Bacteria from the Gut of Australian Termites
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May, 1978, P . 823-828 0099-2240/78/0035-0823$02.00/0 Copyright © 1978 American Society f o r Microbiology Vol. 3 , N o . 5 Printed i n U.S.A. Bacteria from t h e G u t o f Australian Termites M. L . EUTICK, R . W. O'BRIEN,* AND M . SLAYTOR Department o f Biochemistry, The University of Sydney, Sydney, N e w South Wales 2006, Australia Received f o r publication 1 4 October 1977 T h e major g u t bacteria of the worker caste of nine species o f Australian termites, belonging t o four families, were isolated an d identified t o generic level. A l l species were either facultative anaerobes or strict aerobes. A correlation appears t o exist between t h e major g u t bacterium a n d t h e family t o which t h e termite belongs. T h e major from t h e two lowest termites, Mastotermes darwiniensis (family Mastotermitidae) a n d Cryptotermes primus (family Kalo- termitidae), was Streptococcus; from four species belonging t o t h e Rhinotermi- tidae (Heterotermes ferox, Coptotermes acinaciformis, C . lacteus, Schedo- rhinotermes intermedius intermedius) i t was Enterobacter; an d from three species o f t h e Termitidae (Nasutitermes exitiosus, N . graveolus, N . walkeri) i t was Staphylococcus. Enterobacter was a minor symbiont o f M . darwiniensis, C . primus, a n d N. graveolus; Streptococcus was a minor symbiont o f H . ferox, C . lacteus, S . intermedius intermedius, a n d N . exitiosus; a n d Bacillus was a minor symbiont o f C . acinaciformis a n d S . intermedius intermedius. M. darwiniensis possessed another minor symbiont tentatively identified as Flavobacterium. C . acinaciformis from three widely separated locations possessed a similar micro- biota, indicating some form of control on t h e composition o f t h e g u t bacteria. Bacteria, capable o f growth on N-free medium i n t h e presence o f nitrogen gas, were isolated from a l l termites, except N . exitiosus a n d N . walkeri, a n d were identified as Enterobacter. N o cellulose-degrading bacteria were isolated. Studies o n t h e role o f bacteria i n t h e nutrition of termites have largely centered around their ability t o degrade cellulose o r t o f i x nitrogen gas. Cellulose-degrading bacteria have been isolated from some species oftermites (21, 26), b u t many attempts have been unsuccessful ( 8 , 10, 16), a n d other claims ( 3 , 1 7 , 1 8 , 2 3, 2 5 ) need t o b e viewed with some caution since t h e methods used f o r t h e surface sterilization o f t h e termites were either n o t given o r n o t evaluated. Breznak e t a l . ( 6 ) have demonstrated fixation o f nitrogen g a s b y termites a n d have shown that this ability w a s lost when t h e g ut bacteria were removed with antibiotics. Dinitrogen (N2) fixa- tion b y termites h a s also been reported b y B e - nemann ( 4 ) a n d French e t a l . (12), a n d nitrogen- fixing bacteria have been isolated b y French et a l . (12) a n d Potrikus a n d Breznak (22). W e have carried o u t a survey o f a wider scope than previously undertaken, t o isolate a n d iden- tify t h e bacteria present in t h e hindgut o f nine species o f Australian termites, with a view t o a better understanding o f t h e symbiosis between t h e termites a n d their g u t bacteria. MATERIALS AND METHODS Termite species. Th e termite species used were: Coptotermes acinaciformis (Froggatt), Coptotermes lacteus (Froggatt), Cryptotennes primus Hill, Heter- otermes ferox (Froggatt), Mastotennes darwiniensis Froggatt, Nasutitermes exitiosus (Hill), Nasutitermes graveolus (Hill), Nasutitermes walkeri (Hill), a n d Schedorhinotermes intermedius intermnedius (Brauer). C . primus, M . darwiniensis, a n d N . grav- eolus were obtained from t h e Division o f Entomology, CSIRO, Canberra. ferox, N. exitiosus, N . walkeri, a n d S . intermedius were collected within a radius of 2 0 miles o f Sydney. C . acinaciforrnis w a s collected from three locations: ( i ) near Sydney; (ii) Taree, N.S.W.; a n d (iii) Rollingstone, Queensland. C . lacteus w a s collected a t Bilpin, N.S.W. Th e termites were maintained o n Eucalyptus s p . wood i n their own nest material i n polythene bins a n d were examined within 2 4 h o f receipt o r Surface sterilization o f termites an d removal o f gut. Th e termites (worker caste) were surface ster- ilized (21) a n d were then rubbed a n d rolled across t h e surface of a reinforced clostridial medium (RCM) agar plate (Oxoid), which was then incubated i n a i r f o r 7 2 h a t 30°C. I f a n y organisms grew on this plate, t h e termite was considered t o b e nonsterile, and-the e x - periment was repeated with a fresh worker termite. Th e sterilized termites were dissected i n a sterile lam- inar a i r flow cabinet using sterile instruments. Th e head of the termite was severed, a n d a dorsal incision w a s made. Th e g u t was without rupturing, a n d was washed i n sterile Krebs-Ringer phosphate buffer, pH 7.4. Th e guts from five termites were h o - mogenized individually i n 0 . 5 m l o f th e same buffer. Isolation an d counting o f bacteria. Serial dilu- tions o f t h e five g u t suspensions were made a n d spread 8 2 3
Transcript of Bacteria from the Gut of Australian Termites
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A P P L I E D AND ENVIRONMENTAL M I C R O B I O L O G Y , M a y , 1 9 7 8 , P . 8 2 3 - 8 2 8
0 0 9 9 - 2 2 4 0 / 7 8 / 0 0 3 5 - 0 8 2 3 $ 0 2 . 0 0 / 0
C o p y r i g h t © 1 9 7 8 A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y
V o l . 3 5 , N o . 5
P r i n t e d i n U . S . A .
