Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Behavioural endocrinology of the stoplight parrotfish, Sparisoma viride, Scaridae, a protogynous coral… Cardwell, James Robert 1989

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1989_A1 C36_5.pdf [ 12.64MB ]
Metadata
JSON: 831-1.0098250.json
JSON-LD: 831-1.0098250-ld.json
RDF/XML (Pretty): 831-1.0098250-rdf.xml
RDF/JSON: 831-1.0098250-rdf.json
Turtle: 831-1.0098250-turtle.txt
N-Triples: 831-1.0098250-rdf-ntriples.txt
Original Record: 831-1.0098250-source.json
Full Text
831-1.0098250-fulltext.txt
Citation
831-1.0098250.ris

Full Text

BEHAVIOURAL ENDOCRINOLOGY OF THE STOPLIGHT PARROTFISH, SPARISOMA VIRIDE, SCARED AE, A PROTOGYNOUS CORAL REEF FISH By JAMES ROBERT CARD WELL B.Sc, The University of British Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1989 ©James Robert Cardwell, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~2oo\o The University of British Columbia Vancouver, Canada Date V? I\")99 DE-6 (2/88) ii Abstract The behavioural endocrinology of a protogynous coral reef fish, the stoplight parrotfish (Sparisoma viride, Scaridae) was investigated at Glover's Reef, Belize. Detailed behavioural observations in the field were combined with radioimmunoassay of steroids circulating in plasma (11-ketotestosterone, testosterone and 17p-estradiol) and histological examination of gonads to obtain precise correlations of behaviour, colouration and gonadal condition with endocrine status. The size, sex and colour phase distribution, together with histological analysis suggests that some individuals of this species undergo sex change as mature adult females, while others change sex as immature individuals, becoming functional males without passing through a female phase. Furthermore, some individuals change sex and colour phase simultaneously while others retain female-like 'initial phase' (Iph) colouration and function as Iph males before acquiring 'terminal phase' (Tph) colouration. Large Tph males defend permanent, all-purpose territories on which they pair-spawn daily with the females of a harem group. Smaller Tph males (bachelors) neither defend territories, nor spawn, but feed in groups and inhabit overlapping home-ranges. Females also inhabit overlapping home-ranges within the confines of a Tph male's territory. They spawn with the same male every day at high tide. Iph males are rare in this population. They spawn by releasing milt into the gamete cloud left after a pair-spawning event. Iph males also pair-spawn with females in the absence of Tph males. Sex change is correlated with the onset of 11-ketotestosterone production, and a dramatic decrease in plasma levels of estradiol. This is the first report to show that a naturally-occurring androgen increases in plasma concentration during sex change in a protogynous marine species. Administration of 11-ketotestosterone promotes sex and colour change in adult females. Thus, 11-ketotestosterone appears to play a key role in sex and colour phase change in this species. iii Males that retain Iph colouration after sex change have lower levels of 11-ketotestosterone (undetectable) and higher levels of estradiol than Tph males or males with transitional colouration. This suggests that estradiol may suppress colour phase change in Iph males. Bachelor Tph males have lower levels of testosterone and 11-ketotestosterone than territorial males. Bachelors rapidly take over experimentally vacated territories, confinning the hypothesis that they are normally excluded from suitable habitat by territorial males. One week after territory acquisition, 11-ketotestosterone and testosterone increase to levels over and above those in undisturbed territorial males, but by three weeks, androgen levels are not significantly different from those in undisturbed territorial males. Simulated territorial intrusion promoted increased androgen production in Tph males, while access to territories without neighbours did not. Thus, the pattern of androgen production seen after territory acquisition is due to interactions with neighbouring males during territory boundary re-establishment. Increased levels of androgen during territorial challenges may promote increased aggressiveness and territorial vigilance, thereby increasing the chances of successfully defending against the challenge. These findings are discussed in light of recent theory in behavioural endocrinology. iv TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables viii List of Figures ix Acknowledgements xi Chapter One. General Introduction 1 Chapter Two. General Materials and Methods 7 A. Study site 7 B. Identification of individuals 7 C. Documenting behaviour 9 D. Capture of fish 10 E. Sexing fish 10 F. Collection and treatment of blood samples 11 G. Histology 11 Chapter Three. Sex change and colour phase change in the stoplight parrotfish 13 A. Introduction 13 B. Materials and Methods 15 C. Results 15 1. Gonadal Histology 15 a. Anatomy of the gonad 15 b. Histology of the ovary 17 c. Histology of the testis 21 d. Description of intersex gonads 24 2. Colouration 24 3. Size, Sex and Colour Distribution 26 V D. Discussion 30 Chapter Four. The stoplight social and mating system 35 A. Introduction 35 B. Materials and Methods 36 C. Results 38 1. Description of Behaviours 38 a. General maintenance behaviours 38 b. Social behaviours 38 c. Spawning behaviours 39 2. Social Behaviour and the Use of Space 41 a. Tph males 41 b. Iph males 50 c. Females 51 3. The Stoplight Mating System 53 a. Location of spawning 53 b. Time of spawning 53 c. Frequency of spawning 57 d. Which individuals spawn 58 4. Social Control of Sex Change 61 D. Discussion 62 Chapter Five. Hormonal control of sex and colour phase change 66 A. Introduction 66 B. Materials and Methods 68 1. Radioimmunoassay Procedures and Assay Validation 68 a. Chemicals 68 b. Extraction of steroids from plasma 69 c. Radioimmunoassay procedure 70 vi d. Determination of unknown values 71 e. Assay validation 71 2. Analysis 72 3. Hormone Administration 72 C. Results 73 1. Assay Validation 73 2. Hormonal Correlates of Sex and Colour Change 75 a. Sex change 75 b. Colour phase change 75 3. Effects of Androgen on Females 78 D. Discussion 80 Chapter Six. Hormones and social status in Tph males 85 A. Introduction 85 B. Materials and Methods 86 1. Hormonal Correlates of Social Status 86 2. Effect of Access to Vacant Territories 86 3. Simulated Territorial Intrusion 87 a. STI 87 b. Access to singleton reefs 88, 4. Analysis 88 C. Results 88 1. Hormonal Correlates of Territorial and Bachelor Status 88 2. Effect of Removing Territorial Males 90 3. Effect of Simulated Territorial Intrusion and access to singleton reefs 93 D. Discussion 97 Chapter Seven. General Discussion 100 1. The Adaptive Significance and Optimal Timing of Sex Change 100 vii 2. Iph Males: An Alternative Life History 103 3. The Hormonal Control of Reproductive Behaviour 107 4. Behavioural Endocrinology in the Field I l l Chapter Eight. References Cited 114 viii LIST OF TABLES Table Page 1.1 Terminology for patterns of sexuality in hermaphroditic fishes 5 3.1 The relative frequencies of sex and colour types in a sample from Glover's Reef 28 4.1 Time of spawning at w.b. reef, July 6,1985 56 4.2 Frequency of spawning in two harem groups 57 5.1 Validation of RIAs for 11KT, T and E2 measured in parrotfish plasma 74 6.1 Effects of simulated territorial intrusion on the behaviour of Tph males on isolated reefs 94 ix LIST OF FIGURES Figure Page 2.1 Map of Belize, C.A., showing the location of Glover's Reef 8 3.1 Cross-sections of ovaries; pre-vitellogenic stages 16 3.2 Cross-sections of ovaries; vitellogenic stage 18 3.3 Cross-sections of ovaries; mature stage and ovulation 20 3.4 Cross sections of testes; Gross structure and spermatogonia 22 3.5 Cross-sections of testes; spermatocytes, spermatids and spermatozoa 23 3.6 Cross-sections of intersex gonads 25 3.7 Photographs of stoplight colour phases 27 3.8 Size-frequency distribution of stoplights captured at Glover's Reef 29 3.9 Summary of the presumptive life-history pathway of stoplight parrotfish . . . 33 4.1 Illustration of some aspects of stoplight social and reproductive behaviour. . 