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1980

Morphological, histological, and physiological aspects of Morphological, histological, and physiological aspects of

assimilation in larval spot, leiostomus xanthurus lacepede assimilation in larval spot, leiostomus xanthurus lacepede

John Jeffrey Govoni College of William and Mary - Virginia Institute of Marine Science

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Recommended Citation Recommended Citation Govoni, John Jeffrey, "Morphological, histological, and physiological aspects of assimilation in larval spot, leiostomus xanthurus lacepede" (1980). Dissertations, Theses, and Masters Projects. Paper 1539616672. https://dx.doi.org/doi:10.25773/v5-r4nj-ds72

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ft L 035 87

GOVON1, JOHN JEFFREY

MORPHOLOGICAL, HISTOLOGICAL, AND PHYSIOLOGICAL ASPECTS OF ASSIMILATION IN LARVAL SPOT, LErOSTOMUS XANTHURU5 LACEPEDE

ITie C o lle g e o f W illia m and Mary in V i r g in ia

PH.D. 1980

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niversityMicrcxiims

itemationaJ

MORPHOLOGICAL, HISTOLOGICAL( AND PHYSIOLOGICAL

ASPECTS OF ASSIMILATION IN LARVAL SPOT,

LEIOSTOMUS XANTHUftUS LACEPEOE

A D isse r ta t ion

Presented to

The Faculty o f the School o f Marine Science

The College o f W illiam and Mary In V irg in ia

In P a r t ia l F u l f i l lm e n t

of the Requirements fo r the Degree of

Doctor o f Philosophy

by

John J e f f re y Govoni

1980

APPROVAL SHEET

Tills d is s e r ta t io n 1s submitted In p a r t ia l f u l f i l lm e n t o f

the requirements f o r the degree o f

DOCTOR Of PHILOSOPHY- ) / _ > {

jjbhn J e f f re y Go von 1

Approved * April 1980C '

ohn V. H errlner

Crafg l . Smith

’ Richard L. Wetzel

-Z.David S. PetersNational Oceanic and At.nosphertc A d m in is tra t ion National Marine F isheries Service Southeast F isher ies Center Beaufort Laboratory

11

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................................................................................ . v

PROLOGUE......................................................................................................................v1i

LIST OF TABLES..................... , .................................................. . ....................... v111

LIST OF FIGURES................................................................................................ x

ABSTRACT............................................................................................... * xv

INTRODUCTION................................................................................................... . * 2

THE EARLY LIFE HISTORY OFLEI05T0MJS XANTHURUS: A BRIEF REVIEW................................................... 6

ASPECTS OF THE MORPHOLOGICAL, HISTOLOGICAL, AND FUNCTIONAL DEVELOPMENT OF THE ALIMENTARY CANALAND ASSOCIATED STRUCTURES............................................................................ 11

In t ro d u c t io n ................................ , 12

M a te r ia ls and M ethods............................. . .......................................... 13

Results .......................... . . . . . . . 15

D iscuss ion..................................................... 34

EXPERIMENTAL RADIOTRACER STUDIES OF SHORTTEHM CARBON ASSIIMLATION ............................................................................ 39

In tro d u c t io n . ................................ , , . , .......................... 40

Conceptual Framework. . , , ........................................... 44

M a te r ia ls and Methods .............................. . . . . . ...................... 46

R e s u l t s .............................................................. 64

D iscussion ......................................................... 91

1 !1

EPILOGUE

TABLE OF CONTENTS (concluded)

Page

95

APPENDIX.....................................................................................................................97

LITERATURE CITED.............................................................................. « . , , 109

V ITA ........................................................................................................................... 122

i v

ACKNOWLEDGMENTS

I take pleasure In acknowledging Dr. John V. K e rr in e r who in

la rg e part a ffo rded me the o p p o rtu n ity o f fu r th e r graduate study and

who supported me throughout, 1 thank Drs. J . A, Muslck, D. 5. Pe ters ,

C. R. Smith, and R* L, Wetzel f o r th e i r suggestions, c r i t ic is m s , and

review of the manuscript. A dd it io n a l g rad ltude is due Drs* Peters and

Wetiel who endured many long hours of d iscuss ion concerning animal

energetics and rad io tra ce r methodology and who, along w ith Dr. P. L*

Zubko ff, provided necessary la b o ra to ry space and equipment.

Conversations w ith Drs. J . H* S, B la x te r , G. H. B o eh le r t, E. D. Houde,

R. Lasker, G- C* Laurence, and C. P. 0 1Connel were also most h e lp fu l .

This work would have been impossible w ithou t the support o f the

Southeast F ishery Center, N0AA-NMFS, B eau fo rt, North C a ro lin a . I

thank the e n t i re s ta f f fo r t h e i r co rd ia l he lp and assistance* Special

thanks are due D. S. Peters who sponsored my work and A, B. Powell

whose amiable cooperation was in va luab le .

A m u lt itude o f people co n tr ib u te d much to the m ateria l presented

he re in . Among them 1 acknowledge Dr. C, L. Ruddell fo r h is to lo g ic a l

in s t ru c t io n and assistance; J . C. Hoagman and M. H, Peoples fo r

d r a f t in g ; R, T, Doyle fo r photomicroscopy; Dr. J , L* Oupuy, N, T,

Windsor, and A, T. Leggett fo r C h lo re l la and Brachionus p ! 1 c a t i l i s

v

c u l tu re s i J . E. Warrlner f o r techn ica l ass is tance and hand ling o f

Iso topes; J. Colvocoresses fo r computer programing; and A.D. Estes

and H. Powles fo r f ie ld specimens.

I wish to thank Or. R.M. Ibara fo r h is f r ie n d s h ip and continual

In te re s t 1n my progress as a s tudent. I thank my fe l lo w s tuden ts ,

e s p e c ia l ly J. Colvocoresses, B. K l lc h , D* M ark le , L. Mercer, J.

Olney, B. Raschi, J. Ross* G. Sedberry, J. Smith and C, and B. Uenner

f o r t h e i r sympathetic ears*

F in a l ly , and c e r ta in ly most, I an Indebted to Mary jane Govonl fo r

her lo y a l t y , s a c r i f ic e , and enduring support - both m a te r ia l and

mora 1 e .

PROLOGUE

“ I t may be w ortty mentioning here that the o r ig in a l meaning of the

word "organ" is ''working to o l . " This etymology symbolizes the fa c t

that describ ing an organ and studying i t by the techniques o f a l l

science cannot so fa r provide a complete understanding o f i t . The

organ acquires i t s f u l l s ign if icance only when i t fun c t io n s and when

i t s performance is integrated in the l i f e o f the organism."

from " I l lu s io n s o f Understanding"

In Rene Dubos, The Dreams of Reason:

Science and U top ias. 196K

v i i

LIST OF TABLES

Table

1.

2 .

3.

4.

5.

6.

7.

8.

9.

10.

Page

Tooth courts o f la rv a l Lelostomus xanthurus* ■ * * ■ . 26

Gut contents o f la rv a l sp o t, Lelostomus xanthurus ■ . . 28

B loenergetic components o f la rv a l f ishes onvarious d ie ts ..................... 43

Summary o f s t a t i s t i c s c h a ra c te r iz in g development o f d is c re te age cohorts o f la rv a l Lelostomus xanthurus used In carbon a s s im i la t io n experiments * * ♦ * ■ * • * 47

Major n u t r i t io n a l components o f Brachlonus p U c a t l l 1s expressed as a percentage o f to ta l <fry weight - ■ . - ■ 50

Experimental recovery e f f ic ie n c y o f ^CQg.SLBima^ o f re su lts w ith known q u a n t i t ie s o fNaHl4C03 ............................................................................................. 60

Regression table f o r Brachlonus p U c a t iH s body burden re la te d to time 1 n carbon a s s im i la t io n experiments w ith d isc re te age cohorts o f la rv a l Leiostomus xa n th u ru s ........................ 65

Stepwise m u lt ip le regress ion tab le o f uptake re la ted to time, age* leng th * and w e igh t in carbon a s s im ila t io n experiments w ith d isc re te age cohorts o f la rv a l Lei ostomus x a n th u r u s ............................................... 69

Stepwise m u lt ip le regress ion tab le o f re ta ined re la te d to time, age, le n g th , and w e igh t 1 n carbon a s s im ila t io n experiments w ith d isc re te age cohorts o f la rv a l Lelostomus xanthurus * * .................................... * ■ 70

Stepwise m u lt ip le regress ion tab le o f 14c respired re la te d to time, age, le n g th , and w e igh t 1 n carbon a s s im ila t io n experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus ..................................... . . . 71

vi 11

LIST OF TABLES (concluded)

Table

11. Stepwise m u lt ip le regression ta b le o f PQ^C re la ted to t im e, age, length* and weight in carbon a s s im ila t io n experiments w ith d is c re te age cohorts of la rv a l Lelostomus xanthurus.

1 2 . Surrmary of values used to assess carbon a s s im ila t io nin d isc re te age cohorts o f la rv a l Lelostomus xanthurus.

13. Stepwise m u lt ip le regression ta b le o f carbon re te n t io n , the index o f the c o e f f ic ie n t o f u t i l i z a t i o n * re la tedto age, leng th , weight, and c o n d it io n fa c to r of Lelostomus xanthurus la rv a l age cohorts ..............................

14. Stepwise m u lt ip le regression ta b le o f carbon absorp tion , the index o f absorption e f f ic ie n c y , re la ted to age, length, weight, and con d it io n fa c to ro f Lelostomus xanthurus la rva l age co h o rts ..........................

15. Sunmary o f d u a l- t ra ce r values used to ca lcu la ted absorption e f f ic ie n c y by the in d ic a to r r a t io method . .

16. Surnnary of values used to assess carbon a s s im ila t io n 1n age cohorts o f la rva l Lelostomus xanthurus fed reduced Brachlonus p i i c a t i l 1s conce n tra t io n s ......................

ix

Page

. 11

. 76

, IS

. 80

. 85

. B7

LIST OF FIGURES

Figure Page

1 . Morphological development o f the alimentary canalo f Lelostomus xanthurus. In c ip ie n t gut o f a yo lk-sacla rva 2.2 mm M L ............................... 30

2 . Morphological development o f the a limentary canal of Lelostomus xanthurus. In c ip ie n t gut o f a yolk-sacla rva 2.3 ran NL , 30

3. Morphological development of the alimentary canal o f Lelostomus xanthurus. In c ip ie n t gut o f a la rva 2.B nmNL 1n o l l -g lo b u le absorption ...................................... . . . . . 30

4. Morphological development of the alimentary canal of Lelostomus xanthurus. Alimentary canal o f a larva3.0 run NL. ..................................................... 30

5. Morphological development o f the alimentary canal o f Lelostomus xanthurus. Alimentary canal o f a transform ing la rva 7,6 mm SL......................................... ............................................. 30

G, Morphological development of the alimentary canal of Leiostomus xanthurus. Alimentary canal o f an early Juven ile 25.0 m SL. . . . . . . . ........................ 30

7, Photomicrograph o f the In c ip ie n t gut o f a Leiostomusxanthurus yo lk-sac larva 2.3 mm NL. Longitudinalsec tion showing yo lk mass* o i l g lobule, in c ip ie n t gut* l i v e r * and p a n c r e a s ..................... .................................................. 3L

B. Photomicrograph of the in c ip ie n t gut o f a Lei ostonusxanthurus yo lk-sac larva 2.3 nm NL. Cross sectionshowing mucosal ep ithe lium and s t r ia te d border . . . . . . 31

9. Photomicrograph o f the alimentary canal o f a Lelostomusxanthurus larva 4,4 nm NL, Longitudinal section showingfo re g u t, midgut, h indgut, l i v e r * and pancreas ................ . 31

10. Photomicrograph o f the alimentary canal o f a Leiostomusxanthurus larva 4.4 nm NL, Longitudinal sectionshowing two types o f mucous c e l ls in f o r e g u t ............................ 31

x

LIST OF FIGURES (continued)

F igure Page

11. Photomicrograph o f the a lim en ta ry canal o f a Lelostomusxanthurus larva 4 ,4 nm NL. Long itud ina l se c t ion showingm id g u t ..................................... , . , . ...................................................... 32

12. Photomicrograph of the a lim en ta ry canal o f a Lelostomusxanthurus larva 4.4 mm NL, Long itud ina l sec t ion showinghindgut ep ithe lium w ith s t r ia te d border and in t r a c e l lu la r I n c lu s io n s ................................................................................................. 32

13. Photomicrograph o f a Lelostomus xanthurus la rva4.4 im NL. Longitudinal sec tion showing l i v e r ac in iw ith l i v e r s inus, hepatocytes, and glycogen vacuoles . . . 32

14. Photomicrograph o f a Leiostomus xanthurus la rva4.4 nm SL. Long itud ina l se c t ion showing pancreatica c in i and zymogen granules ................................. 32

15. Photomicrograph o f a transform ing Leiostomus xanthurus la rva 12.3 nm SL. Longitud inal section showing ju n c t io n o f bucco-pharynx and esophogus w ith esophageal fo ld s and Increased number o f mucous c e l ls ....................... . . . . . . . 33

16. Photomicrographs o f a trans fo rm ing Lelostomus xanthurus la rva 12,3 mm SL, Longitud ina l section showing the ju n c t io n o f the esophagus and b l in d sac w ith developing g a s tr ic g la n d s ........................................................... 33

17. Photomicrograph of an early ju v e n i le Leiostomus xanthurus 18,1 mm SL. Long itud ina l section o fg a s tr ic corpus o f a f is h 18.1 nm SL showing uniformsecretory c e l ls o f the g a s tr ic g lands. 33

18. Photomicrograph o f an early ju v e n i le Lelostomus xanthurus 33,0 nm SL. Long itud ina l section o fthe In te s t in e showing in t r a c e l l u la r Inc lus ions ..................... 33

19. Photomicrograph o f an e a r ly ju v e n i le Leiostomus xanthurus 33,0 irm 5L. Long itud ina l section o f therectum showing granu lar in t r a c e l l u l a r I n c lu s io n s ................. 33

20. Weight and length regressions f o r Leiostomus xanthuruslarvae used in carbon a s s im i la t io n experiments 43

2 1 . Experimental design o f carbon a s s im i la t io n w ith d isc re teage cohorts la rv a l Leiostomus xanthurus. .................................. 51

xi

LIST OF FIGURES (continued)

Figure Page

22. Schematic diagram o f re te n t io n chambers used 1n carbon a s s im ila t io n experiments w ith age cohorts o f la rv a l Leiostomus xanthurus* 54

1423. Schematic diagram o f b u bb le -trap fo r recovery o f CO, in carbon a ss im ila t io n experiments w ith age cohorts o f la rv a l Lelostomus xanthurus............................................................ 56

24, Decay o f chemllurnlnescent a c t i v i t y in la rvae andSrachlpnus samples* ....................... . . . . . 5a

£5. Quench curves fo r larvae and Brachlonus samples................. 59

£6 . Quench curves fo r larvae and Brachlonus samples fromd u a l- lab e l experiments* - * * * ........................................................62

1427. Biochemical f ra c t io n a l o f c in Brachlonusduring la b e l in g ........................................................................................... 66

£8 . Carbon-14 uptake by la rv a l Lelostomus xanthurusIn carbon a ss im ila t io n experiments* * ........................................... 67

29. Carbon-14 re ta ined, re sp ire d , and lo s t as P014C byla rv a l Lelostomus xanthurus in carbon a s s im i la t io n experiments* * * * * * .................................................................... 73

30. Carbon-14 re ta ined , re s p ire d , and lo s t as PO ^c byla rv a l Leiostomus xanthurus In carbon ass lm f1a t lo n experiments"! ISata pooled from a l l experim ents.......................... 74

31. Carbon re te n t io n , the index o f the c o e f f i c ie n t o f u t i l i z a t i o n , re la ted to development and c o n d it io nfa c to r of la rv a l Leiostomus xanthurus age cohorts - * * * 77

32. Carbon absorption, the index o f absorption e f f ic ie n c y , re la te d to development and con d it io n fa c to r o fla rv a l Lelostomus xanthurus age cohorts* . . . . . . . . 79

33. Percent CO2 respired re la te d to development and cond it ion fa c to r o f la r v a l Lelostomus xanthurus age cohorts......................................................................... 32

34. Condition fa c to r re la ted to age and notochord o r standard length o f la rv a l Leiostomus xanthurus age cohorts used In ca rb o n a s s im i la t io n experiments- * - 33

x l l

LIST OF FIGURES [continued)

Figure Page5135. Carbon-14 and Cr re ten tion and loss by la rva l

Lelostomus xanthurus In a d u a l- t ra ce r experiment w ith a 15 day age c o h o r t * * * .................... * * BA

35. Carbon-14 re ten tion and loss by la rv a l Lelostomus xanthurus 1n carbon ass im ila t ion experiments w ith reduced Brachlonus p U c a t f l l s concentrations* * * • 86

37. Carbon Ingested per dry weight o f la rva l Lelostomus xanthurus fed high and low concentrations o f Brachlonus p l1 c a t1 l is - ■ * * ......................................... 89

38. The number o f r o t i f e r s ( Brachlonus p U c a t lH s )Ingested re la ted to development o f la rva l Lelostomus xanthurus age cohorts....................................................................... 90

39, Length and age re la t ion sh ip s o f labora to ry-rearedLelostomus xanthurus larvae" * * * * * * * .......................... 98

40. Weight and age re la t ion sh ip s o f labora to ry-rearedLeiostomus xanthurus la rva e .......................................................... 99

41. Schematic surnnary o f the development oflabora tory-reared Lelostomus xanthurus la rv a e ...........................100

42. Head length and notochord or standard length re la t ion sh ip s o f labora tory-reared and w ild Lelostomus xanthurus la rvae- * ................................. 101

43. Head he ight and notochord or standard length re la tionsh ips o f labora tory-reared and w ild Leiostomus xanthurus la rva e .................... . • * , ...........................102

44, Eye diameter and notochord or standard length re la tionsh ips o f labora to ry-reared and w i ld Lelostomus xanthurus la rvae .......................................................... 103

45, Eye height and notochord o r standard length re la tionsh ips o f labora tory-reared and w i ld Lelostomus xanthurus la rva e ................................................................104

46. Snout to pectoral length and notochord o r standard length re la tionsh ips o f labora to ry-reared and w ild Lelostomus xanthurus la rvae .......................................................... 105

47. Pectoral depth and notochord or standard length re la t ion sh ip s o f labora tory-reared and w ild Lelostomus xanthurus la rvae- * * ■ * ■ ♦ * ■ * * » * ■ • l og

x i i i

LIST OF FIGURES (concluded)

Figure Page

48, Preanal length and notochord or standard length r e la t io n ­ships o f 1aboratory-reared and w i ld Lelostomus xanthurus l a r v a e .............................................................................................* . * * 107

49. Depth a t anus and notochord o r standard length r e la t io n ­ships o f 1abora tory-reared and w i ld Lelostomus xanthurus larvae , . * , * . . . . , .................................................................... 108

xf v

ABSTRACT

In view o f questions concerning the d ig e s t iv e and a ss im ila t ive a b i l i t i e s o f la rva l marine f is h e s , the changes 1n these processes with development, and th e i r re la t io n s h ip w ith s ta rva t io n - In du ce d m o rta l i ty ; t h i s study has three o b je c t iv e s ; (1 ) to examine the morphological and h is to lo g ic a l development o f the a lim entary canal and associated s tru c tu re s of la rva l spot, Lelostomus xanthurus (P isces: Sciaenldae);(2 ) t o assess short- te rm carbon asslm lTatlon In d is c re te age cohorts o f la rv a l spot; and (3) to re la te any changes In a s s im i la t io n to development. The a lim entary canal and associated s tru c tu re s o f larval spot from hatching through transfo rm ation (metamorphosis) were examined using l ig h t microscopy* These f in d in g s were re la ted to re su lts of e xp e r im e n ta l-ra d io tra ce r studies o f carbon a s s im i la t io n In d iscre te age cohorts o f la rv a l spot.

