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A study of the effects of prolactin and testosterone on the parental behaviour of the male stickleback… Smith, Reginald Jan Frederick 1964

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A STUDY OF THE EFFECTS OF PROLACTIN AND TESTOSTERONE ON THE PARENTAL BEHAVIOUR OF THE MALE STICKLEBACK Gasterosteus aculeatus L. REGINALD JAN B.A., University of by FREDERICK SMITH B r i t i s h Columbia, 1962 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1964 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 of B r i t i s h Columbia, I agree that 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 reference and study- I f u r t h e r agree that per-m i s s i o n f o r extensive 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 granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying 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 gain s h a l l not be allowed without my w r i t t e n permission* Department of Zoology  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 S Canada Date August 24. 1964. ABSTRACT Male stickleback did not show a greater increase i n displacement or parental fanning when injected with p r o l a c t i n than they did when injected with s a l i n e aolvent alone. Displacement fanning was higher i n gonadectomized f i s h with p i t u i t a r y a c t i v i t y suppressed by short photoperiod than i n gonadectomized f i s h with an active p i t u i t a r y under long photoperiods when both groups were treated with the same testosterone concentration. Injection of p i t u i t a r y fractions i n t o the short photoperiod f i s h reduced fanning to the leve l s found under long photoperiods. Normal males under long photoperiod showed s i g n i f i c a n t l y higher fanning than any of the gonadectomized groups Indicating that methyl- testosterone-in concentrations used did not f u l l y replace the effects of the normal gonad. Exposure of males to a wide range of testosterone concentrations indicated that a r e l a t i v e l y high concentration of testosterone was required f o r normal fanning. Low levels of fanning occurred a f t e r gonadectomy of f i s h i n breeding condition i n both "displacement" fanning and parental fanning stages, i n d i c a t i n g the gonad was not required f o r performance of the fanning pattern as such but was required for fanning to reach levels approaching these found i n normal f i s h . i i No evidence was found that p r o l a c t i n i n i t i a t e s or maintains the parental fanning cycle. Testosterone however was necessary for normal fanning l e v e l s . ACKNOWLEDGMENTS I wish to express my gratitude to Dr. W. S. Hoar for suggesting the problem and for his time and valuable suggestions. Special thanks are due to Frank van Neetten who prepared the h i s t o l o g i c a l material on stickleback kidney, and to B i l l Woodall for h i s help i n obtaining f i s h . I wish to acknowledge the f i n a n c i a l support provided by a National Research Council Studentship. TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . 4 I . COLLECTION, MAINTENANCE AND OBSERVATION OF FISH. . 4 I I . EXPERIMENTS TO TEST THE EFFECTS OF PROLACTIN ON FANNING . . . . . . . . . . . . . . . . . . . 9 1. P r o l a c t i n i n j e c t i o n s on parental fanning • . 9 2. P r o l a c t i n injections on "displacement" fanning. . . . . . . 9 3. P r o l a c t i n i n j e c t i o n during the parental fanning cycle. . . . . . . . . . . . . . . 11 I I I . EXPERIMENTS TO TEST THE EFFECTS OF GONADAL AND PITUITARY HORMONES ON FANNING. . 11 1. Photoperiod, p i t u i t a r y and gonadectomy with replacement of testosterone . . . . . 11 2. Range of testosterone dosages. . . . . . . . 13 3. Gonadectomy i n the mature phase. • . . . . .15 RESULTS. .17 I. THE,EFFECTS OF PROLACTIN ON FANNING. . ..... . . . 17 1. Effect of p r o l a c t i n i n j e c t i o n on parental fanning 17 i ) percent fanning . . . . . 17 2. Ef f e c t of p r o l a c t i n on "displacement" fanning. . . . . . . . . . . . . . . . . . 17 i ) percent fanning • • 17 i i ) length of fanning bouts . . . . . . . .19 3. Effects of p r o l a c t i n i n j e c t i o n during the parental fanning cycle 19 i v PAGE I I . EFFECTS OF GONADAL AND PITUITARY HORMONES ON PARENTAL ACTIVITY. . . . . . . . . . . . . . . 23 1. Effects of Photoperiod, p i t u i t a r y and gonadectomy with replacement of testosterone . . • . . . . . . . . . . . 23 i ) nesting dates. . . . . . . . . . . . 23 i i ) percent fanning. . . . . . . . . . . 2 3 i i i ) length of fanning bouts. . . . . . . 26 2. Effects of a range of testosterone dosages. . . . . . . . . . . . . . . . .26 i ) nesting dates 29 i i ) colouration and sand digging . . . . 29 i i i ) percent of time spent fanning. . • .32 iv) average length of fanning bouts. . . 33 v) histology of kidney tubules. . . . . 33 3. Effects of gonadectomy i n the mature phase 37 i ) occurance of fanning . 37 i i ) percent of time spent fanning. . . . 38 i i i ) length of fanning bouts. . . . . . . 38 DISCUSSION . 41 I. PROLACTIN. 41 I I . PITUITARY AND GONADAL HORMONES . . . . . . . . . 48 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . 56 BIBLIOGRAPHY . . . - 58 V LIST OF TABLES TABLE PAGE I. Numbers of Fish Used i n Each Category of Experiment to Test Effect of P r o l a c t i n on Parental Fanning. . . . . . . . . . 10 I I . Summary of Treatments and Numbers of Fish used i n Experiment to Test Effect of P i t u i t a r y A c t i v i t y on "Displacement" Fanning. . . . . . 14 I I I . Effects of P r o l a c t i n i n Aqueous Suspension on Parental Fanning. 18 IV. E f f e c t of P r o l a c t i n I n j e c t i o n on Parental Fanning Showing Means for Groups of Fish Under Different Treatments. . . . . . . . . . 21 V. Differences i n Percent of Time Spent Fanning by Fish Before and A f t e r I n j e c t i o n with P r o l a c t i n and with Kreb's Solution 22 VI. Comparison of Percent Fanning 16-hour and 8-hour Photoperiods and Hormone Treatments. . 25 VII . FSfeSerftf o f Surviving FislM which Showed Nuptial Colouration with a Range of Testosterone Dosages . . . . . . . . . . . . . . 31 V I I I . Average Percent of Time Spent Fanning and Average Lengths of Fanning Bouts i n Fish Gonadectomized or Sham Operated i n the Mature Phase. . . . . . . . . . . . 39 LIST OF FIGURES FIGURE PAGE 1. Effects of P r o l a c t i n on Displacement Fanning . . 20 2. Dates of Nest Building by Fish Under 16-hour and 8-hour Photoperiods with P i t u i t a r y Hormones and Gonadectomy with Replacement of Testosterone. . . . . . . . . 24 3. Mean Percent of Time Spent Fanning Under 16-hour and 8-hour Photoperiods by Gonadectomized Fish with Testosterone and P i t u i t a r y Hormones . . . 27 4. Comparison of Mean Length of Fanning Bouts Under 16-hour and 8-hour Photoperiod with In j e c t i o n of P i t u i t a r y Hormones and Gonadectomy with Replacement of Testosterone. . . . . . . . . . 28 5. Mean Nesting Dates of Stickleback i n a Range of Testosterone Concentrations. . . . . . . . . . 30 6. Percent of Time Spent Fanning i n a Range of Testosterone Concentrations 34 7. Average Length of Fanning Bouts i n a Range of Testosterone Concentrations. . . . . . . . . . 35 8. Average Heights of Granular Cel l s i n Kidneys of Male Stickleback Treated with Four Concen-trations of Testosterone, Normal and Sham Operated Fish. 36 9. Average Percent of Time Spent Fanning on Consecutive Days A f t e r Gonadectomy or Sham Operation by Fish with and without Eggs. . . . 40 INTRODUCTION Care of eggs or young by the parent animal i s a s p e c i a l i z a t i o n which occurs time and again throughout the a n i -mal kingdom. Many of the advances made by the vertabrates i n th e i r evolution have been related to improved care of the young, f o r example, the c l e i d o i c egg allowing colonization of land, or the evolution of mammary glands and placental develop-ment i n mammals* Increasing s u r v i v a l of the young i s the essence of the evolutionary process. The examples above are of physiological mechanisms of parental care but j u s t as important are behavioural mechanisms which permit the i n t e r n a l f e r t i l i z a t i o n required f o r c l e i d o i c eggs or v i v i p a r i t y , or lead to the se l e c t i o n or building of a safe place f o r the young, incubation of the eggs or suckling of the young. Even within one group, such as the te l e o s t s , many mechanisms, physiological and behavioural, may be evolved to increase s u r v i v a l of the young. Some C i c h l i d s and c a t f i s h protect the eggs i n t h e i r mouths, C i c h l i d s (Baerends and Baerends van Roon, 1950) and Sticklebacks (Tinbergen, 1951) may herd and protect schools of newly hatched young, Cottids dig burrow f o r t h e i r eggs (Morris, 1954) and Diseus f i s h Svmphsodon discus nourish