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UBC Theses and Dissertations

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 of Newfoundland, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y . a v a i 1 a b 1 e f o r r e f e r e n c e a nd S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r b y h.i)s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r pub 1 i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a i ABSTRACT Treatment of 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 the l e v e l of prespawn-i n g aggressiveness. Gametogenesis i n the t e s t e s of the t e s t e s of the 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 of treatment most t e s t e s contain spermatozoa and spermatogonia w h i l e the t e s t e s o f the c o n t r o l s are more mature and co n t a i n spermatocytes and spermatozoa or spermatozoa on l y . F o r t y - n i n e days of treatment 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 are completely mature and contain 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 h e i g h t evidence t h a t the I.C.I, compound i s not having 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 are immersed i n a s o l u t i o n of t h i o u r e a . The adenohypophysis of 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 of untreated c o n t r o l s . i i TABLE OF CONTENTS Page I . INTRODUCTION 1 I I . METHODS AND MATERIALS 7 A. Sex Determination 8 B. Behavior Observations 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. Thiourea Immersion 11 F. General C o n d i t i o n of F i s h 13 G. H i s t o l o g y 13 I I I . RESULTS 15 A. Behavior Studies 15 1. Prespawning Aggressiveness .... 15 a) M e t h a l l i b u r e Treatment 15 b) Other Treatments 21 i ) L u t e i n i z i n g Hormone 21 i i ) Thiourea Immersion ..... 23 2. Nest B u i l d i n g 23 B. H i s t o l o g y 25 1. T e s t i s and Kidney 25 a) M e t h a l l i b u r e Treatment on the T e s t i s 32 b) M e t h a l l i b u r e Treatment on the Kidney 35 c) LH Treatment 37 d) Thiourea Treatment 37 e) GSI 38 i i i Page 4. Thyroid 38 a) M e t h a l l i b u r e and Thiourea Treatment 38 5. P i t u i t a r y 40 a) Morphology 40 b) M e t h a l l i b u r e and Thiourea Treatment 42 IV. DISCUSSION 47 A. Prespawning Aggressive Behavior .. 50 B. T e s t i s and Kidney 52 C. P i t u i t a r y 57 V. CONCLUSIONS 60 V I . BIBLIOGRAPHY 61 i v LIST OF TABLES Page I Summary of Experiments Performed 12 I I Amount of Aggressive Behavior i n M e t h a l l i b u r e - t r e a t e d and Contr o l s 20 I I I Number of F i s h used f o r Examination of T e s t i s H i s t o l o g y 44 IV E f f e c t o f M e t h a l l i b u r e , LH and Thiourea on H i s t o l o g y of the T e s t i s and the Kidney 34 V G.S.I. Comparison of E f f e c t of M e t h a l l i b u r e and Thiourea Treatment on Gonosomatic Index 39 VI Mean Thyroid C e l l Height, f o l l o w i n g Thiourea and M e t h a l l i b u r e Treatment 41 V I I Amount of Gra n u l a t i o n i n the Baso 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 Thiourea Immersion 43 V LIST OF FIGURES Figur e 1 Facing Page 5 6 7 8 9 10 - 17 10 11 Aggressive l e v e l s of s t i c k l e b a c k s i n j e c t e d w i t h 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 Aggressive l e v e l s of 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 Aggressive l e v e l s of s t i c k l e b a c k s i n j e c t e d w i t h LH and LH + METH compared wi t h c o n t r o l s Aggressive l e v e l s of 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 Diagrams of the Stages of T e s t i s Maturation Stage 0 Stage I Stage I I Stage I I I Stage IV Photomicrographs o f the Stages of T e s t i s Maturation Stage 0 x 200 Stage 0 x 900 17 18 19 24 26 26 27 27 28 30 30 F i g u r e Facing 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 the 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 C o n t r o l x 200 45 21 C o n t r o l x 900 45 22 Thiourea t r e a t e d x 200 46 23 Thiourea t r e a t e d x 900 46 24 Thiourea t r e a t e d x 200 46 ACKNOWLEDGEMENT I would l i k e to express my g r a t i t u d e to Dr. W.S. Hoar f o r h i s guidance, c r i t i c i s m and encourage-ment throughout the p r o j e c t and during the pr e p a r a t i o n of the manuscript. I am indebted to Dr. J.A. Dodds* f o r the sup p l i e s of I.C.I. 33,828 and to Dr. A.E. W i l h e l m i * * f o r the p u r i f i e d bovine LH. I would l i k e to thank Dr. J.F. Leatherland, Dr. M.S. J a r i a l , Mr. L. Sharman and Mr. C. Grainge f o r t h e i r help w i t h the h i s t o l o g y and Mr. W.R. Black, Mr. P. E l l i c k s o n and Mr. C. Levings f o r t h e i r a s s i s t a n c e s e i n i n g at the 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 to 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 suggestions. The research was made p o s s i b l e through the f i n a n c i a l support of the N a t i o n a l Research C o u n c i l of Canada. •V e t e r i n a r y Medical Department, Ayerst L a b o r a t o r i e s , Montreal, P.Q. **Endocrinology Study Section, Department of Biochemistry, Emory U n i v e r s i t y , A t l a n t a , Georgia 1 I. INTRODUCTION A number of s t u d i e s have helped e l u c i d a t e the c h a r a c t e r i s t i c r e productive behavior of the three-spined s t i c k l e b a c k Gasterosteus aculeatus L. (Tinbergen and t e r P e l k w i j k , 1937; van I e r s e l , 1953, 1959). B r i e f l y the sequence i s as f o l l o w s : In the l a t e w i n t e r or e a r l y s p r i n g the f i s h show a decreased tendency to s c h o o l . 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 of e i t h e r sex although they are p a r t i c u l a r l y aggressive towards other males. During t h i s time the s i l v e r y w i n t e r f i s h develop c h a r a c t e r i s t i c breeding c o l o r a t i o n . The l a t e r a l and d o r s a l surfaces of both males and females darken to a mottled green-b l a c k . The t h r o a t r e g i o n of. the males becomes red and t h e i r eyes change from s i l v e r to b r i g h t b l u e . L a t e r the males c o l l e c t algae and glue i t together to form a nest w i t h a s t i c k y mucous s e c r e t i o n produced by the kidneys at t h i s stage. A f t e r t u n n e l i n g through the nest the male i s ready to court a g r a v i d female. The male leads the female to the nest 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 the male p o i n t s to the nest entrance the female enters and i n response to h i s prods, spawns. The male f o l l o w s through the nest and f e r t i l i z e s the eggs. He then chases the female away. The male continues to defend the t e r r i t o r y and cares f o r the c l u t c h of eggs by fanning water 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 the eggs have hatched the male guards the young f o r a few days. This completes the r e p r o d u c t i v e c y c l e . The preceding d e s c r i p t i o n of reproductive behavior a p p l i e s to the s a l t water form of Gasterosteus which enters f r e s h water to breed. I have used only the freshwater form which l i v e s a l l year i n f r e s h water. Hagen (1967) who stu d i e d both forms i n the Vancouver area, 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 behavior of both very s i m i l a r and he can detect no b e h a v i o r a l b a r r i e r s to i n t e r b r e e d i n g between the two forms. The r o l e of the endocrine system i n t h i s behavior sequence i s e v i d e n t l y complex and at present 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 observations of the e f f e c t s of removal of an endocrine gland and subsequent replacement therapy i n v o l v i n g i n j e c t i o n s of gland e x t r a c t s or s y n t h e t i c hormones. Removal of the gonads of s t i c k l e b a c k s i s a comparatively simple 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 out by a number of workers (Baggerman, 1957; Hoar, 1962 a, b; Wai and Hoar, 1963); removal of the p i t u i t a r y , however, i s d i f f i c u l t and the o p e r a t i o n has not yet been s u c c e s s f u l w i t h Gasterosteus. I n v e s t i g a t o r s have t h e r e f o r e used photoperiod c o n t r o l to r e g u l a t e p i t u i t a r y a c t i v i t y . Baggerman (1957) showed t h a t the seasonal reproductive c y c l e of 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 the changing 3 l i g h t c o n d i t i o n s w h i l e the e f f e c t s of temperature are a d d i t i v e . I t i s assumed t h a t short photoperiod (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 of darkness) suppresses the high gonadotrophin production c h a r a c t e r i s t i c of breeding f i s h , w h i l e long photoperiod (16 hr of l i g h t a l t e r n a t i n g w i t h 8 h r of darkness) r e a d i l y b r i n g s f i s h i n t o reproductive c o n d i t i o n . I n v e s t i g a t o r s of the endocrinology o f s t i c k l e -back reproduction have used the d i f f e r e n c e s i n response, to t v a r i o u s treatments, o f short photoperiod p h y s i o l o g i c a l l y i hypophysectomized f i s h compared w i t h long photoperiod f i s h . Wai and Hoar (1963) used gonadectomy and subsequent treatment w i t h methyl testosterone (a s y n t h e t i c androgen) on long and short photoperiod s t i c k l e b a c k s . They found the high l e v e l of pre-spawning aggressiveness, c h a r a c t e r i s t i c o f t e r r i t o r i a l f i s h p r i o r to nest b u i l d i n g , appeared:in gonadectomized f i s h under long photoperiod but not i n gonadectomized f i s h under short photoperiod. The a d d i t i o n of methyl testosterone d i d not g r e a t l y a f f e c t the aggressiveness o f these c a s t r a t e d f i s h . The androgen treatment d i d , however, r e s t o r e the secondary sex characters (mucous s e c r e t i n g kidney tubules and n u p t i a l c o l o r a t i o n ) which had been l o s t a f t e r c a s t r a t i o n . Castrated f i s h showed n e i t h e r nest b u i l d i n g nor sexual behavior. Methyl t e s t o s t e r o n e treatment, however, r e s u l t e d i n the appearance of both these b e h a v i o r a l sequences i n males under both photoperiod regimes. These behavior p a t t e r n s reappeared more q u i c k l y and i n more o f the long photoperiod f i s h . 