolve b e c a u s e t he hos t w a s a p o s e m a t i c a n d u n p a l a t a b l e ; resu l t s of h e r s t u d y w e r e cons i s t en t w i t h t he l a t t e r h y p o t h e s i s . O n the bas i s of hos t a n d p a r a s i t e biology, M o o r e (1984) c o n c l u d e d t h a t hos t su ic ide w a s unl ike ly to evolve in a r t h r o p o d - A c a n t h o c e p h a l a a s soc ia t ions . Converse ly , in a s y s t e m m o r e a m e n a b l e to k in se lec t ion , b e h a v i o r cons i s t en t w i t h hos t su ic ide w a s d e m o n s t r a t e d (McAl l i s t e r a n d R o i t b e r g , 1987).

Last ly , we sugges t t h a t a c o m p a r a t i v e a p p r o a c h , o p t i m a l l y o n e t h a t cons iders h o s t a n d p a r a s i t e phy logen ie s , m a y b e neces sa ry to fully u n d e r s t a n d t h e evo lu t ion of b e h a v i o r a l a l t e r a t i ons ( J o n e s , 1985; M o o r e a n d Gote l l i , 1990). A b e h a v i o r a l a l t e r a t i o n m a y show u p in a p a r a s i t e / h o s t a s soc i a t i on b e c a u s e it evolved in a n a n c e s t r a l a s soc ia t ion a n d w a s r e t a i n e d in d e s c e n d a n t t a x a ( M o o r e a n d Gote l l i , 1990); benefi ts to d e s c e n d a n t t a x a m i g h t h a v e c h a n g e d or b e a b s e n t a l toge the r . Pa ra s i t e s cou ld benefi t f rom a n evolved h o s t behav io r ; t h a t is, t h e p a r a s i t e m a y elicit a p reex i s t i ng h o s t b e h a v i o r r a t h e r t h a n i n d u c e a novel b e h a v i o r ( M o o r e , 1984). For e x a m p l e , W i c k l e r (1976) sugges t ed t h a t t h e b e h a v i o r s h o w n b y t r e m a t o d e - p a r a s i t i z e d a n t s , t h a t is, a t t a c h i n g themse lves by m a n d i b l e s to t he u p p e r p o r t i o n of a p l a n t , is s imi l a r to a phy logene t i ca l l y old " s l e e p i n g " b e h a v i o r found in o t h e r H y m e n o p t e r a . Fever m a y m a k e hos t s c o n s p i c u o u s , s u c h t h a t t he p a r a s i t e benefi ts v ia d i s s e m i n a t i o n to t he defini t ive hos t . Last ly , t he p a r a s i t e m a y benefi t f rom a h o s t p a t h o l o g i c r e s p o n s e . C r a m (1931) n o t e d t h a t g r a s s h o p p e r s infected w i t h t h e n e m a t o d e Tetrameres americana w e r e d r o o p y ( the p a r a s i t e encys t s in g r a s s h o p p e r m u s c l e ) , h e n c e m i g h t b e v u l n e r a b l e to t h e gal l i form final hos t . A l t h o u g h cho ice of e n c y s t m e n t si te m a y h a v e evolved t h r o u g h se lec t ion favor ing inc reased p r e d a t i o n by t h e defini t ive hos t , th is i n t e r p r e t a t i o n is p r e l i m i n a r y in t h e a b s e n c e of o t h e r i n fo rma t ion .

T h e c o m p a r a t i v e a p p r o a c h c a n t ake o n e of two d i r ec t ions : s t u d y of a s ingle p a r a s i t e t a x o n in a va r i e ty of hos t s , o r s t u d y of a va r i e ty of p a r a s i t e s in a s ingle hos t t a x o n [for a n e x a m p l e of t he former , see J o n e s ' (1985) d i scuss ion a b o u t t h e effects of Chelonus p a r a s i t e s o n m e t a m o r p h o s i s in L e p i d o p t e r a ] . T h e p r e s e n t r ev iew sugges t s t h a t t h e r e is a g r e a t d e a l of c o n v e r g e n c e in t h e b e h a v i o r of p a r a s i t i z e d insec t s . I n d e e d , a n u m b e r of b e h a v i o r a l c h a n g e s n o t e d h e r e have pa ra l l e l s in non - in sec t sy s t ems ( H o l m e s a n d Be the l , 1972; M o o r e , 1983b, 1984; M o o r e a n d Gote l l i , 1990). E l eva t ion - seek ing b e h a v i o r is


p a r t i c u l a r l y w i d e s p r e a d . T h e fact t h a t p a r a s i t e s as d i s t a n t l y r e l a t ed as fungi ,

v i ruses , p a r a s i t o i d s , a n d t r e m a t o d e s i n d u c e this s a m e b e h a v i o r in the i r insec t

hos t s m i g h t sugges t t h a t e leva t ion-seek ing is a gene ra l i zed insec t r e s p o n s e to

p a r a s i t i s m , a n d t h a t a n y benefi ts to t he p a r a s i t e a r e fo r tu i tous . I n s o m e

n o n i n s e c t sys t ems hos t b e h a v i o r (hence benefits?) differs d e p e n d i n g u p o n the

p a r a s i t e (Be the l a n d H o l m e s , 1973; R a u , 1983, 1984; M o o r e a n d Lasswel l ,

1986). C o m p a r i s o n s of hos t b e h a v i o r in a s ingle insec t species p a r a s i t i z e d by

t a x o n o m i c a l l y u n r e l a t e d p a r a s i t e s w o u l d be of va lue .

Acknowledgments

For comments on versions of this manuscript we thank Ann Baker, John Capinera, Deb Kendall, Jim Krysan, Chuck MacVean, and Tom Unruh. Support for this project was received from the National Science Foundation (Grant BSR-8452076), the Monsanto Company, the Burroughs Wellcome Fund, the Exxon Education Foundation, and the Whitehall Foundation, as well as from NSF grant BSR-8817495 to J. Moore and N. Gotelli. Additional support for D. Horton was obtained from the Colorado Agricultural Experiment Station, USDA-ARS, the Washington Tree Fruit Research Commission, Yakima, and the Winter Pear Bureau, Portland, Oregon.

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Chapter 6

^Hfl Redirection of Host JHfl Metabolism and Effects on ^flH Parasite Nutrition

S. N. Thompson Department of Entomology University of California Riverside, California

I. Introduction

II. Insect Parasitism and Host Viability

III. Altered Metabolite Levels in Host Tissues A. Carbohydrates B. Proteins and Amino Acids C. Lipids

IV. Metabolic Alteration in Insect Hosts A. Trichoplusia ni Parasitized by

Hyposoter exiguae B. Manduca sexta Parasitized by Cotesia

congregata

V. Basis for Metabolic Alterations: Hormones, Viruslike Particles, and Parasite Mediation

VI. Nutritional Consequences of Metabolic Redirection and Physiological Alteration

VII. "Host Regulation" and Its Role in Redirecting Host Metabolism and Physiology

VIII. Enantiostastis and Metabolic Regulation

IX. Conclusion References

I. Introduction

T h i s c h a p t e r ou t l ines t he effects of p a r a s i t i s m by p r o t e l e a n insec t p a r a s i t e s o r

p a r a s i t o i d s 1 on t he i n t e r m e d i a r y m e t a b o l i s m of the i r insect hos t s . Successful

p a r a s i t i c r e l a t i onsh ip s invo lv ing k o i n o b i o n t i c p a r a s i t o i d s (Sec t ion I I ) r e q u i r e

t h a t t h e p a r a s i t e be " a d a p t e d to t he p h y s i o c h e m i c a l c o n d i t i o n s of t h e h o s t "

a n d "u t i l i ze hos t n u t r i e n t in a m a n n e r c o m p a t i b l e w i t h hos t s u r v i v a l "

( S m y t h , 1976). T o fulfill t hese r e q u i r e m e n t s , a l t e r a t i ons in t h e hos t ' s phys io l -

l rThe term parasite is used here to describe the activity and behavior of the parasitic larval stages of "protelean insect parasites" (Askew, 1971). Parasitoid is used in reference to the free-living adult stage.

Parasites and Pathogens of Insects Volume I: Parasites 125


126 S. Ν. Thompson

ogy often a r e necessa ry (V inson , 1984). T h i s " r e d i r e c t i o n " of t h e h o s t is a m u l t i d i m e n s i o n a l p h e n o m e n o n usua l ly a c c o m p a n i e d by o b s e r v a b l e c h a n g e s in g r o w t h , d e v e l o p m e n t , a n d b e h a v i o r ( T h o m p s o n , 1990). T h e h o s t r e m a i n s a d i s t inc t a n d v iab le enti ty, b u t its fate is p e r m a n e n t l y a l t e r ed ( T h o m p s o n , 1983a) . T h e p rocess of r ed i rec t ion is cr i t ica l for t h e success of t h e p a r a s i t e a n d h a s i m p o r t a n t c o n s e q u e n c e s in t he evolu t ion of these p a r a s i t e - h o s t re lat i onsh ips .

T h e phys io logy of t he p a r a s i t i z e d hos t reflects c h a n g e s in t he n o r m a l funct ion a n d act ivi ty of t he hos t ' s t i ssues a n d o r g a n sy s t ems . T h e o b s e r v e d effects a r e man i fes t ed t h r o u g h c h a n g e s in m e t a b o l i s m . I n d e e d , m e t a b o l i s m is r e spons ib l e for m a i n t a i n i n g phys io log ica l funct ion a n d a l t h o u g h n o t m a c r o -scopica l ly ev iden t , phys io logica l i n t e g r a t i o n is ach ieved t h r o u g h m e t a b o l i c , t h a t is, b i o c h e m i c a l , r egu la t i on . E v e n the s imples t phys io log ica l a d j u s t m e n t involves c o m p l e x n e t w o r k s of b i o c h e m i c a l r eac t ions a n d the i r a s soc ia t ed r e g u l a t o r y m e c h a n i s m s i n t e g r a t e d t h r o u g h the hos t ' s n e r v o u s a n d e n d o c r i n e sy s t ems . T h e m o l e c u l a r a n d b i o c h e m i c a l bases for t h e r ed i r ec t i on of hos t phys io logy a r e t he sub jec t of i n t ense s tudy. C h a n g e s in i n t e r m e d i a r y m e t a b o l ism t h a t m a y b e a d a p t i v e in r e l a t ion to n o u r i s h m e n t of t h e d e v e l o p i n g p a r a s i t e l a rvae a r e t he sub jec t of th is d i scuss ion .

II. Insect Parasitism and Host Viability

P r o t e l e a n insec t p a r a s i t e s c o m p r i s e t h e la rges t g r o u p of p a r a s i t i c a n i m a l s . However , b e c a u s e t he fate of t he insect hos t is, in m o s t cases , d e a t h , insec t p a r a s i t e s were h is tor ica l ly cons ide red to b e p r i m i t i v e — " the i r feeding b e h a v ior i n t e r m e d i a t e b e t w e e n the p a r a s i t i c a n d p r e d a c e o u s e n d s of t h e b e h a v i o r a l c o n t i n u u m " ( K n u t s o n a n d Berg , 1966). I t is now a p p r e c i a t e d t h a t t h e p a r a sit ic s tages of insect p a r a s i t e s exh ib i t a g r e a t d ivers i ty of " feeding b e h a v i o r . " A s k e w a n d S h a w (1986) d i s t i ngu i shed id iob ion t i c p a r a s i t o i d s , w h i c h p a r a lyze a n d kill t he i r hos t quickly, a n d ko inob ion t i c species , w h i c h deve lop in a h o s t t h a t r e m a i n s al ive a n d c o n t i n u e s to feed. I n t h e l a t t e r case , t h e h o s t a n d p a r a s i t e often exh ib i t a n a r r a y of phys io log ica l , b e h a v i o r a l , a n d d e v e l o p m e n ta l i n t e r ac t i ons .

E s t a b l i s h i n g t h e v iabi l i ty of t he p a r a s i t e - h o s t c o m p l e x is i m p o r t a n t for assess ing the p a t h o l o g i c a l c o n s e q u e n c e s of p a r a s i t i s m to t h e hos t . V iab i l i t y is i nex t r i cab ly l inked w i t h energy. T h e o rde r ly p roces s ing of ene rgy is t h e funct ion of m e t a b o l i s m a n d is ach ieved t h r o u g h the r e g u l a t i o n of b i o c h e m i c a l p a t h w a y s . I n this r e g a r d , t he a d e n y l a t e nuc l eo t i de pool p l ays a u b i q u i t o u s a n d p ivo ta l role . A d e n y l a t e s inf luence t he act ivi ty of m a n y r e g u l a t o r y en z y m e s a n d the i r ra t ios a r e m e a s u r e s of t h e p h o s p h o r y l a t i o n p o t e n t i a l as p r e d i c t e d by the t heo ry of m e t a b o l i c r egu l a t i on t h r o u g h f rac t ional ampl i f ica -

6. Host Metabolism and Parasite Nutrition 127

t ion (see N e w s h o l m e a n d S t a r t , 1973). B e c a u s e m a n y r e g u l a t o r y e n z y m e s a r e affected b y a d e n y l a t e ra t ios r a t h e r t h a n by t h e a b s o l u t e c o n c e n t r a t i o n s of i n d i v i d u a l nuc l eo t ide s , t h e ene rgy c h a r g e r a t i o [ E C = A T P + J A D P ) / A T P + A D P + A M P ) ] h a s p r o v e n to b e of c o n s i d e r a b l e i m p o r t a n c e in m e t a b o l i c r e g u l a t i o n (A tk inson , 1968). C h a p m a n et al. (1971) d e m o n s t r a t e d E C to b e a n overa l l i n d i c a t o r of v iabi l i ty in vivo. E C is a q u a l i t a t i v e m e a s u r e of t h e p o t e n t i a l ene rgy of t h e o r g a n i s m a n d therefore p rov ides a p a r a m e t e r for e v a l u a t i o n t h e overal l m e t a b o l i c s t ress i n d u c e d by p a r a s i t i s m .

S tud i e s b y T h o m p s o n a n d Y a m a d a (1984) o n the a d e n y l a t e n u c l e o t i d e poo l of t h e insec t hos t Trichoplusia ni ( H u b n e r ) p a r a s i t i z e d by t h e so l i t a ry i c h n e u m o n i d Hyposter exiguae (Viereck) c lear ly d e m o n s t r a t e d t h a t t h e e n e r g y s t a t u s of t h e insec t hos t is m a i n t a i n e d following p a r a s i t i z a t i o n d e s p i t e ex t en sive a l t e r a t i o n s in phys io logy a n d behav io r . T h e c o n c e n t r a t i o n s a n d r a t io s of a d e n y l a t e nuc l eo t ides in t h e fat b o d y of four th - ins t a r T. ni w e r e d e t e r m i n e d 3 d a y s pos tov ipos i t ion . T h e level of A M P t e n d e d to i nc rea se a n d A T P d e c reased , b u t t h e E C was on ly s l ight ly a l t e r ed . S u b s e q u e n t in vivo 1 3 P - N M R s tud ie s w i t h Manduca sexta ( L i n n a e u s ) p a r a s i t i z e d by t h e g r e g a r i o u s b r a c o n i d Cotesia congregata ( M a s s o n ) [=Apanteles congregatus (Say) ] d e m o n s t r a t e d t h a t t he overal l e n e r g y s t a t u s of th is insec t hos t a lso w a s m i n i m a l l y affected b y p a r a s i t i s m ( T h o m p s o n et al., 1988).

III. Altered Metabolite Levels in Host Tissues

A. Carbohydrates

D a h l m a n a n d V i n s o n (1975) w e r e t he first to obse rve e l eva ted h e m o l y m p h t r e h a l o s e levels in p a r a s i t i z e d insec ts . T h e m e a n t r eha lo se c o n c e n t r a t i o n in Heliothis virescens (Fabr i c ius ) p a r a s i t i z e d l a te in t h e s econd i n s t a r b y t h e b r a con id Microplitis croceipes (Cres son) i n c r e a s e d from a p p r o x i m a t e l y 5 to 40 mM b y 6 d a y s pos tov ipos i t ion , a n d the rea f te r dec l ined . I n c o n t r a s t , t h e t r e h a l o s e level in h e m o l y m p h of con t ro l insec ts i n c r e a s e d from 4 to 16 mM d u r i n g t h e s a m e p e r i o d . T h e fat b o d y g lycogen level of p a r a s i t i z e d H. virescens a l so i n c r e a s e d fol lowing p a r a s i t i z a t i o n , f rom a m e a n of 81 μ g / m g fresh w e i g h t to a p p r o x i m a t e l y 100 μ g / m g 5 d a y s pos tov ipos i t ion . The rea f t e r , g lycogen level d e c r e a s e d a n d , a t t h e t i m e of p a r a s i t e e m e r g e n c e , t h e fat b o d y of p a r a s i t i z e d l a rvae w a s a l m o s t g lycogen d e p l e t e d ( D a h l m a n a n d V i n s o n , 1980). G l y c o g e n a lso i n c r e a s e d in fat b o d y of con t ro l l a rvae , b u t to a lesser ex t en t . T h e a u t h o r s sugges t ed t h a t g lycogen m o b i l i z a t i o n m a y h a v e c o n t r i b u t e d to t h e e leva t ion in h e m o l y m p h t r eha lo se o b s e r v e d in p a r a s i t i z e d insec t s . E l e v a t e d h e m o l y m p h t r e h a l o s e in H. virescens w a s l a t e r r e p o r t e d by D a h l m a n a n d V i n s o n

128 S. Ν. Thompson

(1976) d u r i n g p a r a s i t i z a t i o n by the i c h n e u m o n i d p a r a s i t o i d Campoletis sonorensis ( C a m e r o n ) .

Trichoplusia ni l a rvae pa r a s i t i z ed la te in t h e th i rd s t a d i u m by H. exiguae d i sp l ayed s h a r p l y e leva ted t i ssue c a r b o h y d r a t e levels d u r i n g the i r deve lop m e n t ( T h o m p s o n , 1982a) . H e m o l y m p h t r eha lose c o n c e n t r a t i o n i n c r e a s e d from 12 to 15 mM a t t he t i m e of ovipos i t ion to a m e a n of a p p r o x i m a t e l y 35 mM d u r i n g the fifth s t a d i u m , 2 to 4 d a y s after p a r a s i t i z a t i o n . T h e t r eha lo se level in con t ro l l a rvae r e m a i n e d a l m o s t c o n s t a n t . T h e g lycogen level in t h e fat b o d y of p a r a s i t i z e d la rvae a lso i nc reased d u r i n g the four th s t a d i u m from a m e a n of a p p r o x i m a t e l y 50 μ g / m g fresh we igh t to 140 μ g / m g b y 2 d a y s pos tov ipos i t ion . The rea f t e r , g lycogen dec l ined . I n c o n t r a s t , t h e g lycogen level in fat b o d y of con t ro l l a rvae dec l ined t h r o u g h o u t t h e four th s t a d i u m from 50 μ g / m g to a p p r o x i m a t e l y 10 μ g / m g , a n d i nc rea sed d u r i n g the fifth s t ad i u m to a p p r o x i m a t e l y 120 μ g / m g .

A l t e r ed hos t t i ssue c a r b o h y d r a t e levels have a lso b e e n r e p o r t e d d u r i n g o t h e r insec t p a r a s i t e - h o s t r e l a t i onsh ips . F u h r e r (1972) r e p o r t e d a n i n c r e a s e d level of g lycogen in fat body, m i d g u t , a n d h y p o d e r m i s of Pieris brassicae L i n -n e a u s p a r a s i t i z e d by Cotesia (=Apanteles) glomeratus ( L i n n e a u s ) . O n the bas i s of h is to logica l s tud ie s , H a w l i t z k y a n d B o u l a y (1986) sugges t ed t h a t t i ssues of Anagasta kuehniella Ze l le r pa r a s i t i z ed by Phanerotoma flavitestacea F i sch . h a d h i g h e r levels of g lycogen t h a n were obse rved in con t ro l l a rvae . T h o m p s o n et al. (1990) obse rved e leva ted g lycogen in h is to logica l sec t ions of Μ. sexta fat b o d y following p a r a s i t i z a t i o n by C. congregata. T h e h e m o l y m p h t r eha lo se level of M. sexta, however , was r e d u c e d by p a r a s i t i s m , verifying ear l i e r r e p o r t s by D a h l m a n (1969, 1975) on this p a r a s i t e - h o s t r e l a t i onsh ip .

B. Proteins and Amino Acids

J u n n i k k a l a (1966) w a s a m o n g the first to r e p o r t a l t e r a t i o n s in n i t r o g e n m e tabo l i t es following p a r a s i t i z a t i o n by a n insec t p a r a s i t o i d . T i s s u e a m i n o ac ids a n d p r o t e i n s were e x a m i n e d in P. brassicae l a rvae p a r a s i t i z e d w h e n o n e d a y old by the g r e g a r i o u s p a r a s i t o i d C. glomerata. Fol lowing p a r a s i t i z a t i o n , t h e to ta l c o n c e n t r a t i o n of n inhyd r in -pos i t i ve c o m p o u n d s in t h e hos t ' s h e m o l y m p h fluctuated a n d was on ly occas iona l ly h i g h e r t h a n t h e levels in h e m o l y m p h of u n p a r a s i t i z e d con t ro l s . D u r i n g the fifth s t a d i u m , however , t h e level of h e m o l y m p h p r o t e i n w a s s ignif icant ly lower in p a r a s i t i z e d l a rvae . T h e p r e d o m i n a n t p ro t e in , d e s i g n a t e d " p e p t i d e 1" a n d p r o b a b l y a s t o r age p r o t e i n , in h e m o l y m p h of u n p a r a s i t i z e d last i n s t a r l a rvae was a b s e n t f rom h e m o l y m p h of p a r a s i t i z e d insec ts .

T h e h e m o l y m p h a m i n o acid compos i t i ons of p a r a s i t i z e d a n d u n p a r as i t ized P. brassicae were qua l i t a t ive ly a n d q u a n t i t a t i v e l y s imi l a r un t i l t h e e n d of t he fifth s t a d i u m . A t t h a t t ime t h e levels of several a m i n o ac ids , i n c l u d i n g


pro l i ne , se r ine , g l u t a m a t e / g l u t a m i n e , a n d g lyc ine , w e r e s ignif icant ly h i g h e r in h e m o l y m p h of p a r a s i t i z e d l a rvae . I n c o n t r a s t , t y ros ine w a s p r e s e n t in h e m o l y m p h from p a r a s i t i z e d l a rvae a t m u c h lower levels t h a n in con t ro l s . T h e c h a n g e s in n i t r o g e n m e t a b o l i t e s a p p e a r e d to reflect t h e a l t e r ed d e v e l o p m e n t a l p a t t e r n of t he p a r a s i t i z e d l a rvae . T h e l a t t e r failed to m e t a m o r p h o s e to t h e p u p a l s t age . T h e a u t h o r p o i n t e d ou t , however , t h a t , d u r i n g its s econd s t a d i u m , t h e p a r a s i t e feeds o n t h e hos t ' s fat body, w h i c h is a l m o s t c o m p l e t e l y a b s e n t b y t he t i m e the p a r a s i t e mo l t s to t he t h i r d i n s t a r a n d e m e r g e s f rom t h e hos t to p u p a t e . S o m e of t he c h a n g e s in n i t r o g e n m e t a b o l i t e s may , therefore , h a v e b e e n d u e to t i ssue d a m a g e .

Severa l o t h e r s tud ies have d e m o n s t r a t e d q u a l i t a t i v e a n d q u a n t i t a t i v e c h a n g e s in h e m o l y m p h p r o t e i n c o m p o s i t i o n in p a r a s i t i z e d insec t hos t s as la rge ly e v i d e n c e d by e l ec t ropho re t i c p a t t e r n s (see V i n s o n a n d I w a n t s c h , 1980; B e c k a g e et al., 1987, 1989; J o n e s , 1989). B e c a u s e t h e n a t u r e a n d funct ions of these p r o t e i n s a r e la rge ly u n k n o w n , a n d the i r p o t e n t i a l roles in i n t e r m e d i a r y m e t a b o l i s m unc lea r , p r o t e i n s will no t b e d i scussed fu r the r h e r e .

B a r r a s et al. (1969) e x a m i n e d h e m o l y m p h a m i n o acid levels in t h e hos t H. virescens d u r i n g p a r a s i t i s m b y t h e so l i t a ry p a r a s i t e Cardiochiles nigriceps V ie r -eck. A l t h o u g h n o q u a l i t a t i v e differences were r e p o r t e d , t h e h e m o l y m p h a m i n o acid c o n c e n t r a t i o n of f ou r th - i n s t a r hos t l a rvae p a r a s i t i z e d in t h e s econd s t a d i u m w a s a p p r o x i m a t e l y ha l f t h a t of h e m o l y m p h f rom u n p a r a s i t i z e d cont ro ls . P a r a s i t e l a rvae were la te first o r ear ly second i n s t a r s a t t h a t s t age of hos t d e v e l o p m e n t . B e c a u s e these p a r a s i t e s tages d e v e l o p w i t h i n t h e hos t h e m o c o e l a n d feed o n h e m o l y m p h , t h e a u t h o r s c o n c l u d e d t h a t t h e d e c r e a s e d a m i n o ac ids o b s e r v e d in t he hos t were t h e resu l t of p a r a s i t e u t i l i za t ion . T h o m p s o n (1986a) a lso obse rved d e c r e a s e d h e m o l y m p h a m i n o acid levels in t h e hos t T. ni p a r a s i t i z e d by H. exiguae. I n c o n t r a s t to these r e su l t s , V i n s o n a n d I w a n t s c h (cf. V i n s o n , 1990b) obse rved a n inc rease in a m i n o acid levels in t he h e m o l y m p h of H. virescens p a r a s i t i z e d b y M. croceipes o r C. sonorensis.

C. Lipids

Severa l s tud ie s sugges t ed t h a t q u a l i t a t i v e a n d q u a n t i t a t i v e c h a n g e s in l ipid m e t a b o l i t e s m a y a c c o m p a n y p a r a s i t i s m . B a r r a s et al. (1970) r e p o r t e d t h a t t h e re la t ive levels of v a r i o u s l ipid classes in t h e h e m o l y m p h a n d solid t i ssues of H. virescens w e r e s l ight ly a l t e r ed following p a r a s i t i z a t i o n b y Cardiochiles nigriceps V ie reck , a l t h o u g h t r ig lycer ide w a s t he m a j o r l ipid of t i ssues of b o t h p a r a s i t i z e d a n d u n p a r a s i t i z e d i n d i v i d u a l s . Differences in t h e re la t ive levels of fat ty ac ids in v a r i o u s l ipid fract ions were a lso ev iden t b e t w e e n con t ro l a n d p a r a s i t i z e d insec t s . T h e t r ig lycer ides of p a r a s i t i z e d H. virescens, for e x a m p l e , c o n t a i n e d s ignif icant ly lower re la t ive a m o u n t s of u n s a t u r a t e d fat ty ac ids .

T h o m p s o n (1982b) r e p o r t e d t h a t t h e c o n c e n t r a t i o n of to ta l l ip id in w h o l e

130 S. Ν. Thompson

fifth-instar l a rvae of T. ni w a s n e a r c o n s t a n t following p a r a s i t i z a t i o n b y H. exiguae, w h e r e a s t he c o n c e n t r a t i o n in con t ro l s i nc r ea sed t h r o u g h o u t t h e fifth s t a d i u m . Moreove r , t he re la t ive level of t r ig lycer ide w a s c o n s t a n t in p a r a si t ized l a rvae a n d the i nc reased l ipid in con t ro l s w a s la rge ly d u e to t r i g lycer ide depos i t i on .

I n h i b i t i o n of l i p o p h o r i n - m e d i a t e d l ipid t r a n s p o r t in h e m o l y m p h of Phi-losamia cynthia ( D r u r y ) d u r i n g p a r a s i t i s m by the d i p t e r o u s p a r a s i t e Blepharipa sericariae C o r n a l i a [=B. zebina (Walke r ) ] w a s d e m o n s t r a t e d b y H a y a k a w a (1986) . A sma l l p e p t i d e secre ted b y the p a r a s i t e l a rva w a s d e m o n s t r a t e d to b e r e spons ib l e . L a t e r , H a y a k a w a (1987) r e p o r t e d t h a t t h e factor i n h i b i t e d l i p o p h o r i n u p t a k e of d iacy lg lycer ide f rom locus t fat body. Recent ly , H o r w o o d a n d H a l e s (1991) r e p o r t e d t h a t t h e l ipid c o n t e n t of t h e o r t h o p t e r a n hos t Chortoicetes terminifera (Walker ) w a s s ignif icant ly e leva ted d u r i n g p a r a s i t i s m b y t h e d i p t e r o u s p a r a s i t e Tnchopsidea oestracea (Wes twood) a n d sugges t ed t h a t th is effect m a y h a v e re su l t ed from a n inh ib i t i on of l ipid t r a n s p o r t .

IV. Metabolic Alteration in Insect Hosts

T h e foregoing s tud ies d e m o n s t r a t i n g t h a t hos t t i ssue m e t a b o l i t e levels a r e a l t e r ed d u r i n g p a r a s i t i s m s t rong ly sugges t t h a t hos t m e t a b o l i s m is d i rec t ly affected b y p a r a s i t i s m . Very few s tud ie s , however , have b e e n c o n d u c t e d to d e m o n s t r a t e t he n a t u r e of those b i o c h e m i c a l les ions . A t t h e p r e s e n t t i m e o u r k n o w l e d g e is l imi ted to a few s tud ies of t he i n t e r m e d i a r y m e t a b o l i s m of c a r b o h y d r a t e s .

A. Trichoplusia ni Parasitized by Hyposoter exiguae

Fol lowing the d e m o n s t r a t i o n by T h o m p s o n (1982a) t h a t e l eva ted h e m o l y m p h t r eha lose c o n c e n t r a t i o n a n d i nc rea sed fat b o d y g lycogen d e p o s i t i o n o c c u r r e d in T. ni following p a r a s i t i z a t i o n b y H. exiguae, T h o m p s o n a n d C o h e n (1984) r e p o r t e d t h a t excre t ion of u r i c ac id by p a r a s i t i z e d l a rvae w a s significan t l y g r e a t e r t h a n t h a t of con t ro l s . T h e a u t h o r s sugges t ed t h a t t h e u r i c ac id p r o d u c t i o n r e su l t ed from a m i n o acid d e a m i n a t i o n a n d t h a t u t i l i za t ion of g lucogen ic a m i n o ac ids for de novo c a r b o h y d r a t e syn thes i s m a y h a v e b e e n r e spons ib l e for t he e leva ted c a r b o h y d r a t e reserves in p a r a s i t i z e d hos t s .

T h o m p s o n a n d B i n d e r (1984) d e m o n s t r a t e d e leva ted g l u c o n e o g e n i c en z y m e act iv i ty in T. ni l a rvae 3 d a y s pos tov ipos i t ion . T h e m a x i m a l e n z y m e veloci ty of b o t h p h o s p h o f r u c t o k i n a s e ( P F K a s e - c a t a l y z i n g glycolyt ic conver s ion of g lucose to p y r u v a t e ) a n d fructose b i s p h o s p h a t a s e ( F B P a s e - c a t a l y z i n g g luconeogenes i s ) w a s s ignif icant ly h i g h e r in fat b o d y i so la ted f rom p a r a si t ized l a rvae . T h e act ivi ty of F B P a s e , however , w a s e leva ted to a g r e a t e r ex ten t , r e su l t i ng in a p o t e n t i a l severalfold inc rease in g l u c o n e o g e n i c flux.


S u b s e q u e n t l y , T h o m p s o n (1986a) r e p o r t e d t h a t t h e to ta l h e m o l y m p h g lucogen ic a m i n o ac id c o n c e n t r a t i o n in p a r a s i t i z e d l a rvae w a s d e c r e a s e d a p p r o x i m a t e l y 3 5 % t h r e e d a y s after p a r a s i t i z a t i o n .

T h e r e g u l a t i o n of F B P a s e in fat b o d y of f ou r th - i n s t a r T. ni w a s e x a m i n e d b y T h o m p s o n (1985) . A d e n o s i n e m o n o p h o s p h a t e ( A M P ) a n d f ruc tose-2 ,6-b i s p h o s p h a t e ( F 2 , 6 B P a t e ) , m a j o r effectors of F B P a s e in l iver (Pilkis et al., 1981), w e r e a lso m o d u l a t o r s of th is e n z y m e in T. ni. T h e i r effects in T. ni w e r e s imi l a r to t hose r e p o r t e d in r a t l iver (Van Schaf t ingen a n d H e r s , 1981). B o t h effectors w e r e i n h i b i t o r y a n d t h e i nh ib i t i on c a u s e d b y each w a s e n h a n c e d in t h e p r e s e n c e of t he o the r . B e c a u s e p r e v i o u s resu l t s d e m o n s t r a t e d t h a t fat b o d y A M P level w a s e leva ted following p a r a s i t i z a t i o n ( T h o m p s o n a n d Y a m a d a , 1984), these f indings r e g a r d i n g A M P m o d u l a t i o n in i t ia l ly a p p e a r e d i ncons i s t en t w i t h t h e conc lus ion t h a t i n c r e a s e d c a r b o h y d r a t e rese rves in p a r a s i t i z e d T. ni r e su l t ed f rom e n h a n c e d g luconeogenes i s . I n fat b o d y p r e p a r a t i o n s f rom p a r a s i t i z e d l a rvae , however , b o t h A M P a n d F 2 , 6 B P a t e w e r e less inh ib i to ry . T h e findings d e m o n s t r a t e d t h a t g luconeogenes i s m a y i nc r ea se d e s p i t e m i n o r inc reases in A M P level, a l t h o u g h t h e bas i s for t h e d e c r e a s e d sens i t iv i ty of F B P a s e to A M P w a s u n e x p l a i n e d .

N u t r i t i o n a l s tud ie s fu r the r i n d i c a t e d t h a t t h e r e g u l a t i o n of de novo c a r b o h y d r a t e syn thes i s in T. ni m a y b e a l t e r ed b y p a r a s i t i z a t i o n ( T h o m p s o n , 1986b) . H e m o l y m p h t r eha lo se level of insec ts r e a r e d o n art if icial m e d i a w i t h inc r ea sed levels of suc rose w a s c o n s t a n t . I n p a r a s i t i z e d l a rvae , however , t h e h e m o l y m p h t r eha lo se level w a s m a r k e d l y e leva ted b y i n c r e a s i n g t h e d i e t a r y c a r b o h y d r a t e level. T h u s , i t w a s c o n c l u d e d t h a t t h e r e g u l a t i o n of c a r b o h y d r a t e b iosyn thes i s in r e s p o n s e to d i e t a r y s u g a r m a y b e a b n o r m a l l y r e g u l a t e d in p a r a s i t i z e d T. ni.

B. Manduca sexta Parasitized by Cotesia congregata

Severa l in vivo Ν M R s tud ie s have b e e n c o n d u c t e d to d i s c e r n t h e m e t a b o l i c bas i s for e l eva ted g lycogen d e p o s i t i o n in M. sexta p a r a s i t i z e d b y C. congregata. M e t a b o l i s m of [ 1 3 C ] 2 - p y r u v a t e b y fifth-ins t a r l a rvae w a s e x a m i n e d b y T h o m p s o n et al. (1988) . T h e l abe led s u b s t r a t e w a s m e t a b o l i z e d m u c h s lower b y p a r a s i t i z e d l a rvae a n d t h e p r o d u c t s i n to w h i c h t h e l abe l w a s i n c o r p o r a t e d differed m a r k e d l y b e t w e e n p a r a s i t i z e d a n d u n p a r a s i t i z e d insec t s . I n con t ro l l a rvae , p y r u v a t e w a s i n c o r p o r a t e d p r i m a r i l y i n t o fat fol lowing o x i d a t i o n to ace ty l -CoA. T h e difference in fat c o n t e n t b e t w e e n con t ro l a n d p a r a s i t i z e d l a rvae w a s s t r ik ing a n d e v i d e n t f rom the i r respec t ive n a t u r a l a b u n d a n c e 1 3 C -N M R s p e c t r a . I n p a r a s i t i z e d l a rvae 1 3 C from p y r u v a t e w a s i n c o r p o r a t e d i n t o a m i n o ac ids after d e c a r b o x y l a t i o n to o x a l o a c e t a t e a n d m e t a b o l i s m t h r o u g h t h e K r e b s cycle . Signif icant g luconeogenes i s f rom p y r u v a t e , however , w a s n o t o b s e r v e d in e i the r con t ro l o r p a r a s i t i z e d l a rvae .

S u b s e q u e n t N M R s tud ies r e p o r t e d b y T h o m p s o n et al. (1990) d e m o n -

132 S. Ν. Thompson

s t r a t e d t h a t i n c o r p o r a t i o n of [ 1 3 C ] 1-glucose in to s t o r a g e m e t a b o l i t e s differed b e t w e e n p a r a s i t i z e d a n d u n p a r a s i t i z e d M. sexta. C o n t r o l insec ts i n c o r p o r a t e d labe l f rom glucose in to fat a n d [ 1 3 C ] 1-trehalose. [ I 3 C ] 1-Trehalose a n d [ 1 3 C ] 1-glycogen were t he m a j o r p r o d u c t s of l abe led g lucose in p a r a s i t i z e d l a rvae . I n c o n t r a s t to con t ro l l a rvae , pa r a s i t i z ed insec ts d id n o t i n c o r p o r a t e l abe l i n to fat. Differences in t he m e t a b o l i s m of p r e f o r m e d (d ie ta ry ) g lucose , therefore , pa r t i a l l y e x p l a i n e d the differences in s t o r a g e m e t a b o l i t e levels o b served b e t w e e n p a r a s i t i z e d a n d u n p a r a s i t i z e d M. sexta.

V. Basis for Metabolic Alterations: Hormones, Viruslike Particles, and Parasite Mediation

Red i rec t ion of hos t phys io logy is often in i t i a t ed in r e s p o n s e to p a r a s i t o i d -de r ived factors injected in to t he hos t w i th t he p a r a s i t e egg d u r i n g ov ipos i t ion . T h u s , effects of p a r a s i t i s m a r e often n o t e d even before t h e p a r a s i t e egg h a t c h e s . Severa l ent i t ies a r e r e spons ib l e (Stol tz , 1986). A l t h o u g h these va r i ous factors m a y in te rac t , often p r o d u c i n g synerg i s t i c effects (see, e.g., T a n a k a a n d V i n s o n , 1991), p e r h a p s t he m o s t n o t a b l e a r e t he p o l y d n a v i r u s e s ( P o l y d n a v i r i d a e — S t o l t z et al., 1984; Whi t f ie ld , 1990), v i rus l ike pa r t i c l e s (see Feder ic i , 1991) o r i g i n a t i n g from t h e ca lyx reg ion of t h e r e p r o d u c t i v e t r ac t of m o s t i c h n e u m o n i d a n d b r a c o n i d p a r a s i t o i d s (Stol tz et al., 1981). N u m e r ous s tud ies have d e m o n s t r a t e d t h a t p o l y d n a v i r u s e s p l a y a cr i t ica l ro le in s u p p r e s s i o n of t he hos t ' s defense r e sponse ( S c h m i d t a n d S c h u c h m a n n -F e d d e r s e n , 1989; V i n s o n , 1990a), as well as m a n y o t h e r phys io log ica l effects.

S o m e effects of p a r a s i t i s m on hos t m e t a b o l i t e levels a l so a p p e a r to be in i t i a t ed by p o l y d n a v i r u s a n d / o r act ivi ty of o t h e r p a r a s i t o i d - d e r i v e d factors ini t ia l ly o c c u r r i n g i n d e p e n d e n t of t he p a r a s i t e itself. C h a n g e s in c a r b o h y d r a t e reserves in H. virescens a n d T. ni in r e sponse to p a r a s i t i s m b y M. croceipes ( D a h l m a n a n d V i n s o n , 1975) a n d H. exiguae ( T h o m p s o n , 1982a) , r e spec tively, we re obse rved i m m e d i a t e l y following p a r a s i t i z a t i o n before p a r a s i t e h a t c h i n g a n d d e v e l o p m e n t . D a h l m a n a n d V i n s o n (1977) d e m o n s t r a t e d t h a t e leva t ion of h e m o l y m p h t r eha lose in H. virescens cou ld b e i n d u c e d b y in jec t ion of ca lyx fluid of M. croceipes.

M a n y phys io log ica l effects of p a r a s i t i s m / p o l y d n a v i r u s ac t iv i ty a r e hor -m o n a l l y m e d i a t e d (Beckage , 1985; L a w r e n c e , 1986). For e x a m p l e , a l ter a t ions of hos t g r o w t h a n d d e v e l o p m e n t a l p a t t e r n s a r e closely r e l a t ed to t i te rs of e c d y s o n e a n d j u v e n i l e h o r m o n e (Dover et al., 1988a) , a n d p r e v i o u s inves t i g a t i o n sugges t ed t h a t t he p r o t h o r a c i c g l a n d s m a y be a m o n g the h o s t t i ssues t h a t p o l y d n a v i r u s i n v a d e s (Dover et al., 1988b) . L i t t l e is k n o w n of t h e effects


of d e v e l o p m e n t a l h o r m o n e s o n i n t e r m e d i a r y m e t a b o l i s m (see Keeley, 1978) a n d the se h o r m o n e s h a v e n o t b e e n d i rec t ly i m p l i c a t e d in t h e m e t a b o l i c c h a n g e s o b s e r v e d in p a r a s i t i z e d insec ts . I n sec t s , however , have a d d i t i o n a l h o r m o n e s t h a t a r e k n o w n to p l a y a f u n d a m e n t a l role in m e t a b o l i c r e g u l a t i o n .

H o r m o n e s w i t h insu l in - a n d g lucagon l ike act ivi t ies have b e e n i so la ted f rom t issues of n u m e r o u s insec ts (Steele , 1983). I n M. sexta l a rvae , for e x a m p le , a d i p o k i n e t i c h o r m o n e o r i g i n a t i n g from the c o r p o r a c a r d i a c a s t i m u l a t e s g lycogen p h o s p h o r y l a s e act iv i ty (Ziegler , 1990) in a m a n n e r s imi l a r to t h a t k n o w n for t h e v e r t e b r a t e h o r m o n e g l u c a g o n . I n this r e g a r d , s t ud i e s b y T h o m p s o n (1985) o n g l u c o n e o g e n i c act iv i ty in T. ni a r e of i n t e r e s t b e c a u s e t h e d e c r e a s e d i n h i b i t o r y r e s p o n s e of f ructose b i s p h o s p h a t a s e to r e g u l a t i o n b y A M P a n d F 2 , 6 B P a t e in p a r a s i t i z e d l a rvae is cons i s t en t w i t h t h e effect of g l u c a g o n o n th is m e t a b o l i c p a t h w a y in l iver (Van Schaf t ingen et al., 1980). A l t h o u g h p r e v i o u s inves t iga t ions have failed to d e m o n s t r a t e a n y effect of factors de r ived from t h e c o r p o r a c a r d i a c a on g luconeogenes i s ( J . E . Steele , p e r s o n a l c o m m u n i c a t i o n ) , Steele et al. (1988) r e p o r t e d t h a t g lycogen p h o s p h o r y l a s e s t i m u l a t i o n a lone is n o t sufficient to exp la in t r e h a l o s e syn thes i s i n d u c e d by c o r p u s c a r d i a c u m h o r m o n e s .

T h e resu l t s of n u t r i t i o n a l inves t iga t ions i n d i c a t i n g t h a t d i e t a r y r e g u l a t i o n of g luconeogenes i s w a s a l t e r ed in p a r a s i t i z e d T. ni ( T h o m p s o n , 1986b; Sect ion IV .A) m a y fu r the r sugges t t h a t a g lucagon l ike r e g u l a t i o n of de novo c a r b o h y d r a t e syn thes i s is involved in e l eva t ing h e m o l y m p h t r e h a l o s e level in p a r a s i t i z e d l a rvae . R o c h a et al. (1973) obse rved m a r k e d l y e leva ted g l u c a g o n levels in sep t i c h u m a n sub jec t s . Subsequen t l y , L o n g et al. (1976) r e p o r t e d t h a t t he r a t e of g luconeogenes i s f rom a m i n o ac ids w a s e l eva ted d u r i n g seps is , a n d t h a t t h e a d m i n i s t r a t i o n of s u g a r failed to m e d i a t e th is r e s p o n s e . T h e a u t h o r s sugges t ed t h a t t h e resu l t s w e r e likely c a u s e d b y a h o r m o n a l i m b a l a n c e . A l t h o u g h inves t iga t ions w i t h v e r t e b r a t e a n i m a l s c a n n o t b e d i rec t ly c o m p a r e d to s tud i e s w i t h insec ts , t h e p o t e n t i a l role of h o r m o n e s in i n d u c i n g the m e t a b o l i c c h a n g e s o b s e r v e d in p a r a s i t i z e d insec ts n e e d s to b e inves t i g a t e d .

A l m o s t n o t h i n g is k n o w n c o n c e r n i n g t h e role of t h e d e v e l o p i n g p a r a s i t e l a rvae in m e d i a t i n g effects of p a r a s i t i s m on hos t physiology. S u c h effects m i g h t b e expec t ed as t he p a r a s i t e l a rvae m a t u r e a n d grow, a n d b e p a r t i c u la r ly e v i d e n t d u r i n g hos t a s soc ia t ions of g r e g a r i o u s species in w h i c h t h e b i o m a s s of p a r a s i t e s is c o n s i d e r a b l e re la t ive to t h a t of t h e hos t . I n d e e d , B e c k a g e a n d R idd i fo rd (1983) d e m o n s t r a t e d t h a t heavi ly p a r a s i t i z e d M. sexta have a l a r g e r hos t m a s s t h a n l ight ly p a r a s i t i z e d l a rvae , i n d e p e n d e n t of t h e c o n t r i b u t i o n b y t h e p a r a s i t e s . T h e resu l t s sugges t t h a t t h e n u t r i t i o n a l p h y s i ology of t h e hos t is carefully s y n c h r o n i z e d w i t h p a r a s i t e d e v e l o p m e n t a n d modif ied in r e l a t ion to p a r a s i t e b u r d e n to satisfy t he n u t r i t i o n a l r e q u i r e m e n t s of t h e d e v e l o p i n g p a r a s i t e s .

134 S. Ν. Thompson

Prev ious inves t iga t ions sugges t t h a t p a r a s i t e r e s p i r a t i o n m a y i n d u c e m e t abo l i c c h a n g e s in t he hos t . F i she r (1963) d e m o n s t r a t e d t h a t t h e 0 2 level in h e m o l y m p h of Ephestia kuhniella H i i b n e r G r a v e n h o r s t d e c r e a s e d d r a m a t i c a l l y d u r i n g p a r a s i t i s m by Venturia (=Nemeritis) canescens G r a v e n h o r s t . B a s e d o n t h e r a t e of oxygen c o n s u m p t i o n of t he deve lop ing p a r a s i t e , it w a s c o n c l u d e d t h a t t he d e c r e a s e d oxygen level in t he hos t ' s h e m o l y m p h w a s d u e to p a r a s i t e r e sp i r a t i on . T h e oxygen c o n s u m p t i o n of newly h a t c h e d p a r a s i t e l a rvae w a s a p p r o x i m a t e l y e q u a l to t he to ta l oxygen c o n t e n t of t h e hos t ' s h e m o l y m p h a t a n y given t ime . B e c a u s e t he t r a chea l sy s t em p rov ides oxygen d i rec t ly to insec t t i ssues , t h e p o t e n t i a l effects of h e m o l y m p h oxygen d e p l e t i o n o n hos t phys io logy a n d m e t a b o l i s m a r e difficult to e v a l u a t e . H o w e v e r , o x y g e n cons u m p t i o n by p a r a s i t i z e d E. kuhniella l a rvae was d e c r e a s e d d u r i n g p a r a s i t i s m . D a h l m a n a n d H e r a l d (1971) d e m o n s t r a t e d t h a t t he cyclic p a t t e r n of 0 2

c o n s u m p t i o n d u r i n g d e v e l o p m e n t of M. sexta w a s d a m p e n e d in p a r a s i t i z e d l a rvae . Moreove r , pa r a s i t i z ed l a rvae d i sp l ayed s ignif icant ly lower 0 2 cons u m p t i o n t h a n the i r u n p a r a s i t i z e d c o u n t e r p a r t s d u r i n g t h e l a t t e r p a r t of each s t a d i u m . D e c r e a s e d h e m o l y m p h 0 2 a n d r e sp i r a t i on of M. sexta p a r a s i t i z e d b y C. congregata w o u l d be cons i s t en t w i t h t he d e c r e a s e d r a t e of g lucose u t i l i za t ion in p a r a s i t i z e d hos t s de sc r ibed in t he foregoing (Sec t ion I V ) .

VI. Nutritional Consequences of Metabolic Redirection and Physiological Alteration

D a h l m a n a n d V i n s o n (1975) sugges ted t h a t a l t e r a t i ons in t h e c h e m i c a l c o m pos i t ion of hos t t issues m a y p l ay a n u t r i t i o n a l role a n d inf luence t h e insec t p a r a s i t e - h o s t r e l a t ionsh ip . T h e s e a u t h o r s p r o p o s e d t h a t t h e e leva ted t r e ha lose c o n c e n t r a t i o n in H. virescens i n d u c e d following p a r a s i t i z a t i o n b y M. croceipes p r o v i d e d n u t r i e n t d i rec t ly to t he deve lop ing p a r a s i t e . T h i s p a r a s i t e species c o m p l e t e s its en t i r e l a rva l d e v e l o p m e n t feeding o n hos t h e m o l y m p h . T h e hos t ' s fat b o d y a n d o t h e r t issues a r e no t c o n s u m e d . Resu l t s a n d conc lu s ions of these e x p e r i m e n t s w i t h M. croceipes w e r e c o n t r a s t e d w i t h t hose of s tud ies involv ing a n o t h e r b r a c o n i d , C. nigriceps. H e m o l y m p h t r eha lo se in H. virescens w a s n o t e leva ted after p a r a s i t i z a t i o n by this spec ies . Cardiochiles nigriceps, however , feeds act ively o n hos t t issues t h r o u g h o u t i ts d e v e l o p m e n t , a n d the a u t h o r s sugges t ed t h a t t he p a r a s i t e l a rvae o b t a i n sufficient c a r b o h y d r a t e as well as l ipid n u t r i e n t b y c o n s u m p t i o n of fat body.

T h e n a t u r e of t h e c h a n g e s in s t o r age c a r b o h y d r a t e levels in T. ni p a r a si t ized b y H. exiguae (Sect ion I I I ) a lso sugges t s t h e p o t e n t i a l for d i r ec t n u t r i t iona l benefi t . T h e p a r a s i t i c l a rvae of this species feed o n h e m o l y m p h d u r i n g the i r ea r ly s t a d i a a n d s u b s e q u e n t l y on fat b o d y a n d o t h e r solid hos t t i ssues .


T h u s , e l eva ted h e m o l y m p h t r eha lo se levels m a y p rov ide a n i m p o r t a n t sou rce of n u t r i e n t for t h e ea r ly i n s t a r s of t h e p a r a s i t e , w h e r e a s h i g h levels of glycogen s to red in t h e fat b o d y a r e c o n s u m e d in the t h i r d s t a d i u m .

N u t r i t i o n a l s tud ie s e m p l o y i n g in vitro c u l t u r e of insec t p a r a s i t e s o n def ined art if icial m e d i a w o u l d p r o v i d e cr i t ica l a n s w e r s r e g a r d i n g t h e p o t e n t i a l n u t r i t i ona l effects of a l t e r ed hos t t i ssue m e t a b o l i t e levels o n p a r a s i t e g r o w t h a n d d e v e l o p m e n t . Unfo r tuna t e ly , s u c h t e c h n i q u e s a r e c u r r e n t l y u n a v a i l a b l e for k i o n o b i o n t i c spec ies . In vitro c u l t u r e m e t h o d s a n d art if icial d ie t s a r e , however , ava i l ab le for r e a r i n g a few id iob ion t i c p a r a s i t e s , a n d the resu l t s of n u t r i t i o n a l i nves t iga t ions sugges t t h a t t h e i nc rea se in h e m o l y m p h c a r b o h y d r a t e t i te r o b s e r v e d in hos t s p a r a s i t i z e d by k o i n o b i o n t i c p a r a s i t e s m a y b e sufficient to p r o v i d e n u t r i t i o n a l benefi t to t he d e v e l o p i n g la rvae . T h o m p s o n (1983b) d e m o n s t r a t e d t h a t i n c r e a s i n g t h e level of d i e t a r y g lucose in a n art if icial m e d i u m from 0.5 to 1 .5%, a n inc rease a p p r o x i m a t e l y e q u a l to t h a t o b s e r v e d in b o t h T. ni fol lowing p a r a s i t i z a t i o n by H. exiguae ( T h o m p s o n , 1982a; Sec t ion I I I ) a n d H. virescens d u r i n g p a r a s i t i s m by M. croceipes ( D a h l m a n a n d V i n s o n , 1975; Sec t ion I I I ) , r e su l t ed in a n i nc rea se in m e a n re la t ive g r o w t h r a t e of l a rvae of t h e so l i t a ry cha lc id p a r a s i t e Brachymeria lasus (Walker ) f rom 200 to 250 m g / g / d a y . A s imi l a r i nc rease in d i e t a r y g lucose in a def ined art i f icial d i e t for t h e i c h n e u m o n i d p a r a s i t e Exeristes roborator (Fabr ic ius ) i n c r e a s e d t h e m e a n g r o w t h r a t e f rom a p p r o x i m a t e l y 5 to 12 m g / g / d a y ( T h o m p s o n , 1982c).

D e s p i t e ev idence sugges t ing n u t r i t i o n a l benefi t for t he a l t e r e d h o s t t i ssue c a r b o h y d r a t e levels obse rved in p a r a s i t i z e d hos t s , t h e conc lu s ion a p p e a r s i ncons i s t en t w i t h t he resu l t s of s tud ies o n s o m e insec t p a r a s i t e - h o s t r e l a t ion sh ips . T h e so l i ta ry p a r a s i t e C. sonorensis, l ike H. exiguae, feeds o n t h e hos t ' s h e m o l y m p h d u r i n g its ea r ly s t a d i a a n d d i rec t ly o n solid hos t t i ssues d u r i n g t h e las t t w o s t a d i a . I n th is case , h e m o l y m p h t r eha lose in t h e h o s t H. virescens w a s e leva ted on ly s l ight ly d u r i n g t h e ea r ly s tages of p a r a s i t e d e v e l o p m e n t , a n d m o r e d r a m a t i c a l l y l a t e r w h e n it w a s unl ike ly t h a t t h e h e m o l y m p h w o u l d s u p p l y s ignif icant n u t r i e n t over t h a t o b t a i n e d b y c o n s u m p t i o n of t h e o t h e r hos t t i ssue ( D a h l m a n a n d V i n s o n , 1976; Sec t ion I I I ) . C o n c l u s i o n s r e g a r d i n g t h e s ignif icance of g lycogen s tores d u r i n g s o m e p a r a s i t e - h o s t r e l a t i o n s h i p s m a y b e s imi la r ly cr i t ic ized. I n M. sexta p a r a s i t i z e d b y C. congregata, for e x a m ple , t h e fat b o d y g lycogen level w a s i n c r e a s e d b u t t h e h e m o l y m p h t r eha lo se level dec l i ned . Cotesia congregata, however , feeds on ly o n the h o s t h e m o l y m p h a n d therefore t h e g lycogen s tores a r e unl ike ly to be of d i r ec t n u t r i t i o n a l benefi t for th is species .

W h e n e v a l u a t i n g t h e p o t e n t i a l n u t r i t i o n a l s ignif icance of hos t m e t a b o l i t e levels for d e v e l o p i n g p a r a s i t e s , c a r e m u s t b e t a k e n to cons ide r t h e overa l l effects of p a r a s i t i s m o n to ta l n u t r i e n t avai labi l i ty . For e x a m p l e , in m a n y cases , i n c l u d i n g H. virescens a n d T. ni p a r a s i t i z e d by M. croceipes a n d H. exiguae, respect ively, hos t g r o w t h is severely r e t a r d e d following p a r a s i t i z a t i o n . T h e

136 S. Ν. Thompson

to ta l n u t r i e n t ava i l ab le for the deve lop ing pa ra s i t e ( s ) may , therefore , b e less, de sp i t e t he finding t h a t t he c o n c e n t r a t i o n of i n d i v i d u a l m e t a b o l i t e s m a y have i nc rea sed . D u r i n g p a r a s i t e - h o s t r e l a t ionsh ips in w h i c h t h e p a r a s i t e c o m ple tes its d e v e l o p m e n t on hos t h e m o l y m p h a lone , for e x a m p l e , M. croceipes, e leva ted levels of h e m o l y m p h n u t r i e n t s may, neve r the les s , b e i m p o r t a n t for o p t i m a l p a r a s i t e g r o w t h a n d d e v e l o p m e n t . However , in cases w h e r e t h e ent i re hos t c o n t e n t s a r e c o n s u m e d a t t he c o m p l e t i o n of p a r a s i t e d e v e l o p m e n t , as is t he case w i t h H. exiguae, e leva ted hos t h e m o l y m p h m e t a b o l i t e levels likely p rov ide n u t r i t i o n a l benefi t on ly for t he ear ly p a r a s i t i c s t ages .

I n a d d i t i o n to a l t e r a t i ons in t he c h e m i c a l c o m p o s i t i o n of hos t t i ssues b r o u g h t a b o u t by c h a n g e s in i n t e r m e d i a r y m e t a b o l i s m , o t h e r p a r a s i t o i d -i n d u c e d c h a n g e s in hos t b e h a v i o r a n d phys io logy m a y p l a y a n in t e rac t ive ro le in in f luenc ing p a r a s i t e n u t r i t i o n . Slow g r o w t h , de l ayed o r p r ecoc ious deve lop m e n t , a n d / o r i n t e r r u p t e d hos t m e t a m o r p h o s i s m a y act to e n s u r e a n a d e q u a t e t i m e pe r iod for p a r a s i t e n o u r i s h m e n t a n d d e v e l o p m e n t , as well as m a x i m a l exp lo i t a t ion of a n often l imi ted food supply . D e c r e a s e d food c o n s u m p t i o n , a c o m m o n b e h a v i o r d u r i n g p a r a s i t i s m , m a y p l a y a role b e c a u s e " n u t r i e n t -d e p r i v e d " insec ts deve lop slower ( T h o m p s o n , 1982a) . M o r e o v e r , p r e v i o u s s tud ies i n d i c a t e t h a t modif ied n u t r i t i o n a l phys io logy r e su l t i ng f rom d e c r e a s e d food c o n s u m p t i o n m a y a lso have s ignif icant c o n s e q u e n c e s . T h o m p s o n (1983c) d e m o n s t r a t e d t h a t d e c r e a s e d food c o n s u m p t i o n d i s p l a y e d by T. ni p a r a s i t i z e d by H. exiguae was a c c o m p a n i e d by a ne t i nc rease in a s s imi l a t ion , g r e a t e r t h a n t h a t obse rved in u n p a r a s i t i z e d pair-fed la rvae . T h i s m a y b e i m p o r t a n t for p r o v i d i n g a d e q u a t e n u t r i e n t to t he hos t a n d t h u s for m a i n t a i n i n g h e m o l y m p h n u t r i e n t levels for deve lop ing p a r a s i t e s . S u b s e q u e n t inves t iga t ions d e m o n s t r a t e d t h a t d i e t a r y c a r b o h y d r a t e level h a d li t t le effect o n t h e h e m o l y m p h t r eha lose level in u n p a r a s i t i z e d T. ni, b u t m a r k e d l y i n c r e a s e d the c o n c e n t r a t ion in p a r a s i t i z e d l a rvae ( T h o m p s o n , 1986b; Sec t ion IV .A) .

VII. "Host Regulation" and Its Role in Redirecting Host Metabolism and Physiology

V i n s o n a n d I w a n t s c h (1980) de sc r ibed the overal l effects of p a r a s i t i s m o n the insec t hos t as reflect ing p a r a s i t o i d r egu l a t i on of hos t physiology. " H o s t r egu l a t i o n " is r e spons ib l e for modi fy ing the hos t ' s phys io logy to e n s u r e p a r a s i t e surv iva l ( V i n s o n , 1984). J o n e s (1989) s u b s e q u e n t l y u sed the t e r m " r e d i r e c t i o n . " Recent ly , in te res t in hos t r egu l a t i on h a s focused o n i nh ib i t i on of t h e hos t ' s defense following p a r a s i t i z a t i o n . Clear ly , " i m m u n o s u p p r e s s i o n " is cr i t ical for successful p a r a s i t i s m a n d p a r a s i t o i d s have evolved a va r i e ty of m e a n s


b y w h i c h th is is a c c o m p l i s h e d ( V i n s o n , 1990a) , in m a n y cases b e i n g in i t i a t ed b y p o l y d n a v i r u s a n d / o r o t h e r p a r a s i t o i d - d e r i v e d factors (Stol tz , 1986). T h u s , v i ewing t h e insec t p a r a s i t o i d - h o s t r e l a t i o n s h i p as o n e in w h i c h t h e p a r a s i t o i d is " in c o n t r o l " ( J o n e s , 1985a ,b) a s a r e su l t of its ab i l i ty to r e g u l a t e t h e phys io logy of t h e hos t is a p p r o p r i a t e . I n d e e d , this p a r a d i g m fo rmed t h e bas i s of D a w k i n s ' (1982) def ini t ion of a p a r a s i t i z e d hos t as a n " e x t e n d e d p h e -n o t y p e " of t h e p a r a s i t e .

I n c o n t r a s t to t h e i nh ib i t i on of hos t defense , t h e r e is a p a u c i t y of exper i m e n t a l ev idence d e m o n s t r a t i n g a n y benefi t to m o s t o t h e r phys io log ica l a n d m e t a b o l i c a l t e r a t i o n s obse rved in p a r a s i t i z e d insec ts . A l t h o u g h it h a s b e e n d e m o n s t r a t e d t h a t m a n y a l t e r a t i o n s a r e b r o u g h t a b o u t b y p a r a s i t o i d - d e r i v e d factors , in fer r ing p a r a s i t o i d r e g u l a t i o n of hos t biology, th is by i tself does n o t d e m o n s t r a t e a n y a d a p t i v e s ignif icance of t h e effects obse rved . I n m o s t cases t h e " h o s t r e g u l a t i o n " p a r a d i g m necess i t a tes a subjec t ive a s s e s s m e n t of p h y s i o logica l a l t e r a t i o n in r e l a t ion to p a r a s i t e surv iva l . T o u n d e r s t a n d phys io log i cal a l t e r a t i o n w i th in t he overal l con t ex t of " h o s t r e g u l a t i o n , " s o m e re s e a r c h e r s have e m p h a s i z e d the necess i ty to exp l a in t he bas i s of a l t e r a t i o n s t h a t fail to m e e t a d e m o n s t r a t e d o r h y p o t h e s i z e d c r i te r ia of benefi t . T h u s , J o n e s et al. (1986) d i s t i n g u i s h e d beneficial effects f rom " i n d i r e c t s t ress effects" ; t h e l a t t e r b e i n g " u n i n t e n t i o n a l " ( J o n e s et al., 1981). A r a t i o n a l ex ten sion w a s t h e s u b s e q u e n t c h a r a c t e r i z a t i o n of p a r a s i t e s as " h o s t r e g u l a t o r s " o r " h o s t c o n f o r m e r s " ( L a w r e n c e , 1986). I n t he l a t t e r case , p a r a s i t i s m fails to resu l t in o b s e r v a b l e d e v e l o p m e n t a l c h a n g e s .

D e s p i t e l imi ted e x p e r i m e n t a l ev idence , it is r e a s o n a b l e to a s s u m e t h a t m a n y a l t e r a t i o n s in hos t phys io logy a r e of s ignif icance for successful p a r a s i t i s m — f o r e x a m p l e , to e n s u r e a su i t ab l e e n v i r o n m e n t a n d n u t r i t i o n a l mi l ieu for t he d e v e l o p i n g p a r a s i t e (Sec t ion V I ) . V i n s o n (1984) s t a t ed : " T h e p r o b l e m of n u t r i t i o n a l su i t ab i l i ty m a y n o t b e lack of specific n u t r i e n t s o r accessory g r o w t h factors in t he hos t , b u t t h e q u a l i t y of a n d t h e ab i l i ty to o b t a i n c e r t a i n n u t r i e n t s p r e s e n t in hos t t i ssues a t t he p r o p e r t i m e a n d to c o m p e t e w i t h hos t t i ssues for ava i l ab le n u t r i e n t s . . . t he p a r a s i t o i d m a y actively affect these las t two c o n d i t i o n s . " V i e w i n g the insec t p a r a s i t e - h o s t i n t e r ac t i on as a p r o d u c t of " h o s t r e g u l a t i o n , " however , cons ide r s a l t e r e d hos t phys io logy in t e r m s of specific p a r a s i t o i d surv iva l s t ra teg ies a n d fails to exp l a in h o w s u c h s t r a teg ies have evolved. Moreove r , it p rov ides l i t t le i n s igh t i n to h o w the r e l a t i o n s h i p c o n t i n u e s to evolve. C o n s i d e r i n g t h e r e l a t i o n s h i p w i t h i n t h e un ive r sa l p a r a d i g m of a d a p t a t i o n p rov ides a n o t h e r pe r spec t i ve a n d n e w ins igh t i n to t h e n a t u r e of these p a r a s i t i c a s soc ia t ions .

T h e benefi t of phys io log ica l a l t e r a t i o n s to p a r a s i t e su rv iva l a r i ses by n a t u ra l se lec t ion t h r o u g h e v o l u t i o n a r y p rocesses . T h i s evo lu t ion w a s d e s c r i b e d b y B a r n a r d (1990) as a n " a r m s r a c e " d u r i n g w h i c h t h e r e o c c u r s c o n s t a n t ac t ion a n d r eac t ion by b o t h s y m b i o n t s in r e s p o n s e to t he act ivi t ies of the i r p a r t n e r s .

138 S. Ν. Thompson

S p r e n t (1969) d i scussed i m m u n o l o g i c a l i n t e r ac t ion w i t h i n th is f r amework . H i s " a d a p t a t i o n t o l e r a n c e " hypo thes i s p rov ides a bas i s for u n d e r s t a n d i n g t h e d y n a m i c n a t u r e of p a r a s i t e - h o s t coevolu t ion . D a w k i n s a n d K r e b s (1979) ou t l i ned the l imi t a t ions a n d costs of s u c h a d a p t i v e i n t e r ac t ion . L i t t l e is u n d e r s t o o d c o n c e r n i n g such i n t e r ac t i ons d u r i n g t h e p a r a s i t i c p h a s e of t h e insec t p a r a s i t o i d - h o s t r e l a t i o n s h i p b e c a u s e t he p a r a s i t e does n o t r e p r o d u c e in t h e hos t . Moreove r , the hos t d ies following p a r a s i t e d e v e l o p m e n t . T h u s , V i n s o n a n d I w a n t s c h (1980) s t a t ed t h a t t he hos t p l ays n o role in t h e evo lu t ion of t he assoc ia t ion . A l t h o u g h phys io log ica l i n t e r ac t ion m a y n o t r e p r e s e n t a s t r o n g coevo lu t iona ry force, it is a cr i t ical c o m p o n e n t of p a r a s i t o i d evo lu t ion . If, for e x a m p l e , t he hos t ' s m e t a b o l i c r e sponse d u r i n g p a r a s i t i s m h a s s ignif icant n u t r i t i o n a l c o n s e q u e n c e s , t h e n the d e g r e e to w h i c h these r e sponses a r e exp res sed d i rec t ly i m p a c t s p a r a s i t e surv iva l a n d , in t u r n , fitness a n d fecund i ty of the a d u l t p a r a s i t o i d . A less su i t ab l e hos t p r o d u c e s a less fit p a r a s i t o i d , a n d converse ly a m o r e su i t ab l e hos t p r o d u c e s a m o r e fit p a r a s i t o i d .

D u r i n g the e s t a b l i s h m e n t of t he p a r a s i t e - h o s t r e l a t i onsh ip , a l t e r a t i o n s in hos t phys io logy reflect r e sponses to va r ious phys io log ica l a n d m e t a b o l i c s t resses i n d u c e d b y p a r a s i t i s m . T h o s e r e sponses p r o v i d e a bas i s u p o n w h i c h n a t u r a l se lect ion ac ts , t h e r e b y e n a b l i n g t he p a r a s i t e to explo i t t he modi f ied phys io logica l s t a t e of t he hos t . C h a r a c t e r i s t i c s of p a r a s i t e s o r factors in jec ted in to the hos t by t he p a r a s i t o i d t h a t i n d u c e c h a n g e s h a v i n g beneficial effects for p a r a s i t e surv iva l a r e selected for. W h e n the phys io log ica l a n d m e t a b o l i c a l t e r a t i ons t h a t o c c u r d u r i n g p a r a s i t i s m a r e e x a m i n e d f rom a n e v o l u t i o n a r y pe r spec t ive , a n d t h e s y m b i o n t s as i n t e r ac t i ng p a r t n e r s , t h e d iv i s ion of p a t h o logical ve r sus beneficial c h a n g e s takes o n a n e w a n d m o r e p r o f o u n d signific a n c e . Effects t h a t m i g h t o the rwi se be cons ide red " d u l l p a t h o l o g i c a l byp r o d u c t s of in fec t ion" ( D a w k i n s , 1982) a r e n o longer i gno red , b u t a r e recogn ized as factors p l a y i n g a po ten t i a l ly s ignif icant role in t h e evo lu t ion of t h e p a r a s i t e - h o s t r e l a t ionsh ip . All effects of p a r a s i t i s m have t h e p o t e n t i a l to inf luence p a r a s i t e fitness b y c o n t r i b u t i n g to t he va r i ab i l i ty u p o n w h i c h select ion c a n act .

T h e evo lu t ion is ev iden t u p o n cons ide r ing the in te rac t ive n a t u r e of p a r a sit ic effects in in f luenc ing p a r a s i t e n u t r i t i o n d e s c r i b e d ea r l i e r (Sec t ion V I ) . D e c r e a s e d food c o n s u m p t i o n , a c o m m o n b e h a v i o r d u r i n g p a r a s i t i s m , m i g h t b e cons ide red a n ind i rec t s t ress effect (see L a w r e n c e , 1988). A l t h o u g h perh a p s n o t r e g u l a t e d by the p a r a s i t e , d e c r e a s e d food c o n s u m p t i o n is n e v e r t h e less i n d u c e d as a r e su l t of p a r a s i t i s m , a n d m a y have s ignif icant c o n s e q u e n c e s for t h e deve lop ing p a r a s i t e by d e l a y i n g d e v e l o p m e n t . E v e r y effect of p a r a s i t i sm t h a t is d e e m e d beneficial for t he p a r a s i t e n e e d n o t b e r e g u l a t e d , a n d every effect cons ide red r e g u l a t e d does n o t necessar i ly p r o v i d e benefi t to t h e p a r a s i t e . B e c a u s e of t he in te rac t ive n a t u r e of p a r a s i t i c effects in in f luenc ing


t h e o u t c o m e of t h e p a r a s i t e - h o s t r e l a t i onsh ip , t he u t i l i ty of " h o s t r e g u l a t i o n " is bes t a p p r e c i a t e d by c o n s i d e r i n g a l t e r ed hos t phys io logy as b e i n g of overal l benefi t to t h e d e v e l o p i n g p a r a s i t e , as imp l i ed by V i n s o n (1975 , 1990b) .

VIII. Enantiostasis and Metabolic Regulation

T h e difficulty in u n d e r s t a n d i n g a n d e v a l u a t i n g t h e s ignif icance of t h e p h y s i o logica l a n d m e t a b o l i c a l t e r a t i o n s o c c u r r i n g in p a r a s i t i z e d insec ts is con f o u n d e d b y t h e e n a n t i o s t a t i c c h a r a c t e r of m a n y processes in insec t s . M a i n t e n a n c e of phys io log ica l func t ion in t h e face of c h a n g i n g e n v i r o n m e n t a l c o n d i t i o n s h a s l ong b e e n r ecogn ized as f u n d a m e n t a l for su rv iva l of a n y org a n i s m . H o m e o s t a s i s , t h e m a i n t e n a n c e of a n e a r - c o n s t a n t i n t e r n a l s t a t e , is c h a r a c t e r i s t i c of m a n y a n i m a l s . Less spec ia l ized a n i m a l s , however , p a r t i c u l a r ly i n v e r t e b r a t e s , typica l ly funct ion over a w i d e r a n g e of i n t e r n a l c o n d i t ions a n d a r e n o t h o m e o s t a t i c . T h e h e m o l y m p h s u g a r level of insec t s , for e x a m p l e , is m a i n t a i n e d on ly w i t h i n a very b r o a d c o n c e n t r a t i o n r a n g e . I n M. sexta t h e h e m o l y m p h s u g a r level fluctuates great ly , a n d a l t h o u g h h o r m o n e s a r e involved in r e g u l a t i n g h e m o l y m p h s u g a r levels in insec ts (Sec t ion V ) , h e m o l y m p h s u g a r m a y d e c r e a s e to n e a r u n d e t e c t a b l e levels w h e n l a rvae a r e s t a r v e d ( D a h l m a n , 1969, 1973; S ieger t , 1987).

P ros se r (1955; see a lso Prosser , 1986) d i s t i n g u i s h e d phys io log ica l ad jus t m e n t o r confo rmi ty from phys io log ica l r e g u l a t i o n . M a g n u m a n d Towle (1977) refer red to t h e c o n d i t i o n of conse rved phys io log ica l func t ion in n o n -h o m e o s t a t i c sy s t ems as enan t i o s t a s i s . E n a n t i o s t a s i s is often c h a r a c t e r i z e d b y b a l a n c i n g a d j u s t m e n t s in o n e phys io log ica l p rocess w i t h c o u n t e r c h a n g e s in o t h e r s . T h u s , c a u t i o n m u s t b e exerc ised w h e n e v a l u a t i n g c h a n g e s in t h e i n t e r n a l mi l i eu of p a r a s i t i z e d insec ts b e c a u s e m a n y a l t e r a t i o n s m a y s i m p l y reflect o p p o s i n g a d j u s t m e n t s in s eeming ly u n r e l a t e d p rocesses . T h e e l eva ted t r e h a l o s e level in T. ni p a r a s i t i z e d b y H. exiguae (Sec t ion I I I . A ) , for e x a m p l e , m a y r e p r e s e n t a c o m p e n s a t o r y a d a p t a t i o n to t h e s i m u l t a n e o u s d e c r e a s e in t h e level of a m i n o ac ids . I n th is m a n n e r t h e s y s t e m is m a i n t a i n e d in a func t iona l s t e a d y s t a t e . O s m o t i c p r e s s u r e m a y reflect t h e specific func t ion p r e s e r v e d .

A l t h o u g h r e s e a r c h e r s h a v e s p e c u l a t e d o n the p o t e n t i a l of e l eva ted h e m o l y m p h t r e h a l o s e to p rov ide n u t r i e n t for d e v e l o p i n g p a r a s i t e s , a m i n o ac ids may , in fact, b e m o r e i m p o r t a n t . A d e c r e a s e d a m i n o acid level o b s e r v e d in s o m e hos t s m a y resu l t f rom r a p i d a b s o r p t i o n b y the p a r a s i t e ( s ) . A m i n o ac ids often o c c u r in insec t b lood a t levels exceed ing t h a t of t r eha lo se . I n u n p a r as i t i zed fifth-instar T. ni, t h e level of g l u t a m i n e a lone is a p p r o x i m a t e l y t h r e e -

140 S. Ν. Thompson

fold t h a t of t r eha lose . Moreove r , in vitro n u t r i t i o n a l s tud ie s o n t h e id iob ion t i c p a r a s i t e s d e s c r i b e d in Sec t ion V I I i nd i ca t e t h a t a m i n o ac ids m a y to ta l ly s p a r e c a r b o h y d r a t e as a n ene rgy sou rce for l a rva l d e v e l o p m e n t ( T h o m p s o n , 1976).

IX. Conclusion

Red i rec t ion of hos t phys io logy cha rac t e r i ze s m a n y insec t p a r a s i t e - h o s t re lat ionsh ips . A l t h o u g h o u r u n d e r s t a n d i n g of the m e t a b o l i c bas i s for these c h a n g e s is still r u d i m e n t a r y , r ed i rec t ion often inc ludes a l t e r a t i o n s in t i ssue levels of l ip ids , c a r b o h y d r a t e s , a n d n i t r ogen m e t a b o l i t e s . M e t a b o l i c s tud ies sugges t t h a t s o m e c h a n g e s reflect expec t ed a d j u s t m e n t s b r o u g h t a b o u t b y t he hos t ' s n o r m a l e n a n t i o s t a t i c r e g u l a t o r y m e c h a n i s m s in r e s p o n s e to t he s t ress of p a r a s i t i s m . I n o t h e r cases , however , t he n o r m a l r e g u l a t o r y m e c h a n i s m s a p p e a r to be a l t e red o r i nope ra t i ve . I n those i n s t ances it m i g h t be c o n c l u d e d t h a t r egu l a t i on of hos t m e t a b o l i s m by the p a r a s i t e h a s s u p e r s e d e d t h e hos t ' s n o r m a l r egu l a to ry func t ions . I n e i the r case the c h a n g e s o b s e r v e d m a y have n u t r i t i o n a l c o n s e q u e n c e s for the deve lop ing p a r a s i t e l a rvae .

T h e va r ious m a n n e r s in w h i c h we v iew insect p a r a s i t i s m reflect m o r e t h a n " s e m a n t i c " differences (Fisher , 1986). T h e y inf luence in a f u n d a m e n t a l w a y o u r u n d e r s t a n d i n g of insec t p a r a s i t i s m (see Goff, 1 9 8 2 ) — o u r i n t e r p r e t a t i o n of e x p e r i m e n t a l d a t a a n d o u r a p p r o a c h to fu r ther r e s e a r c h . T h e c o n c e p t of " h o s t r e g u l a t i o n " h a s a n d c o n t i n u e s to p rov ide a v a l u a b l e f r a m e w o r k w i t h i n w h i c h the n a t u r e of t he insect p a r a s i t e - h o s t r e l a t i o n s h i p c a n b e u n d e r s t o o d . I t is i m p o r t a n t to recognize t he d y n a m i c n a t u r e of t he insec t p a r a s i t e - h o s t i n t e rac t ion a n d t h e evo lu t iona ry bas is for the d e v e l o p m e n t of th is c o m p l e x symbios i s .

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Dahlman, D. L., and Vinson, S. B. (1975). Trehalose and glucose levels in the hemolymph of Heliothis virescens parasitized by Microplitis croceipes or Cardiochiles nigriceps. Comp. Biochem. Physiol. Β 52B:465-468.

Dahlman, D. L., and Vinson, S. B. (1976). Trehalose level in the hemolymph οϊ Heliothis virescens parasitized by Campoletis sonorensis. Ann. Entomol. Soc. Am. 69:523-524.

Dahlman, D. L., and Vinson, S. B. (1977). Effect of calyx fluid from an insect parasitoid on host hemolymph dry weight and trehalose content. J. Invertebr. Pathol. 29:227-229.

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Dawkins, R., and Krebs, J. R. (1979). Arms races between and within species. Proc. R. Soc. London, Ser. 5 205:489-511 .

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Dover, Β. Α., Davies, D. H., and Vinson S. B. (1988b). Degeneration of last-instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus. J. Invertebr. Pathol. 51 :80 -91 .

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Fisch. (Hymenoptera, Braconidae) on the dry weight and chemical composition of its host Anagasta kuehniella Zell. (Lepidoptera, Pyralidae). J. Insect Physiol. 32:269-274.

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144 S. Ν. Thompson

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Chapter 7

Teratocytes: Developmental and Biochemical Characteristics Douglas L Dahlman Department of Entomology University of Kentucky Lexington, Kentucky

S. Bradleigh Vinson Department of Entomology Texas A&M University College Station, Texas

I. Introduction

II. Origins

III. Distribution within Taxonomic Groups

IV. Developmental Characteristics A. Morphology B. Comparisons among Egg, Egg-

Larval, Larval, Larval-Pupal, and Adult Parasitoids

C. Comparisons between Single and Multicell Tissues

V. Physiological and Biochemical Characteristics A. Trophic Functions B. Immunosuppressive Functions C. Secretory Functions D. General Effects on Host Growth and

Development

VI. Interactions of Teratocytes, Venoms, and Polydnavirus

VII. Conclusion and Summary Acknowledgments References

I. Introduction

Scient i s t s h a v e r e p o r t e d u n u s u a l l y l a rge cells d i s p e r s e d t h r o u g h o u t t h e h e m o l y m p h of c e r t a in p a r a s i t i z e d insec t s . T h e s e cells w e r e a s s igned v a r i o u s n a m e s t h a t reflected a p r o p o s e d funct ion a n d / o r e m b r y o l o g i c a l sou rce , b u t they r e m a i n e d a cur ios i ty un t i l t he 1960s, w h e n i n c r e a s e d k n o w l e d g e a b o u t insec t i m m u n o l o g y , physio logy, b iochemis t ry , virology, a n d m o l e c u l a r b io logy, as well as a d v a n c e s in i n s t r u m e n t a t i o n t e c h n i q u e s , m a d e it poss ib le to look b e y o n d desc r ip t ive biology. T h e funct ion of these cells, m o s t c o m m o n l y cal led t e r a tocy t e s , w a s first r ev iewed by Sal t (1968) . T w o i m p o r t a n t rev iews on the i r e m b r y o l o g i c a l o r ig in ( the serosa l m e m b r a n e of t he p a r a s i t o i d e m bryo) s u b s e q u e n t l y a p p e a r e d ( I v a n o v a - K a s a s , 1972; T r e m b l a y a n d C a l -t a g i r o n e , 1973). H a w l i t z k y a n d L a u g e (1979) d i scussed t h e h y p o t h e t i c a l roles of t h e serosa l m e m b r a n e in a s ingle egg- la rva l p a r a s i t o i d a n d V i n s o n



146 Douglas L Dahlman and S. Bradleigh Vinson

a n d I w a n t s c h ' s 1980 rev iew on hos t r egu l a t i on b y insec t p a r a s i t o i d s i n c l u d e d a m a j o r sec t ion o n t e r a tocy te s . I n t e r e s t in t h e ro le of t e r a tocy t e s in t h e r e g u l a t i o n of hos t physiology, b iochemis t ry , endocr ino logy , i m m u n o l o g y , e tc . , h a s acce le ra t ed d u r i n g t h e las t 10 yea r s . D a h l m a n (1990, 1991) p r o v i d e d overv iews of t e r a tocy t e funct ions a n d rev iews b y B e c k a g e (1990) a n d C o u -d r o n (1991) i n c l u d e d sec t ions on t e r a tocy te s .

P r io r to 1980, t e r a tocy te s w e r e genera l ly t h o u g h t to r a p i d l y a b s o r b n u t r i en t s t h a t w e r e c o n s u m e d l a t e r b y t h e deve lop ing p a r a s i t o i d . C l e a r l y t h e v iew t o d a y is t h a t t e r a tocy te s d o m o r e t h a n s imp ly s u p p l y a n a d d i t i o n a l s o u r c e of n u t r i e n t s to t h e deve lop ing p a r a s i t o i d l a rva . I m p o r t a n t i n t e r ac t i ons b e t w e e n t e r a tocy te s a n d o t h e r c o m p o n e n t s of t he p a r a s i t o i d s y s t e m ( v e n o m , accessory g l a n d p r o d u c t s , a n d , in s o m e cases , p o l y d n a v i r u s ) a r e b e i n g inves t iga t ed .

II. Origins

D e v e l o p i n g e m b r y o s of ce r t a in p a r a s i t i c H y m e n o p t e r a a r e enc losed w i t h i n a n e x t e r n a l m e m b r a n e t h a t , for different species , m a y h a v e different o r ig ins a n d fates. T h e p o l a r bod ies a r e cons ide red to b e t h e m o s t l ikely u l t i m a t e sou rce of these e m b r y o n i c m e m b r a n e s . A c c o r d i n g to T r e m b l a y a n d C a l -t a g i r o n e (1973) t h e p o l a r bod ies a r e t h e d a u g h t e r cells of t h e p r i m a r y a n d s e c o n d a r y oocy tes fo rmed d u r i n g egg m a t u r a t i o n . T h e specific t e r m s a s s igned to t he m e m b r a n e s t h a t deve lop f rom these cells v a r y w i t h t h e insec t t a x o n . I n t he B r a c o n i d a e , w h i c h c o n t a i n t he ma jo r i ty of r e p o r t e d i n s t a n c e s of t e r a tocy te s , t h e p o l a r bod ie s give r ise to a serosa l m e m b r a n e t h a t in t u r n d i ssoc ia tes to yield i n d i v i d u a l cells after t h e egg h a t c h e s (Fig . 1).

T r e m b l a y a n d C a l t a g i r o n e (1973) s t a t e d t h a t t h e se rosa l m e m b r a n e envel o p i n g t h e e m b r y o falls in to o n e of t h r e e types : (1) a t r u e se rosa , (2) a d e u t o s e r o s a ( p s e u d o s e r o s a ) , o r (3) a t r o p h a m n i o n . T h e se rosa is de r ived from t h e first c leavage n u c l e u s . Sources of t he t r u e se rosa in b r a c o n i d s m a y b e from e i the r se rosa l d e l a m i n a t i o n o r pro l i fe ra t ion of p o l a r bod ie s ( T r e m b lay a n d C a l v e r t , 1971; T r e m b l a y a n d C a l t a g i r o n e , 1973). A serosa l m e m b r a n e o r i g i n a t i n g by d e l a m i n a t i o n a lso h a s b e e n d e s c r i b e d for a n ich-n e u m o n i d , a l t h o u g h n o ev idence of t e r a tocy tes h a s b e e n r e p o r t e d for th is g r o u p . T h e d e u t o s e r o s a l m e m b r a n e , in c o n t r a s t to a t r u e se rosa , is ex t en sively de r ived f rom t h e e m b r y o p r o p e r b u t a l so c o n t a i n s cells f rom p o o r l y def ined e x t r a e m b r y o n i c t i ssues . T h e t r o p h a m n i o n i c m e m b r a n e h a s n o co r re s p o n d i n g s t r u c t u r e in t h e e m b r y o n i c d e v e l o p m e n t of insec ts in g e n e r a l a n d , in t h e o p i n i o n of T r e m b l a y a n d C a l t a g i r o n e (1973) , t h e t e r m is n o t e m b r y -ological ly a p p r o p r i a t e b u t it h a s b e e n used so f requen t ly " t h a t it s eems a d v i s a b l e to p re se rve i t . "

U n d o u b t e d l y va r ious c o m b i n a t i o n s of these m e m b r a n e s will b e found u p o n closer e x a m i n a t i o n of t h e embryo log ica l d e v e l o p m e n t of v a r i o u s species of

7. Teratocytes: Developmental and Biochemical Characteristics 147

Figure 1 Well-developed (left) and hatching larvae (right) of Cardiochiles nigriceps showing the location of the two types of serosal cells that enclose the embryo. Only Type I I cells form teratocytes when liberated from the embryo. The Type I serosal cells degrade (F. Pennacchio, S. B. Vinson, and E. Tremblay, Texas A & M University, unpublished).

H y m e n o p t e r a . I n fact, a t leas t o n e i n t e r m e d i a t e e x a m p l e is k n o w n w h e r e t h e t r o p h a m n i o t i c m e m b r a n e is de r ived from b o t h p o l a r nuc le i a n d c leavage nuc le i ( T r e m b l a y a n d C a l v e r t , 1971). M e m b r a n e s of t h e t r o p h a m n i o t i c t y p e a lso a r e found in e n d o p a r a s i t i c H y m e n o p t e r a t h a t d e v e l o p p o l y e m b r y o n i c a l l y ( I v a n o v a - K a s a s , 1972).

III. Distribution within Taxonomic Groups

I f t h e s t r ic t def in i t ion of t e r a tocy te s is followed (i .e. , i n d i v i d u a l cells r e l eased in to t h e h o s t h e m o c o e l as t h e r e su l t of t h e d i s soc ia t ion of t h e se rosa l m e m b r a n e of t h e e m b r y o ) , t hese cells a r e r e s t r i c t ed to a few famil ies of t h e H y m e n o p t e r a ( B r a c o n i d a e , P l a t y g a s t e r i d a e , a n d Sce l ion idae) . A s ingle ter-


a t o c y t e p e r p a r a s i t o i d was r e p o r t e d for o n e t r i c h o g r a m m a t i d species (Voegele et aL, 1974), b u t S t r a n d (1986) conv inc ing ly a r g u e d t h a t th is s ingle cell is a p o l a r b o d y a n d shou ld no t be cons ide red a t e r a t o c y t e . H o w e v e r , in a b r o a d e r sense , n o n d i s s o c i a t e d t issues of t he s a m e or ig in (i .e. , p o l a r bod ies ) have b e e n d e s c r i b e d from severa l species of t he supe r fami ly C h a l c i d o i d e a . S p e c u l a t i o n on p r e r equ i s i t e s for c o n t i n u e d act iv i ty of p o s t e m e r g e n c e e x t r a e m b r y o n i c t i ssue wou ld be t h a t t he p a r a s i t o i d egg m u s t be d e p o s i t e d w i t h i n t he hos t so t h a t the t issues have access to h o s t b o d y fluids for s u s t e n a n c e . T h u s , for e x a m p l e , e x a m i n a t i o n of h o m o p t e r a n hos t s p a r a s i t i z e d b y P i p u n -cu l idae ( D i p t e r a ) m i g h t p rov ide a d d i t i o n a l ins igh t i n to t he i m p o r t a n c e of these e x t r a e m b r y o n i c t i ssues . Neve r the l e s s , o t h e r factors m u s t be neces sa ry for success b e c a u s e t e ra tocy tes have n o t been r e p o r t e d f rom m a n y o t h e r families of p a r a s i t i c H y m e n o p t e r a .

IV. Developmental Characteristics

A. Morphology

1. A b u n d a n c e , P e r s i s t e n c e , a n d G r o w t h

S o m e of t he s y n o n y m s used for t e r a tocy te s reflect o b s e r v a t i o n s o n t h e size, funct ion , a n d poss ib le or ig ins of these spec ia l ized e x t r a e m b r y o n i c cells. T h e y h a v e been cal led g i a n t cells, t r o p h i c cells, t r o p h a m n i o n cells, a n d t r o -p h o s e r o s a cells, b u t t he t e r m t e ra tocy te s , o r ig ina l ly sugges t ed by H o l l a n d e (1920) , is c u r r e n t l y m o s t c o m m o n l y used . A l t h o u g h a c o n t i n u u m b e t w e e n n o d i s soc ia t ion a n d to ta l d i s soc ia t ion of t h e e x t r a e m b r y o n i c m e m b r a n e p r o b a bly c a n be found in va r ious species , th is d i scuss ion will b e p r i m a r i l y re s t r ic ted to those m e m b r a n e s t h a t d i s soc ia te .

N e w l y ovipos i ted eggs of e n d o p a r a s i t i c H y m e n o p t e r a typ ica l ly a r e s m a l l w i t h re la t ively l i t t le yolk. D u r i n g e m b r y o g e n e s i s , egg v o l u m e inc reases severa l h u n d r e d f o l d w i t h c o n c o m i t a n t g r o w t h of t he serosa l m e m b r a n e a n d s t r e t c h i n g of t h e cho r ion . Dif ferent ia t ion a m o n g the cells fo rming t h e serosa l m e m b r a n e p r o b a b l y occu r s d u r i n g e m b r y o g e n e s i s . I n Cardiochiles nigriceps t he serosa l m e m b r a n e cells have two fates. T h o s e cells nex t to t he ep i the l i a l cells of t h e e m b r y o beg in to d e g e n e r a t e j u s t p r i o r to l a rva l h a t c h i n g , a l t h o u g h they m a y r e m a i n for a whi le even after h a t c h i n g . S m i t h (1952) d e s c r i b e d a s imi l a r p h e n o m e n o n . However , a n t e r i o r a n d pos t e r io r to t he e m b r y o the se rosa is severa l layers th ick (Fig. 1) a n d w h e n these cells s e p a r a t e f rom t h e e m b r y o , i n s t e a d of d e g e n e r a t i n g , they a r e re leased a n d deve lop in to t h e t e r a tocy t e s ( F . P e n n a c c h i o , S. B . V i n s o n , a n d E . T r e m b l a y , u n p u b l i s h e d ) . T h e s i t u a t i o n in o t h e r species h a s no t b e e n d e s c r i b e d in de ta i l , b u t t h e serosa l m e m b r a n e is t h o u g h t to d i s soc ia te i n to i n d i v i d u a l t e r a tocy te s . T h e n u m b e r of t e r a tocy t e s


r e p o r t e d f rom i n d i v i d u a l p a r a s i t o i d species r a n g e s f rom as few as 8 to as m a n y as 800 ( V i n s o n a n d I w a n t s c h , 1980). O n c e d i s soc ia t ed , t hey d o n o t u n d e r g o fu r the r cell d iv is ion , a l t h o u g h t h e p lo idy level m a y inc rea se m a n y -fold ( S t r a n d a n d W o n g , 1991). A l t h o u g h prec i se de ta i l s a r e species specific, t h e v o l u m e of i n d i v i d u a l t e r a tocy te s usua l ly inc reases manyfo ld d u r i n g the i r r e s idence w i t h i n t h e hos t . In i t i a l d i a m e t e r s r a n g e from 5 to 86 μ π ι w i t h a 5-to 10-fold i nc rea se in d i a m e t e r b e i n g q u i t e c o m m o n ( V i n s o n a n d I w a n t s c h , 1980). O n e e x a m p l e of over a 3000-fold inc rease in size h a s b e e n r e c o r d e d in a n a t u r a l l y p a r a s i t i z e d hos t ( S m i t h , 1952), wh i l e even l a rge r sizes h a v e b e e n o b t a i n e d w h e n t h e t e r a tocy te s w e r e t r ans fe r r ed to n o n p a r a s i t i z e d hos t s ( V i n son , 1970; S t r a n d a n d W o n g , 1991). T e r a t o c y t e s w i t h d i a m e t e r s as g r e a t as 1 m m h a v e b e e n found in Heliothis virescens p u p a e t h a t w e r e in jec ted as fifth-i n s t a r l a rvae w i t h C. nigriceps t e r a tocy te s ( P e n n a c c h i o et al., 1993b) .

I n m o s t r e p o r t e d cases , t he in i t ia l n u m b e r of t e r a tocy te s w a s s ignif icant ly r e d u c e d b y t h e t i m e t h e p a r a s i t o i d c o m p l e t e d its l a rva l d e v e l o p m e n t (see n u m b e r w i t h t i m e for Perilitus coccinellae, F ig . 2). T h e c a u s e for th is loss is r e l a t ed to t h e s t age of hos t p a r a s i t i z e d , t he phys io logy of t h e paras i to id—host i n t e r ac t i on , a n d t h e b e h a v i o r of t h e p a r a s i t o i d . S o m e p a r a s i t o i d l a rvae feed

C. nigriceps 150

1 1 9 119 100 88 , ,

Ο Ο Ο Ο 100^m

O A 6 8 10 12 1A Days after paras i t i zat ion

Figure 2 T h e diameters and numbers of teratocytes for three species of braconids are depicted. The teratocytes of Perilitus coccinellae reach over 450 μπι in diameter whereas those of Apanteles kariyai rarely exceed 100 μπι. Data for Cardiochiles nigriceps provided by S. B. Vinson (Texas A & M University, unpublished), for P. coccinellae provided by K. Kadono-Okuda (Tsukuba University, unpublished), and for A. kariyai provided by T. Okuda (National Institute of Sericulture and Entomological Science, Tsukuba, J a p a n , unpublished).


a l m o s t exclusively on t h e e n l a r g e d t e r a t o c y t e cells (Sch l inge r a n d H a l l , 1960). T h e r e is c lear ev idence of t e r a tocy te s p r e s e n t in t he d iges t ive t r ac t of s o m e p a r a s i t o i d l a rvae (Sluss a n d L e u t e n e g g e r , 1968; A r a k a w a a n d K i t a n o , 1989; S t r a n d a n d W o n g , 1991). I n o t h e r cases it is p r e s u m e d t h a t t h e cells a r e phys ica l ly de s t royed as t he resu l t of t he g r o w t h of t he p a r a s i t o i d w i t h i n t h e l imi ted confines of t h e hos t ( L o a n a n d H o l d a w a y , 1961). I n t hose p a r a s i t o i d species t h a t d o n o t comple t e ly c o n s u m e the i r hos t , it is m o r e c o m m o n t h a t a t leas t s o m e of t h e t e r a tocy te s pers i s t a l ive in t he hos t after t h e p a r a s i t o i d h a s ex i ted (Tawfik, 1961; K i t a n o , 1965). O n the o t h e r h a n d , t h e in i t ia l c o m p l e m e n t of t e r a tocy te s re leased u p o n h a t c h of a Microplitis croceipes egg w a s still found in t h e b o d y cavi ty of i ts hos t after t h e p a r a s i t o i d l a rva ex i ted ( V i n s o n a n d Lewi s , 1973). T h e ava i l ab le d a t a sugges t t h a t t he evo lu t ion of t e r a tocy t e s h a s t a k e n two p a t h w a y s . I n s o m e p a r a s i t o i d - h o s t r e l a t i o n s h i p s t h e tera t o c y t e n u m b e r s d e c r e a s e p r i o r to t h e g e n e r a l c o n s u m p t i o n of h o s t t i ssues b y t h e d e v e l o p i n g p a r a s i t o i d , w h e r e a s in o t h e r s t he t e r a tocy te s e i the r d o n o t s ignif icant ly d e c r e a s e in n u m b e r o r t hey a r e on ly c o n s u m e d w h e n t h e t i ssues of t h e hos t a r e c o n s u m e d . For e x a m p l e , in t h e m i d p o i n t of the i r d e v e l o p m e n t in t h e hos t , t h e n u m b e r of t e r a tocy te s f rom Apanteles kariyai eggs h a s n o t d e c r e a s e d a t all (Fig . 2) . I n c o n t r a s t , 6 % of t h e C. nigriceps t e r a tocy t e s a r e lost by t h e m i d p o i n t of i ts d e v e l o p m e n t a n d a b o u t 2 0 % of t h e t e r a tocy t e s f rom P. coccinellae a r e g o n e b y 7 d a y s .