40 4.2 Map showing use of space by territorial and bachelor males 43 4.3 Map showing changes in territory boundaries following removal of TTph males 44 4.4 Frequency of Chase, Display and Feeding Bites exhibited by territorial and bachelor males 46 4.5 Rates of movement by territorial and bachelor males 48 4.6 Size-distribution of territorial and bachelor Tph males 49 4.7 Map showing female home-ranges 52 4.8 Map showing location of spawning acts at w.b. reef 54 4.9 Time of predicted daytime high tide and time of observed spawning 55 4.10 Gonosomatic indices of females, Iph males, and bachelor and territorial Tph males 59 5.1 Hormonal correlates of sex change 76 5.2 Hormonal correlates of colour phase change 77 5.3 Cross-sections of gonads from control and androgen-treated females 79 6.1 Plasma levels of 11KT and T in bachelor and territorial Tph males 89 6.2 Effect of access to vacant territories on plasma levels of 11KT andT (1985) 91 6.3 Effect of access to vacant territories on plasma androgen levels (1986) 92 6.4 Effect of simulated territorial intrusion on plasma levels of 11KT and T . . . . 96 xi Acknowledgements First, I thank my supervisor, Dr. N. Robin Liley for his patience, understanding, encouragement, support, advice, criticism and field assistance, and for recognizing the potential of this project in the first place. Partial funding was provided by a Grant-in-aid of Research from Sigma-Xi, a McLean-Fraser fellowship and an NSERC postgraduate scholarship to myself, and NSERC operating grants to N.R. Liley. I thank Dr. D.R. Idler for generously donating antibodies to 11KT, and Mr. R. Castellano (Belize Steel Products) who donated liquid nitrogen in 1985.1 thank the Minister of Commerce and Industry, Fisheries and Cooperatives of Belize, Mr. Eduardo Juan, for personally extending our visas in order that we could stay on the island for the duration of each field season, and to the people and government of Belize for allowing us to conduct research in their waters. I am grateful to Drs. J.P. Sumpter, D.M. Lyster and E.S.P. Tan who patiently taught me the ins-and-outs of radioimmunoassay techniques, and to the staff at the West Vancouver Fisheries Laboratory (Dr. E.M. Donaldson, Tillman Benfey and Helen Dye) for technical support, friendly, expert advice and for allowing me to use their facilities from time to time. Similarly, I am grateful to the members of Dr. John Phillips' laboratory for the use of their photo-microscope, computer time, advice and coffee-time chats. Alistair Blachford provided much assistance at the computing center, and kindly wrote the program for the digitizing tablet. I thank Gil and Marcia-Jo Lomont for assistance during our stays on Long Caye. The Lomonts also allowed us to use their outboard engine and other supplies following the theft of some of our equipment in 1987. I thank Ralph and George Jackson, and Carlos, Maruga and Felix Martinez for transporting us and/or supplies to or from the island on several occasions. R. Jackson kindly lent us his fishing net, which contributed greatly to the success of the project. Bill Reid of Redden Net Co. was extremely helpful in acquiring our gill-net, despite its modest size. Several people extended their hospitality to us while passing through Glover's Reef on scientific expeditions or travel, often providing a brief, but much needed respite of civilization, for which I thank them. I am also grateful to the members of my research committee (Drs. E.M. Donaldson, A.R.E. Sinclair, J.D. McPhail and B. Gorzalka) and to Dr. J.N.M. Smith, Dr. S.G. Hoffman, Carlos Brunet and Troy Baird for their thoughtful advice and criticism. Troy Baird also carefully taught me the histological techniques for teleost gonads. Expert and invaluable field assistance, advice, friendship, culinary surprises and laughter were provided by J.S. McKinnon and J.L. Smith (whom I also thank for remembering to bring the net-mending needles). I extend my sincere gratitude to my family and friends, and especially to my parents and parents-in-law (Patricia and Dr. David Cardwell and Gabrielle and Peter Kirby), who provided moral support and encouragement throughout this project. Finally, I thank Jacqueline, my wife and best friend, for her encouragement, advice and moral support, and for two years of field assistance beyond the call of duty. 1 CHAPTER ONE General Introduction With the growing importance of fish as a food resource, increased attention is being focussed on teleost reproductive physiology (see references in Richter and Goos 1982, Idler et al. 1987). In particular, great progress has been made in recent years in the investigation of teleost reproductive endocrinology. However, progress in the behavioural endocrinology of teleost reproduction has been less rapid (reviews in Liley and Stacey 1983, Liley et al. 1987). In females, seasonal spawning cycles are generally correlated with cycles of steroid-dependent changes in gonadal condition (e.g. Scott et al. 1980a, Stacey et al. 1984, Kadmon et al. 1983, review in Liley and Stacey 1983). In addition, ovarian steroids may control female attractivity by acting as pheromones when released into the water (review in Stacey 1987). However, while ovariectomy has been shown to abolish reproductive behaviour in females of a few species (Liley 1969), direct evidence for a role of gonadal steroids in the control of female reproductive behaviour has been demonstrated only in the guppy (Poecilia reticulata, Liley 1972). In males, there is a clear correlation between hormone-dependent cycles of sperm production with periods of reproductive behaviour. Furthermore, reproductive behaviours and secondary sex characteristics are abolished by castration or by treatment with drugs that prevent the production or effects of gonadal steroids, and are restored by androgen replacement therapy (Liley 1969, Liley and Stacey 1983, Moore 1986). Thus, it is generally concluded that all aspects of male teleost reproductive behaviour (territorial defence, nest-building, pairing, spawning, parental care, etc.) are under the control of androgens (reviews in Liley and Stacey 1983, Moore 1986). However, although the classical castration and replacement therapy experiments provide information about whether gonadal hormones are necessary and sufficient for the expression of a 2 given behaviour, they have not addressed the issue of which gonadal steroids are primarily responsible for maintaining reproductive behaviour (Liley and Stacey 1983). Without confirmatory evidence that the hormone undergoes changes in plasma concentration in synchrony with changes in the behaviour being examined, investigators risk the possibility that the observed effects of hormone injection may be pharmacological. One technique for addressing this problem is the use of radioimmunoassays (RIAs) to determine the temporal patterns of circulating hormone concentrations in relation to the onset, maintenance and completion of behaviour. This type of investigation provides precise correlations between behavioural activity and hormone levels. Furthermore, RIAs offer the added advantage of monitoring hormone levels under natural conditions and during ongoing and changing social situations or experimental regimes; such detailed information was previously unavailable through castration and hormone-injection techniques (Liley etal. 1987). Although correlational studies do not in themselves prove that causal endocrine-behaviour relationships exist, they provide investigators with evidence of which hormones may be important in controlling the behaviours in question (e.g. birds: O'Connell etal. 1981, Silverin and Wingfield 1982, Wingfield 1984a,b, 1985; reptiles: Moore 1987, Moore and Marler 1987, Crews et al. 1984; fishes: Kyle et al. 1985, Hannes 1983, Hannes and Franck 1984, Stacey et al. 1984, Scott et al. 1984, Liley et al. 1986a,b, Kobayashi etal. 1988, Linville etal. 1987). This information may then be utilized in manipulative experiments designed to test the suggested relationships (e.g. Silverin 1980, Moore 1984, Searcy and Wingfield 1986, Wingfield 1984c, 1985, Wingfield and Silverin 1986, Moore 1987, Tokarz 1987). Unfortunately, correlational studies have not yet been