4 The evidence from the above s t u d i e s i s t h a t p i t u i t a r y gonadotrophins c o n t r o l pre-spawning aggressiveness, w h i l e gonadal s t e r o i d s c o n t r o l secondary sex characters, and both gonadotrophins and androgen act s y n e r g i s t i c a l l y to produce nest b u i l d i n g and sexual behavior (Hoar, 1965). To o b t a i n a d d i t i o n a l evidence of the endocrine c o n t r o l or pre-spawning behavior and gametogenesis, a pharmacological technique of suppressing gonadotrophin was sought. Hoar, Wiebe and Wai (1967) discussed p r e l i m i n a r y r e s u l t s obtained u s i n g m e t h a l l i b u r e ( I . C . I . 33,828), a non-steroid, non-hormonal compound, on three species of f i s h . 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 surfperch s t r o n g l y i n d i c a t e t h a t m e t h a l l i b u r e s e l e c t i v e l y i n h i b i t s p i t u i t a r y f u n c t i o n . The gonads of the t r e a t e d f i s h showed d i v i s i o n of the primary germ c e l l s but no maturation of these c e l l s . C o n t r o l s e x h i b i t e d normal germ c e l l maturation. The data reported by Hoar et a l . (1967) on s t i c k l e -backs were considered p r e l i m i n a r y . M o r t a l i t y was high i n the t r e a t e d f i s h and the sample s i z e was s m a l l . However, as w e l l as the e f f e c t on gametogenesis, there was an i n d i c a t i o n o f an e f f e c t on gonadal s t e r o i d p r oduction. The c e l l s of the brush border segment of the kidney tubules remained at a s i z e c h a r a c t e r i s t i c of 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 of t h i s segment of the kidney become l a r g e w i t h granular cytoplasm under the i n f l u e n c e of androgen. Wiebe (1968) continued the i n v e s t i g a t i o n on surfperch Cymatogaster aggregata. He found treatment 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, r e g r e s s i o n of the i n t e r s t i t i a l c e l l s and l a c k of increase i n s i z e of the f l e s h y m o d i f i c a t i o n s of the anal f i n . These m o d i f i c a t i o n s o f the anal f i n are a secondary sex s t r u c t u r e which develop under the i n f l u e n c e of 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 detected when the 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 the goitrogen, t h i o u r e a . Wiebe (1967) reported t h a t there was a s i g n i f i c a n t drop i n the repro d u c t i v e behavior of 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 untreated c o n t r o l s . The present study continues the i n v e s t i g a t i o n of the e f f e c t s of m e t h a l l i b u r e on Gasterosteus to f u r t h e r the understanding o f the c o n t r o l of the p i t u i t a r y gonadotrophins on prespawning aggressive behavior and t e s t i s p h ysiology. These two aspects of the study are c l o s e l y i n t e r r e l a t e d . The work to date suggests t h a t m e t h a l l i b u r e acts d i r e c t l y on the p i t u i t a r y gonadotrophs r a t h e r than the t i s s u e s o f the t e s t i s (Leatherland and Pandey, 1968). This 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 by examining s e c t i o n s of the t e s t i s ((.game to genie t i s s u e ) , the height of the kidney tubules (as a measure o f s t e r o i d e f f e c t s ) , and the 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 untreated c o n t r o l s . At present, p i t u i t a r y gonadotrophin i s thought to c o n t r o l d i r e c t l y the increased aggressiveness of male s t i c k l e b a c k s when they set up and maintain d i s c r e t e t e r r i t o r i e s p r i o r to spawning. I f m e t h a l l i b u r e i s b l o c k i n g the a c t i o n of gonadotrophin then presumably t r e a t e d s t i c k l e -6 backs should be l e s s aggressive than untreated c o n t r o l s . In b r i e f , the primary o b j e c t i v e of the study was to i n v e s t i g a t e the e f f e c t s of m e t h a l l i b u r e on pre-spawning aggressive behavior and to i n v e s t i g a t e the r e s u l t s i n terms of the gonadotrophic c o n t r o l over t h i s behavior and t e s t i c u l a r p h y s i o l o g y . 7 I I . METHODS AND MATERIALS Freshwater s t i c k l e b a c k s type B' (Heuts, 1947) were caught at v a r i o u s times throughout the year i n the L i t t l e Campbell R i v e r south of Vancouver, B r i t i s h Columbia. They were kept u n t i l needed f o r the experiments i n l a r g e tanks of running d e c h l o r i n a t e d water. Although the tanks were housed i n the l a b o r a t o r y , the photoperiod was regulated at the n a t u r a l length by a p h o t o c e l l placed i n a south window. The water temperature v a r i e d between 5° and 14° C depending on the time of year. At the outset of an experiment, f i s h were placed i n groups of two or four i n 16 l i t r e g l a s s aquaria (45 l i t r e g l a s s aquaria were used i n one experiment, No. 8 ) . Dechlorinated water f o r t i f i e d w i t h 20 ppm calcium c h l o r i d e was c o n t i n u a l l y aerated and f i l t e r e d through gla s s wool; the f i l t e r s were changed once a week. During the experiments the aquaria were kept i n an a i r - c o n d i t i o n e d room which maintained water temperature at 20 + 1°C. The photoperiod provided 16 hr of l i g h t a l t e r n a t i n g w i t h 8 hr of darkness. I l l u m i n a t i o n (50-55 f t - c ) was provided by f l u o r e s c e n t lamps. A l l aquaria contained sand and water s p r i t e ( C e r a t o p t e r i s sp . ) . The p l a n t s covered about 75% of the water surface. The f i s h were fed frozen b r i n e shrimp (Artemia  s a l i n a ) once a day. As a p r e c a u t i o n against fungal i n f e c t i o n , 2-3 drops of 1% malachite green s o l u t i o n were u s u a l l y added to each aquarium at the beginning of the experiments. 8 A. Sex Determination Male f i s h were used i n the experiments. U s u a l l y , males could be d i s t i n g u i s h e d from females by the greater amount of red pigmentation i n the 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 of c o l o r could be seen, so a t i n y i n c i s i o n was made i n one side of the f i s h . Males were d i s t i n g u i s h e d from females on the b a s i s o f the d i f f e r e n c e i n shape and pigmentation of the gonads. The males were kept i n 50% sea water f o r 48 h r a f t e r t h i s o p e r a t i o n before they were placed i n groups of two i n the gla s s aquaria. A p r e p a r a t i o n c a l l e d Orabase* was used over the i n c i s i o n . This p r e p a r a t i o n adhered to and closed the wound f o r an hour or two, a f t e r which i t dropped o f f . B. Behavior Observations In the behavior s t u d i e s , a l l aquaria were observed f o r periods of f i v e minutes two or three occasions each week. The behavior observed was of two main types: (1) Aggressive behavior and (2) N e s t - b u i l d i n g behavior. Three components of aggressive behavior 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 at another but stops be f o r e reaching i t , t h i s type of behavior i s very common when two or 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 tank; (b) B i t e where one f i s h makes a d a r t at another and b i t e s i t once or s e v e r a l times; (c) Chase where the aggressive f i s h pursues the subordinate one who must * Squibb Oral P r o t e c t i v e Paste 9 seek s h e l t e r . The nest 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 of: (a) Sand-digging where a male, i n a head down posture, p i c k s up a mouthful of sand, turns, swims away and s p i t s i t out; (b) Searching where a male p i c k s up small p ieces of roots or algae and s p i t s them out; (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 of algae 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 over the nest area w h i l e s e c r e t i n g a mucous from the kidney and (e) Fanning where a male adopts a head down p o s i t i o n at 30°-40° to the h o r i z o n t a l at the nest entrance and maintains t h i s p o s i t i o n by the combined forward swimming movements of the t a i l and the backward swimming movements of the p e c t o r a l s . Quantative data were obtained by the use of an Esterline-Angus pen recorder w i t h the keys numbered so t h a t each number corresponded to a p a r t i c u l a r behavior movement. Thus, when a behavior p a t t e r n was observed the appropriate key was depressed. The frequency of the v a r i o u s p a t t e r n s was recorded. The f i s h i n each aquarium were assigned a l e v e l of aggression f o r each observation p e r i o d , based on the f o l l o w -i n g 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 loose group. L e v e l It One or two a t t a c k s per minute (ten or fewer i n f i v e minutes); loose schooling, no s o c i a l order; no evidence of dominance or subordination. 