C yto log lca l evidence suggested tha t the a lim en ta ry canal and associated s truc tu res were func t io n a l In f i r s t - f e e d in g la rvae a fte r the completion of yolk-sac and o i1 -g lob u le absorp tion , and changed l i t t l e p r io r to trans fo rm ation . There 1s no e la b o ra t io n o f t issues during the la rv a l phase. Major developmental changes associated w ith t ra n s ­form ation were accompanied by changes in h a b ita t and feeding regime.

The behavior of carbon-14 ind ica ted th a t food was q u ic k ly digested and carbon ass im ila ted fo l lo w in g th e ingestion o f a un ifo rm ly labeled r a t io n . A percentage o f newly ass im ila ted carbon entered ra p id ly Into sho rt- te rm metabolic processes and ex ited the la rva v ia re sp ira t io n beginning as e a r ly as 1 h a f te r in g e s t io n . Gut evacuation was complete w i th in 6 or 7 h,

Uptake, short-term re te n t io n , and loss o f were used to compute two parameters, carbon re te n t io n and carbon a b so rp t io n , tha t were taken as r e la t iv e ind ices of the c o e f f ic ie n t of u t i l i z a t i o n and absorption e f f ic ie n c y . The re la t io n sh ip s between these two in d ice s o f carbon a s s im ila t io n and measures o f development Ind ica ted th a t ass im ila t ive a b i l i t i e s did not Improve w ith la r v a l development. Because there were no major changes in a lim entary canal t is s u e s , t h is might be expected.

The ind ices o f a ss im ila t io n were nega tive ly re la te d to la rva l c o n d it io n fa c to r and th is re la t io n s h ip has im portant Im p lica tions to la rv a l growth and s u rv iv a l . Condition fa c to r is an In d ic a to r o f larval growth and robustness. Larvae th a t have been feeding well enough to s u rv iv e , but less than o p t im a lly f o r maximum growth, apparently a ss im ila te more carbon from a ra t io n than do larvae w ith a b e t te r growth h is to ry *

These re s u l ts are p la u s ib le in e vo lu t iona ry terms. Larvae may m it ig a te the r is k of a patchy food d is t r ib u t io n by f u l l y u t i l i z in g a v a i la b le resources. Such compensatory mechanisms may be of great adaptive s ig n if ica n ce to pelagic marine la rvae .

xv

MORPHOLOGICAL, HISTOLOGICAL, AMD PHYSIOLOGICAL

ASPECTS OF ASSIMILATION IN LARVAL SPOT,

LEIOSTOMUS XANTHURUS LACEPEDE

INTRODUCTION

Research presented here is intended as one pa rt o f the m anifo ld

approach necessary to understand the re la t io n s h ip between stock and

recru itm ent of marine f ishes w ith pe lag ic eggs and la rvae . Because o f

the high annual v a r ia b i l i t y In re c ru itm e n t, due in pa rt to d i f f e r ­

e n t ia l s ta rva tion- induced m o r ta l i t y , t h is research examines the

a b i l i t y of a la rva to ass im ila te a v a i la b le food resources.

The su rv iva l ra te o f many marine f is h e s through th e i r e a r ly l i f e

h is to ry is extremely low and may be the major determinant o f numerical

yea r-c lass s treng th (Beverton, 1962; Q u lland , 1965; Cushing, 1969),

M o r ta l i t y is o ften near 99,9%, e s p e c ia l ly among h ig h ly fecund f ish e s

w ith pelagic eggs and larvae (see review in Dahlberg, 1979}. To

exp la in surv iva l In terms o f any s in g le fa c to r is Impossible f o r many

s y n e rg is t ic and spec ies -spec if ic fa c to rs in f luence m o r ta l i t y * Yet, a

general p a r t i t io n in g o f a c t ive mechanisms has enhanced an under­

standing o f the problem, Egg and la rvae m o r ta l i t y can be a t t r ib u te d

to adverse environmental cond it ions ( e .g . , Bonnet, 1939; L i l l e lu n d ,

1965; B la x te r , 1969; Dickson, e t al . , 1974; Le tt and Koh le r, 1976;

Rogers, 1976; Rosenthal and A ld e r ic e , 1976; F o r re s te r , 1977), genetic

defects (V lad im irov , 1970), and predation (Lebour, 1925; Murphy, 1961;

F raser, 1969; Hempel , 1965; Dekhnik, et a l . , 1970; L i l le lu n d and

Lasker, 1971; The llacker and Lasker, 1974; Westernhagen and Rosenthal,

2

3

1976; Kuhlmann, 1977; Laurence, et a l * , 1979), Fish larvae experience

a d d it io n a l m o r ta l i ty a t t r ib u ta b le to s ta rv a t io n * As the major causes

of la rv a l m o r ta l i ty {Hempel , 1974; Cushing, 1976; Hunter, 1976),

s ta rv a t io n and predation probably In te ra c t w ith s yn e rg is t ic e f fe c ts ;

the la rva tha t feeds poorly grows poorly and th is abates feeding

success and predator avoidance (Cushing, 1976).

S ta rva tion induced m o r ta l i t y was recognized as e a r ly as 1897

(Fabre-Domergue and 01etr1x) and re la ted research has progressed along

several l in e s . The " c r i t i c a l period concept", hypothesiz ing th a t year

class s treng th is determined by the a v a i l a b i l i t y o f p lank ton ic food

immediately a f te r yolk-sac a b so rp t io n , was f i r s t proposed by H jo r t

{1914; 1926). Research designed to te s t t h is hypothesis inc luded the

ana lys is of su rv iva l curves f o r natura l popu la tions (Marr* 1955), and

the c o r re la t io n o f la rva l gut contents and microzooplenkton abundance

in the sea w ith la rv a l cond it ion and abundance (see reviews by May,

1974). Though not a l l agree th a t c r i t i c a l periods e x is t or th a t food

l im i t a t io n is s ig n i f ic a n t in nature (Dekhnik e t a l , , 1970; Dekhnik and

Singukova, 1976), catastroph ic m o r ta l i t ie s apparen tly associated with

inadequate feeding conditions In the sea occur during the la rv a l stage

( e .g . , t f lb o rg , 1976). Inasmuch as s ta rv a t io n m o r ta l i t y has never been

proven c a te g o r ic a l ly , more evidence 1s needed using d iagnostic

h is to lo g ic a l , morphological, and biochemical c r i t e r i a (E h r l ic h , 1974a,

1974b; llmeda and Ochfai, 1975; E h r l ich et a l . , 1976; O 'Connell, 1976;

T h e i la c ke r , 1978), and experimental bioassay techniques (Lasker,

1975}.

4

Experimental research has accelerated w ith the a b i l i t y to

successfu lly rear la rva l marine f ishes in the la b o ra to ry . Experiments

have assessed the s e n s i t iv i t y o f larvae to delayed f i r s t - f e e d in g , the

e f fe c ts o f food density and small scale d is t r ib u t io n on la rva l sur­

v iva l and growth, and la rva l searching a b i l i t y and feeding e f f ic ie n c y

{see reviews 1n May, 1974; Ah1strom and Moser, 1976; Vlymen, 1977;

Hqude, 1978)* The resu lts o f these studies suggest th a t the in te ­

grated concentration of food organisms in the ocean ( e .g . , Beers and

Stewart, 1967, 1971; A r th u r, 1977) may be in s u f f ic ie n t to support

la rv a l f is h su rv iva l; though concentrations in bays and estuaries may

be adequate (see review 1n Houde, 1978)* The contagious sm all-sca le

d is t r ib u t io n of food organisms in the open sea may a lso be c r i t i c a l

{Lasker, 1975; Vlymen, 1977; Houde and Schekter, 1978), and the

In te ra c t io n o f food a v a i la b i l i t y , growth, and predation remain

obscure.

Most recen tly , phys io log ica l energetic studies have enhanced our

understanding o f s ta rva t io n , growth, and su rv iva l o f marine f is h

la rvae . Researchers have attempted to e s ta b lish the amount o f food

necessary to support la rva l f is h metabolism and growth by estim ating

the terms of energy budget equations ( I v le v , 1939; Wlnberg, 1956;

Palohelmo and D ick ie , 1966a, 1966b; Warren and Davis, 1967). T he ir

re s u l ts ind ica te tha t energy reserves upon yo lk-sac absorption are

inadequate to support metabolism and growth without feed ing (Lasker,

1964; B la x te r , 1969) and tha t natural d e n s it ie s of food organisms are

5

s y n o t fc a l ly Inadequate to support s u rv iv a l (see review 1n Laurence,

1977, Houde, 1978). Vlyroen's ( 1977) model, based 1n p a r t on swlrnirfng

e n e rg e t ics , p red ic ts th a t the contagious m 1crod is tr1button o f food

organisms 1s a c tu a l ly necessary fo r la rv a l s u rv iv a l .

Host phys io log ica l ene rge tic s tud ies on f is h larvae have estimated

equation terms as instantaneous rates a t a s in g le developmental

in te rv a l and have not attempted to reso lve the changes in m atter*

energy u t i l i z a t i o n through e n t i r e la r v a l l i f e . Larval f ishes a re ,

however, developing animals; m atter-energy u t i l i z a t i o n involves

dynamic processes influenced p o ss ib ly by age as w e ll as ra t io n s ize

and environmental fa c to rs .

The amount o f matter-energy a v a i la b le f o r metabolism and growth

1s determined, in p a r t , by the a b i l i t y o f the la rva to d iges t and

a ss im ila te n u tr ie n ts ( P h i l l i p s , 1969). Some have speculated th a t

a s s im i la t io n 1s i n i t i a l l y low but Improves w ith la rv a l age as the

r e s u l t o f the morphological development o f the a lim en ta ry canal

(Rosenthal and Herrtpel, 1970; Nishlkawa, 1975). Besides Laurence

(1971,1977) there 1s l i t t l e q u a n t i ta t iv e evidence to support t h is .

This d is s e r ta t io n has three o b je c t iv e s : (1) to examine the

morphological and h is to lo g ic a l development o f the a lim entary canal and

associa ted s tru c tu re s o f la rv a l sp o t, Leiostomus xanthurus; (2) to

assess sho rt- te rm carbon a s s im i la t io n in d is c re te age cohorts o f

la rv a l spo t; and (3) to re la te any changes in a s s im i la t io n to

development. I t is hoped th a t the in fo rm a tio n here in co n tr ib u te s to

the understanding o f the broader question o f p h y s io lg ic a l energetics

and s u rv iv a l o f la rv a l marine f is h e s .

THE EARLV LIFE HISTORY OF LEIOSTOMUS XANTHURUS

A BRIEF REVIEW

Spot, Lelostomus xanthurus Lacepede, is a h ig h ly fecund,

coastal-marine, sclaenid f 1sh ranging from Cape Codt Massachusetts, to

Bay o f Campeche, Mexico (Dawson, 1958), Populations f lu c tu a te

d ram a tica lly , and year-c lass strength is probably determined o ffshore

during the pelagic egg and la rva l phases (Joseph, 1972),

Spot spawn over the continental sh e lf in la te f a l l and w in te r

(see review 1n Johnson, 1978)* Ova counts range from 70,000 to 90,000

per female (Dawson, 1958)* Ovarian eggs o f several sizes w ith in

s ing le females (Hildebrand and Cable, 1930) and repeated spawning in

the labora tory suggest m u lt ip le spawning in nature. The spawning

period may not be p ro trac ted , however, as p re l im ina ry analyses o f

d a l ly o to l i t h growth increments ind ica te a s in g le , ra the r s h o r t ,

spawning season1. Spot begin the offshore spawning m igra tion

[Pacheco, 1962) a f te r th e i r second suraner (Hildebrand and Cable, 1930;

Pearson, 1929). The spherica l, pelagic eggs range from 0,72 to

0.87 mm 1n diameter and have e ith e r a s in g le , spherica l o i l g lobule or

m u lt ip le o i l globules grouped together w ith in the v i t e l l i n e membrane

(Powell and Gordy, 1980). Oil globules coalesce during development

leaving a single globule in the newly hatched yo lk-sac la rva (Powell

1 Peters, D, S ., J . C. DeYane, M. T, Boyd, L. C, Clements* and A, B. Powell. 1978. Pre lim inary observations on feeding, growth, and energy budget o f la rva l spot ( Leiostomus xanthurus) . Ann, Rept. of the Beaufort Laboratory to the U.S. Dept, o f Energy, pp. 377-397.

7

8

and Gordy* 1980), Hatching occurs in ca. Z days a t 20°C (see

Appendix),

Larvae have been described by many authors (see reviews 1n Fruge

and Truesdale* 1978; Johnson, 1978)* but t h e i r ecology 1s not well

documented, Newly-hatched, yo lk -sac larvae range from 1.6 to 2*5 mm

notochord length (NL). Yolk-sac and o i l - g lo b u le absorption are

complete 1n 4 to 7 days at 20°C (see Appendix). The larvae* now

2*0 to 2.4 run NL* begin feeding as members o f the plankton community

(Part I ) , Spot la rvae , l i k e most marine f is h la rva e , are v isua l

feeders w ith feeding genera lly r e s t r ic te d to d a y l ig h t hours (K je lson

e t al .* 1976; K je lson and Johnson* 1976). Development to

trans fo rm ation (metamorphosis) takes place in waters ove r ly in g the

con t in e n ta l sh e lf ( e .g . , Powles and Stender, 1976). P re lim inary

evidence suggests th a t la rv a l growth Is extremely rap id*

Transformation begins by ca. 7.0 mn standard length (SL) and is

complete by 16,0 to 18,0 mm SL (Powell and Gordy* 1980; also see

Appendix), The average growth ra te suggests th a t transform ing larvae

and e a r ly ju v e n i le s are not l im ite d by food a v a i l a b i l i t y . 1 Food

l im i t a t io n may, however* be an Important fa c to r In the su rv iva l o f

f i r s t - f e e d in g la rvae ; a short spawning period would am plify the

c r i t i c a l coincidence of zooplankton abundance and f i r s t feeding.

Spot, along w ith many other sc ia e n id s , u t i l i z e es tuaries as

nursery grounds [see review 1n Chao and Muslck, 1977), Transforming

larvae enter es tuaries during the f i r s t h a l f o f the year when as small

as 10.0 m SL. Larvae complete trans fo rm a tion w ith in es tuaries and

9

coasta l bays during spring and sunnier o f t h e i r f i r s t year.

Transforming larvae feed i n i t i a l l y on ep lfaunal Inverteb ra tes followed

by Infauna 1 invertebra tes and vegetable d e t r i t u s {P a rt I ) . Growth 1s

rap id during the f i r s t summer, and by December when most have moved

o f fsh o re to over-w in te r, they have grown to about 150 mn SL w ith some

as la rg e as 190 mm SL (Chao and Musick, 1977). Some sm alle r juven iles

o v e r-w in te r in the deeper channels o f es tua r ies (Chao and Musick,

1977). Juveniles may penetrate freshwater as well as hypersaline

lagoons and marshes {see review in Johnson, 1978).

The to lerance of eggs, la rvae , and ju v e n i le spot to temperature

and s a l i n i t y v a r ia t io n 1s not well documented. Eggs are not buoyant

and hatching success is reduced in water less than 26 ° /o o . Likewise,

ha tch ing success and su rv iva l through yo lk -sac absorption is reduced

below 15°C (A,B. Powell* personal communication). Eggs and larvae

g e n e ra l ly occur in 18 to 22flC and 30 to 35 ° /oo water in the South

A t la n t ic B ight (Powles and Stender, 1976); though la rvae and Juveniles

are taken in temperatures from 6.3 to 32.5°C and s a l in i t i e s from 0 to

34.2 ° /o o (see review In Johnson, 1978). Temperatures o f 4 to 5°C may

be le th a l to juven iles (H ildebrand and Cable, 1930; Dawson, 1958).

The d is t r ib u t io n o f larvae and Juven iles 1s apparently l im i te d by

temperature ra ther than s a l in i t y [Parker, 1971). Temperature

preferences can be in fe rred from the seasonal movement o f larvae and

Juven iles In to and out of es tuarine nursery areas. Transforming

10

larvae and early juven iles enter estuaries 1n spring as the

temperature rises above fi°C; ju v e n i le s generally depart 1n f a l l as the

temperature drops below 1Q°C (Parker* 1971; Chao and Musick, 1977),

ASPECTS OF THE MORPHOLOGICAL, HISTOLOGICAL, AND

FUNCTIONAL DEVELOPMENT OF THE ALIMENTARY CANAL

AND ASSOCIATED STRUCTURES

INTRODUCTION

Starvation-Induced m o r ta l i ty o f pelagic marine f is h larvae as a

determ inant of f lu c tu a t io n s in numerical year c lass s treng th has been

the sub jec t o f controversy fo r almost a century (see reviews by

B la x te r , 1969i May, 1974). S ta rva tion has received much a t te n t io n ,

In c lud ing extensive f ie ld and labora to ry s tudy, but the d ig es tive and

a s s im i la t iv e a b i l i t i e s of la rv a l f ishes remain to be determined

(H unter, 1976). While some have assumed th a t these a b i l i t i e s are

I n i t i a l l y very poor but improve with la rva l age as the re s u l t of the

development o f alimentary canal t issues [Rosenthal and Hempel, 1970;

Nishlkawa, 1975; Laurence, 1977), l i t t l e supporting evidence e x is ts .

Changes in d ig e s t io n and a ss im ila t io n should be concomitant w ith

changes in the alimentary canal and associated s t ru c tu re s .

