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Some effects of methallibure (I.C.I. 33828) on the stickleback : Gasterosteus aculeatus L. Carew, Barbara Agnes Mary 1968

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SOME EFFECTS OF METHALLIBURE ( I . C . I . 33,828) ON THE STICKLEBACK, GASTEROSTEUS ACULEATUS L.  by BARBARA AGNES MARY CAREW B.Sc., Memorial U n i v e r s i t y o f Newfoundland, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of Zoology We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1968  In  presenting  for  an  that  advanced  thesis  shall  I further  agree  for scholarly  Department  or by  pub 1 i c a t i o n  without  thesis  degree  the Library  Study.  or  this  my  Department  in partial  at the U n i v e r s i t y  make  that  permission  may  be  h.i)s r e p r e s e n t a t i v e s .  written  thesis  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  granted  Columbia  by  requirements  Columbia,  t h e Head  shall  and  copying  It i s understood  gain  I agree  for reference  for extensive  for financial  permission.  the  of B r i t i s h  i t freely. avai1ab1e  purposes  of this  fulfilment of  of  of  this  my  that  n o t be  copying  allowed  i  ABSTRACT  Treatment o f male s t i c k l e b a c k s w i t h m e t h a l l i b u r e b r i n g s about a marked r e d u c t i o n i n t h e l e v e l o f prespawning aggressiveness.  Gametogenesis  i n the testes o f the  t e s t e s o f t h e t r e a t e d f i s h i s slowed down so t h a t a f t e r 30-38 days o f t r e a t m e n t most t e s t e s c o n t a i n  spermatozoa  and spermatogonia w h i l e t h e t e s t e s o f t h e c o n t r o l s a r e more mature and c o n t a i n spermatocytes and spermatozoa o r spermatozoa o n l y .  F o r t y - n i n e days o f t r e a t m e n t r e s u l t i n  t e s t e s w i t h spermatogonia, spermatocytes and spermatozoa, c o n t r o l t e s t e s a r e c o m p l e t e l y mature and c o n t a i n o n l y spermatozoa.  M e t h a l l i b u r e causes no s i g n i f i c a n t  increase  i n t h y r o i d e p i t h e l i a l c e l l height evidence t h a t the I.C.I, compound i s n o t h a v i n g a d i r e c t g o i t r o g e n i c e f f e c t such as i s found when f i s h a r e immersed i n a s o l u t i o n o f t h i o u r e a . The adenohypophysis o f m e t h a l l i b u r e t r e a t e d f i s h  contains  l e s s b a s o p h i l i c m a t e r i a l than t h a t o f u n t r e a t e d c o n t r o l s .  ii  TABLE OF CONTENTS Page I.  INTRODUCTION  1  II.  METHODS AND MATERIALS  7  III.  A. Sex D e t e r m i n a t i o n  8  B. B e h a v i o r O b s e r v a t i o n s  8  C. M e t h a l l i b u r e I n j e c t i o n and Immersion  10  D. L u t e i n i z i n g Hormone I n j e c t i o n ....  11  E. T h i o u r e a Immersion  11  F. G e n e r a l C o n d i t i o n o f F i s h  13  G.  13  Histology  RESULTS  15 A. B e h a v i o r S t u d i e s  15  1. Prespawning  15  A g g r e s s i v e n e s s ....  a) M e t h a l l i b u r e Treatment  15  b) Other Treatments i ) L u t e i n i z i n g Hormone  21 21  i i ) T h i o u r e a Immersion ..... 2. Nest B u i l d i n g B. H i s t o l o g y 1. T e s t i s and K i d n e y a) M e t h a l l i b u r e Treatment on the T e s t i s b) M e t h a l l i b u r e Treatment on  23 23 25 25 32  the Kidney  35  c) LH Treatment  37  d) T h i o u r e a Treatment  37  e) GSI  38  iii Page 4. T h y r o i d  38  a) M e t h a l l i b u r e and T h i o u r e a Treatment 5. P i t u i t a r y  40  a) Morphology b) M e t h a l l i b u r e and T h i o u r e a Treatment IV.  DISCUSSION A. Prespawning  38  40 42 47  A g g r e s s i v e B e h a v i o r ..  50  B. T e s t i s and K i d n e y  52  C. P i t u i t a r y  57  V.  CONCLUSIONS  60  VI.  BIBLIOGRAPHY  61  iv  LIST OF TABLES Page I  Summary o f E x p e r i m e n t s Performed  II  Amount o f A g g r e s s i v e B e h a v i o r i n M e t h a l l i b u r e - t r e a t e d and C o n t r o l s  III  20  Number o f F i s h used f o r E x a m i n a t i o n of T e s t i s Histology  IV  12  44  E f f e c t o f M e t h a l l i b u r e , LH and T h i o u r e a on H i s t o l o g y o f t h e T e s t i s and t h e Kidney  V  G.S.I.  34 Comparison o f E f f e c t o f  M e t h a l l i b u r e and T h i o u r e a Treatment on Gonosomatic Index VI  Mean T h y r o i d C e l l H e i g h t ,  39 following  T h i o u r e a and M e t h a l l i b u r e Treatment VII  41  Amount o f G r a n u l a t i o n i n t h e B a s o p h i l s (Thyrotrophs and Gonadotrophs i n Mesoadenohypophysis) a f t e r M e t h a l l i b u r e and T h i o u r e a Immersion  43  V LIST OF FIGURES Figure 1  Facing Aggressive l e v e l s of sticklebacks injected with methallibure with  compared  controls  17  Aggressive l e v e l s of sticklebacks immersed i n m e t h a l l i b u r e with  compared  controls  18  Aggressive l e v e l s of sticklebacks i n j e c t e d w i t h LH and LH + METH compared with  controls  19  Aggressive l e v e l s of sticklebacks immersed i n t h i o u r e a and compared w i t h  methallibure  controls  24  Diagrams o f t h e Stages o f T e s t i s Maturation 5  Stage 0  26  6  Stage I  26  7  Stage I I  27  8  Stage I I I  27  9  Stage IV  28  10 - 17  Photomicrographs o f t h e Stages o f Testis Maturation  10  Stage 0  x  200  30  11  Stage 0  x  900  30  Page  Figure  F a c i n g Page  12  Stage I I I x  200  30  13  Stage I I I x  900  30  14  Stage I I  x  200  31  15  Stage IV  x  200  31  16  Stage 0  x  900  31  17  Stage IV  x  900  31  18 - 24  Photomicrographs o f t h e P i t u i t a r y  18  M e t h a l l i b u r e t r e a t e d x 200  45  19  M e t h a l l i b u r e t r e a t e d x 900  45  20  Control  x 200  45  21  Control  x 900  45  22  Thiourea treated  x 200  46  23  Thiourea treated  x 900  46  24  Thiourea treated  x 200  46  ACKNOWLEDGEMENT  I would l i k e t o express my g r a t i t u d e t o Dr. W.S. Hoar f o r h i s guidance, c r i t i c i s m and encouragement t h r o u g h o u t t h e p r o j e c t and d u r i n g t h e p r e p a r a t i o n o f the m a n u s c r i p t .  I am i n d e b t e d t o Dr. J.A. Dodds* f o r t h e  s u p p l i e s o f I . C . I . 33,828 and t o Dr. A.E. W i l h e l m i * * f o r the p u r i f i e d b o v i n e LH.  I would l i k e t o thank Dr. J . F .  L e a t h e r l a n d , Dr. M.S. J a r i a l , Mr. L. Sharman and Mr. C. G r a i n g e f o r t h e i r h e l p w i t h t h e h i s t o l o g y and Mr. W.R. B l a c k , Mr. P. E l l i c k s o n and Mr. C. L e v i n g s  for their  a s s i s t a n c e s e i n i n g a t t h e L i t t l e Campbell R i v e r .  I am  p a r t i c u l a r l y g r a t e f u l t o Mr. D.L. Kramer, Mr. S. Pandey and Dr. J . P . Wiebe f o r t h e i r many h e l p f u l s u g g e s t i o n s . The  r e s e a r c h was made p o s s i b l e through t h e f i n a n c i a l  o f t h e N a t i o n a l Research C o u n c i l o f Canada.  • V e t e r i n a r y M e d i c a l Department, A y e r s t L a b o r a t o r i e s , M o n t r e a l , P.Q. * * E n d o c r i n o l o g y Study S e c t i o n , Department o f B i o c h e m i s t r y , Emory U n i v e r s i t y , A t l a n t a , Georgia  support  1  I.  INTRODUCTION  A number o f s t u d i e s have h e l p e d e l u c i d a t e t h e characteristic  reproductive behavior of the three-spined  s t i c k l e b a c k G a s t e r o s t e u s a c u l e a t u s L. (Tinbergen and t e r P e l k w i j k , 1937; van I e r s e l , 1953, 1959). sequence i s as f o l l o w s :  B r i e f l y the  In the l a t e w i n t e r or e a r l y s p r i n g  t h e f i s h show a d e c r e a s e d tendency  to school.  The males  defend d i s c r e t e t e r r i t o r i e s a g a i n s t i n t r u d e r s o f e i t h e r sex a l t h o u g h t h e y a r e p a r t i c u l a r l y a g g r e s s i v e towards o t h e r males.  D u r i n g t h i s t i m e t h e s i l v e r y w i n t e r f i s h develop  characteristic  breeding coloration.  The l a t e r a l and d o r s a l  s u r f a c e s o f b o t h males and females darken t o a m o t t l e d black.  green-  The t h r o a t r e g i o n of. t h e males becomes r e d and t h e i r  eyes change from s i l v e r t o b r i g h t b l u e .  L a t e r t h e males  c o l l e c t a l g a e and g l u e i t t o g e t h e r t o form a n e s t w i t h a s t i c k y mucous s e c r e t i o n produced b y t h e k i d n e y s a t t h i s stage.  A f t e r t u n n e l i n g through t h e n e s t t h e male i s ready  t o c o u r t a g r a v i d female.  The male l e a d s t h e female t o t h e  n e s t w i t h a d i s t i n c t i v e z i g - z a g swimming motion.  When t h e  male p o i n t s t o t h e n e s t e n t r a n c e t h e female e n t e r s and i n response t o h i s p r o d s , spawns. The male f o l l o w s through t h e n e s t and f e r t i l i z e s t h e eggs. away.  He then chases the female  The male c o n t i n u e s t o defend t h e t e r r i t o r y and c a r e s  f o r t h e c l u t c h o f eggs b y f a n n i n g w a t e r over them w i t h h i s  2  r a p i d l y moving p e c t o r a l s .  When t h e eggs have hatched  male guards the young f o r a few d a y s .  the  T h i s completes the  reproductive cycle. The p r e c e d i n g  d e s c r i p t i o n of reproductive behavior  a p p l i e s t o the s a l t w a t e r form o f G a s t e r o s t e u s w h i c h e n t e r s fresh water to breed.  I have used o n l y the f r e s h w a t e r  which l i v e s a l l year i n f r e s h water.  Hagen (1967)  form  who  s t u d i e d b o t h forms i n the Vancouver a r e a , f i n d s the  aggressive  t e r r i t o r i a l and r e p r o d u c t i v e b e h a v i o r o f b o t h v e r y s i m i l a r and he can d e t e c t no b e h a v i o r a l b a r r i e r s t o i n t e r b r e e d i n g between t h e two The  forms.  r o l e o f the e n d o c r i n e  system i n t h i s  behavior  sequence i s e v i d e n t l y complex and a t p r e s e n t o n l y p a r t i a l l y understood.  Techniques used i n e n d o c r i n o l o g i c a l s t u d i e s  r e l y h e a v i l y on o b s e r v a t i o n s o f the e f f e c t s o f removal o f an e n d o c r i n e g l a n d and subsequent replacement t h e r a p y i n v o l v i n g i n j e c t i o n s o f g l a n d e x t r a c t s o r s y n t h e t i c hormones. Removal o f the gonads o f s t i c k l e b a c k s i s a  comparatively  s i m p l e o p e r a t i o n and has been s u c c e s s f u l l y c a r r i e d o u t by a number o f w o r k e r s (Baggerman, 1957; Wai  Hoar, 1962  a, b;  and Hoar, 1963); removal o f the p i t u i t a r y , however, i s  d i f f i c u l t and t h e o p e r a t i o n has n o t y e t been s u c c e s s f u l with Gasterosteus. photoperiod  I n v e s t i g a t o r s have t h e r e f o r e used  c o n t r o l to regulate p i t u i t a r y  activity.  Baggerman (1957) showed t h a t t h e s e a s o n a l  reproductive  c y c l e o f s t i c k l e b a c k i s l a r g e l y c o n t r o l l e d by t h e changing  3  l i g h t c o n d i t i o n s w h i l e t h e e f f e c t s o f temperature a r e additive.  I t i s assumed t h a t s h o r t p h o t o p e r i o d  (8 h r o f  l i g h t a l t e r n a t i n g w i t h 16 h r o f darkness) suppresses t h e high gonadotrophin production c h a r a c t e r i s t i c o f breeding f i s h , w h i l e long photoperiod  (16 h r o f l i g h t a l t e r n a t i n g w i t h  8 h r o f darkness) r e a d i l y b r i n g s f i s h i n t o condition.  reproductive  I n v e s t i g a t o r s of the endocrinology  of stickle-  b a c k r e p r o d u c t i o n have used t h e d i f f e r e n c e s i n response, t o v a r i o u s treatments, i  o f short photoperiod  t  physiologically  hypophysectomized f i s h compared w i t h l o n g p h o t o p e r i o d Wai  fish.  and Hoar (1963) used gonadectomy and subsequent  treatment w i t h methyl t e s t o s t e r o n e on l o n g and s h o r t p h o t o p e r i o d  (a s y n t h e t i c androgen)  s t i c k l e b a c k s . They found t h e  h i g h l e v e l o f pre-spawning a g g r e s s i v e n e s s ,  characteristic  o f t e r r i t o r i a l f i s h p r i o r t o n e s t b u i l d i n g , appeared:in gonadectomized f i s h under l o n g p h o t o p e r i o d b u t n o t i n gonadectomized f i s h under s h o r t p h o t o p e r i o d .  