fully exploited in fishes; most behaviour-endocrine investigations involving fish have been conducted on laboratory-held subjects. Moreover, the few such investigations that have been conducted in the field have not been combined with detailed behavioural observations that potentially hold clues to the behavioural significance of endocrine conditions. It is well known that in higher vertebrates, hormones present during development have permanent, directing (organizing) effects on the expression of behaviour in adulthood (Phoenix 3 et al. 1959, Hutchison 1978, Leshner 1978, Wilson et al. 1981, MacLusky and Naftolin 1981). These long-term, organizational (or developmental) effects are distinct from the short-term, reversable (activational) effects of hormones (such as those discussed above) that modulate ongoing behaviours (ibid.). While the effects of hormone treatments on sex differentiation in teleost larvae are well documented (reviews in Schreck 1974, Hunter and Donaldson 1983, Adkins-Regan 1986), little research effort has been directed at the effects of such treatments on behavioural development (Billy and Liley 1985, Liley et al. 1987). One report has established that exogenous androgen (methyl testosterone) administered to developing tilapia (Sarotherodon mossambicus) masculinizes behaviour of females when tested as adults (Billy and Liley 1985). However, it is not known in any teleost whether changes in hormone secretion during development correspond to the period of behavioural differentiation (Hurk and Peute 1979, Hunter and Donaldson 1983). Thus, conclusions on whether hormones play a similar role in regulating the development and differentiation of behaviour in both teleosts and mammals are not yet possible. The teleost proclivity for exhibiting various forms of hermaphroditism is well known (Policansky 1982, 1987, Reinboth 1970, Chan 1970, Shapiro 1979, 1987). Two types of sequential sex change have been described: protandry, in which individuals differentiate as males first and then change sex into females, and protogyny, in which individuals mature first as females before eventually becoming males. Teleosts that undergo natural sequential sex change may provide a rare opportunity to examine both developmental and activational effects of hormones within a short period of time. In effect, sequential hermaphroditism provides a natural form of the classical castration and replacement therapy experiment. The primary goals of this investigation were to examine changes in circulating hormone concentrations that accompany female to male sex change in a protogynous marine teleost, (the stoplight parrotfish, Sparisoma viride) and to determine the role of hormones in the initiation and maintenance of reproductive behaviour. 4 Parrotfish are members of the sub-order Labroidei, family Scaridae. They are abundant inhabitants of coral reefs in all tropical oceans, and are well known for their bright colouration and specialized feeding habits. Their teeth are fused into large plates resembling beaks (hence their common name) that are specialized for scraping algae from coral substrates (Randall 1967). Most parrotfish exhibit marked sexual dichromatism, and as a result, early taxonomists classified males and females of many parrotfish as separate species (Winn and Bardach 1957, 1960, Clavijo 1982). In general, the colour pattern exhibited by females is termed initial colour phase (abbreviated as Iph) while that of males is termed terminal colour phase (Tph; see Table 1.1 for a glossary of terms used in this thesis). Reinboth (1968) was the first to suggest that this family exhibits protogynous sex change. As in the closely related Labridae, the pattern of sexual ontogeny in some scarid species is confused by the existence of two types of males (a condition known as diandry; Reinboth 1970); those that begin life as males and never change sex, (termed primary males), and those that result from sex change, (termed secondary males). Furthermore, some protogynous species have secondarily evolved prematurational sex change, in which males result from sex change of immature females (a condition termed secondary gonochorism; Robertson and Warner 1978). Of the two scarid sub-families, only the Scarinae appear to be diandric (Robertson and Warner 1978, Choat and Robertson 1975); the Sparisomatinae (which includes S. viride) are apparently monandric, in that all males are derived from sex changed females. In the Sparisomatinae, there are two kinds of secondary males; those that exhibit female-like colouration (Iph males) and those that exhibit Tph colouration (Tph males). Both kinds of males are found in S. viride (Robertson and Warner 1978). Individuals are occasionally found to exhibit a colour pattern that is intermediate between Iph and Tph. In the present thesis, this colour pattern is termed transitional colour phase. In contrast, individuals whose gonads are intermediate between functional female and functional male are termed intersex fish, regardless of colouration. 5 Table 1.1 Terminology for Patterns of Sexuality in Hermaphroditic Teleosts protogyny Female-first sequential sex change protandry Male-first sequential sex change secondary male Male derived from a sex changed female primary male Individual that is male throughout life (a gonochorist) diandric Protogynous species with two ontogenetic types of males monandric All males are derived from sex changed females secondary gonochore Male derived from a female that changed sex before maturing initial phase Colour pattern associated with females terminal phase Colour pattern associated with large males Iph male Male with female-like colouration Tph male Male with terminal phase colouration transitional Intermediate colour pattern intersex Fish whose gonads contain ovarian and testicular elements The pattern of sexuality in S. viride has been examined previously, although there is confusion as to whether individuals change sex into males as adult females or as immatures. Therefore, I investigate the sexual ontogeny of Sparisoma viride, and present details of parrotfish gonadal histology, which have not been described previously (Chapter Three). A vital pre-requisite to an investigation of the relationship between hormones and behaviour in a free-living species is an understanding of the species' social and mating system. Sparisoma viride is large, easy to trap and blood sample, and is abundant on shallow reefs in tropical waters; these factors provided an unusual opportunity to collect detailed information on 6 this species' elaborate social and mating system (Chapter Four) and to correlate these data with changes in hormone levels (Chapters Five and Six). 7 CHAPTER TWO General Materials and Methods A. Study site Field work was conducted at Glover's reef, Belize, C.A. from May to July 1984, April to August 1985, May to August 1986 and May to July 1987. Glover's reef is located at approximately 87°45'W, 16°45'N in the Caribbean Sea, approximately 45 km south east of the Belizean town of Dangriga, and approximately 22 km east of the barrier reef of Belize (fig. 2.1). Glover's reef is a true coral atoll with a fringing reef surrounding a shallow lagoon (Stoddart 1962). The lagoon contains over seven hundred isolated patch reefs which range in size from 5 to approximately 100 m in longest diameter. Patch reefs are surrounded by a halo of sand and expanses of sea-grass beds (Thalassia spp.; Burke 1982). Most of the study sites were located in the lagoon on about 30 patch reefs, but a small number of sites were on sea grass beds. All study sites were located within two km of Long Caye, one of the three major islands at Glover's Reef, which served as base of operations. B. Identification of individuals A striking feature of terminal phase colouration is the pattern of yellow scales on both sides of the caudal peduncle. The number of yellow scales varies from none to as many as seven, and the pattern is usually different on each side of the fish. Furthermore, individual scales vary in the shape and degree of yellow colouration. These distinctive spot-patterns allowed indentification of individuals which could then be observed repeatedly. Fish with initial phase colouration do not have easily recognized spot patterns, making identification and observation of individuals more difficult. Therefore, we captured and sexed a number of Iph fish (see techniques below) and attached a tag made from coloured plastic to each fish. The tags were cut into shapes that were distinguishable from a distance of 5-10 m, even 8 89°00* 88 c 00* Figure 2.1 The location of Glover's Reef, Belize. Shaded area represents approximate location of the study area at Glovers reef. The inset shows the area represented by the larger map (box). 