2. S E M a n d T E M U l t r a s t r u c t u r e

E a r l y r e p o r t s of t e r a tocy te s in p a r a s i t i z e d hos t s i n c l u d e d l ine d r a w i n g s , p h o t o g r a p h s of w h o l e cells , sec t ions d y e d w i t h va r ious cy to logica l s t a i n s , e tc . ( V i n s o n , 1970). M o r e r ecen t r e p o r t s have i n c l u d e d exce l len t t r a n s m i s s i o n e lec t ron m i c r o s c o p y ( T E M ) p h o t o m i c r o g r a p h s s h o w i n g u l t r a s t r u c t u r e (Sluss a n d L e u t e n e g g e r , 1968; T r e m b l a y a n d I a c c a r i n o , 1971 ; V i n s o n a n d Sco t t , 1974; S t r a n d et al., 1985, 1986; T a n a k a a n d W a g o , 1990; P e n n a c c h i o et al., 1993a; d e B u r o n et al., 1993). I n f o r m a t i o n from s c a n n i n g e lec t ron m i c r o s copy ( S E M ) s tud ies a l so h a s a p p e a r e d ( D a h l m a n , 1991). B o t h T E M a n d S E M s tud ie s s h o w t h a t t e r a tocy te s a r e covered w i t h a d e n s e m a t of microvi l l i (F ig . 3A) . T h i s e x p a n d e d surface a r e a is cons ide r ed to e n h a n c e b o t h t h e p o t e n t i a l a b s o r p t i v e a n d sec re to ry funct ions of t he t e r a tocy t e s . U l t r a s t r u c -t u r a l s t ud i e s show l a rge extens ive ly ramif ied nuc le i , ex tens ive r e t i c u l a r a n d swol len e n d o p l a s m i c r e t i c u l u m (Fig . 3B) , s e q u e s t e r i n g m i t o c h o n d r i a , o r g a -

Figure 3 (A) Scanning electron micrograph of a fixed teratocyte of Microplitis croceipes (5 days after hatching) showing extensive microvillae. (B) Thin section of a fixed teratocyte of Cardiochiles nigriceps showing the extensive swollen vacuoles surrounded by ribosomes and rough endoplasmic reticulum.


nized Golg i , myel in l ike s t r u c t u r e s , p u t a t i v e a u t o p h a g i c vacuoles , a n d ce l lu la r o u t p o c k e t i n g s . M a n y of these fea tures a r e a lso s h a r e d b y n o n d i s s o c i a t e d serosa l m e m b r a n e s ( L a w r e n c e , 1990), t r o p h a m n i o n s (Koscielski et al., 1978), a n d g u t ep i the l ia l cells.

B. Comparisons among Egg, Egg-Larval, Larval, Larval-Pupal, and Adult Parasitoids

All s t ages of t he life cycle of insec ts a r e suscep t ib le to p a r a s i t i z a t i o n by o n e o r m o r e species of p a r a s i t i c insec ts . W i t h t he excep t ion of p a r a s i t o i d s t h a t on ly a t t a ck t he p u p a l s t age , t e ra tocy tes have b e e n obse rved in p a r a s i t i z e d hos t s of all o t h e r life s t ages .

T h r e e species of scel ionid egg p a r a s i t o i d s have b e e n r e p o r t e d to have t e r a tocy te s (Ge r l i ng a n d O r i o n , 1973; S t r a n d et al., 1985; Volkoff a n d C o l -azza , 1992). T e r a t o c y t e s have b e e n d o c u m e n t e d in severa l species of Chelonus (Bracon idae ) ( B u h l e r et al., 1985; J o n e s , 1987), w h i c h a r e e g g / l a r v a l p a r a s i to ids . T h e s ingle r e p o r t of t e r a tocy tes in a p l a t y g a s t e r i d a lso is a n e g g / l a r val p a r a s i t o i d (Hi l l a n d Emery , 1937).

T h e la rva l s t age of hos t s is b y far t he m o s t c o m m o n in w h i c h t e r a tocy t e s have b e e n r e p o r t e d . However , it a lso is poss ib le t h a t th is s imp ly reflects t h e fact t h a t m o s t of t he e x a m p l e s t h u s far s t u d i e d h a v e b e e n l a rva l p a r a s i t o i d s . O r d e r s h o s t i n g p a r a s i t o i d s w i t h t e ra tocy tes i n c l u d e L e p i d o p t e r a , H e m i p t e r a , a n d H o m o p t e r a . A few of t he species p a r a s i t i z i n g l e p i d o p t e r o u s l a rvae a r e g r e g a r i o u s ( K i t a n o , 1965; Beckage , 1990) a n d p a r t i c u l a r l y l a rge n u m b e r s of t e r a tocy te s a r e p r e s e n t in t he h e m o c o e l . N y m p h s ( larvae) of a p h i d s (Spencer , 1926) a n d m i r i d s ( C o h e n a n d D e B o l t , 1984) a r e hos t s for severa l species of b r a c o n i d s t h a t yield t e r a tocy te s , a n d a d u l t s of c e r t a in famil ies of C o l e o p t e r a ( J ackson , 1928; S m i t h , 1952; Sluss , 1968), as well as a d u l t a p h i d s , a r e hos t s for b r a c o n i d s t h a t re lease t e r a tocy te s . Diachasmimorpha (=Biosteres) long-icaudata, t he l a r v a l / p u p a l b r a c o n i d p a r a s i t o i d of t he t e p h r i t i d fruit fly, Anastrepha suspensa, re leases a n in tac t serosa l m e m b r a n e t h a t m a y p l a y a role s imi l a r to t h a t of t he t e ra tocy tes ( L a w r e n c e , 1990). I t is i n t e r e s t i ng to n o t e t h a t we have no t found a n y r eco rds of t e r a tocy te s a s soc ia t ed w i t h p u p a l p a r a s i t o i d s . P e r h a p s t he i n t e r n a l his tolysis of t i ssues a s soc ia t ed w i t h rea l loca t ion of s t r u c t u r a l c o m p o n e n t s obv ia t e s t he need for t e r a tocy te s t h a t m a y serve a l y t i c /d i s soc i a t i on funct ion in o t h e r types of hos t s .

C. Comparisons between Single and Multicell Tissues

As def ined, t e r a tocy te s a r e i n d i v i d u a l cells re leased f rom a n e x t r a e m b r y o n i c m e m b r a n e s u b s e q u e n t to t he h a t c h of t he p a r a s i t o i d egg. As such , they a r e r e s t r i c t ed to a few families of H y m e n o p t e r a . However , f rom a func t iona l


p o i n t of view, e x t r a e m b r y o n i c t i ssue f rom t h e p a r a s i t o i d t h a t pers i s t s in t h e h o s t pos tec los ion m a y b e s imi l a r w h e t h e r o r n o t t he cells of t he m e m b r a n e h a v e d i s soc ia t ed . G i v e n t h a t t h e t e r a tocy t e s a r i se from p o l a r bod ie s , it c a n b e a r g u e d t h a t t i ssues f rom t h e s a m e s o u r c e m i g h t have s imi l a r func t ions . T h e ro le of p o l a r bod ie s in e n d o p a r a s i t i s m h a s b e e n d i scussed ( T r e m b l a y a n d C a l t a g i r o n e , 1973), a n d I v a n o v a - K a s a s (1972) a d d r e s s e d a s o m e w h a t m o r e spec ia l ized a s p e c t of th is sub jec t in a r ev iew o n insect p o l y e m b r y o n y . C l e a r l y a va r i e ty of e x t r a e m b r y o n i c t issues m a y pers i s t w i t h i n t he hos t s a n d these t i ssues m o r e t h a n likely express a d ivers i ty of b io logica l act ivi t ies t h a t cont r i b u t e to t h e success of t h e p a r a s i t o i d .

V. Physiological and Biochemical Characteristics

Since e x p e r i m e n t a l s tud ies on t he func t ions of t e r a tocy te s h a v e on ly r ecen t ly b e e n in i t i a t ed , m u c h r e m a i n s to b e e x p l a i n e d w i t h r e spec t to specific roles p l a y e d by these cells in t h e p a r a s i t o i d - h o s t i n t e r ac t i on . T h i s is especia l ly t r u e w i t h egg p a r a s i t o i d s b u t t h e i n fo rma t ion from even the m o s t f r equen t ly s t u d i e d g r o u p ( l e p i d o p t e r o u s l a rva l p a r a s i t o i d s ) is very l imi ted . His tor ica l ly , t h e p r i m a r y roles of t e r a tocy te s have b e e n r e p o r t e d to b e t r o p h i c , i m m u n o s u p p r e s s i v e , a n d secretory.

A. Trophic Functions

T h e t r e m e n d o u s i nc rea se in v o l u m e of t e r a tocy te s followed by the i r d i s a p p e a r a n c e t o w a r d t h e c o m p l e t i o n of t h e p a r a s i t o i d ' s l a rva l g r o w t h l eads to t h e c o n c l u s i o n t h a t t e r a tocy te s serve as a sou rce of specific n u t r i e n t s , p a r t i c u l a r l y for t h e l a t e r i n s t a r s of t h e p a r a s i t o i d . I n fact, c o m p l e t e cells a n d f r a g m e n t s h a v e b e e n found in t he d iges t ive t r ac t s of s o m e p a r a s i t o i d s (Sluss , 1968; A r a k a w a a n d K i t a n o , 1989; S t r a n d a n d W o n g , 1991). T h e d e n s e m a t of microvi l l i o b s e r v e d o n t e r a tocy te s (Fig . 3A) cou ld b e c o n s i d e r e d a n a d a p t a t ion for a b s o r p t i o n of n u t r i e n t s f rom t h e hos t ' s h e m o l y m p h . A l t h o u g h it is poss ib le t h a t t e r a tocy te s m a y syn thes i ze specific n u t r i e n t s r e q u i r e d for suc cessful p a r a s i t o i d d e v e l o p m e n t , it is a l so poss ib le t h a t t e r a tocy t e s re lease factors t h a t e i the r a l t e r hos t n u t r i e n t s o r inf luence t h e phys io log ica l e n v i r o n m e n t in w h i c h the p a r a s i t o i d deve lops . For e x a m p l e , t h e t e r a tocy t e s of Tele-nomus heliothidis secre te h i s to ly t ic e n z y m e s t h a t d iges t t he c o n t e n t of t h e egg hos t ( S t r a n d et al., 1986).

I n a t l eas t two species , t he success of in vitro r e a r i n g of p a r a s i t o i d l a rvae w a s e n h a n c e d b y i n c l u d i n g t e r a tocy t e s in t h e r e a r i n g m e d i a ( S t r a n d et al., 1988; G r e a n y et al., 1989), p e r h a p s as t he resu l t of t h e p r o d u c t i o n of h i s to ly t ic


TC Μ 669 440

^ - 2 3 2 200

140

/

69"

4 6 ^

A Β Figure 4 (A) Native PAGE (2.5 to 20% gradient) of an 11-day teratocyte homoge-nate of Perilitus coccinellae stained with Coomassie Brilliant Blue (T. Okuda, National Institute of Sericulture and Entomological Science, Tsukuba, J apan , unpublished). (B) Autoradiograph of 3 5 S- labeled proteins from an SDS-PAGE of a 9-day teratocyte homogenate of Cardiochiles nigriceps (A. Mourad and S. B. Vinson, Texas A & M University, unpublished). TC = teratocytes, Μ = marker.

Μ TC


e n z y m e s t h a t h y d r o l y z e c o m p l e x molecu le s found in t h e r e a r i n g m e d i a . I n o n e r e p o r t w h e r e t e r a tocy t e s d i d n o t i m p r o v e in vitro g r o w t h , t h e r e a r i n g m e d i a m a y h a v e b e e n deficient in o n e o r m o r e cr i t ica l c o m p o n e n t s b e c a u s e it s u p p o r t e d on ly m a r g i n a l g r o w t h w i t h o u t t e r a tocy te s ; therefore , it m a y n o t h a v e b e e n poss ib le to d e m o n s t r a t e a n y s u p p l e m e n t a l effects of t e r a tocy t e s ( R o t u n d o et al., 1988). I n c o n t r a s t , in vitro s t ud ie s w i t h C. nigriceps b y P e n n a c -ch io et al. (1992b) d e m o n s t r a t e d b o t h g r o w t h a n d m o l t i n g f rom first to s econd i n s t a r w i t h o u t ac t ive t e r a tocy t e s .

I t is un l ike ly t h a t t e r a tocy t e s a r e e a t e n b y M. croceipes l a rvae b e c a u s e t h e o r ig ina l n u m b e r r e m a i n in t h e h o s t after t h e p a r a s i t o i d h a s ex i ted ( V i n s o n a n d Lewi s , 1973). A s ignif icant p r o p o r t i o n of t h e t e r a tocy t e s f rom Cotesia (=Apanteles) glomerata a l so r e m a i n a t t h e t e r m i n a t i o n of p a r a s i t o i d l a rva l d e v e l o p m e n t ( H a s h i m o t o a n d K i t a n o , 1971). I t h a s b e e n sugges t ed t h a t t h e t e r a tocy t e s m i g h t d i s i n t e g r a t e in t h e hos t ' s h e m o l y m p h a n d t h u s re lease o r r e p l e n i s h n u t r i e n t supp l i e s t h a t a r e t h e n ava i l ab le to t h e p a r a s i t o i d . H o w ever , t h e bes t ev idence for t r u e t r o p h i c func t ion c o m e s f rom T. O k u d a (persona l c o m m u n i c a t i o n ) . H e found t h a t t e r a tocy te s f rom P. coccinellae c o n t a i n o n e m a j o r p r o t e i n b a n d (Fig . 4A) , w h e r e a s t h e r e d u c t i o n in t e r a t o c y t e n u m be r s f rom 600 to less t h a n 50 (see F ig . 2, K . K o d o n o - O k u d a , p e r s o n a l c o m m u n i c a t i o n ) sugges t s t h a t t e r a tocy te s f rom P. coccinellae a r e ac tua l ly cons u m e d b y t h e p a r a s i t o i d . A s m e n t i o n e d ear l ier , t e r a tocy te s co u l d a l so p l a y a t r o p h i c ro le b y d iges t ing hos t h e m o l y m p h p r o t e i n s , e tc .

B. Immunosuppressive Functions

I n 1968 G e o r g e Sa l t s p e c u l a t e d t h a t t e r a tocy te s a id in r e s i s t ance to h o s t defense m e c h a n i s m s . H e a r g u e d t h a t t e r a tocy t e g r o w t h is ach ieved b y a b s o r b i n g n u t r i e n t s f rom h o s t h e m o l y m p h w i t h t h e c o n c o m i t a n t d e p l e t i o n of h e m o l y m p h m a t e r i a l . As a c o n s e q u e n c e , h e m a t o p o i e s i s is r e t a r d e d a n d p o t en t i a l h e m o c y t i c r eac t ions of t h e h o s t to t h e y o u n g p a r a s i t o i d a r e d e p r e s s e d . H e fu r the r a r g u e d t h a t t h e p r e s e n c e of l a rge n u m b e r s of t e r a tocy t e s after t h e p a r a s i t o i d left i ts h o s t " w a s a shee r w a s t e of t h e t e r a tocy t e s if t he i r func t ion w a s to n o u r i s h ; b u t was u n d e r s t a n d a b l e if t hey h a d a l r e a d y p e r f o r m e d the i r func t ion of p r o t e c t i n g t h e y o u n g l a rvae f rom a defense r e a c t i o n . " Sa l t (1971) r e p o r t e d severa l i n t e r e s t i ng o b s e r v a t i o n s in w h i c h eggs a n d l a rvae of t h e i c h n e u m o n i d w a s p Nemeritis canescens, w h i c h does n o t p r o d u c e t e r a t o c y t e s , w e r e i m p l a n t e d in to u n n a t u r a l hos t s . H e o b s e r v e d t h a t t h e eggs a n d l a rvae w e r e r a p i d l y e n c a p s u l a t e d , excep t in severa l hos t s t h a t h a d previous ly , b u t u n k n o w n to Sa l t , b e e n p a r a s i t i z e d b y a b r a c o n i d . I n e ach case h e o b s e r v e d t e r a tocy t e s a n d c o n s i d e r e d this to b e ev idence t h a t t e r a tocy t e s a r e a gene r i c w a y to p r e v e n t h o s t defense r eac t ions . Howeve r , t hese resu l t s m a y t o d a y b e


i n t e r p r e t e d to be d u e to the p r e s e n c e of t he p o l y d n a v i r u s e s ( V i n s o n a n d Stol tz , 1986).

K i t a n o (1969, 1974) a n d V i n s o n (1972) conf i rmed Sal t ' s o b s e r v a t i o n s b y d e m o n s t r a t i n g t h a t p a r a s i t o i d l a rvae were n o t e n c a p s u l a t e d w h e n t e r a tocy t e s were i n t r o d u c e d in to t he hos t ' s h e m o c o e l , even in t h e a b s e n c e of e i the r h o s t v e n o m o r ca lyx fluid (po lydnav i ru s ) . However , s u c h resu l t s m u s t b e in te r p r e t e d w i t h c a u t i o n as p o l y d n a v i r u s D N A s e q u e n c e s a lso o c c u r in t e r a tocy t e s ( D a h l m a n , 1991). I n o t h e r species , t he t e r a tocy te s c o n t r i b u t e to , b u t a r e n o t en t i re ly r e spons ib l e for, i nh ib i t ion of e n c a p s u l a t i o n ( T a n a k a a n d W a g o , 1990). V i n s o n (1972) found t h a t e n c a p s u l a t i o n w a s less effective w h e n the t e r a tocy te s h a d b e e n in t he hos t for severa l d a y s before t h e i n t r o d u c t i o n of t he p a r a s i t o i d l a rva . Genera l ly , y o u n g e r t e ra tocy tes were m o r e effective t h a n o lde r t e ra tocy tes in i nh ib i t i ng e n c a p s u l a t i o n .

Te ra tocy t e s m a y also c o n t r i b u t e to hos t i m m u n o s u p p r e s s i o n by i n h i b i t i n g h e m o l y m p h p h e n o l o x i d a s e ( P O ) activity. T h o u g h d e p r e s s e d P O act iv i ty in p a r a s i t i z e d la rvae was r e p o r t e d by S roka a n d V i n s o n (1978) , t he i n d e p e n d e n t c o n t r i b u t i o n of t e r a tocy tes to this p rocess h a s on ly recen t ly b e e n r e p o r t ed . K i t a n o et al. (1990) found t h a t P O inh ib i t i on w a s g r e a t e r in y o u n g e r p a r a s i t o i d s of C. glomerata (i .e. , y o u n g t e ra tocy tes ) . Howeve r , P O inh ib i t i on m a y no t have a n y re la t ion to p r e v e n t i o n of e n c a p s u l a t i o n b e c a u s e w h e n p r o P O w a s ac t iva ted w i t h in ject ions of z y m o s a n , as ev idenced by s ignif icant m e l a n i z a t i o n fo rma t ion , t he p a r a s i t o i d l a rvae were n o t e n c a p s u l a t e d . I n a n o t h e r species , only o lde r t e ra tocy tes of A. kariyai h a d a n i n h i b i t o r y effect o n hos t P O act ivi ty ( T a n a k a a n d W a g o , 1990). I n this case , t he P O i n h i b i t o ry factor was re leased in to t he m e d i u m w h e n t e r a tocy te s were i n c u b a t e d in Grace ' s m e d i u m for 48 hr .

C. Secretory Functions

E v i d e n c e a c q u i r e d from u l t r a s t r u c t u r a l s tud ies (Fig . 3A) a n d e x p e r i m e n t a l a p p r o a c h e s u s i n g in vitro t e c h n i q u e s c a n be used to d e m o n s t r a t e t h e sec re to ry n a t u r e of t e r a tocy te s . T h e u l t r a s t r u c t u r a l fea tures of t e r a tocy te s i n d i c a t e t h a t t hey a r e c a p a b l e of the syn thes i s a n d secre t ion of p r o t e i n a n d p e r h a p s o t h e r s u b s t a n c e s . M a n y of these s a m e fea tures a r e s h a r e d by t r o p h a m n i o n m e m b r a n e s (Koscielski et al., 1978) a n d nond i s soc i a t ed serosa l m e m b r a n e s (Lawrence , 1990). T h e specific a spec t s of the secre to ry role will va ry a c c o r d i n g to t he phys io logica l r e q u i r e m e n t s d i c t a t e d by the p a r a s i t o i d .

A l t h o u g h on ly o n e species of egg p a r a s i t o i d (Telenomus heliothidis, a. scel ionid) h a s b e e n s t ud i ed in de ta i l ( S t r a n d et al., 1985, 1986; S t r a n d , 1986), a m a j o r role of t e r a tocy tes in th is sys t em is to re lease e n z y m e s t h a t d iges t t h e hos t t issues a n d p rov ide n u t r i e n t s for t h e d e v e l o p i n g p a r a s i t o i d . T h e s e ter-


a tocy te s p r o d u c e l a rge n u m b e r s of e l e c t r o n - d e n s e ves icu la r bod ie s t h a t a r e p r e s u m a b l y re leased in to t he hos t egg. D e t e c t a b l e levels of ac id p h o s p h a t a s e , e s t e ra se , a n d l euc ine a m i n o p e p t i d a s e w e r e found in t e r a t o c y t e ex t r ac t s . I n a d d i t i o n , in vitro m e d i a to w h i c h t e r a tocy te s were a d d e d " c l e a r e d u p , " sug ges t ing a h y d r o l y t i c role of t he t e r a tocy te s o r a t e r a tocy t e sec re to ry p r o d u c t . T o o u r k n o w l e d g e , th is is t he on ly species ' t e r a tocy te s t h a t have b e e n s t u d i e d for sec re to ry e n z y m e s , b u t it s eems likely t h a t t e r a tocy te s of o t h e r egg p a r a s i to ids a l so w o r k in a s imi l a r m a n n e r . I t is unl ike ly t h a t t e r a tocy t e s assoc i a t e d w i t h p a r a s i t o i d s u s i n g l a rva l a n d a d u l t hos t s will secre te e n z y m e s t h a t i n d i s c r i m i n a t e l y d iges t hos t t i ssue b e c a u s e t he in tegr i ty of m o s t hos t s is m a i n t a i n e d by t h e p a r a s i t o i d un t i l t h e p a r a s i t o i d comple t e ly d e v o u r s t h e hos t ' s i n t e r n a l o r g a n s j u s t before t h e c o m p l e t i o n of l a rva l d e v e l o p m e n t . H o w ever, it is poss ib le t h a t t e r a tocy te s re lease e n z y m e s t h a t a t t a c k on ly specific t i ssues , for e x a m p l e , co l lagenese , w h i c h w o u l d a t t a ck t h e co l lagen s h e a t h s u r r o u n d i n g the fat body. S u c h ac t ion w o u l d p e r m i t select ive re lease of fat b o d y cells, w h i c h w o u l d n o t kill t he hos t , b u t w o u l d c o n t r i b u t e e x t r a n u t r i en t s to t h e p a r a s i t i z e d l a rva . T h i s m a y in fact a c c o u n t for t h e o f t en -no ted fr iabi l i ty of fat b o d y a n d t h e o p a l e s c e n c e of p a r a s i t i z e d hos t b lood . I t w o u l d a lso exp l a in e n h a n c e d in vitro t e r a t o c y t e g r o w t h in t he p r e s e n c e of fat b o d y ( G r e a n y et al., 1989) a n d d e c r e a s e d fat b o d y m a s s in p a r a s i t i z e d l a rvae ( D a h l m a n a n d V i n s o n , 1980; Z h a n g et al, 1992).

B r e w e r et al. (1973) , u s i n g na t ive p o l y a c r y l a m i d e gel e l ec t rophores i s , found severa l p r o t e i n s in t e r a tocy te s of C. nigriceps t h a t w e r e a b s e n t in n o n p a r a s i t i z e d h e m o l y m p h , b u t they d id no t c o m p a r e t h e p r o t e i n profile to h e m o l y m p h of n a t u r a l l y p a r a s i t i z e d l a rvae . I t s eems likely t h a t t e r a tocy t e s a r e a s o u r c e of these p r o t e i n s t h a t a p p e a r t o w a r d the e n d of t h e p a r a s i t o i d ' s life w i t h i n its hos t (Brewer et al., 1973; Beckage , 1990; L a w r e n c e , 1990). I n the i r 1980 review, V i n s o n a n d I w a n t s c h m e n t i o n e d t h a t t e r a tocy t e s r a p i d l y a b s o r b e d 1 4 C - l a b e l e d a m i n o ac ids , syn thes i zed p r o t e i n s , a n d sec re ted s o m e of t h e m back i n t o t he m e d i u m (Fig . 4B) . Beckage (1990) , u s i n g s imi l a r t e c h n i q u e s , r e p o r t e d t h a t t e r a tocy te s of Cotesia congregata syn thes i zed p r o t e i n s in vitro t h a t differed from those a s soc ia t ed w i t h t h e p a r a s i t o i d . D a h l m a n (1991) m a d e s imi la r o b s e r v a t i o n s w i t h t e r a tocy te s f rom M. croceipes, a p a r a s i toid of H. virescens a n d Helicoverpa zea.

I n a d d i t i o n to re lease of e n z y m e s , on ly two specific funct ions h a v e b e e n sugges t ed for t h e p r o t e i n a c e o u s t e r a t o c y t e sec re to ry p r o d u c t s . B o t h K i t a n o et al. (1990) a n d T a n a k a a n d W a g o (1990) bel ieve t h a t t e r a tocy t e s a r e r e spons i b le for i n h i b i t i n g P O act iv i ty in t h e h e m o l y m p h . I t is t h o u g h t t h a t P O inh ib i t i on m a y in ter fere w i t h t h e hos t ' s ab i l i ty to defend itself a g a i n s t t h e p a r a s i t o i d l a rva . A second specific ac t iv i ty of t e r a tocy t e s is t h e i n h i b i t i o n of h e m o l y m p h j u v e n i l e h o r m o n e es t e rase ( J H E ) . I t h a s b e e n k n o w n for severa l yea r s t h a t t h e J H E t i ters in H. virescens p a r a s i t i z e d b y M. croceipes w e r e


r e d u c e d ( D a h l m a n et al., 1990). Z h a n g a n d D a h l m a n (1989) a n d Z h a n g et al. (1992) conclus ive ly d e m o n s t r a t e d t h a t in ject ion of t e r a tocy t e s de r ived f rom in vivo sou rces comple t e ly i nh ib i t ed p r o d u c t i o n of J H E w h e n a t l eas t o n e e m b r y o e q u i v a l e n t of M. croceipes t e r a tocy te s w a s u sed . D a h l m a n (1991) found t h a t c u l t u r e m e d i a in w h i c h in vitro M. croceipes t e r a tocy t e s h a d b e e n he ld ( b u t no t con t ro l m e d i a ) c a u s e d l a rva l m o r t a l i t y a n d d e l a y e d g r o w t h w h e n injected i n t o y o u n g fifth-ins t a r H. virescens. I t a l so is i m p o r t a n t to n o t e t h a t H a y a k a w a (1990) pur i f ied a n d r e p o r t e d o n t h e s t r u c t u r e of ( H a y a k a w a , 1991) a 4 . 5 -kD p e p t i d e from t h e h e m o l y m p h of Pseudaletia separata p a r a s i t i z e d b y A. kariyai t h a t r ep re s sed J H E act ivi ty w h e n in jec ted i n t o n o n p a r a s i t i z e d hos t s . I t is r e a s o n a b l e to p o s t u l a t e t h a t it is a t e r a t o c y t e p r o d u c t .

Sec re t ion of n o n p r o t e i n p r o d u c t s by t e r a tocy te s is a l so poss ib le . F u h r e r a n d Elsufty (1979) found a fungis ta t ic s u b s t a n c e in t h e t e r a tocy te s of C. glomerata. J o i n e r et al. (1973) r e p o r t e d j u v e n i l e h o r m o n e ( J H ) ac t iv i ty in ex t rac t s f rom C. nigriceps t e r a tocy te s . E x t r a c t s f rom o lde r t e r a tocy te s h a d g r e a t e r J H act iv i ty in t he Galleria b ioassay. T h e y w e r e n o t su r e , a n d it h a s n o t b e e n d e m o n s t r a t e d in a n y o t h e r sy s t em w h e t h e r t he t e r a tocy t e s syn thes i zed J H o r w h e t h e r t hey s imp ly se rved as a d e p o t for J H poss ib ly a c q u i r e d f rom the hos t . A l t h o u g h Locusta migratoria is n o t a p a r a s i t o i d , t h e fact t h a t i ts serosa l cells syn thes ize J H ( H a r t m a n n et aL, 1987) gives c red ib i l i ty to t h e r e p o r t of J o i n e r et al. (1973) . G r o s s n i k l a u s - B u r g i n a n d L a n z r e i n (1990) sug ges ted t h a t t e r a tocy tes from a Chelonus species r e l eased J H (no ev idence p r o v i d e d ) , w h e r e a s Beckage a n d Ridd i fo rd (1982) found on ly s l ight J H activity in t he ce l lu la r fraction (which w o u l d i n c l u d e t h e t e ra tocy tes ) f rom h e m o l y m p h of Manduca sexta p a r a s i t i z e d b y C. congregata.

D. General Effects on Host Growth and Development

E x p e r i m e n t s t h a t inves t iga ted t he poss ib le role of t e r a tocy te s in t h e a b s e n c e of c o n f o u n d i n g factors such as t h e p a r a s i t o i d l a rva , po i son g l a n d p r o d u c t s , a n d / o r ca lyx fluid (po lydnav i ru s ) were first c o n d u c t e d by V i n s o n (1970) . H e showed t h a t y o u n g e r t e ra tocy tes f rom C. nigriceps d e l a y e d H. virescens l a rva l d e v e l o p m e n t m o r e t h a n o lde r t e r a tocy te s . M o s t of t h e t r e a t e d l a rvae e v e n t u ally p u p a t e d or deve loped a b n o r m a l i t i e s r e su l t i ng from i n c o m p l e t e l a r v a l -p u p a l ecdyses . Z h a n g a n d D a h l m a n (1989) , in the i r s tud ies w i t h M. croceipes, o b s e r v e d b o t h age - a n d d o s e - d e p e n d e n t effects of t e r a tocy t e s . T h e s e effects w e r e exp res sed as de l ayed la rva l g r o w t h a n d m o r t a l i t y a n d i n c o m p l e t e l a r v a l - p u p a l ecdys is . W a n i et al. (1990) found t h a t o lde r t e r a tocy t e s of A. kariyai p r o l o n g e d the l a rva l s t age of i ts hos t , P. separata. Recen t ly P e n n a c c h i o et al. (1992a) c o n d u c t e d a de t a i l ed e x a m i n a t i o n of t h e C. nignceps-H. virescens p a r a s i t e - h o s t c o m p l e x . T h e y showed t h a t h i g h e r doses of t e r a tocy t e s (equiva l en t to 2, 4 , o r 8 e m b r y o equ iva len t s ) h a d a g r e a t e r i m p a c t o n t h e p r e v e n t i o n


of l a r v a l - p u p a l ecdys is . T h e a g e of t h e h o s t a t t h e t i m e of t r e a t m e n t a l so w a s i m p o r t a n t , w i t h o lde r l a rvae b e i n g less r e spons ive . I t a l so w a s i n t e r e s t i n g to n o t e t h a t even t h e a p p a r e n t l y " n o r m a l " p u p a e o b t a i n e d f rom t e r a t o c y t e -t r e a t e d l a rvae failed to deve lop i n t o a d u l t s . Z h a n g a n d D a h l m a n (1989) found t h a t even a 0 .25 e m b r y o e q u i v a l e n t of t e r a tocy te s p r e v e n t e d p u p a t i o n of 8 0 % of t h e t r e a t e d m a t u r e H. virescens l a rvae . Howeve r , a t l eas t s o m e of t h e p u p a e t h a t d id form d e v e l o p e d i n t o a d u l t s . T h e y a lso r e p o r t e d t h a t y o u n g e r hos t s w e r e m o r e sensi t ive a n d t h a t y o u n g e r t e r a tocy te s w e r e m o r e p o t e n t .

Z h a n g a n d D a h l m a n (1989) o b s e r v e d t h a t in jected t e r a tocy t e s of M. croceipes c a u s e d m a n y of t h e s a m e d e v e l o p m e n t a l a b e r r a t i o n s o b s e r v e d in t r u ly p a r a s i t i z e d hos t s . D a h l m a n et al. (1990) r e p o r t e d r e d u c e d levels of ecd y s t e r o i d in p a r a s i t i z e d H. virescens a t t h e t i m e of t h e n o r m a l s u r g e a s soc i a t ed w i t h la rva l—pupal ecdys is . Z h a n g et al. (1992) found a s imi l a r r e d u c t i o n in ecdys t e ro id t i te r in t e r a t o c y t e - t r e a t e d l a rvae . I n c o n t r a s t , in t h e ve ry s a m e h o s t b u t w i t h a different b r a c o n i d p a r a s i t o i d (C. nigriceps), P e n n a c c h i o et al. ( 1 9 9 1 , 1992a) r e p o r t e d a threefold e leva t ion of ecdys t e ro id t i te r in l a rvae rece iv ing two e m b r y o e q u i v a l e n t s of t e r a tocy te s a s n e w fifth i n s t a r s . T h e y a t t e m p t e d to exp la in th is difference b y sugges t i ng i n c r e a s e d p r o d u c t i o n of e cdys t e ro id s as t h e resu l t of in te r fe rence w i t h n o r m a l feedback m e c h a n i s m s t h a t con t ro l t h e r a t e of ecdys t e ro id syn thes i s . T h e y a lso r ecogn ized t h a t inac t ive ecdys t e ro id d e g r a d a t i o n p r o d u c t s m i g h t r e s p o n d in t h e R I A u s e d to quan t i fy ecdys t e ro id t i ter , t h u s g iv ing falsely h igh resu l t s .

I t is c u r i o u s t h a t t he t e r a tocy te s f rom two la rva l e n d o p a r a s i t o i d s u s i n g t h e s a m e h o s t species w o u l d p r o d u c e s u c h different resu l t s in ecdys t e ro id t i ter . H o w e v e r , w h e n t h e hos t is t ru ly p a r a s i t i z e d b y e i the r spec ies , e cdys t e ro id t i t e rs a r e r e d u c e d (Dover et al., 1987; D a h l m a n et al., 1990; P e n n a c c h i o et al., 1991 , 1993b) . I t m u s t b e k e p t in m i n d t h a t t h e m o d e of feeding b y these t w o p a r a s i t o i d s is q u i t e different . Cardiochiles nigriceps feeds o n h e m o l y m p h t h r o u g h t h e first few d a y s following h a t c h b u t t h e n beg ins to feed o n se lec ted h o s t t i ssue un t i l t h e t i m e it e m e r g e s from its hos t a n d c o n s u m e s t h e e n t i r e c a r c a s s ( V i n s o n a n d B a r r a s , 1970). P e n n a c c h i o et al. (1992 , 1993b) p r o p o s e d t h a t t h e t e r a tocy t e s f rom C. nigriceps r e g u l a t e p r o t e i n m e t a b o l i s m , poss ib ly b y i nac t i va t i on of 2 0 - h y d r o x y e c d y s o n e . T h i s resu l t s in i n c r e a s e d t i t e rs of h e m o l y m p h p r o t e i n s a t a t i m e c o r r e l a t e d w i t h r a p i d p a r a s i t o i d l a rva l g r o w t h . T h u s , t h e p a r a s i t o i d m o r e efficiently explo i t s its n a t u r a l food s o u r c e a n d r e a c h e s t h e cr i t ica l size neces sa ry for m o l t i n g a n d ex i t ing f rom its hos t . I n c o n t r a s t , M. croceipes a p p a r e n t l y der ives its n u t r i e n t s f rom its hos t ' s h e m o l y m p h a l t h o u g h t h e hos t ' s fat b o d y fails to p r o p e r l y d e v e l o p ( D a h l m a n a n d V i n s o n , 1980). T h e hos t m a y r e m a i n al ive ( a l t h o u g h inac t ive a n d d e v e l o p i n g n o fu r the r t h a n t h e cell f o r m a t i o n s t age ; W e b b a n d D a h l m a n , 1985) for m o r e t h a n a week after t h e p a r a s i t o i d e m e r g e s ( V i n s o n a n d Lewis , 1973). U n d o u b t e d l y v a r i o u s feedback m e c h a n i s m s m u s t b e involved w i t h b o t h of t hese


p a r a s i t o i d - h o s t i n t e rac t ions wi th respec t to the i r different m e a n s of interfering w i t h t h e h o r m o n a l t i ters of the i r hos t s .

VI. Interactions of Teratocytes, Venoms, and Polydnavirus

A s u b s t a n t i a l n u m b e r of s tud ies have b e e n c o n d u c t e d o n t h e i m p o r t a n c e of i n t e r ac t ions b e t w e e n the p o l y d n a v i r u s e s a n d v e n o m s of p a r a s i t i c w a s p s ( T a n aka , 1987; D o v e r et al., 1987, 1988; Stol tz etal, 1988; S t r a n d a n d Dover , 1991; T a n a k a a n d V i n s o n , 1991). However , re la t ively l i t t le of th is r e s e a r c h h a s i n c l u d e d t e r a tocy te s as a t h i rd c o m p o n e n t in t he tes ts . W a n i et al. (1990) showed t h a t y o u n g t e r a tocy te s from A. kariyai d id n o t i nh ib i t g r o w t h a n d d e v e l o p m e n t of t he hos t . However , w h e n hos t s were first in jec ted w i t h v e n o m a n d ca lyx fluid a n d s u b s e q u e n t l y in jec ted w i t h y o u n g t e r a tocy t e s a t t h e a p p r o x i m a t e t i m e t e ra tocy tes w o u l d n o r m a l l y be re leased in a p a r a s i t i z e d hos t , b o t h l a r v a l - l a r v a l a n d l a r v a l - p u p a l d e v e l o p m e n t w e r e i m p a i r e d . S t r a n d a n d W o n g (1991) found t h a t a n e m b r y o e q u i v a l e n t dose of Microplitis demolitor t e r a tocy tes h a d a n incons i s t en t effect on t he d e v e l o p m e n t of its hos t , Pseudoplusia includens, b u t w h e n the inject ion of t e r a tocy tes w a s p r e c e d e d w i t h a n inject ion of ca lyx fluid o r p o l y d n a v i r u s p lus v e n o m , t h e d e v e l o p m e n t a l a l t e r a t i ons were very s imi la r to those obse rved d u r i n g n o r m a l p a r a s i t i s m . I n fact, D. molitor t e r a tocy tes were e n c a p s u l a t e d w h e n in jec ted in to n o n p a r a s i t i z e d P. includens l a rvae b u t r e m a i n e d u n e n c a p s u l a t e d w h e n in jec ted in to hos t s t h a t h a d p rev ious ly b e e n injected w i t h a m i x t u r e of p o l y d n a v i r u s a n d v e n o m ( S t r a n d a n d N o d a , 1991). P e n n a c c h i o et al. (1992a) sugges t t h a t i nh ib i t i on of ecdys te ro id p r o d u c t i o n by v e n o m a n d p o l y d n a v i r u s exp l a in s w h y ecdys te ro id t i ters a r e low in p a r a s i t i z e d hos t s as c o m p a r e d to t h e h i g h t i ters seen w h e n hos t s a r e on ly t r e a t e d w i th t e r a tocy te s . J o n e s (1987) conc l u d e d t h a t p o l y d n a v i r u s a n d v e n o m ( b u t n o t t e ra tocy tes ) w e r e n e c e s s a r y for t h e p r o d u c t i o n of a factor t h a t c a u s e d p recoc ious m e t a m o r p h o s i s / s u p p r e s s e d p r e p u p a l d e v e l o p m e n t in Trichoplusia ni p a r a s i t i z e d by a Chelonus sp . T a n a k a (1987) found t h a t a b o u t 5 0 % of t h e Microplitis mediator eggs w i t h comple t e ly d e v e l o p e d se rosa l m e m b r a n e s (nond i s soc i a t ed t e ra tocy tes ) w e r e p r o t e c t e d f rom e n c a p s u l a t i o n w h e n injected w i t h ca lyx fluid, w h e r e a s n o n e w e r e p r o tec ted w i t h o u t t he ca lyx fluid.

VII. Conclusion and Summary

Tera tocy t e s a r e u n u s u a l e x t r a e m b r y o n i c cells a s soc ia t ed w i t h ce r t a in g r o u p s of e n d o p a r a s i t i c H y m e n o p t e r a . T h e y p l a y a n i m p o r t a n t , b u t i n c o m p l e t e l y


def ined , role in t h e to ta l i n t e r ac t i on b e t w e e n the l a rva l s t age of t h e p a r a s i t o i d

a n d its hos t . I t is n o t c lea r w h y they a r e r e s t r i c t ed to on ly c e r t a i n famil ies ,

a l t h o u g h it is l ikely t h a t r e l a t ed t i ssues p e r f o r m s imi l a r roles in o t h e r g r o u p s

of p a r a s i t o i d s . Specific funct ions v a r y b e t w e e n p a r a s i t o i d species a n d t h e

d e v e l o p m e n t a l s t age of t he hos t . T r o p h i c , i m m u n o s u p p r e s s i v e , a n d sec re to ry

func t ions h a v e b e e n well d o c u m e n t e d . T i t e r s of t he p r inc ip l e h o r m o n e s in t h e

h o s t m a y b e a l t e r e d by t e r a tocy t e s . I n t e r a c t i o n s a t severa l levels of c o m p l e x

ity a r e poss ib le , n o t on ly b e t w e e n t h e t e r a tocy te s a n d t h e hos t b u t a l so

b e t w e e n o t h e r p a r a s i t o i d - d e r i v e d factors s u c h a s v e n o m a n d n o n p a t h o g e n i c

p o l y d n a v i r u s e s r e s i d e n t w i t h i n b o t h t h e p a r a s i t o i d a n d t h e e x t r a e m b r y o n i c

t i ssues . I t is likely t h a t t he success of t h e p a r a s i t o i d is d e p e n d e n t u p o n each

t ype of i n t e r ac t i on . M u c h r e m a i n s to b e l e a r n e d a n d t h e c o m i n g d e c a d e will

y ie ld c o n s i d e r a b l e i n fo rma t ion o n th is c o m p l e x a n d i n t e r e s t i ng a s p e c t of

insec t pa ras i to logy .

Acknowledgments

We gratefully acknowledge the support of USDA Competitive Research Grant 89-37250-4705 (D.L.D.) and a grant from R.J . Reynolds (D.L.D.). We are grateful for the critical review and constructive suggestions provided by Dr. P. D. Greany. This is paper 91-07-147 of the Kentucky Agriculture Experiment Station, Lexington, KY 40546-0091.

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Chapter 8

The Polydnavirus Life Cycle Donald B. Stoltz Department of Microbiology and Immunology Dalhousie University Halifax, Nova Scotia, Canada

T h e p o l y d n a v i r u s e s c o m p r i s e a g r o u p of r e m a r k a b l e a g e n t s w h o s e life cycles a r e i nex t r i c ab ly l inked w i t h those of ce r t a in p a r a s i t i c H y m e n o p t e r a ( p a r a s i to ids) . T h e n a t u r e of t h a t l i nkage , a n d in p a r t i c u l a r its b io logica l conse q u e n c e s , is t he sub jec t of th is a r t ic le .

T h e s e v i ruses a r e a s s igned to t he family Po lydnav i r i dae p r i m a r i l y o n t h e bas i s of b o t h g e n o m e s t r u c t u r e a n d hos t r a n g e , a l t h o u g h o t h e r factors s u c h as m o r p h o l o g y a n d si te of r ep l i ca t ion a r e equa l l y va l id as d i a g n o s t i c i n d i c a t o r s for t h e family (Stol tz et al., 1984). W h e n ex t r ac t ed f rom pur i f ied v i r ions , t h e p o l y d n a v i r u s g e n o m e is seen to cons is t of a p o p u l a t i o n of d o u b l e - s t r a n d e d c i r cu l a r D N A s ; w i t h i n th is p o p u l a t i o n , t h e r e exist a n u m b e r of different c lasses of mo lecu le s t h a t differ in t e r m s of b o t h m o l e c u l a r w e i g h t a n d gene t i c c o n t e n t (Kre l l a n d Stol tz , 1980; K r e l l et al., 1982; T h e i l m a n n a n d S u m m e r s , 1988; G u z o , 1988). L i n e a r D N A s e q u e n c e s h o m o l o g o u s to e n c a p s i d a t e d v i ra l D N A s a r e cova len t ly l inked to p a r a s i t o i d c h r o m o s o m a l D N A ( F l e m i n g a n d S u m m e r s , 1986; X u a n d Stol tz , 1991; see references in F l e m i n g a n d K r e l l , V o l u m e 1, C h a p t e r 9 ) ; th is a p p e a r s to be a s t a b l e r e l a t i onsh ip , n o w t h o u g h t to be r e q u i r e d for t h e t r a n s m i s s i o n of p o l y d n a v i r u s e s w i t h i n p a r a s i t o i d p o p u l a t ions (Sto l tz , 1990).

T h e p o l y d n a v i r u s family cons is t s of two l a rge g r o u p s of v i ruses , w h i c h

I. Introduction III. Future Directions Acknowledgments References

II. Life Cycle A. Transmission and Replication B. Genetic Colonization of the Host

I. Introduction



8. The Polydnavirus Life Cycle 169

have b e e n def ined o n t h e bas i s of cons i s t en t differences in b o t h m o r p h o l o g y a n d hos t r a n g e . Unofficially, these h a v e b e e n referred to as t h e b r a c o - a n d i chnov i ru s g r o u p s . Bracov i rus pa r t i c l e s cons is t of cy l ind r i ca l n u c l e o c a p s i d s e n v e l o p e d by a s ingle u n i t m e m b r a n e ; t hese have b e e n o b s e r v e d on ly in c e r t a i n species of b r a c o n i d p a r a s i t o i d s . I c h n o v i r u s pa r t i c l e s cons is t of fus i form n u c l e o c a p s i d s s u r r o u n d e d by two u n i t m e m b r a n e enve lopes ; t hese a r e r e s t r i c t ed to t h e I c h n e u m o n i d a e . Typ ica l e x a m p l e s of p o l y d n a v i r u s e s a n d ( e n c a p s i d a t e d ) p o l y d n a v i r u s g e n o m e s a r e i l l u s t r a t ed in F ig . 1.

V i r a l r ep l i ca t ion is r e s t r i c t ed to t h e p a r a s i t o i d ovary. W h e n m a t u r e , virions e n t e r t h e l u m e n of t he r e p r o d u c t i v e t rac t , w h e r e t hey c o m p r i s e t h e p a r t i c u l a t e f ract ion of a so-cal led " ca lyx fluid" ( N o r t o n et al., 1975; S to l tz a n d V i n s o n , 1979a) ; th i s m a t e r i a l , t o g e t h e r w i t h o n e o r m o r e p a r a s i t o i d eggs , is in jec ted i n t o hos t a n i m a l s d u r i n g ovipos i t ion (Stol tz a n d V i n s o n , 1979a) . R e p l i c a t i o n in p a r a s i t i z e d a n i m a l s , however , h a s neve r b e e n o b s e r v e d (Stol tz a n d V i n s o n , 1979a; T h e i l m a n n a n d S u m m e r s , 1986; D o v e r et al., 1989), a n d is there fore a s s u m e d no t to occur .

II. Life Cycle Typical ly , a v i ru s life cycle cons is t s of t r a n s m i s s i o n a n d r ep l i ca t i on w i t h i n a su scep t ib l e hos t ; s t r ic t ly s p e a k i n g , b o t h of these events a r e in t h e case of p o l y d n a v i r u s e s r e s t r i c t ed to ce r t a in p a r a s i t o i d spec ies . T h e p o l y d n a v i r u s life cycle , however , m u s t b e c o n s i d e r e d in a m u c h b r o a d e r con tex t , s ince t r a n s mis s ion a n d r ep l i ca t ion w i t h i n p a r a s i t o i d p o p u l a t i o n s w o u l d n o t be poss ib le in t h e a b s e n c e of a n a d d i t i o n a l e l e m e n t , name ly , t hose insec ts t h a t serve as hos t s for p a r a s i t o i d s . A s w e n o w know, p o l y d n a v i r u s e s a r e i n v a r i a b l y p r e s e n t in hos t a n i m a l s w h e r e they ac t to e n s u r e successful p a r a s i t i s m (see Sec t ion I I . B ) . I n sho r t , t h e ob l i ga to ry l i nkage b e t w e e n p a r a s i t o i d a n d h o s t r e q u i r e s t he p r e s e n c e of p o l y d n a v i r u s in b o t h . I therefore a r g u e t h a t t h e p o l y d n a v i r u s life cycle , un l ike t h a t of o t h e r v i ruses , in fact c o m p r i s e s two a r m s . T h e s e — t r a n s m i s s i o n / r e p l i c a t i o n , a n d w h a t m a y b e referred to as a gene t i c co lon iza t ion of t h e hos t (Stol tz et al., 1 9 8 6 ) — a p p e a r , respect ively, to be m e d i a t e d b y two d i s t i nc t forms of virus-specif ic D N A . Specifically, t r a n s m i s s i o n a n d rep l i ca t i on w o u l d a p p e a r to b e d i r ec t ed b y a p e r m a n e n t l y i n t e g r a t e d v i ra l g e n o m e

Figure 1 Typical examples of polydnavirus particles and genomes extracted therefrom. Below left is an electron micrograph of a bracovirus (from Protapanteles pal-eacritae) and, to the right, an ichnovirus (from Hyposoter exiguae). Above these, agarose gel electrophoretic profiles of DNAs extracted from virus particles are seen after ethidium bromide staining. Left, bracovirus DNA from Cardiochiles nigriceps; right, ichnovirus from H. rivalis.

170 Donald Β. Stoltz

(i .e. , a p rov i rus ) , w h e r e a s a gene t i c co lon iza t ion of t h e h o s t is effected b y c i r cu l a r molecu les de l ivered in e n c a p s i d a t e d form to hos t cells a n d t i ssues d u r i n g ovipos i t ion .

I n w h a t follows, I will dea l briefly w i th t r a n s m i s s i o n , s ince th is sub jec t h a s b e e n recen t ly t r e a t e d in s o m e d e p t h (Stol tz , 1990), a n d even m o r e briefly w i t h r ep l i ca t ion , s ince a l m o s t n o t h i n g is k n o w n a b o u t h o w this o c c u r s ( F l e m ing a n d K r e l l , V o l u m e 1, C h a p t e r 9) . C o n s i d e r a b l y m o r e a t t e n t i o n will b e g iven to t he sub jec t of v i rus -d i r ec t ed act ivi ty in t he hos t , s ince th is a s p e c t of p o l y d n a v i r u s b io logy h a s n o t recen t ly b e e n rev iewed .

A. Transmission and Replication

I t h a s b e e n c lear for s o m e t i m e t h a t p o l y d n a v i r u s t r a n s m i s s i o n o c c u r s w i t h 1 0 0 % efficiency, m a k i n g ver t ica l t ransfer t h r o u g h g e r m l ine t i ssue likely. Neve r the l e s s , it was ini t ia l ly cons ide red poss ib le t h a t d e v e l o p i n g p a r a s i t o i d l a rvae cou ld b e c o m e infected per os, by c o n s u m i n g v i ra l D N A de l ive red to t h e h o s t d u r i n g ovipos i t ion . Moreove r , it is k n o w n t h a t s u c h D N A c a n pers i s t t h r o u g h o u t t h e n a t u r a l course of p a r a s i t i z a t i o n ( T h e i l m a n n a n d S u m m e r s , 1986; Stol tz et al., 1986). T h e avai labi l i ty of isofemale l ines (i .e. , d e r i ved f rom a s ingle female) c a r r y i n g m a r k e r p o l y d n a v i r u s D N A circles p e r m i t t e d t h e des ign of e x p e r i m e n t s a i m e d a t d e t e r m i n i n g w h e t h e r per os t r a n s m i s s i o n d i d in fact occur . I n o n e s u c h e x p e r i m e n t , w a s h e d eggs f rom a Cotesia melanoscela l ine w e r e co- injected a l o n g w i t h b racov i rus from a l ine t h a t c a r r i e d a n e x t r a g e n o m e s e g m e n t ; n o n e of t he a d u l t female w a s p s r e su l t i ng from th is expe r i m e n t ca r r i ed t h a t g e n o m e s e g m e n t , sugges t i ng t h a t l a rvae e i t he r d i d n o t c o n s u m e s u c h D N A , or else cou ld n o t b e c o m e infected b y t h a t r o u t e . I n t h e case of t he i c h n e u m o n i d p a r a s i t o i d Hyposoter fiigitivus, w a s h e d eggs w e r e a b l e to deve lop to m a t u r i t y in Malacosoma americana; a d u l t females still c a r r i e d t h e n o r m a l p o l y d n a v i r u s a s soc ia t ed w i t h t h a t species , sugges t ing that per os infect ion w o u l d p r o b a b l y b e i r r e l evan t to p o l y d n a v i r u s t r a n s m i s s i o n , even if it did o c c u r (Stol tz et al., 1986).

I sofemale l ines were a lso e m p l o y e d in gene t i c c ross ing e x p e r i m e n t s , t h e resu l t s of w h i c h c lear ly d e m o n s t r a t e d t h a t b o t h b r a c o - a n d i chnov i ru s ge n o m e s e g m e n t s s eg rega t e in M e n d e l i a n fashion. I t w a s c o n c l u d e d t h a t t r a n s mis s ion of t he p o l y d n a v i r u s g e n o m e m o s t likely r e q u i r e s a phys i ca l l i nkage to t he p a r a s i t o i d g e n o m e ; a c h r o m o s o m a l m o d e l for p o l y d n a v i r u s t r a n s m i s s i o n h a s b e e n d i scussed b y Stol tz (1990) . M o l e c u l a r ev idence for c h r o m o s o m a l i n t e g r a t i o n of i chnov i rus g e n o m e s e g m e n t s h a d p rev ious ly b e e n d e v e l o p e d b y F l e m i n g a n d S u m m e r s (1986) , a n d m o r e r ecen t o b s e r v a t i o n s n o w s t rong ly sugges t t h a t c h r o m o s o m a l i n t e g r a t i o n is a l m o s t ce r t a in ly a g e n e r a l f ea tu re of t h e p o l y d n a v i r u s family ( X u a n d Stol tz , 1991; F l e m i n g a n d K r e l l , V o l u m e 1, C h a p t e r 9) . I n sho r t , b o t h gene t i c a n d m o l e c u l a r a p p r o a c h e s to t h e q u e s t i o n


of p o l y d n a v i r u s t r a n s m i s s i o n h a v e i n d e p e n d e n t l y led to t h e s a m e conc lus ion : t h e p o l y d n a v i r u s g e n o m e is t r a n s m i t t e d in t h e form of a n e n d o g e n o u s p r o v i r u s .

P o l y d n a v i r u s r ep l i ca t ion is r e s t r i c t ed to t h e p a r a s i t o i d ovary, a n d rep l i ca t ion b e g i n s in t h e p u p a l s t age ( N o r t o n a n d V i n s o n , 1983; T h e i l m a n n a n d S u m m e r s , 1986) a n d c o n t i n u e s t h r o u g h o u t t h e life of t h e a d u l t female . I t h a s b e e n sugges t ed t h a t r ep l i ca t ion of v i ra l D N A m i g h t b e t r iggered b y h o r m o n a l c h a n g e s a s soc i a t ed w i t h m e t a m o r p h o s i s a n d / o r m o r p h o g e n e s i s of t h e ova ry ( N o r t o n a n d V i n s o n , 1983). I n k e e p i n g w i t h th is s u p p o s i t i o n , W e b b a n d S u m m e r s (1992) h a v e r ecen t ly d e m o n s t r a t e d t h a t v i ra l D N A rep l i c a t i on in e x p l a n t e d ovar ies c a n b e i n d u c e d b y t h e a d d i t i o n of e c d y s o n e . T h e ava i lab i l i ty of in vitro s y s t ems s u c h as these s h o u l d very qu ick ly l ead to a n u n d e r s t a n d i n g of t h e m o l e c u l a r bas i s of v i ra l r ep l i ca t ion .

T h e q u e s t i o n of w h i c h form of v i ra l D N A serves as t e m p l a t e for t h e syn thes i s of t h e c i r cu l a r mo lecu le s t h a t a r e d e s t i n e d for e n c a p s i d a t i o n r e m a i n s as ye t u n a n s w e r e d . I n th is r e g a r d , it s h o u l d first b e n o t e d t h a t b o t h l i nea r ( i n t e g r a t e d ) a n d c i r cu l a r ( e x t r a c h r o m o s o m a l ) fo rms of v i r a l D N A a r e t h o u g h t to b e p r e s e n t in m o s t if n o t all p a r a s i t o i d t i ssues ( F l e m i n g a n d S u m m e r s , 1986; Sto l tz et aL, 1986), even in t h e a b s e n c e of v i r a l r ep l i ca t i on . T h u s , it m i g h t r e a s o n a b l y b e a s s u m e d t h a t a t leas t a few e x t r a c h r o m o s o m a l v i ra l D N A s a r e p r e s e n t in o v a r i a n p r i m o r d i a , a n d therefore a r e p o t e n t i a l l y ava i l ab le to serve l a t e r on a s r ep l i ca t ion t e m p l a t e s . Howeve r , T h e i l m a n n a n d S u m m e r s (1986) w e r e u n a b l e to d e t e c t t h e c i r cu l a r form of C s V g e n o m e s e g m e n t Β d u r i n g t h e ea r ly p u p a l s t ages of Campoletis sonorensis; t h e c o g n a t e ( c o r r e s p o n d i n g ) l i nea r c h r o m o s o m a l s e q u e n c e s , o n t h e o t h e r h a n d , w e r e o b s e r v e d . T h i s s t u d y sugges t s t h a t e x t r a c h r o m o s o m a l p o l y d n a v i r u s D N A s a r e n o t p r e s e n t in t h e p a r a s i t o i d ova ry p r i o r to v i ra l D N A rep l i ca t i on , in t u r n sugges t i ng t h a t l i nea r t e m p l a t e s m a y b e u t i l ized . G e n e t i c e x p e r i m e n t a t i o n h a s p r o v i d e d a d d i t i o n a l ev idence to t h e effect t h a t e x t r a c h r o m o s o m a l m o l e cules p l a y n o role in d e t e r m i n i n g t h e c o m p o s i t i o n of t h e p o l y d n a v i r u s gen o m e (Sto l tz , 1990). T h e r e f o r e , s u c h molecu le s a r e e i the r in s o m e m a n n e r e l i m i n a t e d f rom t h e ova ry p r i o r to t h e onse t of v i ra l D N A rep l i ca t ion (or w e r e n e v e r p r e s e n t in t h a t p a r t i c u l a r o r g a n ) o r else d o n o t serve as D N A rep l i ca t ion t e m p l a t e s .

I n a n y case , g iven t h a t p o l y d n a v i r u s g e n o m e s a r e c h r o m o s o m a l l y t r a n s m i t t e d (Stol tz , 1990), it s eems likely t h a t l i nea r c h r o m o s o m a l D N A seq u e n c e s m u s t — a t leas t i n i t i a l l y — r e p r e s e n t a p o i n t of d e p a r t u r e in t h e a m p l i f i c a t i o n / r e p l i c a t i o n of p o l y d n a v i r u s D N A ; h o w th is m i g h t o c c u r r e m a i n s to b e d e t e r m i n e d , b u t severa l poss ibi l i t ies a r e p l a u s i b l e . Fo r e x a m p l e , a m p l i fication of v i ra l D N A cou ld in t h e o r y o c c u r in situ ( i .e. , a t c h r o m o s o m a l loci) , c i r cu l a r v i ra l D N A s s u b s e q u e n t l y b e i n g g e n e r a t e d v ia h o m o l o g o u s r e c o m b i n a t i o n ; a l te rna t ive ly , excised, p r e s u m a b l y c i rcu la r , copies of c h r o m o s o m a l loci m i g h t serve as t e m p l a t e s for D N A rep l i ca t ion . Final ly, s u c h loci m i g h t b e


excised in the i r en t i r e ty p r io r to ampl i f i ca t ion , leaving n o l inea r c h r o m o s o m al copy b e h i n d . As a w o r k i n g h y p o t h e s i s , I p r e s en t l y favor t he first m e c h a n i s m , s ince t h e resu l t s of gene t i c c ross ing e x p e r i m e n t s t e n d to mi l i t a t e a g a i n s t a n i n v o l v e m e n t of c i rcu lar , e x t r a c h r o m o s o m a l D N A t e m p l a t e s in p o l y d n a v i r u s r ep l i ca t ion (unless p o t e n t i a l c i r cu la r r ep l i ca t ion t e m p l a t e s a r e r e m o v e d from the ovary p r i o r to t he onse t of v i ra l D N A rep l i ca t ion ) . Exc i s ion of c i r cu l a r D N A s m u s t in a n y case p r e s u m a b l y o c c u r a t s o m e p o i n t in t h e r ep l i ca t ion cycle, a n d r ecen t s e q u e n c i n g d a t a h a v e b e e n i n t e r p r e t e d b y F l e m ing a n d S u m m e r s (1990) as sugges t ing a poss ib le role for t h e C s V 5 4 0 - b p r e p e a t e l e m e n t in t h a t p rocess . I t s h o u l d be n o t e d t h a t if p o l y d n a v i r u s gen o m e s e g m e n t s a r e g e n e r a t e d from l inea r c h r o m o s o m a l t e m p l a t e s , t h e n a m plif icat ion of a n y s u c h t e m p l a t e s need no t be ex tens ive ; i n s t e a d , excis ion cou ld in s o m e w a y be c o u p l e d to D N A rep l i ca t ion .

T h e so-cal led " o n i o n s k i n " m o d e l for in situ ampl i f i ca t ion (see O s h e i m a n d Mil le r , 1983; S t a r k et al., 1989) m a y n o t b e en t i re ly a p p r o p r i a t e h e r e , a n d w o u l d in a n y case be i n c o m p a t i b l e w i t h d a t a r e p o r t e d by T h e i l m a n n a n d S u m m e r s (1986) to t h e effect t h a t t he a m o u n t of c h r o m o s o m a l p o l y d n a v i r u s -specific D N A r e m a i n s c o n s t a n t d u r i n g p u p a t i o n , w h e n v i ra l r ep l i ca t ion first beg ins .

E a c h of t he r ep l i ca t ion scena r ios l is ted he r e a l lows ce r t a in p r e d i c t i o n s to be m a d e . A n in situ ampl i f i ca t ion m o d e l , for e x a m p l e , w o u l d n o t necessa r i ly r e q u i r e t h a t s e q u e n c e s r e s p o n d i n g to r ep l i ca t ion s igna ls b e loca t ed o n ex t r a -c h r o m o s o m a l D N A s ; they cou ld i n s t e a d b e loca ted w i t h i n flanking c h r o m o s o m a l s e q u e n c e s . O n the o t h e r h a n d , t he d e m o n s t r a t e d pe r s i s t ence of po lyd n a v i r u s D N A in t h e p a r a s i t i z e d hos t (see t he following) m i g h t well r e q u i r e s o m e t h i n g e q u i v a l e n t to a n a u t o n o m o u s l y r ep l i ca t i ng s e q u e n c e .

I have a r g u e d h e r e t h a t t he l inear , i n t e g r a t e d form of v i ra l D N A is neces sa ry for b o t h v i rus t r a n s m i s s i o n a n d D N A rep l i ca t ion ( w h e t h e r o r n o t th is occu r s f rom l inea r o r c i r cu la r t e m p l a t e s ) . W h a t t h e n is t he role of t h e c i r cu l a r form of v i ra l D N A ? A l t h o u g h its funct ion , if any, in t he p a r a s i t o i d itself is p r e sen t ly u n k n o w n , it s eems c lear t h a t c i r cu la r v i ra l g e n o m e s e g m e n t s a r e t r a n s c r i p t i o n a l l y act ive in t he p a r a s i t i z e d hos t , t h e r e b e i n g as ye t n o ev idence for i n t e g r a t i o n of p o l y d n a v i r u s D N A in s u c h a n i m a l s . T h i s a s p e c t of t h e p o l y d n a v i r u s life cycle is cons ide red in t h e nex t sec t ion .

B. Genetic Colonization of the Host

T h e resu l t s of e lec t ron mic roscop i c s tud ies ca r r i ed o u t in t he m i d to l a te 1970s c lear ly i n d i c a t e d t h a t p o l y d n a v i r u s pa r t i c l e s ( t hen c o m m o n l y refer red to as v i r u s l i k e / c a l y x fluid par t ic les ) w e r e d e s t i n e d for e x p o r t i n t o hos t a n i m a l t issues (Stol tz a n d V i n s o n , 1977, 1979b) . A t t he s a m e t ime , it b e c a m e inc reas ing ly c lear t h a t a t least s o m e of t he obv ious c h a n g e s r e su l t i ng f rom


n a t u r a l p a r a s i t i s m cou ld a lso be o b s e r v e d following m a n u a l in jec t ion of ca lyx fluid ( V i n s o n , 1972, 1977; N o r t o n a n d V i n s o n , 1977; D a h l m a n a n d V i n s o n , 1977; V i n s o n et al., 1979); we n o w k n o w t h a t t he b io logica l ac t iv i ty of c r u d e ca lyx fluid c a n , in m a n y cases , b e d u p l i c a t e d u s i n g g r ad i en t -pu r i f i ed p o l y d n a v i r u s pa r t i c l e s . S tud ie s ca r r i ed o u t in two different l a b o r a t o r i e s h a v e clearly d e m o n s t r a t e d t h a t b io logica l ac t iv i ty c a n b e a b r o g a t e d by cova len t c ross -l ink ing of e n c a p s i d a t e d v i ra l g e n o m e s in situ (Cook et al., 1984; G u z o a n d Sto l tz , 1985, 1987; Stol tz a n d G u z o , 1986; Beckage et al., 1987, 1990). T h i s sugges t s t h a t b io logica l ac t iv i ty is for t h e m o s t p a r t n o t d u e to s o m e cy to tox ic s t r u c t u r a l c o m p o n e n t s c a r r i e d i n t o t h e hos t by i n v a d i n g v i ru s pa r t i c l e s . I n s t e a d , ac t iv i ty a p p e a r s to b e d e p e n d e n t u p o n the p r e s e n c e of a v i ab l e v i ra l g e n o m e . S tud i e s c lear ly i n d i c a t e t h a t c i r cu l a r v i ra l D N A s pers i s t t h r o u g h o u t t he n a t u r a l cou r se of p a r a s i t i z a t i o n (Stol tz et al., 1986; T h e i l m a n n a n d S u m m e r s , 1986) a n d a r e t r a n s c r i p t i o n a l l y act ive ( F l e m i n g et al., 1983; B l i s sa rd et al., 1986, 1987, 1989; T h e i l m a n n a n d S u m m e r s , 1988; Stol tz et al, 1988; W e b b a n d S u m m e r s , 1990) in o n e o r m o r e hos t t i ssues . T h u s , t h e r e is a t leas t i nd i r ec t ev idence to sugges t t h a t t r a n s c r i p t i o n — w h e t h e r e n c o d e d o r i n d u c e d b y p o l y d n a v i r u s — i s a p r o b a b l e r e q u i r e m e n t for successful p a r a s i t i s m ( G u z o a n d Sto l tz , 1987).

I n sho r t , t h e b e h a v i o r of p o l y d n a v i r u s D N A in hos t a n i m a l s h a s all t h e e a r m a r k s of a gene t i c co lon iza t ion . T h e t e r m genetic colonization h a s a p a r t i c u la r m e a n i n g in biology, s ince it ca r r i e s t h e i m p l i c a t i o n t h a t t h e p roces s is d i r e c t e d t o w a r d the a c c o m p l i s h m e n t of s o m e goa l t h a t is of benefi t to t h e co lon iz ing (pa ras i t i c ) ent i ty. A c u r s o r y rev iew of t he b io logica l act iv i t ies n o w a t t r i b u t e d to p o l y d n a v i r u s e s (Tab le 1) will sugges t severa l a r e n a s in w h i c h p o l y d n a v i r u s e s cou ld have c ruc ia l s ignif icance for successful p a r a s i t i s m . T h e m o s t o b v i o u s of these , of cou r se , is i m m u n o s u p p r e s s i o n .

1. I m m u n o s u p p r e s s i o n

I n s e c t l a rvae a r e c lear ly q u i t e c a p a b l e of m o u n t i n g effective i m m u n e re sponses a g a i n s t l a rge foreign ob jec t s . Pa ra s i t o id eggs a n d l a rvae , t h e n , m u s t e i t he r avoid s u c h a r e s p o n s e , o r s u p p r e s s it , o r have it s u p p r e s s e d b y m a t e r i a l s ) in jec ted by the female p a r a s i t o i d d u r i n g ovipos i t ion . I n t h e c o n t e x t of p o l y d n a v i r u s biology, t h e e x p e r i m e n t a l a p p r o a c h h a s b e e n la rge ly a i m e d a t d e m o n s t r a t i n g v i r u s - i n d u c e d i m m u n o s u p p r e s s i o n , p a r t i c u l a r l y d u r i n g t h e ea r ly s t ages of p a r a s i t i z a t i o n . S u p p r e s s i o n of ce l lu la r i m m u n i t y ( e n c a p s u l a t ion a n d n o d u l a t i o n ) u s i n g pur i f ied i chnov i rus w a s first d e s c r i b e d b y E d s o n et al. (1981) , a n d s u b s e q u e n t l y b y Stol tz a n d G u z o (1986) ; in each i n s t a n c e , i m m u n o s u p p r e s s i o n w a s in p a r t a t t r i b u t e d to a n i nh ib i t i on of p l a s m a t o c y t e a d h e s i v e a n d s p r e a d i n g capac i t y (Stol tz a n d G u z o , 1986; Dav ie s et al., 1987), a l t h o u g h d e p l e t i o n in t h e n u m b e r s of c i r cu l a t i ng p l a s m a t o c y t e s w a s a lso n o t e d in o n e case ( C s V in Heliothis virescens). As m e n t i o n e d ear l ier , c h a n g e s in


Table 1

Physiological Changes in Host Animals Attributed to the Presence of Polydnavirus

Activity References"

Suppression of cellular immune response

Inhibition of weight gain Changes in hemocyte count

or behavior

Appearance of new hemolymph polypeptides

Inhibition of phenoloxidase activity

Inhibition of protein storage in fat body

Reduction in hemolymph viscosity

Change in hemolymph trehalose concentration

Degeneration of hemopoietic tissue

Pigmentation changes

Degeneration of the prothoracic gland

Prolongation or arrest of development

Perturbation of hormone levels

Vinson (1977); Edson efal. (1981) (V); Guzo and Stoltz (1985, 1987); Stoltz and Guzo (1986) (V); Vinson and Stoltz (1986) (V); Tanaka (1987a); Strand and Noda (1991) (V).

Vinson (1972); Vinson et al. (1979) (V).

Stoltz and Guzo (1986); Guzo and Stoltz (1987); Tanaka (1987b); Davies et al. (1987) (V); Wago and Tanaka (1989); Strand and Noda (1991) (V); D. B. Stoltz and Ν. E. Beckage (V; unpublished data); Stoltz, this report.

Cook et al. (1984) (V); Beckage et al. (1987).

Stoltz and Cook (1983) (V); Beckage et al. (1990) (V); Strand and Noda (1991) (V).

Tanaka (1986)

Davies et al. (1987) (V); Strand and Noda (1991); D. B. Stoltz (unpublished data).

Dahlman and Vinson (1977)

Guzo and Stoltz (1987)

Beckage et al. (1990) (V).

Dover et al. (1988a) (V).

Tanaka et al. (1987); Dover et al. (1988b) (V); Tanaka and Vinson (1991a); K. W. Schleifer and Ν. E. Beckage (unpublished data)

Dover et al. (1987, 1988b) (V); Tanaka et al. (1987); Tanaka and Vinson (1991b)

aV = gradient-purified virus used (otherwise, calyx fluid). Note that venom is required for full activity of some braconid polydnaviruses.

p l a s m a t o c y t e b e h a v i o r a r e likely no t d u e to a d i r ec t cy to tox ic effect c a u s e d b y

v i rus : C s V a d d e d to h e m o c y t e p r e p a r a t i o n s , for e x a m p l e , h a d n o effect o n

p l a s m a t o c y t e a d h e s i o n a n d s p r e a d i n g , w h e r e a s p l a s m a from p a r a s i t i z e d H.

virescens l a rvae d i d (Davies et al., 1987).

C o n s i d e r a b l y less i n fo rma t ion is ava i l ab le on t h e effects of pur i f ied bra-

coviruses; however , a n i nh ib i t i on of p l a s m a t o c y t e s p r e a d i n g h a s n o w b e e n

d e m o n s t r a t e d in two different sys t ems ( S t r a n d a n d N o d a , 1991; D . B . Sto l tz


a n d Ν . E . B e c k a g e , u n p u b l i s h e d ) . Cer ta in ly , ca lyx fluid—comprising p r i m a r i l y p o l y d n a v i r u s p a r t i c l e s — s e e m s to b e r e q u i r e d for successful p a r a s i t i s m in all of t h e b r a c o n i d p a r a s i t o i d / h o s t sy s t ems t h a t h a v e b e e n s t u d i e d to d a t e ( V i n s o n , 1974, 1977; G u z o a n d Sto l tz , 1985, 1987; K i t a n o , 1986; T a n a k a , 1987a; Ν . E . Beckage , ( p e r s o n a l c o m m u n i c a t i o n ) . A s w i t h ich-n o v i r u s / h o s t s y s t e m s , i m m u n o s u p p r e s s i o n is a s soc ia t ed w i t h o b v i o u s h e m a tological c h a n g e s ( G u z o a n d Sto l tz , 1987; T a n a k a , 1987b; W a g o a n d T a n a k a , 1989; D . B . Sto l tz a n d Ν . E . Beckage , u n p u b l i s h e d ) , i n c l u d i n g d e s t r u c t i o n of h e m o p o i e t i c t i ssue ( G u z o a n d Sto l tz , 1987); in o n e case , a d i r ec t effect of ca lyx fluid/venom o n hos t h e m o c y t e s h a s b e e n obse rved ( W a g o a n d T a n a k a , 1989). A r e q u i r e m e n t for v e n o m (for successful p a r a s i t i s m ) c o m p l i c a t e s t h e p i c t u r e in m o s t ( K i t a n o , 1982, 1986; G u z o a n d Stol tz , 1985, 1987; T a n a k a , 1987a) b u t no t all ( V i n s o n , 1977; B e c k a g e et aL, 1987, 1990) b r a c o v i r u s / h o s t s y s t e m s (see t h e following).

I m m u n o s u p p r e s s i o n i n d u c e d by p a r a s i t o i d s m i g h t logical ly b e e x p e c t e d to r e d u c e r e s i s t ance to m i c r o b i a l infect ion in hos t l a rvae , a n d th is h a s n o w b e e n d o c u m e n t e d (Stol tz a n d G u z o , 1986; G u z o a n d Sto l tz , 1987). I n t h e l ong t e r m , t h e n , a gene ra l i zed i m m u n o s u p p r e s s i o n w o u l d be l e tha l to p a r a s i toid as well as hos t un less e i the r (a) s o m e d e g r e e of ce l lu la r i m m u n i t y r e t u r n e d o r (b) o t h e r defense m e c h a n i s m s (e.g. , a n t i b a c t e r i a l p r o t e i n s ) w e r e e i t he r c a p a b l e of m a k i n g u p for t h a t loss o r else w e r e in fact a u g m e n t e d . As yet , very few s tud i e s have b e e n d i r e c t e d t o w a r d a n s w e r i n g th is q u e s t i o n ( b u t see Ross a n d D u n n , 1989). I n t h e case of t h e i chnov i ru s H f V (from Hyposoter

fitgitivus), w h i c h s u p p r e s s e s ce l lu la r i m m u n i t y in Malacosoma disstria, a d e g r e e of ce l lu la r i m m u n i t y r e t u r n s in 1 to 2 d a y s after h a t c h i n g of t he p a r a s i t o i d egg a n d t h e r e s u r g e n t e n c a p s u l a t i o n r e s p o n s e a p p e a r s to b e select ive, s ince p a r a s i toid l a rvae r e m a i n unaffected . S imi l a r e x a m p l e s of th is t ype of i n t e r a c t i o n m a y b e found e l sewhere in t he l i t e r a t u r e (e.g. , V i n s o n , 1972). I t is of cou r se poss ib le t h a t t he surfaces of p a r a s i t o i d l a rvae a r e in s o m e i n s t a n c e s n o t r ecogn ized as foreign, in w h i c h case a c o m p l e t e recovery of ce l lu la r i m m u n e r e s p o n s e s cou ld in t heo ry b e to l e ra t ed by the p a r a s i t o i d .

P a r t i c u l a r l y in t he case of b r a c o v i r u s / h o s t s y s t e m s , v e n o m g l a n d s e c r e t i o n s — i n a d d i t i o n to p o l y d n a v i r u s — m a y be r e q u i r e d for successful p a r a s i t i s m . T h e r e is s o m e q u e s t i o n , however , as to w h e t h e r t h e ro le of v e n o m in s u c h cases s h o u l d bes t b e d e s c r i b e d as p r i m a r y o r accessory. M o s t s t ud i e s t e n d to sugges t a n accessory func t ion . F i r s t , v e n o m a lone h a s n o o b v i o u s b io logica l activity, a t leas t in t he sy s t ems e x a m i n e d to d a t e ; conversely , ca lyx fluid o r p o l y d n a v i r u s e s a r e on the i r o w n fully ac t ive in a va r i e ty of s i t u a t i o n s . T h i s is espec ia l ly t r u e in t he case of t he i chnov i ruses ( E d s o n et aL, 1981; Sto l tz a n d G u z o , 1986), b u t h o l d s as well for a t l eas t two we l l - s tud ied b r a -covi ruses ( V i n s o n , 1977; B e c k a g e et aL, 1987, 1990). I n t h e case of t h e b r a covi rus C m V (from Cotesia melanoscela), v e n o m a p p e a r s to ac t a t a n ea r ly


s t age of v i rus p e n e t r a t i o n (Stol tz et al., 1988), p r o m o t i n g the u n c o a t i n g of v i ra l D N A a n d s u b s e q u e n t gene t i c co lon iza t ion . T h o u g h necessary , th i s funct ion is p r o b a b l y bes t i n t e r p r e t e d as p r o v i d i n g s u p p o r t for t h e m o r e i m p o r t a n t tasks m e d i a t e d by the act ivi ty of p o l y d n a v i r u s D N A .

Obvious ly , very l i t t le is as yet k n o w n c o n c e r n i n g the biological role of p a r a s i t o i d v e n o m g l a n d secre t ions , a n d it is therefore likely t h a t a s m o r e i n fo rma t ion is deve loped , we will find t h a t these p r o d u c t s a r e neces sa ry in ways as yet u n i m a g i n e d . T h i s a r g u m e n t is s u p p o r t e d by t h e r ecen t finding t h a t t he C s V g e n o m e a p p e a r s to have a c q u i r e d a p a r a s i t o i d v e n o m g l a n d g e n e ( W e b b a n d S u m m e r s , 1990); th is gene w o u l d of cou r se be c a r r i e d i n to p a r a si t ized a n i m a l s by C s V par t i c l es in jected d u r i n g ovipos i t ion . T h e r e w o u l d in this case be two p o t e n t i a l sources for v e n o m : the p a r a s i t o i d v e n o m g l a n d itself, a n d gene p r o d u c t s specified by the e n c a p s i d a t e d form of v i ra l D N A .

I t is still no t c lear t h a t m e l a n i z a t i o n p lays a p ivo ta l role in insec t defense r e sponses . S tud ie s w i t h Drosophila m u t a n t s sugges t t h a t t h e m e l a n i z a t i o n a n d h a r d e n i n g of h e m o c y t i c capsu les m a y be s e c o n d a r y to t h e in i t ia l non-se l f r ecogn i t ion even t (Rizki a n d Rizki , 1990). S o m e p o l y d n a v i r u s e s a r e k n o w n to i nh ib i t p h e n o l o x i d a s e act ivi ty (Stol tz a n d Cook , 1983; Beckage et al., 1990), w h e r e a s o t h e r s a p p a r e n t l y d o n o t (Davies et al., 1987). I n t e r e s t i n g , ca lyx fluid from Apanteles kariyai promotes m e l a n i z a t i o n (of fat b o d y cells; T a n a k a , 1986)! A t th is j u n c t u r e , it w o u l d seem r e a s o n a b l e to su spec t t h a t a n i nh ib i t ion of p h e n o l o x i d a s e act ivi ty m a y be r e q u i r e d for successful p a r a s i t i s m in s o m e biologica l sy s t ems , b u t no t o t h e r s . As w i t h p h e n o l o x i d a s e i nh ib i t i on , a n u m b e r of o t h e r p o l y d n a v i r u s - i n d u c e d c h a n g e s a r e of in t r ins i c in t e re s t in the i r o w n r e g a r d , a n d m a y (or m a y no t ) causa l ly r e l a t e to i m m u n o s u p p r e s s ion. T h e s e i n c l u d e t he a p p e a r a n c e of m a j o r n e w p o l y p e p t i d e s (Cook et al., 1984; Beckage et al., 1987, 1990) a n d a r e d u c t i o n in h e m o l y m p h viscosi ty (Davies et al., 1987; S t r a n d a n d N o d a , 1991; D . B . Sto l tz , u n p u b l i s h e d d a t a ) .

I m m u n o s u p p r e s s i o n p rov ides p e r h a p s t he m o s t obv ious a n d r ead i ly u n d e r s t o o d l inkage b e t w e e n the two forms of p o l y d n a v i r u s D N A . Surv iva l of t he linear, c h r o m o s o m a l l y i n t e g r a t e d g e n o m e — a s s u m e d to be c a r r i e d w i t h i n p a r a s i t o i d eggs a n d l a r v a e — d e p e n d s u p o n the p r e s e n c e in hos t t i ssues of t r a n s c r i p t i o n a l l y act ive , circular v i ra l D N A . However , as d e s c r i b e d in t he following, i m m u n o s u p p r e s s i o n is by n o m e a n s t h e on ly neces sa ry func t ion ca r r i ed o u t by t h e la t te r .

2. D e v e l o p m e n t a l R e g u l a t i o n

I t is of cou r se a x i o m a t i c t h a t t he life cycles of e n d o p a r a s i t o i d s m u s t b e well a d a p t e d to those of t he hos t s w i th in w h i c h they res ide . I n a d d i t i o n to a po t en t i a l l y l e tha l i m m u n e r e sponse , t he e n d o p a r a s i t i c life s t ages m u s t p r e s u m ab ly have to c o n t e n d w i t h o t h e r a spec t s of hos t phys io logy t h a t m a y b e equa l l y t h r e a t e n i n g ; of these , t he onse t of m e t a m o r p h o s i s cou ld r e p r e s e n t a se r ious


t h r e a t to successful p a r a s i t i s m . For e x a m p l e , t h e phys io logy of a p u p a l h o s t will be e x p e c t e d to differ g r ea t l y from t h a t of a l a rva l hos t ; a p a r a s i t o i d l a r v a d e v e l o p i n g w i t h i n a hos t l a rva w o u l d p r e s u m a b l y have to m a k e m a j o r ad jus t m e n t s in r e s p o n s e to hos t p u p a t i o n ( p e r h a p s by p u p a t i n g i tself) . I t is a l so w o r t h n o t i n g t h a t t h e p u p a l cu t ic le is, in g e n e r a l , likely to be m u c h t o u g h e r t h a n t h e l a rva l cu t ic le , a n d h e n c e m o r e difficult to p e n e t r a t e b y t h e e m e r g i n g p a r a s i t o i d .

A l t h o u g h m a n y p a r a s i t o i d s h a v e evolved in s u c h a w a y t h a t the i r eggs a n d l a rvae c a n a c c o m m o d a t e e x p o s u r e to two different hos t phys io log ies (e.g. , e g g / l a r v a l p a r a s i t o i d s ) , o t h e r s h a v e deve loped s t ra teg ies t h a t a l low t h e m to c o m p l e t e d e v e l o p m e n t w i t h i n a s ingle t y p e of hos t mi l i eu ( la rva l p a r a s i t o i d s ) . I t h a s b e e n a r g u e d t h a t th is r e q u i r e s t h a t t h e p a r a s i t o i d h a v e t h e capac i t y to m o d u l a t e , o r r e g u l a t e , hos t phys io logy ( V i n s o n a n d I w a n t s c h , 1980). S ince t h e t r a n s i t i o n f rom l a r v a to p u p a is u n d e r h o r m o n a l con t ro l , we m a y r e a s o n a b l y expec t t h a t m a n y p a r a s i t o i d s h a v e d e v e l o p e d t h e capac i t y to affect t h e e n d o c r i n e phys io logy of t he hos t . T h e l i t e r a t u r e is in fact r ep l e t e w i t h e x a m ples of h o s t / p a r a s i t o i d sys t ems in w h i c h t h e m e t a m o r p h o s i s of h o s t l a rvae is e i t he r d e l a y e d , acce le ra t ed , o r a r r e s t e d ; th is l i t e r a tu r e h a s r ecen t ly b e e n r ev iewed b y B e c k a g e (1985) a n d L a w r e n c e (1986) a n d will n o t b e t r e a t e d fu r the r h e r e . I will i n s t e a d confine myse l f to a c o n s i d e r a t i o n of w h a t is k n o w n c o n c e r n i n g t h e role of polydnaviruses in t h e r e g u l a t i o n of hos t e n d o c r i n e syst e m s . T h i s d i scuss ion will b e necessar i ly brief, s ince re la t ive ly few s tud ie s h a v e as yet t a r g e t e d t he p o l y d n a v i r u s e s as p o t e n t i a l m e d i a t o r s of d e v e l o p m e n t a l m o d u l a t i o n .

Po lydnav i ru se s cou ld in t heo ry affect hos t d e v e l o p m e n t in a n u m b e r of w a y s . For e x a m p l e , p r o l o n g a t i o n of t h e l a rva l s t age cou ld b e c a u s e d b y a n i nc r ea se in j u v e n i l e h o r m o n e t i te r o r a d e c r e a s e in e c d y s o n e levels; t h e fo rmer cou ld b e c a u s e d by a d e c r e a s e in j u v e n i l e h o r m o n e ( J H ) e s t e ra se ac t iv i ty (e .g. , H a y a k a w a , 1990), a n d t h e l a t t e r by a n i nh ib i t i on of p r o t h o r a c i c o t r o p i c h o r m o n e ( P T T H ) re lease o r by a d i r ec t effect o n t h e p r o t h o r a c i c g l a n d . T h e r e a r e o t h e r poss ibi l i t ies , m o s t of w h i c h h a v e in fact b e e n a s soc i a t ed w i t h e n d o p a r a s i t i s m in o n e s y s t e m o r a n o t h e r . E v i d e n c e for t he p a r t i c i p a t i o n of ca lyx fluids in h o r m o n a l p e r t u r b a t i o n w a s r e p o r t e d for b o t h a b r a c o v i r u s a n d a n i c h n o v i r u s in 1987 (Dover et al., 1987; T a n a k a et al., 1987). T a k i n g these in t u r n , ca lyx fluid a n d v e n o m from A. kariyai w e r e s h o w n to i nh ib i t a n i nc r ea se in ecdys t e ro id levels r e q u i r e d for p u p a t i o n , t h u s p r o l o n g i n g t h e final i n s t a r in Pseudaletia separata hos t l a rvae . S ince in ject ion of P T T H i n d u c e d p u p a t i o n , it w a s a s s u m e d t h a t t h e p r o t h o r a c i c g l a n d s of p a r a s i t i z e d l a rvae w e r e in t ac t ; it w a s a l so a s s u m e d t h a t t h e p r o t h o r a c i c g l a n d of ca lyx f l u i d / v e n o m - i n j e c t e d l a rvae r e m a i n e d i n t ac t too , a l t h o u g h P T T H injec t ions w e r e n o t specifically c a r r i e d o u t u s i n g these l a rvae . In jec t ions of e i the r ca lyx fluid o r v e n o m a l o n e h a d n o effect. I t s h o u l d be n o t e d t h a t in th is sy s t em, ca lyx f l u i d / v e n o m


m e r e l y de l ay p u p a t i o n , w h e r e a s n a t u r a l l y p a r a s i t i z e d l a rvae c a n n o t p u p a t e . T h u s , o t h e r factors , poss ib ly i n c l u d i n g t e r a tocy te s ( Z h a n g a n d D a h l m a n , 1989), m u s t b e involved in p r e v e n t i n g p u p a t i o n . M o r e recent ly , ca lyx fluid in c o m b i n a t i o n w i t h v e n o m from the b r a c o n i d p a r a s i t o i d Cardiochiles nigriceps w a s s h o w n to d e p r e s s p r o t h o r a c i c g l a n d funct ion in Heliothis virescens ( T a n a k a a n d V i n s o n , 1991a) , a n d inh ib i t i on of ecdys te ro id re lease in r e s p o n s e to P T T H w a s obse rved following in vitro i n c u b a t i o n of p r o t h o r a c i c g l a n d s w i t h ca lyx a n d v e n o m fluids, sugges t ing a direct effect o n th is t i ssue . H o w this m i g h t o c c u r r e m a i n s to b e e luc ida t ed ; p e n e t r a t i o n of t h e p r o t h o r a c i c g l a n d by v i rus pa r t i c l e s h a s no t b e e n obse rved . I t s h o u l d b e n o t e d t h a t t he effect of v e n o m in this sy s t em is a p p a r e n t l y hos t (i .e. , p a r a s i t o i d ) specific: a c o m b i n a t ion of Microplitis croceipes v e n o m w i t h C. nicriceps ca lyx fluid h a d n o effect o n hos t p u p a t i o n ( T a n a k a a n d V i n s o n , 1991b). C a l y x fluid from t h e ich-n e u m o n i d w a s p Campoletis sonorensis h a s a lso b e e n s h o w n to i n d u c e a d e c r e a s e in ecdys t e ro id levels a n d d e v e l o p m e n t a l a r r e s t in l a s t - i n s t a r H. virescens l a rvae (Dover et al., 1987). I n this case , on ly t h e p a r t i c u l a t e fract ion of ca lyx fluid w a s act ive, sugges t i ng t h a t C s V v i r ions were involved; as w i t h o t h e r ich-n e u m o n i d / h o s t sy s t ems , t he re was n o a p p a r e n t r e q u i r e m e n t for w a s p v e n o m . L i g a t i o n e x p e r i m e n t s sugges t ed t h a t t he t a r g e t t i ssue r e s ided in t h e t h o r a x , w h i c h in t u r n sugges t ed t h a t ca lyx fluid m i g h t b e exe r t i ng s o m e effect o n t h e p r o t h o r a c i c g l a n d itself.

S u b s e q u e n t w o r k u s i n g puri f ied C s V h a s now clear ly e s t ab l i shed t h a t th is p o l y d n a v i r u s c a n i n d u c e d e v e l o p m e n t a l a r r e s t in H. virescens l a rvae (Dove r et al., 1988a ,b) . F u r t h e r m o r e , it a p p e a r s t h a t th is is c a u s e d b y a v i r u s - i n d u c e d d e g e n e r a t i o n of t he p r o t h o r a c i c g l a n d (Dover et al., 1988a) , w h i c h m a y in t u r n a c c o u n t for a n obse rved r e d u c t i o n in ecdys t e ro id t i te r (Dover et al., 1988b) . I t s h o u l d be no t i ced t h a t t he effect is d o s e - d e p e n d e n t : in jec t ion of lower doses of C s V resu l t ed in t h e d e g e n e r a t i o n of s o m e p r o t h o r a c i c g l a n d cells, b u t n o t al l , a n d d e v e l o p m e n t a l a r r e s t w a s r ep l aced by a p r o l o n g a t i o n of t h e las t l a rva l ins ta r .

T h e effect of p o l y d n a v i r u s o n the p r o t h o r a c i c g l a n d is p r o b a b l y n o t d i rec t , s ince t he r e is n o ev idence to sugges t t h a t v i rus pa r t i c l e s c a n i n v a d e th is t i ssue , w h i c h in t u r n ra ises the very i m p o r t a n t q u e s t i o n of h o w s o m e cells a r e a b l e to a p p a r e n t l y e scape be ing d a m a g e d . T o m y k n o w l e d g e , on ly o n e o t h e r a t t e m p t h a s b e e n m a d e to assoc ia te pur i f ied p o l y d n a v i r u s w i t h a n e n d o c r i n e funct ion: in th is case , a b racov i rus ( C c V ) from Cotesia congregata h a s b e e n s h o w n to de l ay t he d e v e l o p m e n t of Manduca sexta l a rvae (Ν . E . B e c k a g e , p e r s o n a l c o m m u n i c a t i o n ) , sugges t ing t h a t C c V act iv i ty c a n a p p a r e n t l y b e expres sed in t he a b s e n c e of w a s p v e n o m .

I t c a n b e p r e d i c t e d t h a t a n u m b e r of a d d i t i o n a l p o l y d n a v i r u s e s will in t h e n e a r fu ture b e e x a m i n e d in t e r m s of the i r p o t e n t i a l for in f luenc ing hos t e n d o c r i n e s y s t e m s , a n d it c a n conf ident ly be sugges t ed t h a t m a n y , if n o t


m o s t , will be found to d o so. I t s h o u l d a lso be k e p t in m i n d t h a t o t h e r v i ru s -i n d u c e d c h a n g e s in hos t physiology, p e r h a p s i n c l u d i n g i m m u n o s u p p r e s s i o n , m a y well have a h o r m o n a l bas i s . I t is, for e x a m p l e , well k n o w n t h a t s t r ik ing c h a n g e s in h e m o c y t e m o r p h o l o g y a n d func t ion c a n o c c u r d u r i n g m o l t i n g o r m e t a m o r p h o s i s (e.g. , K u r a t a et aL, 1989), p r e s u m a b l y in r e s p o n s e to c h a n g e s in t h e levels of o n e o r m o r e h o r m o n e s .

3 . C h a n g e s i n N u t r i e n t L e v e l s a n d / o r A c c e s s i b i l i t y

C h a n g e s in n u t r i e n t levels of va r i ous t i ssues h a v e o n severa l occas ions b e e n a t t r i b u t e d to t he act ivi ty of ca lyx fluid o r p o l y d n a v i r u s e s . For e x a m p l e , ca lyx fluid f rom t h e b r a c o n i d Microplitis croceipes is k n o w n to e leva te h e m o l y m p h t r e h a l o s e levels in H. virescens l a rvae ; v e n o m is no t r e q u i r e d ( D a h l m a n a n d V i n s o n , 1977). I t is of in te res t to n o t e t h a t t r eha lose is r ead i ly a b s o r b e d b y M. croceipes l a rvae ( E d s o n a n d V i n s o n , 1977). Pa r t i cu l a r l y in t h e case of h e m o l y m p h feeders , it m a y b e i m p o r t a n t to d ive r t n u t r i e n t s a w a y f rom t i ssues in w h i c h they m i g h t o t h e r w i s e b e s to r ed , a n d h e n c e b e u n a v a i l a b l e to t h e d e v e l o p i n g p a r a s i t o i d . Pseudaletia separata l a rvae p a r a s i t i z e d b y A. kariyai m a y b e a case in po in t : t he fat b o d y t i ssue of p a r a s i t i z e d l a rvae does n o t a c c u m u l a t e t h e p r o t e i n s t o r a g e g r a n u l e s cha rac te r i s t i ca l ly o b s e r v e d in n o r m a l l a rvae . I n h i b i t i o n of p r o t e i n d e p o s i t i o n in th is t i ssue c a n a lso b e d e m o n s t r a t e d following in jec t ion of ca lyx fluid a n d v e n o m ( T a n a k a , 1986). A n o t h e r poss ib le exa m p l e of nu t r i en t diversion m a y be presented by the p h e n o m e n o n of paras i t ized-i n d u c e d c a s t r a t i o n of hos t l a rvae ( R e e d - L a r s e n a n d B r o w n , 1990); conceivably, p a r a s i t o i d l a rvae m a y have to c o m p e t e w i t h hos t g o n a d s for n u t r i e n t s . I t h a s b e e n s u g g e s t e d — b u t as yet n o t d e m o n s t r a t e d — t h a t p o l y d n a v i r u s e s cou ld b e involved in th is p rocess of p r e m a t u r e g o n a d a t r o p h y ( J u n n i k k a l a , 1985).

T h e scope of b io logica l act iv i t ies t h a t c a n be a t t r i b u t e d to p o l y d n a v i r u s e s will l ikely b e found , u l t imate ly , to be m u c h b r o a d e r t h a n is i n d i c a t e d b y t h e list p r o v i d e d in T a b l e 1. H o w e v e r , v i ewed m o r e closely, s o m e of these s e e m ingly d i s p a r a t e p h e n o m e n a cou ld well c o m p r i s e a c a s c a d e of r e l a t ed even t s , p r e c i p i t a t e d b y p o l y d n a v i r u s ac t ing u p o n s o m e p r i m a r y t a rge t . T h e s e u n u s u al v i ruses s h o u l d b e seen as e x t r e m e l y useful tools t h a t , u s e d jud ic ious ly , s h o u l d tell u s m u c h a b o u t n o r m a l h o s t physiology, a n d h o w it m a y b e m a n i p u l a t e d , if n e e d be , to o u r a d v a n t a g e . I n t h e following sec t ion , I p r o v i d e a b r ie f overv iew of h o w s o m e a spec t s of p o l y d n a v i r u s r e s e a r c h m a y b e e x p l o r e d in t h e n o t - t o o - d i s t a n t fu ture .

