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The effects of early and late androgen treatments and induced sex-reversals on the behavior of Sarotherodon… Billy, Allen J. 1982

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THE EFFECTS OF EARLY AND LATE ANDROGEN TREATMENTS AND INDUCED SEX-REVERSALS ON THE BEHAVIOR OF Sarotherodon mossambicus (PISCES: CICHLIDAE) by ALLEN J . BILLY .Sc., U n i v e r s i t y Of B r i t i s h Columbia, Vancouver, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard, THE UNIVERSITY OF BRITISH COLUMBIA October 1982 © A l l e n J . B i l l y , 1982 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 of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the 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 l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree 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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of 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 not be a llowed without my w r i t t e n p e r m i s s i o n . Department of -^~oQ /c The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date fl/pS. /iH DE-6 (2/79) i i ABSTRACT The goal of t h i s t h e s i s was to i n v e s t i g a t e the r e l a t i o n s h i p between e a r l y hormone treatments and the development of behaviour i n a t e l e o s t . Groups of Sarotherodon mossambicus (n = 60 f i s h / g r o u p ) were exposed to androgen treatment at d i f f e r e n t stages of development, and the e f f e c t s of the treatment on gonadal and b e h a v i o u r a l d i f f e r e n t i a t i o n were assessed. The f i r s t experiment examined the e f f e c t s of androgen treatments on gonadal d i f f e r e n t i a t i o n . A l l of the f i s h i n each treatment group were sexed, and group sex r a t i o s were compared to determine which treatment had i n f l u e n c e d gonadal development. Three treatments administered at d i f f e r e n t times d u r i n g the f i r s t 7 weeks of l i f e m a s c u l i n i z e d groups of f i s h , i n d i c a t i n g that gonadal sex d i r e c t i o n c o u l d be i n f l u e n c e d by androgen over a wide p e r i o d i n development. However, gonadal s e n s i t i v i t y to hormonal i n f l u e n c e s changed over time as each type of hormone treatment (immersion or o r a l ) most e f f e c t i v e l y i n f l u e n c e d gonadal sex d i r e c t i o n during a short p e r i o d (one to three weeks) in gonadal development. The second experiment e v a l u a t e d the e f f e c t of androgen treatment on the development of male behaviour. Behavioural d i f f e r e n c e s were d e t e c t e d between treatment groups i n f i v e measures of t e r r i t o r i a l defense and a g g r e s s i o n : i n t r a -t e r r i t o r i a l f i g h t i n g ( d u r a t i o n of f i g h t i n g and frequency of a g g r e s s i v e a c t s i n i t i a t e d d u r i n g f i g h t s ) , i n t e r - t e r r i t o r i a l f i g h t i n g ( d u r a t i o n of f i g h t i n g and frequency of a g g r e s s i v e a c t s i n i t i a t e d d u r i n g f i g h t s ) , and a t t a c k i n g ( f r e q u e n c y ) . Male behaviours were i n f l u e n c e d only by androgen treatments i n i t i a t e d d u r i n g the f i r s t 5 weeks of l i f e . Treatments s t a r t e d a f t e r week 5 d i d not a f f e c t male b e h a v i o u r a l development. The t h i r d experiment examined the e f f e c t s of androgen treatment on the development of female behaviour. Behavioural d i f f e r e n c e s were detected between groups i n two measures of male-female c o u r t s h i p i n t e r a c t i o n s : t i l t s r e c e i v e d from males and b u t t s r e c e i v e d from males. These r e s u l t s i n d i c a t e that the development of a female c o u r t s h i p behaviour (nest approaching) was i n f l u e n c e d by exposure to androgen treatments. An androgen treatment i n i t i a t e d a f t e r the f i f t h week of l i f e produced the most s t r i k i n g i n f l u e n c e on female b e h a v i o u r a l development, suggesting that female b e h a v i o u r a l development f o l l o w s male b e h a v i o u r a l development. Females exposed to androgen before week 5 were e i t h e r sex-reversed and possessed t y p i c a l male behaviour p a t t e r n s , or appeared to be u n a f f e c t e d by treatment and possessed t y p i c a l female behaviour p a t t e r n s . The f o u r t h experiment examined the p o s s i b i l i t y that female f i s h were s e n s i t i z e d to subsequent androgen treatment a f t e r exposure to an e a r l y androgen treatment. Females t e s t e d i n the t h i r d experiment were exposed to a second androgen treatment and t h e i r behaviour p a t t e r n s were r e t e s t e d . Females i n one group were very s e n s i t i v e to a second androgen treatment as they adopted male c o l o u r a t i o n , male behaviour ( t i l t s given and l a t e r a l d i s p l a y s g i v e n ) , and v i s i t e d male nests more f r e q u e n t l y than females i n any other group. Females i n the other treatment groups were l e s s s e n s i t i v e , or not s e n s i t i v e , to the i v second androgen treatment. These r e s u l t s i n d i c a t e that the hormone-dependent mechanisms u n d e r l y i n g the e x p r e s s i o n of a d u l t behaviour are s e n s i t i z e d to androgen e a r l y i n development. The f i f t h experiment was designed to d e t e c t g e n e t i c females sex-reversed by an e a r l y androgen treatment. Sex-reversed g e n e t i c females were det e c t e d by breeding presumptive males from the v a r i o u s treatment groups with untreated females and then sexing a l l of the o f f s p r i n g . Crosses between a g e n e t i c male and a g e n e t i c female produced roughly equal numbers of male and female o f f s p r i n g while c r o s s e s between two g e n e t i c females produced n e a r l y a l l female o f f s p r i n g . Since the behaviour of each male had been t e s t e d in the second experiment, i t was p o s s i b l e t o compare the behaviour of sex-reversed g e n e t i c females to the behaviour of untreated g e n e t i c males. T h i s comparison i n d i c a t e d that behaviour d i f f e r e n c e s e x i s t e d between sex-reversed females and c o n t r o l males in four measures: i n t e r -t e r r i t o r i a l f i g h t i n g , a t t a c k i n g , d i g g i n g and t i l t i n g to females. However, no b e h a v i o u r a l d i f f e r e n c e s were d e t e c t e d between sex-reversed and non sex-reversed f i s h i n the same treatment groups. T h i s f i n d i n g i n d i c a t e s that e a r l y androgen treatments i n f l u e n c e the development of male behaviours i n a s i m i l a r manner in sex-reversed and non sex-reversed f i s h . The f i n a l chapters of t h i s t h e s i s d i s c u s s the c h a r a c t e r i s t i c s of gonadal and b e h a v i o u r a l development in a t e l e o s t i n g r e a t e r depth. I n s i g h t s i n t o the r e l a t i o n s h i p between androgen and b e h a v i o u r a l development in a t e l e o s t are compared to what i s known about hormonal i n f l u e n c e s on V b e h a v i o u r a l development i n other v e r t e b r a t e s . T h i s comparison i n d i c a t e s that androgen i n f l u e n c e s b e h a v i o u r a l development in a s i m i l a r manner in both t e l e o s t s and mammals. v i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS I . INTRODUCTION 1 A. GENERAL INTRODUCTION AND BACKGROUND INFORMATION 1 1. I n t r o d u c t i o n to the Problem 1 2. P r o j e c t O b j e c t i v e s 2 3. Gonadal Development 3 4. Gonadal Hormones and the Regulation of Behaviour . 7 5. Gonadal Hormones and the Development of Behaviour 9 6. P r o j e c t Overview 10 11 . METHODS 12 A. GENERAL METHODS . 12 1. Experimental Animals 12 2. Housing and Maintenance 12 3. F i s h I d e n t i f i c a t i o n 13 4. Treatment Groups 14 5. Treatment A d m i n i s t r a t i o n 15 B. SYNOPSIS OF BEHAVIOUR 18 1 . Overview 18 2. I n t r a - T e r r i t o r i a l Behaviours 19 3. I n t e r - T e r r i t o r i a l Behaviours 22 4. E x t r a - T e r r i t o r i a l Behaviours 23 v i i 5. Behaviour Components 24 C. DATA RECORDING TECHNIQUES 27 1. General T e s t i n g Procedures 27 2. T e r r i t o r y C h a r a c t e r i s t i c s 28 3. F i g h t i n g V i g o r 28 4. Nest V i s i t i n g 29 5. Absenteeism 29 6. Body C o l o u r a t i o n 29 7. Summary 32 I I I . RESULTS 34 EXPERIMENT I. THE EFFECTS OF METHYLTESTOSTERONE ON GONADAL DIFFERENTIATION 34 A. INTRODUCTION 34 B. METHODS 35 1 . Subjects 35 2. Sexing Methods 35 3. S t a t i s t i c a l Treatment of Data 36 C. RESULTS 36 D. DISCUSSION 38 E. SUMMARY 41 EXPERIMENT I I . THE EFFECTS OF METHYLTESTOSTERONE ON THE DEVELOPMENT OF MALE BEHAVIOUR 42 A. INTRODUCTION 4 2 B. METHODS 42 1 . Subjects 42 v i i i 2. Data Co l l e c t i o n 43 3. S t a t i s t i c a l Treatment of Data 43 C. RESULTS 44 1. I n t r a - T e r r i t o r i a l Fighting 44 2. I n t e r - T e r r i t o r i a l Fighting 47 3. Attacking 52 D. DISCUSSION 57 E. SUMMARY 63 EXPERIMENT I I I . THE EFFECTS OF METHYLTESTOSTERONE ON THE DEVELOPMENT OF FEMALE BEHAVIOUR 65 A. INTRODUCTION '. 65 B. METHODS 66 1 . Subjects 66 2. Data Collection 66 3. S t a t i s t i c a l Treatment of Data 66 C. RESULTS 67 1. T i l t s Received by Females 67 2. Butts Received by Females 67 D. DISCUSSION 72 E. SUMMARY 75 EXPERIMENT IV. THE EFFECTS OF A SECOND METHYLTESTOSTERONE TREATMENT ON EARLY-TREATED FEMALES 76 A. INTRODUCTION 7 6 B. METHODS 78 ix i 1 . Subjects 78 2. Data C o l l e c t i o n 78 3. Treatment A d m i n i s t r a t i o n . 78 4. S t a t i s t i c a l Treatment of Data 78 C. RESULTS 79 1. Body C o l o u r a t i o n . 79 2. T i l t s Given by Females 79 3. L a t e r a l D i s p l a y s Given by Females 82 4. Nest V i s i t i n g 82 5. L a t e r a l D i s p l a y s Received by Females from Males 87 6. Androgen Induced Changes i n Female Behaviour .. 87 D. DISCUSSION 95 E. SUMMARY 99 EXPERIMENT V. THE BEHAVIOUR OF SEX-REVERSED FEMALES 101 A. INTRODUCTION 101 B. METHODS 101 1 . Subjects .101 2. Data C o l l e c t i o n 102 3. S t a t i s t i c a l Treatment of Data 102 C. RESULTS AND DISCUSSION 104 1. D e t e c t i o n of Sex-Reversed Females 104 2. Morphology of Sex-Reversed Females 104 3. Behaviour of Sex-Reversed Females 104 D. SUMMARY 115 IV. DISCUSSION 116 A. THE EFFECTS OF METHYLTESTOSTERONE X ON GONADAL DIFFERENTIATION 116 1. S e n s i t i v e Periods i n Gonadal Development 116 2. Threshold L e v e l s i n Gonadal Development 118 3. P a r a d o x i c a l F e m i n i z a t i o n 119 4. I n f l u e n c e s of Hormone Treatment on S e x u a l l y D i f f e r e n t i a t e d Gonads 121 5. Gonadal S e n s i t i z a t i o n to Androgen 123 6 . Summary . 124 B. THE EFFECTS OF METHYLTESTOSTERONE ON BEHAVIOURAL DIFFERENTIATION 1 25 1. S e n s i t i v e P e r i o d s i n Be h a v i o u r a l Development 125 2. Behavioural Development i n Other V e r t e b r a t e s 128 3. Behaviour of Sex-Reversed F i s h 132 4. Be h a v i o u r a l S e n s i t i z a t i o n to Androgen 133 5. A Comparison of Gonadal and Behavioural Development C h a r a c t e r i s t i c s 135 6. Summary 136 V. BIBLIOGRAPHY 137 x i LIST OF TABLES Table I - Treatment Groups 14 Table I I . Experimental Groups 15 Table I I I . Behaviour Measures Recorded 32 Table IV. Treatment Group Sex Ra t i o s 37 Table V. C o r r e l a t i o n C o e f f i c i e n t s f o r F i g h t Duration versus F i g h t Vigor 58 Table VI. Female Body C o l o u r a t i o n 80 Table V I I . A Summary of Androgen Induced Changes i n Female Behaviour 93 Table V I I I . A Summary of Breedings Performed and Sex Ra t i o s Obtained i n a S e r i e s of Crosses I n v o l v i n g Males from V a r i o u s Treatment Groups 105 Table IX. Comparisons of C o n t r o l F i s h (Group VI) with Sex-Reversed F i s h (Groups I and I I I ) 108 Table X. Comparisons of C o n t r o l F i s h (Group VI) with Non Sex-Reversed F i s h (Groups I and I I I ) 111 Table XI. Comparisons of Sex-Reversed with Non Sex-Reversed F i s h (Groups I and I I I ) 113 x i i LIST OF FIGURES F i g u r e 1. Male I n t r a - T e r r i t o r i a l F i g h t i n g 45 F i g u r e 2. Aggressive Acts During I n t r a - T e r r i t o r i a l F i g h t i n g 48 F i g u r e 3. Male I n t e r - T e r r i t o r i a l F i g h t i n g 50 F i g u r e 4. Aggressive Acts During I n t e r - T e r r i t o r i a l F i g h t i n g 53 F i g u r e 5. A t t a c k i n g by Males 55 F i g u r e 6. T i l t s Received by Females 68 F i g u r e 7. Butts Received by Females 70 F i g u r e 8. T i l t s Given by Females 83 F i g u r e 9. L a t e r a l D i s p l a y s Given by Females 85 F i g u r e 10. Nest V i s i t i n g by Females 88 F i g u r e 11. L a t e r a l D i s p l a y s Received by Females 90 F i g u r e 12. S e n s i t i v e Periods During Behavioural Development 128 ACKNOWLEDGEMENTS A Master's t h e s i s i s very s i m i l a r to a sex change o p e r a t i o n . In both cases, a s u c c e s s f u l product depends on c o n t r i b u t i o n s from s e v e r a l people. Some people suggest c e r t a i n b i t s be cut out, others suggest adding new b i t s , others recommend r e l o c a t i o n of b i t s a l r e a d y present, and s t i l l o thers p o i n t out where a l i t t l e n i p and tuck w i l l improve the o v e r a l l shape. To a l l who p a r t i c i p a t e d i n t h i s o p e r a t i o n : thank-you f o r your c o n t r i b u t i o n s . I wish to thank my s u p e r v i s o r , Dr. N.R. L i l e y , f o r h i s pa t i e n c e , a s s i s t a n c e , and c o n s t r u c t i v e c r i t i c i s m s . Part or a l l of the manuscript has b e n e f i t e d from c r i t i c a l reading by members of my s u p e r v i s o r y committee, N.R. L i l e y , D. McPhail, and A. Perks. I would a l s o l i k e to thank Lee Ann Otto, Pam McDonald, Doug Begle, and Wayne Goodey f o r commenting on the manuscript and p a r t i c i p a t i n g i n v a l u a b l e d i s c u s s i o n s as the t h e s i s work progressed. 1 I. INTRODUCTION A. GENERAL INTRODUCTION AND BACKGROUND INFORMATION j_. I n t r o d u c t i o n to the Problem I t i s known that a r e l a t i o n s h i p e x i s t s between e a r l y androgen treatments and the development of behaviour i n mammals. Vario u s s t u d i e s have shown that p r e n a t a l and neonatal androgen treatments a c t to suppress the development of female behaviour p a t t e r n s while s t i m u l a t i n g the development of male behaviour p a t t e r n s ( t o p i c reviewed by Baum, 1979 and Beatty, 1979). In c o n t r a s t to the mammalian s i t u a t i o n , v i r t u a l l y nothing i s known about the r o l e of hormones i n b e h a v i o u r a l development in t e l e o s t s . However, one study does i n d i c a t e that androgen treatments can i n f l u e n c e the development of f i s h behaviour. Lindsay (1974) t r e a t e d guppies ( P o e c i l i a r e t i c u l a t a ) with androgen f o r one week a f t e r p a r t u r i t i o n and observed that the t r e a t e d females possessed normal female behaviour p a t t e r n s and morphology when mature. However, when the a d u l t f i s h were given a second androgen treatment, the behaviour of e a r l y - t r e a t e d females was m a s c u l i n i z e d to a g r e a t e r extent than the behaviour of females not exposed to an e a r l y treatment. T h i s o b s e r v a t i o n suggests that androgen i n f l u e n c e d a hormone-sensitive b e h a v i o u r a l mechanism d u r i n g development. Since the behaviour of e a r l y - t r e a t e d female mammals i s a l s o m a s c u l i n i z e d to a grea t e r degree by androgen l a t e r in l i f e than the behaviour of non-t r e a t e d females (see Baum, 1979 and Beatty, 1979), there i s a 2 p r e l i m i n a r y i n d i c a t i o n that androgen p l a y s a s i m i l a r r o l e i n be h a v i o u r a l development i n both mammals and t e l e o s t s . I t would be of t h e o r e t i c a l i n t e r e s t to pursue t h i s i n i t i a l o b s e r v a t i o n and determine whether s i m i l a r mechanisms and p r i n c i p l e s r e g u l a t e the development of behaviour i n both mammals and t e l e o s t s . Understanding the r e l a t i o n s h i p between e a r l y androgen and a d u l t behaviour would a l s o be of p r a c t i c a l i n t e r e s t to f i s h c u l t u r i s t s . Treatments with androgen are now being used i n aquaculture p r o j e c t s i n attempts to produce u n i s e x u a l stocks ( i n Sarotherodon , Guerrero, 1981), and to s t i m u l a t e growth ( i n salmonids, Fagerlund and McBride, 1977). The beh a v i o u r a l consequences of these treatments have yet to be examined. I f exposure to androgen treatments e a r l y i n development a l t e r s the exp r e s s i o n of a d u l t behaviours, then the f i s h c u l t u r i s t may b e n e f i t from knowing what kind of changes i n behaviour are produced. 2. P r o j e c t O b j e c t i v e s T h i s t h e s i s was undertaken to explore an untouched res e a r c h area of t h e o r e t i c a l and p r a c t i c a l i n t e r e s t - the r e l a t i o n s h i p between e a r l y androgen treatments and the development of re p r o d u c t i v e behaviour i n f i s h . The o b j e c t i v e s of t h i s i n v e s t i g a t i o n were as f o l l o w s : 1. To assess the i n f l u e n c e of androgen treatments on gonadal and behaviour-al development in male and female f i s h . 2. To determine whether androgen produces i t s g r e a t e s t i n f l u e n c e on gonadal and b e h a v i o u r a l c h a r a c t e r i s t i c s d u r i n g 3 s p e c i f i c s e n s i t i v e p e r i o d s i n development. If so, do the s e n s i t i v e p e r i o d s f o r gonadal and b e h a v i o u r a l d i f f e r e n t i a t i o n c o i n c ide? 3. To determine i f the behaviour of a d u l t female f i s h i s i n f l u e n c e d by an e a r l y androgen treatment even though there are no m o r p h o l o g i c a l s i g n s of m a s c u l i n i z a t i o n , and whether such e a r l y - t r e a t e d females are s e n s i t i z e d to subsequent exposure to androgen. 4. To examine the b e h a v i o u r a l and morphological e f f e c t s of hormone-induced s e x - r e v e r s a l on g e n e t i c females. Do sex-reversed g e n e t i c females e x h i b i t the normal complement of male behaviours, or do' they e x h i b i t a mix of male and female behaviours? 3^ . Gonadal Development The endocrine system in t e l e o s t s i s s i m i l a r to that found in other v e r t e b r a t e s and c o n s i s t s of a h y p o t h a l a m u s - p i t u i t a r y -gonad a x i s and f a c t o r s produced by components of the a x i s . T h i s system i s d e s c r i b e d i n d e t a i l by: Donaldson, 1973; Fontaine, 1976; Hoar, 1969; Ozon, 1972a and b; Reinboth, 1972a; and Sundararaj, 1981. Environmental i n f l u e n c e s on the t e l e o s t endocrine system are reviewed by Crim (1982) and by De Vlaming (1974). In g e n e r a l , the endocrine f a c t o r s that have been i d e n t i f i e d in t e l e o s t s are a l s o found in other v e r t e b r a t e s . However, at l e a s t one exception to t h i s g e n e r a l i z a t i o n e x i s t s s i n c e the main gonadal androgen in male t e l e o s t s appears to be 11-ketotestosterone and not t e s t o s t e r o n e as i n male mammals 4 (see, for example, Scott et a l . , 1980). It should be stressed that investigations of the endocrine system in teleosts are largely exploratory and a great deal more work needs to be performed before the d e t a i l s of the system can be determined. While various authors agree that genetic factors underlie gonadal development and sexual d i f f e r e n t i a t i o n in teleosts (for reviews, see: Schreck, 1974a; Persov, 1975; and Yamamoto, 1969), the biochemical events i n i t i a t e d by genetic factors to influence gonadal development have not been adequately described (Harrington, 1974). However, i t is suspected that certain c e l l s in the undifferentiated gonad produce sex steroids early in development which determine sex direction (see Yamamoto, 1969). It is not known which steroids are produced to influence gonadal sex d i r e c t i o n at this stage of development. The early production of sex steroids may be regulated by the developing p i t u i t a r y . While i t remains to be experimentally confirmed that the p i t u i t a r y controls early gonadal development, this model is supported by several independent lines of evidence. F i r s t , i t is clear that sex d i f f e r e n t i a t i o n in the developing teleost gonad can be influenced by early hormone treatments. Androgen treatments cause juvenile f i s h to develop along male l i n e s , while estrogen treatments cause juvenile f i s h to develop along female lines (for reviews, see: Donaldson and Hunter, 1982; Guerrero, 1981; Johnstone et a l . , 1979 and 1978; L i l e y , 1980; Schreck, 1974a; Yamamoto, 1969; and Yamazaki, 1976). Hormone-induced sex-reversal has been accomplished in a number of species, including: S. mossambicus (Clemens and 5 Inslee, 1968, and Nakamura and Takahashi, 1973); Oryzias latipes (Yamamoto, 1968 and 1959a); and Salmo gairdneri (Okada et a l . , 1979) . Hishida (1965 and 1962) demonstrated that the d i f f e r e n t i a t i n g l a r v a l gonad of 0. latip e s a c t i v e l y accumulates radioactively l a b e l l e d steroids whereas the f u l l y d i f f e r e n t i a t e d adult gonad does not. Such studies suggest that the developing gonad is a target organ for c i r c u l a t i n g hormones and that an early interaction occurs between steroids and the developing gonad. Evidence for a relationship between the p i t u i t a r y and the developing gonad comes from studies of juvenile f i s h treated with an antigonadotropic agent (methallibure) and/or sex hormones. Various studies indicate that gonadal development in juvenile teleosts is prevented i f p i t u i t a r y gonadotropin production is blocked (in S. mossambicus , Lanzing, 1978). Other studies indicate that gonadal development in juvenile teleosts i s stimulated i f gonadotropin levels are enhanced (see, for example, Crim and Evans, 1979). Such studies suggest that gonadal development requires p i t u i t a r y involvement. That gonadal development is regulated by the p i t u i t a r y i s also reinforced by histochemical studies which demonstrate that p i t u i t a r y development precedes, and then i s correlated with, gonadal development (in S. mossambicus, see Chiba et a l . , 1978; Sasayama and Takahashi, 1975; and Nakamura and Takahashi, 1973). A large number of studies have concentrated on the relat i o n s h i p between exogenous hormone treatments and their 6 a f f e c t s on gonadal development i n t e l e o s t s . E x c e l l e n t reviews of t h i s t o p i c have been prepared by: Donaldson and Hunter (1982); Guerrero (1981); Johnstone et a l . (1979 and 1978); Schreck (1974); Yamamoto (1969); and Yamazaki (1976). A l i t e r a t u r e survey i n d i c a t e s that gonadal development can be i n f l u e n c e d by a v a r i e t y of hormone a d m i n i s t r a t i o n s . The e f f e c t i v e n e s s of any treatment i n i n f l u e n c i n g gonadal development depends on the o v e r a l l hormone exposure produced by a set of treatment c h a r a c t e r i s t i c s with res p e c t t o gonadal s e n s i t i v i t y to the hormone at a c e r t a i n stage of development. Treatment e f f e c t i v e n e s s can be a l t e r e d by v a r y i n g any of the f o l l o w i n g treatment c h a r a c t e r i s t i c s : the dosage a p p l i e d (Clemens and I n s l