9 after substantial algal growth. To attach the tags, fish were lightly anaesthetized in tricaine-methane sulfonate (MS-222; ca. 0.1 g/1) or 2-phenoxyethanol (Syndel; ca. 0.2 ml/1) and a sterilized needle (16 gauge) was inserted through the dorsal musculature. A nylon clothing tag (Wydoski and Emory 1983) was attached to the identification tag and then passed through the needle. Following attachment of the tag, the fish were quickly returned to the point of capture and released into crevices in the coral as a precaution against predation. The tags usually remained in place over the study period (two to four months), but some worked their way through the soft muscle tissue and fell out. Only one tagged fish was found over more than one field season (see Chapter Four). C. Documenting Behaviour Detailed scale maps of the study reefs were produced using a compass and tape measure. In most cases, coral boulders, outcrops and other distinguishing features were used as landmarks, but where these were insufficient, coloured flagging tape was tied to the substrate. The maps were redrawn on acetate sheets and placed beneath a sheet of mylar or abraded acetate and attached to a plexiglass slate. This apparatus served as an underwater note pad. Distribution of fish, territoriality and use of space were recorded on the pads with a soft pencil, and these were later transcribed onto paper to obtain a permanent record. To document the use of space by Tph males and by females, individual fish were observed for fifteen-minute periods, and their movements were plotted continuously onto the maps. Five such traces (each separated by at least two days) were recorded for each fish under observation. To determine home-range size, replicate maps for a particular fish were superimposed. A continuous line (or "polygon of smallest area") was drawn which encompassed all 5 maps while covering the smallest area possible. Area was calculated with the aid of a digitizing tablet, using underlayed maps of the reefs for determining scale. Mapping observations were conducted at different times of the day, ranging from 0900 to 1800 hrs. Stoplight parrotfish are diurnal animals, becoming dormant at night (Winn and 10 Bardach 1957,1960). Therefore, observations outside the above time period were limited to a few incidental observations. Point-sighting observations were conducted on some occasions. These included identifying an individual and noting its position on a map during brief observations (< 5 minutes) repeated 5 times over 7-14 days. Although this technique was not as accurate as the above mapping technique for determining the location of territory or home range boundaries, it allowed observation of a large number of fish in a short period of time when determination of home-range or territory boundaries was not important (e.g. on small patch reefs with only one Tph male). D. Capture of fish Initially, fish were captured using a speargun. However, since this method resulted in the death of the animal, a netting technique was developed which allowed specific individuals to be captured, examined, bled, and in some cases treated with hormones and/or tagged before being released relatively unharmed. In most cases, the same method was used to capture fish that were to be killed for examination of their gonads. The net was a heavy-gauge (30-40 KG test strength monofilament) gill net with a mesh size (6.6 cm) that was too small to gill most individuals. The net was placed in a strategic location, secured to the substrate with lead line and coral rubble, and held upright by polystyrene floats attached to the top. The technique involved slowly herding a particular fish towards the net. Upon contacting the net, the fish would attempt to push its way through. At that point, the observers removed the parrotfish from the gill net using small hand-held nets, and transfered the fish to the waiting boat. E. Sexing fish The technique for sexing fish was as described by Ross (1982). Briefly, a polyethylene tube (i.d. 1 mm) was inserted into the genital opening and suction applied to the other end. Egg material was sucked into the tube and examined by eye for evidence of ovulation. The presence 11 of free-flowing eggs with transparent cytoplasm indicates that the fish is an ovulated female. Unovulated eggs, on the other hand, appear opaque and are connected to each other by ovarian tissue. As the genital pore in males is too small to allow the tube to pass through, failure to get the tube into the genital tract was considered evidence that the fish was an Iph male and the fish was bled and sacrificed to confirm its sexual status. Several Iph males were identified using this method, however a few fish were mistakenly classified as male, but later turned out to be female or immature. F. Collection and treatment of blood samples Immediately after capture, a blood sample was withdrawn into a 3 ml syringe from the caudal artery by a left lateral puncture using 18 or 20-gauge needles. Syringes were heparinized prior to use with 20 ml of a 1000 i.u./ml heparin solution (Allen and Hanbury's). One to three ml of blood were transferred into glass culture tubes, sealed with parawax film and placed in a thermos flask containing ice. Once back on land (10 min to 2 h later), the tubes were spun in a hand centrifuge (ca. 2000 rpm, 780 x g) to separate blood cells from plasma. Plasma was taken up in a glass pipette, transferred to a numbered polypropylene vial and frozen and stored over liquid nitrogen. For transport to Vancouver, samples were placed over dry-ice in a styrofoam container. At U.B.C., the samples were thawed in an ice-bath, mixed and centrifuged to remove clotted plasma, and 200 ml aliquots were re-frozen at -70°C in an ultra deep freeze until assay. Details of radioimmunoassay procedures are presented in Chapter Five. G. Histology Whole gonads were dissected from freshly-killed specimens, and preserved in Bouin's fluid. For transportation to Vancouver, gonads were stored in tissue paper soaked in Bouin's fluid and sealed in plastic bags. Histological analysis was carried out on blocks of tissue cut from the gonads. Cylindrical blocks were cut anterior to the junction of the two lobes. Blocks were dehydrated in increasing concentrations of ethanol, transferred to benzene, and embedded 12 in paraffin wax ('Parawax +', Fisher). Sections (7-12 urn) were then cut from the blocks and mounted on cleaned, gelatin-subbed microscope slides. The sections were subsequently re-hydrated and stained using Harris's haematoxylin and counterstained with eosin (Culling 1963). Finally, stained sections were dehyrated a second time and cover slips were added and sealed with Permount (Fisher). 