B a c t e r i a f r o m t h e G u t o f A u s t r a l i a n T e r m i t e s
M. L . EUTICK, R . W. O'BRIEN,* AND M . SLAYTOR
D e p a r t m e n t o f B i o c h e m i s t r y , The U n i v e r s i t y o f S y d n e y , S y d n e y , N e w S o u t h Wales 2 0 0 6 , A u s t r a l i a
R e c e i v e d f o r p u b l i c a t i o n 1 4 O c t o b e r 1 9 7 7
Th e m a j o r g u t b a c t e r i a o f t h e w o r k e r c a s t e o f n i n e s p e c i e s o f A u s t r a l i a n
t e r m i t e s , b e l o n g i n g t o f o u r f a m i l i e s , were i s o l a t e d and i d e n t i f i e d t o g e n e r i c l e v e l .A l l s p e c i e s were e i t h e r f a c u l t a t i v e a n a e r o b e s or s t r i c t a e r o b e s . A c o r r e l a t i o n
appears t o e x i s t b e t w e e n t h e m a j o r g u t b a c t e r i u m a n d t h e f a m i l y t o w h i c h t h e
t e r m i t e b e l o n g s . Th e m a j o r b a c t e r i u m f r o m t h e two l o w e s t t e r m i t e s , Mastotermes
d a r w i n i e n s i s ( f a m i l y M a s t o t e r m i t i d a e ) a n d C r y p t o t e r m e s primus ( f a m i l y K a l o -
t e r m i t i d a e ) , was S t r e p t o c o c c u s ; f r o m f o u r s p e c i e s b e l o n g i n g t o t h e R h i n o t e r m i -
t i d a e ( H e t e r o t e r m e s f e r o x , C o p t o t e r m e s a c i n a c i f o r m i s , C . l a c t e u s , S c h e d o -
r h i n o t e r m e s i n t e r m e d i u s i n t e r m e d i u s ) i t was E n t e r o b a c t e r ; and from t h r e es p e c i e s o f t h e T e r m i t i d a e ( N a s u t i t e r m e s e x i t i o s u s , N . g r a v e o l u s , N . w a l k e r i ) i t
was S t a p h y l o c o c c u s . E n t e r o b a c t e r was a m i n o r s y m b i o n t o f M. d a r w i n i e n s i s , C .p r i m u s , a n d N . g r a v e o l u s ; S t r e p t o c o c c u s was a m i n o r s y m b i o n t o f H . f e r o x , C .l a c t e u s , S . i n t e r m e d i u s i n t e r m e d i u s , a n d N . e x i t i o s u s ; a n d B a c i l l u s was a m i n o r
s y m b i o n t o f C . a c i n a c i f o r m i s a n d S . i n t e r m e d i u s i n t e r m e d i u s . M. d a r w i n i e n s i s
p o s s e s s e d a n o t h e r m i n o r s y m b i o n t t e n t a t i v e l y i d e n t i f i e d a s F l a v o b a c t e r i u m . C .a c i n a c i f o r m i s f r o m t h r e e w i d e l y s e p a r a t e d l o c a t i o n s p o s s e s s e d a s i m i l a r m i c r o -
b i o t a , i n d i c a t i n g some f o r m o f c on tr o l on t h e c o m p o s i t i o n o f t h e g u t b a c t e r i a .B a c t e r i a , c a p a b l e o f g r o w t h on N - f r e e medium i n t h e pr e s e n c e o f n i t r o g e n gas,
were i s o l a t e d f r o m a l l t e r m i t e s , e x c e p t N . e x i t i o s u s a n d N . w a l k e r i , a n d were
i d e n t i f i e d as E n t e r o b a c t e r . N o c e l l u l o s e - d e g r a d i n g b a c t e r i a were i s o l a t e d .
S t u d i e s o n t h e r o l e o f b a c t e r i a i n t h e n u t r i t i o no f t e r m i t es h a v e l a r g e l y c e n t e r e d a r o u n d t h e i ra b i l i t y t o d e g r a d e c e l l u l o s e o r t o f i x n i t r o g e n g a s .C e l l u l o s e - d e g r a d i n g b a c t e r i a h a v e b e e n i s o l a t e df r o m some s p e c i e s o f t e r m i t es ( 2 1 , 2 6 ) , b u t manya t t e m p t s h a v e b e e n u n s u c c e s s f u l ( 8 , 1 0 , 1 6 ) , a n do t h e r c l a i m s ( 3 , 1 7 , 1 8 , 2 3 , 2 5 ) n e e d t o b e v i e w e d
w i t h some c a u t i o n s i n c e t h e m e t h o d s u s e d f o rt h e s u r f a c e s t e r i l i z a t i o n o f t h e t e r m i t e s w e r e
e i t h e r n o t g i v e n o r n o t e v a l u a t e d .B r e z n a k e t a l . ( 6 ) h a v e d e m o n s t r a t e d f i x a t i o n
o f n i t r o g e n g a s b y t e r m i t e s a n d h a v e s h o w n t h a t
t h i s a b i l i t y w a s l o s t when t h e g u t b a c t e r i a w e r er e m o v e d w i t h a n t i b i o t i c s . D i n i t r o g e n ( N 2 ) f i x a -t i o n b y t e r m i t e s h a s a l s o b e e n r e p o r t e d b y B e -
nemann ( 4 ) a n d F r e n c h e t a l . ( 1 2 ) , a n d n i t r o g e n -f i x i n g b a c t e r i a h a v e b e e n i s o l a t e d b y F r e n c h e t
a l . ( 1 2 ) a n d P o t r i k u s a n d B r e z n a k ( 2 2 ) .W e h a v e c a r r i e d o u t a s u r v e y o f a w i d e r s c o p e
t h a n p r e v i o u s l y u n d e r t a k e n , t o i s o l a t e a n d i d e n -
t i f y t h e b a c t e r i a p r e s e n t i n t h e h i n d g u t o f n i n e
s p e c i e s o f A u s t r a l i a n t e r m i t e s , w i t h a v i e w t o a
b e t t e r u n d e r s t a n d i n g o f t h e s y m b i o s i s b e t w e e nt h e t e r m i t e s a n d t h e i r g u t b a c t e r i a .