e e , 1968), treatment d u r a t i o n (Simpson, 1976), the mode of s t e r o i d a d m i n i s t r a t i o n ( H i s h i d a and Kawamoto, 1970), the s t e r o i d a p p l i e d ( H i s h i d a and Kawamoto, 1970), the age of the f i s h when treatment i s i n i t i a t e d (Okada, 1964), or environmental c o n d i t i o n s d u r i n g treatment (Hackmann and Reinboth, 1974). A l s o , a s p e c i e s - s p e c i f i c set of response t h r e s h o l d s appears to re g u l a t e t i s s u e responses to hormonal i n f l u e n c e s s i n c e a treatment causing s e x - r e v e r s a l i n one s p e c i e s may be t o t a l l y i n e f f e c t i v e when a p p l i e d to another s p e c i e s . For example, e a r l y m e t h y l t e s t o s t e r o n e (MT) treatments that produce a l l - m a l e o f f s p r i n g when a p p l i e d to j u v e n i l e S. mossambicus (Clemens and I n s l e e , 1968) have no e f f e c t on gonadal development when a p p l i e d to j u v e n i l e grass carp, Ctenopharynqodon i d e l l a (Stanley and Thomas, 1978). M a n i p u l a t i n g any of the a b o v e - l i s t e d v a r i a b l e s may a l t e r 7 the i n f l u e n c e of a hormone treatment on gonadal development. The range of the e f f e c t s produced by d i f f e r e n t hormone treatments on developing t e l e o s t gonads can be arranged on a continuum from low to high e f f e c t i v e n e s s as f o l l o w s : no e f f e c t (very low exposures), development of an o v o - t e s t i s (low exposures), sex-r e v e r s a l , p a r a d o x i c a l f e m i n i z a t i o n (high exposures), androgenic c a s t r a t i o n (very high exposures). T h i s continuum i s a r e l a t i v e s c a l e d e v i s e d by assuming that s h o r t e r treatment d u r a t i o n s and lower treatment doses using weaker s t e r o i d s produce a l e s s e f f e c t i v e treatment than r e l a t i v e l y longer treatment d u r a t i o n s and higher treatment doses using stronger s t e r o i d s . _4. Gonadal Hormones and the R e g u l a t i o n of Behaviour It i s c l e a r that gonadal f a c t o r s p l a y a c e n t r a l r o l e i n the maintenance and r e g u l a t i o n of f i s h behaviour (see reviews by: .Baggerman, 1968; De Vlaming, 1974; Hoar, 1969, 1965, and 1962; and L i l e y , 1969 and 1980). T h i s c o n c l u s i o n has developed p r i m a r i l y from s t u d i e s i n v o l v i n g c a s t r a t i o n and hormone replacement therapy and from s t u d i e s in which gonadal hormones were administered to i n t a c t f i s h . C a s t r a t i o n of a d u l t f i s h u s u a l l y r e s u l t s i n the l o s s or r e d u c t i o n of secondary sexual c h a r a c t e r i s t i c s , i n c l u d i n g sex-s p e c i f i c behaviours. Hormone replacement therapy a p p l i e d to c a s t r a t e s u s u a l l y r e s t o r e s secondary sexual c h a r a c t e r i s t i c s and s e x - s p e c i f i c behaviours to p r e c a s t r a t i o n l e v e l s . For example, Johns and L i l e y (1970) found that c a s t r a t i o n prevents f i n e l o n g a t i o n (a male secondary sexual c h a r a c t e r i s t i c ) and 8 r e p r o d u c t i v e behaviour in the male blue gourami ( T r i c h o q a s t e r  t r i c h o p t e r u s ) , while MT therapy a p p l i e d to c a s t r a t e s produces f i n e l o n g a t i o n and r e s t o r e s normal r e p r o d u c t i v e behaviour. A s i m i l a r approach to the study of hormones and behaviour in other s p e c i e s i n d i c a t e s that gonadal hormones r e g u l a t e v a r i o u s elements of a g g r e s s i v e , r e p r o d u c t i v e , t e r r i t o r i a l , and p a r e n t a l behaviours in male and female t e l e o s t s (see, f o r example, Wapler-Leong and Reinboth, 1974; Reinboth, 1972b; Wai and Hoar, 1963; Hoar, 1962; and Noble and Kumpf, 1936). Gonadal hormone involvement i n the r e g u l a t i o n of behaviour has a l s o been s t u d i e d by a d m i n i s t e r i n g hormones to i n t a c t f i s h and o b s e r v i n g concomitant changes in behaviour. For example, F e r n a l d (1976) observed that t e s t o s t e r o n e (T) i n j e c t i o n s i n c r e a s e d the l e v e l s of approaching and a t t a c k i n g behaviours in male Haplochromis b u r t o n i . F e r n a l d concluded that t e s t o s t e r o n e a f f e c t e d the c o n t r o l of both sexual and a g o n i s t i c behaviours. S i m i l a r work has i m p l i c a t e d androgens in the c o n t r o l of a g g r e s s i v e and n e s t - b u i l d i n g behaviours in H. b u r t o n i (Wapler-Leong and Reinboth, 1974); in p a r e n t a l behaviour, in the blue gourami T. t r i c h o p t e r u s (Kramer, 1972); in the r a t e of swimming and s c h o o l i n g i n t e n s i t y i n chum salmon (Oncorhynchus keta) f r y (Hoar et a l . , 1952); and general a c t i v i t y l e v e l s in g o l d f i s h ( C a r a s s i u s auratus) (Stanley and Tescher, 1931). While the preceding paragraphs i n d i c a t e that many aspects of behaviour are r e g u l a t e d by gonadal hormone l e v e l s , the idea that gonadotropins d i r e c t l y r e g u l a t e elements of behaviour in t e l e o s t s i s under a c t i v e d i s c u s s i o n . T h i s idea has been 9 presented (Fiedler, 1974; Wai and Hoar, 1963) to explain observations that some f i s h display elements of reproductive behaviour for several weeks or months after gonadectomy, when gonadal hormone level s have presumably dropped (for example, i n : Hemichromis bimaculatus, Noble and Kumpf, 1936; Aequidens  l a t i f r o n s , Aronson, 1959; and Gasterosteus aculeatus, Baggerman, 1968). Such observations suggest that certain elements of behaviour are r e l a t i v e l y independent of gonadal hormone control. However, L i l e y (1980) cautions that the evidence supporting the concept of d i r e c t gonadotropin control of behaviour i s weak and requires additional investigation. Therefore, in this thesis, gonadal secretions w i l l be considered the key endocrine factors responsible for regulating f i s h behaviour. 5. Gonadal Hormones and the Development of Behaviour While i t is known that gonadal hormones regulate the expression of behaviour, the role of gonadal hormones in the development of f i s h behaviour is not known. Most investigations of early hormone treatments are primarily concerned with examining the role of hormones in sex determination and gonadal development. Observations of the relationship between early hormonal influences and behavioural development have been of secondary concern and are usually q u a l i t a t i v e in nature ( L i l e y , 1980). However, a relationship between gonadal hormones and behavioural development is established in other vertebrates and is suspected in teleosts (see Lindsay, 1974 and section I-A-1). Insights into the role of gonadal hormones in behavioural 10 development have been obtained i n s t u d i e s of mammals (reviewed by Baum, 1979 and Beatty, 1979) and b i r d s (reviewed by Adkins, 1978). Basi c f e a t u r e s of b e h a v i o u r a l development i n these groups i n c l u d e : 1) gonadal hormones present d u r i n g development i n f l u e n c e the form (male or female) and i n t e n s i t y (frequency and/or d u r a t i o n ) of many a d u l t behaviours; 2) gonadal hormones permanently i n f l u e n c e behaviour when a p p l i e d d u r i n g development but t e m p o r a r i l y i n f l u e n c e behaviour when a p p l i e d d u r i n g adulthood; 3) a behaviour can be i n f l u e n c e d to some degree by gonadal hormones over a broad p e r i o d (weeks) d u r i n g development, but w i t h i n t h i s extended p e r i o d of s e n s i t i v i t y there i s a sh o r t e r p e r i o d (days) of maximum s e n s i t i v i t y to hormonal i n f l u e n c e s ; and 4) d i f f e r e n t behaviours are i n f l u e n c e d to d i f f e r e n t degrees by e a r l y hormone treatments. 6. P r o j e c t Overview The approach used to examine hormone treatment e f f e c t s on beh a v i o u r a l development i s o u t l i n e d below ( a d d i t i o n a l d e t a i l s are provided in s e c t i o n s to f o l l o w ) . F i r s t , groups of f i s h were exposed to androgen treatments at p a r t i c u l a r stages of development. Each experimental group r e c e i v e d e i t h e r an immersion treatment (hormone added to the aquarium water) or an o r a l treatment (hormone added to the d i e t ) . Treatments were a p p l i e d d u r i n g the f o l l o w i n g stages of development: 1 ) d u r i n g the f i r s t 1 2 days of l i f e , from f e r t i l i z a t i o n to 4 - 6 days a f t e r h a t c h i ng; 2) from day 0 (when young are removed from the female's buc c a l pouch 12 days a f t e r f e r t i l i z a t i o n ) to day 21; 3) 11 from day 21 to day 42; or 4) when mature. C o n t r o l f i s h were as s i g n e d to group VI and were r a i s e d without androgen treatment. Once e a r l y - t r e a t e d f i s h had matured, r e p r e s e n t a t i v e s of each group were su b j e c t e d to behaviour t e s t i n g under s t a n d a r d i z e d c o n d i t i o n s . Then, the behaviour p a t t e r n s of experimental and c o n t r o l f i s h were compared for q u a n t i t a t i v e and q u a l i t a t i v e d i f f e r e n c e s i n a g g r e s s i v e , t e r r i t o r i a l , and r e p r o d u c t i v e behaviours. I t was expected that any b e h a v i o u r a l d i f f e r e n c e s observed would be due to d i f f e r e n t exposures to androgen d u r i n g development. A s e r i e s of experiments was designed to e x p l o r e s p e c i f i c a s pects of the r o l e of e a r l y hormonal i n f l u e n c e s in development. Treatment e f f e c t s on gonadal development were assessed i n experiment I a f t e r sexing a l l f i s h . Treatment e f f e c t s on male b e h a v i o u r a l development were examined in experiment II f o l l o w i n g behaviour t e s t i n g of males. Treatment e f f e c t s on female b e h a v i o u r a l development were s t u d i e d in experiment III f o l l o w i n g behaviour t e s t i n g of females. In experiment IV, e a r l y - t r e a t e d females were exposed to a second androgen treatment and then t e s t e d to determine i f e a r l y treatments s e n s i t i z e d females to subsequent androgen exposures. Sex-reversed genetic females were d e t e c t e d i n experiment V and then the behaviour of sex-reversed females was compared with that of untreated g e n e t ic males. 12 11. METHODS A. GENERAL METHODS K Experimental Animals A l l Sarotherodon ( T i l a p i a ) mossambicus Rii p p e l l (taxonomy reviewed i n Trewavas, 1973, and Thys van den Audenaerde, 1971) used i n t h i s study are d e r i v e d from a colony maintained at the Department of Zoology (U.B.C.) f o r at l e a s t f i v e g e n e r a t i o n s . Colony founders are b e l i e v e d to be descendants of Malaysian stock obtained from a l o c a l aquarium shop. 2. Housing and Maintenance F i s h were housed in tanks ranging in volume from 31 to 330 l i t e r s at a s t o c k i n g rat e of roughly 2 cm of f i s h l e n g t h per l i t e r of tank water. As the f i s h grew l a r g e r , they were t r a n s f e r r e d to p r o g r e s s i v e l y l a r g e r storage tanks. Each storage tank was f i l t e r e d and a e r a t e d . Each f i l t e r was f i t t e d with f i b r o u s m a t e r i a l ( f l e x i b l e i n s u l a t i o n , AF300-Type 2, F l e c k Bros., Burnaby, B.C.) and c h a r c o a l . Storage tanks were s u p p l i e d with v e g e t a t i o n ( C e r a t o p t e r i s t h a i i c t r o i d e s and Lemna). A l l tanks were kept i n rooms maintained at 24~26°C. I l l u m i n a t i o n was provided by 40 watt f l u o r e s c e n t tube l i g h t s suspended 15-20 cm above each tank. The photoperiod (12L:12D) was a u t o m a t i c a l l y c o n t r o l l e d by e l e c t r o n i c t i m e r s . F i s h were fed commercially prepared t r o u t chow p e l l e t s 1 3 ( C l a r k ' s New Age F i s h P e l l e t s , Moore-Clark Co., La Conner, Washington) once a day (0900 h o u r s ) . The amount of food s u p p l i e d to f i s h i n each tank v a r i e d and was determined by the amount the f i s h would consume w i t h i n 10 minutes. Each tank was cleaned weekly by r e p l a c i n g one-half of the tank water with f r e s h water and changing the f i l t e r m a t e r i a l . 3^ . F i s h I d e n t i f i c a t i o n F i s h were marked by a t t a c h i n g a unique combination of b r i g h t l y c o l o u r e d beads to each i n d i v i d u a l . One or two beads on the animal's l e f t s i d e i n d i c a t e d the treatment group the f i s h was ass i g n e d to, and one or two beads on the animal's r i g h t s i d e i n d i c a t e d the number assigned to the f i s h . Beads were attached one week p r i o r to t e s t i n g as f o l l o w s . F i r s t , each f i s h was a n a e s t h e t i z e d by immersion in a 1:1000 MS-222 (w/w methane t r i c a i n e s u l f o n a t e ) s o l u t i o n . When the f i s h was n e a r l y motionless, on i t s s i d e , and r e s p i r i n g slowly, i t was removed from the s o l u t i o n and p l a c e d on wet paper towels. A 5 cm l e n g t h of nylon monofilament was then sewn through the d o r s a l musculature of the caudal peduncle using a #22 suture needle. The a p p r o p r i a t e beads were threaded onto each end of the monofilament and the two ends of monofilament were melted together to produce a loop. The f i s h was then returned to water and allowed to recover. 1 4 4 . Treatment Groups Six treatment groups (n = 60 f i s h / g r o u p ) were formed from f i v e separate broods as i n d i c a t e d i n Table I. Table I - Treatment Groups Group I II III IV V VI a, b c d, e e e e Notes: - Each lower case l e t t e r r e p r e s e n t s one of the f i v e broods used - Fry i n groups I and III are from 2 broods (a,b, or d = 30 f i s h ) - n = 60 f i s h / t r e a t m e n t group Each group r e c e i v e d a s p e c i f i c 1 7- <?C - m e t h y l t e s t o s t e r o n e (MT; Sigma Chemical Co., St. L o u i s , M i s s o u r i ) treatment as i n d i c a t e d i n Table I I . Four groups ( I , I I , I I I , and IV) r e c e i v e d treatment at d i f f e r e n t stages of development d u r i n g the suspected p e r i o d of gonadal and b e h a v i o u r a l d i f f e r e n t i a t i o n . One l a t e treatment (V) was a p p l i e d to s e x u a l l y mature i n d i v i d u a l s which had a l r e a d y had r e p r o d u c t i v e experience. One treatment (VI) i s a c o n t r o l treatment and f i s h in t h i s group were not exposed to MT. 15 Table II - Experimental Groups Group Treatment A p p l i e d Length of Treatment Hormone A p p l i c a t ion Dosage I F e r t i l i z a t i o n 12 days to r e l e a s e II Days 0 - 2 1 22 days P o s t - r e l e a s e III Days 0 - 2 1 22 days P o s t - r e l e a s e IV Days 21 - 42 22 days P o s t - r e l e a s e V When mature 40 days (>1 year old) VI C o n t r o l D i s s o l v e d i n e t h a n o l , added to water Added to water In food In food In food 1 mg/1 water 1 mg/1 water 30 jug/g d i e t 30 ug/g d i e t 60 ug/g d i e t 5. Treatment A d m i n i s t r a t i o n Group I: Treatment s t a r t e d w i t h i n three hours of spawning. The male was removed and MT added to the water at a dose of 1 mg/1 of tank water. T h i r t y - o n e mg of MT was d i s s o l v e d i n 15 ml of 75% ethanol and poured i n t o the aquarium. Every three days, one-half of the aquarium water was r e p l a c e d with c o n d i t i o n e d water and a f r e s h MT s o l u t i o n was added. The a d d i t i o n of androgen to the water d i d not appear to a f f e c t the performance of mouthbrooding behaviour in the female. 1 6 Group I I : Treatment s t a r t e d on day 12 a f t e r spawning, when young were v o l u n t a r i l y r e l e a s e d by the mouthbrooding female. The female was removed and MT added to the aquarium water at a dose of 1 mg/1 of tank water. The MT was ground in a t i s s u e homogenizer c o n t a i n i n g a few drops of polyoxyethylene (Tween 80 o i l ) and washed i n t o the aquarium. Every three days, one-half of the aquarium water was r e p l a c e d with c o n d i t i o n e d water and a f r e s h MT p r e p a r a t i o n was a p p l i e d . Groups I I I , IV, and V: F i s h r e c e i v e d MT i n the d i e t d u r i n g p e r i o d s i n d i c a t e d i n Table I I . Fre s h hormone-treated food was prepared f o r each group using the same ba s i c r e c i p e . F i r s t , a MT stock s o l u t i o n was prepared f o r each treatment by d i s s o l v i n g 30 mg of MT i n 600 ml of 75% e t h a n o l . Then, 60 ml of stock s o l u t i o n were added to each 100 grams of the group I I I or group IV d i e t s (30 wg MT/gm of d i e t ) , while 120 ml of stock s o l u t i o n were added to each 100 grams of the group V d i e t (60 /jg MT/gm of d i e t ) . I t was necessary to double the stock s o l u t i o n dose and halve the amount of food used f o r the group V treatment as the food intake rate of a d u l t f i s h i s c o n s i d e r a b l y l e s s than the food intake r a t e of j u v e n i l e f i s h (Rajamani and Job, 1976). T h i s a l t e r a t i o n i n s u r e d that a l l the MT t r e a t e d food was consumed by the group V f i s h . Once the stock s o l u t i o n was thoroughly mixed with the d i e t , the p r e p a r a t i o n was d r i e d in an SO^C oven f o r 4-5 hours to •evaporate the et h a n o l . When dry, the p r e p a r a t i o n was cooled to room temperature and t r a n s f e r r e d to a s e a l e d p l a s t i c c o n t a i n e r . When not i n use, stock s o l u t i o n s and d i e t s were s t o r e d i n a 17 r e f r i g e r a t o r at 7°C. On the f i r s t day of treatment, and once each succeeding week, one-quarter of the f i s h in each group were weighed and the t o t a l weight f o r a l l f i s h was estimated. During the week a f t e r weighing, f i s h were fed at 2 .5% of the t o t a l body weight four times per day ( 0 9 0 0 , 1 1 0 0 , 1400 and 1600 h o u r s ) . The group V f i s h were not weighed weekly past day 100 post-r e l e a s e . These f i s h were weighed again one week before i n i t i a t i n g the l a t e treatment. I t was not necessary to weigh these f i s h on a weekly b a s i s d u r i n g treatment s i n c e a d u l t f i s h reared under c o n f i n e d c o n d i t i o n s are known to gain weight slowly (Mironova, 1 9 6 5 ) . F i s h were weighed immediately a f t e r treatment V terminated, and no weight gains were noted. Group V I : C o n t r o l f i s h r e c e i v e d a d i e t prepared i n the same manner as the group III and group IV d i e t s , except that MT was not added to the stock s o l u t i o n . T h i s d i e t was admi n i s t e r e d from days 0 to 42 p o s t - r e l e a s e and f o r 40 days when the f i s h were s e x u a l l y mature (> 1 year o l d ) . J u v e n i l e group I I I , IV, and VI f i s h were t r e a t e d i n 31 l i t e r tanks while a d u l t group V and group VI f i s h were h e l d i n 330 l i t e r tanks during treatment. 18 B. SYNOPSIS OF BEHAVIOUR J_. Overview The f o l l o w i n g i s a b r i e f d e s c r i p t i o n of the behaviour of S. mossambicus. I have f r e e l y borrowed terminology and, to va r i o u s degrees, modified d e s c r i p t i o n s of behaviour o r i g i n a l l y proposed by other authors ( N e i l , 1964 and Baerends and Baerends van Roon, 1950). Many behaviours are common to both sexes, with q u a n t i t a t i v e r a t h e r than q u a l i t a t i v e d i f f e r e n c e s d i s t i n g u i s h i n g behaviour p a t t e r n s between the sexes. Behaviours r e s t r i c t e d to one sex are s p e c i f i c a l l y i n d i c a t e d . Behaviours are d e s c r i b e d with r e f e r e n c e to the three major contexts in which the behaviours occur ( i n t r a - , i n t e r - , or e x t r a - t e r r i t o r i a l ) . Behaviour p a t t e r n s e x h i b i t e d e x c l u s i v e l y i n a c e r t a i n context are d e s c r i b e d under the a p p r o p r i a t e heading. Behaviour components e x h i b i t e d by both sexes i n more than one context are d e s c r i b e d in the f i n a l s e c t i o n ( I I - B - 5 ) . The n a t u r a l h a b i t a t of S. mossambicus i s the q u i e t e r waters of small l a k e s , overflows, swamps, and e s t u a r i e s a s s o c i a t e d with the c o a s t a l drainage systems of east A f r i c a (Trewavas, 1968; W h i t e f i e l d and Blaber, 1979; B a l a r i n , 1979). S. mossambicus i s a v a l u a b l e food f i s h which has been widely t r a n s p l a n t e d and i s now c i r c u m t r o p i c a l l y d i s t r i b u t e d i n a v a r i e t y of h a b i t a t s , i n c l u d i n g : ponds, r i c e f i e l d s and b r a c k i s h lagoons (Brock, 1954; Chimits, 1955 and 1957). The general s o c i a l o r g a n i z a t i o n of t h i s s p e c i e s can be d i v i d e d i n t o : 1) a s c h o o l i n g s o c i e t y and 2) a t e r r i t o r i a l 19 s o c i e t y (Baerends and Baerends van Roon, 1950). Schools c o n s i s t of s i m i l a r - s i z e d i n d i v i d u a l s of both sexes and are normally found between midwater and the s u r f a c e although schools descend to the bottom to forage. The t e r r i t o r i a l s o c i e t y i s formed by s e x u a l l y mature males that leave the s c h o o l , descend to the bottom, and e s t a b l i s h t e r r i t o r i e s . S e v e r a l males w i l l e s t a b l i s h t h e i r t e r r i t o r i e s i n a c l o s e c l u s t e r , with the number and s i z e of ..the t e r r i t o r i e s depending on the area a v a i l a b l e , and the number, s i z e , and a g g r e s s i v e n e s s of the mature males. The s o c i a l o r g a n i z a t i o n of S. mossambicus has been s t u d i e d under domesticated c o n d i t i o n s ( N e i l , 1964) and under more n a t u r a l c o n d i t i o n s ( l a r g e ponds, N e i l , 1966) and found to be the same in both s i t u a t i o n s . S i m i l a r l y , in t h i s study, groups of S. mossambicus were observed to separate i n t o d i s t i n c t t e r r i t o r i a l and s c h o o l i n g s o c i e t i e s w i t h i n 36 hours of being p l a c e d i n a l a r g e tank. For example, in experiment I I , when 6 males and 4 females/group were placed i n a l a r g e o b s e r v a t i o n tank, the m a j o r i t y of the males (4-6) develop t e r r i t o r i e s while males without t e r r i t o r i e s and females form a s c h o o l . 