13 CHAPTER THREE Sex Change and Colour Phase Change in the Stoplight Parrotfish A. Introduction In his pioneering studies on protogynous labrids, Reinboth (1962a,b, 1967, 1968, 1970) discovered that former ovarian function can be diagnosed from the internal structure of the testis. Testes from males that previously functioned as females (secondary males) have a membrane-lined central cavity (formerly the ovarian cavity) and numerous peripheral sperm ducts. In contrast, testes from primary males, which are male their entire lives, have a single, central sperm duct. Reinboth (1968) examined the structure of gonads from four male stoplight parrotfish. He argued that the four testes were secondary (previously functioned as ovaries) and that, therefore, Sparisoma viride is a protogynous hermaphrodite. Robertson and Warner (1978) examined a larger number of gonads and, like Reinboth (1968), concluded that all males in this species result from sex change by females (that is, 5. viride is monandric - see Reinboth 1970). However, in their study population at San Bias, Panama, Robertson and Warner (1978) reported a high degree of overlap in the size range of males and females. Also, the two fish they found with intersex gonads were both small (110 and 199 mm in standard length) compared with functional females or males. Finally, they found several males that were as small as or smaller than the smallest mature female (< 160 mm SL). These findings led Robertson and Warner (1978) to speculate that this species has secondarily evolved pre-maturational sex change (^ secondary gonochorism). Testes from secondarily gonochoristic teleosts still pass through a juvenile female stage, and therefore, as with testes from post-maturational protogynous species, possess a membrane-lined central cavity (Sadovy and Shapiro, 1987). The crucial distinction between truly protogynous and secondarily gonochoristic individuals is that gonads from the former proceed from immature ovary to mature, functional 14 ovary to mature, functional testis, while gonads from secondary gonochores do not pass through a functional ovarian stage, but change direcdy from an immature ovary into a mature, functional testis. The definitive criterion for ascribing protogyny to a species is histological evidence in the same gonad of both degenerating ovarian tissue and spermatogenic tissue containing spermatocytes, spermatids or spermatozoa (Sadovy and Shapiro 1987, Shapiro 1987). Although Robertson and Warner (1978) found two fish with 'transitional' gonads (i.e. intersex), neither Reinboth (1968) nor Robertson and Warner (1978) provide detailed histological descriptions of intersex S. viride. Thus, there is confusion over whether 5. viride is a truly protogynous species, or if individuals change sex before maturing as females. In this chapter I present evidence from histological analysis, as well as size-sex distribution data that S. viride employs a mixed strategy of true protogyny in some individuals, and pre-maturational sex change in others. Many reef fishes, including scarids, show extreme sexual dimorphism in colour patterns (Thresher, 1984). This has led investigators to misdiagnose males and females as separate species (Winn and Bardach, 1957,1960, found that Sparisoma abildgaairdi and S. viridis were females and males, respectively, of a single species, 5. viride). It is generally thought that sex and colour change are synchronous in most parrotfish species, although there are exceptions to this (Choat and Robertson, 1975, Robertson and Warner 1978). Sparisoma viride exhibits marked sexual dimorphism, and the male and female colour patterns have been described previously (Bohlke and Chaplin 1968, colour photographs in Boschung et al. 1983 and fig. 3.8), but transitional colouration has not. Therefore, I also present here a description of the secondary sex characteristics of female, male and intersex stoplight parrotfish, and present data suggesting that sex and colour change in this species are asynchronous. 15 B. Materials and Methods Procedures for identifying and catching fish, and subsequent histological preparation of their gonads are presented in the general methods section (Chapter Two, above). Haematoxylin and eosin stained sections of gonad material were observed under a light microscope. Gonads from all fish were examined to determine their sex, either by visual examination of gonadal material withdrawn by cannula, or by dissection. A total of 420 gonads, representing over 90% of all fish caught, were dissected and preserved; of these, 117 were examined in detail under light microscopy (60 ovaries, 28 testes, 17 intersex gonads and 12 gonads from hormone or control treated fish). To determine cell diameters, a sample of twenty cells of each type were measured with an optical micrometer first along the widest diameter, and again perpendicular to that axis. The two measurements were then averaged. Photomicrography was conducted using a blue filter for tungsten lighting. Photomicrographs were taken of representative sections of good quality. C. Results 1. GONADAL HISTOLOGY  La. Anatomy of the gonad In general, the stoplight gonad consists of a pair of muscular sacs located in the posterior portion of the peritoneal cavity. The sacs come together at the gonopore where gametes are broadcast into the environment. Connective tissue holds the gonad to the liver (ventrally) and the swim-bladder (dorsally). Branches of the gonadal arteries and veins run along the tunic, and through the gonadal tissues. The tunic of the ovary is a thick, muscular wall, and is involuted to form gonadal lamellae. In the testis, the tunic is thinner, and often breaks during histological preparation. Figure 3.1 Cross-sections of ovaries, (a) Nest of oogonia (oog). (b) Pre-vitellogenic oocyte in the chromatin nucleolus stage (arrow), (c) Pre-vitellogenic oocyte in the peri-nucleolus stage. N, nucleus; No, nucleolus; F, follicle cell. a,b, and c: 1496X. 17 1 .b. Histology of the ovary l.b l . Oogonia: The smallest germ cells found in ovaries (mean diameter +/ SE = 8.5 +/- 0.4 p.m see fig. 3.1a). These oval cells are characterized by large nuclei and a thin layer , of lightly-staining cytoplasm. I.b2- Previtellogenie oocytes: First growth phase oocytes (Nagahama 1983) are more spherical than oogonia, and are characterized by highly basophilic cytoplasm and large, round, lightly-staining nuclei. I identified two types (stages) based on the position and characteristics of nucleoli: (i) Chromatin nucleolus stage (fig. 3.1b): In these oocytes, the nucleus is a large, round body (nuclear diameter = 14.5 +/-1.2 p:m) with wispy, string-like chromatin material visible (chromosomes in prophase of meiosis, Khoo 1975). Few nucleoli are discernible, but if they are, (as in the later stages), they are large, irregular and located centrally. These oocytes are still relatively small, but have undergone a 3-fold increase in diameter over the previous stage (mean diameter = 26.1 +/- 2.2 nm). (ii) Peri-nucleolus stage (fig. 3.1c): Numerous nucleoli appear as a ring around the periphery of the nucleus, which has enlarged to 29.1 +/- 0.9 pm. The nucleoli are much more regular in shape, although somewhat smaller. Early on (mean cell diameter = 54.1 +/- 1.6 |im) the cytoplasm is still uniform and highly basophilic. Towards the end of the stage, however, (cell diameter = 73.5 +/-1.9 (im), the cytoplasm becomes less basophilic (stains lighter), especially near the nucleus (nuclear diameter is now 42.0 +/- 0.7um). I.b3. Yolk Vesicle stage: (i) Early stage (fig. 3.2a): A ring of yolk vesicles appears around the periphery of the cytoplasm. This marks the onset of yolk incorporation into the oocyte (second growth phase, Nagahama 1983). Mean diameter is 112.9 +/- 4.1 pm. As yolk incorporation progresses, yolk vesicles increase in number and size to 18 Figure 3.2 Cross-sections of ovaries, (a) Early yolk vessicle (yv) stage oocyte. 598X. (b) Late yolk vessicle stage (arrow). 239X. (c) Early yolk platelet (yp) stage. 598X. (d) Late yolk platelet stage with migrating nucleus (mig). N, nucleus. 239X. 19 become distributed throughout the outer half of the cytoplasm. Nucleoli are still evident in the periphery of the nucleus (diameter = 56.6 +/- 3.4 \im), but are less well defined. (ii) Late stage (fig. 3.2b): Oocytes are larger, (diameter = 201.2 +/- 3.8 um), and small yolk platelets have formed in the cytoplasm. Cytoplasm is now less basophilic. The nucleus takes up the eosin stain, and has enlarged to 74.1 +/- 2.8 um. I.b4. Yolk Platelet stage (fig. 3.2c): The yolk platelets (= yolk granules, Khoo, 1974) coalesce and fill the inner region of the cytoplasm, displacing the yolk vesicles to the periphery. Diameter is now 282.4 +/- 8.6 um. As the oocytes approach the late stage of maturation, the nucleus (diameter 92.4 +/- 3.2 um) migrates toward the periphery of the cytoplasm (fig. 3.2d). I.b5. Mature oocyte stage (fig. 3.3a): Most of the egg is filled with yolk, which has coalesced into large globules. Mature oocytes are at the end of the yolk-deposition stage, and growth slows down (oocyte diameter = 356.3 +/- 5.9 pm). As the yolk coalesces further, the eggs become transluscent. I.b6- Ovulated oocytes (fig. 3.3b): Due to hardening of the yolk during histological preparation, mature and ovulated oocyte sections are usually of poor quality. Under the microscope, the outlines of ovulated oocytes are irregular, and the only cellular components visible, if any are the vitelline membranes. In freshly-collected specimens which had ovulated eggs in the oviduct, the eggs were completely transparent, becoming nearly invisible in water. I.b7. Atresia : Atresia seems to be a normal part of female function, as atretic bodies are found in many apparently normal ovaries (fig. 3.3c). Atretic bodies are essentially identical to those described by Chan et al. (1967) in the freshwater protogynous Monopterus albus, and by Khoo (1975) in the goldfish. I also found empty follicles in fish that had recently ovulated or which had many mature oocytes (fig. 3.3d). 