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Induction of parental behavior in the blue gourami, Trichogaster trichopterus (Pisces, Belontiidae) Kramer, Donald Lawrence 1971

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THE INDUCTION OF PARENTAL BEHAVIOR IN THE BLUE GOURAMI, TRICHOGASTER TRICHOPTERUS (PISCES, BELONTIIDAE) by DONALD LAWRENCE KRAMER B.S c , Boston College, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1971 In presenting 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 r e q u i r e -ments 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 C o l -umbia, 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 permission 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 permis-s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, B r i t i s h Columbia i i ABSTRACT The blue gourami, Trichogaster t r i c h o p t e r u s , i s a small, freshwater t r o p i c a l f i s h , n a t i v e to Southeast A s i a . P a r e n t a l be-havior i s normally shown only by males a f t e r spawning. Females and non-parental males eat small numbers of t e s t eggs which they are given, while p a r e n t a l males r e t r i e v e them to t h e i r nests. 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 f a c t o r s r e s p o n s i b l e f o r the sudden change i n behavior from egg-eating to p a r e n t a l care which takes place at the time of spawning. In the i n i t i a l s e c t i o n , the p a t t e r n s of p a r e n t a l behavior are described, and q u a n t i t a t i v e data on the development and main-tenance of parental behavior i n male f i s h spawning fo r the f i r s t time are presented. The experimental s t u d i e s r e v e a l that s t i m u l i from the eggs are an important f a c t o r i n the i n d u c t i o n of p a r e n t a l behavior. Many naive males developed parental behavior w i t h i n 0 . 5 - 2 . 5 min when given eggs spawned by other f i s h . This response was depen-dent upon the number of eggs presented: a l a r g e p r o p o r t i o n of f i s h became pa r e n t a l when given 2 0 0 0 - 3 0 0 0 eggs, a smaller propor-t i o n d i d so when given 5 0 0 - 1 0 0 0 eggs, and none d i d so when given 1 0 0 eggs. However, even 1 0 0 eggs e v i d e n t l y a f f e c t e d p a r e n t a l responsiveness because some f i s h e v e n t u a l l y developed complete par e n t a l behavior when repeatedly given 1 0 0 eggs. These r e s u l t s i n d i c a t e that eggs can induce p a r e n t a l behav-i o r , not that they do so i n the n a t u r a l spawning s i t u a t i o n . However, t e s t s of males spawning with females whose oviducts were plugged i n d i c a t e d that exposure to eggs was an important compo-i i i nent i n the n a t u r a l development of p a r e n t a l behavior. Spawning by i t s e l f d i d not induce f i s h to become p a r e n t a l , but i t d i d r e -duce the number of eggs necessary f o r p a r e n t a l behavior to de-velop. Besides spawning and s t i m u l i from the eggs, androgens a l s o play a r o l e i n the development of p a r e n t a l behavior. I t was shown th a t , a f t e r spawning, males performed more parental beTiav-i o r than females, although d e t a i l e d observations of females r e -vealed that they had some capacity to perform p a r e n t a l behavior. The m a s c u l i n i z a t i o n of females by means of methyl testosterone implants allowed them to develop p a r e n t a l behavior i n response to l a r g e numbers of eggs. Whether c a s t r a t i o n of males eliminated t h e i r c a p a c i t y to perform p a r e n t a l behavior could not be deter-mined with c e r t a i n t y . The presence of young f a c i l i t a t e s the maintenance of paren-t a l responsiveness to eggs when males w i t h developing broods are compared with males whose broods have been removed. Apparently, p h y s i c a l contact v/ith the young i s necessary because males lo s e t h e i r p a r e n t a l responsiveness when t h e i r broods are placed i n , baskets which allow only v i s u a l and chemical contact. An important r e s u l t of t h i s study i s the demonstration of the s i g n i f i c a n c e of stimulus strength i n the i n d u c t i o n of paren-t a l responsiveness. This f a c t o r has not often been taken i n t o c o n s i d e r a t i o n i n other s t u d i e s of p a r e n t a l behavior. By v a r y i n g stimulus strength, r e p e t i t i o n of s t i m u l i , and sexual experience, patterns of the i n d u c t i o n of p a r e n t a l behavior were found i n gouramis which were s i m i l a r to those observed i n other s t u d i e s on a v a r i e t y of mammals, b i r d s , and f i s h . That i s , p a r e n t a l i v behavior developed e i t h e r almost immediately or gr a d u a l l y with the p r e s e n t a t i o n of young to non-parental f i s h , or i t was shown only i f the young were presented during the " s e n s i t i v e p e r i o d " a f t e r spawning. The f i n d i n g of such d i f f e r e n t patterns i n a s i n g l e species as a r e s u l t of v a r y i n g the strength of s t i m u l i from the eggs sug-gested that the motiva t i n g e f f e c t s of eggs and young on p a r e n t a l responsiveness may represent a common pa t t e r n underlying the con-t r o l of pa r e n t a l behavior i n a v a r i e t y of vertebrate species. This p o s s i b i l i t y and the r e l a t i o n s h i p between hormones and stim-u l i from the young i n the c o n t r o l of p a r e n t a l behavior are d i s -cussed i n the f i n a l chapter. V TABLE OF CONTENTS PAGE I . INTRODUCTION 1 Chapter 1. I n t r o d u c t i o n to the Problem and Methods. . . 1 A. The Problem 1 B. The Animal 2 C. The Methods 3 1. Culture 3 2 . Maintenance 3 3 . I n t r o d u c t i o n of Spawning 6 LL. C o l l e c t i o n of Eggs 9 5. S u r g i c a l Procedures . 10 6. Behavioral Observations 12 D. Summary 12 Chapter 2. Reproductive Behavior of the Blue Gourami . . 13 A. Q u a l i t a t i v e D e s c r i p t i o n 13 1. Nest B u i l d i n g 13 2. Spawning 14 3 . Parental Behavior 15 LL. D e s c r i p t i o n s and D e f i n i t i o n s of Behavior Used i n Recording , 16 5. Development of the Young. . . . . . 20 B. A Qu a n t i t a t i v e Study of the Natural Induction of P a r e n t a l Behavior 21 1. I n t r o d u c t i o n 21 2 . Methods 21 3 . R e s u l t s and Discussion 23 v i PAGE C. A Q u a n t i t a t i v e Study of the Maintenance of Par e n t a l and Nest B u i l d i n g Behavior 27 1. I n t r o d u c t i o n 27 2. Methods 27 3. R e s u l t s and Dis c u s s i o n 29 D. Summary . . . . . . 34 I I . FACTORS INVOLVED IN THE INDUCTION OF PARENTAL BEHAVIOR. . . . . . . . . . 36 Chapter 3. The Role of Eggs 36 A. Induction of Par e n t a l Behavior v/ith Clutches Spawned by Other F i s h 36 1. I n t r o d u c t i o n 36 2. Methods 37 3. R e s u l t s and Di s c u s s i o n . . . . . . . 38 B. The E f f e c t of the Number of Stimulus Eggs on the Induction of Par e n t a l Behavior. . . . . . . . 40 1. I n t r o d u c t i o n 40 2. Methods 41 3. R e s u l t s 43; 4. Discussion 51 C. Induction of Par e n t a l Behavior by Repeated Exposure to Small Numbers of Eggs 53 1. I n t r o d u c t i o n . . 53 2. Methods 54 3. R e s u l t s and Dis c u s s i o n 56 D. Summary 60 v i i PAGE Chapter L\. The Stimulus f o r the Induction of Par e n t a l Behavior . 6 l 1. I n t r o d u c t i o n 61 2. R e s u l t s and Dis c u s s i o n 62 Chapter 5« The Role of Performance of the Spawning Act 65 A. The Role of Spawning 65 1. I n t r o d u c t i o n 65 2. Methods 66 3. Results . . . . . 68 LL. D i s c u s s i o n 70 B. The E f f e c t of Spawning on the Speed of Develop-ment of Par e n t a l Behavior . 72 1. I n t r o d u c t i o n 72 2. Methods 72 3. R e s u l t s and Discussion 73 C. Summary 76 Chapter 6. The Role of Androgens 77 A. Sexual Dimorphism i n Par e n t a l Behavior 77 1. I n t r o d u c t i o n 77 2. Methods . . 78 3. R e s u l t s and Di s c u s s i o n . 80 B. E f f e c t of Methyl Testosterone on the Pa r e n t a l and Nest B u i l d i n g Behavior and the Secondary Sex Characters of Female Blue Gouramis 85 1. I n t r o d u c t i o n . . 85 v i i i PAGE 2. Methods 86 3. Results and Discussion 89 C. The Ef f e c t s of Castration on the Parental and Nest Building Behavior of Male Gouramis 97 1. Introduction. 97 2. Methods 98 3. Results 101 A. Discussion 103 D. Hormones and the Parental and Nest Building Behavior of Fish 106 E. Summary . . 109 I I I . MAINTENANCE OF PARENTAL BEHAVIOR I l l Chapter 7. The Role of Young i n the Maintenance of Parental Behavior I l l 1. Introduction H I 2. Methods . H I 3. Results 113 if. Discussion 114 5.- Summary . . . . . . . . . .118 IV. GENER& DISCUSSION 119 Chapter 8. General Conclusions About the Control of Parental Behavior i n the Gourami 119 Chapter 9. E f f e c t s of Eggs and Young on Parental Responsiveness i n Vertebrates 124 A. Introduction. . 124 i x PAGE B. Evidence f o r M o t i v a t i o n a l E f f e c t s of Eggs and Young 125 1. Animals I n i t i a l l y Unresponsive: Pa r e n t a l Behavior A f t e r Extended Expo-sure to Young • . . 126 2. Animals I n i t i a l l y Responsive to Young: Changes i n Responsiveness to Weak S t i m u l i A f t e r Exposure to Strong S t i m u l i . . . 130 3. Animals Responsive to Young Only at P a r t i c u l a r Times: S e n s i t i v e P e r i o d s f o r E f f e c t s of Eggs and Young on Responsiveness . 131 i f . Changes i n the I n t e n s i t y of P a r e n t a l Behavior 132 5. Role of Eggs and Young i n the Maintenance of P a r e n t a l Responsiveness 133 C. The Nature of the M o t i v a t i n g S t i m u l i From the Young 136 D. The Mechanism by Which Young A f f e c t Paren-t a l Responsiveness 138 E. The Role of M o t i v a t i o n a l E f f e c t s of the Young i n R e l a t i o n to the Role of Hormones i n the Induction of P a r e n t a l Behavior . 140 F. Suggestion of a Common Pa t t e r n i n the Motiva-t i o n a l E f f e c t s of Young 143 BIBLIOGRAPHY. . . . . . 147 X LIST OF TABLES TABLE PAGE I Number of f i s h b u i l d i n g nests of d i f f e r e n t types before spawning and from 0 to 1 3 days a f t e r spawning . . . . . . . . . 3 2 I I The number of eggs s u r v i v i n g out of 1 0 0 pre-sented i n the overnight t e s t 3 9 I I I Number of f i s h developing p a r e n t a l behavior a f t e r p r e s e n t a t i o n of d i f f e r e n t numbers of stimulus eggs k7 IV The e f f e c t of the number of stimulus eggs on measures of l a t e n c y and strength of p a r e n t a l behavior i n male gouramis 4 8 V The e f f e c t of 5 m o d i f i c a t i o n s of the stimulus f o r the i n d u c t i o n of p a r e n t a l behavior on egg-ea t i n g i n h i b i t i o n i n male gouramis 6 3 VI The e f f e c t of exposure to eggs and performance of the spawning act on the i n d u c t i o n of egg-ea t i n g i n h i b i t i o n i n male gouramis without previous reproductive experience. . . . . 6 9 VTI E f f e c t s of spawning experience on p a r e n t a l behavior i n male gouramis presented with 1.5 ml eggs 7 5 VI I I Sexual dimorphism i n the p a r e n t a l behavior of gouramis 8 1 IX Mean and range of weights i n mg of s t e r o i d implanted i n t o female gouramis . 87 x i table page X P a r e n t a l behavior of female gouramis implanted vrith methyl testosterone or c h o l e s t e r o l and presented with the stimulus of 1.5 ml r e c e n t l y spawned eggs 93 XI S t a t i s t i c a l comparisons of the e f f e c t of the presence of young on the maintenance of p a r e n t a l behavior . .116 x i i LIST OF FIGURES FIGURE PAGE 1 The e f f e c t of the pH of the aquarium water at the time of spawning on egg s u r v i v a l 5 2 Diagrammatic r e p r e s e n t a t i o n of a 40 1 aquarium set up f o r spawning or t e s t i n g of gouramis. I n s e r t s show the p a r t i t i o n and s l i d i n g door separately and the f l o a t i n g p l a s t i c nest support 7 3 The r e l a t i o n s h i p between standard l e n g t h of the female and the number of eggs i n the c l u t c h r e -moved s h o r t l y a f t e r the completion of spawning. . . . 11 4 I n d i v i d u a l records of the development of p a r e n t a l behavior i n naive male gouramis . . . . . 24 5 Latencies to p a r e n t a l behavior f o l l o w i n g the f i r s t f i v e s u c c e s s f u l spavining a c t s i n male gouramis 26 6 Changes i n the frequency of p a r e n t a l and nest b u i l d i n g behavior i n male gouramis v/ith develop-i n g broods of young 30 7 Changes i n nest diameter of 6 male gouramis i n r e l a t i o n to the time of spawning 33 8 The development of r e t r i e v i n g behavior i n naive male gouramis presented with 100, 500, 1000, 2000, or 3000 r e c e n t l y spawned eggs 44 9 Changes i n p a r e n t a l behavior over the course of a 20 min re c o r d i n g i n naive male gouramis which developed strong r e t r i e v i n g 46 x i i i FIGURE PAGE 10 The e f f e c t of the number of stimulus eggs on the number of r e t r i e v i n g a c t s performed i n 20 min by naive male gouramis . k9 11 The e f f e c t of the number of stimulus eggs on the overnight s u r v i v a l of 200 t e s t eggs i n naive male gouramis 50 12 Development of p a r e n t a l behavior i n an i n e x -perienced gourami exposed to repeated presen-t a t i o n s of 100 eggs 57 13 The development of r e t r i e v i n g behavior i n response to 3000 eggs i n male gouramis which were allowed A ) no p r i o r spawning experience, or B) p r i o r spawning experience 7k Ik The r e l a t i o n s h i p between egg v i a b i l i t y (percent hatching of 100 incubated eggs) and male p a r e n t a l success (estimated percent of l i v e eggs eaten) Qk 15 I n d i v i d u a l values f o r k measures of p a r e n t a l behavior i n female gouramis implanted with methyl testosterone or c h o l e s t e r o l and pre-sented with 1.5 ml r e c e n t l y spawned eggs. 91 16 The e f f e c t of methyl testosterone and choles-t e r o l on the development of r e t r i e v i n g behavior i n female gouramis 92 17 The e f f e c t of methyl testosterone and c h o l e s t e r o l implants on the r a t i o of snout-dorsal f i n l e n g t h x i v FIGURE PAGE to standard l e n g t h (S.D.L./S.L.) i n female gouramis 95 18 P a r e n t a l behavior by cas t r a t e d (C) and sham operated (S) male gouramis presented with 2000 f r e s h l y spawned eggs 102 19 Mean nest index of 9 c a s t r a t e d and 11 sham operated male gouramis tested f o r the i n d u c t i o n of p a r e n t a l behavior 101f 20 Parental responsiveness i n male gouramis which had spawned and a) been l e f t v/ith t h e i r brood, b) had t h e i r brood placed i n a f l o a t i n g basket i n the tank, or c) had t h e i r brood removed, or which d) had not r e c e n t l y spawned 115 1 X V ACKNOWLEDGEMENTS I am very g r a t e f u l to Dr. N.R. L i l e y , my supervisor, f o r guidance at every stage of my work, f o r encouragement and patience i n the e a r l y stages and f o r much thoughtful d i s c u s s i o n and c a r e f u l c r i t i c i s m l a t e r on. Many people have contributed to making the Zoology Department of the U n i v e r s i t y of B r i t i s h Columbia a place i n which i t was p o s s i b l e and pleasant to work. In p a r t i c u l a r , I would l i k e to thank Mr. Steven Borden and Mrs. Dolores Lauriente of the Departmental computing f a c i l i t y , Mr. Dave Anderson of the Zoology Workshop, and the s t a f f of the Woodward Biomedical L i b r a r y . Part or a l l of the manuscript has b e n e f i t t e d from c r i t i c a l r eading by P. B a l l i n , J . Bryan, R. Drent, I . E f f o r d , J . Krebs, D. Randall, and A. Perks, as w e l l as N.R. L i l e y . I would a l s o l i k e to thank N. Stacey f o r much e n t h u s i a s t i c d i s c u s s i o n and fo r the ideas which lead to the development of a s u c c e s s f u l oviduct plug f o r female gouramis. The National Research Council of Canada has provided f i -n a n c i a l support f o r t h i s work through an operating grant to N.R. L i l e y and a postgraduate s c h o l a r s h i p to myself. 1 I . INTRODUCTION CHAPTER 1 INTRODUCTION TO THE PROBLEM AND METHODS A. THE PROBLEM The blue gourami, Trichogaster t r i c h o p t e r u s , i s a s m a l l , freshwater t r o p i c a l t e l e o s t f i s h , n a t i v e to Southeast A s i a . Par-e n t a l care of eggs and lar v a e i s shown by the male. When eggs are given to a p a r e n t a l male, he r e t r i e v e s them to h i s nest, but eggs given to a male not i n pa r e n t a l c o n d i t i o n are eaten. During spawning, the male's response to eggs changes from e a t i n g to par-e n t a l care i n a very short period of time. Thus there i s a dra-matic s h i f t i n the beh a v i o r a l response to a constant stimulus. This i s a p a r t i c u l a r case of a very general problem i n bio l o g y : the causation of behavior, or, more s p e c i f i c a l l y , why an ani,mal responds to a stimulus at one time and not at another. I t appeared that the study of the i n d u c t i o n of p a r e n t a l behavior i n the gourami might y i e l d i n s i g h t s i n t o the processes which c o n t r o l changes i n behavior. The present t h e s i s was undertaken to i n v e s -t i g a t e t h i s p o s s i b i l i t y . The study v/as o r i g i n a l l y intended to be an i n v e s t i g a t i o n of the hormonal c o n t r o l of pa r e n t a l behavior. However, one of the f i r s t d i s c o v e r i e s v/as that p a r e n t a l behavior could be induced by exposure to la r g e numbers of eggs without p a r t i c u l a r endocrine manipulation. This f i n d i n g p a r a l l e l e d c e r t a i n observations on several other species of f i s h , b i r d s , and mammals. I t i s sugges-ted that s t i m u l i from the eggs might be important i n the i n d u c t i o n of p a r e n t a l behavior. This f a c t o r i n the c o n t r o l of pa r e n t a l 2 behavior has been much l e s s thoroughly i n v e s t i g a t e d than the r o l e of other f a c t o r s such as hormones and p r i o r experience. I there-fore modified my approach to look at the r o l e of s t i m u l i from eggs i n the i n d u c t i o n of p a r e n t a l behavior. A f t e r an i n t r o d u c t i o n to the f i s h and the methods, the the-s i s begins with a d e t a i l e d d e s c r i p t i o n of the development and main-tenance of p a r e n t a l behavior i n the blue gourami. Subsequent chapters present experiments examining the r o l e s of eggs, p e r f o r -mance of the spawning a c t , and androgens i n these processes. Since the r o l e of eggs i n the i n d u c t i o n of p a r e n t a l behavior could be r e l a t e d to t h e i r r o l e i n i t s maintenance, a f i n a l chap-t e r presents data on the e f f e c t s of the presence of young on the maintenance of p a r e n t a l behavior. The concluding chapters attempt to r e l a t e the i n d i v i d u a l experiments to each other and to work on other species. They present some t e n t a t i v e g e n e r a l i z a t i o n s about the c o n t r o l of p a r e n t a l behavior i n ve r t e b r a t e s . B. THE ANIMAL The blue gourami belongs to the f a m i l y B e l o n t i i d a e i n the suborder Anabantoidei of the order Perciformes (Liem, 1963). The anabantoid f i s h e s are c h a r a c t e r i z e d by a suprabranchial r e s p i r a -t o r y chamber which permits the u t i l i z a t i o n of atmospheric a i r . Information on many aspects of t h e i r b i o l o g y i s provided i n the major monograph of F o r s e l i u s (1957a,b,c). Taxonomic r e l a t i o n -ships and phylogeny of the group have r e c e n t l y been reviewed by Liem (1963). The f i s h used i n t h i s study were the common l i g h t blue race of the blue gourami, r e f e r r e d to by M i l l e r (196i+) as Trichogaster 3 trichopterus sumatranus. The o r i g i n a l stock was purchased from aquarium dealers i n Vancouver. A l l experimental animals were raised i n the laboratory. In order to prevent inbreeding, broods of young to be raised for experiments were always offspring of one of our lab stock mated with a newly purchased i n d i v i d u a l . C. THE METHODS 1. Culture In order to provide experimental f i s h , the offspring of a single spawning were reared together i n a large tank of 140 to 240 1 capacity. As the brood developed, smaller i n d i v i d u a l s were culled out, and f i s h were divided among several large tanks. In order to control reproductive experience, males were separated from the brood as they developed elongated dorsal f i n s , a male secondary sex character. Maturation began at 10 weeks, but many f i s h required longer. Under good conditions f i s h remained i n breeding condition for more than two years. 2. Maintenance The f i s h were kept i n 4-0 1 glass and stainless steel aquaria or larger tanks constructed of fiberglass-coated plywood, which was painted on the inside with "Rustoleum" grey paint. A l l ex-periments were carried out i n 40 1 tanks. Tanks were l i t by cool white fluorescent l i g h t s controlled by automatic timers. Only lov; l e v e l s of natural l i g h t entered the room from p a r t i a l l y shielded windows. The l i g h t period was from 7:00 to 19-30 or from 11:00 to 23:30. 4 The p r i n c i p a l water source was Vancouver c i t y tap water. In newly set up tanks the water was allowed to stand f o r 24 hr be-fore f i s h were added, but no i l l e f f e c t s were observed when up to one t h i r d of the water i n e s t a b l i s h e d tanks was replaced w i t h cold tap water. In the i n i t i a l part of the study approximately 2-3 g of sea s a l t were added per 40 1. Later, about 3 g or more of marble chips were added as w e l l . F i n a l l y , i t was found that egg s u r v i v a l was improved i f about 5 g of sodium bicarbonate (100 ml of a 50 g / l s o l u t i o n ) was added per tank. For the most p a r t , I was not concerned w i t h water q u a l i t y . However, c e r t a i n aspects of i t were considered i n more d e t a i l during two periods i n which the f i s h spawned at much reduced f r e -quencies and with low egg v i a b i l i t y . In the f i r s t p e riod at the s t a r t of my study I obtained i n d i r e c t evidence that tap water was i n h i b i t o r y to spawning i f i t had been i n d i r e c t contact w i t h wood coated with f i b e r g l a s s r e s i n ("Esterex 101") . In a l a t e r period spawning success seemed b e t t e r f o r a time i n water from the de-partmental dechl o r i n a t e d water supply than i n tapwater. In a t e s t of the e f f e c t s of pH I found that spawnings took place at every pH te s t e d from 4 . 0 to above 8 . 0 , but that egg s u r v i v a l was r e -duced at the lower pH values ( F i g . 1 ) . . Gouramis reduced the pH of the water i n which they l i v e d . I t dropped as low as 3*5 i n crowded stock tanks. Though the f i s h survived such low l e v e l s very w e l l , marble chips and bicarbonate were added i n an attempt to improve egg v i a b i l i t y by keeping the pH above 5 . 0 . The p r i n c i p a l food was frozen brine shrimp which was de-f r o s t e d before use. This was the sole food during experiments 5 e>IOO -x o < X LU O or UJ Q . 75 50 -25 " O 6 7 P H 8 FIGURE 1. The e f f e c t of the pH of the aquarium water at the time of spawning on egg s u r v i v a l . Each point rep-resents the percent hatching success of approxi-mately 100 eggs from a d i f f e r e n t spawning. 6 except f o r p l a n t s or small i n v e r t e b r a t e s that the f i s h might f i n d i n the aquaria. F i s h i n stock tanks r e c e i v e d both bri n e shrimp and e i t h e r commercial t r o u t chow or "TetraMin Staple Flake Food." The cheapest and most convenient food f o r young f r y was s t r a i n e d l i v e r ("Heinz" baby food). A small amount of t h i s was suspended i n aquarium water and s t i r r e d i n t o the tank. As the f i s h grew l a r g e r they were g r a d u a l l y switched to f i n e l y ground t r o u t chow and frozen b r i n e shrimp. Temperatures were kept at 25° to 27° by means of i n d i v i d u a l t h e r m o s t a t i c a l l y c o n t r o l l e d heaters. A l l aquaria were equipped with sub-gravel f i l t e r s covered with "DelMonte" white or brown sand. They were planted w i t h an aquatic f e r n c a l l e d water s p r i t e , C e r a t o p t e r i s t h a l i c t r o i d e s . 3. Induction of Spawning Johns (1966) developed a r e l i a b l e way to induce spawning i n gouramis. The procedure which I followed was s l i g h t l y modified from h i s . To prepare tanks f o r spawning the sand was cleaned and f r e s h water added. Water s p r i t e p l a n t s were planted i n each r e a r corner and the tank was d i v i d e d by a black p l e x i g l a s s p a r t i t i o n having a s l i d i n g door. For cover and a framework f o r the nest, s e v e r a l f l o a t i n g water s p r i t e p l a n t s or an a r t i f i c i a l nest sup-port were provided. The l a t t e r was a 21+ x 13 cm sheet of t h i n , green polyethelene w i t h many t r i a n g u l a r spaces, roughly 2-3 cm on a s i d e , cut i n t o i t . Figure 2 shows d i a g r a m a t i c a l l y the com-ponents of the spawning set up. A male was added to one side of a tank set up i n t h i s fash-i o n . One or more days l a t e r when he had a nest, a female was 7 H E A T E R N E S T S U P P O R T AIR S U P P L Y S U B - G R A V E L F I L T E R H A R 6 E E 6 G P R E S E N -TATI ON F R A M E AQUARIUM SET UP FOR SPAWNING OR TESTING c. PLASTIC . NEST SUPPORT B. PARTITION WITH SLIDING DOOR 1 0 C M FIGURE 2. Diagrammatic r e p r e s e n t a t i o n of a 4-0 1 aquarium set up f o r spawning or t e s t i n g of gouramis (A). I n s e r t s show the p a r t i t i o n and s l i d i n g door separately (B), and the f l o a t i n g p l a s t i c nest support (C). A l l are drawn to the same scale as i n d i c a t e d by the 1 0 cm l i n e . - 8 added to the other side of the tank. On the next afternoon the p a r t i t i o n was opened. Spawning u s u a l l y occurred on the f o l l o w i n g day. I f repeated spawnings were d e s i r e d , the male was l e f t a f t e r spawning, and the female was replaced. The p a r t i t i o n was opened one day l a t e r and the c y c l e repeated. From time to time, some of the water was replaced w i t h f r e s h tapwater. In t h i s way spawning could be obtained on a l t e r n a t e days f o r long periods of time. Success was v a r i a b l e , . and 3 or more a c t i v e males were u s u a l l y set up to be c e r t a i n of eggs on a given day. To b r i n g males and females i n t o spawning c o n d i t i o n but pre-vent spawning, the above procedure was followed except that the p a r t i t i o n s were closed about 1 hr a f t e r the l i g h t p eriod began on the day of expected spawning. Few spawnings began t h i s e a r l y , but the f i s h apparently had had s u f f i c i e n t contact to b r i n g them i n t o spawning c o n d i t i o n . P a i r s i n spawning c o n d i t i o n could be d i s t i n g u i s h e d l a t e r i n the day by the darker c o l o r and increased a c t i v i t y of the females. Such females u s u a l l y i n i t i a t e d spawning immediately a f t e r the p a r t i t i o n was opened. I found no evidence of p a i r bonding or of a p a r e n t a l phase i n which sexual behavior was not shown. Thus females could be switched from tank to tank, and males could be expected to spawn even i f they had some eggs or f r y from a previous spawning. The c o n t r o l l i n g f a c t o r i n spawning appeared to be the occurrence of o v u l a t i o n by the females. Most males seemed ready to spawn at any time, but o c c a s i o n a l l y males were seen to respond only aggres-s i v e l y to repeated approaches by r i p e females. 9 k. C o l l e c t i o n of Eggs Many experiments r e q u i r e d the p r e s e n t a t i o n of eggs to f i s h which had not spawned. A f t e r spawning by donor p a i r s , t h e i r eggs were removed with a beaker dipped under the surface near the nest. The eggs were concentrated and separated from p l a n t m a t e r i a l by pouring them through a piece of f i b e r g l a s s screen i n t o a f i n e meshed net held i n a beaker of aquarium water. A squeeze b o t t l e f i l l e d w ith aquarium water was used to wash the eggs to the bot-tom of the net. Then the eggs were r i n s e d i n t o a container of aquarium water. Batches of up to 800 eggs were counted. The eggs were placed i n a p e t r i d i s h over a black background and counted as they were picked up with an eyedropper p i p e t t e . Larger numbers of eggs v/ere u s u a l l y determined v o l u m e t r i c a l l y . A s e c t i o n of a 10 ml p i p e t t e with i t s lower end covered by f i n e n e t t i n g was placed upright i n a beaker of aquarium water. Eggs were added by means of an eyedropper p i p e t t e . A f t e r they had f l o a t e d to the top, the volume was determined, and the eggs were placed i n a 10 ml beaker. From s e v e r a l counts, 1000 eggs were considered equiva-l e n t to 0.5 ml. I n d i v i d u a l counts were subject to e r r o r s due to the eggs clumping together.. Counts were u s u a l l y not double-checked be-cause of the requirements of speed, and because i t seemed that damage to eggs from excessive handling was p o t e n t i a l l y a greater source of e r r o r than miscounting. The volumetric method u s u a l l y d i d not appear to damage the eggs. However, m o r t a l i t y was sometimes greater than expected, e s p e c i a l l y when eggs were not removed from the p i p e t t e as soon 10 as they had reached a s t a b l e volume. The number of eggs which could be expected from each donor p a i r was h i g h l y v a r i a b l e . In one experiment i n which the number of eggs per c l u t c h was counted, the range was about 5°° to 2500 with a mean of about 1500. The number of eggs produced seemed r e l a t e d i n part to female l e n g t h ( F i g . 