III. Future Directions

I n r e cen t yea r s , p o l y d n a v i r u s r e s e a r c h h a s m a d e s ignif icant p r o g r e s s o n a n u m b e r of i m p o r t a n t i ssues , a n d t h e e s t a b l i s h m e n t of a firm b a s e of knowl -


edge u p o n w h i c h to bu i ld fur ther ; d e v e l o p m e n t of th is exc i t ing field s eems likely to a d v a n c e rapidly . I n a d d i t i o n , w i t h t he d e m o n s t r a t i o n t h a t pur i f ied v i rus h a s b iological ac t iv i ty in a va r i e ty of con tex t s , it w o u l d s e e m likely t h a t a n u m b e r of l a b o r a t o r i e s h a v i n g n o in te res t in p o l y d n a v i r u s e s p e r se m a y none the l e s s c o m e to perce ive t h e m as v a l u a b l e tools for s t u d y i n g insec t p h y s iology. Clear ly , g iven the complex i ty of p o l y d n a v i r u s / p a r a s i t o i d / h o s t syst e m s , a mul t i face ted a p p r o a c h will c o n t i n u e to be p r o d u c t i v e a n d s h o u l d b e e n c o u r a g e d w h e r e v e r poss ib le . I n a n y case , we a r e n o w a t a s t age w h e r e it h a s b e c o m e a t leas t poss ib le to f o r m u l a t e , a n d to s o m e ex t en t a d d r e s s , a n u m b e r of f u n d a m e n t a l q u e s t i o n s . A few of these a r e c o n s i d e r e d nex t .

1. I s t h e P o l y d n a v i r u s G e n o m e a G e n e t i c M o s a i c ?

C h r o m o s o m a l t r a n s m i s s i o n of p o l y d n a v i r u s g e n o m e s n a t u r a l l y ra i ses t h e q u e s t i o n of w h e t h e r these ent i t ies a r e i n d e e d v i ruses ( they cou ld j u s t as r ead i ly b e referred to as , say, n u c l e a r secre t ions) . T h i s in t u r n ra i ses t he q u e s t i o n of w h e t h e r it w o u l d have b e e n eas ie r for a p a r a s i t o i d to evolve a n e w " v i r u s " or, a l te rna t ive ly , co-op t t h e services of o n e a l r e a d y in ex i s tence . S u c h q u e s t i o n s a r e b y n o m e a n s idle ones , a n d i n d e e d the p o l y d n a v i r u s p h e n o m e n o n r equ i r e s t h a t s o m e t h o u g h t b e g iven as to w h a t t h e w o r d " v i r u s " rea l ly m e a n s . I n this r e g a r d , it is of in te res t to no t e t h a t def in i t ions for " v i r u s " a r e b o t h sca rce a n d , p e r h a p s for tunate ly , s o m e w h a t n e b u l o u s ; I therefore offer t he following:

virus: infectious entity whose genome, consisting of either DNA or RNA, is minimally packaged within a protein coat designed to facilitate entry into a susceptible host cell (we could add that lacking ribosomes, all viruses must necessarily replicate within cellular hosts).

T h e w o r d " in fec t ion" is used h e r e in t he b r o a d e s t poss ib le sense , so as to i n c l u d e s u c h p h e n o m e n a as oncogenes i s , gene t i c co lon iza t ion , a n d so on ; t h e t e r m " s u s c e p t i b l e " does no t necessar i ly i m p l y t h a t a p r o d u c t i v e r ep l i ca t ion cycle will en sue . I n fact, it m a t t e r s l i t t le w h e t h e r o n e th inks of p o l y d n a v i r u s e s as b e i n g v i ruses o r n u c l e a r secre t ions , s ince e i the r d e s i g n a t i o n w o u l d a p p e a r to b e l eg i t ima te . G i v e n t h a t , is t h e q u e s t i o n b e i n g a d d r e s s e d in this sec t ion rea l ly itself l eg i t imate? P u t a n o t h e r way, is it in fact poss ib le to d i s t i n g u i s h b e t w e e n ce l lu lar a n d v i ra l genes in t he p o l y d n a v i r u s con tex t? ( T h e r e a d e r s h o u l d k e e p in m i n d t h a t all v i ra l genes m u s t or ig ina l ly h a v e b e e n cel lu lar . )

To the q u e s t i o n of w h e t h e r t he r e a r e c lass ical v i ra l genes w i t h i n t h e p o l y d n a v i r u s r epe r to i r e , t he a n s w e r m u s t be : y e s — p r o v i d e d on ly t h a t o u r definit ion for " v i r u s " is r e a s o n a b l e . For e x a m p l e , p o l y d n a v i r u s pa r t i c l e s d e m o n s t r ab ly possess p r o t e i n coa ts t h a t a r e s t r u c t u r a l l y a n d func t iona l ly a n a l o g o u s to v i ra l c aps ids (Stol tz a n d V i n s o n , 1979a) . T h e r e is i n d e e d n o r e a s o n n o t to refer to t h e m as c aps id s , w h e t h e r o r no t t h e genes e n c o d i n g t h e m a r e t h o u g h t of as ce l lu la r o r v i ra l in t e r m s of or ig in . However , m o v i n g b e y o n d a cons ider -


a t i o n of t hose genes involved in pa r t i c l e m o r p h o g e n e s i s , t h e r e is a t p r e s e n t n o easy w a y to def ine o t h e r p a t e n t l y v i ra l genes in t h e p o l y d n a v i r u s con tex t . T h i s , however , s h o u l d n o t p r e c l u d e r e a s o n e d s p e c u l a t i o n , a n d to t h a t e n d I will specifically sugges t t h a t t h e p o l y d n a v i r u s g e n o m e will even tua l l y b e s h o w n to i n c l u d e n o t on ly v i ra l a n d p a r a s i t o i d genes , b u t t races of h o s t genes as well ( i .e. , f rom t h e p a r a s i t i z e d h o s t ) ; p r e s u m a b l y , all t h r e e of t he se en t i t i es have coevolved over mi l l ions of y e a r s . A n u m b e r of h y p o t h e s e s r e l e v a n t to th is i ssue m a y b e sub jec t to scientific inqui ry , t h e resu l t s of w h i c h cou ld b e of c o n s i d e r a b l e in te res t , r ega rd l e s s of w h e t h e r o r n o t t h e h y p o t h e s e s t h e m s e l v e s p rove va l id :

(a) I f t h e p o l y d n a v i r u s e s a r e de r ived f rom class ical v i ruses , t h e n s t r a t e gies e m p l o y e d b y t h e l a t t e r to m o d u l a t e hos t phys io logy (e.g. , t h e b a c u l o v i r u s ecdys t e ro id g lucosyl t r ans fe rase ; O 'Re i l l y a n d Mil le r , 1989) m a y well b e r e p r e s e n t e d w i t h i n p o l y d n a v i r u s g e n o m e s . S u c h genes , if p r e s e n t , wil l p r o b a bly t u r n o u t to have b e e n a s s imi l a t ed f rom t h e hos t o r g a n i s m . I t is of i n t e r e s t in th is r e g a r d to n o t e t h a t a po lydnav i ru s l i ke pa r t i c l e found in Venturia can-escens exh ib i t s i m m u n o l o g i c a l c ross - reac t iv i ty w i t h o n e o r m o r e h o s t (Ephestia kuhniella) p r o t e i n s , i n c l u d i n g h e m o l i n , a k n o w n a n t i b a c t e r i a l p r o t e i n (Berg et al, 1988; S c h m i d t a n d T h e o p o l d , 1991).

(b) Similar ly , genes e m p l o y e d by p a r a s i t o i d s to r e g u l a t e h o s t phys io logy m a y n o w have a c q u i r e d p o l y d n a v i r u s e x c i s i o n / e n c a p s i d a t i o n s igna l s . S u c h genes cou ld in t h e o r y r e p r e s e n t d u p l i c a t e copies of coex is t ing p a r a s i t o i d genes . A n in t e r e s t i ng e x a m p l e of th is m a y a l r e a d y have b e e n d i scovered ; specifically, W e b b a n d S u m m e r s (1990) p r o p o s e t h a t a p a r a s i t o i d v e n o m g l a n d g e n e is e n c a p s i d a t e d w i t h i n C s V pa r t i c l e s . P r e s u m a b l y , c o n t i n u o u s exp res s ion of th is gene in hos t l a rvae w o u l d be of benefi t to t h e d e v e l o p i n g p a r a s i t o i d .

(c) Converse ly , genes n o longer r e q u i r e d in (pa ra s i t i zed ) a n i m a l s , in w h i c h p o l y d n a v i r u s e s d o n o t r ep l i ca t e , m a y have lost t h e capac i t y to excise f rom t h e p a r a s i t o i d g e n o m e ; p r i m e c a n d i d a t e s he r e w o u l d b e genes r e q u i r i n g exp re s s ion on ly in t h e p a r a s i t o i d ova ry (e .g. , t hose e n c o d i n g v i r ion s t r u c t u r a l p r o t e i n s ) . Stil l , it is of i n t e re s t to n o t e t h a t s o m e C s V genes n o t e x p r e s s e d in h o s t l a rvae a r e neve r the le s s e n c a p s i d a t e d ( T h e i l m a n n a n d S u m m e r s , 1988; B l i s sa rd et al, 1989). T h e s ignif icance of s u c h o b s e r v a t i o n s , wh i l e u n d o u b t e d , r e m a i n s to be clarif ied.

(d) I f t h e p o l y d n a v i r u s e s a r e phy logene t i ca l l y r e l a t e d to o n e o r m o r e " c l a s s i ca l " v i ruses , t h e n it m a y be useful in th is c o n t e x t to e x a m i n e v i ruses h a v i n g s imi l a r morpho logy . I t h a s b e e n a r g u e d p rev ious ly t h a t t h e bacu lov i ruses b e a r a t leas t a superf ic ia l r e s e m b l a n c e to t h e b racov i ruses (Stol tz a n d V i n s o n , 1979a) . I t is therefore conce ivab le t h a t genes d e t e r m i n ing t h e s t r u c t u r e / p a c k a g i n g of b r acov i rus n u c l e o c a p s i d s m a y s h a r e h o m o -


logy w i t h c o g n a t e bacu lov i rus genes . I t will be of p a r t i c u l a r i n t e re s t in th is r e g a r d to e x a m i n e a n u n u s u a l bacu lov i rus t h a t r ep l i ca tes in t h e ova ry of t h e i c h n e u m o n i d p a r a s i t o i d Mesoleius tenthredinis (Stol tz , 1981).

2 . W h a t A r e t h e F u n c t i o n a l R o l e s o f P o l y d n a v i r u s G e n e P r o d u c t s ?

T h i s sec t ion shou ld of cou r se be prefaced w i t h a c a u t i o n a r y n o t e to t he effect t h a t w h a t a r e referred to he r e as p o l y d n a v i r u s func t ions c a n j u s t as l eg i t ima te ly be cons ide red to r e p r e s e n t p a r a s i t o i d func t ions . As d i s cus sed in t h e p r e v i o u s sec t ion , t h e r e is n o easy d i s t inc t ion to b e m a d e h e r e ( p e r h a p s , concep tua l ly , n o n e n e e d be m a d e ) . I n a n y case , in k e e p i n g w i t h t h e h y p o t h e sis t h a t p o l y d n a v i r u s e s a r e phy logene t i ca l ly r e l a t ed to c lass ical v i ruses , I will a s s u m e h e r e — a s I have e a r l i e r — t h a t t he r e a r e i n d e e d po lydnav i rus - spec i f i c genes . L ike o t h e r v i ruses , p o l y d n a v i r u s e s p r e s u m a b l y code for b o t h s t r u c t u r a l a n d r e g u l a t o r y p r o t e i n s ; t he fo rmer r e p r e s e n t c o m p o n e n t s of a s s e m b l e d v i r ions , w h e r e a s t he l a t t e r a r e typica l ly r e q u i r e d for r ep l i ca t ive func t ions . However , un l ike o t h e r v i ruses , p o l y d n a v i r u s e s a r e likely to r e q u i r e two t ypes of r e g u l a t o r y p r o t e i n s : those o p e r a t i n g in t h e p a r a s i t o i d (e.g. , D N A rep l i ca t ion , etc.) a n d those expres sed in t he hos t ( funct ions r e q u i r e d for gene t i c co lon iza t ion) . T h e ident i f ica t ion of a t leas t s o m e m a j o r s t r u c t u r a l p r o t e i n s c a n be r ead i ly a c c o m p l i s h e d us ing t e c h n i q u e s t h a t a r e n o w s t a n d a r d in m a n y l abo ra to r i e s ; m a p p i n g these to p a r t i c u l a r g e n o m e s e g m e n t s s h o u l d p rove re la t ively s t r a i gh t fo rwa rd . T h e ident i f ica t ion of r e g u l a t o r y gene p r o d u c t s r e q u i r e d for r ep l i ca t ion m a y p rove to b e a m o r e d e m a n d i n g task , especia l ly s ince t h e u s u a l r e q u i r e m e n t s for s t u d y i n g r e p l i c a t i o n — f o r e x a m p l e , a n abi l i ty to g row t h e v i rus in vitro—are no t m e t . T h u s , p r o g r e s s h e r e m a y o c c u r m o r e slowly. O n the o t h e r h a n d , r a p i d p rog re s s in ident i fy ing p r o t e i n s exp re s sed in hos t a n i m a l s shou ld be expec t ed . I n d e e d , p r e l i m i n a r y w o r k h a s a l r e a d y b e e n r e p o r t e d (Bl issard et al., 1989); in a d d i t i o n , in vitro sy s t ems (e.g. , Stol tz et al., 1988) cou ld be exp lo i ted for this p u r p o s e .

W h a t r e m a i n s to b e a n s w e r e d is t he very i m p o r t a n t q u e s t i o n of d e t e r m i n ing w h a t funct ions v i ra l gene p r o d u c t s a r e p e r f o r m i n g in t h e p a r a s i t i z e d hos t . I n s o m e cases , obv ious a p p r o a c h e s will b e sugges t ed b y the a p p e a r a n c e of o n e o r m o r e m a j o r n e w p o l y p e p t i d e s (e.g. , C o o k et al., 1984; B e c k a g e et al., 1987); these p r e s u m a b l y , b u t no t necessar i ly , will b e e n c o d e d b y p o l y d n a v i r u s genes . I t m a y b e poss ib le to purify t he r e l evan t p r o t e i n a n d t h e n use it d i rec t ly in b iological e x p e r i m e n t s : Wi l l in ject ion of s u c h a p r o t e i n , for e x a m p l e , m i m i c s o m e k n o w n p o l y d n a v i r u s - i n d u c e d c h a n g e in hos t phys io l ogy? I n m o s t cases , however , novel m e t h o d o l o g i e s m a y b e r e q u i r e d in o r d e r to co r r e l a t e b iological act ivi ty w i t h a p a r t i c u l a r p o l y d n a v i r u s g e n o m e segm e n t . Possible a p p r o a c h e s h e r e m i g h t i n c l u d e t he use of non ly t i c bacu lov i ru s express ion vec tors c a r r y i n g i n d i v i d u a l p o l y d n a v i r u s genes o r g e n o m e seg-


meri t s . I t m i g h t a l so b e poss ib le to t ransfec t newly p a r a s i t i z e d h o s t l a rvae u s i n g an t i - s ense c o n s t r u c t s d e s i g n e d to a b r o g a t e t h e expres s ion of genes r e q u i r e d for successful p a r a s i t i s m ; a l te rna t ive ly , s imi l a r c o n s t r u c t s cou ld b e de l ive red i n t o h o s t eggs b y mic ropro jec t i l e b o m b a r d m e n t . A n t i s e r a co u l d b e r a i sed a g a i n s t vi rus-specif ic g e n e p r o d u c t s ident if ied u s i n g c D N A expres s ion l ib ra r i e s , a n d t h e n assessed in t e r m s of the i r ab i l i ty to in ter fere w i t h p o l y d n a v i r u s - i n d u c e d ac t iv i ty in t he hos t a n i m a l ( W e b b a n d S u m m e r s , 1990). N o n e of th i s will be p a r t i c u l a r l y easy, b u t t h e r e w a r d s m a y b e cons id e r a b l e : p o l y d n a v i r u s e s m a y e n c o d e a n u m b e r of p r o d u c t s h a v i n g p o t e n t i a l for p r ac t i c a l a p p l i c a t i o n in t h e b iopes t i c ide indus t ry .

T h e r e a r e of cou r se a n u m b e r of a d d i t i o n a l q u e s t i o n s t h a t r e l a t e to v i r u s -specific exp res s ion in t he p a r a s i t i z e d hos t . W e as yet k n o w n o t h i n g a b o u t t h e t i ssue specificity, if any, of v i ra l g e n e p r o d u c t s affecting h o s t phys io logy. N o r d o we k n o w w h i c h g e n e p r o d u c t s p r o m o t e t he d e v e l o p m e n t of t h e p a r a s i t o i d egg a n d w h i c h t h e l a rva . H o s t specificity, in t e r m s of a r e q u i r e m e n t for p o l y d n a v i r u s e s in successful p a r a s i t i s m , is a lso a la rge ly u n e x p l o r e d a r e a ; of p o t e n t i a l i n t e re s t h e r e is t h e o b s e r v a t i o n t h a t H f V D N A pers i s t s in Lymantria dispar l a rvae , b u t p a r a s i t i s m of t h a t h o s t is never the less unsuccessfu l (Stol tz et aL, 1986).

3 . W h y I s t h e P o l y d n a v i r u s G e n o m e S e g m e n t e d ?

T h i s is o n e of t he m o r e i n t e r e s t i ng q u e s t i o n s c o n c e r n i n g t h e p o l y d nav i ru se s a n d h a s ye t to b e a d d r e s s e d . S e g m e n t a t i o n will p e r h a p s u l t i m a t e l y b e found to h a v e s o m e t h i n g to d o w i t h t h e or ig in of t hese v i ruses ; p u r e l y m e c h a n i s t i c e x p l a n a t i o n s (e .g. , s e g m e n t a t i o n cou ld faci l i tate excis ion , o r r e d u c e m u t a t i o n a l loads ) s e e m overly s impl i s t i c . I t cou ld b e a r g u e d t h a t if p o l y d n a v i r u s e s a r o s e f rom t h e p a r a s i t o i d g e n o m e , t h e n the i r genes w o u l d m o s t l ikely a l r e a d y have b e e n s e p a r a t e d from each o the r , in w h i c h case e x c i s i o n / e n c a p s i d a t i o n s imp ly serves as a m e a n s to b r i n g t h e m t o g e t h e r a s a func t ion ing w h o l e . Al te rna t ive ly , if o n e a s s u m e s t h a t p o l y d n a v i r u s e s a r e r e l a t ed to c lass ical v i ruses , t h e n it b e c o m e s r e a s o n a b l e to s u p p o s e t h a t s e g m e n t a t i o n m u s t have confer red s o m e as ye t u n r e c o g n i z e d benefi t to t h e e s t ab l i sh m e n t a n d c o n t i n u e d evo lu t ion of p o l y d n a v i r u s / p a r a s i t o i d / h o s t c o m p l e x e s . P e r h a p s t h e p o l y d n a v i r u s e s cou ld h a v e a r i s en f rom a n c e s t r a l defect ive in te r fer ing pa r t i c l e s ; D I pa r t i c l e s e n c a p s i d a t e s u b g e n o m i c mo lecu le s a n d in a d d i t ion m a y r e d u c e t h e p a t h o g e n i c i t y of t h e p a r e n t a l v i r ion , w h i c h cou ld well have b e e n a p r e r e q u i s i t e for t h e acqu i s i t i on of p o l y d n a v i r u s p r o g e n i t o r s b y p a r a s i t o i d s . A t p r e s e n t , it is difficult to envis ion h o w this i ssue m i g h t b e a d d r e s s e d expe r imen ta l ly .

I n t h e foregoing d i scuss ions , I have c o n s i d e r e d on ly a few of t h e m a n y i n t e r e s t i n g q u e s t i o n s facing s t u d e n t s of p o l y d n a v i r u s biology. M y e x p l o r a t i o n h a s b e e n la rge ly a n d necessar i ly specu la t ive in n a t u r e s ince , in t h e a b s e n c e of


defini t ive i n fo rma t ion b e a r i n g o n these q u e s t i o n s , p e r h a p s t he bes t t h a t c a n be h o p e d for is a set of w o r k i n g h y p o t h e s e s . I t is t h e a u t h o r ' s h o p e t h a t s o m e of these will p rove sufficiently a t t r ac t ive to s t i m u l a t e fu r the r d i a l o g u e , a n d p e r h a p s even s o m e d i r ec t ed r e sea r ch .

Acknowledgments

I wish to acknowledge the contributions of a number of individuals who have made significant contributions over the years in the capacity of either graduate student (Peter Krell, David Guzo, Deming Xu) or research assistant (Doug Cook, Elizabeth Belland). Original research from my laboratory was funded in part by the Medical Research Council and in part by the Canadian Forestry Service.

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Chapter 9

Polydnavirus Genome Organization Jo-Ann G. W. Fleming Peter J. Krell Department of Animal Science Department of Microbiology Texas A&M University University of Guelph College Station, Texas Guelph, Ontario, Canada

I. Introduction

II. Polydnaviruses as Part of Biological Systems

III. Multipartite Genome Structure

IV. Genome Packaging

V. Intraspecific Genomic Complexity

VI. Transcription and Gene Families A. Functional Organization of the

Genome B. Known CsV Gene Families

C. Temporal Patterns of CsV Gene Expression

D. Significance of the Gene Families

VII. Polydnavirus Transmission, Integration, and Replication

VIII. Polydnavirus Evolution A. Gene Divergence B. Species Specificity C. Virus Origin

IX. Conclusions Acknowledgments References

I. Introduction

Po lydnav i ru se s p l a y a n u n u s u a l role in p a r a s i t o i d life cycles b e c a u s e t h e v i ruses a p p e a r to be symbio t i ca l ly a s soc i a t ed w i t h t h e w a s p b u t i n d u c e signi f icant phys io log ica l c h a n g e s in l e p i d o p t e r a n hos t l a rvae p a r a s i t i z e d b y t h e w a s p s . T h e s e c h a n g e s have b e e n c o n s i d e r e d ev idence of " h o s t r e g u l a t i o n " by t h e p a r a s i t o i d o r factors de r ived f rom it ( V i n s o n a n d I w a n t s c h , 1980). Polydnav i ru se s a r e i m p l i c a t e d as o n e of t he factors t h a t m a y b e n e e d e d to s ecu re t h e successful d e v e l o p m e n t of t h e p a r a s i t o i d in its hos t a n d therefore a r e be l ieved to b e involved in d e t e r m i n i n g t he species specificity of host— p a r a s i t o i d i n t e r ac t i ons . T h i s a s p e c t of t he b io logy of p o l y d n a v i r u s e s is cove red in m o r e de t a i l in t he p r e c e d i n g c h a p t e r by D . Stol tz a n d in o t h e r rev iews (Stol tz a n d V i n s o n , 1979; K r e l l , 1991a ,b ; F l e m i n g , 1992).

T h e m o s t d i s t inc t ive c h a r a c t e r i s t i c of v i ruses in t h e family P o l y d n a v i r i d a e

Parasites and Pathogens of Insects Copyright © 1993 by Academic Press, Inc. Volume 1: Parasites 189 All rights of reproduction in any form reserved.

190 Jo-Ann G. W. Fleming and Peter J. Krell

is t h e u n u s u a l o r g a n i z a t i o n of t he g e n o m e s as col lec t ions of severa l supe r -coiled D N A s (Stol tz et al., 1984; F r a n c k i et al., 1991). T h e s t r u c t u r a l a n d func t iona l o r g a n i z a t i o n of these c o m p l e x v i ra l g e n o m e s is t h e p r i m a r y focus of this review. T h e gene t i c r e l a t i o n s h i p of t h e v i ruses w i t h t h e w a s p h o s t a l so will be cons ide red b e c a u s e of t h e imp l i ca t i ons t h a t r e l a t i o n s h i p h a s for v i ru s r ep l i ca t ion a n d the evo lu t ion of t he v i rus a n d p a r a s i t o i d ( F l e m i n g , 1991 , 1992; Kre l l , 1991a) . T h e d i scuss ion a lso will i n c l u d e s o m e s p e c u l a t i o n s in t h e h o p e t h a t t hey will s t i m u l a t e d e b a t e a n d h e l p to identify p r o b l e m s for fu tu re inves t iga t ions of these i m p o r t a n t v i ruses .

II. Polydnaviruses as Part of Biological Systems

All p o l y d n a v i r u s e s have s e g m e n t e d g e n o m e s of m u l t i p l e c i r cu l a r D N A s (see Sec t ion I I I ) . T w o m a j o r s u b g r o u p s of p o l y d n a v i r u s e s , t he b racov i ruses a n d the i chnov i ruses , a r e r ecogn ized ( F r a n c k i et al., 1991). T h e b racov i ruses have b e e n i so la ted f rom w a s p s in t h e family B r a c o n i d a e , w h e r e a s i chnov i ruses have b e e n i so la ted f rom w a s p s in t he family I c h n e u m o n i d a e . I n a d d i t i o n to differences in the i r hos t r a n g e s , these two s u b g r o u p s c a n b e d i f ferent ia ted o n t h e bas i s of the i r s ignif icant ly different m o r p h o l o g i e s (Stol tz a n d V i n s o n , 1979; K r e l l a n d Bever idge , 1987; Kre l l , 1991a ,b) . T o d a t e t h e i chnov i rus Campoletis sonorensis v i rus ( C s V ) is t he p o l y d n a v i r u s t h a t h a s b e e n m o s t i n t e n sively s t u d i e d a t t h e m o l e c u l a r level. T h e r e m a r k s in th is rev iew a r e b a s e d heavily, b u t not exclusively, o n s tud ies of t h a t v i rus s y s t e m . F u t u r e inves t igat ions of o t h e r p o l y d n a v i r u s e s will b e neces sa ry before it will b e c l ea r w h e t h e r t he phys i ca l a n d gene t i c o r g a n i z a t i o n of C s V is typ ica l of i chnov i ruses a n d how s imi la r b racov i ruses a n d i chnov i ruses a r e a t t h e m o l e c u l a r level.

Po lydnav i ruses a p p e a r to r ep l i ca t e p r imar i ly , a n d poss ib ly exclusively, in t h e w a s p hos t (Stol tz a n d V i n s o n , 1979). Ava i l ab le S o u t h e r n a n d d o t b lo t d a t a sugges t t h a t t he a m o u n t of Campoletis sonorensis v i rus-specif ic D N A re m a i n s a t re la t ively c o n s t a n t levels in p a r a s i t i z e d o r v i rus - in jec ted Heliothis virescens l a rvae , o n e h a b i t u a l hos t of C. sonorensis w a s p s ( T h e i l m a n n a n d S u m m e r s , 1986). I n a d d i t i o n , e lec t ron mic roscop ic s tud ies have n o t r e p o r t e d v i rus pa r t i c l e s in p a r a s i t i z e d hos t t i ssues a t l a t e r t imes after ov ipos i t ion . A s ignif icant level of v i ra l r ep l i ca t ion t h u s does no t a p p e a r to o c c u r in t h e p a r a s i t i z e d hos t , a l t h o u g h in vivo l abe l ing s tud ies a r e n e e d e d to conf i rm t h a t low to m o d e r a t e levels of r ep l i ca t ion d o n o t occu r in o n e o r m o r e t i ssues of t h e l e p i d o p t e r a n hos t . T h e o b s e r v a t i o n s t h a t t he v i rus r ep l i ca tes p r i m a r i l y in t h e w a s p hos t b u t causes pa tho log i ca l c h a n g e s on ly in t h e p a r a s i t i z e d h o s t w e r e t h e first i n d i c a t i o n t h a t p o l y d n a v i r a l genes involved in t h e different func t ions m i g h t b e r e g u l a t e d in a species-specif ic m a n n e r . Ava i l ab le d a t a o n C s V g e n e t r a n s c r i p t i o n a r e cons i s t en t w i t h t h e idea t h a t a t leas t s o m e p o l y d n a v i r a l

9. Polydnavirus Genome Organization 191

genes a r e exp re s sed in a species-specif ic m a n n e r (see Sec t ion V I ) . H o w e v e r , b e c a u s e t h e funct ions of t h e v i ra l g e n e p r o d u c t s a r e n o t ye t k n o w n , it is n o t c lea r to w h a t e x t e n t p a r t i c u l a r p rocesses like r ep l i ca t ion a r e r e s t r i c t ed to on ly o n e of t h e h o s t species of p o l y d n a v i r u s e s , t h a t is, w a s p o r ( pa r a s i t i z ed ) l ep i d o p t e r a n l a rva .

T h e g ross fea tures of v i ra l r ep l i ca t ion a n d m o r p h o g e n e s i s h a v e b e e n s h o w n in n u m e r o u s e lec t ron m i c r o s c o p i c s tud ie s , even t h o u g h t h e m o l e c u l a r m e c h a n i s m s involved a r e n o t k n o w n ( s u m m a r i z e d in Stol tz a n d V i n s o n , 1979; K r e l l , 1991a) . V i r a l D N A syn thes i s a n d v i rus m a t u r a t i o n b e g i n in t h e l a te p h a r a t e female w a s p a n d c o n t i n u e in t h e a d u l t for a t leas t severa l d a y s after ec los ion ( N o r t o n et al., 1975; N o r t o n a n d V i n s o n , 1983; F l e m i n g a n d S u m m e r s , 1986; T h e i l m a n n a n d S u m m e r s , 1986). V i r a l r ep l i ca t i on a p p e a r s to b e r e s t r i c t ed to t h e nuc le i of ep i the l i a l cells in t h e ca lyx r eg ion of t h e ov iduc t s (Stol tz a n d V i n s o n , 1979). T h e newly syn thes i zed v i ra l D N A s a r e p a c k a g e d in s t r u c t u r a l l y c o m p l e x n u c l e o c a p s i d s t h a t a c q u i r e a n enve lope f rom m e m b r a n e s t h a t a p p e a r to be free w i t h i n t he n u c l e u s i tself (Stol tz a n d V i n s o n , 1979). T h i s m e t h o d of e n v e l o p m e n t , w h i c h a lso is seen d u r i n g bacu lov i ru s m a t u r a t i o n , h a s b e e n i n t e r p r e t e d as ev idence for de novo i n t r a n u c l e a r m e m b r a n e syn thes i s (Stol tz et al., 1973; Feder ic i , 1986). N o b io c h e m i c a l ev idence to sugges t n e w p a t h w a y s for m e m b r a n e b iosyn thes i s in t h e n u c l e u s exis ts , b u t s o m e a d d i t i o n a l r e s e a r c h to d e t e r m i n e t h e or ig in of t h e i n t r a n u c l e a r m e m b r a n e s m a y b e w a r r a n t e d s ince th is m e t h o d of e n v e l o p m e n t is n o t gene ra l l y obse rved in o t h e r a n i m a l v i rus famil ies . T h e l e n t i c u l a r i chnov i ru s n u c l e o c a p s i d s a r e i nd iv idua l l y enve loped in th is m e m b r a n e a n d t h e n a c q u i r e a s econd m e m b r a n e as t hey b u d t h r o u g h t h e n u c l e a r enve lope a n d t h e p l a s m a l e m m a ( N o r t o n et al., 1975; Stol tz a n d V i n s o n , 1979; K r e l l a n d S to l tz , 1980; K r e l l , 1987; K r e l l a n d Beve r idge , 1987). I n c o n t r a s t , o n e o r m o r e cy l ind r i ca l b r acov i rus n u c l e o c a p s i d s m a y b e enve loped in each m e m b r a n e found in t h e n u c l e u s . T h e b r acov i ru s v i r ions a r e r e l eased w i t h o u t a c q u i r i n g a d d i t i o n a l m e m b r a n e s w h e n t h e cells lyse ( N o r t o n et al., 1975; K r e l l a n d Sto l tz , 1979; Sto l tz a n d V i n s o n , 1979; K r e l l a n d Beve r idge , 1987). I n b o t h cases , t h e v i r ions a c c u m u l a t e to ve ry h i g h dens i t i e s in t h e l u m e n of t h e l a t e r a l ov iduc t s . T h e v i ruses t h u s a r e idea l ly pos i t i oned b o t h t e m p o r a l l y a n d phys ica l ly for t ransfe r i n t o l e p i d o p t e r a n hos t l a rvae w h e n t h e female w a s p ovipos i t s eggs .

III. Multipartite Genome Structure

T h e u n i q u e fea tu re of all p o l y d n a v i r u s e s is t h e m u l t i p a r t i t e o r s e g m e n t e d n a t u r e of t h e supe rco i l ed g e n o m e . A l t h o u g h o t h e r D N A v i ruses c o n t a i n cova-len t ly c losed c i rcu la r , d o u b l e - s t r a n d e d (ds) D N A (e.g. , o n e D N A m o l e c u l e p e r bacu lov i rus ) o r a r e s e g m e n t e d (e.g. , t h e two s i n g l e - s t r a n d e d , c i r cu l a r


D N A s of the g e m i n i v i r u s s u b g r o u p w i t h b i p a r t i t e g e n o m e s ) , p o l y d n a v i r u s e s a r e u n u s u a l in h a v i n g m u l t i p l e covalent ly closed c i rcu la r , d s D N A s in t h e g e n o m e of a g iven v i rus . V i rus l ike par t i c les r e p o r t e d in s o m e b r a c o n i d a n d i c h n e u m o n i d w a s p s c a n n o t l eg i t imate ly be cons ide red p o l y d n a v i r u s e s un t i l a m u l t i p a r t i t e , superco i l ed D N A g e n o m e h a s b e e n d e m o n s t r a t e d ( rev iewed in F l e m i n g , 1992; Kre l l , 1991a) . Po lydnav i r a l D N A s , w h o s e covalen t ly c losed c i r cu la r n a t u r e h a s b e e n d e m o n s t r a t e d b y m o l e c u l a r a n d b iophys i ca l m e t h o d s , a r e resolved in to superco i l ed a n d re laxed c i r cu la r f ract ions by i sopycn ic cen t r i fuga t ion in ce s ium c h l o r i d e - e t h i d i u m b r o m i d e g r a d i e n t s (Kre l l et al., 1982; T h e i l m a n n a n d S u m m e r s , 1986; Bl i s sa rd et al., 1987). I t is p r o b a b l e t h a t v i r ions c o n t a i n t he supe rhe l i ca l form of t he D N A s b e c a u s e t he super coiled form w o u l d be expec t ed to be favored t h e r m o d y n a m i c a l l y a n d t h e superco i l ed form p r e d o m i n a t e s w h e n n ick ing d u e to h a n d l i n g is m i n i m i z e d .

Po lydnav i ruses from different w a s p species differ in t e r m s of s e g m e n t n u m b e r , D N A size r a n g e , s e g m e n t m o l a r r a t ios , a n d to ta l g e n o m e size. A l t h o u g h these cha rac te r i s t i c s a r e q u i t e r e p r o d u c i b l e for a g iven po lyd n a v i r u s " s p e c i e s " i so la ted from a p a r t i c u l a r w a s p hos t spec ies , t he h e t e r o gene i ty in these cha rac te r i s t i c s is g r e a t e r a m o n g v i ruses in t h e family Polyd-nav i r i dae t h a n a m o n g v i ruses in o t h e r v i rus famil ies w i t h s e g m e n t e d g e n o m e s ( M u r p h y a n d K i n g s b u r y , 1990).

T h e n u m b e r of D N A s e g m e n t s p e r g e n o m e c a n b e e s t i m a t e d f rom t h e n u m b e r of b a n d s de t ec t ed in a g a r o s e gels , b u t s u c h e s t i m a t e s s h o u l d b e r e g a r d e d on ly as a p p r o x i m a t i o n s for severa l r e a s o n s . B o t h supe rhe l i ca l a n d r e l axed c i r cu la r forms of t he D N A s a r e p r e s e n t in a g a r o s e gels a n d a r e resolved f rom o n e a n o t h e r to v a r y i n g ex ten t s , d e p e n d i n g o n t h e r a n g e of D N A sizes, a g a r o s e c o n c e n t r a t i o n , field s t r e n g t h , e tc . T h e two forms often c a n be di f ferent ia ted in reg ions w h e r e t he reso lu t ion is g o o d if t h e supe rco i l ed a n d r e l axed c i r cu la r fract ions a r e r u n in pa ra l l e l . Howeve r , in a r e a s of t h e gel w h e r e t he two forms of different D N A s c o m i g r a t e significantly, th is d i s t i nc t ion is n o t a l w a y s c lear -cu t . As expec t ed for l a rge c i r cu la r mo lecu le s , t h e ex t r ac t ion m e t h o d , s to rage cond i t i ons , a n d e lec t rophores i s m e t h o d s c a n p r o foundly affect t he n u m b e r of b a n d s resolved a n d / o r t h e re la t ive p r o p o r t i o n of supe rco i l ed molecu le s a n d the c o r r e s p o n d i n g re laxed c i r cu l a r forms de t ec t ab l e p e r s a m p l e (Stol tz et al., 1981; J o n e s et al., 1986). T h e to ta l n u m b e r of b a n d s (open c i r cu la r a n d superco i l ed ) d e t e c t a b l e in Hyposoter exiguae v i ru s , for e x a m p l e , i nc reased from a p p r o x i m a t e l y 9 to 30 w h e n t h e ex t r ac t ion p r o tocol w a s modif ied (Stol tz et al., 1981). Similar ly, t he n u m b e r of s e g m e n t s in t he C s V g e n o m e inc reased from a p p r o x i m a t e l y 4 to 25 w h e n l a rge r a m o u n t s of m a t e r i a l we re e x a m i n e d after i so la t ion by m o r e r igo rous m e t h o d s (Stol tz et al., 1981; K r e l l et al., 1982).

E s t i m a t e s a r e fu r ther c o m p l i c a t e d by the fact t h a t t he v a r i o u s D N A segm e n t s a p p e a r to be p r e s e n t in n o n e q u i m o l a r r a t ios . T h e D N A s in t h e C s V


g e n o m e a p p e a r to be p r e s e n t in a t l eas t t h r e e " c l a s s e s " b a s e d o n re la t ive a b u n d a n c e (Fig . 1, a n d Bl i s sa rd et al., 1986a; K r e l l et al., 1982). T h e m o l e c u l a r bas i s a n d b io logica l s ignif icance of t h e n o n e q u i m o l a r i t y of t h e g e n o m i c s e g m e n t s a r e n o t k n o w n . D N A s p r e s e n t in m o r e l imi ted a m o u n t s m a y b e u n d e t e c t a b l e u n d e r c e r t a i n e x p e r i m e n t a l cond i t i ons . T h r e e a d d i t i o n a l D N A s e g m e n t s , A 2 , L 2 , a n d O 2 , w e r e v i sua l i zed in e t h i d i u m b r o m i d e - s t a i n e d gels of t h e C s V g e n o m e after e l ec t rophores i s of h i g h e r a m o u n t s of D N A (Bl i s sa rd et al., 1986a) . M o r e sensi t ive t e c h n i q u e s like a u t o r a d i o g r a p h y c a n resu l t in t h e d e t e c t i o n of o t h e r D N A s p r e s e n t in even lower a m o u n t s , for e x a m p l e , C 2

in C s V ( T h e i l m a n n a n d S u m m e r s , 1987). T h e a p p a r e n t differences in re la t ive a b u n d a n c e m a y be p a r t l y d u e to t h e c o m i g r a t i o n of two o r m o r e u n r e l a t ed D N A s of t h e s a m e size. C o m i g r a t i o n of u n r e l a t e d s u p e r h e l i c a l D N A s c a n resu l t in t he u n d e r e s t i m a t i o n of t h e to ta l n u m b e r of s e g m e n t s a n d m a y be u n d e t e c t e d excep t by c lon ing a n d / o r phys i ca l m a p p i n g . T h e C s V b a n d de s i g n a t e d D N A G (8.4 k b p ) c o n t a i n s two forms t h a t cou ld be d i s t i n g u i s h e d by S a i l a n d H i n d i 11 d iges t ion (Kre l l et al., 1982). T h e s e forms m a y r e p r e s e n t u n r e l a t e d D N A s , b u t b e c a u s e c lon ing , de t a i l ed phys i ca l m a p p i n g , a n d r ec ip roca l h y b r i d i z a t i o n of these two forms of supe rhe l i x G were n o t u n d e r t a k e n , t h e poss ib i l i ty t h a t t h e two forms r e p r e s e n t two res t r i c t ion f r a g m e n t l e n g t h p o l y m o r p h i s m s ( R F L P s ) on essent ia l ly t he s a m e D N A c a n n o t b e e x c l u d e d .

Al te rna t ive ly , t h e v i ra l D N A s p r e s e n t in a p p a r e n t l y " h y p e r m o l a r " a m o u n t s re la t ive to t he o t h e r s e g m e n t s m a y b e s ingle D N A species t h a t a r e syn thes i zed m o r e efficiently. W h e t h e r t he g r e a t e r a b u n d a n c e of t hese D N A s is func t iona l ly s ignif icant is n o t k n o w n , b u t it is t e m p t i n g to s p e c u l a t e t h a t t h e n o n e q u i m o l a r i t y of t he D N A s reflects a gene d o s a g e effect b y w h i c h t h e copy n u m b e r of s e g m e n t s c o n t a i n i n g essen t ia l v i ra l genes for p r o t e i n s re q u i r e d in h i g h a m o u n t s is i nc r ea sed . T h e a p p a r e n t l y " h y p e r m o l a r " C s V D N A W (15.8 k b p ) , for e x a m p l e , c o n t a i n s two genes t h a t a r e exp re s sed in Heliothis l a rvae w i t h i n 2 h r after p a r a s i t i z a t i o n b y C. sonorensis w a s p s a n d t h a t c o n t i n u e to b e a m o n g the m o s t h igh ly exp res sed v i ra l genes for a b o u t 9 d a y s wh i l e t h e w a s p deve lops as a n e n d o p a r a s i t e (Bl i ssard et al., 1986b, 1987). I f t h e n o n e q u i m o l a r i t y is n o t a n ar t i fact , t h e different re la t ive a b u n d a n c e s of t h e D N A s ra i se s ignif icant q u e s t i o n s a b o u t h o w the syn thes i s of t h e D N A s is differential ly r e g u l a t e d o r w h a t p a c k a g i n g s igna ls a r e involved in the i r differen t i a l e n c a p s i d a t i o n .

W h e t h e r t h e r e p o r t e d D N A profiles give a r e p r e s e n t a t i v e p i c t u r e of t h e g e n o m e of a g iven p o l y d n a v i r u s " s p e c i e s " is a lso o p e n to q u e s t i o n . T h e a m o u n t of v i ra l D N A p e r female is u sua l l y s ignif icant ly less t h a n 1 μ g p e r p a i r of l a t e r a l ov iduc t s , for e x a m p l e , 150 n g C s V D N A p e r C. sonorensis f emale ( F l e m i n g a n d S u m m e r s , 1986). M a n y of t h e r e p o r t e d D N A e l e c t r o p h o r e t i c profiles therefore w e r e d e t e r m i n e d w i t h v i ra l D N A i so la ted f rom severa l females in a s ingle l a b o r a t o r y p o p u l a t i o n . B e c a u s e of t h e difficulty of r e a r i n g

RC

SH

- W

- Q - 0 1

- M

-H

-B

CsV Figure 1 CsV DNA. CsV DNA was isolated from sucrose gradient-purified virions from pooled C. sonorensis females and was electrophoresed in its undigested, native form in a 0.7% agarose gel in Tris acetate buffer containing 0.5μg ethidium bromide/ml . The regions of the gel in which the superhelical (SH) and relaxed circular (RC) forms of the DNAs migrate are indicated by arrows. The differences in intensity among the approximately 25 to 28 supercoiled DNAs that migrate in the lower portion of the gel are reproducible. Selected superhelical DNA bands are indicated by letter designations according to the scheme used in the original description of CsV (Krell etaL, 1982). Sizes of the selected DNAs (in kilobase pairs) are: Β (6.7), Η (8.5), Μ (10.4), Ο 1 (11.3), Q (12.2), and W (15.8).


t h e p a r a s i t o i d s , o t h e r r e p o r t e d profiles r e p r e s e n t t h e D N A o b t a i n e d f rom a s ingle feral female (Stol tz et al., 1981). A p a r t f rom t h e p r o b l e m of d e t e c -tab i l i ty w h e r e l imi t ed a m o u n t s of m a t e r i a l w e r e u sed , t h e n u m b e r of insec ts s a m p l e d to p r o d u c e a D N A profile m a y be i m p o r t a n t b e c a u s e gene t i c v a r i a t ion in t h e n u m b e r of s e g m e n t s c a n o c c u r w i t h i n a g iven v i ru s . T h e n u m b e r of D N A s in t h e g e n o m e s of t h e v i ruses from s o m e g e o g r a p h i c a l l y i so la ted p o p u l a t i o n s of Cotesia melanoscela ( C m V ) o r f rom i n d i v i d u a l females f rom l a b o r a t o r y colonies of Hyposoter lymantriae o r C. sonorensis v a r i ed (Stol tz et al., 1986; S to l tz a n d X u , 1990; J . F l e m i n g , u n p u b l i s h e d ) . A l t h o u g h t h e s imi lar i t ies b e t w e e n v i ra l profiles of i so la tes f rom i n d i v i d u a l females w i t h i n t h e s a m e w a s p species exceeded t h e differences, severa l s e g m e n t s w i t h i n a v i ru s " s p e c i e s " w e r e v a r i a b l e in s o m e cases (Stol tz a n d X u , 1990).

T h e o b s e r v e d in t raspeci f ic v a r i a t i o n s sugges t t h a t t h e use of v i ra l D N A from poo led w a s p s m a y resu l t in a n o v e r e s t i m a t i o n of t he g e n o m i c c o m p l e x i ty of t hese v i ruses w h e r e a s use of m a t e r i a l f rom single females m a y c a u s e o n e to u n d e r e s t i m a t e t he p o t e n t i a l complexi ty . T h e v a r i a t i o n s o b s e r v e d in v i ra l D N A s from i n d i v i d u a l females w i t h i n a w a s p species u n d e r s c o r e t h e a p p a r e n t p las t i c i ty of p o l y d n a v i r u s g e n o m e s . T h e a p p a r e n t a b s e n c e of w h o l e v i ra l g e n o m i c s e g m e n t s in s o m e i n d i v i d u a l s w i t h i n a species a l so sugges t s t h a t t h e v i ra l genes o n t h e m i s s i n g s e g m e n t m a y b e n o n e s s e n t i a l for v iab i l i ty in e i t he r t h e w a s p o r t h e p a r a s i t i z e d hos t . Al te rna t ive ly , if a n essen t ia l g e n e w e r e l oca t ed o n a s u p e r h e l i x t h a t is de l e t ed in s o m e s t r a i n s , a d u p l i c a t e o r closely r e l a t e d g e n e loca ted on a n o t h e r v i r a l g e n o m i c s e g m e n t m i g h t s u p p l y t h e neces sa ry gene t i c i n fo rma t ion . T h e p r e s e n c e of v i ra l g e n e famil ies in p o l y d nav i ru se s like C s V a n d t h e m u l t i p a r t i t e s t r u c t u r e of t h e g e n o m e s t h u s cou ld po t en t i a l l y i nc rea se t h e a p p a r e n t p las t i c i ty of p o l y d n a v i r u s g e n o m e s w i t h r e spec t to g e n o m i c s e g m e n t size a n d n u m b e r .

In te r spec i f ic differences in v i ra l D N A profiles gene ra l l y exceed i n t r a specific differences. J o n e s a n d his a ssoc ia tes (1986) p r e s e n t ev idence t h a t t h e p o l y d n a v i r u s e s of t h e c o n g e n e r i c species Chelonus n e a r curvimaculatus a n d C. insularis c a n b e d i s t i n g u i s h e d b y t he se c r i t e r ia . T h e D N A profiles of p o l y d n a v i r u s e s f rom m o r e d i s t a n t l y r e l a t ed w a s p s in different g e n e r a o r famil ies a r e less s imi l a r in t e r m s of t he n u m b e r s a n d sizes of t h e D N A s t h a n t h e v i ruses of t h e t w o Chelonus species a r e . U n l i k e t he l imi t ed r a n g e of 1 0 - 1 2 d s R N A s p e r g e n o m e of a typ ica l r eov i rus , for e x a m p l e , different p o l y d n a v i r u s e s c a n h a v e f rom fewer t h a n 10 (Hyposoter annulipes v i rus ) to m o r e t h a n 25 ( C s V ) c i r cu l a r D N A molecu le s (Stol tz et al., 1981; K r e l l et al., 1982; B l i s sa rd et al., 1986a) . P o l y d n a v i r a l g e n o m e s often h a v e 15 o r m o r e s e g m e n t s (Stol tz et al., 1981; S to l tz a n d X u , 1990; X u a n d Sto l tz , 1991). T h e D N A s t e n d to b e fairly l a rge b u t c a n v a r y w i t h i n a fairly b r o a d size r a n g e . T h e C s V g e n o m e , for e x a m p l e , is c o m p o s e d of D N A s of a p p r o x i m a t e l y 5 - 2 1 k b p , a n d l a r g e r D N A s


a p p e a r to be p r e s e n t in the g e n o m e of Cotesia melanoscela v i rus (Kre l l et al., 1982; Bl i s sa rd et al, 1986a; Stol tz a n d X u , 1990).

T h e va r iab i l i ty in v i ra l D N A n u m b e r , size, a n d m o l a r i t y m a k e s it difficult to e s t i m a t e a n a g g r e g a t e g e n o m e size accu ra t e ly for a n y p o l y d n a v i r u s . Avai l ab l e e s t i m a t e s a r e b a s e d on the s u m s of t he sizes of t h e c i r cu l a r D N A m o l e cules o r all r es t r i c t ion f r agmen t s in d iges t ed v i ra l D N A a n d s h o u l d b e r e g a r d e d on ly as p r e l i m i n a r y e s t ima t e s . Sizes c a n r a n g e f rom 75 k b p for Heliothis exiguae v i rus ( H e V ) to 250 k b p or m o r e for CsV , i n d i c a t i n g t h a t p o l y d n a v i r a l g e n o m e s t h u s t end to be l a rge a n d v a r y w i t h i n a w i d e r a n g e (Kre l l et al, 1982; Kre l l , 1991a) .

A l t h o u g h differences in t he n u m b e r a n d sizes of D N A s in p o l y d n a v i r u s g e n o m e s h is tor ica l ly have b e e n used as a p r e l i m i n a r y m e t h o d of d i s t i n g u i s h ing the v i ruses from different w a s p species , these c r i t e r ia c lear ly a r e i n a d e q u a t e for phy logene t i c c o m p a r i s o n s . B o t h e x p e r i m e n t a l m e t h o d s a n d the level of gene t i c p o l y m o r p h i s m s w i t h i n a p o l y d n a v i r u s " s p e c i e s " (or even a p o p u l a t i o n w i t h i n a " spec ies" ) c a n s ignif icant ly inf luence t h e D N A profile obse rved . M o r e significantly, these t ra i t s d o n o t a d d r e s s t h e gene t i c r e l a t ed -ness of t he v i ra l g e n o m e s in a mean ing fu l way. D N A h y b r i d i z a t i o n a n d nuc l eo t i de s e q u e n c e d a t a will p rov ide a s o u n d e r bas i s for fu tu re c o m p a r i sons .

IV. Genome Packaging

T h e D N A profiles of g iven p o l y d n a v i r u s " s p e c i e s " a r e q u i t e r e p r o d u c i b l e , excep t for t h e fairly l imi ted v a r i a t i o n s in D N A s e g m e n t n u m b e r n o t e d ear l ier . T h i s a p p e a r s to b e t r u e w h e t h e r t h e v i ra l D N A is o b t a i n e d f rom different i n d i v i d u a l females , w a s p g e n e r a t i o n s , or w a s p p o p u l a t i o n s (Kre l l et al., 1982; Stol tz et al., 1986; J . F l e m i n g , u n p u b l i s h e d d a t a ) . T h e r e p r o d u c i b i l i t y sug gests t h a t t he n u m b e r a n d r a t i o of s e g m e n t s in a v i r ion a r e r e g u l a t e d a t t h e level of e i the r D N A rep l i ca t ion a n d / o r p a c k a g i n g . H o w e v e r , b e c a u s e a n e l ec t ropho re t i c profile of a given p o l y d n a v i r a l g e n o m e reflects all t h e D N A s found in a p o p u l a t i o n of v i rus pa r t i c l e s t h a t a r e p r e s e n t in o n e (or m o r e ) ov iduc t s , it is no t c lear w h e t h e r each n u c l e o c a p s i d ( a n d / o r v i r ion) c o n t a i n s all v i ra l D N A s , specific subse t s of t he g e n o m e , r a n d o m col lec t ions of t h e v i ra l D N A s , o r i n d i v i d u a l D N A s . I f each nuc l eocaps id (or v i r ion) w e r e to c o n t a i n less t h a n t h e full c o m p l e m e n t of v i ra l D N A s , t he obse rved r e p r o d u c i b i l i t y w o u l d sugges t t h a t t he r a t i o of different v i ra l subc lasses c o n t a i n i n g v a r y i n g n u m b e r s of D N A s p e r pa r t i c l e a lso is r e g u l a t e d in each female . P r e c e d e n t s for t he va r ious poss ib le p a t t e r n s of p a c k a g i n g exist in o t h e r v i rus famil ies c h a r a c te r ized b y s e g m e n t e d g e n o m e s (Bel loncik, 1989; M u r p h y a n d K i n g s b u r y , 1990; M a t t h e w s , 1991).


A p a r t f rom its p o t e n t i a l in t e res t f rom a m o l e c u l a r b io logica l s t a n d p o i n t , t he p a c k a g i n g s t r a t e g y a d o p t e d b y p o l y d n a v i r u s e s cou ld theore t i ca l ly affect t h e p r o b a b i l i t y t h a t a p a r t i c u l a r v i ra l gene is expres sed in a g iven cell of a p a r a s i t i z e d hos t . Ava i l ab le d a t a sugges t t h a t p o l y d n a v i r a l genes a r e segreg a t e d a m o n g t h e g e n o m i c s e g m e n t s (see Sec t ion V I ) . A s s u m i n g t h a t each v i r ion is e q u a l l y c a p a b l e of infect ing a g iven hos t cell, t h e p r o b a b i l i t y t h a t a p a r t i c u l a r v i ra l g e n e is expres sed in t he cell is theore t i ca l ly g r e a t e r if each infect ing v i r ion c o n t a i n s t he en t i r e v i ra l g e n o m e t h a n if each v i r ion c o n t a i n s on ly a s u b s e t of t h e g e n o m e . I n t h e l a t t e r case , t he p r o b a b i l i t y t h a t a p a r t i c u l a r g e n e is exp res sed in t he cell w o u l d be p r o p o r t i o n a l to t he p e r c e n t a g e of t h e in jec ted i n o c u l u m v i rus pa r t i c l e s t h a t c o n t a i n e d t h e g e n o m i c s e g m e n t o n w h i c h t h e g e n e is l oca ted . T h e p a c k a g i n g s t r a t egy w o u l d b e less i m p o r t a n t f rom th is s t a n d p o i n t if t he gene of i n t e r e s t w e r e d u p l i c a t e d o n m o r e t h a n o n e g e n o m i c D N A s e g m e n t , m o r e t h a n a s ingle v i r ion w e r e to infect each cell , o r specific v i ra l subc la s ses w i t h different affinities for r e cep t o r s o n different hos t cell types w e r e s h o w n to exist .

L i t t l e d i r ec t e x p e r i m e n t a l work h a s a d d r e s s e d t h e p r o b l e m of p o l y d n a v i r a l g e n o m e p a c k a g i n g . T h e r a t i o of t he n u c l e o c a p s i d size (vo lume) to t h e v i ra l g e n o m e size c a n p r o v i d e a r o u g h e s t i m a t e of t h e p r o p o r t i o n of t h e g e n o m e t h a t a typ ica l v i rus pa r t i c l e cou ld c o n t a i n . N u c l e o c a p s i d s of i chnov i ruses a r e of un i fo rm size a n d t e n d to be l a rge , for e x a m p l e , 85 n m X 330 n m for C s V ( N o r t o n et al., 1975; Stol tz a n d V i n s o n , 1979). C o m p a r i s o n of t h e pa r t i c l e v o l u m e : g e n o m e size ra t ios of C s V o r H e V w i t h t he ra t ios for bacu lov i ruses sugges t s t h a t each i chnov i rus v i r ion is sufficiently l a rge to e n c a p s i d a t e t h e en t i r e g e n o m e a n d cou ld poss ib ly c o n t a i n m u l t i p l e copies of a t l eas t s o m e of t h e v i ra l D N A s (Stol tz a n d V i n s o n , 1979; K r e l l a n d Stol tz , 1980; K r e l l et al., 1982). A t t e m p t s to f rac t iona te C s V in to p u t a t i v e subc lasses c o n t a i n i n g differe n t s u b s e t s of D N A s by suc rose o r C s C l g r a d i e n t u l t r acen t r i fuga t ion w e r e unsuccess fu l (Kre l l et al., 1982). T h u s , e i the r subc lasses of v i r ions c o n t a i n i n g different c o m p l e m e n t s of v i ra l D N A d o no t exist for C s V o r these c lasses h a v e dens i t i e s t h a t a r e too s imi l a r to each o t h e r to b e s e p a r a t e d b y t h e t e c h n i q u e s u sed .

Bracov i ruses h a v e r o d - s h a p e d n u c l e o c a p s i d s w i t h fixed d i a m e t e r s b u t v a r i a b l e l e n g t h s , b u t w h e t h e r they a r e m u l t i c o m p o n e n t v i ruses in w h i c h different p o r t i o n s of t he g e n o m e a r e d i s t r i b u t e d a m o n g different n u c l e o c a p sids t h a t a r e phys ica l ly d i s t inc t f rom o n e a n o t h e r is no t c lear . W h e t h e r t he l a r g e r n u c l e o c a p s i d s cou ld c o n t a i n t he en t i r e g e n o m e h a s n o t b e e n de te r m i n e d . I f o n e c o m p a r e s a h i s t o g r a m of t he f r equency of different-s ized n u c l e o c a p s i d s in C m V w i t h t h e f r equency of t h e different sizes of c i r cu l a r C m V D N A s (as d e t e r m i n e d by e l ec t ron m i c r o s c o p y of K l e i n s c h m i d t s p r e a d s ) , t h e two h i s t o g r a m s a r e very s imi l a r (Kre l l a n d Sto l tz , 1979). I n o t h e r w o r d s , t h e p r o p o r t i o n s of long n u c l e o c a p s i d s a n d l a rge D N A s , for e x a m p l e , w e r e s imi -


lar . Sugges t ive a s these d a t a a r e , m o r e de ta i l ed b i o c h e m i c a l a n d e lec t ron mic roscop i c s tud ie s a r e r e q u i r e d to d e t e r m i n e w h e t h e r t h e v i ra l D N A s a r e s ingly e n c a p s i d a t e d .

I f each b racov i rus n u c l e o c a p s i d even tua l ly were s h o w n to c o n t a i n on ly o n e o r a few D N A s , t he p o r t i o n of t he v i ra l g e n o m e p r e s e n t in a n y infected l e p i d o p t e r a n hos t cell w o u l d t h e n d e p e n d on the n u m b e r of v i r ions t h a t infect t h e hos t cell. However , in severa l b racov i rus species , for e x a m p l e , C m V , m o r e t h a n o n e n u c l e o c a p s i d is enve loped in each v i r ion (Stol tz a n d V i n s o n , 1979). A s ingle infect ing v i r ion t h u s cou ld i n t r o d u c e m o r e t h a n o n e g e n o m i c segm e n t even if each n u c l e o c a p s i d c o n t a i n s a s ingle D N A . I t will b e of i n t e r e s t to d e t e r m i n e w h e t h e r b racov i rus n u c l e o c a p s i d s a r e co -enve loped a t r a n d o m or w h e t h e r specific subse t s of n u c l e o c a p s i d s a r e enve loped in p a r t i c u l a r v i ru s pa r t i c l e s , b e c a u s e a n o n r a n d o m e n v e l o p m e n t s t r a t e g y h y p o t h e t i c a l l y w o u l d be a n efficient m e c h a n i s m to e n s u r e t h a t specific subse t s of v i ra l D N A s w o u l d b e p r e s e n t in a g iven infected cell.

V. Intraspecific Genomic Complexity

P r e l i m i n a r y ana lys i s of t he g e n o m e of H e V by S o u t h e r n b lo t h y b r i d i z a t i o n d e m o n s t r a t e d t h a t t he H e V D N A s p r e s e n t in h i g h e r m o l a r a m o u n t s d i d n o t c ros s -hybr id i ze w i th o t h e r H e V D N A s u n d e r t he h i g h s t r i n g e n c y c o n d i t i o n s used (Kre l l a n d Stol tz , 1980). S imi l a r resu l t s w e r e r e p o r t e d for t h e " h y p e r m o l a r " C s V D N A s (Kre l l et al., 1982). T h e s e s tud ies sugges t ed t h a t t h e m o r e a b u n d a n t H e V o r C s V D N A s were u n r e l a t e d to o t h e r D N A s in t h e r e spec tive v i ra l g e n o m e s a n d therefore m i g h t b e u n i q u e . H o w e v e r , s u b s e q u e n t S o u t h e r n b lo t ana lyse s of C s V w i t h c loned v i ra l D N A p r o b e s u n d e r s imi l a r h i g h s t r i ngency cond i t i ons cons i s ten t ly d e t e c t e d s ignif icant c ro s s -hyb r id i za t ion of t he p r o b e D N A s w i t h o n e or m o r e v i ra l D N A s in a d d i t i o n to t h e expec t ed h y b r i d i z a t i o n to t he supe rhe l i ca l a n d r e l axed c i r cu l a r forms of t h e s e g m e n t f rom w h i c h t h e p r o b e D N A s w e r e c loned ( F l e m i n g a n d S u m m e r s , 1986; Bl i s sa rd et al., 1987; T h e i l m a n n a n d S u m m e r s , 1987). E a c h of t h e c loned D N A p r o b e s r e p r o d u c i b l y h y b r i d i z e d w i th on ly a s u b s e t of t h e gen o m i c s e g m e n t s . T h e subse t s of h y b r i d i z i n g v i ra l D N A s differed for t he va r ious p r o b e s tes ted . N o n e of t he p r o b e s tes ted h y b r i d i z e d w i t h all g e n o m i c s e g m e n t s o r d e t e c t e d a " n e s t e d s e t " of s e g m e n t s , t h a t is , a p r o b e t h a t hyb r id i zed w i t h a g iven supe rhe l ix d id n o t h y b r i d i z e w i t h all successively l a rge r s e g m e n t s (Bl i ssard et al., 1986b, 1980; F l e m i n g a n d S u m m e r s , 1986; T h e i l m a n n a n d S u m m e r s , 1987, 1988). T h e h y b r i d i z a t i o n p a t t e r n s a r g u e against C s V b e i n g a defective v i rus . T h e d a t a s u p p o r t t h e i n t e r p r e t a t i o n t h a t t h e m u l t i p l e D N A s in a p o l y d n a v i r u s reflect a s e g m e n t e d g e n o m e .

P a r t of t h e a p p a r e n t c ros s -hybr id i za t ion m a y reflect m u l t i p l e r e l axed cir-


c u l a r fo rms of a g iven c i r cu l a r mo lecu l e , a s h a s b e e n sugges t ed for C s V D N A Β ( T h e i l m a n n a n d S u m m e r s , 1987). M o r e i m p o r t a n t l y , severa l g e n e famil ies exist in t h e C s V g e n o m e (see Sec t ion V I ) , a n d a t leas t in t h e case of t h e 540-b p r e p e a t e l e m e n t g e n e family, t h e m e m b e r s have b e e n s h o w n u n a m b i g u o u s l y to exist on m o r e t h a n o n e supe rhe l i ca l D N A (Bl i ssa rd et aL, 1986b, 1987; T h e i l m a n n a n d S u m m e r s , 1987). Seg rega t ion of t he g e n e family m e m be r s a m o n g t h e g e n o m i c s e g m e n t s sugges t s t h a t fairly ex tens ive i n t r a g e n o m i c r e c o m b i n a t i o n h a s o c c u r r e d d u r i n g t h e evo lu t ion of t h e v i ru s . A t h i r d poss i b le e x p l a n a t i o n , w h i c h h a s n o t yet b e e n tes ted expe r imen ta l ly , m a y be m o r e a p p l i c a b l e to cases w h e r e v i ru s f rom t w o w a s p p o p u l a t i o n s c o n t a i n s l ight ly different n u m b e r s of c ro s s -hyb r id i z ing supe rhe l i ce s , for e x a m p l e , D N A s D a n d G of t h e ( T r u r o ) N o v a Sco t ia a n d C o n n e c t i c u t s t r a i n s of C m V (Stol tz et aL, 1986). D u p l i c a t i o n of a g e n e or i n se r t ion of r a n d o m vi ra l o r w a s p seq u e n c e s i n t o a v i ra l supe rhe l i x cou ld theore t i ca l ly give r ise to two o r m o r e differently s ized g e n o m i c s e g m e n t s t h a t have t he s a m e s e q u e n c e a p a r t f rom t h e i n se r t ed o r d u p l i c a t e d f r a g m e n t s . De l e t i ons cou ld s imi la r ly r e su l t in two closely r e l a t e d molecu le s of different size.

T h e g e n o m i c c o m p l e x i t y of t he C s V g e n o m e w a s c o r r o b o r a t e d by S o u t h e r n c ross -b lo t h y b r i d i z a t i o n ana lyse s in w h i c h each E c o R I f r a g m e n t hyb r id i zed w i t h o n e or m o r e E c o R I f r a g m e n t s in a d d i t i o n to i tself ( T h e i l m a n n a n d S u m m e r s , 1987). T h e d a t a a r e cons i s t en t w i t h t h e ex i s tence of g e n e famil ies a n d / o r repe t i t ive D N A w i t h i n t h e v i ra l g e n o m e . H o w e v e r , b e c a u s e t h e or ig in of each f r a g m e n t in t h e (d iges ted) g e n o m e is n o t k n o w n , a t leas t s o m e of t h e c ro s s -hyb r id i z ing s e q u e n c e s m a y r e p r e s e n t r e s t r i c t ion f r a g m e n t l e n g t h p o l y m o r p h i s m s , w h i c h have b e e n d e t e c t e d in t he C s V g e n o m e ( F l e m ing et aL, 1990; F l e m i n g , 1991; J . F l e m i n g , u n p u b l i s h e d d a t a ) . [ R F L P s a l so h a v e b e e n d e t e c t e d in t h e g e n o m e s of o t h e r i chnov i ruses a n d b racov i ru se s (Stol tz a n d X u , 1990).] B e c a u s e R F L P s reflect t h e g a i n o r loss of a r e s t r i c t ion e n d o n u c l e a s e si te on different copies of a D N A t h a t exist in a p o p u l a t i o n , a g iven p r o b e will h y b r i d i z e w i t h severa l differently sized f r a g m e n t s , p a r t of w h i c h a r e i den t i ca l . T h e ex is tence of R F L P s in a v i rus p o p u l a t i o n t h u s cou ld resu l t in a n e x a g g e r a t e d e s t i m a t e of i n t r a g e n o m i c c o m p l e x i t y in s o m e expe r i m e n t a l de s igns .

VI. Transcription and Gene Families

A. Functional Organization of the Genome

O n e of t h e ear l ies t sugges t ions t h a t p o l y d n a v i r u s g e n o m e s a r e t r a n s c r i p t iona l ly ac t ive w a s t h e o b s e r v a t i o n t h a t u l t rav io le t i r r a d i a t i o n of C. sonorensis "ca lyx fluid" ( the v i rus a n d o t h e r s u b s t a n c e s in t he l a t e r a l ov iduc t s ) r e d u c e d

2 0 0 Jo-Ann G. W. Fleming and Peter J. Krell

its b io logical ac t iv i ty in injected l e p i d o p t e r a n l a rvae in a d o s a g e - d e p e n d e n t m a n n e r ( V i n s o n et al., 1979). To d a t e , t r a n s c r i p t i o n h a s b e e n s t u d i e d in de t a i l on ly in t he C s V sys t em. H y b r i d i z a t i o n of c D N A s of t he p o l y a d e n y l a t e d m R N A s from p a r a s i t i z e d H. virescens l a rvae or C. sonorensis p u p a e to t h e C s V g e n o m e p r o v i d e d t h e first p roo f t h a t p o l y d n a v i r u s genes a r e t r a n s c r i b e d ( F l e m i n g et al., 1983). T h e d a t a i n d i c a t e d t h a t C s V t r a n s c r i p t i o n in t h e p a r a s i t i z e d hos t occu r s w i t h i n 2 h r after t h e female w a s p ovipos i t s eggs a n d p o l y d n a v i r u s a n d c o n t i n u e s a t r ead i ly d e t e c t a b l e levels for a p p r o x i m a t e l y 9 d a y s , w h e n the final C. sonorensis i n s t a r e m e r g e s f rom the m o r i b u n d hos t . S o m e of t he v i ra l g e n e p r o d u c t s a r e bel ieved to a l t e r t h e phys io logy of t h e p a r a s i t i z e d hos t . T h e s e s tud ies a lso i n d i c a t e d t h a t severa l , b u t n o t al l , of t he C s V g e n o m i c s e g m e n t s c o n t a i n s e q u e n c e s t h a t a r e t r a n s c r i b e d in t h e p a r a si t ized hos t a n d sugges t ed t h a t different v i ra l s e q u e n c e s w e r e exp res sed in t h e w a s p a n d the l e p i d o p t e r a n hos t .

L a t e r inves t iga t ions s ignif icant ly e x t e n d e d a n d refined the ea r ly e x p r e s sion s tud ies (Bl i ssard et al., 1986a, 1987, 1989; T h e i l m a n n a n d S u m m e r s , 1987; T h e i l m a n n a n d S u m m e r s , 1988). A t least 12 m R N A s a r e d e t e c t a b l e in H. virescens l a rvae p a r a s i t i z e d by C. sonorensis w a s p s o r injected w i t h pur i f ied CsV, i n d i c a t i n g t h a t n e i t h e r w a s p t issue n o r o t h e r n o n v i r a l factors a r e re q u i r e d for C s V t r a n s c r i p t i o n (Bl issard et al., 1986b) . T h e s e e x p e r i m e n t s a lso p r o v i d e d the s u p p o r t i n g m o l e c u l a r d a t a for t h e p ivo ta l e x p e r i m e n t s of E d s o n a n d h e r coworkers (1981) , w h o d e m o n s t r a t e d t h a t g r ad i en t -pu r i f i ed C s V a lone w a s sufficient to p r e v e n t t he e n c a p s u l a t i o n of C. sonorensis eggs in hos t l a rvae a n d e n d o p a r a s i t i c s tages of t he w a s p s . A l t h o u g h a d d i t i o n a l factors , for e x a m p l e , v e n o m s , a lso a r e necessa ry in s o m e b racov i rus s y s t e m s , t h e d a t a from the C s V sys t em s t rong ly sugges t t h a t t he a l t e r a t i o n of t h e phys io logy of t he p a r a s i t i z e d hos t d e p e n d s p r i m a r i l y on vi ra l gene express ion .

T h e p r o b a b l e loca t ion of t he genes in t he v i ra l g e n o m e a n d t h e size of t h e t r a n s c r i p t s e n c o d e d by p a r t i c u l a r v i ra l D N A f r agmen t s w e r e d e t e r m i n e d by b lo t ana lyses (Bl i ssard et al., 1986a ,b) . C l o n e d v i ra l D N A s c o n t a i n i n g t h e expres sed s e q u e n c e s o r t he flanking, n o n t r a n s c r i b e d s e q u e n c e s w e r e u s e d s e p a r a t e l y as p r o b e s w i t h S o u t h e r n b lo ts of u n d i g e s t e d C s V g e n o m i c D N A a n d N o r t h e r n b lo ts of to ta l p o l y a d e n y l a t e d m R N A s from p a r a s i t i z e d h o s t l a rvae , respect ive ly (Bl issard et al., 1986b) . T h e resu l t s of these e x p e r i m e n t s p r o v i d e d the first i nd i ca t i on of severa l of t he u n u s u a l fea tures of t h e func t ional o r g a n i z a t i o n of p o l y d n a v i r u s g e n o m e s . F i rs t , typ ica l of w h a t o n e m i g h t expec t for a v i rus w i t h a s e g m e n t e d g e n o m e , t he different p r o b e s h y b r i d i z e d w i t h different g e n o m i c s e g m e n t s . However , each p r o b e expec ted ly h y b r i d i z e d w i t h m o r e t h a n o n e v i ra l D N A . U n r e l a t e d p r o b e D N A s h y b r i d i z e d w i t h different subse t s of t he v i ra l g e n o m i c s e g m e n t s , b u t a g iven g e n o m i c s e g m e n t s o m e t i m e s h y b r i d i z e d w i t h m o r e t h a n o n e of t he u n r e l a t e d p r o b e s . I n o t h e r w o r d s , different p r o b e s identif ied different, b u t s o m e t i m e s pa r t i a l l y ove r l ap -


p i n g , s u b s e t s of t he v i ra l g e n o m i c D N A s e g m e n t s . T h e a p p a r e n t c ross -h y b r i d i z a t i o n w a s m u c h too ex tens ive a n d too c o m p l e x to be whol ly exp l a i n e d b y t h e p r e s e n c e of m u l t i p l e topo log ica l forms of t h e c i r cu l a r D N A s . S e c o n d , p r o b e s t h a t w e r e u n r e l a t e d (as d e t e r m i n e d by p h y s i c a l m a p p i n g a n d t h e S o u t h e r n b lo t d a t a ) h y b r i d i z e d w i t h different m R N A s . T h e ma jo r i t y of t h e p r o b e s each h y b r i d i z e d w i t h o n e m R N A ap iece . U n e x p e c t e d l y , two of t h e gene-specif ic p r o b e s each h y b r i d i z e d w i t h two a b u n d a n t v i ra l m e s s a g e s ap i ece , o n e to t r a n s c r i p t s of 1.0 a n d 1.6 k b a n d the o t h e r to m R N A s of 1.1 a n d 1.4 k b . T h e r e l a t ed m R N A s cou ld be i n t e r p r e t e d as t r a n s c r i p t s of m e m be r s of a m u l t i g e n e family, a l t e rna t ive ly spl iced m R N A s , e tc . T h i r d , h y b r i d -se lec ted m R N A s cou ld b e t r a n s l a t e d in vitro, i n d i c a t i n g t h a t t he c o d i n g seq u e n c e s w e r e n o t p s e u d o g e n e s .

T h e m o d e l of t he C s V g e n o m e t h a t e m e r g e d f rom these s t ud i e s is o n e in w h i c h t h e p o l y d n a v i r a l g e n o m e is b o t h func t iona l ly a n d phys ica l ly m u l t i p a r t i te . I n d i v i d u a l genes , i n c l u d i n g m e m b e r s of gene famil ies , a r e s e g r e g a t e d a m o n g t h e g e n o m i c s e g m e n t s . T h e v i ra l supe rhe l i ca l D N A s a r e n o t i den t i ca l b u t m a y s h a r e o n e o r m o r e s e q u e n c e s . Ava i l ab le d a t a f rom s tud ie s of t h r e e C s V g e n e famil ies (d i scussed in t h e following) s u p p o r t th is m o d e l . H o w e v e r , n o n e of t h e C s V g e n o m i c s e g m e n t s h a s b e e n s e q u e n c e d in its ent i re ty . A d d i t iona l m o l e c u l a r c o m p a r i s o n s of different g e n o m i c s e g m e n t s a r e n e e d e d to d e t e r m i n e w h e t h e r each s u p e r h e l i x is a m o s a i c of u n i q u e a n d s h a r e d seq u e n c e s . S u c h c o m p a r i s o n s will d e m o n s t r a t e (1) how ex tens ive t h e s h a r e d s e q u e n c e s a r e , t h a t is, a r e t hey re s t r i c t ed to t h e genes in m u l t i g e n e famil ies o r d o they a lso i n c l u d e o the r , n o n e x p r e s s e d s e q u e n c e s , a n d (2) h o w s imi l a r t h e r e l a t ed s e q u e n c e s on different D N A s a r e .

B. Known CsV Gene Families

T h e p r e l i m i n a r y ident i f ica t ion of t h e genes e n c o d i n g t h e 1.0- a n d 1.6-kb m R N A s as m e m b e r s of a g e n e family w a s conf i rmed by n u c l e o t i d e (n t ) s e q u e n c e ana lys i s of t h e r e l a t ed s e q u e n c e s (Bl i ssa rd et aL, 1987). c D N A s c o r r e s p o n d i n g to t he 1.6- a n d 1.0-kb t r a n s c r i p t s w e r e m a p p e d to genes o n different r eg ions of (c loned) supe rhe l i x W (15.8 k b p ) . I t is of in t e re s t t h a t t h e c D N A for t h e 1.6-kb m R N A h y b r i d i z e d C s V g e n o m i c D N A s W, R (13.3 k b p ) , a n d Μ (10.4 k b p ) , w h e r e a s t h e c D N A for t h e 1.0-kb t r a n s c r i p t hyb r id i zed on ly w i t h D N A W, cons i s t en t w i t h t h e c o n c e p t of t h e s eg rega t i on a n d s e q u e n c e d i v e r g e n c e of r e l a t ed genes in t h e g e n o m e . C o m p a r i s o n of t h e n u c l e o t i d e s e q u e n c e s of t h e c D N A for t he 1.6-kb t r a n s c r i p t a n d t h e h o m o logous g e n o m i c s e q u e n c e on D N A W i n d i c a t e d t h a t t h e t w o s e q u e n c e s a r e iden t i ca l excep t for two i n t r o n s t h a t a r e p r e s e n t in t h e g e n o m i c s e q u e n c e . ( T h e g e n o m i c copy of t h e g e n e for t h e 1.0-kb m R N A h a s n o t ye t b e e n


s e q u e n c e d . ) T h e two c D N A s of t he 1.0- a n d 1.6-kb m R N A s showed t h a t t h e genes s h a r e five reg ions ( a p p r o x i m a t e l y 1 2 0 - 1 7 0 n t each) of s ignif icant seq u e n c e s imi la r i ty ( 6 8 - 8 8 % iden t i ty ) t h a t were i n t e r s p e r s e d w i t h r eg ions of s ignif icant s e q u e n c e d ive rgence . B a s e d o n the p r e d i c t e d a m i n o acid seq u e n c e s , t he a m i n o a n d c a r b o x y t e r m i n i of t he two p r o t e i n s a r e s imi la r . A n iden t i ca l 2 6 - a m i n o acid s e q u e n c e is cen t ra l ly loca ted in each g e n e a n d m a y r e p r e s e n t a funct ional ly i m p o r t a n t d o m a i n . U s i n g t he bacu lov i rus expres s ion vec to r sys t em, it w a s s h o w n t h a t t he 1.0-kb t r a n s c r i p t c o d e d for a 2 4 - k D a p o l y p e p t i d e t h a t was g lycosy la ted to a 2 6 - k D a form w h e r e a s t h e 1.6-kb t r a n s c r i p t coded for a nong lycosy la t ed 2 3 - k D a p o l y p e p t i d e (Bl i ssa rd et al., 1989). B o t h gene p r o d u c t s were sec re ted from Spodoptera frugiperda cells .

M u c h less is k n o w n a b o u t a s econd g e n e family t h a t c o n t a i n s t h e genes for t h e 1.1- a n d 1.4-kb t r a n s c r i p t s t h a t a lso a r e a b u n d a n t in t h e p a r a s i t i z e d hos t (Bl i ssard et al., 1986b) . T h e funct ion of these genes is n o t k n o w n , b u t t h e f r a g m e n t c o n t a i n i n g o n e of t h e two genes w a s recen t ly s h o w n to h y b r i d i z e w i t h C. sonorensis v e n o m g l a n d c D N A s ( W e b b a n d S u m m e r s , 1990b) . W h e t h er these genes code for p ro t e in s r e l a t ed to t he v e n o m p r o t e i n s p e r se o r o t h e r n o n v e n o m w a s p p r o t e i n s found in t he v e n o m g l a n d t issues h a s n o t b e e n d e t e r m i n e d .

A t h i r d C s V g e n e family, t e r m e d the 5 4 0 - b p r e p e a t e l e m e n t g e n e family, w a s identif ied in t he cour se of s tud ies to d e t e r m i n e w h e t h e r repe t i t ive seq u e n c e s were c o m m o n in t he g e n o m e a n d r e spons ib l e for m u c h of t h e o b served c ro s s -hyb r id i za t i on ( T h e i l m a n n a n d S u m m e r s , 1987, 1988). D N A O 1

(11.3 k b p ) h y b r i d i z e d w i t h m o s t of t he v i ra l supe rhe l i ces u n d e r t h e lowest s t r i ngency cond i t i on tes ted w h e r e a s t he o t h e r C s V D N A p r o b e s h y b r i d i z e d w i th m u c h s m a l l e r subse t s of t he g e n o m e (Fig. 2, a n d T h e i l m a n n a n d S u m m e r s , 1987). F u r t h e r ana lyses revea led t h a t the s e q u e n c e s o n D N A O 1 r e spons ib l e for t he h i g h level of i n t r a g e n o m i c h y b r i d i z a t i o n m a p p e d to two d i sc re t e r eg ions of t he supe rhe l ix . Re l a t ed s e q u e n c e s a l so w e r e m a p p e d b y h y b r i d i z a t i o n to d i sc re te f r agmen t s on m o s t , b u t n o t al l , of t h e se lec ted c loned g e n o m i c supe rhe l i ces t h a t were e x a m i n e d in de t a i l ( T h e i l m a n n a n d S u m m e r s , 1987).

N u c l e o t i d e s e q u e n c e ana lys i s of t he c ros s -hybr id i z ing reg ions r evea led t h a t r e l a t ed f r a g m e n t s a r e c o m p o s e d of a n imper fec t ly conse rved , r e p e a t e d s e q u e n c e e l e m e n t of a p p r o x i m a t e l y 540 b p ( T h e i l m a n n a n d S u m m e r s , 1987). A s ingle copy of t he r e p e a t e l e m e n t exists o n D N A Β (6.7 k b p ) w h e r e a s 4 .5 a n d 2.5 copies of it o c c u r as d i r ec t t a n d e m a r r a y s on D N A s Η (8.5 k b p ) a n d O 1 , respect ively. T h e n i n e copies of t he e l e m e n t t h a t have b e e n s e q u e n c e d to d a t e a r e on ave rage a p p r o x i m a t e l y 6 0 - 7 0 % s imi la r a t t h e n u c l e o t i d e level, a l t h o u g h s h o r t p a t c h e s of h i g h e r s imi la r i ty occur .

S u b s e q u e n t c o m p a r i s o n of t he v i ra l g e n o m i c copy a n d a h o m o l o g o u s c D N A conf i rmed t h a t t he r e p e a t e l e m e n t on D N A Β is t r a n s c r i b e d in t h e

9. Polydnavirus Genome Organization 2 0 3

p a r a s i t i z e d hos t , b u t N o r t h e r n b lo t ana lyse s sugges t ed t h a t th is e l e m e n t is n o t exp re s sed in t h e w a s p ' s ov iduc t s ( T h e i l m a n n a n d S u m m e r s , 1988). I n c o n t r a s t , a 5 4 0 - b p r e l a t ed s e q u e n c e ident if ied o n D N A W a p p e a r s to be t r a n s c r i b e d on ly in t h e w a s p (Bl i ssard et al., 1989). N o r t h e r n b lo t ana lyse s i n d i c a t e d t h a t t h e r e p e a t r e l a t ed s e q u e n c e s o n D N A s Η a n d O 1 a r e t r a n sc r ibed in b o t h p a r a s i t i z e d h o s t l a rvae a n d C. sonorensis ov iduc t s ( T h e i l m a n n a n d S u m m e r s , 1988). A l t h o u g h the r e p e a t e l e m e n t r e l a t ed s e q u e n c e s o n C s V D N A s Β a n d Η each h y b r i d i z e d w i t h o n e t r a n s c r i p t in t h e m R N A s of t h e h o s t in w h i c h t h e s e q u e n c e is exp re s sed , t h e r e p e a t e l e m e n t D N A s o n O 1 o r W each h y b r i d i z e d w i t h two o r m o r e t r a n s c r i p t s ( T h e i l m a n n a n d S u m m e r s , 1988; B l i s sa rd et al., 1989). T h e re la t ive a b u n d a n c e of t he four t r a n s c r i p t s d e t e c t e d w i t h t he D N A O 1 r e p e a t e l e m e n t r e l a t ed p r o b e s va r i ed d e p e n d i n g on t h e h o s t a n d t h e p a r t i c u l a r D N A O 1 f r a g m e n t u sed . W h e t h e r s o m e of t h e m u l t i p l e t r a n s c r i p t s d e t e c t e d by t h e D N A O 1 p r o b e s a r e a l t e rna t ive ly sp l iced m R N A s o r reflect c r o s s - h y b r i d i z a t i o n w i t h m R N A s e n c o d e d b y r e l a t ed seq u e n c e s o n o t h e r supe rhe l i ces is n o t k n o w n . W h e t h e r t h e 5 4 0 - b p e l e m e n t r e l a t ed m R N A s c a n b e t r a n s l a t e d h a s n o t yet b e e n d e t e r m i n e d by in vitro o r in vivo s t ud i e s , b u t o p e n r e a d i n g f rames have b e e n ident i f ied ( T h e i l m a n n a n d S u m m e r s , 1987).

A l imi t ed n u m b e r of o t h e r C s V genes have b e e n ident i f ied a n d p a r t i a l l y c h a r a c t e r i z e d . A second g e n e t h a t codes for a spl iced m R N A expre s sed on ly in t h e w a s p was ident i f ied o n D N A Β ( T h e i l m a n n a n d S u m m e r s , 1988). T h i s g e n e does n o t a p p e a r to b e closely r e l a t e d to t h e 5 4 0 - b p r e p e a t e l e m e n t g e n e . F u r t h e r s t u d y of C s V D N A W also ident i f ied t h r e e to five wasp-speci f ic genes in a d d i t i o n to t he two p rev ious ly c h a r a c t e r i z e d genes for t h e 1.0- a n d 1.6-kb t r a n s c r i p t s p r e s e n t in t he p a r a s i t i z e d hos t (Bl i ssard et al., 1989). O n e of t hese wasp-spec i f ic genes m a p s to t h e r eg ion c o n t a i n i n g 5 4 0 - b p r e p e a t r e l a t e d s e q u e n c e s . W h e t h e r t h e o t h e r wasp-speci f ic genes o n D N A W b e l o n g to m u l t i g e n e famil ies is n o t c lea r f rom the l imi ted e x t a n t d a t a .

Very few p o l y d n a v i r a l genes have b e e n s e q u e n c e d , b u t t h e ava i l ab le d a t a sugges t t h a t t h e s t r u c t u r e of p o l y d n a v i r a l genes is typ ica l of e u k a r y o t i c genes in g e n e r a l . A t leas t two of t h e C s V genes a r e spl iced , a n d nuc l eo t i de s cons i s t e n t w i t h t h e 5 ' a n d 3 ' spl ice j u n c t i o n c o n s e n s u s s e q u e n c e s o c c u r a t t h e e x o n - i n t r o n j u n c t i o n s (Bl i ssa rd et al., 1987; T h e i l m a n n a n d S u m m e r s , 1988). S e q u e n c e s r e s e m b l i n g typ ica l t r a n s c r i p t i o n a l a n d t r a n s l a t i o n a l s igna ls a p p e a r to b e used , a n d the s e q u e n c e con t ex t a r o u n d t h e t r a n s l a t i o n a l s t a r t s i te is cons i s t en t w i t h t h e t r a n s l a t i o n in i t i a t ion c o n s e n s u s s e q u e n c e of K o z a k (Bl i ssa rd et al., 1987; T h e i l m a n n a n d S u m m e r s , 1988). T h e t e m p o r a l p a t t e r n of expres s ion of genes involved in v i rus r ep l i ca t ion in t h e female w a s p sug ges ts t h a t fu tu re inves t iga t ions m a y identify u n i q u e r e g u l a t o r y s e q u e n c e s , for e x a m p l e , h o r m o n e - r e s p o n s i v e e l e m e n t s , in t he 5 ' r eg ions of s o m e po lyd nav i r a l genes (see Sec t ion V I I ) .

> CO

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3 ϋ ί in ω

W-

Q -

Μ -

Β -

- Ο 1

Α Figure 2 Distribution of 540-bp repeat e lement related sequences in the CsV genome . (A) Duplicate lanes of undigested CsV DNA were hybridized with 3 2 P-labeled CsV DNA u n d e r h igh (50% formamide) stringency conditions o r with vector-free 540-bp repeat e lement sequences from DNA O 1 (p0 1 -HC1185) u n d e r lower (30% formamide) stringency conditions. Equivalent autoradiographic exposures are shown. Al though 540-bp repeat e lement related sequences are widespread in the genome, not all CsV DNAs hybridize detectably with the 540-bp repeat e lement probe u n d e r the stringency conditions used. T h e genomic distribution of the 540-bp repeat element related sequences detected in these Southern blots is equivalent to that reported in the original description of the gene family by Thei lmann and Summers (1987). (continues)

204

R-Q -Ol M-G-

D -

C1-B -

CsV 1 2

540 3 4

-Α 1 Λ 3

Β Figure 2 (continued) (B) T h e reg ion con ta in ing p redomina te ly superhel ica l forms of t he viral DNAs is en la rged . To il lustrate bo th faint a n d in tense b a n d s m o r e clearly, lands 1 a n d 2 show shor t a n d long p h o t o g r a p h i c exposures , respectively, of t he a u t o r a d i o g r a p h of viral DNA hybridized with t h e CsV DNA p robe . Lanes 3 a n d 4 a r e comparab l e shor t a n d long p h o t o g r a p h i c exposures , respectively, of lanes hybridized with the 540-bp r epea t e l e m e n t p robe . Bo th t he identi ty a n d complexi ty of p robes can significantly affect t he hybridizat ion results in studies of polydnavirus g e n o m e s . Note tha t some CsV DNA bands , for example , A * - A 5

a n d C 2 , a re m o r e readily detectable with t he 540-bp r epea t e l e m e n t p robe t h a n with t he m o r e complex (total) viral genomic DNA p robe . T h e DNA b a n d s designa t ed A 2 - A 5 o r C 2 were identified after t h e initial character izat ion, i l lustrat ing t he difficulty in accurately es t imat ing t he total n u m b e r of DNAs in a polydnavirus g e n o m e . D u r i n g the last decade t he or iginal scheme for des igna t ing b a n d s has b e e n modif ied to a c c o m m o d a t e t he addi t ional b a n d s as they were discovered. Addi tona l DNA b a n d with t he mos t similar size a n d d is t inguished from the DNA b a n d observed in t he initial s tudy by numer ica l superscr ip ts o r subscripts g rea te r t h a n one , for example , C 2 . DNA b a n d s des igna ted by the same let ter a re of similar size b u t a re not necessarily re la ted at t h e molecula r level. As t he complexi ty of polydnavi rus g e n o m e s has b e c o m e m o r e a p p a r e n t with t ime, o the r des ignat ion schemes a re be ing cons idered by investigators in the field.

205


C. Temporal Patterns of CSV Gene Expression

L i m i t e d d a t a a b o u t t he t e m p o r a l r egu l a t i on of C s V genes a r e ava i l ab le . T h e m o s t s t r ik ing fea tu re of t he p re sen t ly ava i l ab le , s e m i q u a n t i t a t i v e N o r t h e r n b lo t d a t a is t he s imi la r i ty of t he s t eady- s t a t e levels of m a n y of t h e t r a n s c r i p t s f rom a p p r o x i m a t e l y 12 h r to a t least 6 d a y s pos tov ipos i t ion (Bl i ssard et al., 1986b) . S o m e m i n o r differences in t he t e m p o r a l exp res s ion of t h e genes for t he 1.0-, 1.6-, a n d a r e l a t ed 1.3-kb m R N A have b e e n n o t e d . H o w e v e r , these differences were no t cons i s t en t in t he severa l s tud ies of th is g e n e family a n d c a n n o t b e c lear ly d i s t i ngu i shed from the differences in t h e a m o u n t s of t h e t h r ee r e l a t ed t r a n s c r i p t s re la t ive to o n e a n o t h e r a t g iven s a m p l i n g t imes (Bl i ssard et al., 1986b, 1987, 1989). T h e 5 4 0 - b p r e p e a t e l e m e n t genes , e spe cially t h e genes loca ted on C s V D N A s Β a n d O 1 , a p p e a r to differ f rom t h e p a t t e r n d e s c r i b e d h e r e . T h e s e genes a r e expres sed m a x i m a l l y a n d a t s imi l a r levels b e t w e e n 2 a n d 24 h r pos tov ipos i t ion a n d t h e n r e m a i n a t s l ight ly d e c reased b u t re la t ively c o n s t a n t levels un t i l levels i n c r e a s e d a t 8 d a y s pos t ovipos i t ion ( T h e i l m a n n a n d S u m m e r s , 1988). W h e t h e r t h e v a r i a t i o n s in t h e m R N A levels n o t e d in t h e p u b l i s h e d s tud ies a r e ev idence of t e m p o r a l r egu l a t ion is u n c l e a r b e c a u s e the obse rved differences were s l ight a n d i n t e r n a l con t ro l s (e.g. , a cons t i tu t ive ly expressed hos t gene) t h a t w o u l d al low u n a m b i g u o u s c o m p a r i s o n s of s a m p l e s t a k e n a t different t i m e p o i n t s w e r e n o t i n c l u d e d .

T h e re la t ively c o n s t a n t s t eady- s t a t e levels sugges t t h a t severa l C s V gene p r o d u c t s m a y be r e q u i r e d a t a p p r o x i m a t e l y s imi l a r levels i m m e d i a t e l y after ov ipos i t ion a n d a t l a t e r t imes to p r e v e n t t he e n c a p s u l a t i o n of t h e w a s p ' s egg a n d l a rva l i n s t a r s . T h i s i n t e r p r e t a t i o n shou ld be c o n s i d e r e d t en t a t i ve un t i l m o r e de t a i l ed q u a n t i t a t i v e s tud ies a r e d o n e b e c a u s e t h e r e p o r t e d work used c o m p l e x p r o b e s (e.g. , to ta l C s V g e n o m i c D N A ) o r e x a m i n e d t h e t r a n s c r i p t ion of m e m b e r s of gene families in a s ingle t i ssue a n d / o r d e v e l o p m e n t a l s t age of t he insec t s . G e n e s in m u l t i g e n e families often a r e c o o r d i n a t e l y a n d d e v e l o p m e n t a l l y r e g u l a t e d , a s a r e t he h e m a g l o b i n or c h o r i o n genes (Li , 1983; references in H i b n e r et al., 1991; X i o n g et al., 1988). G r e a t e r differences in t h e t e m p o r a l p a t t e r n of express ion of s ing le -copy vi ra l genes m i g h t b e e x p e c t e d . W h a t effect p o l y d n a v i r u s e s h a v e on hos t t r a n s c r i p t i o n is n o t k n o w n b u t a lso s h o u l d be cons ide r ed in fu ture s tud ies .

D. Significance of the Gene Families

T h e p r e s e n c e of m o r e t h a n o n e g e n e family in t he C s V g e n o m e is i m p o r t a n t for severa l r e a s o n s . F i r s t , t he C s V g e n e famil ies , especia l ly t h e 5 4 0 - b p r e p e a t e l e m e n t gene family, a r e ev idence for p a s t s e q u e n c e d u p l i c a t i o n a n d in t e r seg m e n t a l r e c o m b i n a t i o n (Bl issard etal, 1987; T h e i l m a n n a n d S u m m e r s , 1987).


T h e m u l t i p l e g e n e copies c a n a l t e rna t ive ly b e i n t e r p r e t e d as poss ib le s i tes for fu tu re i n t e r m o l e c u l a r o r i n t r a m o l e c u l a r crossovers in t h e c o n t i n u e d evo lu t ion of t h e v i ru s . T h e c o n c o m i t a n t s eg rega t ion of r e l a t ed genes w i t h i n a p o l y d nav i r a l g e n o m e is cons i s t en t w i t h t h e m o d e l t h a t each p o l y d n a v i r a l g e n o m i c s e g m e n t m a y b e a c h i m e r a of u n i q u e a n d s h a r e d s e q u e n c e s . A s n o t e d p rev i ously, s u c h a m o d e l cou ld a c c o u n t for m u c h of t h e a p p a r e n t i n t r a g e n o m i c c o m p l e x i t y of C s V . T h e seg rega t ion of r e l a t ed genes o n different v i ra l supe r -he l ica l D N A s cou ld be i m p o r t a n t if less t h a n t h e en t i r e v i ra l g e n o m e w e r e e n c a p s i d a t e d in each v i r ion . T h i s w o u l d b e especia l ly t r u e if t h e v i ra l genes w e r e exp re s sed in a t issue-specif ic m a n n e r .

Second , it m a y b e s ignif icant t h a t t h e four C s V genes ini t ia l ly ident i f ied as b e i n g m e m b e r s of g e n e families a p p e a r to be a m o n g t h e v i ra l genes t h a t a r e t h e m o s t h igh ly exp res sed in hos t l a rvae . G e n e d u p l i c a t i o n is a m e c h a n i s m for i n c r e a s i n g g e n e d o s a g e , t h e r e b y in f luenc ing t h e m R N A levels of genes w i t h cr i t ica l func t ions . T h i s cou ld b e especia l ly i m p o r t a n t for genes t h a t a r e exp re s sed i m m e d i a t e l y after ov ipos i t ion . W h e t h e r t he C s V genes b e i n g d i s cussed p l a y cr i t ica l roles is con jec tu ra l a t th i s t i m e b e c a u s e it is n o t k n o w n w h a t t h e func t ion of any C s V or o t h e r p o l y d n a v i r a l g e n e is .