20 Figure 3.3 Cross-sections of ovaries, (a) Mature oocytes (mo) undergoing yolk coalescence, (b) Ovulated oocytes (ov) in the ovarian lumen (1). (c) Atretic oocytes (ao). (d) Post-ovulatory follicles (pof). a,b,c and d: 120X. 21 I.e. Histology of the testis Testes retain the formerly-ovarian cavity (fig. 3.4a), and, like ovaries, contain lamellar folds. However, the central cavity is not used for sperm transport; that function is taken up by numerous vasa deferentia at the gonad's periphery (Shapiro and Sadovy 1987). Spermatogenesis takes place in nests (see fig. 3.5b and 3.5c). Within each nest, nearly all cells are at the same stage of development. Long, narrow cells which are similar to Leydig cells described in other teleosts {e.g. Chan et al., 1967) are found in the spaces between nests (fig. 3.4c). In testes, the largest cells are primordial germ cells, (PGC's; Henderson 1962). PGC's (diameter = 4.9 +/- 0.1 um) have large, lightly staining nuclei, and very little cytoplasm. Spermatogonia are the same average size as PGC's (4.8 +/- 0.2 um; fig. 3.4b), but have dense chromatin, and stain slighdy darker. Spermatogonia are found in nests on the gonadal lamellae (fig. 3.5c). Following the descriptions of Henderson (1962), Ahsan (1966), Moe (1969), and Warner (1974), I distinguished 4 stages of post-spermatogonial germ cell development: l.ci. Primary spermatocytes (fig. 3.5a): These cells are small (diameter =4.4 +/- 0.1 um), and stain more deeply than gonial cells. They often appear umbrella-shaped, as chromatin material migrates to the end of the cell during meiosis. 1.C2- Secondary spermatocytes (fig. 3.5b): Smaller (diameter =3.0 +/- 0.1 um) and more numerous than in the previous stage. Secondary spermatocytes are also more uniform in colour than primary spermatocytes, staining a deep shade of purple with haematoxylin. I.C3. Spermatids (fig. 3.5b and 3.5c): These cells are dense, (staining almost black), round, and small (2.0 +/- 0.1 um). This is the penultimate stage of spermatogenesis. The cells do not undergo further division, but change shape and become tailed. I.C4. Spermatozoa (fig. 3.5d): The cells are oval to cardioid in shape. Their tails tend to agglutinate within the nests, giving the nests a 'parachute-shaped' appearance (Hendersen 1962, Pollard 1972, Warner 1975b). 22 a Figure 3.4 Cross-sections of testes, (a) Functional secondary testis showing nests of spermatogenic tissue, the former ovarian cavity (oc) and peripheral sperm sinuses (sin). 120X. (b) Primary germ cell (pgc), secondary spermatocytes (sc2) and spermatids (st). 1496X. (c) interstitial cells (i). bv, blood vessel. 1496X. 23 Figure 3.5 Cross-sections of testes, (a) Primary spermatocytes (sci). 1496X. (b) crypts of spermatogenic tissue; sci, primary spermatocytes; sc2, secondary spermatocytes; st, spermatids. 239X. (c) Higher magnification of fig. 3.5b. sg, spermatogonia; other abbreviations as in fig. 3.5b. 596X. (d) Tailed spermatozoa. 1496X. 24 1 .d. Description of intersex gonads Seventeen gonads were diagnosed as undergoing sexual transition (intersex). Early stage intersex gonads (fig. 3.6a) contain a few crypts of spermatocytes and spermatids, and occasionally crypts of tailed spermatozoa (fig. 3.6a). Ovarian tissue in such gonads is abundant, but undergoing atresia. In two cases, resorbing ovulated oocytes had formed a dense, yolky mass within the ovarian cavity. In contrast, late stage intersex gonads contain little ovarian tissue, except for dispersed pre-vitellogenic oocytes (fig. 3.6b). These oocytes contain little or no visible nuclear material (see fig. 3.6b), indicating atresia (Stacey 1977). Yellow-brown bodies are also present in late stage intersex gonads, possibly indicating degenerated vitellogenic oocytes (Khoo 1975,1979, Nagahama 1983, but see caution in Sadovy and Shapiro 1987, who point out that yellow-brown bodies are found in several non-gonadal organs, and can result from non-specific degeneration of a variety of tissues). Spermatogenic tissue in these gonads is abundant and densely packed. Spermatocytes, spermatids and tailed spermatozoa are usually present. As the sperm sinuses did not contain spermatozoa, final maturation and spermiation (Fostier et al. 1983) of the testis was probably incomplete. Ovarian and testicular tissues are not evenly distributed within intersexual gonads. The first signs of spermatogenesis are found at the periphery of the gonadal lamellae. Conversely, atretic oocytes are more common near the ovarian cavity. Also, ovarian and testicular tissues of S. viride are not separated by a membrane as in some protogynous species (Chan and Yeung 1983, Sadovy and Shapiro 1987). Rather, intersexual gonads contain crypts of spermatocytes and spermatids intermingled with degenerating oocytes. However, in one case, one lobe was much further advanced than the other, the former containing no ovarian tissue, and the latter containing little spermatogenic tissue (fig. 3.6c). 2. COLOURATION Females display 'initial phase' (Iph) colouration, characterized by a greyish-olive body and a bright red ventral region. A number of scales along the flanks are white, producing a Figure 3.6 Cross-sections of gonads undergoing sex change, (a) Early stage of pre-maturational sex change showing nests of tailed spermatozoa (sz) previtellogenic oocytes (pvo) and a crypt of spermatocytes (sci). 598X. (b) Later stage intersex gonad showing numerous spermatogenic crypts at various stages of development and degenerating pre-vitellogenic oocytes (pvo). 598X. (c) intersex gonad; one lobe (arrow) shows advanced spermatogenesis compared with the other (sc, spermatocytes; st, spermatids). 239X. 26 spotted pattern (fig. 3.7a). Most males have 'terminal phase' (Tph) colouration, characterized by forest green bodies with blue markings around the mouth and eyes, orange along the posterior margin of the operculae, and light blue on the edges of the fins (fig. 3.7b). The scales have burgundy outlines. Furthermore, Tph males have a distinctive yellow spot on the upper edges of the opercula and larger yellow spots on either side of the caudal peduncle. Finally, the caudal fin of Tph males is deeply lunate, while that of Iph fish is much less so. The change from initial phase to terminal phase colouration first becomes evident on the head, with the facial region taking on a grey-green colour. Also, a faint orange band develops behind the mouth and faint blue patches can be seen around the mouth and eyes. As colour change progresses, the white spots along the flanks become less intense, and the reddish-brown colour of the upper body, as well as the bright red ventral region fade to an olive-green (fig. 3.7c). At the same time, facial markings intensify. The last markings to appear are the distinctive yellow spots on the opercula and caudal peduncle and the bright orange slash on the rear edge of each operculum. The caudal fin becomes more lunate, and develops green, burgundy, and blue markings, as do the dorsal and anal fins. The final stage of colour transition is an essentially terminal phase fish with faint white spots along the sides. At this stage, the spots appear more intense during apparently stressful periods (agonistic encounters, etc.) but fade to blend almost completely with the general body colour when the fish feeds. Fish with fully-developed terminal phase colouration are apparendy unable to revert to the Iph spot pattern (Robertson and Warner 1978, and personal observation). Spots were never seen in larger Tph males, even after violent aggressive attacks or encounters with predators. 