3 ) . Stimulus eggs were presented to a f i s h i n a square f l o a t i n g frame made of foam rubber 10 cm on a side and 2.5 cm deep. This frame f l o a t e d j u s t at the surface so that eggs were prevented from d r i f t i n g about the tank. To keep the eggs from s i n k i n g as they were being added to the tank, they were poured i n t o a small p l a s t i c v i a l cap held j u s t under the surface. This was freed of eggs and then removed. 5. S u r g i c a l Procedures This s e c t i o n concerns some general c o n s i d e r a t i o n s of s u r g i -c a l procedure. D e t a i l s are discussed i n the appropriate chap-t e r s . I n the course of the study I developed the impression that anesthesia was sometimes more s t r e s s f u l f o r the f i s h than the a c t u a l s u r g i c a l operation. For simple operations such as p e l v i c f i n amputation and hormone p e l l e t i m p l a n t a t i o n , the f i s h could be r e s t r a i n e d by wrapping i n a damp paper towel. For measurements of le n g t h , i t was found t h a t , i f excess water were removed from a f i s h by b r i e f l y p l a c i n g i t on a damp paper towel, i t would u s u a l l y remain quiescent when placed on a dry pane of gl a s s . The s a l i n e s o l u t i o n used i n c a s t r a t i o n experiments was de-r i v e d from Cortland's s a l t s o l u t i o n (Wolf, 1963). I t consisted 11 X o K => - J o co CD e> UJ u. o oc U i m 2500, 15 OQ 50QJ E X P E R I E N C E O F F E M A L E # P R E V I O U S E X P E R I E N C E O F I R S T S P A W N I N G O <5>o ° o o o o . o ~ o I 5.0 o o 6.0 7.0 S T A N D A R D L E N G T H O F F E M A L E CC M J FIGURE 3. The r e l a t i o n s h i p between standard l e n g t h of the female (the distance between the t i p of the upper jaw and the crease at the j u n c t i o n of the median caudal f i n rays and the median hypural p l a t e ) and the number of eggs i n the c l u t c h removed s h o r t l y a f t e r the completion of spawning. F i l l e d c i r c l e s represent older females which had spawned pre-v i o u s l y . Open c i r c l e s represent s i x month o l d females spawning f o r the f i r s t time. 12 of the f o l l o w i n g concentrations of s a l t s ( g / l ) : NaCl - 7.25, KC1 - 0.38, C a C l 2 - 0.162, MgSCy7H 20 - 0.23, NaHCO^ - 1.0, NaHgPCy 2H 20 - O.Zfl. Glucose was not added. 6. Behavioral Observations F i s h were observed from a distance of about 1 m or l e s s . A p o s i t i o n s l i g h t l y lower than the tank f a c i l i t a t e d observation of the surface. To record behavior e i t h e r a ^-channel "Rustrak" r e -corder or a 20-channel "Esterline-Angus" event recorder was used. Both ran at a speed of 3 in/min and were equipped with a keyboard arranged so that pens of the recorder were d e f l e c t e d as long as they keys v/ere held down. D e t a i l s of the b e h a v i o r a l d e f i n i t i o n s , l e n g t h of r e c o r d i n g sessions, e t c . , w i l l be given i n the appro-p r i a t e chapters. D. SUMMARY 1. The blue gourami i s a f r e s h water t r o p i c a l f i s h i n which pa r e n t a l behavior i s shown by the male. 2. A r a p i d change i n the response to eggs occurs at the time of spawning. Eggs are eaten i f presented before spawning and r e -t r i e v e d i f presented a f t e r spawning. 3. The t o p i c of t h i s t h e s i s i s the nature of f a c t o r s governing t h i s change. 4. The chapter presents the p r i n c i p a l experimental procedures and methods used f o r the c u l t u r e and maintenance of the f i s h . 1 3 CHAPTER 2 REPRODUCTIVE BEHAVIOR OF THE BLUE GOURAMI This chapter describes the reproductive behavior of the blue gourami w i t h an emphasis on the development and maintenance of p a r e n t a l behavior. " P a r e n t a l behavior" w i l l be used to r e f e r s p e c i f i c a l l y to those p a t t e r n s i n v o l v i n g care of eggs or young. Nest b u i l d i n g w i l l be considered separately. M i l l e r ( 1 9 6 4 ) and Johns ( 1 9 6 6 ) have given d e t a i l e d d e s c r i p t i o n s of sexual, agonis-t i c and nest b u i l d i n g behavior, but they discussed p a r e n t a l be-havior only b r i e f l y . The f o l l o w i n g d e s c r i p t i o n s are based on t h e i r r e p o r t s and extensive observations of my own. S p e c i f i c ; behavior p a t t e r n s w i l l be c a p i t a l i z e d at the time they are f i r s t mentioned. A. QUALITATIVE DESCRIPTION 1 . Nest B u i l d i n g The nest c o n s i s t s of a mound of foam among p l a n t s at the surface of the v/ater and serves as the focus of reproductive ac-t i v i t y . Nest b u i l d i n g i s a prominent a c t i v i t y before spawning, between spawning bouts, and during the p a r e n t a l p e r i o d . The nest i s composed of bubbles which are formed i n two ways. In BUBBLE BLOWING BENEATH THE SURFACE the male takes one or more gulps of a i r at the surface, then swims under the nest where he r e l e a s e s a cloud of f i n e bubbles. In BUBBLE BLOWING AT THE SURFACE the male re l e a s e s a small group of bubbles a f t e r each gulp of a i r while remaining at the surface. This i s u s u a l l y repeated i n long s e r i e s . Bubble blowing at the surface i s l e s s l i k e l y to be o r i e n t e d to a s i n g l e s i t e than i s bubble blowing beneath the sur-face. These two patterns of bubble blowing are d i s t i n g u i s h e d from SURFACE MOUTHING i n which a male repeatedly gulps at the sur-face but does not r e l e a s e bubbles, and from AIR BREATHING, which i n v o l v e s only b r i e f contact w i t h the surface and a prominent move-ment of the f l o o r of the buccal c a v i t y . Nest q u a l i t y was recorded according to the f o l l o w i n g s c a l e : No nest. T r a c e — s m a l l groups of bubbles, none greater than 1 cm • i n diameter. N l — s e v e r a l separate, t h i n patches of foam. N 2 — s i n g l e , l a r g e patch of foam. N 3 —a s i n g l e patch of foam, t h i c k enough so t h a t the upper surface i s r a i s e d above the surface of the water at the center. Sometimes a f i s h b u i l t a nest consisted of a l a r g e patch of foam as w e l l as s e v e r a l smaller patches (N2 or N3 + 1 ) . 2. Spawning In p a i r s of f i s h ready to spawn the female i s u s u a l l y forced i n t o h i d i n g by the aggressiveness of the male. The male spends the m a j o r i t y of h i s time i n nest b u i l d i n g . T y p i c a l l y , i t i s the female that i n i t i a t e s a spawning sequence by l e a v i n g her h i d i n g place, d a r t i n g toward the male, and BUTTING him once or twice i n the s i d e . In response to t h i s the male u s u a l l y darkens and DIS-PLAYS with the medial f i n s spread and the body t i l t e d , heat up-wards. The male slowly moves to a p o s i t i o n j u s t under the nest while the female remains very c l o s e to him. The d i s p l a y gives 15 way to RUBBING i n which the male moves back and f o r t h , c o n t a c t i n g the female's a n t e r o - v e n t r a l surface w i t h h i s mid-dorsal surface. Rubbing leads to ENCIRCLING as the male forms a curve and then cl o s e s the curve to form a t i g h t CLASP. A f t e r a few seconds he begins to QUIVER. Fine v i b r a t i o n s of h i s whole body increase i n amplitude u n t i l , at the peak, the p a i r ROLLS OVER so that t h e i r vents are d i r e c t e d upward toward the nest as eggs are r e l e a s e d . (OVIPOSITION). Presumably, sperm i s a l s o released at t h i s time. The p a i r d r i f t s p a s s i v e l y downward without swimming f o r a few seconds. The male u s u a l l y recovers f i r s t and v i g o r o u s l y BITES and CHASES the female u n t i l she i s once more i n h i d i n g . Sequen-. ces do not always go to completion. And even when they are be-h a v i o r a l l y complete, eggs are not always r e l e a s e d . The female u s u a l l y r e l e a s e s her f u l l complement of eggs i n a s e r i e s of at l e a s t 5 spawning sequences d i s t r i b u t e d over an hour or more. Some aspects of spawning are i l l u s t r a t e d by M i l l e r (1964). 3. P a r e n t a l Behavior A f t e r a spawning a c t , the male r e t u r n s from chasing the f e -male and begins bubble blowing or one of s e v e r a l patterns i n v o l -v i n g contact w i t h the eggs. When the eggs are i n the nest the male spends long periods i n MOUTHING. When mouthing, the male moves about slowly under the nest, p i c k i n g up mouthfuls of eggs and foam and r e l e a s i n g them again. He may perform chewing move-ments while he holds eggs and foam i n h i s mouth. Eggs picked up while mouthing may be released immediately at the surface, or the male may drop down and re l e a s e them under the nest. Releases may be or i e n t e d to one p a r t i c u l a r area and r e s u l t i n the c o n s o l i d a t i o n 16 of the egg mass. The f u n c t i o n of mouthing may be to mix the eggs with the foam i n order to hold them more securely i n the nest, or i t may a l l o w the male to s o r t out dead eggs which are eaten. Males which are mouthing may a l s o begin SPOUTING, that i s , they o r i e n t s t e e p l y upward wi t h the jaws w e l l i n t o the foam of the nest, then with strong opercular movements, they send a spout of water above the surface f o r a b r i e f p e r i o d . The f u n c t i o n of t h i s i s not known. The male RETRIEVES eggs which d r i f t away from the nest or which are not released d i r e c t l y i n t o the foam because of the p o s i t i o n of the spawning p a i r . He u s u a l l y p i c k s up s e v e r a l eggs per t r i p . Once at the nest, he may simply open h i s mouth to r e -lease the eggs as he swims past, or pause under the nest and .blow the eggs upwards, or swim d i r e c t l y to the nest and begin mouthing. A r a p i d opening and c l o s i n g of the jaws i s c h a r a c t e r i s t i c of the r e l e a s e of eggs. The r e t r i e v i n g of eggs xs u s u a l l y accompanied by mouthing. Larvae and f r y are simply r e l e a s e d under the nest. Older f r y t r y to avoid the male and are captured by a vigorous s t r i k e which I have c a l l e d SNAPPING. Only p a r e n t a l males are i n h i b i t e d from e a t i n g eggs and young. Females and non-parental males eat them eagerly. if. D e s c r i p t i o n s and D e f i n i t i o n s of Behavior Used i n Recording The d e s c r i p t i o n s of p a r e n t a l behavior l i s t e d i n the pre-vious s e c t i o n could not always be a p p l i e d d i r e c t l y i n q u a n t i t a -t i v e r e c o r d i n g of a f i s h ' s a c t i v i t y . The problem arose because eggs were not v i s i b l e at a l l times. I f a f i s h ' picked up eggs i n h i s mouth, an observer could not determine whether he was e a t i n g 17 them or performing the i n i t i a l step of egg r e t r i e v a l . I f a f i s h , a f t e r p i c k i n g up eggs, contacted the foam of the nest v/ith h i s mouth, an observer could not determine whether he had actually-released eggs. Thus c e r t a i n behavior p a t t e r n s had to be defined on the b a s i s of the more r e a d i l y observable behavior thai; was u s u a l l y c o r r e l a t e d v/ith the a c t i v i t y of i n t e r e s t . This s e c t i o n d escribes the conventions used i n r e c o r d i n g the p r i n c i p a l pat-t e r n s of p a r e n t a l behavior. a. Mouthing - Mouthing was c h a r a c t e r i z e d by contact of the f i s h ' s jaws with the foam of the nest. Therefore, e i t h e r the frequency or p r o p o r t i o n of time spent i n c o n t a c t i n g the nest were used as measures of mouthing. One p o t e n t i a l inaccuracy was that some nest contact might occur at other times such as i n nest b u i l d i n g . Considering only bouts of nest contact longer than 2 sec e l i m i n a t e d t h i s contamination, but also e l i m i n a t e d many shorter bouts of mouthing. b. R e t r i e v i n g - A t y p i c a l r e t r i e v i n g act consisted of p i c k -i n g up eggs, swimming to the nest, pausing b r i e f l y , o r i e n t i n g upv/ard toward the nest, and r e l e a s i n g eggs v/hile performing a r a p i d opening and c l o s i n g of the jaws or v/hile mouthing. Some-times, however, a f i s h would perform a l l or part of the sequence without a c t u a l l y r e l e a s i n g eggs. Or a f i s h might r e l e a s e eggs as he was p i c k i n g them up, or swim a distance and then r e l e a s e them i n midwater without any o r i e n t a t i o n to the nest. These patterns approximated r e t r i e v i n g , but d i d not appear to be genu-in e r e t r i e v i n g , because they were seen i n f i s h which, upon i n -spec t i o n of the nest, v/ere found to have been e a t i n g eggs. 18 I n i t i a l l y , I considered these " f a l s e r e t r i e v i n g a c t s " to be " a c c i d e n t a l , " i . e . , the f i s h j u s t happened to contact the nest a f t e r p i c k i n g up eggs, or he picked up too many eggs and r e l e a s e d some while t r y i n g to swallow the r e s t . L a t e r observations sug-gested that they should be considered as " i n t e n t i o n movements" or incomplete r e t r i e v i n g a c t s (See Ch. 3C). I was not s u c c e s s f u l i n f i n d i n g a d e f i n i t i o n f o r the record-i n g of r e t r i e v i n g which excluded a l l incomplete r e t r i e v i n g a c t s . In most experiments a f i s h was considered to have r e t r i e v e d i f , a f t e r p i c k i n g up eggs, he approached h i s nest (or a nest support i f no nest was present) and e i t h e r contacted the nest, v i s i b l y r eleased eggs, or performed the jaw movements that u s u a l l y accom-panied egg r e l e a s e . Jaw movements were very r a r e i n f i s h which were l a t e r found to have no eggs i n t h e i r nests. Releases of eggs were not counted as r e t r i e v i n g acts i f they were not accom-panied by o r i e n t a t i o n to a nest, except i n one experiment concern-i n g the e f f e c t s of androgens on females (Ch. 6B). In that ex-periment, any egg release away from the frame where the eggs had been presented was considered a r e t r i e v i n g a c t . c. Egg Contact - In many experiments stimulus eggs were presented to f i s h i n a f l o a t i n g frame made of foam rubber. F i s h approached and began to eat the eggs or p i c k them up to r e t r i e v e . Because I could not d i s t i n g u i s h the two p a t t e r n s by any d i f f e r -ence i n the way the eggs were picked up, I had to record simply contact with the eggs. Since the eggs were sometimes not v i s i b l e because of t h e i r small s i z e or because the frame blocked the view, egg contact was recorded whenever the f i s h was i n the 19 p r e s e n t a t i o n frame w i t h h i s jaws i n contact w i t h the surface of the water. d. Egg-Eating I n h i b i t i o n - Since non-parental males eat eggs while p a r e n t a l males r e t r i e v e them, the s u r v i v a l of a knov/n number of eggs w i t h a male could be used as a measure of p a r e n t a l behavior. A given number of l i v e eggs were placed i n the tank vrith a male. Their s u r v i v a l was determined on the next day by removing the nest and f l o a t i n g p l a n t s and c a r e f u l l y checking f o r the presence of eggs. In some cases a few eggs were recovered from tanks of males that had been e a t i n g eggs. Apparently such eggs had been overlooked because they had become lodged among the leaves of the f l o a t i n g p l a n t s . Therefore, i n some experiments a c r i t e r i o n was set up so that a male was not considered to be showing egg-eating i n h i b i t i o n unless 25 or more eggs were recov-ered. The number of t e s t eggs v a r i e d i n d i f f e r e n t experiments. At f i r s t 100 eggs were used. Later 200 eggs were presented because i t seemed that t h i s would provide a c l e a r e r d i s t i n c t i o n between p a r e n t a l and non-parental males, i n case p a r e n t a l males ate some of the f i r s t eggs they picked up. However, i n both cases the s u r v i v a l r a t e was o f t e n only about 50 per cent, even with males i n p a r e n t a l c o n d i t i o n . P o s s i b l e reasons f o r t h i s are discussed i n Chapter 6A. S p e c i f i c d e t a i l s of the t e s t s are presented i n the i n d i v i d u a l experiments. 20 5. Development of the Young Freshly-spawned eggs are buoyant and c l e a r to l i g h t y e l l o w i n c o l o r . They are n e a r l y round and measure about 0.83 nmi i n the greatest diameter. Because of t h e i r s i z e and c o l o r they are a t f i r s t d i f f i c u l t to d i s t i n g u i s h w i t h i n the foam of the nest, but they become darker as the melanophores develop. Hatching takes place about 2if hr a f t e r f e r t i l i z a t i o n . Hodges and Behre (1953) have provided a d e t a i l e d d e s c r i p t i o n of development to hatching. Newly hatched l a r v a e f l o a t upside down under a l a r g e y o l k sac. They r e s t i n contact v/ith p l a n t s or the lower surface of the nest. Disturbance u s u a l l y r e s u l t s i n a b r i e f period of r a p i d and apparently random swimming followed by a r e t u r n to the sur-face. One day a f t e r hatching the l a r v a e measure about 2.8 mm. They remain i n contact w i t h the nest or p l a n t s . They respond b r i e f l y to a l i g h t disturbance by v/r i g g l i n g b r i e f l y about. In response to stronger s t i m u l a t i o n they swim d i r e c t l y to the bottom where they may remain f o r some minutes before r e t u r n i n g to the surface. By the t h i r d day a f t e r f e r t i l i z a t i o n the f r y are 3*4 mm long and mostly free swimming, but they s t i l l remain near the surface among the f l o a t i n g p l a n t s . They dart away from strange objects and a l s o attempt to avoid the male, but no longer dive to the bottom. From then on the course i s slow development; the f r y move away from the surface g r a d u a l l y u n t i l they may be found at a l l l e v e l s i n the tank. 21 B. A QUANTITATIVE STUDY OF THE NATURAL INDUCTION OF PARENTAL BEHAVIOR 1. I n t r o d u c t i o n The purpose of t h i s study was to r e c o r d the i n i t i a l develop-ment of p a r e n t a l behavior i n gouramis spawning f o r the f i r s t time. I was p a r t i c u l a r l y i n t e r e s t e d i n the amount of sexual be-havior and the d u r a t i o n of exposure to eggs that preceded the on-set of pa r e n t a l behavior and the p a t t e r n of frequency changes i n parental behavior that took place a f t e r the f i r s t p a r e n t a l act had been shown. These measures might y i e l d i n s i g h t s i n t o the r o l e of spawning and eggs i n the i n d u c t i o n of p a r e n t a l behavior. 2. Methods Six 7 month o l d males without previous reproductive e x p e r i -ence were brought i n t o spawning c o n d i t i o n , but prevented from s t a r t i n g to spawn by c l o s i n g the p a r t i t i o n i n the morning before spawning began. Later i n the day, when the f i s h were ready to spawn, a 10 to 15 min r e c o r d i n g of the male's behavior was made. Then the p a r t i t i o n was opened and a continuous record was made of sexual, aggressive, nest b u i l d i n g and p a r e n t a l behavior u n t i l 1 hr a f t e r the f i r s t egg r e l e a s e . The f o l l o w i n g behavior was recorded on the keyboard of a 20 channel E s t e r l i n e Angus recorder with a chart speed of 3 in/min: 1. Butt by female 2. Male d i s p l a y 3. Rubbing 4. E n c i r c l i n g 22 Clasping 6. Quivering 7. R o l l i n g over 8. Ovi p o s i t i o n 9. Chasing and b i t i n g the female 10. Egg contact 11. Egg re l e a s e 12. Nest contact 13. Bubble r e l e a s e at surface 14. Bubble r e l e a s e beneath surface The complete record formed a s e r i e s of bouts of sexual and aggressive behavior separated by i n t e r v a l s of v a r i a b l e l e n g t h i n which the female was h i d i n g . For each sexual-aggressive bout, I noted whether o v i p o s i t i o n had occurred and, i f not, what was the most advanced element of sexual behavior performed i n t h a i bout. For each i n t e r v a l , i n c l u d i n g the rec o r d i n g before opening the p a r t i t i o n , the frequency of bubble blowing beneath the surface, bubble blowing at the surface and nest contact was c a l c u l a t e d separately. In a d d i t i o n , a f t e r sexual bouts y i e l d i n g eggs, I de-termined two measures of the latency from exposure to eggs to the s t a r t of pa r e n t a l behavior. The f i r s t measure was the i n t e r v a l from the s t a r t of o v i p o s i t i o n u n t i l the f i r s t egg contact. (This included the b r i e f period without swimming as w e l l as chasing the female and r e t u r n i n g to the nest.) The second measure was the i n t e r v a l from the f i r s t egg contact to the f i r s t p a r e n t a l act, i . e . , mouthing or egg r e l e a s e . (This i n c l u d e d e a t i n g the eggs and time spent swimming from where the eggs were picked up : to where they were released.) 23 3 . R e s u l t s and D i s c u s s i o n In a l l p a i r s sexual a c t i v i t y began as soon as the p a r t i t i o n was opened ( F i g . 4). In 2 cases t h i s continued to o v i p o s i t i o n . In the other k cases v a r i a b l e amounts of sexual behavior preceded the f i r s t s u c c e s s f u l spawning. P a r e n t a l responses f o l l o w i n g contact w i t h eggs were essen-t i a l l y immediate, a l l f i s h mouthing or r e l e a s i n g eggs w i t h i n 5 sec ( F i g . 5B). There was no evidence of any egg-eating at the f i r s t contact w i t h eggs, though i t was d i f f i c u l t to be c e r t a i n of t h i s , because a f i s h that was apparently mouthing could a l s o be e a t i n g eggs. The o c c a s i o n a l l o n g i n t e r v a l s between egg contact and r e l e a s e f o l l o w i n g l a t e r spawning a c t s might i n d i c a t e that some eggs were being eaten. S u r p r i s i n g l y , males d i d not always appear to immediately de-t e c t eggs i n t h e i r nest a f t e r the f i r s t s u c c e s s f u l spawning. They returned from chasing the female and began to nest b u i l d again, apparently f i r s t c o n t a c t i n g the eggs a c c i d e n t l y . With subsequent spawnings they seemed to "expect" the presence of eggs. This i s r e f l e c t e d i n the shorter i n t e r v a l s bet\veen o v i -p o s i t i o n and egg contact and subsequent spawnings ( F i g . 5A). Mouthing, as measured by the frequency of nest contacts, began w i t h high frequency only a f t e r o v i p o s i t i o n ( F i g . k)• Even spawning a c t s which were complete except f o r the r e l e a s e of eggs di d not a f f e c t the frequency of nest contact. Two males began to show high r a t e s of nest contact a f t e r the f i r s t o v i p o s i t i o n . The others d i d not do so u n t i l a f t e r t h e i r second o v i p o s i t i o n . Even a f t e r c o n t a c t i n g the eggs, they b r i e f l y mouthed them or r e t r i e v e d and then paid l i t t l e f u r t h e r a t t e n t i o n to them. In 24 FIGURE k. I n d i v i d u a l records of the development of p a r e n t a l behavior i n naive male gouramis (A-F). For each f i s h the record was. d i v i d e d i n t o bouts of sexual and aggressive behavior and bouts of nest b u i l d i n g and p a r e n t a l behavior during which the female was h i d i n g . The numbers on the lower l i n e i n d i c a t e , the l e n g t h of bouts i n minutes. Bouts of sexual behavior which l e d to o v i p o s i t i o n are i n d i c a t e d on the next l i n e by f i l l e d squares. For bouts which d i d not l e a d to o v i p o s i t i o n , the most com-p l e t e element of sexual behavior performed i s i n -dic a t e d by a l e t t e r . The groups of v e r t i c a l bars i n d i c a t e the frequency of bubble blowing beneath the surface, bubble blowing at the surface, and nest contact. The f i r s t bout of nest b u i l d i n g and pa r e n t a l behavior i s the one taken before opening the p a r t i t i o n , which i s i n d i c a t e d by a v e r t i c a l l i n e . NUMBER OF ACTS PER MINUTE 26 L E N G T H OF I N T E R V A L 3.0-c o . 2 . 0 -1.0 • A. OVIPOSITION-EGG CONTACT 0 C6.0) 1 C5.C) 0 . 3 • 0 . 2 0.1 B. EGG C O N T A C T -P A R E N T A L BEHAVIOR C0.8) JP—V A 3 4 N U M B E R 5 1 2 3 4 O F S P A W N I N G A C T FIGURE 5. Latencies to pare n t a l behavior f o l l o w i n g each of the f i r s t f i v e s u c c e s s f u l spawning a c t s i n male gouramis, A) I n t e r v a l between o v i p o s i t i o n and subsequent con-t a c t w i t h eggs. B) I n t e r v a l between contact w i t h eggs and the s t a r t of mouthing or r e t r i e v i n g . 27 most cases mouthing occurred at high frequency from the s t a r t . The r a t e was often maximal or n e a r l y so a f t e r the f i r s t or sec-ond o v i p o s i t i o n . Several c o n s i s t e n t changes i n nest b u i l d i n g behavior occur-red. Nest b u i l d i n g was g e n e r a l l y i n f r e q u e n t before opening the p a r t i t i o n and c o n s i s t e d predominantly of bubble blowing at the surface. A f t e r incomplete spawning, high frequencies of bubble blowing beneath the surface were performed ( F i g . /+B,D,E,F). These frequencies were much greater than those observed before opening the p a r t i t i o n or i n the p a r e n t a l phase (See s e c t i o n C). When high r a t e s of nest contact began, bubble blowing beneath the surface was depressed, but v a r i a b l e amounts of bubble blowing at the surface occurred. C. A QUANTITATIVE STUDY OF THE MAINTENANCE OF PARENTAL AND NEST BUILDING BEHAVIOR 1. I n t r o d u c t i o n The purpose of t h i s study was to record q u a n t i t a t i v e changes i n p a r e n t a l and nest b u i l d i n g behavior of male gouramis l e f t with a developing c l u t c h of eggs f o r 13 days. : 2 . Methods The subjects were s i x 15 month o l d adult males without pre-vious reproductive experience. The standard procedure v/as used to induce them to spawn. Only a small number of v i a b l e young r e s u l t e d from the f i r s t spawning of one male, so a second spawn-i n g was allowed. A f t e r spawning had been completed, the female 28 was removed and the p a r t i t i o n was l e f t open. Observations of par-ental and nest building behavior were made before opening the par-t i t i o n on the day before spawning (Prespawning), immediately a f t e r the completion of spawning (Day 0) , on the morning and afternoon of the next two days (Days l a , l b , 2a, and 2b), on the afternoon of Days 3, k, 7, 10, and 13-At each observation period a 15 min continuous record was made of parental and next building behavior, the diameter and quality of the nest was recorded, and a " r e t r i e v i n g teat" v/as given where applicable ( i . e . , a f t e r spawning and before dispersal of the f r y ) . By recording a) contact with the nest (or eggs v/here there was no nest), b) gulping a i r at the surface, c) bubble or egg re-lease beneath the surface, d) bubble release at the surface, and e) snapping, the following behavioral measures could be determined: 1. Frequency of nest contact 2. Frequency of prolonged nest contact (2 sec or longer) 3. Total duration of long nest contact A. Frequency of release beneath the nest a f t e r nest contact 5. Frequency of snapping 6. Frequency of bubble blowing beneath surface 7. Frequency of bubble blowing at surface. For the r e t r i e v i n g test 50 to 100 eggs were displaced from the nest. Usually they were placed on the opposite side of the p a r t i t i o n , but most males ra r e l y swam that far from the nest on Days 0 and 1, and the eggs had to be moved closer. Dispersal 29 of the f r y and c e s s a t i o n of the male's responses to them occurred together, and no r e t r i e v i n g t e s t s were p o s s i b l e from then on. An index of the area covered by the nest was obtained by measurement of the greatest diameter, c o n s i d e r i n g as part of the nest any patch of foam at l e a s t 1 cm across. 3. Results and D i s c u s s i o n The frequency of nest contact was maximal immediately a f t e r spawning and then slowly d e c l i n e d ( F i g . 6A). The frequence of long nest contacts and the dur a t i o n of long nest contacts showed the same pa t t e r n except that the d e c l i n e was more r a p i d and was e s s e n t i a l l y zero by Day 2b . Snapping ( F i g . 6B) was most frequent on Days 3 and LL. Even then i t was r a r e c o n s i d e r i n g the l a r g e numbers of f r y which sur-rounded the male. A f t e r Day LL, the males u s u a l l y showed no overt response to f r y . Before Day 3, the f r y were not s u f f i c i e n t l y mobile to st i m u l a t e snapping. However, males r e t r i e v e d them con-s i s t e n t l y when they were d i s p l a c e d by the experimenter. The few snaps that occurred during that period were due to the a c c i d e n t a l presence of older f r y i n two tanks. One was the f i s h that was given a second spawning because of the low success of the f i r s t . A few f r y from the f i r s t brood had not been removed. In the other case, l+ or 5 young f i s h appeared a few days a f t e r observa-t i o n s s t a r t e d . Apparently these were l e f t from a previous spawn-i n g and had not been removed when the tank was cleaned. Nest b u i l d i n g was infrequent immediately a f t e r spawning as the males were occupied almost e x c l u s i v e l y w i t h mouthing. Bubble blowing beneath the surface ( F i g . 