T h i r d , g e n e famil ies p rov ide a m e c h a n i s m for r e g u l a t i n g g e n e expres s ion in a t issue-specif ic m a n n e r , s u c h as t h e h u m a n co l lagen a n d ch icken t r o p o n i n g e n e famil ies ( B r e i t b a r t a n d N a d a l - G i n a r d , 1989; V u o r i o a n d d e C r o m -b r u g g h e , 1990; M a r c h a n t et aL, 1991). T h e t i s sue specificity of C s V genes is n o t k n o w n . R e l a t e d b u t v a r i a n t forms of a g e n e m a y e n c o d e p r o d u c t s t h a t differ func t iona l ly to v a r y i n g deg rees , for e x a m p l e , t h e co l lagens specific to t h e c o r n e a o r m u s c l e , respect ively. A l t e r e d t i ssue specificities a m o n g m e m b e r s of a g e n e family m a y b e o n e m e c h a n i s m of ref ining a s y s t e m p h y s i ologically. T h i s m a y b e p a r t i c u l a r l y i m p o r t a n t w h e r e m o r e t h a n o n e t i ssue a p p e a r s to b e infected, as in p a r a s i t i z e d hos t s .

F o u r t h , t h e s tud ie s on t h e 5 4 0 - b p r e p e a t e l e m e n t g e n e family conf i rmed t h a t p o l y d n a v i r a l genes in s o m e cases a r e r e g u l a t e d in a species-specif ic m a n n e r , t h a t is, t h e genes a r e exp res sed exclusively in e i the r t h e w a s p o r t h e p a r a s i t i z e d hos t . T h e finding t h a t s o m e v i ra l genes a r e exp res sed in b o t h insec ts , however , w a s s o m e w h a t s u r p r i s i n g in v iew of t h e s ignif icant b io logical differences in t h e two insec ts ( w a s p a n d l e p i d o p t e r a n la rvae) a n d t h e i n t e r ac t i on of t h e v i ru s w i t h t h e t w o spec ies . Po lydnav i r a l g e n e r e g u l a t i o n h a s n o t b e e n inves t iga t ed sys temat ica l ly , b u t fu ture inves t iga t ions of t h e different ia l r e g u l a t i o n of t h e t h r e e subc lasses of v i ra l genes ( w a s p specific, l e p i d o p t e r a n specific, a n d genes exp re s sed in b o t h species) m a y p r o v i d e i n f o r m a t i o n useful to t h e s t u d y of insec t gene r e g u l a t i o n in g e n e r a l . I n t h e case of genes exp re s sed in b o t h hos t spec ies , w h a t b io logica l ac t iv i ty t h e p r o t e i n p r o d u c t ( s ) have (if a n y ) in each species a l so will b e of i n t e re s t .

Fif th, t h e s tud ie s d e m o n s t r a t e d t h a t a g iven g e n o m i c s e g m e n t c a n c o n t a i n


both genes expressed exclusively in t he p a r a s i t i z e d hos t a n d genes t h a t a r e w a s p specific. T h a t genes expressed solely in e i the r t h e w a s p or t h e l e p i d o p t e r a n hos t a r e i n t e r spe r sed on a D N A s e g m e n t a n d a r e no t c lus t e red t o g e t h e r o n different v i ra l g e n o m i c s e g m e n t s is of in te res t w i t h r e g a r d to t h e or ig in a n d evo lu t ion of p o l y d n a v i r u s e s (see Sec t ion V I I I ) . T h e i n t e r spe r s ion of t h e two species-specif ic c lasses of v i ra l genes on a g iven s u p e r h e l i x is of less in te res t f rom the s t a n d p o i n t of g e n e t r a n s c r i p t i o n b e c a u s e d a t a f rom o t h e r e u k a r y o t i c sys t ems sugges t t h a t t i ssue- o r species-specif ic express ion d e p e n d s on specific r e g u l a t o r y e l emen t s of t he genes themse lves , a n d t h e genes t h e r e fore w o u l d b e expec t ed to b e r e g u l a t e d i n d e p e n d e n t l y .

VII. Polydnavirus Transmission, Integration, and Replication

E v i d e n c e from s tud ies of the h o s t - p a r a s i t o i d i n t e r ac t i on s u p p o r t s t h e t h eo ry t h a t p o l y d n a v i r u s e s p l ay a n essent ia l role in d e t e r m i n i n g w h e t h e r t h e eggs a n d i m m a t u r e s tages of ce r t a in i c h n e u m o n i d or b r a c o n i d w a s p s c a n c o m p l e t e e n d o p a r a s i t i c d e v e l o p m e n t in t he l e p i d o p t e r a n hos t . G i v e n t h e cr i t ica l funct ion of p o l y d n a v i r u s e s in the life cycles of these p a r a s i t o i d s a n d t h e gene ra l ly accep t ed no t i on t h a t these v i ruses o c c u r in t he ov iduc t s of all females w i t h i n species in w h i c h a p o l y d n a v i r u s h a s b e e n de t ec t ed , it is i m p o r t a n t to k n o w how the v i ruses a r e t r a n s m i t t e d b e t w e e n w a s p g e n e r a t i o n s w i t h sufficient efficiency to e n s u r e t h a t all p r o g e n y a c q u i r e t he v i rus . T h e i ssue of h o w a g iven p o l y d n a v i r u s is m a i n t a i n e d w i th in a w a s p species b e c o m e s d o u b l y i m p o r t a n t in l ight of t he a p p a r e n t species specificity of t h e v i ruses (see Sect ion V I I I ) . M a i n t a i n i n g the " c o r r e c t " p o l y d n a v i r u s , t h a t is , t h e v i ru s t h a t a p p e a r s to be specific for the g iven w a s p species , cou ld b e cr i t ica l to t h e r e p r o d u c t i v e success of t he p a r a s i t o i d b e c a u s e n o t all p o l y d n a v i r u s e s a r e equa l ly effective in a l t e r ing t he phys io logy of a g iven l e p i d o p t e r a n h o s t ( V i n son , 1977; V i n s o n a n d Stol tz , 1986). I n n a t u r e , l a rva l hos t s c a n b e p a r a si t ized by w a s p s of m o r e t h a n o n e species , a n d t h u s e n d o p a r a s i t o i d s p o t e n t ial ly cou ld c o m e i n t o con t ac t w i t h t h e v i rus f rom m o r e t h a n o n e w a s p species . Pos tu l a t ed m e c h a n i s m s therefore m u s t a c c o u n t for t he efficiency a n d specificity of t r a n s m i s s i o n .

I n sec t v i ruses typica l ly a r e t r a n s m i t t e d by per os acqu i s i t ion o r b y t r a n s -ovar ia l t r a n s m i s s i o n . Bacu lov i ruses a r e we l l -known e x a m p l e s of insec t v i ruses t r a n s m i t t e d by inges t ion of v i rus- infes ted m a t e r i a l . T r a n s o v a r i a l t r a n s m i s s i o n , a m e c h a n i s m in w h i c h ovar ia l t issues a r e infected, h a s b e e n d e m o n s t r a t e d for b u n y a v i r u s e s (e.g. , L a C r o s s e v i rus ) , flaviviruses (e.g. , d e n g u e v i rus ) , a n d r h a b d o v i r u s e s (e.g. , t h e s i g m a v i rus of Drosophila melanogaster) ( G o n z a l e z -S c a r a n o a n d N a t h a n s o n , 1990; M o n a t h , 1990; Rosen , 1984). E i t h e r of these


m e c h a n i s m s a p p e a r e d poss ib le w h e n the s tud ies o n t r a n s m i s s i o n w e r e ini t i a t e d . L a r v a l e n d o p a r a s i t o i d s c o n s u m e hos t t i ssue or h e m o l y p h , e i t he r of w h i c h cou ld be infected or c o n t a m i n a t e d w i t h v i rus in jected by the female w a s p p a r e n t d u r i n g ovipos i t ion . A p a r a s i t i z e d hos t c o n t a i n s a fairly s ignif icant a m o u n t of v i ra l D N A ( T h e i l m a n n a n d S u m m e r s , 1986). I t is unc l ea r , however , w h e t h e r t h e v i ra l D N A p r e s e n t in a p a r a s i t i z e d hos t is in a form t h a t w o u l d b e infect ious for t h e d e v e l o p i n g p a r a s i t o i d . T h e species specificity of v i rus ac q u i r e d per os cou ld b e e x p l a i n e d b y a r g u i n g t h a t t he w a s p t issues w e r e p e r m i s sive for r ep l i ca t ion of on ly t he v i rus typ ica l ly a s soc ia t ed w i t h t h a t w a s p spec ies . ( A l t h o u g h th is a r g u m e n t is v a g u e a n d u l t i m a t e l y unsat is fac tory , it often is i nvoked for lack of a m o r e defini t ive e x p l a n a t i o n d u e to o u r c o m p a r a t i v e l y p o o r u n d e r s t a n d i n g of pe rmis s ivenes s a n d hos t r a n g e in m o s t v i ra l sys tems . ) I n t h e s e c o n d m o d e l ( t r ansova r i a l t r a n s m i s s i o n ) , infect ion of t he egg w o u l d p r o v i d e a r e a d y sou rce of i n o c u l u m t h a t is c o m p a t i b l e w i t h t h e w a s p hos t .

A t h i r d m e c h a n i s m , ver t i ca l t r a n s m i s s i o n t h r o u g h t h e g e r m l i n e a s p rov i n c e s , is i m p o r t a n t in t h e biology of m a n y a n i m a l v i ruses s u c h as r e t r o v i ruses . T h e cova len t l inkage of t h e v i ra l g e n o m e to hos t ce l lu la r D N A seq u e n c e s d i s t i ngu i shes p rov i ra l t r a n s m i s s i o n from t r a n s o v a r i a l t r a n s m i s s i o n in w h i c h the v i ra l g e n o m e is e x t r a c h r o m o s o m a l a n d c a n sel f - repl icate u n d e r a p p r o p r i a t e cond i t i ons . N o p r e c e d e n t for p rov i ra l g e r m l i n e t r a n s m i s s i o n of insec t v i ruses ex is ted . A l t h o u g h re t rov i rus l ike pa r t i c l e s c o n t a i n i n g R N A re l a t ed to copia h a v e b e e n d e t e c t e d in Drosophila melanogaster, th is gene ra l ly is n o t c o n s i d e r e d a n e x a m p l e of g e r m l i n e t r a n s m i s s i o n of a n insec t v i rus ( H e i n e et al., 1980; S h i b a a n d Sa igo , 1983). T h e pa r t i c l e s a r e non in fec t ious , a n d t h e r e t r o t r a n s p o s o n copia, w h i c h lacks a n env ( enve lope) gene , is gene t ica l ly d i s t inc t f rom t r u e r e t rov i ruses (Echa l ie r , 1989).

T h e p h y s i c a l a n d gene t i c d a t a s u p p o r t i n g t h e p r o b a b l e t r a n s m i s s i o n of p o l y d n a v i r u s e s t h r o u g h the g e r m l i n e c o m e f rom t h e i chnov i ruses C s V a n d Hyposoter fiigitivus v i rus (HfV) a n d t h e b racov i rus C m V . V i r i o n s h a v e n o t b e e n d e t e c t e d in m a l e w a s p s o r in n o n o v i d u c t t i ssues of female w a s p s . I t therefore w a s s u r p r i s i n g t h a t each b o d y s e g m e n t , t h a t is, h e a d , t h o r a x , a n d a b d o m e n , of each m a l e o r female C. sonorensis w a s p s a m p l e d c o n t a i n e d low b u t r e p r o d u c i b l e a m o u n t s of CsV-speci f ic D N A ( F l e m i n g a n d S u m m e r s , 1986). S u b s e q u e n t phys i ca l ana lys i s of t h e virus-specif ic D N A in m a l e C. sonorensis w a s p s revea led two differently o r g a n i z e d forms . O n e fract ion w a s iden t i ca l to t he D N A iso la ted f rom v i r ions w h e n a n a l y z e d e i the r in its n a t i v e , u n d i g e s t e d s t a t e o r after d iges t ion w i t h res t r i c t ion e n d o n u c l e a s e s . T h i s ep iso-m a l f ract ion a p p e a r e d to c o n t a i n all t h e s e g m e n t s of t h e v i ra l g e n o m e b u t w a s p r e s e n t in m a l e s o m a t i c t i ssues in s u b s t a n t i a l l y lower a m o u n t s t h a n t h e s e c o n d form.

S o u t h e r n b lo t ana lyse s of d iges ted m a l e w a s p ce l lu la r D N A a n d c o m p a r a bly t r e a t e d C s V D N A d e m o n s t r a t e d t h a t t he second , p r e d o m i n a t e form of


virus-specif ic D N A in w a s p s o m a t i c t issues is cons i s ten t ly d e t e c t a b l e as off-size ( a n o m a l o u s l y s ized) res t r i c t ion f r agmen t s w h e n c o m p a r e d to t h e fragm e n t s in c o m p a r a b l y d iges ted v i ra l D N A con t ro l s (Fig . 3 , a n d F l e m i n g a n d S u m m e r s , 1986). T h e offsize res t r i c t ion f r a g m e n t s d e t e c t e d in d iges t ed cel lul a r D N A a r e r e p r o d u c i b l e w h e n D N A from different i n d i v i d u a l s o r g e n e r a -

Η O1 W

v cf v cf ν c?

Xho I EcoRI Hind III Figure 3 Offsize restriction fragments. Southern blots of cellular DNA from pooled male C. sonorensis wasps or CsV DNA (V) digested with the indicated restriction endonucleases were hybridized with cloned sequences from viral DNAs H, O 1 , or W under stringent conditions. Each lane of male DNA contains one or more offsize restriction fragments not present in the viral DNA control.


t ions is u sed ( J . F l e m i n g , u n p u b l i s h e d d a t a ) . I d e n t i c a l offsize res t r i c t ion f r a g m e n t s a r e d e t e c t a b l e in t he n o n o v i d u c t s o m a t i c t i ssue ( h e a d a n d t h o r a x ) D N A of C. sonorensis f emale w a s p s . W h e n w a s p D N A is d iges t ed w i t h en z y m e s t h a t d o n o t c u t t h e v i ra l supe rhe l i c a l D N A used as a p r o b e , s ingle h i g h - m o l e c u l a r f r agmen t s t h a t exceed t h e size of t h e v i ra l g e n o m i c s e g m e n t u sed a s a p r o b e a r e d e t e c t a b l e in t h e ce l lu la r D N A ( F l e m i n g a n d S u m m e r s , 1986). T h i s is cons i s t en t w i t h resu l t s i n d i c a t i n g t h a t t h e virus-specif ic D N A is a s soc i a t ed w i t h t h e h i g h - m o l e c u l a r - w e i g h t f ract ion of w a s p D N A pur i f ied o n c e s i u m c h l o r i d e - e t h i d i u m b r o m i d e g r a d i e n t s ( F l e m i n g a n d S u m m e r s , 1986). A s ingle , d i sc re te f r a g m e n t on each v i ra l g e n o m i c s e g m e n t t e s ted is cons i s t en t ly h o m o l o g o u s to t he offsize res t r i c t ion f r agmen t s d e t e c t e d b y t h a t p r o b e , a n d t h e r e m a i n d e r of each g e n o m i c s e g m e n t a p p e a r s to b e u n a l t e r e d in w a s p ce l lu la r D N A . T h e s e findings sugges t ed t h a t t he v i ra l D N A s a r e i n t e g r a t e d .

C o m p a r i s o n of c loned w a s p D N A re l a t ed to C s V D N A Β a n d c loned e x t r a c h r o m o s o m a l D N A Β (6.7 k b p ) b y de t a i l ed phys i ca l m a p p i n g , r e c i p r o cal S o u t h e r n b lo t h y b r i d i z a t i o n , a n d n u c l e o t i d e s e q u e n c e ana lys i s i nd i ca t e s t h a t t h e C s V D N A B-specific s e q u e n c e s in C. sonorensis ce l lu la r D N A a r e co l inea r w i t h t h e e x t r a c h r o m o s o m a l s e q u e n c e s . Re la t ive to t h e e x t r a -c h r o m o s o m a l c i r cu l a r mo lecu l e , t he h o m o l o g o u s ce l lu la r copy is l i nea r i zed a t a s i te w i t h i n t h e f r a g m e n t a s soc ia t ed w i t h t h e offsize res t r i c t ion f r a g m e n t s . R e a r r a n g e m e n t of t h e v i ra l mo lecu l e in s o m a t i c t i ssues c a n b e ru l ed o u t as a n e x p l a n a t i o n for t h e offsize res t r i c t ion f r agmen t s . T h e v i ra l D N A in w a s p ce l lu la r D N A is covalent ly l inked to n o n v i r a l s e q u e n c e s , t h a t is, D N A Β is i n t e g r a t e d in t h e w a s p g e n o m e ( F l e m i n g a n d S u m m e r s , 1990, 1991). All t h e C s V s e g m e n t s t h a t have b e e n e x a m i n e d ( to d a t e , six supe rhe l i ce s o r a p p r o x i m a t e l y 2 5 % of t h e C s V g e n o m e ) a lso a r e d e t e c t a b l e as offsize r e s t r i c t ion f r a g m e n t s (Fig . 3 , F l e m i n g a n d S u m m e r s , 1986; J . F l e m i n g , u n p u b l i s h e d d a t a ) . T h e en t i r e C s V g e n o m e t h u s a p p e a r s to b e i n t e g r a t e d , sugges t i ng t h a t C s V m a y b e t h e first d o c u m e n t e d e x a m p l e of a n i n t e g r a t e d D N A v i ru s in insec ts ( F l e m i n g a n d S u m m e r s , 1990, 1991). Phys ica l m a p p i n g a n d S o u t h e r n b lo t d a t a a lso h a v e d e m o n s t r a t e d t h a t HfV-specif ic D N A is i n t e g r a t e d in t h e ce l lu la r D N A of m a l e H.fiigitivus i c h n e u m o n i d w a s p s ( X u a n d Sto l tz , 1991).

T h e s e q u e n c e s a b u t t i n g t h e j u n c t i o n s of t h e v i ra l a n d w a s p s e q u e n c e s a r e s t r u c t u r a l l y c o m p l e x a n d sugges t t h a t t h e i n t e g r a t e d C s V D N A is n o t closely r e l a t e d to t h e c h a r a c t e r i z e d classes of t r a n s p o s a b l e e l e m e n t s . T h e i n t e g r a t e d copy of D N A Β t e r m i n a t e s in s h o r t (59 n t ) imper fec t d i r ec t r e p e a t s , b u t a s ingle copy of t h e t e r m i n a l r e p e a t s e q u e n c e is found in t h e c i rcu la r , e x t r a c h r o m o s o m a l D N A Β mo lecu l e i so la ted f rom v i r ions (Fig . 4 ) . S e q u e n c e s n e a r each t e r m i n u s of i n t e g r a t e d D N A Β form imper fec t i nve r t ed r e p e a t s w i t h s e q u e n c e s in t h e 5 4 0 - b p r e p e a t e l e m e n t t h a t is l oca ted cen t r a l ly o n D N A Β ( F l e m i n g a n d S u m m e r s , 1991). Base p a i r i n g b e t w e e n t h e t e r m i n a l a n d in te r n a l s e q u e n c e s t h a t form these i nve r t ed r e p e a t s a r e h y p o t h e s i z e d to r e su l t in a


Left Jen Right Jen

Extrachromosomal DNA Β

Figure 4 Integrated and extrachromosomal CsV DNA B. The integrated copy of CsV DNA Β (wide bars) flanked by wasp sequences (narrow speckled bars) is shown schematically at the top. The terminal imperfect direct repeats ( D R R or D R L , open or solid squares at the right or left junction, respectively) are immediately flanked by short sequences that are related to each other but contain more mismatches than the D R R or D R L sequences (rectangles with heavy or narrow diagonal lines that are located immediately to the left of D R R or D R L in the figure). Two sets of sequences close to the terminal repeats form imperfect inverted repeats (sequence pairs marked by short or long arrows with one or two asterisks, respectively) with sequences near the 5' and 3 ' termini of the single 540-bp repeat element (small open circle) that is located centrally in integrated CsV DNA B. The inverted repeats have been hypothesized to contribute to the stabilization of a conformation that facilitates recombination between the terminal direct repeats during replication, shown schematically in the middle figure (Fleming and Summers, 1991). The episomal form of DNA Β isolated from purified CsV virions is shown schematically at the bottom and contains a single copy of the direct repeat sequence. Different (cloned) molecules of extrachromosomal DNA Β contain either D R R or D R L . Other viral sequences immediately adjacent to the direct repeat sequences are identical in both episomal DNA Β subclasses.


c o n f o r m a t i o n in w h i c h t h e e n d s of t he virus-specif ic s e q u e n c e s a r e b r o u g h t i n to p rox imi ty . R e c o m b i n a t i o n b e t w e e n t h e d i r ec t r e p e a t s t h e n cou ld o c c u r d u r i n g r ep l i ca t ion ( F l e m i n g a n d S u m m e r s , 1990, 1991).

T h e gene t i c ana ly se s of t he b racov i rus C m V s u p p o r t t h e h y p o t h e s i s of t r a n s m i s s i o n of p o l y d n a v i r u s e s t h r o u g h t h e w a s p s ' g e r m l i n e , b u t t h e m e c h a n i s m ut i l ized by b racov i ruses p r e s e n t l y is unc lea r . T h e in i t ia l a n a l y s e s took a d v a n t a g e of t he p o l y m o r p h i c g e n o m i c s e g m e n t s p r e s e n t in g e o g r a p h i c a l l y i so la ted p o p u l a t i o n s of C. melanoscela. A g e n o m i c s e g m e n t d e s i g n a t e d D is p r e s e n t in t h e v i ru s pa r t i c l e s in t h e C o n n e c t i c u t s t r a i n b u t is a b s e n t f rom t h e v i r ions in t h e ( T r u r o ) N o v a Sco t ia s t r a in . W h e n C o n n e c t i c u t m a l e s w e r e m a t e d w i t h N o v a Scot ia females , D N A D w a s found in t h e D N A iso la ted f rom v i r ions in F l females (Stol tz et aL, 1986). T h e s e d a t a i n d i c a t e t h a t p o l y d n a v i r a l D N A c a n be t r a n s m i t t e d in w a s p s p e r m . W h a t form of C m V D N A is t r a n s m i t t e d in s p e r m , however , is u n c l e a r f rom t h e a c c o m p a n y i n g p h y s i c a l ana ly se s . C m V p r o b e s failed to h y b r i d i z e w i t h t h e C. melanoscela m a l e w a s p c h r o m o s o m a l D N A , a n d on ly e p i s o m a l forms of CmV-spec i f i c D N A w e r e d e t e c t a b l e in m a l e w a s p D N A (Stol tz et aL, 1986). T h e s e d a t a a r e i ncons i s t en t w i t h t h e i n t e g r a t i o n of t h e b racov i rus D N A a n d sugges t ed t h a t t h e v i ra l g e n o m e cou ld be t r a n s m i t t e d as e p i s o m a l D N A s . H o w e v e r , in l a t e r gene t i c s tud ie s of t he s a m e two C. melanoscela p o p u l a t i o n s , t h r e e H i n d i 11 f r a g m e n t s t h a t d i s t i ngu i sh t h e v i ruses in t h e w a s p p o p u l a t i o n s s e g r e g a t e d in r a t io s cons i s t en t w i t h M e n d e l i a n i n h e r i t a n c e , sugges t i ng t h a t t h e v i ra l D N A s a r e i n h e r i t e d a s p a r t of t h e w a s p c h r o m o s o m a l g e n o m e (Stol tz , 1990). F u r t h e r phys i ca l ana lyse s a r e neces sa ry to resolve t he d i s c r e p a n c y b e t w e e n t h e p h y s i c a l a n d gene t i c d a t a f rom t h e b r acov i ru s sys t em.

T h e M e n d e l i a n seg rega t ion ra t ios obse rved in t he b r acov i ru s s y s t e m a r e s t r o n g ev idence a g a i n s t e i the r per os o r t r ansova r i a l t r a n s m i s s i o n . B o t h ep i so m a l a n d i n t e g r a t e d p o l y d n a v i r a l D N A s a r e p r e s e n t in (C. sonorensis) w a s p s o m a t i c t i ssues ( F l e m i n g a n d S u m m e r s , 1986), b u t h o w i m p o r t a n t t h e ep i -s o m e s a r e for v i rus t r a n s m i s s i o n is q u e s t i o n a b l e in v iew of t h e M e n d e l i a n i n h e r i t a n c e p a t t e r n s t h a t h a v e b e e n obse rved for C m V . ( T h e a l t e r n a t i v e i n t e r p r e t a t i o n t h a t t h e c i r cu l a r D N A s in m a l e s r e su l t f rom low levels of v i ru s r ep l i ca t i on f rom t h e i n t e g r a t e d v i ra l D N A t e m p l a t e s is of c o n s i d e r a b l e in te r est f rom t h e s t a n d p o i n t of g e n e r e g u l a t i o n b e c a u s e s ignif icant levels of v i ru s r ep l i ca t i on a r e r e s t r i c t ed to t h e female r e p r o d u c t i v e t rac t . ) I n h e r i t a n c e as ( i n t e g r a t e d ) e n d o g e n o u s p rov i ruses a p p e a r s to b e t he m o s t p r o b a b l e m e c h a n i s m for t h e t r a n s m i s s i o n of p o l y d n a v i r u s e s of i c h n e u m o n i d s , for e x a m p l e , C s V a n d HfV. A d d i t i o n a l work , howeve r m u s t b e d o n e to d e t e r m i n e m o r e c lear ly w h a t m o l e c u l a r m e c h a n i s m is u sed by b racov i ruses like C m V .

T h e gene t i c r e l a t i o n s h i p of t h e p a r a s i t o i d a n d a n e n d o g e n o u s p o l y d n a v i r u s is a close o n e . I t therefore is r e a s o n a b l e to u se p o l y d n a v i r u s D N A s as gene t i c m a r k e r s for in t raspeci f ic gene t i c ana lyses ( rev iewed in F l e m i n g ,


1991). E i t h e r p o l y m o r p h i c g e n o m i c s e g m e n t s o r R F L P s in t h e ex t r a c h r o m o s o m a l D N A in v i rus from females c a n b e u sed . T h e p r e d i c t e d h i g h e r f requency of R F L P s a n d the d e c r e a s e d n u m b e r of ar t i facts d u e to D N A topo logy in d iges ted D N A sugges t t h a t R F L P s will be m o r e useful. T h i s s imp le a p p r o a c h is l imi ted by the fact t h a t v i rus is p r e s e n t in s ignif icant a m o u n t s only in t he female wasp . T h e i n t e g r a t e d v i ra l D N A s , however , a p p e a r to be s t ab ly i n t e g r a t e d in t h e c h r o m o s o m a l D N A of b o t h m a l e a n d female w a s p s a n d d o no t a p p e a r to be repe t i t ive , t h a t is, each v i ra l D N A a p p e a r s to exist a t a s ingle locus o r a t on ly a few loci. T h u s , t he i n t e g r a t e d v i ra l D N A s p r o b a b l y will u l t ima t e ly p rove to be m o r e i m p o r t a n t in t h e gene t i c ana lys i s of p a r a s i t o i d s , b u t m o r e sensi t ive a n d soph i s t i c a t ed m e t h o d s , for e x a m p l e , p o l y m e r a s e c h a i n reac t ion t echno logy a n d p u l s e d field e lec t rophores i s , ( P C R ) , will be r e q u i r e d .

T h e r ep l i ca t ion s t r a t egy of p o l y d n a v i r u s e s is n o t k n o w n , a n d a n u m b e r of m o d e l s c a n be h y p o t h e s i z e d . T w o m o d e l s t h a t exp la in m u l t i p a r t i t e g e n o m e s in s o m e o t h e r sys t ems p r o b a b l y c a n be d i smis sed for p o l y d n a v i r u s e s b e c a u s e n o ev idence for a l a rge " m a s t e r m o l e c u l e " o r a t a n d e m a r r a y of severa l different g e n o m i c s e g m e n t s h a s b e e n found in t he s tud ies of e i the r t he ex t r a c h r o m o s o m a l o r i n t e g r a t e d v i ra l D N A s (Bl issard et al., 1986b; F l e m i n g a n d S u m m e r s , 1991). T h e i n t e g r a t e d C s V D N A B , for e x a m p l e , is flanked b y w a s p s e q u e n c e s , a n d the m a p p i n g d a t a for t he o t h e r v i ra l D N A s sugges t t h a t t h e v i ra l g e n o m i c s e g m e n t s exist a t different, n o n c o n t i g u o u s loci. U n l i k e p l a n t m i t o c h o n d r i a l g e n o m e s , t h e i n d i v i d u a l s e g m e n t s therefore a r e unl ike ly to resu l t f rom the r e c o m b i n a t i o n b e t w e e n r e p e a t e d s e q u e n c e s o n a l a rge c i r cu l a r mo lecu l e c o m p o s e d of severa l s e g m e n t s ( P a l m e r a n d Sh ie lds , 1984). T h e d a t a from the C s V sys t em also sugges t t h a t t h e supe rhe l i ces a r e un l ike ly to be syn thes i zed as p a r t of a long , l inea r c o n c a t a m e r t h a t s u b s e q u e n t l y is c leaved to yield different i n d i v i d u a l g e n o m i c s e g m e n t s .

If, as s eems likely, t he i n t e g r a t e d v i ra l D N A s a r e u sed as t e m p l a t e s , p o s t u l a t ed m e c h a n i s m s m u s t a c c o u n t for b o t h t he h igh level of ampl i f i ca t ion of t h e D N A s a n d the c h a n g e from a l i nea r mo lecu l e to a c i r cu l a r o n e . I t r e m a i n s to b e d e t e r m i n e d w h e t h e r t he v i ra l D N A s a r e excised f rom t h e c h r o m o s o m e a n d w h e t h e r t h e v i ra l D N A s a r e ampl i f ied as e x t r a c h r o m o s o m a l e l e m e n t s . T h e s ingle copy of t he t e r m i n a l d i r ec t r e p e a t s e q u e n c e in C s V e x t r a c h r o m o s o m a l D N A Β sugges t s t h a t t he two d i rec t r e p e a t s a t t h e e n d s of t h e l inea r ( in te g r a t e d ) copy r e c o m b i n e d u r i n g rep l i ca t ion , r e su l t ing in a c i r cu l a r m o l e c u l e (Fig . 4 a n d F l e m i n g a n d S u m m e r s , 1991). R e c o m b i n a t i o n b e t w e e n d i rec t ly r e p e a t e d s e q u e n c e s can resu l t in excis ion of t he i n t e r v e n i n g D N A t h a t w o u l d b e p r e s e n t in t he r e s u l t a n t c i r cu la r molecu le , as h a s b e e n d e m o n s t r a t e d u n a m b i g u o u s l y in in vitro s tud ies of a m o u s e r e t r o t r a n s p o s o n ( E d e l m a n n et al., 1989). Al terna t ive ly , it is a lso poss ib le t h a t t he v i ra l s e q u e n c e s a r e s y n t h e sized b y a copy choice o r o t h e r m e c h a n i s m , a n d t e r m i n i of t he e x t r a -


c h r o m o s o m a l , l i nea r p r o d u c t r e c o m b i n e to fo rm a c i r cu l a r D N A w i t h o u t t h e excis ion of t he g e n o m i c s e q u e n c e s . A l t h o u g h re t rov i rus D N A s g e n e r a t e d b y rever se t r a n s c r i p t i o n p r i o r to i n t e g r a t i o n in re t rov i rus - infec ted cells a r e n o t s t r ic t ly a n a l o g o u s to p o l y d n a v i r a l D N A s , t h e circles w i t h a s ingle long t e r m i n a l r e p e a t ( L T R ) sugges t t h a t h o m o l o g o u s r e c o m b i n a t i o n b e t w e e n t h e t e r m i ni of l i nea r D N A c a n resu l t in a c i r cu l a r mo lecu l e w i t h a s ingle copy of t h e d i r ec t r e p e a t ( V a r m u s a n d B r o w n , 1989).

A b i p h a s i c r ep l i ca t ion s t r a t egy a lso is poss ib le a n d m i g h t b e t t e r e x p l a i n t h e l o g a r i t h m i c i nc r ea se of v i ra l D N A in n e w l y eclosed a d u l t s . V i r a l r ep l i ca t ion beg ins in l a te p h a r a t e females b u t c o n t i n u e s in t h e a d u l t female w a s p for severa l d a y s after eclosion ( N o r t o n a n d V i n s o n , 1983; F l e m i n g a n d S u m m e r s , 1986; T h e i l m a n n a n d S u m m e r s , 1986). T h e in i t ia l even t m a y g e n e r a t e c i r cu l a r mo lecu le s f rom t h e i n t e g r a t e d v i ra l D N A s by o n e of t h e m e c h a n i s m s o u t l i n e d ear l ier . T h e c i r cu la r D N A s g e n e r a t e d in t he first p h a s e m i g h t t h e n act as t e m p l a t e s for D N A syn thes i s in t h e second p h a s e . C o n t i n u e d a m p l i fication of t h e e p i s o m a l D N A s b y a ro l l ing circle o r o t h e r m e c h a n i s m cou ld a c c o u n t for t h e r a p i d inc rease in t h e v i ra l supe rhe l i ca l D N A s in newly eclosed a d u l t females . Different e p i s o m e s p r e s u m a b l y w o u l d b e t e m p l a t e s for t h e syn thes i s of t h e different D N A g e n o m i c s e g m e n t s .

W h a t e v e r r ep l i ca t ion m e c h a n i s m is used , t h e res t r i c t ion of h i g h levels of r ep l i ca t i on to la te p h a r a t e a n d a d u l t female w a s p s sugges t s t h a t r ep l i ca t i on is d e v e l o p m e n t a l l y r e g u l a t e d ( N o r t o n a n d V i n s o n , 1983). A l t e r e d h o r m o n a l s t a t u s in t h e p u p a t i n g female is a n o b v i o u s poss ib le m e c h a n i s m of r e g u l a t i n g t h e w a s p a n d / o r v i ra l genes involved in v i rus r ep l i ca t ion . E c d y s t e r o i d s h a v e b e e n r e p o r t e d to inf luence t h e level of C s V rep l i ca t ion ( W e b b a n d S u m m e r s , 1990a, 1993). W h a t o t h e r (wasp) hos t factors a r e involved a n d w h e t h e r r ep l i ca t i on in t h e p u p a l a n d a d u l t female is s imi la r ly r e g u l a t e d a r e n o t k n o w n .

VIII. Polydnavirus Evolution

A. Gene Divergence

T h e m o s t o b v i o u s fea tures of p o l y d n a v i r u s g e n o m e s , t h e s e g m e n t a t i o n a n d l a rge size, a r e of i n t e r e s t f rom a n e v o l u t i o n a r y p o i n t of view. V i r u s e s h a v e evolved severa l s t r a t eg ies ( a l t e rna t i ve sp l ic ing , f rameshi f t ing , etc.) to inc rease t h e c o d i n g capac i ty of the i r g e n o m e s w i t h o u t s ignif icant c o n c o m i t a n t i nc reases in size. T h e a g g r e g a t e g e n o m e sizes of p o l y d n a v i r u s e s sugges t t h a t t hey a r e a m o n g t h e v i ruses (e .g. , bacu lov i ruses , poxv i ru se s , h e r p e s v i r u s e s , e tc . ) w i t h t he l a rges t g e n o m e s ( M u r p h y a n d K i n g s b u r y , 1990). T h e l a rge g e n o m e s p a r t l y m a y reflect t h e s t r u c t u r a l c o m p l e x i t y of t h e v i r ions [e.g. , 25


p o l y p e p t i d e s in C s V (Kre l l et al., 1982)] a n d t h e m u l t i p l e func t ions t h a t p o l y d n a v i r u s e s m a y p l a y in t he w a s p a n d in t h e o n e or m o r e l e p i d o p t e r a n hos t s of t he w a s p . T h i s e x p l a n a t i o n , however , m a y b e insufficient b e c a u s e b r o a d hos t r a n g e s d o no t necessar i ly r e q u i r e g e n o m e s as l a rge as t hose of p o l y d n a v i r u s e s . F lav iv i ruses , for e x a m p l e , have b o t h i n v e r t e b r a t e a n d m a m m a l i a n hos t s b u t have R N A g e n o m e s of a p p r o x i m a t e l y 10 k b ( M u r p h y a n d K i n g s b u r y , 1990).

T h e m u l t i p a r t i t e n a t u r e of t he g e n o m e m a y have r e d u c e d the se lec t ion p r e s s u r e for sma l l g e n o m e size. A l t h o u g h the a g g r e g a t e g e n o m e s a r e l a rge , each s e g m e n t typica l ly is less t h a n 1 5 - 2 0 k b p a n d cou ld p r e s u m a b l y a c c o m m o d a t e m u l t i p l e inse r t ions w i t h o u t adverse ly affecting t h e efficiency of rep l i ca t ion . T h e i n t e g r a t i o n of t he p o l y d n a v i r a l D N A s a lso m a y have r e d u c e d the select ion for sma l l g e n o m e size b e c a u s e t he v i ra l D N A w o u l d be p r o p a g a t e d as p a r t of t he c h r o m o s o m e s d u r i n g p a r t of t he v i ra l life cycle.

T h e n u m b e r of m u l t i g e n e families a n d the d ive rgence w i t h i n t h e v i ra l gene families a r e no tewor thy . V i r a l gene families a r e n o t u n i q u e to po lyd nav i ruses a n d have b e e n r e p o r t e d for Afr ican swine fever a n d m y x o m a v i ru s ( A l m e n d r a l et al., 1990; G o n z a l e z et al., 1990; U p t o n et al., 1990). M u l t i g e n e famil ies , however , a r e n o t a typ ica l fea ture of v i ra l g e n o m e s , a n d t h e p r e s e n c e of m o r e t h a n two families in a g iven v i rus , t h a t is, CsV , is u n u s u a l . T h e i n t e g r a t i o n of p o l y d n a v i r u s e s m a y have inf luenced b o t h t h e n u m b e r of gene families a n d the a p p a r e n t n u m b e r of m e m b e r s w i t h i n p a r t i c u l a r ( C s V ) m u l t igene famil ies . O h t a (1988b) sugges t s t h a t evo lu t ion t h r o u g h gene d u p l i c a t ion occu r s m o r e r a p i d l y d u r i n g i n t e r c h r o m o s o m a l u n e q u a l c ross ing-over d u r i n g sexua l r e p r o d u c t i o n .

V i r a l g e n e families c a n reflect t he acqu i s i t ion of hos t s e q u e n c e s (e.g. , t he s e rp ins of m y x o m a v i rus) o r t he d u p l i c a t i o n of a v i ra l g e n e w i t h n o d e t e c t a b l e h o m o l o g y wi th a hos t gene ( U p t o n et al., 1990). W e s t e r n b lo t d a t a sugges t t h a t s o m e C s V enve lope p r o t e i n s a n d C. sonorensis v e n o m g l a n d p r o t e i n s s h a r e c o m m o n ep i topes ( W e b b a n d S u m m e r s , 1990b) . T o w h a t ex t en t t h e c ross- reac t ive p ro t e in s a r e evo lu t ionar i ly r e l a t ed is u n c l e a r b e c a u s e n e i t h e r t he w a s p genes for t he v e n o m s n o r t he v i ra l genes for t h e c ross - reac t ive enve lope p r o t e i n s have b e e n ident i f ied. A m i n o acid o r n u c l e o t i d e s e q u e n c e d a t a shou ld h e l p to clarify the evo lu t i ona ry r e l a t i o n s h i p of t he genes , b u t t h e serological c ross- reac t iv i ty of t h e p ro t e in s is a t least cons i s t en t w i t h t h e poss i bi l i ty t h a t C s V a c q u i r e d a n a n c e s t r a l v e n o m gene(s ) d u r i n g t h e coevo lu t ion of t h e w a s p a n d v i rus . T h e poss ib le select ive a d v a n t a g e of a c q u i r i n g w a s p genes w i th biological act ivi ty in t he l e p i d o p t e r a n hos t ( s ) is o b v i o u s .

D u p l i c a t i o n of (viral) genes no t de r ived from t h e w a s p g e n o m e a lso cou ld p rov ide a select ive a d v a n t a g e to t he v i rus by i nc rea s ing t h e copy n u m b e r of a g e n e o r a func t iona l d o m a i n w i th in a gene . T h e t a n d e m l y r e p e a t e d 5 4 0 - b p e l e m e n t s p r o b a b l y r e su l t ed from u n e q u a l c ross ing-over a n d s ignif icant se-


q u e n c e d i v e r g e n c e a m o n g the e l e m e n t s . P u b l i s h e d d o t p lo t a n a l y s e s sugges t t h a t c e r t a i n s e q u e n c e s w i t h i n t h e t a n d e m l y r e p e a t e d 5 4 0 - b p e l e m e n t s o n C s V D N A Η a r e m o r e h igh ly conse rved t h a n t h e o t h e r s e q u e n c e s in t hose e l e m e n t s ( T h e i l m a n n a n d S u m m e r s , 1987). W h e t h e r t h e m o r e h igh ly r e l a t e d s e q u e n c e s w i t h i n t h e D N A Η 5 4 0 - b p r e p e a t s r e p r e s e n t func t iona l d o m a i n s p r e s e n t l y is unc lea r .

G e n e d u p l i c a t i o n c a n b e ind i rec t ly i m p o r t a n t to a n o r g a n i s m b e c a u s e t h e ex i s tence of m u l t i p l e copies of a g e n e p e r m i t s m u t a t i o n s to a c c u m u l a t e in o n e o r m o r e copies of t h e gene w i t h o u t d e t r i m e n t to t he o r g a n i s m (Li , 1983; O h t a , 1988a) . Al te rna t ive ly , t h e d i v e r g e n c e of r e l a t ed genes c a n h e l p to refine t h e phys io logy of a s y s t e m b y e n c o d i n g v a r i a n t p r o t e i n s w i t h s l ight ly m o d ified func t ions o r by c o d i n g for r e l a t ed p e p t i d e s t h a t form m u l t i m e r i c p r o te ins t h a t a r e func t iona l ly s u p e r i o r to t h e m o n o m e r s (Li , 1983). T h e t h r e e s imi l a r r eg ions in t h e p r e d i c t e d a m i n o acid s e q u e n c e s for t h e 1.0- a n d 1.6-kb m R N A s sugges t t h a t t h e p r o t e i n s h a v e s imi la r , b u t n o t necessa r i ly i den t i ca l , b io logica l ac t iv i t ies . W h e t h e r a n y r e l a t ed C s V genes e n c o d e s u b u n i t s of m u l t i m e r i c p r o t e i n s c a n n o t be a d d r e s s e d a t t h e p r e s e n t t i m e b e c a u s e l i t t le is k n o w n a b o u t t h e phys i ca l a n d func t iona l cha rac te r i s t i c s of t he p r o t e i n p r o d uc t s of t h e r e l a t e d genes o r t he i n t e r ac t i on of t h e p r o t e i n s w h e n they a r e co-exp re s sed in cells of t he w a s p o r p a r a s i t i z e d hos t .

T h e level of d i v e r g e n c e a m o n g m e m b e r s of s o m e C s V g e n e famil ies s t u d ied to d a t e a p p e a r s to b e fairly h i g h c o m p a r e d to t h e re la t ive h o m o g e n e i t y w i t h i n g e n e famil ies s u c h as t he Bombyx cho r ion genes ( X i o n g et al., 1988; H i b n e r et al., 1991). B e c a u s e t h e 5 4 0 - b p r e p e a t e l e m e n t s e q u e n c e s w e r e in i t ial ly ident i f ied b y S o u t h e r n b lo t h y b r i d i z a t i o n , a m i n i m u m level of 6 0 - 7 0 % s imi la r i ty cou ld b e p r e d i c t e d f rom t h e h y b r i d i z a t i o n s t r i ngency c o n d i t i o n s t h a t w e r e u s e d . I t is u n c l e a r w h y t h e level of v a r i a t i o n a m o n g t h e m e m b e r s is so un i fo rm . T h e e l e m e n t s o n different g e n o m i c s e g m e n t s a r e a p p r o x i m a t e l y 6 0 - 7 0 % s imi l a r to o n e a n o t h e r ( T h e i l m a n n a n d S u m m e r s , 1987). A d j a c e n t e l e m e n t s in a t a n d e m a r r a y m i g h t b e e x p e c t e d to b e m o r e h igh ly r e l a t ed to o n e a n o t h e r t h a n t h e m o r e d i s t a l e l e m e n t s , b u t t he e l e m e n t s t h a t a r e t a n d e m l y a r r a y e d o n a g iven g e n o m i c s e g m e n t a lso a r e on ly a p p r o x i m a t e l y 60— 7 0 % s imi l a r (or less in a few cases) . T h e obse rved d ive rgence c a n b e in te r p r e t e d a s a n i n d i c a t i o n t h a t s t r o n g se lec t ion p r e s s u r e for v a r i a n t genes exis ted p r i o r to u n e q u a l cross ing-over . As n o t e d ear l ier , sho r t s e q u e n c e s w i t h i n m o s t of t h e e l e m e n t s on C s V D N A Η a p p e a r to b e s imi la r . T h e p a t t e r n s c a n b e a l t e rna t ive ly i n t e r p r e t e d as sugges t i ng t h a t t h e c h a n g e s o u t s i d e of t hese s h o r t r eg ions of s imi la r i ty w e r e select ively n e u t r a l whi le these conse rved reg ions w e r e u n d e r pos i t ive se lec t ion a n d d ive rged less. K r e l l (1991a) h a s p r o p o s e d t h a t t h e 5 4 0 - b p r e p e a t e l e m e n t s r e p r e s e n t r e m n a n t s of d u p l i c a t e d b u t n o n e s s e n t i a l p s e u d o g e n e s t h a t a r e select ively n e u t r a l a n d h e n c e w e r e n o t e l i m i n a t e d d u r i n g v i ra l r ep l i ca t ion . A less e x t r e m e i n t e r p r e t a t i o n is t h a t o n e


or m o r e p s e u d o g e n e s m a y exist in this o r o t h e r p o l y d n a v i r u s m u l t i g e n e families in a d d i t i o n to func t iona l genes , as typica l ly occu r s in o t h e r e u k a r y o -tic g e n e families (Li , 1983). W h i c h of t h e four e x p l a n a t i o n s ap p l i e s to t h e 5 4 0 - b p r e p e a t e l e m e n t g e n e family c a n n o t b e e v a l u a t e d n o w b e c a u s e it is n o t k n o w n w h e t h e r these s e q u e n c e s c a n b e t r a n s l a t e d o r w h a t b io logica l ac t iv i ty t h e p r o d u c t s have . However , inves t iga to rs of p o l y d n a v i r u s g e n o m i c c o m p l e x ity a n d g e n e families in t h e fu ture shou ld cons ide r t he poss ib i l i ty t h a t funct iona l a n d non func t i ona l r e l a t ed s e q u e n c e s c a n coexis t in a g e n o m e .

B. Species Specificity

Po lydnav i ruses genera l ly a r e cons ide red to be species specific w i t h r e spec t to t h e w a s p hos t in w h i c h they rep l i ca te . A given p o l y d n a v i r u s " s p e c i e s , " as j u d g e d by its D N A e l ec t rophore t i c profile, c a n b e r e p r o d u c i b l y i so la ted f rom a g iven w a s p species . T h e s imi lar i t ies in t he e l ec t ropho re t i c profiles of u n d iges ted D N A s from i n d i v i d u a l Hyposoter lymantriae females in a s ingle p o p u la t ion o u t w e i g h e d the obse rved differences (Stol tz a n d X u , 1990). T h e overall s imi la r i ty of t h e e l ec t rophore t i c profiles of t he u n d i g e s t e d g e n o m i c D N A s of i n d i v i d u a l females from geog raph i ca l l y i so la ted p o p u l a t i o n s of C. melanoscela a l so is cons i s t en t w i t h th is conc lus ion , even t h o u g h s o m e polym o r p h i c s e g m e n t s were identif ied (Stol tz et al., 1986). Converse ly , t h e v i ruses from different w a s p species c a n b e d i s t i ngu i shed f rom o n e a n o t h e r b y the i r D N A profiles a n d by the i r b iological act ivi t ies in different l e p i d o p t e r a n spe cies. I n sp i t e of t he p r o b l e m s as soc ia t ed w i t h these c r i t e r ia , t h e i n t e r p r e t a t i o n t h a t t he v i ruses of different w a s p species a r e themse lves d i s t i nc t species a p p e a r s to b e val id for t he p o l y d n a v i r u s e s t h a t have b e e n s t u d i e d so far.

T h e a p p a r e n t species specificity of t he v i ruses a n d t h e m u t u a l i s t i c i n t e r d e p e n d e n c e of w a s p a n d p o l y d n a v i r u s for successful r e p r o d u c t i o n a n d su rv iva l sugges t t h a t t h e w a s p a n d v i rus m a y have coevolved a l o n g pa ra l l e l p a t h s . I n t e g r a t i o n a n d g e r m l i n e t r a n s m i s s i o n of t he v i ruses a r e n o t neces sa ry for t h e p o s t u l a t e d pa ra l l e l coevolu t ion to have o c c u r r e d . Howeve r , t he a p p a r e n t l y d u a l life cycle of t he v i ruses as a l t e rna t e ly e x t r a c h r o m o s o m a l ( a n d ex t race l lu lar) v i r ions a n d e n d o g e n e o u s p rov i ruses p rov ides a n efficient m e c h a n i s m for se lec t ing a n d m a i n t a i n i n g v i ra l v a r i a n t s t h a t resu l t in i m p r o v e d su rv iva l of w a s p progeny . Coevo lu t i on of w a s p a n d v i rus sugges t s t h a t p o l y d n a v i r u s e s from p a r a s i t o i d species t h a t a r e phy logene t i ca l ly r e l a t ed m a y b e m o r e r e l a t e d to each o t h e r t h a n v i ruses from less closely r e l a t ed w a s p s . Hyposoterfiigitivus v i rus g e n o m i c D N A h y b r i d i z e d w i t h v i ra l D N A s of six Hyposoter o r Diadegma species b u t n o t w i t h t h e v i ra l D N A s of Campoletis, Campoplex, o r Glypta species (Kre l l , 1991a) . Stol tz a n d X u (1990) a lso p r e s e n t S o u t h e r n b lo t d a t a sugges t ing t h a t v i ra l D N A s from severa l Hyposoter species a r e r e l a t ed . T h e s e d a t a a n d the l imi ted ava i l ab le serological c o m p a r i s o n s of p o l y d n a v i r u s e s sugges t


t h a t v i ruses of c o n g e n e r i c w a s p species o r species in m o r e closely r e l a t e d g e n e r a a r e m o r e h igh ly r e l a t e d t h a n v i ruses f rom m o r e d i s t a n t l y r e l a t e d w a s p s (Cook a n d Stol tz , 1983).

W h i c h p o l y d n a v i r u s e s r e p r e s e n t d i s t i nc t v i ra l species as o p p o s e d to p a -t h o v a r s o r b i o t y p e s of a s ingle v i ru s a n d h o w species specific p o l y d n a v i r u s e s a r e w i t h r e spec t to t h e w a s p will b e c o m e c lea re r as m o r e d e t a i l e d m o l e c u l a r d a t a b e c o m e ava i l ab le . W h e t h e r t h e va r ious p o l y d n a v i r u s e s h a v e d ive rged a t t h e s a m e r a t e s as t h e w a s p species w i t h i n a g e n u s (or o t h e r t a x o n ) is n o t c lear . F i r s t , e i the r t h e v i ru s o r t h e w a s p m a y b e u n d e r s t r o n g e r se lec t ion t h a n t h e o t h e r s y m b i o n t . Selec t ion for v i ra l v a r i a n t s w o u l d n o t necessa r i ly r e su l t in t h e se lec t ion of w a s p v a r i a n t s , a n d vice ve r sa . S e c o n d , t h e inf luence of t h e l e p i d o p t e r a n h o s t a s a select ive force a lso m u s t b e c o n s i d e r e d w h e n m a k i n g p h y l o g e n e t i c c o m p a r i s o n s of t h e w a s p s a n d t h e v i ruses . W a s p s t h a t p a r a s i t i z e t h e s a m e h o s t o r closely r e l a t ed hos t s p r e s u m a b l y m u s t o v e r c o m e t h e s a m e o r s i m i l a r h o s t defense m e c h a n i s m s . M o r e d i s t a n t l y r e l a t e d hos t s m i g h t b e e x p e c t e d to exer t different se lect ion p r e s s u r e s o n t h e w a s p . B e c a u s e v i ra l genes a p p e a r to b e involved in t h e a b r o g a t i o n of hos t i m m u n i t y , v i ruses of r e l a t ed w a s p s t h a t p a r a s i t i z e t h e s a m e o r r e l a t e d l e p i d o p t e r a n hos t s m a y b e m o r e s imi l a r t h a n v i ruses f rom r e l a t e d w a s p s t h a t have b e c o m e a d a p t e d to m o r e d i s t a n t l y r e l a t ed hos t s .

H o w r e l a t e d two v i ruses (or wasps ) a p p e a r to b e c a n d e p e n d o n w h a t genes a r e s t u d i e d . N o t all genes w i t h i n t h e g e n o m e of e i t he r t h e v i ru s o r t h e w a s p w o u l d b e e x p e c t e d to evolve a t t h e s a m e r a t e . V i r a l genes involved in a n essen t ia l func t ion s u c h as v i ra l D N A syn thes i s m i g h t c h a n g e m o r e slowly t h a n genes for s t r u c t u r a l p r o t e i n s . T h e enve lope genes of h u m a n a n d s i m i a n i m m u n o d e f i c i e n c y v i ruses , for e x a m p l e , a r e no to r ious ly v a r i a b l e re la t ive to s o m e o t h e r genes in t h e respec t ive v i ra l g e n o m e s . ( T h e q u e s t i o n s of w h e t h e r t h e p o l y d n a v i r a l s t r u c t u r a l genes a r e u n d e r s imi l a r se lec t ion p r e s s u r e a n d , if so , w h i c h of t h e m a n y s t r u c t u r a l genes a r e v a r i a b l e a n d w h i c h a r e less sub jec t to c h a n g e a r e c u r r e n t l y u n a n s w e r e d . ) N u c l e o t i d e s e q u e n c e c o m p a r i s o n s of p a r t i c u l a r v i ra l genes of s imi l a r func t ion t h u s a p p e a r m o r e likely to yield useful i n f o r m a t i o n t h a n h y b r i d i z a t i o n ana ly se s u s i n g en t i r e g e n o m i c segm e n t s o r u n c h a r a c t e r i z e d v i ra l D N A f r a g m e n t s . N o t all p o r t i o n s of a g e n e a r e likely to evolve a t s imi l a r r a t e s , a n d th is m u s t b e t a k e n i n t o a c c o u n t w h e n a n a l y z i n g t h e d a t a . F u n c t i o n a l d o m a i n s m a y be m o r e h igh ly c o n s e r v e d if t hey a r e cr i t ica l to t h e func t ion of t h e gene p r o d u c t , as in t h e case of e n z y m e s , o r t h e y m a y b e s o m e w h a t m o r e v a r i a b l e if t hey a r e se lec ted a g a i n s t , a s in t h e case of ep i t opes r ecogn ized b y t h e i m m u n e s y s t e m ( G o o d et al., 1988; H u g h e s , 1991; T h o m a s a n d W i l s o n , 1991).

C o m p a r i s o n s of p o l y d n a v i r u s e s f rom c o n g e n e r i c species o r f rom closely r e l a t ed g e n e r a a r e likely to b e m o r e easi ly i n t e r p r e t a b l e f rom t h e s t a n d p o i n t of s y s t e m a t i c s , b u t c o m p a r i s o n s of v i ruses f rom m o r e d i s t a n t l y r e l a t e d spe -


cies, for e x a m p l e , those in different w a s p subfami l ies o r famil ies , a lso m a y b e useful in t r ac ing the evo lu t ion of p o l y d n a v i r u s e s . A l t h o u g h it is r e a s o n a b l e to expec t t h a t i chnov i ruses a n d b racov i ruses will be s u b s t a n t i a l l y less r e l a t e d to o n e a n o t h e r t h a n v i ruses of congene r i c w a s p species , it c a n n o t b e p r e d i c t e d from e x t a n t d a t a to w h a t ex t en t t he two v i ra l s u b g r o u p s a r e gene t ica l ly r e l a t ed . M o l e c u l a r ana lyses will h e l p to d e t e r m i n e w h e t h e r t h e s imi l a r m u l t i p a r t i t e g e n o m i c o r g a n i z a t i o n of p o l y d n a v i r u s e s in t h e two s u b g r o u p s reflects c o n v e r g e n t evo lu t ion o r evo lu t ion from a c o m m o n a n c e s t r a l v i rus o r w a s p s e q u e n c e .

C. Virus Origin

M o l e c u l a r ana lyse s m a y h e l p to t r ace t he p a t h s of p o l y d n a v i r u s evo lu t ion , b u t t he or ig in of p o l y d n a v i r u s e s is likely to r e m a i n o b s c u r e . T h e o r i e s a b o u t t he or ig in of t h e v i ruses m u s t a c c o u n t for t h e fact t h a t , un l ike m o s t v i ruses , p o l y d n a v i r u s e s have d i s t inc t ly different ecological r e l a t i o n s h i p s w i t h t h e two insec t hos t s . T h e w a s p - v i r u s r e l a t i o n s h i p a p p e a r s to b e m u t u a l i s t i c , w h e r e a s the in t e rac t ion of t he v i rus w i th t he p a r a s i t i z e d hos t c a n b e i n t e r p r e t e d as p a t h o g e n i c . Po lydnav i ruses m i g h t be v iewed as t h e p r o g e n y of a n a n c e s t r a l v i rus o r as w a s p (hos t ) , D N A s e q u e n c e s t h a t have g a i n e d a l imi ted d e g r e e of a u t o n o m y (S t r aus s et al., 1990; Whi t f ie ld , 1990).

Pa thogen i c i t y c a n be cons ide red as selectively d i s a d v a n t a g e o u s to a v i rus if it r esu l t s in t h e p r e m a t u r e d e a t h of t he hos t p r i o r to v i ra l r ep l i ca t ion a n d d i s s e m i n a t i o n ( T e m i n , 1989). F r o m this s t a n d p o i n t , p a r t i a l o r select ive a t t e n u a t i o n of a n a n c i e n t p a t h o g e n i c v i rus m i g h t have b e e n select ively favorab le to it if t he loss of v i ru l ence to one of its hos t s ( the w a s p ) i nc r ea sed the p r o b a bil i ty of v i rus r ep l i ca t ion a n d t r a n s m i s s i o n to p r o g e n y (wasp) hos t s . T h e virus 's v i ru l ence for t he l e p i d o p t e r a n hos t a lso m a y have d e c r e a s e d if s u c h c h a n g e s d e c r e a s e d p r e m a t u r e hos t m o r t a l i t y re la t ive to w a s p e m e r g e n c e . T h i s is cons i s t en t w i t h o b s e r v a t i o n s t h a t in ject ion of p o l y d n a v i r u s e s c a n resu l t in s u p e r n u m e r a r y i n s t a r s o r p s u e d o p a r a s i t i z e d l a rvae a n d r a re ly causes a fatal infect ion in t he habitual hos t in the a b s e n c e of t h e p a r a s i t o i d o r o t h e r m i c r o b i a l infect ions . Al te rna t ive ly , se lect ion for i n c r e a s e d v i ru l ence for t he l e p i d o p t e r a n hos t m a y have o c c u r r e d if deb i l i t a t i on of t h e hos t i n c r e a s e d the surv iva l of t he a l t e r n a t e (wasp) hos t .

I n a second h y p o t h e s i s , w a s p s e q u e n c e s m a y have b e e n t r ans fe r r ed to t he p a r a s i t i z e d hos t . I f t he w a s p D N A s were expres sed a n d the p r o d u c t s w e r e biological ly act ive in t he hos t a n d ind i rec t ly i nc reased t h e fitness of t h e w a s p , t he s e q u e n c e s cou ld have b e c o m e fixed in t he w a s p p o p u l a t i o n . T h e or ig in of t he m u l t i p l e v i ra l g e n o m i c s e g m e n t s c a n b e exp l a ined s imp ly if t hey a r e de r ived from a n c i e n t d i spe r sed repe t i t ive e l e m e n t s . Acqu i s i t i on of t h e ab i l i ty to r ep l i ca t e a u t o n o m o u s l y in selected t issues o r cells of t he w a s p , for e x a m p l e ,


t h e ca lyx ep i the l i a l cells, w o u l d b e a s soc ia t ed w i t h i n c r e a s e d copy n u m b e r , w h i c h w o u l d i nc rea se t h e p r o b a b i l i t y of t r a n s m i s s i o n . A vi ra l o r ig in exp l a in s t h e cons i s t en t t r a n s m i s s i o n of b iological ly ac t ive D N A b e t w e e n t h e two insec t species m o r e s imp ly t h a n a w a s p or ig in , b u t several poss ib le m e c h a n i s m s c a n b e s p e c u l a t e d . A n a l o g o u s to t r a n s f o r m a t i o n , t h e s imples t h y p o t h e t i c a l m e c h a n i s m m a y be lysis of w a s p cells followed by D N A u p t a k e b y cells of t h e p a r a s i t i z e d hos t . I n a s econd scena r io , e x t r a c h r o m o s o m a l w a s p s e q u e n c e s m a y h a v e b e c o m e e n t r a p p e d in vacuoles o r o t h e r u n i t m e m b r a n e s t h a t s u b s e q u e n t l y w e r e s l o u g h e d off f rom the w a s p cell a n d fused w i t h t h e hos t ' s cell m e m b r a n e s .

IX. Conclusions

T o d a y p o l y d n a v i r u s e s a r e inc reas ing ly r ecogn ized as b e i n g r e m a r k a b l y ge ne t ica l ly c o m p l e x biological ent i t ies t h a t o c c u p y a n u n u s u a l ecological n i c h e in t h e insec t wor ld . M a n y q u e s t i o n s a b o u t t h e m a r e p r e s en t l y u n a n s w e r e d , b u t it is b e c o m i n g m o r e a p p a r e n t t h a t t h e funct ion of these v i ruses in t h e h o s t - p a r a s i t o i d i n t e r ac t ion c a n n o t be fully u n d e r s t o o d un t i l t he i r m o l e c u l a r gene t i c s is b e t t e r inves t iga ted . I n a d d i t i o n to the i r i m p o r t a n c e to insec t sc ience , t he i r g e n o m e o r g a n i z a t i o n a n d gene t i c c o m p l e x i t y m a k e po lyd nav i ru se s u n i q u e a m o n g v i ruses , a l so w a r r a n t i n g fu r the r m o l e c u l a r s t u d y of t h e m for w h a t it c a n tell us a b o u t t he d ivers i ty of gene t i c s t r a teg ies t h a t have evolved in t he b io logica l wor ld .

Acknowledgments

We thank Brent Graham and Robert Harrison for valuable discussions and comments on the manuscript and Bruce Webb for providing a preprint of a manuscript on ecdysteroid effects. We thank David Theilmann for providing clone p O ^ H C l 185 and Loyd Sneed for advice on photography. This work was supported by grants from the USDA Competitive Grants Program to J.G.W.F. and from the Natural Sciences and Engineering Research Council of Canada to P.J.K.

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Chapter 10

ΗVenoms of Parasitic Hymenoptera as Investigatory Tools Davy Jones Thomas Coudron Graduate Center for Toxicology Biological Control of Insects Lab University of Kentucky USDA-ARS Lexington, Kentucky Columbia, Missouri

I. Introduction

II. Uses of Venoms in Addressing Questions in Various Scientific Fields A. Inferences on the Bionomics

of the Host and Parasite B. Use of Venoms in Physiological

Studies C. Use of Venoms in Behavioral Studies D. Use of Venoms in Evolutionary

Studies E. Use of Venoms in Taxonomic Studies

F. Use of Venoms to Study Molecular Biology of Proteins

G. Use of Venoms as Pharmacological Probes of Heterologous Systems

H. Use of Venoms in Practical Biological Control

I. Use of Venoms as Classroom Tools

III. Conclusions Acknowledgments References

H y m e n o p t e r a n v e n o m s ho ld g r e a t p r o m i s e as useful a n d effective tools for a d d r e s s i n g f u n d a m e n t a l q u e s t i o n s in m a n y a r e a s of b io logica l sc ience . T h e y a l so h a v e use in p rac t i ca l b io logica l con t ro l a n d c l a s s r o o m i n s t r u c t i o n . I n th is c h a p t e r we rev iew w a y s in w h i c h h y m e n o p t e r a n v e n o m s have o r m a y faci l i tate i nves t iga t ions o n a spec t s of insec t b i o n o m i c s , physiology, evo lu t ion , t a x o n omy, behav io r , p h a r m a c o l o g y , m o l e c u l a r biology, b io logica l con t ro l , a n d cur -r i c u l a r i n s t r u c t i o n . A goa l of th is r ev iew is to foster a t t e n t i o n to t h e n u m e r o u s o p p o r t u n i t i e s a w a i t i n g in t h e u s e of these v e n o m s . E x a m p l e s a r e g iven of new, t e s t ab l e h y p o t h e s e s fol lowing t h e a p p l i c a t i o n of v e n o m s as r e s e a r c h tools .

I. Introduction T h e tox ino logy of v e n o m s is a r a p i d l y g r o w i n g field, especia l ly w i t h t h e a p p l i c a t i o n of m o l e c u l a r t e c h n i q u e s t h a t faci l i tate a n u n d e r s t a n d i n g of t h e

Parasites and Pathogens of Insects Volume 1: Parasites 2 2 7


2 2 8 Davy Jones and Thomas Coudron

t h o u s a n d s of different n a t u r a l l y o c c u r r i n g v e n o m o u s c o m p o u n d s . T h e forces of su rv iva l a n d food p r o c u r e m e n t have p l aced s t r o n g select ive p r e s s u r e o n the c o m p o s i t i o n of t he v e n o m s . As a resul t , t he c o m p o n e n t s of h y m e n o p t e r a n v e n o m s r e p r e s e n t a w ide s p e c t r u m of c h e m i c a l act ivi t ies . A n i n c r e a s e d u n d e r s t a n d i n g of t h e diversi ty, m e c h a n i s m s of ac t ion , a n d s t r u c t u r e s of v e n o m c o m p o n e n t s will p e r m i t the i r use as inves t iga t ive tools in a w i d e va r i e ty of d i sc ip l ines . I n s o m e s i t ua t i ons , u se of v e n o m s m a y offer t h e m e a n s to a d d r e s s f u n d a m e n t a l q u e s t i o n s t h a t have b e e n refractory to o t h e r m e t h o d s .

T h e s t u d y of v e n o m s h a s m u c h to offer in p rac t i ca l b io logica l con t ro l . R e c e n t efforts u t i l iz ing effects of v e n o m s on a w i d e r a n g e of insec ts have d e l i n e a t e d less e m p i r i c a l m e t h o d s for p r e d i c t i n g t he success of g iven biological con t ro l efforts ( J o n e s , 1986; C o u d r o n a n d Pu t t i e r , 1988). I n t h e a r e n a of t he c l a s s room, t he w ide d ivers i ty of h y m e n o p t e r a n v e n o m s offers n u m e r o u s i n s t r u c t i o n a l possibi l i t ies for d e m o n s t r a t i n g va r ious b io logica l , evo lu t ionary , o r m o l e c u l a r p r inc ip l e s .

T h e p o t e n t i a l for m a j o r a d v a n c e s a n d uses of p a r a s i t i c w a s p v e n o m s h a s no t b e e n wide ly rea l ized b e c a u s e m u c h less effort h a s b e e n d e v o t e d to t h e b i o c h e m i s t r y a n d m o l e c u l a r b iology of these a g e n t s , as c o m p a r e d w i t h o t h e r a spec t s of h o s t - p a r a s i t e i n t e r ac t ions . T h e p a u c i t y of case e x a m p l e s forces us to e x a m i n e h y m e n o p t e r a n v e n o m s from a different pe r spec t ive t h a n is u s u ally d o n e in r ev iewing va r ious p a r a s i t e i n t e r ac t ions o r r e g u l a t o r y m e d i a t o r s . Reviews of w a s p v e n o m s b a s e d on g r o u p of w a s p o r k ind of effect c a u s e d in t h e hos t a r e ava i l ab le (Piek a n d Spanje r , 1986; C o u d r o n , 1991). I n this r e view, we assess t h e s t a t u s of r e s e a r c h o n w a s p v e n o m s from t h e pe r spec t i ve of h o w s u c h v e n o m s c a n be used to a d d r e s s q u e s t i o n s in different scientific fields. By following s u c h a fo rma t , we feel t h a t we c a n p r o v i d e t h e r e a d e r w i t h i n fo rma t ion a n d n e w perspec t ives o n how r e sea r che r s c a n a p p l y t he g r o w i n g b o d y of i n fo rma t ion on h y m e n o p t e r a n v e n o m s to the i r o w n r e s e a r c h n e e d s .

II. Uses of Venoms in Addressing Questions in Various Scientific Fields

A. Inferences on the Bionomics of the Host and Parasite

F a u n a l su rveys f requent ly yield n e w species of insec ts a b o u t w h i c h l i t t le is k n o w n , o t h e r t h a n the d a t e a n d p lace of col lect ion. M a n y species of p a r a s i t i c w a s p s a r e k n o w n only from m u s e u m s p e c i m e n s , w i t h l i t t le i n fo rma t ion o n the n a t u r a l b iology of t he w a s p o r t he i den t i t y of its hos t s . I n s u c h cases , t h e inves t iga to r w h o des i res to identify t he u n k n o w n hos t m a y n o t have a bas i s from w h i c h to focus a d d i t i o n a l field s tud ies (see Whi t f ie ld a n d W a g n e r , 1988, for d i scuss ion) . For tuna te ly , it is b e c o m i n g a p p a r e n t t h a t t h e v e n o m s of

10. Venoms of Hymenoptera as Investigatory Tools 2 2 9

different w a s p species a r e t a i lo red to a c c o m m o d a t e t he phys io logy of the i r hos t species . C o m p o n e n t s of v e n o m s a r e b e c o m i n g ident i f ied t h a t a r e d i a g nos t i c of i n t e r ac t i on w i t h specific phys io log ica l p a t h w a y s found in ce r t a in hos t t a x a . T h u s , ana lys i s of t h e v e n o m of a new, o r l i t t le k n o w n , w a s p species c a n ass is t t h e inves t iga to r in focusing ini t ia l r e s e a r c h efforts t o w a r d identifyi n g t h e m o s t likely c a n d i d a t e hos t s .

1. B i o l o g y o f H o s t

I n v e s t i g a t o r s of t he b i o n o m i c s of h o s t - p a r a s i t e i n t e r ac t i ons have l ong r ecogn ized ce r t a in co r re l a t e s b e t w e e n the accessibi l i ty or mob i l i t y of hos t s a n d t h e o c c u r r e n c e of p e r m a n e n t pa r a ly s i s of t h e hos t c a u s e d b y the w a s p . Specific cases of p e r m a n e n t pa r a ly s i s of t he concea l ed hos t i n c l u d e Coeloides spp . (Bracon idae ) t h a t p e r m a n e n t l y p a r a l y z e the i r c ryp t i c c u r c u l i o n i d hos t s ( D e L e o n , 1935), a n d the p e r m a n e n t pa ra ly s i s i n d u c e d in t he cod l ing m o t h by Mastrus carpocapsae ( M c C l u r e , 1933). H o s t s t h a t a r e p e r m a n e n t l y p a r a l y z e d a r e s o m e t i m e s " p r e s e r v e d " in situ b y t h e a g e n t s in jected in to t h e hos t . T h e hos t of M. carpocapsae is p r e se rved u p to 73 d a y s , a n d t h e h o s t of Mellitoba acasta c a n b e p r e s e r v e d u p to 9 m o n t h s (Ste iner , 1986). P r e s e r v a t i o n of exp o s e d hos t s in situ o ccu r s , b u t is m u c h less c o m m o n s ince t h e r e is a g r e a t e r l ike l ihood of p r e d a t i o n on these hos t s c o m p a r e d w i t h a concea l ed hos t . T h e s e c o n s i d e r a t i o n s c a n b e in fo rmat ive in in i t ia l su rveys on h y m e n o p t e r a n f a u n a , as well as in ecological s tud ie s def in ing n i che p a r t i t i o n i n g a n d food w e b s . I f ana lys i s shows t h a t a w a s p s p e c i m e n ' s v e n o m c o n t a i n s c o m p o n e n t s for b o t h pa ra ly s i s a n d p r e s e r v a t i o n , it is m o r e p r o b a b l e t h a t t he w a s p e i t he r (1) i n t e rac t s w i t h a concea l ed hos t o r (2) m a k e s t h e hos t concea l ed after in jec t ing it w i t h these factors . T h u s , c o n s i d e r a t i o n of t h e w a s p ' s v e n o m c o m p o n e n t s will g e n e r a t e t e s t ab l e h y p o t h e s e s a b o u t t h e t a x o n o m i c g r o u p a n d b io logy of t h e w a s p ' s hos t . D i a g n o s t i c c o m p o n e n t s of t h e v e n o m m a y p r o v i d e in i t ia l c lues as to t he n i ches t h a t s h o u l d b e e x a m i n e d first to identify a n u n k n o w n hos t . A s w a s p v e n o m c o m p o n e n t s b e c o m e b e t t e r u n d e r s t o o d , it s h o u l d b e poss ib le to identify specific m o l e c u l a r c o m p o n e n t s t h a t a r e d i r e c t e d t o w a r d p a r t i c u l a r hos t t e m p l a t e s (e.g. , m o t h ve r sus butterf ly, o r L e p i d o p t e r a ve r sus C o l e o p t e r a ) .

S i m i l a r c o n s i d e r a t i o n s c a n b e u sed w i t h r e spec t to sessile hos t s , w h i c h a r e u sua l l y n o t p a r a l y z e d . For e x a m p l e , v e n o m from Chelonus w a s p s (egg- la rva l p a r a s i t e ) does n o t p a r a l y z e t h e hos t (Tay lo r a n d J o n e s , 1989). Nasonia vitripen-nis, a p u p a l p a r a s i t e , does n o t p a r a l y z e its hos t (Ratcliffe a n d K i n g , 1967). I t w o u l d be p r e d i c t e d t h a t in species t h a t s t ing sessile hos t s t h e p a r a l y z i n g c o m p o n e n t s a r e a b s e n t , o r h a v e lost o r c h a n g e d funct ion . F u t u r e r e s e a r c h o n v e n o m s will show w h e t h e r th is h y p o t h e s i s is cor rec t . I f co r rec t , t h e n in t he a b s e n c e of o t h e r i n fo rma t ion t h e p r e s e n c e of a p a r a l y z i n g v e n o m w o u l d i n d i c a t e t h a t t h e (poor ly k n o w n ) w a s p species does n o t a t t a c k a sessile h o s t


(or sessile hos t s t age ) . Po lydnav i ruses o c c u r p r i m a r i l y in w a s p s t h a t a t t a c k l e p i d o p t e r a n s o r s y m p h y t a n s (Stol tz a n d V i n s o n , 1979). T h e p r e s e n c e of b o t h p o l y d n a v i r u s e s a n d a p a r a l y z i n g v e n o m t h e n n a r r o w s t he hos t r a n g e to t h e l a rva l s t age of a l e p i d o p t e r a n o r s y m p h y t a n .

We bel ieve t h a t uses of v e n o m c o m p o n e n t s to d r a w inferences o n p a r a s i t e -h o s t b iology will b e i m p o r t a n t in l imi t ing cr i t ica l s i t ua t i ons s u c h a s d i s a p p e a r i n g t rop ica l r a in forests. I n d e e d , it is likely t h a t t h e r e will b e ex t inc t p a r a s i t e species f rom such a r e a s t h a t will be k n o w n on ly from m u s e u m s p e c i m e n s t a k e n in c u r r e n t f auna l su rveys . O u r p r i m a r y a n d m o s t d i r ec t sou rce of i n fo rma t ion a b o u t the i r hos t s in t he fu ture m a y c o m e from ana lys i s of t he v e n o m t h a t in ter faced t he w a s p w i t h its hos t .

2. B i o l o g y o f Paras i te

V e n o m c o m p o n e n t s m a y p rov ide l eads to the b io logy of t he p a r a s i t e . For e x a m p l e , e n d o p a r a s i t e s s e l d o m p a r a l y z e the i r hos t , or , if t h e y d o , it is u sua l ly t e m p o r a r y . Cotesia congregata, Cotesia glomerata, a n d Microplitis croceipes a r e all e x a m p l e s of e n d o p a r a s i t e s t h a t inject in to t h e m o b i l e hos t a v e n o m t h a t does no t h a v e p a r a l y z i n g act ivi ty ( K i t a n o , 1986; Beckage et al., 1987; T a n a k a a n d V i n s o n , 1991). Converse ly , those p a r a s i t e s t h a t d o p a r a l y z e the i r hos t , per m a n e n t l y o r t empora r i ly , a r e usua l ly e c topa ra s i t e s , especia l ly t hose t h a t c a u s e p e r m a n e n t pa ra ly s i s (e.g. , Bracon hebetor, B e a r d , 1952; Clinocentrus gra-cilipes, Shaw, 1981). T h u s , ana lys i s of t h e v e n o m from field-collected a d u l t w a s p s c an p rov ide c lues on the i r b iology t h a t c a n n o t b e infer red f rom m o r pho log ica l i n fo rma t ion . As t h e b i o c h e m i s t r y of p a r a s i t e v e n o m s b e c o m e s b e t t e r u n d e r s t o o d , it will b e poss ib le to use m o l e c u l a r m a r k e r s t h a t d i s t in gu i sh b e t w e e n v e n o m s t h a t c a u s e t e m p o r a r y ve r sus p e r m a n e n t p a r a l y s i s . I n s i t ua t i ons w h e r e n o w we c a n only say "b io logy u n k n o w n , " i n f o r m a t i o n in t h e fu ture , b a s e d o n v e n o m ana lys i s , m a y p e r m i t inferences s u c h a s : " T h e w a s p m o s t likely p e r m a n e n t l y p a r a l y z e s l e p i d o p t e r a n l a rva l hos t s t h a t o c c u r in, o r w h i c h t he w a s p p u t s in , concea led p laces , in a p re se rved s t a t e (i .e. , t h e hos t l a rva l s t age c a n b e found weeks o r m o n t h s o u t of s eason) , a n d u p o n w h i c h t h e p a r a s i t e l a rvae feed as e c t o p a r a s i t e s . " C o n c e i v a b l y o t h e r a spec t s of t h e p a r a s i t e biology, s u c h as so l i ta ry ve rsus g r ega r ious p a r a s i t i s m , will b e d i a g -n o s a b l e on t he bas i s of key v e n o m c o m p o n e n t s .

B. Use of Venoms in Physiological Studies

1. U s e a s P r o b e s o f N o r m a l H o s t P h y s i o l o g y

a. Timing of developmental events. T h e abi l i ty of v e n o m c o m p o n e n t s to exer t a n effect on ly o n ce r t a in d e v e l o p m e n t a l s t ages of t h e hos t sugges t s c h a n g e s in a spec t s of t he hos t t e m p l a t e t h a t m i g h t o t h e r w i s e r e m a i n u n i n v e s t iga ted . For e x a m p l e , v e n o m p r o t e i n s f rom Chelonus sp . a r e re la t ively s t ab l e

10. Venoms of Hymenoptera as Investigatory Tools 231

d u r i n g t he first 48 h r of hos t e m b r y o n i c d e v e l o p m e n t , b u t a r e r a p i d l y d e g r a d e d by a se r ine p r o t e a s e act iv i ty a p p e a r i n g la te in e m b r y o n i c deve lop m e n t (Le luk a n d J o n e s , 1989). L a r v a e of Paramyelois transitella t h a t a r e p a r a lyzed by t h e v e n o m from t h e be th i l id w a s p Goniozus emigratus i m m e d i a t e l y p r i o r to p u p a t i o n will p r o c e e d to c o m p l e t e t he p u p a t i o n p roces s . H o w e v e r , p a r a s i t i s m ear l ie r in t he l a rva l s t a d i u m causes a hos t pa ra ly s i s t h a t r esu l t s in d e a t h ( G o r d h a n d H a w k i n s , 1981).

A n e x a m p l e of use of v e n o m to uncove r h o r m o n a l l y d r i v e n even t s w a s p r o v i d e d b y C o u d r o n (1991) , w h o showed t h a t in ject ion of v e n o m from Euplectrus plathypenae sufficiently p r i o r to t h e nex t m o l t b locks apolys i s in t h e hos t . M a t e r i a l in jected by female C. gracilipes (B racon idae ) will p r e v e n t t h e hos t f rom m o l t i n g if in jected sufficiently in a d v a n c e of t he nex t m o l t (Shaw, 1981). T e m p o r a r y pa ra lys i s of sp ide r s by w a s p s (e.g. , Polysphincta eximia, C l a u s e n , 1940) c a n a lso be a s soc ia t ed w i t h i nh ib i t i on of m o l t i n g .

T h e s e e x a m p l e s i l lus t ra te h o w t h e ac t ion of v e n o m s c a n revea l d e v e l o p m e n t a l w i n d o w s t h a t o c c u r d u r i n g a r t h r o p o d d e v e l o p m e n t t h a t m a y n o t b e access ible o r d e t e c t a b l e w i t h p r e s e n t tools . I n fact, s o m e v e n o m s p r o d u c e a set of s y m p t o m s in t he hos t t h a t have n o t b e e n p r o d u c e d b y c o n v e n t i o n a l phys io log ica l p r o b e s (e.g. , C o u d r o n , 1991, d e s c r i b e d in t he foregoing) . We p r e d i c t t h a t t hese v e n o m s will be found to in t e r ac t w i t h p a r t s of t h e c o g n a t e phys io log ica l m a c h i n e r y n o t p r e sen t l y k n o w n to exist .

b. Host immune response. M e l a n i z a t i o n of t he h e m o l y m p h is cons id e r ed to b e a p a r t of t h e hos t i m m u n e r e s p o n s e t h a t is s u p p r e s s e d b y t h e p o l y d n a v i r u s of b r a c o n i d a n d i c h n e u m o n i d w a s p s (Beckage et al., 1990). I n a n i n t e r e s t i n g s t u d y by Stol tz et aL (1988) it w a s s h o w n t h a t w i t h o u t coinjec-t ion of t h e v e n o m , t he w a s p ' s {Cotesia melanoscela) p o l y d n a v i r u s d i d n o t u n c o a t a t t h e n u c l e a r p o r e s of hos t t i ssue . L e l u k et al. (1988) showed t h a t in jec t ion of t h e v e n o m from Chelonus, w h i c h a lso h a s a p o l y d n a v i r u s (Che l l i ah a n d J o n e s , 1990), a t t h e p r o p e r t i m e w a s neces sa ry for t h e m e l a n i z a t i o n r e s p o n s e of hos t h e m o l y m p h to b e s u p p r e s s e d . N o k n o w n a g e n t s c a n a r r e s t t h e infect ion p rocess of insec t v i ruses . T h e r e f o r e , e x p e r i m e n t a l use of v e n o m s in sy s t ems s u c h as those c i ted ear l ie r cou ld revea l p a t h w a y s involved in insec t i m m u n e r e s p o n s e a n d v i rus infect ion.

c. Physiology of nervous system. V e n o m c o m p o n e n t s c a n b e very powerful tools in ana lys i s of t h e n e r v o u s s y s t e m of insec ts , a n d as m o r e insec t v e n o m p r o t e i n s b e c o m e well s t u d i e d , the i r u se in s u c h inves t iga t ions will p r o b a b l y e x p a n d . P a r a l y z i n g c o m p o n e n t s f rom p a r a s i t i c w a s p s have a l r e a d y b e e n used to assess p r e - a n d p o s t s y n a p t i c a spec t s of t h e n e r v o u s s y s t e m of the i r hos t s ( W a l t h e r et a l . , 1976). Sco rp ion v e n o m p r o t e i n s h a v e b e e n u s e d to i so la te insec t s o d i u m c h a n n e l c o m p o n e n t s to w h i c h they b i n d (de L i m a et al., 1988) a n d we c a n p r e d i c t t h a t s imi l a r uses will b e found for s o m e p a r a l y z i n g v e n o m s of p a r a s i t i c w a s p s .


2. U s e i n S t u d i e s o n B i o c h e m i c a l P a t h w a y s i n t h e W a s p

T h e v e n o m g l a n d is a specia l ized t i ssue t h a t p r o d u c e s a l imi ted n u m b e r of p r o t e i n s in h i g h re la t ive a b u n d a n c e . T h e s e cha rac te r i s t i c s m a k e t h e v e n o m g l a n d a s imp le a n d a m e n a b l e sy s t em w i t h w h i c h to s t u d y t h e phys io log ica l r e g u l a t i o n of t i ssue activity. For e x a m p l e , J o n e s a n d W o z n i a k (1991) have assessed the o n t o g e n y of b iosyn thes i s of v e n o m c o m p o n e n t s in d e v e l o p m e n t of w a s p p u p a e a n d a d u l t s , a n d have ident if ied specific phys io log ica l even t s co r r e l a t ed w i t h i n d u c e d express ion of t he v e n o m p r o t e i n s . W e b b a n d S u m m e r s (1990) a n a l y z e d p o l y d n a v i r u s s t r u c t u r a l p r o t e i n s a n d v e n o m p r o t e i n s w i t h c o m m o n i m m u n o d e t e r m i n a n t s , a n d fu r the r d e t e r m i n e d t h a t t he exp re s s ion of these a n d o t h e r p o l y d n a v i r u s p r o t e i n s w a s r e g u l a t e d b y p u p a l ecd y s t e r o i d s . D a t a from Piek a n d Span je r (1986) sugges t d e v e l o p m e n t a l l y regu l a t e d p r o d u c t i o n of p a r a l y z i n g act ivi ty in t he v e n o m g l a n d of Venturia canescens. T h u s , w a s p v e n o m p r o t e i n s , in themse lves , m a k e useful m o d e l s in s tud i e s o n phys io log ica l p a t h w a y s a n d r egu l a t i on of t h e d e v e l o p m e n t a l exp ress ion of p r o t e i n s .

C. Use of Venoms in Behavioral Studies

W a s p v e n o m s p rov ide useful pe r spec t ives w i t h w h i c h to s t u d y t h e b e h a v i o r of insec ts . For e x a m p l e , r e s ea r che r s have i n q u i r e d as to t he b e h a v i o r of p a r a s i t i c w a s p s as a d a p t e d for effective use of v e n o m . A s k e w (1971) r e p o r t e d t h a t s o m e b r a c o n i d s p lace the i r eggs in ne rve gang l i a , a n d p r e d a t o r y w a s p s inject v e n o m in to specific gang l i a w h e r e it c a n act . W i l b e r t (1964) d e t e r m i n e d t h a t Aphelinus semiflavus first s t ings t he hos t leg to p a r a l y z e t he hos t , a n d on ly t h e n inser t s its s t ing fu r the r in to t he hos t to oviposi t . Euplectrus s pp . use the i r ov ipos i tor to first s t ing , in ject ing the i r v e n o m in to t he h e m o c o e l of t h e hos t , followed by a series of sha l low p u n c t u r e s of t he hos t cut ic le a n d d e p o s i t i o n of eggs o n each p u n c t u r e site ( C o u d r o n a n d Kelly, 1985).

W a s p behav io r s c a n a lso assis t in b i o c h e m i c a l s tud ies o n v e n o m p r o t e i n s . T h e ov ipos i t iona l b e h a v i o r of a w a s p was ut i l ized in s tud ies o n t h e v e n o m of a Chelonus species . J o n e s (1987) found t h a t Chelonus n e a r curvimaculatus w a s p s prefer to oviposi t i n to y o u n g hos t e m b r y o s m u c h m o r e t h a n in to old e m b r y o s , a n d t h a t ovipos i t ion in to o lde r hos t e m b r y o s resu l t s in t h e d e a t h of t h e e n d o p a r a s i t e . I t w a s h y p o t h e s i z e d t h a t s o m e t h i n g a b o u t t h e t e m p l a t e of o lde r e m b r y o s p r e v e n t e d t he v e n o m from ac t ing . L e l u k a n d J o n e s (1989) tes ted this h y p o t h e s i s a n d in t he p rocess d i scovered a n d c h a r a c t e r i z e d a l a rge i nc rea se in se r ine p r o t e a s e act ivi ty in o lde r e m b r y o s t h a t r a p i d l y d e g r a d e d the v e n o m . T h e d e g r a d a t i o n of t he v e n o m by t h e se r ine p r o t e a s e act iv i ty l eads to t he d e a t h of t he e n d o p a r a s i t e .

O n e c a n a lso modify b e h a v i o r a l even ts of t he p a r a s i t e to p r o b e t h e ac t ion of its v e n o m . For e x a m p l e , Le luk a n d J o n e s (1989) s o u g h t to d e t e r m i n e t h e


o r d e r of e n t r y of severa l p a r a s i t e - d e r i v e d factors d u r i n g ov ipos i t ion . U s i n g female w a s p s c a l i b r a t e d to s p e n d a n ave rage of 20 s econds for ov ipos i t ion , t h e y i n t e r r u p t e d ovipos i t ion a t success ive in t e rva l s . A n t i b o d i e s a g a i n s t t h e v e n o m p r o t e i n s were t h e n used to d e t e r m i n e t h a t t h e female injects t h e v e n o m first d u r i n g ovipos i t ion , followed by the egg a n d p o l y d n a v i r u s . C a r e fully t i m e d d i s r u p t i o n of w a s p ovipos i t ion b e h a v i o r a l lowed t h e a u t h o r s to o b t a i n hos t s in jec ted on ly w i t h n a t u r a l a m o u n t s of v e n o m , a n d n o o t h e r r e g u l a t o r y m a t e r i a l s . T h u s , it w a s poss ib le for these a u t h o r s to p r o b e h o s t b i o c h e m i c a l p rocesses as in f luenced solely by n a t u r a l a m o u n t s of in jec ted v e n o m . S t r a n d et al. (1983) s imi la r ly u sed female ov ipos i t ion b e h a v i o r as a tool in e x a m i n i n g t h e effects of specific in jec ted m a t e r i a l s o n hos t d e v e l o p m e n t . S h a w (1981) u t i l ized t h e b e h a v i o r of a n e c t o p a r a s i t e to assess t h e ac t ion of its r e g u l a t o r y m a t e r i a l s on t h e hos t . T h e p a r a s i t e p l aced its eggs o n t h e surface of t h e hos t after in ject ion of v e n o m , e n a b l i n g the i r s u b s e q u e n t r e m o v a l to o b t a i n a hos t on ly inf luenced b y the in jected m a t e r i a l . T h u s , c o m b i n e d use of b o t h b i o c h e m i s t r y a n d b e h a v i o r c a n p e r m i t e x p e r i m e n t a l m a n i p u l a t i o n s a n d tes ts t h a t cou ld n o t b e a c c o m p l i s h e d w i t h e i t he r a p p r o a c h a lone . A t p r e s e n t , we have l imi ted e x a m p l e s tud ies c o m b i n i n g b e h a v i o r a l a spec t s a n d v e n o m s tud i e s . H o w e v e r , m a n y n e w a n d different w a y s exist in w h i c h t h e d i sc ip l ines of e tho logy a n d b i o c h e m i s t r y c a n b e c o m e c o u p l e d t h r o u g h s t ud i e s o n a n d uses of p a r a s i t e v e n o m s .

D. Use of Venoms in Evolutionary Studies

Paras i t i c w a s p v e n o m s c a n be u sed to a d d r e s s q u e s t i o n s o n t h e e v o l u t i o n a r y forces b e a r i n g u p o n p a r a s i t e s t h a t a r e in a coevo lu t i ona ry s t a t e w i t h t he i r hos t s , a n d in spec i a t i on of p a r a s i t e s to explo i t n e w hos t s . I n m a n y w a s p - h o s t s y s t e m s , t h e v e n o m is t he first c h e m i c a l con t ac t f rom the w a s p t h a t is r e ceived b y t h e hos t . T h e success of t h a t v e n o m in a l t e r i ng t h e h o s t is t h e p ivo ta l p o i n t t h a t d e t e r m i n e s w h e t h e r t h e u t i l i za t ion of t h e h o s t b y t h e p a r a s i t e will p r o c e e d . T h e r e f o r e , o n e w o u l d p r e d i c t t h a t v e n o m s w o u l d b e u n d e r very s t r o n g p r e s s u r e s of m o l e c u l a r evo lu t ion for successful i n t e rces s ion in t h e hos t ' s phys io log ica l p a t h w a y s . T h e r e is essent ia l ly n o b i o c h e m i c a l f r a m e w o r k of p a r a s i t i c w a s p v e n o m s from w h i c h h y p o t h e s e s c a n b e gener a t e d c o n c e r n i n g th is a s p e c t of w a s p - h o s t coevo lu t ion . P r e sen t l y inves t iga t ions c a n n o t p r e - o r p o s t d i c t c o n v e r g e n t evo lu t ion in p a r a s i t e s for a n a l o g o u s effects ve r sus h o m o l o g o u s effects on s imi l a r ve rsus d i s s imi l a r h o s t t e m p l a t e s . Fo r e x a m p l e , t h e r e a r e a n y n u m b e r of p a t h w a y s by w h i c h , say, b l o c k a g e of a m o l t c a n b e c a u s e d b y a v e n o m . T h e v e n o m m a y b lock apolys i s i n d e p e n d e n t of t h e h o s t ecdys t e ro id t i t e r (Kel ly a n d C o u d r o n , 1990; C o u d r o n , 1991), o r t h e p a r a s i t e ' s r e g u l a t o r y m e d i a t o r m a y ac tua l ly s u p p r e s s t h e ecdys t e ro id t i te r ( J o n e s et al., 1986). I t is of f u n d a m e n t a l in t e res t to u n d e r s t a n d h o w select ive


p r e s s u r e s on u n r e l a t e d p a r a s i t e s w o u l d resu l t in c o n v e r g e n t evo lu t ion of two different m e c h a n i s m s of b lock ing hos t m o l t i n g , a n d w h e n t h e h o m o l o g o u s v e n o m molecu les w o u l d be i n d e p e n d e n t l y se lec ted t o w a r d the s a m e func t ion . As spec ia t ion even t s p a r t i t i o n va r ious r e sources (hos t t e m p l a t e s ) , i n fo rmat ion m a y b e e x t r a p o l a t e d c o n c e r n i n g a d v a n t a g e o u s evo lu t i ona ry s t r a t eg ies . For e x a m p l e , does a very l a te l ineage t h a t " e n c o u n t e r s " a t e m p l a t e n i c h e s imi la r to t h a t of a n ear ly l ineage r e a d a p t t he s a m e p r e v i o u s m o l e c u l a r m e c h a n i s m ? Al te rna t ive ly , does t h a t l a te l ineage a l t e r t h e func t ion of a differe n t mo lecu l e used for a different p u r p o s e by a n i m m e d i a t e l y a n c e s t r a l l ineage? Ana lys i s of t he a c t u a l v e n o m c o m p o n e n t s c a u s i n g a p p a r e n t l y h o m o logous effects will reveal w h i c h v e n o m c o m p o n e n t s of u n r e l a t e d o r d i s t a n t l y r e l a t ed p a r a s i t e s have conve rged to have t he s a m e funct ion . E v o l u t i o n a r y r a t e s m a y b e a s c e r t a i n e d by c o m p a r i n g s e q u e n c e d a t a for specific v e n o m c o m p o n e n t s a n d the f requency of conse rved s e q u e n c e s .

A n e x a m p l e from s tud ies on s n a k e v e n o m s i l lus t ra tes h o w s u c h cons ider a t i ons as d e s c r i b e d he r e c a n p rov ide ins igh ts on m o l e c u l a r evo lu t ion . J e n g et al. (1979) found ev idence t h a t o n e type of v e n o m p r o t e i n func t ion ing in h igh -affinity m e m b r a n e b i n d i n g h a d evolved from p h o s p h o l i p a s e A 2 e n z y m e s (also p r e s e n t in s n a k e v e n o m ) , w i t h o u t r e t en t i on a n d expres s ion of p h o s p h o l ipase activity. S imi l a r m e c h a n i s m s m a y o p e r a t e in h y m e n o p t e r a n v e n o m s . Le luk et al. (1989) showed i m m u n o l o g i c a l conse rva t i on of ep i topes in v e n o m s from p a r a s i t i c H y m e n o p t e r a w i th ep i topes in v e n o m p r o t e i n s f rom h i g h e r social o r p r e d a t o r y H y m e n o p t e r a in w h i c h t h e v e n o m serves a different funct ion . W e b b a n d S u m m e r s (1990) showed t h a t ep i topes from w a s p v e n o m p ro t e in s p r o d u c e d in t he v e n o m g l a n d were a lso found o n p r o t e i n s s y n t h e sized in t he ovary t h a t serve as p o l y d n a v i r u s s t r u c t u r a l p r o t e i n s .

W i t h r e spec t to c o m p a r i s o n s across w i d e r p h y l o g e n e t i c d i s t a n c e s , Le luk et al. (1989) found severa l t r e n d s in h y m e n o p t e r a n v e n o m s (Fig . 1). B r a c o n i d

Figure Ί Sodium dodecyl sulfate polyacrylamide gel electrophoresis of venom proteins from different species of Hymenoptera. Lane assignments: (1) Chelonus insularis, (2) Chelonus near curvimaculatus, (3) Dinoponera grandis, (4) Diacamma sp., (5) Paraponera clavata, (6) Ponerine sp., (7) Pogonomyrmex rugosus, (8) Pogonomyrmex maricopa, (9) Dasymutilla foxi, (10) Dasymutilla nogalensis, (11) Pepsis chrysothemis, (12) Sphecius grandis, (13) Apis mellifera, (14) Bombus sonorus, (15) Xylocopa varipuncta, (16) molecular size markers, in kilodaltons, (17) Microplitis sp., (18) Cotesia congregata, (19) Ichneumonidae sp., (20) Ichneumonidae sp., (21) Dasymutilla nogalensis (same as 10), (22) Pepsis chrysothemis (same as 11), (23) Sphecious grandis (same as 12), (24) Polistes arizonen-sis, (25) Paravespula pensylvanica, (26) Vespa luctuosa, (27) Brachygastra mellifica, (28) Syn-oeca septentrionalis, (29) Parachartegus fraternus, (30) Apis mellifera (same as 13), (31) Bombus sonorus (same as 14). Figure is adapted from Leluk et al. (1989), with permission of the publisher.