This study was undertaken to describe c e r ta in aspects o f the

fu n c t io n a l development of the a limentary canal, l i v e r , pancreas, jaws,

and tee th o f spot, Lelostomus xanthurus■ Spat Is a h ig h ly fecund,

s d a e n ld f i s h w ith pelagic eggs and larvae, fe a r-c la s s size

f lu c tu a te s d r a s t ic a l ly , probably due to d i f f e r e n t ia l la rv a l m o r ta l i ty

(Joseph, 1972); s ta rva tion may be a causative fa c to r . Knowledge of

the extent and t im ing of developmental changes in the a lim entary canal

and associated structures should provide a basis f o r eva lua ting

changes In the d igestive and a ss im ila t ive a b i l i t i e s o f spot during I t s

e a r ly l i f e h is to ry .

12

13

MATERIALS AND METHODS

Specimens fo r d issec tion and h is to lo g ic a l study were obtained

from two sources. Eggs were obtained from f is h Induced to spawn

through temperature and photoperiod manipulation and hormone

In je c t io n . Eggs and larvae were reared at 20 + 2°C In 30-35 D/oo

s a l i n i t y seawater and fed sequen tia l ly w ith the d ln o f la g e l la te *

^ymnodlnium splendens, the r o t i f e r , BracHonus p l lc a tH i s . and w ild

plankton- Specimens were f ixed in c h i l le d neutra l bu ffe red

form alin plus ac ro le in , dehydrated 1n methanol. I n f i l t r a t e d w ith

and embedded 1n glycol methacrylate (2-hydroxy-ethyl m e thacry la te ),

and sectioned at 2 and 4 pm according to m odifica tions o f Ruddell

(1971}, For comparison, f i e ld co lle c t io n s of the V i rg in ia In s t i tu te

o f Marine Science (VIMS), f ixed 1n buffered fo rm a lin 1n seawater,

were dehydrated 1n Isopropanol, i n f i l t r a t e d w ith and embedded in

p a ra f f in , and sectioned a t 5 yin. H is to lo g ica l and hlstochemlcal

s ta ins used fo r glycol methacrylate sections were a d d fuchsln -

t o l d d ine blue 0, a lk a l in e blue 6B - neutra l red, and p e r io d ic acid

S c h i f f 's reagent (PAS) - to lu id ln e blue 0. P a ra ff in embedded

specimens were stained in hematoxylin - astra blue - eos in Y.

h e t t l e r , W.F., A.B, Powell, and L.C. Clements. 1978. Laboratory induced spawning o f spo t, Lelostomus xanthurus (Lacepede). Ann. Rept. o f the Beaufort Laboratory to the U.S. Department of Energy- pp. 351-356.

14

The development o f jaws and d e n t i t io n was studied on specimens

cleared and sta ined w ith a l i z a r in red S - a lc ia n blue by a

m o d if ica t io n o f Dlngeraus and Uhler (1977).

Specimens f o r gut content a n a lys is were obtained from f i e l d

c o l le c t io n s o f the South Caro lina W i ld l i f e and Marine Resources

Department, NOAA-NMFS Sandy Hook Laboratory* and VIMS. Food items

were excised from 59 specimens, mounted 1n CMC-5 (T u r to x )* and

id e n t i f ie d to the lowest poss ib le taxon.

Lengths reported herein are notochord lengths (NL) measured

before the complete form ation o f hypural elements and standard

length (SL) measured th e re a f te r .

15

RESULTS

Though development 1s continuous, the a lim entary canal is

discussed w ith in the framework of three phases th a t gen e ra lly

correspond w ith changes 1n gross morphology: the yo lk -sac la rva

{1*6 - 2.5 mm NL), the la rva (2,5 - 7*0 inn NL), and the transforming

la rva (7 ,0 -18,0 ran 5L). A t a rearing temperature o f 20°C these

phases correspond to 2-7, 7-31, and 31-60 days from ha tch ing .

P e r t in e n t d e ta i ls of the e a r ly ju v e n i le a lim en ta ry canal are also

described . While many te rm ino log ies o f f i s h development In te rva ls

e x is t ( e .g . , Hubbs, 1943; Ah ls trom , 1968; Ba lon, 1975), the present

terms a f fo rd s im p l ic i ty and close alignment w ith a lim entary canal

development*

A lim en ta ry Canal

The yo lk-sac la rva . In the yolk-sac la rva the a lim entary canal

1s u n d if fe re n t ia te d along i t s length and not fu n c t io n a l* The mouth is

not open. The bucco-pharynx is l ined w ith squamous e p ithe lium and

sca tte red mucous c e l ls of un iform appearance underla in by f ib ro u s

connective t is s u e . The in c ip ie n t gut 1s a s t ra ig h t tube ly in g in the

dorsa l v isce ra l ca v ity above the yo lk mass and d e f le c t in g v e n t ra l ly to

a closed anus {Figure 1), The narrow lumen is open p o s te r io r ly

(F igu re 7 ) , but is p a r t ia l l y occluded in the a n te r io r h a l f by pressure

from the o i l g lobu le . I t is l ined w ith c lo s e ly packed, columnar

e p i t h e l ia l c e l l s , 12-15 inti t h ic k . The nucle i o f these c e l ls are

16

located basal 1y and are ca. 3 unn 1 n diameter. The cytoplasm 1s

basoph il ic and granular. Mitochondria are not v is ib le . A p ic a l ly ,

the e p i th e l ia l c e l ls have a prominent* a c id o p h i l ic s t r ia te d border w ith

m ic r o v i l l i 3-4 pm deep (F igure 8). A 5 pm laye r o f a re o la r connective

t issue w ith f ib ro b la s ts underlies the ep ithe lium . This l i n in g ,

lack ing rugae, cons titu tes the early mucosa, but a submucosa and

muscularis are not apparent. A th in serosa is d lsce rnab le .

The a limentary canal becomes func t io n a l during the completion o f

yo lk -sac and o l l -g lo b u le absorption. This t ra n s i t io n is ra p id , tak ing

about 3D h at 2G°C, The yo lk is absorbed before the o i l g lobu le .

During I ts absorption 1 t remains as a cap o f yo lk granules enclosed by

the v i t e l l i n e syncytium ly in g over the a n te r io r pa rt o f the o i l

g lobule (F igure 2 ) . The pos te r io r h a l f o f the a lim entary canal w ith a

lumen abuts the o i l globule whereas the a n te r io r a lim entary canal

remains p a r t ia l l y occluded and l ie s between the o i l g lobule and the

dorsal wall o f the v iscera l ca v ity (F igure 2 ), During f in a l yo lk and

o11-globule absorption, the lumen extends the e n t i re length o f the

a lim entary canal, and the alimentary canal In to r ts to form a loop

midway along I t s length. Two c i r c u la r c o n s t r ic t io n s * the p y lo r ic po rt

and the lleocaeca l po rt , develop fu r th e r d iv id in g the a lim en ta ry canal

in to fou r sections: the bucco-pharynx, the fo regu t, m idgut, and

hlndgut (Figure 3). The mouth and anus are now open, and f i r s t

feeding may occur. The number o f PAS p o s i t iv e mucous c e l ls increases

17

along the s ing le la y e r o f squamous e p i th e l ia l c e l ls l in in g the

bucco-pharynx.

The la r v a . The la rv a l bucco-pharynx, f o r e - , m id-, and h lndgut

are d is t in g u ish e d by h is to lo g ic a l d i f fe re n c e s and remain unchanged

u n t i l t rans fo rm a tion . The a lim en ta ry canal length and su rface area,

however. Increase w ith Increased body s iz e .

The w a lls o f the bucco-pharynx are l in e d w ith s t r a t i f i e d squamous

e p ith e l iu m , 3 to 4 deep. Two la y e rs , an Inner 7 to 8 um la y e r of

a re o la r connective t is s u e , and an o u te r 4 m la y e r o f f ib ro u s

connective t is s u e , l i e beneath the ep ith e l iu m . F ib ro b la s ts are seen

1n these connective t issue la y e rs , and a few un iform PAS p o s i t iv e

mucous c e l ls occur In the ep ith e liu m . The v e n tra l w a lls have more

numerous and la rg e r PAS p o s it iv e mucous c e l l s . Taste buds appear in

the dorsa l and v e n tra l w a l ls ; though they lack gus ta to ry pores and are

presumably not fu n c t io n a l . They are more numerous on fo ld s and ridges

o f the ve n tra l w a l l .

The fo regut connects the bucco-pharynx w ith the midgut (F igures

4,9) and d i f fe re n t ia te s from the a n te r io r closed section o f the

In c ip ie n t gut. I t s mucosa cons is ts o f cuboidal e p i th e l ia l c e l l s , 10

to 20 ym th ic k , w ith basoph ilic cytoplasm. The basa lly loca ted nucle i

are a lso b a so p h il ic . Nucleoli are not v is ib le . The surface o f the

mucosa o fte n has jagged p ro jec t io n s th a t are p o ss ib ly remnants o f

sloughed c e l ls . Two types o f mucous c e l l s , d is t in g u ish e d on the basis

o f c e l lu la r morphology and s ta in in g re a c t io n s , are te n ta t iv e ly

id e n t i f ie d In the fo regu t mucosa (F igu re 10). Most numerous (Type 1)

is

are PAS p o s it iv e mucous c e l ls (8-12 pm 1n diameter) which resemble

to p ica l vertebra te goblet c e l ls and are s im i la r to those o f the

bucco-pharynx. They s ta in m etachromatica lly w ith basic dyes

suggesting tha t they contain acid mucopolysaccharides. The second

type {Type 2) are large (20 pm 1n d la m te r) , b a s o p h i l ic , and PAS

negative c e l l s , often aggregated 1n groups. The fo regu t mucosa has

shallow lo n g itud ina l fo ld s . A 30 pm th ic k lamina propria o f fibrous

connective t issue w ith f ib ro b la s ts l ie s subjacent to the ep ithe lium .

There Is no muscular!s mucosa or submucosa. The muscular!s consists

of 6 to 7 pm o f c i r c u la r smooth muscle f ib e rs , and a very th in serosa

is v is ib le .

The m idgut, l im ite d by the p y lo r ic and lleocaecal p o r ts , is

bulbous w ith a much wider lumen than e i th e r the fo re - or hlndgut

(Figure 9 ) , The mucosa becomes h ig h ly convoluted with deep (40-50 pm)

rugae, thus increasing surface area. I t s ep ithe lium co ns is ts of

basoph il ic columnar c e l ls , 25 to 30 pm h igh, w ith a 2 to 3 urn s tr ia te d

border o f a c id o p h i l ic m ic r o v i l l i underla in by a term inal web. Nuclei

are basoph il ic w ith v is ib le n u c le o l i . Numerous supra- and

in fra n u c le a r mitochondria are seen w ith in the cytoplasm. The most

c h a ra c te r is t ic feature of these c e l ls is the presence o f numerous,

supranuclear, vacuolar, in t r a c e l l u la r inc lus ions (Figure 11). The

mucosa lacks mucous c e l ls . A th in (8-10 pm) lamina p ropr ia o f areolar

connective t issue w ith f ib ro b la s ts underlies the ep ith e l iu m . There Is

no d is t in c t muscularls mucosa or submucosa. The muscularis consists

o f a 5 urn th ic k layer of c i r c u la r smooth muscle f ib e rs . A very th in

serosa is v is ib le .

19

The h lndgu t begins a t the l leacaeca l p o r t , ends a t the deve lop ing

anal s p h in c te r (F igure 4, 9 ) , and d i f f e r s from the mldgut m a in ly In

the cy to logy o f i t s mucosal e p i t h e l ia l c e l l s . The h lndgut mucosa has

sha llower (ca . 25 vm) rugae than those o f the m ldgut. I t s . mucosa

cons is ts o f a laye r o f un ifo rm co lu ttnar e p i t h e l ia l c e l l s , s l i g h t l y

s h o r te r (25 ^m) than those o f the m ldgut, w i th a low (1 o r 2 urn)

a c id o p h i l ic s t r ia te d border. There Is no evidence o f a te rm ina l web

beneath the m i c r o v i l l i . The basal cytoplasm o f the e p i t h e l ia l c e l l s

Is b a s o p h i l ic w h ile the a p ica l cytoplasm 1s a c id o p h i l i c . The nucleus

is loca ted b a s a l ly . As In the mldgut e p i th e l iu m , the re are numerous

supra- and in t ra n u c le a r m itochondria and supranuc lear cytop lasm ic

In c lu s io n s , but these in c lu s io n s are g ranu la r and a c id o p h i l ic

(F igure 12). The hlndgut has a th in (8 - 10 ^m) lamina p rop r ia

subjacent to the e p ith e l iu m , but no m uscularis mucosa or submucosa.

The m uscularis cons is ts o f a 1 jim th ic k la y e r o f c i r c u la r smooth

muscle c e l l s . There 1s a th in serosa. E ry th ro cy te s and an assortm ent

o f leucocytes are v is ib le w i th in the mid- and h lndgu t m uscu la r is .

The trans fo rm ing la rva and e a r ly ju v e n i le . The major changes 1n

the a lim en ta ry canal are associa ted w ith tra n s fo rm a t io n

(metamorphosis). The a l im en ta ry canal completes I t s development w i th

the fo rm ation o f the esophagus, stomach, In te s t in e w i th p y lo r ic

caecae, and rectum (F igure 6).

The bucco-pharynx remains unchanged w ith a few exceptions. A

buccal valve develops midway sepa ra t ing the pharynx and the buccal

20

c a v i t ie s . The ta s te buds* now open v ia gustatory pores, become more

numerous, and presumably fu n c t io n a l .

The esophagus d i f fe re n t ia te s from the fo regut and merges

p o s te r io r ly w ith the developing stomach. The esophageal mucosa

cons is ts o f s t r a t i f i e d cuboida! e p i t h e l ia l c e l ls 10 pm th ic k lack ing

c i l i a . The number o f Type 1 mucous c e l ls in the mucosa g rea t ly

increases (Figure 15), hut Type 2 mucous c e l ls were not seen in the

esophagus. The lamina p ropria and submucosa d i f f e r e n t ia te by ca.

13 mm SL. This d is t in c t io n 1s based on the te x tu re o f connective

t is su e fo r no muscularis mucosa develops In the a lim en ta ry canal. The

th in (6 pm) lamina p ropria underlies the e p ith e liu m and consis ts o f

f ib ro u s connective t is s u e . The submucosa, comprised of 15-20 pm o f

a reo la r connective t issu e w ith a few dispersed lo n g itu d in a l muscle

c e l l s , l ie s subjacent to the lamina p ro p r ia . A 10 pm la ye r of

c i r c u la r , smooth muscle f ib e rs w ith a few s t r ia te d muscle c e l ls appear

in add it ion to the inner layer o f lo n g itu d in a l muscle by ca, 13 mn SL.

These layers decussate a t the esophageal g a s tr ic ju n c t io n .

The stomach d i f fe re n t ia te s from the p o s te r io r section o f the

fo re g u t . The stomach anlage appears as a b l in d sac th a t bulges from

the p o s te r io r end of the fo regu t as i t meets the mldgut (F igure 5 ) .

This out pocketing continues to expand and extends as a bulbous sac

dorsal to the ju n c t io n w ith the m ldgut. Uniform secre to ry c e l ls

appear in the b l ind sac by ca. 10 mm SL [F igure 17). These c e l ls

increase 1n number and even tua lly l in e d is t in c t g a s t r ic glands In the

corpus (sensus B a rr ing ton , 1957 ) by IB itm SL (F igure 17). The g a s t r ic

HI

mucosa consists of simple columnar e p i th e l ia l c e l ls w ithou t a s tr ia te d

border- The apical cytoplasm is s l ig h t ly a c id o p h i l ic , perhaps because

o f the presence of mucigen, and contrasts w i th the b a so p h il ic basal

cytoplasm, Mucous c e l ls , s im i la r to Type 1 c e l ls in form, appear

w h ile the mucosa develops deep rugae. A lam ina propria underlies the

developing g a s tr ic ep ithe lium . The stomach has a 15 ym th ic k

submucosa of a reo lar connective t is s u e , A la y e r o f a few c i r c u la r

muscle f ib e rs is seer w i th in the mucosa. The g a s tr ic muscularis

co ns is ts of a 40 urn th ick inner la ye r of smooth, c i r c u la r muscle and a

3 ym ou ter laye r of smooth, lo n g itu d in a l muscle. The serosa is

sometimes v i s ib le . The ju n c t io n o f the esophagus and the stomach is

marked by an abrupt change In ep ithe lium (F ig u re 16}.

The in te s t in e is separated from the stomach by a developing

p y lo r ic sph inc te r and d i f fe re n t ia te s from the midgut, I t is genera lly

J-shaped (Figure 6} and separated from the s t ra ig h t rectum by the

ileocaeca l va lve, Cytol og ica l ly i t s ep ithe lium Is s im i la r to the

mldgut (Figure 16), A few mucous c e l ls , s im i la r to Type 1 c e l ls ,

appear in the mucosa. A d is t in c t io n between the lamina p ro p r ia and

submucosa is not apparent as 1t 1s in the esophagus and stomach.

Ins tead , a gradual t ra n s it io n from inner f ib ro u s to ou te r a reo la r

connective t issue develops, The rugae of th e midgut deepen in the

in te s t in e * The muscularis th ickens greatly and an ou te r laye r o f

lo n g itu d in a l muscle appears by ca. 13 urn SL- P y lo r ic caeca develop as

b l in d outgrowths of an te rio r In te s t in e ju s t p o s te r io r to the p y lo r ic

sph in c te r and are h is to lo g ic a l ly s im i la r to the In te s t in e (F igure 20).

The f i r s t caecum appears a t ca. 10 mm SL. The ju v e n i le complement of

22

seven to e igh t Is complete by 18 mm SL and Is attendant w ith the

completion o f stomach development.

The rectum d if fe re n t ia te s from the hlndgut but shares a s im i la r

epithe lium {Figure 19). As in the In te s t in e , mucous c e l ls appear 1n

the mucosa and Increase In number near the anal sph inc te r. The mucosa

develops secondary fo lds . The lamina propria and submucosa become

d is t ingu ishab le by ca. 23 m) SL, The lamina propria consists o f

aero lar connective tissue whereas the submucosa consis ts of f ib ro u s

co rrec t ive t issue , a reverse of the esophageal, g a s t r ic , and

in te s t in a l cond it ion . U fth ln the muscularis, the inner layer o f

c i r c u la r muscle expands and a th ic k outer layer o f smooth,

lo n g itud ina l muscle appears by ca. 13 mm SL. The d i f f e r e n t i a l l y

s ta in ing cytoplasmic Inc lus ions, so conspicuous 1n the la rva l mid- and

hindgut, are also obvious 1n transform ing larvae and early ju v e n i le s

(Figures 18, 19).