the young with a secretion of the skin (Egamii and L s h i i , 1962). Two of the most common dangers to young f i s h and eggs are predation by the many scavengers of the seas and lakes, and the 2. ever present d i f f i c u l t y of the low oxygen concentration of many natural waters. The f i r s t problem i s often overcome by construction of a "nest" to protect the eggs from predators, and perhaps to keep them from being carried away by water current, beyond the protection of the parent or into unsuitable environments. The construction of a nest, confining eggs to an area of l i m i t e d water c i r c u l a t i o n enhances the second problem, oxygen. This problem i s then often overcome by the parent fanning fresh water with i t s f i n s to c i r c u l a t e i t through the nest i n order to v e n t i l a t e the eggs, or young. This i s found i n a wide v a r i e t y of f i s h , f o r example wrasse (F e i d l e r , 1962), Cottids (Morris, 1954), Badis badis (Barlow, 1964) and the Sticklebacks (van I e r s e l 1953, Morris 1958). A l l these behavioural mechanisms must have some physio-l o g i c a l background related to sense receptors, the central nervous system, the endocrine system or the effector organs. In a few cases some of t h i s background has been explored experi-mentally, as i n the re l a t i o n s h i p between the p i t u i t a r y hormone P r o l a c t i n and "broodiness" i n the jewel f i s h (Noble Kumpf and B i l l i n g s , 1938), testosterone and nest building i n the male threespined stickleback Gasterosteus aculeatus (Wai and Hoar, 1963) and fanning i n the wrasse Grenilabus ocellatus ( F i e d l e r , 1962) which seems to be related again to p r o l a c t i n . 3. The male threespine stickleback shows several patterns of behaviour associated with the care of eggs and young, (van I e r s e l , 1953), I t migrates to a suitable l o c a t i o n , builds a nest, induces the female to lay eggs i n the nest, then fans water over the eggs u n t i l they hatch; a f t e r hatching i t keeps the young i n the region of the nest for a time u n t i l they are free swimming. The object of t h i s study i s to investigate the endocrine background of the fanning behaviour of the male G. aculeatus with s p e c i a l reference to p r o l a c t i n and testosterone. P r o l a c t i n has been implicated i n parental behaviour i n f i s h : wrasse ( F e i d l e r , 1962), jewel f i s h (Noble et a l , 1938) and discus f i s h (Egami and I s h i i , 1962), b i r d s ; pigeons and hens and mammals (Lekman, 1961). Testosterone which controls many a c t i v i t i e s c h a r a c t e r i s t i c of male animals and which has been shown to control an e a r l i e r pattern i n th i s sequence, nest building by the stickleback (Wai and Hoar, 1963) was also investigated with reference to fanning i n the stickleback. 4. MATERIALS AND METHODS I, COLLECTION AND MAINTENANCE OF FISH The stickleback used i n t h i s study were of both the plated and unplated forms (Heuts, 1947) of Gasterosteus  aculeatus L. They were collected by dip net or by l i f t net from drainage ditches on the Musquern Indian Reserve (fresh water) and from f l o a t s i n Coal Harbour (brackish water); both locations are i n Vancouver, B.C. Heavy i n f e s t a t i o n with parasites was sometimes found, e s p e c i a l l y i n Coal Harbour f i s h . Only f i s h which appeared healthy were used i n experiments. Before being used i n experiments the stickleback were sometimes held i n cooled (10° - 15°C) r e c i r c u l a t i n g sea-water tanks under natural photoperiod. However, they were usually placed i n observation tanks immediately a f t e r capture. A l l f i s h whether i n holding or observation tanks were fed exclusively on frozen brine shrimp (Artemia s a l l n a ) . For observation during an experiment, f i s h were placed i n 16 l i t r e glass aquaria with sand, and plants suitable f o r nest building and for sheltering females,subordinate males. The water i n ;the aquaria was continuously f i l t e r e d through glass wool and aerated. The aquaria were illuminated (50 - 55 ft.-C.) by overhead flourescent l i g h t s and the photoperiod was 5. controlled by an e l e c t r i c time switch to s u i t the p a r t i c u l a r experiment. During experiments, a l l f i s h were kept i n fresh dechlorinated water with C a + + ion added as CaCl2 to bring the Ca concentration up to 20ppm. This permitted 100 per cent s u r v i v a l of anadromous f i s h while the Ca concentration does not exceed that found n a t u r a l l y i n many B r i t i s h Columbia streams and lakes. This was required because normally anadromous Gasterosteus aculeatus show a very high mortality when placed i n dechlorinated Vancouver tap water with i t s low ion content (Smith, 1962). Problems of supply necessitated the use of anadromous stickleback during the winter months. The male stickleback were observed f o r periods ranging from 10 to 30 minutes depending on the experiment. A "hide" was found to be necessary to prevent disturbing f i s h over long periods of observation; i t was arranged so as to permit the observer to enter and leave the room without alarming the f i s h . Behaviour was recorded by means of a typewriter, at two-second i n t e r v a l s during an observation. The i n t e r v a l s were timed by a metronome f i t t e d with an e l e c t r i c contact and a buzzer. The metronome was set to t i c k every second but the e l e c t r i c contact was made only at one extreme of the pendulum's arc therefore a c t i v a t i n g the buzzer every two seconds. The t o t a l observation period (e.g. 15 minutes) was timed on a stop watch thus allowing correction of behaviour frequencies or durations i f the metronome varied from the proper i n t e r v a l . Behavioural i n f o r -mation was recorded i n the form of abbreviations, e.g. • • p pu fn fn fn p. indicates that at the f i r s t two i n t e r -vals the f i s h did not show nesting or parental a c t i v i t y ; at the t h i r d i t was i n a p o s i t i o n over the nest and pointing i t s head at the nest; at the fourth i t was pushing the nest with i t s snout, at the f i f t h , s i x t h and seventh observations i t was fanning the nest, etc. These abbreviations were then t o t a l l e d e i t h e r manually or by means of e l e c t r i c contacts on the type-writer keys connected to automatic counters. Several items of behaviour were recorded: 1) Building and Maintenance of Nest tmt testing material f o r building bmt bringing material to the nest rmt removing material from nest d sand digging P pointing at nest with snout from a p o s i t i o n above the nest pu pushing at nest with snout, not distinguished from "boring" (van I e r s e l , 1953) S glueing nest material with g l u e - l i k e kidney secretion 2) Parental fn fanning water over the eggs 7. c - catching young sp - s p i t t i n g young out Apart from "p" these behaviour patterns are described i n the l i t e r a t u r e as d i s t i n c t items (van I e r s e l , 1953), The two major vari a t i o n s between the c r i t e r i a i n t h i s study and those used by van I e r s e l are the f a i l u r e to d i s t i n g u i s h between pushing and "boring" i n the present study, and the use of the term "fanning bout" to ref e r only to the duration of a period of uninterrupted fanning rather than as van I e r s e l (1953) uses i t to re f e r to a period of fanning interrupted only by short periods of nesting a c t i v i t y . Calculations performed on the data were: 1. Per cent of time spent fanning number of 2 second i n t e r v a l s spent fanning x 100 £ a l l 2 second i n t e r v a l s - when ca l c u l a t i n g mean "percent of time fanning" f o r a group of observations: £ Percent of time spent fanning £ Observations 2. Average length of fanning bouts number of 2 second i n t e r v a l s spent fanning number of fanning bouts 8. - when ca l c u l a t i n g average bout length f o r a group of observations: £ Average bout length per observation £ Observations showing fanning* I f the observations which showed no fanning were included i t would d i s t o r t the average bout length. S t a t i s t i c s (Steele and Torrie,1960 ) applied were: 1. Standard deviation of the mean / s2 9 s- = / — — where = variance, n = number of x V n observations. 2. 95 per cent Confidence i n t e r v a l on the mean (s-) ( t for n-1 d.f.) where the confidence X in t e r v a l s of two samples overlap the means, the means are not s i g n i f i c a n t l y d i f f e r e n t . 