3. SIZE. SEX AND COLOUR DISTRIBUTION All females bore Iph colouration, and all Tph fish were males (Table 3.1). Ten intersex fish were indistinguishable in colour pattern from Iph females. Of these, nine were in the early stages and one was in the late stage of gonadal sex change. Seven intersex fish were undergoing Figure 3.7 Photographs showing the colour phases of stoplight parrotfish: (a) Initial phase female, (b) transitional colour phase male (c) later transitional colour phase male (d) Terminal colour phase male. 28 colour transition. There was a statistically significant tendency for early-stage intersex fish to have Iph colouration, and for late-stage intersex fish to have transitional colouration (Fisher exact test, two-tailed, P < 0.001). No females were found with either Tph or Transitional colouration. Nor did any fish with intersex gonads have Tph colouration. On the other hand, several fish with fully-developed testes had either Iph or transitional colouration. These results indicate that sex and colour change are usually, but not always synchronous events. When they are asynchronous, sex change precedes colour change. Sex Female Intersex Male Colour (early) (late) Tph 0 0 0 223 Tr 0 0 7 12 Iph 182 9 1 6 Table 3.1 The frequency of Sex and Colour types. Values represent number of each colour/sexual type in over 400 fish examined at Glover's Reef 1984-1987. Tph - terminal colour phase; Tr - transitional colour phase; Iph - initial colour phase. Mature females ranged in size from 170 to 350 mm (standard length, SL; fig. 3.8). Tph males ranged from 160 to 390 mm SL, but most were larger than 280 mm. Thus, the sexes are bimodally distributed with size; males are generally larger than females. Nevertheless, examination of figure 3.8 reveals that some males occur at the lower end of the size distribution. In fact, as Robertson and Warner (1978) found in the population at San Bias, Panama, some males are smaller than the smallest mature female (<170 mm SL - present study; <160 mm -Robertson and Warner 1978). Iph males and males with transitional colouration were generally limited to small size classes. The smallest male collected (121 mm SL) had transitional colouration and a small testis (<0.1 g). However, several small males (smallest 160 mm SL) had fully developed Tph colouration. 29 Number of Fish 50 - i a 45 - Intersex Fish — * — Tph Males 40 - Females 35 -30 -25 -20 -15 -10 - / , 5 -0 - T T T T*M * I' T I T I 1 1 1 — I' I 'I I i M T f^ T^ f l i t 11 16 Number of Fish 4 21 26 31 Standard length (cm) 36 41 Tr. Col. Phase Males Initial Phase Males b 0 I i f i 1 I 11 26 31 Standard length (cm) I T i T " i I "i " i i"~ i I i "" i i r 36 41 Figure 3.8 Size-frequency distribution (10 mm size classes) of stoplights captured at Glover's Reef: (a) Tph males (n=223), females (n=151) and intersex fish (n=17); (b) Iph males (n=6) and transitional colour phase males (n=12). 30 Intersex fish and males with Tr colour phase cover a broad size range, although Tr males tend to be restricted to lower size classes (fig. 3.8b). The largest intersex fish found in the present study was similar in size to the largest females. On the other hand, Iph males were consistently small. D. Discussion The general anatomy of gonads from Sparisoma viride is similar to the anatomy of gonads from other protogynous species (Reinboth 1962, Roede 1972, Warner 1975b). All testes examined had a structure which suggested previously ovarian organization, each having a central cavity and an infolded tunic which formed lamellae. This confirms the findings of Reinboth (1968), and Robertson and Warner (1978) that Sparisoma viride is monandric. Sperm are not formed in lobules, as in gonochoristic teleosts (Nagahama 1983) but in nests on the gonadal lamellae. Sperm transport is carried out by numerous vasa deferentia around the gonadal periphery. The cellular morphology of ovaries and testes from S. viride is similar in detail to that described in protogynous labroids (e.g. Pimelometopon pulchrum, Warner 1975b; Coris julis, Reinboth 1962; Halichoeres spp., Roede 1972) as well as gonochoristic species (e.g. Galaxias maculatus, Pollard 1972; Carassius auratus, Khoo 1975; Tandanus tandanus, Davis 1977; Couesius plumbus, Ahsan 1966; Salvelinus fontinalis, Hendersen 1962; Salmo gairdneri, Hurk and Peute 1979). Oogenesis follows the group-synchronous pattern (DeVlaming 1983) in which clutches of oocytes at several stages of development occur simultaneously. Both ovaries and testes of Sparisoma viride contain undifferentiated germ cells corresponding to those that, owing to the fact that they are indistinguishable by light microscopy, as well as to a presumed bipotentiality of gonia in protogynous teleosts, Reinboth (1980) labelled deuterogonia. As the size ranges of oogonia and spermatogonia overlap, and since their staining characteristics are similar, they cannot be relied upon for diagnosing the sex of the specimen. The developmental origin of deuterogonia is unknown (see discussion in Chan and Yeung 1983 and Nagahama 1983). 31 The configuration of tissues within the gonad conforms to the undelimited, Epinephelus type described by Sadovy and Shapiro (1987), in which tissues of the secondary sex develop among the degenerating tissues of the primary sex. In some species, such as Coris julis, the origin of the testis can be traced to particular cells on the ovarian lamellae (Reinboth 1970, Chan 1970). However, in Sparisoma viride, male tissues are not identifiable until the formation of spermatocytes from gonial cells. The present histological evidence of functional females undergoing sex change leaves little doubt that some individuals undergo true protogynous sex change. The size distribution of male and female Sparisoma viride both at Glover's reef (present study) and in Panama (Robertson and Warner 1978) corroborates the histological data suggesting that the species is protogynous. However, it is important to note that in both populations, some males are smaller than the smallest mature female. Also, one intersex fish (fish # 27) did not show evidence of previous female function (all oocytes previtellogenic, no "brown bodies" or corpora atretica from vitellogenic oocytes; see fig. 3.6a). Although the evidence is circumstantial, the small size of some males, together with the finding of apparently immature intersex fish strongly suggests that some individuals change sex while immature, rather than as mature females. Thus, Sparisoma viride appears to employ a mixed strategy, in which some individuals change sex early in life, while others change sex after functioning as females. Pre-maturational sex change is not uncommon in scarid species. Choat and Robertson (1975), Robertson and Warner (1978), and Robertson et al. (1982) found secondary males which were smaller than the smallest mature female in nine species from the South Pacific, the Western Atlantic and the Indian Ocean, respectively. Robertson and Warner (1978) and Robertson et al. (1982) speculate that secondarily gonochoristic males in sparisomatinine scarids are functionally equivalent to primary males (Reinboth, 1970) in many scarinine and labrid species. I will address this issue further in Chapter Seven. 