M A T E R I A L S AND M E TH ODS
T e r m i t e s p e c i e s . The t e r m i t e s p e c i e s u s e d w e r e :
C o p t o t e r m e s a c i n a c i f o r m i s ( F r o g g a t t ) , C o p t o t e r m e sl a c t e u s ( F r o g g a t t ) , C r y p t o t e n n e s p r i m u s H i l l , H e t e r -
o t e r m e s f e r o x ( F r o g g a t t ) , M a s t o t e n n e s d a r w i n i e n s i s
F r o g g a t t , N a s u t i t e r m e s e x i t i o s u s ( H i l l ) , N a s u t i t e r m e sg r a v e o l u s ( H i l l ) , N a s u t i t e r m e s w a l k e r i ( H i l l ) , a n d
S c h e d o r h i n o t e r m e s i n t e r m e d i u s i n t e r m n e d i u s( B r a u e r ) . C . p r i m u s , M . d a r w i n i e n s i s , a n d N . g r a v -e o l u s w e r e o b t a i n e d f r o m t h e D i v i s i o n o f E n t o m o l o g y ,C S I R O , C a n b e r r a . H . f e r o x , N . e x i t i o s u s , N . w a l k e r i ,a n d S . i n t e r m e d i u s w e r e c o l l e c t e d w i t h i n a r a d i u s o f
2 0 m i l e s o f S y d n e y . C . a c i n a c i f o r r n i s w a s c o l l e c t e df r o m t h r e e l o c a t i o n s : ( i ) n e a r S y d n e y ; ( i i ) T a r e e ,
N . S . W . ; a n d ( i i i ) R o l l i n g s t o n e , Q u e e n s l a n d . C . l a c t e u sw a s c o l l e c t e d a t B i l p i n , N . S . W . The t e r m i t e s w e r e
m a i n t a i n e d o n E u c a l y p t u s s p . wood i n t h e i r own n e s t
m a t e r i a l i n p o l y t h e n e b i n s a n d w e r e e x a m i n e d w i t h i n2 4 h o f r e c e i p t o r c o l l e c t i o n .
S u r f a c e s t e r i l i z a t i o n o f t e r m i t e s an d r e m o v a lo f g u t . The t e r m i t e s ( w o r k e r c a s t e ) were s u r f a c e s t e r -i l i z e d ( 2 1 ) a n d w e r e t h e n r u b b e d a n d r o l l e d a c r o s s t h e
s u r f a c e o f a r e i n f o r c e d c l o s t r i d i a l medium ( R C M ) a g a r
p l a t e ( O x o i d ) , w h i c h was t h e n i n c u b a t e d i n a i r f o r 7 2
h a t 3 0 ° C . I f a n y o r g a n i s m s g r e w on t h i s p l a t e , t h e
t e r m i t e was c o n s i d e r e d t o b e n o n s t e r i l e , a n d - t h e e x -
p e r i m e n t was r e p e a t e d w i t h a f r e s h w o r k e r t e r m i t e .
The s t e r i l i z e d t e r m i t e s w e r e d i s s e c t e d i n a s t e r i l e l a m -
i n a r a i r f l o w c a b i n e t u s i n g s t e r i l e i n s t r u m e n t s . Th e
h e a d o f t h e t e r m i t e was s e v e r e d , a n d a d o r s a l i n c i s i o nw a s m a d e . The g u t was r e m o v e d , w i t h o u t r u p t u r i n g ,
a n d was w a s h e d i n s t e r i l e K r e b s - R i n g e r p h o s p h a t eb u f f e r , pH 7 . 4 . Th e g u t s f r o m f i v e t e r m i t e s were h o -
m o g e n i z e d i n d i v i d u a l l y i n 0 . 5 m l o f t h e s a m e b u f f e r .I s o l a t i o n an d c o u n t i n g o f b a c t e r i a . S e r i a l d i l u -
t i o n s o f t h e f i v e g u t s u s p e n s i o n s were made a n d s p r e a d
8 2 3
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824 EUTICK, O ' B R I E N , AND S LAY TO R
on d u p l i c a t e RCM agar p l a t e s ( f r e s h l y p r e p a r e d ) .
They were i n c u b a t e d a e r o b i c a l l y f o r 7 2 h a t 3 0 ° C , a n d
t h e b a c t e r i a l c o l o n i e s were c o u n t e d . Each c o l o n y t y p e
f r o m e a c h o f t h e f i v e s e r i a l d i l u t i o n s was p u r i f i e d b yp l a t i n g t h r e e t i m e s i n s equence on RCM agar p l a t e s .S i n c e Gram s t a i n i n g a n d m i c r o s c o p i c e x a m i n a t i o n
s h o w e d t h a t t h e same m o r p h o l o g i c a l t y p e s o f b a c t e r i a
were i s o l a t e d f r o m e a c h s e r i e s o f p l a t e s , one i s o l a t e o f
e a c h t y p e was t h e n s u b j e c t e d t o f u r t h e r b i o c h e m i c a l
t e s t s .Gu t c o n t e n t s were a l s o i n o c u l a t e d i n t o l i q u i d RCM
m e d i u m , r e n d e r e d a n a e r o b i c b y t h e a d d i t i o n o f s t e r i l es o d i u m d i t h i o n i t e , u s i n g m e t h y l e n e b l u e as an i n d i c a -
t o r o f a n a e r o b i o s i s , a n d were i n c u b a t e d f o r 1 4 d a y s a t
3 0 ° C . RCM agar p l a t e c u l t u r e s were a l s o i n c u b a t e d
f o r 7 d a y s a t 3 0 ° C i n an a n a e r o b e j a r , u s i n g a GasPak
a n a e r o b i c s y s t e m ( B a l t i m o r e B i o l o g i c a l L a b o r a t o r y ,D i v i s i o n o f B e c t o n , D i c k i n s o n & C o . , C o c k e y s v i l l e ,
M d . ) t oremove
oxygen. P u t a t i v e N 2 - f i x i n g b a c t e r i awere i s o l a t e d b y p l a ti ng g u t c o n t e n t s on N - f r e e agar
( 1 3 ) i n w h i c h t h e sucrose was r e p l a c e d b y g l u c o s e . Thep l a t e s were p l a c e d i n an a n a e r o b e j a r , w h i c h was
e v a c u a t e d on a w a t e r j e t pump ( v a c u u m , a b o u t 2 0 x
1 0 2 P a ) , t h e n f l u s h e d w i t h n i t r o g e n , a n d i n c u b a t e d a t
3 0 ° C f o r 7 d a y s . A c o n t a i n e r o f 1 M H 2 S O 4 was p l a c e d
i n t h e j a r t o a b s o r b any ammonia p r e s e n t i n t h e
n i t r o g e n . Th e i s o l a t e s were p u r i f i e d b y p l a t i n g f o u r
t i m e s i n s u c c e s s i o n on N - f r e e agar i n c u b a t e d u n d e r
t h e same c o n d i t i o n s . Th e N - f r e e p l a t e s were s t o r e d i na s e a l e d c o n t a i n e r i n t h e presence o f 1 M H 2 S O 4 b e f o r e
use.