6C) reached a peak of about 30 PRElOl I I 2 I 3 I 41' I DAYS FIGURE 6. Changes i n the frequency of parental and nest b u i l d -ing behavior i n male gouramis with developing broods of young. A l l data are from 15 min recordings on 6 f i s h taken before spawning and from 0 to 13 days afterwards. Points indicate means, bars equal two standard errors and l i n e s indicate range of values. A. Nest contact (Mouthing), B. Snapping, C. Bubble blowing beneath the surface, D. Bubble blowing at the surface. 3 1 2.5/min on the next morning, then d e c l i n e d through Day 3. A f t e r that frequencies were h i g h l y v a r i a b l e . Bubble blowing at the surface ( F i g . 6D) was more frequent than bubble blowing beneath the surface even on Day 1. On Day 2 i t reached a peak frequency which was sustained f o r s e v e r a l days. Changes i n nest q u a l i t y (Table I ) and diameter ( F i g . 7) a l s o provide evidence of changes i n nest b u i l d i n g . Before opening the p a r t i t i o n f i s h had N2 or N3 nests, and at the time of spawning a l l f i s h had N3 nests. These decreased i n s i z e w i t h the presence of eggs and i n some cases consisted of l i t t l e more than the mass of eggs themselves. There was considerable v a r i a t i o n on Day 1, but by Day 2 most f i s h had Nl nests which covered a wide area. These extensive areas of foam l a s t e d through Day l+, and then de-c l i n e d . There were great i n d i v i d u a l d i f f e r e n c e s i n nest b u i l d i n g . Some f i s h remained very a c t i v e while others ceased nest b u i l d i n g e n t i r e l y a f t e r Day 1. In order to be c e r t a i n that " i g n o r i n g " the f r y was a genuine p a r e n t a l response, f r y aged 13 days and ol d e r were given to two non-parental males. These males eagerly chased and ate the f r y . A f t e r the completion of be h a v i o r a l observations, t e s t s with eggs on Days 16 to 1 8 were used to determine whether the males of t h i s experiment would r e t r i e v e the eggs. One male f a i l e d to approach the eggs. The other f i v e began r e t r i e v i n g v/ithin a fev; seconds of c o n t a c t i n g the eggs. These observations d i f f e r from those of Johns (1966) v/ho found that h i s males ate t e s t eggs 11+ days a f t e r spawning, even when t h e i r f r y were s t i l l present. P a r e n t a l males were observed to be f a r more aggressive to the observer than non-parental males. They r e a d i l y b i t 32 TABLE I . Number of f i s h b u i l d i n g nests of d i f f e r e n t types be-fore spawning and from 0 to 13 days a f t e r spawning NUMBER OF NESTS BUILT DAY NONE TRACE Nl N2 N2+1 N3_ N3+1 PRE - - - 2 1 3 -0 - - - 6 l a - 1 2 1 1 1 -l b 2 - 1 2 1 2 a - 1 1 1 1 2 -2 b - 1 3 - 2 3 - 1 3 - 2 A - 2 1 - 2 - 1 7 1 2 1 2 1 0 - 2 1 1 - 1 1 1 3 - 3 1 1 - - 1 33 FIGURE 7. Changes i n nest diameter of 6 male gouramis i n r e -l a t i o n to the time of spawning (Day 0 ) . P o i n t s show means, bars equal two standard e r r o r s , l i n e s show range of values. 3k thermometers and f i n g e r s placed i n the tank and sometimes per-formed t h r e a t d i s p l a y s through the f r o n t w a l l of the aquarium. No f i e l d s t u d i e s are a v a i l a b l e which could i n d i c a t e how my observations r e l a t e to the n a t u r a l p a r e n t a l behavior of w i l d f i s h . I do not know what i s the normal number of eggs per nest or when the f r y disperse i f they are not constrained by the w a l l s of an aquarium. Even the r a i s i n g of a s i n g l e brood may not be normal f o r gouramis. Though c i c h l i d s and three-spined s t i c k l e -backs have a p a r e n t a l phase i n which no sexual behavior i s shown, no such period has been found i n the gourami. P a r e n t a l males w i t h young i n the nest may continue to add more eggs when-ever r i p e females are a v a i l a b l e . D. SUMMARY 1. The f i r s t s e c t i o n d escribes nest b u i l d i n g , spawning, and par e n t a l care i n the blue gourami, as w e l l as b r i e f l y summarizing the development of eggs and young. I t a l s o presents the opera-t i o n a l d e f i n i t i o n s used i n o b t a i n i n g q u a n t i t a t i v e records of be-havior. 2. The second s e c t i o n presents q u a n t i t a t i v e data on the devel-opment of p a r e n t a l behavior during spawning i n naive males. 3. P a r e n t a l behavior began w i t h i n 5 sec of the f i r s t contact with the eggs at the f i r s t o v i p o s i t i o n . k» Mouthing was r a r e l y shown u n t i l there were eggs i n the nest and then developed r a p i d l y , u s u a l l y reaching almost maximal l e v e l s i n the i n t e r v a l f o l l o w i n g the f i r s t or second spawning act. 35 5. The t h i r d s e c t i o n describes changes i n the frequency of nest b u i l d i n g and p a r e n t a l behavior during the f i r s t 13 days a f t e r spawning. 36 I I . FACTORS INVOLVED IN THE INDUCTION OF PARENTAL BEHAVIOR CHAPTER 3 ! THE ROLE OF EGGS In some i n i t i a l experiments i n v o l v i n g the pre s e n t a t i o n of eggs to gouramis at d i f f e r e n t stages of spawning, I began to get the impression that the eggs themselves might be inducing paren-t a l responsiveness. Since a s i m i l a r e f f e c t of eggs or young had been observed i n s e v e r a l other species i n c l u d i n g l a b o r a t o r y r a t s (Rosenblatt, 1967), mice ( N o i r o t , 1964a,b,c), domestic fowl (Bur-rows and Byerly, 1938), and three-spined s t i c k l e b a c k s (Van I e r s e l , 1953)> I performed an experiment to determine whether eggs alone could induce p a r e n t a l behavior i n the blue gourami. This was found to be the case. A f t e r s e v e r a l other experiments on the i n t e r a c t i o n of the e f f e c t of eggs with the e f f e c t of spawning, a comparison of my observations i n the gourami w i t h those on other species l e d to me examine i n more d e t a i l p a r e n t a l i n d u c t i o n i n response to eggs alone. Therefore, I studied the e f f e c t s of the number of stimulus eggs and repeated exposure to small numbers of eggs on the development of pa r e n t a l behavior. These two experiments and the i n i t i a l study of the e f f e c t of eggs on pa r e n t a l behavior are included i n t h i s chapter. A. INDUCTION OF PARENTAL BEHAVIOR WITH CLUTCHES SPAWNED BY OTHER FISH 1. I n t r o d u c t i o n The purpose of t h i s experiment was to determine whether eggs 37 spawned by other f i s h could induce p a r e n t a l behavior i n non-pa r e n t a l male gouramis. The measure of p a r e n t a l responsiveness was egg-eating i n h i b i t i o n . 2. Methods The s u b j e c t s were 14 s e x u a l l y experienced a d u l t males not showing egg-eating i n h i b i t i o n at the s t a r t of the experiment. The standard spawning c o n d i t i o n s were used except that contact with the female was prevented by keeping the p a r t i t i o n c l o s e d . Seven f i s h were given an e n t i r e c l u t c h of eggs spawned by another p a i r of f i s h . The other seven at t h i s time r e c e i v e d no-t h i n g . The c l u t c h e s were obtained l e s s than an hour a f t e r the c e s s a t i o n of spawning by r a i s i n g a p e t r i d i s h under the nest of the donor f i s h . The e n t i r e contents of the d i s h were given to the r e c i p i e n t s . In the morning of the day f o l l o w i n g egg presen-t a t i o n any remaining eggs were removed. Later i n the same day a l l f i s h r e c e i v ed what w i l l be r e f e r r e d to as an egg s u r v i v a l t e s t . For t h i s , 100 r e c e n t l y spawned eggs were placed i n the. tank. The s u r v i v a l of these eggs was determined the f o l l o w i n g morning. The c r i t e r i o n f o r r e c o g n i z i n g the i n d u c t i o n of egg-e a t i n g i n h i b i t i o n was the s u r v i v a l of 25 or more eggs out of the 100 presented i n the t e s t . This somewhat a r b i t r a r y c r i t e r i o n was considered to be above the number of eggs that could s u r v i v e by becoming lodged among the f l o a t i n g p l a n t s . Since i t was not p o s s i b l e to o b t a i n 7 c l u t c h e s on the same day, the experiment was spread out over a number of days. Each egg-treated f i s h was p a i r e d with a f i s h which d i d not r e c e i v e eggs. I f both members of a p a i r f a i l e d to reach c r i t e r i o n , they 38 were given a second t r i a l performed i n the same way as the f i r s t . 3. R e s u l t s and Discussion A f t e r the f i r s t t r i a l , the f i s h c l e a r l y f e l l i n t o two groups. Four of the f i s h (E) which had received c l u t c h e s on the preceding day scored between 58 and 64. A l l the f i s h (C) which had not r e -ceived a c l u t c h and the r e s t of the E's scored between 0 and 13 (Table I I ) . A f t e r the second t r i a l of those p a i r s i n which the E male had not developed egg-eating i n h i b i t i o n i n the f i r s t t r i a l , a l l E's scored above 44 and a l l C's below 13. A f t e r d i v i d i n g the E and C groups i n t o those which reached c r i t e r i o n f o r egg-eating i n h i b i t i o n and those which d i d not, these r e s u l t s were analysed by the F i s h e r Exact P r o b a b i l i t y Test ( S i e g e l , 19%) . The proba-b i l i t y of o b t a i n i n g by chance the r e s u l t s of the f i r s t t r i a l was P=0.035» The p r o b a b i l i t y of o b t a i n i n g the d i s t r i b u t i o n of scores obtained a f t e r the f i n a l t r i a l of each p a i r , whether one or two t r i a l s were given v/as P=0.0003. This experiment confirmed that l a r g e numbers of eggs could induce p a r e n t a l behavior i n male gouramis. One hundred t e s t eggs were eaten by males which had not r e c e n t l y spawned, but many v/ere not eaten i f the male had p r e v i o u s l y been exposed to a l a r g e num-ber of eggs. The development of egg-eating i n h i b i t i o n v/as not simple s a t i a t i o n , f o r males continued to feed on other items. Further-more, though i t v/as not s p e c i f i c a l l y recorded, males not only f a i l e d to eat eggs but a l s o r e t r i e v e d them to t h e i r nests. 39 TABLE I I . The number of eggs s u r v i v i n g out of 100 presented i n the overnight t e s t . In each p a i r , f i s h E r e c e i v e d a c l u t c h of eggs on the previous day while f i s h C d i d not. PAIR Number of eggs s u r v i v i n g out of 100 presented i n the overnight t e s t TRIAL 1 E TRIAL 2 E C 1 2 3 4 5 6 7 2 59 58 o 64 60 2 4 3 13 o l l l 45 o 44 56 SUM MEAN 245 35.0 23 3.3 145 48.3 o 0 40 B. THE EFFECT OF THE NUMBER OF STIMULUS EGGS ON THE INDUCTION OF PARENTAL BEHAVIOR 1. I n t r o d u c t i o n The previous experiment i n d i c a t e d a r o l e f o r eggs i n the i n d u c t i o n of a pa r e n t a l s t a t e . Examination of the l i t e r a t u r e on the i n d u c t i o n of par e n t a l behavior by exposure to eggs or young revealed three basic patterns. Domestic mice ( N o i r o t , 1964b, 1969b) and three-spined s t i c k l e b a c k s (Van I e r s e l , 1953) seemed to become pa r e n t a l promptly upon pr e s e n t a t i o n of young. White r a t s (Rosenblatt, 1967) and domestic fowl (Burrows and Byerly, 1938; Ramsay, 1953) on the other hand, re q u i r e d a considerable p e r i o d of exposure to young i f they were not already i n the appropriate p h y s i o l o g i c a l s t a t e . Other species such as goats ( K l o p f e r et a l . , 1964; K l o p f e r and Kl o p f e r , 1968) r e q u i r e d s t i m u l i from the young to come i n t o a pa r e n t a l s t a t e , but they seemed s e n s i t i v e to such s t i m u l i only during a s p e c i f i c , l i m i t e d period of time associated with p a r t u r i t i o n . I t occurred to me that i f I tested gourami males w i t h l a r g e numbers of eggs, I would have found e s s e n t i a l l y continuous r e -sponsiveness to young, as i n the previous experiment, whereas i f I had used smaller numbers of eggs I would have found the males s e n s i t i v e only a f t e r spawning (See Ch. 5)« This suggested that species d i f f e r e n c e s i n responsiveness to young represented d i f -f e r e nt aspects of a common phenomenon r a t h e r than d i f f e r e n t sys-tems f o r the c o n t r o l of par e n t a l behavior. Perhaps wi t h a strong enough stimulus any animal w i l l become p a r e n t a l . With weaker s t i m u l i p a r e n t a l behavior develops only i f the animal i s " s e n s i -41 s i t i z e d " by spawning or p a r t u r i t i o n or i f i t r e c e i v e s extended exposure to the stimulus. To t e s t the i d e a that the d i f f e r e n t p a t t e r n s f o r the induc-t i o n of pa r e n t a l behavior were d i f f e r e n t aspects of a common process, I wanted to confirm that they could a l l be found i n the gourami by appropriate manipulation of the stimulus strength. That i s , I was l o o k i n g f o r the prompt development of p a r e n t a l be-havior at high stimulus l e v e l s and the absence of p a r e n t a l be-havior at low stimulus l e v e l s and increased e f f e c t i v e n e s s of low-er stimulus l e v e l s with spawning or repeated p r e s e n t a t i o n . No other study seems to have examined the response of a d u l t s to young over a range of stimulus l e v e l s i n c l u d i n g those both e f f e c -t i v e and i n e f f e c t i v e f o r i n d u c i n g p a r e n t a l behavior. 2. Methods The general experimental procedure was to present i n d i v i d u a l f i s h w i t h 100, 500, 1000, 2000, or 3000 eggs, record p a r e n t a l responses during the i n i t i a l contact w i t h the eggs and t e s t f o r egg-eating i n h i b i t i o n . The subjects were 35 adult male gouramis, a l l from the same brood and without previous sexual or p a r e n t a l experience. They were approximately 7 months o l d at the time of t e s t i n g . A randomized blocks design was used with 7 blocks and 5 treatments. Two or 3 f i s h were tested each day between 14 :00 and 17:30. Each f i s h was held i n a separate 40 1 aquarium. The f i s h v/as r e s t r i c t e d to one h a l f of the tank by an opaque p a r t i t i o n . Tanks contained a r t i f i c i a l nest supports and f l o a t i n g egg pre-s e n t a t i o n frames. F i s h were placed i n the experimental tank at 42 l e a s t 2 days before t e s t i n g . The tanks were l i t from 7:00 to 19:30 d a i l y . The basic method of o b t a i n i n g and measuring stimulus eggs and p r e s e n t i n g them to the s u b j e c t s has been described i n Chapter 1. A t e s t c o nsisted of p l a c i n g eggs i n the foam rubber frame and observing f o r 20 min a f t e r the f i s h ' s f i r s t contact w i t h the eggs. The f o l l o w i n g behavior was recorded: 1) Egg contact, 2) R e t r i e v e , 3) Release eggs without r e t r i e v i n g , and if) Nest contact. From the r e c o r d i n g c h a r t s the f o l l o w i n g measures were ob-tained : 1. Time from the s t a r t of egg contact to the f i r s t r e -t r i e v i n g a c t . 2. Duration of egg contact preceding the f i r s t r e -t r i e v i n g a c t . 3. The i n t e r v a l separating each of the f i r s t 10 r e t r i e v i n g a c t s . if. Duration of egg contact between the f i r s t 10 r e t r i e v i n g a c t s . 5. T o t a l number of r e t r i e v i n g a c t s i n each 5 min i n t e r v a l . 6. T o t a l duration of nest contact i n each 5 min i n t e r v a l . Measurements of d u r a t i o n were made to the nearest 2 sec, and bouts were considered continuous i f there were no breaks longer than 2 sec. Bouts shorter than 2 sec were c a l c u l a t e d as 2 sec i n d u r a t i o n . A f t e r r e c o r d i n g the response to the eggs, the f i s h were l e f t alone f o r a t o t a l exposure of approximately 90 min ( a c t u a l range, 8 i f - l l8 min). Then the stimulus eggs were a l l removed along with the nest, nest support and p r e s e n t a t i o n frame. The 43 nest support was r i n s e d and returned, and the eggs were counted. Then 200 l i v e eggs were placed i n the tank to determine whether egg-eating i n h i b i t i o n p e r s i s t e d overnight. The s u r v i v o r s of these 200 v/ere removed and counted the f o l l o w i n g morning between 10:00 and 11:00. Since small numbers of s u r v i v i n g eggs might have been overlooked by the f i s h , the c r i t e r i o n f o r egg-eating i n h i b i t i o n was recovery of 25 or more eggs. The e f f e c t of stimulus strength on the number of f i s h be-coming p a r e n t a l v/as tested by the Cochran Q Test. The e f f e c t on the measures of l a t e n c y and i n t e n s i t y of p a r e n t a l behavior was tes t e d by the Friedman A n a l y s i s of Variance (both t e s t s from S i e g e l , 1956). 3. R e s u l t s a. Development of P a r e n t a l Behavior - P a r e n t a l behavior de-veloped i n response to eggs i n a number of sub j e c t s . There was a sudden switch i n behavior from egg contact to a r e g u l a r s e r i e s of r e t r i e v i n g t r i p s between the pre s e n t a t i o n frame and the nest ( F i g . 8 ) . One or more r e t r i e v i n g a c t s were recorded f o r a l l but four f i s h . But the pa t t e r n w i t h which r e t r i e v i n g developed c l e a r l y d i v i d e d the subjects i n t o two groups. In one group, v/hich I have c a l l e d "strong r e t r i e v e r s , " the f i r s t r e t r i e v i n g act was followed very q u i c k l y by a second, and subsequent a c t s were only separated by b r i e f periods i n which eggs were picked up. The other group, "weak r e t r i e v e r s , " performed fewer r e t r i e v i n g a c t s separated by long i n t e r v a l s of egg contact i n which eggs were e v i d e n t l y eaten. kk i r 1 1 5 10 15 20 M I N U T E S FIGURE 8. The development of r e t r i e v i n g behavior i n naive male gouramis presented with i , 1 0 0 ; i i , 5 0 0 ; i i i , 1 0 0 0 ; i v , 2 0 0 0 ; and v, 3 0 0 0 recently spawned eggs. The abscissa gives time from the f i r s t egg contact i n min. The ordinate give cumulative number of r e t r i e v -ing acts. The l e t t e r s a-g indicate the f i s h from blocks 1 - 7 , respectively. I f fewer than 1 0 r e t r i e v -ing acts were performed, the f i n a l one i s indicated by a large f i l l e d c i r c l e . Only two f i s h , both i n the 500 egg treatment, d i d not f a l l c l e a r -l y i n t o the s t r o n g or weak group. They s t a r t e d s t r o n g l y but then began to spend longer periods i n egg contact. Since a f i s h could reach the c r i t e r i o n f o r r e t r i e v i n g by f o l l o w i n g egg contact w i t h nest contact and since some nest con-t a c t i s shown even by non-parental f i s h (Ch. 2 ) , some or a l l of the " r e t r i e v i n g a c t s " by the weak r e t r i e v e r s may have been a r t i -f a c t s of the o p e r a t i o n a l d e f i n i t i o n of r e t r i e v i n g . However, they could a l s o be i n d i c a t i o n s of a weak r e t r i e v i n g tendency. Most of the strong r e t r i e v e r s began r e t r i e v i n g between 0.5 and 2 .5 min of t h e i r f i r s t egg contact. One delayed n e a r l y 7 min. Egg contact occupied O.k to 1 .9 min of the time preceding the f i r s t r e t r i e v i n g a c t . Over the course of the 20 min r e c o r d i n g the r a t e of r e t r i e v -i n g d e c l i n e d while the r a t e of mouthing increased ( F i g . 9)« When the nests were removed a f t e r 90 min, there was a s i g n i f i c a n t r e -covery of eggs from f i s h which had shown strong r e t r i e v i n g , though i t was seldom 100 per cent of the eggs presented. F i s h which showed week r e t r i e v i n g ate as many as 1Z+00 eggs. The 90 min exposure to the stimulus eggs was s u f f i c i e n t to cause some f i s h to develop egg-eating i n h i b i t i o n which l a s t e d overnight. Only f i s h which had shown strong r e t r i e v i n g showed s i g n i f i c a n t overnight s u r v i v a l of the 200 t e s t eggs. However, not a l l strong r e t r i e v e r s developed p e r s i s t e n t p a r e n t a l responsiveness, and some ate most or a l l of the t e s t eggs. b. E f f e c t s of Stimulus Strength - Stimulus s t r e n g t h had a s i g n i f i c a n t e f f e c t on the number of f i s h developing strong 46 in 0)10-1 i -o < UJ cc I-LU CC I— o < o o (-£0 LU Z U_ o z 5 1 -B . 0 - 5 F I V E 6 : 1 0 11 -1 5 1 6 - 2 0 M I N U T E I N T E R V A L S FIGURE 9. Changes i n par e n t a l behavior over the course of a 20 min r e c o r d i n g i n naive male gouramis which developed strong r e t r i e v i n g behavior i n response to 500 (n=2), 1000 (n=2), 2000 (n=6), or 3000 eggs (n=5). The f i g u r e shows A) mean number of r e t r i e v i n g a c t s per 5 min, and B) mean dura t i o n of nest contact per 5 min i n each of four successive 5 min i n t e r v a l s a f t e r the f i r s t con-t a c t with the stimulus eggs. TABLE I I I . hi Number of f i s h developing p a r e n t a l behavior a f t e r p r e s e n t a t i o n of d i f f e r e n t numbers of stimulus eggs. Number of F i s h Developing P a r e n t a l Behavior Number of Stimulus Eggs n Strong R e t r i e v i n g Egg-Eating I n h i b i t i o n 100 500 1000 2000 3000 7. 7 7 7 7 0 2 2 6 5 o 0 2 5 3 Q DF P Cochran Q Test 12.60 h <.02 11.25 k <.05 TABLE IV. The e f f e c t of the number of stimulus eggs on measures of la t e n c y and strength of p a r e n t a l behavior i n male gouramis. Egg Contact T o t a l Duration R e t r i e v i n g Preceding Total R e t r i e v i n g of Nest Stimulus Latency R e t r i e v i n g R e t r i e v i n g P l u s Re- Contact S u r v i v a l of Eggs (Min)• (Min) Acts l e a s i n g Acts (Min) 200 Eggs 100 7.79+3.24 2.9111.19 2.71±0.89 3.0010.97 0.0910.03 0.5710.42 500 2.88+1.35 1.51±0.55 9.00+3.28 9.5713.07 1.8510.89 3.2812.64 1000 8.00+3.21 4.46+1.60 7.1413-40 13.7112.89 1.9511.23 35.71122.44 2000 2.69+1.03 I.42+O.46 26.2815.89 27.5715.33 2.4111.03 63.42124.67 3000 1.60+0.28 0.98+0.17 27.57l6.66 37.5715.86 2.0910.93 57.00124.95 Friedman A n a l y s i s of Variance 4 5.81 7.98 14.14 18.74 5.10 14.55 DF 4 4 4 4 4 4 P < .30 < .10 < .01 <.001 < .30 < .01 49 60 -i o < >40 e i-u DC k . O ffi20 -I O o 3 £ A A 8 A A A A C 1 0 0 5 0 0 1 0 0 0 2 0 0 0 N U M B E R O F S T I M U L U S E G G S A A A A A O O 3 0 0 0 FIGURE 10. The e f f e c t of the number of stimulus eggs on the number of r e t r i e v i n g a c t s performed i n 20 min by naive male gouramis. T r i a n g l e s i n d i c a t e f i s h which developed "strong r e t r i e v i n g . " C i r c l e s i n d i c a t e f i s h which developed "weak r e t r i e v i n g . " 50 200n co O O UJ 150 I-co UJ o o ™ 100-< > ac 50H Z) CO 100 500 N U M B E R O F agge ££e oSe ^ A A O 1000 2 0 0 0 3 0 0 0 S T I M U L U S E G G S FIGURE 11. The e f f e c t of the number of stimulus eggs on the overnight s u r v i v a l of 200 t e s t eggs i n naive male gouramis. T r i a n g l e s i n d i c a t e f i s h which developed "strong r e t r i e v i n g . " C i r c l e s i n d i c a t e f i s h which developed "weak r e t r i e v i n g . " 51 r e t r i e v i n g and egg-eating i n h i b i t i o n (Table I I I ) . The e f f e c t s of stimulus strength on s i x measures of the l a t e n c y and i n t e n s i t y of p a r e n t a l behavior are summarized i n Table IV. Stimulus strength had a s i g n i f i c a n t e f f e c t on frequency of r e t r i e v i n g , frequency of r e t r i e v i n g p l u s r e l e a s i n g , and overnight egg s u r v i v a l , but not on e i t h e r measure of l a t e n c y or on the t o t a l d u r a t i o n of nest con-t a c t . The s i g n i f i c a n t e f f e c t of stimulus strength on the number of r e t r i e v i n g a c t s seemed to be due to an increase i n the number of strong r e t r i e v e r s as w e l l as the number of r e t r i e v i n g a c t s which each performed ( F i g . 10). The e f f e c t of stimulus strength on egg s u r v i v a l scores appeared to be an all-or-none e f f e c t ( F i g . 11). 4. D i s c u s s i o n Exposure to l a r g e numbers of eggs induced p a r e n t a l responses i n non-parental f i s h . This process occurred i n f i s h which had never p r e v i o u s l y been p a r e n t a l . R e t r i e v i n g and mouthing beha-v i o r , as w e l l as egg-eating i n h i b i t i o n , were shown. The induc-t i o n process was very r a p i d , r e q u i r i n g l e s s than 2 min of contact v/ith eggs and sometimes t a k i n g place w i t h i n 0.5 min of the s t a r t of egg contact. A f t e r 90 min exposure to a strong stimulus, some f i s h continued to show p a r e n t a l responses to a much weaker stimu-l u s . The observations of r e t r i e v i n g and egg-eating i n h i b i t i o n suggest that f i s h which have developed p a r e n t a l responsiveness are q u a l i t a t i v e l y d i f f e r e n t from those that have not. However, there i s some evidence that the two s t a t e s are not a b s o l u t e l y d i s t i n c t . Non-parental males may show incomplete r e t r i e v i n g 52 a c t s , and perhaps i s o l a t e d complete r e t r i e v i n g a c t s . Males i n the p a r e n t a l s t a t e eat some eggs. Many overnight s u r v i v a l scores were lower than expected even a l l o w i n g f o r some n a t u r a l egg mor-t a l i t y . Males which i n i t i a l l y appeared to be p a r e n t a l a l s o r e -verted to the non-parental s t a t e . This was seen i n the two males i n the 500 egg treatment which began strong r e t r i e v i n g but then returned to e a t i n g eggs. Several males which had shown strong r e t r i e v i n g and egg-eating i n h i b i t i o n during the i n i t i a l 90 min f a i l e d to maintain egg-eating i n h i b i t i o n overnight. F i s h spawning n a t u r a l l y perform t h e i r f i r s t p a r e n t a l act w i t h i n 5 sec of the f i r s t contact with eggs, whereas even the most r a p i d l y responding f i s h i n the present experiment r e q u i r e d more than 20 sec. Most re q u i r e d between AO sec and 2.5 min. The d i f f e r e n c e cannot be accounted f o r by the f a c t that the f i s h i n t h i s experiment had a greater distance to r e t r i e v e , since t r a v e l from frame to nest r e q u i r e d only 6 sec or l e s s . In the present study the time from the f i r s t egg contact to the f i r s t r e t r i e v i n g act was longer than the i n t e r v a l between egg contact and r e t r i e v -i n g i n subsequent t r i p s . I t seems l i k e l y that a r a p i d develop-ment of pa r e n t a l responsiveness was t a k i n g place d u r i n g the i n i -t i a l longer p e r i o d of egg contact which preceded the s t a r t of r e t r i e v i n g . I n c r e a s i n g number of stimulus eggs induced a greater propor-t i o n of the f i s h to develop complete p a r e n t a l behavior. Only at the lowest stimulus l e v e l of 100 eggs d i d n o . f i s h become paren-t a l . With the maximal stimulus some f a i l e d to become p a r e n t a l . Among the f i s h that became p a r e n t a l there was r e l a t i v e l y l i t t l e e f f e c t of stimulus s t r e n g t h on the speed of i n d u c t i o n or the 53 i n t e n s i t y of p a r e n t a l behavior other than the frequency of r e -t r i e v i n g . C. INDUCTION OF PARENTAL BEHAVIOR BY REPEATED EXPOSURE TO SMALL NUMBERS OF EGGS 1 . I n t r o d u c t i o n The purpose of t h i s i n v e s t i g a t i o n was to determine whether par e n t a l responsiveness could be induced by long-term exposure to a stimulus too small to induce behavior immediately. The r a -t i o n a l e f o r t h i s was discussed i n the i n t r o d u c t i o n to the previous s e c t i o n . I hypothesized that the p a t t e r n f o r i n d u c t i o n of paren-t a l behavior observed i n other species could be found i n the gourami by appropriate v a r i a t i o n of stimulus strength. Since long-term exposure to a weak stimulus was impossible because, the eggs were r a p i d l y eaten, I decided to t r y repeated p r e s e n t a t i o n of the stimulus, r e p l a c i n g i t as i t was eaten. In the previous experiment, f i s h which d i d not develop r e g u l a r p a r e n t a l behavior sometimes showed components of r e -t r i e v i n g . They approached or contacted the nest without r e l e a s -i n g eggs, or they r e l e a s e d eggs without approaching the nest. Such behavior could be unrelated to p a r e n t a l responsiveness, or i t could i n d i c a t e weak p a r e n t a l motivation. Therefore, i n the present experiment r e t r i e v i n g was d i v i d e d i n t o components so that p o s s i b l e i n d i c a t i o n s of i n c r e a s i n g p a r e n t a l motivation could be recorded. I had i n i t i a l l y hoped to t e s t f i s h w ith and without p r i o r p a r e n t a l experience. However, I was unable to complete the ex-54 periment as planned. A f t e r t e s t i n g only two experienced and two inexperienced f i s h , I had performed 277 t r i a l s i n 24 days and used eggs from 73 spawnings. The great amount of time r e q u i r e d f o r each f i s h and i n c r e a s i n g d i f f i c u l t i e s i n o b t a i n i n g enough f r e s h eggs f o r t e s t i n g caused me to terminate the experiment. No conclusions can be drawn about e f f e c t s of p r i o r experience, but observations on the development of p a r e n t a l behavior add to the understanding of t h i s process. 