17 18 19 20 21 22 23 24 25 26 2 7 28 29 30 31

- 116

- 97

- 68

- 45

- 24

- 18 - 14

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16

-116 - 9 7

- 6 6

- 4 5

-24

-18


a n d i c h n e u m o n i d v e n o m s have a n a b u n d a n c e of h i g h - m o l e c u l a r - w e i g h t p r o te ins over 100 k D a , w h e r e a s m o s t of such p r o t e i n s a r e mi s s ing from t h e v e n o m s of h i g h e r H y m e n o p t e r a s u c h as bees a n d p r e d a t o r y w a s p s . F u r t h e r , t he v e n o m s of b r a c o n i d s a n d i c h n e u m o n i d s a r e mi s s ing t h e very sma l l , b u t a b u n d a n t p r o t e i n s found in v e n o m s of bees a n d p r e d a t o r y w a s p s s u c h as me l i t t i n (Fig. 1). We have now e x a m i n e d a p a r a s i t i c w a s p f rom t h e family E u l o p h i d a e a n d find t h a t it is a l so mi s s ing m o s t of t h e a b u n d a n t , h igh -m o l e c u l a r - w e i g h t p r o t e i n s t h a t typify b r a c o n i d a n d i c h n e u m o n i d w a s p s (F ig . 2) . A n o t h e r p a r a m e t e r involved in this c o m p a r i s o n is t h a t t h e v e n o m p r o t e i n s t h a t were e x a m i n e d p rev ious ly from identif ied w a s p s w e r e all f rom e n d o p a r a s i t i c species , w h e r e a s Euplectrus plathypenae is a n e c t o p a r a s i t e .

V e n o m p ro t e in s a r e c lear ly r e spons ive to , a n d c a n serve as sub jec t s of s t u d y of, forces of m o l e c u l a r evo lu t ion . T h e field of m o l e c u l a r evo lu t ion of v e n o m s is in its infancy. G iven the t ens of t h o u s a n d s of p a r a s i t i c H y m e n o p t e ra t h a t a r e t h o u g h t to exist , a n d the even g r e a t e r n u m b e r of hos t s a g a i n s t w h i c h the i r v e n o m s act , t h e r e c lear ly exists a h u g e a r e a of m o l e c u l a r r e s e a r c h t h a t is essent ia l ly a n o p e n field for inves t iga to r s i n t e re s t ed in m o l e c u l a r evo lu t ion .

E. Use of Venoms in Taxonomic Studies

M a n y h y m e n o p t e r a n phy logen ie s a r e unc lea r , w i t h t h e t a x o n o m i c p l a c e m e n t of m a n y g r o u p s u n c e r t a i n . A l t h o u g h n o s ingle fea tu re of a n insec t c a n serve a s t h e defini t ive t a x o n o m i c c r i t e r ion , t h e i n t ense se lec t ion p r e s s u r e t h a t t h e v e n o m is u n d e r (d i scussed in t he foregoing) sh o u l d m a k e it a n ex t r eme ly sensi t ive i n d i c a t o r of t he phy logen i c d i s t a n c e b e t w e e n spec ies . S ince c h a n g e in a s ingle p r o t e i n in t he c o m p l e x m i x t u r e of v e n o m p r o t e i n s (Fig . 1) c a n conce ivab ly confer access to a n a l t e r ed hos t r a n g e , t h e v e n o m cou ld b e a sensi t ive d e t e c t o r of s ib l ing species . A g a i n , r igo rous use of m o l e c u l a r ana lys i s of v e n o m p r o t e i n s for such t a x o n o m i c p u r p o s e s a t th is ear ly s t age h a s very few e x a m p l e s . However , on those occas ions in w h i c h p r o p e r t i e s of v e n o m s h a v e b e e n tes ted in d r a w i n g p h y l o g e n e t i c inferences , it h a s y ie lded useful r esu l t s . For e x a m p l e , Whi t f ie ld (1992) found a useful a p p l i c a t i o n in t he use of t h e p r e s e n c e or a b s e n c e of a p e r m a n e n t l y p a r a l y z i n g v e n o m as a t a x o n o m i c c h a r a c t e r w i t h i n t h e R o g a d i n i . V a n A c h t e r b e r g (1988) a n d Q u i c k e a n d v a n A c h t e r b e r g (1990) found t h a t t he m o r p h o l o g y of t he v e n o m a p p a r a t u s itself w a s a useful c h a r a c t e r t o w a r d so lu t ions to such t a x o n o m i c q u e s t i o n s .

Le luk a n d J o n e s (1989) found t h a t t he v e n o m of species in t h e g e n u s Chelonus h a s a cha rac t e r i s t i c g r o u p of h i g h - m o l e c u l a r - w e i g h t , ye l low-s ta in ing p r o t e i n s (Fig . 1). O t h e r g e n e r a in t he subfami ly C h e l o n i n a e (e .g. , Ascogaster) s h a r e m a n y i m m u n o l o g i c a l l y conse rved v e n o m ep i topes w i t h Chelonus (F ig . 2; J o n e s et al., 1990). O n e g r o u p w h o s e r e l a t i o n s h i p to t he C h e l o n i n a e o n t h e


5 2 -4 7 -

3 3 -

SILVER IMMUNOBLOTS STAIN anti-total anti-33kD

venom proteins η-terminal peptide Figure 2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis of venom proteins from Chelonus near curvimaculatus (Cc), Ascogaster quadridentata (Aq), and Euplectrus plathypenae (Ep). Left, the proteins visualized by silver staining; right, two panels show the result of immunoblott ing similar gels (see Leluk et al., 1989, for methods) using antiserum against total venom proteins from C. near curvimaculatus and antiserum against an N-terminal octapeptide for the 33-kDa protein from C. near curvimaculatus conjugated to keyhole limpet hemocyanin. These results show strong cross-reactivity in general between the venom proteins of C. near curvimaculatus and A. quadridentata. T h e venom from E. plathypenae, which blocks host apolysis independently of ecdysteroid suppression (Coudron, 1991), did not show detectable cross-reactivity with either antiserum.

bas i s of c lass ical m o r p h o l o g i c a l c h a r a c t e r s w a s for m a n y yea r s u n c e r t a i n is Sigalphus ( M u e s e b e c k et al., 1951; K r o m b e i n et al., 1979). Species in th is g r o u p a r e very r a r e , a n d very l i t t le b i o n o m i c i n fo rma t ion a b o u t t h e m is ava i l ab le t h a t m i g h t o t h e r w i s e ass is t in the i r t a x o n o m i c p l a c e m e n t . H o w e v e r , t he resu l t s o b t a i n e d on ye l low-s ta in ing p r o t e i n s in b o t h O l d a n d N e w Wor ld Chelonus, a n d the very s t r o n g i m m u n o l o g i c a l c o n s e r v a t i o n of ep i topes be tween Chelonus a n d Ascogaster, sugges t t h a t s u c h tes ts o n Sigalphus c a n resolve q u e s t i o n s o n its p h y l o g e n e t i c a s soc ia t ion w i t h Chelonus. S h o u l d t h e v e n o m of Sigalphus possess t h e h i g h - m o l e c u l a r - w e i g h t , i m m u n o r e a c t i v e ye l low-s ta in ing

kD Cc Aq Ep Cc Aq Ep Cc Aq Ep 131 —


p r o t e i n s , t he resu l t s w o u l d favor t h e i n t e r p r e t a t i o n t h a t Sigalphus is closely r e l a t ed to t he C h e l o n i n a e in gene ra l , a n d Chelonus in p a r t i c u l a r . T h e p o t e n t i a l for ex tens ion of this k ind of a p p r o a c h to t a x o n o m i c q u e s t i o n s on o t h e r g r o u p s of p a r a s i t i c w a s p s is i m m e d i a t e l y ev iden t .

F. Use of Venoms to Study Molecular Biology of Proteins

A classic e x a m p l e of the c o n t r i b u t i o n s t h a t s tud ies o n h y m e n o p t e r a n v e n o m s c a n m a k e to m o l e c u l a r b iology is t he w o r k on t h e express ion of h o n e y b e e me l i t t i n . S tud ie s on me l i t t i n p rocess ing from p r e p r o m e l i t t i n to p r o m e l i t t i n to final sec re ted me l i t t i n w e r e p i o n e e r i n g efforts t h a t led to gene ra l ly a p p l i c a b l e c o n c e p t s on p r o t e i n secre t ion (Kre i l et al., 1980). W i t h r e spec t to m o l e c u l a r a r c h i t e c t u r e itself, t he ear ly s tud ies on me l i t t i n led to its d e s c r i p t i o n in t h e M e r c k i n d e x ( W i n d h o l z , 1983) as t he "first p o l y p e p t i d e w h o s e b io logica l effects c a n b e u n d e r s t o o d on the bas i s of i ts p r i m a r y s t r u c t u r e . "

T h e r e have b e e n few p u b l i s h e d r e p o r t s of t he s e q u e n c e of a n y v e n o m p r o t e i n from p a r a s i t i c w a s p s . T h u s , insec t v e n o m o l o g y h a s n o t yet h a d a n o p p o r t u n i t y to c o n t r i b u t e to gene ra l p r inc ip les in m o l e c u l a r b io logy a n d g e n e express ion . R e c e n t s tud ies on c lon ing a n d s e q u e n c i n g of v e n o m p r o t e i n s in Chelonus p a r a s i t i c w a s p s ( J o n e s et al., 1992) a n d o t h e r n o n p a r a s i t i c H y m e n o p t e r a ( V l a s a k et al., 1983; V l a s a k a n d Kre i l , 1984; F a n g et al., 1988) have led to t he rea l i za t ion t h a t s ignal p e p t i d e s in h y m e n o p t e r a n v e n o m p r o t e i n s often have t he s t a n d a r d bas i c a m i n o ac ids a t each e n d r ep l aced w i t h o r followed by g l u t a m i c acid r e s idues ( J o n e s et al., 1992). T h e s e resu l t s sugges t t he t e s t ab le hypo thes i s t h a t spec ia l ized t issues of t h e h y m e n o p t e r a n v e n o m g l a n d use a form of s igna l p e p t i d e m a c h i n e r y t h a t is different t h a n t h e c lass ical m e c h a n i s m . I n a d d i t i o n , those a u t h o r s found t h a t a 3 3 - k D a v e n o m p r o t e i n w a s p r i m a r i l y c o m p o s e d of a u n i q u e series of 12 t a n d e m r e p e a t s . T h e s e a n d o t h e r d a t a p r o m p t e d the i r h y p o t h e s i s t h a t s o m e cr i t ica l v e n o m p ro t e in s evolved by d u p l i c a t i o n / d e l e t i o n of such r e p e a t s , d u r i n g spec ia t ion or m o l e c u l a r coevo lu t ion . T h a t t he first s e q u e n c i n g of c loned c D N A s for p a r a s i t e v e n o m p ro t e in s wou ld yield such i n t r i g u i n g o u t c o m e s , a n d g e n e r a t e s u c h h y p o t h e s e s , p rov ides g r e a t o p t i m i s m t h a t fu ture r e s e a r c h in to th is a r e a will b e p a r t i c u l a r l y fruitful. T h u s , v e n o m p ro t e in s offer m o d e l sy s t ems for e luc ida t i on of b o t h genera l ly a p p l i c a b l e m o l e c u l a r p r inc ip l e s a n d u n i q u e excep t ions .

G. Use of Venoms as Pharmacological Probes of Heterologous Systems

H y m e n o p t e r a n v e n o m s from bees a n d p r e d a t o r y w a s p s h a v e b e e n very useful as e x p e r i m e n t a l p r o b e s of he t e ro logous sys t ems . U s e of he t e ro logous n e u r o -

ΊΟ. Venoms of Hymenoptera as Investigatory Tools 2 3 9

tox ins from bees a n d p r e d a t o r y w a s p s were u sed to assess b i o c h e m i c a l p a t h w a y s in n o n t a r g e t insec ts ( Q u i s t a d et al., 1988). H o n e y b e e me l i t t i n h a s b e e n u s e d as a p r o b e of m e m b r a n e s t r u c t u r e ( B a n k s a n d Sh ipo l in i , 1986). A r ecen t r e p o r t de sc r ibes t h e use of me l i t t i n to des t ab i l i ze cell m e m b r a n e s in t h e i so la t ion of cell nuc le i ( S m i t h et al., 1988). S c h m i d t (1986a ,b ) found it useful to u se v e n o m c o m p o n e n t s to s t u d y m e c h a n i s m s b y w h i c h a l l e rgen ic r eac t ions a r e e l ic i ted. W i t h r e spec t to p a r a s i t i c w a s p s , Piek a n d S p a n j e r (1986) u s e d a v e n o m n e u r o t o x i n f rom o n e w a s p to select ively b lock exc i t a to ry n e u r o m u s c u la r t r a n s m i s s i o n , p r i o r to p r o b i n g t h e ne rve p r e p a r a t i o n w i t h t h e v e n o m of a different w a s p . We c a n a n t i c i p a t e t h a t p a r a s i t i c w a s p v e n o m s will a l so be c o m e useful for m e d i c i n a l s t ud i e s . T h e v e n o m of Microbracon h a s effects t h a t pa ra l l e l t h e b o t u l i s m tox in (Thesleff, 1960), t he l a t t e r of w h i c h h a s b e e n a p o p u l a r p h a r m a c o l o g i c a l p r o b e in s tud ie s of v e r t e b r a t e s y s t e m s .

T h e n u m e r o u s a d v a n c e s t h a t have b e e n m a d e u s i n g tox ins a n d v e n o m s a t t e s t to t h e p o w e r of s u c h he t e ro logous p r o b e s in d i s sec t ing m o l e c u l a r p h e n o m e n a (e.g. , a c t i n o m y s i n , c o r d y c e p i n , t e t r a d o t o x i n , e tc . ) . We c a n a n t i c i p a t e t h a t as t h e p o t e n t i a l of p a r a s i t i c w a s p v e n o m s b e c o m e s b e t t e r u n d e r s t o o d a n d inves t iga t ed , these a g e n t s will a lso b e c o m e p o p u l a r a n d effective p h a r maco log ica l p r o b e s . M a n y of these efforts will b e ass i s ted w i t h s tud ies of t h e effect of v e n o m c o m p o n e n t s o n cells in c u l t u r e .

H. Use of Venoms in Practical Biological Control

V e n o m s h a v e uses in b iological con t ro l f rom a n u m b e r of different p e r s p e c t ives . For e x a m p l e , genes for s co rp ion tox ins t h a t a r e select ively ac t ive a g a i n s t a r t h r o p o d s a r e b e i n g tes ted as a m e a n s to i nc rea se t h e effectiveness of bacu lov i ruses ( C a r b o n e l l et al., 1988; D e e et al., 1990; C o u d r o n , 1991). T h e t r e m e n d o u s d ivers i ty of b o t h p a r a s i t i c w a s p v e n o m s a n d t h e h o s t t e m p l a t e s o n w h i c h they ac t offers a n u n p a r a l l e l e d r e sou rce of s ing le -gene p r o d u c t s for e n g i n e e r i n g of insec t p a t h o g e n s (see a l so B e a r d , 1971). T h e s e a r e genes w h o s e p r o d u c t s a r e a l r e a d y evo lu t ionar i ly d e s i g n e d to i n t e r c e d e in t h e b io c h e m i c a l p a t h w a y s of o u r m o s t i m p o r t a n t pes t insec ts ( J o n e s , 1986). Bacu lov i ruses c a n ac t as t h e vec to r to de l iver toxins to t a r g e t e d insec ts t h a t a r e n o t n a t u r a l hos t s of a v e n o m - p r o d u c i n g p r e d a t o r o r p a r a s i t e . For e x a m ple , t h e v e n o m from Euplectrus plathypenae is effective o n over 50 fact i t ious hos t s ( C o u d r o n a n d Pu t t i e r , 1988). T h u s , t h e l imi ts o n p a t h o g e n i c i t y of a s low-act ing bacu lov i rus d u e to n a t u r a l l imi ts o n hos t r a n g e c a n b e o v e r c o m e b y in se r t i on of t he gene for th is v e n o m p r o t e i n in to t h e v i ru s . S imu l t aneous ly , t h e v i ru s p rov ides a m e a n s of e n t r y of t he v e n o m i n t o hos t s t h a t t h e p a r a s i t e w o u l d n o r m a l l y n o t s t ing .

I n d e v e l o p i n g a less e m p i r i c a l bas i s for p r e d i c t i n g t h e success of s o m e bio logica l con t ro l efforts, it is neces sa ry to be a b l e to p r e d i c t w h e t h e r t h e


p a r a s i t e will be ab l e to r egu la t e , a n d t h u s surv ive in , t he phys io logy of a n e w t a r g e t hos t ( J o n e s , 1986). T h e v e n o m p lays a m a j o r role in successful u t i l izat ion of t he hos t by s o m e g r o u p s of p a r a s i t e s . T h e r e f o r e , as a d d i t i o n a l s tud ie s define the specific v e n o m c o m p o n e n t s t h a t r e g u l a t e specific phys io log ica l p a t h w a y s , it will b e c o m e poss ib le to p r e d i c t w h e t h e r a p r o p o s e d b io logica l con t ro l effort will fail d u e to inab i l i ty of t he pa r a s i t e ' s v e n o m to r e g u l a t e t h e p o t e n t i a l hos t .

F r o m a different a p p r o a c h , it h a s b e e n p r o p o s e d t h a t a n u m b e r of u n u s u a l hos t r eco rds o c c u r r i n g in over 70 yea rs of b iological con t ro l l i t e r a t u r e a r i se from "ob l i ga t e m u t u a l p a r a s i t i s m , " in w h i c h the s t i ng ing of a hos t by o n e w a s p species enab l e s t he surv iva l of a second w a s p species in t h a t o t h e r w i s e n o n p e r m i s s i v e hos t ( G u z o a n d Stol tz , 1985). T h e bas i s of th is p h e n o m e n o n h a d no t b e e n p rev ious ly u n d e r s t o o d , a n d t h u s b iological con t ro l r e s e a r c h e r s have no t h a d a m e c h a n i s m to a n t i c i p a t e w h i c h hos t r e co rds a r e of th is t ype . I t is now a p p a r e n t t h a t t he v e n o m from o n e w a s p species c a n e n a b l e t he surv iva l of a s econd w a s p species in a n o the rwi se i n h o s p i t a b l e phys io log ica l e n v i r o n m e n t ( G u z o a n d Stol tz , 1985; V i n s o n a n d Sto l tz , 1986). N o w t h a t it is k n o w n t h a t t he v e n o m p a r t i c i p a t e s in i m m u n o s u p p r e s s i o n of t h e hos t a n d p a r a s i t e su rv iva l in a n u m b e r of sys t ems ( K i t a n o , 1986; Tay lo r a n d J o n e s , 1989; T a n a k a a n d V i n s o n , 1991; o t h e r c h a p t e r s in th is v o l u m e ) , a n d t h a t u n d e r ce r t a in cond i t i ons the ac t ion of t he v e n o m in a n o r m a l l y pe rmis s ive hos t c an b e a b r o g a t e d , the theore t i ca l f r amework is p r e s e n t to deve lop p r e d ic t ive ru les on r eco rds of ob l iga t e m u t u a l p a r a s i t i s m .

I n t he fu ture , g e r m l i n e t r a n s f o r m a t i o n p r o c e d u r e s for H y m e n o p t e r a will i nev i t ab ly be deve loped . A theore t i ca l f r amework e s t ab l i shed o n t h e ac t ion of p a r a s i t i c w a s p v e n o m s will p e r m i t se lect ion of t h e a p p r o p r i a t e v e n o m gene( s ) t h a t will e n a b l e t h e t r a n s f o r m e d w a s p to r ed i rec t t he phys io logy of n o n p e r miss ive hos t s t h a t a r e no t affected by the w a s p ' s n a t u r a l v e n o m .

I. Use of Venoms as Classroom Tools

V e n o m s from H y m e n o p t e r a have a n u m b e r of a p p l i c a t i o n s in t he c l a s s r o o m or l a b o r a t o r y exercises for i l lus t ra t ion or exercise in s t u d y i n g b io logica l , evo lu t ionary , o r m o l e c u l a r p r inc ip le s . For e x a m p l e , me l i t t i n is a r e a g e n t u s e d to lyse r ed b l o o d cell m e m b r a n e s , a n d c a n b e easi ly u s e d to i l l u s t r a t e p r o p e r ties of cell m e m b r a n e s . C o m m e r c i a l l y ava i lab le sp ide r v e n o m c a n b e u sed to i l l u s t r a t e neu rophys io log i ca l p r inc ip les in phys io logy l a b o r a t o r y exerc ises . I n m e d i c a l school i n s t ruc t ion , v e n o m s of social h y m e n o p t e r a n species a r e u s e d to i l lus t ra te p r inc ip les in a l le rgenic r eac t ions . I t is c o m m o n for l a b o r a t o r y exercises on insect phys io logy to use l iga t ion of va r ious b o d y reg ions to i l lus t ra te phys io log ica l p r inc ip les . Piek (1966) a n d C o u d r o n (1991) showed h o w this f o rma t c a n be easi ly e x t e n d e d to use of p a r a s i t i c w a s p v e n o m s w i t h

ΊΟ. Venoms of Hymenoptera as Investigatory Tools 241

different ac t ions v ia different b o d y p a r t s . We have a lso u t i l ized w a s p v e n o m s in o u r o w n c l a s s r o o m i n s t r u c t i o n in biological con t ro l top ics . Fo r e x a m p l e , t h e ab i l i ty to obse rve a n d m a n i p u l a t e Chelonus s t i ng ing b e h a v i o r for v e n o m entry , a n d to o b s e r v e w h e t h e r t he hos t s b e c o m e t ru ly p a r a s i t i z e d ( su rv iv ing p a r a s i t e ) o r p s e u d o p a r a s i t i z e d ( d e a d p a r a s i t e ) , h a s e n a b l e d s t u d e n t s in l a b exercises to coup l e t h e d isc ip l ines of insec t b e h a v i o r a n d insec t physiology. T h e s e rea l e x a m p l e s p rov ide conf idence t h a t t he m a n y a d d i t i o n a l poss i bil i t ies t h a t c a n be env i s ioned cou ld easi ly be a d a p t e d for va r ious i n s t r u c t i o n al p u r p o s e s .

III. Conclusions

I n this c h a p t e r we have a r t i c u l a t e d a n u m b e r of w a y s in w h i c h v e n o m s of p a r a s i t i c H y m e n o p t e r a c a n serve as effective tools in inves t iga t ions in d ive r se scientific d i sc ip l ines . As i l lu s t r a t ions of s u c h use , we e x a m i n e d a va r i e ty of r e s e a r c h a r e a s f rom the different pe r spec t ives offered by u t i l i za t ion of w a s p v e n o m s . A n u m b e r of new, t e s t ab le h y p o t h e s e s w e r e g e n e r a t e d as a conse q u e n c e of s u c h pe r spec t ives . For e x a m p l e , we have p r o p o s e d t h a t w a s p veno m s will b e found to c o n t a i n c o m p o n e n t s t h a t a r e d i a g n o s t i c of t a x a of hos t s w i t h d i s t inc t ive b i o n o m i c s a n d / o r inferable physiology. We a lso p r o p o s e t h a t c e r t a in v e n o m p r o t e i n s have evolved d u r i n g h y m e n o p t e r a n evo lu t ion b y d u p l i ca t ion o r de l e t ion of a core t a n d e m r e p e a t . We also h y p o t h e s i z e t h a t t h e ce l lu la r m a c h i n e r y involved in p r o t e i n secre t ion by t h e v e n o m g l a n d is differe n t f rom, o r is a d i s t inc t modi f i ca t ion of, t h e o r t h o d o x m a c h i n e r y u sed in p r o t e i n sec re t ion from o t h e r t i ssues . We also p r e d i c t t h a t , as a g e n e r a l ru l e , t he v e n o m s of h i g h e r H y m e n o p t e r a c o n t a i n few p r o t e i n s w i t h a m o l e c u l a r we igh t above 100 k D a , w h e r e a s t he v e n o m s of lower, p a r a s i t i c H y m e n o p t e r a c o n t a i n m a n y re la t ive ly a b u n d a n t p r o t e i n s above t h a t size.

As t h e b i o c h e m i s t r y a n d m o l e c u l a r b io logy of v e n o m s b e c o m e b e t t e r u n d e r s t o o d , w e a n t i c i p a t e t h a t t h e list of e x a m p l e s of s u c h uses of p a r a s i t e v e n o m s will i nc rease d r ama t i ca l l y . T h e re la t ively sma l l size of t h e list a t p r e s e n t a t t e s t s b o t h to h o w m u c h this a r e a h a s b e e n here tofore over looked a n d to t h e luc ra t ive r e s e a r c h p o t e n t i a l t h a t awai t s inves t iga to r s a t t r a c t e d to t h e s t u d y of p a r a s i t i c w a s p v e n o m s . T h e r e is every r e a s o n t h a t m a j o r a d v a n c e s a r e in t he offing for this field in itself, a n d for o t h e r d i sc ip l ines t o w a r d w h i c h p a r a s i t e v e n o m s c a n be e x p e r i m e n t a l l y a p p l i e d .

Acknowledgments

The authors gratefully recognize Drs. M. Chippendale, W. Steiner, and D. Stoltz for the review of an early draft. This work was supported, in part, by NIH GM 33995 and a Biomedical


Research Support Grant (D. Jones), the USDA ARS (T. Coudon), and the Kentucky Agricultural Experiment Station (D. Jones).

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I. Introduction

II. Immune Mechanisms in Mosquitoes A. Non-Self Recognition B. Cellular Aspects C. Humoral Aspects D. Biochemical Aspects

III. Genetic Control of Susceptibility/Refractoriness A. Historical Perspective B. Genes and Gene Products C. Molecular Approaches in Vector

Control: Future Directions Acknowledgments References

I. Introduction

M o s q u i t o - b o r n e d i seases a r e a m a j o r c a u s e of m o r b i d i t y a n d m o r t a l i t y in h u m a n p o p u l a t i o n s t h r o u g h o u t t he wor ld , b u t a r e especia l ly i m p o r t a n t in t h e t rop ics a n d s u b t r o p i c s of b o t h h e m i s p h e r e s . M a l a r i a , l y m p h a t i c f i lariasis , yel low fever, a n d o t h e r a rbov i r a l d i seases infect a s ignif icant p e r c e n t a g e of t h e wor ld ' s p o p u l a t i o n a n d p lace a n even l a rge r p e r c e n t a g e a t r isk. Plasmodium, t h e c a u s a t i v e a g e n t of m a l a r i a , is t r a n s m i t t e d b y Anopheles species a n d infects over 200 mi l l ion i n d i v i d u a l s each year , w i t h y o u n g c h i l d r e n suffering t h e g r e a t e s t mor ta l i ty . T h e filarioid n e m a t o d e s , Wuchereria bancrofli, Brugia malayi, a n d B. timori, a r e t r a n s m i t t e d to h u m a n s by severa l species of m o s q u i toes , p r i m a r i l y in t h e g e n e r a Culex, Aedes, Mansonia, a n d Anopheles, a n d c a u s e d e b i l i t a t i n g l y m p h a t i c filariasis in n e a r l y 100 mi l l ion peop l e . I n a d d i t i o n , yel low fever, d e n g u e fever, J a p a n e s e encepha l i t i s , a n d o t h e r m o s q u i t o - b o r n e a r b o v i r u s e s infect t h o u s a n d s of p e o p l e t h r o u g h o u t t he wor ld . D e s p i t e t he e c o n o m i c a n d h e a l t h - r e l a t e d i m p a c t t hese d i seases h a v e o n t h e wor ld ' s p o p u -

Parasites and Pathogens of Insects Copyright © 1993 by Academic Press, Inc. Volume 1: Parasites 2 4 5 All rights of reproduction in any form reserved.

Bruce Μ. Christensen David W. Severson Department of Animal Health and Biomedical Sciences University of Wisconsin Madison, Wisconsin

Biochemical and Molecular Basis of Mosquito Susceptibility to Plasmodium and Filarioid Nematodes

Chapter 11

2 4 6 Bruce Μ. Christensen and David W. Severson

l a t ion , re la t ively li t t le a t t e n t i o n h a s b e e n given to r e s e a r c h a i m e d a t clarifying t he c o m p l e x r e l a t i onsh ips t h a t exist b e t w e e n these p a t h o g e n s a n d the m o s q u i t o e s r e q u i r e d for the i r d e v e l o p m e n t , m a i n t e n a n c e , a n d s u b s e q u e n t t r a n s m i s s i o n to h u m a n s . A l t h o u g h p r i m a r y con t ro l of these d i seases h a s b e e n a c c o m p l i s h e d t h r o u g h m o s q u i t o con t ro l p r o g r a m s , r a p i d l y i n c r e a s i n g res ist a n c e to c h e m i c a l insec t ic ides , t oge the r w i t h a n a w a r e n e s s of t h e e n v i r o n m e n t a l insu l t s c a u s e d b y these chemica l s , h a s s ignif icant ly r e d u c e d t h e effect iveness of these t r a d i t i o n a l con t ro l efforts.

I t is well k n o w n t h a t only select species , o r even select s t r a in s of a g iven species , of m o s q u i t o e s funct ion as n a t u r a l vec tors of t he a b o v e - m e n t i o n e d p a t h o g e n s . T h e ma jo r i ty of species d o n o t s u p p o r t p a r a s i t e d e v e l o p m e n t to t h e infective s t age . We define a suscep t ib l e m o s q u i t o as o n e t h a t s u p p o r t s t h e c o m p l e t e d e v e l o p m e n t of t he p a r a s i t e . A r e s i s t an t m o s q u i t o species is o n e t h a t kills t he i n v a d i n g p a r a s i t e by act ive i m m u n e m e c h a n i s m s , w h e r e a s a refractory m o s q u i t o is def ined as o n e in w h i c h the re s eems to b e a phys io log i cal i n c o m p a t i b i l i t y b e t w e e n the p a r a s i t e a n d the p o t e n t i a l vector . I n refracto ry s i t ua t ions , t he p a r a s i t e m a y successfully i n v a d e t h e h e m o c o e l e n v i r o n m e n t of t he hos t a n d r each the a p p r o p r i a t e t i ssue si te for d e v e l o p m e n t , b u t t h e n s imp ly fails to deve lop . A t h o r o u g h u n d e r s t a n d i n g of t h e m e c h a n i s m s con t ro l l ing suscept ib i l i ty , r e s i s t ance , a n d ref rac tor iness of m o s q u i t o e s for t h e p a r a s i t e s they t r a n s m i t is cr i t ica l for t he d e v e l o p m e n t of n e w a n d innova t ive vec to r con t ro l s t ra teg ies , as well as to ga in a c o m p l e t e u n d e r s t a n d i n g of t he ep idemio logy of these m o s q u i t o - b o r n e d iseases ; however , l i t t le r e s e a r c h effort h a s b e e n d i r ec t ed t o w a r d p rov id ing this f u n d a m e n t a l i n fo rma t ion .

A s ignif icant b o d y of l i t e r a tu re , however , is b e g i n n i n g to a c c u m u l a t e re g a r d i n g the i m m u n e r e s p o n s e of insects a n d o t h e r a r t h r o p o d s a g a i n s t b o t h m i c r o b i a l a n d m e t a z o a n p a t h o g e n s . T h e n u m b e r of books , s y m p o s i a p u b l i c a t ions , a n d rev iew ar t ic les on i n v e r t e b r a t e i m m u n i t y over t he las t five yea r s s u p p o r t th is s t a t e m e n t (Brehe l in , 1986; D u n n , 1986, 1990; G u p t a , 1986; Lackie , 1986; B o m a n a n d H u l t m a r k , 1987; N a p p i a n d C h r i s t e n s e n , 1987; C h r i s t e n s e n a n d N a p p i , 1988; Lackie , 1988; C h r i s t e n s e n a n d Tracy , 1989; L a w a n d Wells , 1989; K a r p , 1990; S u g u m a r a n , 1990). T h e s e r e s e a r c h findings , t o g e t h e r w i th t he u p d a t e d m a t e r i a l rev iewed e l sewhere in th is v o l u m e , have a d d e d s ignif icant ins igh t to o u r u n d e r s t a n d i n g of r e s i s t ance p h e n o m e n a in select a r t h r o p o d s . B u t m u c h of these d a t a m a y b e l imi ted b e c a u s e t h e ma jo r i ty of s tud ies have c o n c e r n e d on ly a l imi ted n u m b e r of insec t spec ies , w i t h a m a j o r e m p h a s i s on select species of l e p i d o p t e r a n s . I t c a n b e ser ious ly q u e s t i o n e d h o w a p p l i c a b l e d a t a from l e p i d o p t e r a n l a rvae real ly a r e to clarifying o u r u n d e r s t a n d i n g of m e c h a n i s m s o p e r a t i n g in a d u l t , h e m a t o p h a g o u s d i p t e r a n s ; therefore , t he a m o u n t of d a t a p r e sen t ly ava i l ab le r e g a r d i n g t h e biological i n t e rp l ay b e t w e e n m o s q u i t o e s a n d the p a r a s i t e s they t r a n s m i t is q u i t e l imi ted in re la t ion to o t h e r m o d e l sy s t ems b e i n g used in insec t i m m u -

11. Mosquito Susceptibility to Plasmodium and Filarioids 2 4 7

ni ty r e s e a r c h . T h i s lack of d a t a is even m o r e a p p a r e n t for t h e m e d i c a l l y i m p o r t a n t p s y c h o d i d s , s imu l i id s , a n d c e r a t o p o g o n i d s .

R e c e n t even t s , however , have s t i m u l a t e d a n i n c r e a s e d r e s e a r c h effort a i m e d a t u n d e r s t a n d i n g t h e c o m p l e x r e l a t i onsh ip s t h a t d e t e r m i n e c o m p a t i bi l i ty a n d i n c o m p a t i b i l i t y in v e c t o r - p a r a s i t e m o d e l s . O n e r e a s o n for th is effort u n d o u b t e d l y involves t he pub l i c i zed in te res t of p r i v a t e , n a t i o n a l , a n d i n t e r n a t i o n a l fund ing agenc ies in t h e vec to r b iology a r e n a . I n a d d i t i o n , t h e fai lure of h igh-vis ib i l i ty p r o g r a m s des igned to p r o d u c e effective vacc ines , t o g e t h e r w i t h t he c o n t i n u i n g evo lu t ion of d r u g r e s i s t ance by v e c t o r - b o r n e p a r a s i t e s , h a s r e n e w e d in te res t in t h e vec to r as a logical focus for n e w con t ro l s t r a t eg ies . B u t p e r h a p s m o r e i m p o r t a n t l y , t h e d e v e l o p m e n t of c o n t e m p o r a r y t echno log ies h a s p r o v i d e d the m e a n s w h e r e b y soph i s t i c a t ed m o l e c u l a r , cel lular , a n d b i o c h e m i c a l s t ud i e s c a n be p e r f o r m e d successfully u s i n g e x p e r i m e n ta l a n i m a l s of ex t r eme ly sma l l size. O n e h o p e s these incen t ives will encou r a g e s o m e of t h e o u t s t a n d i n g r e s e a r c h e r s p re sen t ly u s i n g m o d e l s y s t e m s of l i t t le e c o n o m i c a n d n o p u b l i c h e a l t h s ignif icance to tes t t he findings f rom these m o d e l s y s t e m s in vec tors t h a t c a u s e m o r b i d i t y a n d m o r t a l i t y for mi l l ions of h u m a n s t h r o u g h o u t t h e wor ld .

T h e r e h a v e b e e n s o m e a d v a n c e s in o u r u n d e r s t a n d i n g of t he b i o c h e m i c a l a n d m o l e c u l a r bas i s of m o s q u i t o suscep t ib i l i ty to p a r a s i t e s d u r i n g t h e las t severa l yea r s , a n d th is rev iew will a t t e m p t to u p d a t e s o m e specific f indings d u r i n g t h e las t 3 - 4 yea r s . A d d i t i o n a l i n f o r m a t i o n is ava i l ab le in severa l o t h e r r ev iew ar t ic les d e a l i n g specifically w i t h t h e r e s p o n s e of m o s q u i t o e s to filarial w o r m infect ion ( C h r i s t e n s e n , 1986; T o w n s o n a n d C h a i t h o n g , 1991), m o s q u i to inf luences o n suscep t ib i l i ty to a rbov i ru se s (B i shop a n d Beaty , 1986), a n d i m m u n e m e c h a n i s m s in a r t h r o p o d vec to rs ( C h r i s t e n s e n a n d Tracy , 1989). T h e first sec t ion of th is rev iew will c o n c e r n s o m e of t h e ce l lu la r a n d b io c h e m i c a l a spec t s of t he i m m u n e r e s p o n s e of m o s q u i t o e s a g a i n s t p a r a s i t e s a n d t h e fol lowing sec t ion will a d d r e s s o u r c u r r e n t s t a t e of k n o w l e d g e r e g a r d i n g the m o l e c u l a r bas i s for suscep t ib i l i ty a n d / o r ref rac tor iness in m o s q u i t o vecto r s . B e c a u s e of t he p a u c i t y of d a t a ava i l ab le c o n c e r n i n g e i the r t h e gene t i c con t ro l of suscep t ib i l i ty o r t h e defensive r e sponses a g a i n s t a r b o v i r u s e s in m o s q u i t o e s , th is r ev iew will c o n c e r n on ly t h e r e l a t i onsh ips t h a t m o s q u i t o e s h a v e w i t h Plasmodium a n d filarioid p a r a s i t e s .

II. Immune Mechanisms in Mosquitoes

A. Non-Self Recognition

M o s q u i t o e s possess a n a r s e n a l of ce l lu la r a n d h u m o r a l c o m p o n e n t s os tens i b ly d e s i g n e d to r e t a i n t he in tegr i ty of self, a n d these c o m p o n e n t s c a n b e ve ry


effective in ident i fy ing a n d des t roy ing a m u l t i t u d e of foreign insu l t s . Ac t ive i m m u n e r e sponses of m o s q u i t o e s a g a i n s t m a l a r i a p a r a s i t e s (Col l ins et al., 1986) a n d filarioid n e m a t o d e s (Bee rn t sen et al., 1989) gene ra l ly a r e c h a r a c ter ized by the e n c a s e m e n t of t he p a r a s i t e in a m e l a n o t i c c a p s u l e . A l t h o u g h " m e l a n i z a t i o n " o r " m e l a n o t i c e n c a p s u l a t i o n " h a s b e e n t h e sub jec t of i n t en sive s t u d y in severa l species of insec ts r e p r e s e n t i n g severa l o r d e r s (see rev iews in G u p t a , 1986; C h r i s t e n s e n a n d N a p p i , 1988; N a p p i a n d C h r i s t e n s e n , 1987; Lack ie , 1988; C h r i s t e n s e n a n d Tracy, 1989; S u g u m a r a n , 1990; K a r p , 1990), very l i t t le specific i n fo rma t ion is ava i lab le c o n c e r n i n g t h e m e c h a n i s m s cont ro l l ing these r eac t ions , o r t he cell b iology a n d b i o c h e m i s t r y involved . Vi r tua l ly n o t h i n g is k n o w n c o n c e r n i n g s igna l r ecogn i t ion a n d t r a n s d u c t i o n re q u i r e d for i m m u n e recogn i t ion of non-se l f c o m p o n e n t s a n d the in i t i a t ion of effector m e c h a n i s m s r e spons ib l e for p a r a s i t e d e s t r u c t i o n (see C h r i s t e n s e n a n d Tracy, 1989). T h e sugges t ion t h a t t he p r o p h e n o l ox idase ac t iva t ing syst e m p lays a m a j o r role in i m m u n e recogn i t ion (Ratcliffe et al., 1984; S o d e r h a l l a n d S m i t h , 1986) is n o t s u p p o r t e d by a p p r o p r i a t e e x p e r i m e n t a l d a t a . Alt h o u g h this c a s c a d e of reac t ions s eems cr i t ical for t h e p r o d u c t i o n of m e l a n o tic c o m p o u n d s ( A s h i d a a n d Y a m a z a k i , 1990; S u g u m a r a n , 1990), t h e r e is n o d i r ec t ev idence t h a t a n i m m u n e r e s p o n s e is i n i t i a t ed by or d e p e n d e n t o n c o m p o n e n t s of t he p r o p h e n o l ox idase ac t iva t ing c a s c a d e (Lack ie , 1988; B a y n e , 1990). R e c e n t s tud ies u s ing tyros ine-def ic ient m u t a n t s of Drosophila melanogaster c lear ly showed th is d i p t e r a n to b e c a p a b l e of r ecogn i t i on a n d e n c a p s u l a t i o n of p a r a s i t o i d s even t h o u g h they were i n c a p a b l e of fo rming m e l a n o t i c capsu le s (Rizki a n d Rizki , 1990). B a y n e (1990) p rov ides a n excellen t rev iew of phagocy tos i s a n d non-se l f r ecogn i t ion in i n v e r t e b r a t e s t h a t a d d r e s s e s m a n y of t he p r o b l e m s i n h e r e n t in s tud ies d e s i g n e d to d e t e r m i n e m e c h a n i s m s of s igna l r ecogni t ion . I t is a p p a r e n t , however , t h a t ce l lu la r c o m p o n e n t s p l ay key roles in b o t h recogn i t ion a n d d e s t r u c t i o n of p a r a s i t e s in i m m u n o r e s p o n s i v e m o s q u i t o hos t s .

B. Cellular Aspects

A l t h o u g h n u m e r o u s r epo r t s exist r e g a r d i n g h u m o r a l m e l a n i z a t i o n of foreign i n v a d e r s w i t h i n t he h e m o c o e l of ce r t a in d i p t e r a n s , w i t h o u t t h e p a r t i c i p a t i o n of h e m o c y t e s (Go tz , 1986), u l t r a s t r u c t u r a l a n d in vitro s t ud ie s w i t h m o s q u i toes p rov ide conv inc ing ev idence t h a t h e m o c y t e s a r e cr i t ica l for effective r ecogn i t ion a n d s u b s e q u e n t m e l a n o t i c e n c a p s u l a t i o n ( C h e n a n d L a u r e n c e , 1985, 1987; F o r t o n et al., 1985; C h r i s t e n s e n a n d F o r t o n , 1986). I n a d d i t i o n , h e m o c y t e p o p u l a t i o n s have b e e n s h o w n to inc rease s ignif icant ly d u r i n g i m m u n e r e sponses , w i th th is i nc rease likely d u e to m i to t i c act ivi ty in c i r cu l a t i ng cells ( C h r i s t e n s e n et al., 1989). C o n c o m i t a n t w i t h t he inc rease in h e m o c y t e n u m b e r s , surface c h a n g e s on i m m u n e - a c t i v a t e d cells have b e e n ident i f ied


u s i n g fluorescein-labeled w h e a t g e r m a g g l u t i n i n ( W G A ) ( N a p p i a n d C h r i s t e n s e n , 1986; Li a n d C h r i s t e n s e n , 1990). Resu l t s f rom these s tud ie s , a n d s tud ie s of D. melanogaster (Rizki a n d Rizki , 1983; N a p p i a n d Si lvers , 1984), sugges t t h a t surface b i n d i n g of W G A prov ides a va l id i n d i c a t o r of i m m u n o c o m p e t e n c e a n d / o r i m m u n e ac t iva t ion in h e m o c y t e p o p u l a t i o n s . Be c a u s e t h e i den t i t y of h e m o c y t e surface g lycopro te ins b i n d i n g W G A , o r t h e p o t e n t i a l ro le these molecu le s m i g h t p l a y in r ecogn i t ion m e c h a n i s m s , is n o t k n o w n , o u r l a b o r a t o r y h a s in i t i a t ed s tud ie s d e s i g n e d to identify a n d c h a r a c ter ize h e m o c y t e surface m e m b r a n e molecu le s t h a t m i g h t b e a s soc i a t ed w i t h i m m u n e ac t iva t ion or effector m e c h a n i s m s .

H e m o c y t e s f rom mic ro f i l a r i ae - inocu la ted ( i m m u n e - a c t i v a t e d ) , sa l ine -i n o c u l a t e d ( w o u n d i n g con t ro l ) , a n d u n i n o c u l a t e d A. aegypti w e r e c o m p a r e d u s i n g s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l ec t rophores i s ( S D S -P A G E ) , 1 2 5 I - l a b e l i n g , a n d W G A b i n d i n g t e c h n i q u e s ( S p r a y a n d C h r i s t e n s e n , 1991). B o t h i m m u n e ac t iva t ion a n d w o u n d i n g i n d u c e d a s ignif icant i nc r ea se in h e m o c y t e p o l y p e p t i d e syn thes i s a n d r e su l t ed in t he express ion of a 200-k D a p o l y p e p t i d e t h a t w a s n o t seen in cells from u n i n o c u l a t e d m o s q u i t o e s . B e t w e e n 15 a n d 20 h e m o c y t e surface p r o t e i n s w e r e l abe led w i t h 1 2 5 I , w i t h a s ignif icant i nc r ea se in in tens i t i es seen w i t h h e m o c y t e s f rom i n o c u l a t e d m o s q u i t o e s . O n e of t h e l abe led surface p r o t e i n s c o r r e s p o n d e d to t h e u n i q u e 200-k D a p o l y p e p t i d e v i sua l ized w i t h S D S - P A G E . T w o - d i m e n s i o n a l e lec t rophores i s a l so revea led severa l p o l y p e p t i d e s u n i q u e to h e m o c y t e s f rom i m m u n e - a c t i v a t e d o r w o u n d e d m o s q u i t o e s , b u t ce r t a in p o l y p e p t i d e s w e r e cha rac t e r i s t i c on ly of cells f rom m o s q u i t o e s u n d e r g o i n g a m e l a n o t i c e n c a p su l a t i on r eac t ion . R a d i o l a b e l e d W G A ident i f ied t h r e e surface p r o t e i n - W G A c o m p l e x e s of 59 , 73 , a n d 107 k D a o n t h e surface of h e m o c y t e s f rom all t h r e e g r o u p s of A. aegypti. T h i s s t u d y is t h e first to e v a l u a t e p o l y p e p t i d e syn thes i s a n d surface p r o t e i n c h a n g e s in ac t iva t ed h e m o c y t e s from a n y insec t species , a n d resu l t s o b t a i n e d p rov ide a firm s t a r t i n g p o i n t for t he iden t i f ica t ion of specific h e m o c y t e surface molecu le s t h a t m i g h t b e involved in w o u n d h e a l i n g a n d i m m u n e r e sponse s . C h a r a c t e r i z a t i o n of these surface mo lecu le s s eems v i ta l to o u r u n d e r s t a n d i n g of t h e in i t ia l even t s of t he i m m u n e r e s p o n s e in m o s q u i t o e s , a n d p r o d u c t i o n of a b a n k of m o n o c l o n a l a n t i b o d i e s a g a i n s t i m m u n e - a c t i v a t e d h e m o c y t e s w o u l d likely p rov ide t h e tools neces sa ry to ach ieve th is e n d . L ikewise , t h e d e v e l o p m e n t of c u l t u r e t e c h n i q u e s for h e m o cytes w o u l d g rea t ly facil i tate these types of s t ud i e s ( C h r i s t e n s e n a n d Tracy , 1989).

C. Humoral Aspects

A l t h o u g h u n d e r s t a n d i n g ce l lu la r a spec t s of i m m u n e r e sponses in m o s q u i t o e s is s t rong ly jus t i f ied if we a r e to clarify r ecogn i t i on a n d effector even t s , t h e r e


a r e u n d o u b t e d l y h u m o r a l c o m p o n e n t s t h a t p l ay equa l l y cr i t ica l ro les . As it b e c o m e s m o r e a p p a r e n t t h a t t he i m m u n e r e sponses of m o s q u i t o e s a n d o t h e r insec ts r e p r e s e n t h igh ly soph i s t i ca t ed a n d c o m p l e x ce l lu la r a n d b i o c h e m i c a l even t s , t he d y n a m i c i n t e rp l ay of h u m o r a l a n d ce l lu la r c o m p o n e n t s m u s t b e cons ide r ed w h e n e v a l u a t i n g e i the r of these h e m o l y m p h c o m p a r t m e n t s in s tud ies of insec t i m m u n i t y . D e s p i t e t he ident i f ica t ion of severa l " i m m u n e p r o t e i n s " t h a t funct ion to p ro t ec t hos t s f rom m i c r o b i a l insu l t (e .g. , l y sozyme , d ip t e r i c in s , cec rop ins , a t t a c in s , defens ins , h e m o l i n , s a r co tox ins , p h o r m i c i n s , a n d e i cosano ids , see D u n n , 1990; S u n et al., 1990; S t a n l e y - S a m u e l s o n et al., 1991), few s tud ies have identif ied p r o t e i n c h a n g e s a s soc ia t ed w i t h m e l a n o t i c e n c a p s u l a t i o n or t he effect of specific " i m m u n e p r o t e i n s " o n m o s q u i t o -t r a n s m i t t e d p a r a s i t e s .

U s i n g in vivo l abe l ing t e c h n i q u e s w i th [ 3 5 S ] m e t h i o n i n e , we recen t ly r e p o r t ed t h a t a n 8 4 - k D a h e m o l y m p h p l a s m a p o l y p e p t i d e is p re fe ren t ia l ly exp res sed in A. aegypti d u r i n g w o u n d h e a l i n g o r m e l a n o t i c e n c a p s u l a t i o n r eac t ions , a n d t h a t t h e express ion is g r e a t e r a n d pers i s t s for a l onge r t i m e following i m m u n e ac t iva t ion (Bee rn t s en a n d C h r i s t e n s e n , 1990). D a t a f rom these s tud ies a lso sugges t t h a t th is p o l y p e p t i d e is closely a s soc i a t ed w i t h h e m o c y t e s , is n o t i n d u c e d in r e sponse to bac te r i a l c o n t a m i n a t i o n , a n d is n o t a s t r ess - re la ted (hea t shock) p ro t e in . I n e x p e r i m e n t s ye t to be p u b l i s h e d , th is p o l y p e p t i d e was e l ec t rob lo t t ed o n t o a po lyv iny l idene d i f luor ide m e m b r a n e for d i r ec t s e q u e n c i n g . T w o i n t e r n a l s e q u e n c e s of 15 a n d 8 a m i n o ac ids w e r e o b t a i n e d , b u t n o conc lus ions cou ld be d r a w n r e g a r d i n g t h e n a t u r e of this p r o t e i n following ana lys i s w i th t he N B R F p r o t e i n d a t a b a s e s e q u e n c e sys t em. T o o b t a i n e n o u g h m a t e r i a l for c lon ing , we c o n s t r u c t e d d e g e n e r a t e olig o n u c l e o t i d e p r i m e r s to each e n d of t h e 15 -amino acid s e q u e n c e a n d cond u c t e d P C R ampl i f i ca t ion as p e r Lee et al. (1988) , u s i n g A. aegypti g e n o m i c D N A as a t e m p l a t e . A c lone w a s o b t a i n e d a n d the d e d u c e d s e q u e n c e m a t c h e s t he d a t a o b t a i n e d from o u r d i r ec t m i c r o s e q u e n c i n g . A ful l - length c lone shou ld p rov ide v a l u a b l e i n fo rma t ion as to t h e iden t i ty a n d func t ion of th is 8 4 - k D a p o l y p e p t i d e . T h e s e n e w technologies n o w m a k e it poss ib le to o b t a i n d a t a for ident i f ica t ion a n d c h a r a c t e r i z a t i o n of p o l y p e p t i d e s ava i l ab le on ly in ex t r eme ly l imi ted quan t i t y , a n d therefore e n a b l e worke r s to a n s w e r q u e s t i o n s in h e m a t o p h a g o u s d i p t e r a n s t h a t cou ld n o t b e a d e q u a t e l y a d d re s sed on ly a few yea r s ago . I n s e c t hos t size is n o longer a se r ious d e t r i m e n t to s tud ies t h a t a d d r e s s m e c h a n i s m s of v e c t o r - p a r a s i t e compa t ib i l i ty .

I n a d d i t i o n to o u r s tud ies (Bee rn t sen a n d C h r i s t e n s e n , 1990), a r e p o r t b y G w a d z et al. (1989) conf i rms t h a t ident i f ica t ion a n d / o r e v a l u a t i o n of t hese insec t i m m u n e molecu les in m o s q u i t o e s cou ld p rov ide s ignif icant i n s igh t i n t o m e c h a n i s m s of r e s i s t ance . G w a d z a n d coworkers in jec ted cec rop ins i n to Anopheles m o s q u i t o e s p rev ious ly infected w i t h Plasmodium a n d d e t e r m i n e d t h a t cec rop ins p r e v e n t e d s p o r o g o n y by a b o r t i n g t he n o r m a l d e v e l o p m e n t of oo-

Ί1. Mosquito Susceptibility to Plasmodium and Filarioids 251

cysts . A l t h o u g h these resu l t s w e r e de r ived f rom a n art if icial s i t u a t i o n , t hey p r o v i d e s o u n d jus t i f i ca t ion for s t ud i e s d e s i g n e d to assess p r o t e i n a l t e r a t i o n s in m o s q u i t o s t r a i n s a n d species b o t h r e s i s t an t a n d suscep t ib l e to t h e p a r a s i t e s t hey t r a n s m i t .

A l o n g - s t a n d i n g q u e s t i o n in insec t i m m u n i t y r e g a r d s t h e p o t e n t i a l for o p s o n i c factors in h e m o l y m p h p l a s m a ( B a y n e , 1990), a n d t h e ro le lec t ins m i g h t p l a y in th is p rocess ( R e n w r a n t z , 1986). A l t h o u g h d a t a r e g a r d i n g h e m a g g l u t i n i n ac t iv i ty in insec t b lood a r e equ ivoca l , t h e series of s t ud i e s b y O g u r a a n d coworker s (1985; O g u r a , 1986, 1987) w i t h Armigeres subalbatus a n d Brugia microf i la r iae sugges t t h a t a h e m a g g l u t i n i n in t h e p l a s m a of th is m o s q u i t o m i g h t func t ion as a m e d i a t o r for m e l a n o t i c e n c a p s u l a t i o n r eac t ions . T h i s is especia l ly r e l evan t b e c a u s e A. subalbatus funct ions as a n a t u r a l vec to r for B. pahangi, b u t kills B. malayi microf i la r iae in b o t h t h e h e m o c o e l a n d t h o r a c i c m u s c u l a t u r e b y m e l a n o t i c e n c a p s u l a t i o n ( Y a m a m o t o et al., 1985; B e e r n t s e n et al., 1989). T h e d i sc re t e specificity of this r e s p o n s e c o u l d b e d i s r u p t e d b y t h e a d d i t i o n of specific s u g a r s a g a i n s t t he h e m a g g l u t i n i n o r b y t h e e x p o s u r e of microf i la r iae to h e m o l y m p h s a m p l e s from p u p a e , w h e r e t h e h e m a g g l u t i n a t i n g act iv i ty is h i g h e r t h a n in a d u l t m o s q u i t o e s ( O g u r a , 1986). T h e A. subalbatus-Brugia s pp . m o d e l s y s t e m is d e s e r v i n g of fu r the r s t u d y a n d cou ld p rove v a l u a b l e for a d d r e s s i n g f u n d a m e n t a l q u e s t i o n s r e g a r d i n g recogn i t ion p h e n o m e n a in m o s q u i t o e s .

D. Biochemical Aspects

B e c a u s e " m e l a n i z a t i o n " is t h e p r i m a r y m e c h a n i s m r e spons ib l e for t h e d e s t r u c t i o n of filarial w o r m la rvae a n d m a l a r i a l o r g a n i s m s in r e s i s t an t m o s q u i toes , t h e b i o c h e m i s t r y r e spons ib l e for t he p r o d u c t i o n of m e l a n o t i c m a t e r i a l s h a s rece ived c o n s i d e r a b l e a t t e n t i o n in r e c e n t yea r s . C r i t i c a l to t h e success of these s tud i e s h a s b e e n the d e v e l o p m e n t of h igh ly sensi t ive a n d a c c u r a t e a s says for m e a s u r i n g t h e act iv i ty of e n z y m e s involved in m e l a n o t i c e n c a p s u l a t i o n r eac t ions a n d t h e a s s e s s m e n t of q u a n t i t a t i v e c h a n g e s in levels of s u b s t r a t e s a n d i n t e r m e d i a t e s r e q u i r e d for t h e p r o d u c t i o n of p r o t e i n - p o l y p h e n o l c o m p l e x e s . I t h a s b e c o m e a p p a r e n t in t h e las t severa l yea r s t h a t t h e p r o d u c t i o n of m e l a n o t i c capsu l e s is a c o m p l e x b i o c h e m i c a l p rocess invo lv ing c a t e c h o l a m i n e s , c a t e c h o l a m i n e de r iva t ives , c a t e c h o l a m i n e - m e t a b o l i z i n g enz y m e s , a n d likely o t h e r i n d u c i b l e h e m o l y m p h c o m p o n e n t s ( C h r i s t e n s e n a n d Tracy , 1989).

P h e n o l ox ida se ( ty ros inase ) is a c o p p e r - c o n t a i n i n g o x y g e n a s e t h a t c a t a lyzes t h e h y d r o x y la t ion of ty ros ine to 0 - d i p h e n o l s ( m o n o p h e n o l ox ida se act ivi ty) , a n d t h e o x i d a t i o n of o-d iphenols to the i r c o r r e s p o n d i n g o -qu inones ( d i p h e n o l ox ida se ac t iv i ty) . T h e o -qu inones a r e chemica l ly reac t ive a n d c a n b o t h p o l y m e r i z e , if s t r u c t u r a l l y a p p r o p r i a t e , a n d b i n d cova len t ly to n u c l e o -


phi l ic p r o t e i n r e s idues to form p r o t e i n - p o l y p h e n o l c o m p l e x e s , c o m m o n l y referred to as " m e l a n i n " o r " s c l e ro t i n " ( C h r i s t e n s e n a n d Tracy , 1989; Li a n d N a p p i , 1991). C o n s e q u e n t l y , p h e n o l ox idases have b e e n the m o s t in tens ive ly s t u d i e d c o m p o n e n t of ce l lu lar i m m u n e r e sponses in a r t h r o p o d s . B e c a u s e it is cr i t ica l to assess t he a u g m e n t a t i o n of e n z y m e act iv i ty in vivo to c lear ly de l in ea t e t he funct ion of these molecu les in t he p r o d u c t i o n of m e l a n o t i c c o m p o u n d s , n e w a p p r o a c h e s were necessa ry in o r d e r to accu ra t e ly m e a s u r e p h e nol ox idase act ivi ty in t he m i n u t e h e m o l y m p h s a m p l e s ava i l ab le f rom m o s q u i t o e s . Previously, p h e n o l ox idase act ivi ty was m e a s u r e d b y m o n i t o r i n g fo rma t ion of d o p a c h r o m e from L - 3 , 4 - d i h y d r o x y p h e n y l a l a n i n e ( L - D o p a ) a t 475 n m . A l t h o u g h useful for in vitro a s says w i t h l a rge a m o u n t s of s a m p l e , th i s a s say is fairly insens i t ive b e c a u s e of t he low m o l a r a b s o r p t i o n coefficient of d o p a c h r o m e . I n a d d i t i o n , d o p a c h r o m e is n o t a n e n d p r o d u c t a n d is fu r the r ox id ized to o t h e r i n t e r m e d i a t e s a n d even tua l ly to m e l a n i n , t h e r e b y r e su l t i ng in e r roneous ly low e s t i m a t i o n s of e n z y m a t i c ac t iv i ty ( V a c h t e n h e i m et al., 1985).

N a p p i et al. (1987) u sed a r a d i o m e t r i c assay, o r ig ina l ly d e s c r i b e d by T o w n s e n d et al. (1984) , to assess m o n o p h e n o l ox idase act iv i ty in h e m o l y m p h p l a s m a from i m m u n e - r e a c t i v e A. aegypti. T h i s a s say is b a s e d on t h e q u a n t i t a t ion of t r i t i a t ed w a t e r fo rmed d u r i n g the h y d r o x y l a t i o n of L - [ 3 , 5 - 3 H ] t y r o s i n e to L - d o p a . Subsequen t ly , Li et al. (1989) u sed this a s say to e v a l u a t e m o n o -p h e n o l ox idase act ivi ty in h e m o c y t e s col lected from i m m u n e - r e a c t i v e a n d con t ro l A. aegypti a n d Aedes trivittatus. T h e y were ab l e to a s say e n z y m e act ivi ty from h e m o c y t e s perfused from only t h r ee m o s q u i t o e s , a n d r e p o r t e d t h a t e n z y m e act ivi ty in h e m o c y t e s likely p lays a s ignif icant role in t h e p r o d u c t i o n of m e l a n o t i c c apsu l e s a r o u n d microf i lar iae .

A l t h o u g h m a m m a l i a n ty ros inase c a n c lear ly ca t a lyze t h r e e r eac t ions in t he p a t h w a y of m e l a n i n b i o s y n t h e s i s — t h e h y d r o x y l a t i o n of ty ros ine , t h e d e h y d r o g e n a t i o n of L - d o p a , a n d the d e h y d r o g e n a t i o n of 5 , 6 -d ihyd roxy indo l e ( K o r n e r a n d Pawelek, 1 9 8 2 ) — d i s t i n c t m o n o p h e n o l a n d d i p h e n o l ox idases have b e e n r e p o r t e d in D. melanogaster (Rizki a n d Rizki , 1983; Pen tz et al., 1986). To accu ra t e ly assess d i p h e n o l ox idase activity, we deve loped a n i m p roved a s say b a s e d on the r e p o r t by S u g u m a r a n (1986) t h a t m u s h r o o m ty ros inase ca ta lyzes t he ox ida t ive d e c a r b o x y l a t i o n of 3 , 4 - d i h y d r o x y m a n d e l i c acid to form 3 , 4 - d i h y d r o x y b e n z a l d e h y d e (Li et al., 1990). B e c a u s e t he se a r e ca techo l s a n d the p r o d u c t of t he r eac t ion is s t ab le , a q u a n t i t a t i v e a s say w a s deve loped us ing h i g h - p r e s s u r e l iqu id c h r o m a t o g r a p h y w i t h e l e c t r o c h e m i c a l de t ec t i on ( H P L C - E C D ) , a n d the app l i cab i l i t y of this a s say for m e a s u r i n g d i p h e n o l ox idase act ivi ty in m i c r o g r a m q u a n t i t i e s of c r u d e , cell-free h e m o l y m p h from m o s q u i t o e s was d e m o n s t r a t e d (Li et al., 1990).

T h e ava i lab i l i ty of h igh ly sensi t ive a n d specific a s says for b o t h m o n o p h e n o l a n d d i p h e n o l ox idase act ivi ty e n a b l e d Li et al. (1992) to cr i t ical ly


assess these ca t a ly t i c act ivi t ies in h e m o l y m p h c o m p a r t m e n t s of A. aegypti d u r i n g m e l a n o t i c e n c a p s u l a t i o n r eac t ions . N o s ignif icant differences in e i the r m o n o p h e n o l o r d i p h e n o l ox ida se ac t iv i ty in h e m o c y t e s , cell-free p l a s m a , o r c o m p l e t e h e m o l y m p h ( p l a s m a + h e m o c y t e s ) were n o t e d b e t w e e n s a m p l e s from u n i n o c u l a t e d (naive) o r s a l i ne - inocu la t ed ( w o u n d e d ) m o s q u i t o e s , a l t h o u g h base l i ne levels of b o t h act ivi t ies were d e t e c t e d . B o t h e n z y m e act ivi t ies w e r e s ignif icant ly e leva ted in h e m o c y t e a n d c o m p l e t e h e m o l y m p h s a m p l e s fol lowing i m m u n e ac t iva t ion b y the i nocu l a t i on of microf i la r iae , b u t n o signi f icant i nc r ea se in e n z y m e act iv i ty w a s d e t e c t e d in cell-free p l a s m a . I n c reases in t he act ivi ty of b o t h e n z y m e s w e r e p r o p o r t i o n a l in all s a m p l e s , t h e r e b y sugges t i ng t h a t a s ingle e n z y m e m i g h t be r eac t i ng w i t h b o t h m o n o -p h e n o l s a n d o-d iphenols w i t h i n t h e h e m o l y m p h of this m o s q u i t o . T h e s e s tud ie s a lso sugges t ed t h a t t he i n c r e a s e d p h e n o l ox idase act iv i ty n o t e d d u r ing m e l a n o t i c e n c a p s u l a t i o n reac t ions is a s soc ia t ed p r i m a r i l y w i t h t he h e m o cytes . T h i s a u g m e n t e d e n z y m e act iv i ty w a s a resu l t of in vivo a c t iva t ion b y p a r a s i t e s , a n d this ac t iv i ty cou ld n o t be i nc r ea sed by the a p p l i c a t i o n of t h e c o m m o n l y used p h e n o l ox ida se ac t iva to r s z y m o s a n a n d l a m i n a r i n (Li et al., 1992).

A m o n p h e n o l ox ida se a s say recen t ly w a s deve loped t h a t is m u c h m o r e sensi t ive t h a n the specific r a d i o e n z y m a t i c a s say (Li a n d N a p p i , 1991). T h i s a s say a lso uses H P L C - E C D , a n d it m e a s u r e s t he h y d r o x y l a t i o n of t y ros ine u s i n g JV-ace ty ldopamine as cofactor a n d a s c o r b a t e a s a r e d u c i n g a g e n t . T h e a s c o r b a t e m a i n t a i n s t he level of L - d o p a p r o d u c e d d u r i n g ty ros ine h y d r o x y l a t ion b y r e d u c i n g d o p a q u i n o n e back to t he o -d iphenol n o n e n z y m a t i c a l l y . T h e ut i l i ty of th is a s say w a s d e m o n s t r a t e d b y Li a n d N a p p i (1991) u s i n g mic ro l i te r q u a n t i t i e s of h e m o l y m p h from the l a rvae of D. melanogaster.

W i t h t h e ava i lab i l i ty of these h igh ly sensi t ive r a d i o m e t r i c a n d H P L C -E C D assays for b o t h m o n o p h e n o l a n d d i p h e n o l ox idase activity, r e s e a r c h e r s w o r k i n g w i t h a n y insec t s y s t e m s h o u l d be a b l e to m o r e a c c u r a t e l y assess t h e role t he se e n z y m e s m i g h t p l a y in h o s t - p a t h o g e n as soc ia t ions . C o n t r a r y to m a n y o t h e r insec t m o d e l sy s t ems w h e r e va r ious m e a n s of p h e n o l o x i d a s e ac t iva t ion m u s t be e m p l o y e d in vitro to m e a s u r e act ivi ty by t h e spec t r o p h o t o m e t r y m e t h o d , e n z y m e act iv i ty a n d its a u g m e n t a t i o n d u e to foreign insu l t c a n b e assessed w i t h o u t ac t iva t ion u s i n g these n e w p ro toco l s . U s i n g the r a d i o e n z y m a t i c a s say for m o n o p h e n o l ox idase , Beckage et al. (1990) w e r e a b l e to m e a s u r e act ivi ty d i rec t ly in Manduca sexta h e m o l y m p h s a m p l e s w i t h o u t u s i n g a n ac t iva tor . B e c a u s e of t h e l imi t a t i ons of t he s p e c t r o p h o t o m e t r i c p h e n o l ox ida se a s say c o m m o n l y used ( G a r c i a - C a n o v a s et al., 1982; G a r c i a -C a r m o n a et al., 1982; T o w n s e n d et al., 1984; V a c h t e n h e i m et al., 1985), t h e u s e of m o r e specific a n d sensi t ive H P L C - E C D assays s h o u l d b e e n c o u r a g e d for r e s e a r c h in t h e field of a r t h r o p o d i m m u n i t y .

A l t h o u g h h e m o c y t e s s e e m to c o n t r i b u t e s ignif icant p h e n o l ox ida se ac t iv i ty


for t he m e l a n o t i c e n c a p s u l a t i o n of microf i lar iae w i t h i n t h e h e m o c o e l e n v i r o n m e n t of m o s q u i t o e s (Li et al., 1992), t he e n c a s e m e n t of ook ine tes of Plasmodium o n t he surface of the m i d g u t in A. gambiae likely does no t involve t h e d i r ec t p a r t i c i p a t i o n of c i r cu l a t i ng cells. Fol lowing the se lect ion of a r e s i s t an t s t r a i n of A. gambiae t h a t me lan izes t he ook ine te b e t w e e n t h e b a s a l m e m b r a n e l a b y r i n t h a n d ba sa l l a m i n a of t he m i d g u t (Col l ins et al., 1986), Paskewi tz et al. (1988 , 1989) r e p o r t e d t h a t r e s i s t ance m i g h t be d u e to i n c r e a s e d p h e n o l ox idase act ivi ty w i t h i n m i d g u t cells. I n c u b a t i o n of suscep t ib l e a n d r e s i s t an t m i d g u t s w i t h L - D o p a o r d o p a m i n e re su l t ed in s ignif icant differences in t h e p a t t e r n of m e l a n i n depos i t i on , w i th t he refractory s t r a in s h o w i n g i n t ense depos i t i on of m e l a n o t i c m a t e r i a l s a r o u n d e n c a p s u l a t e d p a r a s i t e s a n d w i t h i n m i d g u t cells in t h e i m m e d i a t e p r o x i m i t y of t h e ook ine te . T h e r e is n o ev idence to sugges t , however , t h a t differences in p h e n o l o x i d a s e act iv i ty b e t w e e n these s t r a in s reflect differences in t he genes r e spons ib l e for t h e express ion of th is e n z y m e .

E v e n t h o u g h a key role for p h e n o l ox idase in i m m u n e effector m e c h a n i s m s of m o s q u i t o e s a n d o t h e r insects is a p p a r e n t , t he specific s u b s t r a t e s involved , a n d the b i o c h e m i c a l p a t h w a y s by w h i c h they a r e conve r t ed to m e l a n o t i c s u b s t a n c e s , have n o t b e e n c h a r a c t e r i z e d for a n y insec t species ( C h r i s t e n s e n a n d Tracy , 1989). U n d o u b t e d l y , o t h e r c a t e c h o l a m i n e - m e t a b o l i z i n g e n z y m e s in a d d i t i o n to p h e n o l ox idases a lso a r e involved in m e l a n o t i c e n c a p s u l a t i o n reac t ions . S tud ie s of d o p a d e c a r b o x y l a s e c o n d u c t e d in t he l a b o r a t o r y of Dr . A . J . N a p p i (Loyola U n i v e r s i t y of C h i c a g o ) w e r e r e p o r t e d a t t h e A m e r i c a n Socie ty of Paras i to log is t s a n n u a l m e e t i n g in M a d i s o n , W i s c o n s i n , in A u g u s t , 1991. U s i n g a t e m p e r a t u r e - s e n s i t i v e d o p a d e c a r b o x y l a s e m u t a n t s t r a i n of D. melanogaster a n d the p a r a s i t o i d Leptopilina, this l a b o r a t o r y showed t h a t t h e ce l lu la r a n d m e l a n o t i c i m m u n e r e sponse m e c h a n i s m s w e r e severely c o m p r o mi sed in hos t s w i t h r e d u c e d levels of th is e n z y m e . L ikewise , we h a v e u n p u b l i s h e d d a t a from o u r s tud ie s w i t h A. aegypti t h a t sugges t d o p a d e c a r b o x y l ase m i g h t a lso be a factor p a r t i c i p a t i n g in t he m e l a n o t i c e n c a p s u l a t i o n p rocess .

S tud ie s by M u n k i r s et al. (1990) identif ied ty ros ine , d o p a m i n e , a n d JV-β-a l a n y l d o p a m i n e in h e m o l y m p h p l a s m a from A. aegypti u s i n g H P L C - E C D . Fol lowing i m m u n e ac t iva t ion by the i n t r a t h o r a c i c i nocu l a t i on of hea t -k i l l ed microf i la r iae , a s ingle m a j o r p e a k (cal led p e a k I ) was seen o n the c h r o m a t o -g r a m t h a t w a s no t p r e s e n t in na ive h e m o l y m p h s a m p l e s . W o u n d i n g b y the i n o c u l a t i o n of sa l ine w i t h o u t microf i lar iae resu l t ed in h e m o l y m p h s a m p l e s t h a t c o n t a i n e d on ly 5 % of t he p e a k I seen in i m m u n e - r e a c t i v e p l a s m a . T h i s u n k n o w n c a t e c h o l a m i n e a lso w a s de t ec t ed after t r e a t m e n t of h e m o l y m p h p l a s m a w i t h mi ld a lka l ine cond i t i ons , i n d i c a t i n g t h a t it is n o r m a l l y p r e s e n t as a n e l ec t rochemica l ly ine r t form. Peak I , however , d id n o t c o c h r o m a t o -


g r a p h w i t h a n y of t h e c a t e c h o l a m i n e s i m p l i c a t e d in m e l a n o t i c e n c a p s u l a t i o n r eac t ions .

N o o t h e r p u b l i s h e d a c c o u n t s a d d r e s s c h a n g e s in c a t e c h o l a m i n e s u b s t r a t e s a s soc i a t ed w i t h m e l a n o t i c e n c a p s u l a t i o n reac t ions in insec t s . F u r t h e r m o r e , t h e r e a r e n o r e p o r t s e v i d e n t t h a t e v a l u a t e c a t e c h o l a m i n e - m e t a b o l i z i n g en z y m e s , s u c h as d e c a r b o x y l a s e s , t r ans fe rases , i somera se s , o r s y n t h e t a s e s , r e g a r d i n g the i r p o t e n t i a l ro le in t h e p r o d u c t i o n of p r o t e i n - p o l y p h e n o l c o m plexes r e q u i r e d for p a r a s i t e d e s t r u c t i o n . Severa l ear l ie r rev iews s t rong ly sugges t t h a t m o r e de t a i l ed s tud ie s of o t h e r e n z y m e s a n d the i r s u b s t r a t e s in i m m u n e - r e a c t i v e insec ts a r e v i ta l for o u r u n d e r s t a n d i n g of t h e b i o c h e m i c a l p a t h w a y s involved in t h e effector m e c h a n i s m s of m o s q u i t o e s u s e d to kill p a r a s i t e s in r e s i s t an t hos t s ( N a p p i a n d C h r i s t e n s e n , 1987; C h r i s t e n s e n a n d N a p p i , 1988; C h r i s t e n s e n a n d Tracy , 1989). Clear ly , t h e r e r e m a i n s a n e e d to a d e q u a t e l y a d d r e s s this cr i t ica l a r e a of r e s e a r c h .

III. Genetic Control of Susceptibility/Refractoriness

A. Historical Perspective

I t h a s b e e n rea l ized for s o m e t i m e t h a t suscep t ib i l i ty to p a r a s i t i c infect ion va r ies b e t w e e n different m o s q u i t o species a n d even a m o n g different geog r a p h i c a l s t r a i n s of t he s a m e species , a n d ear ly s tud ie s w i t h m o s q u i t o -Plasmodium m o d e l s were t h e first to d e m o n s t r a t e t h a t suscep t ib i l i ty of m o s q u i t o e s cou ld b e i nc r ea sed b y se lec t ion (Huff, 1929; T r a g e r , 1942; M i c k s , 1949). L ikewise , R o u b a u d (1937) r e p o r t e d a g r e a t e r suscep t ib i l i ty of c e r t a i n s t r a i n s of A. aegypti to infect ion w i t h t he filarial w o r m Dirofilaria immitis a n d sugges t ed t h a t suscep t ib i l i ty w a s a n i n h e r i t e d cha rac t e r . K a r t m a n (1953) c o m p a r e d suscept ib i l i t i es of va r ious s t r a in s of A. aegypti, A. albopictus, Culex pipiens, a n d C. p. quinquefasciatus to D. immitis a n d was a b l e to o b t a i n , b y m a s s se lec t ion , s t r a i n s of A. aegypti m o r e refractory a n d m o r e suscep t ib l e to th i s p a r a s i t e .

I n a c lass ic series of s tud ie s , M a c d o n a l d (1962a ,b , 1963) clarified t h e m o d e of i n h e r i t a n c e of suscep t ib i l i ty of A. aegypti to s e m i p e r i o d i c B. malayi. F r o m a n in i t ia l p o p u l a t i o n of m o s q u i t o e s s h o w i n g a 17—31% suscep t ib i l i ty to th is p a r a s i t e , M a c d o n a l d o b t a i n e d by famil ia l se lect ion t h r o u g h 15 g e n e r a t ions a s t r a i n w i t h a m e a n suscep t ib i l i ty of 8 4 % . T h r o u g h a ser ies of c rosses a n d backcrosses b e t w e e n th is h igh ly suscep t ib l e s t r a in a n d un i fo rmly refracto ry s t r a i n s , h e d e m o n s t r a t e d t h a t suscep t ib i l i ty w a s con t ro l l ed b y a sex-l inked recess ive g e n e t h a t h e d e s i g n a t e d fm for filarial w o r m suscept ib i l i ty , B.


malayi. A l t h o u g h fm is t he ma jo r con t ro l l ing factor, M a c d o n a l d (1963) a lso d e m o n s t r a t e d t h a t th is gene d id no t con t ro l suscep t ib i l i ty en t i re ly a n d t h e r e fore c o n c l u d e d t h a t modi fy ing genes a lso m u s t p l ay a ro le . M a c d o n a l d a n d R a m a c h a n d r a n (1965) s u b s e q u e n t l y showed t h a t fm a l so con t ro l l ed s u s c e p t ibi l i ty of A. aegypti to pe r iod ic B. malayi, B. pahangi, Wuchereria bancrofti, a n d s u b p e r i o d i c W. bancrofti, b u t h a d n o inf luence on suscep t ib i l i ty to D. immitis o r D. repens. B e c a u s e fm h o m o z y g o t e s a r e suscep t ib le to all m u s c l e - i n h a b i t i n g filarial w o r m s tes ted , B a r r (1975) sugges ted t h a t se lec t ion for t he fm h o m o -zygote d i s r u p t e d a bas i c defense m e c h a n i s m of t he m o s q u i t o .

S u b s e q u e n t s tud ies by R a g h a v e n et al. (1967) , Zie lke (1973) , a n d M c G r e e v y et al. (1974) c lear ly showed t h a t suscep t ib i l i ty of A. aegypti to D. immitis a lso w a s con t ro l l ed by a sex- l inked recessive gene , b u t no t fm. M c G r e e v y et al. (1974) d e s i g n a t e d this gene f1, a n d sugges t ed t h a t genes con t ro l l i ng t he suscep t ib i l i ty of A. aegypti to filarial w o r m species d i rec t ly affect t he hos t t i ssue s u p p o r t i n g p a r a s i t e d e v e l o p m e n t r a t h e r t h a n the p a r a si te itself. S tud i e s by N a y a r et al. (1988) a lso sugges t t h a t genes in f luenc ing suscep t ib i l i ty of m o s q u i t o e s to D. immitis a r e expres sed in t he t i ssue si te of p a r a s i t e d e v e l o p m e n t . By t r a n s p l a n t i n g D. immitis-'mfected M a l p i g h i a n tu bu les from a refractory m o s q u i t o l ine to t he h e m o c o e l of un in fec ted suscep t i ble l ines , a n d by c o n d u c t i n g t he rec ip roca l t r a n s p l a n t s , t hey r e p o r t e d t h a t t he success o r fai lure of p a r a s i t e d e v e l o p m e n t was d e t e r m i n e d b y t h e gen o t y p e of the M a l p i g h i a n t u b u l e s . T h e g e n o t y p e of t h e h e m o l y m p h b a t h i n g the M a l p i g h i a n t u b u l e s h a d n o inf luence on p a r a s i t e d e v e l o p m e n t . Howeve r , K o b a y a s h i et al. (1986) used p a r a b i o t i c t w i n n i n g w i t h B. malayi su scep t ib l e a n d refractory s t r a ins of A. aegypti to d e m o n s t r a t e t h a t d e v e l o p m e n t of l a rvae i n h a b i t i n g t he tho rac ic m u s c u l a t u r e was i nh ib i t ed in suscep t ib l e m o s q u i t o e s w h e n they were t w i n n e d w i t h a refractory m o s q u i t o . T h e y sugges t ed t h a t h e m o l y m p h factors m i g h t be r e spons ib l e for t he refractory c o n d i t i o n of A. aegypti for th is filarial w o r m species .

A l t h o u g h Huf f (1929) , T r a g e r (1942) , a n d W a r d (1963) r e p o r t e d a gene t i c bas i s for v a r i a t i o n in suscept ib i l i ty of severa l different m o s q u i t o species for Plasmodium infect ions, t he first c lear gene t i c ev idence was p r o v i d e d b y K i l a m a a n d C r a i g (1969) . T h e y d e m o n s t r a t e d t h a t P. gallinaceum s u s c e p t ibi l i ty in A. aegypti is d e t e r m i n e d p r i m a r i l y by a s ingle a u t o s o m a l d o m i n a n t gene , pis. T h e i r d a t a , however , a lso sugges t t h a t o t h e r gene t i c factors a r e involved in Plasmodium suscept ib i l i ty in this vector , b e c a u s e u p to six deve lop ing oocytes w e r e f requent ly i so la ted from the m i d g u t s of i n d i v i d u a l m o s q u i toes t h a t they classified as comple t e ly refractory. T h e y a r b i t r a r i l y se lec ted a c o u n t of t en deve lop ing oocytes as t he p o i n t of d e m a r c a t i o n b e t w e e n refracto ry a n d suscep t ib l e i nd iv idua l s . L ikewise , W a r d (1963) w a s u n a b l e to deve l o p a 1 0 0 % refractory s t r a in of A. aegypti even after 26 g e n e r a t i o n s of se lec t ion , sugges t i ng a m u l t i g e n i c m o d e of i n h e r i t a n c e . M o r e recent ly, a s t r a i n of A.


gambiae, a p r i n c i p a l vec to r of h u m a n m a l a r i a , h a s b e e n se lec ted for r e s i s t ance to severa l species of Plasmodium b y Col l ins et al. (1986) . G e n e t i c ana lys i s of Plasmodium r e s i s t ance in A. gambiae h a s sugges t ed t h a t two u n l i n k e d genes con t ro l t h e express ion of suscep t ib l e a n d r e s i s t an t p h e n o t y p e s (Vern ick et al., 1989).

E v e n t h o u g h th is gene t i c bas i s for suscep t ib i l i ty a n d ref rac tor iness of cert a i n m o s q u i t o e s to b o t h filarial w o r m a n d Plasmodium infect ions h a s b e e n k n o w n for severa l d e c a d e s , very l i t t le i n fo rma t ion h a s b e e n g e n e r a t e d conc e r n i n g the genes o r g e n e p r o d u c t s con t ro l l i ng these c o n d i t i o n s in a n y m o s q u i t o spec ies .

B. Genes and Gene Products

R e c e n t b io t echno log ica l a d v a n c e s h a v e s p u r r e d a r a p i d l y d e v e l o p i n g re s e a r c h effort a i m e d a t a d v a n c i n g o u r knowledge in t h e a r e a of v e c t o r -p a r a s i t e r e l a t i o n s h i p s a n d vec to r gene t i cs , as ev idenced b y a m e e t i n g in

J a n u a r y , 1991 , in T u c s o n , A r i z o n a , o n t he " P r o s p e c t s for M a l a r i a C o n t r o l b y G e n e t i c M a n i p u l a t i o n of i ts V e c t o r s " o r g a n i z e d b y t h e W H O Spec ia l P r o g r a m for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Diseases a n d t h e M a c A r t h u r F o u n d a t i o n [Wor ld H e a l t h O r g a n i z a t i o n ( W H O ) , 1991] . H o w e v e r , t h e ge ne t i c i n f o r m a t i o n p re sen t ly ava i l ab le as a bas i s for this r e s e a r c h effort is l imi t ed for t h e ma jo r i t y of m o s q u i t o vec to r s .

Aropheles gambiae h a s rece ived c o n s i d e r a b l e a t t e n t i o n b e c a u s e of i ts i m p o r t a n c e in t h e t r a n s m i s s i o n of h u m a n m a l a r i a a n d t h e se lec t ion of a r e s i s t an t s t r a i n t h a t successfully des t roys ook ine tes on t he m i d g u t surface (Col l ins et al., 1986). I n a d d i t i o n to t h e d a t a sugges t ing t h a t t w o u n l i n k e d genes con t ro l suscep t ib i l i ty in this species (Vernick et al., 1989), Vern ick a n d Co l l in s (1989) p r o v i d e ev idence t h a t o n e of these loci exh ib i t s a t igh t l inkage a s soc ia t ion w i t h a p o l y m o r p h i c a u t o s o m a l e s t e ra se locus . T h e effector m e c h a n i s m s re s p o n s i b l e for t h e ki l l ing of ook ine tes involve t he m e l a n o t i c e n c a s e m e n t of t h e p a r a s i t e ; consequen t ly , s tud ie s b y Paskewi tz et al. (1989) h a v e sugges t ed t h a t differences in p h e n o l ox idase ac t iv i ty m i g h t a c c o u n t for differences in s u s c e p t ibi l i ty b e t w e e n s t r a in s of A. gambiae. Howeve r , R o m a n s et al. (1991) cond u c t e d backcrosses b e t w e e n two i n b r e d l ines of A. gambiae to d e t e r m i n e l inka g e a s soc ia t ions b e t w e e n a p r e s u m e d d i p h e n o l ox ida se g e n e ( D o x - A 2 de r ived f rom Drosophila) a n d the e s t e rase locus l inked w i t h re f rac tor iness . Resu l t s i n d i c t e d t h a t t h e t w o genes s e g r e g a t e d i n d e p e n d e n t l y . F r o m d a t a p r e s en t l y ava i l ab le , it is difficult to assess t h e roles t h a t these e n z y m e s ac tua l ly p l a y in in f luenc ing Plasmodium suscep t ib i l i ty in th is vector . B e c a u s e of t h e lack of ident i f ied gene t i c m a r k e r s for A. gambiae, efforts to def ine t h e p rec i se gene t i c bas i s for r e s i s t ance a r e l imi ted .

T h e m o s t de t a i l ed i n f o r m a t i o n c o n c e r n i n g the phys i ca l l oca t ion of genes


con t ro l l ing suscept ib i l i ty is k n o w n for A. aegypti, b e c a u s e th is is t h e on ly species for w h i c h ex tens ive gene t i c l inkage d a t a a r e ava i l ab le . A gene t i c l inkage m a p , b a s e d on a b o u t 70 m o r p h o l o g i c a l m u t a n t a n d i sozyme m a r k e r s (cons i s t ing of t h r e e l inkage g r o u p s ) , h a s b e e n deve loped for th is species ( M u n s t e r m a n n a n d C r a i g , 1979; M u n s t e r m a n n , 1990). C o n s e q u e n t l y , de te r m i n i n g t h e g e n o m e pos i t ion of genes of in te res t c a n b e a c c o m p l i s h e d t h r o u g h l inkage ana lys i s w i th these ident if ied m a r k e r loci. K i l a m a a n d C r a i g (1969) a n d M a c d o n a l d (1962b) used this t e c h n i q u e to d e t e r m i n e t h a t pis is l oca t ed on l inkage g r o u p I I a n d fm is l oca ted on l inkage g r o u p I . T h e s e v e c t o r -p a r a s i t e m o d e l sys t ems (A. aegypti-P. gallinaceum a n d A. aegypti-Brugia spp . ) therefore a r e p a r t i c u l a r l y i m p o r t a n t b e c a u s e i m m e d i a t e reference p o i n t s for genes of in te res t a r e ava i l ab le for r e sea r ch efforts d e s i g n e d to i so la te a n d cha rac t e r i z e genes con t ro l l ing suscept ibi l i ty . T rad i t i ona l ly , however , a l a rge n u m b e r of s eg rega t ing p o p u l a t i o n s a r e r e q u i r e d to d e v e l o p l inkage assoc ia t ions for genes of in te res t , b e c a u s e on ly a l imi ted n u m b e r of loci s eg rega t e in each cross . B e c a u s e res t r i c t ion f r a g m e n t l eng th p o l y m o r p h i s m ( R F L P ) m a r k e r s r e p r e s e n t a n e w class of m o l e c u l a r m a r k e r s t h a t p e r m i t t h e d e v e l o p m e n t of de t a i l ed gene t i c m a p s from a l imi ted n u m b e r of crosses ( L a n d r y a n d M i c h e l m o r e , 1987), we have t a k e n th is a p p r o a c h in o u r in i t ia l efforts to i sola te t he genes inf luenc ing suscep t ib i l i ty of A. aegypti to P. gallinaceum a n d B. malayi.

We have c o n s t r u c t e d a R F L P gene t i c l inkage m a p cons i s t ing of 50 c lones t h a t identify 53 loci cover ing 134 m a p un i t s across t h e A. aegypti g e n o m e (Severson et al., 1993). T h e R F L P m a r k e r s i n c l u d e 42 r a n d o m c D N A c lones , 3 r a n d o m g e n o m i c D N A clones , a n d 5 c D N A clones of k n o w n genes . O u r s eg rega t i ng p o p u l a t i o n s cons is ted of F 2 p r o g e n y o b t a i n e d from crosses be tween the L ive rpoo l a n d R E D or B Z s t r a ins of A. aegypti. T h e R E D s t r a i n ca r r i es t h e red-eye (re) locus on l inkage g r o u p I , t h e s p o t - a b d o m e n (s) locus on l inkage g r o u p I I , a n d the b l ack - t a r sus (bit) locus o n l inkage g r o u p I I I . T h e B Z s t r a in ca r r ies t he re locus a n d the b ronze -cu t i c l e (bz) locus o n l inkage g r o u p I . D e t e r m i n a t i o n of l inkage assoc ia t ions b e t w e e n o u r R F L P m a r k e r s a n d severa l m u t a n t m a r k e r loci a l lowed us to i n t e g r a t e t h e m in to t he t h r e e p rev ious ly ident if ied l inkage g r o u p s ( M u n s t e r m a n n , 1990).

A l t h o u g h the R F L P m a r k e r s a r e r a n d o m l y d i s t r i b u t e d t h r o u g h o u t t he A. aegypti g e n o m e , a c o n s i d e r a b l e p r o p o r t i o n ( 3 0 % ) of t h e m fitted a 1:1 seg rega t ion r a t i o in a t leas t o n e of o u r m a p p i n g p o p u l a t i o n s . T h i s reflects p r o g e n y of a m a t i n g b e t w e e n a h e t e r o z y g o u s i n d i v i d u a l a n d a h o m o z y g o u s i n d i v i d u a l for each of these loci. The re fo r e , de sp i t e m a n y g e n e r a t i o n s of m a i n t e n a n c e as i n d e p e n d e n t colonies , o u r s eg rega t ion d a t a i nd i ca t e t h a t l a b o r a t o r y s t r a i n s of A. aegypti r e m a i n h igh ly h e t e r o z y g o u s . T h i s h a s i m p o r t a n t i m p l i c a t i o n s for fu ture m a p p i n g efforts, s ince crosses involv ing h e t e r o z y g o u s i n d i v i d u a l s c a n l imi t t h e n u m b e r of d i a g n o s t i c loci ident i f iable w i th a g iven c ross .


T h e ava i lab i l i ty of a s a t u r a t e d R F L P m a p p rov ides a v a l u a b l e m e c h a n i s m to a d d r e s s a spec t s of vec to r b io logy a t t he i r m o s t f u n d a m e n t a l level. T h i s l i nkage m a p cou ld facil i tate t he i so la t ion of genes of in t e res t w h o s e g e n e p r o d u c t is u n k n o w n (i.e. , filarial w o r m o r m a l a r i a suscep t ib i l i ty ) by p rov id ing p o i n t s for c h r o m o s o m e w a l k i n g . I n a d d i t i o n , m a n y i m p o r t a n t p h e n o t y p i c c h a r a c t e r s , s u c h as filarial w o r m a n d Plasmodium suscept ib i l i ty , a r e a conse q u e n c e of t h e j o i n t ac t ion of two or m o r e i n d i v i d u a l genes . T h e s e c h a r a c t e r s a r e gene ra l ly refer red to as m u l t i g e n i c o r q u a n t i t a t i v e t r a i t s , a n d exp res s ion of a p a r t i c u l a r p h e n o t y p e d e p e n d s u p o n a b a l a n c e b e t w e e n i n d i v i d u a l g e n e effects a n d the i r in te rac t ive effects w i t h each o ther . A s a t u r a t e d R F L P gene t i c l i nkage m a p p rov ides a useful m e c h a n i s m for t h e r e so lu t ion of c o m p l e x p h e n o t y p i c t r a i t s i n to the i r i n d i v i d u a l gene t i c c o m p o n e n t s ( N i e n h u i s et al., 1987; O s b o r n et al., 1987; P a t e r s o n et al., 1988; Tanks l ey a n d H e w i t t , 1988; L a n d e r a n d Bo t s t e in , 1989), by ident i fy ing all i n t e rva l s b e t w e e n R F L P m a r k ers ( referred to as q u a n t i t a t i v e t r a i t loci, Q T L s ) t h a t c o n t a i n a gene ( s ) affecting a c h a r a c t e r t h r o u g h o u t t h e g e n o m e . E a c h Q T L c a n t h e n b e e x a m ined as a d i sc re t e gene t i c en t i ty a n d its i n d i v i d u a l a n d in te rac t ive p r o p e r t i e s m e a s u r e d . T h e s e R F L P m a r k e r s a lso c a n b e u s e d to e x a m i n e c h r o m o s o m a l c o n t e n t a n d l inea r g e n e o r d e r a m o n g r e l a t ed species ( B o n i e r b a l e et al., 1988; T a n k s l e y et al., 1988). T h i s will l ikely p rove v a l u a b l e for o t h e r m o s q u i t o species b e c a u s e a n ear l i e r e x a m i n a t i o n of l inkage r e l a t i o n s h i p s of p h e n o t y p i c c h a r a c t e r s a m o n g h i g h e r d i p t e r a n species sugges t s t h a t t h e m a j o r l i nkage g r o u p s a r e la rge ly conse rved (Fos te r et al., 1981).

S ince t h e ba s i c c h r o m o s o m e n u m b e r is conse rved a m o n g m o s q u i t o species (n = 3), o n e c a n p r e d i c t t h a t c h r o m o s o m a l c o n t e n t a n d l i nea r g e n e o r d e r a r e h igh ly conse rved a m o n g m o s q u i t o spec ies . T h i s s h o u l d a l low the u t i l i za t ion of t h e w e a l t h of i n f o r m a t i o n c o n c e r n i n g t h e gene t ics of A. aegypti to r a p i d l y d e v e l o p a de t a i l ed R F L P gene t i c l i nkage m a p for a gene t i ca l ly u n c h a r a c -te r ized b u t r e l a t ed m o s q u i t o species . T h i s s cena r io a p p e a r s q u i t e p r o b a b l e as we a r e successfully u s ing o u r R F L P m a r k e r s to e x a m i n e l inkage w i t h i n segr e g a t i n g p o p u l a t i o n s of Aedes albopictus. P r e l i m i n a r y resu l t s i n d i c a t e t h a t t h e l i nea r o r d e r for t h r e e of o u r R F L P m a r k e r s o n l inkage g r o u p I is conse rved b e t w e e n A. aegypti a n d A. albopictus (Y. M o r i , D . W. Severson , a n d Β . M . C h r i s t e n s e n , u n p u b l i s h e d d a t a ) . We have a lso successfully h y b r i d i z e d m a n y of o u r R F L P m a r k e r s to g e n o m e b lo ts of o t h e r m o s q u i t o species i n c l u d i n g : Aedes togoi, Anopheles gambiae, Armigeres subalbatus, a n d severa l subspec i e s of t h e Culex pipiens c o m p l e x (C.p. molestus, C.p. pallans, C.p. pipiens, C.p. quin-quefasciatus).

N o d i r e c t ev idence is p r e s e n t l y ava i l ab le , however , t h a t identif ies a g e n e p r o d u c t r e s p o n s i b l e for suscep t ib i l i ty o r ref rac tor iness of a n y m o s q u i t o spe cies for a p a r t i c u l a r p a r a s i t e , b u t r ecen t ly we have ident i f ied severa l i n d u c e d p o l y p e p t i d e s t h a t a r e s t rong ly a s soc i a t ed w i t h A. aegypti s t r a i n s t h a t a r e


ref rac tory for t h e d e v e l o p m e n t of B. malayi ( W a t t a m a n d C h r i s t e n s e n , 1992). P o l y p e p t i d e syn thes i s in tho rac ic t i ssue was assessed , u s i n g in vivo r a d i o l a b e l -ing t e c h n i q u e s (Bee rn t s en a n d C h r i s t e n s e n , 1990), in suscep t ib l e a n d refracto ry s t r a in s of A. aegypti a n d a refractory s t r a in newly i so la ted f rom a s u s c e p t ible s t r a i n . Six p o l y p e p t i d e differences w e r e r ecogn ized b y S D S - P A G E a n d t w o - d i m e n s i o n a l e l ec t rophores i s . T h e s e p o l y p e p t i d e s were seen on ly in t h e refractory s t r a ins for 48 h r following a b lood m e a l . A seven th p o l y p e p t i d e w a s p r e s e n t in those refractory m o s q u i t o e s t h a t h a d inges ted suc rose , b u t inc r eased in in tens i ty following b loodfeed ing . T h e p r e s e n c e of microf i la r iae in t he b l o o d m e a l w a s n o t necessa ry to s t i m u l a t e t he syn thes i s of these m o l e cules . A l t h o u g h these differences in b l o o d m e a l - i n d u c e d p o l y p e p t i d e p r o d u c t ion w e r e s t rong ly co r r e l a t ed w i t h v a r i a t i o n in suscept ib i l i ty , d i r ec t ev idence is n o t yet ava i l ab le r e g a r d i n g the role these p o l y p e p t i d e s m i g h t p l a y in m e d i a t i n g t he gene t ica l ly d e t e r m i n e d v a r i a t i o n in suscep t ib i l i ty ( W a t t a m a n d C h r i s t e n s e n , 1992).