10 L e v e l I I : Three or more attack s per minute, f i s h of equal or near equal s t a t u s , no dominance or sub-o r d i n a t i o n . L e v e l 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 no p a r t i c u l a r dominance, one f i s h has a p r e f e r r e d area and b i t e s f i s h e n t e r i n g t h i s area but does not f i g h t v i g o r o u s l y or subordinate other f i s h . L e v e l IV: A dominance-subordinate r e l a t i o n s h i p i s w e l l marked; the dominant f i s h a t t a c k s other f i s h v i g o r o u s l y , d r i v e s them i n t o corners and sometimes k i l l s them. When more than one dominant i s i n the tank the t e r r i t o r i a l boundaries are sharp. The number of aggressive acts i s u s u a l l y g r e a t l y reduced at L e v e l IV, s i n c e a tank o f t e n contains a s i n g l e dominant male w i t h a l l subordinates i n h i d i n g , so t h a t encounters between f i s h are few. Consequently, i n most experiments once a week, a "standard" female or j u v e n i l e f i s h was used. The "standard" f i s h was placed i n s i d e a glass tube (6.5 cm i n diameter) and tubes w i t h f i s h placed i n the middle region of a l l tanks which were ra t e d L e v e l IV d u r i n g the previous observation p e r i o d . U s u a l l y the "standard" f i s h 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. The number of att a c k s on the tube i n f i v e minutes was recorded. C. M e t h a l l i b u r e I n j e c t i o n and Immersion Several dosages and frequencies of dose of M e t h a l l i b u r e were t e s t e d (Table I ) . In most experiments a 11 f i n e suspension of m e t h a l l i b u r e i n d i s t i l l e d water or f r e s h -water t e l e o s t s a l i n e * was prepared w i t h a small amount (one drop per 5 ml) of the w e t t i n g agent "Tween 80". E i t h e r 0.05 or 0.025 ml of suspension was i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h a 26 G. needle. Successive i n j e c t i o n s were made on a l t e r n a t e s i d e s of the f i s h . In the f i r s t experiments there was some leakage of i n j e c t e d suspension so "Orabase" was a p p l i e d to the hole made by the needle. In experiments No. 6 and 7 the use of "Orabase" was considered unnecessary because the i n j e c t e d volume was so s m a l l . In three of the e i g h t experiments m e t h a l l i b u r e was added to the ambient water of a group o f experimental f i s h . D e t a i l s of the amount added are i n Table 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 of LH i n 0.025 ml d i s t i l l e d water twice a week. A second group i n No. 6 and 7 was i n j e c t e d w i t h a mixture of 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 of f i s h was t r e a t e d w i t h t h i o u r e a by immersion w i t h 1.5 gm of t h i o u r e a added to the 45 l i t r e tanks every week f o r seven weeks. *1 l i t r e of freshwater t e l e o s t s a l i n e contains 5.50 gm NaCl, 0.14 gm KC1, 0.12 gm C a C l 2 i n d i s t i l l e d water. **NIH-LH-B5 Bovine to * * Ul CTl ft' f+ CD ID (X) CD oo tr r t O > to H 00 00 d r t O I—1 I-1 to o to o to Ul CO 00 o to Ul CO 00 o IO o to U l (jO to o to Ul CO CO CTl n r t 0 S oo P> Ul if U l if CO It) M tr) 1-. II O II tf tf r t r t 0 0 £ to g oo 0) o c O M O M (D CD O O r t r t O 0 O to D H CD to CD to O O 3 0 < o (D to r t 0 to to to O 00 00 O to Ul Ul Ul U l U l o to Ul o Ul o Ul o Ul o U l o U l CO o o 00 00 00 o to U l o Ul o U l o U l o U l o U l CO 00 00 to o to o to Ul CO 42 o o to Ul co CO o * o * U l Ul U l U l 00 U l td § 3 H. &> r t H -o 3 No. f i s h Arat. mg V o l . ml Days bet. i n j . No. f i s h V o l . ml Days bet. i n j . No. F i s h No f i s h Amt. mg Vol. ml Days bet. No. f-i-sh Amt. mg. V o l . ml Days bet. i n j . No. f i s h Amt. ma V o l . ml No days per week i s h Amt. gm No days p e r week. H 3 CD O r t CD S CD H Q j d CD H 3 oo I—I. Q J CD h-1 O H-r t 3 CD (D 8S 1 9£ H 3 (D r 1 o a r t CD 3 (D H r t 3 3 1 —1.(1) CD H* O H r t H ' (D H CD i-3 td > S td Si d p> n «< o Hi td CD H H" 3 CD 3 r t 01 T) CD Hi 0 H 3 CD & 8 F td H 13 F. General C o n d i t i o n of F i s h Only f i s h which appeared h e a l t h y were used f o r experiments. However, there were m o r t a l i t i e s among both experimental and c o n t r o l f i s h i n most experiments. In some cases the f i s h developed fungal i n f e c t i o n but i n others the cause of death was not apparent. The i n i t i a l i n j e c t i o n doses of m e t h a l l i b u r e may have been too high but 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 equal i n experimental and c o n t r o l groups. The exceptions were groups i n j e c t e d w i t h LH alone or i n combinations w i t h m e t h a l l i b u r e . The higher m o r t a l i t y i n these groups was probably due to the j o i n t e f f e c t s of LH and long photoperiod ( n a t u r a l gonadotrophin at h i g h l e v e l s ) s i n c e the dose of LH was the same as t h a t used by Hoar (1962b) on s t i c k l e b a c k s kept under short photoperiod, when i t had no i l l e f f e c t s . G. H i s t o l o g y A l l f i s h were k i l l e d by an overdose of t r i c a i n e methane s u l f o n a t e (Sandoz MS 222), the gonad and body weight recorded and t h e i r t i s s u e s f i x e d i n Bouin's p i c r i c a c i d -f o r m a l - a c e t i c a c i d s o l u t i o n . 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 procedure and s t a i n e d u s u a l l y w i t h haematoxylin and eosin, 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 b l u e . Sections of the v a r i o u s t i s s u e s were cut at 5-lOji. In experiments No. 1 to 4 o n l y the gonads were sectioned, i n No. 5 both gonads and kidneys and i n No. 6-8 gonads, kidneys and t h y r o i d s were sectioned. In experiment No. 8 14 p i t u i t a r i e s were a l s o sectioned and s t a i n e d w i t h a l c i a n blue-PAS-orange G. 15 I I I . RESULTS A s e r i e s o f e i g h t experiments was c a r r i e d out between October 1966 and A p r i l 1968. The d e t a i l s are summarized i n Table I . In each of the experiments one or two groups of 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 of 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 three groups were immersed i n a d i l u t e suspension of the compound. Two groups of f i s h were 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 mixture of equal amounts of LH and m e t h a l l i b u r e . A s i n g l e group of f i s h was immersed i n a d i l u t e s o l u t i o n of t h i o u r e a . The f i r s t two experiments form a p r e l i m i n a r y study. They helped f a m i l i a r i z e me w i t h the v a r i o u s behavior patterns of the s t i c k l e b a c k s , the general care o f the f i s h and the technique of i n j e c t i o n . The experiments No. 3 to 8 formed the b a s i s of the d e t a i l e d study (Table I) . A. Behavior Studies 1. Prespawning Aggressiveness a) M e t h a l l i b u r e Treatment Since two methods of treatment 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 important to determine i f eq u i v a l e n t r e s u l t s were obtained w i t h both methods. Therefore, data from experiments No. 6, 7 and 8 on the l e v e l s of aggressive:.behavior were evaluated f o r both methods and i t was found t h a t both r e s u l t e d i n a marked drop 16 i n the l e v e l of aggressiveness a f t e r four weeks of m e t h a l l i b u r e treatment ( F i g . 1 ) . The aggressive l e v e l of the c o n t r o l s increased over the same four week p e r i o d . To determine the e f f e c t s of i n j e c t i o n , data were combined from experiments No. 6 and 7 ( F i g . 1 ) . At the beginning (Week 0), before i n j e c t i o n s were begun, four tanks were at Lev e l I (two experimental, two c o n t r o l ) and twelve tanks were at Level s I I I and IV (seven experimental, f i v e c o n t r o l ) . A f t e r two weeks of treatment the change i n aggressiveness was already apparent w i t h an increase i n the c o n t r o l s and a decrease i n the experimentals. E i g h t of the experimental tanks were between Levels 0 and I I w i t h no t e r r i t o r i a l i t y present, w h i l e o n l y one c o n t r o l tank was between these l e v e l s . The remaining s i x c o n t r o l tanks and the one experimental were between L e v e l s I I I and IV. A f t e r four weeks of treatment the 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 at Levels 0 and I, w i t h a maximum of ten aggressive acts i n f i v e minutes. A l l the c o n t r o l s were at L e v e l s I I I and IV. From the second week to the f o u r t h week there was a s l i g h t drop i n l e v e l of two of the 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 behavior w h i l e none of the experimentals d i d . When data from experiments No. 7 and 8 were combined almost the same r e s u l t s were obtained a f t e r four weeks of immersion i n m e t h a l l i b u r e . In t h i s case ( F i g . 2) before treatment four tanks of f i s h were at L e v e l I (three c o n t r o l , one experimental) w h i l e e i g h t were at Levels I I I and IV 17 Figur e 1 Aggressive l e v e l s o f s t i c k l e b a c k s i n j e c t e d w i t h 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. before the treatment was begun B. two weeks a f t e r the f i r s t i n j e c t i o n C. four weeks a f t e r the f i r s t i n j e c t i o n • Open bars, m e t h a l l i b u r e i n j e c t e d ; bars w i t h s l a n t e d l i n e s , c o n t r o l . 6 A 7i I 7 •'i i O I 11 III IV A G G R E S S I V E L E V E L 7\ .1. I / n ; O I II III IV A G G R E S S I V E L E V E L B * 4 z Z 3 1 O | II III IV A G G R E S S I V E L E V E L 18 F i g u r e 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. before the treatment was begun B. two weeks a f t e r the f i r s t a d d i t i o n C. four weeks a f t e r the f i r s t a d d i t i o n Bars w i t h dots, m e t h a l l i b u r e t r e a t e d ; bars w i t h s l a n t e d l i n e s c o n t r o l s . 