2. Methods The basic procedure was to record p a r e n t a l responses a f t e r each of a s e r i e s of presentations of 100 eggs. This stimulus, presented alone, was known from the previous experiment to be i n -s u f f i c i e n t to induce p a r e n t a l behavior. The experimental design c a l l e d f o r 10 t r i a l s per day, every day u n t i l complete p a r e n t a l behavior developed, but there were not always s u f f i c i e n t eggs f o r t h i s . The subjects were four 11 month old adu l t male gouramis, i n i t i a l l y without sexual or p a r e n t a l experience. Two v/ere given parental experience by pre s e n t a t i o n with 2000 l i v e eggs. They were allowed to care f o r these eggs f o r 24 hr. Pa r e n t a l e x p e r i -ence was given 16 and 35 days, r e s p e c t i v e l y , before the s t a r t of t e s t i n g i n the two experienced f i s h which were studied. The experimental aquaria were set up as i n the previous experiment with p l a s t i c nest supports and foam rubber egg pre-s e n t a t i o n frames. A p a i r e d design was used, and one experienced and one inexperienced f i s h were te s t e d at the same time. . Testing was c a r r i e d out i n the afternoon a f t e r spawning by 55 the egg donor f i s h . For each t r i a l 100 eggs were placed i n the pre s e n t a t i o n frame, and the f i s h was observed u n t i l i t had made 3 contacts with the eggs. The dur a t i o n of the i n i t i a l contact with the eggs was recorded, and whether any of the f o l l o w i n g components of r e t r i e v i n g occurred a f t e r c o n t a c t i n g the eggs. 1 . Release eggs without o r i e n t a t i o n to the nest 2. Approach nest 3. Pause under nest k» Orient upwards toward nest 5 . Open and clo s e jaws while o r i e n t e d to nest 6. Observed re l e a s e of eggs 7. Nest contact. The two members of a p a i r were tes t e d a l t e r n a t e l y . The frame was inspected before each new t r i a l to be c e r t a i n that the eggs from the previous t r i a l had been removed by the f i s h . I n t e r -t r i a l i n t e r v a l s depended upon the speed wi t h which f i s h approached the frame and ate the eggs. As they became more f a m i l i a r with the procedure t h i s time decreased. In the f i r s t p a i r the i n t e r -v a l s t a r t e d at about 6 min and dec l i n e d to 2 min. The second pair-went from 15 to 10 min i n t e r v a l s . I n t e r v a l s were longer be-tween the f i f t h and s i x t h t r i a l s while more eggs were being counted. A f t e r the f i n a l t r i a l of the day, the nest was removed and the eggs i n i t were counted. Any other eggs i n the tank,.e.g., i n the pr e s e n t a t i o n frame, were removed sepa r a t e l y . Then the nest support was replaced, and 200 eggs were presented. The s u r v i v o r s of these eggs could be d i s t i n g u i s h e d upon removal of the eggs f o l l o w i n g the next day's t r i a l by t h e i r darker c o l o r , 56 even i f they had not yet hatched. On days when i n s u f f i c i e n t eggs were produced, eggs were f i r s t reserved f o r the 200 egg t e s t s . Then any eggs l e f t were used i n as many t r i a l s as p o s s i b l e . 3. R e s u l t s and Discussion A l l f i s h showed some components of r e t r i e v i n g : egg r e l e a s e , approaches to the nest, pausing, o r i e n t i n g , and nest contact. Three of the four, two experienced and one inexperienced, devel-oped complete r e t r i e v i n g , c h a r a c t e r i z e d by the appearance of s i g n i f i c a n t numbers of eggs i n the nest on the second and f i f t h days of t e s t i n g . The appearance of eggs i n the nest was c o r r e -l a t e d with a decrease i n the d u r a t i o n of the i n i t i a l egg contact, as the f i s h r e t r i e v e d i n s t e a d of e a t i n g the eggs, and with the appearance of jaw movements while o r i e n t e d to the nest. Jaw movements were r a r e l y seen except i n cases where there was e v i -dence that eggs were a c t u a l l y r e l e a s e d . The other components appeared i r r e g u l a r l y , but t h e i r frequency and completeness appeared to increase s l i g h t l y toward the s t a r t of complete r e -t r i e v i n g . This suggests that incomplete r e t r i e v i n g a c t s r e f l e c t low l e v e l s of p a r e n t a l tendency. The development of p a r e n t a l behavior i n the one inexper-ienced f i s h i s presented i n F i g . 12. The two experienced f i s h were quite s i m i l a r . Jaw movements f i r s t appeared on the second day of t e s t i n g i n the two experienced f i s h and on the f i f t h day i n the inexperienced f i s h . S p e c i f i c a l l y t h i s was on the 13th, 18th and 38th prese n t a t i o n s of 100 eggs and a f t e r 1, 1, and k overnight t e s t s with 200 eggs, r e s p e c t i v e l y . On the day i n , which they appeared, jaw movements were f i r s t shown on the t h i r d , 57 FIGURE 12. Development of p a r e n t a l behavior i n an i n e x p e r i -enced gourami exposed to repeated p r e s e n t a t i o n s of 100 eggs. Successive days of t e s t i n g are i n d i c a t e d along the a b s c i s s a . Each 100 egg t r i a l i s i n d i c a -ted by a c i r c l e on the a b s c i s s a , and each 200 egg overnight s u r v i v a l t e s t by a t r i a n g l e . The upper graph p l o t s the d u r a t i o n of the i n i t i a l contact with eggs i n each t r i a l . (Open c i r c l e s i n d i c a t e that the contact with eggs was not observed.) The second graph i n d i c a t e s by a short bar behavior o c c u r r i n g i n each t r i a l . The t h i r d and f o u r t h graphs present the percentage of t r i a l eggs recov-ered from the nest and the 2if hr s u r v i v a l of 200 t e s t eggs, r e s p e c t i v e l y . Abbreviations of behavior p a t t e r n s : R - re l e a s e eggs without o r i e n t a t i o n to the nest, AN - approach nest, P - pause, under nest, 0 - o r i e n t upwards toward nest, J - jaw movements while o r i e n t e d to nest, RN - re l e a s e eggs at nest, NC - contact vrith nest. 9£ 59 eighth and t h i r d egg p r e s e n t a t i o n s . In two cases, r e t r i e v i n g was shown c o n s i s t e n t l y a f t e r the f i r s t complete r e t r i e v i n g a c t . In one experienced f i s h complete r e t r i e v i n g appeared i n l a t e t r i a l s on two d i f f e r e n t days before i t appeared c o n s i s t e n t l y . F i s h which showed c o n s i s t e n t r e t r i e v i n g a l s o seemed to eat some eggs. Even on days i n which complete r e t r i e v i n g was con-s i s t e n t l y performed i n a l l t r i a l s , only about 50 to 60 per cent of the eggs presented were recovered from the nest. Egg s u r v i v a l overnight was very low even a f t e r r e g u l a r r e t r i e v i n g was shown i n the 100 egg t r i a l s . I n two cases, t h i s improved g r a d u a l l y with successive days of t e s t i n g , but i n one experienced f i s h no more than 30 of 200 eggs ever s u r v i v e d . The r e l a t i v e i n e f f i c i -ency of r e t r i e v i n g i s s u r p r i s i n g . But t h i s may e x p l a i n why i n so many overnight egg s u r v i v a l t e s t s , many fewer eggs are recovered than expected on the b a s i s of known or estimated egg m o r t a l i t y . The r e s u l t s of t h i s experiment appear to provide evidence that small numbers of eggs a f f e c t the s e n s i t i v i t y of the f i s h to f u r t h e r egg presentations and that t h i s s e n s i t i z a t i o n i s cumula-t i v e . However, there i s a d i f f i c u l t y inherent i n t h i s method of t e s t i n g . I f the e f f e c t i v e stimulus of the egg i s something r e -leased i n t o the water r a t h e r than something which r e q u i r e s ac-t u a l contact by the f i s h , then the e f f e c t i v e stimulus could be accumulating i n the water r a t h e r than " i n the f i s h . " However, other experiments gave no i n d i c a t i o n that any stimulus except the eggs themselves was e f f e c t i v e (See Ch. k). Taken together with the r e s u l t s of the previous experiments, t h i s study shows that eggs may have important motivating e f f e c t s on p a r e n t a l responsiveness. However, i t does not show what 60 s i g n i f i c a n c e these motivating e f f e c t s may have i n the development of p a r e n t a l behavior during normal spawning. This question i s explored i n more d e t a i l i n Chapter 5« D. SUMMARY 1. In the f i r s t experiment, experienced males developed egg-ea t i n g i n h i b i t i o n when, given c l u t c h e s spawned by other f i s h , but males not given c l u t c h e s ate the t e s t eggs. 2. In the second experiment, naive males were observed during t h e i r i n i t i a l contact with 100, 500, 1000, 2000, or 5000 eggs. 3. There was a p o s i t i v e e f f e c t of the number of stimulus eggs on the number of f i s h developing p a r e n t a l behavior. LL. Most f i s h which developed p a r e n t a l behavior began to r e -t r i e v e w i t h i n 0 .5-2.5 min a f t e r they f i r s t contacted eggs and then continued t o . r e t r i e v e c o n s i s t e n t l y and to show other e l e -ments of pa r e n t a l behavior. 5. In the t h i r d experiment 3 of LL male gouramis ev e n t u a l l y de-veloped p a r e n t a l responses when repeatedly exposed to 100 eggs, a stimulus too weak to i n i t i a l l y induce p a r e n t a l behavior. 61 CHAPTER Zf THE STIMULUS FOR THE INDUCTION OF PARENTAL BEHAVIOR 1. I n t r o d u c t i o n and Methods Although t h i s t h e s i s places l i t t l e emphasis on the nature of the stimulus f o r the i n d u c t i o n of pa r e n t a l behavior or the cues by which a p a r e n t a l a d u l t recognizes eggs or young, a few small experiments have i n v e s t i g a t e d these questions. In the f i r s t experiment on the i n d u c t i o n of p a r e n t a l behavior by pr e s e n t a t i o n of c l u t c h e s spawned by other f i s h (Ch. 3A), the eggs were not the only f a c t o r of p o s s i b l e s i g n i f i c a n c e . The goal of t h i s e x p e r i -ment v/as to determine whether the foam of the nest and v/ater from a tank i n which a spawning had taken place or the presence of a female i n the tank (though separated from the male by a par-t i t i o n ) were important f o r the development of p a r e n t a l behavior. Therefore, the procedure used i n Ch. 3A v/as modified by the e l i m i n a t i o n of c e r t a i n components. The o r i g i n a l procedure i n -volved t r a n s f e r r i n g a whole c l u t c h of eggs i n a p e t r i d i s h to a male set up f o r spawning but prevented from access to the female. The m o d i f i c a t i o n s were as f o l l o w s : Group i - No female i n the tank Group i i - Eggs removed, but nest m a t e r i a l and donor tank water added Group i i i - Nest m a t e r i a l and donor tank water removed, but eggs added Group i v - Nest m a t e r i a l and donor tank water separated from eggs as i n groups i i and i i i , but both added to the tank 62 Group v - No female present, only tapwater added as a stimulus. The subjects were s e x u a l l y experienced males which had spawned two to three weeks p r e v i o u s l y . A l l f i s h were given a p r e l i m i n a r y overnight t e s t w i t h 100 eggs and then assigned at random to one of the f i v e treatments. Treatments were performed i n the afternoon, as soon as p o s s i b l e a f t e r the f i s h p r o v i d i n g the stimulus c l u t c h e s ceased spawning. S u r v i v i n g eggs were removed the next morning, and a 100 egg over-night t e s t was performed on that afternoon. I f fewer than 25 eggs survived, the treatment and overnight t e s t were repeated. The scores v/ere separated i n t o those greater than or equal to 25 and those l e s s than 25 and analysed by the F i s h e r Exact Proba-b i l i t y Test ( S i e g e l , 1956). 2. R e s u l t s and Di s c u s s i o n The complete treatment (Group i v ) induced egg-eating i n h i b i -t i o n i n a l l 1+ f i s h on the f i r s t p r e s e n t a t i o n (Table V). In the absence of the female 3 of if f i s h s t i l l developed egg-eating i n -h i b i t i o n . When the eggs were r i n s e d before p r e s e n t a t i o n , only 2 of Li f i s h responded to the f i r s t treatment, but the others r e -sponded a f t e r the second treatment. E l i m i n a t i n g only the eggs prevented development of pa r e n t a l behavior. None of the f i s h given only tapwater developed p a r e n t a l behavior. The number of f i s h w ith 25 or more eggs s u r v i v i n g overnight d i d not d i f f e r s i g n i f i c a n t l y between groups i i and v or among groups i , i i i , and i v . But i i i and i v d i f f e r e n t from i i and v (P=0.01if). The p r o b a b i l i t y that group i v/as not d i f f e r e n t from 63 TABLE V. The e f f e c t of 5 m o d i f i c a t i o n s of the stimulus f o r the i n d u c t i o n of p a r e n t a l behavior on egg-eating i n h i b i t i o n i n male gouramis. F i s h r e c e i v e d d i f f e r e n t combinations of three p r i n c i p a l s t i m u l i : a) the eggs, b) nest mater-i a l and water from the egg donor tank, c) the presence of a female i n the tank but separated from the male by an opaque p a r t i t i o n . The t a b l e shows f o r each t r e a t -ment the number of f i s h which reached the c r i t e r i o n of 25 out of 100 eggs s u r v i v i n g i n an overnight t e s t . F i s h were tested before and a f t e r the f i r s t t r i a l . Those which d i d not become pa r e n t a l on the f i r s t t r i a l were given a second t r i a l w ith the same treatment. The f i n a l column shows the t o t a l number of f i s h which de-veloped egg-eating i n h i b i t i o n . STIMULUS NUMBER OF FISH REACHING CRITERION A f t e r A f t e r Eggs Nest Female n Before 1st 2nd T o t a l i + + - 4 0 3 0 3 i i - + + if 0 0 0 0 i i i + - + if 0 2 2 if i v + + + i f O i f - . A v - - - A O 0 0 0 64 i i or v i s 0.071. Thus eggs appear to be the key f a c t o r i n the i n d u c t i o n of p a r e n t a l behavior. A small number of t e s t s i n d i c a t e d that eggs k i l l e d by heat-i n g or f r e e z i n g would not induce p a r e n t a l behavior. The e f f e c -t i v e n e s s of eggs does not seem to be r e s t r i c t e d to eggs j u s t a f t e r spawning. I have induced p a r e n t a l behavior i n s e v e r a l f i s h by p r e s e n t a t i o n of 1 day old eggs. And more recent work i n our l a b o r a t o r y (Blythe Chang, personal communication) has i n d i c a t e d that even week o l d f r y are e f f e c t i v e . The s t i m u l i by which a f i s h already i n the p a r e n t a l s t a t e recognizes eggs may or may not be the same as those which are r e -sponsible f o r the i n d u c t i o n of the p a r e n t a l s t a t e . A small num-ber of t r i a l s w ith p a r e n t a l f i s h have i n d i c a t e d that they w i l l r e t r i e v e eggs d a r k l y stained w i t h Trypan Blue or C h l o r a z o l Black as long as the eggs are not k i l l e d . Eggs k i l l e d d u r i n g s t a i n i n g with Neutral Red were eaten. Eggs of the congeneric species, T. l e e r i , were r e t r i e v e d . Eggs of the paradise f i s h , Macropodus  o p e r c u l a r i s , a member of the same fa m i l y , were r e t r i e v e d i n some cases and eaten i n others. L i t t l e i s known about the sensory b a s i s of the i n d u c t i o n of p a r e n t a l behavior. Attempts to block i t by d e s t r u c t i o n of the o l f a c t o r y capsule or amputation of the p e l v i c f i n s (which possess t a s t e buds, Scharrer et a l . , 1947) were unsuccessful. 65 CHAPTER 5 THE ROLE OF PERFORMANCE OF THE SPAWNING ACT A. THE ROLE OF SPAWNING 1. I n t r o d u c t i o n The i n d u c t i o n of p a r e n t a l behavior i n naive f i s h by expo-sure to l a r g e numbers of eggs corresponds to observations i n severa l other species. But a major question remains: What r o l e do eggs have i n the n a t u r a l i n d u c t i o n of pa r e n t a l behavior at the time of spawning? There are s e v e r a l reasons f o r t h i n k i n g that exposure to eggs at the f i r s t complete spawning i s not the only f a c t o r i n v o l v e d i n the i n d u c t i o n of par e n t a l behavior. For one t h i n g , the numbers of eggs required to c o n s i s t e n t l y induce p a r e n t a l responses are very l a r g e . Since the female doesn't r e l e a s e her whole c l u t c h i n one spawning a c t , i t would seem p o s s i b l e that a spawning male could eat the separate batches of eggs without becoming p a r e n t a l . Also, even with very l a r g e numbers of eggs a c e r t a i n p r o p o r t i o n of non-spawning f i s h f a i l to become p a r e n t a l . Such p a r e n t a l f a i l -ures are seldom, i f ever, observed i n n a t u r a l spawnings. F i -n a l l y , though the l a t e n c i e s observed f o r the i n d u c t i o n of paren-t a l responses are short, they are not as short as i n n a t u r a l spawnings. A l l of these observations suggest that the performance of the spawning act may play some r o l e i n the i n d u c t i o n of paren-t a l behavior. I t could be that spawning i t s e l f induces the par-e n t a l s t a t e . Or spawning could i n some way f a c i l i t a t e the r e -sponse to eggs. Another p o s s i b i l i t y i s that spawning i t s e l f i s not s i g n i f i c a n t , but the eggs immediately a f t e r a spawning act 66 are a more potent stimulus than when they are c o l l e c t e d a f t e r the completion of a l l spawning. This experiment was designed to t e s t these hypotheses. 2. Methods The su b j e c t s were 35 s e x u a l l y inexperienced males, a l l from the same brood, and approximately 6 months o l d . The general ex-perimental procedure was to b r i n g a l l f i s h i n t o spawning condi-t i o n , then to present them with one of the f o l l o w i n g stimulus s i t u a t i o n s : -Spawning experience without exposure to eggs. -Exposure to eggs without performance of spawning. -Spawning experience followed by exposure to eggs. -Neither spawning experience nor exposure to eggs. A f t e r at l e a s t 1 1/2 hr any eggs or nest i n the tank were removed and a l l f i s h were given an overnight t e s t w i t h 200 eggs. The f i s h were brought i n t o spawning c o n d i t i o n by the s t a n -dard method, but the p a r t i t i o n s , i n s t e a d of being l e f t open u n t i l spawning occurred, were closed e a r l y i n the morning of the spawn-i n g day so that the time of the f i r s t spawning act could be con-t r o l l e d . P a i r s which came i n t o spawning c o n d i t i o n could be r e -cognized by the darker c o l o r a t i o n and increased a c t i v i t y of the females. For the groups which received "spawning experience" the oviduct of the female was plugged and the p a r t i t i o n opened to allow three complete spawning a c t s with the male. A f t e r each spav/ning act a check was made to a s c e r t a i n that no eggs had been released past the plug. A spawning act was considered complete 67 only when the f i s h showed the b r i e f swimming i n h i b i t i o n that normally f o l l o w s the r e l e a s e of gametes. To make oviduct plugs f o r the females, a piece of f i n e poly-ethylene tubing (outside diameter = 1.2 mm) was pushed i n t o a s e c t i o n of s o f t foam rubber to form a core. The foam surrounding the tube was trimmed i n t o a cone approximately 10 mm long and If mm wide at the base. When a female was ready to spawn, she was removed from the aquarium and held v e n t r a l side up i n a folded, damp paper towel. The moistened p l u s v/as i n s e r t e d i n t o the ano-g e n i t a l opening and pushed i n the p o s t e r i o r and d o r s a l d i r e c t i o n u n t i l only i t s base was exposed. The female was gently returned to the tank and u s u a l l y showed normal spawning behavior when tested 30 min l a t e r . The plug could be e a s i l y removed at the end of a t e s t . To provide eggs most of the foam was removed from the nest s i t e s of the donor f i s h before spawning so that the eggs could be c o l l e c t e d by d i p p i n g the edge of a p e t r i d i s h j u s t under the sur-face. Eggs were counted as they were picked up by eyedropper p i p e t t e from the p e t r i d i s h and t r a n s f e r r e d to a small beaker. They v/ere rel e a s e d i n t o the experimental tank by gently d i p p i n g the beaker under the surface so that they f l o a t e d f r e e . "Expo-sure to eggs" consisted of p r e s e n t a t i o n of e i t h e r 200 or 800 eggs. The 200 eggs were l e s s than 20 min o l d . I t v/as not p o s s i b l e to meet the same age c r i t e r i o n with 800 eggs. A l l were more than 15 and l e s s than 60 min o l d . "Spawning experience followed by exposure to eggs" was achieved by two methods. In one method the f i s h were given the "spawning experience" immediately followed by the "exposure to 68 eggs" as described above. In the other, a f t e r two spawning a c t s with a female bearing an oviduct plug, a t h i r d was allowed w i t h the female a f t e r the plug was removed or w i t h another normal f e -male. I t was hoped that t h i s would all o w a comparison of natu-r a l l y released eggs vrith the counted eggs used i n the other treatments. In summary, the experiment c o n s i s t e d of b r i n g i n g a l l f i s h i n t o spawning c o n d i t i o n and t e s t i n g them wi t h 200 eggs a f t e r ex-posure to one of the f o l l o w i n g stimulus s i t u a t i o n s : i . 200 eggs, i i . 800 eggs, i i i . 3 spawning a c t s , no eggs, i v . 3 spawning a c t s followed by 200 eggs. v. 3 spawning a c t s followed by 800 eggs, v i . 3 spawning a c t s , 2 of which were with a female, bearing an oviduct plug, and the t h i r d with a normal female or one vrith the oviduct plug r e -moved (the number of eggs removed from these tanks at the end of the stimulus period ranged from 20 to 2527). v i i . No spawning a c t s , no eggs. Five f i s h were used i n each group. 3. R e s u l t s The only f i s h developing egg-eating i n h i b i t i o n were those which received both eggs and spawning experience (Table V I ) . Only one of the f i v e r e c e i v i n g 200 eggs plus spawning showed egg-e a t i n g i n h i b i t i o n whereas f i v e r e c e i v i n g 800 eggs and spawning 69 TABLE VI. The e f f e c t of exposure to eggs and performance of the spawning act on the i n d u c t i o n of egg-eating i n h i b i t i o n i n male gouramis without previous reproductive e x p e r i -ence. The t a b l e shows the number of spawning ac t s and the number of eggs which comprised the stimulus f o r each group, as w e l l as two measures of the r e s u l t s of an overnight t e s t w i t h 200 eggs: mean egg s u r v i v a l , and number of f i s h reaching the c r i t e r i o n f o r egg-eating i n h i b i t i o n . GROUP N STIMULUS TEST RESULTS Number of Number Number Number Mean F i s h with of of of S u r v i v a l 25 or more Spawning Eggs Test of Test Eggs Sur-Acts Presented v i v i n g i 5 0 200 200 0.2 0 i i 5 0 800 200 1.8 0 i i i 5 3 0 200 1.8 0 i v 5 3 200 200 21.2 1 V 5 3 800 200 163.0 5 v i 5 3 Natural Spawning 200 109.6 4 v i i 5 0 0 200 1.4 0 70 d i d so. Four of those allowed n a t u r a l spawnings showed egg-e a t i n g i n h i b i t i o n . By the F i s h e r Exact P r o b a b i l i t y Test f i s h r e c e i v i n g spawning experience p l u s 800 eggs were d i f f e r e n t from those r e c e i v i n g only 800 eggs, only spawning experience, or n e i t h e r (P=0.00A0) and from those r e c e i v i n g 200 eggs p l u s spawn-i n g experience (P=0.0238). 4. Discussion This experiment showed that when moderate numbers of eggs are presented, spawning experience i s r e q u i r e d f o r p a r e n t a l r e -sponsiveness to develop. Spawning by i t s e l f was i n s u f f i c i e n t , and even 200 eggs immediately a f t e r spawning r a r e l y induced egg-e a t i n g i n h i b i t i o n . On the other hand, egg-eating i n h i b i t i o n was c o n s i s t e n t l y induced when spawning v/as followed by 800 eggs.. Eight hundred eggs without spawning were i n s u f f i c i e n t to induce egg-eating i n h i b i t i o n . Since previous experiments have shown that greater numbers of eggs can induce egg-eating i n h i b i t i o n , i t appears that the e f f e c t of spawning i s to somehow s e n s i t i z e the f i s h to the s t i m u l i from the eggs. In another experiment 500 or 1000 eggs were e f f e c t i v e i n i n -ducing p a r e n t a l behavior i n a small proportion of f i s h without spawning experience. In t h i s experiment 800 eggs alone d i d not seem to have t h i s e f f e c t . However, egg-eating i n h i b i t i o n was the only measure of p a r e n t a l behavior i n t h i s experiment. Also, absolute comparisons of e f f e c t i v e egg numbers betv/een t h i s and other experiments may not be v a l i d because i n t h i s experiment the eggs v/ere simply released i n t o the tank near the nest, whereas i n other experiments they were presented i n a group i n the foam 71 rubber frame. I t had been hoped that group v i , i n which the eggs were "presented" by a n a t u r a l l y spawning female, would allow a com-pari s o n of the e f f e c t i v e n e s s of experimental and n a t u r a l egg pre-s e n t a t i o n s . I t seemed p o s s i b l e that n a t u r a l l y released eggs would prove to be a more potent stimulus f o r the i n d u c t i o n of par e n t a l behavior than counted eggs, but, i n order to demonstrate t h i s , n a t u r a l l y released eggs would have to have induced egg-eati n g i n h i b i t i o n when an equal or greater number of counted eggs f a i l e d to do so. Thus i n t h i s experiment the number of n a t u r a l l y released eggs would have to have been 200 or l e s s . But i n the 1+ cases i n which egg-eating i n h i b i t i o n was induced i n group v i , the number of eggs recovered was more than 200. Thus no conclusions can be drawn about the r e l a t i v e e f f e c t i v e n e s s of eggs of d i f f e r -ent ages or the e f f e c t of the counting procedures. These r e s u l t s are s i m i l a r to a number of s t u d i e s of mammals i n which f a c t o r s associated w i t h pregnancy and p a r t u r i t i o n have been shown to f a c i l i t a t e responses to young. K l o p f e r et a l . (1964) showed that female goats were s e n s i t i v e to young only i n the immediate post-partum p e r i o d . Terkel and Rosenblatt (1968) decreased the la t e n c y f o r the i n d u c t i o n of p a r e n t a l behavior i n r a t s by i n j e c t i n g plasma from p a s t - p a r t u r i e n t females i n t o v i r -g i n s . Pregnancy has al s o been shown to i n f l u e n c e maternal r e -sponsiveness to pups i n hamsters (Richards, 1966b) and r a t s ( L o t t and Rosenblatt, 1969). 72 B. THE EFFECT OF SPAWNING ON THE SPEED OF DEVELOPMENT OF PARENTAL BEHAVIOR 1. I n t r o d u c t i o n Spawning was shown i n the previous experiment to f a c i l i t a t e the development of p a r e n t a l responsiveness w i t h moderate numbers of eggs. The purpose of the present experiment v/as to determine whether spawning had a detectable e f f e c t on the development of pa r e n t a l behavior when s u f f i c i e n t eggs v/ere given to induce par-e n t a l behavior without spawning experience. I t was a p r e l i m i n -ary i n v e s t i g a t i o n and v/as terminated when i t was c l e a r that the treatments had l i t t l e e f f e c t . 2. Methods The subjects were 9 month o l d a d u l t male gouramis without previous reproductive experience. The standard spawning pro-cedures were used with f l o a t i n g p l a s t i c nest supports and foam rubber egg p r e s e n t a t i o n frames. The experimental design was s i m i l a r to that of the previous experiment i n that f i s h v/ere brought i n t o spawning c o n d i t i o n , then allowed no spawning or 3 spawning a c t s with a female bear-i n g an oviduct p l u g and presented w i t h stimulus eggs. However, in s t e a d of 200 or 800 stimulus eggs, the very strong stimulus of 1 . 5 ml (about 3000 eggs) was used. The behavior of the f i s h dur-i n g i t s exposure to the strong and l o c a l i z e d p a r e n t a l stimulus was recorded. A f t e r approximately 1 hr, the stimulus eggs were removed and 200 eggs were given. S u r v i v a l of these 200 v/as de-termined the f o l l o w i n g morning. 73 Methods f o r b r i n g i n g the f i s h i n t o spawning readiness, a l l o w -i n g spawning without egg experience and o b t a i n i n g , measuring and presenting eggs have been described p r e v i o u s l y . Contact with eggs and r e t r i e v i n g were recorded f o r 20 min f o l l o w i n g the f i r s t contact with eggs. From the recorder c h a r t s I measured the time from the f i r s t egg contact to the f i r s t r e t r i e v i n g act and the i n t e r v a l s between each of the f i r s t 10 r e t r i e v i n g a c t s . I a l s o measured the amount of time i n each of these i n t e r v a l s a c t u a l l y spent c o n t a c t i n g eggs. This experiment was performed before the one reported i n Ch. 3B. Thus the short l a t e n c i e s f o r the s t a r t of p a r e n t a l be-havior were s u r p r i s i n g . In order to determine whether the pre-spawning exposure to the female r a t h e r than the a c t u a l spawning experience was the s i g n i f i c a n t f a c t o r i n the r a p i d i n d u c t i o n of p a r e n t a l behavior, two f i s h were t e s t e d without p r i o r exposure to the female. 