2. I n t r a - T e r r i t o r i a l Behaviours A) Digging A male e s t a b l i s h e s a t e r r i t o r y by d i g g i n g a nest p i t i n the s u b s t r a t e . Digging occurs when a . f i s h swims head down i n t o the s u b s t r a t e , secures a mouthful of ,sand, swims a short d i s t a n c e from the center of the p i t , and s p i t s out the m a t e r i a l . The d i s p l a c e d sand i s deposited on the edge of a t e r r i t o r y and, as 2 0 i t accumulates, forms a r a i s e d rim around the nest. T h i s r a i s e d rim d e f i n e s t e r r i t o r i a l boundaries. L o c a l i z e d d i g g i n g produces a p i t which a male occupies and defends from i n t r u d e r s while attempting to a t t r a c t spawning p a r t n e r s . Digging i s used to maintain the nest rim and to remove d e b r i s from the p i t . Each male d i g s throughout i t s re s i d e n c y i n a t e r r i t o r y , with the frequency of d i g g i n g at a peak when the t e r r i t o r y i s being e s t a b l i s h e d . Females are a l s o observed d i g g i n g , but only i n the l a t e r stages of c o u r t s h i p p r i o r to spawning. B) Aggressive I n t e r a c t i o n s I n t r a - t e r r i t o r i a l f i g h t i n g normally occurs between a t e r r i t o r i a l male and an i n t r u d e r w i t h i n an e s t a b l i s h e d t e r r i t o r y . Each f i g h t u s u a l l y i n v o l v e s two males, although neighbouring t e r r i t o r i a l males and s c h o o l i n g males f r e q u e n t l y j o i n i n once a f i g h t has s t a r t e d . Up to s i x f i s h have been observed f i g h t i n g s i m u l t a n e o u s l y . I t i s not unusual f o r two or three f i s h to f i g h t while one or two f i s h r e s t and observe the i n t e r a c t i o n from the edge of the t e r r i t o r y . As a f i s h leaves the f i g h t to r e s t , one of the r e s t i n g f i s h e n t e r s the f i g h t . I n t r a -t e r r i t o r i a l f i g h t i n g between the same i n t r u d e r and r e s i d e n t may occur i n frequent bouts over s e v e r a l hours, although each f i g h t i n g bout u s u a l l y l a s t s no longer than 1-2 minutes. I n t r a - t e r r i t o r i a l f i g h t s i n v o l v i n g female f i s h are a l s o observed. If c o u r t s h i p between a r e s i d e n t male and a female i s i n t e r r u p t e d by an .intruder • s h o r t l y before spawning, the female p a r t i c i p a t e s i n i n t r a - t e r r i t o r i a l f i g h t i n g in e f f o r t s to oust 21 the i n t r u d e r . When the i n t r u d e r l e a v e s , the female resumes c o u r t s h i p a c t i v i t i e s with the r e s i d e n t male. Short bouts (<1 minute) of i n t r a - t e r r i t o r i a l f i g h t i n g are o c c a s i o n a l l y observed when two f o r a g i n g females meet i n an unoccupied t e r r i t o r y . Such i n t e r a c t i o n s are b r i e f and end a b r u p t l y as both females leave the t e r r i t o r y . The behaviour components used i n i n t r a - t e r r i t o r i a l f i g h t i n g a r e : c i r c l i n g , l a t e r a l d i s p l a y , t a i l w a g g i n g , b u t t i n g , a t t a c k i n g , and f r o n t a l d i s p l a y . Only c i r c l i n g i s e x h i b i t e d d u r i n g every i n t r a - t e r r i t o r i a l f i g h t i n g bout. V a r i o u s combinations of the remaining behaviour components are e x h i b i t e d during any p a r t i c u l a r f i g h t i n g bout. C i r c l i n g i n v o l v e s two f i s h o r i e n t i n g a n t i p a r a l l e l to each other, with one f i s h f o l l o w i n g the t a i l of i t s opponent in a c i r c l e w i t h i n the t e r r i t o r y . Once i n i t i a t e d , c i r c l i n g f i s h may t r a v e l beyond t e r r i t o r i a l boundaries for short p e r i o d s of time. A l l median f i n s are e r e c t e d and the opercula are r a i s e d d u r i n g c i r c l i n g . F i s h may exchange t i l t s , l a t e r a l d i s p l a y s , t a i l w a g s , b u t t s , or a t t a c k s at any time while c i r c l i n g . Each i n t r a -t e r r i t o r i a l f i g h t i n g bout ends when c i r c l i n g stops and one f i s h l e a ves the t e r r i t o r y . C) Reproductive I n t e r a c t i o n s Female prespawning behaviour c o n s i s t s of frequent approaches and b r i e f v i s i t s t o a number of n e s t s . As a female r i p e n s , the amount of nest approaching and nest v i s i t i n g a c t i v i t y i n c r e a s e s and becomes ; d i r e c t e d towards a p a r t i c u l a r t e r r i t o r y . A r i p e female w i l l r e p e a t e d l y enter the same 22 t e r r i t o r y , d i g i n the nest, and a s s i s t the r e s i d e n t male i n r e p e l l i n g i n t r u d e r s . Females i n or near a t e r r i t o r y r e c e i v e frequent t i l t s , b u t t s , t a i l w a g s , and l a t e r a l d i s p l a y s from the r e s i d e n t male. Spawning occurs a f t e r the male and female have engaged i n s e v e r a l bouts of c i r c l i n g and d i g g i n g . The female passes over the c e n t e r of the t e r r i t o r y , r a p i d l y v i b r a t e s her body, and r e l e a s e s about a dozen eggs. The male f o l l o w s the female over the eggs, v i b r a t e s h i s body, and r e l e a s e s m i l t . The female r e t u r n s to the eggs a f t e r they have been f e r t i l i z e d , sucks them i n t o her mouth, and s t a r t s mouthbrooding. A s e r i e s of a l t e r n a t i n g spawning a c t s and c i r c l i n g bouts occur u n t i l the female i s spent and leaves the t e r r i t o r y . 3 . I n t e r - T e r r i t o r i a l Behaviours I n t e r - t e r r i t o r i a l f i g h t i n g occurs between t e r r i t o r i a l males durin g boundary d i s p u t e s . Opposing males rush at each other with mouths open, f i n s and o p e r c u l a r a i s e d , ending t h e i r charges at the common boundary (nest r i m ) . Males then o s c i l l a t e back and f o r t h i n synchrony, with one male ( f i n s c o l l a p s e d ) r e t r e a t i n g while i t s opponent ( f i n s r a i s e d ) advances a few c e n t i m e t e r s . T h i s back and f o r t h motion i s completed s e v e r a l times i n r a p i d s u c c e s s i o n , a f t e r which males separate or a t t a c k . Males f r e q u e n t l y exchange t i l t s and f r o n t a l d i s p l a y s before, d u r i n g , and a f t e r i n t e r - t e r r i t o r i a l f i g h t i n g bouts. The d u r a t i o n of back and f o r t h o s c i l l a t i o n s was recorded as an i n t e r - t e r r i t o r i a l f i g h t i n g bout. 23 4. E x t r a - T e r r i t o r i a l Behaviours A) Mouthbrooding During spawning a female S. mossambicus p i c k s up eggs immediately a f t e r f e r t i l i z a t i o n and c a r r i e s them i n her mouth. The eggs develop w i t h i n a buc c a l pouch which the female g e n t l y f l e x e s in a "mouthing" a c t i o n to pass aerated water over the eggs and to remove wastes produced by the developing young (Oppenheimer, 1970). Eggs hatch approximately 4-6 days a f t e r spawning. A f t e r h a t c h i n g , the l a r v a e are maintained i n the bucca l pouch and "mouthed" r e g u l a r l y . On or about day 12 post-spawning, the female r e l e a s e s free-swimming f r y from her mouth. During the ensuing 1-2 weeks, the f r y remain near the female and seek temporary s h e l t e r i n the bucc a l pouch when s t a r t l e d . Mouthbrooding females do not feed, and become a g g r e s s i v e towards other f i s h between days 9-12 of mouthbrooding. During t h i s p e r i o d , mouthbrooding females darken, e x h i b i t l a t e r a l d i s p l a y s , f r o n t a l d i s p l a y s , and b u t t s to discourage the approach of other f i s h . Before day 9 of mouthbrooding, the female w i l l f l e e from other f i s h . B) S c h o o l i n g Members of a school normally p a r t i c i p a t e i n the same a c t i v i t y ( f o r a g i n g , swimming, or r e s t i n g ) at the same time. Schooling f i s h f r e q u e n t l y pass over the c l u s t e r of t e r r i t o r i e s and are courted, butted, and chased by the males. I n d i v i d u a l females leave the school when r i p e and enter the c l u s t e r of t e r r i t o r i e s to court with r e s i d e n t males. I f d r i v e n away from the t e r r i t o r i e s by a male, the female w i l l r e j o i n the 24 s c h o o l . If spawning occurs, the female leaves the t e r r i t o r y , does not r e t u r n to the s c h o o l , and hides from a l l other f i s h while mouthbrooding. I n d i v i d u a l males leave the school when mature and enter the c l u s t e r of t e r r i t o r i e s to compete with t e r r i t o r i a l males f o r e i t h e r e s t a b l i s h e d t e r r i t o r i e s or space to e s t a b l i s h a t e r r i t o r y . When d r i v e n away from a t e r r i t o r y , a male j o i n s the s c h o o l . 5. Behaviour Components T h i s s e c t i o n p r o v i d e s d e t a i l e d d e s c r i p t i o n s of the behaviour components i n v o l v e d in a v a r i e t y of i n t r a - , i n t e r - , and e x t r a - t e r r i t o r i a l i n t e r a c t i o n s . A) L a t e r a l D i s p l a y A l a t e r a l d i s p l a y occurs when a f i s h p r e s e n t s the l a t e r a l aspect of i t s body to an opponent while a r c i n g the t a i l and head towards the opponent. A l l median f i n s are e r e c t e d and the opercula are r a i s e d . While i n the l a t e r a l d i s p l a y p o s i t i o n , a f i s h w i l l f l i c k i t s t a i l at a r i v a l and w i l l n i p at the r i v a l ' s t a i l . B) Tailwagging L a t e r a l d i s p l a y s may be followed immediately by t a i l w a g g i n g . Tailwagging i n v o l v e s a f i s h c o l l a p s i n g a l l f i n s , t u r n i n g away from a r i v a l , and using the caudal f i n to v i o l e n t l y fan water over an opponent. Tailwagging i s used as a t h r e a t s i g n a l when d i r e c t e d by a t e r r i t o r i a l male at an i n t r u d i n g male. A male r e c e i v i n g a t a i l w a g w i l l e i t h e r leave the t e r r i t o r y or p a r t i c i p a t e i n i n t r a - t e r r i t o r i a l f i g h t i n g . Tailwagging i s used 25 as a c o u r t s h i p s i g n a l by a t e r r i t o r i a l male when d i r e c t e d at a female e n t e r i n g a t e r r i t o r y . When used i n a c o u r t s h i p context, t a i l w a g g i n g occurs two or three times f o l l o w i n g a s e r i e s of t i l t s . C) F r o n t a l D i s p l a y A f r o n t a l d i s p l a y occurs when a f i s h adopts a head-on o r i e n t a t i o n to an opponent, e r e c t s a l l f i n s , and r a i s e s the o p e r c u l a . F r o n t a l d i s p l a y s normally precede and f o l l o w i n t e r -t e r r i t o r i a l f i g h t i n g or a t t a c k i n g between males. D) T i l t i n g A t i l t i n g f i s h adopts a head-down a t t i t u d e , c o l l a p s e s a l l f i n s , and holds the body at an angle between 30-40° below the h o r i z o n t a l ( t i l t i n g u s u a l l y i n v o l v e s a head-down posture although two males always t i l t e d by assuming a head-up p o s t u r e ) . T i l t i n g by males occurs i n two contexts, depending on whether a male or a female approaches the nest. If a male approaches, a t i l t i s presented as a t h r e a t s i g n a l and the approaching male u s u a l l y veers away. If the approaching male e n t e r s the t e r r i t o r y , the r e s i d e n t male switches to p r e s e n t i n g l a t e r a l d i s p l a y s , t a i l w a g s , and may i n i t i a t e i n t r a - t e r r i t o r i a l f i g h t i n g . If a female approaches, the male leaves h i s t e r r i t o r y , meets the female, t i l t s , and r e t u r n s to h i s t e r r i t o r y while m a i n t a i n i n g the t i l t . If the female f o l l o w s and e n t e r s the t e r r i t o r y , the male w i l l engage in more advanced c o u r t s h i p a c t i v i t y i n v o l v i n g t i l t s , b u t t s , and t a i l w a g s . Females t i l t at approaching f i s h during the l a t e r stages of mouthbrooding and while i n a male's t e r r i t o r y j u s t p r i o r to 2 6 spawning. E) B u t t i n g B u t t i n g occurs when a f i s h swims towards a nearby f i s h and d i s p l a c e s i t with a bump or a nudge. Butts were a l s o recorded when the d i r e c t e d approach of a f i s h caused a nearby f i s h to dart away and when one f i s h chased another. Both males and females butt although b u t t s are normally i n i t i a t e d by a t e r r i t o r i a l male and d i r e c t e d towards f i s h approaching a t e r r i t o r y . F) A t t a c k i n g A t t a c k i n g occurs when one f i s h charges and v i g o r o u s l y s t r i k e s an opponent. Each a t t a c k may i n v o l v e a b i t i n g a t t a c k and/or head-slamming the opponent i n t o the s u b s t r a t e . An a t t a c k i s a v i o l e n t p h y s i c a l i n t e r a c t i o n between f i s h which f r e q u e n t l y terminates a g g r e s s i v e i n t e r a c t i o n s and d r i v e s one f i s h away. O c c a s i o n a l l y , a t t a c k s have r e s u l t e d i n f i n amputation and body s c a r r i n g . G) Submissive A t t i t u d e When extremely f r i g h t e n e d or appeasing an a t t a c k e r , a f i s h w i l l c o l l a p s e a l l f i n s , p a l e , and adopt a head-up a t t i t u d e with the body maintained between 20 and 30° above the h o r i z o n t a l . 27 C. DATA RECORDING TECHNIQUES J_. General Testing Procedures A l l f i s h were tested under standardized conditions. Each test group was placed in a 330 l i t e r observation tank maintained at 26°C in a temperature-controlled environmental chamber. A black p l a s t i c blind was attached to both the c e i l i n g and the floor 75 cm in front of the test tank. After a one week s e t t l i n g - i n period, behaviour observations were conducted by viewing the f i s h through a small s l i t (3 x 15 cm) in the p l a s t i c . Fish in observation tanks were fed da i l y at 0900 hours, unless otherwise s p e c i f i e d , and behaviour recordings were i n i t i a t e d at least one hour after feeding. The behaviours described in section B were recorded on a 20 channel Esterline Angus event recorder set at a chart speed of 7.6 cm/minute. The recorder was equipped with a keyboard c o n t r o l l i n g pen action. Ten keys were used to encode observed behaviour while the remaining keys were assigned to individual f i s h . Depressing sets of keys provided a record of every action given and received by a f i s h , as well as an indication of which fish(es) interacted with the f i s h under observation. Fish were observed sequentially from the lowest to highest numbered male, and then from the lowest to highest numbered female. Normally, half of the f i s h were tested between 1000-1200 hours .while the other half of the f i s h were tested between 1300-1700 hours. If the f i s h were s t a r t l e d by any external disturbance, a l l observations were terminated for the rest of 28 the day. The remainder of the t e s t i n g s e s s i o n would then be completed the next day. Data f o r each f i s h i n a t e s t group were c o l l e c t e d i n 5 f i f t e e n minute o b s e r v a t i o n p e r i o d s d u r i n g a 16 day t e s t p e r i o d . E f f o r t s were made to conduct a f u l l t e s t i n g s e s s i o n (one ob s e r v a t i o n p e r i o d f o r each f i s h i n the t e s t group) on days 3, 6, 9, 12, and 15 of the 16 day t e s t p e r i o d . 2. T e r r i t o r y C h a r a c t e r i s t i c s A map of the t e r r i t o r i e s was prepared d a i l y and updated as changes o c c u r r e d . Information c o l l e c t e d f o r each map i n c l u d e d : area defended by each t e r r i t o r i a l male, i n t r u d e r males ( i f any) competing f o r a t e r r i t o r y , occupancy changes (turnovers) s i n c e the l a s t o b s e r v a t i o n , and whether the t e r r i t o r y area i n c r e a s e d or decreased s i n c e the l a s t o b s e r v a t i o n . 3. F i g h t i n g V i g o r The t o t a l number of a g g r e s s i v e a c t s performed d u r i n g a l l i n t r a - or i n t e r - t e r r i t o r i a l f i g h t s was determined f o r each group. T h i s information was used to assess the r e l a t i v e v i g o r of f i g h t i n g a c t i v i t y in f i s h from d i f f e r e n t groups. In g e n e r a l , I expected a g g r e s s i v e f i s h to e x h i b i t a higher mean frequency of a g g r e s s i v e a c t s i n i t i a t e d d u r i n g f i g h t s than p a c i f i c f i s h . The t o t a l number of a g g r e s s i v e acts i n i t i a t e d d u r i n g i n t r a -t e r r i t o r i a l f i g h t i n g i s the sum of a l l p h y s i c a l c o n t a c t s ( a t t a c k s , b u t t s ) and t h r e a t s i g n a l s ( l a t e r a l d i s p l a y s , t i l t s , 2 9 t a i l w a g s ) observed in a l l i n t r a - t e r r i t o r i a l f i g h t s . The t o t a l number of a g g r e s s i v e a c t s i n i t i a t e d during i n t e r - t e r r i t o r i a l f i g h t i n g i s the sum of a l l p h y s i c a l c o n t a c t s ( a t t a c k s , b u t t s ) and t h r e a t s i g n a l s ( f r o n t a l d i s p l a y s , t i l t s ) observed i n a l l i n t e r - t e r r i t o r i a l f i g h t s . 4 . Nest V i s i t i n g A r e c o r d was maintained of how long each f i s h remained in a male's t e r r i t o r y . T h i s i n f o r m a t i o n was used to evaluate competition f o r a t e r r i t o r y (male v i s i t s ) or m o t i v a t i o n to p a r t i c i p a t e i n c o u r t s h i p (female v i s i t s ) . Leaving a t e r r i t o r y f o r a p e r i o d g r e a t e r than one minute i s used to d e l i m i t separate v i s i t s . 5_. Absenteeism T h i s measure records how long a t e r r i t o r i a l male i s absent from h i s t e r r i t o r y . A male w i l l leave a t e r r i t o r y to forage, c o u r t , v i s i t a neighbouring t e r r i t o r y , or when d i s p l a c e d from i t s t e r r i t o r y by an i n t r u d e r . Residence in the home t e r r i t o r y f o r a p e r i o d g r e a t e r than one minute i s used to d e l i m i t separate bouts of absenteeism. 6 . Body C o l o u r a t i o n A record was maintained d u r i n g each o b s e r v a t i o n .period of the l e n g t h of time each f i s h possessed c e r t a i n c o l o u r p a t t e r n s . A d d i t i o n a l l y , frequent d a i l y checks of a l l f i s h under 30 o b s e r v a t i o n allowed a l i s t of a l l f i s h p o s s e s s i n g dark c o l o u r a t i o n to be prepared. Each body c o l o u r a t i o n l a b e l (pale, dark 2, dark 3, and dark 4) was d e f i n e d as a c e r t a i n region of a continuum ranging from pale to uniform b l a c k . M a i n t a i n i n g body c o l o u r a t i o n records p r o v i d e d a general i n d i c a t i o n of a f i s h ' s s o c i a l m o t i v a t i o n ( f o r example, dark a g g r e s s i v e versus pale non-a g g r e s s i v e males, dark c o u r t i n g versus pale non-courting females, e t c . ) . Pale c o l o u r a t i o n i s c h a r a c t e r i s t i c of females engaged in f e e d i n g , s c h o o l i n g , and r e s t i n g or of n o n - t e r r i t o r i a l males a s s o c i a t e d with s c h o o l i n g females. In both cases, the body i s a uniform d u l l grey. Dark 2 f i s h are dark v e n t r a l l y and p a l e d o r s a l l y . The v e n t r a l body c o l o u r a t i o n i s a patchwork of l i g h t and dark areas. Dark 2 c o l o u r a t i o n i s t y p i c a l of a n o n - t e r r i t o r i a l male e n t e r i n g a t e r r i t o r y and s t a r t i n g to compete f o r the t e r r i t o r y with the r e s i d e n t . Dark 2 c o l o u r a t i o n i s a l s o adopted by females in the e a r l y stages of c o u r t s h i p or i n the e a r l y stages of mouthbrooding. Dark 3 c o l o u r a t i o n i s s i m i l a r to dark 2 except the body i s u n i f o r m l y dark below the m i d - l i n e . Males p o s s e s s i n g dark 3 c o l o u r a t i o n are normally n o n - t e r r i t o r i a l males i n v o l v e d in i n t e n s e competition with a r e s i d e n t male f o r a t e r r i t o r y . Females adopt dark 3 c o l o u r a t i o n j u s t before spawning and during the l a s t few days of mouthbrooding. Dark 4 c o l o u r a t i o n i s c h a r a c t e r i s t i c of t e r r i t o r i a l males a c t i v e l y c o u r t i n g females, and r e p e l l i n g male i n t r u d e r s . The 31 body i s u n i f o r m l y j e t - b l a c k , a d i s t i n c t white c h i n patch i s present, and the caudal and medial f i n s are trimmed i n red. A d d i t i o n a l d e t a i l s f o r each c o l o u r p a t t e r n and c o r r e l a t i o n s between c o l o u r p a t t e r n s and behaviour are provided i n N e i l (1964) . 32 ]_. Summary Table I I I . Behaviour Measures Recorded Measure Record Frequency Duration Given Received I n t r a - t e r r i t o r i a l F i g h t i n g I n t e r - t e r r i t o r i a l F i g h t i n g Aggressive Acts During I n t r a - t e r r i t o r i a l F i g h t i n g A ggressive A cts During I n t e r - t e r r i t o r i a l F i g h t i n g A t t a c k s T i l t s Butts Digging Nest V i s i t i n g Body C o l o u r a t i o n + + + + * * + + * + + + * + * + * + * + + * + + + + + * TaiIwags F r o n t a l D i s p l a y s Submissive A t t i t u d e s Absenteeism Mouthbrooding T e r r i t o r y Residency T e r r i t o r y Turnovers T e r r i t o r y S i z e Q u a l i t a t i v e Notes ( 1 ) ( 1 ) + + + + + + + + + Notes: + - i n d i c a t e s t h i s measure was recorded * - i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s between groups were dete c t e d in these measures ( 1 ) - i n d i c a t e s data a n a l y s i s was not performed on these measures The data r e c o r d i n g techniques used in t h i s study produced a comprehensive q u a n t i t a t i v e and q u a l i t a t i v e record of an i n d i v i d u a l ' s overt s o c i a l i n t e r a c t i o n s . The data c o l l e c t e d a l s o 3 3 p r o v i d e d i n f o r m a t i o n on the context of each a c t i o n p l u s an overview of the s o c i a l s i t u a t i o n i n the t e s t tank. Table III p r o v i d e s a complete l i s t i n g of the behaviour measures used and other data c o l l e c t e d d u r i n g t h i s study. 3 4 I I I . RESULTS EXPERIMENT I_. THE EFFECTS OF METHYLTESTOSTERONE ON GONADAL DIFFERENTIATION A. INTRODUCTION Th i s experiment examines the e f f e c t s of d i f f e r e n t MT treatments on gonadal d i f f e r e n t i a t i o n . The o b j e c t i v e was to determine whether gonadal d i f f e r e n t i a t i o n i s s e n s i t i v e to MT throughout a prolonged p e r i o d (the f i r s t two months of l i f e ) or dur i n g a r e s t r i c t e d p e r i o d (a one to three week p e r i o d during the f i r s t two months of l i f e ) . A prolonged p e r i o d of hormone s e n s i t i v i t y would be i n d i c a t e d i f MT i n f l u e n c e d gonadal d i f f e r e n t i a t i o n at d i f f e r e n t stages of development. A r e s t r i c t e d p e r i o d of hormone s e n s i t i v i t y would be i n d i c a t e d i f MT i n f l u e n c e d .gonadal d i f f e r e n t i a t i o n at a s i n g l e stage of development. An a d d i t i o n a l o b j e c t i v e of t h i s experiment was to assess the e f f e c t i v e n e s s of d i f f e r e n t hormone treatments on gonadal d i f f e r e n t i a t i o n . The e f f e c t i v e n e s s of a p a r t i c u l a r treatment would be determined by the p r o p o r t i o n of sex-reversed f i s h produced in each treatment group. An e f f e c t i v e treatment was expected to produce more males in a treatment group than an i n e f f e c t i v e treatment. Four treatments were a p p l i e d to developing young dur i n g the f i r s t two months of l i f e when gonadal d i f f e r e n t i a t i o n i s suspected to occur. One treatment was a p p l i e d to mature a d u l t s with f u l l y d i f f e r e n t i a t e d gonads to determine i f s e x - r e v e r s a l 35 c o u l d be induced i n a d u l t f i s h . B. METHODS J_. Subjects The s u b j e c t s of t h i s study were the 360 f i s h o btained from the 5 broods o u t l i n e d i n Table I. 2. Sexinq Methods A sex r a t i o was determined f o r each treatment group by sexing a l l i n d i v i d u a l s i n that group. Most f i s h were sexed as a d u l t s a f t e r a l l behaviour t e s t were completed. F i s h that d i e d d u r i n g e a r l y treatment or while i n storage were sexed as soon a f t e r death as p o s s i b l e . D i f f e r e n t techniques were used to sex immature and mature f i s h . F i s h younger than s i x months were c o n s i d e r e d immature and were sexed by d i s s e c t i n g out the gonads, a p p l y i n g acetocarmine s t a i n , and examining the gonads under a microscope. The c h a r a c t e r i s t i c s of s t a i n e d j u v e n i l e male and female Sarotherodon gonads are d e s c r i b e d in Guerrero and Shelton (1974). F i s h o l d e r than s i x months were c o n s i d e r e d s e x u a l l y mature and were sexed a f t e r d i s s e c t i o n by v i s u a l l y examining the gonads a f t e r d i s s e c t i o n . The c h a r a c t e r i s t i c s of mature male and female Sarotherodon gonads are d e s c r i b e d i n Dadzie (1969; 1974). P r i o r to s a c r i f i c e and d i s s e c t i o n , an attempt was made to sex a d u l t s on the b a s i s of d o r s a l and anal f i n shape (long and po i n t e d i n males, short and rounded i n females), e x t e r n a l 36 g e n i t a l i a ( B a l a r i n , 1979) and body c o l o u r a t i o n ( N e i l , 1964). B e h a v i o u r a l n o t e s a n d b r e e d i n g r e c o r d s were m a i n t a i n e d f o r e a c h a d u l t f i s h a n d were a l s o u s e d a s a i d s i n s e x i n g i n d i v i d u a l s . 