3. " t " test on difference of means 3 = 5Li - X2 / t = -4- s- = / s2 n L +• n 2 S d d V " l " 2 When p i t u i t a r y hormones were used they were injected ( a f t e r anaesthetizing f i s h i n 0.01 per cent Tricaine methane sulph-onate, Sandoz) by means of a 0.25 ml. tuberculin syringe, through a 30 guage needle into the body cavity. When several injections were to be given to the same f i s h the i n j e c t i o n s i t e was alternated between r i g h t and l e f t sides. I I . EXPERIMENTS TO TEST THE EFFECTS OF PROLACTIN ON FANNING Because of widespread effects of p r o l a c t i n on both the A behaviour and physiology of parental a c t i v i t y i n vertabrates, V i t was decided to test t h i s hormone as a possible c o n t r o l l i n g agent for the fanning of the male stickleback. 1. P r o l a c t i n i n j e c t i o n s on parental fanning. The f i r s t experiment was designed to show whether regular i n j e c t i o n of a suspension of p r o l a c t i n i n water would increase the normal parental fanning of the male f i s h . Male stickleback were kept, one f i s h per tank i n observation aquaria under 16* ^ hour photoperiod (16 hours l i g h t , 8 hours dark). When a f i s h b u i l t a nest i t was given an i n j e c t i o n of ei t h e r p r o l a c t i n or d i s t i l l e d water every t h i r d day u n t i l the experiment ended, unless i t was an uninjected control f i s h . F ish under a l l three treatments were allowed to f e r t i l i z e eggs, then were observed twice a week f o r 10 minutes each. Numbers of f i s h used are summarized i n Table I . P r o l a c t i n dosage was: 15 mg./ i n j e c t i o n given suspended i n .025 ml. d i s t i l l e d water ( P a n l i t a r l o t #R10109, Armour Drug Co.). Fish i n both injected classes received up to 8 in j e c t i o n s (depending on date of nest b u i l d i n g ) . 2. P r o l a c t i n i n j e c t i o n s during "displacement" fanning. The second group experiments served as a control.of Che sal i n e solvent i n which p r o l a c t i n was dissolved, and u t i l i z e d a 10. a somewhat higher dosage of p r o l a c t i n . I f p r o l a c t i n i n i t i a t e d parental fanning i t s i n j e c t i o n might lead to an increase i n "displacement" fanning to a l e v e l resembling early parental fanning. (Displacement fanning has been defined by van I e r s e l , 1953, as fanning which occurs a f t e r the nest i s completed but before eggs are present i n the nest.) The f i s h which had a l l b u i l t nests (but had not spawned) were injected as above, with \] either prolactin/of j 0.67 Kreb's saline solution. P r o l a c t i n ( P a n l i t a r l o t #R10109) was dissolved i n the Kreb's solution and frozen i n small amounts to be thawed as needed. Dosage was 25 mg. i n .025 ml. of solution. Six f i s h were used i n i t i a l l y , one per tank; three were given p r o l a c t i n and three were given in j e c t i o n s of Kreb's soluti o n . Behaviour was recorded for two 10-minute periods ( f o r a t o t a l of 20 minutes per day) on days that the 10.a TABLE I Numbers of Fish Used i n Each Category of Experiment to Test Effect of P r o l a c t i n on Parental Fanning Treatment No. of Fish Used No. of Fert. Eggs P r o l a c t i n 6 4 D i s t i l l e d water 5 5 No i n j e c t i o n 10 6 11, f i s h was not injected. Surviving f i s h received a t o t a l of s i x i n j e c t i o n s . One p r o l a c t i n f i s h and one Kreb's sol u t i o n f i s h were permitted to f e r t i l i z e and ra i s e eggs before the experiment but eggs had completed development or died before experiment began. 3. P r o l a c t i n i n j e c t i o n during the parental fanning cycle. A t h i r d series of p r o l a c t i n experiments tested the e f f e c t of a single large i n j e c t i o n during the regular cycle of paren-t a l fanning; f i s h were injected once on the t h i r d or fourth day a f t e r egg laying. They were observed d a i l y one and one-half hours per day throughout the cycle with several observations on the day of i n j e c t i o n , both before and a f t e r the f i s h was injected . P r o l a c t i n dosage was 0.20 mg. given i n 0.01 ml of Kreb«s solu t i o n (National I n s t i t u t e of Health, ovine, l o t #23116). Seven f i s h were used, four with p r o l a c t i n and three with solvent. Later two f i s h were tested with a needle prick substituted f o r an i n j e c t i o n ; two normal f i s h were also observed during undis-turbed fanning cycles. I I I . EXPERIMENTS TO TEST THE EFFECTS OF GONADAL AND PITUITARY HORMONES ON FANNING 1. Photoperiod, p i t u i t a r y and gonadectomy with replacement of testosterone The object of t h i s experiment was to test the ef f e c t of the p i t u i t a r y gland as a whole, through the influence of photo-12. period and crude anterior p i t u i t a r y extract. Lu t e i n i z i n g hormone and p r o l a c t i n alone were also tested. Gonadectomy with replacement of testosterone was used i n an attempt to standardize the l e v e l of gonadal hormone i n the two photoperiods. Two males were kept i n each tank. P i t u i t a r y hormones were injected three times per week by the same methods as p r o l a c t i n i n the above experiments. When f i s h b u i l t nests they were observed for periods of twenty minutes each on days that they were not injected. Gonadectomy was performed, a f t e r anaestheti-zing i n M.S. 222, by cutting 1-2 mm s l i t s i n the body w a l l , j u s t over the gonads, which were then pulled out with forceps and cut free w e l l down the seminal duct. In sham operated f i s h gonads were held with forceps but not cut free, then replaced i n f i s h . A l l operations were done under a dissecting microscope (10X). Males were e a s i l y distinguished from females by the.' darker pigmentation and smaller s i z e of the t e s t i s . A f t e r gonadectomy the operated f i s h were kept i n sea water f o r a week. Only two instances of regeneration were found i n close to 200 f i s h gonadectomized i n th i s manner and re-examined l a t e r . M o r t a l i t y was very low i n immature f i s h . Testosterone was 17-a-methyl testosterone U.S.P. (Sigma Chemical Co. l o t M102B-083). I t was dissolved i n absolute e t h y l alcohol and measured into tanks with a .025 ml tuberculin. 13. syringe. The dosage was equivalent to 1 part/4 X10^ parts of water (Wai and Hoar, 1963). This was given once a week i n 0.1ml. of alcohol. Crude anterior p i t u i t a r y extract (Bovine, Armour l o t #305) l u t e i n i z i n g hormone (N.I.H.-LH-S-7 ovine) and p r o l a c t i n (N.I.H. ovine l o t #23116) were each injected i n 0.2mg. doses dissolved i n .05 ml. 0.67 Kreb's so l u t i o n . Treatments and numbers of f i s h are summarized i n Table I I . 2. Range of testosterone dosages. In t h i s series of experiments a wide range of testoster-one levels were tested on "displacement" fanning. The f i s h used were i n i t i a l l y immature. A l l were kept under 16 hour days, two f i s h per tank. Gonadectomies and sham operations were performed as above. Testosterone, as above, was d i l u t e d i n absolute alcohol and administered once a week i n concentrations to give the following i n i t i a l concentrations i n the water: Testosterone No. Fish Code 1: 4 X 10 6 14 A 1: 4 x 10'7i 14 B 1: 4 x l o f 14 C 1: 4 x 10 9 14 D Normal 18 N Sham op. 10 S The larger number of normal f i s h was to compensate for the fact that immature stickleback are d i f f i c u l t to sex by eye, hence, i n a l l p r o b a b i l i t y some females would Inadvertantly f i n d 14. TABLE I I Summary of Treatments and Numbers of Fish Used i n Experiment to Test Effect of P i t u i t a r y A c t i v i t y on "Displacement" Fanning Photoperiod Operation Hormones No. of Fish Used Code 16 hr. Normal 0 14 16 N Gonadect. Testosterone 14 16 G 8 hr. Normal 0 14 8 N Sham op. 0 14 8 S Gonadect. Testosterone 14 8 Opit Gonadect. Test. •+ LH 14 8 LH Gonadect. Test. + LtH 14 8 LtH Gonadect. Test. + Ant.Pit. 14 8Ant.Pit. 15. t h e i r way into the experiment. The lower number of sham operated f i s h was due to last-minute m o r t a l i t y ; i t was found that operated and sham operated f i s h would not stand f u l l sea water f o r recovery, t h i s l a t e i n the season ( A p r i l ) so 12 parts/thou-sand: sea water was used f o r the l a s t three days of recovery. Fish which b u i l t nests were then observed for 20 minutes each three days per week. Kidney histology: at the termination of t h i s experiment the kidneys of the experimental f i s h were fixed i n Bouin's, dehydrated and imbedded i n p a r a f f i n . Sections of 10 j*. were mounted and stained i n EhrItch's haematoxylin and 2 per cent aqueous eosin so l u t i o n . The height of granular e p i t h e l i a l c e l l s of the conooluted tubules was measured with an o p i t i c a l micro-meter under high power (10x45). Periodic observations on a l l f i s h were carr i e d out to ascertain presence of n u p t i a l colouring and sand digging. 