The a d d i t i o n  of methyl t e s t o s t e r o n e d i d not g r e a t l y a f f e c t the aggressiveness treatment  o f these c a s t r a t e d f i s h .  The androgen  d i d , however, r e s t o r e t h e secondary sex c h a r a c t e r s  (mucous s e c r e t i n g k i d n e y t u b u l e s and n u p t i a l c o l o r a t i o n ) w h i c h had been l o s t a f t e r c a s t r a t i o n . C a s t r a t e d f i s h showed n e i t h e r nest b u i l d i n g nor sexual behavior. t e s t o s t e r o n e treatment,  Methyl  however, r e s u l t e d i n t h e appearance  o f b o t h t h e s e b e h a v i o r a l sequences i n males under b o t h photoperiod  regimes.  These b e h a v i o r p a t t e r n s reappeared  more q u i c k l y and i n more o f t h e l o n g p h o t o p e r i o d  fish.  4  The  e v i d e n c e from t h e above s t u d i e s i s t h a t p i t u i t a r y  g o n a d o t r o p h i n s c o n t r o l pre-spawning a g g r e s s i v e n e s s ,  while  gonadal s t e r o i d s c o n t r o l secondary sex c h a r a c t e r s , and b o t h g o n a d o t r o p h i n s and androgen a c t s y n e r g i s t i c a l l y t o produce n e s t b u i l d i n g and s e x u a l b e h a v i o r  (Hoar, 1965).  To o b t a i n a d d i t i o n a l e v i d e n c e o f the e n d o c r i n e o r pre-spawning b e h a v i o r technique  and gametogenesis, a  o f s u p p r e s s i n g g o n a d o t r o p h i n was  Wiebe and Wai  pharmacological  sought.  Hoar,  (1967) d i s c u s s e d p r e l i m i n a r y r e s u l t s  using methallibure  control  obtained  ( I . C . I . 33,828), a n o n - s t e r o i d ,  hormonal compound, on t h r e e s p e c i e s o f f i s h .  non-  These e x p e r i -  ments, u s i n g s t i c k l e b a c k s , g o l d f i s h and s u r f p e r c h s t r o n g l y indicate that methallibure selectively i n h i b i t s p i t u i t a r y function.  The  gonads o f the t r e a t e d f i s h showed d i v i s i o n o f  t h e p r i m a r y germ c e l l s b u t no m a t u r a t i o n C o n t r o l s e x h i b i t e d normal germ c e l l  of these  cells.  maturation.  The d a t a r e p o r t e d b y Hoar e t a l . (1967) on b a c k s were c o n s i d e r e d p r e l i m i n a r y .  M o r t a l i t y was  t h e t r e a t e d f i s h and the sample s i z e was  small.  w e l l as the e f f e c t on gametogenesis, t h e r e was o f an e f f e c t on gonadal s t e r o i d p r o d u c t i o n .  sticklehigh i n  However, as an  The  indication c e l l s of  t h e b r u s h b o r d e r segment o f the k i d n e y t u b u l e s remained a t a s i z e c h a r a c t e r i s t i c o f immature f i s h i n t r e a t e d s t i c k l e backs.  The c e l l s o f t h i s segment o f t h e k i d n e y become  l a r g e w i t h g r a n u l a r c y t o p l a s m under the i n f l u e n c e o f androgen. Wiebe (1968) c o n t i n u e d t h e i n v e s t i g a t i o n on Cymatogaster aggregata.  He  surfperch  found t r e a t m e n t w i t h m e t h a l l i b u r e  5  r e s u l t e d i n t e s t i s atrophy,  regression of the i n t e r s t i t i a l  c e l l s and l a c k o f i n c r e a s e i n s i z e o f t h e f l e s h y m o d i f i c a t i o n s o f t h e a n a l f i n . These m o d i f i c a t i o n s o f t h e a n a l f i n a r e a secondary sex s t r u c t u r e w h i c h d e v e l o p under t h e i n f l u e n c e o f male gonadal s t e r o i d .  No marked a n t i -  t h y r o i d e f f e c t was d e t e c t e d when t h e m e t h a l l i b u r e t r e a t e d f i s h were compared w i t h a group t r e a t e d w i t h t h e g o i t r o g e n , thiourea.  Wiebe (1967) r e p o r t e d t h a t t h e r e was a s i g n i f i c a n t  drop i n t h e r e p r o d u c t i v e b e h a v i o r o f m e t h a l l i b u r e t r e a t e d f i s h compared w i t h u n t r e a t e d c o n t r o l s . The p r e s e n t s t u d y c o n t i n u e s t h e i n v e s t i g a t i o n o f t h e e f f e c t s o f m e t h a l l i b u r e on G a s t e r o s t e u s understanding  to further the  o f the c o n t r o l o f t h e p i t u i t a r y gonadotrophins  on p r e s p a w n i n g a g g r e s s i v e b e h a v i o r and t e s t i s  physiology.  These two a s p e c t s o f t h e s t u d y a r e c l o s e l y i n t e r r e l a t e d .  The  work t o d a t e suggests t h a t m e t h a l l i b u r e a c t s d i r e c t l y on t h e p i t u i t a r y gonadotrophs r a t h e r t h a n t h e t i s s u e s o f t h e t e s t i s ( L e a t h e r l a n d and Pandey, 1968).  T h i s has been i n v e s t i g a t e d  h i s t o p h y s i o l o g i c a l l y b y examining s e c t i o n s o f t h e t e s t i s ((.game t o genie t i s s u e ) , t h e h e i g h t o f t h e k i d n e y t u b u l e s (as a measure o f s t e r o i d e f f e c t s ) , and t h e p i t u i t a r y ( f o r b a s o p h i l s ) i n t r e a t e d f i s h and u n t r e a t e d c o n t r o l s . At present, p i t u i t a r y gonadotrophin i s thought t o c o n t r o l d i r e c t l y the increased aggressiveness  o f male  s t i c k l e b a c k s when t h e y s e t up and m a i n t a i n d i s c r e t e t e r r i t o r i e s p r i o r t o spawning.  I f methallibure i s blocking  t h e a c t i o n o f g o n a d o t r o p h i n t h e n presumably t r e a t e d s t i c k l e -  6  b a c k s s h o u l d be l e s s a g g r e s s i v e than u n t r e a t e d c o n t r o l s . b r i e f , t h e p r i m a r y o b j e c t i v e o f the s t u d y was  to investigate  t h e e f f e c t s o f m e t h a l l i b u r e on pre-spawning a g g r e s s i v e b e h a v i o r and t o i n v e s t i g a t e t h e r e s u l t s i n terms o f the gonadotrophic physiology.  c o n t r o l over t h i s b e h a v i o r and  In  testicular  7  II.  METHODS AND MATERIALS  F r e s h w a t e r s t i c k l e b a c k s t y p e B'  (Heuts, 1947) were  caught a t v a r i o u s t i m e s t h r o u g h o u t t h e y e a r i n t h e L i t t l e Campbell R i v e r south o f Vancouver, B r i t i s h Columbia.  They  were k e p t u n t i l needed f o r the experiments i n l a r g e t a n k s of running dechlorinated water.  A l t h o u g h t h e t a n k s were  housed i n t h e l a b o r a t o r y , the p h o t o p e r i o d was r e g u l a t e d a t the  n a t u r a l l e n g t h by a p h o t o c e l l p l a c e d i n a south window.  The w a t e r temperature v a r i e d between 5° and 14° C depending on t h e t i m e o f y e a r .  A t t h e o u t s e t o f an experiment,  fish  were p l a c e d i n groups o f two o r f o u r i n 16 l i t r e g l a s s a q u a r i a (45 l i t r e g l a s s a q u a r i a were used i n one experiment, No. 8 ) . D e c h l o r i n a t e d w a t e r f o r t i f i e d w i t h 20 ppm c a l c i u m c h l o r i d e was c o n t i n u a l l y a e r a t e d and f i l t e r e d through g l a s s w o o l ; the  f i l t e r s were changed once a week.  During the experiments  the  a q u a r i a were k e p t i n an a i r - c o n d i t i o n e d room which  m a i n t a i n e d w a t e r temperature a t 20 + 1°C.  The p h o t o p e r i o d  p r o v i d e d 16 h r o f l i g h t a l t e r n a t i n g w i t h 8 h r o f d a r k n e s s . Illumination  (50-55 f t - c ) was p r o v i d e d by f l u o r e s c e n t lamps.  A l l a q u a r i a c o n t a i n e d sand and w a t e r s p r i t e sp.).  (Ceratopteris  The p l a n t s c o v e r e d about 75% o f t h e w a t e r s u r f a c e . The f i s h were f e d f r o z e n b r i n e shrimp ( A r t e m i a  s a l i n a ) once a day.  As a p r e c a u t i o n a g a i n s t f u n g a l  infection,  2-3 drops o f 1% m a l a c h i t e green s o l u t i o n were u s u a l l y added t o each aquarium a t t h e b e g i n n i n g o f t h e e x p e r i m e n t s .  8  A.  Sex D e t e r m i n a t i o n Male f i s h were used i n t h e e x p e r i m e n t s .  Usually,  males c o u l d be d i s t i n g u i s h e d from females b y t h e g r e a t e r amount o f r e d p i g m e n t a t i o n i n t h e t h r o a t r e g i o n . . However, i n one experiment  (No. 5) no t r a c e o f c o l o r c o u l d b e seen,  so a t i n y i n c i s i o n was made i n one s i d e o f t h e f i s h .  Males  were d i s t i n g u i s h e d from females on t h e b a s i s o f t h e d i f f e r e n c e i n shape and p i g m e n t a t i o n o f t h e gonads. males were k e p t i n 50% s e a w a t e r f o r 48 h r a f t e r  The  this  o p e r a t i o n b e f o r e they were p l a c e d i n groups o f two i n t h e glass aquaria. the i n c i s i o n .  A p r e p a r a t i o n c a l l e d Orabase* was used  over  T h i s p r e p a r a t i o n adhered t o and c l o s e d t h e  wound f o r an h o u r o r two, a f t e r w h i c h i t dropped o f f . B.  Behavior Observations I n t h e b e h a v i o r s t u d i e s , a l l a q u a r i a were observed f o r p e r i o d s o f f i v e minutes two o r t h r e e o c c a s i o n s each week.  The b e h a v i o r observed was o f two main t y p e s :  (1) A g g r e s s i v e b e h a v i o r and (2) N e s t - b u i l d i n g b e h a v i o r . Three components o f a g g r e s s i v e b e h a v i o r were d i s t i n g u i s h e d : (a) Approach where one f i s h d a r t s a t another b u t s t o p s before reaching i t ,  t h i s t y p e o f b e h a v i o r i s v e r y common  when two o r more f i s h have e s t a b l i s h e d t e r r i t o r i e s i n a s i n g l e t a n k ; (b) B i t e where one f i s h makes a d a r t a t a n o t h e r and b i t e s i t once o r s e v e r a l t i m e s ; (c) Chase where t h e a g g r e s s i v e f i s h p u r s ues t h e s u b o r d i n a t e one who must * Squibb O r a l P r o t e c t i v e P a s t e  9  seek s h e l t e r .  The n e s t b u i l d i n g  (van I e r s e l ,  1953)  c o n s i s t e d o f : (a) S a n d - d i g g i n g where a male, i n a head down p o s t u r e , p i c k s up a m o u t h f u l o f sand, t u r n s , swims away and s p i t s i t o u t ; (b) S e a r c h i n g where a male p i c k s up s m a l l p i e c e s o f r o o t s o r a l g a e and s p i t s them o u t ; (c) B r i n g i n g m a t e r i a l where a male b r i n g s p i e c e s o f a l g a e or p l a n t to the n e s t - s i t e ;  (d) G l u i n g where a male swims  o v e r t h e n e s t a r e a w h i l e s e c r e t i n g a mucous from t h e k i d n e y and  (e) F a n n i n g where a male adopts a head down p o s i t i o n a t  30°-40° t o t h e h o r i z o n t a l a t t h e n e s t e n t r a n c e and  maintains  t h i s p o s i t i o n b y t h e combined f o r w a r d swimming movements o f t h e t a i l and t h e backward swimming movements o f the pectorals. Q u a n t a t i v e d a t a were o b t a i n e d b y the use o f an E s t e r l i n e - A n g u s pen r e c o r d e r w i t h t h e k e y s numbered so t h a t each number corresponded  t o a p a r t i c u l a r b e h a v i o r movement.  Thus, when a b e h a v i o r p a t t e r n was key was was  depressed.  observed t h e a p p r o p r i a t e  The f r e q u e n c y o f t h e v a r i o u s p a t t e r n s  recorded. The  f i s h i n each aquarium were a s s i g n e d a l e v e l o f  a g g r e s s i o n f o r each o b s e r v a t i o n p e r i o d , based on t h e f o l l o w ing c r i t e r i a  (Hoar, 1962b).  L e v e l 0:  No a t t a c k s , f i s h f r e q u e n t l y swim around i n  a l o o s e group. Level It  One o r two a t t a c k s p e r minute ( t e n o r  fewer i n f i v e m i n u t e s ) ; l o o s e s c h o o l i n g , no  social  o r d e r ; no e v i d e n c e o f dominance o r s u b o r d i n a t i o n .  