3>. S t a t i s t i c a l T r e a t m e n t o f D a t a A g o o d n e s s o f f i t t e s t ( G - t e s t , w i t h Y a t e s ' c o r r e c t i o n f o r c o n t i n u i t y ; S o k a l a n d R o h l f , 1969) was u s e d t o compare t h e o b s e r v e d s e x r a t i o o f e a c h t r e a t m e n t g r o u p w i t h t h e e x p e c t e d 1:1 s e x r a t i o i n an u n t r e a t e d g r o u p . C. RESULTS T a b l e I V i n d i c a t e s t h a t g o n a d a l d i f f e r e n t i a t i o n i s i n f l u e n c e d by some o f t h e hormone t r e a t m e n t s . T h r e e e a r l y t r e a t m e n t g r o u p s ( I : i m m e r s i o n , f e r t i l i z a t i o n t o r e l e a s e ; I I I : MT i n d i e t , d a y s 0-21 p o s t - r e l e a s e ; a n d I V : MT i n d i e t , d a y s 2 1 -42 p o s t - r e l e a s e ) have se x r a t i o s t h a t d i f f e r s i g n i f i c a n t l y f r o m t h e a p p r o x i m a t e l y 1:1 s e x r a t i o o b t a i n e d f o r t h e c o n t r o l g r o u p ( V I ) a n d t h e g r o u p r e c e i v i n g MT f o r 40 d a y s a s a d u l t s ( V ) . One e a r l y hormone t r e a t m e n t ( I I : i m m e r s i o n , d a y s 0-21 p o s t - r e l e a s e ) a p p a r e n t l y d i d n o t i n f l u e n c e g o n a d a l d i f f e r e n t i a t i o n . S i n c e i n g r o u p s I , I I I , a n d I V t h e s e x r a t i o i s skewed i n t h e m a l e d i r e c t i o n , t h e s e g r o u p s p r o b a b l y c o n t a i n s e x - r e v e r s e d f i s h . G r o u p s I I , V, a n d V I ( c o n t r o l ) p o s s e s s a s e x r a t i o c l o s e t o 1:1 and p r o b a b l y do n o t c o n t a i n s e x - r e v e r s e d i n d i v i d u a l s . T h e s e p r e d i c t i o n s were t e s t e d i n ' e x p e r i m e n t I V t h r o u g h a b r e e d i n g p r o g r a m d e s i g n e d t o d e t e c t s e x - r e v e r s e d f i s h . F u r t h e r 3 7 Table IV. Treatment Group Sex R a t i o s Group Number of Females Number of Males (%) Sex R a t i o I II III IV V VI 1 2 2 5 2 2 0 28 2 9 4 8 ( 8 0 ) 3 5 ( 5 8 ) 58 ( 9 7 ) 4 0 ( 6 7 ) 32 ( 5 3 ) 31 ( 5 2 ) 0 . 2 5 0 . 7 0 . 0 3 0 . 5 0 . 9 0 . 9 < 0 . 0 0 5 * > 0 . 1 (NS) < 0 . 0 0 5 * < 0 . 0 0 5 * > 0 . 9 (NS) > 0 . 9 9 5 (NS) Notes : a - based on G-test, («C = 0 . 0 5 , Sokal and Rohlf 2 - t a i l e d f o r sex r a t i o / 1:1 '* - i m p l i e s a s i g n i f i c a n t d e v i a t i o n from a 1:1 sex r a t i o (NS)- i m p l i e s no s i g n i f i c a n t d e v i a t i o n from a 1:1 sex r a t i o 1 9 6 9 ) , comments on sex-reversed f i s h are reserved f o r experiment V. Treatments I, I I I , and IV a p p a r e n t l y i n f l u e n c e d gonadal development to v a r y i n g degrees. T h i s i s i n d i c a t e d by the d i f f e r e n t percentages of males i n each group (Table I V ) . Treatment III i s the most e f f e c t i v e treatment s i n c e i t produced 9 7 % males. A ranking of the e f f e c t i v e n e s s of treatments in m a s c u l i n i z i n g developing f i s h i s as f o l l o w s : 111>I>IV>II=V=VI. 38 D. DISCUSSION The four e a r l y treatments can be d i v i d e d i n t o two c l a s s e s : a p a i r of immersion treatments (I and I I ) , and a p a i r of o r a l treatments (III and I V ) . Each treatment c l a s s shared a common f e a t u r e . The e a r l i e s t treatment w i t h i n each p a i r (I and I I I ) produced a g r e a t e r e f f e c t than the l a t e r treatment (II or IV). Treatment by a d d i t i o n of MT to the water f o r 12 days a f t e r f e r t i l i z a t i o n (I) a f f e c t e d gonadal development whereas a s i m i l a r immersion treatment s t a r t e d 12 days a f t e r f e r t i l i z a t i o n and a p p l i e d f o r 22 days (II) was i n e f f e c t i v e i n i n f l u e n c i n g gonadal d i f f e r e n t i a t i o n . Thus, gonadal development in Sarotherodon f r y appears to be a f f e c t e d by MT immersion treatment only d u r i n g the f i r s t 12 days of development. E c k s t e i n and S p i r a (1965) immersed 4-5 week o l d Sarotherodon aureus f r y in MT ( c o n c e n t r a t i o n s from 0.05 to 1.0 mg/1 f o r 5-6 weeks) and repo r t e d no e f f e c t s on gonadal sex d i r e c t i o n . C o n s i d e r a t i o n of the E c k s t e i n and S p i r a study and the i n e f f e c t i v e n e s s of treatment II suggests that immersion treatments a d m i n i s t e r e d a f t e r the f i r s t 12 days of l i f e have no i n f l u e n c e on gonadal d i f f e r e n t i a t i o n in Sarotherodon. Treatments by a d d i t i o n of MT to the food (III and IV) a l s o produce d i f f e r e n t i n f l u e n c e s on gonadal development. The o r a l treatments were i d e n t i c a l except I I I was a p p l i e d from r e l e a s e to day 21 p o s t - r e l e a s e whereas IV was a p p l i e d d u r i n g days 21-42 p o s t - r e l e a s e . Treatment III produced a ' h i g h l y skewed sex r a t i o while treatment IV produced only a s l i g h t l y skewed sex r a t i o . Thus, gonadal development in Sarotherodon f r y appears to be more 3 9 s e n s i t i v e to MT ingested with the food during the f i r s t 21 days a f t e r r e l e a s e than d u r i n g the next 21 days a f t e r r e l e a s e . T h i s f i n d i n g complements other s t u d i e s of o r a l MT treatments to S. mossambicus f r y (Nakamura, 1975, and Clemens and I n s l e e , 1968). Nakamura fed f r y 50 ug MT/gm of d i e t f o r 19 days a f t e r h a tching and produced a l l male o f f s p r i n g . Clemens and I n s l e e a l s o fed MT to f r y (10, 20, 30, or 40 pg/gm of d i e t f o r the f i r s t 69 days of l i f e ) and produced a l l male o f f s p r i n g i n each group. Both treatment II (immersion; 1 mg/1) and treatment III ( i n d i e t ; 30 ug/gm of d i e t ) were admi n i s t e r e d to f r y durin g the f i r s t 21 days a f t e r r e l e a s e . Treatment II had no a f f e c t on gonadal development, but 97% of the f i s h i n treatment III developed as males. T h i s suggests that immersion treatments are l e s s e f f e c t i v e than o r a l treatments i n i n f l u e n c i n g gonadal development. This o b s e r v a t i o n i m p l i e s that developing f r y must be h i g h l y s e n s i t i v e to MT i n f l u e n c e s very e a r l y in development si n c e 80% of the t r e a t e d f i s h exposed to a r e l a t i v e l y i n e f f e c t i v e immersion treatment ( I , f i r s t 12 days a f t e r f e r t i l i z a t i o n ) developed as males. One f i s h with an o v o - t e s t i s was de t e c t e d i n t h i s study. T h i s sex-reversed f i s h (IIIB4, see Table V I I I ) possessed gonads ( t o t a l gonad length = 45 mm) that were t h r e e - q u a r t e r s t e s t i c u l a r t i s s u e ( c e p h a l i c s e c t i o n ) and one-quarter o v a r i a n t i s s u e (caudal s e c t i o n ) . The diameter of the o v a r i a n s e c t i o n (5 mm) was n e a r l y twice the diameter of the t e s t i c u l a r s e c t i o n . Nakamura (1975) d e s c r i b e d s i m i l a r o v o - t e s t e s i n three male S. mossambicus 40 treated with MT (1000 /iig/gm of diet for 44 days starting 7 days after hatching). It is not known whether these f i s h were sex-reversed. Since a high dose of MT was applied in Nakamura's study, the ova-testis may be a product of paradoxical feminization (discussed in section IV-A-3). However, ovo-testes in the two studies share a common feature in that MT did not d e c i s i v e l y influence sex d i r e c t i o n in a l l gonadal tissues. Treatment of adult f i s h (V) did not influence the sex r a t i o . This suggests that sex d i r e c t i o n in the mature gonad i s not susceptible to influences produced by a prolonged oral MT administration. 41 E. SUMMARY 1. Two treatment groups (II,V) and the c o n t r o l group (VI) possessed a sex r a t i o c l o s e to 1:1. T h i s suggests that treatment by immersion from days 0-21 (II) or by feed i n g as an a d u l t (V) had no s i g n i f i c a n t e f f e c t on sexual d i f f e r e n t i a t i o n of the gonads. 2. Three treatment groups e x h i b i t e d sex r a t i o s that d i f f e r e d s i g n i f i c a n t l y from 1:1, i n d i c a t i n g these treatments d i d i n f l u e n c e gonadal .sex ( I : immersion, f e r t i l i z a t i o n to r e l e a s e ; I I I : MT i n d i e t , days 0-21 p o s t - r e l e a s e ; IV: MT in d i e t , days 21-42 p o s t - r e l e a s e ) . 3. Treatment III was the most e f f e c t i v e of the treatments in i n f l u e n c i n g sex d i f f e r e n t i a t i o n as 97% of the t r e a t e d f i s h developed as males. 4. Treatments I, I I I , and IV were a p p l i e d at d i f f e r e n t stages of development and each treatment produced a skewed sex r a t i o . T h i s i n d i c a t e s that the developing gonad i s s e x u a l l y p l a s t i c and s e n s i t i v e to hormonal i n f l u e n c e s throughout e a r l y development. 5. Treatments a p p l i e d d u r i n g the f i r s t month of l i f e (I and I I I ) were more e f f e c t i v e in i n f l u e n c i n g gonadal development than a treatment a p p l i e d d u r i n g the second month of l i f e ( I V ) . T h i s o b s e r v a t i o n suggests that the gonad i s most s e n s i t i v e to hormonal i n f l u e n c e s e a r l y i n l i f e , with hormone s e n s i t i v i t y d e c r e a s i n g with age. 42 EXPERIMENT J_I_. THE EFFECTS OF METHYLTESTOSTERONE ON THE DEVELOPMENT OF MALE BEHAVIOUR A. INTRODUCTION T h i s experiment examines the i n f l u e n c e of androgen on the development of male behaviour by comparing the behaviour of males exposed to d i f f e r e n t MT treatments. Since c e r t a i n e a r l y hormone treatments induce m o r p h o l o g i c a l and b e h a v i o u r a l sex-r e v e r s a l s (see r e f e r e n c e s c i t e d i n s e c t i o n I-A-4), and si n c e Lindsay (1974) found that an e a r l y androgen treatment i n f l u e n c e d the development of a hormone-sensitive mechanism u n d e r l y i n g behaviour, I expected d i f f e r e n t hormone treatments to produce d i f f e r e n t e f f e c t s on the the development of behaviour. I f d i f f e r e n c e s between groups were detected, then i t should be p o s s i b l e to comment on what elements of male behaviour are i n f l u e n c e d by androgen dur i n g development, how these elements are a f f e c t e d , and whether s e n s i t i v e p e r i o d s e x i s t i n b e h a v i o u r a l d i f f e r e n t i a t i o n . B. METHODS  Subjects Eighteen males and s i x females from each of the s i x treatment groups were used as t e s t animals. 43 2. Data C o l l e c t i o n The b e h a v i o u r a l data, presented f o r each treatment group were c o l l e c t e d in. o b s e r v a t i o n s of three separate t e s t groups (A, B, and C). Each t e s t group c o n s i s t e d of s i x males and two females from the same treatment group, and two untreated stimulus females, maintained and t e s t e d under the standard c o n d i t i o n s d e s c r i b e d i n s e c t i o n I-C-1. _3. S t a t i s t i c a l Treatment of Data The data f o r each behaviour measure (see Table I I I , s e c t i o n I-C-7) was analyzed using a K r u s k a l - W a l l i s one-way a n a l y s i s of v a r i a n c e ( ° C = 0.05; df = 5; S i e g e l , 1956). When a s i g n i f i c a n t d i f f e r e n c e between groups was d e t e c t e d , a simultaneous t e s t procedure based on the Mann-Whitney U-test (°C= 0.05; Sokal and Rohlf, 1969) was used to s p e c i f y which d i f f e r e n c e s were s i g n i f i c a n t . In the f i g u r e s that f o l l o w s i g n i f i c a n t d i f f e r e n c e s between groups are i n d i c a t e d by c o n t r a s t i n g data bar shading p a t t e r n s . A d i f f e r e n c e does not e x i s t between groups s h a r i n g the same shading p a t t e r n . Unshaded data bars i n d i c a t e that d i f f e r e n c e s between the i n d i c a t e d group and any other group were not s i g n i f i c a n t at the p < 0.05 l e v e l . 44 C. RESULTS J_. I n t r a - T e r r i t o r i a l F i g h t i n g A) Du r a t i o n The mean d u r a t i o n of i n t r a - t e r r i t o r i a l f i g h t i n g a c t i v i t y f o r group III f i s h i s at l e a s t three times longer than the mean d u r a t i o n f o r e i t h e r group II or group V f i s h . F i g u r e 1 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group III and e i t h e r group II or group V. D i f f e r e n c e s between group III and groups I, IV, or VI ( c o n t r o l ) are not s i g n i f i c a n t . Although group I and group IV f i s h appear to e x h i b i t more i n t r a - t e r r i t o r i a l f i g h t i n g a c t i v i t y than group VI ( c o n t r o l ) f i s h , t h i s d i f f e r e n c e . i s not s i g n i f i c a n t . S i m i l a r l y , group II and group V f i s h appear to e x h i b i t l e s s i n t r a - t e r r i t o r i a l f i g h t i n g than group VI ( c o n t r o l ) f i s h , but t h i s d i f f e r e n c e i s not s i g n i f i c a n t . 45 F i g u r e 1. Male I n t r a - T e r r i t o r i a l F i g h t i n g . The Mean Duration (+ Standard E r r o r ) of I n t r a - T e r r i t o r i a l F i g h t i n g by Each Male During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 46 MEAN DURATION OF INTRA-TERRITOR IAL FIGHTING BY EACH MALE (in seconds) 200 J 150 A 100 -I 50 -I GROUP IE n =18 fish/group H I 3ZI ETOH SOLN A Q . SOLN I N DIET IN DIET IN DIET CONTROL FERT DAYS DAYS DAYS > 1 TO 0-21 0-21 21-42 YEAR RELEASE 4 7 B) Frequency of Aggressive Acts The mean frequency of a g g r e s s i v e a c t s performed d u r i n g i n t r a - t e r r i t o r i a l f i g h t i n g i s at l e a s t twice as high f o r group III or group VI ( c o n t r o l ) f i s h compared to group V f i s h . F i g u r e 2 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between groups III or VI and group V. Although f i s h i n groups I, I I , and IV appear t o perform fewer a g g r e s s i v e a c t s d u r i n g i n t r a - t e r r i t o r i a l f i g h t i n g than group VI ( c o n t r o l ) f i s h , these d i f f e r e n c e s are not s i g n i f i c a n t . 2. I n t e r - T e r r i t o r i a l F i g h t i n g A) Duration The mean d u r a t i o n of i n t e r - t e r r i t o r i a l f i g h t i n g a c t i v i t y f o r group I and group III f i s h i s at l e a s t f i v e times longer than the mean d u r a t i o n f o r group V f i s h . F i g u r e 3 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between e i t h e r group I or group III and group V. D i f f e r e n c e s between e i t h e r group I or group III and groups I I , IV, and VI ( c o n t r o l ) are not s i g n i f i c a n t . Although group II and group IV f i s h appear to e x h i b i t more i n t e r - t e r r i t o r i a l f i g h t i n g a c t i v i t y than group VI ( c o n t r o l ) f i s h , t h i s d i f f e r e n c e i s not s i g n i f i c a n t . S i m i l a r l y , group V f i s h appear to e x h i b i t l e s s i n t e r - t e r r i t o r i a l f i g h t i n g than group VI ( c o n t r o l ) f i s h , but t h i s d i f f e r e n c e i s not s i g n i f i c a n t . B) Frequency of Aggressive Acts The mean frequency of ag g r e s s i v e a c t s performed during i n t e r - t e r r i t o r i a l f i g h t i n g i s at l e a s t three times higher f o r group I or group III f i s h compared to group V or group VI 48 F i g u r e 2. Aggressive Acts During I n t r a - T e r r i t o r i a l F i g h t i n g . The Mean Frequency (+ Standard E r r o r ) of A g g r e s s i v e Acts Performed by Each Male i n I n t r a - T e r r i t o r i a l F i g h t s During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. • / . . . M E A N F R E Q U E N C Y OF A G G R E S S I V E ACTS INITIATED BY E A C H M A L E 40 -I 30 H 20 -i 10 GROUP lil u Mi m w n= 18 f ish/group 31 E T O H A Q . IN IN IN S O L N S O L N DIET DIET DIET C O N T R O L FERT. DAYS D A Y S D A Y S >1 T O 0 - 2 1 0-21 2 1 - 4 2 Y E A R RELEASE 5 0 F i g u r e 3. Male I n t e r - T e r r i t o r i a l F i g h t i n g . The Mean Duration (+ Standard E r r o r ) of I n t e r - T e r r i t o r i a l F i g h t i n g by Each Male During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 51 M E A N DURATION OF I N T E R - T E R R I T O R I A L F I G H T I N G BY E A C H M A L E (in seconds) 25 A 20 H 15 H 10 -I 5 -4 I n = 18 f i s h / group G R O U P n m E T O H S O L N A Q . S O L N I N DIET IN DIET I N DIET C O N T R O L FERT. TO R E L E A S E D A Y S 0-21 DAYS 0 - 2 1 DAYS 21-42 > 1 Y E A R 52 (control.) f i s h . F i g u r e 4 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between groups I or III and groups V or VI. Although f i s h i n groups II and IV appear to perform more ag g r e s s i v e a c t s d u r i n g i n t e r - t e r r i t o r i a l f i g h t i n g than group VI ( c o n t r o l ) f i s h , these d i f f e r e n c e s are not s i g n i f i c a n t . 3_. A t t a c k i n g The mean frequency of a t t a c k s i n i t i a t e d i s at l e a s t twice as high f o r group I f i s h compared to e i t h e r group I I , V, or VI f i s h . F i g u r e 5 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group I and e i t h e r group I I , V, or VI. D i f f e r e n c e s between group I and groups III and IV are not s i g n i f i c a n t . Although group III and group IV f i s h appear to a t t a c k more f r e q u e n t l y than group VI ( c o n t r o l ) f i s h , these d i f f e r e n c e s are not s i g n i f i c a n t . S i m i l a r l y , group II and group V f i s h appear to attack l e s s f r e q u e n t l y than group VI ( c o n t r o l ) f i s h , but these d i f f e r e n c e s are not s i g n i f i c a n t . gure 4. Aggressive Acts During I n t e r - T e r r i t o r i a l F i g h t i n g . The Mean Frequency (+ Standard E r r o r ) of Aggressive Acts Performed by Each Male in I n t e r - T e r r i t o r i a l F i g h t s During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 54 M E A N F R E Q U E N C Y O F A G G R E S S I V E A C T S IN IT IATED B Y E A C H M A L E n=- 18 f i sh/group m 12 "EL E T O H A Q . IN IN IN S O L N S O L N D I ET D I ET DIET C O N T R O L FERT. DAYS DAYS TO . 0-21 0-21 R E L E A S E D A Y S > 1 21-42 Y E A R 55 F i g u r e 5. A t t a c k i n g by Males. The Mean Frequency (± Standard E r r o r ) of A t t a c k s I n i t i a t e d by Each Male During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 57 D. DISCUSSION There were s i g n i f i c a n t d i f f e r e n c e s between groups i n f i v e measures of t e r r i t o r i a l defense and a g g r e s s i o n : i n t r a -t e r r i t o r i a l f i g h t i n g ( f i g h t i n g d u r a t i o n and frequency of a g g r e s s i v e a c t s ) , i n t e r - t e r r i t o r i a l f i g h t i n g ( f i g h t i n g d u r a t i o n and frequency of a g g r e s s i v e a c t s ) , and a t t a c k i n g ( f r e q u e n c y ) . F i s h immersed in a MT s o l u t i o n from f e r t i l i z a t i o n to r e l e a s e (I) d i s p l a y high i n t e r - t e r r i t o r i a l f i g h t i n g and a t t a c k i n g s c o r e s . F i s h fed MT i n the d i e t from r e l e a s e to day 21 p o s t - r e l e a s e ( I I I ) d i s p l a y high i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g s c o r e s . Adult f i s h fed MT i n the d i e t f o r 40 days (V) d i s p l a y low scores in a l l measures of t e r r i t o r i a l defense and a g g r e s s i o n . Table V i n d i c a t e s that a s i g n i f i c a n t c o r r e l a t i o n e x i s t s between the d u r a t i o n of f i g h t i n g a c t i v i t y and the number of a g g r e s s i v e a c t s i n i t i a t e d d u r i n g f i g h t s f o r f i s h i n a l l of the groups (n = 18 f i s h / g r o u p ) . In other words, f i s h engaged in prolonged f i g h t s are not f i g h t i n g at low i n t e n s i t i e s ( i n i t i a t i n g a small number of a g g r e s s i v e a c t s ) and f i s h engaged in b r i e f f i g h t s are not f i g h t i n g at high i n t e n s i t i e s ( i n i t i a t i n g a l a r g e number of a g g r e s s i v e a c t s ) . D i f f e r e n c e s i n i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g occur between treatment groups but not between the c o n t r o l group and any treatment group. S p e c i f i c a l l y , d i f f e r e n c e s i n i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g were det e c t e d i n comparisons of groups I or III with groups II and/or V. C o n t r o l scores (VI) in both i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g are intermediate between two groups with high scores ( I , I I I ) and two groups with 56 M E A N F R E Q U E N C Y OF ATTACKS INITIATED BY EACH M A L E 5 -I n= 18 f i sh/group GROUP 3ZJ ETOH S O L N AQ. SOLN I N DIET I N DIET IN DIET C O N T R O L FERT. TO RELEASE DAYS 0-21 DAYS 0-21 DAYS 21 -42 > 1 Y E A R 58 Table V. C o r r e l a t i o n C o e f f i c i e n t s f o r F i g h t Duration versus F i g h t V i g o r . Spearman Rank C o r r e l a t i o n C o e f f i c i e n t s f o r the R e l a t i o n s h i p between the Duration of F i g h t i n g A c t i v i t y and the Frequency of Aggressive Acts Performed in F i g h t s . Measure I n t r a - T e r r i t o r i a L F i g h t i n g I n t e l — T e r r i t o r i a I F i g h t i n g Group I II III IV V VI I II III IV V VI r (1) .859* .829* .313* .803* .921* .714* .894* .965* .653* .910* .997* .865* s t . . . r (2) 26.89 23.72 5.27 21.50 37.81 16.33 31.95 58.64 13.79 35.13 196.12 27.53 Notes: * - i n d i c a t e s that a s i g n i f i c a n t r e l a t i o n s h i p e x i s t s between the du r a t i o n of f i g h t i n g a c t i v i t y and the frequency of aggressive acts performed i n f i g h t s . (1) - Spearman Rank C o r r e l a t i o n C o e f f i c i e n t (°C= 0.05; S i e g e l , 1956), 1 - t a i l e d f o r a p o s i t i v e c o r r e l a t i o n between f i g h t i n g duration and the frequency of aggressive acts performed i n f i g h t s . (2) - the s i g n i f i c a n c e of obtaining a value as large as the observed r g value i s determined by c a l c u l a t i n g the t value associated with the r g value and then determining the s i g n i f i c a n c e of the t value. - t (n = 18; degrees of freedom = 16; 1 - t a i l e d t e s t ) = 1.74 ui 60 low scores ( I I , V ) . These o b s e r v a t i o n s suggest that treatments I and III enhance male a g g r e s s i v e n e s s while treatments II and V i n h i b i t male a g g r e s s i v e n e s s . These enhanced and i n h i b i t e d l e v e l s of a c t i v i t y g e n e r a l l y do not d i f f e r s i g n i f i c a n t l y from the c o n t r o l l e v e l of a c t i v i t y . However, the combined e f f e