3. Gonadectomy i n the mature phase. This experiment was designed to test the dependence of fanning, both with and without eggs, on gonadal hormones. For t h i s purpose male stickleback were gonadectomized i n the parental phase. The gonads are larger at t h i s time and the f i s h bleeds more f r e e l y . So, modifications i n the gonadectomy procedure were required: each opening was closed with one suture, 16. ( s u r g i c a l s i l k was used so removal of sutures was unnecessary) and the f i s h were placed i n 12 •%<>: e-lit sea water a f t e r the operation, then returned to t h e i r tanks several hours before the f i r s t post-operational observation on the day a f t e r the operation. M o r t a l i t y was 50 per cent i n s p i t e of these pre-cautions. The stickleback were observed f o r 20 minutes per day, from one day before gonadectomy i n f i s h without eggs or the day a f t e r egg laying i n those with eggs, u n t i l s i x days a f t e r gonadectomy. In f i s h with eggs the operation or sham operation was performed on the second or t h i r d day a f t e r the eggs were layed. A f t e r the experiment was terminated, surviving f i s h were examined for gonad regeneration. In those f i s h which died before examination the heat 23 - 24°C of the water led to rapid decomposition precluding accurate determination of regeneration. c 17. RESULTS I. THE EFFECT OF PROLACTIN ON FANNING 1. Effect of p r o l a c t i n i n j e c t i o n on parental fanning. Due to the f a i l u r e of most of the f i s h to raise eggs successfully no r e a l conclusions can be drawn with regard to the ef f e c t s of p r o l a c t i n on parental fanning (Table I I I ) . The data suggests that a decrease may occur i n the treated f i s h , but the difference i s w e l l within normal v a r i a t i o n or could be due to the effects of i n j e c t i o n as such, or the solvent, 2. Effect of p r o l a c t i n on "Displacement" Fanning. To obtain r e s u l t s that were more comparable average values were calculated f or both percent of time spent fanning and aver-age length of fanning bouts; t h i s was done f o r a l l observations from the time of the second i n j e c t i o n ( i n j e c t i o n s every t h i r d day). By t h i s time the two f i s h with eggs had either raised the young or the f i s h had died. I t also gave some time for the hormone to act, i ) Percent fanning. Fish which received p r o l a c t i n fanned less than those receiving a control i n j e c t i o n of saline solvent (Figure 1). While both the numbers of f i s h and the observed differences were small, these r e s u l t s indicate that p r o l a c t i n does not increase fanning, and may decrease the per-centage of time spent fanning by males without eggs. 18. TABLE I I I Effects of P r o l a c t i n i n Aqueous Suspension on Parental Fanning Treatment No. Fish No. Fert. No. Raising Av. % Observed Eggs Eggs Fanning Successfully Days 1-8 LtH i n j e c t i o n 6 4 1 25.74% Water i n j e c t i o n 5 5 0 0 Uninjected 10 6 1 34.47% 19. i i ) Length of fanning bouts. Differences i n the average length of fanning boats p a r a l l e l e d those found i n the average percent of time spent fanning (Figure 1)• One cannot say from t h i s experiment whether the change i n bout length was a cause or a r e s u l t of the difference i n t o t a l fanning. 3. Effects of p r o l a c t i n i n j e c t i o n during the parental fanning cycle Here each f i s h was injected once, on day two or three of i t s parental fanning cycle. I t appears from the data i n Table IV that p r o l a c t i n increases percent fanning over that found i n solvent injected normal f i s h , from day two through to day f i v e . This i s , however, due to i n d i v i d u a l v a r i a t i o n within the small number of f i s h used. When the differences between i n d i v i d u a l f i s h before and a f t e r i n j e c t i o n are compared (Table V) i t appears that while both p r o l a c t i n and solvent i n j e c t i o n s lead to increased fanning the increases are not s i g n i f i c a n t l y d i f -ferent, with the solvent leading to a s l i g h t l y greater increase than the p r o l a c t i n . Needle pricks alone did not lead to s i g n i -f i c a n t increases i n fanning so the increases are presumably i n response to the i n j e c t i o n of s a l i n e . Figure 1. Effects of P r o l a c t i n on Displacement Fanning 0 = mean % fanning A * mean length of fanning bouts ( N ^ = number of observations U = standard deviation of the mean T = 95% confidence intervals < > U UJ «" 3 O l u_ o o I ol 9 Z. 8 6 5 Z 3 - 2 ° I o I FIG. I (3 7) I C O (41) (2 8) S O L V E N T P R O L A C T I N TABLE IV Effe c t of P r o l a c t i n Injection on Parental Fanning Showing Means for Groups of Fish under Different Treatments Day Treatment No. Obs. Mean %-Fanning 1 Normal 7 11.6 2 Normal 9 21.2 2 P r o l a c t i n 3 30.7 2 Solvent 2 18.0 3 Normal 6 23.8 3 P r o l a c t i n 2 30.9 3 Solvent 1 23.7 4 Normal 4 32.3 4 P r o l a c t i n 4 39.3 4 Solvent 2 29.8 5 Normal 3 45.1 5 ", P r o l a c t i n 3 34.5 5 Solvent 2 41.4 6 -hatching date 22. TABLE V Differences i n Percent of Time Spent Fanning by Individual Fish Before and After Injection with P r o l a c t i n and with Kreb's Solution P R O L A C T I N S O L V E N T % of time spent fanning % of time spent fanning Tank Av. Before Av. After D i f f . Tank Av.Before Av. A f t e r D i f f . 40 18.1 25.9 + 7.8 25 12.2 12.1 - .1 59 14.3 8.4 - 5.9 49 22.8 42.5 +14.7 49 28.2 33.9 + 5.7 34 22.8 28.9 + 6.1 39 23.2 41.2 Av. +18.0 6.4 Av. 6.9 23. I I . EFFECTS OF GONADAL AND PITUITARY HORMONES ON PARENTAL ACTIVITY IN THE MALE STICKLEBACK 1. Effect of photoperiod, p i t u i t a r y and gonadectomy with replacement of testosterone In t h i s experiment f i s h were kept under 8-hour and 16-hour days while being subjected to gonadectomy and replacement with testosterone, and under 8 hours while being treated with crude anterior p i t u i t a r y extract, l u t e i n i z i n g hormone and p r o l a c t i n . i ) Nesting dates. The dates on which f i s h completed nests were recorded (Figure 2). There were no obvious differences i n the time from the beginning of the experiment to mean date of nest bui l d i n g between the d i f f e r e n t groups that b u i l t nests. Normal and sham operated f i s h under 8 hours did not b u i l d nests, i n d i c a t i n g suppression of p i t u i t a r y gonadotrophins: i n accor-dance with the findings of Baggerman (1957) and Hoar (1962). i i ) Percent fanning. The differences found i n the percent of time spent fanning are summarized i n Table VI and Figure 3. The normal 16-hour f i s h fanned s i g n i f i c a n t l y ( 0.001) more than any of the other groups. Fish injected with p i t u i t a r y hormones showed less fanning than 8-hour f i s h without p i t u i t a r y Figure 2. Dates of Nest Building by Fish Under 16-hr. and 8-hr. Photoperiods with P i t u i t a r y Hormones and Gonadectomy with Replacement of Testosterone 0 = mean nesting date f o r group ! = range of nesting dates = number of f i s h FIG. 2 16 N 8 0PIT. 166 8ANT. PIT. 8 LH 8 LtH O o o 18 20 25 DECEMBER 30 I 5 JANUARY GO o - a I O -(4) "(3) ST (3) 15 25. TABLE VI Comparison of % Fanning 16-hr. and 8-hr. Photoperiods and Hormone Treatments (Opit = gonadectomy and testosterone replacement but no p i t u i t a r y i n j e c t i o n ) Photoperiod Treatment No. Obs. Mean % t p Hours Fanning 16 Normal 29 4.10 5.19 0.001*** 16 Gonadect. 30 0.47 - - -16 Normal 29 4.10 8 Opit. 31 1.22 7.27 0.001*** 16 Gonadect. 31 0.47 8 Opit. 30 1.22 . 1.67 0.1 8 Opit. 30 1.22 8 Ant. p i t . 12 0.02 1.90 0.1 8 Opit. 30 1.22 8 LH 23 0.12 2.34 0 .05* 8 Opit. 30 1.22 8 IML 13 0.28 0.23 0.55 8 LfeH 13 0.28 8 LH 23 0.12 0.89 0.4 8 N LtH 13 0.28 8 Ant.pit. 0.02 1.24 0.2 8 v LtH 13 0.28 16 Gonadect. 31 0.47 0.54 0.5 8 LH 23 0.12 16 Gonadect. 31 0.47 1.40 0.2 8 Ant.pit. 12 0.02 16 Gonadect. 31 0.47 1.32 0.2 26. injections (but treated with testosterone) but not s i g n i f i c a n t l y less than 16-hr. gonadectomized f i s h which would presumably have high p i t u i t a r y a c t i v i t y but otherwise be the same as the 8-hour f i s h . There were no s i g n i f i c a n t differences between the three p i t u i t a r y i n j e c t i o n s . These results suggest an i n h i b i t o r y e f f e c t of the p i t u i t a r y on fanning as w e l l as an excitatory e f f e c t by the i n t a c t gonad which was not provided by the re-placement testosterone. i i i ) Length of fanning bouts. In t h i s experiment the length of the fanning bouts was e s s e n t i a l l y the same i n both 16-hour groups and i n the 8-hour 0 p i t u i t a r y group but was s i g n i f i c a n t l y lower i n the f i s h receiving p r o l a c t i n and l u t e i n i z i n g hormone (fFigure 4 ). Only one observation on f i s h receiving anterior p i t u i t a r y extract showed fanning, too few f o r s t a t i s t i c a l analysis. The difference between the three groups of injected f i s h and the 16-hour gonadectomized group may indicate that the difference i s due to the i n j e c t i o n treatment rather than to the p i t u i t a r y f r a c t i o n s . 