T h e r e a r e n u m e r o u s phys io log ica l even ts t h a t t ake p lace w i t h i n vec to r m o s q u i t o e s t h a t cou ld inf luence t he su i tab i l i ty of m o s q u i t o e s as hos t s for p a r a s i t e s . I t is n o t k n o w n , for i n s t a n c e , w h a t ro le sa l ivary g l a n d c o m p o n e n t s m i g h t p l a y in in f luenc ing suscep t ib i l i ty a n d / o r re f rac tor iness . Ea r l i e r s tud ie s by R o s e n b e r g (1985) c lear ly showed t h a t sporozo i tes m a y n o t b e c a p a b l e of r ecogn iz ing the a p p r o p r i a t e m e m b r a n e r ecep to r s o n sa l iva ry g l a n d s of refractory Anopheles in ce r t a in mosquito-Plasmodium m o d e l s y s t e m s . B e c a u s e of t h e i m p o r t a n c e of sa l ivary g l a n d s in t he t r a n s m i s s i o n of severa l p a t h o g e n s by m o s q u i t o e s , r e s e a r c h e r s in J a m e s ' l a b o r a t o r y a t t h e U n i v e r s i t y of Ca l i fo rn ia , I r v ine , have u n d e r t a k e n a m o l e c u l a r ana lys i s of t h e sa l ivary g l a n d s of A. aegypti ( J a m e s et al., 1989, 1991). I t is poss ib le t h a t t he i so la t ion a n d c h a r a c t e r i za t ion of genes expressed specifically in t h e sa l ivary g l a n d s cou ld p r o v i d e n e w ins igh t r e g a r d i n g phys io log ica l m e c h a n i s m s inf luenc ing suscep t ib i l i ty a n d / o r ref rac tor iness . I n a s imi la r m a n n e r , inves t iga to r s in Ra ikhe l ' s l a b o r a tory a t M i c h i g a n S t a t e Univers i ty , E a s t L a n s i n g , have b e e n assess ing the m o l e c u l a r b io logy of vi te l logenesis w i t h t he u l t i m a t e goa l of finding n e w a n d innova t ive m e a n s of d i s r u p t i n g r e p r o d u c t i o n in vec to r m o s q u i t o e s (Ra ikhe l , 1992; R a i k h e l a n d D h a d i a l l a , 1992). I t w o u l d be ex t r eme ly v a l u a b l e if o t h e r r e s e a r c h g r o u p s w o u l d m a k e s imi l a r c o m m i t m e n t s to a m o l e c u l a r ana lys i s of specific t i ssue sites of p a r a s i t e d e v e l o p m e n t w i t h i n select m o s q u i t o spec ies , s u c h as m i d g u t , t ho rac i c m u s c u l a t u r e , a n d M a l p i g h i a n t u b u l e s .

C. Molecular Approaches in Vector Control: Future Directions

T h e gene t i c p las t ic i ty of m o s q u i t o vec tors h a s r e su l t ed in t h e e m e r g e n c e of mu l t i p l e -pes t i c ide - r e s i s t an t vec to r p o p u l a t i o n s , t h e r e b y l imi t ing t h e success of n u m e r o u s vec to r con t ro l p r o g r a m s in a r e a s e n d e m i c for m a l a r i a a n d fil-

11. Mosquito Susceptibility to Plasmodium and Filarioids 261

ar ias i s . A n a l t e r n a t e s t r a t egy for vec to r con t ro l w o u l d be to ut i l ize e l e m e n t s of t h e o b s e r v e d gene t i c p las t ic i ty w i t h i n vec to r p o p u l a t i o n s to d i s r u p t p a r a si te t r a n s m i s s i o n , b y m a n i p u l a t i n g those a spec t s of vec to r c o m p e t e n c e u n d e r gene t i c con t ro l . T h e s teps o u t l i n e d ( W H O , 1991) for t he e n g i n e e r i n g a n d re lease of ref ractory m o s q u i t o e s involve (1) o b t a i n i n g a t h o r o u g h u n d e r s t a n d i n g of t h e m o l e c u l a r bas i s of vec to r c o m p e t e n c e , (2) t he d e v e l o p m e n t of n e c e s s a r y m o l e c u l a r a n d gene t i c a p p r o a c h e s r e q u i r e d to e n g i n e e r ref rac tory m o s q u i t o e s , a n d (3) a m o r e c o m p l e t e u n d e r s t a n d i n g of t h e p o p u l a t i o n dyn a m i c s neces sa ry to effectively a p p l y these t echnolog ies in field s i t u a t i o n s . I t is a p p a r e n t f rom t h e p r e v i o u s d i scuss ion in th is rev iew t h a t s tud ie s d e s i g n e d to e l u c i d a t e t h e m o l e c u l a r bas i s of vec to r c o m p e t e n c e a r e in the i r infancy. L ikewise , p rog re s s in t he d e v e l o p m e n t of t r a n s g e n i c t echno logy in m o s q u i toes h a s b e e n l imi ted .

S t a b l e t r a n s f o r m a t i o n of m o s q u i t o e s h a s b e e n d e m o n s t r a t e d in A. gambiae (Mi l l e r et al., 1987), Aedes triseriatus ( M c G r a n e et al., 1988), a n d A. aegypti ( M o r r i s et al., 1989), a l t h o u g h i n t e g r a t i o n in each of these species o c c u r r e d b y r e c o m b i n a t i o n t h a t w a s i n d e p e n d e n t of Ρ e l e m e n t s . For a t h o r o u g h assess m e n t of s o m e of t he p r o b l e m s a s soc ia t ed w i t h t he p r o d u c t i o n of t r a n s g e n i c m o s q u i t o e s , a n d t h e p o t e n t i a l for u s i n g eng i n ee r ed m o s q u i t o e s in vec to r con t ro l s t r a t eg ie s , severa l r ecen t rev iews a r e r e c o m m e n d e d ( C r a m p t o n et al., 1990; Egg le s ton , 1991; W H O , 1991). I t is a p p a r e n t , however , t h a t t e c h n i q u e s for efficient t r a n s f o r m a t i o n sy s t ems for m o s q u i t o e s p r e s en t l y a r e n o t avai l a b l e .

A l t h o u g h t h e c o n c e p t of r e l eas ing e n g i n e e r e d m o s q u i t o e s w i t h r e d u c e d vec tor ia l c a p a c i t y a n d / o r i nc r ea sed r e s i s t ance to pes t ic ides m i g h t b e a n u l t i m a t e goa l t h a t is n e v e r rea l ized , t h e d e v e l o p m e n t of this t e chno logy w o u l d h a v e a n e n o r m o u s i m p a c t o n o u r m o s t f u n d a m e n t a l u n d e r s t a n d i n g of g e n e r e g u l a t i o n a n d express ion in vec to r s . T h e defini t ive d e t e r m i n a t i o n of g e n e func t ion b e c o m e s poss ib le if t r a n s f o r m a t i o n sys t ems for m o s q u i t o e s b e c o m e as efficient as t hose deve loped for Drosophila.

Acknowledgments

This work was supported in part by National Institutes of Health Grants A l 19769, A l 28781, and Al 33127. We thank Dr. Jianyong Li for his critical review of this manuscript.

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Chapter 12

ΒCellular Defense Responses of Insects: Unresolved Problems Norman A. Ratcliffe Biomedical and Physiological Research Group School of Biological Sciences University College of Swansea Swansea, Wales, United Kingdom

I. Introduction

II. Main Events in Insect Immune Reactivity A. Immediate and Inductive Stage B. Mainly Cellular and Synthetic Stage C. Final Humoral/Cellular Stage

III. Specific Problems in Insect Immunity A. Classification, Origins, and

Manipulation of Insect Blood Cells

B. Recognition of Non-Self Invaders C. Cell-Cell Communication

in Immunity D. Interrelationship of Cellular

and Humoral Immunity E. Future Areas for Study


I. Introduction

I n r e c e n t y e a r s , t h e r e have b e e n a n u m b e r of i m p o r t a n t d e v e l o p m e n t s in o u r u n d e r s t a n d i n g of insec t i m m u n e defense r eac t ions . For e x a m p l e , as far as h u m o r a l factors a r e c o n c e r n e d , i n d u c i b l e i m m u n e p r o t e i n s h a v e b e e n seq u e n c e d a n d c h a r a c t e r i z e d a n d s o m e of t h e genes for the i r p r o d u c t i o n c loned (e.g. , B o m a n a n d H u l t m a r k , 1987; H o f f m a n n a n d H o f f m a n n , 1990; Fay e a n d H u l t m a r k , V o l u m e 2, C h a p t e r 2), t h e role of t h e p r o p h e n o l o x i d a s e s y s t e m (e.g. , Ratcliffe et al., 1984; S u g u m a r a n a n d K a n o s t , V o l u m e 1, C h a p t e r 14) a n d t h e h e m o l y m p h lect ins ( P e n d l a n d et al., 1988) in t h e r ecogn i t i on of foreign a n t i g e n s h a v e b e e n conf i rmed , a n d t h e h e m o l y m p h lec t ins too have b e e n i so la ted , s e q u e n c e d , a n d c D N A clones p r o d u c e d for o n t o g e n e t i c s tud ie s (e .g. , T a k a h a s h i et al., 1985, 1986). A s r e g a r d s ce l lu la r i m m u n i t y , i m p o r t a n t a d v a n c e s h a v e a lso b e e n m a d e , s u c h as t h e de t a i l ed c h a r a c t e r i z a t i o n of cel lul a r reac t iv i ty ( rev iewed in Ratcliffe a n d Rowley, 1979; G o t z a n d B o m a n ,

Parasites and Pathogens of Insects Copyright © 1993 by Academic Press, Inc. Volume I: Parasites 2 6 7 All rights of reproduction in any form reserved.

2 6 8 Norman A. Ratcliffe

1985), i n c l u d i n g a l logenic r ecogn i t ion of grafts ( G e o r g e et aL, 1987) a n d the d e m o n s t r a t i o n of cell—cell coope ra t i ve events ( S c h m i t a n d Ratcliffe, 1977) u t i l iz ing , m o r e recent ly, pur i f ied p o p u l a t i o n s of b lood cells ( A n g g r a e n i a n d Ratcliffe, 1991).

D e s p i t e this p rog res s in o u r k n o w l e d g e of insec t i m m u n i t y , m a n y p r o b l e m s r e m a i n u n a n s w e r e d . T h e p u r p o s e of this br ief overview is to identify a t leas t s o m e of these un reso lved q u e s t i o n s a n d to i n d i c a t e poss ib le d i r ec t i ons for fu ture e n d e a v o r s . A t t e n t i o n will be focused p a r t i c u l a r l y o n the insec t ce l lu la r defenses b u t , s ince ev idence is now a c c u m u l a t i n g for a c o m p l e x i n t e r p l a y b e t w e e n the ce l lu la r a n d h u m o r a l c o m p o n e n t s in t he p r o d u c t i o n of t h e overall i m m u n e react iv i ty of insec ts , such d iv is ions a r e p u r e l y a rb i t r a ry .

II. Main Events in Insect Immune Reactivity

F i g u r e 1 (modif ied from Ratcliffe a n d Rowley, 1987) s u m m a r i z e s the m a i n p h a s e s a n d in t e rac t ions o c c u r r i n g d u r i n g the insec t r e s p o n s e to m i c r o b i a l a n d m a c r o b i a l invas ion . T h e q u e s t i o n m a r k s in this figure i n d i c a t e s o m e , b u t no t all , of t he un re so lved p r o b l e m s in o u r u n d e r s t a n d i n g of t h e p rocesses involved . For conven ience , the insect i m m u n e defenses c a n be d i v i d e d i n t o t h r ee i n t e r a c t i n g p h a s e s (see a lso C h a d w i c k a n d A s t o n , 1978):

(a) A n i m m e d i a t e a n d induc t ive s t age (b) A m a i n l y ce l lu la r a n d syn the t i c s t age (c) A final h u m o r a l / c e l l u l a r s t age

A. Immediate and Inductive Stage

I n s e c t i m m u n i t y is i n d u c e d by w o u n d i n g or by p a r a s i t i c invas ions t h r o u g h the gu t , i n t e g u m e n t , o r t r acheae . H e m o l y m p h e x t r u d e d t h r o u g h t h e in ju ry u p o n e x p o s u r e to t he a i r r a p i d l y coagu la t e s to seal t h e w o u n d . T h e c o a g u l a t ion p rocess involves t h e d e g r a n u l a t i o n of b lood cells ( hemocy te s ) t e r m e d g r a n u l a r cells, cys tocytes , a n d / o r p l a s m a t o c y t e s , w h i c h i n t e r ac t w i t h p l a s m a c o m p o n e n t s to form a n inso lub le gel ( B o h n , 1986). T h i s in i t i a t ion of i m m u n e reac t iv i ty poses a n u m b e r of un reso lved p r o b l e m s in insec t i m m u n i t y , a n d it is very no t i ceab l e t h a t in Fig . 1 m o s t of t he q u e s t i o n m a r k s o c c u r d u r i n g this first p h a s e .

T h e si te of or ig in a n d m o d e of d e v e l o p m e n t of t he b lood cells involved in i m m u n i t y in m o s t insect species a r e t h u s still u n k n o w n . A s imi l a r p r o b l e m re la tes to t h e ident i f ica t ion of t he i m m u n e reac t ive cells w i t h a m u l t i t u d e of n a m e s p u b l i s h e d in t h e l i t e r a tu r e for on ly six to e ight ba s i c h e m o c y t e t ypes .

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I n a d d i t i o n , a v i ta l i m m u n e p rocess o c c u r r i n g ear ly o n in t he insec t ' s re s p o n s e to e x p o s u r e to non-se l f a n t i g e n s is t he r ecogn i t ion of fore ignness a b o u t w h i c h m u c h h a s ye t to b e l e a r n e d . For e x a m p l e , is th i s p roces s cell a n d / o r p l a s m a m e d i a t e d ? W h i c h cell types a n d molecu les a r e involved? H o w is it con t ro l l ed a n d is it b r o a d s p e c t r u m o r h igh ly specific? H o w does t h e r ecogn i t ion p rocess l ead to l a t e r even ts in t he i m m u n e r e sponse? T h e in te rac t ive n a t u r e of t he ce l lu la r a n d h u m o r a l c o m p o n e n t s s h o u l d a lso b e c o n s i d e r e d a t this s t age . T h e s e a r e j u s t a few q u e s t i o n s t h a t have b e e n p o s e d . O t h e r u n r e solved p r o b l e m s in this ear ly p h a s e re la te to o u r p o o r u n d e r s t a n d i n g of t h e c o a g u l a t i o n p rocess itself in insec ts a n d w h e t h e r , as in c r u s t a c e a n s , t he p r o p h e n o l o x i d a s e ( p P O ) c o m p l e x is involved .

T h e e n d resu l t of t he first p h a s e t h e n is t he sea l ing of t h e w o u n d si te o r p o r t a l of invas ion , r ecogn i t ion of t h e invader , a n d ac t iva t ion of t h e i m m u n e sys t em. I n m a n y cases , invas ion is h a l t e d a t th is s t age b y a n a r r a y of a n t i p a r a s i t i c s u b s t a n c e s such as n a t u r a l l y o c c u r r i n g a n t i m i c r o b i a l factors in t h e h e m o l y m p h (e.g. , Crof t et al., 1982; J o n e s et al., 1989), l y sozyme a n d lysosom a l e n z y m e s re leased from the d e g r a n u l a t i n g b lood cells ( Z a c h a r y a n d H o f f m a n n , 1984), a n d a n t i m i c r o b i a l s u b s t a n c e s g e n e r a t e d by ac t iva t ion of t he p P O c o m p l e x (Rowley et al., 1990). Pa ra s i t e s a n d p a t h o g e n s , however , have evolved m a n y s t ra teg ies for avo id ing the insec t i m m u n e defenses so t h a t p rog re s s ion to t he second p h a s e , w h i c h is c h a r a c t e r i z e d b y ce l l -med ia t ed reac t ions a n d the syn thes i s of t he i m m u n e p r o t e i n s , m a y b e cu r t a i l ed . O n l y recen t ly have we l e a r n e d a n y t h i n g a b o u t t h e a v o i d a n c e s t ra teg ies of i n v a d i n g m i c r o b i a l a n d m a c r o b i a l p a r a s i t e s (e.g. , Brehe l in , 1990).

B. Mainly Cellular and Synthetic Stage

I f t h e i n v a d i n g o r g a n i s m surv ives t h e in i t ia l o n s l a u g h t b y t h e insec t i m m u n e defenses b u t is r ecogn ized as foreign, t h e n it m a y r a p i d l y b e p h a g o c y t o s e d a n d / o r e n c a p s u l a t e d by the c i r cu l a t i ng b lood cells. T h e t ype of r eac t ion el ici ted d e p e n d s u p o n the n u m b e r s a n d size as well as t h e p a t h o g e n i c i t y of t h e i n v a d i n g o r g a n i s m . I n Galleria mellonella, p h a g o c y t o s i s a l o n e c a n d e a l w i t h 102— 1 0 3 b ac t e r i a p e r mic ro l i t e r h e m o l y m p h whi le l a rge r n u m b e r s a r e r e m o v e d by n o d u l e fo rma t ion (Wal te rs a n d Ratcliffe, 1983).

T h e r e is c o n s i d e r a b l e d i s a g r e e m e n t as to t he cell t ype ( s ) m a i n l y r e spons i b le for phagocy tos i s in insec t s . Ratcliffe a n d Rowley (1979) r e p o r t e d t h a t t h e p l a s m a t o c y t e s a r e t he m o s t i m p o r t a n t p h a g o c y t i c cells, w h e r e a s Brehe l in a n d Z a c h a r y (1986) bel ieve t h a t t h e g r a n u l a r h e m o c y t e s m a y b e t h e m a i n effector cells. T h e s e differences p r o b a b l y resu l t f rom p r o b l e m s in classifying insec t b lood cells a n d f rom the la rge v a r i a t i o n s o c c u r r i n g in h e m o c y t e types even b e t w e e n closely r e l a t ed species . H o w the r e l evan t p h a g o c y t i c b lood cell r ec -

12. Cellular Defense Responses of Insects 271

ognizes t h e foreign i n v a d e r is a l so a p r o b l e m m e n t i o n e d previous ly . I n insec ts , a n d in m a n y o t h e r a r t h r o p o d s too , a t leas t t w o types of mo lecu le s s e e m to b e r e s p o n s i b l e for non-se l f r ecogn i t i on b y t h e b lood cells, name ly , t h e h e m o l y m p h a g g l u t i n i n s a n d factors g e n e r a t e d d u r i n g ac t iva t ion of t h e p P O c a s c a d e . S ince b o t h types of mo lecu le s m a y b e p r o d u c e d o r s to red b y t h e s a m e h e m o c y t e t ype (Ratcliffe, 1991), t h e n t h e in t e rac t ive n a t u r e of t hese factors s e e m s to b e h igh ly p r o b a b l e a n d ev idence for th is poss ib i l i ty is p r e s e n t e d in Sec t ion 11 L B . De ta i l s of m o l e c u l a r even t s o c c u r r i n g a t t h e h e m o cyte m e m b r a n e a n d s u b s e q u e n t l y in t h e c y t o p l a s m of t he se cells fol lowing b i n d i n g of t h e i n v a d e r o r non-se l f mo lecu le s a r e a lso v i r tua l ly u n k n o w n .

W h a t is b e c o m i n g inc reas ing ly ev iden t , w i t h t he d e v e l o p m e n t of cell s ep a r a t i o n t e c h n i q u e s a n d t h e pur i f i ca t ion of reac t ive mo lecu le s a s soc i a t ed w i t h t h e p P O sys t em, is t h a t ce l lu la r i m m u n i t y in a r t h r o p o d s involves cell- to-cell c o o p e r a t i o n t h a t is carefully o r c h e s t r a t e d b y p r o d u c t s of t h e ac t i va t ed cells t hemse lves ( J o h a n s s o n a n d S o d e r h a l l , 1989a; Ratcliffe, 1991). T h u s , i n t e r a c tive i m m u n e c o m p e t e n t cells a r e n o t conf ined to t h e v e r t e b r a t e i m m u n e s y s t e m .

T h i s s econd s t a g e of insec t i m m u n i t y is a l so c h a r a c t e r i z e d b y t h e syn thes i s of over 15 i m m u n e p r o t e i n s ( B o m a n a n d H u l t m a r k , 1987; Faye a n d H u l t m a r k , V o l u m e 2, C h a p t e r 2) . T h e q u e s t i o n ar i ses a s to w h e n a n d h o w t h e s y n t h e t i c m a c h i n e r y in t he fat body, h e m o c y t e s , a n d o t h e r t i ssues is t u r n e d o n b y t h e i n v a d i n g o r g a n i s m s ? T h e r e is n o w s o m e t e n t a t i v e e v i d e n c e t h a t t h e non-se l f r ecogn i t ion even t s in insec ts ac t iva te t h e r e l e v a n t i m m u n e p r o t e i n genes (Faye , 1990). I n a d d i t i o n , it h a s r ecen t ly b e e n s h o w n t h a t e n d o t o x i n ( L P S ) a n d l a m i n a r i n (a P~l ,3-glucan) , w h i c h a r e b o t h c o m p o n e n t s of m i c r o b i a l cell wal ls a n d ac t iva to r s of t h e p P O recogn i t i on sy s t em, a lso i n d u c e t h e express ion of cec rop ins (one g r o u p of t he i n d u c e d a n t i b a c te r ia l p r o t e i n s in insects) in a n insec t cell l ine (Samakov l i s et al., 1990; see Sec t ion I I I . D ) . Final ly , if these i m m u n e p r o t e i n s a r e s u c h i m p o r t a n t c o m p o n e n t s of t h e insec t defenses , t h e n w h y a r e s o m e insec ts a p p a r e n t l y i n c a p a b l e of p r o d u c i n g t h e m ?

C Final Humoral/Cellular Stage

D u e to t h e efficiency of t h e first t w o s tages in insec t i m m u n i t y , p r o b a b l y on ly a m i n o r i t y of i n v a d i n g o r g a n i s m s surv ive un t i l t h e final p h a s e , w h i c h resu l t s in e i t he r h o s t recovery o r d e a t h . T h i s s t age occu r s a few h o u r s u p to severa l d a y s after i nvas ion of t h e insec t a n d is m a i n l y m e d i a t e d by t h e newly s y n t h e s ized i m m u n e p r o t e i n s . Pa ra s i t e s a n d p a t h o g e n s r e s i s t an t to t h e ki l l ing m e c h a n i s m s w i t h i n t h e b lood cells a n d capsu l e s will , however , b r e a k o u t of t hese


ce l lu la r s t r u c t u r e s d u r i n g this p h a s e a n d elicit a fu r the r r o u n d of p h a gocytos is a n d e n c a p s u l a t i o n by the h e m o c y t e s .

To d a t e , t he insec t i m m u n e p ro t e in s , w h i c h i n c l u d e t h e cec rop ins , a t -t ac ins , d ip t e r i c in s , defens ins , s a rco tox ins , a p i d a e c i n s , a n d ly sozyme ( B o m a n a n d H u l t m a r k , 1987; Cas t ee l s et al., 1990; H o f f m a n n a n d H o f f m a n n , 1990), have all b e e n s h o w n to be a n t i b a c t e r i a l . D o e s th is m e a n t h a t n o s u c h final h u m o r a l s t age is m o u n t e d a g a i n s t o t h e r m i c r o b i a l a n d m a c r o b i a l p a r a s i t e s ? T h i s s eems unl ike ly especia l ly following t h e r e p o r t b y H a m et al. (1991) t h a t a n t i - i m m u n e p r o t e i n a n t i b o d i e s b lock the n o r m a l l y s t r o n g microf i la r ic ida l act ivi ty of t he h e m o l y m p h of Aedes aegypti a n d Simulium ornatum. I n a d d i t i o n , a t t e n t i o n s h o u l d be d r a w n to t he " a n t i b o d y l i k e " r e s p o n s e of Periplaneta ameri-cana d u r i n g w h i c h a l a rge 700 -kDa molecu le is i n d u c e d ( G e o r g e et al., 1987; K a r p , 1990, a n d V o l u m e 1, C h a p t e r 13), w h i c h m a y well be a b l e to n e u t r a l i z e a r a n g e of m i c r o b i a l i n v a d e r s .

A n o t h e r p h e n o m e n o n t h a t cha rac t e r i ze s this final s t age is t h e c o m p l e t i o n of w o u n d hea l i ng p r o b a b l y as a resu l t of t he syn thes i s a n d r e p a i r of t h e b a s e m e n t m e m b r a n e by the h e m o c y t e s ( K n i b i e h l e r et al., 1987). T h e rec ru i t m e n t of h e m o c y t e s a r o u n d capsu les a n d the r e p l a c e m e n t by h e m o p o i e s i s of b lood cells u t i l ized in t h e ce l lu la r defenses cease too . I t h a s b e e n sugges t ed t h a t the h e m o c y t e s in capsu le s m a y secre te b o t h " r e c r u i t i n g " a n d "ce s sa t i on fac to rs" a n d t h a t t he r e c r u i t m e n t of b lood cells m a y d e p e n d u p o n t h e re la t ive a m o u n t s of these s u b s t a n c e s (Lackie , 1988). I n ea r ly c apsu l e s , t he r e c r u i t i n g s igna l w o u l d b e s t r o n g b u t w o u l d w e a k e n w i t h t he a d d i t i o n of m o r e cell l ayers . As r e c r u i t m e n t slows, t he h e m o c y t e s wou ld have m o r e o p p o r t u n i t y to secre te t he cessa t ion factors , w h i c h m a y b e t h e h o m o g e n e o u s , gly-c o s a m i n o g l y c a n s r e p o r t e d coa t i ng m a n y m a t u r e capsu l e s a n d poss ib ly r e l a ted to b a s e m e n t m e m b r a n e m a t e r i a l (Lackie , 1988).

R e g a r d i n g the r e p l a c e m e n t of cells u t i l ized in t h e ce l lu la r defenses , t hese m a y o r i g ina t e from specific h e m o p o i e t i c t i ssue a n d / o r by re lease of sessile p o p u l a t i o n s a t t a c h e d to t he i n t e r n a l o r g a n s . N o d o u b t h u m o r a l factors , s u c h as t he " p l a s m a t o c y t e dep l e t i on fac tor" of Galleria mellonella ( C h a i n a n d A n d e r s o n , 1983), a r e involved, b u t t he w h o l e sub jec t of h e m o p o i e s i s a n d the con t ro l of h e m o c y t e n u m b e r s in insects is no t on ly neg lec ted b u t s o m e w h a t confused. A fasc ina t ing ar t ic le by P a t h a k (1986) i nd i ca t e s t h a t t h e e n d o c r i n e g l a n d s of insec ts p r o b a b l y p l ay a key role in t he con t ro l of b lood cell n u m b e r s a n d cell types as well as in m e d i a t i o n of the i m m u n e s y s t e m itself. T h e subjec t of t h e in t e rac t ion of t he insect e n d o c r i n e a n d i m m u n e s y s t e m s is d e a l t w i t h briefly in Sec t ion 111. A .

T h i s final p h a s e resu l t s e i the r in t he recovery of t h e insec t hos t d u e to t h e efficacy of t he i m m u n e sys t em or else t he i n v a d e r p reva i l s a n d the h o s t is ki l led. T h e ki l l ing of bac t e r i a by t he i m m u n e p ro t e in s is well d o c u m e n t e d a n d h a s b e e n m e n t i o n e d prev ious ly (see Sec t ion I I ) b u t l i t t le is k n o w n of t h e

12. Cellular Defense Responses of Insects 2 7 3

kil l ing m e c h a n i s m of t he h e m o c y t e s t hemse lves t h a t have inges t ed or surr o u n d e d i n v a d e r s . A n d e r s o n et al. (1973) d e s c r i b e d t h e ki l l ing of b a c t e r i a b y Blaberus craniifer h e m o c y t e s a n d W a l t e r s a n d Ratcliffe (1983) showed t h a t w h e n E. colt w e r e s e q u e s t e r e d in n o d u l e s they were r a p i d l y kil led (Wal te r s a n d Ratcliffe, 1983) (Tab le 1). Blaberus h e m o c y t e s too have b e e n s h o w n to c o n t a i n e n d o g e n o u s p e r o x i d a s e ( H . M u l l e t t , u n p u b l i s h e d o b s e r v a t i o n s ) . I n a d d i t i o n , insec t h e m o c y t e s h a v e b e e n r e p o r t e d to c o n t a i n acid p h o s p h a t a s e (e.g. , Cross ley, 1968; Rowley a n d Ratcliffe, 1979), β - g l u c u r o n i d a s e a n d β - g l u c o s a m i n i d a s e (Wal te r s a n d Ratcliffe, 1981), l y sozyme (e.g. , Z a c h a r y a n d H o f f m a n n , 1984), cec rop ins a n d a t t a c in s (T renczek , 1986), a n d t h e p P O s y s t e m , w h i c h h a s recen t ly b e e n s h o w n to have l imi ted a n t i m i c r o b i a l ac t iv i ty (Rowley et al, 1990).

Final ly , m a n y m i c r o b i a l a n d m a c r o b i a l p a r a s i t e s have evolved m e c h a n i s m s for a v o i d a n c e or d e p r e s s i o n of t he insec t hos t r e s p o n s e s (Tab le 2) . T h i s sub jec t h a s b e e n rev iewed extens ive ly (e.g. , Ratcliffe et al., 1985; G o t z a n d B o m a n , 1985; Ratcliffe a n d Rowley, 1987; V i n s o n , 1988; B r e h e l i n , 1990; S c h m i d t a n d T h e o p o l d , 1991) a n d is of e x t r e m e i m p o r t a n c e as it n o t on ly revea ls c lues as to t h e m e c h a n i s m s con t ro l l ing insec t i m m u n i t y , b u t a l so a l lows p a r a s i t e s to su rv ive for long p e r i o d s in the i r hos t s , w h i c h t h e n ac t as t h e vec to rs of t h e m o s t d e v a s t a t i n g d i seases (see Sec t ion I I I . E ) .

III. Specific Problems in Insect Immunity

T h e a f o r e m e n t i o n e d br ief s cena r io h a s ident i f ied a l a rge n u m b e r of u n r e solved p r o b l e m s in o u r u n d e r s t a n d i n g of insec t i m m u n i t y . I n t he r e m a i n d e r of th is c h a p t e r , s o m e of t he m o r e i m p o r t a n t of these u n a n s w e r e d q u e s t i o n s a r e c o n s i d e r e d in m o r e de ta i l . T h e sub jec t s se lec ted a r e t hose t h a t fo rm t h e bas i s of con t ro l a n d c o o r d i n a t i o n of t h e insec t i m m u n e s y s t e m a n d i n c l u d e t h e following:

(a ) T h e classif icat ion, o r ig ins , a n d m a n i p u l a t i o n of insec t b lood cells (b) T h e r ecogn i t i on of non-se l f i n v a d e r s (c ) C e l l - c e l l c o m m u n i c a t i o n in i m m u n i t y (d) T h e i n t e r r e l a t i o n s h i p of ce l lu la r a n d h u m o r a l i m m u n i t y (e ) F u t u r e a r e a s for s t u d y

T h e final t op ic is i n c l u d e d s ince r ecen t ly t h e r e h a s b e e n a d d i t i o n a l i n t e r e s t in t he i m m u n e sy s t ems of i n v e r t e b r a t e s f rom m o r e a p p l i e d a s p e c t s , i n c l u d i n g t h e poss ib le i m p o r t a n c e of t h e hos t defenses as d e t e r m i n a n t s of infect ivi ty in vec to r species ( C h r i s t e n s e n a n d Severson , V o l u m e 1, C h a p t e r 11) a n d t h e use of c h a n g e s in i m m u n e reac t iv i ty as m o n i t o r s of e n v i r o n m e n t a l po l lu t i on .

Tab

le 1

Perc

enta

ge R

ela

tive

Via

bili

ty0

(% R

.V.)

of

E. c

oli

and

B.

ce

reus

in N

od

ule

s fr

om

Ga

lleria

me

llone

lla a

nd i

n C

orr

esp

ond

ing

H

aem

olym

ph

or

Gra

ce's

Ins

ect

Med

ium

Inc

ubat

ed C

ont

rols

B. c

ereu

s E

. col

i

Tim

e (h

r po

stin

ject

ion)

E

xper

imen

tal

(in

nodu

les)

H

emol

ymph

G

race

's

Exp

erim

enta

l (i

n no

dule

s)

Hem

olym

ph

Gra

ce's

4 10

.11

± 4.

65*

558.

7 ±

273.

5 13

2.9

± 40

.5

16.1

4 ±

8.12

51

3.0

± 29

1 32

7.9

+ 12

2

6 65

.66

± 41

.6

2087

.6

± 45

1 12

95.0

± 6

48

4.10

± 3

.07

1152

.2 ±

319

31

7.8

112

9 21

0.0

± 86

.8

2334

.2 ±

489

14

09.9

± 5

11

3.99

± 2

.05

1207

.0 ±

436

47

5.0

± 17

4

12

510.

1 ±

153

2874

.0 ±

586

20

65.1

± 7

15

3.29

± 2

.5

1661

.2 ±

730

50

2.9

± 17

6

15

1270

.0 ±

283

25

98.5

±

520

1895

.2

500

3.10

2.

1 11

47.4

± 3

78

538.

2 ±

218

18

Dea

th

—

1.74

1.

38

1156

.5 ±

520

37

7.3

± 15

3

21

—

1.64

1.

27

1216

.0

455

572.

7 ±

215

24

—

1.75

± 1

.28

1367

.1 ±

419

57

3.0

181

aP

erce

ntag

e re

lati

ve v

iabi

lity

at t

ime

"0"

= 10

0%.

* Sta

ndar

d er

ror.

Mod

ified

fro

m W

alte

rs a

nd R

atcl

iffe

(198

3) w

ith p

erm

issio

n of

Per

gam

on P

ress

.


Table 2 Some Avoidance and Resistance Strategies Adopted by Parasites and Pathogens against Invertebrate Host Defense Mechanisms

A. Passive avoidance 1. Organs colonized out of reach of blood

cells 2. Low-reactivity regions of host occupied 3. Immature hosts invaded

B. Active mechanisms 1. Refractory envelopes/cell walls, etc. 2. Mimicry of host tissues 3. Acquisition of host antigens 4. Lysis and abrogation of leukocytes 5. Inhibition of immune recognition factors 6. Inhibition of chemotaxis 7. Inhibition of phagocytosis 8. Inhibition of nodule formation/

encapsulation 9. Provision of alternative targets

10. Utilization of host response 11. Abrogation of immune proteins

C. No obvious mechanism 1. Host swamped by numbers or rate of

growth of parasite/pathogen?

Modified from Ratcliffe et al. (1985).

A. Classification, Origins, and Manipulation of Insect Blood Cells

T h e c h a r a c t e r i z a t i o n of insec t b lood cell t ypes is a con t rovers i a l t op i c t h a t h a s b e e n r ev iewed extens ively in t h e las t t w e n t y yea r s (e .g. , J o n e s , 1962; P r i ce a n d Ratcliffe, 1974; G u p t a , 1979; Rowley a n d Ratcliffe, 1981; B r e h e l i n a n d Z a c h a r y , 1986). T h e p r o b l e m s in t h e classif icat ion of insec t b lood cells a r e d u e m a i n l y to t h e shee r d ivers i ty in insec t fo rms , w h i c h is ref lected in t h e g r e a t v a r i a t i o n in t h e s t r u c t u r e of t h e b lood cells p r e s e n t . T h e s i t u a t i o n is e x a c e r b a t e d b y a vas t r a n g e of different n a m e s in t h e l i t e r a t u r e for six to e igh t bas i c types of cells . T a b l e 3 , f rom Rowley a n d Ratcliffe (1981) , l ists s o m e of t h e s y n o n y m s used in p u b l i c a t i o n s for t h e six ba s i c types of insec t h e m o c y t e s ident i f ied in t h a t s tudy. I n a d d i t i o n , incons i s t enc ies in n o m e n c l a t u r e h a v e a r i s en d u e to t h e use of insec ts in different d e v e l o p m e n t a l s t ages a n d as a r e su l t of t h e u t i l i za t ion of a r a n g e of e x p e r i m e n t a l t e c h n i q u e s . A t t e m p t s h a v e

Tabl

e 3

List o

f Var

ious

Syn

onym

s fo

r In

sect

Hem

ocyt

es

Cla

ssif

icat

ion

Aut

hor'

s eq

uiva

lent

cla

sses

to:

sche

me 0

Pr

ohem

ocyt

es

Plas

mat

ocyt

es

Gra

nula

r ce

lls

Cys

tocy

tes

Sphe

rule

cel

ls

Oen

ocyt

oids

Hol

land

e (1

911)

pr

oleu

cocy

tes

phag

ocyt

es

gran

ular

leu

cocy

tes

adip

oleu

cocy

tes*

sp

heru

le c

ells

oe

nocy

toid

s

Met

alni

kov

(192

4)

lym

phoc

ytes

ce

ll sp

heru

leus

es

prol

euco

cyte

s <

leuc

ocyt

es

> C

amer

on

(193

4)

lym

phoc

ytes

le

ucoc

ytes

le

ucoc

ytes

? sp

heru

le c

ells

oe

nocy

tes

Yea

ger

(194

5)

prol

euco

cyto

ids

plas

mat

ocyt

es

podo

cyte

s'

verm

ifor

m c

ells

'

chro

mop

hilic

cel

ls?

cyst

ocyt

es

sphe

roid

ocyt

es

oeno

cyte

like

cells

Wig

gles

wor

th

(195

9)

prol

euco

cyte

s ph

agoc

ytic

am

oebo

cyte

s ph

agoc

ytic

am

oebo

cyte

s?

oeno

cyto

ids

Jone

s (1

962)

; A

rpr

ohem

ocyt

es

plas

mat

ocyt

es

gran

ular

hem

ocyt

es

cyst

ocyt

es

sphe

rule

cel

ls

oeno

cyto

ids

nold

(19

74)

podo

cyte

s'

verm

ifor

m c

ells

'

adip

ohem

ocy

tes *

Bre

helin

et a

l. (1

978)

pr

ohem

ocyt

es

plas

mat

ocyt

es

gran

uloc

ytes

? th

rom

bocy

toid

s'

podo

cyte

s'

gran

uloc

ytes

co

agul

ocyt

es

sphe

rule

cel

ls

oeno

cyto

ids

Gup

ta

(197

9)

proh

emoc

ytes

pl

asm

atoc

ytes

gr

anul

ocyt

es

coag

uloc

ytes

sp

heru

locy

tes

oeno

cyto

ids

"Ref

eren

ces

cite

d in

Row

ley

and

Rat

cliff

e (1

981)

. *S

ome

wor

kers

cla

ss a

dipo

hem

ocyt

es a

s gr

anul

ar c

ells

. 'I

n so

me

inst

ance

s th

ese

may

be

clas

sfie

d w

ith

the

plas

mat

ocyt

es.


even b e e n m a d e a lso to classify t h e b lood cells j u s t o n t he u l t r a s t r u c t u r a l a p p e a r a n c e of t he i r g r a n u l e s . S o m e v a r i a t i o n in g r a n u l e s t r u c t u r e is to be expec t ed in a g r o u p c o n t a i n i n g 1-3 mi l l ion species! A c o m m o n e r r o r m a d e b y w o r k e r s n e w to th is field is to a d o p t v e r t e b r a t e m e t h o d s in t h e p r e p a r a t i o n of insec t b lood cells for m ic roscop i ca l e x a m i n a t i o n . N o t h i n g is g u a r a n t e e d m o r e to d i s to r t a n d lyse t h e fragile h e m o c y t e s of insec ts t h a n s m e a r i n g t h e m on a m i c r o s c o p e s l ide. A final p r o b l e m c e r t a i n to confuse i n e x p e r i e n c e d w o r k e r s is t h e r e t e n t i o n of v e r t e b r a t e t e r m s s u c h as " g r a n u l o c y t e " for d e s c r i b ing insec t b lood cells w h e n t h e r e is l i t t le ev idence for h o m o l o g y of a n y cell t ypes in these two g r o u p s of a n i m a l s .

B rehe l in a n d Z a c h a r y (1986) h a v e a lso d i scussed in de t a i l s o m e of t h e p r o b l e m s in ca t ego r i z ing insec t b l o o d cells a n d s t ressed t h e p o i n t t h a t des ig n a t i o n s b a s e d p u r e l y o n h e m o c y t e func t ions a r e a lso u n r e l i a b l e . T h u s , in Locusta migratoria a n d Melolontha melolontha, c apsu le s a r e fo rmed b y g r a n u l a r cells in t h e fo rmer a n d by p l a s m a t o c y t e s in t he la t te r . F u n c t i o n a l l y these two cell t ypes a r e iden t i ca l b u t m o r p h o l o g i c a l l y they a r e q u i t e d i s t inc t . T h e y p r o p o s e d t h a t s tud ie s of insec t b lood cells s h o u l d i n c l u d e u l t r a s t r u c t u r a l , func t iona l , a n d e n z y m a t i c a spec t s in o r d e r for a c lear def in i t ion to b e de r ived (Brehe l in a n d Z a c h a r y , 1986). T o th is list s h o u l d a lso b e a d d e d p h a s e -c o n t r a s t o b s e r v a t i o n s of unf ixed cells. T h i s t e c h n i q u e is p a r t i c u l a r l y useful as it revea ls n o t on ly m o r p h o l o g i c a l cha rac t e r i s t i c s b u t a lso o t h e r p r o p e r t i e s of t h e cells s u c h as the i r s tab i l i ty a n d b e h a v i o r in vitro.

Recent ly , cell types have b e e n ident i f ied by t h e n a t u r e of t he i r cel l -surface d e t e r m i n a n t s as d e t e c t e d by s t a i n i n g w i t h lec t ins a n d m o n o c l o n a l a n t i b o d i e s ( m A b ) . I n Drosophila, for e x a m p l e , t h e h e m o c y t e s r e spons ib l e for t h e e n c a p su l a t i on of a b e r r a n t t i ssues a n d p a r a s i t e s , as well as for t h e re jec t ion of foreign t i s sue i m p l a n t s , have b e e n s h o w n to b i n d fluorescein-labeled w h e a t g e r m a g g l u t i n i n (Rizki a n d Rizki , 1982; N a p p i a n d Si lvers , 1984). Similar ly , in Aedes aegypti t h e r e is a fivefold i nc r ea se in t he p e r c e n t a g e of cells b i n d i n g w h e a t g e r m a g g l u t i n i n following i n o c u l a t i o n w i t h Dirofilaria immitis m i c r o filaria ( N a p p i a n d C h r i s t e n s e n , 1986). L e c t i n - b i n d i n g c a n a lso d i s t i n g u i s h b e t w e e n c e r t a i n h e m o c y t e types in t h e p h a s m i d Extatosoma tiaratum, a l t h o u g h t h e r e is c o n s i d e r a b l e s t a i n i n g c ross - reac t iv i ty w i t h t h e different cell types ( R i c h a r d s et al., 1989), w h i c h l imi t s i ts usefulness for func t iona l work . A n a l t e r n a t i v e a p p r o a c h b y T r e n c z e k a n d B e n n i c h (1992) h a s b e e n to ra i se m A b a g a i n s t specific s u b p o p u l a t i o n s of p l a s m a t o c y t e s a n d g r a n u l a r cells f rom c e c r o p i a m o t h s . M u l l e t t et al. (1993) have l ikewise p r o d u c e d m A b a g a i n s t Blaberus discoidalis a n d Galleria mellonella h e m o c y t e s so t h a t t he se w o r k e r s n o w h a v e powerful tools w i t h w h i c h to p r o b e b lood cell i n t e r r e l a t i o n s h i p s a n d i n t e r a c t i o n s . O n e d r a w b a c k of m A b is t h a t t hey m a y b e genus-spec i f ic (Tab l e 4) a n d therefore unl ike ly to p rov ide a m e a n s of d e r i v i n g a unif ied classificat ion of insec t b lood cells across a r a n g e of insec t o r d e r s .


Table 4 Cross-reactivity of two Blaberus discoidolis Monoclonal Antibodies against the Hemocytes of a Number of Insect Species*

Monoclonal

Species B5 l H b

Blaberus discoidalis + + + + + + Blaberus craniifer + + + + Gromphadorhina sp. + + Periplaneta americana - + Galleria mellonella - -" + + + / + + / + / — = intensity of staining by FITC labeling.

From Mullett et al. (1993).

T h e m e s s a g e is c lear for insect h e m a t o l o g i s t s a n d o t h e r sc ient is t s in th is field. Very useful classif icat ion s c h e m e s for insect h e m o c y t e s a r e ava i l ab le in t he l i t e r a t u r e (e.g. , J o n e s , 1962; Pr ice a n d Ratcliffe, 1974; Brehe l in a n d Zacha ry , 1986). T h e s e s c h e m e s p rov ide t he bas is for ca t egor i z ing t h e h e m o cytes of m o s t insec t species b u t b e p r e p a r e d for t h e u n u s u a l (e .g. , F ig . 2) . I n a d d i t i o n , as a first s tep , e x a m i n e the cells carefully u n d e r p h a s e - c o n t r a s t op t ics b u t b e w a r e of ar t i facts t h a t r ead i ly o c c u r w i t h th is t e c h n i q u e (see A z a m b u j a et al., 1991).

T h e a p p a r e n t d ivers i ty of b lood cells in insec ts is l ikewise reflected in t h e v a r i a t i o n in loca t ion a n d s t r u c t u r e of t he h e m o p o i e t i c t i ssues in these a n i m a l s (e.g. , J o n e s , 1970; G u p t a , 1979; Rowley a n d Ratcliffe, 1981). I n s o m e d i p -t e r a n s s u c h as Musca domestica, t hey m a y b e fo rmed by diffuse col lec t ions of cells, w h e r e a s in t he o r t h o p t e r a n s Gryllus bimaculatus a n d Locusta migratoria, t hey m a y be h igh ly deve loped w i t h s o m e signs in t h e fo rmer species of d i f ferent ia t ion in to va r ious zones , as in h i g h e r a n i m a l g r o u p s ( H o f f m a n n et al., 1979). H e m o c y t e s a r e a lso p r o b a b l y de r ived by t h e mi to t i c d iv i s ion of t h e c i r cu l a t i ng cells. J o n e s a n d L iu (1968) showed t h a t in t h e w a x m o t h Galleria mellonella, a l t h o u g h the mi to t i c r a t e w a s on ly 1 % , th is is sufficient to m a i n t a i n l a rva l h e m o c y t e n u m b e r s a n d p r e c l u d e t he need for h e m o p o i e t i c t i s sue . A n o t h e r sou rce of c i r cu l a t i ng h e m o c y t e s is b y t he re lease of sessile cells loosely a t t a c h e d to t he i n t e r n a l o r g a n s . Recent ly , for e x a m p l e , it h a s b e e n s h o w n t h a t t he t r e a t m e n t of Tipula paludosa l a rvae w i t h e t h e r a n d acet ic ac id v a p o r s i n c r e a s e d the to ta l h e m o c y t e c o u n t s by 2.8-fold d u e to t h e re lease of sessile cells ( G r e e n a n d C a r t e r , 1991).


Figure 2 Giant cell of Rhodnius robustus with ingested granular cells. Scale bar is 10 μπι. From Azambuja et al. (1991) with permission of Memorias Do Instituto Oswaldo Crus.

D e s p i t e these o b s e r v a t i o n s , we still h a v e on ly l imi ted k n o w l e d g e a b o u t t h e i n t e r r e l a t i o n s h i p s of t he va r ious h e m o c y t e types a n d t h e m e c h a n i s m s involved in t he con t ro l of t he d e v e l o p m e n t a n d l i be ra t i on of t h e b lood cells i n to t h e c i r cu l a t i on (Lack ie , 1988). U n f o r t u n a t e l y , few sc ient is t s a r e a t t r a c t e d to th is a r e a of c o m p a r a t i v e hema to logy .

M o s t w o r k e r s r ecogn ize t h e ba s i c b lood cell t ype , t h e p r o h e m o c y t e , f rom w h i c h t h e o t h e r h e m o c y t e types m a y b e de r ived (Lack ie , 1988). M a n y insec t h e m a t o l o g i s t s bel ieve t h a t t he c o m m o n l y d e s c r i b e d b lood cells, t h a t is, t h e p l a s m a t o c y t e s , g r a n u l a r cells, cys tocy tes , s p h e r u l e cells, a n d o e n o c y t o i d s , m a y b e s t ages in d e v e l o p m e n t f rom o n e o r two bas i c cell types (Rowley a n d Ratcliffe, 1981). S h r i v a s t a v a a n d R i c h a r d s (1965) showed in a n a u t o r a d i o g r a p h i c s t u d y of G. mellonella t h a t a d e v e l o p m e n t a l ser ies exis ts p a s s i n g f rom p r o h e m o c y t e s to p l a s m a t o c y t e s to g r a n u l a r cells, w h e r e a s t h e l e p i d o p t e r a n oenocy to id m a y h a v e a s e p a r a t e l i neage ( B e e m a n et aL, 1983). O n e poss ib le , b u t specu la t ive , d e v e l o p m e n t a l s c h e m e is s h o w n in F ig . 3 for t he b lood cells of t h e st ick insec t Clitumnus extradentatus, w i t h t h e m a i n o n t o g e n e t i c p a t h w a y o c c u r r i n g from the p r o h e m o c y t e ( s t em cell) to t h e cys tocy te ( R a t cliffe a n d Rowley, 1987).

R e g a r d i n g t h e con t ro l of t he d e v e l o p m e n t a n d l i be ra t i on of t h e h e m o c y t e s i n to t h e c i r cu l a t i on , t h e r e is ev idence t h a t t h e h e m o g r a m , a n d h e n c e t h e insec t i m m u n e sy s t em, is u n d e r e n d o c r i n e con t ro l ( rev iewed in P a t h a k , 1986). L i g a t u r e e x p e r i m e n t s to i sola te t h e a n t e r i o r e n d o c r i n e g l a n d s show


CYSTOCYTE

Figure 3 Diagrammatic, but speculative, scheme for hemocyte differentiation in the stick insect Clitumnus extradentatus. Modified from Ratcliffe and Rowley (1987) with permission of CRC Press, Inc.

t h a t these s t r u c t u r e s g rea t ly inf luence t he i n t e r conve r s ion of h e m o c y t e types , i nc rease t he mi to t i c ind ices , re lease t he h e m o c y t e s from t h e h e m o p o i e t i c o r g a n s , a n d mob i l i ze t h e sessile cells (e.g. , H i n k s a n d A r n o l d , 1977). S t u d i e s involv ing in ju r ing , b l eed ing , a n d ce l lu la r r eac t ions s u c h as e n c a p s u l a t i o n a lso sugges t t h a t w o u n d s a n d / o r p a r a s i t e s p r o d u c e s o m e factor(s) c a u s i n g the n e u r o e n d o c r i n e sys t em to p r o d u c e m o r e h o r m o n e s t h a t t h e n inf luence t he mi to t i c ind ices a n d re lease of h e m o c y t e s ( P a t h a k , 1986). T h e fact t h a t m a n y p a r a s i t e s , such as t he p a r a s i t o i d s , i n d u c e r a d i c a l hos t e n d o c r i n e c h a n g e s (Beckage , 1990) m u s t have a p r o f o u n d effect o n t h e i m m u n e p o t e n t ial of t h e hos t insect . Final ly, a b l a t i o n a n d i m p l a n t a t i o n s tud ie s of e n d o c r i n e g l a n d s i n d i c a t e too t h a t these o r g a n s g rea t ly affect h e m o c y t e n u m b e r s (Pat h a k , 1983).

I n o r d e r to s t u d y the i n t e r r e l a t i onsh ip s of insec t b lood cells, a s well as the i r funct ions in i m m u n e reactivi ty, t e c h n i q u e s m u s t b e d e v e l o p e d for m a n i p u l a t i n g t he cells a n d s t u d y i n g t h e m in vitro. I n sec t h e m o c y t e s a r e n o t o r ious ly difficult to m a n i p u l a t e a n d this is reflected in t he fact t h a t it w a s n o t un t i l 1974 t h a t p h a g o c y t o s i s w a s first s t u d i e d in vitro (Ratcliffe a n d Rowley, 1974) a n d on ly in 1991 d id it b e c o m e poss ib le to s e p a r a t e t he m a i n i m m u n e reac t ive cells a n d o b t a i n func t iona l m o n o l a y e r s in vitro ( A n g g r a e n i a n d R a t cliffe, 1991). T h e ex ten t of th is p r o b l e m is i l l u s t r a t ed by a t t e m p t s to s e p a r a t e


t h e different h e m o c y t e types of two Blaberus spec ies . We r e p o r t e d p rev ious ly t h e successful s e p a r a t i o n of t h e b lood cells of B. craniifer u s i n g c o n t i n u o u s g r a d i e n t s of Percol l ( M e a d et al., 1986). Howeve r , a t t e m p t s to i so la te t h e h e m o c y t e s of B. discoidalis h ave failed even t h o u g h t h e cells a p p e a r i den t i ca l to t hose of B. craniifer u n d e r t h e l ight m i c r o s c o p e a n d t h e p r o t o c o l a d o p t e d w a s i den t i c a l to t h a t u s e d for t h e B. craniifer cells ( H . M u l l e t t , u n p u b l i s h e d o b s e r v a t i o n s ) . Possibly, as in Schistocerca gregaria ( H u x h a m a n d Lack ie , 1988), t h e B. discoidalis cells m a y c o m p r i s e a m o r e finely g r a d e d r a n g e of g r a n u l e -c o n t a i n i n g cells t h a n those of B. craniifer, w h i c h m a k e s s e p a r a t i o n o n c o n t i n u o u s g r a d i e n t s i m p o s s i b l e .

T h u s , to d a t e , on ly in o n e insec t species , G. mellonella, h a v e s e p a r a t e d a n d i m m u n e reac t ive h e m o c y t e s b e e n o b t a i n e d in vitro (Tab le 5) ( A n g g r a e n i a n d Ratcliffe, 1991). O n e of t h e m a i n p r o b l e m s is t h e a l m o s t i n s t a n t a n e o u s coag u l a t i o n a n d ge l a t ion of t h e h e m o l y m p h following b l eed ing . T h i s c o a g u l a t i o n is d u e to t h e fragile n a t u r e of t he g r a n u l a r cells o r cys tocy tes (des igna t ion d e p e n d s u p o n species) . T h e s e cells a r e p r i m e d to d i s c h a r g e the i r cont en t s u p o n c o n t a c t w i t h m i n u t e q u a n t i t i e s of m i c r o b i a l p r o d u c t s s u c h as β - l j S - g l u c a n s a n d e n d o t o x i n ( L e o n a r d et al., 1985b; Ratcliffe et al., 1991), w h i c h a r e p r e s e n t in a b u n d a n c e on all g l a s s w a r e a n d so lu t ions un les s specific p r e c a u t i o n s a r e t a k e n . Now, for tunate ly , th is p r o b l e m c a n b e p a r t i a l l y overc o m e b y u s i n g a n ice-cold i so ton ic , a n t i c o a g u l a n t so lu t ion c o n t a i n i n g E D T A a n d a t low p H to s tab i l ize t h e cells ( M e a d et a/,^1986). E v e n so , however , t h e

Table 5 Percentage of Galleria mellonella Plasmatocytes and Granular Cells Alone or Mixed, from Washed Cell Preparations Phagocytosing Bacillus cereus after 1 hr Incubation of Monolayers at 25°C

Percentage of cells phagocytic*1

Type of monolayer Cell type counted Control* Experimental*

Plasmatocytes alone Plasmatocytes 5.3 ± 1.3 13.4 ± 2.1*

Granular cells alone Granular cells 0 0

Mixed Plasmatocytes 8.7 ± 2.7 30.1 ± 3.4*/**

Granular cells 0 0

a M e a n value of 18 monolayers from six gradients ± SD. * Monolayers were overlaid with B. cereus suspended in Tris overlay buffer. 'Monolayers were overlaid with B. cereus suspended in Tris overlay buffer containing 0.1 mg m l - 1 laminarin.

* Significantly different compared with control (P < 0.05). **Significantly different compared with plasmatocytes alone (P < 0.005).

Modified from Anggraeni and Ratcliffe (1991) with permission of Pergamon Press.


a m o u n t of m a n i p u l a t i o n poss ib le in t e r m s of cen t r i fuga t ion a n d p i p e t t i n g is s t r ic t ly l imi ted a n d d e p e n d s very m u c h u p o n t h e species u n d e r s tudy. T h u s , G. mellonella b lood cells a r e eas ie r to h a n d l e t h a n those of Locusta migratoria o r Schistocerca gregaria, w h i c h in t u r n a r e m o r e s t ab l e t h a n t hose of s o m e cockroaches a n d p h a s m i d s such as B. discoidalis a n d Extatosoma tiaratum. M u c h a d d i t i o n a l work is r e q u i r e d i n t o t h e s t ab i l i za t ion a n d s e p a r a t i o n of t h e b l o o d cells of a r a n g e of insec t species in o r d e r for a d d i t i o n a l in vitro m o d e l s to b e ava i l ab le for s tudy.

B. Recognition of Non-Self Invaders

T h e bas i s of a n y i m m u n e r e s p o n s e is t h e ab i l i ty to r ecogn ize as foreign a n y o r g a n i s m s i n v a d i n g the body. In sec t s have b e e n s h o w n to b e very effective a t c l ea r ing foreign i n v a d e r s of t he h e m o c o e l by p h a g o c y t o s i s a n d c a p s u l e fo rmat ion (Ratcliffe a n d Rowley, 1987), so t h e y c a n c lear ly d i s c r i m i n a t e self f rom non-self. T h e level of th is d i s c r i m i n a t i o n m a y a lso b e finely t u n e d s ince K a r p a n d his co l leagues ( rev iewed in K a r p , 1990) h a v e r e p o r t e d t h e p r e s e n c e of al lograf t re jec t ion of b o t h in t e g u m e n t a l grafts a n d i m p l a n t s by Periplaneta americana. As m e n t i o n e d p rev ious ly (Sect ion I I . B ) , two poss ib le factors m a y act as non-se l f r ecogn i t ion molecu les in insec ts , t h a t is, t h e h e m o l y m p h a g g l u t i n i n s (often t e r m e d lect ins b y m a n y a u t h o r s a l t h o u g h n o t to ta l ly syno n y m o u s ) a n d c o m p o n e n t s of t h e p r o p h e n o l o x i d a s e c a s c a d e . To these recogn i t ion factors m a y a lso b e a d d e d the i m m u n e p r o t e i n h a e m o l i n (p4) , w h i c h b i n d s to bac t e r i a a n d m a y in i t i a t e t he syn thes i s of t he i m m u n e p r o t e i n s (Faye , 1990) (see Sec t ion I I I . D ) .

T h e a g g l u t i n i n s a r e p r e s e n t in t he h e m o l y m p h a n d in t i ssues , s u c h as t h e gu t , of m o s t insec ts . T h e h e m o l y m p h lect ins a r e syn thes i zed b y b o t h t h e h e m o c y t e s (Fig . 4) a n d the fat b o d y ( rev iewed b y Rowley et al., 1986). T h e o c c u r r e n c e a n d func t ion ing of these molecu le s a r e , however , c o m p l e x . T h u s in s o m e species , s u c h as t h e flesh fly, Sarcophagaperegrina ( K o m a n o et al., 1980), P. americana, a n d S. gregaria ( I n g r a m et al., 1984), t hey a r e i n d u c i b l e , w h e r e a s in o t h e r s , i n c l u d i n g Spodoptera exigua ( P e n d l a n d a n d B o u c i a s , 1986), t hey a r e a p p a r e n t l y no t . I n a d d i t i o n , t h e h e m o l y m p h of t h e c o c k r o a c h B. discoidalis c o n t a i n s n o t o n e b u t t h r e e a g g l u t i n i n s e a c h w i t h i ts o w n c h a r a c ter i s t ic s u g a r - b i n d i n g specificity ( C h e n et al., 1993).

T h e r e is a lso d i s a g r e e m e n t r e g a r d i n g t h e role of t h e a g g l u t i n i n s in i m m u n e r ecogn i t ion . A n d e r s o n et al. (1973) a n d Rowley a n d Ratcliffe (1980) r e p o r t e d t h a t bac t e r i a o r e r y t h r o c y t e s p resens i t i zed w i t h w h o l e h e m o l y m p h of B. craniifer, P. amencana, o r Clitumnus extradentatus w e r e n o t i n t e r n a l i z e d a n y m o r e effectively by t h e h e m o c y t e s t h a n w e r e u n t r e a t e d tes t pa r t i c l e s . L ike wise , B r a d l e y et al. (1989) showed t h a t t he pur i f ied lec t in f rom t h e g r a s s h o p p e r Melanoplus differentialis failed to e n h a n c e t he in vitro a s soc ia t ion of Bacillus


LYSOZYME (bacterial killing)

CECROPINS (bacterial killing)

\ / P H A G O C Y T O S I S ^ ^ NODULES and

C A P S U L E S

COAGULOGENS (hemolymph coagulation)

PROPHENOLOXIDASE (recognition)

AGGLUTININS (recognition)

Figure 4 Diagram illustrating the multifunctional role of insect granule-containing cells ( = granular cells or cystocytes). Modified from Ratcliffe and Rowley (1987) with permission of C R C Press, Inc.

thuringiensis, Nosema locustae s po re s , o r e r y t h r o c y t e s w i t h t h e h e m o c y t e s of th is insec t . I n c o n t r a s t to these s t ud i e s , P e n d l a n d et al. (1988) , w o r k i n g w i t h S. exigua, i so la ted a D-galac tose-spec i f ic h e m o l y m p h lect in t h a t e n h a n c e d b o t h t h e c l e a r a n c e of fungal spo res from t h e b o d y a n d the i r a s soc i a t i on w i t h t h e h e m o c y t e s , w h e r e a s R i c h a r d s a n d Ratcliffe (1990) s h o w e d t h a t t h e pur i f ied a g g l u t i n i n f rom t h e p h a s m i d Extatosoma tiaratum in t h e p r e s e n c e of C a 2 +

s ignif icant ly i n c r e a s e d t h e ro se t t i ng of tes t e r y t h r o c y t e s a r o u n d t h e h e m o cytes of th is species (Tab le 6) . D r i f a n d B r e h e l i n (1989) r e p o r t e d , too , t h a t t h e a g g l u t i n i n in t h e h e m o l y m p h of Locusta migratona ac ts as a n o p s o n i n to e n h a n c e e r y t h r o c y t e u p t a k e b y t h e h e m o c y t e s . T h e s e a p p a r e n t c o n t r a d i c t ions for a role for insec t a g g l u t i n i n s in i m m u n e r ecogn i t i on a r e confus ing . T h i s s i t u a t i o n is c o n f o u n d e d b y t h e r e c e n t s tud ie s of K u b o et al. (1990) a n d K a w a g u c h i et al. (1991) , w h o have s h o w n t h a t , in P. americana a n d S. peregnna, t h e h e m o l y m p h lec t ins m a y b e invo lved n o t on ly in defense b u t a l so in d e v e l o p m e n t a n d r e g e n e r a t i o n p roces ses .

N o d o u b t s o m e , b u t n o t al l , of t h e func t iona l differences r e p o r t e d for insec t a g g l u t i n i n s c a n be e x p l a i n e d b y s u b o p t i m a l e x p e r i m e n t a l c o n d i t i o n s . F o r e x a m p l e , g r e a t c a r e m u s t b e exerc i sed in u s i n g v e r t e b r a t e e r y t h r o c y t e s a s tes t pa r t i c l e s as t hey f requen t ly c l u m p following sens i t i za t ion w i t h lec t in so i m p e d i n g the i r u p t a k e b y t h e h e m o c y t e s (Rowley a n d Ratcliffe, 1980). O t h e r

Tab

le 6

Influ

ence

of

Ca

2+

and

Pur

ifie

d E

xta

toso

ma

tia

ratu

m (

Et)

Lect

in o

n Sp

ont

ane

ous

Ro

setti

ng o

f Ra

bb

it Er

ythr

oc

yte

s (R

E) a

roun

d L

ive

and

Fi

xed

Et

Hem

ocyt

e M

ono

laye

rs

Liv

e he

moc

ytes

0 F

ixed

hem

ocyt

es*

Tre

atm

ent

1 T

reat

men

t 2

PL

S

G c

ells

O

vera

ll P

L

SG

cel

ls

Ove

rall

1. T

BS

/CA

2+

RE

/Ca

2+

11.3

± 2

.7*

57.9

±

4.0

18.2

± 4

.1

9.4

2.3*

48

.5

3.7

14.8

2.

6

2.

TB

S/N

o C

a2

+ R

E/N

o C

a2

+ 3.

8 ±

1.2

24.6

±

2.7

6.8

± 2.

7 2.

0 0.

6 12

.0

1.3

4.2

± 1.

3 3.

E

t le

ctin

/Ca2

+ R

E/C

a2

+ 43

.7 ±

5.4

74

.2

± 5.

7 45

.0

± 5.

3 40

.9

5.9

73.3

5.

1 42

.6 ±

5.5

4.

Et

lect

in/N

o C

a2

+ R

E/C

a2

+ 16

.1 ±

4.6

61

.0

± 5.

8 24

.0

± 5.

6 15

.1 ±

4.2

60

.8 ±

5.6

22

.4 ±

4.1

5.

E

t le

ctin

/Ca2

+ 20

0 m

M D

-gal

acto

se

RE

/Ca2

+ N

D'

ND

N

D

10.4

± 3

.6

48.7

± 5

.2

15.4

± 4

.7

Cat

egor

ies

coun

ted:

pla

smat

ocyt

es (

PL),

spre

adin

g gr

anul

ar (

SG

) cel

ls,

all

cell

type

s (O

vera

ll).

*Mea

n of

8 v

alue

s ±

SD

. 'N

D =

not

don

e.

Mod

ified

fro

m R

icha

rds

and

Ratc

liffe

(19

90)

with

per

miss

ion

of P

erga

mon

Pre

ss.


factors t h a t g rea t ly inf luence test p a r t i c l e - h e m o c y t e a s soc ia t ion i n c l u d e w h e t h e r t h e test pa r t i c l e o r t he h e m o c y t e m o n o l a y e r s a r e p re sens i t i zed w i t h lect in a n d the e x t e n t to w h i c h t h e h e m o c y t e s a r e flattened in t h e m o n o l a y e r s . R i c h a r d s a n d Ratcliffe (1990) found t h a t t h e r e is a g r e a t e r o p s o n i c effect w h e n t h e h e m o c y t e s r a t h e r t h a n t h e tes t pa r t i c l e s a r e sens i t ized w i t h t h e lec t in , wh i l e excessively flattened h e m o c y t e s m a y have too few r e c e p t o r s p e r u n i t surface a r e a to b i n d test pa r t i c l e s effectively.

If, as s eems likely, insect h e m o l y m p h a g g l u t i n i n s d o func t ion a s r ecogn i t ion mo lecu le s , t he q u e s t i o n of h o w they i n t e r ac t w i t h t h e h e m o c y t e surface to form b r i d g i n g molecu les a t t a c h i n g t h e foreign pa r t i c l e s to t h e b lood cells n e e d s to b e a d d r e s s e d . N a i v e insec ts c o n t a i n h e m o l y m p h a g g l u t i n i n s a n d yet these mo lecu le s r e m a i n in t h e c i r cu l a t i on r a t h e r t h a n a t t a c h i n g to t h e b lood cells. S u b s e q u e n t e x p o s u r e a n d a t t a c h m e n t to foreign a n t i g e n s m u s t i n d u c e s o m e sor t of modi f i ca t ion in t he lect in so t h a t it c a n i n t e r ac t w i t h c a r b o h y d r a t e moie t i e s on t h e h e m o c y t e surface. Al te rna t ive ly , t h e modif ied lect in m a y expose a p e p t i d e s e q u e n c e for w h i c h the cell h a s a r ecep to r . T h e w o r k of K o m a n o et al. (1981) on t h e lect in i n d u c e d in in ju red S. peregnna l a rvae m a y b e r e l evan t h e r e . T h e y r e p o r t e d t h a t t he lect in consis ts of α a n d β s u b u n i t s a n d p o s t u l a t e d t h a t s o m e of t he α s u b u n i t s a r e c o n v e r t e d to t he β s u b u n i t s by p a r t i a l p ro teo lys i s to form the act ive lect in w i t h t he s t r u c t u r e of α 4 β 2 . T h i s ac t ive m o l e c u l e m a y poss ib ly be a b l e to b i n d to t he o u t s i d e of t h e h e m o c y t e s a n d t h e p ro teo lys i s involved in p r o d u c i n g t h e act ive m o l e c u l e m a y resu l t f rom t h e re lease of se r ine p r o t e a s e s involved in ac t iva t ion of t h e p P O c a s c a d e ( rev iewed in A s h i d a a n d Y a m a z a k i , 1990; Ratcliffe, 1991).

R e g a r d i n g t h e role of t h e p P O c a s c a d e in i m m u n e r ecogn i t i on a n d o t h e r a s p e c t s of i m m u n e r egu l a t i on , it is m u c h m o r e difficult to o b t a i n defini t ive p r o o f for t h e func t ion ing of p P O in insec t i m m u n i t y b e c a u s e of t h e c o m p l e x ity of t h e sys t em. T h e p P O s y s t e m cons is t s of a c a s c a d e of e n z y m e s a n d a s soc i a t ed factors s u c h as va r ious p l a s m a r e c e p t o r s , se r ine p r o t e a s e s , a n d i n h i b i t o r s (Fig . 5) ( J o h a n s s o n a n d S o d e r h a l l , 1989a; A s h i d a a n d Y a m a z a k i , 1990; Ratcliffe, 1991), w h i c h a r e often difficult to purify in a s t ab l e fo rm.

T h e p P O s y s t e m h a s b e e n r e p o r t e d f rom b o t h t he b lood cells ( A s h i d a et aL, 1988) a n d p l a s m a (Sau l et al., 1987). T h i s difference p r o b a b l y r e su l t s f rom t h e ins tab i l i ty of t he h e m o c y t e s of m a n y insec ts t h a t r ead i ly d i s c h a r g e the i r c o n t e n t s following b l eed ing . I t is a l so s ignif icant t h a t t h e g r a n u l a r cell t ype ( = cys tocy te in s o m e species) m a y c o n t a i n n o t on ly a g g l u t i n i n s b u t a l so c o m p o n e n t s of t h e p P O s y s t e m (Fig . 4) ( rev iewed in Rowley et al., 1986). T h i s cell t y p e r a p i d l y d e g r a n u l a t e s in con t ac t w i t h non-se l f m a t e r i a l s ( R a t cliffe a n d Rowley, 1979) to re lease t h e non-se l f r ecogn i t i on mo lecu l e s . I n Bombyx mori, however , t h e oenocy to id s a n d p l a s m a t o c y t e s c o n t a i n t h e major i ty of t h e p P O a n d t h e g r a n u l a r cells d o n o t have a p p r e c i a b l e a m o u n t s of th is e n z y m e ( A s h i d a et al., 1988). T h e s e differences a r e difficult to reconc i le ,


PLASMA CELL •RECEPTORS" MEMBRANE? COAGULOGEN COAGULIN

LPS • • W

B1.3-GLUCANS— X

PEPTIDOGLYCAN - Y

CAPSULAR Ζ POLYSACCHARIDES

SPONTANEOUS ACTIVATION

(eg. low Ca2+,heat etc.)

• / • /

OPSONISATION KILLING CELL DEGRANULATION IMMUNE HAEMOPOIESIS? MOVEMENTS PROTEINS

Figure 5 Hypothetical scheme for activation of arthropod prophenoloxidase (pPO). LPS = lipopolysaccharide; P O = phenoloxidase. Based largely on the work of Soderhall, Ashida, and colleagues. See text for references. Modified from Ratcliffe (1991) with permission of CRC Press Inc.

a l t h o u g h t h e ma jo r i t y of insec t species do not c o n t a i n oenocy to id s a n d w o u l d r e q u i r e a n a l t e r n a t i v e si te for s y n t h e s i s / s t o r a g e of p P O .

E v i d e n c e for p a r t i c i p a t i o n of p P O in insect i m m u n i t y is r ev iewed elsew h e r e (Ratcliffe, 1991). T h e m o s t conv inc ing p roof is p r o v i d e d by a ser ies of in vitro p h a g o c y t o s i s e x p e r i m e n t s s imi l a r t o t hose d e s c r i b e d b y S m i t h a n d S o d e r h a l l (1983) for c r u s t a c e a n h e m o c y t e m o n o l a y e r s over la id w i t h a m a r i n e bac te r i a l i so la te , Moraxella. H e m o c y t e m o n o l a y e r s of Galleria mellonella, Leuco-phaea maderae, a n d Blaberus craniifer w e r e set u p a n d over la id w i t h t h e b a c t e r i u m Bacillus cereus, p l u s p - l , 3 - g l u c a n , d e x t r a n , l i p o p o l y s a c c h a r i d e ( L P S ) , a n d a p o t e n t i n h i b i t o r of ser ine p r o t e a s e ( p - N P G B ) . T h e P - l ,3 -g lucan a n d L P S , w h i c h a r e b o t h m i c r o b i a l cel l -wall c o m p o n e n t s a n d ac t iva to r s of p P O , e n h a n c e d p h a g o c y t o s i s of B. cereus five- to sixfold (Tab l e 7) (Ratcliffe et al., 1984; L e o n a r d et al., 1985a) . T h e s e e x p e r i m e n t s s e e m to i n d i c a t e t h a t s ince p P O g e n e r a t e s s t icky p r o t e i n s t h a t a d h e r e to foreign i n v a d e r s ( S o d e r h a l l et al., 1979), t h e n the tes t bac t e r i a b e c o m e coa t ed in s t icky o p s o n i c m a t e r i a l t h a t is r ecogn ized as foreign by t h e p h a g o c y t i c cells. T h e s e resu l t s , however , c o n t r a s t w i t h t h e w o r k of D u l a r a y a n d Lack ie (1985) , w h o failed to i n d u c e a n e n c a p s u l a t i o n r e s p o n s e in t he locus t Schistocerea gregaria, in w h i c h S e p h a r o s e b e a d s w e r e p r e c o a t e d w i th p h e n o l o x i d a s e a n d a t leas t four a d d i t i o n a l p r o te ins g e n e r a t e d b y t h e p P O c a s c a d e . A g a i n , t h e r e is n o r e a d y e x p l a n a t i o n for this c o n t r a d i c t i o n in e x p e r i m e n t a l resu l t s excep t t h a t t h e p u t a t i v e o p s o n i n


Table 7 Effect of Laminarin on Phagocytosis of Bacillus cereus by Hemocytes from Three Different Insect Species

Treatment % Hemocytes

phagocytic % Plasmatocytes

phagocytic

% Cystocytes/ Number of granular cells bacteria/100

phagocytic hemocytes

G. mellonella Laminarin (1 mg m l " 1 ) 8.0 ± 1.8** 14.1 ± 3.7* 4.3 ± 1.4* 17.4 ± 3.1* GIM control' 1.2 ± 0.7 2.0 ± 0.6 0.7 ± 0.5 1.8 ± 0.9 0.01 mA//>NPGB/ 1.3 ± 0.5 2.4 ± 1.0 0.7 ± 0.4 2.2 ± 1.1 Laminarin + />NPGB 1.5 ± 0.6 2.8 ± 1.6 0.5 ± 0.4 2.2 ± 0.8 Dextran (1 mg m l " 1 ) 1.9 ± 0.6 2.7 ± 1.1 1.0 ± 0.5 2.6 ± 0.8

L. maderae Laminarin (1 mg m l " 1 ) 9.8 ± 1.6*>< 15.9 ± 4.3* 4.7 ± 1.6* 26.8 ± 6.8* L-J contro l 1.5 ± 0.7 2.2 ± 1.1 0.9 ± 0.8 2.2 ± 1.9 0 .01mM/>NPGB 0.9 ± 0.5 1.2 ± 0.6 0.6 ± 0.4 1.2 ± 0.9 Laminarin + />NPGB 1.4 ± 0.7 0.7 ± 0.6 1.2 ± 0.8 2.3 ± 1.7

B. craniifer Laminarin (1 mg m l " 1 ) 4.6 ± 0.6*^ 9.6 ± 1.4* 1.2 ± 0.4* 10.7 ± 1.8* Carlson's control 0.7 ± 0.3 1.2 ± 0.4 0.6 ± 0.4 1.5 ± 0.6 0.01 mAfj&NPGB 0.9 ± 0.2 1.2 ± 0.4 0.8 ± 0.4 1.3 ± 0.5 Laminarin + />NPGB 1.1 ± 0.2 1.5 ± 0.6 0.8 ± 0.3 1.6 ± 0.5

"Mean value of 20 monolayers (from a total of 28 insects in four experiments) ± SD. bP ^ 0.05 compared with controls. c Mean value of 10 monolayers (from a total of 14 insects in two experiments) ± SD. ^Mean value of 10 monolayers (from a total of 8 insects in two experiments) ± SD. 'GIM = Grace's insect medium. //>NPGB = />-nitrophenyl-/>'-guanidinobenzoate. *L-J = Landureau and Jolles medium.

From Leonard et al. (1985a) with permission of Pergamon Press.

m a y b e ve ry shor t - l ived a n d lost o r i nac t i va t ed b y t h e e x p e r i m e n t a l p r o c e d u r e a d o p t e d b y D u l a r a y a n d Lack ie (1985) .

T h e mu l t i f unc t i ona l ro le of t h e insec t g r a n u l a r h e m o c y t e s ( = cys tocytes ) m u s t a l so b e c o n s i d e r e d s ince these cells c o n t a i n n o t on ly factors a s soc i a t ed w i t h t h e p P O s y s t e m b u t a lso a g g l u t i n i n s (Fig . 4) (Rowley et al., 1986). D e g r a n u l a t i o n of t h e h e m o c y t e s in r e s p o n s e to i n v a d i n g o r g a n i s m s will t h u s re lease c o m p o n e n t s of t h e p P O c a s c a d e as well as t h e a g g l u t i n i n s t h a t h a v e b e e n s h o w n to ac t as r ecogn i t i on mo lecu le s (see t h e foregoing) . E v e n t h e e x p e r i m e n t s d e s c r i b e d p rev ious ly u t i l i z ing p r e sens i t i z a t i on of h e m o c y t e s o r tes t pa r t i c l e s w i t h pur i f ied lect in ( R i c h a r d s a n d Ratcliffe, 1990) d o n o t p r o -


v ide defini t ive p r o o f for a n o p s o n i c role for h e m o l y m p h a g g l u t i n i n s s ince they cou ld b e i n t e r p r e t e d in t e r m s of t he lect in s t i m u l a t i n g t he re lease of t h e p P O to e n h a n c e t he u p t a k e of t he foreign pa r t i c l e s . A d d i t i o n a l w o r k is t h u s urgen t ly r e q u i r e d , p re fe rab ly us ing puri f ied molecu les , to d e t e r m i n e t he re la tive roles of t he p P O a n d agg lu t in in s as well as surface c h a r g e ( rev iewed in Lack ie , 1988) in t h e i m m u n e reac t iv i ty of insec ts .

Recent ly , c lues to t he re la t ive roles of p P O a n d h e m o l y m p h a g g l u t i n i n s have b e c o m e ava i l ab le . F i rs t , R i c h a r d s a n d Ratcliffe (1990) s h o w e d in t h e p h a s m i d Extatosoma tiaratum t h a t s imi l a r p e r c e n t a g e s of e r y t h r o c y t e r o se t t i ng a r o u n d the h e m o c y t e s were i n d u c e d by the pur i f ied p h a s m i d lect in in b o t h live a n d g lu t a ra ldehyde - f ixed m o n o l a y e r s (Tab le 6) . I n o t h e r w o r d s , a t t a c h m e n t of foreign pa r t i c l es to t he h e m o c y t e s was n o t d e p e n d e n t u p o n t h e re lease of s t icky p P O p ro t e in s from these cells. T a b l e 6 a lso shows t h e i m p o r t a n c e of C a 2 + in th is in i t ia l b i n d i n g o r r ecogn i t ion p rocess (for a d e t a i l e d d i scuss ion of poss ib le inf luence of C a 2 + in l e c t i n - m e d i a t e d r ecogn i t i on in i n v e r t e b r a t e s , see R i c h a r d s a n d R e n w r a n t z , 1991).

Second , H . D u r r a n t , N . A. Ratcliffe, a n d C . C . C h e n ( u n p u b l i s h e d obser va t ions ) r e p o r t e d t h a t lec t ins i so la ted from the h e m o l y m p h of Blaberus discoidalis s ignif icant ly e n h a n c e d the p h e n o l o x i d a s e ac t iva t ion of h e m o c y t e lysa te p r e p a r a t i o n s ( H L S ) of this cockroach by the m i c r o b i a l a c t i va to r l a m i n a r i n (a 3 - l , 3 -g lucan) (Tab le 8) . O n e of t he lec t ins , t h e so-cal led p l a s m a factor ( P F ) , was P - l ,3-glucan-specif ic , w h e r e a s t h e o t h e r w a s g a l a c t o s e / glucose-specif ic . T h e P F w a s r e p o r t e d p rev ious ly to e n h a n c e l a m i n a r i n act i va t i on of Blaberus p h e n o l o x i d a s e (Sode rha l l et aL, 1988), b u t on ly by m o d ification of t he pur i f ica t ion p r o c e d u r e was its a g g l u t i n i n ac t iv i ty r evea led ( C . C . C h e n , N . A . Ratcliffe, a n d A. F . Rowley, u n p u b l i s h e d resu l t s ) . I n t e r estingly, p r e i n c u b a t i o n of t he two Blaberus lect ins w i t h the i r specific l i g a n d s u g a r s failed to a b r o g a t e the i r p h e n o l o x i d a s e - a c t i v a t i n g p r o p e r t i e s . Possibly, these lect ins have m u l t i p l e d o m a i n s , as d e s c r i b e d for t he Limulus factor C ( M u t a et aL, 1991), w i th t he s u g a r - b i n d i n g a n d p h e n o l o x i d a s e - a c t i v a t i n g d o m a i n s b e i n g s e p a r a t e . I n a d d i t i o n , v a r i o u s he t e ro logous p l a n t lec t ins s u c h as c o n c a n a v a l i n A , Tetragonolobus purpureas, a n d Bandeiraea simplicifolia a lso e n h a n c e d l a m i n a r i n ac t iva t ion of the Blaberus p h e n o l o x i d a s e , w h e r e a s severa l con t ro l p ro t e in s s u c h as t h y r o g l o b u l i n , a ldo lase , l y sozyme , a n d bov ine s e r u m a l b u m i n d id n o t ( H . D u r r a n t , N . A. Ratcliffe, a n d C . C . C h e n , u n p u b l i s h e d resul t s ) (Tab le 8) .

T h e l a t t e r e x p e r i m e n t s , a l t h o u g h on ly p re l imina ry , a r e i n t e r e s t i n g as t hey h e l p to exp la in t he a p p a r e n t p r e s e n c e of two i m m u n e r ecogn i t ion s y s t e m s in insec ts a n d o t h e r a r t h r o p o d s . I n fact, t h e r e m a y be on ly o n e non-se l f r ecogn i t ion scena r io w i t h ea r ly even t s con t ro l l ed b y the h e m o l y m p h a g g l u t i n i n s , as sugges t ed or ig ina l ly in Fig . 1, a n d l a t e r even ts involv ing the p P O c o m p l e x .


Table 8 Enhancement of Activation of Blaberus discoidalis Prophenoloxidase by Laminarin in the Presence of Various Lectins

Maximum phenoloxidase activity" at optimal Activation mixture lectin/protein concentration ΔΑ490 10 m i n - 1

HLS* + cac. buffer 0.040 ± 0.012

HLS + lam< 0.111 ± 0.022

HLS + L2d 0.100 0.015

HLS + L2 + lam 0.184 ± 0.029

HLS + pf< 0.119 ± 0.016

HLS + pf + lam 0.206 ± 0.033

HLS + con A 0.087 ± 0.017

HLS + con A + lam 0.157 ± 0.022

HLS + lysozyme/ 0.033 ± 0.015

HLS + lysozyme + lam 0.115 ± 0.020

"Assay carried out in multiwell trays and read on a Biorad EIA reader. *Hemocyte lysate supernatant at protein concentration of 2.0 mg m l - 1 . cLaminarin 1.0 mg m l - 1 . dh2 = purified B. discoidalis serum lectin. 'pf = purified B. discoidalis plasma factor (a β-1,3^1υΰ3η-8ρεαίΐΰ lectin). /Protein control.

From H. Durrani, N. A. Ratcliffe, and C. C. Chen (unpublished results).

T h e de t a i l s of t he i n t e r ac t i on of these t w o sets of mo lecu le s awa i t fu r the r inves t iga t ion .

T h i r d , a d d i t i o n a l ev idence for t h e i n t e r ac t i on of t h e h e m o l y m p h lec t ins a n d t h e p P O s y s t e m is p r e s e n t in t h e l i t e r a t u r e . T h u s M i n n i c k et al. (1986) pur i f ied a glucose-specif ic lec t in f rom t h e t o b a c c o h o r n w o r m Manduca sexta, t e r m e d Μ 1 3 , a n d showed t h a t w h e n a d d e d to h e m o l y m p h of t h e s a m e species it p r o v o k e d a c o a g u l a t i o n r e s p o n s e . T h e p P O sys t em m a y well b e involved in c o a g u l a t i o n r e s p o n s e s in insec ts as in s o m e o t h e r a r t h r o p o d s . Also s igni f icant w a s t h e w o r k of D u n p h y a n d G h a d w i c k (1989) w i t h G. mellonella h e m o c y t e s in vitro. T h e y s t u d i e d t h e inf luence of v a r i o u s c a r b o h y d r a t e s o n t h e a d h e s i o n of t h e b a c t e r i a Pseudomonas aeruginosa a n d Proteus mirabilis to t h e h e m o c y t e s a n d n o t e d t h a t c e r t a i n s u g a r s e n h a n c e d b o t h bac t e r i a l a d h e s i o n a n d p P O ac t iva t ion . As a resu l t of t hese e x p e r i m e n t s , D u n p h y a n d C h a d w i c k (1989) sugges t ed t h a t b o t h t he lec t ins a n d t h e p P O s y s t e m a r e o p s o n i c a n d e n h a n c e t h e b i n d i n g of b a c t e r i a to t h e b lood cells.


C Cell—Cell Communication in Immunity

A n o t h e r a r e a of insec t i m m u n i t y in n e e d of fu r the r c lar i f ica t ion is t h e de te r m i n a t i o n of t h e re la t ive roles a n d in t e r ac t i ons of t h e i m m u n e reac t ive cells . T h e w a x m o t h , Galleria mellonella, h a s p roved to b e a n idea l sub jec t for th is w o r k b e c a u s e of t h e ease of ident i f ica t ion a n d re la t ive s tab i l i ty of t h e b l o o d cells. I n Galleria, a n d p r o b a b l y o t h e r insec ts too , t h e t w o m a i n b lood cell types r e spons ib l e for t he ce l lu la r defenses a r e t he g r a n u l a r cells ( = cys tocy tes of s o m e species) a n d t h e p l a s m a t o c y t e s , a l t h o u g h t h e oenocy to id s a r e a lso a si te of s t o r a g e / s y n t h e s i s of p P O ( S c h m i t et aL, 1977). T h e g r a n u l a r cells (see F ig . 4) c o n t a i n b o t h p P O a n d a lso p r o b a b l y a g g l u t i n i n s (Ratcliffe a n d Rowley, 1987), a r e n o n p h a g o c y t i c , u n s t a b l e in vitro, a n d c lear ly d i s t i n g u i s h a b l e f rom the p l a s m a t o c y t e s , w h i c h a r e m a i n l y a g r a n u l a r , p h a g o c y t i c , a n d h igh ly s t ab l e in vitro. T h e fact t h a t t he Galleria p l a s m a t o c y t e s a r e u sua l ly a g r a n u l a r c o n t r a s t s w i t h m a n y o t h e r insec t species (e.g. , cock roaches , locus t s , o t h e r l e p i d o p t e r a n s , etc .) in w h i c h t h e p r e s e n c e of n u m e r o u s g r a n u l e s often m a k e s it difficult to d i s ce rn t h e m from g r a n u l a r cells a n d cys tocy tes . I n a d d i t i o n , g r a n u l e d i s c h a r g e f rom t h e p l a s m a t o c y t e s e n h a n c e s t h e difficulty of u n d e r s t a n d i n g the even t s involved d u r i n g ce l lu la r i m m u n i t y in insec t s .

Ea r l i e r s tud ie s by Ratcliffe a n d G a g e n (1977) a n d S c h m i t a n d Ratcliffe (1977) n o t e d t h a t in Galleria l a rvae , d u r i n g n o d u l e formation a n d e n c a p s u l a t ion in r e s p o n s e to b a c t e r i a a n d foreign i m p l a n t s , respect ively, t h e first a n d i m m e d i a t e (wi th in a few seconds usua l ly ) r e s p o n s e to foreign a n t i g e n s is t h e d e g r a n u l a t i o n of t h e fragile g r a n u l a r cells. P l a s m a a g g l u t i n i n s too m a y well b e involved in th is in i t ia l foreign b o d y - g r a n u l a r cell r ecogn i t i on even t (see Sec t ion I I I . B ) . T h e g r a n u l a r cells t h u s re lease the i r p u t a t i v e non-se l f r ecogn i t ion molecu le s , t h a t is, t he p P O factors a n d a g g l u t i n i n s , o n t o t h e surface of t h e foreign m a t e r i a l , w h i c h often t h e n beg ins to m e l a n i z e . W i t h i n 1 0 - 1 5 m i n t h e s econd i m m u n e reac t ive cell t ype in Galleria, t h e p l a s m a t o c y t e s , a r r ives a t t h e scene a n d forms a mu l t i c e l l u l a r s h e a t h a r o u n d t h e c e n t r a l m e l a n o t i c core of d e g r a n u l a t e d a n d lysed g r a n u l a r cells. E v e n t s s imi l a r to these have a lso b e e n obse rved in t h e st ick insec t Clitumnus extradentatus, t h e locus t Schistocerca gregaria, a n d the bee t le Tenebrio molitor (Ratcliffe a n d Rowley, 1979), so t h a t t hey m a y b e w i d e s p r e a d in insec ts . T h u s , cell—cell c o o p e r a t i o n is involved d u r i n g ce l lu la r reac t iv i ty a n d p r o b a b l y occu r s d u r i n g b o t h c a p sule f o rma t ion a n d p h a g o c y t o s i s in insec ts (Fig . 6) (see t h e fol lowing).

M o r e recent ly, a d d i t i o n a l s tud ie s on ce l l - ce l l c o o p e r a t i o n h a v e b e e n u n d e r t a k e n w i t h pur i f ied p o p u l a t i o n s of Gallena g r a n u l a r cells a n d p l a s m a t o c y t e s ( A n g g r a e n i a n d Ratcliffe, 1991). T h e h e m o c y t e s w e r e pur i f ied o n c o n t i n u o u s Percol l g r a d i e n t s a n d m o n o l a y e r s p r e p a r e d f rom t h e s e p a r a t e d p l a s m a t o c y t e s o r g r a n u l a r cells o r from a m i x t u r e of t h e two . T h e m o n o l a y e r s


w e r e t h e n over la id w i t h hea t -k i l l ed Bacillus cereus e i t he r s u s p e n d e d in buffer a l o n e (cont ro l s ) o r w i t h l a m i n a r i n (a 3 - l , 3 -g lucan ac t iva to r of p P O — expe r i -m e n t a l s ) a n d i n c u b a t e d for 1 h r to al low p h a g o c y t o s i s to occur . T h e resu l t s con f i rmed p l a s m a t o c y t e s a s t h e m a i n p h a g o c y t i c cell t y p e in Galleria; n o inges t ion a t all w a s r e c o r d e d for t he g r a n u l a r cells. P h a g o c y t o s i s b y t h e p l a s m a t o c y t e s i n c r e a s e d f rom 5.3 to 8 . 7 % for con t ro l s (i .e. , w i t h o u t l a m i n a r i n ) a n d f rom m o r e t h a n 13.4 to 3 0 % for e x p e r i m e n t a l s ( i .e . , w i t h l a m i n a r i n ) u p o n a d d i t i o n of g r a n u l a r cells back to t h e i so la t ed p l a s m a t o c y t e s ( T a b l e 5) . A n inc r ea se in p h a g o c y t o s i s f rom 5.3 to 1 3 . 4 % b y p l a s m a t o c y t e s u p o n a d d i t i o n of l a m i n a r i n w a s p r o b a b l y d u e to a s m a l l c o n t a m i n a n t p o p u l a t ion of g r a n u l a r cells. T h e s e resu l t s c lear ly show t h a t t he g r a n u l a r cel ls , a l t h o u g h n o t p h a g o c y t i c t hemse lves , c o n t a i n s o m e factor(s) ( p r o b a b l y p P O a n d / o r a g g l u t i n i n ) essen t ia l for o p t i m a l func t ion ing of t h e p l a s m a t o c y t e s d u r i n g ce l lu la r react ivi ty.

S i m i l a r ce l l - ce l l coope ra t i ve even t s h a v e b e e n d e s c r i b e d d u r i n g ce l lu la r i m m u n e even t s w i t h c r u s t a c e a n b lood cells in vitro (Sode rha l l et al., 1986; Pe r s son et al., 1987) (Fig . 6) a n d t h e role of a 7 6 - k D a p r o t e i n i n ce l lu la r c o m m u n i c a t i o n even t s h a s b e e n ident i f ied ( J o h a n s s o n a n d Soderhall, 1985, 1989a; K o b a y a s h i et al., 1990). T h i s 7 6 - k D a p r o t e i n is a s soc i a t ed w i t h t h e p P O s y s t e m a n d is mu l t i f unc t i ona l s ince it ac ts as a cell a d h e s i o n factor for crayf ish s e m i g r a n u l a r a n d g r a n u l a r cells ( J o h a n s s o n a n d S o d e r h a l l , 1988), c a n d e g r a n u l a t e b o t h s e m i g r a n u l a r a n d g r a n u l a r cells b y a r e g u l a t e d exo-cytos is ( J o h a n s s o n a n d Soderhall, 1989b) , a n d a l so func t ions to p r o m o t e e n c a p s u l a t i o n ( K o b a y a s h i et al., 1990). I n insec ts , l i t t le is k n o w n a b o u t m o lecu l a r even t s con t ro l l i ng ce l l - ce l l c o m m u n i c a t i o n , a l t h o u g h R a n t a m a k i et al. (1991) p a r t i a l l y pur i f ied a 9 0 - k D a p r o t e i n f rom Blaberus craniifer h e m o c y t e s t h a t c ros s - r eac t ed w i t h a monospec i f i c a n t i s e r u m a g a i n s t t h e 7 6 - k D a p r o t e i n f rom crayf ish b lood cells. T h i s 9 0 - k D a p r o t e i n , like t h e 7 6 - k D a m o l e c u l e in crayf ish , a l so func t ions to e n h a n c e a d h e s i o n of t h e cock roach h e m o c y t e s to t h e s u b s t r a t u m a n d to i n d u c e d e g r a n u l a t i o n of these cells .

O t h e r factors , a p a r t f rom t h e 9 0 - k D a p r o t e i n , a r e likely to inf luence c e l l -cell c o m m u n i c a t i o n a n d t h e b e h a v i o r of insec t h e m o c y t e s d u r i n g i m m u n e r eac t ions . S o m e of these s u b s t a n c e s have b e e n d e s c r i b e d in d e t a i l b y Lack i e (1988) a n d i n c l u d e a n e n c a p s u l a t i o n - p r o m o t i n g factor ( E P F ) ( R a t n e r a n d V i n s o n , 1983; D a v i e s et al., 1988), c h e m o k i n i n s ( H u x h a m a n d Lack ie , 1988)j a n d v a r i o u s w o u n d factors (e.g. , C h e r b a s , 1973). All of these s u b s t a n c e s a r e p r o b a b l y involved in t h e in i t i a t ion of i m m u n i t y by w o u n d i n g b u t , a p a r t f rom E P F , l i t t le is k n o w n a b o u t t hese mo lecu le s . E P F h a s b e e n p a r t i a l l y pur i f ied f rom t h e p l a s m a of Heliothis virescens a n d is a s m a l l 3 .5 -kDa , h e a t - s t a b l e p e p t i d e poss ib ly o r i g i n a t i n g f rom the h e m o c y t e s . T h e func t ion of E P F is to p r o m o t e t h e a d h e s i v e b e h a v i o r a n d e n c a p s u l a t i o n ab i l i ty of p l a s m a t o c y t e s in

Insects C r u s t a c e a n s

S T A G E 1 1. Microbial contact

with granular cells

ζ^^>~ bacterium

2. Degranulation and opeonieation by 90kDa protein

bacterium

S T A G E 2

3. Ingestion by phagocyte

S T A G E 1 1. Microbial contact with

semigranular cells

2. Degranulation

c m

S T A G E _ 2

3. Degranulation of granular(a) and further semigranular(b) cells and opsonisation by 76kDa protein

S T A G E

4 . Ingestion by Phagocyte


vitro (Dav ies et al., 1988). E P F m a y poss ib ly b e p r o d u c e d b y ac t iva t ion of t h e p P O c a s c a d e , as m a y the c h e m o k i n i n s (Lackie , 1988).