c t of these d i v e r g i n g i n f l u e n c e s u n d e r l i e s the d e t e c t i o n of d i f f e r e n c e s between treatment groups (between groups I or I I I and groups II and/or V ) . C e r t a i n general trends i n the data should a l s o be c o n s i d e r e d i n a s s e s s i n g the i n f l u e n c e of treatments on behaviour. For example, f i s h exposed to immersion treatment from f e r t i l i z a t i o n to r e l e a s e (I) are g e n e r a l l y more a g g r e s s i v e than f i s h exposed to immersion treatment on days 0-21 p o s t - r e l e a s e ( I I ) . S i m i l a r l y , f i s h exposed to o r a l treatment on days 0-21 p o s t - r e l e a s e ( I I I ) are g e n e r a l l y more a g g r e s s i v e than f i s h exposed to o r a l treatment on days 21-42 p o s t - r e l e a s e (IV). Within each p a i r of immersion or o r a l treatments, the e a r l i e r treatment appears to i n f l u e n c e the development of components of male a g g r e s s i v e behaviours to a g r e a t e r extent than the l a t e r treatment. The r e s u l t s of t h i s experiment show that a d m i n i s t e r i n g the same treatment at d i f f e r e n t times d u r i n g development does not produce the same e f f e c t on behaviour. T h i s suggests that c e r t a i n components of a g g r e s s i v e behaviour may be a s s o c i a t e d with hormone-sensitive p e r i o d s d u r i n g development. Treatments administered d u r i n g a s e n s i t i v e p e r i o d can i n f l u e n c e b e h a v i o u r a l c h a r a c t e r i s t i c s while treatments administered o u t s i d e the s e n s i t i v e p e r i o d produce l e s s of an e f f e c t , i f any, 61 on b e h a v i o u r a l development. While a t t a c k i n g i s not the only b e h a v i o u r a l element expressed d u r i n g i n t r a - t e r r i t o r i a l f i g h t i n g ( l a t e r a l d i s p l a y s , b u t t s , t i l t s , and t a i l w a g s a l s o occur) or i n t e r - t e r r i t o r i a l f i g h t i n g ( f r o n t a l d i s p l a y s , b u t t s , and t i l t s a l s o o c c u r ) , i t was the only element to be performed at an enhanced frequency a f t e r treatment I. The d u r a t i o n of a t t a c k i n g i n t e r a c t i o n s was not a f f e c t e d . A l l other components of i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g were expressed at c o n t r o l l e v e l s of a c t i v i t y . T h i s o b s e r v a t i o n i n d i c a t e s that hormone treatments can a f f e c t the e x p r e s s i o n of behaviour by i n f l u e n c i n g the c h a r a c t e r s t i e s of complex p a t t e r n s of behaviour ( i n t r a - or i n t e r - t e r r i t o r i a l f i g h t i n g ) and by i n f l u e n c i n g i n d i v i d u a l components of behaviour ( a t t a c k i n g ) w i t h i n a complex p a t t e r n . Hormone treatments i n f l u e n c e the development of a g g r e s s i v e behaviour by shaping the c h a r a c t e r i s t i c s (frequency or d u r a t i o n ) of s e v e r a l s p e c i f i c elements of a g g r e s s i v e behaviour. The o b s e r v a t i o n that a l l elements of a g g r e s s i v e behaviour are not i n f l u e n c e d i n the same manner by a p a r t i c u l a r treatment can be e x p l a i n e d by p o s t u l a t i n g that d i f f e r e n t a g g r e s s i v e behaviours are shaped by hormonal i n f l u e n c e s at d i f f e r e n t times d u r i n g development. The idea that s e n s i t i v e p e r i o d s f o r d i f f e r e n t behaviour components are temporally separated i s r e i n f o r c e d by the o b s e r v a t i o n that treatments a p p l i e d at d i f f e r e n t stages of development i n f l u e n c e d i f f e r e n t components of behaviour. For example, treatment I ( a p p l i e d d u r i n g the f i r s t 12 days of l i f e ) i n f l u e n c e s a t t a c k i n g , while treatment III ( a p p l i e d days 21-42 p o s t - r e l e a s e ) i n f l u e n c e s 62 i n t r a - t e r r i t o r i a l f i g h t i n g . Adult f i s h fed MT in the diet (V) display low levels of aggressive behaviour. This inhi b i t o r y influence i s l i k e l y due to MT treatment aff e c t i n g the hormonal state underlying the expression of aggressive behaviour, since group V f i s h did not receive a hormone treatment during development. Baggerman (1968) has suggested that the hormonal mechanism c o n t r o l l i n g aggressive behaviour in male f i s h involves a synergistic action between gonadal and gonadotropic hormones. Peter and Crim (1979) have argued that gonadotropin secretion in teleosts i s responsible for maintaining gonadal androgen production and that c i r c u l a t i n g androgen leve l s regulate gonadotropin secretion through a negative feedback mechanism. Therefore, low levels of aggressive behaviour in group V may be explained in terms of the MT treatment disrupting gonadotropin and gonadal hormone production. That i s , the MT treatment increased c i r c u l a t i n g androgen levels which, through the feedback mechanism, inhibited gonadotropin production. A decrease in gonadotropin production would be followed by a decrease in gonadal hormone production. Reduction of both gonadal and gonadotropic hormone levels would interfere with maintenance of the hormonal state required for the normal expression of aggressive behaviours and result in low aggressive behaviour scores in group V f i s h . 63 E. SUMMARY 1. There were d i f f e r e n c e s between groups in f i v e measures of t e r r i t o r i a l defense and a g g r e s s i o n : i n t r a - t e r r i t o r i a l f i g h t i n g ( f i g h t i n g d u r a t i o n and frequency of a g g r e s s i v e a c t s d u r i n g f i g h t i n g ) , i n t e r - t e r r i t o r i a l f i g h t i n g ( f i g h t i n g d u r a t i o n and frequency of a g g r e s s i v e a c t s d u r i n g f i g h t i n g ) and a t t a c k i n g ( f r e q u e n c y ) . 2. Group I males (immersion, f e r t i l i z a t i o n to r e l e a s e ) d i s p l a y e d high l e v e l s of i n t e r - t e r r i t o r i a l f i g h t i n g and a t t a c k i n g while group III males (MT i n d i e t , days 0-21 post-r e l e a s e ) d i s p l a y e d high l e v e l s of i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g . 3. Group V f i s h (fed MT as a d u l t s ) possessed low a g g r e s s i v e behaviour s c o r e s , presumably because treatment d i s r u p t e d the hormonal s t a t e c o n t r o l l i n g the normal expr e s s i o n of a g g r e s s i v e behaviour. 4. B e h a v i o u r a l d i f f e r e n c e s were most f r e q u e n t l y d e t e c t e d between treatment groups I or III and treatment groups II and/or V. F i s h i n groups I and III were s l i g h t l y more a g g r e s s i v e than group VI ( c o n t r o l ) f i s h , while f i s h i n groups II and V were s l i g h t l y l e s s a g g r e s s i v e than group VI ( c o n t r o l ) f i s h . These d i v e r g i n g tendencies u n d e r l i e the d i f f e r e n c e s i n behaviour d e t e c t e d between treatment groups. 5. S i m i l a r treatments (immersion: I and I I ; o r a l : III and IV) a p p l i e d at d i f f e r e n t p e r i o d s during development produce d i f f e r e n t e f f e c t s on b e h a v i o u r a l development. T h i s o b s e r v a t i o n suggests that there are s e n s i t i v e p e r i o d s d u r i n g b e h a v i o u r a l 64 development. Treatments a p p l i e d d u r i n g the s e n s i t i v e p e r i o d produce more of an i n f l u e n c e on b e h a v i o u r a l development than treatments a p p l i e d o u t s i d e the s e n s i t i v e p e r i o d . 6. Treatments which i n f l u e n c e b e h a v i o u r a l development do so by a f f e c t i n g only one, r a r e l y two, of s e v e r a l components of a g g r e s s i v e behaviour. A l s o , treatments a p p l i e d at d i f f e r e n t stages of development i n f l u e n c e d i f f e r e n t elements of behaviour. These o b s e r v a t i o n s suggest that the s e n s i t i v e p e r i o d s a s s o c i a t e d with d i f f e r e n t elements of behaviour may be temporally separated. 7 . Hormonal i n f l u e n c e s act to shape c e r t a i n c h a r a c t e r i s t i c s (frequency or d u r a t i o n ) of s p e c i f i c components of a g g r e s s i v e behaviour. The i n f l u e n c e s produced by a p a r t i c u l a r treatment do not act e q u a l l y on a l l elements of a g g r e s s i v e behaviour. 6 5 EXPERIMENT I I I . THE EFFECTS OF METHYLTESTOSTERONE ON THE DEVELOPMENT OF FEMALE BEHAVIOUR A. INTRODUCTION T h i s experiment examines the i n f l u e n c e of androgen on the development of female behaviour by comparing the behaviour of females exposed to d i f f e r e n t e a r l y MT treatments. As i n the preceding experiment (the development of male behaviour), I expected d i f f e r e n t hormone treatments to produce d i f f e r e n t e f f e c t s on the development of behaviour. If d i f f e r e n c e s between groups were detected, then i t should be p o s s i b l e to comment on what elements of female behaviour are i n f l u e n c e d by androgen d u r i n g development, how these elements are a f f e c t e d , and whether s e n s i t i v e p e r i o d s e x i s t i n b e h a v i o u r a l d i f f e r e n t i a t i o n . In the preceding study, male behaviour was s t u d i e d by observing f i s h in predominantly male t e s t groups ( 6 males, 4 females). Males in these t e s t groups always formed ex t e n s i v e t e r r i t o r i a l s o c i e t i e s which occupied the e n t i r e tank bottom. Most of the b e h a v i o u r a l i n t e r a c t i o n s observed i n t h i s s i t u a t i o n were between males. I assumed that an e f f e c t i v e method of studying female behaviour would be to reduce the number of male-male i n t e r a c t i o n s and i n c r e a s e the number of i n t e r a c t i o n s i n v o l v i n g females. The r e f o r e I a l t e r e d the t e s t group sex r a t i o in t h i s experiment to 6 females and 2 males. Test groups were now c h a r a c t e r i z e d by a l l - f e m a l e s chools, with two males forming a small t e r r i t o r i a l s o c i e t y occupying only a smal l s e c t i o n of the tank bottom. Most of the b e h a v i o u r a l i n t e r a c t i o n s observed 66 in t h i s s i t u a t i o n i n v o l v e d females. I t was not p o s s i b l e to t e s t s i x females from group III as 58 of 60 f i s h developed as males. Group V females were not examined i n t h i s study as 6 h e a l t h y females were not a v a i l a b l e fo r t e s t i n g . B. METHODS J_. Subjects Six females from each of four treatment groups ( I , I I , IV, and VI) were used as t e s t animals. 2. Data C o l l e c t ion The b e h a v i o u r a l data presented f o r each treatment group was obtained from o b s e r v a t i o n s of s i x females in one t e s t group. Each t e s t group c o n s i s t e d of s i x t r e a t e d females and two u n t r e a t e d s t i m u l u s males maintained and t e s t e d under the standard c o n d i t i o n s d e s c r i b e d i n s e c t i o n I-C-1. 3 . Stat i s t i c a l Treatment of Data The data f o r each behaviour measure were analyzed using a K r u s k a l - W a l l i s one-way a n a l y s i s of v a r i a n c e ( ° C = 0.05; df = 3 ; S i e g e l , 1956). If a s i g n i f i c a n t d i f f e r e n c e between groups was i n d i c a t e d , then a l l p o s s i b l e p a i r s of groups were compared (K o l s t o e , 1973) using the Mann-Whitney U-test ( oc= 0.05; 2-t a i l e d t e s t , S i e g e l , 1956). 6 7 C. RESULTS J_. T i l t s Received by Females The mean frequency of t i l t s r e c e i v e d by females i s lowest f o r group IV f i s h , with f i s h i n other groups r e c e i v i n g at l e a s t four times as many t i l t s d u r i n g the t e s t p e r i o d . F i g u r e 6 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group IV and groups I, I I , or VI ( c o n t r o l ) . Group I and group II f i s h r e c e i v e d fewer t i l t s than group VI ( c o n t r o l ) f i s h , but these d i f f e r e n c e s were not s i g n i f i c a n t . 2 . Butts Received by Females The mean frequency of bu t t s r e c e i v e d by females i s lowest f o r group IV f i s h , with f i s h i n other groups r e c e i v i n g at l e a s t four times as many butt s d u r i n g the t e s t p e r i o d . F i g u r e 7 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group IV and groups I, I I , or VI ( c o n t r o l ) . Group I and group II f i s h r e c e i v e d fewer but t s than group VI ( c o n t r o l ) f i s h , and these d i f f e r e n c e s are a l s o s i g n i f i c a n t . Group I f i s h r e c e i v e d fewer b u t t s than group II f i s h , but t h i s d i f f e r e n c e was not s i g n i f i c a n t . 68 F i g u r e 6. T i l t s Received by Females. The Mean Frequency (+ Standard E r r o r ) of T i l t s Received from Males by Each Female During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 69 M E A N F R E Q U E N C Y TILTS R E C E I V E D BY E A C H F E M A L E I 00 - , O F 50 -A n = 6 f i s h / g r o u p 4 G R O U P U is: 37J E T O H S O L N A Q . S O L N I N DIET C O N T R O L F E R T T O R E L E A S E D A Y S 0-21 D A Y S 21-42 ure 7. Butts Received by Females. The Mean Frequency (+ Standard E r r o r ) of Butts Received from Males by Each Female During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 71 M E A N F R E Q U E N C Y O F BUTTS R E C E I V E D BY EACH F E M A L E 30 -J 20 -I 10 - | G R O U P = •V I n ET ETOH A Q . IN S O L N S O L N D I E T F E R T DAYS D A Y S T O 0-21 21 -42 R E L E A S E n = 6 f i s h / g r o u p C O N T R O L 72 D-. DISCUSSION S i g n i f i c a n t d i f f e r e n c e s e x i s t between groups i n two measures of male-female c o u r t s h i p i n t e r a c t i o n : t i l t s r e c e i v e d from males, and b u t t s r e c e i v e d from males. Group IV females (MT in d i e t , days 21-42 p o s t - r e l e a s e ) r e c e i v e fewer t i l t s and bu t t s from t e r r i t o r i a l males than females i n the c o n t r o l group (VI) or in the other treatment groups ( I : immersion, f e r t i l i z a t i o n to r e l e a s e ; I I : MT i n d i e t , days 0-21 p o s t - r e l e a s e ) . Females i n groups I and II r e c e i v e b u t t s (but not t i l t s ) from males l e s s f r e q u e n t l y than group VI ( c o n t r o l ) females and more f r e q u e n t l y than group IV females. Since females r e c e i v e t i l t s and butts, from t e r r i t o r i a l males only when they leave the school and approach a t e r r i t o r y , i t i s c l e a r that group IV females approach male t e r r i t o r i e s l e s s f r e q u e n t l y than females i n any other group. Nest approaching occurs when a female leaves a school, approaches the c l u s t e r of t e r r i t o r i e s , and b r i e f l y encounters one or more t e r r i t o r i a l males. A f t e r r e c e i v i n g t i l t s and/or b u t t s from one or more males, the female r e j o i n s the s c h o o l . Nest approaching may enable a female to assess p r o s p e c t i v e mates p r i o r to spawning and to monitor the l o c a t i o n s of d i f f e r e n t males. T h i s idea i s r e i n f o r c e d when one notes that females in a l l groups f r e q u e n t l y descend and i n t e r a c t with males even though the females are not in spawning c o n d i t i o n . Therefore, female f i s h are not p a s s i v e p a r t n e r s i n c o u r t s h i p s i n c e they a c t i v e l y seek out males and, by t h e i r presence, s t i m u l a t e males to i n i t i a t e c o u r t s h i p . Kramer (1971) a l s o observed that the 7 3 female blue gourami (T. t r i c h o p t e r u s ) t y p i c a l l y i n i t i a t e s a spawning sequence by approaching and b u t t i n g the male, which s t i m u l a t e s the male to p a r t i c i p a t e i n c o u r t s h i p . Nest approaching i s d i s t i n c t from nest v i s i t i n g (see s e c t i o n I-C-3 ) s i n c e the female r a r e l y enters a t e r r i t o r y when nest approaching. Nest approaching i s a l s o d i s t i n c t from f o r a g i n g descents s i n c e nest approaching i s a s o l o a c t i v i t y i n v o l v i n g a d i r e c t e d approach towards a t e r r i t o r y . Groups of females normally forage at a d i s t a n c e from the c l u s t e r of t e r r i t o r i e s . T h e r e f o r e , treatment IV i n h i b i t e d the development of nest approaching behaviour while treatments I, I I , and VI had l i t t l e , i f any, i n f l u e n c e on nest approaching behaviour. Low l e v e l s of nest approaching a c t i v i t y are r e f l e c t e d i n the low f r e q u e n c i e s of t i l t s and b u t t s r e c e i v e d from males. T i l t s r e c e i v e d and b u t t s r e c e i v e d scores f o r group I and group II females were intermediate between scores f o r females i n group VI ( c o n t r o l ) and group IV (lowest s c o r e s ) . Group I and group II females r e c e i v e d s i g n i f i c a n t l y fewer b u t t s (but not s i g n i f i c a n t l y fewer t i l t s ) from males than group VI ( c o n t r o l ) females. These o b s e r v a t i o n s suggest that treatments I and II a l s o i n h i b i t e d the development of nest approaching behaviour, although t h i s i n h i b i t o r y i n f l u e n c e i s l e s s c l e a r i n groups I and II than i n group VI. An a l t e r n a t e h y p o t h e s i s , that low f r e q u e n c i e s of t i l t s and b u t t s r e c e i v e d from males are due to i n h i b i t e d iocomotory a c t i v i t y in the t r e a t e d females, seems u n l i k e l y . T h i s hypothesis 74 i s r e j e c t e d s i n c e no obvious d i f f e r e n c e i n locomotory a c t i v i t y was n o t i c e d ( q u a l i t a t i v e assessment) and s i n c e no other aspect of female behaviour ( t i l t s g i ven, b u t t s g i v e n , etc.) was expressed at reduced l e v e l s ( q u a n t i t a t i v e assessment) by the group IV females. Nest approaching was s i g n i f i c a n t l y i n f l u e n c e d by treatment IV, the treatment a p p l i e d l a t e s t d u r i n g development (days 21-42 p o s t - r e l e a s e ) . T h i s o b s e r v a t i o n i s c o n s i s t e n t with a concept developed i n the preceding experiment. That i s , treatment IV produced the most s t r i k i n g i n f l u e n c e on the development of nest approaching behaviour as i t was the only treatment a p p l i e d d u r i n g the s e n s i t i v e p e r i o d a s s o c i a t e d with the development of nest approaching. Treatments I and II were a p p l i e d before t h i s s e n s i t i v e p e r i o d and produced l e s s of an e f f e c t on nest approaching behaviour (as i n d i c a t e d by lower b u t t s r e c e i v e d s c o r e s , but not lower t i l t s r e c e i v e d s c o r e s ) . It i s i n t e r e s t i n g to compare MT i n f l u e n c e s on the development of male and female behaviours. In t h i s experiment, MT acted to i n h i b i t ( m a s c u l i n i z e ? ) a female b e h a v i o u r a l c h a r a c t e r i s t i c . In experiment II (the development of male behaviour), MT enhanced the e x p r e s s i o n of male a g g r e s s i v e behaviours. These o b s e r v a t i o n s suggest that e a r l y androgenic i n f l u e n c e s i n h i b i t the development of female behaviours and s t i m u l a t e the development of male behaviours. 75 E. SUMMARY 1. B e h a v i o u r a l d i f f e r e n c e s were detected between groups i n two measures of male-female c o u r t s h i p i n t e r a c t i o n : t i l t s r e c e i v e d from males and b u t t s r e c e i v e d from males. 2. Group IV females i n t e r a c t with t e r r i t o r i a l males l e s s f r e q u e n t l y than females i n groups I, I I , or VI (as i n d i c a t e d by low t i l t s r e c e i v e d and b u t t s r e c e i v e d s c o r e s ) . T h i s e f f e c t i s l e s s pronounced i n group I or group II females as they r e c e i v e fewer b u t t s (but not fewer t i l t s ) from males than group VI ( c o n t r o l ) females. 3. The low f r e q u e n c i e s of t i l t s and b u t t s r e c e i v e d from males by the group IV females i s due to the low frequency of nest approaching behaviour d i s p l a y e d by these females. 4. Nest approaching i s the only female behaviour a f f e c t e d by MT treatment. Treatment from days 21-42 p o s t - r e l e a s e (IV) had the g r e a t e s t i n f l u e n c e on the development of nest approaching behaviour suggesting that there i s a s e n s i t i v e p e r i o d a s s o c i a t e d with the development of t h i s behaviour. Treatments I and II had l e s s of an e f f e c t on the development of nest approaching behaviour presumably because they were a p p l i e d before the s e n s i t i v e p e r i o d f o r nest approaching ( f e r t i l i z a t i o n to r e l e a s e and days 0-21 p o s t - r e l e a s e , r e s p e c t i v e l y ) . 5. A comparison of the i n f l u e n c e of MT on the development of male and female behaviours suggests that MT i n h i b i t s the development of female behaviour and s t i m u l a t e s the development of male behaviour. 7 6 EXPERIMENT IV. THE EFFECTS OF A SECOND METHYLTESTOSTERONE TREATMENT ON EARLY-TREATED FEMALES A. INTRODUCTION Baum (1979) and Beatty (1979) review mammalian s t u d i e s that i n d i c a t e t hat exposure to androgen d u r i n g development a f f e c t s female s e n s i t i v i t y to androgen l a t e r i n l i f e . In g e n e r a l , these s t u d i e s show that animals exposed to an e a r l y androgen treatment are more responsive to androgen in adulthood than animals not exposed to an e a r l y androgen treatment. T h e r e f o r e , the development of androgen-dependent mechanisms u n d e r l y i n g the expres s i o n of a d u l t behaviour appears to be i n f l u e n c e d by e a r l y androgen treatments. Lindsay (1974) observed that e a r l y - t r e a t e d female guppies (P. r e t i c u l a t a ) were more responsive to androgen as a d u l t s than female guppies not exposed to an e a r l y treatment. This study examines the hypothesis that a s i m i l a r phenomenon occurs in S. mossambicus . I assumed that a standard androgen treatment would produce d i f f e r e n t degrees of b e h a v i o u r a l m a s c u l i n i z a t i o n i n a d u l t females p o s s e s s i n g d i f f e r e n t androgen s e n s i t i v i t i e s . Furthermore, I expected d i f f e r e n c e s i n androgen s e n s i t i v i t i e s to be e a s i e s t to detect when the behaviour of females undergoing normal development ( c o n t r o l group VI) i s compared to the behaviour of females t r e a t e d with androgen dur i n g development (treatment groups I, I I , and IV). T h i s experiment i s designed to explore s e v e r a l r e l a t e d q u e s t i o n s . F i r s t , are elements of the c e n t r a l nervous system 7 7 (CNS) s e n s i t i z e d to androgenic i n f l u e n c e s encountered l a t e r i n l i f e as a r e s u l t of exposure to androgen d u r i n g development? I f so, are these CNS elements s e n s i t i z e d to androgen d u r i n g a r e s t r i c t e d p e r i o d i n development (a one to three week p e r i o d d u r i n g the f i r s t two months of l i f e ) , or at d i f f e r e n t times over a wide p e r i o d d u r i n g development (throughout the f i r s t two months of l i f e ) ? T h i s study a l s o examines whether behaviour i n g e n e r a l , or only c e r t a i n components of behaviour are a f f e c t e d by the a d u l t MT treatment. If the second MT treatment m a s c u l i n i z e s a l l behaviour p a t t e r n s , then a l l of the b e h a v i o u r - c o n t r o l l i n g CNS elements have been s e n s i t i z e d by the f i r s t MT treatment. However, i f the second MT treatment m a s c u l i n i z e s only some components of behaviour, then presumably only some elements of the CNS have been s e n s i t i z e d by the f i r s t MT treatment. The former r e s u l t would i n d i c a t e that the neuromolecular mechanism u n d e r l y i n g a l l elements of behaviour develop d u r i n g the same p e r i o d of development. The l a t t e r r e s u l t would i n d i c a t e that the neuromolecular mechanism u n d e r l y i n g d i f f e r e n t elements of behaviour develop at d i f f e r e n t p e r i o d s d u r i n g development. 