A t t e m p t s t o i s o l a t e c e l l u l o s e - d e g r a d i n g b a c t e r i a
were made b y i n c u b a t i n g g u t c o n t e n t s i n t h e t h r e ec e l l u l o s e m e d i a o f A a r o n s o n ( 1 ) a n d i n a medium o f
t h e f o l l o w i n g c o mp o s i t i o n ( g r a m s pe r l i t e r o f d i s t i l l e dw a t e r ) : K 2 H P O 4 , 1 . 0 ; MgSO4 7 H 2 0 , 1 . 0 ; ( N H 4 ) 2 S O 4 ,2 . 0 ; K C I , 0 . 5 ; C a C 1 2 ( a n h y d r o u s ) , 0 . 0 2 ; F e S O 4 7 H 2 0 ,0 . 0 1 ; MnSO4 4 H 2 0 , 0 . 0 0 1 ; a n d a s t r i p ( 5 b y 1 c m ) o f
Whatman n o. 1 f i l t e r paper. A l l o f t h e c u l t u r e s were
i n c u b a t e d a t 3 0 ° C f o r 1 4 d a y s u n d e r a e r o b i c c o n d i -
t i o n s .M o r p h o l o g i c a l c h a r a c t e r i s t i c s . B a c t e r i a l f o r m
a n d Gram s t a i n were d e t e r m i n e d on c e l l s grown i nl i q u i d RCM m e d i u m ( 2 4 ) .
F l a g e l l a t i o n was d e t e r m i n e d on c u l t u r e s grown f o r2 4 h on p e p t o n e - y e a s t e x t r a c t a gar p l a t e s . The c e l l s
were e m u l s i f i e d i n w a t e r , a d r o p o f t h e e m u l s i o n was
a p p l i e d t o a c a r b o n / n i t r o c e l l u l o s e - c o a t e d copper g r i d ,a n d t h e c e l l s were a l l o w e d t o s e t t l e f o r 1 m i n . A d r o p
o f 1% ( w t / v o l ) p h o s p h o t u n g s t i c a c i d ( p H 6 . 5 ) was
a d d e d , a n d t h e c u l t u r e s were a l l o w e d t o s t a n d f o r a
f u r t h e r 1 5 s b e f o r e excess l i q u i d was r e m o v e d w i t hb l o t t i n g paper. The s p e c i m e n s were e x a m i n e d b y d i -r e c t o b s er v a t i o n , u s i ng a P h i l i p s EM300 e l e ct r o n m i -
croscope a t a m a g n i f i c a t i o n o f x 2 5 , 0 8 0 .
B i o c h e m i c a l c h a r a ct e r i s t i c s . B i o c h e m i c a l t e s t s
a n d t e s t s o f t h e a b i l i t y o f t h e i s o l a t e s t o grow on
c e r t a i n m e d i a were c a r r i e d o u t a t 3 0 ° C as d e s c r i b e d
b y S k e r m a n ( 2 4 ) , w i t h t h e f o l l o w i n g e x c e p t i o n s , w h i c h
were c a r r i e d o u t a c c o r d i n g t o t h e c i t e d m e t h o d s : g l u -
cose a n d l a c t o s e o x i d a t i o n a n d f e r m e n t a t i o n ( 2 ) ; m a l -o n a t e a n d g l u c o n a t e u t i l i z a t i o n ( 1 4 ) ; a c i d a n d gas
p r o d u c t i o n f r o m s u g a r a l c o h o l s u s i n g m e d i u m 2 , con-
t a i n i n g 1% ( w t / v o l ) o f t h e s u b s t r a t e ( 9 ) . Th e f o r m a t i o no f D- a n d L - l a c t a t e f r o m g l uc o s e f e r m en t at i o n was
d e t e r m i n e d q u a l i t a t i v e l y on t h e c l a r i f i e d , s p e n t RCM
medium b y a d d i n g 2 . 5 , u m o l o f n i c o t i n a m i d e a d e n i n e
d i n u c l e o t i d e a n d 5 U o f D - o r L - l a c t a t e d e h y d r o g e n a s e( E C 1 . 1 . 1 . 2 7 a n d E C 1 . 1 . 1 . 2 8 ) ( S i g m a ) i n t h e p r e s e n c e
o f 0 . 5 . mM g l y c i n e - h y d r a z i n e b u f f e r , pH 9 . 0 , a n d o b -
s e r v i n g t h e r e d u c t i o n o f n i c o t i n a m i d e a d e n i n e d i n u c l e -
o t i d e a t 3 4 0 n m . A l l i s o l a t e s w e r e i d e n t i f i e d t o g e n e r i c
l e v e l u s i n g S k e r m a n ' s k e y ( 2 4 ) .E s t i m a t i o n o f t e r m i t e g u t s i z e . A n e s t i m a t e o f
t h e s i z e o f t h e g u t o f e a c h t e r m i t e s p e c i e s was made
b y f i t t i n g t h e abdomen i n t o c a l i b r a t e d c a p i l l a r y t u b e s .