Associated Structures

The l i v e r . The l i v e r 1s d i f fe re n t ia te d a t hatching and l ie s

dorsal to the yolk mass (Figure 7 ) . Hepatocytes are compact,

g ranu la r, basoph il ic , and lack o f vacuoles In yo lk -sac larvae. In

larvae the l i v e r l ie s beneath and p a r t ia l l y surrounding the a lim entary

canal (Figure 9 ) . I t increases in size and becomes b ilobed. Large

(12 um in diameter) hepatocytes surround d is t in c t c a n a l ic u l i . These

c e l ls contain voluminous (4 urn 1n diameter) PAS p o s it iv e vacuoles

(Figure 13) tha t are presumably the s ite s o f glycogen storage (no

diastase con tro ls were used). The g a l l bladder appears on the r ig h t

l i t e r a l lobe o f the la rv a l l i v e r by ca. 3,0 mm NL. The l i v e r in e a r ly

ju v e n i le s occupies more space in the v isce ra l c a v i ty than any o the r

period in the s p o t 's e a r ly l i f e h is to ry . I ts two lobes extend around

the p o s te r io r loop o f the in te s t in e .

The pancreas. The exocrine pancreas, an in s u la r organ in a d u lt

spo t, is d i f fe re n t ia te d a t hatching w ith well developed b a soph il ic

ac ina r c e l ls . Pancreatic ducts and zymogen granules are not ye t

apparent. The la rv a l pancreas l i e s p r im a r i ly along the r ig h t side o f

the mldgut but sends disseminated branches In to the l i v e r . Pancreatic

ducts appear soon a f t e r yo lk abso rp t ion , and conspicuous a c id o p h i l ic

zymogen granules appear w ith in the pancreatic a c in i (F igure 14). The

pancreas remains unchanged during tra n s fo rm a tio n .

Jaws. At hatch ing anlagen o f the upper and lower jaws are formed

as p ro c a r t i la g e w ith basoph ilic chondrocytes ly in g w ith in a th in

m a tr ix o f chondromucen. At f i r s t feeding (ca , 2.0 mm NL) the upper

jaw is comprised o f pa ired prem axillae w ith the premaxi 11 a completely

excluding the m a x il la from the gapei the lower jaw is comprised o f

pa ired Meckel's c a r t i la g e s . Dentarles and a r t i c u la r s w ith angular

processes are d is ce rra b le as separate lower-jaw elements by ca. 4 .0 mm

NL* C a lc i f ic a t io n o f the jaw elements begins by ca. 0 .5 mm SL in the

a n te r io r shafts and ascending processess o f the p rem ax il lae , the

a n te r io r shafts o f the m a x il lae , and the p o s te r io r flanges o f the

a r t i c u la r s . The a n te r io r t i p o f the den ta rles begins to c a lc i f y by

12 Jim SL. C a lc i f ic a t io n proceeds posteriad along the axes o f the

p rem ax il lae , m a x i l la e , and denta rles and spreads in to the la te ra l

24

flanges o f these elements by IS rim SL. Meckel's c a r t i la g e s p e rs is t in

Juven iles as carti lagenous shafts enclosed w ith in each a r t i c u la r .

Caution must be exercised in the in te rp re ta t io n of s ta in in g

re a c t io n s * Premaxillae* m a x i l la e , d e n ta r le s , and a r t i c u la r s are

dermal bones not preformed as c a r t i la g e , ye t they o ften s ta in w ith

a lc ia n blue* A lc ian blue reacts w ith many a d d mucopolysaccharides

In c lu d in g chondromucin (Pearse, L9G8)* H is to lo g ic a l ly , hya line

c a r t i la g e w ith chondrocytes completely f i l l i n g lacunae embedded w ith in

a th ic k chondromudn m a tr ix , predominates 1n endochondral elements of

larvae and early ju v e n i le s . Perichondral bone appears on the

periphery of some elements by ca. 23 mm SL; though the a l iz a r in

re a c t io n is v is ib le In elements o f much sm a lle r specimens* A l iz a r in

red $ che la tes Ca ions (Pearse, 1968} and thus may in d ic a te the

presences o f c a lc i f ie d c a r t i la g e as w e ll as t ru e o s s i f ie d bone*

The gape, terminal in larvae and trans fo rm ing la rvae , becomes

p rog ress lve ly in fe r io r in p o s it io n during the e a r ly Ju ven ile stage.

D e n t l t io n * Developing teeth are f i r s t seen 1n the a reo la r

connective t issue underly ing the bucco-pharyngeal e p ith e l iu m o f the

la rva e , Retrorse conical tee th erupt on the prem axillae a t ca, 4.0 mu

NL and on the dentaries by 5 ,2 nm NL (Table 1), At ca. 16 rm a second

h o r izon ta l row of recurved v i l l i form tee th erupts 1 inquad on the

p rem axillae and dentaries* This row o f v i l l i f o r m teeth replaces

la rv a l tee th as add itiona l rows of recurved v i l l i f o r m teeth erupt

1 inquad forming tooth rows. At complete t ra n s fo rm a tio n , the

polyphyodont d e n t i t io n (Peyer, 1968) o f spot 1s comprised o f three

25

to o th rows, Premaki 11 a ry and dentary te e th w i th in a to o th fa m i ly

(Peyer, 1968) are sh o rte r toward the U nqua l aspect.

V i l l i f o r m pharyngeal tee th begin to erup t on the paired medial

c e r ta to b ra n c h ia ls of b ranch ia l arch 5 and on the pharyngobranch ia ls o f

arches 2 , 3 , and 4 by 5,5 mm NL (Table 1 ) , The complete complement o f

pharyngeal teeth is reached by complete t ra n s fo rm a t io n w ith v i l l i f o r m

tee th on the a n te r io r aspect of the ce ra to b ra n ch ia ls and

pharyngobranchials 3 grading to con ica l te e th toward th e p o s te r io r

aspect,

Gut Contents

Labora tory reared specimens show no evidence o f feed ing as y o lk -

sac la rvae . Green chyme, comprised presumably o f d in o f la g e l1ates and

a lga l c e l l s , is seen d u r ing o i l - g lo b u le a b s o rp t io n . R o t i fe rs and

copepod nau p lH are taken soon a f te r the absorp tion o f the o i l

g lo b u le . The d ie t of w i ld larvae c o n s is ts p r im a r i ly o f copepod

n a u p l i i and copepotMtes w ith some ju v e n i le pelecypods and c ladocerans.

The s ize o f preferred food organisms Increases w ith the s ize o f the

larvae [K je lso n et a l . , 1975; Table 2 ) , and, as the la rv a grows, a d u lt

calanolds e n te r the d ie t . During t ra n s fo rm a t io n , e p ib e n th ic

h a rpac tico id copepods (Ph.vl 1 opodopsvl lus s p p , ) dominate the d ie t

(Table 2 ) . Juveniles feed on d e t r i tu s and ep ifaunal in v e r te b ra te s ,

mainly anne lids (Parker, 1971; Peters and K je ls o n , 1975; Chao and

Musick, 1977; Sheridan, 1978).

26

Table 1, Tooth counts o f la rva l Lelostomus xanthurus. P re m ax llU ry and dentary teeth were counted on the l e f t s ide and counts m u l t ip l ie d by two; pharyngeal tee th were observed but not counted.

Notochord/Standard Premaxi 1 la ry Denta ry Epibranchial Ceratobr;

Age Length Teeth Teeth Teeth Teel

4 2,4 _ _ _

7 2.4 - - -2.7 - - - -3.0 - - -3.'’ - - _ •

4.0 2 - - -

23 4.5 10 - - -28 4,3 B - -28 5.2 10 2 - -30 5.2 10 - - -35 5.5 14 - + +35 6,4 12 fi - -36 5,1 14 2 - -36 10.0 30 22 +41 5.4 12 - - -41 G.9 20 18 + +42 10.5 30 16 + +43 5.B 12 8 - -50 6.6 18 12 - -54 8.0 IB 16 + +54 8.2 16 14 + +

12.6 32 26 + +12.6 44 38 + +12,9 40 32 + +12.9 34 50 + +13.2 52 58 + +13.9 30 48 + +14.5 90 62 + t17.7 108 76 +18.5 116 78 + +19.1 108 72 + +19,4 116 38 + +20.0 108 86 +20.0 76 72 + +20,1 96 76 + +20.3 106 78 +20.5 68 80 +21.0 106 86 + +

27

Table 1. ( cone 1 tided)

Notochord/'Standard P rem axllla ry Dentary Ep ibranch ia l Ceratobranchlal

Age Length Teeth Teeth Teeth Teeth

21.0 ee 85 + +21.5 102 98 + +22 , 9 70 54 + +23.9 64 62 + +24.2 64 74 + +

Table

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Figure 1 -6 . Morphological development o f the a l im e n ta ry canal o f

Lelostomus xanthurus. 1. In c ip ie n t g u t o f a yo lk-sac

larva 2 . 2 wn NL, 2. In c ip ie n t gut o f a yo lk-sac larva

2.3 ran NL. 3. In c ip ie n t gut o f a la rv a 2,8 inn NL in

o i l -g lo b u le abso rp t ion . A. A lim entary canal o f a larva

3.0 ran NL, 5, A lim entary canal o f a transform ing larva

7.6 ran SL. 6, A lim entary canal o f an e a r ly ju v e n ile

25.0 mm SL. Abbrev ia tions : BP, bucco-pharynx; BS, b l in d

sac; E, esophagus; FG, fo re g u t ; HG, h lndgu t; I ,

In te s t in e ; IG* in c ip ie n t g u t; MG, m ldgut; 0Gt o i l

g lobu le ; PC, p y lo r ic ceaca; R, rectum; S, stomach; YM,

yolkmass.

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31

Figures 7-8. Photomicrographs o f the in c ip ie n t gut o f a Leiostomus

xanthurus yo lk -sac la rva 2 .3 mm NL. 7. Long itud ina l

section showing y o lk mass, o i l g lobu le , In c ip ie n t gu t,

l i v e r , and pancreas. 8. Cross sec tion showing mucosal

ep ithe lium and s t r ia te d .

Figures 9-10. Photomicrographs o f the a lim en ta ry canal o f a

Leiostomus xanthurus la rva 4.4 mn NL. 9. Long itud ina l

section showing fo re g u t , m ldgut, h lndgu t, l i v e r , and

pancreas. 10. L o n g itud ina l section showing two types

o f mucous c e l ls 1n fo re g u t . Abbrev ia tions: CT,

connective t is s u e ; FG, fo re g u t ; EP, e p ith e l iu m ; HG,

h lndgut; IG, in c ip ie n t g u t ; L, lumen; L I* l i v e r ; MG*

midgut; MCI, mucous c e l l type 1* MC2, mucous c e l l type

2; OG, o i l g lobu le ; P, pancreas; SB, s t r ia te d border;

YM. yolkmass.

' I , ' _. ...,..

\

•

e e

T

. . .

32

Figures 11-14, Photomicrographs o f the a lim entary canal o f a

Lelostomus xanthurus larva 4 ,4 mm NL. 11.

Longitudinal section showing m idgut, 12,

Longitudinal section showing h lndgut ep ithe lium w ith

s t r ia te d border and In t r a c e l lu la r In c lus ions . 13,

Longitudinal section showing l i v e r ac in i w ith l i v e r

s inus , hepatocytes, and glycogen vacuoles, 14,

Longitudinal section showing pancreatic ac in i and

zymogen granules. Abbreviations: EP, e p ith e l iu m ; GJ,

glycogen vacuoles; H, hepatocytes; IC I, i n t r a c e l lu la r

Inc lus ions ; LS, l i v e r sinus; PA, pancreatic a c in i ; SB,

s t r ia te d border; ZG, zymogen granules.

~· 0 I

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-~ . ' ..

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33

F ig u re s 15-

Figure 17*

Figures 18-

16. Photomicrographs o f a trans fo rm ing Lelostomus

xanthurus la rva 12*3 m SL. 15* Long itud ina l

section showing ju n c t io n o f bueco-pharynx and

esophagus w ith esophageal fo ld s and Increased

number o f mucous c e l ls . 16* L o n g itud ina l section

showing the Junction o f the esophagus and b l in d sac

w ith developing g a s tr ic glands*

Photomicrograph o f an e a r ly ju v e n i le Lelostomus

xanthurus 18.1 mn SL* L o n g itud ina l sec tion o f

gas tr ic corpus o f a f is h 18.1 mn SL showing

uniform secre to ry c e l ls o f the g a s t r ic glands.

. Photomicrographs o f an e a r ly ju v e n i le Lelostomus

xanthurus 33.0 mti 5L. 18- Long itud ina l sec tion

o f the In te s t in e showing I n t r a c e l lu la r inc lus ions*

19. Long itud ina l section o f the rectum showing

granular I n t r a c e l lu la r In c lu s io n s . Abbreviations:

BP, bucco-pharynx; BS, b l in d sac; E, esophagus;

GG, g a s tr ic g lands; IC l t i n t r a c e l l u l a r in c lu s io n s ;

MU, mucosa.

34

DISCUSSION

The a b i l i t y to acquire , d ig e s t , and ass im ila te n u t r ie n ts should

be maximized by adaptation throughout the development o f a f i s h . The

numerous mitochondria and I n t r a c e l lu la r Inc lus ions in th e mid- and

hlndgut ep ith e liu m , as well as the h is to lo g y of the l i v e r and

pancreas, suggest th a t the a lim en ta ry canal becomes fu n c t io n a l du r ing

o i l -g lo b u le absorption when f i r s t feeding may occur. There Is no

e labora tion of c e l l types from f i r s t feed ing u n t i l t ra n s fo rm a t io n ,

though the surface area Increases. The l i v e r expands r a p id ly in the

la rva and is a c t iv e in glycogen storage. E h r l lc h (1974a, b) has found

th a t p ro te in and carbohydrate are t ra n s fe r re d to body t is s u e s

p r e fe r e n t ia l ly over t r lg y lc e r id e s in la rv a l Cluoea harengus and

Pleuronectes p la te ss a . Growth is o f prime importance to the pelagic

marine larva 1n enhancing predator avoidance and feeding success.

P ro te in deposit ion is undoubtedly manifested as la rva l growth, wh ile

high carbohydrate storage as hepatic glycogen poss ib ly has a sparing

e f fe c t on p ro te in catabolism; Cowey e t al . (1975) demonstrated such an

e f fe c t in a d u lt P. p la te ssa . The bulbous midgut perhaps serves a

fu n c t io n s im ila r to a morphological stomach in a llow ing the Ingestion

and storage of many food items, an adaptation which is advantageous to

a pelagic marine larva confronted w ith a patchy food supp ly .

The major changes In the development o f the a lim en ta ry canal and

associated s tru c tu re s are accompanied by changes in h a b i ta t and

35

feed ing regimen. The re tro rs e , c o n ic a l, la rv a l te e th are we’l l suited

fo r seizure o f pelagic copepod naupHi and copepodites while the

mucous ce lls o f the bucco-pharynx and fo regu t aid in Inges tion , The

movement of transform ing larvae in to es tua rine nursery areas (Chao and

Musick, 1977) Is concomitant w ith the d i f f e r e n t ia t io n of the

esophagus, stomach. In te s t in e , and rectum, as well as the appearance

o f the adult complement o f v I lH fo r tn jaw and m o la rlfo rm pharyngeal

te e th . This corresponds w ith a d ie t s h i f t to e p i- and Infaunal

in v e r te b ra te s . The fu n c t io n a l development o f ta s te buds must aid in

the se lec t ion o f these food items while the pharyngeal teeth and the

Increase in mucous c e l ls aid ir> m astication and in g e s t io n . Complete

development o f gas tr ic glands presumably a id w ith p re l im ina ry

p ro te o lys i s .

The sequence of the a lim entary canal development in spot is

s im i la r to tha t described fo r o ther marine f ish e s (Harder, 1960;

Fukusho, 1972; others below) w ith some notable s p e c ia l iz a t io n s . The

I n t o r t ion o f the a lim entary canal to form a loop i s common among

paracarthopteryg lan and acanthopteryglan la rvae (elopomorph and

protacanthopterygfan larvae have a s t ra ig h t g u t ) ; but i t Is not always

simultaneous with yo lk-sac or o i l -g lo b u le absorp tion (e .g . , the case

o f Trachurus symmetric us: The llacke r, 1970), Goblet or mucous ce lls

are present in the bucco-pharynx and foregut In spot as well as most

o th e r larvae (Tanaka 1969a, b; Tanaka 1971; Vu 1976), In co n tra s t,

O'Connell (1976} found them only a t the pharyngeal p o r t in Engraulls

mordax and Vu (1976) found goblet c e l ls in the mid- and hindgut o f

36

la rv a l D1centrarchus la b ra x . The occurrence o f two types o f mucous

c e l ls in the la rv a l fo re g u t concurs w i th R e lfe l and T r a v l l l (1977) who

described s ix d i f f e r e n t esophageal mucous c e l l types 1n a d u lts w i th

pa irs o f types associated w i th f iv e p a r t ic u la r fa m i l ie s o f f reshw a te r

te le o s ts . The apparent absence o f Type 2 mucous c e l ls 1n J u ve n ile

spot suggests th a t these c e l l s are e i t h e r t ra n s ie n t la r v a l s t ru c tu re s

o r s imply Type 1 c e l ls th a t have e xp e lle d t h e i r contents and are be ing

sloughed o f f . Taste buds appear 1n the bucco-pharynx o f la r v a l sp o t,

but are presumed not fu n c t io n a l u n t i l the e a r ly Ju v e n i le phase. In

many te le o s ts w ith demersal eggs, however, tas te buds are found 1n

yo lk -sac la rvae (Tanaka, 1969a). The c o n tro v e rs ia l r o d le t o r

pear-shaped c e l l (see reviews 1n Kapoor e t a l . , 1975; H arr ison and

Odense, 1978), a lso I d e n t i f ie d as the sporozoan Rhadbospora the lo h a n i

( e .g . , B a n n is te r , 1966), has been found In the a lim e n ta ry canal o f many

fishes In c lu d in g the young o f la b o ra to ry cu ltu re d Scophthalmus maximus

(Anderson and Roberts, 1976). Smallwood and Smallwood (1931) re fe r re d

to In te s t in a l secretory c e l l s , p o ss ib ly Id e n t ic a l to these pear-shaped

c e l ls (B a r r in g to n , 1957), in la rv a l Cyprinus c a rp io . A lk a l in e b lue 6B

is p a r t i c u la r l y useful in dem onstrating these p a ra s ite s (R u d de ll ,

personal conm unlcation), y e t they were n o t found 1n la rv a l o r e a r ly

Juven ile spo t.