78 B. METHODS J_. S u b j e c t s The females t e s t e d i n experiment III (see preceding chapter) were used as s u b j e c t s i n t h i s experiment. 2 . Data C o l l e c t i o n Test group composition and data c o l l e c t i o n procedures are the same as i n experiment I I I . 3^ . Treatment A d m i n i s t r a t i o n The l a t e MT treatment a p p l i e d to f i s h i n groups I, II,IV and VI i s i d e n t i c a l to the treatment a p p l i e d to group V f i s h i n experiment I ( 6 0 jug/gm of d i e t f o r 40 days when mature). Stock s o l u t i o n s and batches of hormone t r e a t e d food were prepared, s t o r e d , and administered as d e s c r i b e d i n s e c t i o n I'I-A-5. F i s h i n each group were exposed to the second MT treatment approximately 2 0 months a f t e r the f i r s t MT treatment. _4. S t a t i s t i c a l Treatment of Data Data a n a l y s i s was conducted i n two st e p s . Step one was designed to detec t s i g n i f i c a n t b e h a v i o u r a l d i f f e r e n c e s between groups and the procedure used i s the same as o u t l i n e d in experiment .111 . Step two compares the b e h a v i o u r a l data obtained in experiment III to the data obtained i n experiment IV i n a 79 s e r i e s of Wilcoxon Matched-Pairs Signed-Ranks t e s t s ( ° C = .025; 1 - t a i l e d t e s t ; S i e g e l , 1956). T h i s step determines i f behaviour scores o btained i n experiment IV i n c r e a s e d or decreased from experiment III l e v e l s under the i n f l u e n c e of the l a t e hormone treatment. T h i s a n a l y s i s a l s o i n d i c a t e s whether s i g n i f i c a n t changes i n behaviour scores occur i n a l l treatment groups, or were r e s t r i c t e d to c e r t a i n groups. C. RESULTS J_. Body C o l o u r a t i o n The l a t e MT treatment induced dark body c o l o u r a t i o n (a male secondary sexual c h a r a c t e r i s t i c ; see s e c t i o n II-C-6) i n a l l e a r l y - t r e a t e d females (groups I, II and IV) but not i n c o n t r o l females (group VI; see Table V I ) . Furthermore, dark body c o l o u r a t i o n was adopted at l e a s t three times more o f t e n by group I females than by females i n e i t h e r group II or group IV. 2. T i l t s Given by Females T i l t s are normally d i s p l a y e d by c o u r t i n g males to females and are r a r e l y e x h i b i t e d by untreated females. Group VI ( c o n t r o l ) females d i d not d i s p l a y t i l t s d uring the t e s t p e r i o d whereas t r e a t e d females i n groups I, I I , and IV d i d d i s p l a y t i l t s to other f i s h . F i g u r e 8 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group VI ( c o n t r o l ) and groups I and I I . Although group IV females appear to t i l t more f r e q u e n t l y 8 0 Table VI. Female Body C o l o u r a t i o n . The Number of Females Adopting Dark Body C o l o u r a t i o n Each Day of a 40 Day Hormone Treatment. Group Total I II IV V * VI 3 3 1 2 4 4 2 3 3 3 4 5 4 6 6 4 1 3 _ 5 5 2 4 3 1 4 5 3 6 4 4 4 _ _ 5 2 1 1 4 4 5 4 4 5 1 1 1 2 2 2 2 1 3 3 94 26 29 20 0 5 i 10 i t i t 15 20 25 30 Days of Hormone Treatment i 35 40 Notes: indicates no dark females were observed that day. The group V data presented here was obtained from 6 control females exposed to the late treatment in experiment II. Note that the social environment in experiment II (6 males, 4 females in each test group) is quite different from the social environment in experiment III (2 males, 6 females in each test group), indicating body colouration is regulated by social factors as well as endocrine factors. oo 8 2 than group I and group VI females, these d i f f e r e n c e s are not s i g n i f i c a n t . The r e l a t i v e l y h i g h mean frequency of t i l t s given by group IV females r e s u l t s from one f i s h performing an unu s u a l l y l a r g e number of t i l t s (x = 7 7 t i l t s g i v e n ) . 3 . L a t e r a l D i s p l a y s Given by Females L a t e r a l d i s p l a y s are normally an aspect of male a g g r e s s i v e behaviour and are r a r e l y e x h i b i t e d by untreated females. The mean frequency of l a t e r a l ' d i s p l a y s given i s lowest f o r group VI ( c o n t r o l ) f i s h . F i g u r e 9 i n d i c a t e s that a s i g n i f i c a n t d i f f e r e n c e e x i s t s between group VI ( c o n t r o l ) and group I. Group II and group IV females appear to e x h i b i t l a t e r a l d i s p l a y s more f r e q u e n t l y than group VI ( c o n t r o l ) females, but these d i f f e r e n c e s are not s i g n i f i c a n t . 4. Nest V i s i t i n g The mean d u r a t i o n of each female v i s i t to a male's nest was g r e a t l y extended by the l a t e treatment in e a r l y - t r e a t e d females. Females i n groups I, I I , and IV would darken, enter a male's t e r r i t o r y , and r e s t on the nest bottom f o r unusually long p e r i o d s of time. F i g u r e 10 i n d i c a t e s that the mean d u r a t i o n of nest v i s i t i n g a c t i v i t y f o r females i n groups I, I I , and IV i s much gr e a t e r than the mean d u r a t i o n for group VI ( c o n t r o l ) females. F i g u r e 10 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group VI ( c o n t r o l ) and e i t h e r group I or group I I . A s i g n i f i c a n t d i f f e r e n c e a l s o e x i s t s between group I and group IV. 83 F i g u r e 8. T i l t s Given by Females. The Mean Frequency (+ Standard E r r o r ) of T i l t s Given by Each Female During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 84 M E A N F R E Q U E N C Y O F TILTS G I V E N BY E A C H F E M A L E 3 0 2 0 -1 1 0 n = 6 f i s h / group G R O U P E T O H S O L N JX A Q S O L N I E I N D I E T C O N T R O L F E R T . T O R E L E A S E D A Y S 0 - 2 1 D A Y S 2 1 - 4 2 8 5 F i g u r e 9. L a t e r a l D i s p l a y s Given by Females. The Mean Frequency (+. Standard E r r o r ) of L a t e r a l D i s p l a y s Given by Each Female During a 16 Day Test P e r i o d . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 86 M E A N F R E Q U E N C Y O F L A T E R A L D I S P L A Y S G I V E N B Y E A C H F E M A L E 1 5 1 0 - \ n= 6 f ish/group G R O U P I E 37J E T O H S O L N A Q : S O L N I N D I E T C O N T R O L F E R T . T O R E L E A S E D A Y S 0 - 2 1 D A Y S 2 1 - 4 2 87 S i g n i f i c a n t d i f f e r e n c e s do not e x i s t between group IV and e i t h e r group VI ( c o n t r o l ) or group I I . 5. L a t e r a l P i sp l a y s Received by Females f rom Males The mean nest v i s i t i n g scores f o r females can be ranked in the f o l l o w i n g order: I > II > IV > VI (see F i g u r e 10). The same ranking of group scores, and a s i m i l a r p a t t e r n of d i f f e r e n c e s between group scores, i s observed i n the measure of the mean number of l a t e r a l d i s p l a y s r e c e i v e d by females from males. F i g u r e 11 i n d i c a t e s that s i g n i f i c a n t d i f f e r e n c e s e x i s t between group I and e i t h e r group IV or group VI ( c o n t r o l ) . These f i n d i n g s suggest that darkened females were regarded as i n t r u d e r males by r e s i d e n t males, and not as females, s i n c e darkened females r e c e i v e d an i n c r e a s e d number of a g g r e s s i v e s i g n a l s ( l a t e r a l d i s p l a y s ) while c o u r t s h i p ( t i l t s r e c e i v e d ) d i d not i n c r e a s e . G e n e r a l l y , the grea t e r the d u r a t i o n of female v i s i t s to male nests, the gr e a t e r the number of l a t e r a l d i s p l a y s r e c e i v e d from males. 6. Androgen Induced Changes i n Female Behaviour The b e h a v i o u r a l data c o l l e c t e d f o r each group of f i s h i n . t h i s experiment (IV) was compared to the b e h a v i o u r a l data c o l l e c t e d f o r the same f i s h i n experiment I I I . Table VII summarizes the data a n a l y s i s and i n d i c a t e s which elements of behaviour, i n which groups, were a f f e c t e d by the second MT treatment. A l l other elements of behaviour (see s e c t i o n IT-C-7), 88 F i g u r e 10. Nest V i s i t i n g by Females. The Mean Duration (+ Standard E r r o r ) of Nest V i s i t i n g A c t i v i t y E x h i b i t e d by Each Female During a 16 Day Test P e r i o d . Note: A s i g n i f i c a n t d i f f e r e n c e does not e x i s t between II and IV i n t h i s f i g u r e . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 89 M E A N DURATION O F N E S T VISITING BY E A C H F E M A L E (in seconds) 1000 500 H n = 6 f ish j group G R O U P I n E T O H AQ. S O L N S O L N F E R T . DAYS TO 0-21 R E L E A S E m IN DIET DAYS 21-42 i n C O N T R O L 9 0 F i g u r e 11. L a t e r a l D i s p l a y s Received by Females. The Mean Frequency (+ Standard E r r o r ) of L a t e r a l D i s p l a y s Received from Males by Females During a 16 Day Test P e r i o d . Note: A s i g n i f i c a n t d i f f e r e n c e does not e x i s t between II and IV i n t h i s f i g u r e . Data bar shading p a t t e r n s are e x p l a i n e d on page 43. 9 1 M E A N F R E Q U E N C Y O F L A T E R A L D I S P L A Y S R E C E I V E D BY E A C H F E M A L E 10 H 5 A n = 6 f ish / group GROUP E T O H S O L N u A Q . S O L N T5T IN DIET C O N T R O L F E RT. TO R E L E A S E D A Y S 0 - 2 1 DAYS 21 - 4 2 9 2 f o r a l l other groups, were d i s p l a y e d at s i m i l a r l e v e l s i n both experiments. The second MT treatment had the g r e a t e s t i n f l u e n c e on group I females ( a f f e c t i n g 4 behaviour measures), a l e s s e r i n f l u e n c e on group II females ( a f f e c t i n g 3 behaviour measures), and had no i n f l u e n c e on group IV or group VI females. A l l of the observed changes i n behaviour represent i n c r e a s e d m a s c u l i n i z a t i o n of behaviour. 9 3 Table V I I . A Summary of Androgen Induced Changes i n Female Behaviour. Be h a v i o u r a l Data from Experiment III Compared to Beh a v i o u r a l Data from Experiment IV. Behaviour Measure Group(s) Affected Experiment III - Experiment IV D i r e c t i o n Score (x ± S.E.) Score (x ± S.E.) of Change L a t e r a l Displays Given by Females 1.2 ± 1.2 11.8 ±.3.8 increase < 0.025 T i l t s Given by Females I II 0 0 .6.2 ± 2.8 21.2 ± 12.0 increase increase < 0.025 < 0.025 Female Nest V i s i t i n g Ac t i v i t y I II 32.8 ± 15.2 24.3 ± 16.0 800.8 ± 233.1 485.5 ± 81.3 increase increase < 0.025 < 0.025 L a t e r a l Displays Received by Females 2.7 + 1.3 7.8 ± 1.4 increase < 0.025 Notes: * - based on Wilcoxon Matched-Pairs Signed-Ranks test (Siegel, 1956), 1-t a i l e d f o r l a t e treatment > early treatment. 9 5 D. DISCUSSION The r e s u l t s of t h i s experiment i n d i c a t e t hat e a r l y androgen treatments s e n s i t i z e female f i s h to a second androgen treatment adm i n i s t e r e d l a t e r i n l i f e . A l s o , d i f f e r e n t e a r l y androgen treatments appear to i n f l u e n c e the development of hormone-s e n s i t i v e b e h a v i o u r a l mechanisms to d i f f e r e n t degrees, s i n c e groups were m a s c u l i n i z e d to d i f f e r e n t degrees by the same l a t e MT treatment. Responsiveness to the second MT treatment by females i n the v a r i o u s groups can be . ranked i n the f o l l o w i n g manner: I>II>IV>VI. Group I (immersion, f e r t i l i z a t i o n to r e l e a s e ) females were more responsive to the l a t e androgen treatment than females i n any other group, i n d i c a t i n g these f i s h were h i g h l y s e n s i t i z e d to androgen d u r i n g development. Group I females respond to the l a t e treatment by adopting male c o l o u r a t i o n , u t i l i z i n g elements of male behaviour ( t i l t s given and l a t e r a l d i s p l a y s g i v e n ) , and v i s i t i n g male nests at higher l e v e l s than females in group IV and/or group VI. Group II females ( t r e a t e d by immersion on days 0 - 2 1 p o s t -r e l e a s e ) were i n f l u e n c e d by the l a t e treatment to a l e s s e r degree than group I females, but to a greater degree than group VI females. Group II females responded to the l a t e treatment by adopting male c o l o u r a t i o n , t i l t i n g and v i s i t i n g male nests at higher i n t e n s i t i e s than group VI ( c o n t r o l ) f i s h . Thus, while treatment II d i d not i n f l u e n c e gonadal development (see experiment I ) , i t d i d i n f l u e n c e b e h a v i o u r a l development by s e n s i t i z i n g an androgen-dependent mechanism u n d e r l y i n g the 9 6 e x p r e s s i o n of behaviour. Group IV females (MT i n d i e t , days 21-42 p o s t - r e l e a s e ) were i n f l u e n c e d by the l a t e treatment to a l e s s e r degree than group II females, but to a g r e a t e r degree than group VI females. Group IV females responded to the l a t e treatment by adopting male c o l o u r a t i o n more f r e q u e n t l y than group VI f i s h . The r e s u l t s of t h i s experiment i n d i c a t e that f i s h are s e n s i t i z e d to androgen e a r l y i n development, with the degree of s e n s i t i z a t i o n d e c r e a s i n g as development progresses (I>II>IV>VI). T h i s o b s e r v a t i o n suggests that s e n s i t i z a t i o n does not occur duri n g a short-term c r i t i c a l p e r i o d i n development. Rather, i t appears that elements of the developing CNS can be s e n s i t i z e d at d i f f e r e n t times dur i n g the f i r s t two months of l i f e . I t i s a l s o i n t e r e s t i n g to note that f i s h are s e n s i t i z e d p r i m a r i l y d u r i n g the p e r i o d i n which the hormonal mechanism u n d e r l y i n g male behaviours a p p a r e n t l y develops (see experiment I I : the development of male behaviour i s a f f e c t e d by MT between f e r t i l i z a t i o n to day 21 p o s t - r e l e a s e ) . Only the mechanism u n d e r l y i n g body c o l o u r a t i o n i s s e n s i t i z e d to androgen d u r i n g the p e r i o d i n which female behaviour ap p a r e n t l y develops (see experiment I I I : the development of female behaviour i s a f f e c t e d by MT between days 21-42 p o s t - r e l e a s e ) . T h i s o b s e r v a t i o n may be a p r e l i m i n a r y i n d i c a t i o n that the developing CNS becomes l e s s s e n s i t i v e to androgen as development progresses (perhaps female b e h a v i o u r a l elements need to develop f r ee of androgen). T h e r e f o r e , the p o s s i b i l i t y e x i s t s that male and female behaviours (and a s s o c i a t e d CNS elements) develop at d i f f e r e n t 97 times. A comparison of the b e h a v i o u r a l data c o l l e c t e d i n experiments III ( e f f e c t s of e a r l y androgen treatment on female behaviour) and IV ( e f f e c t s of a second androgen treatment on the behaviour of e a r l y - t r e a t e d females) i n d i c a t e s that the l a t e MT treatment does not completely m a s c u l i n i z e female behaviour. Only a few male c h a r a c t e r i s t i c s were adopted by the t r e a t e d females (group I: t i l t s given, l a t e r a l d i s p l a y s g i v e n , nest v i s i t i n g ; group I I : t i l t s given, nest v i s i t i n g ; groups IV and VI: no changes in b e h a v i o u r ) . These o b s e r v a t i o n s imply that d i f f e r e n t elements of behaviour can be s e n s i t i z e d to androgen at d i f f e r e n t times d u r i n g development. For example, treatments I and II were a p p l i e d d u r i n g the p e r i o d when the CNS elements r e g u l a t i n g t i l t i n g c o u l d be i n f l u e n c e d ( s e n s i t i z e d ) by hormone. Treatment IV was a p p l i e d a f t e r t h i s s e n s i t i v e p e r i o d and CNS elements r e g u l a t i n g t i l t i n g were not s e n s i t i z e d to androgen by t h i s treatment. Lanzing and Bower (1974) and N e i l (1964) have s t u d i e d the f a c t o r s r e s p o n s i b l e f o r dark body c o l o u r a t i o n i n S. mossambicus and noted that the melanophores are h e a v i l y i n n e r v a t e d . A l s o , changes i n body c o l o u r a t i o n are r a p i d and f a d i n g or darkening can occur w i t h i n seconds. These o b s e r v a t i o n s suggest that dark body c o l o u r a t i o n i s c o n t r o l l e d by the nervous system and that a l l of the e a r l y treatments ( I , I I , and IV) i n f l u e n c e d the development of nervous elements r e g u l a t i n g dark body c o l o u r a t i o n . Note however, that c o u r t s h i p c o l o u r a t i o n i s a secondary sexual c h a r a c t e r i s t i c which can be e l i m i n a t e d by 98 castration (see reviews by Baggerman, 1968; de Vlaming, 1974; Hoar, 1969, 1965, and 1962; and Yamamoto, 1968), suggesting colouration i s also influenced by gonadal hormone l e v e l s . Dark body colouration also appears to be regulated by s o c i a l factors. Two groups of previously untreated females (n = 6 fish/group) received the same late MT treatment while f i s h in each group were in d i f f e r e n t s o c i a l environments (see Table VI; compare group V* with group VI). Treated females in group V* adopted dark colouration more frequently than females in group VI. At present, while i t i s not clear how nervous, hormonal, and so c i a l factors interact to regulate body colouration (see various hypotheses reviewed by F u j i i , 1969), i t is clear that the early MT treatments e f f e c t i v e l y sensitized the mechanism regulating body colouration to later androgenic influences. 9 9 E. SUMMARY 1. E a r l y androgen treatments can s e n s i t i z e female f i s h to androgen l a t e r i n l i f e . 2 . Androgen s e n s i t i v i t y was assessed by a d m i n i s t e r i n g the same l a t e MT treatment to groups of e a r l y - t r e a t e d females and then o b s e r v i n g the degree of b e h a v i o u r a l m a s c u l i n i z a t i o n produced i n females i n each group. D i f f e r e n t e a r l y androgen treatments produced d i f f e r e n t degrees of androgen s e n s i t i v i t y i n female f i s h (I>II>IV>VI). 3 . Group I females were very responsive to the l a t e androgen treatment as they adopted male c o l o u r a t i o n , male behaviour ( t i l t s g i ven, l a t e r a l d i s p l a y s given, and nest v i s i t i n g ) , more f r e q u e n t l y than females i n group IV and/or group VI ( c o n t r o l ) . 4 . Group II females were more responsive to the l a t e androgen treatment than group VI ( c o n t r o l ) females. Group II females adopt male c o l o u r a t i o n and male behaviour ( t i l t s g iven, nest v i s i t i n g ) more f r e q u e n t l y than group VI ( c o n t r o l ) females. 5 . Group IV females adopt male c o l o u r a t i o n , but not male behaviours, more f r e q u e n t l y than group VI ( c o n t r o l ) females. 6 . Group VI ( c o n t r o l ) females were not a f f e c t e d by the l a t e MT treatment. 7. Female f i s h appear to be s e n s i t i z e d to androgen by treatments (I and II) a d m i n i s t e r e d before day 21 p o s t - r e l e a s e . The behaviour of female f i s h exposed to an e a r l y androgen treatment (IV) administered a f t e r day 21 p o s t - r e l e a s e was not a f f e c t e d by the l a t e MT treatment. Thus, s e n s i t i z a t i o n to 100 androgen appears to occur throughout a prolonged p e r i o d in the f i r s t two months of l i f e , r a t h e r than d u r i n g a s p e c i f i c s h o r t -term (one to three week) p e r i o d i n development. 8. The developing CNS appears to be most s e n s i t i v e to androgenic i n f l u e n c e s d u r i n g the f i r s t month of l i f e . The CNS a l s o appears to become l e s s s e n s i t i v e to androgen as development p r o g r e s s e s . 101 EXPERIMENT V. THE BEHAVIOUR OF SEX-REVERSED FEMALES A. INTRODUCTION T h i s experiment was designed to dete c t female f i s h that were sex-reversed by an e a r l y hormone treatment and to compare the behaviour of sex-reversed g e n e t i c females to the behaviour of u n t r eated genetic males. Sex-reversed f i s h were detected by breeding t r e a t e d "males" (genetic males and g e n e t i c females) with u n treated females. Crosses between two g e n e t i c females should produce only female o f f s p r i n g (Clemens and I n s l e e , 1968), while c r o s s e s between a genetic male and a g e n e t i c female should produce roughly equal numbers of male and female o f f s p r i n g . B. METHODS J_. S u b j e c t s A f t e r o b s e r v a t i o n s of behaviour i n Experiment I I , a male was p a i r e d with an untreated female i n an 82 l i t e r p l e x i g l a s s tank (21 x 60 x 65 cm). Each female was checked twice d a i l y (0900 and 1600 hours) f o r signs of mouthbrooding a c t i v i t y . Young were removed from the female a f t e r twelve days of mouthbrooding, or e a r l i e r i f the female v o l u n t a r i l y e j e c t e d the young. Fry were p l a c e d i n r e a r i n g tanks stocked at one f i s h per l i t e r of tank water. Rearing tank volumes ranged from 20 to 40 l i t e r s . I t was not p o s s i b l e to breed a l l of the t e s t e d males due to l i m i t a t i o n s imposed by the numbers of breeding and r e a r i n g tanks 1 02 a v a i l a b l e at any one time. N e v e r t h e l e s s , at l e a s t s i x males from each treatment group were bred. 2. Data C o l l e c t ion Breeding records were maintained f o r each f i s h used i n t h i s study. The date of spawning, d u r a t i o n of mouthbrooding, number of young produced, and egg and young m o r t a l i t i e s were recorded. A sex r a t i o was determined f o r the o f f s p r i n g produced i n each c r o s s by sexing a l l s u r v i v i n g young between the ages of 35 to 75 days p o s t - r e l e a s e (as d e s c r i b e d in s e c t i o n I I I - B - 2 ) . B e h a v i o u r a l data f o r each f i s h bred i n t h i s experiment was obtained from the behaviour records prepared i n Experiment I I . 3. S t a t i s t i c a l Treatment of Data Behaviour data f o r sex-reversed f i s h (n = 5) were compared to the data c o l l e c t e d f o r c o n t r o l males (n = 18) in a s e r i e s of Mann-Whitney U - t e s t s ( « = 0.05; 2 - t a i l e d t e s t ; S i e g e l , 1 9 5 6 ) . If s i g n i f i c a n t d i f f e r e n c e s were detected, two a d d i t i o n a l comparisons were made to determine i f b e h a v i o u r a l d i f f e r e n c e s were r e s t r i c t e d to sex-reversed f i s h or were common to a l l f i s h exposed to the same e a r l y hormone treatment. A second s e r i e s of Mann-Whitney U - t e s t s (°C = 0.05; 2 - t a i l e d t e s t ; S i e g e l , 1956) compared the behaviour data f o r non sex-reversed f i s h ( i n groups I and I I I ) to the scores of c o n t r o l males (group V I ) . If s i g n i f i c a n t d i f f e r e n c e s were detected, then the behaviour of both sex-reversed and non sex-reversed f i s h 1 0 3 d i f f e r s from the behaviour of c o n t r o l f i s h . Such a f i n d i n g would i n d i c a t e a general treatment e f f e c t on a l l e a r l y - t r e a t e d f i s h i n groups I and,III. I f s i g n i f i c a n t d i f f e r e n c e s were not d e t e c t e d between non sex-reversed f i s h and the c o n t r o l s , then behaviour d i f f e r e n c e s would be l i m i t e d to sex-reversed f i s h . A t h i r d s e r i e s of Mann-Whitney U - t e s t s ( ° C = 0 . 