C a l c u l a t i o n s o f t h e g u t v o l u m e w e r e ma de o n t h e
a s s u m p t i o n s t h a t t h e g u t w a s a p p r o x i m a t e l y c y l i n d r i -c a l a n d t h a t i t o c c u p i e d t h e w h o l e v o l u m e o f t h e
a b d o m e n . T h i s g u t v o l u m e w a s t h e n u s e d t o c a l c u l a t e
t h e number o f b a c t e r i a l c e l l s p e r m i l l i l i t e r o f t e r m i t e
g u t .
R ESU L TS
E a c h t e r m i t e s p e c i e s p o s s e s s e d o n e m a j o r b a c -t e r i a l s p e c i e s a t c o nc en t r a t i o n s o f 5 x 1 0 ' t o 3
x 1 0 7 c e l l s p e r m l o f t e r m i t e g u t ( T a b l e 1 ) . O t h e rb a c t e r i a l s p e c i e s w e r e p r e s e n t , i n l e s s e r n u m b e r s ,i n a l l t e r m i t e s , e x c e p t N . w a l k e r i . I t i s c o n c e i v -
a b l e t h a t s t i l l o t h e r s p e c i e s o f b a c t e r i a a r e p r e s -e n t i n t h e g u t b u t w e r e n o t d e t e c t e d o n t h e RCMp l a t e s ( s e e b e l o w o n t h e i s o l a t i o n o f N 2 - f i x i n go r g a n i s m s ) . B a c t e r i a p r e s e n t i n l o w n u m b e r sw o u l d b e d i s g u i s e d o n p l a t e s b y t h e o v e r w h e l m -
i n g n u m b e r s o f t h e m a j o r b a c t e r i u m , m i g h t b ew a s h e d o u t d u r i n g s e r i a l d i l u t i o n , o r m i g h t n o t
g r o w o n RCMa g a r .
The l o w e s tb a c t e r i a l
c o u n t
w a s a b o u t 1 0 5 c e l l s p e r m l o f t e r m i t e g u t f o rs t r a i n MD3. N o s t r i c t a n a e r o b e s w e r e i s o l a t e d ;o r g a n i s m s g r o w i n g i n a n a e r o b i c m e d i u m , o r o na n a e r o b i c p l a t e s , w e r e i d e n t i c a l t o t h o s e g r o w i n go n a e r o b i c p l a t e s . The b i o c h e m i c a l c h a r a c t e r i s -t i c s o f t h e b a c t e r i a l i s o l a t e s a r e s h o w n i n T a b l e2 . F o r e a s e o f c o m p a r i s o n , t h e b a c t e r i a h a v eb e e n g r o u p e d a c c o r d i n g t o g e n u s r a t h e r t h a na c c o r d i n g t o t h e t e r m i t e f r o m w h i c h t h e y w e r ei s o l a t e d .
S t r a i n s C A 1 , C A 2 , C N 1 , C L i , C P 2 , H F 1 , MD2,N G 2 , a n d S 1 3 ( T a b l e 2 ) w e r e i d e n t i f i e d a s E n -
t e r o b a c t e r ( 7 ) s i n c e t h e y w e r e g r a m - n e g a t i v e ,p e r i t r i c h o u s l y f l a g e l l a t e d r o d s c a p a b l e o f u t i l i z -i n g c i t r a t e a s a s o l e c a r b o n s o u r c e . A l l w e r e a b l et o g r o w i n c y a n i d e b r o t h , w h i c h d i s t i n g u i s h e st h e m f r o m S h i g e l l a , E d w a r d s i e l l a , Y e r s i n i a ,a n d E s c h e r i c h i a s p e c i e s ( 7 ) , a n d a l l w e r e m e t h y lr e d n e g a t i v e a n d V o g e s - P r o s k a u e r p o s i t i v e ,w h i c h d i s t i n g u i s h e s t h e m f r o m t h e g e n u s C i t r o -b a c t e r ( 7 ) . S t r a i n d i f f e r e n c e s a p p e a r t o e x i s t , a ss h o w n b y t h e p a t t e r n o f a r g i n i n e , o r n i t h i n e , a n dl y s i n e d e c a r b o x y l a s e s . The c h a r a c t e r s s h o w n b ys t r a i n s C A 1 , C A 2 , a n d CN 1 w e r e i d e n t i c a l ; CAlw a s i s o l a t e d f r o m C . a c i n a c i f o r m i s c o l l e c t e d i nQ u e e n s l a n d , CA2 w a s i s o l a t e d f r o m C . a c i n a c i -
f o r m i s c o l l e c t e d n e a r S y d n e y , a n d CN1 w a s i s o -l a t e d f r o m C . a c i n a c i f o r m i s c o l l e c t e d a t T a r e e .
S t r a i n s N E L , N G 1 , a n d NW ( T a b l e 2 ) w e r eg r a m - p o s i t i v e , n o n m o t i l e c o c c i , c h a r a c t e r i s t i -
A P P L . E N V I R O N . M I C R O B I O L .