2. E f f e c t of a range of testosterone dosages In t h i s experiment the f i s h were a l l kept under 16-hour photoperiod. Normal and sham operated f i s h were compared with gonadectomized f i s h exposed to four d i f f e r e n t dosages of testost-Figure 3. Mean Percent of Time Spent Fanning Under 16-hour and 8-hour Photoperiods by Gonadectomized Fish with Testosterone and P i t u i t a r y Hormones O = mean j j = one standard deviation of the mean T = 95% confidence i n t e r v a l on the mean LU Q_ CO o i O I FIG. 3 I I I I6N I6G 80 PIT. 8ANT. PIT 8LH 8LtH TREATMENTS Figure 4. Comparison of Mean Length of Fanning Bouts Under 16-hour and 8-hour Photoperiod with In j e c t i o n of P i t u i t a r y Hormones and Gonadectomy with Replacement of Testosterone 0 = mean [j = standard deviation of the mean T "L = 95% confidence i n t e r v a l on mean = significance of difference between bracketed means on a t test = number of observations (N) FIG. 4 ° s < > z 3 LU O-l o d o N.S. 24) at •sr P <0.05 A  N^ S. P <O.OOI A (12) C7) T CO C7)J C2) 16 N 16 G 8 OPIT. 8 ANT. PIT. 8 LH 8 L-bH TREATMENTS 29. 6 9 erone ranging from 1:4X10 to 1:4X10 . Fish that b u i l t nests were observed three times a week for 20 minutes each day. i ) Nesting dates. The dates on which f i s h completed nests under the d i f f e r e n t treatments were recorded and compared. -Only normal, sham operated and f i s h i n the two highest dosages of testoster-one (A and B) b u i l t nests. A c l e a r separation between the f i s h treated with testosterone and the f i s h with i n t a c t gonads (normal and sham operated f i s h ) i s shown i n Figure 5. This may have been due to the treated f i s h receiving a r e l a t i v e l y high amount of testosterone as soon as treatment started whereas i t took some time for the long photoperiod to stimulate hormone production i n the gonads of the normal and sham-operated f i s h . i i ) Colouration and sand digging. Periodic checks were made on the colouration of a l l the f i s h i n t h i s experiment. At the same time the amount of sand digging as shown by the sand i n the aquarium was noted. Table VII shows that there were no clear trends and that a majority of f i s h i n a l l groups showed n u p t i a l colouration, both blue eyes and red throat throughout most of the experiment. This would seem to indicate that the lowest testosterone dosage given (1:4X10 ) was more than s u f f i c i e n t to cause these colour changes. The colouring of the male stickleback has d e f i n i t e l y Figure 5. Mean Nesting Dates of Stickleback i n a Range of Testosterone Concentrations N = normal unoperated S = Sham operated A = 1:4x10 Testosterone 7 B = 1:4x10 Testosterone 0 = mean, corrected to nearest day n u T J. standard deviation of mean 95% Confidence i n t e r v a l s on mean significance of difference between bracketed means on t test number of observations lO 0 CO QJ O o d "V" in o i n >-< o in 04 O m < < t/> Z S1N3W1V3>J1 31. - TABLE VII Percent of Surviving Fish Which Showed Nuptial Colouration with a Range of Testosterone Dosages A p r i l 11 A p r i l 29 Mav 20 Mav 27 eyes throat eyes throat eyes throat eyes thro* N 23% 7% 36% 27% 62% 62% 88% 100% 3 43% 14% 43% 43% 60% 60% 80% 60% A 52% 48% 69% 81% 80% 80% 67% 67% B 38% 31% 55% 45% 80% 80% 100% 100% C 67% 25% 83% 42% 71% 42% 67% 67% D 36% 43% 50% 43% 100% 56% 88% 88% 32. been shown to be under control of gonadal hormones (Ikeda, 1933). In sand digging, a behaviour pattern which i s part of agonistic behaviour as w e l l as nest b u i l d i n g , was more frequent although not s i g n i f i c a n t l y so i n group C f i s h than those i n group D. i i i ) Percent of time spent fanning. The average percent of time spent fanning by f i s h with i n t a c t gonads (normal and sham-operated) and gonadectomized f i s h treated with 1:4x10^ testosterone are compared i n Figure 6. No fanning was observed by f i s h i n lower concentrations of testosterone. The stickleback i n testosterone fanned s i g n i f i -cantly less than those with gonads, though the difference was not so great as i n the previous experiment (Figure 3.) where the 16-hour normal and 16-hour gonadectomized f i s h are comparable to normal and group A f i s h i n t h i s experiment. The difference between the r e s u l t s of the two experiments l i e s i n the higher fanning by 16-hour normal f i s h i n the f i r s t experiment; the f i s h i n 1:4x10 testosterone fanned at e s s e n t i a l l y the same levels i n both experiments. The difference i s , therefore, probably due to a d i f f e r e n t i a l stimulus to the gonads of the normal f i s h , i n s p i t e of the fact that both sets of f i s h were kept i n the same room, under the same photoperiods and temperatures although at d i f f e r e n t seasons(Experiment 11(1) December-January, Experiment 33. 11(2) April-May). This conclusion i s further supported by the onset of nest building i n Experiment 11(2) normal f i s h which was l a t e r r e l a t i v e to testosterone treated f i s h than i n Experiment 11(1). i v ) Average length of fanning bouts. There were no s i g n i f i c a n t differences between the lengths of the fanning bouts i n the three groups of f i s h that b u i l t nests and fanned (figure 7.). v) Histology of kidney tubules. Kidneys from two f i s h under each treatment were preserved i n Bouin's solution then sectioned and stained. The height of the granulated c e l l s i n the convoluted tubules of the stickleback kidney has been shown (Wai and Hoar, 1963) to be dependant on testosterone dosage. So, t h i s procedure was used to check on the dosage received by f i s h under d i f f e r e n t treatments. Fish i n the d i f f e r e n t concentrations of testoster-one did respond d i f f e r e n t l y i n the manner to be expected (Figure 8.). The fact that f i s h with i n t a c t gonads showed somewhat lower c e l l height than those i n 1:4x10^ of testosterone although they fanned more indicates that fanning i s not e n t i r e l y dependent on testosterone l e v e l , or that the synthetic testosterone does not duplicate natural product from the f i s h . Figure 6. Percent of Time Spent Fanning i n a Range of Testosterone Concentrations (16-hr.) normal unoperated f i s h Sham operated 1: 4x10, v Tes tos terone cone. mean one standard deviation of the mean (N)-95% confidence i n t e r v a l on the mean pro b a b i l i t y difference i n means not due to chance, t te s t . number of observations FIG. 6 0 5 £ 3 00 o 5 s O P.<005 — A I 3 I N.S. C27> N.S. X CI2!> N S A TREATMENT Figure 7. Average Length of Fanning Bouts i n a Range of Testosterone Concentrations normal unoperated f i s h Sham operated 1:4x10 Tes tos terone concentration mean one s tandard deviation of the mean T _ (N)-95% confidence i n t e r v a l on the mean Pr o b a b i l i t y difference i n means not due to chance number of observations (only those which showed fanning were used) FIG. 7 N S A TREATMENT Figure 8. Average Heights of Granular C e l l s i n Kidneys of Male Stickleback Treated with Four Concentrations of Testosterone, Normal and Sham Operated Fish O = mean n U - standard deviation of the mean i = 95% confidence i n t e r v a l I I - range 25 20 o 15 u IO O N S A TREATMENTS FIG. 8 9 B I 37. 3. Effects of Gonadectomy i n the mature phase Stickleback with eggs were gonadectomized or sham operated on the second or t h i r d day of t h e i r egg r a i s i n g cycle, f i s h without eggs were observed then gonadectomized on one day then observed the next and following days, i ) Occurance of fanning. Baggerman (1957) quotes van I e r s e l and Burggraaf (personal communication) as saying that stickleback gonadectomized while r a i s i n g eggs continue "normally though at a lower i n t e n s i t y " and that stickleback gonadectomized i n the mature phase, with a nest b u i l t without eggs, do not fan. The occurance of fanning i n the current experiment i s summarized below: Out of 9 f i s h gonadectomized with eggs and l i v i n g over 6 days, 8 removed eggs on day 3-4 from nest and e s s e n t i a l l y stopped fanning. 