3. R e s u l t s and Discussion F i s h allowed to spawn began to r e t r i e v e s l i g h t l y e a r l i e r and r e t r i e v e d s l i g h t l y more r a p i d l y than f i s h not allowed to spawn ( F i g . 13). However, observation of the f i s h suggested that t h i s was because f i s h which had been allowed to spawn were bolder and returned more promptly to the eggs a f t e r each r e t r i e v i n g a c t . In order to e l i m i n a t e t h i s e f f e c t , the r e s u l t s were analysed i n terms of the amount of egg contact preceding r e t r i e v i n g and . occupying the i n t e r v a l s between r e t r i e v i n g a c t s . The two-groups were nearly i d e n t i c a l when examined i n t h i s f a s h i o n . Spawning experience had no detectable e f f e c t on the number of stimulus 7k M I N U T E S FIGURE 13. The development of r e t r i e v i n g behavior i n response to 3000 eggs i n male gouramis which were allowed A) no p r i o r spawning experience, or B) prior spawn-ing experience. The graph shows the time of occur-rence of each of the f i r s t 10 r e t r i e v i n g acts except for one f i s h which performed only k r e t r i e v i n g acts during the recorded time. 75 TABLE VTI. E f f e c t s of spawning experience on p a r e n t a l behavior i n male gouramis presented w i t h 1.5 ml eggs. The ta b l e shows f o r each i n d i v i d u a l the le n g t h of time i t was exposed to the stimulus eggs, the volume of those eggs eaten and the s u r v i v a l of 200 eggs i n an over-night t e s t . TREATMENT No spawning No spawning No spawning No exposure to female No exposure to female DURATION EXPOSURE TO STIMULUS FISH ' (MIN) a 47 b 47 c 85 d 60 e 58 VOL. EATEN (ML) 0.1 0.1 0.4 0.2 0.1 OVERNIGHT SURVIVAL OF 200 EGGS 8 253 207 174 173 3 spawnings f 3 spawnings g 3 spawnings h 48 45 52 0.1 0.3 0.3 235 182 148 76 eggs eaten or on overnight egg s u r v i v a l (Table V I I ) . Egg-eating i n h i b i t i o n p e r s i s t e d overnight w i t h as l i t t l e as W? min exposure to the l a r g e number of eggs. The scores above 200 probably i n d i c a t e f a i l u r e to remove a l l the stimulus eggs be-fore p r e s e n t a t i o n of the 200 t e s t eggs. C. SUMMARY 1. P a r e n t a l behavior developed i n f i s h which were allowed 3 spawning a c t s and exposure to 800 eggs, but not i n f i s h which r e -ceived e i t h e r of these s t i m u l i alone. 2. Since i n other experiments males developed p a r e n t a l beha-v i o r without spawning when given l a r g e r numbers of eggs, spawn-i n g must s e n s i t i z e the f i s h i n some way to the i n f l u e n c e of the eggs. 3. Spawning had only a s l i g h t e f f e c t , i f any, on the r a t e of development of p a r e n t a l behavior i n f i s h given l a r g e numbers of eggs. 77 CHAPTER 6 THE ROLE OF ANDROGENS A. SEXUAL DIMORPHISM IN PARENTAL BEHAVIOR 1 . I n t r o d u c t i o n Most of my observations and those of M i l l e r ( 1 9 6 4 ) i n d i c a -ted that p a r e n t a l behavior i n blue gouramis was performed e x c l u -s i v e l y by the male. The female u s u a l l y has no opportunity to contact the eggs because of the male's aggressiveness. But when she can approach the nest, f o r example, a f t e r removal of the male, she u s u a l l y begins to eat the eggs. However, there have been b r i e f r e p o r t s of pa r e n t a l behavior by females of other species of the family B e l o n t i i d a e ( F o r s e l i u s , 1 9 5 7 c ) . And I have occasion-a l l y observed r e t r i e v i n g a c t s by female gouramis given l a r g e num-bers of eggs. The purpose of t h i s experiment was to examine i n more d e t a i l the sexual dimorphism i n the pa r e n t a l behavior of the blue gourami. The basic design of the experiment was to remove the eggs a f t e r spawning and replace a known number i n the tank a f t e r one member of the p a i r had been removed. R e t r i e v i n g and s u r v i v a l of eggs i n the tank were the measures of pa r e n t a l behavior. The de-si g n of the experiment a l s o allowed me to gain more inf o r m a t i o n about the maintenance of pa r e n t a l behavior. The c o n s i s t e n t l y low egg s u r v i v a l i n overnight t e s t s i n v a r i o u s experiments (about 50% at best) suggested that p a r e n t a l males ate some of the l i v e eggs which they were given. I t was p o s s i b l e that the missing eggs died and then were eaten, but egg v i a b i l i t y u s u a l l y appeared to be higher than t h i s . I t was a l s o p o s s i b l e that some aspect of 78 my experimental s i t u a t i o n was inadequate f o r normal maintenance of p a r e n t a l behavior. For example, i t may not p e r s i s t as w e l l when induced by eggs without spawning, or when the number of eggs present v/as very low, or when the t e s t eggs were from an unfamil-i a l female. P a r e n t a l behavior may a l s o have been di s r u p t e d by the disturbance of removing the nest and eggs. In t h i s e x p e r i -ment I used males which had spawned normally and tested them with l a r g e r numbers of t h e i r own eggs. I observed them immediately a f t e r r e p l a c i n g the eggs to see i f r e t r i e v i n g began promptly. And I incubated a sample of the eggs separately to see i f the egg l o s s v/as greater than that expected by n a t u r a l m o r t a l i t y . The experiment was performed i n three separate groups of t e s t s . In the second and t h i r d groups I v a r i e d some aspects of v/ater q u a l i t y (source, pH and p r i o r i n h a b i t a t i o n by gouramis) and female q u a l i t y (previous experience, r e a r i n g c o n d i t i o n s ) i n order to attempt to s o r t out the reasons f o r a period of low spawning success and low egg v i a b i l i t y . The e f f e c t s of water q u a l i t y have already been mentioned (Ch. I B ) . Tests of males and females were balanced w i t h i n the v a r i o u s treatments, so that i n the present s e c t i o n only r e s u l t s r e l e v a n t to sexual dimor-phism of p a r e n t a l behavior and to egg-eating by p a r e n t a l males w i l l be presented. 2. Methods The subjects i n the f i r s t group were adu l t gouramis w i t h previous breeding experience. In the second and t h i r d groups the males were also experienced, but the females were spawning f o r the f i r s t time. The inexperienced females were a l l from the 79 same brood but those i n the second group were r a i s e d i n the or-dinary c o n d i t i o n s of our l a b o r a t o r y , while the t h i r d group was r a i s e d on a d i e t of commercial t r o u t chow i n another l a b o r a t o r y . F i s h were set up to spawn using the standard procedure, with p l a s t i c nest supports s u b s t i t u t e d f o r f l o a t i n g p l a n t s . E i t h e r tap water or water from the departmental d e c h l o r i n a t e d water sup-p l y or a mixture was used. Some tanks were completely cleaned before the experiment; others were simply topped up wi t h f r e s h water. In the third.group of t e s t s , the pH of the tanks was checked d a i l y and i n three groups was adjusted to approximately 4.0, 6.5, or 8.0 by means of sodium bicarbonate and hy d r o c h l o r i c a c i d s o l u t i o n s . When a spawning was completed, the nest, eggs and nest sup-port were dipped out of the tank w i t h a l l beaker. One member of-the p a i r was removed from the tank. The nest support was ri n s e d and replaced,and the p a r t i t i o n was l e f t open. In order to prevent p o s s i b l e damage to the eggs, they were not concentrated by the usual method of pouring the c l u t c h i n t o a fine-meshed net. A f t e r removing most of the water from the beaker with a p i p e t t e , the eggs were poured i n t o a p e t r i d i s h with the remaining water and counted as they were picked up with an eyedropper. Approximately 100 l i v e eggs were placed i n a 100 ml beaker w i t h some water from the spawning tank. This was f l o a t e d i n the tank as a t e s t f o r egg v i a b i l i t y . Then approxi-mately 800 l i v e eggs, or the remainder of the c l u t c h i f l e s s than 800, were counted i n t o a second beaker f o r the t e s t . The 800 eggs were released i n t o the tank near the nest sup-p o r t . The t e s t f i s h was observed f o r 5 min from the f i r s t egg 80 contact. The t o t a l number of r e t r i e v i n g a c t s w i t h i n that p e r i o d was recorded. On the f o l l o w i n g afternoon at about the time of hatching, the eggs were removed from the tank. The number of l i v e and dead eggs remaining i n the tank and i n the f l o a t i n g beaker was counted. From the s u r v i v a l of eggs i n the beaker, an expected s u r v i v a l f o r the tank was c a l c u l a t e d . An estimate of the number eaten was ob-tained by s u b t r a c t i n g the a c t u a l s u r v i v a l from the expected sur-v i v a l . From t h i s the estimated per cent of l i v e and dead eggs eaten was c a l c u l a t e d . Altogether 29 spawnings were recorded, wi t h 14 males and 15 females being t e s t e d . 3. R e s u l t s and D i s c u s s i o n The experiment confirmed the sexual dimorphism i n the ten-dency to perform p a r e n t a l behavior as measured by frequency of r e t r i e v i n g , egg s u r v i v a l , and d i s c r i m i n a t i o n between l i v e and dead eggs (Table V I I I ) . But the d i f f e r e n c e between the sexes was not absolute. Some females performed p a r e n t a l behavior, and the pa r e n t a l care by males was f a r from p e r f e c t . The d i f f e r e n c e between the frequency of r e t r i e v i n g by males and females was very great. A l l males r e t r i e v e d d u r i n g the 5 min p e r i o d . Four females r e t r i e v e d during the 5 min observation, and two more were seen to r e t r i e v e l a t e r . Two others performed incomplete r e t r i e v i n g a c t s — i . e . , o r i e n t a t i o n to the surface i n one area of the tank, but no egg r e l e a s e . Only one of the f e -males r e t r i e v e d at frequencies comparable to those observed f o r males. Males and females d i d not d i f f e r as g r e a t l y i n egg-eating 81 TABLE V I I I . Sexual dimorphism i n the p a r e n t a l behavior of gour-amis. A. Comparison of males and females vrith respect to egg-eating i n h i b i t i o n and the frequency of r e t r i e v i n g shown i n response to 800 of t h e i r own eggs. B. Comparison of the estimated percent eaten of l i v e and dead eggs by males and females. A. Males vs. Females (Mann-Whitney U Test, 1 - t a i l e d ) MEAN+S.E. Male(n=14) Female(n=15) U P R e t r i e v i n g a c t s / 5 min 11.36+1.26 0.67+0.46 3.5 « .001 Percent l i v e eggs eaten 73.59+8.56 96.62+1.83 48.5 <-01 B. Percent l i v e vs. percent dead eggs eaten (Wilcoxon Matched-P a i r s Signed Ranks Test, 1 - t a i l e d ) MEAN+S.E. n Dead L i v e T P Males 14 89.22+7.01 73.59±8.56 21 .025 Females 15 95.39+2.64 96.62+1.83 42 N.S. 82 as they d i d i n r e t r i e v i n g . The p r o p o r t i o n of l i v e eggs eaten was very high f o r a l l females and f o r a l l but f i v e males. F i v e a d d i -t i o n a l males ate most of the eggs, but the number s u r v i v i n g (16-76) was s t i l l greater than could be explained by the eggs being overlooked i n the r e l a t i v e l y simple environment of the t e s t tank. None of the females showed egg-eating i n h i b i t i o n at a l e v e l com-parable to the 5 highest males. But 6 gave some i n d i c a t i o n of egg-eating i n h i b i t i o n with 16 to 184 eggs s u r v i v i n g . F i v e of these females had a l s o r e t r i e v e d or performed incomplete r e t r i e v -i n g . At the time of egg removal f i v e of the females w i t h s i g n i -f i c a n t numbers of eggs present a l s o had small patches of foam under the nest support as d i d one other female. P a r e n t a l beha-v i o r was more frequent among females spawning f o r the f i r s t time than among experienced females. The explanation f o r the apparent high r a t e of egg-eating by " p a r e n t a l " males remains p u z z l i n g . The high r a t e s of egg l o s s v/ere comparable to those observed i n other experiments such as those showing i n d u c t i o n of p a r e n t a l behavior by exposure to eggs alone (Ch. 3B). These high egg l o s s e s occurred i n s p i t e of pre-sumably more favorable c o n d i t i o n s . A l o s s of p a r e n t a l behavior because of the disturbance of the procedures does not seem to provide a s u f f i c i e n t explanation. Some males i n d i c a t e d d i s r u p -t i o n of p a r e n t a l behavior apparently e a t i n g the f i r s t eggs they picked up or some of those picked up between r e t r i e v i n g a c t s , but a l l males d i d r e t r i e v e repeatedly while there were s t i l l many eggs l e f t . A l s o, eggs v/ere eaten even by males that began r e t r i e v i n g very promptly. The method used assumes that m o r t a l i t y , other than that 83 caused by males e a t i n g eggs, i s the same i n the tank as i n the beaker. The apparent high egg l o s s e s might be explained by an er r o r i n t h i s assumption. I t i s p o s s i b l e that there were other egg predators i n the tank, though there i s no evidence of t h i s , or that the male damages the eggs i n r e t r i e v i n g or mouthing and then eats the dead eggs. The only other p o s s i b l e explanations of the low overnight egg s u r v i v a l suggest that even "normal" parental behavior i s not very e f f i c i e n t . Males w i t h c l u t c h e s of low v i a b i l i t y could eat l i v e eggs along w i t h dead ones. But the data do not support t h i s hypothesis ( F i g . 14). Some eggs may be swallowed during r e t r i e v i n g . This would e x p l a i n why a s i m i l a r f r a c t i o n of eggs s u r v i v e s whether 100 (Ch. 3A), 200 (Ch. 3B),; or 800 eggs are given. Corroborating evidence a l s o comes from the experiment reported i n Ch. 3C. There, males that r e t r i e v e d i n response to each of a s e r i e s of presentations of 100 eggs, had only about h a l f of the eggs i n the nest at the end of the s e r i e s . Males may al s o eat some of the eggs i n the nest, e i t h e r a c c i d e n t l y , or as a necessary stimulus to maintain them i n the p a r e n t a l s t a t e . Perhaps t h i s i s f a c i l i t a t e d i n the absence of the nest. Some p r e l i m i n a r y s t u d i e s on paradise f i s h , Macropodus  o p e r c u l a r i s , confirmed e a r l i e r r e p o r t s c i t e d by F o r s e l i u s (1957c) that female as wel l . a s male paradise f i s h can perform p a r e n t a l behavior. Paradise f i s h of both sexes (3 males and 2 females) showed l e s s e a t i n g of l i v e eggs than d i d gouramis when tested i n a s i m i l a r f a s h i o n . The d i f f e r e n c e between paradise f i s h and gouramis r a i s e s some i n t e r e s t i n g questions. One i s how d i f f e r e n c e s i n pa r e n t a l behavior r e l a t e to a v a r i e t y of d i f f e r -ences i n the co u r t s h i p of the two species. A second question Sk Ul 100 -i OD Z Ul V-< Ul CO <D C9 Ul Ul > 7 5 -5 0 -2 5 -Ui u cc Ul a 1~ 2 5 5 0 I 7 5 1 0 0 P E R C E N T H A T C H I N G I N I N C U B A T I O N FIGURE lk. The r e l a t i o n s h i p between egg v i a b i l i t y (percent hatching of 100 incubated eggs) and male p a r e n t a l success (estimated percent of l i v e eggs eaten). 85 concerns the r o l e of gonadal hormones. I f androgens play a r o l e i n the dimorphic p a r e n t a l behavior of gouramis, what r o l e do go-nadal hormones play i n the more monomorphic p a r e n t a l behavior of paradise f i s h ? The reasons f o r egg-eating by p a r e n t a l male gouramis are not yet understood. But t h i s study has provided ample support f o r a widely accepted aphorism, which recommends the post-hatching per-i o d as the most appropriate time f o r the enumeration of young fowl. B. EFFECT OF METHYL TESTOSTERONE ON THE PARENTAL AND NEST BUILDING BEHAVIOR AND THE SECONDARY SEX CHARACTERS OF FEMALE BLUE GOURAMIS 1. I n t r o d u c t i o n The observation that p a r e n t a l behavior i s performed pre-dominantly by males suggests that androgens may play a r o l e i n the i n d u c t i o n of p a r e n t a l behavior. As a f i r s t step i n t e s t i n g t h i s hypothesis I decided to implant females w i t h p e l l e t s of methyl testosterone (MT) and compare t h e i r responses to a l a r g e number of eggs w i t h those of c o n t r o l females implanted w i t h c h o l -e s t e r o l (C). Implanting females with androgens was chosen over c a s t r a t i o n and androgen treatment of males because of i t s tech-n i c a l s i m p l i c i t y and because i t avoided the problems of hidden regeneration of androgenic t i s s u e (Johns and L i l e y , 1970). In a d d i t i o n to p a r e n t a l behavior, nest b u i l d i n g and two morphologi-c a l c h a r a c t e r i s t i c s were studied. These were the l e n g t h of the d o r s a l f i n , a secondary sex character, and the d e n s i t y of melano-86 phores on the s c a l e s , which was thought to be r e l a t e d to the more pronounced darkening of males duri n g sexual or aggressive a r o u s a l . 2. Methods Sixteen a d u l t female gouramis r a i s e d i n t h i s l a b o r a t o r y were used. A l l had spawned 2 to 15 days before the s t a r t of the ex-periment. One MT implanted female died a f t e r 30 days. The data from t h i s i n d i v i d u a l are not inc l u d e d i n the a n a l y s i s . The f i s h were held i n kO 1 aquaria each d i v i d e d i n t o 4 com-partments by p a r t i t i o n s of p l e x i g l a s s and f i b e r g l a s s window screen. MT and C t r e a t e d f i s h v/ere held i n d i f f e r e n t aquaria to minimize the r i s k of exposing the C group to androgens. P e l l e t s v/ere made by melting MT (Mann Research L a b o r a t o r i e s ) or C (Calbiochem) then repeatedly d i p p i n g one end of a short piece of braided s i l k thread i n t o the c o o l i n g s t e r o i d u n t i l a bead of s u f f i c i e n t s i z e v/as formed. Each female received two im-p l a n t s . The f i r s t was implanted' at the s t a r t of the experiment (Day 1) and the second on Day 17. The weights of s t e r o i d i n the tv/o implants are given i n Table IX. P e l l e t s were i n s e r t e d i n t o the body c a v i t y through a small s l i t i n the body w a l l . The f i s h were t e s t e d f o r the i n d u c t i o n of p a r e n t a l behavior i n random order between 25 and 38 days a f t e r the i n i t i a l s t e r o i d implant. For t h i s the subjects were t r a n s f e r r e d to separate AO 1 glass and s t a i n l e s s s t e e l aquaria maintained under c o n d i t i o n s s i m i l a r to the ho l d i n g compartments. Each contained rooted and f l o a t i n g water s p r i t e p l a n t s and an egg p r e s e n t a t i o n frame. Each aquarium served f o r tv/o subjects (from the same treatment group) 87 TABLE IX. Mean and range of weights i n mg of s t e r o i d implanted i n t o female gouramis. The values are given f o r the f i r s t implant (Day 1) and the second (Day 17) and f o r the t o t a l r e c e i v ed by each animal. WEIGHT OF STEROID (Mg) F i r s t Implant Second Implant T o t a l Mean Range Mean Range Mean Range Methyl testosterone 3.6 2.4-5.1 2.8 2.2-3.1 6.4 4.9-7-7 C h o l e s t e r o l :. .3.8 2.8-5.0 2.6 2.1-2.9 6.3 4.9-7-9 88 which were separated by a p a r t i t i o n of black p l e x i g l a s s . A f i s h was placed i n one h a l f of the t e s t aquarium at l e a s t 2 days be-fore the s t a r t of t e s t i n g . Stimulus eggs were obtained as described p r e v i o u s l y . A f t e r adding 1.5 ml (about 3000) eggs to the p r e s e n t a t i o n frame, egg contact and r e t r i e v i n g behavior were recorded f o r 20 min. from the f i r s t contact w i t h eggs. In t h i s experiment any release of eggs away from the p r e s e n t a t i o n frame was counted as a r e t r i e v i n g a c t . One to 1 1/2 hr a f t e r the f i r s t egg contact, a l l eggs r e -maining were removed and t h e i r volume determined. In order to t e s t the p e r s i s t e n t e f f e c t s of exposure to the stimulus eggs, 200 l i v e eggs were added and allowed to f l o a t over the surface of the tank. S u r v i v a l of the 200 was determined at about 9:30 the f o l -lowing morning. From the behavioral record the f o l l o w i n g measures were ob-t a i n e d : 1. T o t a l number of r e t r i e v i n g a c t s . . 2. The time from the f i r s t egg contact to the f i r s t r e t r i e v i n g a c t . 3. The duration of egg contact before the f i r s t r e t r i e v i n g a c t . A. The l e n g t h of the i n t e r v a l s between each of the f i r s t 10 r e t r i e v i n g a c t s . 5« The amount of egg contact between each of the f i r s t 10 r e t r i e v i n g a c t s . Holding compartments and t e s t i n g tanks were inspected; each morning f o r signs of nest b u i l d i n g , and the q u a l i t y of the nest was rated according to the scale p r e v i o u s l y described (Ch. 2 ) . 8 9 The f i s h were inspected d a i l y f o r the presence of a black rim on the t i p of the d o r s a l f i n . This had been observed i n pre-l i m i n a r y i n v e s t i g a t i o n s to be one of the f i r s t e f f e c t s of andro-gens i n female gouramis. The f i e h were measured on Days 1, 8 , 1 6 , 2 3 , and 1+3 to the nearest 0.1 mm using d i a l - r e a d i n g c a l i p e r s . The f o l l o w i n g measurements were made: Standard l e n g t h - Tip of upper jaw to crease at j u n c t i o n of median caudal f i n rays and median hypural p l a t e . , Snout-Dorsal f i n l e n g t h - Tip of upper jaw to t i p of d o r s a l f i n . The r a t i o of snout-dorsal f i n l e n g t h to standard l e n g t h was c a l -culated to give a measure of the r e l a t i v e d o r s a l f i n l e n g t h . At the end of the experiment (Day Zf5) 1 0 or more s c a l e s were removed i n d i v i d u a l l y with f i n e foreceps from the v e n t r a l caudal r e g i o n j u s t below the l a t e r a l l i n e and a n t e r i o r to the caudal spot. They were placed on a microscope s l i d e i n a few drops of a d r e n a l i n c h l o r i d e s o l u t i o n ( 1 : 1 0 0 0 ) which caused aggre-gati o n of the pigment. The density of the melanophores on 1 0 s c a l e s from each f i s h was determined a few minutes l a t e r . The pigmented area of each s c a l e was centered i n the microscope f i e l d at low power. Then under high m a g n i f i c a t i o n ( 2 7 0 X) the t o t a l number of melanophores i n the f i e l d was determined w i t h the a i d of a g r i d type g r a t i c u l e . 3. R e s u l t s and D i s c u s s i o n MT t r e a t e d f i s h showed more r e t r i e v i n g a c t s and ate fewer eggs during the stimulus period than c o n t r o l s . They a l s o ate 90 fewer of the 200 eggs i n the overnight t e s t . There was very l i t -t l e d i f f e r e n c e between the two groups i n the time to the f i r s t r e t r i e v i n g a c t , but MT t r e a t e d f i s h spent s i g n i f i c a n t l y l e s s time c o n t a c t i n g eggs before t h e i r f i r s t r e t r i e v i n g act ( F i g . 15 and Table X). As i n the previous experiment, r e t r i e v i n g a c t s were r e c o r -ded f o r some c o n t r o l females. Often they d i d not i n c l u d e a l l the components of normal male r e t r i e v i n g — p a u s e , o r i e n t a t i o n , and jaw movements. However, three females showed some components of nor-mal male r e t r i e v i n g . One c o n t r o l f i s h shov/ed signs of egg-eating i n h i b i t i o n . Twenty s i x of the 200 eggs survived overnight with t h i s i n d i v i d u a l . Though a low number, i t does seem more than could have survived by being overlooked among the f l o a t i n g p l a n t s . Several e f f e c t s of androgen treatment are shown i n the de-velopment of r e t r i e v i n g behavior i n r e l a t i o n to the d u r a t i o n of egg contact ( F i g . 16). Masculinized females began to r e t r i e v e a f t e r l e s s egg contact. In f i v e of seven cases the f i r s t r e -t r i e v i n g act was r a p i d l y followed by a r e g u l a r s e r i e s of r e -t r i e v i n g a c t s separated by short i n t e r v a l s spent p i c k i n g up eggs. This p a t t e r n was very s i m i l a r to that of normal males given 1.5 ml eggs (Ch. 3B). A p a t t e r n of moderately r e g u l a r r e t r i e v i n g was achieved by four of the eight c o n t r o l females. But these d i f -fered' not only i n the form of the egg r e l e a s e s , as described above, but also i n that they d i d not s t a r t abruptly at the maxi-mal r a t e and i n v o l v e d more egg contact, apparently egg-eating, i n the i n t e r v a l s . I t i s i n t e r e s t i n g that four of the eight con-t r o l females showed some r e g u l a r i t y i n r e t r i e v i n g , even though 9 1 O < §40 -1 LU E 3 0 - | y-U J CC LU 2 I OH a. ! © MT C A AA A O A o 0 0 O A4 M l.5n I.O -LU t— < LU CO e> LU iuo.5-O > © • OOO O o O O AA A A A AA CO cc H i o L U ~ cc o -LU — CQ CD L _ 2 2 8 " O < y— 2 O O o CD LU LU 2-b. co ^200-O LU cl ' 50 < > > QC Z3 CO IOO-50" MT O o & o O M A A O M T C M MT C M FIGURE 15. I n d i v i d u a l values f o r 4 measures of p a r e n t a l beha-v i o r i n female gouramis implanted vrith methyl t e s -tosterone ( ©) or c h o l e s t e r o l (O ) presented w i t h 1.5 ml r e c e n t l y spawned eggs. For comparison, data are included from another experiment (Ch. 3B) i n which p r e v i o u s l y inexperienced male gouramis (A) were exposed to a s i m i l a r t e s t i n g procedure. 92 FIGURE 16. The e f f e c t of methyl testosterone and c h o l e s t e r o l on the development of r e t r i e v i n g behavior i n female gouramis. For each f i s h the occurrence of each of the f i r s t 10 r e t r i e v i n g a c t s i s p l o t t e d i n r e l a t i o n to the cumulative amount of time spent i n contact with eggs. Large c i r c l e s i n d i c a t e the f i n a l r e t r i e v -i n g act performed during the recording session.. 93 TABLE X. Parental behavior of female gouramis implanted v/ith methyl testosterone (n=7) or cholesterol (n=8) and presented v/ith the stimulus of 1.5 ml recently spawned eggs. Note: When the variances of the tv/o groups were not equal, degrees of freedom was appropriately re-duced according to Welch's solution to the Behrens-Fisher problem (See Brovmlee, 1965, pp. 299-303). Measure Methyl Testosterone Mean+S.E. Cholesterol Mean+S.E. t test ( 1-tailed) t DF P Total Retrieving Acts i n 20 Min Time (Min) From F i r s t Egg Con-tact to F i r s t Retrieving Act Duration (Min) of Egg Contact Before F i r s t Retrieving Act Volume of Stimulus Eggs Eaten (Ml) Overnight Survival of 200 Eggs 2A.14+A.61 3.62+1.67 0.A8+0.13 0.17+0.05 8.37+2.94 4.76+2.22 3.17+0.98 1.01+0.08 114.43+27.62 8.87+2.63 2.95 13 < . o i 0.40 13 N.S. 2.72 7 <.025 8.80 13 <.ooo5 3.80 6 <.Q05 9k they v/ere e a t i n g eggs i n between. They were more r e g u l a r than were the "weak r e t r i e v e r s " of the experiment reported i n Ch. 3B. Note, however, that i n that experiment, simple r e l e a s e s were not recorded as " r e t r i e v i n g , " These r e s u l t s nevertheless suggest that even c o n t r o l females had a c e r t a i n r e t r i e v i n g tendency which was stimulated by androgens, a f f e c t i n g the frequency, complete-ness, and l a t e n c y to the s t a r t of r e t r i e v i n g . While none of the 8 C implanted f i s h b u i l t nests, 5 of 7 MT t r e a t e d females showed nest b u i l d i n g during the course of the ex-periment. Only one b u i l t a l e v e l N3 nest, but a l l 5 showed the N2 l e v e l of nest b u i l d i n g on at l e a s t one day. Only 2 f i s h showed any s i g n of nest b u i l d i n g i n the h o l d i n g compartments before being placed i n the t e s t i n g tanks. One f i s h began 1 3 days a f t e r the s t a r t of treatment and the other 7 days l a t e r . The other 3 f i s h b u i l t only during t h e i r stay i n the t e s t i n g tanks. There v/as a tendency f o r the f i s h which v/ere t e s t e d l a t e r to show more nest b u i l d i n g than f i s h which v/ere t e s t e d e a r l i e r . D a i l y observations of the f i s h i n d i c a t e d the presence of a dark rim on the t i p of the d o r s a l f i n i n a l l MT t r e a t e d f i s h 3 days a f t e r i m p l a n t a t i o n of the s t e r o i d p e l l e t s . This dark area p e r s i s t e d f o r 5 to 1 2 days. A f t e r only 8 days the two treatments showed a h i g h l y s i g n i f i c a n t d i f f e r e n c e i n t h e i r e f f e c t on d o r s a l f i n l e n g t h ( F i g . 1 7 ) . The r a t i o of snout-dorsal f i n l e n g t h to standard l e n g t h i n the MT t r e a t e d females was very s i m i l a r to that of normal males by the end of the experiment. The greater length of the d o r s a l f i n of the male gourami has been described i n the popular aquarium l i t e r a t u r e (e.g., Innes, 1 9 6 6 ) and quan-t i f i e d by J u l i a n o ( 1 9 6 5 ) . Johns and L i l e y ( 1 9 7 0 ) showed;the 9 5 1.050-{ J Q CO CHOLESTEROL^ 900-0 10 20 30 DAYS AFTER STEROID IMPLANTATION T 40 FIGURE 17. The effect of methyl testosterone and cholesterol implants on the r a t i o of snout-dorsal f i n length to standard length (S.D.L./S.L.) i n female gouramis. The points show means and v e r t i c a l l i n e s indicate 95% confidence l i m i t s . For comparison the values from 10 normal males are included. The hatched bar indicates the days on which a dark rim was seen on the t i p of the dorsal f i n of methyl testosterone treated f i s h . 