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B A C T E R I A FR OM G U T OF T E R M I T E S 8 2 5
TABLE 1 . D i s t r i b u t i o n o f g u t b a c t e r i a amo ng t h e t e r m i t e f a m i l i e s
B a c t e r i a l g e n e r a i d e n t i f i e d
F a m i l y S u b f a m i l y S p e c i e s S t r e F l a v o -s S r p t o - E n t e r o - S t a p h y l o - B a c i l l u s > o
C O C C U S b a c t e r c o c c u s b a c t e r i u m
M a s t o t e r m i t i d a e M. d a r u w i n i e n s i s +++" + +K a l o t e r m i t i d a e C . p r i m u s +++ +
R h i n o t e r m i t i d a e H e t e r o t e r m i t i n a e H . f e r o x + +++
C o p t o t e r m i t i n a e C . a c i n a c i f o r m i s ++++ ++
C . l a c t e u s + ++++
R h i n o t e r m i t i n a e S . i n t e r m e d i u s i n - ++ ++++ +
t e r m e d i u s
T e r m i t i d a e N a s u t i t e r m i t i n a e N . e x i t i o s u s + ++++
N . g r a v e o l u s ++ ++++
N . u w a l k e r i ++++
Numbers i n p a r e n t h e s e s a r e b a c t e r i a l c e l l s x 1 0 ' p e r m i l l i l i t e r o f t e r mi t e g u t : ++++, ( 1 t o 3 ) ; +++, ( 0 . 5 t o 0 . 9 ) ; + + , ( 0 . 1 t o0 . 4 ) ; + ( 0 . 0 1 t o 0 . 0 3 ) .
c a l l y d i v i d i n g i n t w o p l a n e s t o f o r m t e t r a d s a n d
p a c k e t s o f c e l l s , a n d w e r e i d e n t i f i e d a s S t a p h y -l o c o c c u s ( 7 ) . S t r a i n d i f f e r e n c e s a r e a p p a r e n t i nt h e i r d i f f e r i n g a b i l i t i e s t o f o r m a c e t o i n o r a c i d
f r o m g l u c o s e , l a c t o s e , a n d m a n n i t o l .
S t r a i n s C L 2 , C P 1 , H F 2 , MD1, N E 2 a n d S 1 2( T a b l e 2 ) w e r e i d e n t i f i e d a s S t r e p t o c o c c u s ( 7 )s i n c e t h e y w e r e g r a m - p o s i t i v e , n o n m o t i l e c o c c ic h a r a c t e r i s t i c a l l y f o r m i n g p a i r s , o r c h a i n s , o f
c e l l s a n d p r o d u c i n g o n l y L - l a c t a t e , b u t n o g a s ,f r o m g l u c o s e f e r m e n t a t i o n .
S t r a i n s CN 2 a n d S I l ( T a b l e 2 ) w e r e i d e n t i f i e da s B a c i l l u s ( 7 ) s i n c e t h e y w e r e g r a m - p o s i t i v e ,
m o t i l e , s p o r e f o r m i n g r o d s . B o t h w e r e s t r i c ta e r o b e s . S I l a p p e a r e d t o u t i l i z e t h e n i t r o g e n o u s
c o m p o n e n t s o f t h e RCM f o r g r o w t h , s i n c e i t d i d
n o t g r o w o n g l u c o s e .S t r a i n MD3 was a g r a m - n e g a t i v e , n o n s p o r i n g
m o t i l e r o d . The b i o c h e m i c a l c h a r a c t e r i s t i c s ( T a -
b l e 2 ) o f t h e i s o l a t e , p l u s t h e f a c t t h a t t h e c o l o -n i e s w e r e y e l l o w a n d t r a n s l u c e n t , i n d i c a t e d t h a t
i t w a s a F l a v o b a c t e r i u m ( 7 ) .I s o l a t i o n o f p u t a t i v e N 2 - f i x i n g b a c t e r i a w a s
i n i t i a l l y a t t e m p t e d u s i n g t h e m e t h o d o f S k e r m a n( 2 4 ) , b u t t h e r e s u l t s w e r e d i f f i c u l t t o i n t e r p r e t .The r e p o r t b y F r e n c h e t a l . ( 1 2 ) o f t h e p r e s e n c e
o f N 2 - f i x i n g b a c t e r i a i n t h r e e o f t h e s p e c i e s o f
t e r m i t e s u s e d i n t h i s s t u d y ( C . l a c t e u s , M. d a r -
w i n i e n s i s , a n d N . e x i t i o s u s ) p r o m p t e d u s t o r e -e x a m i n e t h e g u t o f o u r t e r m i t e s f o r N 2 - f i x i n gb a c t e r i a . The g u t c o n t e n t s o f a l l o f t h e t e r m i t e s ,e x c e p t N . e x i t i o s u s a n d N . w a l k e r i , y i e l d e d b a c -
t e r i a c a p a b l e o f g r o w t h o n N - f r e e m e d i u m . Then u m b e r s w e r e n o t d e t e r m i n e d b e c a u s e o f t h e
s m a l l s i z e o f t h e c o l o n i e s . A l l o f t h e i s o l a t e s w e r e
i d e n t i f i e d a s E n t e r o b a c t e r s i n c e t h e y w e r e g r a m -n e g a t i v e , m o t i l e r o d s , c a p a b l e o f u t i l i z i n g c i t r a t ea s a s o l e c a r b o n s o u r c e a n d c a p a b l e o f g r o w i n g
i n c y a n i d e b r o t h . A l l w e r e m e t h y l r e d n e g a t i v e ,V o g e s - P r o s k a u e r p o s i t i v e , a n d c a t a l a s e a n d b e n -
z i d i n e p o s i t i v e a n d w e r e c a p a b l e o f r e d u c i n gn i t r a t e t o n i t r i t e . The E n t e r o b a c t e r s t r a i n s o f C .p r i m u s a n d M. d a r w i n i e n s i s w e r e d e t e c t e d o n l y
o n t h e N - f r e e medium a n d n o t o n RCM. The N -
f r e e medium w o u l d f a v o r t h e g r o w t h o f t h eE n t e r o b a c t e r s p e c i e s a n d w o u l d p r e v e n t g r o w t ho f t h e S t r e p t o c o c c u s s p e c i e s ( s t r a i n s CP1 a n dM D 1 ) . A l l o f t h e E n t e r o b a c t e r s t r a i n s , w h e t h e ri s o l a t e d o n RCM a g a r o r N - f r e e a g a r , w e r e c a -p a b l e o f g r o w t h o n N - f r e e a g a r u n d e r a i r i n t h e
p r e s e n c e o f H 2 S O 4 , i n d i c a t i n g t h a t t h e y d i d n o t
r e q u i r e a n a e r o b i c o r m i c r o a e r o p h i l i c c o n d i t i o n s
t o g r o w o n N - f r e e a g a r . The o t h e r b a c t e r i a li s o l a t e s f o u n d o n RCM a g a r w e r e u n a b l e t o g r o w
u n d e r t h e s e c o n d i t i o n s , i n d i c a t i n g t h a t t h e N -f r e e a g a r w a s f r e e o f n i t r o g e n o u s c o m p o u n d s .