1 fanned at low l e v e l 5 days then stopped, eggs i n nest but undeveloped. Out of 3 f i s h with sham operation with eggs and l i v i n g over 6 days, 3 fanned through to day 6 or 7. Out of 7 f i s h gonadectomized without eggs and l i v i n g over 6 days, 1 never fanned again; 4 stopped fanning a f t e r 2 days; 1 fanned for four days; 1 fanned f o r s i x days. 38. i i ) Percent of time spent fanning. The average amounts of time spent fanning under the d i f f e r e n t treatments are summarized i n Table VIII and i l l u s t r a t e d with confidence i n t e r v a l s i n Figure 9. The main points to be noted are that: a) f i s h gonadectomized while r a i s i n g eggs fanned much less than sham operated f i s h even during the second and t h i r d days when t h e i r eggs were s t i l l i n the nest; b) from the fourth day on the fanning of f i s h gonadectomized, with eggs, was not s i g n i f i c a n t l y d i f f e r e n t from the very low l e v e l found i n f i s h gonadectomized without eggs, and c) although fanning i n the sham operated f i s h was much higher than i n gonadectomized f i s h up u n t i l the seventh day (aft e r the hatching date of the eggs) i t was s t i l l w e l l below that expected of a normal f i s h , from the second day on (van I e r s e l , 1953). i i i ) Length of fanning bouts. There were too few observations with fanning to draw meaningful conclusions about differences i n bout length. Scanning Table VIII indicates they were probably not d i f f e r e n t . 39. TABLE VIII Average Percent of Time Spent Fanning and Average Lengths of Fanning Bouts i n Fish Gonadectomized or Sham Operated i n Mature Phase + performed Gonadectomy _ ^  p e r f o m e d Eees + P r e s e n t e s - not present (eggs layed on day 0 i n f i s h with eggs) Day Gonadect. Eggs No. of Mean % No. of Mean Bout Observations Fanning Observations Length 1 - 25 9.21 25 5.2 1 - - 9 4.82 9 5.6 2 - + 3 23.48 3 5.5 2 + + 9 4.49 7 7.5 2 + - 7 0.67 4 3.4 2 S + 3 23.97 3 6.9 3 + 9 1.08 6 4.8 3 + — 7 1.14 5 4.8 3 s + 3 21.73 3 9.7 4 + + 9 0.51 3 5.7 4 + - 7 0.83 2 12.9 4 s + 3 17.52 3 10.4 5 + 9 0.17 1 9.0 5 + •a 7 0.45 2 9.5 5 s + 3 17.19 3 6.0 6 + 9 0 0 0 6 + - 7 0 0 0 6 s + 3 16.61 3 5.5 7 + 9 0.02 1 0.9 7 + - 7 0.14 1 5.7 7 s + 3 1.46 1 6.5 40. Figure 9. Average Percent of Time Spent Fanning on Consecutive Days After Gonadectomy or Sham Operation by Fish With and Without Eggs O A = mean = 95% confidence i n t e r v a l s With Eggs • = normal ® = sham operated O = Gonadectomized Without Eggs A = normal A = Gonadectomized °7o O F T I M E S P E N T F A N N I N G _ r o O Ln O Ln O I • 1 1 © HCH -OH -< > U OH 31 Oi LH -<> O ^ © 41. DISCUSSION I. PROLACTIN There was good reason to think as did Baggerman (1957) that the fanning of the male stickleback might be influenced by P r o l a c t i n . This anterior p i t u i t a r y hormone i s associated with parental care and more s p e c i f i c a l l y with maternal care throughout the vertabrates. P r o l a c t i n (luteotrophic hormone, LtH) i s a protein hor-mone secreted by the anterior p i t u i t a r y i n t e l e o s t s , birds and mammals (Beach, 1948). I t has been linked with parental be-haviour and physiology i n several widely separate groups, as w e l l as with an assortment of other factors e.g. migration to water i n Diemvctilus viridescens (Grant and Grant, 1958), melanophore control i n f i s h (Pickford and Kosto, 1957) or osmoregulation of Mvxine (Chester-Jones et a l , 1962). Prolactin's e f f e c t on parental phenomena seems to be e l i c i t e d two ways: 1. i n h i b i t i o n of reproductive behaviour and physiology during the brooding phase; 2. stimulation of parental aspects of physiology and behaviour. Aggression, controlled by gonadal hormones, i s an i n t e -g r a l part of the mating behaviour of many animals, usually i t i s directed against members of the same sex and juveniles of 42. the species. Thus when an animal i s i n i t s mating phase i t may attack and k i l l young animals. This tendency must be suppressed a f t e r mating i f the animal i s to ra i s e i t s own young success-f u l l y . A substance, l i k e p r o l a c t i n , which tends to suppress gonadotrophic a c t i v i t y w i l l a id parental care. In the cock Nablvandov (1945) found that p r o l a c t i n increased broodiness, but so did other gonadal i n h i b i t o r s such as blindness and alcohol. Apparently p r o l a c t i n merely i n h i b i t s F ,S H secretion i t does not seem to cause broodiness but to f a c i l i t a t e i t by repressing sexual aggressiveness. Riddle (1938) found a s i m i l a r FSH i n h i b i t i n g action i n pigeons but p r o l a c t i n also led to increased parental behaviour. So too with the Jewel Fish where p r o l a c t i n and gonadal hormones seem to work antagonis-t i c a l l y . Castration greatly f a c i l i t a t e s the induction of brooding behaviour by p r o l a c t i n and th i s e f f e c t i s n u l l i f i e d by i n j e c t i o n of FSH into an i n t a c t f i s h (Noble et a l , 1938). Noble et a l (1938) showed that p r o l a c t i n was the most e f f e c t i v e of several hormones i n stimulating brooding behaviour i n the Jewel F i s h , Hemichromis bimaculatus. This f i s h builds a nest, fans the eggs to aerate them and herds i t s young for a while a f t e r hatching, f a i l u r e to attack young f i s h was taken to indicate "broodiness". I t i s not clear whether other components of brooding behaviour were influenced or not. I t must be noted that other substances including proluton, corpus luteum extract, 43. a n t u i t r i n - S , phenol, thyroxin, sodium hydroxide and alcohol also produced broodiness i n some f i s h . Whether these act through p r o l a c t i n release, an unknown hormonal facto r , or the central nervous system was not investigated. No experiments on hypo-physectomized f i s h were carried out. More recently F i e d l e r (1962) has shown that p r o l a c t i n induces fanning even i n the absence of a nest i n the Wrasse Crenilabrus ocellatus. In s t i l l another teleost the Discus F i s h , Svmphvsodon discus, a mucous secretion "discus milk" i s produced by the skin and fed to the f r y , t h i s seems to be stimulated by p r o l a c t i n (Egami and I s h i i , 1962). An i n t e r e s t i n g e f f e c t i s described by I s h i i (1960,1961). Apparently p r o l a c t i n i s important i n maintaining gestation i n viviparous t e l e o s t s , Ditrema and Neoditrema as w e l l as i n the top-minnow Gambusia. He also reported ( i n Egami, 1962) that p r o l a c t i n showed no observable e f f e c t on the gonad or i t s "corpora lutea" i n Lebistes r e t i c u l a t u s . So, we have a hormone which, i f we accept the idea that the same basic hormones have been adapted to d i f f e r e n t functions i n the course of evolution (Medawar, 1953), has controlled functions f a c i l i t a t i n g care of the young i n a wide area of the vertebrate subphylum (Eisner, 1961, Lehrman, 1961). I t has also been related to other functions such as osmoregulation or water need i n Fundulus (Pickford and Atz, 1957) and Diemyctilus (Grant and Grant, 1958) and melanophore contraction i n f i s h (Pickford and Atz, 1957). Once a hormone has acquired some 44. control of one phase of l i f e e.g. parental reactions, i t seems l o g i c a l that l a t e r developments i n the physiology and behaviour of t h i s phase might f a l l under i t s control e.g. corpus luteum and mammary gland. Even the sketchy reports of osmoregulatory and chromatophore effects might be correlated to th i s when we remember the importance of osmotic medium and colour change i n the reproduction of many f i s h and amphibians. Although p r o l a c t i n -l i k e substances have been found i n many lower vertebrates e.g. f i s h (Pickford and Atz, 1957), r e l a t i v e l y few investigations of i t s function have been carried out. And, i n many of these investigations mammalian p r o l a c t i n was used though i t i s not cle a r whether or not the hormone i s chemically s i m i l a r i n a l l groups (Gerschwind, 1958). P r o l a c t i n has been related to parental behaviour i n two f i s h (see above); i n f a c t , i t i s the only hormone which has ac t u a l l y been linked to a parental behaviour pattern such as fanning i n a teleo s t . The lslationship between p r o l a c t i n and parental behaviour i s , however, a tenuous one. In the Jewel Fish where the e f f e c t seems to be antigonadal, many other substances w i l l e l i c i t very nearly the same e f f e c t . In the wrasse, the only other f i s h i n which a behaviour change associ-ated with p r o l a c t i n has been found, i t seems f a i r l y clear-cut although the dosage l e v e l used, 1 to 6mg. f o r f i s h with an 45. average length of 8.4 cm. was high. Dosages of this order proved f a t a l to stickleback. The high dosage required may, of course, r e f l e c t an i n s e n s i t i v i t y of the wrasse to mammalian p r o l a c t i n . I t must also be noted that p r o l a c t i n , l i k e other p i t u i t a r y proteins, i s not a pure substance. Feidler's (1962) hormone may have acted through the ef f e c t of a contaminant, or through feed-back from another gland. I t seemed that p r o l a c t i n was the most l i k e l y endocrine candidate f o r control of the