96 dependence of t h i s character upon androgens i n males. The pre-sent study confirms these f i n d i n g s and p o i n t s out the p o t e n t i a l of the female f o r r a p i d development of t h i s masculine t r a i t . The MT t r e a t e d f i s h appeared much darker than the C tr e a t e d 2 f i s h . C tr e a t e d f i s h averaged 111.09 melanophores per mm . MT increased t h i s to an average of 502.93. This d i f f e r e n c e was h i g h l y s i g n i f i c a n t ( 1 - t a i l e d t t e s t , P<0.0005). A group of 10 normal males measured f o r comparison averaged only 121.85, much c l o s e r to the c o n t r o l females. In summary, the present i n v e s t i g a t i o n has shown that the i n d u c t i o n of par e n t a l behavior i s f a c i l i t a t e d by androgens. Com-parison with the r e s u l t s of s i m i l a r t e s t s on normal males shows that MT t r e a t e d females gave about the same l e v e l of response as normal males. In both groups some i n d i v i d u a l s f a i l e d to respond to the stimulus eggs wi t h high l e v e l s of r e t r i e v i n g and p e r s i s -tent egg-eating i n h i b i t i o n . And among those t h a t d i d respond, the frequency of r e t r i e v i n g was h i g h l y v a r i a b l e . Since normal males do not show pa r e n t a l behavior when presented w i t h s m a l l numbers of eggs, androgens apparently do not induce p a r e n t a l be-havior, but s e n s i t i z e the f i s h i n some way to the eggs which are the i n d u c t i v e stimulus. Further d i s c u s s i o n of the r o l e of go-nadal hormones i n the p a r e n t a l behavior of f i s h w i l l be reserved u n t i l the f o l l o w i n g s e c t i o n . 97 C. THE EFFECTS OF CASTRATION ON THE PARENTAL AND NEST BUILDING BEHAVIOR OF MALE GOURAMIS 1. I n t r o d u c t i o n The previous experiment provided evidence that androgens had an e f f e c t on the capacity of gouramis to develop p a r e n t a l beha-v i o r . These r e s u l t s d i f f e r e d from those of some workers on the three-spined s t i c k l e b a c k , a f i s h that i s s i m i l a r to the gourami i n that only the male performs p a r e n t a l behavior and i n that p a r e n t a l behavior can be induced by presenting eggs to males which have not spawned (Van I e r s e l , 1953). Baggerman (1957) c i t e d unpublished work by Van I e r s e l and Burggraaf which showed that c a s t r a t i o n d i d not prevent the development of p a r e n t a l be-havior i n response to eggs, though the fanning shown was at a "somewhat lower l e v e l . " Baggerman reported that she had repeated these experiments w i t h the same r e s u l t s . On the other hand, Smith and Hoar (1967) c a s t r a t e d males on the second and t h i r d days a f t e r spawning and found t h a t 8 of 9 males ate the eggs on the t h i r d or f o u r t h day. These r e s u l t s do not correspond to those of De Jong-van der Heij ( c i t e d by Baggerman, 1968) who c a s t r a t e d 8 males on the t h i r d and f o u r t h days of the p a r e n t a l c y c l e and found no e f f e c t on fanning. The l a c k of e f f e c t of c a s t r a t i o n sometimes observed i n , s t i c k l e b a c k s could i n d i c a t e a d i f f e r e n t r o l e of androgens from that i n gouramis. But another p o s s i b i l i t y i s that androgens are necessary to develop the capacity f o r p a r e n t a l behavior but not necessary to maintain i t . Thus females r e q u i r e androgen t r e a t -ment, but males are unaffected by c a s t r a t i o n . To compare 98 gouramis w i t h s t i c k l e b a c k s , I decided to c a s t r a t e male gouramis and attempt to induce p a r e n t a l behavior by p r e s e n t a t i o n of a l a r g e number of eggs. In a p r e l i m i n a r y experiment l+ experienced males were cas-t r a t e d and tested 9-13 days a f t e r c a s t r a t i o n . A l l performed some r e t r i e v i n g a c t s , and 2 performed as w e l l as the c o n t r o l s . One f i s h , t ested 3 1 days a f t e r gonadectoray, performed l i t t l e r e t r i e v -i n g . The r e s u l t s were complicated by low v i a b i l i t y of the stimu-l u s eggs and by the f a c t that most of the f i s h e v e n t u a l l y resumed nest b u i l d i n g i n d i c a t i n g the presence of androgens. However, i n a l l cases nest b u i l d i n g was at the 0 or t r a c e l e v e l at the time of t e s t i n g . These r e s u l t s at l e a s t suggested that experienced males could perform p a r e n t a l behavior a short time a f t e r andro-gen l e v e l s and nest b u i l d i n g had been g r e a t l y reduced. There-f o r e , I decided to determine whether inexperienced f i s h , c a s t r a -ted f o r a longer period of time, could a l s o r e t a i n the c a p a c i t y to perform p a r e n t a l behavior. 2. Methods The intended procedure was to c a s t r a t e or perform a sham operation upon inexperienced a d u l t male gouramis, and a f t e r 5 0 days to t e s t f o r the i n d u c t i o n of p a r e n t a l behavior. This plan had to be modified because only a very small p r o p o r t i o n of sham operated f i s h developed p a r e n t a l behavior on the f i r s t t r i a l . I thought that t h i s might be because they had had t h e i r nest r e -moved before t e s t i n g ( i n order to make t h e i r experimental s i t u -a t i o n more s i m i l a r to the c a s t r a t e d f i s h ) . R e - t e s t i n g the shams provided no evidence that t h i s was a major f a c t o r , but a l a r g e r i 99 p r o p o r t i o n developed p a r e n t a l behavior. The c a s t r a t e s were then r e - t e s t e d as w e l l , but by t h i s time elongated d o r s a l f i n s and the r e - i n i t i a t i o n of nest b u i l d i n g i n many suggested regeneration of androgenic t i s s u e . The c a s t r a t i o n procedure was adapted from that used by Johns (Johns and L i l e y , 1970). Before operations f i s h were held at l e a s t overnight i n i n d i v i d u a l s e c t i o n s of a AO 1 aquarium i n a t e l e o s t s a l i n e s o l u t i o n (See Ch. 1 ) . Instead of MS 222 c o l d v/as used as the a n e s t h e t i c . For an operation, a f i s h was removed and placed i n 700 ml. of s a l i n e i n a 1 1 beaker. This v/as placed on a bed of crushed i c e so that i t would cool from 26° to 15°C i n about 15-20 min. At t h i s temperature the f i s h was almost com-p l e t e l y anesthetized and was wrapped i n a c o l d , wet paper towel v/ith only the middle part of the body exposed. A s e m i - c i r c u l a r cut was made f o l l o w i n g the p o s t e r i o r curve of the body c a v i t y . The lower end of t h i s curve was brought very close to the vent. Care was taken to remove the whole t e s t i s . At the d i s t a l end small pieces of t e s t i c u l a r t i s s u e may l i e along blood v e s s e l s . At the proximal end the juncture of t e s t i s v/ith the v e n t r a l body w a l l was cut wit h f i n e forceps. Sham operated f i s h r e c e i v e d the same i n c i s i o n , but the t e s t i s was l e f t i n t a c t . The v/ound was closed with a s i n g l e suture, and the f i s h was returned to the hol d i n g tank. A r t i f i c i a l r e s p i r a t i o n by means of an eyedropper p i p e t t e was sometimes re q u i r e d . For the next few days the s a l i n e was g r a d u a l l y d i l u t e d with tap water. A f t e r about 1 week the f i s h were placed i n groups i n AO 1 aquaria of f r e s h water. Forty days a f t e r the operation 22 c a s t r a t e s were s u r v i v i n g i n good h e a l t h . Of these, 9 had lon g d o r s a l f i n s and v/ere judged to have regenerated t e s t e s . The other 13 had short d o r s a l f i n s , and, along vrith 11 sham operated f i s h , were placed i n i n d i v i d u a l aquaria f o r t e s t i n g . F i s h were tes t e d i n ifO 1 aquaria using the standard set up vrith a r t i f i c i a l nest supports. The only m o d i f i c a t i o n was the a d d i t i o n of 5 g of sodium bicarbonate to each tank to improve egg v i a b i l i t y . Egg donor tanks v/ere s i m i l a r l y t r e a t e d . The presence of nests was recorded d a i l y . Three c a s t r a t e d males began low l e v e l s of nest b u i l d i n g w i t h i n the f i r s t 10 days and were r e -j e c t e d . The d e s c r i p t i o n s of nest types were given i n Ch. 2. To com-pute a nest b u i l d i n g index, point values were a l l o t e d f o r nest q u a l i t y as f o l l o w s : Nest P o i n t s None 0 Trace 1 Nl 2 N2 3 N3 k ' For each group a mean nest index was computed f o r blocks of 1+ days s t a r t i n g 1-2 days a f t e r the f i s h were introduced i n t o the t e s t i n g aquaria. The method of o b t a i n i n g , c o l l e c t i n g and measuring t e s t eggs was the same as described p r e v i o u s l y . The stimulus was 1.0 ml (approximately 2000) f r e s h l y spawned eggs. In the f i r s t t r i a l , shams had t h e i r nests removed an hour, or more before t e s t i n g to c o n t r o l f o r e f f e c t s of the nest on p a r e n t a l behavior. Castrates had t h e i r nest supports s i m i l a r l y d i s t u r b e d . In the second t r i a l 101 • only h a l f the shams had t h e i r nests removed. The number of r e -t r i e v i n g a c t s i n 15 min a f t e r the f i r s t contact vrith eggs was r e -corded. In the f i r s t t r i a l , eggs were removed approximately 1 hr (range 55-100 min) a f t e r they had been presented. In the second t r i a l eggs v/ere l e f t i n the tanks and removed a f t e r 24 hr. The f i r s t t r i a l was given 50-57 days and the second t r i a l 57-80 days a f t e r the operations. But, as noted above, most of the sham operated f i s h r e c e i v ed the second t r i a l a shorter time (5-6 days) a f t e r the f i r s t t r i a l than the c a s t r a t e s d i d (8-19 days). A f t e r t e s t i n g was completed, f i s h were autopsied to check f o r evidence of t e s t i c u l a r regeneration. 3. R e s u l t s In the f i r s t t r i a l few r e t r i e v i n g a c t s were performed e i t h e r by c a s t r a t e s or sham operated f i s h . By the second t r i a l consider' able numbers of c a s t r a t e s were beginning to show signs of the presence of androgens—nest b u i l d i n g and elong a t i n g d o r s a l f i n s — yet they f a i l e d to show more than s l i g h t i n d i c a t i o n s of pa r e n t a l behavior development while about h a l f the sham operated f i s h d i d so. The f i r s t t r i a l d i d not correspond v/ell to previous e x p e r i -ments. F i r s t , l i t t l e r e t r i e v i n g was done by sham operated f i s h . Secondly, both groups of t e n picked up and released eggs without r e t r i e v i n g , and considerable numbers of eggs s t i l l remained un-eaten and not r e t r i e v e d at the end of the t e s t period ( F i g . 18B). In the second t r i a l , the shams v/ere t e s t e d , h a l f vrith and h a l f v/ithout nests. This d i d not appear to have any e f f e c t on the p r o b a b i l i t y of occurrence of par e n t a l behavior ( F i g . 18). 102 CO I-o < CD 5 0 -> 2 0 H UJ OC U l or A. i t e J I i—JiU. FIGURE 18. Parental behavior by castrated (C) and sham operated (S) male gouramis presented with 2000 freshly spawned eggs. Each f i s h received two t r i a l s on d i f f e r e n t days. Nests were removed before testing of a l l shams i n the f i r s t t r i a l , but i n the second t r i a l nests were l e f t with half the shams (S+) and removed from half (S-). A. Number of r e t r i e v i n g acts performed i n 15 min. B. Survival of 2000 eggs. In the f i r s t t r i a l , eggs were removed.after approximately 60 min. In the second t r i a l , eggs v/ere l e f t overnight. 103 Only r a r e r e t r i e v i n g a c t s and low overnight egg s u r v i v a l were shown by the c a s t r a t e d f i s h . Only 3 c a s t r a t e s , l a t e r r e j e c t e d from the experiment, d i d any nest b u i l d i n g during the f i r s t 1 0 days i n the t e s t i n g tanks. This was i n strong c o n t r a s t to the shams which, f o r the most part , c o n s i s t e n t l y b u i l t N2 or N3 l e v e l nests. However, a f t e r 1 0 days one a f t e r another of the c a s t r a t e s began to produce t r a c e s of foam and l a t e r Nl or N2 l e v e l nests. N3 nests were very r a r e . Nest b u i l d i n g seemed to s t a b i l i z e at a lov/er l e v e l i n c a s t r a t e s than i n the shams ( F i g . 1 9 ) . Dorsal f i n s a l s o showed elongation at the same time as nest b u i l d i n g began. Autopsy r e s u l t s f a i l e d to turn up any c l e a r evidence of t e s t i c u l a r t i s s u e , even i n the 3 f i s h which showed nest b u i l d i n g soon a f t e r being placed i n the t e s t i n g tanks. Three f i s h had small pieces of t i s s u e which could not be i d e n t i f i e d . In one case the t i s s u e was somewhat s i m i l a r to normal t e s t i s . k. Discussion This experiment has r a i s e d more questions than i t has an-swered. There i s a suggestion that e l i m i n a t i o n or r e d u c t i o n of c i r c u l a t i n g androgens i n t e r f e r e s w i t h the i n d u c t i o n of p a r e n t a l behavior. However, t h i s must remain a t e n t a t i v e c o n c l u s i o n be-cause eggs given i n the f i r s t t r i a l could have i n f l u e n c e d r e r sponslveness i n the second t r i a l , and the i n t e r v a l between t r i a l s was shorter f o r shams than c a s t r a t e s . The f a c t that f i s h i n the p r e l i m i n a r y experiment were able to develop p a r e n t a l behavior while those castrated longer were not, could be due to the p r i o r experience of the f i s h i n the p r e l i m i n a r y experiment, to t h e i r 4 8 12 16 20 24 28 34 D A Y S FIGURE 19. Mean nest index of 9 castrated and 11 sham operated male gouramis tested for the induction of parental behavior. Each point represents the mean nest b u i l d -ing index for the preceding k days. 105 shorter period of c a s t r a t i o n or to regeneration of androgenic t i s s u e . F a i l u r e of the shams to develop p a r e n t a l behavior i n the f i r s t t r i a l remains unexplained. There was no support f o r the hypothesis that removal of nests i n t e r f e r e d w i t h the development of p a r e n t a l behavior. I t i s p o s s i b l e that the stock of f i s h used was simply l e s s responsive to eggs f o r some reason. Or i t may be that r a i s i n g the pH of the aquarium water has some d i r e c t e f f e c t on the i n d u c t i o n of p a r e n t a l behavior or an i n d i r e c t e f f e c t v i a an e f f e c t on the eggs. Though i t seems u n l i k e l y that treatments which increase egg v i a b i l i t y could decrease t h e i r stimulus value f o r i n d u c t i o n of pa r e n t a l behavior, t h i s remains a p o s s i b i l i t y . Androgen treatment of females i n the previous experiment provided c l e a r evidence that nest b u i l d i n g and d o r s a l f i n l e n g t h are androgen-dependent c h a r a c t e r i s t i c s . This was supported by the present experiment i n which nest b u i l d i n g v/as g r e a t l y r e -duced and d o r s a l f i n l e n g t h decreased a f t e r c a s t r a t i o n . By the same token, the subsequent increase i n d o r s a l f i n l e n g t h and nest b u i l d i n g give a strong i n d i c a t i o n of a recovery of a source of androgens. Autopsy r e s u l t s cannot confirm complete absence of t e s t i c u l a r t i s s u e , but they c e r t a i n l y showed that there was no major regeneration of t e s t i s . As suggested by Johns and L i l e y (1970) the most l i k e l y explanation seems to be that there i s an extra-gonadal androgen source, perhaps the i n t e r r e n a l s . 106 D. HORMONES AND THE PARENTAL AND NEST BUILDING BEHAVIOR OF FISH The r o l e of hormones i n the p a r e n t a l and nest b u i l d i n g be-havior of f i s h has r e c e n t l y been reviewed by L i l e y (1969). The three experiments reported i n t h i s chapter have provided evidence of a r o l e f o r androgens i n the p a r e n t a l behavior of the gourami. Males perform more par e n t a l behavior than females. P a r e n t a l r e -sponsiveness to eggs i s increased i n females implanted with methyl testosterone. C a s t r a t i o n may reduce the c a p a c i t y to develop parental behavior i n males. The s t i c k l e b a c k i s the only other species which shows sexual dimorphism i n p a r e n t a l behavior and which has been stu d i e d . As discussed i n the i n t r o d u c t i o n to the previous s e c t i o n , c a s t r a t i o n may (Smith and Hoar, 1967) or may not (De Jong-van der H e i j , c i t e d by Baggerman, 1968) d i s r u p t e s t a b l i s h e d p a r e n t a l behavior, and may not prevent the establishment of p a r e n t a l behavior (Baggerman, 1957). Though there may be a d i f f e r e n c e between the c o n t r o l . o f p a r e n t a l behavior i n gouramis and s t i c k l e b a c k s , the p r i o r exper-ience of the s u b j e c t s or time since c a s t r a t i o n could a l s o account f o r the f a i l u r e of c a s t r a t i o n to prevent the development of par-e n t a l behavior of s t i c k l e b a c k s . Whether the d i f f e r e n c e between Smith and Hoar's and De Jong-van der H e i j ' s r e s u l t s on mainten-ance of p a r e n t a l behavior are due to d i f f e r e n c e s i n technique or population d i f f e r e n c e s i n s t i c k l e b a c k s remains to be determined. The only other report of e f f e c t s of gonadectomy on the r e -sponses of f i s h to eggs or young i s a study by Noble, Kumpf and B i l l i n g s (1938) on the response of jewel f i s h , Hemichromis bima-c u l a t u s , to f r y . In t h i s species both sexes show p a r e n t a l care. 107 Gonadectomy of e i t h e r sex d i d not a f f e c t the c a p a c i t y of e x p e r i -enced f i s h to respond to hormone treatments and the pre s e n t a t i o n of f r y . What i s the r o l e of gonadal hormones i n the p a r e n t a l beha-v i o r of f i s h ? In both gouramis and s t i c k l e b a c k s the presence of gonadal hormones i s not s u f f i c i e n t f o r development of p a r e n t a l behavior. Normal males of both species do not show p a r e n t a l be-havior unless they spawn or are given eggs. But i n gouramis, at l e a s t , androgens seem to a f f e c t the a b i l i t y of the f i s h to r e -spond to the stimulus eggs with p a r e n t a l behavior. The occasional p a r e n t a l responses performed by post-spawning females and by c h o l e s t e r o l implanted females i n two experiments suggest that even without androgenic i n f l u e n c e s , there i s a basic l e v e l of r e -sponsiveness to eggs which i s increased i n some way by androgens. However, gonadal hormones are not n e c e s s a r i l y the only hor-mones to i n f l u e n c e p a r e n t a l behavior. Noble, Kumpf and B i l l i n g s ( 1 9 3 8 ) found that i n j e c t i o n s of p r o l a c t i n and corpus luteum ex-t r a c t were the most e f f e c t i v e of many substances t r i e d i n promo-t i n g the development of p a r e n t a l responses to t e s t f r y . Blum ( 1 9 6 8 ) c i t e d a study by Bartmann i n which both estrogen and pro-l a c t i n were s a i d to be r e q u i r e d f o r the development of p a r e n t a l behavior i n female mouthbrooding c i c h l i d s . I n d i r e c t evidence of another r o l e f o r hormones i n the par-ental' behavior of gouramis comes from the study of the e f f e c t of spawning on the development of p a r e n t a l behavior (Ch. \5). I t seems l i k e l y that gouramis are more s e n s i t i v e to eggs a f t e r spawn-i n g because of the r e l e a s e of some hormone, but there i s no ex-perimental evidence to support t h i s . 1 0 8 I t i s noteworthy that the "spawning f a c t o r , " l i k e androgens, does not induce p a r e n t a l behavior, but s e n s i t i z e s the f i s h to the i n f l u e n c e of eggs. Noble, Kumpf, and B i l l i n g s ' ( 1 9 3 8 ) study seems s i m i l a r i n that p a r e n t a l behavior was induced by d a i l y i n j e c t i o n s and t e s t s with f r y . Since s i g n i f i c a n t numbers of c o n t r o l f i s h developed p a r e n t a l behavior, there may have been an e f f e c t of r e -peated exposure to f r y which was f a c i l i t a t e d by c e r t a i n of the hormone treatments. However, some s t u d i e s on the e f f e c t s of hormones on p a r e n t a l behavior cannot be i n t e r p r e t e d as changes i n responsiveness to eggs or young. In contrast to the r e l a t i v e l y few s t u d i e s on the e f f e c t of hormones on the i n t e r a c t i o n of a d u l t and young, there have been quite a number of i n v e s t i g a t i o n s on the e f f e c t s of hor-mones on "parental behavior" i n the absence of young. In s e v e r a l species p r o l a c t i n i n j e c t i o n s have been found to e l i c i t fanning be-havior s i m i l a r to that of p a r e n t a l i n d i v i d u a l s . The species i n -clude the c i c h l i d s , Symphysodon a e q u i f a s c i a t a a x e l r o d i (Blum and F i e d l e r , 1 9 6 4 ) and Pterophyllum s c a l a r e (Blum and F i e d l e r , 1 9 6 5 ; Blum, 1 9 6 8 ) and the wrasse C r e n i l a b r u s o c e l l a t u s ( F i e d l e r , 1 9 6 2 ) . But Smith and Hoar ( 1 9 6 7 ) found l i t t l e e f f e c t of p r o l a c t i n on fanning at an empty nest i n s t i c k l e b a c k s . Both Smith and Hoar (.1967) and Baggerman ( 1 9 6 8 ) found that fanning at an empty nest v/as much reduced by c a s t r a t i o n . And the former authors showed that t h i s behavior v/as r e s t o r e d by methyl testosterone. These r e s u l t s are of general i n t e r e s t because they seem to be one of the few cases of p a r e n t a l care i n the complete absence of eggs or young. The experiments reported i n t h i s chapter have confirmed the 109 conclusions of Johns and L i l e y (1970) that nest b u i l d i n g i n gour-amis i s dependent upon androgens. As i n t h e i r study, c a s t r a t i o n g r e a t l y reduced or elim i n a t e d nest b u i l d i n g . In a d d i t i o n , nest b u i l d i n g v/as stimulated by methyl testosterone implants i n f e -males. In a r e l a t e d species, C o l i s a l a l i a , testosterone p r o p i -onate has been reported to induce nest b u i l d i n g i n females (For-s e l i u s , 1957c). However, Machemer and F i e d l e r (1965) used com-bined treatment v/ith p r o l a c t i n and methyl testosterone to induce nest b u i l d i n g i n females of Macropodus o p e r c u l a r i s , another spe-c i e s of the fa m i l y B e l o n t i i d a e . I n males t h i s treatment was more e f f e c t i v e than e i t h e r methyl testosterone or p r o l a c t i n alone (Machemer and F i e d l e r , 1965; Machemer, 1971). In other species i n which the male b u i l d s the nest, t h i s behavior has been shov/n to depend upon androgens. Such species i n c l u d e the three-spined s t i c k l e b a c k (Hoar, 1962; Wai and Hoar, 1963; Smith and Hoar, 1967; Baggerman, 1968) and tv/o Centrarchid species, Lepomis meg-a l o t i s and L. gibbosus (Smith, 1969). In a d d i t i o n , Wai and Hoar (1963) t r e a t e d gonadectomized female s t i c k l e b a c k s with methyl testosterone and induced nest b u i l d i n g i n a small p r o p o r t i o n of the f i s h . Both sexes of the c i c h l i d T i l a p i a macrocephala p a r t i -c i p a t e i n nest b u i l d i n g , but the female b u i l d s more than the male. In t h i s species there appears to be l i t t l e e f f e c t of c a s t r a t i o n on nest b u i l d i n g by males. But ovariectomy reduces female nest b u i l d i n g to the low male l e v e l (Aronson, 1951). E. SUMMARY 1. Males and females were compared with respect to r e t r i e v i n g behavior and egg-eating i n h i b i t i o n a f t e r spawning. 1 1 0 2. Males showed s i g n i f i c a n t l y more r e t r i e v i n g and l e s s egg-eati n g than females, although they also ate some of t h e i r own eggs. 3. Some females demonstrated low l e v e l s of r e t r i e v i n g and egg-ea t i n g i n h i b i t i o n . 4. A f t e r i m p l a n t a t i o n of p e l l e t s of methyl testosterone, f e -males responded with masculine l e v e l s of pa r e n t a l behavior to the stimulus of 3 0 0 0 eggs. 5. C h o l e s t e r o l implanted females responded to 3 0 0 0 eggs with only occasional p a r e n t a l a c t s . 6. , There was some evidence that inexperienced castrated males were l e s s l i k e l y to develop p a r e n t a l behavior i n response to 2 0 0 0 eggs than sham operated c o n t r o l s were. 7. Studies on the hormonal c o n t r o l of p a r e n t a l and nest b u i l d i n g behavior i n f i s h were reviewed. I l l I I I . MAINTENANCE OF PARENTAL BEHAVIOR CHAPTER ? THE ROLE OF YOUNG IN THE MAINTENANCE OF PARENTAL BEHAVIOR 1. I n t r o d u c t i o n The e f f e c t of young on the maintenance of p a r e n t a l behavior seems to be a w e l l known but l i t t l e studied phenomenon. However, i t i s commonly believed that p a r e n t a l behavior continues i n many species u n t i l the young become independent but d e c l i n e s prema-t u r e l y i f the young are removed. Experimental evidence i s a v a i l -able to support t h i s view f o r a few species i n c l u d i n g the l a b o r a -t o r y r a t (Rosenblatt and Lehrman, 1963) and the three-spined s t i c k l e b a c k (Van I e r s e l , 1953)- Since the r o l e of young i n the maintenance of p a r e n t a l behavior could be r e l a t e d to the role^ of young i n the i n d u c t i o n of p a r e n t a l behavior, I hoped to determine whether p a r e n t a l behavior was maintained longer i n the presence of young than i n t h e i r absence i n gouramis. I f t h i s occurred, I a l s o hoped to determine whether p h y s i c a l contact w i t h the young was necessary to s u s t a i n p a r e n t a l responsiveness, or whether ; v i s u a l and chemical cues were s u f f i c i e n t . 2. Methods The basic procedure was to a l l o w f i s h to spawn and to r e -t a i n t h e i r brood u n t i l the f o l l o w i n g morning. Then a l l the eggs were removed and e i t h e r a) replaced, b) not replaced, or c) r e -placed w i t h i n a f l o a t i n g container which allowed chemical and v i s u a l contact, but prevented p h y s i c a l contact between parent 112 and young. Six to eight days l a t e r the f i s h were tes t e d f o r t h e i r response to 2 successive p r e s e n t a t i o n s of 100 eggs. Since some f i s h which had had t h e i r eggs removed showed p a r e n t a l be-havior at the time of t e s t i n g , a f o u r t h group (d) was added to determine the response to the t e s t of f i s h which had not r e c e n t l y spawned. The subjects were 28 a d u l t males with previous breeding ex-perience. The standard spawning set up was used with p l a s t i c nest supports s u b s t i t u t e d f o r f l o a t i n g p l a n t s . The water of ex-perimental and egg donor tanks was buffered by the a d d i t i o n of 5 g of sodium bicarbonate i n order to improve egg v i a b i l i t y . Spawning was induced by the usual procedure. I f i t d i d not occur one or two days a f t e r opening the p a r t i t i o n , f u r t h e r attempts were made with new females u n t i l spawning was achieved. • F i s h were randomly assigned to one of the treatments (a-c) i n the or-der i n which they spawned. The subjects of the f o u r t h group were s i m i l a r i n age and previous experience to those of the other three, but they had not spawned w i t h i n at l e a s t 2 months of the time of t e s t i n g . The "baskets" that prevented p h y s i c a l contact with the young were c y l i n d r i c a l p l a s t i c containers 9-5 cm i n diameter and 7 cm deep. Two 5 cm square openings were cut i n t o the s i d e s and a 1+ cm square was cut i n t o the bottom. These were covered with f i n e nylon screening held i n place by " S i l a s t i c " glue. F i v e r e c t a n -gular styrofoam blocks (2 X 1 X 2 cm) were glued onto the outer rim to provide f l o a t a t i o n . F i s h i n groups "b" and "c" were checked twice each day f o r the presence of young, and any found were removed. At t h i s time 113 t h e b a s k e t s w e r e r a i s e d a n d l o w e r e d s e v e r a l t i m e s t o r e p l a c e t h e w a t e r a n d i n s u r e d i s p e r s i o n o f a n y s p e c i f i c o d o r s . F r y w e r e f e d f r o m t h e t i m e t h e y v / e r e J> or k d a y s o l d v / i t h a s u s p e n s i o n o f s t r a i n e d b e e f l i v e r , s o m e o f w h i c h w a s a l s o p l a c e d i n g r o u p " a " t a n k s . N e s t q u a l i t y v/ a s r e c o r d e d d a i l y 1 h r a f t e r t h e l i g h t s c a m e o n . P a r e n t a l b e h a v i o r w a s t e s t e d 7, 8, o r 9 d a y s a f t e r s p a w n i n g . O n e h u n d r e d f r e s h l y s p a w n e d e g g s w e r e p l a c e d i n a p r e s e n t a t i o n f r a m e , a n d a A m i n r e c o r d w a s m a d e o f e g g c o n t a c t a n d r e t r i e v i n g , s t a r t i n g f r o m t h e i n i t i a l c o n t a c t . A s e c o n d s i m i l a r t r i a l v / a s g i v e n i m m e d i a t e l y a f t e r t h e f i r s t . O n e f i s h l e f t w i t h i t s b r o o d w a s v e r y f r i g h t e n e d , p r e v e n t i n g b e h a v i o r a l o b s e r v a t i o n , b u t t h i s f i s h d i d t r a n s f e r t h e e g g s t o h i s n e s t a f t e r t h e o b s e r v e r l e f t t h e r o o m . A f t e r 28 t o 188 m i n , t h e n e s t a n d a n y e g g s p r e s e n t v / e r e r e m o v e d a n d t h e e g g s v / e r e c o u n t e d . T h e r e w a s n o e v i d e n c e t h a t t h e i n t e r v a l l e n g t h h a d a n e f f e c t o n e g g s u r v i v a l . ; T h e t w o m e a s u r e s o f p a r e n t a l b e h a v i o r v / e r e t h e t o t a l n u m b e r o f r e t r i e v i n g a c t s i n t h e t v / o i f m i n t r i a l s c o m b i n e d a n d t h e s u r -v i v a l o f t h e 200 t e s t e g g s . T r e a t m e n t s a - c w e r e a n a l y s e d b y t h e K r u s k a l - W a l l i s o n e - w a y a n a l y s i s o f v a r i a n c e . T h e M a n n - W h i t n e y U T e s t w a s u s e d t o c o m p a r e t h e f o u r t h g r o u p , w h i c h h a d b e e n a d d e d a f t e r t h e s t a r t o f t h e e x p e r i m e n t , t o t h e o t h e r t h r e e , a n d t o c o m p a r e p a i r s o f t r e a t m e n t s a m o n g t h e f i r s t t h r e e . B o t h t e s t s w e r e f r o m S i e g e l (1956). 