The mechanism o f l i p i d and p ro te in d ig e s t io n in la r v a l and

ju v e n i le f is h e s may w arrent f u r t h e r research. The e p i t h e l i a l c e l ls o f

spo t 's la r v a l mid- and h in d g u t are s im i la r c y to lo g ic a l ly to those o f

s ix species described by Iwai (1969). He concluded th a t the vacuo la r

37

Inc lus ions of the midgut conta in l i p id s , resynthesized a f te r

hydro lys is and abso rp t ion , and th a t the g ranu la r in c lu s io n s o f the

hindgut contain p ro te in , the re s u l t o f p lnocy tos ls from the lumen.

Kawai and Ikeda (1973) and Tanaka et a l . (1972) reported weak pep tic

a c t i v i t y In larvae before the d i f f e r e n t ia t io n o f g a s tr ic glands while

t r y p t i c a c t i v i t y appeared s h o r t ly a f t e r hatch ing and increased

g radua lly w ith growth. The pancreas o f spot and other larvae (e .g .

Urneda and O ch ia i, 1973; O’C onne ll, 1976; Vu, 1976) contains

conspicuous zymogen granules presumably comprised o f t r y p s in , T ryp t ic

a c t i v i t y alone might not completely hydrolyze p ro te ins to amino a d d s

w ithout p re l im ina ry peptic d ig e s t io n , and Yamamoto (1966) and Stroband

(1977) have made specu la tive c o r re la t io n s between the lack o f peptic

d iges tion 1n stomachless adu lt te le o s ts and the incidence of

p ln o c y c to t ic protelnaceous, in t r a c e l l u la r in c lu s io n s . A cco rd ing ly ,

p inocytos is of p ro te in compensates fo r Incomplete p ro te o ly s is as might

be the case 1n la rv a l f ish e s ( Iw a i , 1969). While the lo ca t io n o f

absorption agrees w ith adu lt f ishes (Fange and Grove, 1979), one might

suspect complete p ro te o lys is a f te r the development o f g a s t r ic g lands.

Yamamoto (1966) has suggested th is based on the lack o f p ln o c y to t ic

invag inations and granular in t r a c e l lu la r Inc lus ions in a d u lt Salmo

1r id e u s . Yet, a c id o p h i l ic in t r a c e l l u la r in c lu s io n s occur in ea r ly

ju v e n i le spot even a f te r complete stomach d i f f e r e n t ia t io n , O'Connell

(1976) also reports f in d in g s th a t co n tras t Iw a i ’ s hypothesis.

The alim entary canal and associated s t ru c tu re s o f spo t, s im i la r

to tha t o f o ther marine te le o s ts , becomes fu n c t io n a l at f i r s t feeding

38

and change l i t t l e during the la rva l phase. Other than the expansion

o f a lim en ta ry canal surface area, there are no morphological or

h is to lo g ic a l changes th a t could e f fe c t major changes in d igestion o r

a s s im i la t io n p r io r to trans fo rm ation . The complete development o f a

stomach w ith functional g a s t r ic glands d u r in g trans fo rm a tion may

in f lu e n ce d igestion and a s s im ila t io n and is accompanied by changes in

h a b ita t and feeding regimen.

EXPERIMENTAL RADIOTRACER STUDIES OF THE

SHORT-TERM CARBON ASSIMILATION

INTRODUCTION

Our understanding of la rv a l f is h s ta rv a t io n and growth and the

re la t io n s h ip of these processes to stock recru itm ent has been re ce n t ly

advanced by the a p p l ic a t io n o f p h ys io log ica l energe tic p r in c ip le s . In

phys io log ica l energetic models developed by Iv le v (1939) and Wlnberg

(1956) and la te r re fined by Paloheimo and D ick ie (1966a,b) and Warren

and Davis (1967), a f i s h 's energy budget can be expressed as:

Qf = Q* + Q' + Q_ (1)

where = Q+ = energy of food consumed (m ges ta )

Q* - energy of waste products in feces and u r in e (egesta and

excre ta )

Q‘ - energy o f growth

Q_ = energy o f metabolism.

Rearranging g ives:

Q+ - Q* = Q' + q_ (2 )

or

bq+ = Q' + q__ (3)

(symbols fo l lo w in g Laurence, 1977). The c o e f f i c ie n t , b , represents

a s s im i la t io n and is gen e ra lly expressed as e i th e r a c o e f f ic ie n t , the

c o e f f ic ie n t o f u t i l i z a t i o n (Wlriberg, 1956); o r as a percentage,

absorption e f f ic ie n c y (Kapoor et a l . , 1975). A s s im i la t io n , defined as

40

41

th a t pa rt of the food In take which 1s u t i l i z e d (B re t t and Groves,

1979) 1s d i f f i c u l t to measure d i r e c t l y in aquatic animals la rgely

because i t is d i f f i c u l t to separate egesta from excreta (Johannes and

5atom1, 1967i Conover, 1978), The c o e f f i c ie n t o f u t i l i z a t i o n

represents the fra c t io n o f m etabolizable energy a v a i la b le a fte r losses

In feces (the resu lt o f Incomplete absorp tion from the alimentary

canal) and in urine or through the g i l l s ( la rg e ly the resu lt o f

deamination of p ro te in s ) . Absorption e f f i c ie n c y , the d iffe rence

between the matter-energy In Ingesta and egesta expressed as a

percentage, has been cus tom arily used synonomously w ith ass im ila t ion

e f f ic ie n c y . Such estimates might be b iased , however, i f some egesta

is confounded w ith exc re ta . The c o e f f ic ie n t o f u t i l i z a t i o n d i f fe rs

from absorption e f f ic ie n c y and is e as ie r to measure as defecatory and

excre to ry losses are combined. Because te le o s ts are amnonotelic

[F o rs te r and Goldstein, 1969), a s s im i la t io n should be the dominant

process encompassed by the c o e f f ic ie n t , e s p e c ia l ly 1f carbon 1s used

as a measure.

As o r ig in a l ly fo rm u la ted , these equations used energy units

( c a lo r ie s ) , but others have subs titu ted t o t a l wet o r dry weight,

weights of carbohydrates, fa ts , and p ro te in s , or weights of to ta l

carbon and nitrogen (see reviews in B re t t and Groves, 1979; Conover,

1978). H a tte r and energy are thus used in te rchangeab ly , though d ire c t

analogies are not always v a l id [ e .g . , W legert, 1968).

Important progress has been made in understanding the b io log ica l

processes represented by th e various terms o f ene rge tic models with

42

la rv a l f ish e s (Table 3 ) , but most o f these stud ies d e a lt w ith

freshwater species w ith precocious la rv a e . Furthermore, few attempts

have estimated energe tic parameters o f marine larvae or assessed

changes in these terms during la rv a l development; though i t 1s well

known th a t r e s p i ra t io n and e xc re t io n ra te s , and convers ion

e f f ic ie n c ie s can change w ith body weight (see review by B re t t and

Groves, 1979}, Laurence (1977), an excep tion , estimated ra te s o f

In g e s t io n , metabolism, and growth In i la r v a l marine f is h and used

these estimates to s imulate changes In a s s im i la t io n w ith development.

Rosenthal and Hempel (1970) and Nlshikawa (1975) suggested th a t

d ig e s t io n Improves w ith la rva l age as the re su lt o f the m orphological

development o f the a lim entary cana l, but the re 1s l i t t l e d i r e c t

evidence to support t h is .

This study was undertaken to estim ate short- te rm carbon

a s s im ila t io n in d is c re te age cohorts o f la rv a l spo t, Leiostomus

xanthurus, to assess changes in a s s im i la t io n , and to r e la te such

changes to development. The c o e f f ic ie n t o f u t i l i z a t i o n and abso rp tion

e f f ic ie n c y were used here in to assess carbon a s s im i la t io n .

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44

CONCEPTUAL FRAMEWORK

Radiotracer methodology, u t i l i z in g carbon-14 was used to compute

two parameters that were taken as r e la t i v e ind ices o f the c o e f f ic ie n t

of u t i l i z a t i o n and absorp tion e f f ic ie n c y . Carbon re te n t io n , the Index

o f the c o e f f ic ie n t o f u t i l i z a t i o n , was computed from the body

burden retained in la rvae and corrected f o r re s p i ra to ry loss a f te r

in g e s t io n and complete a lim entary canal evacuation. Carbon

abso rp t ion , the Index o f absorption e f f i c ie n c y , was computed from l^c

ingested and egested as p a r t ic u la te s . Computations fo llowed Sorokin

(1966):

C = a * r (4)where

C - to ta l carbon tra n s fe r re d (mg C)

a = re c ip ro c a l of the food substra te sp e c if ic

a c t i v i t y

r = r a d io a c t iv i t y in la rvae or p a r t ic u la te egesta

j DPM ,,^mgDW'

Carbon retention and carbon absorption were considered re la t ive

ind ices because use o f Equation (4) to estim ate carbon tra n s fe r has

some methodological problems. Carbon-14 can en te r several

phys io lo g ica l processes w+th d i f fe re n t ra tes w ith in an animal and be

lo s t through re sp ira t io n or excre tion . I f th is occurs during

45

Ingestion o r Alimentary canal evacuation, estimates o f to ta l carbon

ingested o r retained ca lcu la ted w ith Equation (4) w i l l be biased

(Conover and Francis, 1973). To co rrec t t h is b ias, Lampert (1977)

co llec ted respired ^CQg and added these DPM to the body burden o f h is

animals. This co rrec t ion 1s not e n t i r e ly adequate, however, because

the s p e c if ic a c t iv i t y o f carbon lo s t as CQg f lu c tu a te s during o f the

experiment: I t increases w ith time a f te r Ingestion as more

ass im ila ted . Because the sp e c if ic a c t i v i t y o f carbon lo s t 1s unknown,

the absolute amount of t o ta l carbon reta ined w ith in an animal cannot

be ca lcu la ted . Such ca lcu la t io n s requ ire multlcampartmerital analys is

(Robertson, 1956; A tk ins, 1969; Jacquez, 1972); but data necessary to

support these models are d i f f i c u l t to c o l le c t (Conover and Franc is ,

1973), Nonetheless, estimates of carbon ass im ila t io n using equations

s im ila r to Equation (4 ), compare well w ith c a lo r lm e tr lc estim ates,

esp e c ia l ly i f estimates are adjusted fo r re sp ira to ry loss (Lampert,

1977), Moreover such estimates o f carbon a s s im ila t io n are comparable

among age cohorts i f any b ias is constant.

Use o f Equation (4) to estimate and assess changes in these

ind ices is ju s t i f i e d by the fo l low ing assumptions, a l l addressed

herein: ( 1 ) the food substrate Is un iform ly labeled, i . e . , the

biochemical f ra c t io n a t io n o f 1s constant; ( 2 ) the s p e c if ic

a c t iv i t y o f the food substra te is constant during the feeding

In te rv a l ; (3 ) resp ira to ry loss does not occur during the feeding

In te rva l (no corrections f o r such bias could be made); (4) the

estimates o f carbon re ten tion and carbon absorption were taken at

complete alimentary canal evacuation; and (5) any biases are constant

among age cohorts.

46

MATERIALS AND METHODS

Experimental Animals

Spot larvae were obtained from labo ra to ry cu ltu red popu la t ions^ .

A r t i f i c i a l l y spawned and f e r t i l i z e d eggs were cu ltu red in tanks at

20 + 2“C and 30-35 ° /o o s a l i n i t y . This approximated cond it io n s

encountered in nature (see E ar ly L i f e H is to ry s e c t io n } . A f te r

hatching larvae were maintained at concentra tions o f < 1 0 /1 w ith a

photoperiod o f 12 h l i g h t and dark and fed se q u e n tia l ly w ith the

d ln o f la g e l la te , Qymnodlnlum splendens. the r o t i f e r Brachionus

p i i c a t i l 1 s . and w ild plankton a t d e n s it ie s o f 5 to 25 p a r t ic le s /m l .

Larvae were removed from the c u l tu re tanks the evening before an

experiment, placed 1n an experimental feeding ta n k , and fas ted

ove rn ig h t. S ta t is t ic s sumnarlzlng development o f la rva l spot age

cohorts are presented In Table 4. Condition fa c to r , o f te n used as an

in d ic a to r o f la rv a l growth and robustness (Hempel and B la x te r , 1961;

B la x te r , 1971), varied among co h o rts . The growth exponents o f weight

and length regressions o f larvae used 1n experiments (F igure 20) are

somewhat lower than those reported by Laurence (1979). Larvae were

ge n e ra lly hea lthy , however, and showed no h is to lo g ic a l signs

d ia g n o s t ic of s ta rv a t io n (Umeda and O ch la i, 1973; O 'C onne ll, 1976;

T h e ilacke r, 1978),

47

Table 4. Sumnary of s t a t i s t i c s ch a ra c te r iz in g development of d iscre te age cohorts o f Tarval Lelostomus xanthurus used 1n carbon a s s im ila t io n experim ents.

Age (days from hatch lng)

Mean Notochord or Standard Length

[mm)

Mean dry weight

(mg)C ond it i ona

Factor

7 1 .8 0 . 0 2 2 37,7

e 2.5 0.050 32.0

9 2,2 0.034 31,9

14 2 , 8 0.075 34,1

15 3*1 0.048 16.1

16 3,2 0.140 42.7

31 0 .6 1.330 46.3

34 6 . 8 1,505 47.9

43 1 1 .4 4.426 29.9

47 9.2 2,320 29.0

* ^ o n a it .o n fa c to r . ^ £ ^ , 3 x 10*

48

Figure 20* Weight and length regressions fo r Leiostomus xanthurus

larvae used In carbon a s s im ila t io n experiments. Upper

l in e fo r postf lex ion la rva e ; lower l in e fo r p re f le x io n

larvae (Ahlstrom, 1968).

20 00

I 0.00- 000- 6 00 -

2 0 0 -

( 00 -

w t => 0,00021

f = 089

n * 2 5 8

o 0 60- c0 40-

u* O 2 0 -»-a.o

0 I 0-o <:m3 -

0 0 4

w t = 0.00280 Lt 3 061 ri

r - 0,78

4 6 S 10o IOO

N O TO C H O ftD / S TANOAHD l E N O T H ( m r i )

49

Brachlonus p l lc a t l1 1 s was chosen as the experimental food

organism because i t is amenable to mass c u ltu re in the laboratory

(The l!acker and HcMaster* 1971J and 1s an adequate n u t r i t io n a l source

f o r la rva l fishes [May* 1971; Solangl and Ogle, 1977J. Prelim inary

experiments suggested tha t i t could be re a d i ly labeled w ith and

th a t la rva l spot consumed them from f i r s t feeding through

trans fo rm a tion ; though the behavioral mode of ingestion changed with

development* This allowed the use o f the same food substrate among

experiments with d i f f e r in g age cohorts o f la rvae . The major

n u t r i t io n a l components o f Brachlonus. cu ltu re d by the fo l low ing

p ro to c o l, were determined and values compared w ith the l i te ra tu re

(Table 5), Total p ro te in and l i p i d e x tra c t io n s fo llow ed Lowry et a l . ,

(1951), B ligh and Dyer (1959), and Hanson and 01 ley (1963),

Carbohydrates were extrac ted by p re c ip i ta t in g p ro te ins from Brachlonus

homogenates with t r i c h lo ro a c e t ic acid, d is s o lv in g l ip id s 1n ethanol,

and p re c ip i ta t in g carbohydrates w ith NaCl. E x trac t ions were assayed

fo l lo w in g Lowry et al ■* (1951), Barnes and Blackstock (1973), and a

m o d if ica t io n o f the anthrone method (S tr ic k la n d and Parsons* 1972).

Experimental Design

The experimental design Included la b e l in g o f Brachlonus and

m onitoring changes in i t s s p e c if ic a c t i v i t y , es tim ating carbon

ingested by feeding la rv a e , and es tim ating carbon re ta ined and lost

during alimentary canal evacuation [F igu re 21). Experiments were

conducted in 20 + LaC* 30-35 ° /oo s a l in i t y * 0.45 pm f i l t e r e d seawater

“ s t e r i l i z e d '1 by u l t r a v io le t i r r a d ia t io n . Experiments began at the

50

Table 5* Major n u t r i t io n a l components of Brachionus p l i c a t lH s expressed as a percentage o f to ta l dry weight.

ComponentPresent

StudyEhrlIch

(1972)Scott and

Baynes (1978)

Oglno and Watanabe

(1978)

Pro te in 38.9 53,6 b 58

L ip id 1.7 4.6a 1 1 . 2 b 10

Carbohydrate 1.9 8 ,6 6 ,Qb 6

Total carbon 41.7 33.1

Total nitrogen 9,0 0 .0

C/N ra t io 4.6 4.2

Ash 7,0 8 .6

a Determined as to ta l t r ig ly c e r id e s .

b Calculated as grand rtean of th e ir experimental values.

51

Figure 21. Experimental design o f carbon a s s im i la t io n w ith age

cohorts o f la r v a l Leiostomus xanthurus.

Ctifartlfw

Labeling

Bw chiortu t

Labeling

Sieving, Resuspension, and got clearance U 2 hl

Brachionus Samples for liqu id sc in tilla tion counting

Sieving and ResuSpenSion in feeding chamber

Feeding Chamber ( 14 C uptake)

1, 2 & 4 h

Transfer tu retention chambers

R etention Chambers ( 1 4 C retention)

Brachionus Samples tor liq u id sc in tilla tion counting

L a r v a e sam ples fo r l i q u i d sc in lilla tion counting

L a r v a e sam ples fo r l i q u i d2,4,0,7 Si 9 h sc in tilla tion counting

1 4

1 4 CO2 TfaP —

ICO 2 bubble trap

Fi ter

' 4 C0? tra P samples tor liqu id sc in tilla tion counting

1 4 COz bubble traps for liqu id scin tilfa tion counting

P O 1 4 C res idue for l i q u i d Scintillation counting

52

beginning o f the 12 h l ig h t photoperiod* Larvae were fed Brachtonus

at high dens it ies (250*10QQ/ml) to a llow the a lim en ta ry canal to f i l l

and provide p h ys io lo g ica lly equ iva lent ra tion s among age cohorts .

Brachlonus was labeled fo l lo w in g Sorokin (1966). Two 1 cu ltu re s

(ca, 3.0 x 10? c e l ls /m l) o f the u n ce llu la r algae Chlorel la s j). ( s t ra in

Va 52) were innoculated w ith 200 pel o f NaHl*C0 3 and cu ltu re d under

constant I l lu m in a t io n fo r 2 weeks to allow maximum popu la tion growth

and isotope f ix a t io n (ca. 8.0 x 10® c e l ls /m l; 3 .0 x 10*^ CPM/cel 1).

The cu ltu re medium (Dupuy et al . t 1977 ) was adjusted to pH 9 w ith

NaGH. Generally, Brachlonus was fed high concentralons (ca . 8 .0 x 10®

c e l ls /m l) o f Chlorel la fo r 5 days o r through 2 to 3 generations before

use in an experiment. P r io r to an experiment, Brachionus was Iso la ted

by gentle sieving on a 35 pm n ltex screen, washed w ith and resuspended

in seawater, and fasted fo r 12 h to c lear t h e i r guts of labeled algae.