fanning i n the male stickleback. Baggerman (1957) quotes van I e r s e l and Burggraaf (personal communication) as finding that when males castrated i n the sexual phase were given eggs they started a normal fanning cycle though at a somewhat lower l e v e l , thus showing the gonads were not e s s e n t i a l f or parental fanning. Baggerman's own result s confirmed t h i s , and she speculated that parental behaviour might be "induced and maintained by p r o l a c t i n secreted by the p i t u i t a r y . " The findi n g of van I e r s e l (1953) that a peak i n fanning was noted on the normal hatching date even when the eggs were removed from the nest of the male stickleback, indicated the presence of an " i n t e r n a l factor" c o n t r o l l i n g i n part, the expression of the fanning cycle. This also encouraged the view 46. that a hormone controlled fanning. On these bases, experiments were undertaken to examine the effects of p r o l a c t i n on fanning i n the male stickleback. Male f i s h with eggs were given a series of i n j e c t i o n s of p r o l a c t i n at what seemed to be a reasonable, physiological dosage based on studies on small mammals. I f p r o l a c t i n maintained the fanning cycle there would be a higher l e v e l of fanning shown i n those f i s h receiving p r o l a c t i n than i n uninjected f i s h or those injected with sali n e . M o r t a l i t y of eggs and f i s h invalidated the resul t s of t h i s experiment (only one p r o l a c t i n and one normal f i s h yielded r e s u l t s , the normal f i s h fanning more than the p r o l a c t i n i n -jected f i s h ) . Next, stickleback without eggs were given a series of p r o l a c t i n i n j e c t i o n s . I f p r o l a c t i n induced the parental cycle then i t should cause an increase i n the fanning of f i s h without eggs to a l e v e l above the normal "displacement" fanning. This time the experiment went w e l l but again p r o l a c t i n showed no excitatory e f f e c t on fanning. The hormone injected f i s h fanned less than solvent injected f i s h (Figure 1). I t was thoughts that this might be due to a delete.tous e f f e c t of frequent i n j e c t i o n , too low a dosage or the need of some " p r i -ming" eff e c t of the eggs (Sevenster-Bol, 1962). Therefore f i s h were given a higher dosage i n a single i n j e c t i o n a f t e r they had begun to raise eggs. This did cause increased fanning 47. but not as much of an increase as i n j e c t i o n of the solvent used to dissolve the hormone alone (Table V). Later i n the course of an experiment designed to test several factors, p r o l a c t i n was given i n a series of i n j e c t i o n s to f i s h under 8-hour days which were brought into breeding condition with testosterone. I t was hoped that with the p i t u i t a r y suppressed by the short photoperiod, any effects of the injected p r o l a c t i n which might, have been masked by the active p i t u i t a r y i n e a r l i e r experiments would show up. As before, p r o l a c t i n injected f i s h fanned at a lower l e v e l than control f i s h . I f one were forced to form a conclusion from these experiments on the e f f e c t of p r o l a c t i n on fanning, i t could only be that p r o l a c t i n has a s l i g h t l y i n h i -b i t o r y action on parental and "displacement" fanning the male stickleback. The work of Feidler (1962) on the wrasse Crenilabrus o c e l l a t u s . of course, shows that t h i s conclusion can not be generalized to include other f i s h which show parental fanning. I t can also be pointed out however that Noble et a l (1938) report no e f f e c t of p r o l a c t i n on the fanning of the Jewel Fish, although p r o l a c t i n did seem to influence the "broodiness" of the f i s h towards i t s young. Possibly i n the stickleback too p r o l a c t i n i s related to the short period of care f o r the newly hatched young, rather than to the fanning behaviour. 48. I I . PITUITARY AND GONADAL HORMONES The r e s u l t s of the experiments with p r o l a c t i n raised several questions about the hormonal control of parental be-haviour i n the male stickleback. Is fanning under the control of the p i t u i t a r y ? Are gonadal hormones necessary f o r i t s expression? Or, does removal or i n h i b i t i o n of the gonads lead to an increase i n parental behaviour as i n the Jewel Fish (Noble et a l 1938) or the rooster (Nalvandov, 1945)? The f i r s t question could be answered most s a t i s f a c t o r i l y with hypophysectomized animals. Hypophysectomy of male stickleback could not, however, be performed with s u f f i c i e n t recovery to permit observations. Therefore photoperiod was used to achieve "physiological hypophysectomy" by keeping f i s h under al t e r n a t i n g periods of 8-hours of l i g h t and 16-hours of darkness. This photoperiod has been found by Baggerman (1957) and Wai and Hoar (1963) to suppress development of mating behaviour and aggression i n male stickleback, presumably by suppressing the secretion of p i t u i t a r y gonadotrophins. This was further confirmed i n the current experiment by the f a i l u r e of f i s h with i n t a c t gonads (both unoperated and sham operated) to reach maturity under 8-hour days. Gonadectomized f i s h under 8-hour days were treated with methyl testosterone to bring them into breeding condition, t h e i r levels of "displacement" 49. fanning were then compared to those of f i s h gonadectomized and treated with testosterone i n the same way, but held under 16-hour days. These f i s h were gonadectomized to prevent the e f f e c t of p i t u i t a r y a c t i v i t y on the gonads from influencing the r e s u l t s . In t h i s way both groups of f i s h received the same amount of testosterone presumably only the a c t i v i t y of the p i t u i t a r y gland i t s e l f was changed. The r e s u l t s (Figure 3.) show that fanning was performed less (though not s i g n i f i -cantly) i n the f i s h under 16-hour days. Apparently without the benefit of the i n t a c t gonad, the p i t u i t a r y does not stim-ulate increased fanning. This i s indicated too i n the same figure by the fact that three groups of 8-hour f i s h , which d i f f e r e d from the two above groups only i n that they received in j e c t i o n s of p i t u i t a r y f r a c t i o n s , crude anterior p i t u i t a r y extract, l u t e i n i z i n g hormone (thought to increase aggression i n sticklebacks:£Wai and Hoar, 1963) or p r o l a c t i n , also showed N generally lower levels of fanning. The higher amount of time spent fanning by normal f i s h under 16-hour days may indicate an output of hormones by the p i t u i t a r y stimulated gonads that was higher than the amount of testosterone picked up from the medium by treated f i s h , or i t may indicate a d i f f e r e n t i n a c t i -v i t y between the hormones of the i n t a c t gonad and methyl testosterone. 50. From the above experiment i t seemed l o g i c a l to investigate the r e l a t i o n s h i p between fanning and the gonads. Unfortunately, the next experiment was planned with a p i t u i t a r y e f f e c t rather than a gonadal one i n mind. Gonadectomized f i s h under 16-hour photoperiods were placed i n a range methyl testosterone con-centrations from 1:4x10 , the same as the previous experiment, 9 down to 1:4x10 . This experiment was intended to produce two or more groups of f i s h which b u i l t nests but had d i f f e r e n t levels of gonadal hormone. I f , as i n the case of the Jewel Fish, (Noble et a l 1938) the gonadal hormones were antagonistic to parental care then there should be more parental care shown^in the group with the lower testosterone dosage. Two such groups were produced. As would be expected from the r e s u l t s of previous experiements, no increase i n fanning due to unmasking of a p i t u i t a r y e f f e c t was found. In-stead the group i n the lower concentration showed no fanning while those i n the higher concentration did show fanning, though less than that shown by 16-hour normal f i s h (Figure 6.). I f testosterone dosage above, as w e l l as below those used pre-v i o u s l y had been used, a clearer trend might have been found i n the relationship between testosterone and fanning. One i n t e r e s t i n g r e s u l t of experiment was the fact that the lowest concentration (l/1000th of the highest) s t i l l led to a high degree of colouration and to some sand digging; the next 51. highest concentration led to more sand digging, the higher one than that to some rough nest building and the highest l e v e l to we l l formed nests and"displacement" fanning. This suggests that the temporal relationship of some components of the sexual and breeding behaviour i . e . colouration, sand digging, and nest building and displacement