10  Level I I :  Three o r more a t t a c k s p e r minute,  fish  o f e q u a l o r near e q u a l s t a t u s , no dominance o r subordination. Level I I I : T e r r i t o r i a l r e l a t i o n s h i p but p a r t i c u l a r dominance, one  f i s h has  no  a preferred  and b i t e s f i s h e n t e r i n g t h i s area b u t does n o t v i g o r o u s l y or subordinate L e v e l IV:  other  A dominance-subordinate r e l a t i o n s h i p i s  v i g o r o u s l y , d r i v e s them i n t o c o r n e r s and  fish  sometimes  When more than one dominant i s i n the  t a n k the t e r r i t o r i a l b o u n d a r i e s are The  fight  fish.  w e l l marked; the dominant f i s h a t t a c k s o t h e r  k i l l s them.  area  number o f a g g r e s s i v e  sharp.  acts i s u s u a l l y greatly  reduced a t L e v e l IV, s i n c e a t a n k o f t e n c o n t a i n s a s i n g l e dominant male w i t h a l l s u b o r d i n a t e s e n c o u n t e r s between f i s h are few.  i n h i d i n g , so t h a t  C o n s e q u e n t l y , i n most  e x p e r i m e n t s once a week, a " s t a n d a r d " was  used.  The  "standard"  f i s h was  female o r j u v e n i l e f i s h  p l a c e d i n s i d e a g l a s s tube  (6.5 cm i n d i a m e t e r ) and tubes w i t h f i s h p l a c e d i n t h e m i d d l e r e g i o n o f a l l t a n k s w h i c h were r a t e d L e v e l IV d u r i n g  the  previous observation period.  fish  U s u a l l y the "standard"  would a c t i v e l y swim up and down i n s i d e the g l a s s tube. number o f a t t a c k s on the tube i n f i v e minutes was C.  M e t h a l l i b u r e I n j e c t i o n and S e v e r a l dosages and M e t h a l l i b u r e were t e s t e d  The  recorded.  Immersion f r e q u e n c i e s o f dose o f  (Table I ) .  I n most experiments a  11  f i n e suspension o f m e t h a l l i b u r e i n d i s t i l l e d water o r f r e s h w a t e r t e l e o s t s a l i n e * was p r e p a r e d w i t h a s m a l l amount (one drop p e r 5 ml) o f t h e w e t t i n g agent "Tween 80". 0.05 o r 0.025 ml o f s u s p e n s i o n was i n j e c t e d p e r i t o n e a l l y w i t h a 26 G. n e e d l e . made on a l t e r n a t e  Either  intra-  S u c c e s s i v e i n j e c t i o n s were  sides o f the f i s h .  In the f i r s t  experiments  t h e r e was some l e a k a g e o f i n j e c t e d s u s p e n s i o n so "Orabase" was a p p l i e d t o t h e h o l e made b y t h e n e e d l e .  I n experiments  No. 6 and 7 t h e u s e o f "Orabase" was c o n s i d e r e d u n n e c e s s a r y because t h e i n j e c t e d volume was so s m a l l . I n t h r e e o f t h e e i g h t experiments m e t h a l l i b u r e was added t o t h e ambient w a t e r o f a group o f e x p e r i m e n t a l f i s h . D e t a i l s o f t h e amount added a r e i n T a b l e I . p. L u t e i n i z i n g Hormone I n j e c t i o n * * In experiments No. 6 and 7, one group o f f i s h was i n j e c t e d w i t h 0.20 mg o f LH i n 0.025 ml d i s t i l l e d t w i c e a week.  A second group i n No. 6 and 7 was  water injected  w i t h a m i x t u r e o f m e t h a l l i b u r e and LH (0.20 mg LH + 0.20 mg methallibure) . E.  Thiourea  Immersion In experiment No. 8, a group o f f i s h was t r e a t e d  w i t h t h i o u r e a b y immersion w i t h 1.5 gm o f t h i o u r e a added t o the 45 l i t r e t a n k s e v e r y week f o r seven weeks. *1 l i t r e o f f r e s h w a t e r t e l e o s t s a l i n e c o n t a i n s 5.50 gm N a C l , 0.14 gm KC1, 0.12 gm C a C l i n d i s t i l l e d water. 2  **NIH-LH-B5 B o v i n e  CTl  (X) CD oo d tr rt rt O O > to I— I- to H  1  1  Ul  if  M tr) 1-. II O II tf tf  rt 0  S oo P>  rt 0  £ to g oo 0) o c  O M O M  (D O  rt  CD O  rt 0  3 0 < rt 0  to  O  00  Ul  Ul  Ul  Ul  Ul  o  o  o  o  o  o  Ul  Ul  Ul  Ul  o  CO  00  CO  o  00  00  o  o  o  Ul  Ul  Ul  to CO  00  to  Ul  rt H-  o 3  No. fish Arat. mg Vol. ml  Ul  o 00  o  o  o  o  Ul  Ul  Ul  Ul  Days b e t . inj. No. fish Vol. ml Days b e t . inj.  CO  00  &>  00  O to to  H.  to O O to D H o to CD to CD to (D O O  to to Ul  00  rt 0  td §3  CO  if  It)  n  to o  Ul  No. Fish  00  o  o  IO  to  No fish Amt. mg  o  o  Ul  Ul  Vol. ml  to  to  to (jO  to  No. f-i-sh Amt. mg.  o  Ul  Ul  to  * * Ul CTl  o  o  CO  to co CO  00 CO  o * o *  ft' f+ CD ID Ul 00  Ul  Ul  3  CD O H rt CD Qj d CD H 3  oo QJ  I—I.  Ul  Ul  ish Amt. gm No days  per  week.  1  9£ 8S  p> n «< o  1  i-3 td >  H 3  (D r o a rt CD  3 (D  H rt 3 3 —1.(1)  1  CD H* O H Vol. rtH' (D ml H Days b e t . CD inj. No. fish Amt. ma Vol. ml No days per week  d  CD hO Hrt 3 CD (D  Days b e t .  CO  42 to  S H CD  1  S td Si  Hi  td CD H H" 3 CD 3 rt 01  T) CD Hi 0 H 3 CD &  8 F td H  13  F.  General Condition o f F i s h O n l y f i s h w h i c h appeared h e a l t h y were used f o r experiments.  However, t h e r e were m o r t a l i t i e s among b o t h  e x p e r i m e n t a l and c o n t r o l f i s h i n most experiments.  I n some  cases t h e f i s h d e v e l o p e d f u n g a l i n f e c t i o n b u t i n o t h e r s t h e cause o f d e a t h was n o t apparent.  The i n i t i a l i n j e c t i o n doses  o f m e t h a l l i b u r e may have been t o o h i g h b u t i n l a t e r e x p e r i ments m o r t a l i t i e s were about e q u a l i n e x p e r i m e n t a l and c o n t r o l groups.  The e x c e p t i o n s were groups i n j e c t e d w i t h  LH a l o n e o r i n c o m b i n a t i o n s w i t h m e t h a l l i b u r e .  The h i g h e r  m o r t a l i t y i n t h e s e groups was p r o b a b l y due t o t h e j o i n t e f f e c t s o f LH and l o n g p h o t o p e r i o d  ( n a t u r a l gonadotrophin  a t h i g h l e v e l s ) s i n c e t h e dose o f LH was t h e same as t h a t used b y Hoar (1962b) on s t i c k l e b a c k s k e p t under s h o r t photoperiod, G.  when i t had no i l l  effects.  Histology A l l f i s h were k i l l e d b y an overdose o f t r i c a i n e methane s u l f o n a t e (Sandoz MS 222), recorded  t h e gonad and body w e i g h t  and t h e i r t i s s u e s f i x e d i n B o u i n ' s p i c r i c a c i d -  formal-acetic acid solution.  The t i s s u e s were embedded i n  p a r a f f i n u s i n g r o u t i n e p r o c e d u r e and s t a i n e d u s u a l l y w i t h haematoxylin blue.  and e o s i n , l e s s f r e q u e n t l y w i t h p i c r o - a n i l i n e  S e c t i o n s o f t h e v a r i o u s t i s s u e s were c u t a t 5-lOji.  I n e x p e r i m e n t s No. 1 t o 4 o n l y t h e gonads were s e c t i o n e d , i n No. 5 b o t h gonads and k i d n e y s kidneys  and i n No. 6-8 gonads,  and t h y r o i d s were s e c t i o n e d .  I n experiment No. 8  14  p i t u i t a r i e s were a l s o s e c t i o n e d and s t a i n e d w i t h blue-PAS-orange G.  alcian  15  III.  RESULTS  A s e r i e s o f e i g h t experiments was c a r r i e d o u t between October 1966 and A p r i l 1968. summarized i n T a b l e I .  The d e t a i l s a r e  I n each o f t h e experiments one o r  two groups o f f i s h were t r e a t e d w i t h m e t h a l l i b u r e .  In a l l ,  f i v e groups o f f i s h were i n j e c t e d w i t h m e t h a l l i b u r e and t h r e e groups were immersed i n a d i l u t e s u s p e n s i o n o f t h e compound.  Two groups o f f i s h w e r e i n j e c t e d w i t h L u t e i n i z i n g  Hormone (LH) and two groups w i t h a m i x t u r e o f e q u a l amounts o f LH and m e t h a l l i b u r e .  A s i n g l e group o f f i s h was immersed  in a d i l u t e solution of thiourea. The f i r s t two experiments form a p r e l i m i n a r y s t u d y . They h e l p e d f a m i l i a r i z e me w i t h t h e v a r i o u s b e h a v i o r p a t t e r n s of  t h e s t i c k l e b a c k s , t h e g e n e r a l c a r e o f t h e f i s h and t h e  technique o f i n j e c t i o n .  The experiments No. 3 t o 8 formed  the b a s i s o f t h e d e t a i l e d s t u d y (Table I) . A. B e h a v i o r S t u d i e s 1. Prespawning A g g r e s s i v e n e s s a)  Methallibure  Treatment  S i n c e two methods o f t r e a t m e n t w i t h m e t h a l l i b u r e were used  (immersion and i n j e c t i o n ) i t was i m p o r t a n t t o  d e t e r m i n e i f e q u i v a l e n t r e s u l t s were o b t a i n e d w i t h b o t h methods. the  T h e r e f o r e , d a t a from experiments No. 6, 7 and 8 on  l e v e l s o f a g g r e s s i v e : . b e h a v i o r were e v a l u a t e d f o r b o t h  methods and i t was found t h a t b o t h r e s u l t e d i n a marked drop  16  i n the l e v e l of aggressiveness m e t h a l l i b u r e treatment  a f t e r f o u r weeks o f  ( F i g . 1 ) . The a g g r e s s i v e l e v e l o f  t h e c o n t r o l s i n c r e a s e d over t h e same f o u r week p e r i o d . To d e t e r m i n e t h e e f f e c t s o f i n j e c t i o n , d a t a were combined from experiments No. 6 and 7 ( F i g . 1 ) . beginning  At the  (Week 0 ) , b e f o r e i n j e c t i o n s were begun, f o u r tanks  were a t L e v e l I (two e x p e r i m e n t a l , two c o n t r o l ) and t w e l v e t a n k s were a t L e v e l s I I I and IV (seven e x p e r i m e n t a l , control).  A f t e r two weeks o f t r e a t m e n t  five  t h e change i n  a g g r e s s i v e n e s s was a l r e a d y apparent w i t h an i n c r e a s e i n t h e c o n t r o l s and a d e c r e a s e i n t h e e x p e r i m e n t a l s .  Eight o f the  e x p e r i m e n t a l tanks were between L e v e l s 0 and I I w i t h no t e r r i t o r i a l i t y p r e s e n t , w h i l e o n l y one c o n t r o l tank was between t h e s e l e v e l s .  The r e m a i n i n g  s i x c o n t r o l t a n k s and  the one e x p e r i m e n t a l were between L e v e l s I I I and IV. A f t e r f o u r weeks o f t r e a t m e n t t h e groups i n j e c t e d w i t h m e t h a l l i b u r e were a l l a t L e v e l s 0 and I , w i t h a maximum o f ten aggressive acts i n f i v e minutes. at L e v e l s I I I and IV.  A l l t h e c o n t r o l s were  From t h e second week t o t h e f o u r t h  week t h e r e was a s l i g h t drop i n l e v e l o f two o f t h e c o n t r o l tanks although a l l c o n t r o l tanks e x h i b i t e d t e r r i t o r i a l b e h a v i o r w h i l e none o f t h e e x p e r i m e n t a l s d i d . When d a t a from e x p e r i m e n t s No. 7 and 8 were combined a l m o s t t h e same r e s u l t s were o b t a i n e d a f t e r f o u r weeks o f immersion i n m e t h a l l i b u r e . treatment  I n t h i s case ( F i g . 2) b e f o r e  f o u r t a n k s o f f i s h were a t L e v e l I ( t h r e e c o n t r o l ,  one e x p e r i m e n t a l ) w h i l e e i g h t were a t L e v e l s I I I and IV  17  Figure 1 Aggressive l e v e l s o f sticklebacks m e t h a l l i b u r e compared w i t h  •  injected with  controls.  A.  b e f o r e t h e t r e a t m e n t was begun  B.  two weeks a f t e r t h e f i r s t  C.  f o u r weeks a f t e r t h e f i r s t  injection injection  Open b a r s , m e t h a l l i b u r e i n j e c t e d ; b a r s w i t h lines,  control.  slanted  A  6  7 7i  •'i  I  O  i  I  11  AGGRESSIVE  III  IV  I  LEVEL  /  7\ .1.  O  I  II  AGGRESSIVE  * z Z  n ; III  LEVEL  4 3  O  |  II  A G G R E S S I V E  IV  III  1 IV  LEVEL  B  18  Figure 2 Aggressive  l e v e l s o f s t i c k l e b a c k s immersed  i n m e t h a l l i b u r e compared w i t h c o n t r o l s . A.  b e f o r e t h e t r e a t m e n t was begun  B.  two weeks a f t e r t h e f i r s t a d d i t i o n  C.  f o u r weeks a f t e r t h e f i r s t a d d i t i o n  Bars with dots, methallibure treated; bars with slanted lines controls.  19 (three c o n t r o l , f i v e experimental). treatment  A f t e r two weeks o f  t h e r e had been o n l y a s l i g h t change i n l e v e l o f  aggressiveness.  