To assess label u n ifo rm ity the d is t r ib u t io n o f in biochemical

frac t ions in Brachlonus was determined, Brachionus was labeled as

described and samples co llec ted a f te r 2, 4, 6 , and 8 days o f la b e l in g .

Total p ro te ins , l i p id s , and carbohydrates were ex trac ted from these

samples and the CPM of these ex trac ts were expressed as ra t io s o f

to ta l extracted CPtf,

The spec if ic a c t i v i t y o f Brachionus was monitored by ho ld ing 1 1

o f labeled Brachlonus 1n a beaker, sampling i n i t i a l l y , and 1, 2 , and

4 h th e re a f te r . T r ip l ic a te a l iq u o ts were f l l t e r d through ta red*

g la s s - f lb e r f i l t e r s , and washed tw ice with 0 .5 N HCl and d i s t i l l e d

53

water. They were then frozen , Tyoph ll ized , and stored w ith in a

des icca to r in a fre e ze r.

Carbon-14 uptake by la rvae was estimated by adding labeled

Brachlonus to a feeding tank and sampling larvae at 1 , 2 , and 4 h.

T r ip l i c a te samples were su ffocated on a n ite x screen, placed on ta re d ,

g la s s - f ib e r f i l t e r s , and trea ted as described fo r Brachionus samples.

Dead or moribund larvae were d iscarded.

Carbon-14 re te n t io n and loss by la rvae was estimated by

t ra n s fe r r in g larvae fed labeled Brachionus fo r 1 , 2 , and 4 h to a

series o f re te n t io n chambers (F igure 22) and sampling at 2, 4 , 6 , 7

and 9 h. Retention chambers were sealed w ith rubber stoppers f i t t e d

w ith ^ C 02 traps suspended in the atmosphere above the sea water.

Traps consisted o f a wel 1 w ith a 2.5 cm fo lded g la s s - f ib e r f i l t e r and

two in je c t io n ports (F igure 22}. Sampling involved In je c t in g 200 pi

o f phenethylamine in to the tra p well and a llow ing any respired ^ C 0 2

in the atmosphere above the sea water to react fo r 5 minutes; the

chamber was then opened and the la rvae sampled as described. A f te r

removing la rvae , the chambers were resealed and 0,3 ml o f concentrated

H2 5 O4 was in jec ted in to the w ater. P re lim ina ry experiments ind ica ted

tha t t h is a c id i f ic a t io n lowered the pH below 4 and thus released

H^C0 3 as gaseous to be co l le c te d in the suspended t ra p .

A c id i f ie d chambers then were reopened. The phenethylamine and f i l t e r

were removed, the trap well washed w ith 200 ul o f 95% methanol, and the

phenethylamine, f i l t e r , and washings placed 1n a s c i n t i l l a t i o n v ia l f o r

coun ting . To c o l le c t any remaining ^COg* the chambers were then

54

Figure 22, Schematic diagram of re te n t io n chambers used 1n carbon

a ss im ila t io n experiments w ith age cohorts o f la rv a l

Leiostomus xanthurus.

□ r . ,

P H E N E THYLAM IN E — I N J E C T I O NI N J E C T I O N

CQ, TRAP

55

f i t t e d to a bubble tra p co n s is t in g o f a series o f two s c in t i l l a t i o n

v ia ls con ta in ing 5 ml o f t ra p s o lu t io n (Woeller 1961) and bubbled w ith

COg-free a i r fo r 20 mln to recover any remaining (F igu re 23).

Following recovery o f the re te n t io n chamber sea water was

assayed fo r egested as p a r t ic u la te organic carbon (PO^C) by

f i l t e r i n g through glass f i b e r f i l t e r s . F i l t e r samples were washed as

described above.

Blanks, designed to assess contamination o f re te n t io n

chambers and la rvae , showed th a t contamination was un im portant.

Blanks consisted o f a se r ies o f re te n t io n chambers to which the

approximated maximum volume of water ca rr ie d over during the t ra n s fe r

o f larvae was added. Blanks were sampled fo r and PQl^C as

described. To assess uptake o f d isso lved and p a r t ic u la te from the

water, blank larvae were exposed f o r 1 h to feeding chamber water from

which labeled Brachionus had been seived at the end o f several

experiments and sampled as described. Analysis of blanks showed th a t

contamination o f samples was va r ia b le but s l ig h t and averaged 9.7%

(^C Q jj) , 8,7% (PO ^C), and 0.3% ( la rv a e ) . Because estim ates were

approximate and probably in f la te d , these e rro rs were ignored.

Sample Processing

Samples o f ly cp h lH ze d Brachlonus and la rvae were removed from

the f re e ze r , brought to room temperature w ith in the d e s icca to r ,

weighed to the nearest vg on an e le c troba lance , placed in a glass

s c in t i l l a t i o n v ia l , wetted w ith 300 y l o f d i s t i l l e d water, and

56

Figure 2 3 1 Schematic diagram o f bubb le -trap fo r recovery o f 14C0£ 1n

carbon a s s im ila t io n experiments w ith age cohorts o f la rv a l

Leiostomus xanthurus.

UJCOCO

tft _l

O 5I- Z 3 o

o 5l/» 3O — Z I-£ 5£L O< to £ *

O 0=

z “w 3H < LU XX O

o:u

o - |

ol/l

L

57

digested in 2 ml o f NCS (Nuclear Chicago t issue s o lu b i l i s e r ) f o r 1 to

Z days at 50°C. P a r t ic u la te organic carbon-14 samples were t re a te d

s im i la r ly , but were not weighed. A f te r d ig e s t io n samples were then

cooled to room temperature before adding s c i n t i l l a t i o n c o c k ta i l ( 6 g

POP. 0.05 g P0P0P/1 to lu e n e }. Brachlonus. la rvae , PQ^C, t ra p ,

and ^COg bubble-trap samples were combined w ith 15 ml o f

s c i n t i l l a t i o n c o c k ta i l , held a t room temperature in the dark f o r 36 h

to decay chemlluminescence (F igure 24}, and counted in a l iq u id

s c in t i l l a t i o n counter fo r the e n t i re pulse height spectrum a t 2%

e r ro r . Counts per minute (CPM) were corrected fo r counting e f f ic ie n c y

and converted to d is in te g ra t io n s per minute (DPM) using quench curves

(Figure 25). Mean counting e f f ic ie n c ie s were used to c o rre c t t ra p

( 8 6 .0%) and bubble tra p (85.0%} samples.

Trap samples (DPM) were summed and corrected fo r recovery

e f f ic ie n c y to give to ta l resp ired by la rva e . Recovery of to ta l

1*C0 2 # determined in p re l im in a ry experiments w ith known q u a n t i t ie s of

Na2 ^ 0 3 , was 854 e f f i c i e n t (Table 6 ) .

Notochord length was taken as the mean o f a subsample in

experiments w ith young age cohorts and as the mean of experimental

samples fo r o lder coho rts .

Considerable v a r i a b i l i t y was observed m the dry weight o f larvae

samples, esp e c ia l ly in samples of sm aller la rvae . In experiments w ith

larvae weighing <0 .1 Mg. the mean dry weight o f the age co h o rt ,

ca lcu la ted by w in so r iza t io n , was used in body-burden c a lc u la t io n s * In

other experiments in d iv id u a l sample weights were used w ith values

58

Figure 24, Decay o f chemil u mine scent a c t i v i t y in larvae and

Brachionus samples.

100

8 0 -

70

d

lo 60

<t -

U.O

I-z 40“kiJU<ELlIa.

LARVAE

BRACHIONUSIO -

20100 30~I 40

T f M E ( h)

59

Figure £5. Quench curves fo r la rvae and Braehi onus samples. Curves

f i t t e d by polynomial regress ion.

COUN

TING

EF

FICI

ENCY

(%

}

100

LARVAE/ £ F F = 91163 - 0 ISBWt - 0 002 Wl9 0

8 0

BRACHIONUS EFF=89 9 3 3 - 0 3 55W t - 0 0 0 2 W1

6 0

5 0 — O IO 3020 4 0 6050 70

DRY W E I G H T ( mg )

60

Table 6. Experimental recovery e f f ic ie n c y o f ^CQg. Summary o f resu lts w ith known q u a n t i t ie s o f Nag^COj.

DPM DPM DPM DPM Total RecoveryNa2 ^CG 3 re ten tion bubble tra p bubble tra p DPM e f f ic ie n c y

added chamber tra p 1 2 recovered {%)

138453,0 46544.7 51294.4 14231,4 112070.5 80.954781,6 50874.9 9414.2 115070.7 83.151466,7 53350.6 14717.5 119534.9 86.3

mean 83.4

69226.5 23068,2 29966.1 8350.5 61384.8 83.922386.9 29369.1 6303.a 5B059.8 S3.913063.8 32035.4 7936.9 53036.1 76,7

mean 83,1

34613.2 14162.9 13599.6 3193.9 30956.4 89.412053.1 11290.0 5899.9 26243,0 75.813620.1 11627,8 2818.7 27866,6 80.5

mean 81.9

6922.6 2154.9 3042.5 1074.9 6272,3 90.62132.1 4265.1 579.9 6977.1 100.02734.2 3186.2 347.2 6267,6 90.5

mean 93.7

Mean of a l l observations 05.1

61

pred ic ted front the weight and length regressions used fo r the few

missing o r suspect weights*

Mean to ta l carbon content o f Brachlonus* (41.7%, Table 4 )* used

to c a lc u la te s p e c i f ic a c t i v i t y (a o f Equation 4 ) , was determined by

gas chromatography.

Data Processing

Carbon-14 uptake, re ta in e d , and lo s t , as well as carbon re te n t io n

and absorp tion were re la te d to development by s te p w ise -m u lt ip le

reg ress ion . Percentage scores (transformed to the arcsine 1n degrees)

were regressed on time [ lo g transformed or polynomial fu n c t io n s ) , age,

leng th , and weight (measures of development}, and co n d it io n fa c to r .

In some cases the number o f observations was low compared w ith the

number o f independent v a r ia b le s , but the m atrix o f simple c o r re la t io n

c o e f f ic ie n ts allowed assessment o f assoc ia t ions .

A n c i l la ry Experiments

D u a l- tra ce r experiments were conducted to compare the behavior o f

an ass im ila ted and metabolized t ra c e r ( ^ C ) against tha t o f an

unassim ila ted and in e r t t ra c e r ( ^ C r ) . This design also allowed an

a lte rn a te c a lc u la t io n o f absorption e f f ic ie n c y , Carbon-14 labeled

Brachionus was suspended in 1 1 o f seawater con ta in ing ca, 200 y d

5^CrCl3 and were allowed to f i x S^Cr f o r 12 h. Experiments were

conducted fo l lo w in g general p ro to c o l. Samples were counted by the

Isotope exc lus ion method (Kobayashi and Maudsley, 1970; Sheppard and

Marlow, 1971), Quench curves fo r and 51[> are shown in F igure 26.

62

Figure 26. Quench curves fo r larvae and Brachlonus samples from dual

label experiments* Curves f i t t e d by eye*

COUN

TING

EF

FIC

IENC

Y (%

)

2-0 n

1.5 -

GO

40 -

LAHVAE30-

Channel

20 ■

Channel I =13.77* Channel 2 = 0.07 7(

4030 3020 0 2010 40

DRY WEIGHT (mg)

63

Results were used to c a lc u la te carbon re te n t io n , carbon abso rp t ion ,

and absorption e f f ic ie n c y by the In d ic a to r - r a t io method (Calow and

F le tche r, 1972; Wlghtman, 1975; Caimen, 1977).

Several expertments were conducted with a reduced, more

environm enta lly r e a l i s t i c food concentra tion , because d ig e s t io n and

a ss im ila t io n might p o ss ib ly be less e f f i c ie n t under maximal feed ing .

Experiments were conducted as described above w ith several age cohorts

using a Brachionus concen tra tion of 1-10 r o t i f e r s / 1 .

A comparison was made between the number o f r o t i f e r s Ingested

(ca lcu la ted using the to ta l carbon per dry weight o f Brachionus.

values o f the weight o f an in d iv id u a l r o t i f e r , and values o f carbon

ingested) and v isual counts o f the number of r o t i f e r s in a lim entary

canals excised from la rv a e . Values used fo r the dry weight o f an

in d iv id u a l r o t i f e r were 0,16 yg (The ilacker and McMaster, 1971) and

0.75 yg per r o t i f e r (S co tt and Baynes, 1978). The number of r o t i f e r s

ingested per larva was estimated as fo l lo w s :

mg C Ingested/la rvae_____mg C/mg DW r o t i fe r s

Number o f r o t i f e r s =(c a lc u la te d by Equation [ 4 ] )

mg D lrf?rotifer

These estimates were compared w ith counts of r o t i f e r s excised from a

subsample o f larvae thus serv ing as a check o f experimental accuracy.

64

RESULTS

Carbon-14 Uptake. Retention, and lo ss

Specific a c t i v i t y estim ates o f Brachionus were c o n s is te n t ly high

among experiments and h ig h ly v a r ia b le both w ith in and among

experiments (Table 6 ) . Regressions o f Brachionus body burden on time

indicated that the slope was never s ig n i f i c a n t ly d i f fe re n t from zero

(Table 7), Thus, the s p e c if ic a c t i v i t y o f Brachionus was considered

constant; the mean of a l l Brachlonus s p e c i f ic a c t iv i t y estim ates

w ith in each experiment was used in c a lc u la t io n s of carbon Ingested,

re ta ined, and egested,

Experiments to assess the biochemical f ra c t io n a t io n o f in

Brachlonus ind ica te d tha t maximum la be l f i x a t io n is complete w ith in

2 days of la b e lin g and tha t the f r a c t io n o f in to ta l ex trac ted

p ro te in , l ip id s , and carbohydrates remains constant th e re a f te r

(Figure 21 ), Each experiment u t i l i z e d Brachionus labeled f o r 4 to

11 days (a t le a s t 3 population d o u b lin g s ) , thus the food su b s tra te was

considered un ifo rm ly labe led .

The magnitude o f uptake by la rva l spot varied among

experiments due to d if fe re n ce s in Brachionus sp e c if ic a c t i v i t y , but

the pattern of uptake showed some trends among age cohorts

(Figure 28), M u l t ip le regression o f uptake (expressed as a

Tabl

e 7.

Reg

ress

ions

of

Brac

hion

us

body

bu

rden

(D

PM/m

g DW

) re

late

d to

time

(min

) in

carb

on

assi

mila

tion

expe

rimen

ts

with

di

scre

te

age

coho

rts

of la

rval

Le

iost

omus

xa

nthu

rus.

65

4 O QQ CM O L fl a r - l in CM- 0 —f vo a v CM r--. O r— O in

* p—■+ r - r -- CO O VO O i VO Oin # a * -A ■A r *■ A B

r—1 CO CM r-«. M" <r» 00 $ fO mm in CM r*-

r-1—• i n

<b■at-3 —■c a 3dn cn O E

Ec a . in a

o t vi <1> -C «TCft O t3<£ <_)■—'

o CM 8O v ** =0

CQ lc V0 CM m tT iso > DO m n *

A A m a A A n Ao €0 o i—1 D oa t n 03 r - . CM ** U 3 rOCM CM U T

O CTv 1—* CM 03

1j0 o o O 3 ;a VO in o o m

Lu 4 A » •* 4 4

a o a o o o

o Q o

VO »“H r-—

■c B «■ A A *1

1—1 r~l p----1 •—P 1-----P

C +J□ c

flj

*J <- CO CM «—4 f—1 P—* 1—P

« (J o m CM O o CM4 * ■ * ■ft A

di n- o o o o o at- Vt-

)- a*o oO V_1

£i'4— I %t— £ I—

1— •r—CO I— 1—

o Oi o in

<M cn to LO o Ol

c r\j *- *3- VO <r *

a 4 o■ CM CM Ln

■ i— ■—| r * 4 rf

-U CM CM *T t-H 1-H A

«3 1 1 i + + i

O"UJ CJ O' O CO i—p

VO CTi CM o (M

c rn cn CM ro

o *■ m » 4i *■ B

CM ro Q O m

iA o CM o> VO 10lfl <M n ■-M

a?t- II ii II ■I II N

O1&> 3 3 3 3Q£ □ O tl a Cl Q

i 1 C "£ w D1

Eat

E

3 : TZ £ £a .a £ a.

O % o.a &

O ')O'I--,

ALO

U0<SI

oo

VC

voo

4CM

CO■

roi

3

esx□

CMi/1<M

LflNNJ

N

ffiinco

Cvir*v.

tjiE

&

cO

66

Figure 27. Biochemical f ra c t io n a t io n o f in Brachlonus during

label1ng,

Ra

tio

POPULATION DOUBLINGS

1,5 35 4.5 9.5

CARBOHYDRATEOOl

7A5 5 aoT I M E ( 4 4 y t }

67

Figure Z8. Carbon-14 uptake by la rv a l Lelostomus xanthurus in carbon

a s s im i la t io n experiments* Values expressed as a

percentage o f body burden (DPM/mg DW) a t 1 b feed ing -

% BO

DY

BURD

EN

JDP

M/m

gDW

)

MC UPTAKE

3 0 0

200

A P -

100 -

i— t f0 1 2 3 4

TIME thrsl

68

percentage o f the 1 h estim ate) on log t im e, length , M ig h t , and age

Ind icated th a t uptake was re la te d to time and length (P<0,10;

Table 8 ) . Among a l l age cohorts uptake was rapid fo r the f i r s t hour

and then slowed (F igu re Z8); the marked in f le c t io n in uptake at 60 mln

o f feeding 1s probably due to the onset o f egestion, Visual

observations o f feeding behavior and defecation agreed w ith th is

p a t te rn . Because estimates o f ing e s t io n based on uptake would be

confounded I f egestlon has occurred, 14C re ten tion and loss was

followed fo r the 1 h feeding se r ie s o n ly .

Carbon-14 re ta ined and lo s t a f te r 1 h of feeding showed a

cons is ten t p a tte rn w ith t im e, bu t no trends with development (F igure

£9). Pooled data fo r a l l experiments are shown In Figure 30.