3. R e s u l t s T h e r e v/as a s i g n i f i c a n t e f f e c t o f t r e a t m e n t s o n e g g s u r v i -v a l , a n d the e f f e c t o n r e t r i e v i n g a p p r o a c h e d s i g n i f i c a n c e ( F i g . 11 v 20; Table X I ) . The f i s h l e f t w i t h t h e i r own broods showed more r e t r i e v i n g and higher egg s u r v i v a l than any of the other groups. The f i s h whose broods were l e f t i n the f l o a t i n g baskets were not d i f f e r e n t from those whose broods were removed. In both^groups some f i s h continued to show p a r e n t a l responses equivalent to those of males l e f t w ith t h e i r broods. But most f i s h showed l i t t l e p a r e n t a l behavior. None of the f i s h which had not r e c e n t l y spawned showed high l e v e l s of p a r e n t a l behavior i n the t e s t . In tv/o cases the recovery of eggs was considerably greater than the c o r r e c t number of t e s t eggs. This was apparently due to e r r o r s i n counting as a r e s u l t of eggs clumping together. I t v/as found to be very d i f f i c u l t to remove a l l the eggs on the f i r s t attempt from tanks of the two treatments which demanded t h i s . I nspections revealed small numbers of f r y e s p e c i a l l y i n the f i r s t few days a f t e r removal. However, the r e s u l t s i n d i c a t e d no c o r r e l a t i o n betv/een p a r e n t a l responsiveness i n the egg t e s t and e i t h e r the number of f r y removed or the l a t e s t day on which f r y were present. There was no i n d i c a t i o n of any e f f e c t of the number of days between spawning and t e s t i n g . if. D i s c u s s i o n To summarize these r e s u l t s i t may be s a i d that gouramis r e -tained t h e i r p a r e n t a l responsiveness to eggs and f r y more e f f e c -t i v e l y f o r 6 to 8 days i f they were l e f t i n contact v/ith t h e i r developing brood than i f t h e i r brood v/as removed or placed i n a f l o a t i n g basket v/hich prevented d i r e c t p h y s i c a l contact. But not a l l males l e f t w ith t h e i r broods v/ere immediately responsive to eggs, nor had a l l males whose broods had been removed l o s t 115 cc o 20 16 . CO I-o < ± 12 > UJ E e . 4 . A. co (9 o 200 -C. 150 -> z> CO 100 -90 • <»(230) <»(244; m—»t w B. a b e d T R E A T M ENTS FIGURE 20. Parental responsiveness i n male gouramis which had spawned and a) been l e f t with t h e i r brood, b) had t h e i r brood placed i n a f l o a t i n g basket i n the tank, or c) had t h e i r brood removed, or which d) had not r e c e n t l y spawned. The measures of pa r e n t a l behavior are A) t o t a l number of r e t r i e v i n g a c t s i n two 4 min t r i a l s with 100 eggs, and B) s u r v i v a l of the 200 eggs f o r 28-188 min. 1 1 6 TABLE XI. S t a t i s t i c a l comparisons of the e f f e c t of the presence of young on the maintenance of p a r e n t a l behavior. The treatments were a) prood present, b) brood i n a f l o a t i n g basket, c) brood removed, and d) no recent spawning. The value i n ( ) d i f f e r e d i n the opposite d i r e c t i o n from that p r e d i c t e d . K r u s k a l -W a l l i s One-Way A n a l y s i s Mann-Whitney U Test, 1 - t a i l e d P Values of Variance a vs b a vs c a vs d b vs c b vs d c vs d R e t r i e v i n g < . 1 0 .037 .017 . 0 0 1 ( . 2 6 7 ) .027 - 0 0 3 Egg S u r v i v a l < .02 . 0 1 3 . 0 0 3 . 0 0 0 . 3 5 5 .027 .082 117 t h e i r p a r e n t a l responsiveness by 6 to 8 days. Thus, young had a p o s i t i v e e f f e c t on p a r e n t a l responsiveness. But since some males l e f t vrith t h e i r broods ate considerable numbers of eggs, pa r e n t a l responsiveness to f r y does not seem to be i d e n t i c a l to p a r e n t a l responsiveness to eggs. P l a c i n g the brood i n f l o a t i n g baskets el i m i n a t e d i t s e f f e c -t i v e n e s s i n maintaining p a r e n t a l responsiveness. This seems to imply that the sensory modality by which f r y maintain p a r e n t a l responsiveness i s not v i s i o n or o l f a c t i o n . One might argue that the eggs and newly hatched l a r v a e were not s u f f i c i e n t l y v i s i b l e while they f l o a t e d at the surface or that a chemical stimulus d i d not d i f f u s e s u f f i c i e n t l y from the basket. However, the most l i k e l y explanation of these r e s u l t s i s that more d i r e c t contact vrith f r y i s r e q u i r e d . The male may have to p i c k up f r y i n h i s mouth i n order to remain p a r e n t a l l y responsive. I f so, e i t h e r t a s t e or t a c t i l e s t i m u l i could be s i g n i f i c a n t . I t might even be necessary f o r the male to eat some f r y . I t i s s u r p r i s i n g that such close contact i s necessary because the male with older f r y seems to p i c k them up so r a r e l y . The occurrence of high l e v e l s of p a r e n t a l behavior i n some of the f i s h which had had t h e i r broods removed suggests that par-e n t a l behavior may p e r s i s t f o r some time a f t e r the removal of the f r y . Perhaps there i s a gradual d e c l i n e i n responsiveness a f t e r ' t h e removal of f r y so that a p r o g r e s s i v e l y greater number of eggs i s required to e l i c i t p a r e n t a l behavior. Note that the observation of a d e c l i n e i n responsiveness i s dependent upon t e s t i n g with a small number of eggs. As demonstrated i n Ch. 3> t e s t i n g with a l a r g e number of eggs could have e l i c i t e d p a r e n t a l 118 behavior whether or not the f r y had been removed a f t e r spawning. 5. Summary 1. Male gouramis l e f t w i t h t h e i r broods of young and tested with eggs 7-9 days a f t e r spawning v/ere more l i k e l y to show pare n t a l behavior than males which had not r e c e n t l y spawned or which had r e c e n t l y spawned but had t h e i r broods removed a f t e r one day. 2. When broods were placed i n baskets which prevented p h y s i c a l contact with the young while a l l o w i n g v i s u a l and chemical contact, males l o s t p a r e n t a l responsiveness as i f the broods had been r e -moved completely. 119 CHAPTER 8 GENERAL CONCLUSIONS ABOUT THE CONTROL OF PARENTAL BEHAVIOR IN THE GOURAMI The purpose of the present chapter i s to summarize the major f i n d i n g s of t h i s study and r e l a t e them to each other, and to point out the multitude of questions that remain unanswered. Normally, gouramis tes t e d with small numbers of eggs do not show par e n t a l behavior unless they have r e c e n t l y spawned. Obser-v a t i o n s of spawning males showed that p a r e n t a l behavior began very promptly a f t e r the f i r s t o v i p o s i t i o n by the female. Once de-veloped, i t continued i n the presence of young f o r a t l e a s t 2 weeks (Chs. 2C, 7 ) . This study has provided evidence that s t i m u l i from the eggs are an important f a c t o r i n the development and maintenance of pare n t a l behavior. One i n d i c a t i o n of t h i s i s that p a r e n t a l be-havior can be induced i n males which have not spawned by exposure to a la r g e number of eggs f o r a short period of time (Ch. 3A,B) or by extended exposure to a small number of eggs (Ch. 3 0 ) . These r e s u l t s only i n d i c a t e that eggs can induce p a r e n t a l behavior, not that they do so i n the n a t u r a l s i t u a t i o n . However, t e s t s of spawning males i n which exposure to eggs v/as c o n t r o l l e d by plugging the oviducts of the females i n d i c a t e d that exposure to eggs was an important component i n the n a t u r a l development of parental behavior (Ch. 5). Spawning by i t s e l f d i d not induce f i s h to become p a r e n t a l , but i t d i d reduce the number of eggs necessary f o r p a r e n t a l behavior to develop. Besides spawning and s t i m u l i from the eggs, androgens were 120 also found to be an important f a c t o r i n the development of paren-t a l behavior. I t was shown that, a f t e r spawning, males performed more pa r e n t a l behavior than females (Ch. 6A), and that the mas-c u l i n i z a t i o n of females by means of methyl testosterone implants allowed them to develop pa r e n t a l behavior i n response to l a r g e numbers of eggs (Ch. 6B). Whether c a s t r a t i o n of males eliminated t h e i r capacity to perform p a r e n t a l behavior could not be deter-mined with c e r t a i n t y (Ch. 6C). D e t a i l e d observations of females revealed that they had some capacity to perform pa r e n t a l behavior (Ch. 6A,B). I t was u n l i k e l y that t h i s c a pacity was dependent upon endogenous androgens, be-cause the d o r s a l f i n l e n g t h i s so s e n s i t i v e to androgenic i n f l u -ences that i t should have i n d i c a t e d the presence of androgens i n females. I t may be s i g n i f i c a n t that the r o l e of both spawning and androgens seemed to be to a l t e r responsiveness to the moti-v a t i n g i n f l u e n c e s of the eggs, r a t h e r than to cause a period of p a r e n t a l responsiveness independent of the m o t i v a t i n g i n f l u e n c e of the eggs. I t was also found that the presence of young s i g n i f i c a n t l y f a c i l i t a t e d the p e r s i s t e n c e of p a r e n t a l responsiveness to eggs when males with developing broods were compared with males whose broods had been removed. Apparently, p h y s i c a l contact v/ith the young was necessary because baskets which allowed only v i s u a l and chemical contact with the young prevented the maintenance of p a r e n t a l responsiveness. No doubt there are many other f a c t o r s of s i g n i f i c a n c e i n the p a r e n t a l behavior of gouramis. P r i o r reproductive experience i s almost c e r t a i n l y one of these. However, the only experimental 121 study of t h i s f a c t o r (Ch. 3C) had to be terminated before conclu-sive answers were obtained. There may a l s o be e f f e c t s of hormones other than those suggested by s t u d i e s of sexual dimorphism and the spawning f a c t o r . The t h e s i s attempted to take a broad look at a v a r i e t y of f a c t o r s r e l a t e d to p a r e n t a l behavior, r a t h e r than to concentrate on any one f a c t o r . While t h i s l e f t many important questions un-answered, i t a l s o suggested i n t e r r e l a t i o n s h i p s that might not have been noticed had I worked on only one aspect. An important r e s u l t of t h i s study was the demonstration of the s i g n i f i c a n c e of stimulus strength i n the i n d u c t i o n of p a r e n t a l behavior. Animals s e n s i t i z e d by spawning or p r i o r expsoure to s m a l l numbers of eggs required fewer eggs to induce parental behavior than animals not so s e n s i t i z e d . However, l a r g e r numbers of eggs could compensate f o r lower l e v e l s of s e n s i t i v x t y (Ch. 3B). Stimulus strength i s a f a c t o r which has not often been taken i n t o c o n s i d e r a t i o n i n s t u d i e s of the c o n t r o l of p a r e n t a l behavior. I t s r e l a t i o n s h i p to other f a c t o r s i n the c o n t r o l of parental behavior may have im-p l i c a t i o n s f o r the study of parental behavior i n other species. This w i l l be discussed i n more d e t a i l i n the next chapter. But i t may be noted here that by varying stimulus s t r e n g t h and the experimental procedure I found i n a s i n g l e species r e s u l t s such a s ; t h e gradual or sudden development of p a r e n t a l behavior and the development of p a r e n t a l behavior only during " s e n s i t i v e per-i o d s " which p r e v i o u s l y had seemed to be c h a r a c t e r i s t i c of d i f -ferent species. Though the study has given some p r e l i m i n a r y answers to the question of what f a c t o r s c o n t r o l the development of p a r e n t a l 122 behavior, i t has l e f t many more unanswered. The most b a s i c and most i n t e r e s t i n g i s the nature of the mechanism by which the eggs increase p a r e n t a l responsiveness. The mechanism must be able to account f o r both the sudden development of p a r e n t a l behavior i n l e s s than a minute with a strong stimulus and the gradual d e v e l -opment of p a r e n t a l behavior i n response to a weak stimulus. The f a c t that repeated p r e s e n t a t i o n s of a weak stimulus e v e n t u a l l y r e s u l t i n p a r e n t a l responses suggest some so r t of summation. There are a l s o many i n t e r e s t i n g questions about the way that the p h y s i o l o g i c a l f a c t o r s f a c i l i t a t e the response to eggs. Do they a f f e c t p e r i p h e r a l s e n s i t i v i t y to s t i m u l i from eggs? Or i s t h e i r e f f e c t on a c e n t r a l system f o r p a r e n t a l responsiveness? In a d d i t i o n to these questions of general i n t e r e s t , i t i s c l e a r that many questions remain s p e c i f i c a l l y about pa r e n t a l ;be-havior i n gouramis. The p h y s i o l o g i c a l nature of the "spawning f a c t o r " has.not been studied. Why were the c o n t r o l f i s h i n the f i n a l c a s t r a t i o n experiment so unresponsive to eggs? Why do "par-e n t a l " males continue to eat a s i g n i f i c a n t number of eggs? What i s the r e l a t i o n s h i p between hunger l e v e l and egg-eating i n h i b i -t i o n ? What are the s p e c i f i c s t i m u l i that are r e q u i r e d to induce and maintain p a r e n t a l behavior? Can p a r e n t a l behavior be induced by the eggs of other species? Are there d i f f e r e n c e s i n the s t a t e s of p a r e n t a l responsiveness to older f r y and to eggs? F i n a l l y , i t would be very i n t e r e s t i n g to know whether i n other species, as i n the gourami, the r a p i d or gradual develop-ment of p a r e n t a l behavior can be c o n t r o l l e d by varying the strength of s t i m u l i from the young, and whether the r o l e of var-ious -physiological f a c t o r s i s the "causation" of a p a r e n t a l 123 s t a t e or the a l t e r a t i o n of s e n s i t i v i t y to m o t i v a t i n g s t i m u l i from the young. 124 CHAPTER 9 EFFECTS OF EGGS AND YOUNG ON PARENTAL RESPONSIVENESS IN VERTEBRATES A. INTRODUCTION The p r i n c i p a l theme of t h i s t h e s i s i s the r o l e of eggs and young i n r e l a t i o n to other f a c t o r s i n the i n d u c t i o n and mainten-ance of pa r e n t a l behavior. F r e s h l y spawned eggs, i t was discov-ered, may induce gouramis which have never spawned to develop par e n t a l behavior. Eggs and young a l s o seem to be important i n the n a t u r a l development and maintenance of par e n t a l behavior. Since these r e s u l t s appear to p a r a l l e l those on some other v e r t e -brates, i t seems :worthwhile to review the e f f e c t s which s t i m u l i from o f f s p r i n g may have on pa r e n t a l motivation. This may y i e l d i n s i g h t s i n t o the g e n e r a l i t y of m o t i v a t i o n a l e f f e c t s of young on parental behavior, and suggest explanations of i t s f u n c t i o n a l s i g n i f i c a n c e . Some aspects of t h i s t o p i c have been reviewed. Lehrman : ( 1 9 6 1 ) , mainly concerned with the r o l e of hormones i n par e n t a l be-havior, surveyed evidence f o r s t i m u l a t i o n of pa r e n t a l behavior by eggs and young i n b i r d s and mammals. Richards ( 1 9 6 7 ) gave consid-erable a t t e n t i o n to e f f e c t s of young i n h i s d i s c u s s i o n of f a c t o r s a f f e c t i n g p a r e n t a l behavior of r a t s , mice, hamsters, and r a b b i t s . Most r e c e n t l y , Rosenblatt ( 1 9 7 0 ) has surveyed the m o t i v a t i o n a l e f f e c t s of s t i m u l i from the young i n st u d i e s on the l a b o r a t o r y r a t . More s i g n i f i c a n t l y , he has i n t e g r a t e d such s t u d i e s i n t o the context of the whole process of the development and maintenance of p a r e n t a l behavior. 125 A preliminary point concerns terminology and the. evidence which we can use i n the analysis of the motivating e f f e c t s of eggs and young on parental responsiveness. My thoughts on these i s -sues have been much aided by the discussions of Hinde (1970). It i s important for the following discussion to make a d i s t i n c t i o n between the "motivating" and "releasing" e f f e c t s of a stimulus. When an animal responds to a stimulus, we can speak of the stimu-lus as e l i c i t i n g or "releasing" the response. When the effect of the stimulus i s to cause a change i n responsiveness, we can speak of the motivating e f f e c t s of the stimulus. In the study of the role of young i n parental behavior, incremental changes i n respon-siveness are of pa r t i c u l a r i n t e r e s t . Evidence that such a moti-vational change has occurred may be a) that a given response to the same stimulus becomes more l i k e l y , b) that the same stimulus e l i c i t s a response greater i n inte n s i t y , or c) that a weaker stim-ulus i s s u f f i c i e n t to e l i c i t the same response. The following discussion deals with a situ a t i o n i n which the same stimulus, eggs or young, may have both motivating and releasing e f f e c t s on parental behavior. B. EVIDENCE FOR MOTIVATIONAL EFFECTS OF EGGS AND YOUNG , Several types of experiment have provided evidence for mo-t i v a t i o n a l effects of stimuli from eggs and young. 1) Although animals which are not parents usually f a i l to show parental be-havior toward young, some species w i l l begin to show parental re-sponses i f kept i n contact with young for a period of time.. 2) Non-parents of some species w i l l respond promptly to young with parental behavior, but i t may be seen that exposure to young has 126 caused changes i n responsiveness because the animals respond to weaker s t i m u l i which were p r e v i o u s l y i n s u f f i c i e n t . 3) Sometimes an animal i s only responsive to young at one time, f o r example, immediately a f t e r spawning or p a r t u r i t i o n , but m o t i v a t i o n a l ef-f e c t s of young r e v e a l themselves i n that responsiveness i s l o s t unless exposure to young takes place during the " s e n s i t i v e per-i o d . " Thus the e f f e c t of eggs and young on pa r e n t a l responsive-ness i s that responsiveness i s not decreased, r a t h e r than that i t i s increased. Even a f t e r p a r e n t a l behavior i s e s t a b l i s h e d moti-v a t i o n a l e f f e c t s of the young may be found: k) Animals may show i n c r e a s i n g frequency or i n t e n s i t y of pa r e n t a l behavior a f t e r long-er periods i n contact with eggs or young. 5) Maintenance of es-ta b l i s h e d p a r e n t a l behavior may occur only i n the presence of eggs or young. The f o l l o w i n g s e c t i o n s consider each of these types i n t u r n . 1. Animals I n i t i a l l y Unresponsive: P a r e n t a l Behavior A f t e r Extended Exposure to Young Evidence f o r development of responsiveness to young with pro-longed or repeated exposure to them i s . a v a i l a b l e f o r l a b o r a t o r y r a t s , mice, and hamsters, domestic fowl, and the blue gourami. The l a b o r a t o r y r a t has been most thoroughly studied, and recent reviews have been published by Richards (1967) and Rosenblatt (1970). Most v i r g i n female r a t s are i n i t i a l l y unresponsive to young, but i n c r e a s i n g numbers become responsive w i t h successive days of exposure to pups (Wiesner and Sheard, 1933; Cosnier, 196.3; Cosnier and Co u t u r i e r , 1966; Rosenblatt, 1967). Though not a l l workers have found t h i s e f f e c t ( L o t t and Fuchs, 1962), i t appears 127 to occur i n most female r a t s i f the presence of young i s main-tained long enough (Rosenblatt, 1967). In some s t u d i e s , l a b o r a t o r y mice seem to be very responsive to young, high proportions of i n d i v i d u a l s showing p a r e n t a l beha-v i o r w i t h i n a 5 min t e s t period (e.g. N o i r o t , 1969c). However, i n other s t u d i e s v i r g i n female mice may not respond immediately to the stimulus of young, but do so a f t e r 1-if days exposure (Le-blond, 1938). Richards (1966a) reported that v i r g i n female ham-s t e r s would often a t t a c k 6-10 day o l d pups, but then begin to show maternal care. S i m i l a r l y , pregnant females sometimes at t a c k -ed newborn young at f i r s t , then suddenly switched to par e n t a l be-havior (Richards, 1966b). In another study (Noirot and Richards, 1966), v i r g i n females given young i n two t e s t s separated by a 2 day i n t e r v a l were more l i k e l y to show maternal behavior i n the second t e s t , whether or not they had done so i n the f i r s t . There are two r e p o r t s of in c u b a t i o n induced i n b i r d s by con-t a c t with eggs. One i s a report by T a i b e l l (1928, c i t e d by Lehr-man, 196l , and Poulsen, 1953) that broodiness was induced i n a turkey cock by t y i n g i t to a nest w i t h eggs. In the other, Poul-sen (1953) reported that he had induced broodiness out of season i n turkey hens and domestic fowl by c o n f i n i n g them i n a narrow space with p l a s t e r eggs. A number of authors have reported i n d u c t i o n of par e n t a l care i n hen's confined with baby c h i c k s (Burrows and Bye r l y , 1938; C o l -l i a s , 1946; Ramsay, 1953; Maier, 1963a,b), and bob-white q u a i l respond s i m i l a r l y (Stanford, 1952). This e f f e c t has also been reported i n capons and cocks (Goodale, 1916; Saeki and Tanabe, 1955; Kovach, 1967). 1 2 8 The present study has demonstrated a s i m i l a r phenomenon i n blue gouramis. Four males i n i t i a l l y ate eggs i f they were given i n batches of 1 0 0 but a f t e r s e v e r a l presentations per day f o r s e v e r a l days, three began to r e t r i e v e and show other elements of p a r e n t a l care. Most r e p o r t s on the i n d u c t i o n of p a r e n t a l behavior have given very few d e t a i l s of behavioral changes t a k i n g place during t h i s process. R e s u l t s are u s u a l l y given i n terms of p r o p o r t i o n of animals showing a given response or mean la t e n c y i n days to a given response (Rosenblatt, 1 9 6 7 ) or, even l e s s i n f o r m a t i v e , as a group index (Cosnier, 1 9 6 3 ) . The most complete d e s c r i p t i o n of the i n d u c t i o n of maternal behavior i n l a b o r a t o r y r a t s v/as pub-l i s h e d r e c e n t l y by Rosenblatt ( 1 9 7 0 ) . He stated that "the ; v i r g i n i s i n i t i a l l y a t t r a c t e d to the young pups and makes repeated con-t a c t s v/ith them. The s t i m u l a t i o n received during these contacts apparently mounts to the point where l i c k i n g , r e t r i e v i n g , e t c . , are gradually aroused, a l l four items of maternal behavior, appear-i n g w i t h i n a short time of one another" (p. 4 9 8 ) . In another paper d e a l i n g v/ith i n d u c t i o n of p a r e n t a l behavior i n v i r g i n f e -male r a t s i n j e c t e d with blood from post-partum females, Terkel and Rosenblatt ( 1 9 6 8 ) reported that parental behavior v/as f i r s t shown " i n attenuated f a s h i o n " and that l a t e r the females became " f u l l y maternal." R e t r i e v i n g was the l a s t item of maternal be-havior to appear. A f t e r i t s f i r s t appearance, p a r e n t a l behavior was f a i r l y c o n s i s t e n t l y shov/n (Rosenblatt, 1 9 6 7 ; Terkel and Rosen-b l a t t , 1 9 6 8 ) . Richards ( 1 9 6 6 a ) gave a b r i e f d e s c r i p t i o n of r a p i d develop-ment of maternal behavior i n v i r g i n female hamsters when presented 129 with pups. Pups were often attacked at f i r s t , but then l i c k e d and, i f necessary, r e t r i e v e d . Various types of ambivalent beha-v i o r v/ere sometimes shown i n e a r l y p a r t s of the t e s t . The nurs-i n g p o s i t i o n , once adopted, was u s u a l l y maintained f o r the dura-t i o n of the t e s t . In s i m i l a r l y t e s t i n g pregnant females, R i c h -ards (1966b) described the changeover from a t t a c k i n g to p a r e n t a l behavior as very sharp, sometimes t a k i n g place w i t h i n seconds. In chickens broody behavior u s u a l l y developed gradually,, both i n terms of the number of d i f f e r e n t elements of p a r e n t a l be-havior o c c u r r i n g and i n terms of the completeness of the behavi-o r a l responses (Ramsay, 1953; Burrows and B y e r l y , 1938). Once pare n t a l behavior developed, i t continued f o r some time. In studying the gourami, I found that some of the i n d i v i d u a l components of r e t r i e v i n g appeared before the complete behavior. There were i n d i c a t i o n s that these components increased i n f r e -quency and completeness with repeated presentations of eggs. However, complete r e t r i e v i n g began quite suddenly and then,was u s u a l l y c o n s i s t e n t l y performed. Overnight egg s u r v i v a l continued to improve a f t e r the appearance of c o n s i s t e n t r e t r i e v i n g . In summary, i t appears that where the development of paren-t a l behavior r e q u i r e s se v e r a l days of exposure to young, responses to the young are at f i r s t performed i n a weak and incomplete fashion. An increase i n i n t e n s i t y and number of d i f f e r e n t pat-t e r n s shown i s u s u a l l y gradual, but r a t h e r sudaen changes may als o occur. Once they have begun, pa r e n t a l responses continue to appear r e g u l a r l y f o r some time. A more d e t a i l e d understanding of changes i n responsiveness and i n s i g h t s i n t o the processes underlying these changes might come about through the use of more 1 3 0 s e n s i t i v e measures of behavior i n s t e a d of simply whether or not a given response occurred. 2. Animals I n i t i a l l y Responsive to Young: Changes i n Respon-siveness to Weak S t i m u l i A f t e r Exposure to Strong S t i m u l i The previous s e c t i o n d e a l t w i t h the development of pa r e n t a l responsiveness i n animals which d i d not i n i t i a l l y perform paren-t a l behavior when presented with eggs or young. Although most species are non-responsive to young unless they are in . t h e p h y s i -o l o g i c a l s t a t e at which t h e i r young normally appear, there are some exceptions. D e t a i l e d s t u d i e s by Noirot (1964a,b,c, 1965, 1969a,b,c) have shown that house mice, males as w e l l as v i r g i n females, have a high degree of responsiveness to young. The same seems to be true of j u v e n i l e hamsters though i t i s not true of adults (Rowell, 1961). The present study found that gouramis pre sented w i t h a l a r g e number of eggs would r a p i d l y begin p a r e n t a l behavior. In s t i c k l e b a c k s , p a r e n t a l fanning begins to develop when eggs are added to a male's nest (Van I e r s e l , 1953). I f an animal without previous experience responds q u i c k l y with p a r e n t a l behavior upon pres e n t a t i o n of young, one might think that there i s no opportunity f o r an increase i n responsive-ness as a r e s u l t of exposure to young. N o i r o t ' s extensive stud-i e s on mice and the present study of the gourami i n d i c a t e that there may nevertheless be such incremental changes, since a f t e r exposure to a stimulus e l i c i t i n g p a r e n t a l behavior, animals w i l l respond to a weaker stimulus than they would p r e v i o u s l y . Noirot found that a l a r g e p r o p o r t i o n of naive v i r g i n female mice showed par e n t a l behavior w i t h i n 5 min of pre s e n t a t i o n of a 131 newborn young ( N o i r o t , 1969b). Though inexperienced females were l e s s responsive to a dead pup, t h e i r responsiveness was increased by a 5 min exposure to a l i v e pup 2-8 days before t e s t i n g ( N o i r o t , 196ifb, 1969a). In another study, exposure to a 1 day o l d pup als o increased responsiveness to the weaker stimulus of a 10 day old pup (No i r o t , 1965). The present study showed that 100-200 eggs were l e s s l i k e l y to e l i c i t p a r e n t a l behavior i n naive male gouramis than 2000-3000 eggs were. However, a f t e r contact w i t h the l a r g e number of eggs which r e s u l t e d i n the r a p i d development of pa r e n t a l benavior, males a l s o responded to the small number. 