19 (three c o n t r o l , f i v e experimental). A f t e r two weeks of treatment there had been o n l y a s l i g h t change i n l e v e l of aggressiveness. A f t e r four weeks of treatment there had been a more marked change, and three tanks were at Levels  0 and I ( a l l experimental), s i x c o n t r o l tanks were a t Le v e l s I I I and IV and two experimental were at L e v e l I I I . The immersion technique was not 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 slower to show i t s e f f e c t s but a f t e r four weeks of treatment no tanks 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 su b o r d i n a t i o n . Thus, treatment of 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 four weeks can be seen to b r i n g about a decrease i n aggressive behavior, w h i l e = at the same time, the untreated f i s h show an in c r e a s e i n aggressive behavior. The data from experiment No. 7 were used to compare the number of aggressive a c t s , performed i n the t e s t p e r i o d , of the 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 the c o n t r o l s (Table I I ) . The amount of aggression f o r each tank was the t o t a l number of b i t e s , approaches and chases observed i n f i v e minutes. Each tank was observed twice a week. From the four tanks at each treatment a weekly average (+ the Standard E r r o r of the mean) was computed and i s given i n Table I I . From the second through to the 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 the g l a s s tube i n a l l aquaria t h a t were a t L e v e l IV the previous observation p e r i o d . TABLE I I Amount of Aggressive Behavior i n M e t h a l l i b u r e - t r e a t e d and Con t r o l s WEEK T R E A T M E N I Con t r o l 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 values based on average amount of aggressive behavior i n 5 min + S.E. of 4 tanks each observed twice per week *Tubes w i t h standard f i s h were used i n one of two weekly observations - 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 at 0.05 among means f o r any week 21 These data show t h a t the number of aggressive acts i s r e l a t i v e l y low i n the m e t h a l l i b u r e i n j e c t i o n group throughout the f i v e weeks of treatment 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 of aggressive acts during the f i r s t three weeks of treatment. A f t e r t h i s the number of at t a c k s g r a d u a l l y decreases to 4.0 + 2.8 and f i v e weeks of treatment. 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 of aggressive acts d u r i n g the f i r s t three weeks. The high number i s maintained d u r i n g weeks three and f i v e but drops dur i n g week four. This drop i s found i n a l l groups d u r i n g the f o u r t h week but o n l y the c o n t r o l group r i s e to high number 19.8 + 6.8 durin g the f i f t h week. However, due to the small sample s i z e and the v a r i a b i l i t y between tanks the mean of 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 not s i g n i f i c a n t l y d i f f e r e n t from the mean of the c o n t r o l group f o r any week (at 0.05 l e v e l u s i n g the ' 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 ' ) . 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 alone and i n combination w i t h equal concentrations of 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 of these treatments on l e v e l s of aggressive behavior are compared w i t h the c o n t r o l s i n F i g . 3. Before i n j e c t i o n s were begun, four tanks were at Levels  0 and I (three LH and one c o n t r o l ) ; the remaining twenty 22 F i g u r e 3 Aggressive l e v e l s of s t i c k l e b a c k s i n j e c t e d w i t h LH and LH + METH compared w i t h c o n t r o l s . A. befo r e the treatment was begun B. two weeks a f t e r the f i r s t i n j e c t i o n C. four weeks a f t e r the f i r s t i n j e c t i o n bars w i t h open c i r c l e s , LH i n j e c t e d ; bars 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 w i t h s l a n t e d l i n e s , c o n t r o l s . 23 tanks ( f i v e LH, s i x c o n t r o l and nine m e t h a l l i b u r e and LH) were at Levels I I I and IV. A f t e r two weeks of i n j e c t i o n both the 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 of aggression but the LH + METH group showed a drop i n l e v e l . S i x of the seven c o n t r o l tanks were at Leve l IV, as were f i v e of the seven LH tanks. Only three of the nine LH + METH were at L e v e l IV. A f t e r four weeks of treatment a number o f f i s h had died but of those which remained a l l the c o n t r o l s and LH tanks were at Levels I I I and IV as were three of the f i v e LH + METH. These data i n d i c a t e t h a t treatment w i t h LH + METH r e s u l t s i n a l e v e l of aggression intermediate between the high l e v e l s w i t h LH alone and the low l e v e l s w i t h m e t h a l l i b u r e alone. i i ) Thiourea Immersion A group of f i s h was immersed i n t h i o u r e a i n experiment No. 8 and the l e v e l of aggressive behavior compared w i t h the c o n t r o l s and the me t h a l l i b u r e t r e a t e d group ( F i g . 4 ) . Over the four week p e r i o d the t h i o u r e a group maintained a high l e v e l of aggression. During t h i s time one group of c o n t r o l s increased i n l e v e l and both groups of 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 . Thus i t i s concluded t h a t treatment 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 the l e v e l of aggression was high i n many F i g u r e 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. before treatment was begun B. two weeks a f t e r the f i r s t a d d i t i o n C. four weeks a f t e r the f i r s t a d d i t i o n bars w i t h dots, m e t h a l l i b u r e t r e a t e d ; bars w i t h cross hatching, t h i o u r e a t r e a t e d ; bars w i t h s l a n t e d l i n e s , c o n t r o l s . £ 0 o 30 rn — 1/1 < — m — < m T A N K S — K» W wi T _ M T A N K S > O O 70 m —i </> C O < _ m —1 m < m O SSI 7ZZ2 - T " > o c m — vt < _ m _ m < — W \ ' I 1 3 3 T A N K S ui ui o DO tanks and d i s c r e t e areas were defended, nest b u i l d i n g was i n f r e q u e n t l y observed. In a l l experiments o n l y three c o n t r o l f i s h , four LH, three LH + METH, two th i o u r e a , one me 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 n e s t s . H i s t o l o g y 1. T e s t i s and Kidney The c y c l i c a l changes which annually occur i n the 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 by Ahsan and Hoar (1963) i n t o four d i s t i n c t stages o f maturation and are 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 the postspawning p e r i o d d u r i n g August and September. Spermatogonia are most prominent i n the seminiferous tubules but there are a l s o a few nests of primary spermatocytes. The i n t e r s t i t i a l t i s s u e i s i n d i s t i n c t or absent. A few sperm from previous c y c l e s may be present. I I S l i g h t or M i l d A c t i v i t y ( F i g . 7 & 14) found i n nature from October through December. Primary and secondary spermatocytes predominate; spermatogonia are l e s s common; there are a few i n t e r s t i t i a l c e l l s . I I I Moderate A c t i v i t y ( 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 January through March. The e a r l y stages of spermatogenesis are s t i l l evident but spermatids and spermatozoa are conspicuous and the i n t e r s t i t i a l c e l l s are more numerous. 26 F i g u r e 5 - Diagram of Stage 0 This stage i s c h a r a c t e r i z e d by seminiferous tubules ringed w i t h spermatogonia two or three c e l l s t h i c k . The centers of the tubules are f i l l e d w i t h spermotozoa or spermatids. Few spermatocytes or 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 of Stage I Spermatogonia are most prominent i n the seminiferous tubules but there are a l s o a few primary spermatocytes. 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 absent. 27 F i g u r e 7 - Diagram of Stage I I Primary and secondary spermatocytes predominate; spermatogonia are l e s s common; there are a few i n t e r s t i t i a l c e l l s . F i g u r e 8 - Diagram of Stage I I I The e a r l y stages of spermatogenesis are s t i l l p resent but spermatids and spermatozoa are 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 of Stage IV The seminiferous tubules are packed w i t h spermatids and spermatozoa. E a r l i e r stages are 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 are numerous. 9 I V 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 of the breeding season from m i d - A p r i l to e a r l y August. The seminiferous tubules are packed w i t h spermatids and spermatozoa. E a r l i e r stages are d i f f i c u l t to f i n d and i n t e r s t i t i a l c e l l s are numerous. In the present study, a f i f t h stage, defined as Stage 0 ( F i g . 5, 10, 11 & 15), was found i n many m e t h a l l i b u r e t r e a t e d f i s h . This stage was c h a r a c t e r i z e d by seminiferous tubules ringed w i t h spermatogonia two or three c e l l s t h i c k . The centers of the tubules were f i l l e d w i t h spermatozoa or spermatids. Very few, i f any, spermatocytes were present and few i n t e r s t i t i a l c e l l s were evident. In the present study t e s t e s were cat e g o r i z e d according to one of the above l e v e l s of maturation by examination of seven or 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 the middle region of the organs. I t can be seen from Table I, the experiments of 31 to 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 out at d i f f e r e n t seasons of the year. The f i s h were there-f o r e a t d i f f e r e n t stages of t e s t i s maturation depending on the time of year. However, Baggerman (1957, 1966) f i n d s t h a t a t any time of year s t i c k l e b a c k s exposed to 16 hr of l i g h t and 20°C w i l l a t t a i n f u l l breeding c o n d i t i o n i n 2 - 4 weeks. Also, I f i n d most of the c o n t r o l f i s h are mature or maturing i n a l l experiments a f t e r 30 - 38 days exposure to long photoperiod and 20°C. Perhaps the f i s h may vary i n t h e i r responsiveness to m e t h a l l i b u r e treatment at d i f f e r e n t 30 F i g u r e 10 - Stage 0 Photomicrograph of the t e s t i s of 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 31 days x 200 Figur e 11 - Stage 0 Same t e s t i s as F i g . 10 x 900 Figure 12 - Stage I I I Photomicrograph o f the t e s t i s of 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 Figur e 13 - Stage I I I Same t e s t i s as F i g . 