D. Interrelationship of Cellular and Humoral Immunity

I t is a p p a r e n t f rom p r e v i o u s sec t ions t h a t t he d iv is ion of insec t i m m u n i t y i n t o ce l lu la r a n d h u m o r a l c o m p o n e n t s is a m a t t e r of c o n v e n i e n c e r a t h e r t h a n of fact. T h u s , p l a s m a a g g l u t i n i n s a r e de r ived from the fat b o d y a n d / o r h e m o cytes ( T a k a h a s h i et al., 1985; Rowley et al., 1986) a n d re leased i n t o t h e p l a s m a , w h e r e t hey u n d e r t a k e the i r i m m u n e r e g u l a t o r y role t h a t r esu l t s in t h e b i n d i n g of foreign a n t i g e n s a n d a s soc ia t ed a g g l u t i n i n s o n t o t h e h e m o c y t e surface (see Sec t ion I I I . B ) . L ikewise , t he p P O s y s t e m is, a t l eas t par t ia l ly , s y n t h e s i z e d in t h e h e m o c y t e s a n d re leased b y d e g r a n u l a t i o n in to t h e p l a s m a . V a r i o u s i nh ib i t o r s a n d ac t iva to r s con t ro l l i ng t he p P O s y s t e m in a r t h r o p o d s have , however , b e e n i so la ted f rom the p l a s m a ( H e r g e n h a h n et al., 1987, 1988; S o d e r h a l l et al., 1988) so t h a t a c o m p l e x i n t e r p l a y b e t w e e n p l a s m a a n d ce l lu la r c o m p o n e n t s con t ro l s t h e p P O c a s c a d e . Add i t iona l ly , t h e a n t i b a c te r ia l p r o t e i n s have recen t ly b e e n s h o w n in Drosophila a n d o t h e r species to b e syn thes i zed no t on ly in t he fat b o d y b u t a l so in t he h e m o c y t e s (D ick in son et al., 1988; M a t s u y a m a a n d N a t o r i , 1988; Samakov l i s et al., 1990) for re lease i n t o t h e h e m o l y m p h to form t h e h u m o r a l c o m p o n e n t of t h e i m m u n e re sponse .

T h e r e is c o m p e l l i n g ev idence p r o v i d e d by h e m o c y t e t ransfe r e x p e r i m e n t s w i t h Galleria mellonella l a rvae for a role for insec t b lood cells in i n d u c t i o n of a h u m o r a l a n t i b a c t e r i a l r e s p o n s e (de V e r n o et al., 1983, 1984). I n t h e first of these s t u d i e s , h e m o c y t e s f rom Galleria p rev ious ly i m m u n i z e d w i t h Pseu-domonas aeruginosa e n d o t o x i n ( L P S ) w e r e in jected i n t o na ive l a rvae before c h a l l e n g i n g these w i th live P. aeruginosa a t va r i ous t i m e in t e rva l s . G o o d p r o -

Figure 6 Two hypothetical models for cell—cell cooperation in arthropod immunity. T h e insect model is based on observations with Galleria mellonella in which it is proposed that non-self recognition is carried out by the granular cells and ingestion by the plasmatocytes (phagocytes). The crustacean model differs from the insect scheme in having an amplification step at stage 2 in which adjacent granular and semigranular cells are induced to discharge their p P O system. Since most insects have many more granule-containing cells than Galleria, the crustacean scheme may well be present in such species. Circulating agglutinins are probably involved in enhancing contact and binding of the microbial invaders to the surfaces of the granular /semigranular cells at stage 1. Based on the work of Ratcliffe et al. (1984), Soderhall and Smith (1986), Johansson and Soderhall (1988), and colleagues. See text for additional references. Modified from Ratcliffe (1991) with permission of CRC Press, Inc.


t ec t ion w a s afforded b y the t r ans fe r red h e m o c y t e s p r o v i d e d t h a t t hey w e r e in jected in to t h e l a rvae w i t h i n 4 h r of L P S inocu l a t i on of t h e d o n o r s . H e m o cytes from n o n - L P S - i m m u n i z e d l a rvae p r o v i d e d n o p r o t e c t i o n to r ec ip i en t s cha l l enged w i t h P. aeruginosa. D e V e r n o et al. (1983) bel ieve these resu l t s show t h a t t h e h e m o c y t e s a r e act ive in t he i n d u c t i o n of h u m o r a l i m m u n i t y ea r ly in t h e i m m u n e r e s p o n s e a n d m a y b e re leas ing factor(s) to s t i m u l a t e t h e fat b o d y cells to p r o d u c e a n t i b a c t e r i a l p r o t e i n s . Faye a n d W y a t t (1980) a n d A b u -H a k i m a a n d Faye (1981) a lso no t i ced a n i n t i m a t e a s soc ia t ion in vitro b e t w e e n h e m o c y t e s c o n t a i n i n g bac t e r i a a n d the fat b o d y cells f rom Cecropia p u p a e , a n d sugges t ed t h a t t he p h a g o c y t i c g r a n u l a r cells m o v e to a n d d i s i n t e g r a t e in t h e reg ion of t he fat body. I n s u b s e q u e n t e x p e r i m e n t s , D e V e r n o et al. (1984) u s i n g Galleria fat b o d y in vitro a lso showed t h a t m a x i m a l p r o d u c t i o n of a n t i bac t e r i a l act ivi ty w a s s t i m u l a t e d by the p r e s e n c e of L P S a n d / o r h e m o c y t e s . T h e y sugges t ed t h a t t he L P S causes t he re lease of h e m o c y t e factor(s) resu l t ing in e n h a n c e d p r o d u c t i o n of a n t i b a c t e r i a l p r o t e i n s by t h e fat body.

S imi l a r e x p e r i m e n t s u t i l iz ing t he fat b o d y of Manduca sexta ( D u n n et al., 1985) a n d Hyalophora cecropia (T renczek a n d Faye , 1988) p r o d u c e d a d d i t i o n a l s u p p o r t for a role for t he h e m o c y t e s in i n d u c i n g t he syn thes i s of a n t i b a c t e r i a l p r o t e i n s . I n Manduca, D u n n et al. (1985) sugges t ed t h a t f r a g m e n t s of p e p -t idog lycan l i be ra t ed by d iges t ion of t he bac t e r i a by p l a s m a ly sozyme or following phagocy tos i s by t he h e m o c y t e s m a y serve as el ic i tors of p r o t e i n p r o d u c t i o n . T h i s conc lus ion m a y differ s l ight ly f rom t h a t j u s t d e s c r i b e d for Galleria, as t he el ici tor is de r ived f rom the b a c t e r i a a n d n o t f rom t h e h e m o cytes , a l t h o u g h d e V e r n o et al. (1984) d id r e p o r t t h a t a n t i b a c t e r i a l ac t iv i ty cou ld a lso b e s t i m u l a t e d by L P S a lone . Similar ly, w i t h Hyalophora fat b o d y cu l t u r e s , T r e n c z e k a n d Faye (1988) r e p o r t e d t h a t a n t i b a c t e r i a l p r o t e i n p r o d u c t i o n w a s s t i m u l a t e d by L P S a n d bac te r i a . T h e y a lso showed , however , t h a t h e m o c y t e s from in ju red or i m m u n i z e d p u p a e a n d n o t t hose f rom u n t r e a t e d insec ts h a d a n el ici tor effect.

M o s t of t he s tud ie s d e s c r i b e d c lear ly i n d i c a t e a role for t h e h e m o c y t e s in t he i n d u c t i o n of t h e h u m o r a l a n t i b a c t e r i a l p r o t e i n s . T h e fact t h a t t h e p rocess of i n ju r ing t he insec ts d u r i n g d i ssec t ion m a y elicit a n t i b a c t e r i a l p r o t e i n synthesis shows , however , t h a t s ignals o t h e r t h a n those p r o d u c e d t h r o u g h h e m o c y t e - p r o c e s s e d bac te r i a l s u b s t a n c e s m u s t b e involved ( T r e n c z e k a n d Faye , 1988; Samakov l i s et al., 1990). I t m a y be of s ignif icance t h a t pep t idog ly -c a n , L P S , a n d p- l , 3 -g lucan n o t on ly i n d u c e a n t i b a c t e r i a l p r o t e i n p r o d u c t i o n ( D u n n et al., 1985; Samakov l i s et al., 1990) b u t a lso ac t iva te t h e p P O s y s t e m . T h e l a t t e r too is e n h a n c e d b y h e m o l y m p h lect ins ( H . D u r r a n t , N . A. R a t cliffe, a n d C . C . C h e n , u n p u b l i s h e d resul t s ) so t h a t a n e x t r e m e l y c o m p l i c a t e d n e t w o r k is g r a d u a l l y e m e r g i n g t h a t m a y involve m i c r o b e s , a g g l u t i n i n s , p P O , h e m o c y t e s , d a m a g e d t issues , a n d fat b o d y cells. T h e s i t u a t i o n is fu r the r


c o m p l i c a t e d by t h e poss ib le i n v o l v e m e n t of t h e e n d o c r i n e s y s t e m m e d i a t i n g i m m u n e reac t iv i ty even t s d u r i n g t h e life of t h e insec t (Sh i ra i sh i a n d N a t o r i , 1989).

Final ly , Faye (1990) h a s r e p o r t e d a poss ib le c o n n e c t i o n b e t w e e n t h e r ecogn i t ion of b a c t e r i a a n d the ac t iva t ion of t h e i m m u n e p r o t e i n genes in Hy-alophora. T h i s p rocess involves t he i m m u n e p r o t e i n h a e m o l i n , p r ev ious ly ca l led P4 , w h i c h is p r e s e n t in n o r m a l h e m o l y m p h a n d b i n d s to t h e b a c t e r i a l surface in vitro to form a c o m p l e x w i t h a n o t h e r h e m o l y m p h p r o t e i n w i t h a m a s s of a b o u t 125 k D a ( S u n et al., 1990). T h i s c o m p l e x m a y func t ion to de l iver a r ecogn i t i on s igna l to t h e cells r e spons ib l e for t h e syn thes i s of t h e i m m u n e p r o t e i n s . T h e r e a s o n h a e m o l i n is be l ieved to func t ion in i m m u n e r ecogn i t i on a n d ac t iva t ion is, first, b e c a u s e it b i n d s to t h e bac t e r i a l surface a n d , s econd , b e c a u s e it h a s four i m m u n o g l o b u l i n d o m a i n s a n d s h a r e s 3 8 % h o m o l o g y w i t h t he i m m u n o g l o b u l i n p a r t of t h e insect cell r ecogn i t i on m o l e cu le , n e u r o g l i a n (Faye , 1990).

E. Future Areas for Study

F r o m this br ie f overview, it is a p p a r e n t t h a t , w i t h t he a p p l i c a t i o n of m o d e r n b i o c h e m i c a l a n d m o l e c u l a r t e c h n i q u e s , p rog re s s in o u r u n d e r s t a n d i n g of insec t i m m u n i t y h a s b e e n acce l e ra t ing . T h i s p r o g r e s s will n o d o u b t c o n t i n u e in t h e a r e a s o u t l i n e d in t h e foregoing. I n a d d i t i o n , as k n o w l e d g e h a s a c c r u e d of h o w i n v e r t e b r a t e s d e a l w i t h i n v a d i n g p a r a s i t e s a n d p a t h o g e n s , a t t e n t i o n is b e g i n n i n g to focus on m o r e a p p l i e d a spec t s of t h e i m m u n e syst e m s of i n v e r t e b r a t e s . T h u s , t h e poss ib le i m p o r t a n c e of t h e hos t defenses as d e t e r m i n a n t s of infectivi ty in v e c t o r - p a r a s i t e e n c o u n t e r s a n d t h e u s e of c h a n g e s in i m m u n e reac t iv i ty as m o n i t o r s of e n v i r o n m e n t a l po l l u t i on a r e n o w b e i n g e x a m i n e d .

R e g a r d i n g v e c t o r - p a r a s i t i c i n t e r ac t i ons , ev idence h a s b e e n a c c u m u l a t i n g for t h e i m p o r t a n c e of t he vec to r hos t defenses in d e t e r m i n i n g t h e o u t c o m e of s u c h a s soc ia t ions ( rev iewed in M o l y n e u x et al., 1986; Ratcliffe a n d Rowley, 1987; K a a y a , 1989). For e x a m p l e , Co l l ins et al. (1986) r e p o r t e d t h a t in s t r a i n s of Anopheles gambiae ref ractory a n d suscep t ib l e t o w a r d Plasmodium cynomolgi, t h e r e w a s a different ial ab i l i ty of t h e hos t m o s q u i t o e s to e n c a p s u l a t e t h e ook ine t e s in t h e m i d g u t wal l . T h e refractory s t r a in e n c a p s u l a t e d t h e p a r a s i t e s in a l ayer of m e l a n i n l i k e s u b s t a n c e w h e r e a s in suscep t ib l e insec t s on ly a r e d u c e d r eac t ion o c c u r r e d (Paskewi tz et al., 1989). I t w a s sugges t ed t h a t re f rac tor iness is d u e to a g r e a t e r ab i l i ty of t he hos t to r ecogn ize t h e l iv ing p a r a s i t e s . I t is poss ib le t h a t t h e m e l a n i n l i k e s u b s t a n c e r e su l t ed f rom en h a n c e d levels of p P O in t he ref rac tory s t r a in . F u r t h e r m o r e , in s t r a i n s of t h e t se tse flies Glossina palpalis palpalis a n d Glossina morsitans morsitans, w h i c h a r e


ref rac tory a n d suscep t ib l e to Trypanosoma brucei rhodesiense, respect ively, p P O act iv i ty in t h e h e m o l y m p h a n d m i d g u t h a s a lso b e e n s h o w n to b e far g r e a t e r in t h e r e s i s t an t flies. E v e n m o r e s ignif icant were differences b e t w e e n t h e sexes of G. m. morsitans in w h i c h the females show 7 % a n d m a l e s 2 7 % sa l iva ry g l a n d infect ions ( I . M a u d l i n a n d S. C . W e l b u r n , p e r s o n a l c o m m u n i c a t i o n ) . T h e females h a d m u c h h i g h e r levels t h a n the m a l e s of p P O ac t iva t ion of t he h e m o l y m p h w h e n p a r a s i t e s w e r e u sed as el ici tors (Tab le 9) ( N i g a m a n d Ratcliffe, 1991; Y. N i g a m , N . A . Ratcliffe, I . M a u d l i n , a n d S. C . W e l b u r n , u n p u b l i s h e d obse rva t i ons ) . T h e c o n t r i b u t i o n , if any, of t h e h e m o c y t e s to these resu l t s h a s n o t yet b e e n d e t e r m i n e d . A d d i t i o n a l work is c lear ly r e q u i r e d on the role of p P O i m m u n e recogn i t ion in these vec to r spec ies .

T h e i m p o r t a n c e of lect ins in t he g u t a n d h e m o l y m p h of t se tse flies in t h e t r a n s m i s s i o n of t r y p a n o s o m e s h a s a lso b e e n r e p o r t e d (Pere i ra et al., 1981; I b r a h a m et al., 1984; M o l y n e u x et al., 1986; M a u d l i n a n d W e l b u r n , 1987, 1988a ,b ; I n g r a m a n d M o l y n e u x , 1988; W e l b u r n a n d M a u d l i n , 1989, 1990; W e l b u r n et al., 1989). T h e s e worke r s have d e t e c t e d a g g l u t i n i n s for t r y p a n o s o m e s in t h e g u t a n d h e m o l y m p h of tse tse flies a n d bel ieve t h a t t hese m o l e cules a r e i m p o r t a n t for t h e d e v e l o p m e n t of t he p a r a s i t e in t he vec to r insec t . T h u s , a ref rac tory l ine of G. m. morsitans k i l l ing 5 0 % of t r y p a n o s o m e s in 170 h r h a d h i g h e r t i te rs of m i d g u t lec t ins t h a n a suscep t ib l e l ine ki l l ing on ly 1 0 % of p a r a s i t e s in th is t i m e ( W e l b u r n et al., 1989). T h e y c o n c l u d e d t h a t t h e lect in p r e v e n t e d t r y p a n o s o m e e s t a b l i s h m e n t in t he m i d g u t p e r h a p s by lysis of t h e p a r a s i t e s . T h e fact t h a t b lock ing the lect in w i t h its s u g a r l i gand , D -g lu -c o s a m i n e , s ignif icant ly i nc r ea sed m i d g u t infect ion r a t e s of Trypanosoma con-

Table 9 Phenoloxidase Activity of Male and Female Flies of Susceptible G. morsitans morsitans 1/6 Strain

Treatment of Incubation time (hr)

hemolymph 0.25 0.5 1.0 2.0 4.0 6.0 8.0

Control 4 1 male 0 . 2 5 ^ 0.34 0.79 1.10 1.16 1.26 1.33

Control* female 1.37 2.19 2.85 3.02 3.18 3.49 3.57

Procyclics*" male 0.17 0.32 0.90 1.23 1.29 1.29 1.33

Procyclics*" female 1.31 2.23 2.98 3.50 3.73 4.09 4.28

a Controls contained buffer, hemolymph, and L-dopa. ^Measured as units of phenoloxidase where 1U — 1A492 m g - 1 protein. c Means of three experiments but standard deviations not given here.

''Procyclics of Trypanosoma brucei rhodesiense in buffer incubated with hemolymph and L-dopa.

From Y. Nigam, N. A. Ratcliffe, I. Maudlin, and S. C. Welburn (unpublished results).


golense a n d T. brucei rhodesiense s u p p o r t s th is i dea ( M a u d l i n a n d W e l b u r n , 1987). C o n c o m i t a n t l y , however , it h a s b e e n r e p o r t e d t h a t t h e m i d g u t lec t in a n d a h e m o l y m p h lect in in t se tse a r e involved in m a t u r a t i o n of t r y p a n o s o m e s ( W e l b u r n a n d M a u d l i n , 1989, 1990). T h i s a p p a r e n t d u a l ro le for t h e lec t ins i n d i c a t e s t h e c o m p l e x i t y of this v e c t o r - p a r a s i t e a s soc ia t ion .

A d d i t i o n a l ev idence for a role of t h e vec to r i m m u n e s y s t e m as a d e t e r m i n a n t of infect ivi ty is p r o v i d e d b y h e m o l y m p h t ransfe r e x p e r i m e n t s w i t h s im-ul i ids (blackflies) ( H a m , 1986) a n d b y n u m e r o u s r e p o r t s of t h e e n c a p s u l a t i o n of p a r a s i t e s in m o s q u i t o e s ( rev iewed in Ratcliffe, 1982; K a a y a , 1989). H a m (1986) d e s c r i b e d t h e p r e s e n c e of a p a r a s i t e - i n d u c e d or -de r ived factor in t h e h e m o l y m p h of s imul i id s infected w i t h Onchocerca lienalis t h a t confers p r o t e c t ion w h e n t r ans fe r r ed to na ive flies. E x a m p l e s of e n c a p s u l a t i o n in m o s q u i t o e s i n c l u d e a r e p o r t b y C h r i s t e n s e n a n d F o r t o n (1986) t h a t t h e in i t ia l s t a g e in e n c a p s u l a t i o n of Dirofilana immitis microf i la r iae in jected in to Aedes aegypti w a s t h e lysis of h e m o c y t e s a t o r n e a r t h e surface of t h e p a r a s i t e p r i o r to d e p o s i t i o n of a m e l a n i n l i k e p i g m e n t , a n d the o b s e r v a t i o n of C h e n a n d L a u r e n c e (1985) t h a t t h e e n c a p s u l a t i o n of Brugia pahangi microf i la r iae in Anopheles quadri-maculatus involved b o t h h u m o r a l a n d ce l lu la r even t s .

O n c e t h e de ta i l s of t he vec to r i m m u n e reac t ions a r e fully u n d e r s t o o d , it m i g h t b e poss ib le to m a n i p u l a t e t h e m a n d inc rea se t h e re f rac tor iness of t h e h o s t insec t s to p a r a s i t i c i nvas ion . T h i s m i g h t involve t h e i n se r t i on of specific genes to i nc r ea se t h e p o t e n c y of t h e vec to r i m m u n e r e s p o n s e to p r o d u c e ref rac tory s t r a i n s of insec ts ( K a a y a , 1989).

Final ly , t h e fact t h a t t h e i m m u n e sy s t ems of i n v e r t e b r a t e s a r e exquis i t e ly sens i t ive a n d c o n s t a n t l y s a m p l i n g a n d r e s p o n d i n g to m i n u t e q u a n t i t i e s of e n v i r o n m e n t a l mo lecu le s m e a n s t h a t a n y c o n t a m i n a n t s p r e s e n t a r e likely to modify i m m u n i t y in s o m e way. I f s u c h modi f i ca t ions c a n b e careful ly d o c u m e n t e d t h e n it m a y b e poss ib le to u se t h e i n v e r t e b r a t e i m m u n e s y s t e m s as m o n i t o r s of e n v i r o n m e n t a l po l l u t i on . For e x a m p l e , M i o s k y et al. (1989) in m o l l u s k s h a v e d e m o n s t r a t e d t h e v a l u e of m o n o c l o n a l a n t i b o d i e s in m o n i t o r i n g t h e i n c r e a s e d i n c i d e n c e of a l eukemic l ike c o n d i t i o n in t h e soft-shell c l a m , Mya arenaria, co l lec ted f rom p o l l u t e d w a t e r s of M a i n e . W i t h insec ts , un fo r tuna te ly , l i t t le is k n o w n of t h e ac t ion of xenob io t i c s o n t h e i m m u n e s y s t e m . Feir (1979) briefly r ev i ewed i n f o r m a t i o n o n insec t i m m u n e r e sponses to tox ic s u b s t a n c e s s u c h as insec t ic ides , wh i l e S a x e n a a n d S a x e n a (1985) d e s c r i b e d c y t o p a t h o l o g i -cal c h a n g e s in t h e h e m o c y t e s of Periplaneta americana after e x p o s u r e to t h e pes t i c ide , m a l a t h i o n . M o r e recent ly , J o n e s et al. (1989) a lso showed t h a t t h e a n t i b a c t e r i a l ac t iv i ty of t h e h e m o l y m p h of t h e l e p i d o p t e r a n Pieris brassicae w a s s ignif icant ly s u p p r e s s e d b y e x p o s u r e to 2 ,4 ,5 - t r i ch lo rophenoxyace t i c ac id (2 ,4 ,5 -T) . Clear ly , m o r e w o r k is r e q u i r e d in o r d e r to d e t e r m i n e t h e effects of e n v i r o n m e n t a l in su l t s o n a r a n g e of i m m u n e p a r a m e t e r s .


I am grateful to the Nuffield Foundation, The British Council, and The Science and Engineering Research Council (Grants GR/F/17421 , GR/G/60857 , and GR/G/40224) for financial support.

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Chapter 13

The Response to Foreign Tissue Transplants in Insects Richard D. Karp Department of Biological Sciences University of Cincinnati Cincinnati, Ohio

I. Introduction: What Is the Significance of Studying Graft Rejection in Insects?

II. Historical Perspective A. The Encapsulation Response

to Foreign Objects B. The Response to the Transplantation

of Foreign Tissue C. The Issue of Immunologic Memory

III. Mediation and Regulation of the Roach Graft Response A. Inhibition of Graft Rejection

by Gamma Irradiation B. The Effects of Eicosinoids on the

Xenograft Response

IV. Discussion and Conclusions: Where Do We Go from Here?


I. Introduction: What Is the Significance of Studying Graft Rejection in Insects?

W h e n o n e p e r u s e s t he c o n t e m p o r a r y l i t e r a tu r e , it b e c o m e s q u i t e e v i d e n t t h a t t h e use of insec ts as m o d e l s for t he s t u d y of evo lu t ion of i m m u n i t y is i n c r e a s ing a t a t r e m e n d o u s r a t e . T h e i m p o r t a n c e of insec ts in r e s e a r c h is n o mys te ry , s ince they a r e t he d o m i n a n t a n i m a l life-form o n the p l a n e t , a n d therefore r e p r e s e n t o u r m a j o r c o m p e t i t o r in t he ecosys t em. T h e y no t on ly c o m p r i s e a c o n s t a n t t h r e a t to o u r food supply , b u t a l so ac t as vec to rs for m a n y m a j o r p a r a s i t i c d i seases t h a t d e v a s t a t e h u m a n s a r o u n d the wor ld , p a r t i c u l a r l y in t h i r d wor ld c o u n t r i e s . T h u s , a n y t h i n g we c a n l ea rn a b o u t h o w they func t ion , a n d in p a r t i c u l a r defend themse lves a g a i n s t t he m a n y insu l t s of n a t u r e , m i g h t give us s o m e ins igh t s as to h o w to deve lop m e t h o d s for the i r con t ro l . F r o m a p u r e l y b a s i c scientific p o i n t of view, insec ts n o t on ly r e p r e s e n t t h e m a j o r g r o u p of e x t a n t i n v e r t e b r a t e s , b u t a l so c o m p r i s e o n e of t h e few inve r t e -



3 0 6 Richard D. Karp

b r a t e r ep re sen t a t i ve s t h a t c a n be ra i sed successfully in t h e l a b o r a t o r y in n u m b e r s sufficient to b e used to des ign mean ing fu l m o l e c u l a r a n d gene t i c s tud ies to u n r a v e l s ignif icant b iological q u e s t i o n s . T h u s , t hey a r e a n o b v i o u s r e sou rce for d e v e l o p m e n t a l a n d c o m p a r a t i v e s tud ies w h o s e focus is to b e t t e r u n d e r s t a n d t he or ig ins of a d a p t i v e i m m u n i t y . O n e a p p r o a c h for s u c h s tud ie s is t he t r a n s p l a n t a t i o n of foreign t i ssue . Gra f t ing h a s b e c o m e a very a c c e p t e d w a y of s t u d y i n g ce l l -med ia t ed i m m u n i t y in i n v e r t e b r a t e s , a n d t h u s h a s b e e n wide ly used in e x p e r i m e n t a t i o n p r o b i n g t h e l imi ts of a d a p t i v e i m m u n i t y in these a n i m a l s . I t w a s on ly n a t u r a l t h a t such t e c h n i q u e s w o u l d b e a p p l i e d to insec ts in a n effort to l e a rn t he ex t en t of the i r defense capab i l i t i e s . W h a t follows in this c h a p t e r is t he i n t e r e s t i ng h i s to ry of t h e s t u d y of graft r e s p o n siveness in insec ts a n d the conc lus ions t h a t c a n be d r a w n from the resu l t s .

II. Historical Perspective

Insec t s a r e k n o w n to have several m a j o r ce l lu la r defense m e c h a n i s m s for d e a l i n g w i t h foreign i n v a d e r s . T h e s e i n c l u d e p h a g o c y t o s i s , n o d u l e f o r m a t i o n , a n d e n c a p s u l a t i o n . N o d u l e fo rma t ion is a s t r a t egy u s e d b y m a n y inve r t e b r a t e s to c o n t a i n l a rge n u m b e r s of bac t e r i a by e n t r a p p i n g t h e m w i t h h e m o cytes , w h e r e a s e n c a p s u l a t i o n is t h e use of h e m o c y t e s to wal l off objec ts t h a t a r e too l a rge to b e p h a g o c y t o s e d , such as n e m a t o d e p a r a s i t e s (as r ev iewed by Dav ie s a n d Siva-Jothy, 1991). T h e la t te r , a l o n g w i t h t h e t r a n s p l a n t a t i o n of foreign t i ssue , h a s b e e n used as a m e a n s of a s s ay in g for t h e abi l i ty of a n insec t to recognize non-se l f a n d r e s p o n d to this cha l l enge in a specific m a n n e r . T h e d iscovery of such respons iveness w o u l d be t a k e n as ev idence for t he ex i s tence of a t r u e i m m u n e r e s p o n s e in these a n i m a l s . Severa l l a b o r a t o r i e s have b e e n in t e re s t ed in this q u e s t i o n t h r o u g h t he yea r s . T h e following is a br ie f s u m m a ry of s o m e of t he va r ious a p p r o a c h e s t h a t have b e e n used to tes t w h e t h e r specific ce l l -med ia t ed re spons iveness exists in insec ts , a n d t h e conflicts t h a t a rose from the i n t e r p r e t a t i o n of t h e resu l t s g e n e r a t e d by these s t ud i e s .

A. The Encapsulation Response to Foreign Objects

T h e e n c a p s u l a t i o n r e sponse is a h e m o c y t e - m e d i a t e d act ivi ty t h a t essent ia l ly wal ls off l a rge pa r t i c l es f rom the res t of t h e h e m o c o e l a n d its c o n t e n t s . S ince it is c o m m o n l y a s s u m e d t h a t s o m e type of r ecogn i t ion even t in i t i a tes t he p r o cess , s o m e worke r s have ut i l ized e n c a p s u l a t i o n as a m e a n s of d e t e r m i n i n g if insec ts c a n recogn ize a n d reac t to foreign t i ssue . A class ic e x a m p l e of t h e u s e of this t e c h n i q u e is r e p r e s e n t e d by the s t u d y of C a r t o n (1976) in w h i c h insec t eggs of v a r y i n g fore ignness were i m p l a n t e d in to t he h e m o c o e l s of Pimpla

13. The Response to Foreign Tissue Transplants in Insects 3 0 7

instigator l a rvae . T h e m o s t impres s ive e n c a p s u l a t i o n r eac t ions w e r e i n d u c e d by t h e eggs t h a t w e r e t h e m o s t x e n o g e n e i c re la t ive to t he hos t . Al logene ic eggs a lso i n d u c e d a s ignif icant r e s p o n s e , b u t o n e t h a t w a s n o t a s i n t e n s e as t h a t for x e n o g e n e i c eggs . I t h a s b e e n a r g u e d t h a t t he r e s p o n s e to a l logene ic eggs w a s d u e to t he p r e s e n c e of foreign secre t ions o r v i rus l ike pa r t i c l e s coa t ing t h e eggs f rom p a r a s i t i z e d d o n o r s (Lack ie , 1983b) . Howeve r , t h e eggs u s e d in t h e C a r t o n s t u d y were d i s sec ted from Pimpla s t ock -cu l t u r ed a n i m a l s , a n d t h u s d i d n o t c o m e from p a r a s i t i z e d a n i m a l s . E v e n if t h a t w e r e n o t t h e case , severa l s t ud i e s have c lear ly s h o w n t h a t a s soc ia t ed v i rus l ike pa r t i c l e s o r p o l y d n a v i r u s e s t h a t a r e injected in to t h e hos t a l o n g w i t h t h e p a r a s i t o i d eggs ac tua l ly i n h i b i t t h e e n c a p s u l a t i o n r e s p o n s e r a t h e r t h a n s t i m u l a t e its i n d u c t ion (Sal t , 1964, 1965; E d s o n et aL, 1980; Rizki a n d Rizki , 1984; See o t h e r c h a p t e r s in this v o l u m e ) .

A l t h o u g h t h e use of e n c a p s u l a t i o n as a m e a n s of a s s a y i n g for t h e r ecogn i t ion of fore ignness w o u l d a p p e a r to b e a very s t r a i g h t f o r w a r d p roces s , n u m e r o u s r e p o r t s i n d i c a t e t h a t t he r e a r e m a n y va r i ab l e s t h a t exist t h a t m a y c loud t h e i n t e r p r e t a t i o n of t he d a t a . Severa l s tud ies have i n d i c a t e d t h a t t he ab i l i ty to m o u n t a n e n c a p s u l a t i o n r e s p o n s e c a n b e g rea t ly affected by species differences (Sal t , 1963; Lack ie , 1979; Lack ie et aL, 1985; Tackle , 1988). V a r i a t i o n s in r e spons iveness c a n b e d u e to differences in t h e n o r m a l n u m b e r of c i r cu la t ing h e m o c y t e s in a species (Lackie , 1979), t h e role of gene t i c h e t e r o g e n e i t y w i t h i n a species ( C a r t o n a n d B o u l e t r e a u , 1985), t h e d e g r e e of surface c h a r g e (Lack ie , 1983a; C h r i s t e n s e n et aL, 1987), a n d we t t ab i l i t y of t he i m p l a n t e d s u b s t a n c e s (Lack ie , 1983a) . T h u s , t h e inab i l i ty to g e n e r a t e a n e n c a p s u l a t i o n r e s p o n s e to foreign m a t e r i a l m a y b e d u e to i n n a t e species cha rac t e r i s t i c s t h a t h a v e n o t h i n g to d o w i t h t he p r e s e n c e o r a b s e n c e of a d a p t i v e i m m u n e m e c h a n i s m s .

Sco t t (1971) r e p o r t e d on a s t u d y in w h i c h e n c a p s u l a t i o n w a s u s e d to m o n i t o r t h e r ecogn i t i on of xenogene i c , a l logeneic , a n d syngene i c n e r v e co rd i m p l a n t s in t h e A m e r i c a n cockroach , Periplaneta americana. X e n o g e n e i c i m p l a n t s w e r e heavi ly e n c a p s u l a t e d , b u t a l logene ic i m p l a n t s w e r e e n c a p s u l a t e d on ly if t hey h a d b e e n exposed to p ro t eo ly t i c e n z y m e s p r i o r to i m p l a n t a t i o n . O n e i n t e r p r e t a t i o n of these resu l t s cou ld be t h a t t he a l l o a n t i g e n s p r e s e n t o n t h e ne rve co rd w e r e c ryp t i c a n d n o t ava i l ab le for r ecogn i t i on u n t i l e x p o s e d to t h e e n z y m a t i c t r e a t m e n t . I f th i s is t h e case , t h e n lack of r e spons iveness does n o t necessa r i ly m e a n t h a t t he c o r r e s p o n d i n g i m m u n e m e c h a n i s m does n o t exist .

T h e r e is n o q u e s t i o n t h a t t he e n c a p s u l a t i o n r eac t ion is a n essen t ia l w e a p on in t h e defensive a r s e n a l possessed by insec ts a n d serves as a h igh ly effective m e a n s of d e a l i n g w i t h i n v a d i n g o r g a n i s m s . Howeve r , b a s e d o n the re la tively long list of va r i ab l e s t h a t c a n affect its p e r f o r m a n c e , e n c a p s u l a t i o n leaves m u c h to b e des i r ed as a re l i ab le m e a n s of m o n i t o r i n g specific r ecogn i -

3 0 8 Richard D. Κα φ

t ion of fore ignness in s tud ies d e s i g n e d to d e t e r m i n e if a d a p t i v e ce l l -med ia t ed i m m u n i t y exists in insec ts .

B. The Response to the Transplantation of Foreign Tissue

A different a p p r o a c h t h a t h a s b e e n used w i t h s o m e r egu l a r i t y in t h e s t u d y of ce l l -med ia t ed i m m u n i t y in i n v e r t e b r a t e s is t he r e s p o n s e to foreign t i s sue grafts . T h e graf t ing s tud ies in insec ts have no t b e e n w i t h o u t the i r s h a r e of controversy. For i n s t a n c e , a l t h o u g h m o s t worke r s ag r ee t h a t insec ts a r e a b l e to r ecogn ize xenogra f t s , t h e r e is s o m e difference of o p i n i o n as to w h e t h e r these a n i m a l s c a n d i s c r i m i n a t e b e t w e e n a l logene ic differences. T h e r e h a s a lso b e e n a w ide r a n g e of e x p e r i m e n t a l a p p r o a c h e s e m p l o y e d to de t ec t recogn i t ion a n d respons iveness to foreign t i ssue , s o m e of w h i c h have b e e n q u e s t ioned as to the i r effectiveness a n d accuracy. T h e d a t a f rom these a s says have b e e n g a t h e r e d e i the r b y m a c r o s c o p i c o b s e r v a t i o n o r b y t h e u s e of h i s to logica l t e c h n i q u e s . T h i s h a s a d d e d to t h e confusion s ince t he l a t t e r is far m o r e sensi t ive t h a n the former , a n d t h u s one c a n a r r ive a t very different conc lu s ions d e p e n d i n g on h o w the d a t a a r e col lec ted.

I n t he ea r ly 1980s, severa l l abo ra to r i e s p u b l i s h e d s tud ie s r e p o r t i n g o n the t r a n s p l a n t a t i o n of i n t e g u m e n t b e t w e e n insec ts . T h o m a s a n d Ratcliffe (1982) graf ted va r ious species of insec ts a n d followed the i r p rog re s s by g ross obser va t ion of t h e t r a n s p l a n t s . C o l o r a t i o n , m e l a n i z a t i o n , a n d d i s i n t e g r a t i o n w e r e used as c r i te r ia for sco r ing re jec t ion. B a s e d o n those p a r a m e t e r s , xenogra f t s a p p e a r e d to b e r a p i d l y re jected a n d m e l a n i z e d , w h e r e a s al lograf ts a n d a u t o grafts were no t . However , w h e n a few of t he grafts were e x a m i n e d h i s to logically, it w a s found t h a t t he al lograf ts h a d s t i m u l a t e d ce l lu la r inf i l t ra t ion in t he graft b e d . T h u s , g ross o b s e r v a t i o n of t he al lograf ts in th is s t u d y m a y no t have b e e n sensi t ive e n o u g h to reveal a n a c c u r a t e p i c t u r e of w h a t w a s ac tua l ly o c c u r r i n g in t he hos t a n i m a l .

T h e t r a n s p l a n t a t i o n s tud ies of J o n e s a n d Bell (1982) re l ied solely o n h i s to logical e v a l u a t i o n of t he grafts . Periplaneta americana w a s u sed as t h e r ec ip i en t of i n t e g u m e n t a r y grafts f rom d o n o r s of v a r y i n g p h y l o g e n e t i c d i s t a n c e s f rom Periplaneta. Graf t s were s a m p l e d after va r ious pe r iods of t i m e a n d p r e p a r e d his to logical ly for e x a m i n a t i o n . T h e grafts w e r e scored b y s ta t i s t ica l ly a n a l y z ing the n u m b e r of h e m o c y t e s inf i l t ra t ing each graft s i te , a n d c o m p a r i n g th is to t he a m o u n t of inf i l t ra t ion u n d e r au togra f t con t ro l s . Periplaneta americana vigorous ly re jected xenograf t s f rom Blaberus, Nauphoeta, a n d Leucophaea. O f p a r t i c u l a r in te res t w a s t he fact t h a t t he m o r e sensi t ive h is to logica l p r o c e d u r e revea led a s ignif icant r e sponse to a closely r e l a t ed species , Periplaneta brunnea. J o n e s a n d Bell a l so c o m p a r e d the r e s p o n s e to al lograf ts as c o m p a r e d to au togra f t s , b u t found on ly a s l ight difference t h a t w a s no t s ta t i s t ica l ly signific a n t .

13. The Response to Foreign Tissue Transplants in Insects 3 0 9

Lack ie (1983b) a lso e m p l o y e d P. americana in h e r graf t ing s tud i e s b u t w a s u s i n g a q u i t e different a p p r o a c h for e v a l u a t i n g r e spons iveness . T h e expe r i m e n t a l de s ign w a s to graft foreign i n t e g u m e n t o n t o n y m p h s a n d follow acc e p t a n c e o r re jec t ion by d e t e r m i n i n g w h e t h e r t h e d o n o r t i ssue su rv ived t h e nex t m o l t of t he hos t . I f t h e d o n o r w a s n o t r ecogn ized as foreign, t h e u n r e -j e c t e d e p i d e r m a l l ayer u n d e r l y i n g t h e cut ic le w o u l d p r o d u c e m o r e cu t ic le of t h e d o n o r t y p e . T h i s w a s d e t e r m i n e d by o b s e r v i n g t h e color of t h e t r a n s p l a n t fol lowing t h e mol t : if t h e color w a s d a r k b r o w n ( the color of t h e Blatta orientalis d o n o r cut ic le) , t h e graft w a s scored as accep ted ; if t h e color w a s r e d d i s h b r o w n ( the color of t he Periplaneta americana hos t ) , t h e graft w a s scored as re jec ted . A l t h o u g h it w a s r e p o r t e d t h a t t h e x e n o g e n e i c i n t e g u m e n t f rom Blaberus craniifer w a s re jec ted , c u t i c u l a r t r a n s p l a n t s f rom t h e m o r e closely r e l a t e d Blatta orientalis w e r e no t . I t w a s c o n c l u d e d t h a t t h e ab i l i ty of t h e insec t to r ecogn ize non-se l f w a s r a t h e r weak . H o w e v e r , these resu l t s w e r e in d i r ec t conflict w i t h those of J o n e s a n d Bell (1982) , w h o conv inc ing ly d e m o n s t r a t e d , u s i n g t h e m o r e sensi t ive h is to logica l assay, t h a t P. americana cou ld signific a n t l y (0 .001 > Ρ > 0.005) reject i n t e g u m e n t a r y grafts f rom Bl. orientalis d o n o r s . O t h e r c i r c u m s t a n c e s t h a t m i g h t affect t h e i n t e r p r e t a t i o n of t h e re su l t s of t h e m o l t i n g a s say a r e : (1) As m u c h as 9 0 % of t he m o l t e d cut ic le m a y b e r e s o r b e d following e n z y m a t i c d iges t ion , a n d even tua l ly be i n c o r p o r a t e d in to t h e n e w cut ic le ( H e p b u r n , 1985). T h i s cou ld obv ious ly have a n effect o n t h e color of t he n e w cut ic le r ega rd l e s s of t h e o r ig in of t he e p i d e r m a l l ayer (2) T h e r e w e r e n o au tog ra f t con t ro l s p e r f o r m e d to e n s u r e t h a t Periplaneta cu t ic le w o u l d n o t t u r n d a r k b r o w n d u e to m e l a n i z a t i o n c a u s e d b y t h e t r a u m a of t h e gra f t ing p r o c e d u r e , t h u s g iv ing false pos i t ive resu l t s .

O u r in i t ia l s tud ie s on t r a n s p l a n t a t i o n reac t iv i ty in Periplaneta americana, u s i n g t h e quan t i f i c a t i on of inf i l t ra t ing h e m o c y t e s as a m e a s u r e of r e spons ive ness ( s imi la r to t h e p ro toco l of J o n e s a n d Bell , 1982), r evea led s t r o n g t r a n s p l a n t a t i o n i m m u n i t y a g a i n s t xenogra f t s , b u t a g a i n t h e r e w a s n o c lea r -cu t ev idence for t he p r e s e n c e of al lograf t i m m u n i t y ( G e o r g e et al., 1984). I t w a s o u r bel ief t h a t t h e p a r t i c u l a r a s s a y w e were u s i n g w a s n o t sens i t ive e n o u g h to d e t e r m i n e if a l lograf t i m m u n i t y exis ted in t h e roach , b e c a u s e it w a s b a s e d o n q u a n t i f y i n g t h e n u m b e r s of h e m o c y t e s in t h e graft b e d w i t h n o i n d i c a t i o n of w h e t h e r o r n o t t hey w e r e func t ion ing as i m m u n o c y t e s . W e n e e d e d a m o r e d i r ec t a p p r o a c h for t h e o b s e r v a t i o n of ac t iv i ty a g a i n s t foreign a l lograf ts . After r ev i ewing t h e d a t a from t h e p r e v i o u s s tudy , we o b s e r v e d t h a t al l t h e x e n o grafts w e r e m i s s i n g t h e s u b c u t i c u l a r e p i d e r m a l layer , w h i c h is la rge ly r e s p o n sible for t h e p r o d u c t i o n of t h e exoske le ton (Fig . 1A). H o w e v e r , au tog ra f t con t ro l s , even t h o u g h they m i g h t have s o m e h e m o c y t i c inf i l t ra te p r e s e n t , a l w a y s showed in t ac t e p i d e r m a l layers u n d e r n e a t h t h e cut ic le (Fig . IB ) .

A n e w gra f t ing s t u d y w a s u n d e r t a k e n to test for t h e ex i s tence of a l lograf t i m m u n i t y u s i n g t h e n e w c r i t e r ion of o b s e r v i n g t h e fate of t h e s u b c u t i c u l a r

310 Richard D. Κα φ

Figure 1 Scoring of graft rejection based on the fate of the subcuticular epidermal layer. (A) Five-day xenograft from Eublaberus distanti on Periplaneta americana. (B) Five-day autograft control from the same recipient as above. Note the absence of the subcuticular epidermal layer in the xenograft, whereas the epidermis is fully intact in the autograft control. C = cuticle; Ε = epidermal layer; F = fat body. Bar = 20 μπι.

13. The Response to Foreign Tissue Transplants in Insects 311

e p i d e r m a l l ayer as a d e t e r m i n a t i o n of w h e t h e r o r no t t h e t i ssue h a d b e e n re jec ted . T h e resu l t s of these s tud ie s ( G e o r g e et al., 1987) c lear ly i n d i c a t e d t h a t a l lograf ts w e r e r ecogn ized as foreign, s ince 9 2 % of t he a l logene ic ep ide r m a l l ayers h a d b e e n des t royed by d a y 7 p o s t t r a n s p l a n t a t i o n ve r sus on ly 8 % in au tog ra f t con t ro l s (Tab le 1). A l t h o u g h the al lograf t r e s p o n s e c a n b e cons ide red to be a c u t e , it is re la t ively s lower t h a n the r e s p o n s e to xenogra f t s (Tab le 1), w h i c h is t he s a m e p a t t e r n t h a t is obse rved in m a m m a l s . A s e c o n d s t u d y u s i n g i m p l a n t s seeded w i th a l logene ic h e m o c y t e s l abe led w i t h [ 3 H ] thym i d i n e con f i rmed n o t on ly t h a t a l lograf t i m m u n i t y exis ted in t h e r o a c h , b u t t h a t t h e r e s p o n s e to t he foreign t i ssue w a s cy to tox ic in n a t u r e (Howcrof t a n d K a r p , 1987). T h u s , t h e d i rec t o b s e r v a t i o n of t he fate of l iv ing foreign t i ssue t r a n s p l a n t e d i n t o t he A m e r i c a n cockroach i n d i c a t e d t h a t th is insec t w a s fully c a p a b l e of r ecogn iz ing a n d r eac t ing to a l logene ic differences.

C The Issue of Immunologic Memory

A long-e s t ab l i shed def ini t ion of t r u e a d a p t i v e i m m u n i t y is t h a t s u c h re sponses d i s p l a y b o t h specificity a n d i m m u n o l o g i c m e m o r y . We therefore w e r e i n t e r e s t ed in d e t e r m i n i n g if these i m p o r t a n t c r i t e r ia w e r e p r e s e n t in t h e r o a c h al lograf t r e s p o n s e . Fo r this p u r p o s e a n i m a l s rece ived filter p a p e r i m p l a n t s s eeded w i t h a l logene ic d o n o r h e m o c y t e s as a first-set graft . T h e filter p a p e r t e c h n i q u e w a s e m p l o y e d so t h a t t h e s a m e d o n o r cou ld be u s e d a g a i n . Seven d a y s la ter , t h e r ec ip ien t s rece ived a second-se t i n t e g u m e n t a r y al lograf t f rom

Table 1 Rejection of Foreign Integumentary Grafts by Periplaneta americana

Days posttransplantation

Percentage of graft s rejected a

Days posttransplantation Xenografts Autografts Allografts Autografts

1 94 10 38 15

3 100 16 60 9

5 100 6 80 9

7 100 11 92 8

10 — — 95 5

20 — — 93 7

"Scoring or rejection is based on the presence or absence of the subcuticular epidermal layer as determined by histological examination of the grafts. Values are an average of at least two trials with total sample sizes exceeding 30 animals (except for allograft days 10 and 20, which had sample sizes of 23 and 15, respectively). Data summarized from George et al. (1987).

312 Richard D. Κα φ

t h e s a m e d o n o r as well as a n o t h e r a l logeneic graft f rom a n u n r e l a t e d d o n o r as a t h i r d - p a r t y con t ro l . T h u s , we cou ld test for t he p r e s e n c e of b o t h specificity a n d m e m o r y . Graf t s were r e m o v e d after va r i ous p e r i o d s of t i m e a n d scored his to logical ly for t he p r e s e n c e o r a b s e n c e of t he s u b c u t i c u l a r e p i d e r m a l layer . T h e resu l t s ( H a r t m a n a n d K a r p , 1989) i n d i c a t e d t h a t t he second-se t a l lografts w e r e re jec ted in a n acce le ra t ed fashion, s ince p e a k reac t iv i ty oc c u r r e d in 3 d a y s , w h e r e a s t h i r d - p a r t y con t ro l s followed first-set re jec t ion k ine t ics . T h e s e resu l t s i n d i c a t e d t h a t no t on ly w a s t h e r e a t leas t s h o r t - t e r m m e m o r y p r e s e n t , b u t t h a t t he r e sponse w a s h igh ly specific s ince t h e r ec ip i en t r eac t ed to t he second-se t a n d t h i r d - p a r t y grafts in a differential m a n n e r . T h e rec ip ien t w a s t h u s ab l e to d i s t i ngu i sh b e t w e e n t w o different a l logene ic d o n o r s . T h u s , t h e al lograf t r e sponse in t h e A m e r i c a n cock roach d i sp l ays t h e h a l l m a r k s of t r u e a d a p t i v e i m m u n i t y .

III. Mediation and Regulation of the Roach Graft Response

We have successfully e s t ab l i shed t h a t t he A m e r i c a n cockroach c a n r ecogn ize a n d reject xenograf t s a n d al lograf ts . T h e nex t s t ep in o u r s tud ie s is to find ways to d e t e r m i n e h o w these r e sponses a r e m e d i a t e d a n d r e g u l a t e d . We h a v e b e e n deve lop ing s o m e tools t h a t m a y prove to b e p r o m i s i n g in th is r e g a r d .

A. Inhibition of Graft Rejection by Gamma Irradiation

I t is c o m m o n l y k n o w n t h a t t he use of X i r r a d i a t i o n will i nh ib i t a d a p t i v e i m m u n i t y in m a m m a l s d u e to t he fact t h a t these r e sponses h a v e a ce l lu la r bas i s . S ince gross o b s e r v a t i o n of graft b e d s in t r a n s p l a n t e d roaches revea led t h e p r e s e n c e of h e m o c y t e inf i l t ra tes , we w o n d e r e d if i r r a d i a t i o n w o u l d have a s imi l a r effect on t he roach r e sponse to foreign i n t e g u m e n t . In i t i a l exper i m e n t s revea led t h a t w h e n roaches were sub jec ted to 5000 r a d s of g a m m a i r r a d i a t i o n , t he n u m b e r of c i r cu l a t i ng h e m o c y t e s w a s r e d u c e d to o n e - t h i r d of n o r m a l . O n the bas i s of th is , we exposed rec ip ien t s to 5000 r a d s of r a d i a t i o n a n d t h e n graf ted t h e m w i t h xenograf t s a n d au togra f t s , e i the r o n t h e s a m e d a y as t h e r a d i a t i o n t r e a t m e n t o r 24 h r la ter . T h e grafts w e r e t h e n scored for re jec t ion after v a r i o u s pe r iods of t i m e p o s t t r a n s p l a n t a t i o n . T h e resu l t s i nd i ca t ed t h a t a n i m a l s rece iv ing t r a n s p l a n t s 24 h r after i r r a d i a t i o n , w h e n h e m o cyte n u m b e r s w e r e dep l e t ed , h a d the m a x i m u m r e s p o n s e to xenograf t s d e layed for 10 d a y s as c o m p a r e d to u n t r e a t e d con t ro l s ( E p p e n s t e i n e r a n d K a r p , 1989). T h e r e was a d i r ec t co r re l a t ion b e t w e e n t h e recovery of h e m o c y t e n u m b e r s a n d the recovery of t he full capab i l i t y to reject foreign t i ssue . T h i s ,


t a k e n w i t h p r e v i o u s d a t a i n d i c a t i n g t h a t l abe led h e m o c y t e s a r e n o t kil led w h e n i n c u b a t e d w i t h a l logene ic cell-free h e m o l y m p h (Howcrof t a n d K a r p , 1987), i n d i c a t e d t h a t graft re jec t ion in t h e r o a c h is c e l l -med ia t ed in n a t u r e . I n a d d i t i o n , t hese s tud ie s offer a n e w a p p r o a c h for d e t e r m i n i n g w h i c h t y p e of h e m o c y t e m e d i a t e s graft re jec t ion in t he roach . R o a c h e s cou ld b e i r r a d i a t e d a n d , 24 h r la ter , r e c o n s t i t u t e d w i t h different s u b p o p u l a t i o n s of c i r c u l a t i n g h e m o c y t e s f rom b o t h sens i t ized a n d nonsens i t i zed a n i m a l s to see w h i c h cell t y p e will fully r e s to re t h e r e s p o n s e to foreign graf ts . T h i s w o u l d p r o v i d e d i r ec t ev idence for t he ex i s tence of i m m u n o c y t e s in th is insec t .

B. The Effects of Eicosinoids on the Xenograft Response

P r o d u c t s of e icos inoid b i o s y n t h e t i c p a t h w a y s , s u c h as t he p r o s t a g l a n d i n s , a r e well k n o w n to have powerful effects on m a m m a l i a n i m m u n e r e s p o n s e s ( S t r o m et al., 1977; P l a u t , 1987). I t w a s of in t e res t to us to d e t e r m i n e if e icos inoids h a d s imi l a r effector o r r e g u l a t o r y effects o n t h e r o a c h i m m u n e r e s p o n s e . T h e a p p r o a c h w a s to t r e a t a n i m a l s w i t h d e x a m e t h a s o n e (dex) , w h i c h is a gene ra l i zed i n h i b i t o r of e icos inoids s ince it in terferes w i t h ar -a c h a d o n i c acid re lease , a n d obse rve w h a t effect t he i n h i b i t o r h a d o n x e n o graft re jec t ion in t he roach . A n i m a l s w e r e t r e a t e d w i t h two different doses of d e x e i t he r 24 h r p r i o r to graf t ing , s i m u l t a n e o u s l y w i t h graf t ing , o r 12 h r pos tg ra f t ing . A n i m a l s in jected w i t h 0.2 m g / m l of d e x showed a severely r e d u c e d c a p a b i l i t y for re jec t ing xenogra f t s , n o m a t t e r w h e n they rece ived t h e d e x ( K a r p et al., 1993). P re in jec ted a n i m a l s h a d on ly a 3 5 % r a t e of re jec t ion , s i m u l t a n e o u s h a d on ly 1 7 % , a n d pos t in jec ted h a d on ly 2 1 % , w h e r e a s u n t r e a t e d con t ro l s showed n o r m a l r a t e s of re jec t ion. H o w e v e r , a t a c o n c e n t r a t ion of 0.02 m g / m l , d e x showed a different p a t t e r n of resu l t s . W h e n t h e low d o s e w a s g iven pregraf t , d e x ac tua l ly e n h a n c e d graft re jec t ion w i t h a r a t e of 8 8 % . W h e n t h e low dose of d e x w a s g iven s i m u l t a n e o u s l y w i t h graf t ing o r 12 h r pos tgraf t , t h e effects w e r e s imi l a r to t h a t of t he h igh dose of d e x , in t h a t re jec t ion w a s o n c e m o r e i nh ib i t ed , w i t h r a t e s fall ing to 30 a n d 3 3 % , r e spec tively. By t h e use of d i r ec t cell c o u n t i n g , we k n o w t h a t d e x is n o t d i r ec t ly cy to tox ic to h e m o c y t e s a n d t h u s is n o t cu r t a i l i ng t h e graft r e s p o n s e in th is way. R a t h e r , it w o u l d s e e m t h a t d e x is s h u t t i n g d o w n the graft r e s p o n s e in s o m e u n d e t e r m i n e d r e g u l a t o r y way. T h e use of m o r e specific i nh ib i t o r s in t h e fu tu re will a l low us to identify w h e t h e r it is t he p r o d u c t s of t h e cyclooxy-g e n a s e o r l i poxygenase p a t h w a y s (or b o t h ) t h a t a r e involved in th is p h e n o m e n o n . I n a d d i t i o n , t h e differential effects of t h e low dose of d e x m i g h t a l low us to d issec t t h e r e s p o n s e i n t o its v a r i o u s c o m p o n e n t s (i .e. , r ecogn i t i on ve r sus effector func t ion) so t h a t w e m i g h t even tua l ly u n d e r s t a n d h o w insec t i m m u nocy te s work .

314 Richard D. Karp

IV. Discussion and Conclusions: Where Do We Go from Here?

T h e resu l t s f rom severa l different s tud ies conv inc ing ly i n d i c a t e t h a t long-lived insec ts , s u c h as cockroaches , have t he abi l i ty to r ecogn ize a n d reject xenograf t s . O u r ex tens ive s tud ies m a k e it very c lear t h a t roaches a lso possess t he abi l i ty to recognize a l logeneic differences a n d r e s p o n d to t h e m v ia m e c h a n i s m s t h a t d i sp l ay t he cha rac te r i s t i c s of t r ue a d a p t i v e ce l l -med ia t ed i m m u ni ty ( G e o r g e et aL, 1987; Howcrof t a n d K a r p , 1987). T h e rea l s ignif icance of these f indings goes b e y o n d the o b s e r v a t i o n t h a t th is a n i m a l h a s t he ab i l i ty to reject t r a n s p l a n t e d a l logeneic t i ssue . M o r e i m p o r t a n t l y , it impl ies t h a t roaches have evolved a su rve i l l ance m e c h a n i s m w h e r e b y they c a n recogn ize a n t i g e n i c c h a n g e s on the i r cells o r t i ssues t h a t have c o m e a b o u t t h r o u g h m u t a t i o n or s o m e o t h e r t ype of u n w a n t e d modi f ica t ion . S ince s u c h modi f icat ions m i g h t cons t i t u t e a t h r e a t to t he wel l -be ing of t h e o r g a n i s m , a func t ioning su rve i l l ance sys t em wou ld recognize these c h a n g e s a n d s t i m u l a t e defensive c o u n t e r m e a s u r e s to r emove t h e m .

O u r fu ture s tud ies will focus on the m e c h a n i s m s involved in t he c o c k r o a c h graft r e sponse . We need to identify t he cell type t h a t ac tua l ly m e d i a t e s t h e r e s p o n s e , a s well as define t he m o l e c u l a r c o m p o n e n t s , s u c h as r e c e p t o r s , t h a t a r e r e spons ib l e for r ecogn i t ion of fore ignness . T h e use of r a d i a t i o n c h i m e r a s a n d pass ive t ransfer s tud ie s , as m e n t i o n e d ear l ier , will hopeful ly p rove useful in ident i fy ing t h e i m m u n o c y t e t h a t is r e spons ib l e for graft re jec t ion in t he roach . T h e fact t h a t s u c h cells c a n specifically reject foreign grafts obv ious ly m e a n s t h a t a cell-surface r ecep to r m u s t b e involved in d i s t i n g u i s h i n g self f rom non-self. I n th is r e g a r d , r ecen t s tud ies in o u r l a b i n d i c a t e t h a t x e n o -graf ted roaches show a n inc reased i n d u c t i o n of a 102-kDa h e m o l y m p h p r o tein 14 d a y s p o s t t r a n s p l a n t a t i o n ( H a r j u a n d K a r p , 1993). T h e 102-kDa p r o tein b a n d is t he s a m e o n e t h a t is so closely a s soc ia t ed w i t h t h e a d a p t i v e h u m o r a l r e sponse in t he roach t h a t is i n d u c e d by t h e inject ion of so lub le p r o t e i n s ( D u w e l - E b y et aL, 1991). T h i s ra ises t h e poss ib i l i ty t h a t t h e m o l e cule a s soc ia t ed w i th h u m o r a l i m m u n i t y in t he roach m a y a lso serve as a cell-surface r e c e p t o r in ce l l -med ia t ed r e sponses . Fol low-up s tud ie s , n o w in p r o gress , will be ab l e to m a k e those d i s t inc t ions .

W i t h t h e p r o s p e c t s of so m a n y i m p o r t a n t issues n e e d i n g to b e a d d r e s s e d , t he fu ture looks very b r i g h t for i n v e r t e b r a t e t r a n s p l a n t a t i o n i m m u n o l o g y . We have excel lent a n i m a l m o d e l s a t o u r d i sposa l , such as insec ts , w h i c h c a n b e c u l t u r e d in the l a b , t h u s avo id ing the v ic iss i tudes of s t u d y i n g a n i m a l s from the wi ld . T h e t echno logy t h a t h a s b e e n deve loped a t t he cell a n d m o l e c u l a r levels is n o w fully access ible for u se in s tud ie s o n i n v e r t e b r a t e s . T h u s , w e s t a n d po ised to ask s o m e very soph i s t i ca t ed q u e s t i o n s c o n c e r n i n g t h e evolu-


t ion of a d a p t i v e i m m u n i t y , w i t h t h e e x p e c t a t i o n of s o m e very exc i t ing a n d

r evea l ing resu l t s to c o m e .

Acknowledgments

The work described in this chapter that has emanated from our laboratory was supported by NSF research grants PCM 8316140 and DCB 8702382, N I H research grant G M 39398, and the University of Cincinnati Foundation Fund for Comparative Immunology.

References

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Chapter 14

Regulation of Insect Hemolymph Phenoloxidases Manickam Sugumaran Department of Biology University of Massachusetts at Boston Harbor Campus Boston, Massachusetts

Michael R. Kanost Department of Biochemistry Kansas State University Manhattan, Kansas

I. Introduction

II. Prophenoloxidase Activation A. Prophenoloxidase B. Prophenoloxidase Activating Enzyme C. Prophenoloxidase Cascade D. Triggering of the Cascade E. Lipids as Modulators F. Compartmentalization G. Is the Prophenoloxidase System

a Recognition Mechanism?

III. Control Mechanisms of the Cascade A. Introduction B. Protease Inhibitors C. Prophenoloxidase Inhibitor D. Dopachrome Conversion Factor E. Quinone Isomerase

IV. Conclusion References

In sec t s d o n o t possess c o m p l i c a t e d i m m u n o g l o b u l i n s to tackle i n v a d i n g p a r a sites a n d o t h e r m i c r o o r g a n i s m s , b u t s e e m to have a w i d e s p e c t r u m of o t h e r defense m e c h a n i s m s . D u r i n g the defense reac t ion , t h e a t t a c k i n g foreign o b j e c t is often found e n c a p s u l a t e d a n d d a r k e n e d . T h e d a r k p i g m e n t is i n v a r i a bly a s s u m e d to b e m e l a n i n . T h e r e f o r e , m e l a n i n a n d the e n z y m e s t h a t a r e r e s p o n s i b l e for i ts syn thes i s , name ly , p h e n o l o x i d a s e s , a r e c o n s i d e r e d a s essent ial c o m p o n e n t s of t h e insec t ' s defense m e c h a n i s m . P h e n o l o x i d a s e u s u a l l y o c c u r s as a n inac t ive p r o e n z y m e in t h e h e m o l y m p h a n d is ac t iva t ed d u r i n g a n i m m u n e r e s p o n s e . S o m e a u t h o r s have even invoked p r o p h e n o l o x i d a s e as p a r t of t h e non-se l f r ecogn i t i on m e c h a n i s m s of insec t s . G i v e n s u c h a n i m p o r t a n t ro le in t h e insec t ' s defense s y s t e m , it is essen t ia l to u n d e r s t a n d t h e m o l e c u l a r m e c h a n i s m s of p h e n o l o x i d a s e ac t iva t ion , t h e fate of ac t i va t ed enz y m e , a n d the cou r se of its r eac t ions .

U p o n r ecogn i t i on of fore ignness , a specific, as yet un iden t i f i ed factor in t h e h e m o l y m p h s e e m s to be ac t iva t ed , w h i c h in t u r n ac t iva tes a n inac t ive se r ine



318 Manickam Sugumaran and Michael R. Kanost

p r o t e a s e . T h i s se r ine p r o t e a s e ac t iva tes a n o t h e r se r ine p r o t e a s e t h a t serves as t he d i r ec t ac t iva to r of inac t ive p r o p h e n o l o x i d a s e b y c leaving a p e p t i d e of MT

= 5000 f rom it. T h e ac t iva t ion of p r o p h e n o l o x i d a s e c a n a lso b e ach ieved b y c u t i c u l a r p ro t ea se ( s ) a n d by a n a l t e r n a t e r o u t e invo lv ing p h o s p h o l i p i d s . T h e nega t ive con t ro l s eems to be exe r t ed by se r ine p r o t e a s e i nh ib i t o r s a n d o t h e r less c h a r a c t e r i z e d b iochemica l s . Ac t i va t ed p h e n o l o x i d a s e g e n e r a t e s p r i m a r i ly q u i n o n e s by ac t ing on d i p h e n o l i c s u b s t r a t e s found in t h e h e m o l y m p h . Q u i n o n e s , b e i n g h igh ly cy to toxic , c a n d i rec t ly kill t he foreign o r g a n i s m o r u n d e r g o p o l y m e r i z a t i o n to form p a r t of t he c a p s u l e . N e w l y d i scovered iso-m e r a s e s , namely , q u i n o n e i s o m e r a s e a n d d o p a c h r o m e t a u t o m e r a s e , s e e m to con t ro l the q u i n o n o i d react iv i ty by conve r t i ng q u i n o n e s to q u i n o n e m e t h i d e s a n d p rov ide t h e m e i the r for de toxi f ica t ion o r for o t h e r r eac t ions . Reac t i ons of q u i n o n e s a n d q u i n o n e m e t h i d e s g e n e r a t e m e l a n i n a n d / o r sc lero t in t h a t forms p a r t of t he capsu l e . T h e b i o c h e m i s t r y of these p rocesses is briefly d i scussed .

I. Introduction

In sec t s a n d o t h e r a r t h r o p o d s d o no t possess i m m u n o g l o b u l i n s to tackle inv a d i n g p a r a s i t e s a n d o t h e r objec ts , yet t hey a r e a b l e to defend themse lves a g a i n s t foreign o r g a n i s m s by a va r i e ty of r eac t ions , s u c h as p h a g o c y t o s i s , e n c a p s u l a t i o n , n o d u l e fo rma t ion , a g g l u t i n a t i o n , a n d syn thes i s of a n t i b a c ter ia l p r o t e i n s ( W h i t c o m b et al., 1974; C o o m b e et al., 1984; G o t z a n d B o m a n , 1985; Ratcliffe et al., 1985; G o t z , 1986; C h r i s t e n s e n a n d N a p p i , 1988; G u p t a , 1988; Lack ie , 1988; B o m a n et al., 1991; S c h m i d t a n d T h e o p o l d , 1991). T h e c o m m o n defense reac t ion a g a i n s t p a r a s i t e s a n d o t h e r foreign objec ts t h a t a r e too l a rge to b e p h a g o c y t o s e d in t he insect h e m o c o e l is e n c a p s u l a t i o n by h e m o c y t e s . C e l l u l a r capsu le s t h a t a r e fo rmed by the h e m o c y t e s a r e often found d a r k e n e d a n d h a r d e n e d ( G u p t a , 1988). S imi l a r o b s e r v a t i o n s d u r i n g h u m o r a l e n c a p s u l a t i o n have a lso b e e n m a d e (Go tz , 1986). T h e c h e m i c a l n a t u r e of t he d a r k p i g m e n t w i th in t he c a p s u l e is yet to b e e s t ab l i shed , b u t i n v a r i a b l y r e sea r che r s a s s u m e t h a t th is s u b s t a n c e is m e l a n i n p r o d u c e d b y t h e p o l y m e r i z a t i o n of p h e n o l o x i d a s e - g e n e r a t e d q u i n o n e s ( G u p t a , 1988). T h e r e fore m e l a n i n a n d the e n z y m e t h a t is r e spons ib l e for its b iosyn thes i s , t h a t is , p h e n o l o x i d a s e , a r e cons ide red as i n t eg ra l p a r t s of t h e insec t ' s i m m u n e syst e m . In te res t ing ly , p h e n o l o x i d a s e s a r e p r e s e n t in b o t h cu t ic le a n d h e m o l y m p h a n d a r e k n o w n to p l ay a c ruc ia l role n o t on ly in m e l a n i z a t i o n , b u t a l so in sc le ro t iza t ion of insec t cut ic le ( S u g u m a r a n , 1988, 1991a) a n d w o u n d hea l ing (La i -Fook , 1966).

N u m e r o u s rev iew ar t ic les o n t h e role of p h e n o l o x i d a s e s in insec t i m m u n i t y have a p p e a r e d in t he l i t e r a tu re . T h e excel lent m o n o g r a p h b y G u p t a (1988)

14. Regulation of Insect Hemolymph Phenoloxidases 319

s u m s u p t h e l i t e r a t u r e un t i l 1988, after w h i c h p e r i o d t h e r e a d e r s c a n refer to t h e a r t i c les by S u g u m a r a n (1990) a n d A s h i d a a n d Y a m a z a k i (1990) . B e c a u s e t h e sub jec t m a t t e r of th is book focuses o n insec ts , p i o n e e r i n g s tud i e s c a r r i e d o u t b y S o d e r h a l l a n d his assoc ia tes ( J o h a n s s o n a n d S o d e r h a l l , 1989) o n t h e p a r t i c i p a t i o n of p h e n o l o x i d a s e in c r u s t a c e a n i m m u n i t y a r e n o t r ev iewed h e r e .

II. Prophenoloxidase Activation

A. Prophenoloxidase

T h e r e a r e t h r e e k i n d s of p h e n o l o x i d a s e s : m o n o p h e n o l m o n o o x y g e n a s e , o -d ipheno lox idase , a n d jb -d iphenolox idase ( laccase) . M o n o p h e n o l m o n o o x y -g e n a s e s , s u c h as t y ros inase , a lso possess t h e o -d ipheno lox idase activity, b u t t h e o -d ipheno lox idase does n o t necessar i ly possess m o n o p h e n o l m o n o o x y g e n a s e activity. Final ly , laccases , w h i c h show pre fe rence for />-diphenols , c a n a lso r ead i ly a t t a c k t h e o-d iphenols . T h e r e f o r e , t hey possess o -d ipheno lox idase ac t iv i ty as well . W h i l e t e s t ing t h e p h e n o l o x i d a s e activity, if o -d iphenols s u c h as d o p a o r d o p a m i n e a r e used as s u b s t r a t e s , it is infer red t h a t t h e o-d i p h e n o l o x i d a s e act iv i ty of all t h r e e e n z y m e s will b e d e t e c t e d . S u c h a s says will n o t d i s t i n g u i s h t h e t h r e e different p h e n o l o x i d a s e act iv i t ies . B e c a u s e of these c o m p l i c a t i o n s , we will use on ly t he c o m m o n t e r m p h e n o l o x i d a s e t h r o u g h o u t this text .

P h e n o l o x i d a s e u sua l ly o c c u r s as a n inac t ive p r o e n z y m e form in t h e h e m o l y m p h of m o s t of t he insec ts t es ted . E v i d e n c e for t h e p r e s e n c e of p r o p h -eno lox idases in t h e h e m o l y m p h of Locusta migratona (Brehe l in et al., 1989), Tenebrio molitor ( H e y n e m a n , 1965), Bombyx mori ( A s h i d a , 1971), Antheraea per-nyi ( E v a n s , 1967), Manduca sexta (Aso et al., 1985), Hyalophora cecropia (An-d e r s s o n et al., 1989), Galleria mellonella (Pye , 1974), Sarcophaga barbarta ( H u g h e s , 1976), Calliphora vicina ( T h o m s o n a n d Sin, 1970), Musca domestica ( T s u k a m o t o et al., 1986), Drosophila melanogaster (Seybo ld et al., 1975), a n d Aedes aegypti ( A s h i d a et al., 1990) is well d o c u m e n t e d . P r o p h e n o l o x i d a s e s f rom B. mori ( A s h i d a , 1971), Calliphora ( P a u a n d Eag le s , 1975), M. domestica ( T s u k a m o t o et al., 1986), a n d T. molitor ( H e y n e m a n , 1965) h a v e b e e n h igh ly pur i f ied . I n sp i t e of i ts key ro le in insec t i m m u n i t y , c i r cu l a r sc le ro t i za t ion , a n d w o u n d hea l i ng , it is r a t h e r s u r p r i s i n g t h a t t h e s tud ie s o n th is e n z y m e a r e so l imi t ed . H o w e v e r , u n d e r s t a n d a b l y , t h e difficulties a s soc i a t ed w i t h t h e p u r i fication of th is p r o e n z y m e h a v e severely h a m p e r e d p r o g r e s s in th is l ine of r e s e a r c h . Bes ides , p r o b l e m s a r i s ing from s p o n t a n e o u s ac t iva t ion of t h e en z y m e , i ts a b s o r p t i o n to g lass a n d o t h e r surfaces i n c l u d i n g gels a n d o t h e r m a t e r i a l s u s e d for c h r o m a t o g r a p h y , r a p i d d e g r a d a t i o n , a n d e v e n t u a l i nac -

3 2 0 Manickam Sugumaran and Michael R. Kanost

t iva t ion have d e t e r r e d severa l inves t iga to rs f rom u n d e r t a k i n g a d e t a i l e d s t u d y on th is e n z y m e . M u c h of t he de ta i l ed b i o c h e m i s t r y of p r o p h e n o l o x idase c o m e s from the m o n u m e n t a l work of A s h i d a a n d coworkers ( A s h i d a a n d Y a m a z a k i , 1990).

B. Prophenoloxidase Activating Enzyme

O h n i s h i (1954) first d e m o n s t r a t e d t h a t in Drosophila, a p r o t e i n factor cou ld ac t iva te p r o p h e n o l o x i d a s e activity. S u b s e q u e n t work by Schwe ige r a n d K a r l -son (1962) n o t on ly conf i rmed th is o b s e r v a t i o n b u t a lso p r o v i d e d ev idence t h a t t he ac t iva t ion of the p r o e n z y m e is ach ieved by l imi ted p ro teo lys i s . L a t e r , s imi la r resu l t s were r e p o r t e d in B. mori ( A s h i d a a n d D o h k e , 1980), D. melanogaster (Seybold et al., 1975), S. barbarta ( H u g h e s , 1976), S. bullata (Sau l a n d S u g u m a r a n , 1988), M. sexta (Aso et al., 1985; S a u l a n d S u g u m a r a n , 1987), a n d Blaberus craniifer ( L e o n a r d et al., 1985b) . W o r k i n g w i t h t he silkw o r m B. mori, A s h i d a a n d assoc ia tes successfully pur i f ied t h e p r o p h e n o l o x idase from the h e m o l y m p h a n d d e m o n s t r a t e d t h a t t h e ac t iva t ion of p r o p h eno lox idase is ach ieved by p ro teo ly t i c c leavage of t h e p r o e n z y m e ( A s h i d a a n d D o h k e , 1980). T h e ac t iva t ing p r o t e a s e from t h e l a rva l cu t ic le of t he s a m e o r g a n i s m w a s i so la ted a n d c h a r a c t e r i z e d by D o h k e (1973) , a n d t h e p r e s e n c e of a s imi l a r e n z y m e in t he h e m o l y m p h h a s b e e n d e m o n s t r a t e d b y A s h i d a a n d Yosh ida (1988) . Avai labi l i ty of pur i f ied p r o e n z y m e a n d its a c t i va to r f rom the s a m e o r g a n i s m a i d e d the e s t a b l i s h m e n t of t he p ro t eo ly t i c ac t iva t ion m e c h a n i s m ( A s h i d a a n d D o h k e , 1980). Ac t iva t ion by the c u t i c u l a r p r o t e a s e severed a p e p t i d e of Mr = 5000 from the p r o e n z y m e , conf i rming u n e q u i v o c a l l y t he p a r t i c i p a t i o n of " l imi t ed p ro t eo ly s i s " as a key m e c h a n i s m s of p r o p h e n o l ox idase ac t iva t ion . I n m o s t o t h e r insec ts tes ted , th is m o d e ce r t a in ly s eems to o p e r a t e as ev idenced by the i nh ib i t i on of ac t iva t ion by se r ine p r o tease inac t iva to r s such as d i i sop ropy l p h o s p h o n o f l u o r i d a t e , jb-nitrophenyl-/?-g u a n i d o b e n z o a t e , o r pheny lme thy l su l fony l fluoride. A s h i d a ' s g r o u p a lso d e m o n s t r a t e d t he o c c u r r e n c e of this r eac t ion in t h e h e m o l y m p h ( A s h i d a a n d Yoshida , 1988). In te res t ing ly , t he p r o p h e n o l o x i d a s e ac t i va t i ng e n z y m e from the h e m o l y m p h itself s eems to be p r e s e n t as a p r o e n z y m e , t h e r e b y i n d i c a t i n g t he p r e s e n c e of a c a s c a d e of e n z y m e act ivi t ies a s soc ia ted w i t h p h e n o l o x i d a s e (Yoshida a n d A s h i d a , 1986).

C. Prophenoloxidase Cascade

A l t h o u g h they d i d no t define each act ivi ty specifically, Seybo ld et al. (1975) first r e p o r t e d t he p r e s e n c e of a c a s c a d e of e n z y m e act ivi t ies a s soc i a t ed w i t h p h e n o l o x i d a s e ac t iva t ion . Yoshida a n d A s h i d a (1986) o b s e r v e d t h e m i c r o b i a l ac t iva t ion of two ser ine p ro t ea se s p r io r to p r o p h e n o l o x i d a s e ac t iva t ion in t h e


s i l kworm h e m o l y m p h . O n e of t h e p r o t e a s e s w a s ident i f ied to be t h e p r o p h eno lox ida se ac t iva t ing e n z y m e , w h e r e a s t he o t h e r p r o t e a s e h y d r o l y z i n g b e n -zoy la rg in ine e thy l es te r w a s t e r m e d a b e n z o y l a r g i n i n e e thy l e s t e rase . I n t e r estingly, ac t iv i ty of b e n z o y l a r g i n i n e e thy l e s t e rase a p p e a r e d in t h e p l a s m a p r i o r to t h e a p p e a r a n c e of p r o p h e n o l o x i d a s e ac t i va t ing e n z y m e (or p h e n o l o x idase ac t iv i ty) . T h u s it s eems t h a t m i c r o b i a l p r o d u c t s t r igger even tua l l y in t h e o r d e r benzoy l a r g i n i n e e thy l e s t e rase , p r o p h e n o l o x i d a s e ac t i va t i ng en z y m e , a n d p h e n o l o x i d a s e (Fig . 1). T h e r e f o r e , it is fairly ce r t a in t h a t p r o p h e n o l o x i d a s e ac t iva t ion o c c u r s in a c a s c a d e of r eac t i ons . E v i d e n c e for t h e p r e s e n c e of a s imi l a r c a s c a d e in M. sexta (Sau l a n d S u g u m a r a n , 1987) a n d S. bullata (Sau l a n d S u g u m a r a n , 1988) h a s b e e n p r o v i d e d by S u g u m a r a n ' s

FOREIGN SUBSTANCES

ZYMOGEN ACTIVE E N Z Y M E & C a 2 + MOBILIZATION

/

PRO-BAEE BAEE Λ PRE-PPAE PPAE CUTICULAR PROTEASE

X PROPHENOLOXIDASE PHENOLOXIDASE Λ

DIPHENOL QUINONE

\ \

MELANIN CAPSULE

Figure Ί T h e prophenoloxidase cascade. LPS, peptidoglycan, β-l,3-glucans, a n d / o r other molecules present on foreign substances trigger the prophenoloxidase system of the host by a cascade of reactions. The less-defined initial reaction leads to mobilization of calcium and the activation of enzyme activity that triggers the pro-benzoyl arginine ethyl esterase (PRO-BAEE) to active enzyme. This serine protease in turn activates preprophenoloxidase activating enzyme (PRE-PPAE) to the active form. The active PPAE proteolytically activates prophenoloxidase. Prophenoloxidase can also be activated by the cuticular protease directly. Active phenoloxidase catalyzes the conversion of diphenols to quinones and provides them for melanin capsule formation.


g r o u p . I n b o t h of these o r g a n i s m s , a t leas t o n e se r ine p r o t e a s e s eems to b e ac t iva t ed p r io r to t he ac t iva t ion of p r o p h e n o l o x i d a s e . A n d e r s s o n et al. (1989) have s h o w n the p r e s e n c e of two ac t iva to r p r o t e i n s in H. cecropia.

D. Triggering of the Cascade

A t th is p o i n t it is no t c lear how the first p r o t e a s e in t he s i l kworm t h a t s eems to be p r e s e n t in t he inac t ive p r o e n z y m e form is ac t iva ted b y m i c r o b i a l p r o d uc t s . B u t t he re is n o d o u b t t h a t p r o p h e n o l o x i d a s e is t r igge red b y different m i c r o b i a l p r o d u c t s . For such t r igger ing , d iva l en t c a l c i u m seems to b e essent ial ( L e o n a r d et al., 1985a; Yosh ida a n d A s h i d a , 1986; S a u l a n d S u g u m a r a n , 1987, 1988; A s h i d a a n d Yosh ida , 1988; Brehe l in et al, 1989; B r o o k m a n et al, 1989; A s h i d a a n d Y a m a z a k i , 1990; S u g u m a r a n , 1990). T h e r e f o r e , it is p r o b a ble t h a t s o m e c a l c i u m - d e p e n d e n t e n z y m e act iv i ty a p p e a r s u p o n i n t e r ac t i on of t he foreign objec ts w i t h t he insec t h e m o l y m p h c o m p o n e n t s . Severa l m i c r o bia l p r o d u c t s , s u c h as bac te r i a l , fungal , a n d yeas t cel l-wall c o m p o n e n t s , c a u s e p r o p h e n o l o x i d a s e ac t iva t ion in t h e h e m o l y m p h (see Fig . 1).

I t is well k n o w n a m o n g r e sea r che r s w o r k i n g in t he a r e a of insec t i m m u n i t y t h a t it is essent ia l to m a i n t a i n pyrogen-f ree cond i t i ons w h e n h a n d l i n g insec t b lood cells. Yet, it is s u r p r i s i n g to see severa l a u t h o r s r e p o r t i n g t h e fa i lure of l i popo lysaccha r ide ( L P S , a cel l-wall c o m p o n e n t of g r a m - n e g a t i v e bac t e r i a ) to ac t iva te p r o p h e n o l o x i d a s e . I n B. mori (Yoshida a n d A s h i d a , 1986), G. mellonella ( B r o o k m a n et al, 1989), Schistocerca gregaria ( D u l a r a y a n d Lack ie , 1985), a n d S. bullata (Sau l a n d S u g u m a r a n , 1988), for e x a m p l e , L P S does n o t s e e m to ac t iva te t he p r o p h e n o l o x i d a s e . However , in M. sexta (Sau l a n d S u g u m a r a n , 1987), r a p i d ac t iva t ion of p r o p h e n o l o x i d a s e b y L P S h a s b e e n re p o r t e d .

O n e of us p o i n t e d o u t t h a t L P S - m e d i a t e d p r o p h e n o l o x i d a s e ac t iva t ion is t r a n s i e n t in n a t u r e (Sau l a n d S u g u m a r a n , 1987) a n d o n e c a n easi ly miss t h e activity. A l t h o u g h it is no t c lear w h y th is p h e n o m e n o n o c c u r s , it h a s b e e n i n d e p e n d e n t l y obse rved in a n o t h e r insect as well (Brehe l in et al, 1989). S ince ac t iva ted p h e n o l o x i d a s e is very sticky, it c a n a d h e r e to foreign objec ts a n d o t h e r cell surfaces a n d g e n e r a t e cy to tox ic q u i n o n o i d c o m p o u n d s to kill t h e i n t r u d e r . S u c h toxic p r o d u c t s of this e n z y m e act iv i ty a r e a lso d a n g e r o u s for t h e hos t . T h e r e f o r e , it is likely t h a t t h e ac t iva ted p h e n o l o x i d a s e is shor t - l ived (Sau l a n d S u g u m a r a n , 1987), s imi la r to s o m e of t he c o m p l e m e n t p a t h w a y c o m p o n e n t s found in t he b lood of h i g h e r a n i m a l s . T h e r e f o r e , use of d i s con t i n u o u s a s says c a n c a u s e lack of de t ec t i on of p h e n o l o x i d a s e activity. I n a d d i t ion , o t h e r factors c a n a lso c a u s e m i s l e a d i n g resu l t s as exempl i f ied b y t h e s tud ie s in locus t s . Ini t ial ly, Ratcliffe's g r o u p ( B r o o k m a n et al, 1989) r e p o r t e d a lack of L P S ac t iva t ion in locus t s , w h e r e a s Brehe l in et al. (1989) d e m o n s t r a t e d t h a t L P S c a u s e d p r o p h e n o l o x i d a s e ac t iva t ion in t h e s a m e o r g a n i s m .


Since t h e l a t t e r w o r k e r s d i rec t ly col lec ted t he h e m o l y m p h w i t h o u t t h e use of a n y a n t i c o a g u l a n t s , w h e r e a s B r o o k m a n et al. (1989) u sed a n a n t i c o a g u l a t i o n buffer cons i s t ing of e t h y l e n e d i a m i n e t e t r a - a c e t a t e , c i t r a t e , a n d g lucose , it is likely t h a t s o m e of t h e c o m p o n e n t s of a n t i c o a g u l a n t buffer in te r fe red w i t h t h e ac t iva t ion t r igge r ing m e c h a n i s m s a n d i n h i b i t e d t he p r o p h e n o l o x i d a s e ac t ivat ion . N e v e r t h e l e s s , u p o n re inves t iga t ion in t h e s a m e o r g a n i s m , Ratcliffe 's g r o u p d i d obse rve t h a t L P S c a u s e d p r o p h e n o l o x i d a s e ac t iva t ion in w h o l e b lood (Ratcliffe et al., 1991).

T h e L P S ac t iva t ion m u s t be m e d i a t e d b y s o m e sor t of a r ecogn i t i on m e c h a n i s m . Accord ing ly , a n L P S b i n d i n g p r o t e i n from t h e h e m o l y m p h of t h e A m e r i c a n cock roach h a s b e e n i so la ted by N a t o r i ' s g r o u p ( J o m o r i et al., 1990), b u t its r e l a t i o n s h i p w i t h t he p r o p h e n o l o x i d a s e c a s c a d e h a s n o t yet b e e n def ined. C o n s i d e r a b l y m o r e b i o c h e m i c a l work h a s b e e n d o n e o n t h e t r igge r ing of t he c a s c a d e by g r a m - p o s i t i v e bac t e r i a a n d yeas t ce l lu la r c o m p o n e n t s . S ince a de t a i l ed a c c o u n t of these resu l t s is p r e s e n t e d in t h e a r t i c le b y A s h i d a a n d Y a m a z a k i (1990) , on ly a s h o r t a c c o u n t is g iven h e r e . T h e t r iggering s u b s t a n c e from g r a m - p o s i t i v e b a c t e r i a s eems to b e p e p t i d o g l y c a n (Yoshida a n d A s h i d a , 1986; B r o o k m a n et al., 1989). A s l i t t le as a few n a n o g r a m s p e r mil l i l i ter of p e p t i d o g l y c a n c a n ac t iva te t h e p r o p h e n o l o x i d a s e cas c a d e very efficiently in t he s i l kworm h e m o l y m p h . A s h i d a ' s g r o u p a lso e s t a b l i shed t h a t t he β -1 ,3^1υχαη r e c e p t o r s a n d the p e p t i d o g l y c a n r e c e p t o r s for t r i gge r ing p r o p h e n o l o x i d a s e a r e p r e s e n t as s e p a r a t e en t i t ies in th is o r g a n i s m (Yoshida et al., 1986). T h e β - l , 3 -g lucan b i n d i n g p r o t e i n f rom B. craniifer ( S o d e r h a l l et al., 1988) a n d B. mori ( O c h i a i a n d A s h i d a , 1988) h a s b e e n pur i f ied a n d c h a r a c t e r i z e d , b u t t h e c h a r a c t e r i z a t i o n of a p e p t i d o g l y c a n b i n d ing p r o t e i n is ye t to be r e p o r t e d . Also n o t c lea r a t th is p o i n t is w h e t h e r L P S r e c e p t o r s a r e t h e s a m e as those for p e p t i d o g l y c a n o r p- l , 3 -g lucan r e c e p t o r s o r i n d e p e n d e n t r e cep to r s . O b v i o u s l y m u c h w o r k n e e d s to b e d o n e in th is a r e a .

B a s e d o n t h e foregoing d i scuss ion , a t en t a t i ve s c h e m e c o n t a i n i n g al l t h e c o m p o n e n t s of t he c a s c a d e c a n be dev i sed as s h o w n in F ig . 1. U p o n recogn i t ion of foreign ob jec t s , c e r t a in c o m p o n e n t s in t he h e m o l y m p h s e e m to b e c o m e ac t iva t ed , w h i c h in t u r n t r igger in success ion two se r ine p r o t e a s e s a n d p r o p h e n o l o x i d a s e . A c t i v a t e d p h e n o l o x i d a s e a t t acks i ts s u b s t r a t e a n d p r o d u c e s cy to tox ic q u i n o n e s to kill t h e i n t r u d e r . Al te rna t ive ly , t h e q u i n o n e s c a n p o l y m e r i z e a n d form t h e c a p s u l e . T h e con t ro l m e c h a n i s m s of th is c a s c a d e a r e d i scussed in Sec t ion I I I .

E. Lipids as Modulators

A c t i v a t i o n of insec t p r o p h e n o l o x i d a s e h a s b e e n extens ive ly r ev iewed b y B r u n e t (1980) . T w o types of ac t iva t ion p rocesses have b e e n c h a r a c t e r i z e d so


far. O n e is t he p r o t e a s e - m e d i a t e d ac t iva t ion e l a b o r a t e d in ear l i e r p a g e s . T h e second is t he s p o n t a n e o u s a n d s o m e t i m e s u n a c c o u n t a b l e ac t iva t ion b y p h y s i cal t e c h n i q u e s . As ea r ly as 1945, B o d i n e d e s c r i b e d the ac t iva t ion of l a t e n t p h e n o l o x i d a s e f rom t h e d i a p a u s i n g eggs of t h e g r a s s h o p p e r Melanoplus differ-entialis by surface-act ive r e a g e n t s , h e a t , a n d heavy m e t a l s . L a t e r F u n a t s u a n d I n a b a (1962) ou t l i ned the ac t iva t ion οϊ Musca p r o p h e n o l o x i d a s e by s o d i u m d o d e c y l sulfate , w h e r e a s H e y n e m a n a n d V e r c a u t e r e n (1964) d e m o n s t r a t e d t h e ac t iva t ion of l a t en t p h e n o l o x i d a s e f rom T. molitor b y s o d i u m o lea te . A m o n g the fat ty ac ids tes ted , o lea te w a s t he m o s t p o t e n t ac t iva to r of Tenebrio p h e n o l o x i d a s e ( H e y n e m a n a n d V e r c a u t e r e n , 1968). S o d i u m d o d e c y l sulfate as well as o lea te a lso c a u s e d the ac t iva t ion of Lucilia cuprina p r o e n z y m e ( H a c k m a n n a n d G o l d b e r g , 1967), b u t s u b s e q u e n t work by n u m e r o u s re s e a r c h e r s led to t he e s t a b l i s h m e n t of p r o t e a s e s as t he t r u e ac t iva to r s of p r o p h eno lox idase . As a resu l t , t he ear ly s tud ies on ac t iva t ion m e c h a n i s m s involving phys i ca l t r e a t m e n t s w e r e comple t e ly i gno red for phys io log ica l p u r p o s e s . Howeve r , very recent ly, wh i l e e x a m i n i n g t h e p r o p e r t i e s of l a t e n t p r o p h eno lox idase f rom the left col le ter ia l g l a n d of Periplaneta americana, S u g u m a r a n a n d N e l l a i a p p a n (1990) found t h a t th is p r o e n z y m e is specifically ac t i va t ed by t r e a t m e n t w i th d e t e r g e n t s such as s o d i u m dodecy l sulfate be low the i r cr i t ica l mice l l a r c o n c e n t r a t i o n s . T h e y cou ld no t find a n y ev idence for a n e n d o g e n o u s p r o t e a s e ac t iva to r in th is sys t em. A t h o r o u g h s e a r c h led to t h e d iscovery t h a t low c o n c e n t r a t i o n s of p h o s p h o l i p i d s se rved as effective ac t ivators of n o t on ly cockroach p r o p h e n o l o x i d a s e , b u t a lso t h e p r o p h e n o l o x i d a s e from a va r i e ty of insec ts a n d c r u s t a c e a n s ( M . S u g u m a r a n a n d K . N e l l a i a p p a n , 1990, 1991; M . S u g u m a r a n , u n p u b l i s h e d resu l t s ) . A m o n g t h e p h o s p h o l i p i d s tes ted , lysolec i th in p roved to b e t he m o s t p o t e n t ac t iva to r of l obs t e r p r o p h eno lox idase ( S u g u m a r a n a n d N e l l a i a p p a n , 1991). I t a p p e a r s t h a t c h e m i c a l d e t e r g e n t s s u c h as s o d i u m dodecy l sulfate o r b io logica l d e t e r g e n t s s u c h as fat ty ac ids a n d p h o s p h o l i p i d s a t very low c o n c e n t r a t i o n s b i n d to t h e p r o p h eno lox idase a n d a l t e r its c o n f o r m a t i o n so as to a l low t h e e n z y m e to expose its act ive si te for its s u b s t r a t e s . S u c h accessibi l i ty is p e r h a p s b r o u g h t a b o u t by p ro t eo ly t i c e n z y m e s by sp l i t t ing off t he " i n h i b i t o r y p e p t i d e " r eg ion f rom t h e p r o e n z y m e . T h u s , i r respec t ive of w h a t t r e a t m e n t it is, p r o p h e n o l o x i d a s e ac t iva t ion m a y b e a t t a i n e d by c o n f o r m a t i o n a l c h a n g e s t h a t l e ad to t h e e x p o s u r e of t he act ive si te of t he e n z y m e .

Physiological ly , t he l ipid ac t iva t ion of p r o p h e n o l o x i d a s e h a s i m p o r t a n t c o n s e q u e n c e s . D u r i n g invas ion , m a n y r e sea r che r s have d e m o n s t r a t e d t h e p r e s e n c e of d a m a g e d h e m o c y t e s on o r a r o u n d the foreign ob jec t s . (Ratcliffe et al., 1985). C e l l u l a r d a m a g e c a n c a u s e t he re lease of p h o s p h o l i p a s e A 2 , w h i c h in t u r n l ibe ra te s lysolec i th in a n d free fat ty ac ids . T h e re l eased lyso lec i th in , w h i c h is o n e of t he m o s t p o t e n t b iological d e t e r g e n t s , c a n funct ion as a d i r ec t ac t iva to r of p h e n o l o x i d a s e , whi le t he free fat ty ac ids g e n e r a t e d c a n b e u s e d for t he syn thes i s e f a r a c h i d o n i c acid a n d e i cosano ids , w h i c h cou ld t r igger

14. Regulation of Insect Hemolymph Phenoloxidases 3 2 5

fu r the r i m m u n e reac t ions as d e m o n s t r a t e d b y o t h e r s ( B l o m q u i s t et al., 1991 ; S t a n l e y - S a m u e l s o n et al., 1991).

F. Compartmentalization

Since p r o p h e n o l o x i d a s e cons t i t u t e s a n i m p o r t a n t c o m p o n e n t of t h e insec t ' s defense m e c h a n i s m , it is i m p o r t a n t to e x a m i n e its ce l lu la r loca l i za t ion . E a r l ier s t ud i e s b y E v a n s (1967) in G. mellonella a n d Pye (1974) in A. pernyi i n d i c a t e d t h e p r e s e n c e of t he p r o p h e n o l o x i d a s e in t h e p l a s m a fract ion of h e m o l y m p h . Subsequen t ly , A s h i d a (1981) , u s i n g c a n e s u g a r a s a s t ab i l i z ing a g e n t , r e p o r t e d t h e p r e s e n c e of this e n z y m e in t h e p l a s m a fract ion of B. mori. H o w e v e r , L e o n a r d et al. (1985b) q u e s t i o n e d t h e su i tab i l i ty of th is p r o c e d u r e a n d , u s i n g a n a n t i c o a g u l a t i o n buffer cons i s t ing of e t h y l e n e d i a m i n e t e t r a a c e t a t e , g lucose , a n d c i t r a t e to s e p a r a t e t h e h e m o c y t e s f rom t h e p l a s m a fract ion, c l a i m e d t h a t t he p r o p h e n o l o x i d a s e is p r e s e n t in t h e h e m o c y t e s a n d n o t in t h e p l a s m a fract ion of B. craniifer. B u t , I w a m a a n d A s h i d a (1986) , d u r i n g the i r s tud ie s on t he b iosyn thes i s of p r o p h e n o l o x i d a s e , d e m o n s t r a t e d t h a t o e n o c y t o i d s syn thes i zed this e n z y m e specifically a n d r e l eased it i n t o m e d i u m . H e n c e , it is m o s t likely t h a t p r o p h e n o l o x i d a s e is syn thes i zed b y oenocy to id s a n d sec re ted in to t he p l a s m a . Accord ing ly , u s i n g i m m u n o l o c a l -i za t ion , A s h i d a et al. (1988) conclus ive ly d e m o n s t r a t e d t h e p r e s e n c e of p r o p h e n o l o x i d a s e in t he p l a s m a u n d e r n o r m a l phys io log ica l c o n d i t i o n s . I n b o t h S. bullata a n d M. sexta, o n e of u s (Sau l a n d S u g u m a r a n , 1987, 1988; S a u l et al., 1987) d e m o n s t r a t e d t h a t t h e ma jo r i t y of p r o p h e n o l o x i d a s e is loca l ized in t he p l a s m a a n d n o t in t he cells. T h o u g h Ratcliffe 's g r o u p a s c e r t a i n e d t h e p r e s e n c e of t h e p r o p h e n o l o x i d a s e s y s t e m in t h e ce l lu la r f ract ion of locus t s ( B r o o k m a n et al., 1989), B rehe l in et al., (1989) d e m o n s t r a t e d i ts p r e s e n c e in b o t h t h e p l a s m a a n d ce l lu la r fract ions of t h e s a m e o r g a n i s m . T h e difference in t he se resu l t s is n o d o u b t d u e to t h e differences in e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d b y t he se worke r s . A s s t a t e d ear l ier , Ratcliffe 's g r o u p in jec ted t h e e t h y l e n e d i a m i n e t e t r a - a c e t a t e , c i t r a t e , g lucose buffer i n to t h e o r g a n i s m s before co l lec t ing t he h e m o l y m p h , w h e r e a s Brehe l in ' s g r o u p d i rec t ly col lec ted t h e h e m o l y m p h for f rac t iona t ion s t ud i e s . I t a p p e a r s t h a t t h e a n t i c o a g u l a t i o n buffer in ter feres w i t h t h e ce l lu la r loca l i za t ion s tud i e s . T h i s as well as d i s c r e p anc ie s in L P S ac t iva t ion ce r t a in ly q u e s t i o n s t he w i s d o m of u s i n g a n t i c o a g u l a n t so lu t ions in t he s t u d y of t h e p r o p h e n o l o x i d a s e s y s t e m .