0 5 ; 2 - t a i l e d t e s t ; S i e g e l , 1 9 5 6 ) compared the scores of sex-reversed f i s h with the scores of non sex-reversed f i s h i n groups I and I I I . I f s i g n i f i c a n t d i f f e r e n c e s were de t e c t e d , then the behaviour of sex-reversed f i s h d i f f e r s from the behaviour of non sex-reversed f i s h i n the same treatment groups. 1 04 C. RESULTS AND DISCUSSION K Detect ion of Sex-Reversed Females An examination of Table VIII i n d i c a t e s that f i v e f i s h from groups I and III produced o f f s p r i n g with predominantly female sex r a t i o s . These f i s h are assumed to be sex-reversed g e n e t i c females. Although group IV d e v i a t e s from an expected 1:1 sex r a t i o (see Table IV), suggesting gonadal development was a f f e c t e d i n a small number of f i s h , no sex-reversed f i s h were det e c t e d i n group IV. 2. Morphology of Sex-Reversed Females A comparison of the sex-reversed f i s h with c o n t r o l males (group VI) i n d i c a t e s that sex-reversed f i s h are m o r p h o l o g i c a l l y i d e n t i c a l to c o n t r o l males. 3_. Behaviour of Sex-Reversed Females Comparisons between c o n t r o l male f i s h (n = 18) and sex-reve r s e d f i s h (n = 5 ) i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s in four behaviour measures: i n t e r - t e r r i t o r i a l f i g h t i n g , a t t a c k i n g , d i g g i n g , and t i l t s given to females. The mean score f o r sex-reve r s e d f i s h i s at l e a s t twice as high as the mean score f o r c o n t r o l f i s h in each measure (see Table IX). A comparison of c o n t r o l (group VI.) scores with the scores of non sex-reversed f i s h (groups I and I I I ) i n d i c a t e s that non 1 0 5 Table V I I I . A Summary of Breedings Performed and Sex R a t i o s Obtained i n a S e r i e s of Crosses I n v o l v i n g Males from V a r i o u s Treatment Groups. 106 Breeding Number Females Males M o r t a l i t y Sex Pair Sexed Ratio IC6 x £ 4 7 62 61 IB7 x ? 2 7 40 39 IB9 x £21 61 .59 IIIB4 x ?20 51 49 IB3 x ?73 104 93 IIIA2 x $3 160 128 IIB2 x $ 62 30 20 IIIB5 x ? 3 134 86 IIA7 x ? 70 70 40 VC4 x ?62 72 40 VIB23 x ?23 100 51 IIIB1 x ? 3 50 25 VIC3 x ?76 36 18 VIA3 x £ 70 98 49 VIC5 x ?3 56 27 VC1 x ? 6 4 52 25 IIA1 x $84 56 25 IVB1 x ?20 121 53 IB1 x ? 7 0 57 32 IIB2 x £62 72 - 41 VB3 x $24 63 36 IB2 x ?45 70 30 IIC3 x ?23 92 39 IIB4 x $ 3 78 32 VA3 x £ 3 97 39 VIB2 x ?38 55 22 IVA5 x$85 48 •19 VB7 x £ 3 100 37 VA10 x £3 100 37 1 18 61 : 1 1 0 39 : 1 2 8 29.5 : 1 2 0 24.5 : 1 4 7 23.2 : 1 32 19 4 : 1 10 0 2 : 1 48 0 1-7 : 1 30 0 1.33 : 1 32 26 1.25 : 1 49 ' 44 1.04 : 1 25 36 1 1 18 0 1 . 1 49 1 1 1 29 10 0.99 1 27 7 0.92 1 31 0 0.80 1 68. 0 0.78 1 25 5 0.78 1 31 11 0.76 1 27 55 0.75 1 40 19 0.75 1 53 21 0.74 1 46 10 0.69 : 1 58 32 0.67 : 1 33 6 0.67 : 1 29 0 0.65 : 1 63 1 - 0.6 : 1 63 30 0.6 : 1 107 Breeding Number Females Males M o r t a l i t y Sex Pa i r Sexed Ratio IV A4 x £ 7 6 35 13 IIIB6 x £59 100 36 IIIB2 x ?34 42 14 VIA2 x $68 36 11 IIIC5 x $62 100 63 IVA7 x $20 20 5 22 1 0.59 : 1 64 20 0.56 : 1 28 1 0.5 : 1 25 2 0.44 : 1 27 14 0.43 : 1 15 1 0.33 : 1 Notes: Roman numeral indicates treatment group. A, B, or C indicates Experiment II te s t group. 108 Table IX. Comparisons of C o n t r o l F i s h (Group VI) with Sex-Reversed F i s h (Groups I and I I I ) . 109 Behaviour Measure Control Sex-Reversed p * Fish Fish (n = 18) (n = 5) Inter-Territorial Duration Fighting (in seconds) A t t a c k i n g Frequency D i g g i n g Frequency T i l t s Given Frequency to Females Notes: * - based on Mann-Whitney U-test (c<= 0.05; Siegel, 1956), 2-tailed for "X control ^ sex-reversed, a — mean b - range 5.7 25.2 < 0.05 b 0 - 46 1 - 54 1.1 0 - 5 11.8 2 - 41 < 0.05 23.3 0 - 117-113.2 19 - 258 < 0.05 24.6 0 - 145 69.4 17 - 140 < 0.05 1 10 sex-reversed f i s h engage i n i n t e r - t e r r i t o r i a l f i g h t i n g and a t t a c k i n g at s i g n i f i c a n t l y higher l e v e l s than c o n t r o l f i s h (see Table X). T h e r e f o r e , two of the four b e h a v i o u r a l d i f f e r e n c e s d e t e c t e d between c o n t r o l f i s h and sex-reversed f i s h ( i n t e r -t e r r i t o r i a l f i g h t i n g and a t t a c k i n g ) appear to be due to a general e f f e c t produced by treatments I and III on a l l t r e a t e d f i s h while two be h a v i o u r a l d i f f e r e n c e s ( d i g g i n g and t i l t i n g to females) appear to be r e s t r i c t e d to sex-reversed f i s h . I f t r u e , then I expected to detect s i g n i f i c a n t d i f f e r e n c e s when the d i g g i n g and t i l t i n g to females scores of sex-reversed and non sex-reversed f i s h were compared. However, Table XI i n d i c a t e s that no s i g n i f i c a n t d i f f e r e n c e s were det e c t e d between sex-reversed and non sex-reversed f i s h i n groups I and I I I . Therefore, high d i g g i n g and t i l t i n g to female scores (compared to c o n t r o l s ) should not be regarded as b e h a v i o u r a l c h a r a c t e r i s t i c s l i m i t e d to sex-reversed f i s h . T h i s c o n c l u s i o n i m p l i e s that treatments I and III i n f l u e n c e the development of male behaviour i n a s i m i l a r manner i n sex-reversed and non sex-reversed f i s h . 111 Table X. Comparisons of C o n t r o l F i s h (Group VI) with Non Sex-Reversed F i s h (Groups I and I I I ) . 1 1 2 Behaviour Measure Control Non Sex-Reversed p * Fis h (n = 18) Fis h (n = 8) I n t e r - T e r r i t o r i a l Duration 5.7 14.0 < 0.05 Fighting ( i n seconds) 0 - 4 6 0 - 2 6 A t t a c k i n g Frequency 1.1 0 - 5 8.8 < 0.05 0 - 2 6 D i g g i n g . Frequency 23.3 0 - 117 53.5 > 0.05 (NS) 0 - 127 T i l t s Given to Females Frequency 24.6 0 - 145 ;42.0 > 0.05 (NS) 0 ^ 85 Notes: * - based on Mann-Whitney U-test (»C= 0-05; S i e g e l , 1956), 2 - t a i l e d for non sex-reversed ^ c o n t r o l . a — mean b - range 1 1 3 Table XI. Comparisons of Sex-Reversed with Non Sex-Reversed F i s h (Groups I and I I I ) . 114 Behaviour Measure Sex-Reversed Non Sex-Reversed p * F i s h F i s h (n =5) (n = 8) I n t e r - T e r r i t o r i a l F i g h t i n g Duration ( i n seconds) 25.2 d 1 - 54 14.0 >0.05 (NS) 0 - 2 6 A t t a c k i n g Frequency 11.8 2 - 4 1 8.8 >0.05 (NS) 0 - 2 6 D i g g i n g Frequency 113.2 19 - 258 53.5 >0.05 (NS) 0 - 127 T i l t s Given to Females Frequency 69.4 .17 - 140 42.0 >0.05 (NS) 0 - 85 Notes: * _ based on Mann-Whitney U-test (°C= 0.05; S i e g e l , 1956), 2 - t a i l e d f o r sex-reversed / non sex-reversed. mean b - range 1 1 5 D. SUMMARY 1. F i v e sex-reversed g e n e t i c females were det e c t e d i n c r o s s e s with untreated g e n e t i c females (4 f i s h from group I, 1 f i s h from group I I I ) . 2. Sex-reversed g e n e t i c females are m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from untreated g e n e t i c males. 3. Sex-reversed females i n groups I and III d i f f e r s i g n i f i c a n t l y from c o n t r o l males i n four behaviour measures: i n t e r - t e r r i t o r i a l f i g h t i n g , a t t a c k i n g , d i g g i n g , and t i l t i n g to females. Non sex-reversed f i s h i n groups I and III d i f f e r s i g n i f i c a n t l y from c o n t r o l males in measures o f : i n t e r -t e r r i t o r i a l f i g h t i n g and a t t a c k i n g . T h i s i n d i c a t e s that treatments I and III i n f l u e n c e the development of agg r e s s i v e behaviours i n a s i m i l a r manner i n sex-reversed and non sex-reversed f i s h . 4. Since b e h a v i o u r a l d i f f e r e n c e s c o u l d not be detected between sex-reversed and non sex-reversed f i s h i n groups I and I I I , e a r l y hormone treatments appear to a f f e c t the development of male behaviours in a s i m i l a r manner in sex-reversed and non sex-reversed f i s h . High d i g g i n g and t i l t i n g to female scores d i d not appear to be r e s t r i c t e d to sex-reversed f i s h . 1 16 IV. DISCUSSION A. THE EFFECTS OF METHYLTESTOSTERONE ON GONADAL DIFFERENTIATION J_. S e n s i t ive Per iods in Gonadal Development Experiment I examined the e f f e c t s of MT on gonadal d i f f e r e n t i a t i o n and i n d i c a t e d that e a r l y androgen treatments can i n f l u e n c e gonadal d i f f e r e n t i a t i o n at d i f f e r e n t stages of development. Immersion treatment I ( f e r t i l i z a t i o n to r e l e a s e ) i n f l u e n c e d gonadal d i f f e r e n t i a t i o n at an e a r l i e r stage of development than o r a l treatment III (days 0-21 p o s t - r e l e a s e ) . These o b s e r v a t i o n s i n d i c a t e t hat the developing gonad i s s e x u a l l y p l a s t i c and s e n s i t i v e to hormonal i n f l u e n c e s throughout the f i r s t two months of l i f e . Gonadal d i f f e r e n t i a t i o n does not appear to be i n f l u e n c e d by androgen treatment d u r i n g a s p e c i f i c r e s t r i c t e d p e r i o d (one to three weeks) in development. Other workers have a l s o shown that developing gonads are s e n s i t i v e to hormonal i n f l u e n c e s over a wide p e r i o d d u r i n g development. To i l l u s t r a t e , note that H i s h i d a (1964 and 1965) produced groups of sex-reversed 0. l a t i p e s by a p p l y i n g treatments to eggs immediately a f t e r f e r t i l i z a t i o n or to groups of l a r v a e at d i f f e r e n t times d u r i n g a one month p e r i o d a f t e r h a t c h i n g . Experiment I a l s o i n d i c a t e s that the e f f e c t i v e n e s s of a p a r t i c u l a r treatment in i n f l u e n c i n g gonadal development changes as the gonad ages and becomes l e s s s e n s i t i v e to hormonal 1 1 7 i n f l u e n c e s . Treatment by immersion from f e r t i l i z a t i o n to r e l e a s e f o r 12 days (I) i n f l u e n c e d gonadal development while a s i m i l a r immersion treatment a p p l i e d from days 0-21 p o s t - r e l e a s e (II) d i d not i n f l u e n c e gonadal development. Applying an o r a l treatment d u r i n g days 0-21 p o s t - r e l e a s e ( I I I ) i n f l u e n c e d gonadal development, while a p p l y i n g the i d e n t i c a l treatment dur i n g days 21-42 p o s t - r e l e a s e (IV) had l i t t l e , i f any, i n f l u e n c e on gonadal development. S i m i l a r l y , Hackmann and Reinboth (1974) determined that the gonads of Hemihaplochromis m u l t i c o l o r f r y were most s e n s i t i v e to a p a r t i c u l a r immersion treatment (0.5 mg MT/1 f o r 42-45 hours) when the treatment was a p p l i e d between days 14-16 days a f t e r h a t c h i n g . The same treatment a p p l i e d before or a f t e r t h i s p e r i o d does not i n f l u e n c e gonadal development. Reinboth's study suggests that a p a r t i c u l a r hormone treatment w i l l be most e f f e c t i v e d u r i n g a s i n g l e short-term p e r i o d and not d u r i n g s e v e r a l d i f f e r e n t p e r i o d s i n development. The preceding o b s e r v a t i o n s suggest that each hormone treatment i s a s s o c i a t e d with a short p e r i o d of e f f e c t i v e n e s s i n i n f l u e n c i n g gonadal development. A treatment a p p l i e d during t h i s p e r i o d i n f l u e n c e s gonadal development while a treatment a p p l i e d o u t s i d e t h i s p e r i o d produces l i t t l e or no i n f l u e n c e on gonadal development. The o v e r a l l p e r i o d of gonadal s e n s i t i v i t y to hormonal i n f l u e n c e s (the f i r s t two months of l i f e ) may c o n s i s t of s e v e r a l s h o r t e r p e r i o d s (a few days or weeks) duri n g which s p e c i f i c treatments can i n f l u e n c e development. Assuming d i f f e r e n t hormone treatments are most e f f e c t i v e i n i n f l u e n c i n g gonadal sex d i r e c t i o n at d i f f e r e n t stages of development, i t may 1 18 be p o s s i b l e to i n f l u e n c e sex d i r e c t i o n throughout the f i r s t two months of l i f e by manipulating treatment c h a r a c t e r i s t i c s (see sect ion I-A-3). Note that determining the p e r i o d i n which a p a r t i c u l a r treatment most e f f e c t i v e l y i n f l u e n c e s gonadal development does not n e c e s s a r i l y i n d i c a t e that the normal p e r i o d of sex d i f f e r e n t i a t i o n during development has been d e t e c t e d (as assumed by Hackmann and Reinboth, 1974). D e t e c t i o n of such a p e r i o d simply i n d i c a t e s when a p a r t i c u l a r exogenous treatment s u c c e s s f u l l y o v e r r i d e s the normal course of development and i n f l u e n c e s gonadal sex d i r e c t i o n . At present, i t i t not known when, and i f , endogenous hormone production i n f l u e n c e s gonadal d i f f e r e n t i a t i o n . 2. T h r e s h o l d L e v e l s i n Gonadal Development Immersion treatment I ( f e r t i l i z a t i o n to r e l e a s e ) produced a group of f i s h c o n s i s t i n g of 80% males and 20% females (n = 60). T h e r e f o r e , i t i s c l e a r that treatment I was not e f f e c t i v e i n i n f l u e n c i n g gonadal development i n s e v e r a l females even with prolonged treatment. However, s i n c e a number of g e n e t i c females were sex-reversed (see Table V I I I ) , treatment I e f f e c t i v e l y i n f l u e n c e d sex d i r e c t i o n in some f i s h . S i m i l a r l y , treatment III (MT i n d i e t , days 0-21 p o s t - r e l e a s e ) produced a group of f i s h c o n s i s t i n g of 97% males and 3% females. Both treatments I and III d i d not i n f l u e n c e gonadal development i n some females. The presence of sex-reversed and non sex-reversed f i s h in the same treatment group suggests that gonadal sex d i r e c t i o n in 119 developing f i s h i s r e g u l a t e d by gonadal s e n s i t i v i t y to androgen at a p a r t i c u l a r stage of development. If androgen l e v e l s surpass a s p e c i f i c t h r e s h o l d l e v e l , the gonads develop along male l i n e s . If androgen l e v e l s are below a s p e c i f i c t h r e s h o l d l e v e l , the gonads develop along female l i n e s . V a r i a b l e responses to the same treatment are l i k e l y due to a combination of the f o l l o w i n g f a c t o r s . F i r s t , d i f f e r e n t females were probably exposed to d i f f e r e n t hormone l e v e l s d u r i n g treatment ( f o r example, group III females probably ate d i f f e r e n t amounts of hormone t r e a t e d food). A l s o , g e n e t i c d i f f e r e n c e s would cause j u v e n i l e f i s h to develop at d i f f e r e n t r a t e s , r e s u l t i n g i n f i s h of the same age p o s s e s s i n g gonads with d i f f e r e n t androgen s e n s i t i v i t i e s . Treatment II (immersion, days 0-21 p o s t - r e l e a s e ) was i n e f f e c t i v e i n i n f l u e n c i n g gonadal sex d i r e c t i o n . Treatment IV (MT in d i e t , days 21-42 p o s t - r e l e a s e ) may have i n f l u e n c e d gonadal sex in only a few, i f any, f i s h (see Table I V ) . However, since sex-reversed f i s h were not d e t e c t e d i n group IV and s i n c e sex r a t i o s in Sarotherodon broods f r e q u e n t l y d e v i a t e from a 1:1 sex r a t i o (see Table V I I I ) , treatment IV i s c o n s i d e r e d to produce no e f f e c t on gonadal sex. 3_. P a r a d o x i c a l F e m i n i z a t i o n Some s t u d i e s have i d e n t i f i e d a second t h r e s h o l d l e v e l when very high doses of androgen a r e ' a d m i n i s t e r e d to developing f i s h . If a s p e c i f i c androgen treatment m a s c u l i n i z e s developing f i s h , a s i m i l a r treatment a d m i n i s t e r i n g a higher androgen dosage feminizes developing gonads ( p a r a d o x i c a l f e m i n i z a t i o n ) . For 1 20 example, Nakamura (1975) found that low doses of MT (50 /jg/gm of d i e t f o r 19 days) e f f e c t i v e l y m a s c u l i n i z e s S. mossambicus f r y , while high doses (1000 jug/gm of d i e t f o r 19 or 44 days) produces p a r a d o x i c a l f e m i n i z a t i o n (n = 3). S i m i l a r l y , Donaldson and Hunter (1982; c i t i n g a p e r s o n a l communication from V. Bye, 1980) r e p o r t e d that feeding rainbow t r o u t (S. g a i r d n e r i ) a 3 mg MT/gm of d i e t ( f o r 700 degree-days at 9-12°C) produced a l l - m a l e groups of f i s h while higher doses of MT caused p a r a d o x i c a l f e m i n i z a t i o n (the number of f i s h a f f e c t e d was not g i v e n ) . While l i t t l e i s known of the b i o c h e m i c a l processes u n d e r l y i n g the p r o d u c t i o n of an e s t r o g e n - l i k e e f f e c t from a high-dose androgen treatment in t e l e o s t s , there i s s p e c u l a t i o n that androgen i s a c t u a l l y converted to estrogen ( p o s s i b l e b i o s y n t h e t i c pathways f o r androgen conver s i o n to estrogen i n mammals are o u t l i n e d i n B r a i n , 1979). The s i g n i f i c a n c e of the above s t u d i e s i s twofold. F i r s t , support i s provided f o r the idea that hormonal i n f l u e n c e s may be dominant over g e n e t i c i n f l u e n c e s i n r e g u l a t i n g gonadal d i f f e r e n t i a t i o n . A l s o , support i s p r o v i d e d f o r the concept that gonadal sex d i r e c t i o n i s a f f e c t e d by androgen l e v e l s . If androgen l e v e l s pass a lower t h r e s h o l d l e v e l , then development i s along male l i n e s . If androgen l e v e l s are very high and above a second t h r e s h o l d l e v e l , then p a r a d o x i c a l f e m i n i z a t i o n occurs and development i s along female l i n e s . 121 4. I n f l u e n c e s of Hormone Treatment on S e x u a l l y D i f f e r e n t i a t e d  Gonads While a l a r g e number of s t u d i e s have examined the e f f e c t s of s t e r o i d s on the j u v e n i l e ( s e x u a l l y u n d i f f e r e n t i a t e d ) gonad, l i t t l e i s known of the e f f e c t s of s t e r o i d s on the a d u l t ( s e x u a l l y d i f f e r e n t i a t e d ) gonad ( B i l l a r d et a l . , 1981). In t h i s study, MT treatments (60 ^ig/gm of d i e t f o r 40 days) administered to a d u l t S. mossambicus ( i n experiment I I , group V, 6 males and 4 females; and i n experiment IV, groups I, I I , and IV, 6 males and 18 females) d i d not a f f e c t the s t r u c t u r e of the gonads in any of the f i s h . No d i f f e r e n c e s c o u l d be d e t e c t e d when gonads from t r e a t e d and untreated f i s h were compared by v i s u a l i n s p e c t i o n . These comparisons were conducted by s a c r i f i c i n g t r e a t e d f i s h and examining t h e i r gonads with a d i s s e c t i n g microscope. F i s h were s a c r i f i c e d at v a r i o u s times between 1 and 14 months a f t e r completing the hormone treatment. The s t r u c t u r e of u n t r e a t e d S. mossambicus o v a r i e s and t e s t e s are d e s c r i b e d i n Dadzie (1974 and 1969, r e s p e c t i v e l y ) . A number of s t u d i e s have shown that c e r t a i n s t e r o i d treatments a f f e c t the f u n c t i o n of the a d u l t gonad by i n h i b i t i n g gametogenesis and/or d e s t r o y i n g germ c e l l s (C. auratus or 5. qa i rdner i t r e a t e d with 4 - c h l o r o t e s t o s t e r o n e , Hirose and H i b i y a , 1968a and b, r e s p e c t i v e l y ; S. g a i r d n e r i t r e a t e d with MT, Yamazaki, 1972 and B i l l a r d et a l . , 1981; S. g a i r d n e r i t r e a t e d with e i t h e r MT, t e s t o s t e r o n e , or e s t r a d i o l \1-J3 , B i l l a r d and R i c h a r d , 1981; and Oncorhynchus gorbuscha or 0. k i s u t c h t r e a t e d with MT, Fagerlund and McBride, 1977). I n h i b i t i o n of 1 22 gametogenesis and gonadal suppression i s a l s o observed when s t e r o i d s are administered to subadult f i s h e s with s e x u a l l y d i f f e r e n t i a t e d gonads (Xiphophorus maculatus, t r e a t e d with e i t h e r pregninolone or e s t r a d i o l benzoate, Cohen, 1946; 0. gorbuscha or 0. k i s u t c h t r e a t e d with MT, Yamazaki, 1972). H i s h i d a (1965) found that low doses of l a b e l l e d estrogens (estrone or s t i l b e s t r o l ) are ex c r e t e d by 0. l a t i p e s w i t h i n a few days without a f f e c t i n g the s t r u c t u r e or f u n c t i o n of the a d u l t gonad. B i l l a r d et a l . (1981) found t h a t , even when gametogenesis i s i n h i b i t e d by a high s t e r o i d dose, the s t r u c t u r e of the gonad i s not permanently a f f e c t e d . A l s o , no cases of gonadal sex-r e v e r s a l r e s u l t i n g from s t e r o i d treatments of a d u l t f i s h have been repo r t e d ( e x c l u d i n g work performed on hermaphroditic f i s h e s ) . At best, i t has been p o s s i b l e to t e m p o r a r i l y induce o v o - t e s t i s i n a d u l t f i s h (as i n 0. l a t i p e s , see Okada, 1943). The preceding o b s e r v a t i o n s , p l u s examination of post-treatment a d u l t gonads, suggest that s t e r o i d treatments a f f e c t a d u l t gonads p r i m a r i l y by i n h i b i t i n g t h e i r f u n c t i o n (gametogenesis) r a t h e r than a f f e c t i n g t h e i r s t r u c t u r e . T h i s e f f e c t appears to be r e v e r s i b l e upon withdrawal of the hormone treatment. The e f f e c t s of treatment V on the a d u l t gonad are c o n s i s t e n t with the f i n d i n g s of s t u d i e s o u t l i n e d in the preceding paragraphs. That i s , treatment V i s con s i d e r e d to be i n e f f e c t i v e i n i n f l u e n c i n g sex d i r e c t i o n i n the a d u l t gonads as i t produced no e f f e c t (or only undetected temporary i n f l u e n c e s ) on the s t r u c t u r e of the gonad. However, based on the s t u d i e s presented above, i t i s reasonable to assume the f u n c t i o n of the gonad was i n h i b i t e d 1 23 d u r i n g treatments of a d u l t f i s h . T h i s i n h i b i t i o n of gonadal f u n c t i o n may be e x p l a i n e d by p o s t u l a t i n g that high c i r c u l a t i n g androgen l e v e l s blocked gonadotropin p r o d u c t i o n ( r e q u i r e d f o r normal gonadal f u n c t i o n ) through a negative feedback mechanism (Peter and Crim, 1979). T h i s e x p l a n a t i o n i s c o n s i s t e n t with the argument presented to e x p l a i n low a g g r e s s i v e behaviour scores i n group V f i s h (see D i s c u s s i o n , Experiment I I ) . 5. Gonadal S e n s i t i z a t i o n to Androgen C e r t a i n s t u d i e s i n d i c a t e that e a r l y hormone treatments can s e n s i t i z e gonadal t i s s u e s to subsequent hormone treatments l a t e r in l i f e . That i s , an e a r l y androgen treatment can i n f l u e n c e gonadal t i s s u e without producing a d i s c e r n i b l e e f f e c t on development. Then, when a subsequent hormone treatment i s a p p l i e d the gonad i s found to be very s e n s i t i v e to hormonal i n f l u e n c e s . T h i s concept i s supported by Goetz et a l . (1979) who determined that o r a l a d m i n i s t r a t i o n s of MT (20 pg/gm d i e t f o r 10 weeks) to coho salmon (0. ki sutch) produce a more profound e f f e c t on gonadal d i f f e r e n t i a t i o n i f the developing f i s h has been p r e v i o u s l y immersed i n a MT s o l u t i o n (during the egg and eyed a l e v i n stages; 25-400 vq MT/1). A d m i n i s t e r i n g MT v i a the d i e t i n the l a t e r j u v e n i l e stages only, without p r i o r immersion in MT, produces l e s s of an e f f e c t on gonadal d i f f e r e n t i a t i o n . T h i s i m p l i e s that l a t e r developmental stages were s e n s i t i z e d to MT by the e a r l i e r MT immersion. T h i s i n f o r m a t i o n i s presented to i l l u s t r a t e that a s i m i l a r phenomenon occurs in both gonadal and be h a v i o u r a l development (see experiment IV, the e f f e c t s of a 124 second androgen treatment on the behaviour of e a r l y - t r e a t e d females). In both cases, e a r l y androgen treatments sensit'ize f i s h to androgen treatments a d m i n i s t e r e d l a t e r in l i f e . 