A f t e r f o u r weeks o f t r e a t m e n t t h e r e had  been a more marked change, and t h r e e tanks were a t L e v e l s 0 and I ( a l l e x p e r i m e n t a l ) , s i x c o n t r o l tanks were a t L e v e l s I I I and IV and two e x p e r i m e n t a l were a t L e v e l I I I . The immersion t e c h n i q u e was n o t q u i t e as e f f e c t i v e as i n j e c t i o n and i t was s l o w e r t o show i t s e f f e c t s b u t a f t e r f o u r weeks o f t r e a t m e n t no t a n k s t r e a t e d w i t h m e t h a l l i b u r e showed dominance and s u b o r d i n a t i o n .  Thus, t r e a t m e n t o f  s t i c k l e b a c k s w i t h m e t h a l l i b u r e f o r f o u r weeks can be seen t o b r i n g about a d e c r e a s e i n a g g r e s s i v e b e h a v i o r , w h i l e = a t t h e same time, t h e u n t r e a t e d f i s h show an i n c r e a s e i n aggressive behavior. The d a t a from experiment No. 7 were used t o compare t h e number o f a g g r e s s i v e a c t s , performed i n t h e t e s t p e r i o d , o f t h e two m e t h a l l i b u r e t r e a t e d groups w i t h t h e c o n t r o l s (Table I I ) . The amount o f a g g r e s s i o n f o r each tank was t h e t o t a l number o f b i t e s , approaches and chases observed i n f i v e minutes.  Each tank was observed  t h e f o u r t a n k s a t each t r e a t m e n t Standard  t w i c e a week.  From  a w e e k l y average (+ t h e  E r r o r o f t h e mean) was computed and i s g i v e n i n  Table I I .  From t h e second through  t o t h e f i f t h week, one  of the weekly observation periods included observations w i t h the "standard"  f i s h i n t h e g l a s s tube i n a l l a q u a r i a t h a t  were a t L e v e l IV t h e p r e v i o u s o b s e r v a t i o n p e r i o d .  TABLE I I Amount o f A g g r e s s i v e  Behavior  i n M e t h a l l i b u r e - t r e a t e d and C o n t r o l s  T R E A T  WEEK  M E N I  Control  Meth. I n j .  Meth. Imm.  0  3.8 + 0.9  5.3 + 1.1  12.3 + 5.3  1  9.3 + 3.8  4.5 + 0.3  4.5 + 2.6  2*  22.3 + 10.2  6.8 + 2.8  26.5 + 4.8  3*  22.0 + 3.2  9.8 + 9.1  17.5 + 8.4  4*  5.3 + 1.7  1.3 + 0.9  8.0 + 6.4  5*  19.8 + 6.8  1.5 + 0.7  4.0 + 2.8  A l l v a l u e s based on average amount o f a g g r e s s i v e b e h a v i o r i n 5 min + S.E. o f 4 t a n k s each observed t w i c e p e r week *Tubes w i t h s t a n d a r d f i s h were used i n one o f two w e e k l y o b s e r v a t i o n s - weeks 2 - 5 No s i g n i f i c a n t d i f f e r e n c e a t 0.05 among means f o r any week  21  These d a t a show t h a t t h e number o f a g g r e s s i v e a c t s i s r e l a t i v e l y low i n t h e m e t h a l l i b u r e i n j e c t i o n group t h r o u g h o u t t h e f i v e weeks o f t r e a t m e n t  e s p e c i a l l y i n the  l a s t two weeks (1.3 + 0.9 and 1.5 + 0 . 7 ) . The group immersed i n m e t h a l l i b u r e show an i n c r e a s i n g number o f a g g r e s s i v e a c t s d u r i n g t h e f i r s t t h r e e weeks o f t r e a t m e n t . A f t e r t h i s t h e number o f a t t a c k s g r a d u a l l y d e c r e a s e s t o 4.0 + 2.8 and f i v e weeks o f t r e a t m e n t .  The c o n t r o l s a l s o  e x h i b i t an i n c r e a s i n g number o f a g g r e s s i v e a c t s d u r i n g t h e f i r s t t h r e e weeks.  The h i g h number i s m a i n t a i n e d  weeks t h r e e and f i v e b u t drops d u r i n g week f o u r .  during T h i s drop  i s found i n a l l groups d u r i n g t h e f o u r t h week b u t o n l y t h e c o n t r o l group r i s e t o h i g h number 19.8 + 6.8 d u r i n g t h e f i f t h week.  However, due t o t h e s m a l l sample s i z e and t h e  v a r i a b i l i t y between tanks t h e mean o f e i t h e r m e t h a l l i b u r e t r e a t e d group i s n o t s i g n i f i c a n t l y d i f f e r e n t from t h e mean o f t h e c o n t r o l group f o r any week ( a t 0.05 l e v e l u s i n g t h e 'least significant difference'). b) Other Treatments i)  L u t e i n i z i n g Hormone F i s h were i n j e c t e d w i t h LH a l o n e and i n c o m b i n a t i o n  w i t h e q u a l c o n c e n t r a t i o n s o f m e t h a l l i b u r e i n experiments No. 6 and 7.  The e f f e c t s o f these t r e a t m e n t s on l e v e l s o f  a g g r e s s i v e b e h a v i o r a r e compared w i t h t h e c o n t r o l s i n F i g . 3.  B e f o r e i n j e c t i o n s were begun, f o u r tanks were a t L e v e l s  0 and I ( t h r e e LH and one c o n t r o l ) ; t h e r e m a i n i n g  twenty  22  Figure 3 Aggressive l e v e l s o f sticklebacks LH and LH + METH compared w i t h  injected  with  controls.  A.  b e f o r e t h e t r e a t m e n t was begun  B.  two weeks a f t e r t h e f i r s t i n j e c t i o n  C.  f o u r weeks a f t e r t h e f i r s t i n j e c t i o n  b a r s w i t h open c i r c l e s , LH i n j e c t e d ; b a r s w i t h h o r i z o n t a l l i n e s , LH -f- METH i n j e c t e d ; bars with  slanted l i n e s ,  controls.  23  tanks  ( f i v e LH, s i x c o n t r o l and n i n e m e t h a l l i b u r e and LH)  were a t L e v e l s I I I and IV.  A f t e r two weeks o f i n j e c t i o n  both  t h e c o n t r o l s and the LH group showed.a r i s e i n l e v e l o f a g g r e s s i o n b u t t h e LH + METH group showed a drop i n l e v e l . S i x o f t h e seven c o n t r o l tanks were a t L e v e l IV, as were f i v e o f t h e seven LH t a n k s .  Only t h r e e o f t h e n i n e LH +  METH were a t L e v e l I V . A f t e r f o u r weeks o f t r e a t m e n t a number o f f i s h had d i e d b u t o f those w h i c h remained a l l t h e c o n t r o l s and LH t a n k s were a t L e v e l s I I I and IV as were t h r e e o f t h e f i v e LH + METH. These d a t a i n d i c a t e t h a t t r e a t m e n t w i t h LH + METH r e s u l t s i n a l e v e l o f a g g r e s s i o n i n t e r m e d i a t e between t h e h i g h l e v e l s w i t h LH a l o n e and t h e low l e v e l s w i t h methallibure alone. ii)  Thiourea  Immersion  A group o f f i s h was immersed i n t h i o u r e a i n experiment No. 8 and t h e l e v e l o f a g g r e s s i v e  behavior  compared w i t h t h e c o n t r o l s and t h e m e t h a l l i b u r e t r e a t e d group ( F i g . 4 ) .  Over t h e f o u r week p e r i o d t h e t h i o u r e a  group m a i n t a i n e d  a high l e v e l o f aggression.  During  this  time one group o f c o n t r o l s i n c r e a s e d i n l e v e l and b o t h o f m e t h a l l i b u r e t r e a t e d f i s h dropped i n l e v e l . concluded  groups  Thus i t i s  t h a t t r e a t m e n t w i t h t h i o u r e a has no e f f e c t on  l e v e l o f aggression. 2.  Nest B u i l d i n g Although  t h e l e v e l o f a g g r e s s i o n was h i g h i n many  Figure 4 Aggressive  l e v e l s o f s t i c k l e b a c k s immersed  i n t h i o u r e a and m e t h a l l i b u r e compared w i t h c o n t r o l s . A.  b e f o r e t r e a t m e n t was begun  B.  two weeks a f t e r t h e f i r s t a d d i t i o n  C.  f o u r weeks a f t e r t h e f i r s t a d d i t i o n  bars with dots, methallibure treated; bars with cross hatching, thiourea treated; bars with slanted l i n e s , controls.  —  K»  W  wi  _  o 30 rn 1/1 <  m <  M  —  -T"  T  £  T A N K  T A N K S  T A N K S  > O  0  O O 70 m </>  —  >  SSI  —i  <  _  m  —  7ZZ2  —  <  _  m <  m  o  m vt m  1  m < m  —  o  c  CO  —  \ '  DO  _  33  ui  W I  1  S  ui  t a n k s and d i s c r e t e a r e a s were defended, n e s t b u i l d i n g was i n f r e q u e n t l y observed. control fish,  I n a l l experiments o n l y t h r e e  f o u r LH, t h r e e LH + METH, two t h i o u r e a , one  m e t h a l l i b u r e immersion and no m e t h a l l i b u r e i n j e c t e d b u i l t nests. Histology 1. T e s t i s and K i d n e y The c y c l i c a l changes w h i c h a n n u a l l y o c c u r i n t h e s t i c k l e b a c k t e s t i s were c l a s s i f i e d b y Ahsan and Hoar  (1963)  i n t o f o u r d i s t i n c t s t a g e s o f m a t u r a t i o n and a r e as f o l l o w s : I I n a c t i v e ( F i g . 6) i s c h a r a c t e r i s t i c o f t h e p o s t s p a w n i n g p e r i o d d u r i n g August and September.  Spermatogonia  a r e most  prominent i n the seminiferous t u b u l e s b u t t h e r e a r e a l s o a few n e s t s o f p r i m a r y s p e r m a t o c y t e s . t i s s u e i s i n d i s t i n c t o r absent.  The i n t e r s t i t i a l  A few sperm from p r e v i o u s  c y c l e s may be p r e s e n t . II Slight or Mild Activity  ( F i g . 7 & 14) found i n n a t u r e  from October t h r o u g h December.  P r i m a r y and secondary  spermatocytes predominate; spermatogonia a r e l e s s common; t h e r e a r e a few i n t e r s t i t i a l I I I Moderate A c t i v i t y  cells.  ( F i g . 8, 12 & 13) seen under n a t u r a l  c o n d i t i o n s from J a n u a r y t h r o u g h March.  The e a r l y s t a g e s o f  spermatogenesis a r e s t i l l e v i d e n t b u t s p e r m a t i d s and spermatozoa a r e c o n s p i c u o u s and t h e i n t e r s t i t i a l c e l l s a r e more numerous.  26  F i g u r e 5 - Diagram o f Stage 0 T h i s s t a g e i s c h a r a c t e r i z e d by  seminiferous  t u b u l e s r i n g e d w i t h spermatogonia two o r t h r e e c e l l s The  thick.  c e n t e r s o f the t u b u l e s a r e f i l l e d w i t h spermotozoa o r  spermatids.  Few  spermatocytes o r i n t e r s t i t i a l c e l l s  are  present.  F i g u r e 6 - Diagram o f Stage I Spermatogonia a r e most p r o m i n e n t i n the t u b u l e s b u t t h e r e a r e a l s o a few p r i m a r y I n t e r s t i t i a l c e l l s are i n d i s t i n c t  or  seminiferous  spermatocytes.  absent.  27  F i g u r e 7 - Diagram o f Stage I I P r i m a r y and secondary spermatocytes predominate; spermatogonia a r e l e s s common; t h e r e a r e a few  interstitial  cells.  F i g u r e 8 - Diagram o f Stage I I I The e a r l y s t a g e s o f spermatogenesis a r e s t i l l p r e s e n t b u t s p e r m a t i d s and spermatozoa a r e more numerous. More i n t e r s t i t i a l  c e l l s are present.  7  II  28  F i g u r e 9 - Diagram o f Stage IV The s e m i n i f e r o u s t u b u l e s a r e packed w i t h s p e r m a t i d s and spermatozoa.  E a r l i e r stages a r e  d i f f i c u l t t o f i n d and i n t e r s t i t i a l c e l l s a r e numerous.  9  IV  IV F u l l y A c t i v e ( F i g . 9, 15 & 17) c h a r a c t e r i s t i c o f t h e b r e e d i n g season from m i d - A p r i l t o e a r l y August.  The  s e m i n i f e r o u s t u b u l e s a r e packed w i t h s p e r m a t i d s and spermatozoa.  E a r l i e r s t a g e s a r e d i f f i c u l t t o f i n d and  i n t e r s t i t i a l c e l l s a r e numerous. I n t h e p r e s e n t study, a f i f t h s t a g e , d e f i n e d as Stage 0 ( F i g . 5, 10, 11 & 1 5 ) , was found i n many m e t h a l l i b u r e treated f i s h .  T h i s s t a g e was c h a r a c t e r i z e d b y s e m i n i f e r o u s  t u b u l e s r i n g e d w i t h spermatogonia  two o r t h r e e c e l l s  thick.  The c e n t e r s o f t h e t u b u l e s were f i l l e d w i t h spermatozoa o r spermatids.  V e r y few, i f any, spermatocytes were p r e s e n t  and few i n t e r s t i t i a l c e l l s were e v i d e n t . I n t h e p r e s e n t s t u d y t e s t e s were c a t e g o r i z e d a c c o r d i n g t o one o f t h e above l e v e l s o f m a t u r a t i o n b y e x a m i n a t i o n o f seven o r e i g h t l o n g i t u d i n a l s e c t i o n s through t h e m i d d l e r e g i o n o f t h e organs. I t can be seen from T a b l e I , t h e experiments o f 31 t o 38 days i n d u r a t i o n , i . e . No. 3, 5, 6 & 7, were c a r r i e d o u t a t d i f f e r e n t seasons o f t h e y e a r .  The f i s h were t h e r e -  f o r e a t d i f f e r e n t s t a g e s o f t e s t i s m a t u r a t i o n depending on the time o f year.  