12 x 900 1 spcyt. -2 spcyt. -sp. duct--spgon. spzoa. s p t i d . spermatogonia spermatozoa spermatid primary spermatocyte secondary spermatocyte main sperm duct 31 F i g u r e 14 - Stage I I Photomicrograph of the t e s t i s of a c o n t r o l f i s h under long photoperiod 32 days x 200 Fig u r e 15 - Stage IV Photomicrograph o f the t e s t i s of a c o n t r o l f i s h under long photoperiod 49 days showing main sperm duct and s e v e r a l branches to seminiferous tubules x 200 Fig u r e 16 - Stage 0 Photomicrograph of the t e s t i s of a m e t h a l l i b u r e t r e a t e d f i s h showing r e g r e s s i o n of the i n t e r s t i t i a l c e l l s x 900 Fig u r e 17 - Stage IV Photomicrograph of the t e s t i s of a c o n t r o l f i s h showing w e l l developed i n t e r s t i t i a l c e l l s x 900 32 times of year but since the h i s t o l o g y of the 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 considered j u s t i f i a b l e to combine the r e s u l t s from these experiments. The number of f i s h examined from each experiment i s given i n Table 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 Table IV. a) M e t h a l l i b u r e Treatment on the T e s t i s Samples of three or four f i s h were examined a f t e r 12 to 22 days of immersion i n , or 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 a c t i v e t e s t e s c h a r a c t e r i s t i c of breeding f i s h . 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 12 days were a l s o at Stage IV as were four of the s i x f i s h examined a f t e r 22 days of treatment. The remaining two f i s h were at Stage 0. I t was concluded t h a t m e t h a l l i b u r e treatment brought 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 the f i r s t 12 days of treatment but two of the s i x f i s h examined show a treatment e f f e c t a f t e r 22 days'* of treatment. A f t e r 30 - 38 days of treatment there was a very 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 . In the t e s t e s of most c o n t r o l f i s h gametogenesis was w e l l underway or complete and 20 of the 22 f i s h examined were at Stages I I I and IV. However, gametogenesis i s s e v e r e l y retarded 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 only 3 of the 23 examined are at Stages I I I and IV. The l a r g e s t group of 15 are at Stage 0, showing maturation of about h a l f the germ c e l l s to TABLE I I I Number of F i s h Used f o r Examination  of T e s t i s H i s t o l o g y Treatment Exp. Duration No. Of T o t a l No. i n Days F i s h 4a 12 3 3 4b 22 3 3 C o n t r o l 3 31 6 ) I n j . & U n i n j . 5 a & b 36 & 38 7 ) 22 6 32 3 ) 7 35 6 ) 8 49 8 8 4a 12 3 3 4b 22 4 4 Meth. I n j . 3 31 4 ) 5 a & b 36 & 38 8 ) 23 6 32 5 ) 7 35 6 ) 4a 12 4 4 Meth. Imm. 4b 22 1 1 7 35 7 7 8 49 7 7 L.H. I n j . 6 32 1 ) 7 35 4 ) 5 L.H. + Meth. 6 32 1 ) I n j . 7 35 1 ) 2 TABLE IV E f f e c t of M e t h a l l i b u r e , LH and Thiourea on  H i s t o l o g y o f the T e s t i s and the Kidney Staaes of T e s t i s Brush Border Seg. Kidney Duration S t i m u l a t i o n No Mucous C e l l s Mucous C e l l s Treatment of Treat, i n Days No. F i s h 0 I I I I I I IV No. F i s h * * C e l l Height in/tc No. F i s h ** C e l l Height i n ^ C o n t r o l 12 3 0 0 0 0 3 22 3 0 0 0 0 3 *30 22 0 0 2 11 9 11 10.24 + 0.51 2 23.63 + 1.76 49 8 0 0 0 0 8 1 8.29 4 28.26 + 2.82 Meth. I n j . 12 3 0 0 0 0 3 22 4 1 0 0 0 3 *30 23 15 0 5 1 2 12 9.14 + 0.29 0 Meth. Imm. 12 4 0 0 0 0 4 22 2 1 0 0 0 1 *30 7 4 3 0 0 0 4 9.69 + 0.29 0 49 7 1 0 0 6 0 5 12.04 + 1.17 0 LH *30 5 1 1 0 0 3 1 9.69 4 17.25 + 1.50 Meth. + LH *30 2 0 0 0 0 2 1 13.44 1 23.49 Thiourea 49 8 0 0 0 0 8 2 13.32 + 0.92 3 22.75 + 3.12 * 30 to 38 days ** mean + S.E. 34 35 spermatozoa and the remainder at the spermatogonia l e v e l . R e s ults were very s i m i l a r w i t h the immersion technique. No f i s h were at Stages I I I and IV and four of the seven examined were at Stage 0. A f t e r 49 days of immersion i n m e t h a l l i b u r e there were s t i l l d i f f e r e n c e s between t r e a t e d and c o n t r o l f i s h . A l l c o n t r o l f i s h were completely mature and at Stage IV. 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 of the seven examined were at Stage I I I . In these t e s t e s about h a l f the germ c e l l s were mature sperm and the r e s t i n v a r i o u s intermediate stages (primary and secondary spermatocytes and spermatogonia). The b l o c k i n gametogenesis appears to have been l e s s complete a f t e r 49 days of treatment than a f t e r 30 - 38 days. b) M e t h a l l i b u r e Treatment on the Kidney The h e i g h t of t h e . c e l l s of the brush border segment of the kidney increases two or three times when male s t i c k l e -backs become mature and s t a r t t o b u i l d n e s ts. The height o f these c e l l s increases w i t h high l e v e l s of androgen i n the t e s t i s . ( W a i and Hoar, 1963). To determine the e f f e c t s of m e t h a l l i b u r e on s t e r o i d production, samples of kidney from f i s h t r e a t e d f o r 30 or 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 kidney was computed from the minimum c e l l , h eight of the brush border segment of 25 kidney tubules. The kidneys were c l a s s i f i e d i n t o two groups, those w i t h and those without mucous c e l l s i n the c o l l e c t i n g t u b u l e s . The mean f o r each group and each treatment (+ the 36 Standard Error) was computed. The presence of c l e a r mucous c e l l s i n the c o l l e c t i n g tubule as w e l l as the granular appearance of the convoluted tubules i n d i c a t e s t e r o i d s t i m u l a t i o n . A f t e r 30 days of m e t h a l l i b u r e treatment mucous c e l l s were not present i n any of the kidneys of t r e a t e d f i s h but they were o n l y present i n two of the c o n t r o l f i s h . The c e l l h e i g h t of c o n t r o l s without mucous c e l l s and t h a t of the m e t h a l l i b u r e 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 . These date i n d i c a t e low s t e r o i d l e v e l s i n both t r e a t e d and c o n t r o l groups a f t e r 30 days. However, a f t e r 49 days four of the f i v e c o n t r o l 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 kidney tubules (28.26 + 2.82). There are no mucous c e l l s present i n any of the m e t h a l l i b u r e t r e a t e d group. These data i n d i c a t e h i gher s t e r o i d l e v e l s i n c o n t r o l f i s h . In summary, treatment w i t h m e t h a l l i b u r e e i t h e r by immersion or i n j e c t i o n r e t a r d s gametogenesis i n the t e s t i s a f t e r 30 - 38 days of treatment. There seems to be a b l o c k i n the m e i o t i c transformation of spermatogonia i n t o spermatocytes. Most of the t r e a t e d f i s h e x h i b i t t e s t e s w i t h germ c e l l s which have completely undergone meiosis (spermatozoa) or c e l l s which have not s t a r t e d the m e i o t i c process (spermatogonia). However, a f t e r 49 days of treatment the b l o c k appears to have been l e s s e f f e c t i v e and germ c e l l maturation has o n l y slowed down so t h a t w h i l e the c o n t r o l s 37 are mature the m e t h a l l i b u r e t r e a t e d group are maturing. Steroidogenesis 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 of the kidneys of 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 of a c t i v e l y breeding f i s h . C o n t r o l s a f t e r 49 days under experimental c o n d i t i o n s have kidneys which show signs of s t i m u l a t i o n and mucous production. 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. Of the sample of f i v e , three had f u l l y a c t i v e t e s t e s Stage IV w h i l e the other two were l e s s a c t i v e . There was a l s o evidence of increased s t e r o i d production s i n c e four of the f i v e kidneys examined had mucous c e l l s and granular c e l l s w i t h a height o f 17.25 + 1.50. This i n d i c a t e s a higher s t e r o i d production i n the LH group than the c o n t r o l s where o n l y two of the 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 at Stage IV of t e s t i s s t i m u l a t i o n as were the c o n t r o l group. Three of the f i v e f i s h had mucous c e l l s present i n the kidneys and a mean c e l l height of 22.75 + 3.12 which i s c h a r a c t e r i s t i c of f i s h s e c r e t i n g mucus. The s t a t e s o f gametogenesis and steroidogenesis are the same i n the t h i o u r e a t r e a t e d group as they are i n the c o n t r o l group. I t i s , t h e r e f o r e , concluded 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 steroidogenesis or gametogenesis. e) GSI The gonosomatic index or GSI (Gonad Wt X 100/Total 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 of the 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 s i g n i f i c a n t -l y lower than the GSI of the c o n t r o l f i s h i n two experiments (No. 5 and 6) at the 0.05 l e v e l . In two l a t e r experiments (No. 7 and 8) the GSI of 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 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 . So although there are 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 , the weight of the t e s t i s of the 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 of 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 which i s not 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 the c o n t r o l s or the 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 and Thiourea Treatment The t h y r o i d of Gasterosteus i s composed of a number of f o l l i c l e s found s c a t t e r e d under the pharynx at the roots of the a f f e r e n t b r a n c h i a l a t t e r i e s . Ten unbroken f o l l i c l e s were chosen a t random f o r each f i s h . The lowest and the 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 h e i g h t ) . This same convention of computing