parental behaviour, may be due to a gradual increase i n production of gonadal hormones as the season progresses. The results of the experiment above indicated that fanning might be under the influence of testosterone, despite sketchy reports to the contrary (van I e r s e l and Burggraaf, Baggerman, 1957) which give no i n d i c a t i o n of the number of f i s h used, the conditions of the experiment or the levels of "par-ental behaviour" obtained. Males with and without eggs were gonadectomized i n the mature phase and compared with sham operated f i s h (Figure 9). I t was found that removal of the gonads led to a very marked decrease i n both parental and "displacement" fanning, and to removal of eggs from the nest by a majority of the f i s h gona-dectomized with eggs i n the nest. Fanning occurred occasionally even i n f i s h without eggs, as d i f f e r i n g from the results of Hoar (1962) though i t was very low,UO percent or le s s , and decreased gradually over the seven days or le s s . The longer 52. observation period (20 minutes rather than 10 minutes used by Hoar, 1962) probably accounts for the observation of this very low frequency of fanning during the f i s t few days a f t e r gonad-ectomy. The decline i n fanning a f t e r the t h i r d day i n f i s h which i n i t i a l l y had eggs may be i n part due to the lack of stimulus from the eggs a f t e r the f i s h had removed them from the nest. This i s indicated by the one gonadectomized f i s h which did not remove i t s eggs; i t continued to fan at a low l e v e l f o r the f u l l seven days. The removal of the eggs i n the f i r s t place may be d i r e c t l y due to the absence or reduction of gonadal hormones, or a secondary eff e c t of death of the eggs due to i n s u f f i c i e n t aeration. Under the conditions of t h i s experiment, eggs l e f t i n a nest a f t e r the male had died f a i l e d to develop. In any case i t i s p l a i n the gonadal hormones are required f o r a normal l e v e l of fanning, although the behaviour pattern i t s e l f can occur i n the absence of gonads. This r e s u l t does not necessarily show that testosterone has a d i r e c t e f f e c t on fanning, i t may w e l l be that fanning does not normally occur unless the f i s h i s confronted with the stimulus of the nest, nest entrance or eggs. I f testosterone controls nest building a c t i v i t y , (Wai:. and Hoar, 1963) i t i s possible that with less testosterone the f i s h approaches the nest less often thus finding i t s e l f i n the "releasing s i t u a t i o n " f o r fanning less 53. frequently. In the case of egg rearing f i s h , the lower l e v e l of fanning would retard or prevent development of the eggs, th i s would decrease the effectiveness of the releasing s i t u -ation and further decrease fanning. I t i s p l a i n only that under normal circumstances the gonadal hormones are necessary for a normal fanning cycle. There i s no i n d i c a t i o n of a separate c o n t r o l l i n g mechanism f o r parental and "displacement" fanning. One l i n e of reasoning may indicate that testosterone i s more l i k e l y to increase parental behaviour i n teleosts, and p a r t i c u l a r l y i n the stickleback, than i t i s i n birds or mammals. Testosterone i s a male hormone. In a majority of birds and to an even greater extent i n mammals parental care i s the perogative of the female (Lehrman, 1961). The male seldom helps to care for the young and i n some cases i n birds where the male does help, this i s accompanied by decreased development of the t e s t i s at the time of parental a c t i v i t y (Lehrman, 1961). In teleosts, on the other hand, parental behaviour i s most often carried out by the male. Barlow (1964) suggests that t h i s tendency may have evolved from a s i t u a t i o n i n which a t e r r i t o r i a l male continued to defend his t e r r i t o r y f o r the pur-poses of further breeding, a f t e r the f e r t i l i z e d eggs of the f i r s t mating were present i n the t e r r i t o r y . These eggs might l a t e r become the focus of the t e r r i t o r y , any increase i n care 54. or protection of the brood giving a s e l e c t i v e advantage. In stickleback i n p a r t i c u l a r , aggression which often must be suppressed i n male animals before they can show parental be-haviour, i s under the influence of the p i t u i t a r y (Hoar, 1962) rather than the gonad. A relationship between t e s t i s and parental care seems more l i k e l y where parental behaviour i s the prerogative of the male and aggression i s related to p i t u i t a r y rather than gonadal a c t i v i t y as i n the stickleback. I t has already been shown (Hoar 1962) that the gonadal hormone i s probably the major factor involved i n nest building and sexual behaviour. However Sevenster-Bol has shown that the sight of eggs leads to a short temporary decrease i n sexual a c t i v i t y ( z i g zaging) i n the male stickleback and van I e r s e l (1953) reported long range decline i n sexual a c t i v i t y with increasing number of broods. The r e l a t i o n s h i p between sexual behaviour and fanning seems to be mutual i n h i b i t i o n (van I e r s e l 1953). Wai and Hoar (1962) showed that both testosterone and male genetic background were necessary f o r sexual behaviour, although they did not demonstrate a quantitative relationship between testosterone dosage and i n t e n s i t y of sexual behaviour. The experiments reported above i n this paper seem to show that testosterone i s required f o r normal expression of fanning. The only explanation which seems to reconcile these two results 55. with the mutual i n h i b i t i o n found by van I e r s e l (1953) would be that testosterone raised the general reproductive a c t i v i t y of the f i s h . This a c t i v i t y would then take the form most appropriate to the stimulus s i t u a t i o n i n which the f i s h found i t s e l f . I f there were no eggs present sexual a c t i v i t y would occur, increasing stimulus from eggs would lead to higher parental a c t i v i t y and lower sexual a c t i v i t y i n a rel a t i o n s h i p resembling competetive i n h i b i t i o n . 56 SUMMARY The relationships between fanning and p r o l a c t i n and testosterone i n the male stickleback were investigated. The results are summarized below. 1) P r o l a c t i n administered i n a series of injections to f i s h i n the ••displacement1" fanning phase did not lead to an increase i n fanning but rather to a s l i g h t decrease i n percent of time spent fanning and i n the length of fanning bouts, i n d i c a t i n g p r o l a c t i n alone does not i n i t i a t e "parental" fanning. 2) P r o l a c t i n administered i n a single i n j e c t i o n during the parental fanning cycle did not lead to an increase i n fanning greater than that caused by i n j e c t i o n of the solvent i n which the pr o l a c t i n dissolved, i n d i c a t i n g that the l e v e l of parental fanning i s not d i r e c t l y related to the l e v e l of pro-l a c t i n i n the f i s h . 3) Fish gonadectomized under 8-hour photoperiod fanned more than gonadectomized f i s h under 16-hour photoperiod which received the same concentration of testosterone. Injection of p i t u i t a r y extracts into f i s h under 8-hour photoperiod re-duced fanning to levels comparable to those under 16-hours, in d i c a t i n g that the effe c t of the p i t u i t a r y on fanning may be s l i g h t l y i n h i b i t o r y . 57. 4) In stickleback which were placed i n a wide range of testosterone concentrations under 16-hours only f i s h i n the two highest concentrations b u i l t nests. Of these only those i n the highest concentration fanned, although less than normal f i s h , i n d i c a t i n g that a r e l a t i v e l y high concentration of tes-tosterone i s required for normal fanning. Measurement of height of kidney c e l l s showed that f i s h i n the highest testos-terone concentration had higher mean c e l l height than the normal f i s h i n this experiment i n d i c a t i n g that methyl testosterone i s not as e f f e c t i v e i n replacing the behavioural effects of the gonad as i t i s i n replacing the ef f e c t on c e l l height i n the kidney. 5) Gonadectomizing male stickleback i n the "displacement" fanning and parental fanning stages led to a substantial decrease i n fanning as compared with sham operated f i s h , although i n neither group was fanning completely eliminated. Gonadal hormone may be necessary for normal fanning l e v e l s , but not f o r the expression of fanning as such. 58. BIBLIOGRAPHY Baerends, G. P. and J . Baerends van Roon (1950). An introduction to the ethology of C i c h l i d fishes. Behaviour, Suppl., 1:1-243. Baggerman, B. (1957). 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