However, Baggerman (1957, 1966) f i n d s  t h a t a t any time o f y e a r s t i c k l e b a c k s exposed t o 16 h r o f l i g h t and 20°C w i l l a t t a i n f u l l b r e e d i n g c o n d i t i o n i n 2 - 4 weeks.  A l s o , I f i n d most o f t h e c o n t r o l f i s h a r e mature  o r m a t u r i n g i n a l l experiments  a f t e r 30 - 38 days  exposure  t o l o n g p h o t o p e r i o d and 20°C.  Perhaps t h e f i s h may v a r y i n  t h e i r responsiveness t o m e t h a l l i b u r e treatment a t d i f f e r e n t  30  F i g u r e 10 - Stage 0 Photomicrograph o f t h e t e s t i s o f a f i s h treated with methallibure for 31 days x 200  F i g u r e 11 - Stage 0 Same t e s t i s as F i g . 10 x 900  F i g u r e 12 - Stage I I I Photomicrograph o f t h e t e s t i s o f a f i s h treated with methallibure for 49 days x 200  F i g u r e 13 - Stage I I I Same t e s t i s as F i g . 12 x 900  spgon. spzoa. sptid. 1 spcyt. 2 spcyt. sp. d u c t - -  spermatogonia spermatozoa spermatid p r i m a r y spermatocyte secondary spermatocyte main sperm d u c t  31  F i g u r e 14 - Stage I I Photomicrograph o f t h e t e s t i s o f a c o n t r o l f i s h under l o n g 32 days x  photoperiod  200  F i g u r e 15 - Stage IV Photomicrograph o f t h e t e s t i s o f a c o n t r o l f i s h under l o n g  photoperiod  49 days showing main sperm d u c t and s e v e r a l branches t o tubules x  F i g u r e 16 - Stage 0  seminiferous  200  Photomicrograph o f t h e t e s t i s o f a methallibure treated f i s h  showing  r e g r e s s i o n o f the i n t e r s t i t i a l c e l l s x  900  F i g u r e 17 - Stage IV Photomicrograph o f the t e s t i s o f a c o n t r o l f i s h showing w e l l developed interstitial cells x  900  32  t i m e s o f y e a r b u t s i n c e t h e h i s t o l o g y o f t h e t r e a t e d group was a l s o q u i t e c o n s i s t e n t i t was c o n s i d e r e d j u s t i f i a b l e t o combine t h e r e s u l t s from t h e s e e x p e r i m e n t s .  The number o f  f i s h examined from each experiment i s g i v e n i n T a b l e I I I and the  r e s u l t s o f the h i s t o l o g i c a l examination are given i n  T a b l e IV. a)  M e t h a l l i b u r e Treatment on t h e T e s t i s Samples o f t h r e e o r f o u r f i s h were examined  after  12 t o 22 days o f immersion i n , o r i n j e c t i o n w i t h m e t h a l l i b u r e . A l l 12 and 22 day c o n t r o l t e s t e s were a t Stage IV showing f u l l y active testes characteristic of breeding f i s h .  Fish  t r e a t e d w i t h m e t h a l l i b u r e f o r 12 days were a l s o a t Stage IV as were f o u r o f t h e s i x f i s h examined a f t e r 22 days o f treatment.  The r e m a i n i n g two f i s h were a t Stage 0.  I t was  c o n c l u d e d t h a t m e t h a l l i b u r e t r e a t m e n t b r o u g h t about no d e t e c t a b l e change i n t e s t i s h i s t o l o g y d u r i n g t h e f i r s t  12  days o f t r e a t m e n t b u t two o f t h e s i x f i s h examined show a t r e a t m e n t e f f e c t a f t e r 22 days'* o f t r e a t m e n t . A f t e r 30 - 38 days o f t r e a t m e n t t h e r e was a v e r y marked d i f f e r e n c e between t r e a t e d and c o n t r o l f i s h . t e s t e s o f most c o n t r o l f i s h gametogenesis was w e l l  In the underway  o r complete and 20 o f t h e 22 f i s h examined were a t Stages I I I and IV.  However, gametogenesis i s s e v e r e l y r e t a r d e d i n  f i s h i n j e c t e d w i t h m e t h a l l i b u r e and o n l y 3 o f the 23 examined are  a t Stages I I I and IV.  The l a r g e s t group o f 15 a r e a t  Stage 0, showing m a t u r a t i o n o f about h a l f t h e germ c e l l s t o  TABLE I I I Number o f F i s h Used f o r E x a m i n a t i o n of T e s t i s  Treatment  Control Inj. & Uninj.  Meth. I n j .  Exp. No.  Histology  Duration i n Days  No. Of Fish  Total  4a 4b 3 5 a & b 6 7 8  12 22 31 36 & 38 32 35 49  3 3 6 7 3 6 8  3 3  4a 4b 3 5 a & b 6 7  12 22 31 36 & 38 32 35  3 4 4 8 5 6  ) ) ) )  ) ) ) )  22 8 3 4 23  4a 4b 7 8  12 22 35 49  4 1 7 7  4 1 7 7  L.H. I n j .  6 7  32 35  1 ) 4 )  5  L.H. + Meth. Inj.  6 7  32 35  1 ) 1 )  2  Meth.  Imm.  TABLE IV E f f e c t o f M e t h a l l i b u r e , LH and T h i o u r e a on H i s t o l o g y o f t h e T e s t i s and t h e K i d n e y  Treatment  Duration of Treat, i n Days  No. Fish  Staaes o f T e s t i s Stimulation 0 I I I I I I IV  B r u s h B o r d e r Seg. K i d n e y Mucous C e l l s No Mucous C e l l s ** C e l l No. **Cell No. Height i n ^ H e i g h t in/tc Fish Fish  12  3  0  0  0  0  3  22  3  0  0  0  0  3  *30  22  0  0  2  11  9  11  49  8  0  0  0  0  8  1  12  3  0  0  0  0  3  22  4  1  0  0  0  3  *30  23  15  0  5  1  2  12  4  0  0  0  0  4  22  2  1  0  0  0  1  *30  7  4  3  0  0  49  7  1  0  0  LH  *30  5  1  1  Meth. + LH  *30  2  0  49  8  0  Control  Meth. I n j .  Meth.  Imm.  Thiourea  2  23.63 + 1.76  8.29  4  28.26 + 2.82  12  9.14 + 0.29  0  0  4  9.69 + 0.29  0  6  0  5  12.04 + 1.17  0  0  0  3  1  9.69  4  17.25 + 1.50  0  0  0  2  1  13.44  1  23.49  0  0  0  8  2  13.32 + 0.92  3  22.75 + 3.12  * 30 t o 38 days  10.24 + 0.51  ** mean + S.E. 34  35  spermatozoa and t h e remainder a t t h e spermatogonia l e v e l . R e s u l t s were v e r y s i m i l a r w i t h the immersion technique.  No f i s h were a t S t a g e s I I I and IV and f o u r o f t h e  seven examined were a t Stage 0.  A f t e r 49 days o f immersion  i n m e t h a l l i b u r e t h e r e were s t i l l d i f f e r e n c e s between and c o n t r o l f i s h . and a t Stage IV.  treated  A l l c o n t r o l f i s h were c o m p l e t e l y mature M e t h a l l i b u r e t r e a t e d f i s h , however, were  l e s s mature and s i x o f t h e seven examined were a t Stage I I I . In t h e s e t e s t e s about h a l f t h e germ c e l l s were mature  sperm  and the r e s t i n v a r i o u s i n t e r m e d i a t e s t a g e s (primary and s e c o n d a r y s p e r m a t o c y t e s and s p e r m a t o g o n i a ) .  The b l o c k i n  gametogenesis appears t o have been l e s s complete a f t e r 49 days o f t r e a t m e n t than a f t e r 30 - 38 d a y s . b)  M e t h a l l i b u r e Treatment on t h e K i d n e y The h e i g h t o f t h e . c e l l s o f t h e b r u s h b o r d e r segment o f t h e k i d n e y i n c r e a s e s two o r t h r e e t i m e s when male s t i c k l e b a c k s become mature and s t a r t t o b u i l d n e s t s .  The h e i g h t o f  t h e s e c e l l s i n c r e a s e s w i t h h i g h l e v e l s o f androgen i n t h e t e s t i s . ( W a i and Hoar, 1963).  To d e t e r m i n e t h e e f f e c t s o f  m e t h a l l i b u r e on s t e r o i d p r o d u c t i o n , samples o f k i d n e y from f i s h t r e a t e d f o r 30 o r 49 days w i t h m e t h a l l i b u r e were examined and compared w i t h c o n t r o l s  (Table I I I ) .  A mean c e l l h e i g h t f o r each k i d n e y was computed from the  minimum c e l l , h e i g h t o f t h e b r u s h b o r d e r segment o f 25  kidney tubules.  The k i d n e y s were c l a s s i f i e d  i n t o two groups,  t h o s e w i t h and t h o s e w i t h o u t mucous c e l l s i n t h e c o l l e c t i n g tubules.  The mean f o r each group and each t r e a t m e n t (+ t h e  36  S t a n d a r d E r r o r ) was  computed.  The  p r e s e n c e o f c l e a r mucous  c e l l s i n t h e c o l l e c t i n g t u b u l e as w e l l as the  granular  appearance o f the c o n v o l u t e d t u b u l e s i n d i c a t e s t e r o i d stimulation. A f t e r 30 days o f m e t h a l l i b u r e  t r e a t m e n t mucous c e l l s  were not p r e s e n t i n any o f the k i d n e y s o f t r e a t e d t h e y were o n l y p r e s e n t i n two  o f the c o n t r o l f i s h .  h e i g h t o f c o n t r o l s w i t h o u t mucous c e l l s and methallibure  fish The  that of  cell  the  group were not s i g n i f i c a n t l y d i f f e r e n t .  d a t e i n d i c a t e low  but  These  s t e r o i d l e v e l s i n b o t h t r e a t e d and  control  groups a f t e r 30 d a y s . However, a f t e r 49 days f o u r o f the f i v e  control  f i s h sampled e x h i b i t a marked s t i m u l a t i o n o f the k i d n e y t u b u l e s (28.26 + 2.82). i n any o f the m e t h a l l i b u r e  There are no mucous c e l l s p r e s e n t t r e a t e d group.  higher steroid levels i n control  These d a t a i n d i c a t e  fish.  In summary, t r e a t m e n t w i t h m e t h a l l i b u r e immersion o r i n j e c t i o n r e t a r d s  gametogenesis i n the  a f t e r 30 - 38 days o f t r e a t m e n t . i n the m e i o t i c  either  by  testis  There seems t o be a b l o c k  t r a n s f o r m a t i o n o f spermatogonia i n t o  spermatocytes. Most o f the t r e a t e d  f i s h exhibit testes with  c e l l s w h i c h have c o m p l e t e l y undergone m e i o s i s o r c e l l s w h i c h have not s t a r t e d the m e i o t i c (spermatogonia).  (spermatozoa)  process  However, a f t e r 49 days o f t r e a t m e n t  b l o c k appears t o have been l e s s e f f e c t i v e and m a t u r a t i o n has  germ  the  germ c e l l  o n l y slowed down so t h a t w h i l e the  controls  37 are mature t h e m e t h a l l i b u r e t r e a t e d group a r e  maturing.  S t e r o i d o g e n e s i s has a l s o been a f f e c t e d by m e t h a l l i b u r e treatment  and the c e l l s o f t h e k i d n e y s o f the  t r e a t e d groups never a t t a i n a h e i g h t c h a r a c t e r i s t i c o f a c t i v e l y breeding experimental  fish.  C o n t r o l s a f t e r 49 days under  c o n d i t i o n s have k i d n e y s w h i c h show s i g n s o f  s t i m u l a t i o n and mucous p r o d u c t i o n . c)  LH Treatment F i s h were t r e a t e d f o r 30 days w i t h LH. o f f i v e , t h r e e had  Of the sample  f u l l y a c t i v e t e s t e s Stage IV w h i l e  o t h e r two were l e s s a c t i v e .  There was  the  a l s o evidence of  i n c r e a s e d s t e r o i d p r o d u c t i o n s i n c e f o u r o f the f i v e  kidneys  examined had mucous c e l l s and g r a n u l a r c e l l s w i t h a h e i g h t o f 17.25  + 1.50.  This indicates a higher steroid  production  i n the LH group t h a n the c o n t r o l s where o n l y two o f t h e  20  examined show mucous c e l l s . d)  Thiourea  Treatment F i s h t r e a t e d f o r 49 days w i t h t h i o u r e a were a l l a t  Stage IV o f t e s t i s s t i m u l a t i o n as were t h e c o n t r o l group. Three o f the f i v e f i s h had mucous c e l l s p r e s e n t kidneys  and a mean c e l l h e i g h t o f 22.75 + 3.12  c h a r a c t e r i s t i c o f f i s h s e c r e t i n g mucus.  The  i n the which i s  states of  gametogenesis and s t e r o i d o g e n e s i s are t h e same i n the t h i o u r e a t r e a t e d group as t h e y are i n the c o n t r o l group. t h e r e f o r e , concluded  It is,  t h a t t h i o u r e a has no e f f e c t on e i t h e r  s t e r o i d o g e n e s i s o r gametogenesis.  e)  GSI The  gonosomatic i n d e x o r GSI (Gonad Wt X 1 0 0 / T o t a l  Body Wt) was c a l c u l a t e d f o r m e t h a l l i b u r e ,  t h i o u r e a and  c o n t r o l f i s h f o r experiments No. 5, 6, 7 and 8 (Table V ) . The  GSI o f t h e m e t h a l l i b u r e  t r e a t e d f i s h was j u s t  significant-  l y l o w e r than t h e GSI o f t h e c o n t r o l f i s h i n two experiments (No.  5 and 6) a t t h e 0.05 l e v e l .  I n two l a t e r experiments  (No.  7 and 8) t h e GSI o f m e t h a l l i b u r e  f i s h are not s i g n i f i c a n t l y d i f f e r e n t .  t r e a t e d and c o n t r o l So a l t h o u g h  there  a r e d i f f e r e n c e s i n h i s t o l o g y between t r e a t e d and c o n t r o l f i s h , t h e w e i g h t o f t h e t e s t i s o f t h e t r e a t e d f i s h does not c o n s i s t e n t l y r e f l e c t t h i s d i f f e r e n c e .  