mean f o l l i c l u l a r c e l l height has been used by Eales (1965) on Salmo gairdner, and Wiebe (1968) on Cymatogaster aggregata. TABLE V G. S.I. Comparison of E f f e c t o f M e t h a l l i b u r e and Thiourea Treatment on Gonasomatic Index Exp. C o n t r o l M e t h a l l i b u r e I n j e c t i o n M e t h a l l i b u r e Immersion Thiourea Immersion 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 0.21 + 0.06 0.39 + 0.04 8 0.38 + 0.06 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 Values + Standard E r r o r of Mean Seven f i s h from each treatment - th i o u r e a , m e t h a l l i b u r e and untreated c o n t r o l s - were examined and a grand mean f o r each treatment computed from the mean f o l l i c l e c e l l h e i g h t of each f i s h . These values are given i n Table 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) of the th 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 the means of the m e t h a l l i b u r e t r e a t e d (5.28u) and the c o n t r o l (4.92ju) groups. There was marked t h y r o i d hypertrophy i n three o f the 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 smaller than those of the c o n t r o l s and had g r e a t l y increased e p i t h e l i a l c e l l h e i g h t s and decreased c o l l o i d content. The other four t h i o u r e a t r e a t e d f i s h showed l i t t l e s i g n of increased t h y r o i d a c t i v i t y compared w i t h the 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 of response the d i f f e r e n c between means of the t h i o u r e a t r e a t e d group and the 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 of 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 the m e t h a l l i b u r e t r e a t e d 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 obvious signs of t h y r o i d hypertrophy and the mean f o l l i c u l a r c e l l h e i g h t was not 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 the 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 obvious g o i t r o g e n i c e f f e c t s . P i t u i t a r y a) Morphology The p i t u i t a r y i s made up of four main p a r t s , one TABLE VI Mean Thyroid C e l l Height F o l l o w i n g Thiourea 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 Height pu.) C o n t r o l 4.92 M e t h a l l i b u r e 5.28 Thiourea 10.53* * S t a s t i c a l l y s i g n i f i c a n t 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 s i g n i f i c a n t d i f f e r e n c e 42 nervous the neurohypophysis and three glandular, the pro-adenohypophysis, the meso-adenohypophysis and the meta-adenohypophysis. In the p i t u i t a r y of Gasterosteus, the neurohypophysis i n t e r d i g i t a t e s w i t h the adenohypophysis ( F i g . 17). The t r o p h i c hormones secreted by the p i t u i t a r y are elaborated 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 of the adenohypophysis. Both the gonadotrophin(s) and t h y r o i d s t i m u l a t i n g hormone (TSH) are produced by b a s o p h i l s l o c a t e d i n the mesoadenohypophysis ( P i c k f o r d and Atz, 1957). b) M e t h a l l i b u r e and Thiourea Treatment Gonadotrophs and thyrotrophs are s c a t t e r e d through-out the mesoadenohypophysis. With the s t a i n combination used ( a l c i a n b l u e , PAS, Orange G) they take on a l c a i n b l u e and more or l e s s PAS. This gives them a deep purple 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 types are found i n the same area and have very 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 these two b a s o p h i l s . The e f f e c t s of m e t h a l l i b u r e and t h i o u r e a treatments on the g r a n u l a t i o n of both c e l l types w i l l be considered together. The amount of b a s o p h i l i c m a t e r i a l was r a t e d on a s c a l e of + to ++++. Seven or e i g h t f i s h were examined i n each group. The score f o r each group was based on the 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 of the p i t u i t a r y . The r e s u l t s are t a b u l a t e d i n Table V I I . TABLE V I I Amount of Gr a n u l a t i o n i n the Baso 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 Thiourea Immersion Amount of Granulation i n the Baso p h i l s of the Adenohypophysis + ++ +++. ++++ M e t h a l l i b u r e 3 4 Thiourea 2 3 3 Co n t r o l 2 6 44 The m e t h a l l i b u r e t r e a t e d group contained very l i t t l e b a s o p h i l i c m a t e r i a l i n the mesoadenohypophysis. A l l f i s h were ra t e d e i t h e r + or +-f ( F i g . 18 & 19) . 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 fo the f i s h examined were rated ++++ w h i l e two were rated +++ ( F i g . 20 & 21; Table V I ) . Since m e t h a l l i b u r e treatment was found to r e t a r d gametogenisis and s t e r o i d o g e n i s i s i n the t e s t i s but not increase f o l l i c u l a r c e l l h e i g h t i n the t h y r o i d , most of the degranulated c e l l s are probably gonadotrophs. Thiourea treatment r e s u l t s i n degranulation i n some f i s h . This group i s ra t e d ++,+++ and ++++. I t i s s i g n i f i c a n t t h a t the f i s h r a ted ++ ( F i g . 22) e x h i b i t e d the gr e a t e s t increase 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 the f i s h r a t e d ++++ ( F i g . 24) the l e a s t . Therefore, i t i s concluded t h a t t h i o u r e a r e s u l t s i n degranulation of the thyrotrophs w h i l e m e t h a l l i b u r e r e s u l t s i n degranulation of mainly the gonadotrophs. 45 F i g u r e 18 - Photomicrograph of the p i t u i t a r y of 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 Figure 19 - High power of p a r t of the f i s h of F i g . 18 showing + of b a s o p h i l i c m a t e r i a l x 900 Figure 20 - Photomicrograph o f the p i t u i t a r y of an untreated c o n t r o l f i s h x 200 Figu r e 21 - High power of p a r t of the meso-adenohypophysis of the f i s h of F i g . 20 showing ++++ of b a s o p h i l i c m a t e r i a l x 900 46 F i g u r e 22 - Photomicrograph of the p i t u i t a r y of 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 - High power of p a r t of the meso-adenohypophysis of the f i s h of F i g . 22 showing ++ of b a s o p h i l i c m a t e r i a l x 900 Fig u r e 24 - Photomicrograph of 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 showing ++++ b a s o p h i l i c m a t e r i a l x 900 47 IV. DISCUSSION Brown (1963), Harper (1964), Hemsworth, Jackson and Walpole (1968), Paget et 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 the formation of gonadotrophin of mammals. This non-steroid, non-hormonal compound has the f o l l o w i n g chemical formula: CH =CH.CH.NH.CS.NH.NH.CS.NH.CH^ 2 i J CH 3 Hoar et a l . (1967) were the f i r s t to use i t on t e l e o s t s . They rep o r t t h a t treatment of s t i c k l e b a c k s , g o l d f i s h and surfperch w i t h m e t h a l l i b u r e 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 . Before d i s c u s s i n g the main e f f e c t s of m e t h a l l i b u r e on the 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 to consider the 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 of these i s the somewhat t o x i c e f f e c t o f the compound and the second the p o s s i b l e d i r e c t e f f e c t of m e t h a l l i b u r e on the th y r o i d s o f the t r e a t e d f i s h . The p r e l i m i n a r y study by Hoar e t a l . (1967) and the present one both show t h a t m e t h a l l i b u r e treatment i s to some degree t o x i c to s t i c k l e b a c k s . In my e a r l y experiments up to 50% of the experimental group d i e d during the course of treatment. In 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 only 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 treatment. 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 to m e t h a l l i b u r e s i n c e a t h i r d of c o n t r o l s d i e d 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 out, the very 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 per se was not a prime cause of death i n the e a r l i e r t e s t s . Secondly, s i n c e m e t h a l l i b u r e contains the t h i o u r y l e n e group, i t might be expected to block the synthesis o f thyroxine w i t h r e s u l t a n t increased t h y r o t r o p h i c a c t i v i t y of the p i t u i t a r y and enlargement and h y p e r p l a s i a of the t h y r o i d . Walpole (1965) found a d m i n i s t r a t i o n of l a r g e doses of m e t h a l l i b u r e to r a t s caused a s l i g h t increase i n t h y r o i d weight but normal t h y r o i d h i s t o l o g y . T u l l o c h et a l . 131 (1963) found m e t h a l l i b u r e decreased the uptake of I by the t h y r o i d s of t r e a t e d r a t s and mice. Wiebe (1968) reported no change i n the t h y r o i d h i s t o l o g y of the surfperch Cymatogaster a f t e r 40 days of m e t h a l l i b u r e treatment. Leatherland and Pandey (1968), however, d e s c r i b e an increase 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 of m e t h a l l i b u r e treatment on P o e c i l i a . The r e s u l t s obtained i n the present s e r i e s of experiments are s i m i l a r to those found w i t h Cymatogaster? 