G. Is the Prophenoloxidase System a Recognition Mechanism?

T h e s p e c u l a t i o n t h a t p r o p h e n o l o x i d a s e sy s t ems cou ld serve as a r ecogn i t i on m e c h a n i s m of non-se l f m a t t e r in a n a t t r ac t i ve b u t u n s u b s t a n t i a t e d o n e ( S o d e r h a l l , 1982; Ratcliffe et al., 1984; L e o n a r d et al., 1985a) . T h e a s soc ia t ion


of p h e n o l o x i d a s e w i t h defense reac t ions of insects is well e s t ab l i shed , however, t h e r e is h a r d l y a n y ev idence in t he l i t e r a tu r e to a t t r i b u t e a role for th is e n z y m e in non-se l f r ecogn i t ion . A l t h o u g h ac t iva t ion of t he p r o p h e n o l o x i d a s e sys t em in t he w a x m o t h G. mellonella by a m i c r o b i a l p r o d u c t e n h a n c e d t h e r ecogn i t ion of non-se l f m a t e r i a l (Ratcliffe et al., 1984; L e o n a r d et al., 1985a) , th is does no t serve as p roo f for its role in non-se l f r ecogn i t ion . As s t a t e d ear l ier , n o n e of t he m i c r o b i a l p r o d u c t s tes ted d i rec t ly ac t iva tes p r o p h e n o l o x idase ; r a t h e r they seem to b i n d to s o m e c o m p o n e n t s in p l a s m a (or o n t h e cell surface) a n d t r igger the p r o p h e n o l o x i d a s e c a s c a d e . T h e r e f o r e , if o n e accep t s t he p r e m i s e t h a t p r o p h e n o l o x i d a s e ac t iva t ion is ach ieved by a c a s c a d e of r eac t ions in t he insect ' s h e m o l y m p h , t h e n n a t u r a l l y t he role of p r o p h e n o l o x idase in r ecogn i t ion of non-se l f m a t t e r b e c o m e s q u e s t i o n a b l e . However , t h e r e is n o d o u b t t h a t p r o p h e n o l o x i d a s e ac t iva t ion is a c o n s e q u e n c e of non-se l f r ecogn i t ion . Accord ingly , Rizki a n d Rizki (1990) have successfully d e m o n s t r a t e d t h a t D. melanogaster l a rvae lacking h e m o l y m p h p h e n o l o x i d a s e act iv i ty cou ld read i ly e n c a p s u l a t e a foreign o r g a n i s m injected in to its b lood b u t cou ld n o t m e l a n i z e it. T h e r e f o r e , t he hypo thes i s t h a t p r o p h e n o l o x i d a s e serves as a m e c h a n i s m for r ecogn i t ion of fore ignness shou ld b e r e e v a l u a t e d .

III. Control Mechanisms of the Cascade

A. Introduction

C o - o c c u r r e n c e of p r o p h e n o l o x i d a s e , its s u b s t r a t e ( s ) , a n d ac t iva to r s in t he h e m o l y m p h w o u l d c a u s e u n d e s i r e d ac t iva t ion of p r o p h e n o l o x i d a s e a n d gen e r a t i o n of cy to tox ic q u i n o n e s . S u c h reac t ions a r e de l e t e r ious to t h e o r g a n i s m a n d h e n c e a p p r o p r i a t e con t ro l m e c h a n i s m s m u s t exist in t he o r g a n i s m s to avoid s u c h u n w a n t e d reac t ions . K e e p i n g the e n z y m e s in the i r inac t ive p r o e n z y m e forms, t i m e c o n s t r a i n t s on t he ac t iva ted c o m p o n e n t s , p r o t e c t i n g t h e s u b s t r a t e s f rom reac t i ng w i th the i r e n z y m e s , secre t ion of i nh ib i t o r s , a n d c o m p a r t m e n t a l i z a t i o n of different c o m p o n e n t s a r e a few of t h e w a y s to achieve these goa ls . Ava i lab le ev idence ind ica t e s t h a t all of t hese m e c h a n i s m s a r e effectively u sed in insec ts .

E v i d e n c e for t he p r e s e n c e of (a) p r o e n z y m e forms, (b) t i m e c o n s t r a i n t s o n p h e n o l o x i d a s e activity, a n d (c) c o m p a r t m e n t a l i z a t i o n h a s a l r e a d y b e e n p r e sen t ed . A m p l e e x a m p l e s can b e g iven for t he p r o t e c t i o n of s u b s t r a t e s . T h e ca techo l i c s u b s t r a t e s a r e s to red in t h e h e m o l y m p h as p h o s p h a t e , sulfa te , o r g lycos ide con juga tes t h a t d o n o t serve as s u b s t r a t e s for p h e n o l o x i d a s e ( K r a m e r a n d H o p k i n s , 1987). U p o n e n z y m a t i c d e b l o c k i n g of t h e p ro t ec t ive g r o u p s , free ca techo l s c a n be g e n e r a t e d a t des i r ed p laces . H y d r o l y t i c enz y m e s s u c h as g lycos ides , p h o s p h a t a s e s , a n d sulfa tases a r e u sua l ly c o m p a r t -


m e n t a l i z e d in cells. D u r i n g invas ion by a foreign o r g a n i s m , ce l lu la r c o n t e n t s c a n b e d i s c h a r g e d to e n s u r e m i x i n g of these e n z y m e s w i t h the i r s u b s t r a t e s a n d t h e l i b e r a t e d free 0 - d i p h e n o l s c a n serve as s u b s t r a t e s for p h e n o l o x i d a s e . I n a d d i t i o n , i nh ib i t o r s s e e m to p l a y a c ruc ia l role in con t ro l l ing t h e c a s c a d e .

B. Protease Inhibitors

I t is a c o m m o n p rac t i c e to a d d h e a t - i n a c t i v a t e d h e m o l y m p h to insec t h e m o cyte c u l t u r e s to p r e v e n t d a r k e n i n g reac t ions ( W y a t t , 1956). B a s e d o n t h e e x t r e m e t h e r m a l s tab i l i ty of se r ine p r o t e a s e i nh ib i t o r s of Bovine P a n c r e a t i c T r y p s i n I n h i b i t o r ( B P T I ) c lass , a n d the l imi ted p ro teo ly t i c m o d e of ac t ivat ion of p r o p h e n o l o x i d a s e , it is logical to expec t t he p a r t i c i p a t i o n of t h e r m o s t ab l e se r ine p r o t e a s e i nh ib i t o r s in con t ro l l i ng t he act iv i ty of p r o t e a s e s in volved in p r o p h e n o l o x i d a s e c a s c a d e . T h e r e f o r e , s tud ies w e r e u n d e r t a k e n to i so la te h e a t - s t a b l e p r o t e a s e i nh ib i t o r s f rom t h e h e m o l y m p h of M. sexta a n d S. bullata ( S u g u m a r a n et al., 1985; S a u l a n d S u g u m a r a n , 1986; R a m e s h et al., 1988). T h e s e a t t e m p t s led to t he i so la t ion of t h r e e B P T I t ype se r ine p r o t e a s e i n h i b i t o r s . T w o of these were i so la ted from M. sexta a n d t h e o t h e r w a s i so la ted f rom S. bullata. S u g u m a r a n et al. (1985) d e m o n s t r a t e d for t h e first t i m e d r a s t ic i n h i b i t i o n of p r o t e a s e - m e d i a t e d p r o p h e n o l o x i d a s e ac t iva t ion by al l t h r e e i n h i b i t o r s . I n M. sexta, o n e of these se r ine p r o t e a s e i nh ib i t o r s cou ld i nh ib i t t h e c u t i c u l a r p r o t e a s e - m e d i a t e d ac t iva t ion of p r o p h e n o l o x i d a s e , con f i rming t h e p r o p o s e d phys io log ica l role of th is i n h i b i t o r (Sau l a n d S u g u m a r a n , 1986). B rehe l i n et al. (1991) h a v e d e m o n s t r a t e d t h e p r e s e n c e of a se r ine p r o t e a s e i n h i b i t o r t h a t con t ro l s t h e p r o p h e n o l o x i d a s e ac t iva t ion in t he h e m o l y m p h of L. migratoria.

Manduca i n h i b i t o r A is a h o m o d i m e r w i t h a m o l e c u l a r w e i g h t of 14,000, w h e r e a s Manduca i n h i b i t o r Β is a m o n o m e r w i t h a m o l e c u l a r w e i g h t of 8000 . Sarcophaga i n h i b i t o r is m u c h s m a l l e r a n d closer to t h e m o l e c u l a r we igh t of B P T I . T h e p r i m a r y a m i n o acid s e q u e n c e s (pa r t i a l ) of these i n h i b i t o r s a r e s h o w n in F ig . 2. T h e act ive si te P I r e s idue for all t h r e e i n h i b i t o r s is a r g i n i n e as o p p o s e d to lys ine in B P T I ( R a m e s h et al., 1988; S u g u m a r a n , 1990). I n t e r est ingly, t h e ad jacen t r e s i due a t t h e act ive si te of Manduca i n h i b i t o r s a n d B P T I is a l a n i n e , w h e r e a s lys ine serves th is p u r p o s e in t h e Sarcophaga i nh ib i tor. T h r e e c h y m o t r y p s i n i nh ib i t o r s of t h e B P T I class h a v e b e e n i so la ted f rom B. mori a n d s e q u e n c e d (Sasak i , 1984, 1988). T h e s e i nh ib i t o r s h a v e p h e n y l a l a n i n e as t h e P I r e s idue . A role for these i nh ib i t o r s in r e g u l a t i o n of p r o p h e n o l o x i d a s e ac t iva t ion h a s n o t b e e n e s t ab l i shed .

Se r ine p r o t e a s e i n h i b i t o r s f rom t h e s e rp in g e n e supe r fami ly (Ca r r e l l a n d Boswel l , 1986) have b e e n ident i f ied a n d c h a r a c t e r i z e d in t w o l e p i d o p t e r a n insec t s , M. sexta a n d B. mori. Se rp in s a r e p r o t e i n s of Mr = 4 0 , 0 0 0 - 6 0 , 0 0 0 t h a t , in v e r t e b r a t e s , a p p e a r to func t ion p r i m a r i l y as r e g u l a t o r s of e n d o g e n o u s


Inhibitor 1 10 20

Manduca A A G L Υ Κ Ρ Ρ Ν Ν I Ε S Ε Ν Ε V Υ Τ G Ν Manduca Β Ε D Sarcophaga D V Κ S BPTI R Ρ D

21 30 40

Manduca A I C F L Ρ L Ε V G V C * R Α L F F R Υ G Υ

Manduca Β I C S L Ρ Ρ Ε V G Ρ C R Α G F L Κ # Α Υ Sarcophaga A C L Q Ρ Κ Ε V G Ρ C R Κ S D F V F F Υ BPTI F C L Ε Ρ Ρ Υ Τ G Ρ C Κ Α R I I R Υ F Υ

41 50 60

Manduca A D Ρ A I κ Α C Χ Ε F Μ Υ G G Χ Q - - - -Manduca Β Y S Ε L Ν Κ C Κ L F θ Υ G G C Q G Ν Ε Ν Sarcophaga Ν D A Τ Κ Α C Ε Ε F L Υ G G C R G Ν D Ν BPTI Ν A Κ A G L C Q Τ F V Υ G G C R Α Κ R Ν

61 70 80

Manduca A Manduca Β N F E T L Q A C X Q A Sarcophaga R F Ν Τ Κ Ε Ε c Ε Κ L C L BPTI Ν F Κ S Α Ε D C Μ R Τ C G G Α

# : F or Υ (position "38" of Manduca inhibitor Β ) @ : Τ or G (position "51" of Manduca inhibitor B )

Figure 2 Alignment of the protein sequences of Manduca protease inhibitors, Sarcophaga protease inhibitor, and Bovine Pancreatic Trypsin Inhibitor (BPTI) . T h e PI residue of the reactive site region is indicated by an asterisk.

se r ine p r o t e a s e s . For e x a m p l e , a n t i t h r o m b i n - I I I a n d C l - i n h i b i t o r f rom h u m a n p l a s m a a r e involved in r egu l a t i on of b lood c lo t t ing a n d c o m p l e m e n t ac t iva t ion , respectively. L ike p r o p h e n o l o x i d a s e ac t iva t ion , these a r e p a t h ways in w h i c h a c a s c a d e of se r ine p r o t e a s e s l eads to ac t iva t ion of a n e n z y m e t h r o u g h c leavage of a p r o e n z y m e .


A t ryps in i n h i b i t o r (Mr = 42 ,000) a n d a c h y m o t r y p s i n i n h i b i t o r (Mr = 43,000) f rom the s e rp in supe r fami ly have b e e n i so la ted f rom t h e h e m o l y m p h of B. mori (Sasak i a n d K o b a y a s h i , 1984; E g u c h i a n d S h o m o t o , 1985). T h e s e p r o t e i n s h a v e a reac t ive si te n e a r the i r C - t e r m i n a l e n d s , a t w h i c h they fo rm a c o m p l e x w i t h se r ine p r o t e a s e s . T h e c o m p l e x is s t ab l e in s o d i u m d o d e c y l sulfa te b u t n o t a t h i g h p H (Sasak i , 1985; Sasak i et al., 1987), p r o p e r t i e s in c o m m o n w i t h we l l - cha rac te r i zed h u m a n se rp ins . F o u r s e rp ins h a v e b e e n i so la ted f rom h e m o l y m p h of M. sexta ( K a n o s t , 1990a) . T w o of these p r o t e i n s a r e c h y m o t r y p s i n i nh ib i t o r s , o n e is a t r yps in inh ib i to r , a n d t h e o t h e r is a n e l a s t a se inh ib i to r , all of a p p r o x i m a t e l y Mr = 47 ,000 . A l t h o u g h these four p r o t e i n s a r e t h e m o s t a b u n d a n t s e rp ins p r e s e n t in M. sexta h e m o l y m p h , t w o -d i m e n s i o n a l gel e l ec t rophores i s a n d i m m u n o b l o t t i n g ident i f ied a p p r o x i m a t e l y t en p r o t e i n s of Mr = 4 5 , 0 0 0 - 4 8 , 0 0 0 t h a t r eac t w i t h a n t i b o d i e s to t h e e l a s t a se i n h i b i t o r ( K a n o s t , 1990b) .

c D N A clones for M. sexta e l a s tase i n h i b i t o r a n d the B. mori t r y p s i n i nh ib i to r h a v e b e e n i so la ted a n d s e q u e n c e d ( K a n o s t et al. , 1989; T a k a g i et al., 1990). T h e d e d u c e d p r o t e i n s e q u e n c e s a r e 5 6 % iden t i ca l w i t h each o t h e r (Fig . 3) a n d each insec t p r o t e i n is 2 5 - 3 0 % iden t i ca l w i t h v e r t e b r a t e s e rp in seq u e n c e s . I n t h e reac t ive si te n e a r t h e C - t e r m i n u s , w h i c h d e t e r m i n e s t h e specificity of t h e i n h i b i t o r s , t he two insec t s e rp in s e q u e n c e s a r e q u i t e differen t . T h i s is cons i s t en t w i t h t he o b s e r v a t i o n t h a t in m a m m a l i a n s e rp in genes , t h e reg ion e n c o d i n g the reac t ive si te h a s d ive rged m u c h m o r e r a p i d l y t h a n t h e r e m a i n d e r of t h e gene , r e su l t i ng in a family of i nh ib i t o r s w i t h a fairly c o n s t a n t f r a m e w o r k b u t v a r i a b l e reac t ive sites r e su l t i ng in d ive r se func t ions (Hi l l a n d H a s t i e , 1987). T h e B. mori t r yps in i n h i b i t o r h a s a lys ine a t t h e P I r e s idue of t h e reac t ive si te , w h i c h d e t e r m i n e s its specificity for t r y p s i n , w h e r e as t h e M. sexta e l a s t a se i n h i b i t o r h a s a n a l a n i n e as t h e P I r e s idue , l e a d i n g to specificity for e l a s t a se i nh ib i t i on . A c c o r d i n g to a c o n v e n t i o n of n a m i n g serp i n s b a s e d o n the i r P I r e s i d u e (Ca r r e l l a n d Boswel l , 1986), these t w o p r o te ins m a y b e referred to as B. mori lysse rp in a n d M. sexta a l a s e r p i n .

S e r p i n s h a v e n o t yet b e e n i so la ted f rom n o n l e p i d o p t e r a n insec t s . H o w e v e r , h e m o l y m p h from insects in seven different o r d e r s c o n t a i n a b u n d a n t se r ine p r o t e a s e i n h i b i t o r ac t iv i ty a n d p r o t e i n s of Mr = 4 0 , 0 0 0 - 6 0 , 0 0 0 ( M . K a n o s t , u n p u b l i s h e d d a t a ) . P r e l i m i n a r y e x p e r i m e n t s a lso sugges t t h a t s e rp in s a r e p r e s e n t in h e m o l y m p h of D. melanogaster ( K a n o s t , 1990b) .

T h e h y p o t h e s i s t h a t s e rp ins func t ion as r e g u l a t o r s of p r o p h e n o l o x i d a s e ac t iva t ion is b a s e d o n mos t l y c i r c u m s t a n t i a l ev idence . L ike b l o o d c lo t t ing in v e r t e b r a t e s , p r o p h e n o l o x i d a s e ac t iva t ion in insec ts m u s t b e a r a p i d , local , a n d t r a n s i e n t r e s p o n s e t h a t does no t s p r e a d far f rom t h e o r ig ina l s i te . T h e fact t h a t insec t h e m o l y m p h c o n t a i n s se rp ins s imi l a r to t hose t h a t r e g u l a t e t h e se r ine p r o t e a s e s of ac t iva t ion c a s c a d e s in v e r t e b r a t e s p o i n t s t o w a r d t h e poss i bi l i ty of a c o m p a r a b l e ro le in t he p r o p h e n o l o x i d a s e c a s c a d e . I n s u p p o r t of

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th is s p e c u l a t i o n , in ject ion of a n t i b o d i e s a g a i n s t M. sexta a l a s e r p i n i n t o M. sexta l a rvae resu l t s in i n c r e a s e d p h e n o l o x i d a s e activity, sugges t i ng t h a t t h e a n t i b o d i e s d i s r u p t r e g u l a t i o n of t h e ac t iva t ion p a t h w a y ( K a n o s t , 1990b) . F u r t h e r e x p e r i m e n t s w i t h i so la ted c o m p o n e n t s of t h e p a t h w a y will b e r e q u i r e d to conf i rm the role of s e rp ins as r e g u l a t o r s of p r o p h e n o l o x i d a s e ac t iva t ion .

C. Prophenoloxidase Inhibitor

S u p p r e s s i o n of hos t p h e n o l o x i d a s e ac t iv i ty by i n v a d i n g p a r a s i t e s is a n a t t r a c t ive m e c h a n i s m for con t ro l of hos t defense r eac t ion exh ib i t ed by w a y of p r o p h e n o l o x i d a s e ac t iva t ion (Sal t , 1970).

S to l tz a n d C o o k (1983) d e m o n s t r a t e d t h e s u p p r e s s i o n of h o s t p h e n o l o x idase ac t iv i ty by h y m e n o p t e r a n p a r a s i t o i d s . T h e p a r a s i t o i d s i n t r o d u c e p o l y d n a v i r u s pa r t i c l e s i n t o t h e hos t t h a t a p p e a r to i nh ib i t p h e n o l o x i d a s e ac t iv i ty (Stol tz et al., 1984; B e c k a g e et al., 1990). T h i s resu l t c lear ly i n d i c a t e s t h a t t he p a r a s i t e s a r e e i the r a t t e m p t i n g to e v a d e or d i s r u p t t h e h o s t defense r eac t ion for the i r o w n su rv iva l . H a r d e n i n g of capsu l e s b y p h e n o l o x i d a s e r eac t ion p r o d u c t s c a n po ten t i a l ly l imi t t h e g r o w t h a n d d e v e l o p m e n t of t h e p a r a s i t e s a n d h e n c e p a r a s i t e s m i g h t h a v e a d a p t e d t h e i n h i b i t i o n of p r o p h eno lox idase ac t iva t ion as a s t r a t egy to c i r c u m v e n t this p r o b l e m . T h u s , in a h o s t - p a r a s i t e i n t e r ac t i on t h e i nh ib i t i on of p r o p h e n o l o x i d a s e ac t iv i ty p l ays a c ruc ia l ro le . T h e i nh ib i t i on , w h i c h cou ld o c c u r theore t i ca l ly a t different s t ages in t h e c a s c a d e , n e e d s to b e e x a m i n e d . T h e poss ib le p r e s e n c e of a p h e n o l o x i d a s e i n h i b i t o r in s u c h sys t ems is likely, b u t fu r the r s tud i e s a r e essen t ia l to e v a l u a t e this c o n t e n t i o n .

D. Dopachrome Conversion Factor

T y r o s i n e a n d its h y d r o x y l a t e d p r o d u c t , d o p a , serve as t h e p r e c u r s o r s for e u m e l a n i n b iosyn thes i s . E u m e l a n i n p i g m e n t s a r e wide ly d i s t r i b u t e d in t h e a n i m a l k i n g d o m as skin a n d h a i r p i g m e n t s . B e c a u s e of the i r free r a d i c a l n a t u r e , m e l a n i n s p r o t e c t a n i m a l s f rom d a m a g i n g so la r r a d i a t i o n ( P r o t a , 1988). A b n o r m a l i t i e s in m e l a n i n b iosyn thes i s a r e a s soc i a t ed w i t h vi t i l igo as well a s m e l a n o m a cance r . N a t u r a l l y , n u m e r o u s worke r s have d e v o t e d m u c h a t t e n t i o n to s t u d y the m e c h a n i s m of m e l a n i n b iosyn thes i s . T h e in i t ia l s t eps for m e l a n i n b iosyn thes i s w e r e e s t ab l i shed by the c lass ical w o r k of R a p e r a n d M a s o n a n d h e n c e it is ca l led t he R a p e r - M a s o n p a t h w a y ( M a s o n , 1955). T h e key r eac t ions of t h e R a p e r - M a s o n p a t h w a y a r e : (a) h y d r o x y l a t i o n of t y ros ine to d o p a , (b) o x i d a t i o n of d o p a to d o p a q u i n o n e , (c) i n t r a m o l e c u l a r cyc l iza t ion of d o p a q u i n o n e a n d s u b s e q u e n t g e n e r a t i o n of d o p a c h r o m e , (d) conve r s ion of d o p a c h r o m e to 5 ,6 -d ihydroxy indo le s , a n d (e) ox ida t ive p o l y m e r i z a t i o n of


5 ,6 -d ihydroxy indo le s . T h o u g h the first two reac t ions a r e ca t a lyzed b y u b i q u i tous ly p r e s e n t t y ros inase (a p h e n o l o x i d a s e ) , t h e res t of t h e r eac t ions a r e be l ieved to p r o c e e d nonenzymat i ca l ly , as c h e m i c a l o x i d a t i o n of d o p a cou ld g e n e r a t e m e l a n i n p i g m e n t s w i t h o u t t he need for a n y e n z y m e ( s ) . H o w e v e r , s tud ies d o n e in t he las t d e c a d e paved t h e w a y to t h e ident i f ica t ion of a s econd e n z y m e , namely , d o p a c h r o m e convers ion factor, as a n e w r e g u l a t o r of m e l a n o g e n e s i s (Pawelek et aL, 1980; L e o n a r d et al., 1988; A r o c a et al., 1989, 1991; A s o et al., 1989, 1990; Pawelek, 1990; S u g u m a r a n a n d S e m e n s i , 1991). D o p a c h r o m e convers ion factor f rom m a m m a l i a n sources ca ta lyzes t h e gener a t i on of 5 ,6 -d ihydroxy indo le -2 -ca rboxy l i c acid ( A r o c a et al., 1989, 1991; Pawelek, 1990), w h e r e a s t he e n z y m e i so la ted from insec ts s eems to p r o d u c e 5 ,6 -d ihyd roxy indo le from the s a m e s u b s t r a t e (Aso et al., 1990; S u g u m a r a n a n d S e m e n s i , 1991). R e c e n t inves t iga t ions on the m e c h a n i s m of th is conver sion revea l t h a t t he reac t ion p r o c e e d s t h r o u g h a q u i n o n e m e t h i d e i n t e r m e d i a t e ( S u g u m a r a n a n d Semens i , 1991; S u g u m a r a n , 1991b) . F i g u r e 4 shows t h e modif ied R a p e r - M a s o n p a t h w a y for t he b iosyn thes i s of m e l a n i n (Sugu m a r a n , 1991b) . D o p a c h r o m e in t he a b s e n c e of m e t a l ions h a s a defini te life t i m e a t n e u t r a l p H . The re fo r e , p r e s e n c e of d o p a c h r o m e conver s ion factor in insec ts e n s u r e s t he faster g e n e r a t i o n of m e l a n i n p i g m e n t by r ead i ly g e n e r a t ing the m e l a n i n p r ecu r so r , 5 ,6 -d ihydroxy indo le .

E. Quinone Isomerase

H e m o l y m p h of insec t l a rvae c o n t a i n s n u m e r o u s ty ros ine a n d d o p a de r iva t ives, m a i n l y in s t o r age form for l a t e r use as c u t i c u l a r t a n n i n g p r e c u r s o r s ( K r a m e r a n d H o p k i n s , 1987). I n p a r t i c u l a r , de r iva t ives of JV-ace ty ldopamine a n d Τν - β - a l a n y l d o p a m i n e a r e wide ly found in insec t s . S ince p h e n o l o x i d a s e s a r e nonspeci f ic ( B r u n e t , 1980), t hey will a t t ack i n d i s c r i m i n a t e l y a n y of t he ca techo l i c c o m p o u n d s . N a t u r a l a b u n d a n c e of JV-ace ty ldopamine a n d Ν-β-a l a n y l d o p a m i n e in t he h e m o l y m p h m a k e s it likely t h a t these c o m p o u n d s will be ox id ized by p h e n o l o x i d a s e in pre fe rence to o t h e r s . T h e q u i n o n e s gener a t e d from these c o m p o u n d s d o n o t p o l y m e r i z e i n s t a n t a n e o u s l y u n d e r p h y s i ological cond i t i ons , b u t u n d e r g o slow t r a n s f o r m a t i o n s . As a resu l t , t hey diffuse slowly from t h e site of g e n e r a t i o n a n d c a n reac t w i t h th iols a n d o t h e r nuc leoph i l e s in t h e h e m o l y m p h . S u c h reac t ions c a n have de l e t e r ious effects no t on ly on t h e i n v a d i n g p a r a s i t e s b u t a lso on t h e hos t . To p r e v e n t d a m a g e to self t i ssue , insec ts m u s t have a m e a n s to deac t iva t e t he q u i n o n o i d mo lecu l e s . O n e s u c h m e c h a n i s m h a s b e e n d iscovered (Sau l a n d S u g u m a r a n , 1989a ,b , 1990). Q u i n o n e i s o m e r a s e (Sau l a n d S u g u m a r a n , 1990), a n e n z y m e exh ib i t ing w i d e s u b s t r a t e specificity t o w a r d its s u b s t r a t e s , 4 -a lky l -o -benzoqu inones , conve r t s t he long-l ived q u i n o n e s to shor t - l ived 2-hydroxy-jfr-quinone m e t h ides . Q u i n o n e m e t h i d e s , be ing h igh ly u n s t a b l e , r ead i ly a n d r a p i d l y u n d e r g o


s \

IMINOQUINONE QUINONE QUINONE METHIDE

MELANOCHROME —*- MELANIN Figure 4 Modified R a p e r - M a s o n pathway of melanogenesis. Tyrosinase catalyzes the oxidation of tyrosine to dopa and dopa to dopaquinone. The latter undergoes rapid intramolecular cyclization to form leucochrome. Oxidation of leucochrome generates dopachrome. Dopachrome conversion factor tautomerizes this compound to the quin-one methide, which can either lose carbon dioxide to form 5,6-dihydroxyindole (DHI) or simply isomerize to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Oxidation of these indoles to corresponding quinonoid species and their subsequent polymerization ensure the production of melanin via melanochrome.

M i c h a e l - 1 , 6 - a d d i t i o n reac t ions w i t h s u r r o u n d i n g nuc l eoph i l e s w i t h t h e r egen e r a t i o n of ca t echo l s . T h i s r eac t ion p l ays a c ruc ia l ro le in t h e sc le ro t i za t ion of insec t cu t ic le ( S u g u m a r a n , 1988, 1991a) . T h e role of q u i n o n e i s o m e r a s e w i t h r e spec t to c u t i c u l a r t a n n i n g is s u m m a r i z e d in F ig . 5.

Sc le ro t i za t ion he lps t he sof t -bodied insec t to p r o t e c t itself f rom t h e h a r s h e n v i r o n m e n t by c r e a t i n g a f o r m i d a b l e ba r r i e r . Func t iona l ly , th i s r eac t ion c a n b e u sed in insec t i m m u n i t y in two a d v a n t a g e o u s w a y s . W h e n g e n e r a t e d a t t h e


NADA

HO-

DEHYDRO NADA DEHYDRO NADA QUINONE

Η N Y

ο

HO

3

Υ

NADA QUINONE DEHYDRO NADA QUINONE METHIDE

NADA QUINONE METHIDE

c ^ ^

QUINONE METHIDE SCLEROTIZATION So ! c

i QUINONE TANNING

Figure 5 Mechanisms of cuticular tanning. Sclerotizing precursors such as 7V-acetyl-dopamine (NADA) are oxidized by phenoloxidases (1) to their corresponding quinones. These quinones participate in quinone tanning or serve as substrates for quinone isomerase (2). Quinone methides generated by the action of quinone isomerase participate in quinone methide sclerotization or are converted into dehydro dopamine derivatives by quinone methide tautomerase (3). Further oxidation of dehydro NADA generates quinone and quinone methide derivatives that participate in sclerotization. These reactions are used widely to harden insect cuticle and can also be used during encapsulation reactions. C = Chemical reactions.

site of a foreign object , t he q u i n o n e m e t h i d e cou ld a t t a c h to t he i n t r u d e r ' s surface ( S u g u m a r a n , 1990). T h e ca t echo l a d d u c t s t h u s fo rmed cou ld serve as a b a s e for fu r the r r eac t ions i n c l u d i n g depos i t i on of m e l a n i n a n d sc le ro t in . Second , if t h e q u i n o n e m e t h i d e s a r e g e n e r a t e d a w a y from foreign ob jec t s , d u e to the i r i n h e r e n t reac t iv i ty w i t h wa te r , they c a n yield s ide -cha in h y d r o x y l a t e d ca techo l s t h a t a r e n o t as toxic as q u i n o n e s o r q u i n o n e m e t h i d e . T h e r e f o r e , t h e p r e s e n c e of q u i n o n e i s o m e r a s e in insec t h e m o l y m p h cou ld serve to detoxify t h e q u i n o n e s a n d to depos i t t he q u i n o n e m e t h i d e s o n foreign objec ts (Fig . 6) . Q u i n o n e i s o m e r a s e m i g h t a lso be used efficiently d u r i n g h o s t - p a r a s i t e in te r ac t ions . I f t he h o s t g e n e r a t e s a l k y l q u i n o n e s , t he p a r a s i t e s c a n a d a p t t h e s t r a t egy of u s i n g t h e q u i n o n e i s o m e r a s e to e i the r detoxify these q u i n o n e s o r u se t h e m to m a k e t h e c a p s u l e to p r o t e c t t hemse lves from fu r the r defensive r eac t ions of t h e hos t .

To pe r fo rm its task efficiently, it is a lso a d v a n t a g e o u s for t h e q u i n o n e i s o m e r a s e to c o m p l e x w i th p h e n o l o x i d a s e . E v i d e n c e for t h e ex i s t ence of a q u i n o n e i s o m e r a s e - p h e n o l o x i d a s e c o m p l e x comes from m o l e c u l a r sieve c h r o -


I I DEPOSITION ON

FOREIGN MATTER

Figure 6 Use of quinone methide in insect immunity. 4-Alkylquinones generated by the action of activated phenoloxidase (A) on catecholamine derivatives such as 7V-acetyldopamine (R = C H 3 ) or Λ^-β-alanyldopamine (R = C H 2 C H 2 N H 2 ) are converted by quinone isomerase (B) to quinone methides. Both quinones and quinone methides can be deposited on foreign objects to encapsulate them. Quinone isomerase in the hemolymph can also aid in destroying quinones, as the quinone methides formed react with water instantly to form nontoxic compounds.

m a t o g r a p h y (Sau l a n d S u g u m a r a n , 1989b) as well as i nh ib i t i on s tud i e s . T h e q u i n o n e i s o m e r a s e i so la ted from u n a c t i v a t e d h e m o l y m p h of S. bullata e lu tes w i t h a n a p p a r e n t MT of 80 ,000 , w h e r e a s t h e s a m e e n z y m e f rom ac t i va t ed h e m o l y m p h c o - c h r o m a t o g r a p h s w i t h p h e n o l o x i d a s e a t a h i g h e r m o l e c u l a r w e i g h t (Mr > 200 ,000) , i n d i c a t i n g poss ib le c o m p l e x f o r m a t i o n b e t w e e n these t w o p r o t e i n s . T h i s does n o t c o m e as a su rp r i s e b e c a u s e ac t iva t ed p h e n o l o x i d a s e is k n o w n to b e s t icky a n d b i n d s to g lass , S e p h a d e x gels , e tc . B u t w h a t is s u r p r i s i n g is t h e ab i l i ty of q u i n o n e i s o m e r a s e to con t ro l t h e ac t iv i ty of p h e n o l o x i d a s e . O n c e c o m p l e x e d , t h e p h e n o l o x i d a s e act ivi ty is i n h i b i t e d b y a s m u c h as 5 0 % b y q u i n o n e i s o m e r a s e (Fig . 7). T h u s , q u i n o n e i s o m e r a s e s e e m s to serve as a nega t ive con t ro l m e c h a n i s m for p h e n o l o x i d a s e as well .

IV. Conclusion

I n c o r p o r a t i o n of t hese con t ro l m e c h a n i s m s in to t he p r i m a r y p r o p h e n o l o x i d a s e c a s c a d e l eads to t h e overa l l s c h e m e of r eac t ions d e p i c t e d in F ig . 8. A c c o r d i n g to th is s c h e m e , u p o n e n t r y of foreign s u b s t a n c e s , r ecogn i t i on mo lecu le s (des ign a t e d ve ry b r o a d l y as z y m o g e n ) b i n d to t h e m a n d t r igger a va r i e ty of hos t -defense s y s t e m s i n c l u d i n g c a l c i u m m o b i l i z a t i o n . T h e b i o c h e m i c a l a s p e c t s of th is s t age a r e n o t ye t clarified in de ta i l , b u t s o m e of t h e a s p e c t s have b e e n


0.5 1.0 TIME (min)

Figure 7 Inhibition of phenoloxidase by quinone isomerase. A reaction mixture containing 1 mM JV-acetyldopamine and 10 μg of mushroom tyrosine in 1 ml of 0.1 Μ sodium phosphate buffer, p H 6.0, was incubated at 30°C and oxygen consumed during the reaction was continuously measured using a Clark type oxygen electrode (curve A). At the indicated time about 250 units of purified quinone isomerase from S. bullata was added (Ai). In curve B, quinone isomerase was present from the beginning of the reaction.

r ev iewed by S c h m i d t a n d T h e o p o l d (1991) . W h a t follows th is p roces s r e g a r d ing p r o p h e n o l o x i d a s e ac t iva t ion is fairly well e s t ab l i shed . T h e ac t iva t ion of p r o - b e n z o y l a r g i n i n e e thy l ester , p r e p r o p h e n o l o x i d a s e ac t iva t ing e n z y m e , a n d p r o p h e n o l o x i d a s e occu r s in s e q u e n c e . T h e ce l lu la r d a m a g e a n d c a l c i u m m o b i l i z a t i o n c a n ac t iva te p h o s p h o l i p a s e A 2 , w h i c h spl i ts off a r a c h i d o n i c acid from t h e ce l lu la r p h o s p h o l i p i d s . E i c o s a n o i d s p r o d u c e d from a r a c h i d o n i c acid t r igger fu r the r i m m u n e reac t ion , whi le l y o p h o s p h o l i p i d s a n d p h o s p h o l i p i d s fo rmed d u r i n g ce l lu la r d a m a g e a n d p h o s p h o l i p a s e ac t ion d i rec t ly ac t iva t e p r o p h e n o l o x i d a s e . P r o t e a s e i nh ib i t o r s p rov ide nega t ive con t ro l b y p r e v e n t i n g p r o p h e n o l o x i d a s e ac t iva t ion , w h e r e a s q u i n o n e i s o m e r a s e pa r t i a l l y con t ro l s p h e n o l o x i d a s e activity. Posit ive con t ro l for m e l a n i n a n d / o r sc le ro t in fo rmat ion is p r o v i d e d by b o t h q u i n o n e i s o m e r a s e a n d d o p a c h r o m e conve r s ion factor. By un iden t i f i ed m e c h a n i s m ( s ) p a r a s i t e s a n d o t h e r p r e d a t o r s s e e m to i nh ib i t t he p r o p h e n o l o x i d a s e c a s c a d e (no t s h o w n in F ig . 8) .

T h i s s c h e m e r e p r e s e n t s on ly t h e con t ro l m e c h a n i s m s k n o w a t th is t i m e a n d h e n c e b y n o m e a n s c a n it b e cons ide red as c o m p l e t e . As a d d i t i o n a l r e s e a r c h is c o m p l e t e d , we a r e su re t h a t th is s c h e m e m i g h t b e c o m e m o r e c o m p l e x . A t this p o i n t we a lso like to c a u t i o n t he r e a d e r s r e g a r d i n g c e r t a i n


FOREIGN SUBSTANCES Λ ,N A(

Λ ZYMOGEN ACTIVE ENZYME & C a 2 + MOBILIZATION

PRO-BAEE BAEE PRO PLA 2

TRIGGER IMMUNE SYSTEM

\ EICOSANOIDS

•

" PLA2 ARACHIDONIC ACID

\ PHOSPHOLIPIDS

PRE-PPAE PPAE (& CUTICULAR PROTEASE)

LYSOPHOSPHOLIPIDS

PROTEASE INHIBITORS

PROPHENOLOXIDASE

QUINONE ISOMERASE

DIPHENOL

PHENOLOXIDASE

QUINONE

QUINONE ISOMERASE

DOPACHROME CONVERSION FACTOR •

MELANIN AND/OR SCLEROTIN

Figure 8 The prophenoloxidase cascade and its control mechanisms. Foreign substances activate prophenoloxidase cascade as outlined in Fig. 1. Protease inhibitors inhibit serine proteases, thus preventing them from activating the prophenoloxidase. Cellular damage and calcium mobilization can also trigger the inactive pro-phospholipase A 2 (PRO PLA 2 ) to active P L A 2 . Lipids generated during cellular damage as well as P L A 2 action can directly bind to prophenoloxidase and activate'it. Quinone isomerase can control the activity of active phenoloxidase negatively. In addition, this enzyme can play a positive role by providing the quinone methides for sclerotin formation. Dopachrome conversion factor, by facilitating the production of dihydroxyindoles, participates positively in melanin capsule formation. Phenoloxidase inhibitor could constitute another control mechanism (not shown in figure). T h e reactions of quinones and quinone methides lead to the production of melanin a n d / o r sclerotin capsule.

c o m p l i c a t i o n s . A s p o i n t e d o u t in t h e I n t r o d u c t i o n , t h e r e a r e a t leas t t h r e e different p h e n o l o x i d a s e s k n o w n in va r ious sy s t ems . T h e s e e n z y m e s s e e m to serve t h r e e different func t ions in insec ts , namely , w o u n d h e a l i n g , c u t i c u l a r sc le ro t i za t ion , a n d i m m u n e r e s p o n s e . B e c a u s e of t h e p r o b l e m s in a s s ign ing a n y p a r t i c u l a r role for o n e o r m o r e of t h e p h e n o l o x i d a s e s , it is r a t h e r difficult


to say prec ise ly w h e t h e r o r n o t all t h e m e c h a n i s m s o u t l i n e d in F ig . 8 a r e

a p p l i c a b l e to all insec ts . I t is poss ib le t h a t on ly p a r t of t h e r eac t ions o p e r a t e

in o n e or m o r e insec t s . A s for fu ture d i r ec t ions , p rac t i ca l ly t h e w h o l e a r e a is

w i d e o p e n for a de t a i l ed s tudy. S ince p h e n o l o x i d a s e p a r t i c i p a t e s a s a key

c o m p o n e n t n o t on ly in insec t i m m u n i t y b u t a lso in sc le ro t iz ing m a c h i n e r y

a n d w o u n d - h e a l i n g m e c h a n i s m s , e x a m i n i n g t h e de t a i l ed b i o c h e m i s t r y of th i s

v i ta l e n z y m e s y s t e m a n d its con t ro l m e c h a n i s m s w a r r a n t s c o n s i d e r a b l e ef

fort. S u c h s tud i e s m a y even tua l ly h a v e g r e a t c o m m e r c i a l a n d l ife-saving

p o t e n t i a l s if novel vec to r con t ro l m e a s u r e s a n d insec t ic ides c a n b e m a d e

b a s e d o n th is work .

References

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Index to Volumes 1 and 2 Volume numbers are boldfaced, separated from the first page reference with a colon. Subsequent references to the same material are separated by commas.

Abundance endotoxins in bacter ium/nematode

complexes, 2:12 teratocytes, 1 :148-152

JV-Acetyl galactosamine, BT endotoxin receptor, 2:67

AcNPV, see Autographa californica nuclear polyhedrosis virus

Actin polymerization, in AcNPV cells during gene expression, 2:118

Activity levels changes in parasitized insects, 1:110—

111 host, effect on parasite dissemination,

1:112 Adaptat ion tolerance hypothesis, 1:138 Adenylate cyclase pool, role in host via

bility, 1 :127-128 Adhesion

deuteromycete entomopathogens, 2:212-214

outer membrane protein role, 2:9-10 proteinaceous appendages role, 2:9-

10 Xenorhabdus to hemocytes, 2:8-10

Aedes aegypti hemolymph plasma protein expressed

during wound healing, 1:250 phenol oxidase activity during encap

sulation, 1 :252-253 Plasmodium gallinaceum-'mfected, effects

on reproduction, 1 :88-89

Agglutinins, see also Lectins non-self recognition molecules,

1 :282-285 Τν '-β-Alanyldopamine, in A. aegypti he

molymph plasma, 1:254 Allantonematid nematodes, effects on

bark beetle reproduction, 1:94 Allatostatins, 1:61 Allatotropins, 1:61 Allelochemicals, in host food, effect on

endoparasites, 1:68 Amino acid composition, after parasi

tization, 1 :127-128 Amino acid sequence, cecropins, 2:28-

29 Amino acids, nutrient role for develop

ing parasites, 1 :139-140 Amplification, in situ, polydnavirus,

1:172 Anacridium aegyptium, M. calloti infec

tions, 1 :99-100 Androctonus australis Hector neurotoxin,

2:187-188 Andropin, structure and action, 2:30 Anopheles gambiae, strain susceptibility

differences, 1:257 Antibacterial peptides, origins, 2:43 Antibacterial proteins, induction, 2:6-7 Antibacterial response, humoral , blood

cell role, 1 :293-295 Antigens, hostlike, of parasites, 1:26 Antigen shedding, by parasites, 1:50

3 4 3

3 4 4 Index to Volumes 1 and 2

Antihormones, effect on endoparasite development, 1:69

Antimicrobials, produced by symbiont bacteria, 2 :13-14

Aphidius ervi, development and growth, 1 : 6 -7

Appressoria, deuteromycete ento-mopathogens, 2 :216-218

Arylphorins parasitism effects, 1:44 posttranscriptional regulation, 1:44

Ascovirus cytopathology, 2:86 pathogenesis, 2 :85-89 tissue tropism, 2:84 transmission mode, 2:88

Assassin genes, in nonpermissive hosts, 1:40

Attacin G domain, 2 :33-34 structure and actions, 2 :32-34

Attraction, motility component, 2:3 Autographa californica nuclear polyhedro-

sis virus co-occlusion system, 2:189 cytopathic effects of in vitro infections,

2:114 entry

microfilaments, 2 :114-115 microtubules, 2:115

extracellular virus form, 2:113 gene expression, 2:184 genetic engineering, 2 :97-98 genome

new gene acquisition, transposon-related, 2 :172-173

nonessential regions, 2 :150-151 tolerance to size changes, 2:150 transposon gene disruptions,

2 :169-170 infection cycle, 2 :149-150 morphogenesis, 2 :119-120 polyhedra-derived form, 2:113 replication

nucleosomal structure during, 2 :117-118

pathways of entry, 2 :113-114 in vitro, 2:114, 183 in vivo, 2:182

transcription, nucleosomal structure during, 2 :117-118

Avirulence genes, in plant pathogens, 1 :29-30

Azadiractin, effect on endoparasite development, 1:68

Bacillus thuringiensis characterization, 2:56 crystal protein genes, classification,

2:57 Cyt toxins, mechanism of action,

2 :65-66 endotoxins

Cry toxin mechanism of action, 2 :62-65

Cyt toxin mechanism of action, 2 :65-66

domains affecting specificity, 2:73 δ-endotoxin, 2 :187-188 hydrophobic regions, 2:63 primary structure, 2 :56-58 proteolytic processing and activa

tion, 2 :60-61 receptor characterization, 2 :66-67 resistance to, 2 : 69 -71 , 73 tertiary structure, 2 :58-60

Bacillus thuringiensis aizawai I C 1 , insec-ticidal crystal protein, 2:60

Bacteria, see also specific bacteria attachment to hemocytes, 2 :9-10 cell membrane lysis by cecropins,

2:27 cell wall, degradation by lysozymes,

2 :35-36 gram-positive, triggering of

prophenoloxidase cascade, 1:323 hydrophobicity, 2 : 8 -9 symbionts

independent killing of G. mellonella, 2:12

specificity, 2:17

Index to Volumes 1 and 2 3 4 5

Baculovirus classification, 2:182 enhancin genes, cloning, 2 :140-141 entry, cytoskeleton involvement,

2 :115-116 genetic modification

alternative gene promoters, 2 :184-185

multiple expression vectors, 2 : 185 -186

very late gene promoters, 2 :184-185

genome as interspecies shuttle for lipidop-

teran elements, 2:174 mutagenesis, host transposon role,

2 :168-173 structure, 2:182 transposon-related new gene acqui

sition, 2 :172-173 insecticides

genetic manipulation, 2 :181-182 host range, 2:180 persistence, 2:180, 186-188 production, 2:181 recombinant, 2 :186-191 speed of action, 2:181 target range, 2 :180-181

latent, potential of genetic transfer with recombinant virus insecticides, 2:189

Oryctes rhinoceros, 2:94 pathogenesis

granulosis virus, 2 :90-91 nuclear polyhedrosis virus, 2 :89-90

replication in vitro, 2:183 in vivo, 2:182

symbiotic interactions with host transposons, 2 :173-175

tissue tropism, 2:84 transposon insertions, virus proper

ties compatible with, 2 :148-151 transposons, identification, 2 : 152-

154 uncoating, 2:116

Bark beetle, reproduction, al-lantonematid nematode effects, 1:94

BBMV, see Brush border membrane vesicles

Bee peptides, proline-rich, 2 :34-35 Behavior, see also specific types of behavior

activity level, 1:110-111 microhabitat preference, 1:108-110 reproductive, by castrated insects,

1:111 use of venoms, 1 :232-233

Behavioral fever, 1:114 Benzoylarginine ethyl esterase, 1:321 Bioassays

with H P L C for ecdysteroid quantification, 1:77

insect, enhancement with Trichoplusia ni GV enhancin, 2 :137-140

Biochemical pathways, wasp, venom studies, 1:232

Biological control classical agents, 2:92-93 inoculative agents, 2:92 strategies for viruses, 2 :92-93 venoms for, 1:239-240 viral insecticides, 2 :92-93

Bioluminescence, role in nematode/ bacterium symbiosis, 2 :14-15

Bionomics, host-parasi te , inferred from venoms, 1:228-230

Blood cells characterization, 1:275-280 classification, 1:275-279 hemopoietic tissue diversity, 1:278 manipulation techniques, 1:280-282 prohemocytes, 1:279 role in humoral antibacterial re

sponse, 1:293-295 Body temperature, effect on host sur

vival, 1:114 Braconids, larval parasites, effects on

host endocrine system, 1:709 Bracovirus, nucleocapsids, 1:197-198 Brugia malayi, A. aegypti strains refrac

tory for, 1:260


Brush border membrane vesicles, midgut

binding assays for endotoxin receptors, 2 :66-67

Cry toxin binding, 2 :62-63 BT, see Bacillus thuringiensis Buthus epeus, insectotoxin 1, 2 :187-188

Calcium effect on erythrocyte spontaneous

rosetting, 1:284, 288 in prophenoloxidase cascade, 1:322

Calyx fluid effects on host, 1 :75-76 nutrient level changes due to, 1:179 requirement for parasitization, 1:175

Campoletis sonorensis virus DNA

interspecific differences, 1:195 intraspecies differences, 1 : 1 9 3 -

195 nonequimolarity, 1:193 structure, 1 :192-196

gene expression, temporal pattern, 1:206

gene families characterization, 1 :201-205 significance, 1 :206-208

genome functional organization, 1 :199-201 intraspecific genomic complexity,

1 :198-199 packaging, 1 :196-198 structure, 1 :191-196

Carbohydrases, effect on Xenorhabdus adhesion to hemocytes, 2:8

Carbohydrates in bacterial surface modification sys

tem, 2:7 metabolite levels in host tissues,

1 :127-128 storage alterations, 1:134—135 synthesis, Trichoplusia ni, 1:131

Carbon dioxide, at tractant to host insects, 2:3

Castration, host effects on parasite survival, 1:113 enhancement of parasite dissemina

tion, 1:112 in insect-insect association, 1:98 reproductive behavior after, 1:111

Catecholamines, substrates for melanotic encapsulation reactions, 1:255

Cecropia immunoresponsive factor binding to κΒ-like motifs, 2 :42-43 similarity to transcription factor

N F - K B , 2 :42-43 Cecropins

induction by endotoxin and β-15 3-glucan,

1:271 in response to nonpathogenic bac

teria injection, 2 :6 -7 structure and action, 2 :27-30 synthesis and processing, 2:30

Cell adhesion molecule, hemolin similarity, 2 :38-39

Cell-cell communication, in immunity, 1 :290-293

Cell manipulation techniques, 1:280— 282

Cell rounding, AcNPV-related, 2 : 116-117

Cell-surface determinants, cell type identification, 1:277

Cell-to-cell cooperation, in immune reactivity, 1:271

Cellular immune response early and intermediate cellular stage,

1 :270-271 final humoral/cellular stage, 1:271 —

273 immediate stage, 1 :268-270 inductive stage, 1 :268-270 interrelationship with humoral im

munity, 1 :293-295 mosquitos, 1 :248-249 to nematode entry, 2:6 protein synthesis stage, 1 :270-271

Cessation factors, secretion by hemocytes, 1:272


Cestodes, host reproductive disturbances caused by, 1 :96-98

CI factor, binding affinity to κΒ-like motifs, 2 :42-43

Chelonus egg-larval parasites, effects on host endocrine system, 1:70-71

Chemical resistance, comparison with viral resistance, 2 :205-206

Chitinase, requirement for, 2:221 Chitin synthesis inhibitors, effect on en

doparasites, 1:69 Chymoelastase, 2 :220-221 Chymotrypsin inhibitors, prophenolox

idase cascade, 1:329 C I F , see Cecropia immunoresponsive

factor Cloning, enhancin gene, 2 :140-141 Coccygomimus turionellae, development

and growth, 1:5—6 Coevolution, see also Evolution

adaptat ion tolerance hypothesis, 1:138

gene-for-gene, 1 :29-30 studies, wasp venoms for, 1 :233-

236 Coleoptericin, isolation, 2:34 Conformers

combined conformer-regulator strategy, 1:60

definition, 1:17, 60 Conidia, adhesion, 2 :212-214 Co-occlusion system, for AcNPV, 2:189 Corpora allata, effects of nematode in

fections, 1 :95-96 Cotesia congregata

-Manduca sexta system metabolic alterations, 1:131-132 parasitism characteristics, 1:31—33 polydnavirus induction of

parasitism-specific proteins, 1 :75-76

parasitism of sphingid hosts, 1:38 Coumarins , availability to endo

parasites, 1:68 Crab , parasitism-specific proteins,

1:49

Cry toxins binding characteristics to BBMV,

2:70 mechanism of action, 2 :62-65 primary structure, 2 :56-58 tertiary structure, 2 :56-58

CsV, see Campoletis sonorensis virus Cuticle, penetration by deuteromycete

fungal pathogens adhesion events, 2 :212-214 cuticle-degrading enzymes, 2 :219-

221 major components, schematic, 2:213 prepenetration growth, 2 :214-216 structural adaptations for penetra

tion, 2 :216-219 Cuticle-degrading enzymes

characterization, 2 :219-221 regulation, 2 :219-221

Cuticle tanning, 1:333-334 Cytokines, dual roles in mammals ,

1:27 Cytopathology, major insect viruses,

2:86 Cytoplasmic polyhedrosis virus

cytopathology, 2:86 pathogenesis, 2 :91-92 tissue tropism, 2:84 virulence, 2:95

Cytoskeleton interactions with viral particles

entry into cell, 2 :105-107 nucleocapsid transport, 2:107 particle binding, 2:105 uncoating, 2 :107-108

role in favoring viral over host synthesis, 2:118

Cytoskeleton/nuclear matrix in AcNPV transcription and replica

tion, 2 :117-119 stages of baculovirus replication asso

ciated with, 2 :115-120 Cyt toxins

mechanism of action, 2 :65-66 structure and biological activity,

2:58


Defense response, melanization role, 1:176

Defensins, structure and actions, 2 : 30 -34

Deletion mutants , pathogenicity improvement with, 2:98

Designer genes, parasite, 1:29 Deuteromycete fungal pathogens, see

Fungal pathogens Development

event timing, venoms as probes for, 1 :230-231

host alteration effects on parasite nutri

tion, 1:136 teratocyte effects, 1 :158-160

parasite, see Parasite development regulation, polydnavirus-related,

1 :176-179 Developmental strategies, definition,

1:2 Dexamethasone, effect on xenograft re

jection, 1:313 Diapause, parasite, 1 :9 -10 Diflubenzuron, effect on endoparasite

development, 1:69 Diphenol oxidase, H P L C - E C D , 1:252 Diptericin

G domain, 2 :33-34 gene expression, 2:33 structure and actions, 2 :32-34

Dissemination, parasite, enhancement by behavioral alterations, 1:111 — 112

Diuretic hormone, in baculovirus insecticide system, 2:186

Diurnal behavior, nocturnal insect hosts, 1:110

DNA baculovirus, transposon-associated al

terations, 2 :168-170 Campoletis sonorensis virus

interspecific differences, 1:195 intraspecies variations, 1 :193-195

Heliothis exiguae virus, 1:196 linear and circular forms in para

sitoid tissue, 1:171

polydnavirus genetic colonization of host, 1 : 1 7 2 -

173 multiple covalently closed circular

form, 1:192 persistence in host cells, 1:50 superhelical and relaxed circular

forms, 1:192 Dopachrome, role in mosquito immune

response, 1:252 Dopachrome conversion factor, 1 : 3 3 1 -

332 Dopamine, in A . aegypti hemolymph

plasma, 1:254 Drosophila melanogaster

cecropin levels, 2:28 genomic transposon sequences, 2:151 neuroglian, similarity to hemolin,

2 :38-39 Duplication, viral genes, 1 :216-217

Ecdysone, functions, 1:61 Ecdysone 20-monooxygenase, parasit

ism effects, 1:47 Ecdysteroids

host, effects of parasitoids, 1 :72-73 larval parasite effects, 1:70 metabolism, 1:63 parasite levels relative to host levels,

1 :64-66 production, 1:61 quantification methods, 1:77 quantification with RIA with

chromatography, 1:77 structures, 1:62

Eclosin hormone, 1:61 Efficacy, viral insecticides, 2:82, 96 Egg-larval parasites, effects on host en

docrine system, 1 :70-71 Egg production

cestode infection effects, 1:97 nematode infection effects, 1:96

Egg viability cestode infection effects, 1:97 definition, 1:91


Eicosinoids, effects on xenograft rejection, 1:313

Elastase inhibitors, prophenoloxidase cascade, 1:329

Elevation-seeking behavior effects on parasite dissemination,

1:112 in parasitized insects, 1:108-109

Enantiostasis definition, 1:139 in metabolic regulation, 1 :139-140

Encapsulation catecholamine substrates, 1:255 enzymes in, 1 :251-255 hemolymph plasma protein expressed

during wound healing (A. aegypti), 1:250

melanotic, see Melanotic encapsulation

parasitized larvae, inhibition by teratocytes, 1:156

plasma hemagglutinin function, 1:251

response to foreign objects, 1 :306-308

steinernematids, 2 :5 -6 variables affecting, 1 :307-308

Encapsulation-promoting factor, role in immunity, 1:291-295

Endocrine system host

effect on endoparasites, 1 :66-69 effects of endoparasites, 1 :70-74 involvement with microsporidia-

larval insect hosts, 1:91 parasite interactions, 1:99

immune system control by, 1 :279-280

limitations in studies of, 1:77 Endoparasit ic insects, reproduction dis

turbances caused by, 1 :98-100 Endotoxins

Bacillus thuringiensis binding, 2:62 Cry toxin mechanism of action,

2 :62-65

Cyt toxin mechanism of action, 2 :65-66

hydrophobic regions, 2:63 ion regulation, 2 :67-69 membrane interaction, 2:62 primary structure, 2 :56-58 proteolytic processing and activa

tion, 2 :60-61 tertiary structure, 2 :58-60

induction of cecropin, 1:271 Energy charge ratio, for viability in vivo,

1:127 Enhancin

gene cloning, 2 :140-141 Pseudaletia unipuncta

binding to midgut epithelium, 2:130

enhancement of viral infections in vitro, 2 :131-133

localization and mode of action, 2 :129-130

nucleotide sequence, 2 :138-139 synergistic activity, 2 :129-130

Trichoplusia ni GV effect on peritropic membranes,

2 :133-137 enhancement of insect bioassays,

2 :137-140 nucleotide sequence, 2 : 138 -

139 Entomopoxvirus

cytopathology, 2:86 pathogenesis, 2:91 tissue tropism, 2:84

Enzyme-linked immunosorbent assay, for parasitized insects in host insect population, 1:421

Enzymes cuticle-degrading

characterization, 2 :219-221 regulation, 2 :221-222

host hemolymph, parasitism effects, 1:28

insect-specific, in baculovirus insecticide system, 2 :186-187

secretion by teratocytes, 1 :156-158


Ephedrus californicus, development and growth, 1:7

Epithelium, midgut, see Midgut epithelium

Evolution, see also Coevolution polydnavirus, 1 :215-221 studies, use of venoms, 1 :233-236

Extracellular form, nuclear polyhedrosis virus, 2:113, 149

Extraembryonic tissue, persistence in host, 1:153

Fat body glycogen levels in parasitized

H. virescens, 1:127 production of early proteins, 1:38 protein turnover, nematode effects,

1:95 Fatty acids, levels in lipid fractions after

parasitization, 1 :129-130 Fecundity

microsporidia effects, 1:91 nematode infection effects, 1:96

Few-polyhedra mutants , transposon-related, 2 :152-153

Foraging sites, parasitism effects, 1:110 Fructose bisphosphatase

in H. exiguae-mfectza Trichoplusia ni, 1:130

inhibitory response in parasitized larvae, 1:133

Fungal pathogens, insect cuticle invasion

adhesion events, 2 :212-214 cuticle-degrading enzymes, 2:219—

221 prepenetration growth, 2 :214-216 structural adaptations for penetra

tion, 2 :216-219

Galleria mellonella granular cells and plasmocytes,

cell—cell communication, 1:290— 292

immune reactive hemocytes, separation in vitro, 1 :281-282

plasmatocyte depletion factor, 1:272 Gamma radiation, inhibition of graft

rejection, 1 :312-313 Gas chromatography-mass spectrome

try, for ecdysteroid quantification, 1:77

G domains, diptericin, attacin, and sar-coendotoxin I I , 2 :33-34

Gene expression AcNPV, 2:184 Campoletis sonorensis virus, 1:206 diptericin, 2:33 host, parasitism effects, 1:43 transposon effects, 2 :170-172

Gene-for-gene coevolution, 1 :29-30 Genes

avirulence, in plant pathogens, 1 : 2 9 -30

Campoletis sonorensis virus characterization, 1 :201-205 significance, 1 :206-208

cyt, structure and biological activity, 2:58

designer, of parasites, 1:29 enhancin, cloning, 2 :140-141 env, T E D resemblance, 2 :158-161 gag, T E D resemblance, 2 :158-161 host, regulation factors, 1:51 pol, T E D resemblance, 2 :158-161 polydnavirus

divergence, 1 :215-218 sequencing, 1:203

viral divergence, 1:216 duplication, 1 :216-217

Genetically engineered viruses, 2 :97-98 Genetically manipulated organisms

legislative procedure concerning planned release, 2 :191-193

risk assessment, 2 :188-190 Genetic analyses

intraspecific, polydnavirus DNA as genetic marker, 1 :213-214

S.frugiperda resistance to NPV, 2:204

Index to Volumes 1 and 2 351

Genetic engineering baculovirus

alternative gene promoters, 2:185 insecticides, 2 :181-182 multiple expression vectors, 2 : 185 -

186 risk assessment, 2 :188-190 very late gene promoters, 2 :184-

185 legislative procedure concerning

planned release, 2 :191-193 potential modifications for improved

insect control, 2 :98-99 Genetic linkage maps, A. aegypti, 1 : 2 5 8 -

259 Genetic mosaics, polydnavirus genome,

1 :180-182 Gene transfer, to exploit genes encoding

parasitism-specific proteins, 1:42 Germination, deuteromycete ento-

mopathogens, 2 :214-215 β-15 3-Glucan, induction of cecropin,

1:271 1-Glucose, 1 3 C-labeled, incorporation

into storage metabolites by parasitized M. sexta, 1:132

Glycogen deposition by M. sexta parasitized by

C. congregata, 1 :131-132 fat body, in H. exiguae-inkctea Tri

choplusia ni, 1:130 levels in host fat body, 1:128 storage during parasite—host interac

tion, 1:135 Glycosidated residues, role in pa ras i t e -

host interaction, 1:37 Graft rejection, inhibition by gamma

radiation, 1 :312-313 eicosinoid effects, 1:313

Gram-positive bacteria, triggering of prophenoloxidase cascade, 1:323

Granulosis virus cytopathology, 2:86 Harrisina brillians, 2:94 as inoculative agent, 2:94 pathogenesis, 2 :90-91

Trichoplusia ni, enhancin effect on peritropic membranes,


2 :137-140 gene cloning, 2 :140-141 nucleotide sequence, 2 :138-139

uncoating, 2:116 as viral insecticide, 2 :95-97

Growth host

alteration effects on parasite nutrition, 1:136

teratocyte effects, 1 :158-160 parasite, host altered metabolism ef

fects, 1:135 teratocytes, 1 :148-152

Growth factors, dual roles in mammals , 1:27

Harrisina brillians granulosis virus, 2:94 Heliothis exiguae virus, DNA, 1:196 Heliothis virescens, BT resistance, 2:71 Hemagglutinin, plasma, in melanotic

encapsulation, 1:251 Hemocoel, nematode entry, 2 : 4 -5 Hemocytes

cell-cell communication of Galleria granular cells and plasmocytes, 1 :290-292

granular, multifunctional role, 1 : 2 8 7 -288

immune reactive, separation in vitro, 1 :281-282

induction of humoral antibacterial proteins, 1 :294-295

killing mechanisms, 1:273 monoclonal antibodies against, 1:277 mosquito, role in immune response,

1 :248-249 phagocytosis, laminarin effects,

1 :286-287 polypeptide synthesis, 1:249 production of early proteins, 1:38 prohemocytes, 1:279


role in final wound healing, 1:272 secretion of recruiting and cessation

factors, 1:272 surface protein changes, 1:249 synonyms for, 1:276 types in immune reactivity, 1 :268-270 Xenorhabdus adhesion, carbohydrase

effects, 2:8 Hemolin

function in immune recognition, 1:295

similarity to Drosophila neuroglian, 2 :38-39

structure and actions, 2 :37-39 Hemolymph

agglutinins, 1 :282-285 host, nutrients, 1 :68-69 insect

nematodes in, 2 :5 -7 Xenorhabdus spp. in, 2:7—10

juvenile hormone esterase, inhibition by teratocytes, 1 :157-158

phenoloxidase, inhibition by teratocytes, 1 :156-157

plasma, opsonic factors, 1:251 plasma protein expressed during

wound healing, 1:250 protein composition after parasitiza

tion, 1 :41-42, 128 serpin levels, 1:46 transfer experiments, 1 :293-294,

297 trehalose

in H. exiguae-mkctza Trichoplusia ni, 1:130

levels after parasitization, 1 : 128 -129

as parasite nutrient, 1:134 Hemopoietic tissues, diversity in in

sects, 1:278 HeV, see Hyposoter exiguae virus High-performance liquid chromatogra

phy with electrochemical detection

diphenol oxidase activity, 1:252 monophenol oxidase activity, 1:253

tyrosine, dopamine, and Ν-β-Alanyldopamine detection, 1:254

with RIA for ecdysteroid quantification, 1:77

Hormones, see also specific hormones host, direct effect on parasite devel

opment, 1 :67-68 insect development-related, 1 :61-64 insect-specific, in baculovirus insec

ticide system, 2 :186-187 role in metabolic alterations, 1 : 132 -

133 Host conversion, to nutrients for nema

todes, 2 :12-13 Host death, premature, 2 :233-238 Host finding, by nematodes, 2 : 2 -4 Host -paras i to id-pa thogen interactions

beneficial parasitism effect on host suscepti

bility, 2 :251-253 parasitoids vectors, 2 :253-259

deleterious direct infection of parasitoids,

2 :242-248 host ovipositionally unattractive,

2 :239-240 hosts altered nutritionally or physi

ologically, 2 :240-241 parasitoid death, pathogen toxin-

related, 2 :238-239 preclusion of parasitoid persis

tence, 2 :248-251 premature death of host, 2 : 2 3 3 -

238 Host quality, definition, 1:1:1:2 Host range, baculovirus insecticides,

2:180 Host regulation paradigm, 1:29, 136—

139 Host response, manipulation by para

sites, 1:26 Host suicide, 1:113 Host suitability

definition, 1:2 endocrine effects on endoparasites,

1 :66-69


H P L C , see High-performance liquid chromatography

Humoral immune response antibacterial, blood cell role, 1 : 2 9 3 -

295 interrelationship with cellular immu

nity, 1 :293-295 in mosquitos, 1 :249-251 to nematode entry, 2:6 origins of, 2:43

Hydrophobicity, bacterial, 2 : 8 -9 Hydrophobic regions, BT endotoxins,

2:63 Hymenolepis diminuta infections

Tenebrio molitor, 1:97 Tribolium castaneum, 1:97

Hypoproteinemia, host, factors affecting, 1:45

Hyposoter exiguae development and growth, 1 : 7 - 8 infections, Trichoplusia ni, 1:128, 130-

131 intraspecific genomic complexity,

1 :198-199

Ichnoviruses, nucleocapsids, 1:197 Idiobionts

developmental strategies, 1 :12-16 egg parasites, 1 : 4 - 5 host-exploitation strategy, 1 : 3 - 4 pupal parasites, 1 : 5 - 6

Immune proteins attacins, 2 :32-34 cecropins and related peptides, 2 : 2 7 -

30 defensins, 2 :30-34 definition, 2:27 diptericins, 2 :32-34 genes

C I F binding to κΒ-like motif, 2 :40-42

κΒ-like motif, 2 :40-42 sequence elements, 2 :40-42

hemolin, 2 :37-39 lysozymes, 2 :35-36

overview of source, size, and function, 2:26

P4, see Hemolin proline-rich bee peptides, 2 :34-35

Immune response cell-cell communication, 1 :290-293 cellular, see Cellular immune re

sponse humoral , see Humoral immune re

sponse immunologic memory, 1 :311-312 mosquitos

biochemical aspects, 1 :251-255 cellular aspects, 1 :248-249 humoral aspects, 1 :249-251 non-self recognition, 1 :247-248

nonclonal form of recognition, 2:44 venoms as probes for, 1:230

Immunorecognition, prevention by integral membrane proteins of parasite surfaces, 1:50

Immunosuppression polydnavirus-initiated, 1 :136-137,

173-176 by teratocytes, 1 :155-156

Inoculative agents baculoviruses for, 2:94 for biological control, 2:92

Insecticidal crystal protein (B. thuringiensis aizawai IC1), 2:60

Insecticides, baculovirus genetic manipulation, 2 :181-182 host range, 2:180 persistence, 2:180 production, 2:181 recombinant, 2 :186-188 speed of action, 2:181 target range, 2 :180-181

Insecticyanin, blood levels, parasitism effects, 1:46

Insertion elements gene disruptions caused by, 2:169-170 Spodoptera frugiperda

IFP1.6, 2 :167-168 IFP2.2, 2:168 plaque isolate E, 2 :167-168


Trichoplusia ni IFP2, 2 :164-166 T F P 3 , 2 :166-167

Insertion mutants , baculovirus, 2:152 Integrated pest management programs,

use of viruses in, 2 :96-97 Integration, polydnavirus, 1 :208-215 Intermediate filaments, viral assembly

on, 2 :110-111 Interspecies transfer, transposons,

2 :173-174 Ion channels

activation after BT endotoxin exposure, 2:65

regulation by BT endotoxins, 2 :67-69 Iridovirus

cytopathology, 2:86 pathogenesis, 2 :83-85 tissue tropism, 2:84

Juvadecene, effect on endoparasite development, 1:68

Juvenile hormone, 1:61 distribution, 1:63 functions, 1 :62-63 host, effects of parasitoids, 1 :72-73 link with serosal membrane /

teratocytes, 1 :74-75 metabolism, 1:63 parasite levels relative to host levels,

1 :64-66 quantification, 1 :78-79 structures, 1:62

Juvenile hormone esterase expression in recombinant

baculovirus, 2:187 host, effects of parasitoids, 1 :72-73 inhibition by teratocytes, 1 :157-158 larval parasite effects, 1:70 parasitism effects, 1:47

Juvenile hormone esterase suppressive factor, 1 :47-48

KB-like motif cecropia immunoresponsive factor

binding, 2 :42-43

structure, 2 :40-42 Koinobionts

aphid wasps, 1 : 6 -7 developmental strategies, 1 :12-16 host-exploitation strategy, 1 : 3 - 4 Hyposoter exiguae, 1 : 7 - 8 Microplitis species, 1:8

Laminarin effect on hemocyte phagocytosis,

1 :286-287 induction of cecropin, 1:271

Larval parasites, effects on host endocrine system, 1:70

Lectins, see also Agglutinins binding, cell type identification,

1:277 effect on erythrocyte spontaneous

rosetting, 1:284, 288 interactions with prophenoloxidase

system, 1 :288-289 plasma factor, 1:288 structure and actions, 2 :36-37

Legislative procedures, for planned release of engineered viruses, 2 : 1 9 1 -193

Ligand-blot analyses, BT endotoxin receptors, 2 :66-67

Light reactions, parasitized insects, 1:108, 110

Lipids activation of prophenoloxidase cas

cade, 1 :323-325 metabolite changes after parasitiza

tion, 1 :129-130 transport, lipoprotein-mediated,

1:130 Lipophorin, blood levels, parasitism ef

fects, 1:46 Lipopolysaccaride-binding protein, 2:37 Lipopolysaccharide-mediated proph

enoloxidase activation, 1 :322-323 Lymphocytic choriomeningitis virus, se

lective effects, 1:50 Lysolecithin, activation of prophenolox

idase, 1:324


Lysozyme role in insect antibacterial response,

2:7 structure and actions, 2 :35-36

Magainins, structure and action, 2:30 Makisterone A, 1 :61-62 Malamoeba locustae, effects on desert lo

cust oocyte development, 1:89 Mammals , parasitism-induced protein

alterations, 1:48 Manduca sexta

C. congregata infections, metabolic alterations induced by, 1:131-132

early and late proteins, 1 :31-33 ecdysteroids, 1 :61-62 hemolymph proteins, 1:46

Melanin biosynthesis dopachrome conversion factor, 1:332 R a p e r - M a s o n pathway, 1:331-332

Melanization mechanisms, 1:248 role in insect defense responses,

1:176 venom role in, 1:230

Melanotic encapsulation, see also Encapsulation

catecholamine substrates, 1:255 enzymes in, 1 :251-255 hemolymph plasma protein expressed

during wound healing (A aegypti), 1:250

plasma hemagglutinin function, 1:251

Membrane permeabilization by cecropins, 2:28 by defensins, 2:31

Memory, immunologic, 1:311—312 Mermis nigrescens infections, Schistocerca

gregaria, 1:95 Mermithid infections, effects on host re

production, 1:95 Metabolic regulation, enantiostasis,

1 :139-140 Metabolism, host, redirection by para

sites, 1 :136-139

Metabolites, alteration in host tissues amino acids, 1:128-129 carbohydrates, 1:127-128 lipids, 1:129-130 M. sexta parasitized by C. congregata,

1:131-132 nutritional significance to parasites,

1:134-136 proteins, 1:128-129 Τ ni parasitized by H. exiguae, 1 :130-

131 Metacemyia calloti infections, Anacridium

aegyptium, 1:99-100 Metamorphosis

hormonal changes during, 1:63 host, alteration effects on parasite nu

trition, 1:136 precocious initiation, parasite-

induced, 1:71 Microbial agents, enhancement, 2:128—

129 Microbial insecticides

insect pathogens for, 2:82 virus efficacy, 2:82

Microfilaments in entry of AcNPV, 2:115 viral assembly and release, 2 : 1 1 1 -

112 Microhabitat

effect on parasite survival, 1 :112-113

preference in parasitized insects, 1:108-110

Microplitis species, development and growth, 1:8

Microsporidia effects on host population dynamics,

1 :92-93 horizontal transmission between

hosts, 1:92 host infections, parasitoids suscepti

ble to, 2 :246-247 infections, effect on host reproduc

tion, 1 :89-93 as microbial control agents, 1:93 transovarial transmission, 1:92 transovum transmission, 1:92


vertical transmission to succeeding generations, 1:92

Microtubules in entry of AcNPV, 2 :115-116 viral assembly on, 2:111

Midgut epithelium BBMVs, Cry toxin binding, 2 :62-63 binding of En-Pu, 2:130 BT endotoxin receptors, 2 :66-67 loss of ion regulation, 2 :67-69 viral replication in, 2 :94-95

Mimicry, host defenses, 1:26 Molecular markers, for genes affecting

A . aegypti susceptibility, 1 :258-259 Molecular sieve chromatography, quin

one isomerase-phenoloxidase complexes, 1 :334-335

Molt, juvenile hormone effects, 1 : 6 2 -63

Monoclonal antibodies, cell type determination with, 1:277

Monophenol oxidase, H P L C - E C D , 1:253

Morphogenesis AcNPV, 2 :119-120 Campoletis sonorensis virus, 1:191

Morphology, teratocytes, 1 :148-152 Mosquito, see also Aedes aegypti

immune response biochemical aspects, 1 :251-255 cellular aspects, 1 :248-249 humoral aspects, 1 :249-251 non-self recognition, 1 :247-248

susceptibility/refractoriness to Plasmodium and nematode infections, 1 :255-260

Motility, attraction component, 2:3 Mutagenesis, baculovirus genome, host

transposon role, 2 :168-173

Nematode/bac ter ium-hos t complexes antimicrobial production, 2:13—14 bioluminescence role, 2 :14-15 host conversion to nutrients, 2 :12-13 phase variations, 2 :15-16

pheromones, 2:16—17 physiological and biochemical asso

ciations, 2 :5-12 Nematodes

damage scales, 1 :93-94 effects on fat body protein turnover,

1:95 entry into host

behavioral barriers, 2 :4 -5 physical barriers, 2:4

host finding, 2 : 2 -4 host reproductive disturbances

caused by, 1 :93-96 in insect hemolymph, 2 :5 -7 nutrient deprivation caused by, 1:95 pathogenic in absence of Xenorhabdus

symbiont, 2 :11-12 Nervous system, venoms as probes for

physiology, 1:230 Neuroglian, Drosophila, similarity to he

molin, 2 :38-39 Nicotine, in host food, effect on endo

parasites, 1:68 Nitrogen, metabolites after parasitiza

tion, 1:128 Nomenclature, blood cells, 1:275—

276 Nonclonal recognition, 2:44 Nonoccluded viruses, 2:83 Non-self recognition

apparent presence of two immune recognition systems, 1:288

evasion by nematodes, 2:6 mosquitos, 1 :247-248 prophenoloxidase cascade role,

1 :325-326 recognition factors, 1 :282-289

Nosema spp., infections, effect on host reproduction, 1 :89-91

Nuclear matrix AcNPV association during transcrip

tion, replication, and assembly, 2:117

viral assembly on, 2 :109-110 in viral replication and transcription,

2 :108-109


Nuclear polyhedrosis virus Autographa californica, see Autographa

californica nuclear polyhedrosis virus

biological control with, 2:93—94 cytopathology, 2:86 entry, microtubule involvement,

2:115 extracellular form, role in virus trans

mission, 2:149 pathogenesis, 2:89—90 polyhedral form, role in virus trans

mission, 2:149 S. frugiperda resistance, case history,

2 :201-205 tobacco hookworm host, 1 :34-37 uncoating, 2:116 as viral insecticide, 2 :95-97 virulence, 2:95

Nucleocapsids bracoviruses, 1 :197-198 ichnoviruses, 1:197 transport, 2:107

Nucleosomes, AcNPV, structure, 2 :117-118

Nutrients changes due to calyx fluid/poly-

dnavirus, 1:179 conversion of host by nematodes,

2 :12-13 deprivation, nematode-induced, 1:95 host alterations, 2 :240-241

Nutrition, parasite effects of altered host metabolite lev

els, 1:135 host growth patterns, 1:136

Nutritional physiology, host, syncrony with parasite development, 1:133

Nutritional status, host, effect on endo-parasite development, 1 :67-68

Oenocytoids, prophenoloxidase synthesis, 1:325

Oleate, activation of phenoloxidase, 1:324

Oocyte development, in Metacemyia calloti-'mkctea Anacridium aegyptium, 1 :99-100

Opius concolor-C. capitata parasi te-host system, 1:67

Opius melleus-R. pomonella parasi te-host system, 1:67

Opius tyroni-C. capitata parasi te-host system, 1:67

Opsonic factors, in hemolymph plasma, 1:251

Oryctes rhinoceros baculovirus, 2:94 Outer membrane proteins, role in adhe

sion, 2 :9-10 Oviposition

parasitism effects, 1:110 unattractive, 2 :239-240

Parasite development host altered metabolism effects, 1:135 host ecology-related

arrestment, 1:10 diapause, 1 :9 -10 temperature requirements, 1:9

host nutrition effects, 1 :10-11 host starvation, 1:12 superparasitism, 1 :11-12

idiobionts, 1 : 4 - 6 koinobionts, 1 : 6 - 8 strategies, models of, 1 :12-16

Parasitism benefits to host

behavioral fever, 1:114 kin survival, 1 :113-114

benefits to parasites dissemination, 1 :111-112 survival, 1 :112-113

mediating effects on host physiology, 1:133

specific proteins, 1 :27-28 Parasitoids

death, pathogen toxin-related, 2 : 238-239

definition, 1:60 direct infection, 2 :242-248

3 5 8 Index to Volumes Ί and 2

effects on host development and endocrinology, 1 :72-73

hostlike antigens, 1:26 persistence, preclusion, 2 :248-251 susceptible to host microsporidia in

fections, 2 :246-247 unable to complete development

due to host death, 2:234 due to nondigestible spore accu

mulation, 2:241 as vectors of pathogens, 2 :253-259

Pathogen vectors, parasitoids as, 2 : 253 -259

Peptidoglycan, triggering of prophenoloxidase cascade, 1:323

Periplaneta americana graft rejection, 1:311

eicosinoid effects, 1:313 inhibition by gamma radiation,

1 :312-313 transplantation reactivity, 1 :309-311

Peritropic membranes, Trichoplusia ni GV enhancin effects, 2 :133-137

Permeabilization, membrane by cecropins, 2:28 by defensins, 2:31

Persistence baculovirus insecticides, 2:180 parasitoid, preclusion of, 2 :248-251 recombinant baculoviruses, 2:189 teratocytes, 1 :148-152

pH, alkaline, for BT endotoxins, 2:60 Phagocytosis

cell types responsible for, 1 :270-271 by hemocytes, laminarin effects,

1 :286-287 Pharmacology, venom probes of hetero

logous systems, 1 :238-239 Phase-contrast studies, blood cells,

1:277 Phase variation, Xenorhabdus spp., 2 : 1 5 -

16 Phenoloxidase

biochemical pathways, 1:254 hemolymph activity, inhibition by

teratocytes, 1 :156-157

host, parasitism effects, 1 :46-47 role in mosquito immune response,

1 :251-252 substrates, 1:254

Pheromones, produced by nematodes, 2 :16-17

Physiology, host adaptation tolerance hypothesis,

1:138 adjustment versus regulation, 1:139—

140 alterations, 2 :240-241 polydnavirus-related changes, 1:174 redirection by parasites, 1 :136-139 venoms as probes of, 1 :230-231

Phytoecdysteroids, effect on endoparasite development, 1:68

Phytohormones, effect on endoparasite development, 1:68

Pimpla turionellae, development and growth, 1 : 5 - 6

Plaque isolate Ε insertion element, 2 :167-168

Plasmatocyte depletion factor, 1:272 Plasmocytes, phagocytosis by, 1 : 270 -

271 Plasmids, pAcUW2B expression vector,

2 :185-186 Plasmodium gallinaceum

A. aegypti infections, effects on reproduction, 1 :88-89

A. aegypti susceptibility, genes affecting, 1:257

Polydnavirus amplification in situ, 1:172 in biological systems, 1 :190-191 developmental regulation, 1 :176-179 DNA

as genetic markers for intraspecific genetic analyses, 1 :213-214

multiple covalently closed circular form, 1:192

DNA persistence in host cells, 1:50 effects on prothoracic gland, 1 : 177 -

178 family group, 1 :167-169


gene divergence, 1 :215-218 gene products, functional roles,

1 :182-183 gene sequencing, 1:203 genetic colonization of host, 1:172-173 gene transcripts in host insects, 1:31 genome

functional organization, 1:199-201 genetic mosaic nature, 1 :180-182 packaging, 1 :196-198 segmentation, 1 :183-184 structure, 1 :191-196

immunosuppression, 1:136-137, 173 -176

induction of parasitism-specific proteins in host, 1 :75-76

integration, 1 :208-215 modulatory roles in target insects,

1:50 nutrient level changes due to, 1:179 origin, 1:220-221 replication, 1 :169-172, 208-215

developmental regulation, 1:215 strategy, 1:214

role in metabolic alterations, 1:132 species differences, 1:192 species specificity, 1 :218-220 teratocyte-polydnavirus interactions,

1:160 transmission, 1 :170-172, 208-215

Polyhedra-derived form, nuclear poly-hedrosis virus, 2:113, 149

Polypeptides, associated with A . aegypti strains refractory for B. malayi, 1:260

Polypeptide synthesis, in activated hemocytes, 1:249

Population dynamics, host cestode infection effects, 1:97 microsporidia transmission mode ef

fects, 1 :92-93 Pores, t ransmembrane, BT endotoxin-

related, 2:62, 66, 68 Postlarval protein, isolation, 2:37 Posttranscriptional regulation,

arylphorins, 1:44

Potassium permeability, BT toxin-related increases, 2:68

Precocene, effect on endoparasite development, 1:68

Premature death, host, 2 :233-238 Prohemocytes, development, 1:279 Proline-rich bee peptides, 2:34—35 Promoters, for genetic modification of

baculoviruses, 2 :184-185 Prophenoloxidase

compartmentalization, 1:325 occurrence, 1:319-320 synthesis, 1:325 types, 1:319

Prophenoloxidase activation enzyme, characterization, 1:320

Prophenoloxidase cascade characterization, 1:320-322 control mechanisms

dopachrome conversion factor, 1:331-332

general, 1:326-327 prophenoloxidase inhibitor, 1:331 protease inhibitors, 1:327-331 quinone isomerase, 1 :332-335

lipopolysaccharide-mediated, 1 :322-323

non-self recognition molecule, 1:285— 289

role in non-self recognition, 1:325-326 triggering, 1:322-323

Prophenoloxidase complex interaction with lectins, 1:288-289 role in early immune reactivity, 1:270

Protease inhibitors, prophenoloxidase cascade, 1:327-331

Proteases, in bacterial surface modification system, 2:7

Proteinaceous appendages, role in adhesion, 2 :9-10

Proteins, see also specific proteins antibacterial

induction by hemocytes, 1:294—295 induction by injection of non

pathogenic bacteria, 2 :6 -7 origins, 2:43


early, 1 : 31 -33 , 38-41 endogenous in host, parasitism-

induced modifications, 1 :28-29 late, 1 :31-33 molecular biology, use of venoms,

1:238 parasitism-induced

polydnavirus gene transcripts, 1:31 roles of, 1:37

parasitism-specific, 1 :27-28 Protein synthesis

host, 1 :28-29 parasitism effects, 1:43

in immune reactivity, 1:271 Protein turnover, fat body, nematode

effects, 1:95 Proteolysis, limited, 1:320 Proteolytic processing, BT endo-

endotoxins, 2 :60-61 Prothoracic gland, polydnavirus effects,

1 :177-178 Prothoracicotropic hormones, 1:61

host, effects of parasitoids, 1 :72-73 release during metamorphosis, 1:63

Pseudaletia unipuncta, enhancin binding to midgut epithelium, 2:130 gene cloning, 2 :140-141 enhancement of viral infections

in vitro, 2 :131-133 localization and mode of action,

2 :129-130 nucleotide sequence, 2:138—139 synergistic activity, 2 :129-130

Pyemotes tritici TxP-1 neurotoxin, 2 :187-188

Quinone isomerase, complexation with phenoloxidase complex, 1 :332-335

Radioimmunoassay, with H P L C for ecdysteroid quantification, 1:77

Radiommetry, monophenol oxidase in A . aegypti hemolymph plasma, 1:252

Rape r -Mason pathway, 1 :331-332 Recognition

nonclonal form, 2:44 non-self, see Non-self resognition steps for deuteromycete en-

tomopathogens before cuticle penetration, 2 :215-216

Recombinant insecticides baculovirus, 2 :186-188

effects, 2 :186-188 prospects for, 2 :190-191

potential for genetic transfer with latent baculoviruses, 2:189

Recruiting factors, secretion by hemocytes, 1:272

Refractoriness, A . aegypti strains, 1:260

Regulators combined conformer-regulator strat

egy, 1:60 definition, 1:17, 60

Replication, viral, see Viral replication Reproduction, disturbances

cestode-related, 1 :96-98 endoparasitic insect-related, 1 : 9 8 -

100 microsporidia-related, 1 :89-93 nematode-related, 1 :93-96

Reproductive behavior, castrated insects, 1:111

Resistance, viral, see Viral resistance Respiration, parasite, induction of

metabolic changes in host, 1:134 Restriction fragment length poly

morphism markers, genes affecting A . aegypti susceptibility, 1 : 2 5 8 -259

Retrotransposons, see also Transposons characterization, 2:151 transposable element-D

characterization, 2:153, 156 gene functions, 2 :158-161 genomic structure and organiza

tion, 2 :156-158 mechanism of transposition,

2 :161-163

Index to Volumes Ί and 2 361

transposable element-D (TED), characterization, 2:153, 156

Retroviruses, C-type, assembly, 2:112 R H 5849, effect on endoparasite devel

opment, 1:69 Risk assessment, with genetically ma

nipulated organisms, 2 :188-190 Royalisin, isolation, 2:31 Rutin, in host food, effect on endo

parasites, 1:68

Salivary glands, role in mosquito susceptibility/ refractoriness, 1:260

Sapecin, structure and actions, 2 :30-31 Sarcophaga, immune proteins, 2 :36-37 Sarcotoxin I I , G domain, 2 :33-34 Scanning electron microscopy, terato

cyte ultrastructure, 1 :151-152 Schistosomin, factors affecting, 1:48—49 Secretory enzymes, teratocyte, 1 : 156 -

158 Segmentation, polydnavirus genome,

1 :183-184 Serine protease inhibitors, prophenol

oxidase cascade, 1 :327-329 Serosal membrane

substances secreted by, 1:74—75 types, 1:146

Serpins functions in prophenoloxidase activa

tion, 1 :329-331 hemolymph levels, 1:46

Sodium dodecyl sulfate, activation of prophenoloxidase, 1:324

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polypeptides of A . aegypti strains refractory for B. malayi, 1:260

Southern blot analyses enhancin, 2:141 H e V and CsV intraspecific genomic

complexity, 1 :198-199 Species differences

encapsulation response, 1:307 polydnaviruses, 1:192

Species specificity bacterial symbionts, 2:17 BT endotoxin domains, 2:73 polydnavirus, 1 :218-220

Spodoptera frugiperda insertion elements

IFP1.6, 2 :167-168 IFP2.2, 2:168 plaque isolate E, 2 :167-168

viral susceptibility, case history, 2 :201-205

Spores deuteromycete entomopathogens, ad

hesion, 2 :212-214 nondigestible, accumulation, 2:241

Starvation, host, 1:12 Superparasitism, 1 :11-12 , 60 Surface proteins, changes in activated

hemocytes, 1:249 Survival

host kin, parasitism benefits, 1 : 1 1 3 -114

parasite effects of host behavioral alter

ations, 1 :112-113 host regulation paradigm, 1 : 136 -

139 Susceptibility

A . aegypti, 1 :258-260 A . gambiae, 1 :258-259 host

to nematodes, 2 : 4 -5 parasitism effects, 2 :251-253

Tachyplesins, structure and actions, 2:32

Target range, baculovirus insecticides, 2 :180-181

Taxonomic studies, use of venom for, 1 :236-238

T E D , see Transposable element-D Telenomus heliothidis, enzyme secretion by

teratocytes, 1 :156-157 Temperature

body, parasitism effects, 1:114


elevated, effects on host survival, 1:115 parasite mortality, 1:115

preference changes in parasitized insects, 1:110

requirements for parasites development, 1 :9

Teratocytes abundance, 1 :148-152 distribution, 1 :147-148 effect on

host growth and development, 1 :158-160

juvenile hormone system, 1:75 growth, 1 :148-152 during host life stages, 1:152 immunosuppressive functions, 1 : 1 5 5 -

156 morphology, 1 :148-152 origins, 1 :146-147 persistence, 1 :148-152 polydnavirus—teratocyte interactions,

1:160 secretory functions, 1 :156-158 substances secreted by, 1:74—

75 trophic functions, 1 :153-155 ultrastructure, 1 :151-152 venom interactions, 1:160

Tissue tropisms, major virus types, 2:84

Toxins, see also specific toxins insect-specific, in baculovirus insec

ticide system, 2 :187-188 pathogen-produced, parasitoid death

due to, 2 :238-239 Xenorhabdus spp., 2 :10-12

Transcription factor NF-κΒ, similarity to C I F , 2 :44-45

Transcription, viral, see Viral transcription

Transmembrane pores, BT endotoxin-related, 2:62, 66, 68

Transmembrane potential, deu-teromycete entomopathogens, 2:218

Transmission germline, insect viruses, 1 :209-213 per os

insect viruses, 1 :208-209 iridoviruses, 2:85

polydnavirus, 1 :170-172, 208-215 transovarial

insect viruses, 1 :208-209 microsporidia, 1:92

transovum, microsporidia, 1:92 Transmission electron microscopy, ter

atocytes ultrastructure, 1 :151-152 Transplantation

foreign tissue, 1 :308-311 reactivity in Periplaneta americana,

1 :309-311 Transposable element-D

characterization, 2:153, 156 gene functions, 2 :158-161 genomic structure and organization,

2 :156-158 mechanism of transposition, 2:161 -163

Transposons, see also Retro transposons baculovirus-associated, 2:152-154 classes and properties, 2 :151-152 host

insertion into baculoviruses, 2 : 148-151

role in baculovirus genome mutagenesis, 2 :168-173

symbiotic interactions with baculoviruses, 2 :173-175

Trehalose, hemolymph levels in H. exiguae-'mfected Trichoplusia ni,

1:130 in host tissues, 1 :127-128 as parasite nutrient, 1:134

Trichogramma species, development and growth, 1 : 4 - 5

Trichoplusia ni carbohydrate synthesis alterations,

1:131 endocrine parameters in parasitized

and nonparasitized larvae, 1:71 — 74

gluconeogenic activity, 1:133


Hyposoter exiguae-'mfected, 1:128 metabolic alterations, 1:130-131

Trichoplusia ni GV, enhancin effect on peritropic membranes,


2 :137-140 gene cloning, 2 :140-141 nucleotide sequence, 2 :138-139

Trophic functions, teratocytes, 1 :153-155

Tropism, tissue, major virus types, 2:84 Trypsin inhibitors, prophenoloxidase

cascade, 1:329 Two-dimensional gel electrophoresis,

polypeptides of A. aegypti strains refractory for B. malayi, 1:260

Tyrosine, in A. aegypti hemolymph plasma, 1:254

Ubiquit in, in AcNPV-infected cells after viral replication, 2:119

Uncoating baculoviruses, 2:116 viral particles, 2 :107-108

Vaccinia virus, assembly, 2:112 Vectors

ascoviruses, 2:88 expression, for genetic modification of

baculovirus, 2 :185-186 pathogens, parasitoids as, 2 :253-259

Venom for behavioral studies, 1 :232-233 for biochemical pathway studies,

1:232 in biological control, 1 :239-240 as classroom tools, 1:240-241 in evolution studies, 1 :233-236 for hos t -paras i te bionomics, 1 :228-

230 for molecular biology studies, 1:238 as probes for heterologous systems,

1 :238-239

as probes of normal host physiology, 1:230-231

proteins, as model systems, 1:238 in taxonomic studies, 1:236—238 teratocyte—venom interactions, 1:160

Venom gland, secretions biological role, 1:176 requirement for parasitization,

1:175-176 synergism with viruses, 1:76

Viability energy charge ratio indicator, 1:127 parasi te-host complexes, 1:126-127

Viral assembly AcNPV, association with nuclear ma

trix during, 2:117 on cytoskeletal filaments, 2 :110-112

intermediate filaments, 2 :110-111 microfilaments, 2 :111-112 microtubules, 2:111

on nuclear matrix, 2 :109-110 Viral insecticides, 2 :92-93

efficacy, 2:96 granulosis viruses, 2 :95-97 integrated pest management pro

grams, 2 :96-97 nuclear polyhedrosis viruses, 2:95—97

Viral particles binding, 2:105 cell activation by, 2:105 entry into cells, 2 :105-107 nucleocapsid transport, 2:107 uncoating, 2 :107-108

Viral replication AcNPV

association with nuclear matrix during, 2:117

nucleosomal structure during, 2 :117-118

in vitro, 2:114, 183 in vivo, 2:182

Campoletis sonorensis virus, 1:191 on intermediate filaments, 2 :110-111 in midgut epithelium, 2 :94-95 nuclear matrix role, 2 :108-109 polydnavirus, 1 :169-172, 208-215


developmental regulation, 1:215 strategy, 1:214

Viral resistance, insect to BT endotoxins, 2 :69-71 , 73 comparison with chemical resistance,

2 :205-206 cross-resistance, 2:203, 205 evidence for, 2 :198-201 in field population, 2:200 inheritance mechanisms, 2 :200-201 multiple resistance, 2:203, 206 reversion, 2 :202-203 S. frugiperda viral susceptibility, case

history, 2 :201-205 Viral transcription

AcNPV association with nuclear matrix

during, 2:117 nucleosomal structure during,

2 :117-118 nuclear matrix role, 2 :108-109 transposon effects, 2 :170-172

Virogenic stroma, development, 2:114 Virulence

cytoplasmic polyhedrosis virus, 2:95 genes in plant pathogens, 1 :29-30 nuclear polyhedrosis virus, 2:95

Viruses, see also specific viruses genetically engineered, 2:97—98 nonoccluded, 2:83 occluded, 2:83 pathogenesis

ascoviruses, 2 :85-89 iridoviruses, 2 :83-85

synergism with venom gland secretions, 1:76

tissue tropisms, 2:84

Viruslike particles proteins, mimicry by, 2:38 role in metabolic alterations, 1:132

Vitellogenesis cestode-induced disturbance, 1 :97-98 in Metacemyia calloti-infected An-

acridium aegyptium, 1:99—100

Wasp biochemical pathways, 1:232 venom, see Venom

Western blot analyses, enhancin, 2:141 Western slot blots, for parasitized in

sects in host insect population, 1:421

Wound factors, role in immunity, 1:291 Wound healing

completion, 1:272 plasma protein expressed during,

1:250

Xenografts, rejection, eicosinoid effects, 1:313

Xenorhabdus spp. antimicrobial production, 2 :13-14 endotoxins, 2 :10-12 in insect hemolymph, 2:7—10 mutants , hydrophobicity and nega

tive charge, 2:8 phase variations, 2 :15-16 symbiont specificity, 2:17

Yeast cellular components, in prophenoloxidase cascade, 1:323

Parasites and Pathogens of Insects. Parasites

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