The group o f f i s h  t r e a t e d w i t h t h i o u r e a have a GSI w h i c h i s n o t s i g n i f i c a n t l y d i f f e r e n t from e i t h e r t h e c o n t r o l s o r t h e m e t h a l l i b u r e t r e a t e d group. Thyroid a) M e t h a l l i b u r e The  and T h i o u r e a Treatment  t h y r o i d o f G a s t e r o s t e u s i s composed o f a number  o f f o l l i c l e s found s c a t t e r e d under t h e pharynx a t t h e r o o t s of the afferent b r a n c h i a l a t t e r i e s . were chosen a t random f o r each f i s h .  Ten unbroken f o l l i c l e s The l o w e s t and t h e  t a l l e s t e p i t h e l i a l c e l l s i n each f o l l i c l e were measure and the mean c e l l h e i g h t computed (lowest + t a l l e s t / 2 - mean c e l l height).  T h i s same c o n v e n t i o n o f computing mean  f o l l i c l u l a r c e l l h e i g h t has been used b y E a l e s Salmo g a i r d n e r ,  (1965) on  and Wiebe (1968) on Cymatogaster a g g r e g a t a .  TABLE V  G. S . I . Comparison o f E f f e c t o f M e t h a l l i b u r e and T h i o u r e a Treatment on Gonasomatic Index  Exp.  Control  Methallibure Injection  5  0.53 + 0.05  0.33 + 0.09*  6  0.45 + 0.08  0.21 + 0.02*  7  0.31 + 0.03  0.21 + 0.10  8  0.38 + 0.06  Methallibure Immersion  Thiourea Immersion  0.21 + 0.06  0.39 + 0.04  0.41 + 0.06  0.39 + 0.04  * S i g n i f i c a n t d i f f e r e n c e a t .05 l e v e l Mean V a l u e s + S t a n d a r d E r r o r o f Mean  Seven f i s h from each t r e a t m e n t - t h i o u r e a , and u n t r e a t e d  methallibure  c o n t r o l s - were examined and a grand mean f o r  each t r e a t m e n t computed from t h e mean f o l l i c l e c e l l o f each f i s h .  height  These v a l u e s a r e g i v e n i n T a b l e V I .  The mean f o l l i c l u l a r c e l l h e i g h t  (10.53u) o f t h e  t h i o u r e a t r e a t e d group i s s i g n i f i c a n t l y d i f f e r e n t from t h e means o f t h e m e t h a l l i b u r e  treated  (5.28u) and t h e c o n t r o l  (4.92ju) groups. There was marked t h y r o i d h y p e r t r o p h y i n t h r e e o f t h e t h i o u r e a t r e a t e d f i s h examined.  The f o l l i c l e s  were s m a l l e r t h a n t h o s e o f t h e c o n t r o l s and had g r e a t l y i n c r e a s e d e p i t h e l i a l c e l l h e i g h t s and d e c r e a s e d c o l l o i d content.  The o t h e r f o u r t h i o u r e a t r e a t e d f i s h showed  little  s i g n o f i n c r e a s e d t h y r o i d a c t i v i t y compared w i t h t h e c o n t r o l s However, even w i t h t h i s v a r i a b i l i t y o f response t h e d i f f e r e n c between means o f t h e t h i o u r e a t r e a t e d group and t h e c o n t r o l group i s 5.62u compared w i t h a l e a s t s i g n i f i c a n t d i f f e r e n c e o f l . l l u a t P<0.05. The t h i o u r e a t r e a t e d group a l s o has a s i g n i f i c a n t l y d i f f e r e n t mean from t h e m e t h a l l i b u r e  treated  group a t P<0.05. The m e t h a l l i b u r e t r e a t e d group showed no o b v i o u s s i g n s o f t h y r o i d h y p e r t r o p h y and t h e mean f o l l i c u l a r  cell  h e i g h t was n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t o f t h e c o n t r o l s i n d i c a t i n g t h a t m e t h a l l i b u r e has no o b v i o u s goitrogenic  effects.  Pituitary a)  Morphology The p i t u i t a r y i s made up o f f o u r main p a r t s , one  TABLE V I Mean T h y r o i d C e l l H e i g h t F o l l o w i n g T h i o u r e a and M e t h a l l i b u r e Treatment  Mean F o l l i c l e C e l l H e i g h t pu.) Control  4.92  Methallibure  5.28  Thiourea  * Stastically significant d i f f e r e n c e from c o n t r o l s and m e t h a l l i b u r e t r e a t e d a t P 0.05 u s i n g l e a s t significant difference  10.53*  42 nervous t h e neurohypophysis  and t h r e e g l a n d u l a r , t h e p r o -  adenohypophysis,  t h e meso-adenohypophysis and t h e meta-  adenohypophysis.  I n t h e p i t u i t a r y o f G a s t e r o s t e u s , the  neurohypophysis (Fig.  17).  i n t e r d i g i t a t e s w i t h the  adenohypophysis  The t r o p h i c hormones s e c r e t e d by t h e p i t u i t a r y  are e l a b o r a t e d by v a r i o u s c e l l types l o c a t e d i n d i f f e r e n t p a r t s o f t h e adenohypophysis.  Both the  and t h y r o i d s t i m u l a t i n g hormone (TSH)  gonadotrophin(s)  a r e produced  by  b a s o p h i l s l o c a t e d i n t h e mesoadenohypophysis ( P i c k f o r d Atz, b)  and  1957).  M e t h a l l i b u r e and T h i o u r e a Treatment Gonadotrophs and t h y r o t r o p h s a r e s c a t t e r e d t h r o u g h o u t the mesoadenohypophysis. used  ( a l c i a n b l u e , PAS,  With t h e s t a i n  combination  Orange G) t h e y t a k e on a l c a i n b l u e  and more o r l e s s PAS.  T h i s g i v e s them a deep p u r p l e hue  w i t h p i n k i s h overtones  ( F i g . 19, 21 & 23 B l a c k ) .  Since both  c e l l t y p e s a r e found i n t h e same a r e a and have v e r y s i m i l a r s t a i n i n g r e a c t i o n s , i t was  not p o s s i b l e to d i f f e r e n t i a t e  between t h e s e two b a s o p h i l s .  The e f f e c t s o f m e t h a l l i b u r e  and t h i o u r e a t r e a t m e n t s on t h e g r a n u l a t i o n o f b o t h c e l l  types  w i l l be c o n s i d e r e d t o g e t h e r . The amount o f b a s o p h i l i c m a t e r i a l was s c a l e o f + t o ++++. each group.  r a t e d on a  Seven o r e i g h t f i s h were examined i n  The s c o r e f o r each group was based on t h e  examination o f at l e a s t s i x s a g i t t a l s e c t i o n s o f the pituitary.  The r e s u l t s a r e t a b u l a t e d i n T a b l e V I I .  TABLE V I I Amount o f G r a n u l a t i o n i n t h e B a s o p h i l s (Thyrotrophs and Gonadotrophs i n Mesoadenohypophysis) a f t e r M e t h a l l i b u r e and T h i o u r e a Immersion  Amount o f G r a n u l a t i o n i n t h e B a s o p h i l s o f t h e Adenohypophysis +  Methallibure Thiourea Control  3  ++  +++.  ++++  3  3  2  6  4 2  44  The m e t h a l l i b u r e t r e a t e d group c o n t a i n e d v e r y b a s o p h i l i c m a t e r i a l i n t h e mesoadenohypophysis. were r a t e d e i t h e r + o r +-f ( F i g . 18 & 19) .  little  A l l fish  The c o n t r o l  group, however, had much more b a s o p h i l i c m a t e r i a l and s i x f o t h e f i s h examined were r a t e d ++++ w h i l e two were r a t e d +++ ( F i g . 20 & 21; T a b l e V I ) . S i n c e m e t h a l l i b u r e t r e a t m e n t was found t o r e t a r d g a m e t o g e n i s i s and s t e r o i d o g e n i s i s i n t h e t e s t i s b u t n o t i n c r e a s e f o l l i c u l a r c e l l h e i g h t i n t h e t h y r o i d , most o f t h e degranulated  cells  are probably  Thiourea treatment some f i s h .  gonadotrophs.  results i n degranulation i n  T h i s group i s r a t e d ++,+++ and ++++.  It is  s i g n i f i c a n t t h a t t h e f i s h r a t e d ++ ( F i g . 22) e x h i b i t e d t h e g r e a t e s t i n c r e a s e i n t h y r o i d f o l l i c u l a r c e l l h e i g h t and t h e f i s h r a t e d ++++ ( F i g . 24) t h e l e a s t . concluded  Therefore,  it is  that thiourea results i n degranulation of the  thyrotrophs while methallibure r e s u l t s i n degranulation o f m a i n l y t h e gonadotrophs.  45  F i g u r e 18 - Photomicrograph o f t h e p i t u i t a r y o f a f i s h t r e a t e d w i t h m e t h a l l i b u r e f o r 49 days x 200  F i g u r e 19 - H i g h power o f p a r t o f t h e f i s h o f F i g . 18 showing + o f b a s o p h i l i c m a t e r i a l x 900  F i g u r e 20 - Photomicrograph o f t h e p i t u i t a r y o f an untreated control f i s h x 200  F i g u r e 21 - H i g h power o f p a r t o f t h e  meso-adenohypophysis  o f t h e f i s h o f F i g . 20 showing ++++ o f basophilic material x 900  46  F i g u r e 22 - Photomicrograph o f t h e p i t u i t a r y o f a f i s h t r e a t e d w i t h t h i o u r e a f o r 49 days x  200  F i g u r e 23 - H i g h power o f p a r t o f t h e mesoadenohypophysis o f t h e f i s h o f F i g . 22 showing ++ o f b a s o p h i l i c m a t e r i a l x  900  F i g u r e 24 - Photomicrograph o f the p i t u i t a r y o f a f i s h t r e a t e d w i t h t h i o u r e a f o r 49 days ++++ b a s o p h i l i c m a t e r i a l x  900  showing  47  IV.  DISCUSSION  Brown (1963), Harper (1964), Hemsworth, J a c k s o n and Walpole (1968), Paget e t a l . (1961) and Walpole (1965) provide evidence that m e t h a l l i b u r e ,  dithiocarbamoylhydrazine  ( I . C . I . 33,828) b l o c k s t h e f o r m a t i o n o f g o n a d o t r o p h i n o f mammals.  T h i s n o n - s t e r o i d , non-hormonal compound has t h e  f o l l o w i n g chemical  formula:  CH =CH.CH.NH.CS.NH.NH.CS.NH.CH^ 2 i CH J  3  Hoar e t a l . (1967) were t h e f i r s t t o u s e i t on t e l e o s t s . They r e p o r t t h a t t r e a t m e n t  o f s t i c k l e b a c k s , g o l d f i s h and  surfperch with methallibure i n t e r f e r e s w i t h - p i t u i t a r y gonadotrophin f u n c t i o n . B e f o r e d i s c u s s i n g t h e main e f f e c t s o f m e t h a l l i b u r e on t h e s t i c k l e b a c k , i t may be o f i n t e r e s t t o c o n s i d e r t h e p o s s i b l e s i d e e f f e c t s o f the treatment.  The most  important  o f these i s t h e somewhat t o x i c e f f e c t o f t h e compound and the second t h e p o s s i b l e d i r e c t e f f e c t o f m e t h a l l i b u r e on t h e thyroids o f the treated f i s h . The p r e l i m i n a r y study b y Hoar e t a l . (1967) and t h e p r e s e n t one b o t h show t h a t m e t h a l l i b u r e t r e a t m e n t some degree t o x i c t o s t i c k l e b a c k s . up t o 50% o f t h e e x p e r i m e n t a l of treatment.  i s to  I n my e a r l y e x p e r i m e n t s  group d i e d d u r i n g t h e c o u r s e  I n l a t e r experiments s u r v i v a l was b e t t e r and  i n experiment No. 8 o n l y one t r e a t e d f i s h d i e d d u r i n g seven  48  weeks o f t r e a t m e n t .  Not a l l m o r t a l i t y can be a t t r i b u t e d t o  methallibure since a t h i r d o f controls died i n the e a r l y experiments.  Although  a t o x i c e f f e c t cannot be r u l e d o u t ,  t h e v e r y low m o r t a l i t y i n l a t e r experiments suggests t h a t t h i s f a c t o r p e r se was n o t a p r i m e cause o f d e a t h i n t h e earlier  tests. Secondly, s i n c e m e t h a l l i b u r e c o n t a i n s t h e  t h i o u r y l e n e group, i t might be expected t o b l o c k t h e s y n t h e s i s of thyroxine with r e s u l t a n t increased thyrotrophic a c t i v i t y o f t h e p i t u i t a r y and enlargement and h y p e r p l a s i a o f t h e thyroid.  W a l p o l e (1965) found a d m i n i s t r a t i o n o f l a r g e  doses o f m e t h a l l i b u r e t o r a t s caused a s l i g h t i n c r e a s e i n t h y r o i d w e i g h t b u t normal t h y r o i d h i s t o l o g y .  Tulloch et a l . 131  (1963) found m e t h a l l i b u r e d e c r e a s e d t h e uptake o f I t h e t h y r o i d s o f t r e a t e d r a t s and mice.  by  Wiebe (1968) r e p o r t e d  no change i n t h e t h y r o i d h i s t o l o g y o f t h e s u r f p e r c h Cymatogaster a f t e r 40 days o f m e t h a l l i b u r e t r e a t m e n t .  L e a t h e r l a n d and  Pandey (1968), however, d e s c r i b e an i n c r e a s e i n f o l l i c u l a r c e l l h e i g h t a f t e r 56 days o f m e t h a l l i b u r e t r e a t m e n t Poecilia.  The r e s u l t s o b t a i n e d  on  i n the present s e r i e s o f  e x p e r i m e n t s a r e s i m i l a r t o t h o s e found w i t h Cymatogaster? 49 days o f t r e a t m e n t  caused no s i g n i f i c a n t i n c r e a s e i n t h e  mean t h y r o i d f o l l i c u l a r c e l l h e i g h t o f G a s t e r o s t e u s The Gasterosteus  (Table V I ) .  