49 days o f treatment caused no s i g n i f i c a n t increase i n the 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 of Gasterosteus (Table V I ) . The e f f e c t s of m e t h a l l i b u r e on the t h y r o i d o f Gasterosteus were compared w i t h those obtained when a group of f i s h was t r e a t e d w i t h the goitrogen t h i o u r e a . Thiourea acts by b l o c k i n g the i o d i n i a t i o n of t y r o s i n e which i s one 49 step i n the s y n t h e s i s of thyroxine (Hoar 1966). Thiourea treatment has been used on a number of species of f i s h and u s u a l l y r e s u l t s i n an increase i n f o l l i c u l a r e p i t h e l i a l 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) Cymatogaster). Treatment of 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 increase 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 of s t i m u l a t i o n between i n d i v i d u a l s would suggest t h a t the concentration of t h i o u r e a used (0.003%) was o n l y j u s t e f f e c t i v e . Kinnear (1960) repo r t s t h a t a s o l u t i o n of 0.0025% t h i o u r e a i s the 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 P l a t i c h t h y s . Thiourea has been found by Chambers (1953) to r e s u l t i n l o s s of weight, r e t a r d a t i o n of growth and d e p l e t i o n of l i v e r glycogen i n Fundulus. B a r r i n g t o n and Matty (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 spermatogenesis i s a r r e s t e d i n the t e s t i s at the spermatocyte stage. No s i d e e f f e c t s are observed u s i n g a s o l u t i o n of t h i o u r e a on Gasterosteus. 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 of both 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 the dosages used by Chambers and other e a r l y workers were about ten times greater than those used i n the present study. In summary, the e f f e c t s of m e t h a l l i b u r e and t h i o u r e a on Gasterosteus are very d i f f e r e n t . M e t h a l l i b u r e b r i n g s about r e t a r d a t i o n i n t e s t i s gametogenesis and no s i g n i f i c a n t 50 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 h e i g h t . Thiourea, however, has no observable e f f e c t on the gametogenetic process but b r i n g s about a 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 h e i g h t . M e t h a l l i b u r e i s concluded to have no observable 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 the p o s s i b l e t o x i c e f f e c t , the use of m e t h a l l i b u r e i s considered to 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 the hormonal e f f e c t s of the gonadotrophins on the behavior and v a r i o u s t i s s u e s 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 agressive behavior i n Gasterosteus i s under d i r e c t c o n t r o l of the p i t u i t a r y v i a an L H - l i k e gonadotrophin. Hoar (1962 a, b) demonstrated t h a t both 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 short photoperiod, ' p h y s i o l o g i c a l l y hypophysectomized 1 e x h i b i t e d a low l e v e l of aggressiveness but normal and c a s t r a t e d males under long photoperiod e x h i b i t e d a high l e v e l of aggressiveness. I n j e c t i o n of the short photoperiod f i s h w i t h mammalian LH or c h o r i o n i c gonadotrophin r e s u l t e d i n an increase i n l e v e l of aggressive-ness. Wai and Hoar (1963) concluded t h a t treatment of c a s t r a t e d long and short photoperiod 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 not a f f e c t t h e i r l e v e l of aggressive-ness. Treatment of Gasterosteus 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 of aggressiveness of the c o n t r o l s increased ( F i g . 1 & 2). The drop i n aggressiveness 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 behavior on the p a r t of the t r e a t e d f i s h and an increase i n the number of c o n t r o l f i s h which defend a d i s c r e t e area. The amount of b i t i n g and chasing i s c o n s i s t e n t l y l e s s (than i n the c o n t r o l s ) i n the 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 of the l a s t three 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 mixture of m e t h a l l i b u r e and mammalian LH the l e v e l of aggressiveness i s higher 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 alone ( F i g . 1, 2 & 3) but not as high as t h a t of the c o n t r o l s . The treatment combination of LH + METH d i d not seem to be t o l e r a t e d at a l l w e l l by the f i s h so t h a t the e f f e c t s on the behavior by t h i s replacement therapy must be considered h i g h l y suggestive r a t h e r than c o n c l u s i v e . The study 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 treatment on long photoperiod f i s h r e s u l t s i n the same k i n d of redu c t i o n i n aggressiveness which i s c h a r a c t e r i s t i c of f i s h kept 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 of aggressiveness intermediate between the me t h a l l i b u r e t r e a t e d and the c o n t r o l groups. I t i s very d i f f i c u l t to determine whether m e t h a l l i b u r e a f f e c t s only prespawning aggressive behavior s i n c e nest b u i l d i n g and reproductive behavior occur i n only a few f i s h - whether experimental or c o n t r o l . The height of the c e l l s of the kidney tubules of 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 but so i s the h e i g h t of the kidney c e l l s of the 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 probably low i n both groups. Then i t i s not s u r p r i s i n g t h a t the sequences of reproductive behavior which are thought to be under s t e r o i d or s t e r o i d + gonadotrophin c o n t r o l are f o r the most p a r t absent. However, a lowering i n the l e v e l of gonadotrophins by m e t h a l l i b u r e could l o g i c a l l y be expected to lower s t e r o i d production and i n d i r e c t l y a f f e c t the whole c y c l e of reproductive behavior. Wiebe (1967) f i n d s such a r e s u l t w i t h Cymatogaster. As w i t h Gasterosteus, c e r t a i n elements of re p r o d u c t i v e behavior appear to be under d i r e c t gonadotrophic c o n t r o l and others under d i r e c t gonadal s t e r o i d c o n t r o l . 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 r e p r o d u c t i v e behavior s i n c e i t blocks the gonadotrophin which i n t u r n regulates the gonadal s t e r o i d s . B. T e s t i s and Kidney Spermatogenesis and steroidogenesis i n the t e s t i s of 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 of the gonadotrophin of the p i t u i t a r y and the androgens of the t e s t i s . Much has been learned about the r o l e of the gonadotrophins by the removal of the p i t u i t a r y and sub-sequent replacement of the various hormones. Dodd (1965) summarizes the r e s u l t s of hypophysectomy by a number of workers and concludes t h a t the p i t u i t a r y through the gonadotrophins i s necessary f o r the i n i t i a t i o n and maintenance of spermatogenesis i n the lower vertebrates (except the cyclostomes) but i t s c o n t r o l i s probably l i m i t e d t o phophase of the f i r s t m e i o t i c d i v i s i o n . There i s much evidence, he f i n d s , f o r the maintenance of spermatogenesis i n the absence of the p i t u i t a r y . He suggests t h a t gonadotrophin acts i n d i r e c t l y by s t i m u l a t i n g the s e c r e t i o n of s t e r o i d . F u rther evidence comes from the known a b i l i t y of a v a r i e t y o f s t e r o i d s to s t i m u l a t e c e l l d i v i s i o n . M e t h a l l i b u r e has been found to have marked e f f e c t s on spermatogenesis and steroidogenesis by Hemsworth et::.al. (1968) on r a t s and by Hoar et a l . (1967) on three species of f i s h . Treatment of Gasterosteus 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 days. A f t e r 22 days of treatment o n l y two of the s i x experimental f i s h showed a treatment e f f e c t . The other four t r e a t e d and a l l c o n t r o l f i s h were completely mature. A f t e r 30 - 38 days of m e t h a l l i b u r e treatment the h i s t o l o g y of c o n t r o l and t r e a t e d groups was very 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 treatment b r i n g s about a depression i n gonadotrophic a c t i v i t y . 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 to occur 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 the 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 gonadotrophin would most a f f e c t the spermatogonia to spermatocyte transformation s i n c e t h i s stage appears from work on hypophysectomized f i s h to be the most dependent on gonadotrophin. Therefore, i t i s p o s t u l a t e d t h a t any c e l l s which were spermatocytes before the treatment would proceed at the normal r a t e to become spermatozoa 54 and c e l l s which were spermatogonia would remain at t h i s stage or undergo the i n i t i a l phase of meiosis a t a much slower r a t e than normal. I t appears to take about 30 days of m e t h a l l i b u r e treatment to o b t a i n a widespread 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, during the 49 days of m e t h a l l i b u r e treatment i t i s p o s t u l a t e d t h a t enough gonadotrophin i s present to allow most spermatogonia i n the t r e a t e d f i s h to become spermatocytes. The germ c e l l s of the c o n t r o l s , however, have completely undergone spermatogenesis and are mature sperm. Gonadotrophins a l s o r e g u l a t e the development of the i n t e r s t i t i a l c e l l s i n the t e s t i s . Since these c e l l s develop i n seasonal f i s h d u r i n g the breeding season, are sudanophilic and c h o l e s t e r o l p o s i t i v e , they are thought to produce s t e r o i d (Marshall, 1960). Ahsan (1966), L o f t s , P i c k f o r d and Atz (1966), Sundararaj 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 not have i n t e r s t i t i a l c e l l s ) regress a f t e r 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 treatment would regress the i n t e r s t i t i a l t i s s u e of Gasterosteus. The evidence w i t h respect to t h i s theory i s i n d i r e c t . In male s t i c k l e b a c k s d u r i n g the breeding season, the i n t e r s t i t i a l t i s s u e develops, red c o l o r a t i o n appears i n the s k i n and the c e l l s of the brush border segment of the kidney tubules become f i l l e d w i t h s e c r e t o r y granules 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 ubule. These change are c o n t r o l l e d by androgen elaborated i n the i n t e r s t i t i a l c e l l s . 