No c e l l u l o s e - d e g r a d i n g o r g a n i s m s w e r e i s o -l a t e d f r o m a n y o f t h e t e r m i t e s , b u t i s o l a t e s C A 1 ,C A 2 , C L 1 , C N i , C P 1 , H F 1 , N E l , N G 1 , N G 2 ,NW, a n d S 1 2 w e r e c a p a b l e o f u s i n g c e l l o b i o s e a s
a c a r b o n s o u r c e . C e l l u l o s e - d e g r a d i n g o r g a n i s m sw e r e r e a d i l y i s o l a t e d when t h e d i f f e r e n t c e l l u l o s em e d i a w e r e i n o c u l a t e d w i t h s o i l .
DISCUSSION
T e r m i t e s a r e c l a s s i f i e d i n t o t w o m a j o r p h y l o -g e n e t i c g r o u p s , t h e l o w e r t e r m i t e s a n d t h e h i g h e rt e r m i t e s . The d i s t r i b u t i o n p a t t e r n o f t h e b a c t e -r i a l g e n e r a i d e n t i f i e d i n e a c h t e r m i t e ( T a b l e 1 )s h o w s t h a t t h e r e i s a c o r r e l a t i o n b e t w e e n t h e
m a j o r g u t b a c t e r i u m a n d t h e f a m i l y t o w h i c h
t h e t e r m i t e b e l o n g s . T h u s i n t w o s p e c i e s o f t e r -
m i t e , M. d a r w i n i e n s i s ( f a m i l y M a s t o t e r m i t i d a e )a n d C . p r i m u s ( f a m i l y K a l o t e r m i t i d a e ) , b e l o n g -i n g t o t h e t w o l o w e s t f a m i l i e s o f t h e l o w e r t e r -m i t e s , S t r e p t o c o c c u s was t h e m a j o r b a c t e r i u m ,w h e r e a s i n f o u r s p e c i e s o f t e r mi t e s b e l o n g i n g t o
t h e R h i n o t e r m i t i d a e , w h i c h i s t h e h i g h e s t f a m i l yo f t h e l o w e r t e r m i t e s , E n t e r o b a c t e r wa s t h e m a-
j o r g u t b a c t e r i u m . The T e r m i t i d a e , o r h i g h e rt e r m i t e s , h a s f o u r s u b f a m i l i e s ( 2 0 ) . W e e x a m i n e d
t h r e e s p e c i e s b e l o n g i n g t o o n e o f t h e s e s u b f a m -i l i e s ( N a s u t i t e r m i t i n a e ) a n d f o u n d t h a t t h e i rm a j o r b a c t e r i u m wa s S t a p h y l o c o c c u s .K r a s i l ' n i k o v a n d S a t d y k o v ( 1 9 ) i s o l a t e d B a c -
t e r i u m a e r o g e n e s ( = K l e b s i e l l a a e r o g e n e s ? ) ,
V O L . 3 5 , 1 9 7 8
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8 2 6 E U T I C K , O ' B R I E N , AND S LAY TO R A P P L . E N V I R O N . M I C R O B I O L .
C:::g I+ + + II+ + I+ III+ I+ Z O*O*e
w ; 1+ + + + ++ +
]+ + I + + °°°°°°1L
:4~~~~~~~ zzzz||0| < < .*<
x |+ + + + + E
S| + + + + + + IC;C C C I e E B
.q~~~~ ~~~e+++d+ + + I+ + + + C<< _
-it _Q=WxC
° E> > = ZC ;3 e, );gs ^
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B A C T E R I A FROM GUT O F T E R M I T E S
B a c t e r i u m c l o a c a e ( = E n t e r o b a c t e r c l o a c a e ? ) ,
a n d S t r e p t o c o c c u s f a e c a l i s f r o m two s p e c i e s o f
A n a c a n t h o t e r m e s ( f a m i l y H o d o t e r m i t i d a e ) .
T h i s f a m i l y o f t e r m i t e s i s i n t e r m e d i a t e b e t w e e n
t h e K a l o t e r m i t i d a e a n d t h e R h i n o t e r m i t i d a e ,
a n d t h u s i t appears t h a t t h e g u t b a c t e r i a o f
A n a c a n t h o t e r m e s o v e r l a p t h e s e two f a m i l i e s .S t r e p t o c o c c u s s p . a n d E n t e r o b a c t e r a g g l o m e r -
ans h a v e b e e n i s o l a t e d f r o m R e t i c u l i t e r m e s f l a -v i p e s a n d C o p t o t e r m e s f o r m o s a n u s , b o t h o f
w h i c h are i n t h e R h i n o t e r m i t i d a e ( 5 , 2 2 ) .I n a s t u d y o f t h i s n a t u r e i t i s i m p o r t a n t t o
know w h e t h e r t h e g u t b a c t e r i a are c h a r a c t e r i s t i co f t h e t e r m i t e s , i r r e s p e c t i v e o f t h e i r g e o g r a p h i c a l
l o c a t i o n . S p e c i m e n s o f C . a c i n a c i f o r m i s c o l -l e c t e d i n t h r e e w i d e l y s e p a r a t e d l o c a t i o n s i nA u s t r a l i a a l l p o s s e s s e d E n t e r o b a c t e r as t h e i r
m a j o r g u t b a c t e r i u m . S a m p l e s o f t h e o t h e r t e r -
m i t e s c o l l e c t e d f r o m t h e . o n e g e n e r a l are a over a
l o n g p e r i o d a l s o h a d a c o n s t a n t m i c r o b i o t a .