3. Animals Responsive to Young Only at P a r t i c u l a r Times: Sensi-t i v e Periods f o r E f f e c t s of Eggs and Young on Responsiveness In a v a r i e t y of animals " s e n s i t i v e p e r i o d s " have been found i n which animals, due to i n t e r n a l changes, are more responsive to s t i m u l i from the young than at other times. Such s e n s i t i v e per-i o d s seem to be r e l a t e d to events associated with the n a t u r a l appearance of young—pregnancy and p a r t u r i t i o n i n mammals, approach of egg l a y i n g i n b i r d s , spawning i n f i s h . Although the p h y s i o l o -g i c a l events which a f f e c t responsiveness at t h i s time are o b v i -ously very important, the a c t u a l development of p a r e n t a l behavior seerns to be due to the combined i n f l u e n c e of the p h y s i o l o g i c a l s e n s i t i z a t i o n and motivat i n g i n f l u e n c e s from the young. Female goats are normally very responsive to k i d s immediately a f t e r p a r t u r i t i o n . However, i f they are prevented from contac-t i n g t h e i r young f o r as l i t t l e as one hour a f t e r p a r t u r i t i o n , they l o s e maternal responsiveness, demonstrating that n e i t h e r the 132 p h y s i o l o g i c a l events of p a r t u i r i t i o n nor contact w i t h a young k i d i s by i t s e l f a s u f f i c i e n t stimulus f o r the development of mater-n a l behavior. Just 5 minutes of contact w i t h a k i d immediately a f t e r p a r t u r i t i o n a l lows maternal responsiveness to develop nor-mally and p e r s i s t through an hour or more of separation (Kl o p f e r et a l . , 1964; K l o p f e r and K l o p f e r , 1968). In the present study gouramis were shown to develop p a r e n t a l behavior when given 800 eggs a f t e r spawning, but not when given 800 eggs or spawning, experience without exposure to eggs. Thus wit h t h i s number of stimulus eggs, behavior developed only with the combined i n f l u e n c e of eggs and the p h y s i o l o g i c a l f a c t o r s of spawning. In both of these cases the eggs or young have motiva-t i n g i n f l u e n c e s because p a r e n t a l responsiveness, which i s high during the s e n s i t i v e period, r e q u i r e s exposure to the young at t h i s time f o r i t s normal development or maintenance. h. Changes i n the I n t e n s i t y of P a r e n t a l Behavior When pa r e n t a l behavior begins under n a t u r a l circumstances or a f t e r p r e s e n t a t i o n of young, there may be changes i n i n t e n s i t y , frequency, and duration of p a r e n t a l a c t i v i t i e s . Example of t h i s i n c l u d e many b i r d s i n which f u l l i n c u b a t i o n begins sometime a f t e r the l a y i n g of the f i r s t egg (Beer, 1962; Drent, 1970; Lehrman, 1961) and f i s h i n which the p r o p o r t i o n of time spent fanning gra-d u a l l y i n c r e a s e s (Van I e r s e l , 1953). These changes suggest that e i t h e r the nature of the stimulus i s changing or that exposure to the eggs or young or the performance of p a r e n t a l behavior i n -creases p a r e n t a l responsiveness. One d i f f i c u l t y i n i n t e r p r e t i n g changes i n the i n t e n s i t y of 133 p a r e n t a l behavior i n terms of increased responsiveness i s that the stimulus i t s e l f i s o f t e n changing. In b i r d s , i n c r e a s i n g a t t e n t i v e n e s s often occurs at the same time as more eggs are being added to the c l u t c h . In f i s h , developmental processes may change the stimulus p r o p e r t i e s of the eggs. However, there i s some evidence that p a r e n t a l i n t e n s i t y may change even when the stimulus i s constant. Drent (1970) pointed out that nest a t t e n -t i v e n e s s and tendency to atta c k human i n t r u d e r s both continue to increase a f t e r the c l u t c h i s completed i n h e r r i n g g u l l s , and he c i t e d other references to in c r e a s e s i n a t t e n t i v e n e s s a f t e r com-p l e t i o n of the c l u t c h i n other b i r d s . Beer (1962) added eggs to the incomplete c l u t c h e s of black-headed g u l l s during the l a y i n g p eriod and removed them from complete c l u t c h e s during the i n c u -bation period. His observations i n d i c a t e d that both the number of eggs and an increase i n i n c u b a t i o n tendency were re s p o n s i b l e f o r an increase i n the i n t e n s i t y of in c u b a t i o n behavior from the l a y i n g to the in c u b a t i o n phase. Taking an empty nest as a stan-dard stimulus, Van l e r s e l (1953) found that removing eggs r e s u l t e d i n l e s s of a decrement i n p a r e n t a l fanning i n s t i c k l e b a c k s l a t e r i n the p a r e n t a l phase. Thus there do seem to be genuine increases i n p a r e n t a l responsiveness during the course of the par e n t a l c y c l e . 5.. Role of Eggs and Young i n the Maintenance of Pa r e n t a l Responsiveness : There are two l i n e s of evidence that eggs and young play a motivating r o l e i n the maintenance of par e n t a l responsiveness. One i s that p a r e n t a l responsiveness d e c l i n e s sooner than expected 13k when offspring are removed. The other i s that i t i s maintained longer than expected when the presence of offspring continues. Evidence for a premature decline i n parental responsiveness comes from some studies s p e c i f i c a l l y of parental behavior, but more often from observations that gonadal a c t i v i t y begins again i n those animals which have mutually exclusive sexual and paren-t a l phases. This phenomenon-seems to be widely known, but sur-p r i s i n g l y l i t t l e studied. It has been recorded for laboratory rats (Rosenblatt and Lehrman, 1 9 6 3 ; Moltz et a l . , 1 9 6 9 ) , for many birds (Seubert, 1 9 5 2 ; P e t t i n g i l l , 1 9 7 0 ) , and the three-spined stickleback (Van I e r s e l , 1 9 5 3 ) . Lehrman ( 1 9 6 1 ) c i t e s several other references. The most detailed observations of changes i n actual behavior are those of Rosenblatt and Lehrman ( 1 9 6 3 ) on the r a t . They showed that removal of the pups soon after b i r t h resulted i n the elimination of maternal behavior i n k days. When the separation began several days after p a r t u r i t i o n , maternal behavior s t i l l de-clined, but not as frequently, and i t began again after the res-toration of young i n a higher proportion of females. Findlay and Roth ( 1 9 7 0 ) found that i n three rabbits parental behavior v/as l o s t after 7 days i f conscious contact with the young v/as prevented but l a c t a t i o n was maintained by suckling while under anesthesia. In the r i n g dove, Lehrman and Brody ( 1 9 6 4 ) showed that incubation behavior was maintained by 1 2 days i n contact with eggs, but l o s t i f . the birds v/ere separated from their eggs for 1 2 days. In-sticklebacks, removal of eggs may lead to a gradual decline of parental fanning or to the i n i t i a t i o n of a new reproductive cycle (Van I e r s e l , 1953). Weber ( 1 9 7 0 ) studying a c i c h l i d f i s h , 1 3 5 Cichlasoma nigrofasciatum, found indications that fanning beha-vior declined more rapidly when eggs were removed afte r spawning than when eggs were present. In the present study gouramis usu-a l l y l o s t parental responsiveness within 6-8 days i f young v/ere removed one day after spawning, but usually retained i t i f l e f t with their developing broods. Parental responses may be maintained longer than normal i f presence of the young i s extended, i . e . , i f by substitution, . young are prevented from developing into i n e f f e c t i v e s t i m u l i . Most studies seem to indicate that parental care can be prolonged for a certain period i n t h i s fashion, but that aft e r a time par-ental care i s l o s t i n spite of the presence of eggs or young. Holcomb ( 1 9 7 0 ) has recently reviewed studies of prolonged incuba-tion behavior i n birds v/ith eggs which do not hatch. According to C o l l i a s ( 1 9 4 6 ) , hens could be kept broody for long periods of time i f developing chicks were replaced by young ones. Weber ( 1 9 7 0 ) showed that fanning behavior could be s i g n i f i c a n t l y pro-longed i n his c i c h l i d f i s h by presentation of a r t i f i c i a l eggs which did not hatch. In rat s maternal behavior may be extended by replacing developing l i t t e r s with young pups (Wiesner and Sheard, 1 9 3 3 , c i t e d by Rosenblatt and Lehrman, 1 9 6 3 ) . Additional references for mammals and birds may be found i n Lehrman ( 1 9 6 1 ) . Although I have so far discussed the maintenance Of parental responsiveness as i f i t were a unitary property of an animal, i t should be noted that t h i s i s not the case. Many animals show very di f f e r e n t types of parental care to t h e i r young at d i f f e r e n t stages of development. When such animals are tested with young of d i f f e r e n t .stages from th e i r own, i t i s often found that t h e i r 136 capacity to respond properly i s reduced as test young d i f f e r more s i g n i f i c a n t l y from th e i r own. See, for example, Rowell's (I960) study on hamsters and Emlen and M i l l e r ' s ( 1 9 6 9 ) i n v e s t i g a t i o n of r i n g - b i l l e d g u l l s . The observation that parental responsiveness ceases prema-turely a f t e r a period of separation from the young leads to the conclusion that there i s a tendency for a decline i n responsive-ness even during the normal parental period. This i s opposed i n some way by the presence of young. But many parental animals are not continuously i n contact with t h e i r young. They may leave eggs or young for excursions of varying duration. What keeps, parental responsiveness from declining during these periods? One p o s s i b i l i t y i s that parental responsiveness doesn't start to de-c l i n e u n t i l a f t e r a longer period of separation than usually occurs. Another i s that parental responsiveness starts to de-cl i n e during each absence but i s increased again during contact with the young. C. THE NATURE OF THE MOTIVATING STIMULI FROM THE YOUNG Although there i s considerable work on the stimuli and sen-sory modalities by which parents recognize young, there i s rather l i t t l e on the stimuli from the young necessary for the induction of parental responsiveness. However, the stimulus value of ham-ster pups changes with age (Richards, 1 9 6 6 a ; Noirot and Richards, 1 9 6 6 ) . The amount of contact between adult and young also seems very important.. Enforced proximity i n a narrow cage reduced the latency for parental induction i n rats (Terkel and Rosenblatt, cited by Rosenblatt, 1 9 7 0 ) . Reducing contact by placing the 1 3 7 young i n wire baskets at the side of the female's cage retarded development of p a r e n t a l behavior (Roth, 1 9 6 7 ) . The tv/o r e p o r t s on i n d u c t i o n of i n c u b a t i o n i n b i r d s were s i t u a t i o n s where the b i r d was forced to s i t on the eggs or at l e a s t remain very close to them. This may be s i g n i f i c a n t because b i r d s presented w i t h eggs before they have l a i d t h e i r own may often show f r i g h t or aggressive behavior (Beer, 1 9 6 3 ; Dykstra, 1 9 6 8 ) and thus avoid contact i f they are not i n i t i a l l y responsive. . Maier ( 1 9 6 3 b ) showed that domestic hens were much l e s s l i k e l y to become broody i f contact with c h i c k s was r e s t r i c t e d by p l a c i n g hens and c h i c k s i n adjacent cages or by a n e s t h e t i z i n g the v e n t r a l r e g i o n of the hen. E a r l i e r s t u d i e s (e.g., Burrows and Byerly, 1 9 3 8 ) suggested that development of broodiness was f a c i l i t a t e d i n s m a l l , dark coops. The acceptance of c h i c k s by cocks under the i n f l u e n c e of ethanol i n a study by Kovach ( 1 9 6 7 ) may a l s o have been f a c i l i t a t e d by the close i n i t i a l contact between cock and c h i c k s which t h i s treatment i n v o l v e d . A few authors have explored the s t i m u l i r e q u i r e d to maintain p a r e n t a l responsiveness. Moltz et a l . ( 1 9 6 9 ) found that the e f f e c t of young i n d e l a y i n g the resumption of e s t r u s took place i n female r a t s without n i p p l e s (and consequently without the stim-ulus of s u c k l i n g ) . F i n d l a y and Roth ( 1 9 7 0 ) , as mentioned above, found that even i f the c o n d i t i o n of the mammary glands was- main-tained by s u c k l i n g i n r a b b i t s , maternal behavior was l o s t i f the female was under anesthesia during s u c k l i n g and thus had no con-scious contact .with the young. A few s t u d i e s have i n d i c a t e d that p a r e n t a l behavior i s not maintained as w e l l when a r t i f i c i a l eggs are s u b s t i t u t e d which 1 3 8 d i f f e r e x c e s s i v e l y from normal s i z e i n b i r d s (Holcomb, 1 9 7 0 ) and f i s h (Weber, 1 9 7 0 ) . The present study of gouramis showed that p h y s i c a l separation from young g r e a t l y reduced t h e i r e f f e c t i v e n e s s i n maintaining p a r e n t a l behavior, even though v i s u a l and chemical cues were s t i l l present. And Maier ( 1 9 6 3 b ) found that, as wi t h i n d u c t i o n , maternal behavior was not maintained w e l l i f the hen was anesthetized i n the v e n t r a l region or c h i c k s were placed i n cages adjacent to the hen in s t e a d of i n the same cage. In mice and gouramis i n which changes i n responsiveness, occur a f t e r exposure to s t i m u l i which e l i c i t prompt p a r e n t a l r e -sponses, the m o t i v a t i o n a l e f f e c t s of pr e s e n t a t i o n of the young could be due e i t h e r to exposure to the stimulus or performance of the response. Noirot has explored some aspects of t h i s question i n mice. The f a c i l i t a t i o n of responsiveness to a weak stimulus occurred when females were exposed only to o l f a c t o r y and auditory cues from young ( N o i r o t , 1 9 6 9 a ) , or e i t h e r of these alone (Noirot, 1 9 6 9 c ) , even though such cues had l i t t l e i n common with those pro-vided by the weak stimulus of a dead pup. By p l a c i n g l i v e stimu-l u s pups i n the nest, she found that r e t r i e v i n g of a dead pup was f a c i l i t a t e d even i f i t had not occurred during exposure to the strong stimulus ( N o i r o t , 1 9 6 4 c ) . In the gourami, on the other hand, i t appeared that only f i s h which had developed p a r e n t a l behavior i n response to a l a r g e number of eggs showed respon-siveness to small numbers. D. THE MECHANISM BY WHICH YOUNG AFFECT PARENTAL RESPONSIVENESS There has been very l i t t l e experimental i n v e s t i g a t i o n of the mechanism by which exposure to young could increase p a r e n t a l 139 responsivenss. Such a mechanism or mechanisms must be able to account f o r the r a p i d development of p a r e n t a l behavior during exposure to a strong stimulus and i t s gradual development during exposure to a weak stimulus. The e f f e c t s of one stimulus must p e r s i s t f o r some time, on the order of days, i n the absence of the stimulus. And the e f f e c t s of repeated s t i m u l a t i o n must be cumulative. Exposure to a stimulus must have an e f f e c t on paren-t a l responsiveness even i f no overt p a r e n t a l behavior i s e l i c i t e d . I t i s not impossible that an endocrine mechanism could account f o r these e f f e c t s i f exposure to young caused the r e l e a s e of a hormone which f a c i l i t a t e d p a r e n t a l behavior. This seems p a r t i -c u l a r l y reasonable since c e r t a i n other p h y s i o l o g i c a l processes of p a r e n t a l animals i n which the presence of the young p l a y s a r o l e — e . g . , i n h i b i t i o n of e s t r u s c y c l i n g , maintenance of l a c t a -t i o n , and maintenance of brood patches—do depend upon hormonal mechanisms. I t could be that the same hormonal mechanisms are i n v o l v e d i n p a r e n t a l behavior and p a r e n t a l physiology. Lehrman and Brody ( 1 9 6 4 ) have found evidence f o r t h i s i n r i n g doves. The i n c u b a t i o n tendency could be r e t a i n e d i n doves separated from eggs i f and only i f they were given p r o l a c t i n i n j e c t i o n s i n the meantime. However, t h i s does not seem to be the whole p i c t u r e . Rosen-b l a t t ( 1 9 7 0 ) reviewed s e v e r a l l i n e s of evidence f o r the l a c k of involvement of hormones i n the maintenance of p a r e n t a l behavior i n the r a t . In t h i s species p a r e n t a l behavior was maintained i n the absence of the p i t u i t a r y and o v a r i e s . When young were re-r moved f o r 1 0 days, p r o l a c t i n and oxytocin i n j e c t i o n s f a i l e d to maintain maternal responsiveness. 1/fO And there i s a l s o evidence that, at l e a s t i n r a t s and mice, the e f f e c t of young on the i n d u c t i o n of p a r e n t a l behavior occurs i n s p i t e of major endocrine d e f i c i t s . Rosenblatt (1967) observed that exposure to f i v e 5-10 day ol d pups f o r an average of 6-7 days r e s u l t e d i n p a r e n t a l behavior i n hypophysectomized females as w e l l as i n i n t a c t and gonadectomized male and female r a t s . Cosnier and Coutur i e r (1966) a l s o found no e f f e c t of ovariectomy. P a r e n t a l responses may be developed by gonadectomized and normal males and by hypophysectomized male and female mice (Leblond and Nelson, 1937; Leblond, 1938). Thus i t seems l i k e l y that the e f f e c t s of exposure to young i n these species are not based on an endocrine mechanism, or at l e a s t not on one i n v o l v i n g p i t u i t a r y or gonads. E. THE ROLE OF MOTIVATIONAL EFFECTS OF THE YOUNG IN RELATION TO THE ROLE OF HORMONES IN THE INDUCTION OF PARENTAL BEHAVIOR Young may be able to increase p a r e n t a l responsiveness i n the absence of major endocrine i n f l u e n c e s . But hormones are also known to play an important r o l e i n the p a r e n t a l behavior of many species. How are the two f a c t o r s r e l a t e d ? More s p e c i f i c a l l y , do motivati n g e f f e c t s of young have a r o l e i n the normal i n d u c t i o n of p a r e n t a l behavior i n those species which u s u a l l y show pa r e n t a l behavior only when they have eggs or young of t h e i r own? In t h i s s i t u a t i o n hormonal f a c t o r s have o f t e n been thought to c o n t r o l the changes i n p a r e n t a l responsiveness. At f i r s t s i g h t there seems to be no need to postulate a r o l e f o r m o t i v a t i n g e f f e c t s of young i n the i n d u c t i o n of pa r e n t a l behavior. The p h y s i o l o g i c a l 1 4 1 and p h y s i c a l processes associated with p a r t u r i t i o n or spawning seem s u f f i c i e n t to account f o r i t s development. When young f i r s t appear, the parent often seems to respond immediately, apparently l e a v i n g l i t t l e opportunity f o r i n d u c t i v e e f f e c t s of exposure to young. Though there i s l i t t l e q u a n t i t a t i v e data on t h i s p o i n t , d e s c r i p t i o n s of p a r t u r i t i o n i n r a t s (Rosenblatt and Lehrman, 1 9 6 3 ) , and sheep and goats (Hersher et a l . , 1 9 6 3 ) , f o r example, c e r t a i n l y imply immediate responsiveness to young. In Aronson's ( 1 9 4 9 ) study of T i l a p i a macrocephala, and i n the present study of the gourami, p a r e n t a l care o f t e n began l e s s than a minute a f t e r the eggs were l a i d , and even t h i s delay was p a r t i a l l y due to competing a c t i v i t i e s such as chasing away the female or f e r t i l i z i n g the eggs. In some s t u d i e s of the hormonal c o n t r o l of behavior, a s i m i l a r s i t u a t i o n seems to hold. For example, Lehrman ( 1 9 5 8 b ) and Lehrman and Wortis ( i 9 6 0 ) found that experienced r i n g doves i n j e c t e d w i t h progesterone would begin to incubate w i t h i n a few minutes of having been given eggs. On the other hand, there i s evidence from a few s t u d i e s -> that one way hormones act i s by s e n s i t i z i n g the animals to the moti v a t i n g i n f l u e n c e of young. The evidence from K l o p f e r and h i s co-workers on the maternal behavior of goats and the present study of the r o l e of spawning i n the gourami have already been • discussed i n the s e c t i o n on " s e n s i t i v e p e r i o d s . " In a d d i t i o n , evidence has r e c e n t l y been accumulating i n r a t s that pregnancy and p a r t u r i t i o n i n f l u e n c e p a r e n t a l behavior i n a s i m i l a r fashion. Rosenblatt ( 1 9 7 0 ) reported that by the 1 6 t h day of pregnancy, the latency f o r the i n d u c t i o n of p a r e n t a l behavior by exposure to pups was s i g n i f i c a n t l y reduced. L o t t and Rosenblatt ( 1 9 6 9 ) 1/+2 found that i f pregnancy was terminated by hysterectomy or cae-sarean s e c t i o n before presentation of the pups, the l a t e n c y v/as f u r t h e r shortened, and a progressive r e d u c t i o n i n l a t e n c y was found with the approach of term. Terkel and Rosenblatt (1968) found that v i r g i n females developed p a r e n t a l responsiveness more q u i c k l y than c o n t r o l s when i n j e c t e d v/ith 3-4 ml of plasma removed from females w i t h i n 48 hr a f t e r p a r t u r i t i o n . Recent progress i n determining the hormonal b a s i s of these e f f e c t s has been made by Moltz et a l . (1970) who s i g n i f i c a n t l y reduced the la t e n c y to i n -duction of par e n t a l behavior i n ovariectomized females w i t h a: s e r i e s of i n j e c t i o n s of e s t r a d i o l benzoate, progesterone, and p r o l a c t i n . These authors suggested a hypothesis of responsiveness to .young brought about by e f f e c t s of p r o l a c t i n and estrogen on a c e n t r a l nervous mechanism s e n s i t i z e d by progesterone withdrawl. These s t u d i e s i n d i c a t e that endocrine f a c t o r s of pregnancy.and p a r t u r i t i o n i n f l u e n c e the r a t e at which the animals responded to the motivating i n f l u e n c e s of the young, and suggest the p o s s i -b i l i t y that t h i s i s the way they act at the time of normal par-t u r i t i o n . In other s t u d i e s on the hormonal c o n t r o l of par e n t a l beha-v i o r , one can f i n d i n d i c a t i o n s that young as v/ell as hormones are having important e f f e c t s on pa r e n t a l responsiveness. An example comes from the work of Lehrman and h i s co-workers on the p a r e n t a l behavior of r i n g doves. Although Lehrman (1958a) found no e v i -dence that even very long exposure to stimulus eggs could induce in c u b a t i o n unless the b i r d also r e c e i ved hormonal treatment, and Lott and Comerford (1968) found only one case of p a r e n t a l beha-v i o r e l i c i t e d by exposure to squabs without hormonal treatment, 143 other studies indicated some motivating e f f e c t s of young. It v/as found that birds took longer to begin to incubate eggs or to feed young after the introduction of the stimulus i f they had been given l e s s adequate hormonal treatment (Stern and Lehrman, 196.5) or i f they had not had previous parental experience (Lehrman and Wortis, I 9 6 0 ; Lott and Cornerford, 1 9 6 8 ) . Why should these; ani-mals s t i l l eventually show parental behavior but take longer to do so? One reasonable explanation i s that the stimuli from the eggs or .young are having a motivating influence which compensates for the d e f i c i e n c i e s i n pr i o r experiences or hormonal treatment. Thus i t seems reasonable to consider that at least one way that hormones act i s by a l t e r i n g the r e c e p t i v i t y to the motivating influences of the young. Such a hypothesis may lead to new ways of looking at how hormones affect behavior. F. SUGGESTION OF A COMMON PATTERN IN THE MOTIVATIONAL EFFECTS OF YOUNG Evidence reviewed i n the previous sections has indicated motivating effects of stimuli from eggs and young i n species ; varying widely i n taxonomic position and parental behavior pat-terns. Although there i s l i t t l e reason to believe that the mechanisms for the control of parental care should be similar i n mammals, birds and f i s h , t h i s might suggest the p o s s i b i l i t y of p a r a l l e l systems for the control of parental behavior. However, the ways i n which motivational effects of young are shown i n the various species seem to be very d i f f e r e n t . In some, parental be-havior develops gradually after long exposure to young. In others, i t i s shown very quickly whenever young are presented. In s t i l l others i t seems to develop only v/ith the pre s e n t a t i o n of young during p a r t i c u l a r s e n s i t i v e periods. These v a r i a t i o n s seem to bear no r e l a t i o n s h i p to taxonomic p o s i t i o n . Rats and chickens show s i m i l a r p a tterns of gradual de-velopment of par e n t a l behavior during exposure to young, while mice often respond immediately. Sometimes more than one of these patterns i s found i n a s i n g l e species. For example, r a t s u s u a l l y develop p a r e n t a l behavior g r a d u a l l y when presented v/ith young, but i f t h i s p r e s e n t a t i o n occurs a f t e r caesarean s e c t i o n near the normal time of p a r t u r i t i o n , responses may begin promptly (Moltz et a l . , 1966). In the present study of the gourami, t h i s has been more c l e a r l y demonstrated. When small numbers of eggs are used i n a s i n g l e t e s t , f i s h respond only a f t e r normal spawning and develop-ment of par e n t a l behavior. But i f the f i s h are repeatedly ex-posed to small numbers of eggs, they may begin to show p a r e n t a l behavior. When test e d v/ith a moderate number of eggs, naive f i s h , do not show pa r e n t a l behavior. However, i f they are allowed.to perform the spawning act before exposure to the eggs, they do develop p a r e n t a l behavior. With l a r g e numbers of t e s t eggs, many f i s h begin p a r e n t a l behavior w i t h i n the f i r s t few minutes of exposure. I f such d i f f e r e n t patterns can be observed i n a s i n g l e species as a r e s u l t of va r y i n g the strength of the stimulus, perhaps the apparent i n t e r s p e c i f i c d i f f e r e n c e s i n c o n t r o l of pare n t a l behavior r e f l e c t d i f f e r e n t f a c e t s of a common phenomenon. This basic process may be that exposure to young increases r e -sponsiveness to young, whether or not' p a r e n t a l behavior i.s 145 induced. In animals r e l a t i v e l y i n s e n s i t i v e to young or presented w i t h r e l a t i v e l y weak s t i m u l i , responsiveness could g r a d u a l l y i n -crease w i t h continued exposure u n t i l p a r e n t a l behavior was e l i -c i t e d . In animals more s e n s i t i v e or presented with stronger s t i m u l i p a r e n t a l behavior could begin q u i c k l y . The p h y s i o l o g i c a l changes of spawning, p a r t u r i t i o n , e t c . could increase s e n s i t i v i t y so that young would be more e f f e c t i v e at mo t i v a t i n g p a r e n t a l r e -sponsiveness at the re q u i r e d time. The r o l e of young i n the maintenance of pa r e n t a l behavior might be another aspect of the same process. I f many species possess a mechanism whereby eggs and young have incremental e f f e c t s on p a r e n t a l responsiveness, we can ask what i s the b i o l o g i c a l s i g n i f i c a n c e of such a system. Much of the evidence f o r m o t i v a t i o n a l e f f e c t s of young comes from e x p e r i -ments i n very a r t i f i c i a l s i t u a t i o n s v/here non-parental animals are c l o s e l y confined vrith young. What i s the r o l e of the pheno-menon i n the l i f e of the animal? Some of the evidence f o r m o t i v a t i o n a l e f f e c t s of young comes from s i t u a t i o n s v/here the b i o l o g i c a l u t i l i t y i s c l e a r . The value of m o t i v a t i n g e f f e c t s of young i n the maintenance of p a r e n t a l behavior seems obvious enough. In species i n which sexual and pare n t a l phases are mutually e x c l u s i v e , i t may be b e n e f i c i a l f o r an animal to become non-parental soon a f t e r i t s young are l o s t so that a new brood may be q u i c k l y s t a r t e d . But then the young, while present must be able to counteract t h i s tendency. I t i s •possible that the i n d u c t i o n of p a r e n t a l behavior a f t e r long ex-posure to young r e s u l t s from the a c t i v a t i o n of the system by which young c o n t r o l the maintenance of parental behavior. 1 4 6 The e f f e c t s of young on par e n t a l motivation may al s o have obviou.s f u n c t i o n a l s i g n i f i c a n c e i n s o c i a l mammals, i n v/hich par-e n t a l care of young may be shared among members of a group and not be performed s o l e l y by the mother. Such behavior has been shown among mammals i n primates (Powell et a l . , , 1 9 6 4 ; Alexander, 1 9 7 0 ) , c arnivores (Estes and Goddard, 1 9 6 7 ) , rodents ( D i e t e r l i n , 1 9 6 2 ; Gandelman et a l . , 1 9 7 0 b ) , and others, as w e l l as some b i r d s (Brown, 1 9 7 0 ) . (See Beach, 1 9 6 7 , and Raphael 1, 1 9 7 0 , f o r other references and a more general d i s c u s s i o n of the phenomenon.) 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