e f f e c t s o f m e t h a l l i b u r e on t h e t h y r o i d o f were compared w i t h those o b t a i n e d when a group  o f f i s h was t r e a t e d w i t h t h e g o i t r o g e n t h i o u r e a .  Thiourea  a c t s b y b l o c k i n g t h e i o d i n i a t i o n o f t y r o s i n e w h i c h i s one  49 step i n the s y n t h e s i s of thyroxine  (Hoar 1966).  Thiourea  t r e a t m e n t has been used on a number o f s p e c i e s o f f i s h u s u a l l y r e s u l t s i n an i n c r e a s e i n f o l l i c u l a r  and  epithelial  c e l l h e i g h t and i n some cases marked h y p e r p l a s i a (Atz (1953) Astyanax, Chambers (1953) Fundulus, Wiebe (1968) C y m a t o g a s t e r ) . Treatment o f s t i c k l e b a c k s w i t h t h i o u r e a f o r 49 days r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n mean f o l l i c u l a r c e l l h e i g h t . The v a r i a b i l i t y i n amount o f s t i m u l a t i o n between i n d i v i d u a l s would s u g g e s t t h a t the c o n c e n t r a t i o n o f t h i o u r e a used (0.003%) was  only just effective.  Kinnear  (1960) r e p o r t s  t h a t a s o l u t i o n o f 0.0025% t h i o u r e a i s t h e most d i l u t e t h a t prevents  any s y n t h e s i s o f t h y r o x i n e by the  flounder  Platichthys. T h i o u r e a has been found by Chambers (1953) t o r e s u l t i n l o s s o f w e i g h t , r e t a r d a t i o n o f growth and d e p l e t i o n o f l i v e r glycogen  i n Fundulus.  B a r r i n g t o n and M a t t y (1952)  found t h a t minnows immersed i n t h i o u r e a do not feed w e l l and t h a t s p e r m a t o g e n e s i s i s a r r e s t e d i n the t e s t i s a t t h e spermatocyte s t a g e .  No s i d e e f f e c t s are o b s e r v e d u s i n g a  s o l u t i o n o f t h i o u r e a on G a s t e r o s t e u s .  The  f i s h feed w e l l  and o n l y spermatozoa are found i n the t e s t e s o f b o t h t h i o u r e a t r e a t e d and c o n t r o l f i s h .  I t may  be s i g n i f i c a n t t h a t t h e  dosages used b y Chambers and o t h e r e a r l y w o r k e r s were about t e n times g r e a t e r t h a n those used i n the p r e s e n t  study.  I n summary, t h e e f f e c t s o f m e t h a l l i b u r e and on G a s t e r o s t e u s  are v e r y d i f f e r e n t .  thiourea  Methallibure brings  about r e t a r d a t i o n i n t e s t i s gametogenesis and no  significant  50  increase i n thyroid e p i t h e l i a l c e l l height. however, has no o b s e r v a b l e e f f e c t on t h e  Thiourea,  gametogenetic  p r o c e s s b u t b r i n g s about a s i g n i f i c a n t i n c r e a s e i n t h y r o i d e p i t h e l i a l c e l l height.  M e t h a l l i b u r e i s c o n c l u d e d t o have  no o b s e r v a b l e g o i t r o g e n i c a c t i o n .  Therefore, i n s p i t e of  t h e p o s s i b l e t o x i c e f f e c t , t h e use o f m e t h a l l i b u r e i s c o n s i d e r e d t o have y i e l d e d u s e f u l i n f o r m a t i o n about t h e hormonal e f f e c t s o f t h e gonadotrophins  on t h e b e h a v i o r  and  various tissues of Gasterosteus. A.  Prespawning  Aggressive Behavior  A l l the present evidence i n d i c a t e s t h a t  prespawning  a g r e s s i v e b e h a v i o r i n G a s t e r o s t e u s i s under d i r e c t c o n t r o l of  t h e p i t u i t a r y v i a an L H - l i k e g o n a d o t r o p h i n .  a, b) demonstrated  1  (1962  t h a t b o t h c a s t r a t e d and normal male  s t i c k l e b a c k s h e l d under s h o r t p h o t o p e r i o d , hypophysectomized  Hoar  'physiologically  e x h i b i t e d a low l e v e l o f a g g r e s s i v e n e s s  b u t normal and c a s t r a t e d males under l o n g p h o t o p e r i o d exhibited a high l e v e l of aggressiveness.  I n j e c t i o n of the  s h o r t p h o t o p e r i o d f i s h w i t h mammalian LH o r c h o r i o n i c gonadotrophin ness.  Wai  r e s u l t e d i n an i n c r e a s e i n l e v e l o f a g g r e s s i v e -  and Hoar (1963) concluded t h a t t r e a t m e n t o f  c a s t r a t e d l o n g and s h o r t p h o t o p e r i o d s t i c k l e b a c k s w i t h methyl t e s t o s t e r o n e d i d n o t a f f e c t t h e i r l e v e l o f a g g r e s s i v e ness. Treatment o f G a s t e r o s t e u s w i t h m e t h a l l i b u r e r e s u l t e d i n a marked drop i n l e v e l o f a g g r e s s i v e n e s s o f the c o n t r o l s increased  (Fig. 1 & 2).  The drop i n a g g r e s s i v e n e s s i s  51 a s s o c i a t e d w i t h a l o s s o f t e r r i t o r i a l b e h a v i o r on t h e p a r t o f t h e t r e a t e d f i s h and an i n c r e a s e i n t h e number o f c o n t r o l f i s h w h i c h defend a d i s c r e t e a r e a . and c h a s i n g i s c o n s i s t e n t l y l e s s  The amount o f b i t i n g  (than i n t h e c o n t r o l s )  i n t h e f i s h i n j e c t e d w i t h m e t h a l l i b u r e and l e s s , i n two o f t h e l a s t t h r e e weeks, i n f i s h immersed i n m e t h a l l i b u r e (Table I I ) . When a group o f f i s h i s t r e a t e d w i t h a m i x t u r e o f m e t h a l l i b u r e and mammalian LH t h e l e v e l o f a g g r e s s i v e n e s s i s h i g h e r than t h a t i n f i s h t r e a t e d w i t h m e t h a l l i b u r e a l o n e (Fig.  1, 2 & 3) b u t n o t as h i g h as t h a t o f t h e c o n t r o l s .  The t r e a t m e n t c o m b i n a t i o n o f LH + METH d i d n o t seem t o b e t o l e r a t e d a t a l l w e l l b y t h e f i s h so t h a t t h e e f f e c t s on t h e b e h a v i o r b y t h i s replacement  t h e r a p y must b e c o n s i d e r e d  h i g h l y s u g g e s t i v e r a t h e r than c o n c l u s i v e . The  s t u d y i n d i c a t e s t h a t m e t h a l l i b u r e t r e a t m e n t on  l o n g p h o t o p e r i o d f i s h r e s u l t s i n t h e same k i n d o f r e d u c t i o n i n a g g r e s s i v e n e s s w h i c h i s c h a r a c t e r i s t i c o f f i s h k e p t under short photoperiod.  Treatment w i t h LH + METH r e s u l t s i n a  l e v e l o f a g g r e s s i v e n e s s i n t e r m e d i a t e between t h e m e t h a l l i b u r e t r e a t e d and t h e c o n t r o l  groups.  I t i s v e r y d i f f i c u l t t o determine  whether  m e t h a l l i b u r e a f f e c t s o n l y prespawning a g g r e s s i v e b e h a v i o r s i n c e n e s t b u i l d i n g and r e p r o d u c t i v e b e h a v i o r o c c u r i n o n l y a few f i s h - whether e x p e r i m e n t a l o r c o n t r o l .  The h e i g h t  o f t h e c e l l s o f the k i d n e y t u b u l e s o f f i s h t r e a t e d w i t h m e t h a l l i b u r e f o r 30 - 38 days i s low b u t so i s the h e i g h t o f t h e k i d n e y c e l l s o f t h e c o n t r o l s (Table I V ) .  These r e s u l t s  52  i n d i c a t e t h a t s t e r o i d l e v e l s are p r o b a b l y low i n b o t h groups.  Then i t i s not s u r p r i s i n g t h a t the sequences of  r e p r o d u c t i v e b e h a v i o r which are thought t o be under s t e r o i d o r s t e r o i d + g o n a d o t r o p h i n c o n t r o l a r e f o r the most p a r t absent.  However, a l o w e r i n g i n the l e v e l o f g o n a d o t r o p h i n s  by m e t h a l l i b u r e c o u l d l o g i c a l l y be expected t o lower p r o d u c t i o n and  steroid  i n d i r e c t l y a f f e c t the whole c y c l e o f  reproductive behavior. w i t h Cymatogaster.  Wiebe (1967) f i n d s such a r e s u l t  As w i t h G a s t e r o s t e u s ,  c e r t a i n elements  o f r e p r o d u c t i v e b e h a v i o r appear t o be under d i r e c t gonadotrophic control.  c o n t r o l and o t h e r s under d i r e c t gonadal s t e r o i d  Treatment w i t h m e t h a l l i b u r e e l i m i n a t e s a l l  reproductive behavior  s i n c e i t b l o c k s the g o n a d o t r o p h i n which  i n t u r n r e g u l a t e s the gonadal B.  T e s t i s and  steroids.  Kidney  Spermatogenesis and s t e r o i d o g e n e s i s i n the  testis  o f f i s h are c o n t r o l l e d by the i n t e r a c t i o n o f the g o n a d o t r o p h i n o f the p i t u i t a r y and the androgens o f testis.  Much has been l e a r n e d about the r o l e o f  the  g o n a d o t r o p h i n s by the removal o f the p i t u i t a r y and sequent replacement o f t h e v a r i o u s hormones.  the  sub-  Dodd (1965)  summarizes the r e s u l t s o f hypophysectomy by a number o f w o r k e r s and concludes  t h a t the p i t u i t a r y through t h e  gonadotrophins i s necessary  f o r the i n i t i a t i o n  maintenance of spermatogenesis i n t h e lower  and  vertebrates  (except the c y c l o s t o m e s ) b u t i t s c o n t r o l i s p r o b a b l y  limited  t o phophase o f t h e f i r s t m e i o t i c d i v i s i o n .  There i s much  e v i d e n c e , he f i n d s , f o r t h e maintenance o f spermatogenesis i n t h e absence o f t h e p i t u i t a r y .  He s u g g e s t s t h a t  g o n a d o t r o p h i n a c t s i n d i r e c t l y by s t i m u l a t i n g t h e s e c r e t i o n of s t e r o i d .  F u r t h e r e v i d e n c e comes from t h e known a b i l i t y  of a v a r i e t y of steroids to stimulate c e l l  division.  M e t h a l l i b u r e has been found t o have marked e f f e c t s on spermatogenesis and s t e r o i d o g e n e s i s by Hemsworth et::.al. (1968) on r a t s and b y Hoar et a l . of  (1967) on t h r e e s p e c i e s  fish.  Treatment o f G a s t e r o s t e u s w i t h m e t h a l l i b u r e r e s u l t e d i n no changes i n h i s t o l o g y a f t e r 12 d a y s .  A f t e r 22 days o f  t r e a t m e n t o n l y two o f t h e s i x e x p e r i m e n t a l f i s h showed a treatment e f f e c t .  The o t h e r f o u r t r e a t e d and a l l  f i s h were c o m p l e t e l y mature.  control  A f t e r 30 - 38 days o f  m e t h a l l i b u r e t r e a t m e n t t h e h i s t o l o g y o f c o n t r o l and t r e a t e d groups was v e r y d i f f e r e n t i n d i c a t i n g t h a t m e t h a l l i b u r e t r e a t m e n t b r i n g s about a d e p r e s s i o n i n g o n a d o t r o p h i c activity.  A r e d u c t i o n r a t h e r than a complete b l o c k i s  p o s t u l a t e d s i n c e some spermatogenesis seems t o o c c u r i n t r e a t e d f i s h e s p e c i a l l y i n t h e group t r e a t e d f o r 49 days. Any r e d u c t i o n i n g o n a d o t r o p h i n would most a f f e c t t h e spermatogonia t o spermatocyte t r a n s f o r m a t i o n s i n c e t h i s s t a g e appears from work on hypophysectomized f i s h t o be the most dependent on g o n a d o t r o p h i n .  Therefore, i t i s postulated  t h a t any c e l l s which were spermatocytes b e f o r e t h e t r e a t m e n t would p r o c e e d a t the normal r a t e t o become spermatozoa  54 and c e l l s which were spermatogonia would remain a t t h i s s t a g e o r undergo t h e i n i t i a l phase o f m e i o s i s a t a much s l o w e r r a t e t h a n normal. I t appears t o t a k e about 30 days o f m e t h a l l i b u r e t r e a t m e n t t o o b t a i n a w i d e s p r e a d d i f f e r e n c e i n h i s t o l o g y between t r e a t e d and c o n t r o l f i s h .  However, d u r i n g t h e 49 days o f  m e t h a l l i b u r e t r e a t m e n t i t i s p o s t u l a t e d t h a t enough g o n a d o t r o p h i n i s p r e s e n t t o a l l o w most spermatogonia i n t h e t r e a t e d f i s h t o become s p e r m a t o c y t e s .  The germ c e l l s  of t h e c o n t r o l s , however, have c o m p l e t e l y undergone spermatogenesis and a r e mature sperm. Gonadotrophins the i n t e r s t i t i a l  a l s o r e g u l a t e t h e development o f  c e l l s i n the t e s t i s .  