6. Summary Gonadal development i n S. mossambicus has the f o l l o w i n g c h a r a c t e r i s t i c s : 1) the developing gonad i s s e x u a l l y p l a s t i c and s e n s i t i v e to androgen throughout the f i r s t two months of l i f e ; 2) gonadal sex d i r e c t i o n appears to be determined by androgen l e v e l s with respect to gonadal s e n s i t i v i t y to androgen at a p a r t i c u l a r stage of development; 3) gonadal s e n s i t i v i t y to androgen appears to decrease with age, si n c e e f f e c t i v e androgen treatments (I and III) were l e s s e f f e c t i v e in i n f l u e n c i n g sex d i r e c t i o n when administered l a t e r i n development (II and IV, r e s p e c t i v e l y ) ; 4) hormone treatments can a f f e c t gonadal sex d i r e c t i o n only in immature, s e x u a l l y u n d i f f e r e n t i a t e d f i s h ; and 5) gonadal t i s s u e s may be s e n s i t i z e d to subsequent androgen treatments by exposure to androgen e a r l y in l i f e (as suggested by s t u d i e s conducted with other s p e c i e s ) . 1 25 B. THE EFFECTS OF METHYLTESTOSTERONE ON BEHAVIOURAL DIFFERENTIATION J_. S e n s i t i v e P e r i o d s i n B e h a v i o u r a l Development E a r l i e r s t u d i e s have c o n c e n t r a t e d on examining the s h o r t -term b e h a v i o u r a l consequences of hormone treatments a d m i n i s t e r e d to a d u l t f i s h e s (see s e c t i o n I-A-4). T h i s study c o n c e n t r a t e s on examining the long-term b e h a v i o u r a l consequences of hormone treatments a p p l i e d to developing f i s h . The h i g h l i g h t s of t h i s study are o u t l i n e d below. Treatments I (immersion, f e r t i l i z a t i o n to r e l e a s e ) and III (MT i n d i e t , days 0-21 p o s t - r e l e a s e ) were admi n i s t e r e d d u r i n g the f i r s t 4-5 weeks of l i f e and have a s i g n i f i c a n t e f f e c t on the development of male a g g r e s s i v e behaviour ( i n t r a - and i n t e r -t e r r i t o r i a l f i g h t i n g , and a t t a c k i n g ) . Treatment IV (MT i n d i e t , days 21-42 p o s t - r e l e a s e ) was a d m i n i s t e r e d a f t e r the f i r s t 4-5 weeks of l i f e and had a s i g n i f i c a n t i n f l u e n c e on the development of female nest approaching behaviour. Treatments III and IV are i d e n t i c a l except III was a p p l i e d d u r i n g the f i r s t 3 week p e r i o d f o l l o w i n g r e l e a s e while IV was a p p l i e d d u r i n g the second 3 week p e r i o d . Treatment III a f f e c t e d only the development of male a g g r e s s i v e behaviours (enhanced i n t r a - and i n t e r - t e r r i t o r i a l f i g h t i n g scores) while treatment IV a f f e c t e d only the development of female nest approaching behaviour (as i n d i c a t e d by low t i l t s r e c e i v e d and b u t t s r e c e i v e d s c o r e s ) . A comparison of the b e h a v i o u r a l e f f e c t s produced by the d i f f e r e n t hormone treatments, r e v e a l s e v e r a l i n t e r e s t i n g 126 f e a t u r e s of b e h a v i o u r a l development i n S. mossambicus. Treatments that a f f e c t b e h a v i o u r a l development do so by i n f l u e n c i n g only one or two components of behaviour and not e n t i r e c l a s s e s of behaviour (that i s , the f u l l range of a g g r e s s i v e , t e r r i t o r i a l , or c o u r t s h i p b e h a v i o u r s ) . T h i s o b s e r v a t i o n suggests that the development of c e r t a i n behaviour components may be a s s o c i a t e d with d i f f e r e n t hormone-sensitive p e r i o d s e a r l y i n l i f e . A l s o c o n s i d e r that treatment I a f f e c t s only a t t a c k i n g behaviour while treatment III a f f e c t s only i n t r a -t e r r i t o r i a l f i g h t i n g behaviour. The o b s e r v a t i o n that both treatments I and III r e s u l t e d i n i n c r e a s e d i n t e n s i t i e s of i n t e r -t e r r i t o r i a l f i g h t i n g i m p l i e s that e i t h e r the s e n s i t i v e p e r i o d f o r t h i s behaviour o v e r l a p s both treatment p e r i o d s , or each treatment independently c r e a t e s s u i t a b l e c o n d i t i o n s for i n f l u e n c i n g the development of i n t e r - t e r r i t o r i a l f i g h t i n g . S i m i l a r l y , treatment IV a f f e c t e d only the development of female nest approaching behaviour, making group IV females l e s s l i k e l y to p a r t i c i p a t e i n c o u r t s h i p i n t e r a c t i o n s with males. The preceding o b s e r v a t i o n s can be e x p l a i n e d by c o n s i d e r i n g d i f f e r e n t components of behaviour to be i n f l u e n c e d by hormone treatments d u r i n g s e n s i t i v e p e r i o d s at d i f f e r e n t stages of development. Arranging the treatment-induced i n f l u e n c e s on behaviour i n sequence from e a r l i e s t to l a t e s t treatment, one n o t i c e s that male behaviours are i n f l u e n c e d by the e a r l i e r treatments while a female behaviour i s i n f l u e n c e d by a l a t e r treatment (see F i g u r e 1 2 ) . T h i s o b s e r v a t i o n may be a p r e l i m i n a r y i n d i c a t i o n that s e n s i t i v e p e r i o d s i n the development of c e r t a i n male behaviours 127 precede s e n s i t i v e p e r i o d s f o r development of c e r t a i n female v behaviours. T h i s c o n c l u s i o n i s t e n t a t i v e as there i s not enough evidence at present to d e f i n i t e l y s t a t e that each behaviour component i s a s s o c i a t e d with a temporally d e l i m i t e d s e n s i t i v e p e r i o d d u r i n g development. Since no attempt was made i n t h i s study to d e f i n e the l i m i t s of the suspected s e n s i t i v e p e r i o d s i n be h a v i o u r a l development, i t i s only p o s s i b l e to say that c e r t a i n behaviour components appear to be most s e n s i t i v e to hormonal i n f l u e n c e s at r e l a t i v e l y d i f f e r e n t stages of development. In summary, the r e l a t i o n s h i p between e a r l y androgen treatments and b e h a v i o u r a l development in a t e l e o s t appears to have the f o l l o w i n g c h a r a c t e r i s t i c s : 1) e a r l y androgen treatments can a f f e c t the development of a d u l t male and female b e h a v i o u r a l c h a r a c t e r i s t i c s ; 2) c e r t a i n components of behaviour appear to be most s e n s i t i v e to androgen d u r i n g component-specific s e n s i t i v e p e r i o d s i n development; 3) s e n s i t i v e p e r i o d s f o r d i f f e r e n t components of behaviour occur at d i f f e r e n t stages of development; and 4) s e n s i t i v e p e r i o d s f o r male behaviours appear to precede s e n s i t i v e p e r i o d s f o r female behaviours. The above paragraph o u t l i n e s the suspected r e l a t i o n s h i p between e a r l y androgen and b e h a v i o u r a l development i n a t e l e o s t . While t h i s model of b e h a v i o u r a l development i s based on a l i m i t e d amount of data, i t i s supported by two a d d i t i o n a l l i n e s of evidence. S t u d i e s of be h a v i o u r a l development i n mammals and b i r d s , p l u s behaviour s t u d i e s of sex-reversed f i s h , lend support to the proposed model of behav i o u r a l development i n f i s h and w i l l be d i s c u s s e d i n s e c t i o n s to f o l l o w . 1 28 F i g u r e 12. S e n s i t i v e P e r i o d s During B e h a v i o u r a l Development x - f e r t i l i z a t i o n A • • • • • • B C D • • • • • • I n c r e a s i n g Time of Development —> Notes: ... - Dotted l i n e s i n d i c a t e the temporal l i m i t s a s s o c i a t e d with each behaviour component s e n s i t i v e p e r i o d have not been d e f i n e d . - S o l i d l i n e s i n d i c a t e the r e l a t i v e stage of development during which a behaviour component was a f f e c t e d by a MT treatment. A - a t t a c k i n g B - i n t e r - t e r r i t o r i a l f i g h t i n g C - i n t r a - t e r r i t o r i a l f i g h t i n g D - nest approaching behaviour 2. B e h a v i o u r a l Development in Other V e r t e b r a t e s Behavioural development i s being explored i n other v e r t e b r a t e groups and such s t u d i e s are r e l e v a n t to, c o n s i s t e n t with, and a i d in understanding the model of the development of f i s h behaviour presented i n t h i s d i s c u s s i o n . The r e l a t i o n s h i p 129 between gonadal hormones and be h a v i o u r a l development has been s t u d i e d e x t e n s i v e l y i n mammals ( p r i m a r i l y rodents and primates, reviewed by Baum, 1979 and Beatty, 1979) and, to a l e s s e r extent, i n b i r d s (reviewed by Adkins, 1978). These s t u d i e s have shown that male or female behaviour p a t t e r n s r e s u l t from CNS development along e i t h e r male or female l i n e s , as determined by hormonal i n f l u e n c e s e a r l y i n l i f e . Once the developing CNS has passed beyond the p e r i o d of hormone s e n s i t i v i t y (considered to be the f i r s t few months of l i f e ) and d i f f e r e n t i a t e d i n t o a male or female nervous system, nervous impulses passed through the system s t i m u l a t e the ex p r e s s i o n of s e x - s p e c i f i c behaviour p a t t e r n s . Many s t u d i e s have shown that c e r t a i n b r a i n c e l l s a s s o c i a t e d with the c o n t r o l of c e r t a i n behaviours (as determined i n accessory b r a i n l e s i o n and/or s t i m u l a t i o n s t u d i e s - see above review a r t i c l e s f o r r e f e r e n c e s ) s e l e c t i v e l y i n c o r p o r a t e s t e r o i d s d u r i n g development (Whitsett et a l . , 1972; M o r r e l l and P f a f f , 1978). T h i s o b s e r v a t i o n i m p l i e s that these b r a i n c e l l s are t a r g e t s f o r c i r c u l a t i n g hormones. There i s a l s o evidence that the s t r u c t u r e and f u n c t i o n of b e h a v i o u r a l l y important b r a i n c e l l s are shaped by e a r l y hormonal i n f l u e n c e s while b r a i n c e l l s not a s s o c i a t e d with s e x u a l l y dimorphic behaviours do not e x h i b i t morphological d i f f e r e n c e s ( f o r example, see Ar n o l d and S a l t i e l , 1979). Reports of sexual dimorphism i n b r a i n areas r e g u l a t i n g behaviour have i d e n t i f i e d d i f f e r e n c e s between males and females i n : the number of synapses present (Raisman and F i e l d , 1973 and 1971; T o r a n - A l l e r a n d , 1976; 130 Greenough et a l . , 1977); neuronal c e l l body s i z e s (Gregory, 1975; Dorner and Staudt, 1969 and 1968); neuronal o r g a n e l l e f e a t u r e s ; s y n a p t i c v e s i c l e f e a t u r e s ; and d e n d r i t i c branching p a t t e r n s ( T o r a n - A l l e r a n d , 1978). Other works have i n d i c a t e d that these morphological d i f f e r e n c e s are produced by gonadal hormones e a r l y i n development ( P f a f f , 1966; Wh i t s e t t et a l . , 1972) and cannot be produced by t r e a t i n g a d u l t s with v a r i o u s hormone treatments (Gorski et a l . , 1978). Note that an exce p t i o n to t h i s g e n e r a l i z a t i o n i s presented by Nottebohm (1980), who induced m o r p h o l o g i c a l and f u n c t i o n a l changes i n b r a i n c e l l s a s s o c i a t e d with song production in c a n a r i e s (Serinus c a n a r i u s ) by t r e a t i n g a d u l t s with t e s t o s t e r o n e ( T ) . The above o b s e r v a t i o n s suggest that male and female t e l e o s t s may be d i s t i n g u i s h e d by s e x u a l l y dimorphic nervous systems s i n c e s e x u a l l y dimorphic behaviour p a t t e r n s are present in many s p e c i e s . The c h a r a c t e r i s t i c s of s e n s i t i v e p e r i o d s in be h a v i o u r a l development appear to be s i m i l a r i n both mammals and f i s h (see s e c t i o n IV-B-1). Mammalian s t u d i e s i n d i c a t e that the development of c e r t a i n behaviour components may be a s s o c i a t e d with hormone-s e n s i t i v e p e r i o d s in development For example, while v a r i o u s p r e n a t a l and p e r i n a t a l androgen treatments can s l i g h t l y i n f l u e n c e the development of a g g r e s s i v e behaviour i n female mice (Mus musculus), treatments a d m i n i s t e r e d w i t h i n a few hours of b i r t h produce the g r e a t e s t i n f l u e n c e on the development of ag g r e s s i v e behaviour (see Vom S a a l , 1979, and Whitsett et a l . , 1972). Mammalian s t u d i e s a l s o suggest that the s e n s i t i v e p e r i o d s 131 a s s o c i a t e d with d i f f e r e n t components of behaviour occur at d i f f e r e n t stages of development. For example, c o p u l a t o r y , open-f i e l d , and a c t i v e avoidance behaviours i n the male r a t (Rattus norvegicus) are a l l i n f l u e n c e d by hormone treatments administered e a r l y i n l i f e . However, p r e n a t a l treatments g r e a t l y i n f l u e n c e the development of c o p u l a t o r y behaviour while s i m i l a r neonatal treatments g r e a t l y i n f l u e n c e the development of open-f i e l d and a c t i v e avoidance behaviours (see M e i s e l et a l . , 1979). C e r t a i n authors s t a t e that evidence e x i s t s i n d i c a t i n g that the male nervous system develops e a r l i e r than the female nervous system ( i n r a t s , Gregory, 1975; i n humans, W i t e l s o n , 1976). Such a f i n d i n g i s c o n s i s t e n t with t h i s study as e a r l y hormone treatments i n f l u e n c e d the development of male behaviours, while a l a t e r hormone treatment i n f l u e n c e d the development of a female behaviour i n S. mossambicus. Since male behaviours are a f f e c t e d e a r l i e r than female behaviours, i t i s p o s s i b l e that the n e u r a l mechanisms c o n t r o l l i n g male behaviours develop e a r l i e r than those c o n t r o l l i n g female behaviour. T h e r e f o r e , b e h a v i o u r a l development in a t e l e o s t appears to p a r a l l e l b e h a v i o u r a l development in mammals. In both cases, d i f f e r e n t behaviour components appear to be i n f l u e n c e d by e a r l y hormone treatments d u r i n g v a r i o u s s e n s i t i v e p e r i o d s in development. A l s o , male b e h a v i o u r a l d i f f e r e n t i a t i o n appears to precede female b e h a v i o u r a l d i f f e r e n t i a t i o n i n both groups. 132 3. Behaviour of Sex-Reversed F i s h Comments on the behaviour of sex-reversed f i s h are rare and are g e n e r a l l y presented as minor p o i n t s f o l l o w i n g a . d i s c u s s i o n of whether or not the sex-reversed gonad i s f u n c t i o n a l . If a sex-reversed f i s h breeds, i t i s assumed the f i s h d i s p l a y e d normal c o u r t s h i p and mating behaviours (as re p o r t e d f o r sex-reversed medaka, 0. l a t i p e s , Yamamoto, 1959b, 1958, 1957, 1953, and Yamamoto and Matsuda, 1963). On the other hand, i f sex-reversed f i s h do not breed, attempts are made to e x p l a i n t h i s r e s u l t i n terms of morp h o l o g i c a l d e f i c i e n c i e s i n the gonad or a s s o c i a t e d r e p r o d u c t i v e s t r u c t u r e s rather than i n terms of behaviour ( i n rainbow t r o u t , S. g a i r d n e r i , Johnstone et a l . , 1979; and i n the guppy, P. r e t i c u l a t a , Takahashi, 1975a and b; D z w i l l o , 1962, and Clemens et a l . , 1966). G e n e r a l l y , an in-depth study of the behaviour of sex-reversed f i s h has not been conducted. To my knowledge, only one other study e x i s t s which attempts to examine the behaviour of sex-reversed f i s h in d e t a i l . Lindsay (1974) r e p o r t s that the sexual behaviour of sex-reversed genetic female guppies (P. r e t i c u l a t a , n = 3) i s the same as the sexual behaviour of untreated g e n e t i c males (n = 12). My examination of the behaviour of sex-reversed g e n e t i c female S. mossambicus (n = 5) i n d i c a t e d d i f f e r e n c e s between c o n t r o l f i s h (n = 1 8 ) and sex-reversed f i s h , in measures o f : i n t e r - t e r r i t o r i a l f i g h t i n g , a t t a c k i n g , d i g g i n g , and t i l t i n g to females. However, no be h a v i o u r a l d i f f e r e n c e s c o u l d be d e t e c t e d between sex-reversed and non sex-reversed f i s h (n = 8 ) from the same treatment 133 groups. T h i s f i n d i n g suggests that b e h a v i o u r a l d i f f e r e n c e s between sex-reversed and c o n t r o l f i s h are due to a general treatment e f f e c t common to sex-reversed and non sex-reversed f i s h . Since many sex-reversed f i s h s u c c e s s f u l l y mated with untreated females, there are i n d i c a t i o n s that sex-reversed f i s h d i s p l a y normal or near-normal c o u r t s h i p and mating behaviour. T h i s i m p l i e s that the hormone treatment causing gonadal sex-r e v e r s a l i n f l u e n c e s the development of behaviour along male or female channels. The p r o d u c t i o n of sex-reversed f i s h a l s o i n d i c a t e s that f r y possess the p o t e n t i a l to develop e i t h e r male or female b e h a v i o u r a l c h a r a c t e r i s t i c s . In t h i s study, sex-reversed g e n e t i c females do not d i s p l a y a s i n g l e element of behaviour at female f r e q u e n c i e s and d u r a t i o n s ; a l l ex p r e s s i o n s of behaviour are at l e a s t at normal male f r e q u e n c i e s and d u r a t i o n s . The preceding o b s e r v a t i o n s suggest that hormonal i n f l u e n c e s e a r l y i n l i f e act as a switch mechanism to channel b e h a v i o u r a l development along male or female l i n e s . 4. Beha v i o u r a l S e n s i t i z a t i o n to Androgen T h i s study suggests that d e v e l o p i n g b r a i n c e l l s can be s e n s i t i z e d to l a t e r hormonal i n f l u e n c e s , much l i k e d e v eloping gonadal c e l l s can be s e n s i t i z e d to l a t e r hormone a c t i o n (see Goetz et a l . , 1979 and s e c t i o n IV-A-5). Experiment IV (the e f f e c t s of a second androgen treatment on the behaviour of e a r l y - t r e a t e d females) i n d i c a t e s that the behaviour of e a r l y -t r e a t e d females was m a s c u l i n i z e d to a gr e a t e r extent than 1 34 females not exposed to any e a r l y treatment. Note that only c e r t a i n behaviour components were enhanced by the second hormone treatment ( t i l t s g i ven, l a t e r a l d i s p l a y s given, nest v i s i t i n g ) , suggesting that only c e r t a i n s e t s of b r a i n c e l l s were s e n s i t i z e d by the f i r s t hormone treatment. T h i s , i n t u r n , suggests that d i f f e r e n t s e t s of b r a i n c e l l s mature at d i f f e r e n t stages of development. S i m i l a r r e s u l t s have been obtained i n mammalian s t u d i e s . For example, Vom Saal et a l . (1976) found that the a g g r e s s i o n -i n d u c i n g a c t i o n of T was s i g n i f i c a n t l y enhanced in a d u l t female mice i f the mice had p r e v i o u s l y been exposed to a p r e n a t a l T treatment. Mice not exposed to T d u r i n g development were s i g n i f i c a n t l y l e s s a g g r e s s i v e than p r e n a t a l l y t r e a t e d mice when the second T treatment was a d m i n i s t e r e d . These r e s u l t s can be e x p l a i n e d by p o s t u l a t i n g that e a r l y androgen treatments s e n s i t i z e b r a i n c e l l s to l a t e r androgen i n f l u e n c e s . S e n s i t i z e d b r a i n c e l l s are p i c t u r e d as p o s s e s s i n g a lower t h r e s h o l d to hormone a c t i o n than n o n - s e n s i t i z e d b r a i n c e l l s . If the same hormone l e v e l i s c i r c u l a t i n g i n both s e n s i t i z e d ( t r e a t e d ) and n o n - s e n s i t i z e d (non-treated) animals, the behaviour c o n t r o l l e d by s e n s i t i z e d b r a i n c e l l s w i l l be s t i m u l a t e d to a g r e a t e r extent (enhanced frequency or d u r a t i o n ) than the behaviours c o n t r o l l e d by n o n - s e n s i t i z e d b r a i n c e l l s . 1 35 5 . A Comparison of Gonadal and B e h a v i o u r a l Development  C h a r a c t e r i s t i c s A comparison of gonadal and b e h a v i o u r a l development r e v e a l s c e r t a i n s i m i l a r i t i e s and d i f f e r e n c e s between the two pro c e s s e s . F i r s t , both gonadal and b e h a v i o u r a l c h a r a c t e r i s t i c s are s e n s i t i v e to hormonal i n f l u e n c e s during the f i r s t two months of l i f e . In both cases, the presumptive e a r l y hormone a c t s as a switch mechanism to channel f u r t h e r development along male or female l i n e s . T h e r e f o r e , i t appears that s e n s i t i v e p e r i o d s i n gonadal and b e h a v i o u r a l development o v e r l a p . However, a major d i f f e r e n c e i s noted i n the s e n s i t i v e p e r i o d c h a r a c t e r i s t i c s a s s o c i a t e d with each type of development. Gonadal development appears to be s e n s i t i v e to hormonal i n f l u e n c e s throughout the f i r s t two months of l i f e as sex d i r e c t i o n c o u l d be i n f l u e n c e d at v a r i o u s stages of development by d i f f e r e n t treatments. However, the o v e r a l l hormone-sensitive p e r i o d a s s o c i a t e d with b e h a v i o u r a l (CNS) development may be su b d i v i d e d i n t o s h o r t e r s e n s i t i v e p e r i o d s a s s o c i a t e d with the development of d i f f e r e n t components of behaviour (s i n c e d i f f e r e n t components of behaviour appeared to be most s e n s i t i v e to hormonal i n f l u e n c e s at d i f f e r e n t times d u r i n g development). F i n a l l y , both gonadal and CNS t i s s u e s can be s e n s i t i z e d to subsequent androgen treatments by an androgen treatment a d m i n i s t e r e d d u r i n g development. 136 6. Summary Behavi o u r a l development i n S. mossambicus has the f o l l o w i n g c h a r a c t e r i s t i c s : 1) e a r l y hormone treatments a f f e c t both the form (male or female) and the i n t e n s i t y (frequency or d u r a t i o n ) of c e r t a i n a d u l t behaviours; 2) the development of c e r t a i n behaviours appear to be i n f l u e n c e d to the g r e a t e s t extent by hormone treatments d u r i n g s e n s i t i v e p e r i o d s i n development; 3) s e n s i t i v e p e r i o d s f o r male behaviours appear to precede s e n s i t i v e p e r i o d s f o r female behaviours; 4) neural mechanisms c o n t r o l l i n g behaviour can be s e n s i t i z e d to subsequent androgen treatment by an androgen treatment administered e a r l y i n l i f e ; and 5) d i f f e r e n t behaviours are i n f l u e n c e d to d i f f e r e n t , degrees by e a r l y hormone treatments. 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