A l l o f t h e t e r m i t e s , e x c e p t N . e x i t i o s u s a n d N .
w a l k e r i , h a r b o r e d E n t e r o b a c t e r spp. c a p a b l e o f
g r o w i n g on N - f r e e m e d i u m . I n t h o s e t e r m i t e s
w i t h E n t e r o b a c t e r as t h e m a j o r b a c t e r i u m t h e i rnumbers were very h i g h , s u g g e s t i n g t h e i r i m p o r -
t a n c e i n f i x i n g a t m o s p h e r i c n i t r o g e n t o p r o d u c e
a source o f f i x e d n i t r o g e n f o r t h e t e r m i t e . T wo
t e r m i t e s ( N . e x i t i o s u s a n d N . w a l k e r i ) f a i l e d t o
y i e l d any p u t a t i v e N 2 - f i x i n g b a c t e r i a . F r e n c h e t
a l . ( 1 2 ) f o u n d t h a t N . e x i t i o s u s w o u l d n o t f i xn i t r o g e n when f e d on w o o d , b u t w o u l d when f e d
on f i l t e r paper; no e x p l a n a t i o n was o f f e r e d f o rt h i s f i n d i n g . F r e n c h e t a l . ( 1 2 ) i s o l a t e d N 2 - f i x i n gb a c t e r i a f r o m f o u r o f t h e t e r m i t e s p e c i e s sur-
v e y e d i n t h i s s t u d y a n d i d e n t i f i e d a l l o f t h e i r
i s o l a t e s as C i t r o b a c t e r f r e u n d i i .Th e f u n c t i o n o f t h e o t h e r b a c t e r i a ( S t a p h y l o -
coccus, S t r e p t o c o c c u s , B a c i l l u s , and F l a v o b a c -
t e r i u m s p p . ) was n o t d i s c o v e r e d , b u t t h e y d i d
n o t appear t o b e i n v o l v e d i n c e l l u l o s e u t i l i z a t i o n ,s i n c e we were u n a b l e t o i s o l a t e a n y c e l l u l o s e -
d e g r a d i n g o r g a n i s m s . T h i s was n o t s u r p r i s i n g i n
t h e l o w e r t e r m i t e s , where t h e p r o t o z o a a re con-
s i d e r e d t o b e main a g e n t s o f c e l l u l o s e d e g r a d a -
t i o n ( 1 5 ) , b u t d o e s l e a v e op e n t h e q u e s t i o n o f
h ow t h e h i g h e r t e r m i t e s , which are d e v o i d o f
p r o t o z o a , d i g e s t c e l l u l a s e . T r a c e y an d Youatt
( 2 7 ) h a v e r e p o r t e d t h e presence o f c e l l u l a s e i n
N . e x i t i o s u s , b u t t h e y d i d n o t d e t e r m i n e whether
i t was o f m i c r o b i a l or t e r m i t e o r i g i n .T h e g u t o f t h e t e r m i t e s f a i l e d t o y i e l d any
s t r i c t l y a n a e r o b i c b a c t e r i a . I n a d d i t i o n , t h e En -
t e r o b a c t e r i s o l a t e s were a b l e t o grow on N - f r e e
m e d i u m u n d e r a i r . These r e s u l t s a re c o n s i s t e n t
w i t h t h e f i n d i n g t h a t t h e g u t s o f b o t h C . l a c t e u sa n d N . e x i t i o s u s were a e r o b i c b u t o f l o w E h ( 1 1 ) .
ACKNOWLEDGMENTS
We t h a n k L . I. S l y , D e p a r t m e n t o f M i c r o b i o l o g y , U n i v e r s i t y
o f Q u e e n s l a n d , f o r t h e f l a g e l l a t i o n d e t e r m i n a t i o n s an d f o r
ma ny h e l p f u l c o m m e n t s o n t a x o n o m i c t e s t i n g . W e a r e i n -d e b t e d t o J . A . L . W a t s o n , CSIRO D i v i s i o n o f E n t o m o l o g y ,
C a n b e r r a , f o r i d e n t i f y i n g a n d s u p p l y i n g l i v e t e r m i t e s .M . L . E . w a s t h e r e c i p i e n t o f a n A u s t r a l i a n G o v e r n m e n t
P o s t g r a d u a t e S t u d e n t s h i p .
L I T E R A T U R E CITED
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y l o c o c c i a n d m i c r o c o c c i , p . 5 9 - 6 4 . I n B . M. G i b b s a n d
F . A . S k i n n e r ( e d . ) , I d e n t i f i c a t i o n m e t h o d s f o r m i c r o -
b i o l o g i s t s , p a r t A . A c a d e m i c P r e s s I n c . , L o n d o n .
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t e r m i t e s a n d t h e i r i n t e s t i n a l m i c r o b i o t a , p . 5 5 9 - 5 8 0 . I n
D . H . J e n n i n g s a n d D . L . L e e ( e d . ) , S y m b i o s i s ( S o c i e t y
f o r E x p e r i m e n t a l B i o l o g y S y m p o s i u m S e r . , n o . 2 9 ) .C a m b r i d g e U n i v e r s i t y P r e s s , C a m b r i d g e .
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g e y ' s m a n u a l o f d e t e r m i n a t i v e b a c t e r i o l o g y , 8 t h e d . The
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F . M . W e e s n e r ( e d . ) , B i o l o g y o f t e r m i t e s , v o l . 2 . A c a -
d e m i c P r e s s I n c . , N e w Y o r k .
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I I . An a n a e r o b i c c e l l u l o s e d e c o m p o s i n g a c t i n o m y c e t e ,
M i c r o m o n o s p o r a p r o p i o n i c i n . s p . J . B a c t e r i o l .5 1 : 5 1 - 5 6 .
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8 2 7O L . 3 5 , 1 9 7 8
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828 EUTICK, O'BRIEN, AND S L A Y T O R
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