Since these c e l l s  d e v e l o p i n s e a s o n a l f i s h d u r i n g t h e b r e e d i n g season, a r e s u d a n o p h i l i c and c h o l e s t e r o l p o s i t i v e , t h e y a r e thought t o produce s t e r o i d  ( M a r s h a l l , 1960).  Ahsan (1966), L o f t s ,  P i c k f o r d and A t z (1966), S u n d a r a r a j and Nayyar (1967) and others f i n d t h a t these c e l l s  (or l o b u l e boundary c e l l s i n  f i s h which do n o t have i n t e r s t i t i a l  cells)  regress after  hypophysectomy and i t might, t h e r e f o r e , be expected t h a t m e t h a l l i b u r e t r e a t m e n t would r e g r e s s t h e i n t e r s t i t i a l  tissue  of Gasterosteus. The e v i d e n c e w i t h r e s p e c t t o t h i s t h e o r y i s  indirect.  I n male s t i c k l e b a c k s d u r i n g the b r e e d i n g season, t h e interstitial  t i s s u e d e v e l o p s , r e d c o l o r a t i o n appears i n t h e  s k i n and t h e c e l l s o f t h e b r u s h b o r d e r segment o f the k i d n e y t u b u l e s become f i l l e d w i t h s e c r e t o r y g r a n u l e s and c l e a r  mucous c e l l s appear i n the c o l l e c t i n g t u b u l e . a r e c o n t r o l l e d by androgen e l a b o r a t e d i n the  These change interstitial  cells. F i s h t r e a t e d w i t h m e t h a l l i b u r e never showed the i n c r e a s e d c e l l h e i g h t o r g r a n u l a r c y t o p l a s m i n the c e l l s o f the b r u s h b o r d e r segment o f the k i d n e y t u b u l e .  The mean  v a l u e s a r e i n t h e range o f v a l u e s found by Wai and Hoar (1963) t o be t y p i c a l o f mature b u t s e x u a l l y q u i e s c e n t males The c o n t r o l f i s h v a r i e d somewhat i n t h e i r response photoperiod.  When one group was  exposed t o t h i s  to long light  regime f o r 30 - 38 days o n l y 2 o f the 13 examined e x h i b i t k i d n e y h i s t o l o g y t y p i c a l o f b r e e d i n g males b u t when another group was  exposed t o l o n g p h o t o p e r i o d f o r 49 days f o u r o f  f i v e f i s h examined showed e v i d e n c e o f mucus p r o d u c t i o n by the kidney  (Table I V ) .  The mean c e l l h e i g h t o f t h i s  latter  group i s i n t h e range c h a r a c t e r i s t i c o f b r e e d i n g males  (Wai  and Hoar, 1963). M e t h a l l i b u r e t r e a t e d males r e t a i n e d t h e i r r e d c o l o r a t i o n throughout  the experiments.  T h i s would  suggest  t h a t androgen i s p r o b a b l y b e i n g produced a t a low l e v e l by the t r e a t e d f i s h and may  i m p l y t h a t a c e r t a i n amount o f  gonadotrophin  i s also present.  gonadotrophin  do not appear t o be h i g h enough t o promote  spermatogenesis  However, l e v e l s o f  a t the r a t e found i n c o n t r o l f i s h o r t o  l e a d t o the l e v e l s o f androgen n e c e s s a r y t o b r i n g about the increase i n kidney tubule c e l l height c h a r a c t e r i s t i c of b r e e d i n g males.  56 When hypophysectomized  f i s h are treated with r e -  p r o d u c t i v e hormones s t i m u l a t i o n o f gametogenesis and steroidogenesis r e s u l t s .  Ahsan (1966) f i n d s b o t h p r o c e s s e s  s t i m u l a t e d i n hypophysectomized  l a k e chub t r e a t e d w i t h e i t h e r  mammalian LH o r a crude e x t r a c t o f whole salmon L o f t s ^ t a l . (1966) r e p o r t t h a t m e t h y l  testosterone  s t i m u l a t e s b o t h p r o c e s s e s i n hypophysectomized However, S u n d a r a r a j and Nayyar  pituitaries.  (1967) f i n d  Fundulus.  testosterone  p r o p i o n a t e t r e a t m e n t r e s t o r e s spermatogenesis i n hypophysectomized atropic.  catfish  b u t the i n t e r s t i t i a l c e l l s are  Human c h o r i o n i c g o n a d o t r o p h i n i n d u c e s  significant  dose dependent w e i g h t i n c r e m e n t s i n t h e t e s t e s and r e s t o r e s spermatogenesis and t h e s e c r e t o r y a c t i v i t y o f t h e interstitial  cells.  I n t h e p r e s e n t s t u d y b o v i n e LH was used i n an attempt t o c o u n t e r a c t t h e e f f e c t s o f m e t h a l l i b u r e . Unfortunately,  a l a r g e number o f t h e t r e a t e d f i s h d i e d  d u r i n g t h e c o u r s e o f t h e experiment.  However, t h r e e o f t h e  f i v e s u r v i v i n g LH i n j e c t e d f i s h were a t Stage IV o f t e s t i s m a t u r a t i o n and f o u r o f t h e f i v e kidneys, examined showed c l e a r mucous c e l l s and g r a n u l a r b r u s h b o r d e r c e l l s .  Only  two f i s h i n j e c t e d w i t h LH + METH were examined, b o t h were a t Stage IV o f t e s t i s  stimulation.  One f i s h showed k i d n e y  t u b u l e s w i t h mucous c e l l s , one d i d n o t .  The s m a l l sample  i n d i c a t e s t h a t METH + LH i s more l i k e t h e c o n t r o l group and seems t o promote t e s t i s m a t u r a t i o n and s t e r o i d o g e n e s i s ; any c o n c l u s i o n must remain t e n t a t i v e a t t h i s s t a g e .  57 C.  Pituitary The mesoadenohypophysis o f t e l e o s t s appears t o c o n t a i n a l l t h e c e l l types t h a t a r e a s s o c i a t e d w i t h the mammalian p a r s a n t e r i o r .  There a r e a t l e a s t two types  of  b a s o p h i l s and t h e s e have been i d e n t i f i e d as gonadotrophs and t h y r o t r o p h s  ( P i c k f o r d and A t z , 1957).  (1959) g i v e s h i s t o l o g i c a l e v i d e n c e and FSH  Van  Mullem  f o r the p r e s e n c e o f LH  i n t h e mesoadenohypophysis o f G a s t e r o s t e u s b u t  d i f f e r e n t i a t i o n of c e l l s producing the each o t h e r and t h o s e p r o d u c i n g FSH  gonadotrophinsfrom  i s not  definite.  A f t e r 49 days o f m e t h a l l i b u r e t r e a t m e n t Gasterosteus  t h e r e was  the  in  extensive d e g r a n u l a t i o n of the  b a s o p h i l s o f the mesoadenohypophysis (Table IV and F i g . 17 & 1 8 ) .  The  e f f e c t s on gametogenesis and  steroidogenesis  i n the t e s t i s  (Table IV) a r e s u g g e s t i v e o f l e s s  gonadotrophin  as a r e t h e l o w e r l e v e l s o f  aggressive  b e h a v i o r c h a r a c t e r i s t i c o f the m e t h a l l i b u r e t r e a t e d f i s h (Fig. 1 & 2).  This explanation implies that degranulation  o f the b a s o p h i l s means h y p o - - a c t i v i t y o r a b l o c k i n t h e e l a b o r a t i o n of gonadotrophin.  Such an e x p l a n a t i o n has been  p u t f o r t h b e f o r e t o e x p l a i n d e g r a n u l a t i o n e s p e c i a l l y the c y c l i c degranulation associated with reproduction. example, S o k o l  (1961) f i n d s conspicuous  For  c y c l i c a l changes i n  the b a s o p h i l s o f t h e mesoadenohypophysis o f F u n d u l u s . c e l l s a r e s m a l l and d e g r a n u l a t e d  These  d u r i n g the q u i e s c e n t w i n t e r  p e r i o d and l a r g e and f u l l y g r a n u l a t e d i n the b r e e d i n g S o k o l c o n s i d e r s t h e m a j o r i t y o f these c e l l s t o be  season.  58 gonadotrophs. gonadotrophic  A d d i t i o n a l evidence f o r the  e f f e c t s o f m e t h a l l i b u r e on the  comes from work by Brown (1963) on r a t s . s i g n i f i c a n t l y l e s s FSH  He  pituitary finds  i n t h e p i t u i t a r i e s o f animals  w i t h m e t h a l l i b u r e than i s p r e s e n t control  anti-  treated  i n the p i t u i t a r i e s  of  animals. Some o f t h e d e g r a n u l a t e d  c e l l s i n the meso-  adenohypophysis o f m e t h a l l i b u r e t r e a t e d G a s t e r o s t e u s  may  be  t h y r o t r o p h s s i n c e t h y r o t r o p h s cannot be d i s t i n g u i s h e d from gonadotrophs on t h e b a s i s o f t h e s t a i n c o m b i n a t i o n P o s s i b l e e v i d e n c e t h a t b o t h b a s o p h i l s may  used.  be a f f e c t e d comes  from e x p e r i m e n t s o f L e a t h e r l a n d and Pandey (1968).  They  f i n d t h a t the gonadotrophs o f m e t h a l l i b u r e t r e a t e d guppies a r e l e s s n u m e r o u s , s i g n i f i c a n t l y s m a l l e r and l e s s aldehyde f u c h s i n p o s i t i v e t h a n those o f t h e c o n t r o l s ; the t h r o t r o p h s , on t h e o t h e r hand, are s i g n i f i c a n t l y l a r g e r and more aldehyde f u c h s i n p o s i t i v e t r a n those o f the c o n t r o l s . c o n c l u d e t h e gonadotrophs are h y p o - a c t i v e w h i l e t h y r o t r o p h s are h y p e r - a c t i v e .  increase  Such an i n c r e a s e i n t h y r o i d  c e l l h e i g h t i s n o t found i n G a s t e r o s t e u s methallibure.  the  However, t h e y a l s o f i n d t h a t  m e t h a l l i b u r e t r e a t m e n t b r i n g s about a s i g n i f i c a n t in thyroid e p i t h e l i a l height.  They  treated with  These c o n s i d e r a t i o n s l e a d me t o c o n c l u d e t h a t  most o f t h e d e g r a n u l a t e d s t i c k l e b a c k s are p r o b a b l y Degranulation  basophils i n methallibure treated gonadotrophs.  o f b a s o p h i l s a l s o o c c u r s when a group  of f i s h i s treated with thiourea.  The  amount o f  degranulation  59 i s v a r i a b l e but i t i s c o r r e l a t e d with the increase i n thyroid e p i t h e l i a l c e l l height. most d e g r a n u l a t i o n  Thus, f i s h e x h i b i t i n g t h e  (++ i n T a b l e V I I ) a l s o show t h e g r e a t e s t  thyroid e p i t h e l i a l c e l l height.  Therefore,  c e l l s are very probably thytotrophs.  the degranulated  A number o f o t h e r  w o r k e r s have a l s o found d e g r a n u l a t i o n o f t h e t h y r o t r o p h s i n f i s h treated with thiourea or thiouracil  ( A t z , 1953 on  A s t y a n a x ; B a r r i n g t o n and Matty, 1955 on P h o x i n u s and Grosso, 1961 on L e b i s t e s ) .  60 V.  CONCLUSIONS  Treatment o f s t i c k l e b a c k s w i t h m e t h a l l i b u r e r e s u l t s in: (1)  A marked r e d u c t i o n i n t h e l e v e l o f prespawning  aggressiveness (2)  i n treated f i s h .  A decrease i n the r a t e o f transformation  spermatogonia  t o spermatocytes so t h a t a f t e r 30'- 38 days most o f t h e t e s t e s o f t h e t r e a t e d f i s h c o n t a i n e d spermatogonia and spermatozoa w h i l e t h e t e s t e s o f t h e c o n t r o l s c o n t a i n e d s p e r m a t o c y t e s and spermatozoa o r spermatozoa o n l y . 49 days, t h e t e s t e s o f t h e t r e a t e d group  After  contained  spermatogonia, s p e r m a t o c y t e s and spermatozoa w h i l e those o f t h e c o n t r o l s c o n t a i n e d o n l y spermatozoa. (3)  No e v i d e n c e o f s t i m u l a t i o n o f t h e c e l l s o f t h e b r u s h  b o r d e r segment o f t h e k i d n e y t u b u l e s o f t r e a t e d f i s h under long (4)  photoperiods. A marked d e c r e a s e i n t h e g r a n u l a t i o n o f t h e p i t u i t a r y  basophils. (5)  Some degree o f t o x i c i t y b u t no e v i d e n c e o f a d i r e c t  e f f e c t o f t h e treatment  on t h e t h y r o i d comparable t o t h a t  of thiourea. In b r i e f , i t i s concluded p a r t i a l l y b l o c k s gonadotrophic  that methallibure  a c t i v i t y probably a t the  l e v e l o f the p i t u i t a r y .  T h i s t r e a t m e n t : a f f e c t s prespawn-  ing aggressive behavior  and t e s t i s p h y s i o l o g y i n a manner  s i m i l a r t o an exposure t o s h o r t  photoperiods.  61 VI.  BIBLIOGRAPHY  Ahsan, S.N. (1966). E f f e c t s o f g o n a d o t r o p i c hormones on male hypophysectomized l a k e chub, Couesius plumbeus. Can. J . Z o o l . 44: 703-717. Ahsan, S.N., and W.S. Hoar (1963). Some e f f e c t s o f g o n a d o t r o p i c hormones on t h e t h r e e s p i n e s t i c k l e b a c k , G a s t e r o s t e u s a c u l e a t u s . Can. J . Z o o l . 4 1 : 10451053. A t z , E.H. (1953). 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