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 increased c e l l h e i g h t or granular cytoplasm i n the c e l l s of the brush border segment of the kidney tubule. The mean values are i n the range of values found by Wai and Hoar (1963) to be t y p i c a l of mature but s e x u a l l y quiescent 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 to long photoperiod. When one group was exposed to t h i s l i g h t regime f o r 30 - 38 days only 2 of the 13 examined e x h i b i t kidney h i s t o l o g y t y p i c a l of breeding males but when another group was exposed to long photoperiod f o r 49 days four of f i v e f i s h examined showed evidence of mucus production by the kidney (Table I V ) . The mean c e l l h eight of t h i s l a t t e r group i s i n the range c h a r a c t e r i s t i c of breeding 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 red c o l o r a t i o n throughout the experiments. This would suggest t h a t androgen i s probably being produced at a low l e v e l by the t r e a t e d f i s h and may imply t h a t a c e r t a i n amount of gonadotrophin i s a l s o present. However, l e v e l s of gonadotrophin do not appear to be high enough to promote spermatogenesis at the r a t e found i n c o n t r o l f i s h or to lead to the l e v e l s of androgen necessary to b r i n g about the increase i n kidney tubule c e l l h eight c h a r a c t e r i s t i c of breeding males. 56 When hypophysectomized f i s h are t r e a t e d w i t h r e -produ c t i v e hormones s t i m u l a t i o n of gametogenesis and steroidogenesis r e s u l t s . Ahsan (1966) f i n d s both processes sti m u l a t e d i n hypophysectomized la k e chub t r e a t e d w i t h e i t h e r mammalian LH or a crude e x t r a c t of whole salmon p i t u i t a r i e s . L o f t s ^ t a l . (1966) r e p o r t t h a t methyl testosterone s t i m u l a t e s both processes i n hypophysectomized Fundulus. However, Sundararaj and Nayyar (1967) f i n d testosterone propionate treatment r e s t o r e s spermatogenesis i n hypophysectomized c a t f i s h but the i n t e r s t i t i a l c e l l s are a t r o p i c . Human c h o r i o n i c gonadotrophin induces s i g n i f i c a n t dose dependent weight increments i n the t e s t e s and r e s t o r e s spermatogenesis and the sec r e t o r y a c t i v i t y of the i n t e r s t i t i a l c e l l s . In the present study bovine LH was used i n an attempt to counteract the e f f e c t s of m e t h a l l i b u r e . Unfortun a t e l y , a l a r g e number of the tr e a t e d f i s h d i e d d u r i n g the course of the experiment. However, three of the 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 at Stage IV of t e s t i s maturation and four of the f i v e kidneys, examined showed c l e a r mucous c e l l s and granular brush border 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, both were at Stage IV of t e s t i s s t i m u l a t i o n . One f i s h showed kidney tubules w i t h mucous c e l l s , one d i d not. The small sample i n d i c a t e s t h a t METH + LH i s more l i k e the c o n t r o l group and seems to promote t e s t i s maturation and ste 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 at t h i s stage. 57 C. P i t u i t a r y The mesoadenohypophysis of t e l e o s t s appears to c o n t a i n a l l the c e l l types t h a t are associated w i t h the mammalian pars a n t e r i o r . There are at l e a s t two types of b a s o p h i l s and these have been i d e n t i f i e d as gonadotrophs and thyrotrophs ( P i c k f o r d and Atz, 1957). Van Mullem (1959) gives h i s t o l o g i c a l evidence f o r the presence of LH and FSH i n the mesoadenohypophysis of Gasterosteus but the d i f f e r e n t i a t i o n of c e l l s producing the gonadotrophinsfrom each other and those producing FSH i s not d e f i n i t e . A f t e r 49 days of m e t h a l l i b u r e treatment i n Gasterosteus there was extensive degranulation of the b a s o p h i l s of the mesoadenohypophysis (Table IV and F i g . 17 & 18). The e f f e c t s on gametogenesis and steroidogenesis i n the t e s t i s (Table IV) are suggestive of l e s s gonadotrophin as are the lower l e v e l s of aggressive behavior c h a r a c t e r i s t i c of the m e t h a l l i b u r e t r e a t e d f i s h ( F i g . 1 & 2). This explanation i m p l i e s t h a t degranulation of the b a s o p h i l s means hypo--activity or a b l o c k i n the e l a b o r a t i o n of gonadotrophin. Such an explanation has been put f o r t h before to e x p l a i n degranulation e s p e c i a l l y the c y c l i c degranulation a s s o c i a t e d w i t h reproduction. For example, Sokol (1961) f i n d s conspicuous c y c l i c a l changes i n the b a s o p h i l s of the mesoadenohypophysis of Fundulus. These c e l l s are small and degranulated during the quiescent w i n t e r p e r i o d and l a r g e and f u l l y granulated i n the breeding season. Sokol considers the m a j o r i t y of these c e l l s to be 58 gonadotrophs. A d d i t i o n a l evidence f o r the a n t i -gonadotrophic e f f e c t s of m e t h a l l i b u r e on the p i t u i t a r y comes from work by Brown (1963) on r a t s . He f i n d s s i g n i f i c a n t l y l e s s FSH i n the p i t u i t a r i e s of animals t r e a t e d w i t h m e t h a l l i b u r e than i s present i n the p i t u i t a r i e s of c o n t r o l animals. Some of the degranulated c e l l s i n the meso-adenohypophysis of m e t h a l l i b u r e t r e a t e d Gasterosteus may be thyrotrophs s i n c e thyrotrophs cannot be d i s t i n g u i s h e d from gonadotrophs on the b a s i s of the s t a i n combination used. P o s s i b l e evidence t h a t both b a s o p h i l s may be a f f e c t e d comes from experiments of Leatherland and Pandey (1968). They f i n d t h a t the gonadotrophs of m e t h a l l i b u r e t r e a t e d guppies are 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 smaller and l e s s aldehyde f u c h s i n p o s i t i v e than those of the c o n t r o l s ; the throtrophs, on the other 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 of the c o n t r o l s . They conclude the gonadotrophs are hypo-active w h i l e the thyrotrophs are h y p e r - a c t i v e . However, they a l s o f i n d t h a t m e t h a l l i b u r e treatment b r i n g s about a 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 h e i g h t . Such an increase i n t h y r o i d c e l l h e i g h t i s not found i n Gasterosteus t r e a t e d w i t h m e t h a l l i b u r e . These c o n s i d e r a t i o n s lead me to conclude that most of the degranulated b a s o p h i l s i n m e t h a l l i b u r e t r e a t e d s t i c k l e b a c k s are probably gonadotrophs. Degranulation of b a s o p h i l s a l s o occurs when a group of f i s h i s t r e a t e d w i t h t h i o u r e a . The amount of 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 w i t h the increase i n t h y r o i d e p i t h e l i a l c e l l h e i g h t . Thus, f i s h e x h i b i t i n g the most degranulation (++ i n Table VII) a l s o show the greatest t h y r o i d e p i t h e l i a l c e l l h e i g h t . Therefore, the degranulated c e l l s are very probably thytotrophs. A number of other workers have a l s o found degranulation of the thyrotrophs i n f i s h t r e a t e d w i t h t h i o u r e a or t h i o u r a c i l (Atz, 1953 on Astyanax; B a r r i n g t o n and Matty, 1955 on Phoxinus and Grosso, 1961 on L e b i s t e s ) . 60 V. CONCLUSIONS Treatment of 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 i n : (1) A marked red u c t i o n i n the l e v e l of prespawning aggressiveness i n t r e a t e d f i s h . (2) A decrease i n the r a t e of transformation spermatogonia to spermatocytes so t h a t a f t e r 30'- 38 days most o f the t e s t e s of the t r e a t e d f i s h contained spermatogonia and spermatozoa w h i l e the t e s t e s of the c o n t r o l s contained spermatocytes and spermatozoa or spermatozoa only. A f t e r 49 days, the t e s t e s of the t r e a t e d group contained spermatogonia, spermatocytes and spermatozoa w h i l e those of the c o n t r o l s contained o n l y spermatozoa. (3) No evidence of s t i m u l a t i o n of the c e l l s of the brush border segment of the kidney tubules o f t r e a t e d f i s h under long photoperiods. (4) A marked decrease i n the g r a n u l a t i o n of the p i t u i t a r y b a s o p h i l s . (5) Some degree of t o x i c i t y but no evidence o f a d i r e c t e f f e c t of the treatment on the t h y r o i d comparable to tha t o f t h i o u r e a . In b r i e f , i t i s concluded t h a t m e t h a l l i b u r e p a r t i a l l y b l o c k s gonadotrophic a c t i v i t y probably at the l e v e l o f the p i t u i t a r y . This t r e a t m e n t : a f f e c t s prespawn-i n g aggressive behavior and t e s t i s physiology i n a manner s i m i l a r to an exposure to short photoperiods. 61 VI . BIBLIOGRAPHY Ahsan, S.N. (1966). E f f e c t s o f gonadotropic hormones on male hypophysectomized l a k e chub, Couesius plumbeus. Can. J . Zool. 44: 703-717. Ahsan, S.N., and W.S. Hoar (1963). Some e f f e c t s of gonadotropic hormones on the threespine s t i c k l e b a c k , Gasterosteus aculeatus. Can. J . Zool. 41: 1045-1053. Atz, E.H. (1953). 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Perry, Eds., B i o l o g i c a l C o u n c i l Symposium on agents a f f e c t i n g f e r t i l i t y . C h u r c h i l l , London. Wiebe, J.P. (1967). The reproductive physiology of the v i v i p a r o u s sea perch, Cymatogaster aggregata Gibbons. Ph.D. Thesis. Univ. B r i t i s h Columbia. Wiebe, J.P. (1968). I n h i b i t i o n of p i t u i t a r y gonadotropic a c t i v i t y i n the v i v i p a r o u s sea perch, Cymatogaster  aggregata Gibbons by a d i t h i o c a r b a m o y l -hydrazine d e r i v a t i v e ( I . C . I . 33,828). Can. J . Zool., 46: (In press) 

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