M u lt ip le regressions o f re ta in e d , resp ired , and lo s t as

p a r t ic u la te s (expressed as a percentage o f the 1 h estim ate) on t im e ,

time squared, le n g th , weight, and age Indicated tha t these processes

were re la ted to time squared and tim e, but not to any measure o f

development (P>.QQ1; Tables 9, 10, and 11). Though l^C loss is a

composite of losses v ia egestlon , re s p ira t io n , and e x c re t io n , the

pa tte rn of re te n t io n and PD^C production ind ica ted th a t egestlon

dominates th is loss during the e a r ly minutes fo llow ing t ra n s fe r o f

larvae to a food fre e environment. Egestlon was complete by 6 or 7 h

a f te r feeding stopped (F igure 3 0 ). A percentage o f new ly-ass im ila ted

entered q u ic k ly in to sh o rt- te rm , metabolic processes and was lo s t

as ^C02* I t appears from these data tha t re sp ira to ry loss o f the

labe l began as e a r ly as 1 h fo l lo w in g inges tion ; thus any bias of

carbon Ingestion c a lc u la t io n s due to re sp ira to ry loss o f labe l during

69

Table 8 . Stepwise m u lt ip le regression tab le of l^ c uptake (1) re la te d to time (ra in), age (days), length (ran), and weight (mg) In carbon a ss im ila t io n experiments with d is c re te age cohorts of la rva l Leiostonus xanthurus.

Regression Equation S1mp1 e r d f P a r t ia l F M u l t ip le F

Arcsine Percent Uptake =

+9.620 + 36.212 Lg Time 0.84 1,41 94.81 (<.001)

+0,074 + 37,126 Lg Time 102,05 « . 0 0 l )

+ 1,812 Length 0.03 2,40 2.93 (<.1Q) 51.09 (<.001)

+0,621 + 37,179 Lg Time 99.66 (<.001)

+ 2,950 Length 0.43 (<*75)

+ 0.252 Age 0.04 3,39 0,07 (>■75)

ChCMtfO (< .001)

-14.481 + 37.259 Lg Time 98.36 (<.001)

+ 14,202 Length 0.56 (<■75}

- 1,469 Age 0.44 (>.7&)

- 14,176 Weight 0.02 4,38 0.37 0 . 7 6 ) 24.66 (<.001)

70

Table 9* Stepwise m u l t ip le regression ta b le o f re ta ined ( * ) re la ted to time (m ln ), age (days), leng th {mm), and weight (mg) In carbon a s s im i la t io n experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus.

Regression Equation Simple r d f P a r t ia l F M u lt ip le F

Arcsine Percent Retained -

-31.000 + 1.202 Time -0.35 65,60{<.001)

- 0.004 T ime^ -0.30 63,43(<.001)

- 0.000 Time^ -0.33 3,64 59,51(< .001) 27.25{< ,001)

-32.704 + 1.200 Time 65.59(< .001)

- 0.417 Time^ 63,43(< .001)

+ 0.394 Time^ 59,4 8 (< .001)

+ i .746 Weight 0,11 4,63 1.32(< .75) 20.87{<.001)

-25.303 +■ 1,198 Time 65.30(< .001)

- 0.004 Time^ 63.15{<.001)

+ 0.000 T ime^ 59.26(< .001)

+ 7.528 Weight 1.43(< .25)

+ 2,645 Length 0.08 5,62 0,89 (< . 50) 16,8 4 {<.001)

-10.560 + 1,202 Time 65,74{< .001)

+ 0,004 Time^ 63,44(< .001)

+ 0,000 Hme^ 59.84(< ,001)

+21,910 Weight 1,8 7 {< .10)

+15,067 Length 1,34(< .25)

+ 1,446 Age 0.06 6,61 0,95(< .50) 14 .ia (< .001 )

71

Table 10, Stepwise m u lt ip le regress ion tab le o f resp ired ( * )re la ted to time (m1n), age (days), length (ran), and weight (mg) in carbon a s s im i la t io n experiments with d is c re te age cohorts o f la rva l Lelostomus xanthurus.

Regression Equation S im pler d f P a r t ia l F M u lt ip le F

Arcsine Percent Respired *

m ,Z a i-7 Z .Q 3 6 Log Time -0 .5 8 1 ,6 8 35.GA(<.001)

F - Level is in s u f f i c ie n t fo r f u r t h e r c a lc u la t io n .

72

Table 11. Stepwise m u lt ip le regression tab le o f P014C (%) re la te d to time (m1n)t age (days ), length (nm), and weight (mg) in carbon ass im ila t ion experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus.

Regresson Equation Simple r df P a r t ia l F M u lt ip le F

Arcsine Percent P014C -

216,ASS - 74,176 Lg Time -0.71 1,40 39,63(< .001)

211,882 - 74,176 Lg Time 39 ,42(< .00U

+ 0,190 Age 0,10 2,39 0 .7 8 (< .50) 20,09(<,001)

212,284 - 74.176 Lg Time 38.71(< .001)

- 0.612 Age Q.59(< .75)

- 1.962 Length 0.08 3,38 0.30(> ,75) 13 .26 (< .001)

247.214 - 74.176 Lg Time 39 ,20 (< ,001)

3.286 Age l . 9 8 « . 2 5 )

- 27.628 Length 1,67(<,25)

32.436 Height 0.07 4,37 1 ,4 6 (<,25) 10.44(<.001)

73

Figure £9. Carbon-14 re ta in e d , re sp ire d , and lo s t as PG^C by la rva l

Lelostomus xanthurus 1n carbon a s s im i la t io n experiments.

Values expressed as percentage o f body burden (DPM/mg DW)

at 1 h feed ing.

Si.Q

irn>-aoA

4UuItJa

^ 7" C R E TA IM E O

vfc? -<i

C RESPIRED

5 0

100 —I

5 0

0

—

o ID

/

/

--■■ JE?

100

50

0 ✓ * + ? f i j * i^r -t?o s >o

T IM E 1 hr -s 1

- - - J ^

74

Figure 30. Carbori'14 re ta in e d , resp ired , and lo s t as PO^C by la rv a l

Lelostomus xanthurus In carbon a s s im ila t io n experiments.

Data pooled from a l l experiments; brackets in d ica te 95%

confidence in te rv a ls . Values expressed as a percentage of

body burden (DPM/rng DW) a t 1 h feed ing .

100

90

«0

TO

RETAINED

' 50 a

■a.a

xUJo

2.0IE301

10-

t-xUJ

aUJ

£0H

(0

- "T — T t i 1-----1-----1-----r

RE SPIRED

/ 1 !■ 1T 1-----1-----T-----1-----1----- 1---- 1-----1-----

P O C

10 -

t p 1---------------1----------------r-----------t --------------1---------------1-£ 3 4 5 6 7 9 90 I

T IM E I ft J

75

feeding is probably m in im al. The ra te of p roduction , i n i t i a l l y

very rap id* slows a f te r several hours (Figure 30). C a lcu la t io n s o f

carbon re ta ined and egested were made by pooling data from the 6 and

7 h re te n t io n in te rv a ls .

Carbon A s s im i la t io n and i t s R e la tion to Development

Carbon re te n t io n (Table 12)t the r e la t iv e Index o f the

c o e f f ic ie n t o f u t i l i z a t i o n * was not re la ted to development but was

negative ly re la te d to co n d it io n fa c to r (F igure 3L). Cond ition fa c to r

showed the highest c o r re la t io n fo llowed by weight In m u lt ip le

regression o f re te n t io n on leng th , weight, age, and c o n d it io n fa c to r .

Although the p a r t ia l regression c o e f f ic ie n ts were not s ig n i f ic a n t ,

carbon re te n t io n decreases sharply w ith increasing co n d it io n fa c to r

(the simple regression o f carbon re te n t io n on cond it ion fa c to r

suggested th a t the p r o b a b i l i t y o f a zero slope is <0.25) (Table 13).

Carbon absorption (Table 12), the index of absorption e f f ic ie n c y ,

was nega tive ly re la te d to age as well as condition fa c to r (F igure 32).

M u lt ip le regression ind ica ted that the p a r t ia l regressions coe f­

f ic ie n ts o f age and con d it io n fa c to r were both s ig n i f ic a n t (P<0*10;

Table 14).

No age-re la ted biases in the Indices of a ss im ila t io n were evident

o r . I f present, they were constant. Carbon re ten tion was corrected

fo r re sp ira to ry loss o f but th is co rre c t io n might I t s e l f be

biased. The percentage of ingested l^C i ost through re s p i ra t io n was

not associated w ith age and on ly weakly associated w ith co n d it ion

Tabl

e 12

. Su

mmar

y of

valu

es

used

to

asse

ss

carb

on

assi

mila

tion

in ag

e co

hort

s of

larv

al

Leio

stom

us

xant

huru

s

76

E +*□ C lja l h °4 i/l LJ -Q <

OC -P - 2 * *

I s/4 /Jt_] 4i O '

v cn o itr t .|— /g 0> 0 / > cr> 5 -----

LlJ CO

fO L * — O T ! V f O t - 1_

5 2L

'S +.>C £

- I - CTi 4Jrc -r- lO+j gi >CO « g i

c t11""—■O T J 4 - £ } Q t - 4.

■8— -CLO Pt (1)a j —

I—N

C u 5' O T5 4t- 5-

3 8 .

rO>U ■ <g gi

t / lSi ^£T /g <t -O

CM,

<71Ol

f * 'i n

ooC5

o* rC 3

CM ■--1P’Lo

00

m

r~-LfJ &

C\J

i s

S 2o o

tO lT)

CO cr>

a i m

i fooa

c n

oo

CMar oa

LT>

a i*

CM00

CM CO» * »

P i O i1X1 m

ao

nc ooo

CO

t o

a iooo

CO

oo

CM»

■r-,4 0

CT)

S 3

CO

o■

o

o*r vo—t oo o

O cn to * n o a o

n -■no-

r o■

CXt

r--

r u

r ooo

IDIDoo

r*^

Mean

42

.9

37,6

77

Figure 31* Carbon re te n t io n , the Index o f the c o e f f ic ie n t of

u t i l i z a t i o n , re la te d to development (A} and c o n d it io n

fa c to r (B) o f la rv a l Lelostomus xanthurus age cohorts*

Regressions ca lcu la te d w ith a rcs ine ( In degrees)

transformed percentages.

H Q 1 1 U 3 H H N Q S IJV3 iN 3 3 U 3 cJ 3 N IS 3U Y

otty

o&-LI1

o

O

D

QIM

M0I1M313U NOflMVD 1N33U34

AGE

(dO

Ki)

C

ON

DIT

ION

FA

CTO

R

76

Table 13. Stepwise m u lt ip le regression ta b le o f carbon re te n t io n (1 ) , the Index of the c o e f f ic ie n t o f u t i l i z a t i o n , re la te d to age (days), length (mm), weight (mg), and co n d it io n fa c to r o f Lelostomus xanthurus la rv a l age coho rts .

Regression Equation Simple r d f P a r t ia l F M u lt ip le F

Arcsine Percent Carbon Retention =■

61.666 - 0.608 Condition Factor -0.54 1*7 2 ,89(< ,25)

60.003 - 0,609 Condition Factor 2 .6 4 (< ,25)

+ 1,555 Weight 0.21 2,6 D,40(<.75) 1.52(< .50)

71.357 - 0.504 Cond it ion Factor 1 ,9 2 (<.25}

+13.560 Weight 2 ,02 (< ,25)

- 5.421 Length 0,07 3.5 1 ,68(< ,50) 1.69(< .60}

83.970 - 0.525 Condition Factor 1,7 4 (<,50)

+24.979 Weight 0 ,9 5 (<.50 J

-15.437 Length 0,54(<.75 J

- 1.186 Age 0,05 4,4 0 ,2 4 0 .7 5 ) 1 .13(<.50)

79

Figure 32. Carbon absorp tion , the Index o f absorp tion e f f ic ie n c y ,

re la te d to development (A) and co n d it io n fa c to r (B) o f

la rv a l Lelostomus xanthurus age co ho rts . Regressions

ca lcu la te d w ith a rcs ine ( in degrees) transformed

percentages.

I DO

NOuauossv NOayu'3 ±N33N3d inisdhv

OO O O O O D O O* * * s is in <7 t f j t j _

o

m

- o

K)

NQliBBOSBV NOBUVO lN33U3d

AGE

tdiiT

*)

CO

ND

ITIO

N

80

Table 14. Stepwise m u lt ip le regression tab le o f carbon absorp tion , the Index o f absorption e f f ic ie n c y , re la ted to age (days), length (urn), weight (mg), and cond it ion fa c to r o f Lelostomus xanthurus la rva l age cohorts .

Regression Equation Simple r d f P a r t ia l F M u l t ip le F

Arcsine Carbon Absorption -

87,542 - 0-648 Age -0,79 1,4 6,57{< .10}

110,530 - 0,596 Age 16,41(<,05)

- 0-693 Condition Factor -0,62 2,3 8,96(<.1C) 14.31{< ,05}

108,894 - 0,212 Age 0 ,8 6 {< .75)

- 0-762 Condition Factor 19,34(<0-5)

- 4,467 Weight -0.75 3,2 3.60{<,25) *

79,086 - 2.803 Age 1.64(< ,50)

- 0.737 Condition Factor 4 .21{<-25)

-31 ,795 weight 1,9 0 (< .25)

+22.958 Length -0.78 4,1 1.47{<-50) 3,19(<-25)

* F - leve l in s u f f ic ie n t fo r fu r th e r c a lc u la t io n .

61

fa c to r (F igure 33); thus any bias 1n carbon re te n t io n was considered

constant. An age-re la ted bias might be Inherent in re la t io n s h ip s

between carbon a s s im i la t io n Indices and co n d it io n fa c to r fo r the

cond it ion fa c to r o f la rv a l f ishes genera lly increases w ith length

a f te r yo lk -sa c absorp tion (e .g . , Sameoto, 1972; Sekiguchi, 1975;

E h r l ich e t al . , 1976). Condition fa c to r o f the age cohorts used,

however, showed no assoc ia tion w ith age (F igure 34) and the

c o r re la t io n o f co n d it io n fa c to r w ith length was even weaker.

A n c i l la ry Experiments

The p a tte rn o f 51Cr re ta ined and lo s t was s im i la r to th a t o f

w ith some exceptions (F igu re 35). A residue o f 51Cr remained w ith in

the la rva l a lim entary canal longer than the experiment. Taken as a

whole, however, these re s u l ts support the in fe rence th a t egestlon is

complete w i th in G or 7 h fo l lo w in g feed ing . Carbon abso rp t ion ,

ca lcu la ted by d i f fe re n c e according to general experimental procedures,

was 99% (15 day age c o h o rt , Table 12); a s im i la r value was ca lcu la ted

using the in d ic a to r - r a t io method (Table 15).

The pa tte rn o f re ta ined and lo s t In experiments w ith reduced

Brachionus concentra tions was s im ila r to the main experiments w ith

high concentra tions and maximal feeding (F igu re 36), but estimates of

carbon re te n t io n were h igher (Table 16), Results o f these experiments

were h igh ly va r ia b le and on ly two o f the s ix were successful .

V a r ia b i l i t y was la rg e ly due to va r iab le feeding success. The mean

c o e f f ic ie n t o f v a r ia t io n o f uptake samples was 78,0 compared to a

mean o f 62.8 fo r experiments w ith high Brachionus concen tra t ions .

82

Figure 33. Percent resp ired re la ted to development (A) and

c o n d it io n fa c to r (B) o f la rva l Lei ostonus xanthurus age

cohorts . Regressions ca lcu lated w ith a rcs ine {1n degrees)

transformed percentages.

30

£□L il

O.Vt

zo<D<E

20 -

10-

r ? 0 OS

~i— 10

T ~l—3020

AGE < d o r i )

—|— 40

30

20

100

50

uiyKUJa_

0.46

a so

3 0

I30

■ r ' 40

2 0

100

50CONDITION FACTOR

ARC

SINE

PE

RC

ENT

CARB

ON

- 14

RE

SF

iRE

C(%

|

83

Figure 34. C ond it ion fa c to r re la ted to age (A) and notochord or

standard length (B) of la rv a l Lelostomus xanthurus age

cohorts used 1n carbon a s s im i la t io n experiments. Lines

(B) in d ic a te p red ic ted c o n d it io n fa c to r from weight and

leng th regress ions.

COND

ITIO

N fA

CTO

R

20

10-

20 6040

20

r = 0 .0 610 -

2 ao 4 G 12

NOTOCHORD f STANDARD LENGTH (mm)

84

Figure 35, Carbon-14 and 5lC r re ta ined and lo s t by la rva l Lelostomus

xanthurus 1n a d u a l- t ra c e r experiment w ith 15 day age

co h o rt. Values expressed as a percentage o f body burden

(DPM/mg DW) at 1 h feeding.

100

* ,

roo

f i

*

aL I 3a 3

u£inuft

t “c40 -F -z X

U J U Ju ut t 1CUJ L*la . a .

fi

a**

ii

I

r

<vF s

- rj

oO) o<0

0■+

On oM

<MQ Au / W^ Q I N30U0B A008 1W30M3J

Table

15

. Su

mnar

y of

dual

-tra

cer

valu

es

used

to

calc

ulat

ed

abso

rptio

n ef

ficie

ncy

by th

e in

dic

ato

r-ra

tio

met

hod.

65

ca > >

f ( J+ J c ID 41

in 4 - v t U J

<

COo.

4 0CT»

4 - U1 O A )

q k J-P - 4 — + J 'v .

o■4-

<SI0

* 1 P

H— 10 □ L -

d j O LJ

'P - iQ+ J U- IO

QC

O 4 0 . .

i f fOCVI CVJ

L .o

LD

4-o -— -

c a tb

* o o > a - £

CO t :

■ g *3CO

r v i—- U > 4 0 4 0 VO 4 0 luO U 1 4 0 O O rO rO*—i p t

oT f

4 -o •—

C Q <U

D D )* - E

D -

f stO

r - .CO o iS o r o ^

s ^

y>a *

S QJU . Ll .

86

Figure 36. Carbon-14 re ta ined and lo s t by la rv a l Lelostomus xanthurus

1n carbon a ss im ila t io n experiments w ith reduced Brachionus

p l l c a t H ls concentra tions (5-10/ml )* Values expressed as

a percentage o f body burden {DPM/mg DW) a t 1 h feeding.

BODY

BU

RDEN

ID

PM

/mq

D

W)

,4C RETAINED

7—r- f t r r y

IOO -I

C R E S P I R E D

J &

87

Table 16, Summary o f values used to asses carbon a s s im i la t io n 1n age cohorts o f la rv a l Leiostomus xanthurus fed w ith reduced Brachlonus concentra tions.

Age{days)

Carbon Ingested per dry weight

of la rvae [mg)

Carbon Retained per dry weight

o f larvae (mg)

Carbon Retenti on

<*>

9 0.000903 0.000556 61*5

15 0*002578 0.002222 86.2

Mean 73.8

88

The q u a n t ity o f carbon fngested per dry weight o f larvae 1n

experiments w ith reduced Brachlonus concentrations compared w ith

carbon Ingested a t high concentrations was ge n e ra lly an order o f

magnitude lower (Figure 37).

14The nuirtber o f r o t i f e r s ingested by a la r v a * ca lcu la ted from C

uptake data* Increased e x p o n t ta l ly w ith age and compares well w ith

courts o f the number of r o t i f e r s excised from a lim entary canals

(F igure 38). The count o f r o t i f e r s th a t f a l l s outs ide o f the range

o f ca lcu la ted values was based on a s in g le specimen ra th e r than a

mean o f three counts. The method o f es tim ating Ingestion used

herein Is v a l id -

89

Figure 37, Carbon ingested per dry weight of la rv a l Leiostomus

xanthurus fed high (25Q-lQDG/m1) and low (5 -10 /m l)

concentra tions of Brachlonus p l i c a t i l i s *

CARB

ON

INGE

STED

PE

R W

EIGH

T OF

LA

RVAE

[m

g D

W}

LQ* IO_l - i

IJOm I0"2 -

LOm I0"3

* 250-1000/mf

a M O / m l

LQ* I0-4 T "

10 20“T"30 40

n50

AGE (Dayi)

90

Figure 38. The nunker o f r o t i f e r s ( Brachlonus p l i c a t i l i s ) ingested

re la ted to development o f la rv a l Leiostotnus xan thurus . A.

Regression ca lcu la ted using dry weight value of The ilacker

and McMaster (1971)* B. Regression ca lcu la te d using

maximum dry weight value o f Scott and Baynes (1979).

Stars in d ica te mean number o f r o t i f e r s counted in

a lim entary canals excised from la rva e .

NUM

BER

ot R

OTI

FER

S IN

GE

STE

D

1 0 0 0 - i9 0 0 - 8 00 *

TOO - GOO -

500 -

300

ZOO

Intarcept = 5-6

8 8 :80 - 70 - 60 - 50 -40 -

30 -

2 0 -

r * 0.89

LQ0IO Number of Rotifers Ingested -

LotJto Intercept + 0.048 Log(0 Age

GO4 0 so10 30oAGE ( day* }

91

DISCISSION

C o n f l ic t in g evidence ex is ts concerning the presence o f carbon

pools w ith rapid tu rn o v e r rates 1n a q u a t ic . po1ki1otherm ic animals

[see review in Conover and Franc is , 1973; also S t r e K , 1975; Lampert,

1977; U e tz e l, 1976), In la rva l spot, carbon is a ss im ila te d ra p id ly

a f t e r ingestion and a t least some o f i t enters a s h o r t - te rm , m etabolic

poo l. Sorokin and Panov (1966) reported rapid carbon less through

re s p ira t io n fo l lo w in g Ingestion by la rv a l freshwater bream, Abramjs

brama. Larval spot evidenced the same pa tte rn .

Mean carbon re te n t io n , 42.9%, was somewhat lower than the

expected c o e f f ic ie n t o f u t i l i z a t io n fo r ju ve n ile and a d u lt f is h e s ,

about 70* (Ware, 1975), This d if fe re n c e may be due to a rea l s h i f t 1n

the c o e f f ic ie n t of u t i l i z a t i o n a f te r transfo rm ation o r poss ib le b ias

associated w ith t ra c e r methodology. Carbon re te n t io n , as estimated

he re in , is a r e la t iv e index of the c o e f f ic ie n t of u t i l i z a t i o n because

of the bias associated w ith re s p ira to ry loss of newly ass im ila ted

carbon. A l te r n a t iv e ly , carbon re te n t io n could be reduced at high

feeding le v e ls . The re s u l ts o f experiments with reduced Brachlonus

concentra tions were h ig h ly va r ia b le , but values fo r carbon re te n t io n

[7 3 ,6 ) were close to th a t expected fo r adu lt f ishes* Using a

ra d io tra c e r design and envlronm enta lly r e a l i s t i c food c o n ce n tra t io n s .

92

Sorokin and Panov (1966) found close agreement between carbon

re ten tion and carbon absorp tion .

Mean carbon absorp tion , 87 .6S, approximated a s s im ila t io n

e f f ic ie n c ie s reported fo r ju v e n i le and a d u lt f ishes (see review in

Conover, 1978; B re tt and Groves, 1979) as we ll as those ca lcu la te d f o r

la rv a l f ishes (Table 3). Brachlonus lo r lc a s conta in e i th e r

in d ig e s t ib le c h t t ln (Danner, 1966) o r s c le ro p ro te in (Hyman, 1951) and

comprise a la rge port ion o f the p a r t ic u la te egesta o f la rvae. Carbon

absorption may improve fo l lo w in g transfo rm ation w ith the d ie ta ry

s h i f t th a t Includes more s o f t bodied Inve rteb ra tes (P a rt I ) .

A ss im ila t io n , as assessed here in , does not appear to improve w ith

the la rva l development o f spot p r io r to trans fo rm a tion . This 1s no t

su rp r is ing f o r there are no major changes in the a lim en ta ry canal o r

associated organs tha t could e f fe c t such a change (P a rt I ) * Surface

area o f the a lim entary canal appears to Increase, bu t t h is might

simply keep pace w ith the s ize increase o f the la rvae . Sorokin and

Panov (1966) found no major increase in a s s im i la t io n during the la r v a l

development o f A. brama. Yet Laurence's (1971, 1977) ene rge tic models

fo r H. salmoldes and P. americanus s im ulate Increases 1n a s s im ila t io n

w ith increasing body weight o f f i r s t - f e e d in g la rvae.

Is the negative re la t io n s h ip between carbon absorption and

development rea l o r a r t i f a c t? W e igh t-spec if ic absorption e f f ic ie n c y

might a c tu a l ly decrease w ith la rv a l age i f the percentage o f carbon

absorbed Is constant or increases a t a slower ra te than the Increase

1r la rva l weight. The negative re la t io n s h ip might be an a r t i f a c t i f

93

younger la rvae f i l l t h e i r a lim entary canal and begin egestion sooner

than o ld e r la rvae w ith a grea te r a lim entary canal c a p a c ity .

Experiments w i th la rva l Anchoa lamprotaenia and Clupea harengus

(C h i t t y , 1900; Werner and B la x te r , 1900), larvae w ith a

m orpho log ica lly s t ra ig h t ra the r than looped a lim entary cana l, suggest

tha t egestion may begin w i th in minutes a f t e r in g e s t io n . I f t h is

occurred in experiments w ith young spot la rva e , estimates of Ingestion

and consequently absorption would be In f la te d due to p a r t ia l d igestion

and a s s im i la t io n o f food as i t traversed the a lim entary ca n a l.

D issections o f la rvae , however, Ind ica ted th a t d ig e s t io n o f Brachlonus

takes longer than 1 h, and egested c u t lc u la r lo r lc a s were f i r s t

observed several hours a f te r the t ra n s fe r o f larvae to re te n t io n

chambers. The accuracy o f carbon absorp tion values ca lcu la te d by

d if fe re n ce was also supported by re s u lts o f the d u a l- la b e l experiment.

The in d ic a to r - r a t io method avoids problems associated w ith estimates

of In g e s tion , ye t gave a s im i la r ly high value fo r absorp tion

e f f1 d e n c y .

The negative re la t io n s h ip between a s s im i la t io n and cond it ion

fa c to r has Important im p lica t io n s to la rv a l growth and s u rv iv a l .

Larvae tha t have been feeding well enough to su rv ive , but less than

tha t requ ired f o r maximum growth, apparently ass im ila te more n u tr ie n ts

from a ra t io n than do larvae w ith a b e t te r growth h is to ry . Perhaps a

larva w ith a lower co n d it ion fa c to r Is b iochem ica lly o r c y to lo g fc a l ly

b e tte r primed f o r a ss im ila t io n o f n u t r ie n ts . Koslomarova (1962)

reported th a t la rv a l fsox 1ucius made up fo r growth delays by more

e f f i c ie n t u t i l i z a t i o n of a va ila b le food.

The present r e s u l ts , suggesting that f i r s t - f e e d in g la rvae are

f u l l y capable o f d ig e s t in g and absorbing n u t r ie n ts , and th a t larvae

w ith a poor growth h is to ry are more e f f i c ie n t 1n a s s im ila t in g

a v a i la b le food, are p la u s ib le in evo lu tionary te rm s. Larvae may

m it ig a te the r is k o f a patchy food d is t r ib u t io n by f u l l y u t i l i z i n g

a v a i la b le resources. Such compensatory mechanisms may be o f great

adaptive s ig n if ic a n c e to pe lag ic marine larvae.

EPILOGUE

The alim entary canal o f spot becomes fu n c t io n a l during the

completion o f o i l -g lo b u le absorption when f i r s t feeding may occur, a

period co inc identa l w ith H Jo rt 's " c r i t i c a l pe riod " (see In t ro d u c t io n )*

Host pelagic marine larvae begin to feed during yo lk absorption ( e .g . ,

B laxter and Hempel, 1963; B la x te r and E h r l ic h , 1974; Houde, 1974),

Whether these larvae are capable o f d iges tion and a ss im ila t io n before

yolk-sac absorption is not known. I f they are, they may s to re energy

fo r u t i l i z a t io n should they encounter inadequate food at f i r s t

feeding. There Is also evidence of thermal optima fo r e f f i c i e n t yo lk

u t i l i z a t io n (Ryland and N icho ls , 1967; Laurence, 1973); i f yo lk is

absorbed too ra p id ly larvae die even in the presence o f adequate food.

Perhaps a lack o f coincidence between f i r s t feed ing and func t iona l

alimentary canal development Is the cause. P re lim inary experiments on

the p o fn t-o f-n o -re tu rn (see review 1n May, 1974) in la rva l spot

Ind ica te tha t the d u ra t ion o f the " c r i t i c a l pe r iod " is about two days

fo llow ing yo lk-sac and o i l - g lo b u le abso rp t ion , a f te r which

i r re v e rs ib le s ta rva t io n occurs3 . Spot la rv a e , reared a t 20°C, appear

to be f u l l y equipped to d igest and absorb a v a ila b le food resources

during the " c r i t i c a l p e r iod ."

3 Powell, A, B, 1979, E ffec ts o f delayed feeding on f i r s t feeding spot ( Lelostomus xanthurus) larvae: Surv iva l and morphologicalchanges. Ann. Rept * o f the Beaufort Laboratory to the u ,S. Dept. o f Energy, pp. 411-421,

95

96

Two l in e s o f evidence suggest th a t the a s s im i la t iv e a b i l i t i e s of

la rv a l Leiostomus xanthurus does not Improve s ig n i f i c a n t l y p r io r to

the completion o f t ra n s fo rm a t io n . Taken separa te ly the ahsence of

both major developmental changes in the a lim entary canal and

d is c e rn ib le changes In carbon a s s im i la t io n are equivocal , but taken

together they are Im p l ic a t iv e .

The development o f the a lim entary canal i s q u ite s im i la r among

te le o s ts w ith no major morphological o r h is to lo g ic a l changes occurring

p r io r to trans fo rm a tion or metamorphosis. Among pelagic marine

la rvae , clupeomorph and protacanthopteryg ian la rvae have a s t ra ig h t

a lim en ta ry cana l; paracanthopteryglans and acanthopteryglans have a

looped a lim en ta ry cana l. The a lim en ta ry canal 1s h is to lo g ic a l ly

p a r t i t io n e d in a s im i la r pa tte rn in both groups (see review 1n Part

I ) . While these groups might d i f f e r in the way food traverses the

a lim en ta ry cana l* they probably d o n 't d i f f e r in the mechanism o f

d ig e s t io n and a s s im i la t io n ; though some c o n f l i c t in g evidence remains

concerning these mechanisms. Thus unchanging a s s im i la t iv e a b i l i t i e s

o f la rv a l Leiostomus xanthurus may be a general phenomenon o f

te leos tean development.

Comparative s tud ies w ith o the r species, p r in c ip a l ly those w ith a

s t ra ig h t and looped a lim entary cana ls , and experiments th a t o f fe r

con tro l over co n d it io n fa c to r may be the next step 1n the ana lys is of

s ta rv a t io n induced m o r ta l i ty in the e a r ly l i f e h is to ry o f f is h e s .

97

APPENDIX

Because labo ra to ry reared f-fsh larvae o f te n d i f f e r b iochemical 1yp

b e h a v io ra l ly , and m crphom etr lca lly from w ild la rv a e { e .g . , B a lb o n t in

et a l . , 1973; B la x te r and E h r l lc h , 1974; A r th u r , 1976), one may expect

p hys io lo g ica l d if fe re n ce s as w e l l . Herein are data on the development

o f Leiostomus xanthurus a t 2Q°C in the labo ra to ry w ith comparisons

with f i e l d caught specimens. S ta t is t ic a l comparisons were not made

because o f the p o s s ib i l i t y o f d i f f e r e n t ia l shrinkage from

p rese rva t io n .

98

Figure 39. Length and age re la t io n s h ip of lab o ra to ry reared

Leiostomus xanthurus la rvae: Bars in d ica te ranges.

■AGE COHORTS USED IN CARBON A SSI Ml LAT ION EXPERIMENTS

a OTHER ESTIMATES

i " 1 i i50 GO 70 60 90

{ Days)

99

Figure 40. Weight and age re la t io n s h ip s of la b o ra to ry reared

Lelostoinus xanthurus larvae: Bars In d ic a te ranges.

DRY

WEI

GHT

{m

g)

a 51,4

20 —

15

J O -

5 -

0 - -0

* AGE COHORTS USED IN CAROON ASSIMILATION EXPERIMENTS

a OTHER ESTIM ATES

-J ]| .Ai

~ r~30

T ~4010 20 30 60

AGE ( D o y t )

100

Figure 41. Schematic sumnary o f the development of la b o ra to ry reared

Leiostomus xanthurus la rvae .

EE

2y

£azf fCD□£iuo

>•

o

o

iq

>>Da

uio<

101

Figure 42. Head length and notochord o r standard length re la t io n s h ip s

o f labora to ry-rea red and w ild Leiostomus xanthurus la rva e .

Head length was taken as the d istance from the t i p o f the

snout to the In te rse c t io n o f the c le ith rum and the

notochord.

• v .

to

£D

ro CM

•t •

(uiui) 0V3H

NO

TOC

HO

RD

/STA

ND

AR

D

LENG

TH

(mm

)

102

Figure 43. Head height and notochord or standard length re la t io n s h ip s

o f la b o ra to ry reared and w ild Leiostomus xanthurus la rva e .

Head height was measured fo l lo w in g EhrHch e t a l . (1976).

I.

i CD

<0~T~

liD"T"<■

T ”fO

" r T~to

tUD

* *

m

-O

a>

-u>

m

{u rn ) 1 HGI3 H 0 V 3 H

NOTO

CHOR

D /

STAN

DARD

LE

NGTH

(m

m)

103

Figure 44, Eye diameter and notochord o r standard length

re la t io n s h ip s o f labo ra to ry reared and w ild Leiostomus

xanthurus la rv a e . Eye diameter was measured fo l lo w in g

Richardson and Joseph (1973).

<n

CD

00

- Q

(?>

a>

oaa

tn

□tr01o

u>

tn

- fO

ew — <\l

(uiui) d313W VlO 3A3

104

Figure 45. Eye height and notochord or standard length re la t io n sh ip s

o f labo ra to ry reared and w ild Leiostomus xanthurus la rvae .

Eye height was measured fo l lo w in g E h r ltch e t a l , (1976).

r C

.1

ic

ID

1

fO

CJ

- (T>

- CO

u>

- tfl

u- OJ

(- ■“l-m

n ------- r~tv —

r t —r t tvi — O

(UiOi) 1H0I3H 3A3

NOTO

CHO

RD

/STA

ND

AR

D

LENG

TH

(mm

)

105

Figure 46. Snout to pecto ra l length and notochord or standard length

re la t io n s h ip s o f laboratory reared and w ild Lelostomus

xanthurus la rv a e . Snout to pecto ra l was measured

fo l lo w in g Mayo (1973).

SNOU

T TO

PECT

ORAL

LE

NGTH

(m

m)

7

54

32

07 -

6 - 5 4

32 H I 0

r f 1 '

1--------1--------- 1 ■ ~ i-------- r------- t ---------r ------ I " i t --------1--------- r --------1---------1---------r-------- 1----1— --------------1

a

.t

rf.r

■% i1 T 1---------1--------T- T ------ T------- I 1---------- T------- 1--------1-------- T ------ 1 ---------1---------1---------1---------1----------1

I Z 3 4 5 6 7 0 9 1 0 II 12 13 W B 16 17 18 19

NOTOCHORD /STANDARD HEIGHT {mm)

106

Figure 47. Pectoral depth and notochord or standard length

re la t io n s h ip s o f 1abora tory-reared and w ild Leiostomus

xanthurus la rvae. Pectora l depth was measured fo l lo w in g

Mayo (1973),

<f

K) o

£?

- <£

- r--

£

_ tr

K>

_ (M

* Q

at

- CD

m

u>

* If)

- ir

ro

- OJ

r~m

—r T “rO

T "<\J - O

(ww) Hidaa ivdoioid

NOTO

CHOR

D /

STAN

DARD

LE

NGTH

(m

m)

107

Figure 48. Preanal length and notochord or standard length

re la t ion sh ip s o f labora to ry-rea red and w ild Lelostomus

xanthurus la rv a e . Preanal length was measured fo l lo w in g

Mayo (1973).

9e?6

54

32

i

087

854

3

2

I

0

A

* *

• A■»— i— t— t— f— i— i— I f

Q

t i t i r - r — i— i— r— i— i— i— i— i— i— t— i— t— iI 2 3 4 5 6 7 8 9 10 II 12 1} 14 15 16 17 10 19

NOTOCHORD /STANDARD LENGTH (m m )

108

F igure 49. Depth at anus and notochord or standaivi length

re la t io n s h ip s o f 1aboratory-reared and w i ld Lelostomus

xanthurus la rvae . Depth at anus was measured fo l lo w in g

Mayo (1973).

• 4

oc\l

- to

CM o

EE

acc<.QZ<I—1/1

QCCOXoo

{ w u l ) snNV I V Hld3G

109

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VITA

John J e ffre y Govoni

Born in viareharn, Massachusetts, 2 August 1948, Educated through

secondary school in Bourne Public School System, Bourne,

Massachusettsj graduated from Bourne High School in June, 1966,

Received A,B. in b io lo g y from St. Anselm* s College, Manchester, New

Hampshire in June, 1970. Entered Southeastern Massachusetts

U n iv e rs i ty , North Dartmouth, Massachusetts, 1971; served as a Graduate

Teaching A s s is ta n t ; took M. S. in bto logy May, 1973, Employed as

Research A s s is ta n t , Marine Research, In c . , Woods Hole, Massachusetts,

June 1972 to August 1974, Entered the School of Marine Science of The

College o f W il l ia m and Mary September, 1974; served as a Graduate

Research A s s is ta n t; admitted to candidacy f o r the Ph.D. August, 1977.