32 The size distributions of intersex fish, Iph and transitionally coloured males in Belize are consistent with Robertson and Warner's (1968) findings concerning the size range of S. viride in Panama. The latter authors report 2 intersex fish (both with full Tph colouration) which were 110 and 190 mm SL, respectively, 23 Iph males ranging in size from 130 to 210 mmSL and 6 Tr males which ranged in size from 130 to 200 mm SL. Randall and Randall (1963) report the capture of three transitionally coloured fish (sex was not determined) at the U.S. Virgin Islands. These transitional fish ranged in length from 176 to 180 mm SL. The tendency for early-stage intersex fish to have Iph colouration, and for late-stage intersex fish to have transitional colouration suggests that colour change begins as sex change nears the later stages. The majority of fish probably change Colour soon after commencing sex change. However, it is not clear whether this relationship is causal. Some fish maintain Iph colouration after sex change, becoming female-mimics (Iph males), and appear to function as such for some time. These findings indicate that sex and colour change are not simultaneous events, and may be controlled by separate mechanisms. Buckman and Ogden (1973) drew a similar conclusion for the striped parrotfish, Scams iserti (=croicencis). Iph males eventually change into Tph males, as is suggested by the presence of fully functional males with transitional colouration. This is further supported by the small size of Iph males. Restriction of Iph males to small size classes appears to be a general trend among sparisomatinine parrotfishes (Robertson and Warner 1978). The reason for this is unknown, but may be related to peculiarities of sparisomatinine social and mating systems (Robertson and Warner 1978). Robertson and Warner (1978) interpreted the large size of some females as evidence for non sex-changing females (primary females). However, the large size of some sex changers (up to 302 mm SL), and the fact that a number of males are larger than the largest females suggests that all females may eventually change sex, assuming they live long enough. 33 Large Terminal Phase Male Functional Initial Phase Small (Iph) Female Male Tph Male Immature Female Larva Figure 3.9 Summary of the proposed life-history pathways of stoplight parrotfish. 34 Only a small proportion (1-5%) of Iph fish are males. In fact, considerable effort was spent actively searching for Iph males, so the estimate that they constitute one to five percent of the population is probably too high. However, it is possible that Iph males are more abundant elsewhere. In Panama, Robertson and Warner (1978) found that 10% of Iph fish (23/228) were males. As larvae of parrotfish (and other reef fishes, see Thresher 1984) are pelagic, they are probably distributed over large areas of the Caribbean and tropical Atlantic (Johannes 1978). Local demography may thus be a reflection of stochastic processes operating on larval settlement. Furthermore, the study area at Glovers reef is unlikely to represent the entire range of habitat types in which stoplight parrotfish are found. If becoming an Iph male depends on variable environmental parameters (as seems to be the case in other labroid species; Shapiro 1981) perhaps conditions at Glover's reef do not favour the production of high proportions of Iph males in the population (see general discussion, chapter seven). In summary, the stoplight life-history appears to involve several ontogenetic pathways (summarized in figure 3.9). Histological analysis confirms that all male stoplight parrotfish are sex changed hermaphroditic females. Although some fish change sex as functional females, at least some individuals (secondary gonochores) change sex as immature fish. Following sex change, some males take on Tph colouration. Others retain Iph colouration while still relatively small, but eventually change their colour pattern, becoming Tph males. There is insufficient evidence to determine whether all Iph males result from pre-maturational sex change. However, a few Iph males were smaller than the smallest mature female, suggesting that they changed sex before maturing as females. Finally, while it is possibile that some females never change sex (primary females, sensu Robertson and Warner 1978), this is difficult to establish, and ascribing such a strategy to S. viride would be premature. 35 CHAPTER FOUR The Social and Mating System of Stoplight Parrotfish A. Introduction Coral reef fishes exhibit a diverse array of mating systems, ranging from monogamous pair spawning to polygamous associations involving group-spawning by hundreds of individuals in mixed sex groups (review by Thresher 1984). Recendy, a number of studies have documented the social and mating systems of tropical labroid species. Investigators have attempted to relate the labroid proclivity for evolving polygynous mating systems to factors believed to influence the ability of large males to monopolize females, such as the utilization and distribution of resources important to females (food, hiding spaces, spawning sites; Robertson and Hoffman 1977, Robertson 1981, Tribble 1982) or of females themselves (Thresher 1979, Victor 1987; see also Baird 1989 for general discussion). However, while many detailed investigations have been conducted on wrasses (Labridae; reviews in Robertson 1981, Thresher 1984, Warner 1984a), the social and mating systems of parrotfish have received only cursory or superficial attention (Randall and Randall 1963, Buckman and Ogden 1973, Ogden and Buckman 1973, Barlow 1974, Choat and Robertson 1975, Warner and Downs 1977, Robertson and Warner 1978, Dubin 1981, Clavijo 1982). Based on limited observational evidence, Robertson and Warner (1978) suggest that male stoplight parrotfish do not form permanent territories, but that temporary territorial behaviour may occur during spawning. However, no data are presented which support this claim, nor were data collected on the timing, duration or location of territorial behaviour. The latter authors also assert that this species is one of the few scarids which does not form haremic mating groups. The only other information available on the social and mating system of the stoplight parrotfish is the observation by Randall and Randall (1963) of pair-spawning involving a Tph male and an Iph female. In the present chapter I describe the social and mating system of stoplight parrotfish at Glover's Reef, Belize. In particular, I present evidence that large Tph males defend permanent, 36 all-purpose territories on which they pair-spawn daily with a group of females. Females spawn nearly every day with the same male. Smaller Tph males neither spawn nor defend territories, but feed in groups during the day and occupy non-defended home ranges. On the other hand, Iph males spawn occasionally by streaking or by pair-spawning with females following removal of Tph males. Finally, since changes in local social conditions (removal of the dominant male) have been shown to instigate sex change in at least 17 hermaphroditic teleosts from a variety of families (reviews by Shapiro 1987, Warner 1984a), I examined the possible social control of sex change in S. viride. I present evidence that females do not change sex following removal of territorial males from local groups. B. Materials and Methods I documented the use of space by Tph males on medium-sized patch reefs with two or three males each, on large patch reefs with 6 to >25 males each, and at a non-reef location inhabited by over 40 males. Observations were conducted primarily for 15-minute periods between 09:00 and 17:00 h, but were occasionally earlier and for longer duration, e.g. 06:00 -08:15 h on June 29 and 06:20 - 09:20 h on June 30 when the time of spawning was expected to return to early morning. Point sightings, in which the positions of identified individuals were noted on maps, were conducted on 17 males on 3 large reefs in 1986. In addition, point sightings were conducted in 1984 on 6 males on reefs inhabited by one Tph male and a number of females and juveniles ('singleton reefs'). Detailed 15-minute observations of behaviours were conducted on 18 males on singleton reefs in 1986 as part of a separate experiment (see chapter 5). Because they could not be individually identified by recognizable markings or colour patterns, observations of individual females were limited to tagged individuals on a large reef. This included point sightings of two tagged females in 1984, and 32 mapping observations of 12 females tagged in 1985. 37 To calculate the distance males travel over a fifteen minute observation period, ten territorial and ten non-territorial Tph males were chosen at random, and traces from them were measured using a digitizing tablet. Two replicate maps were used for each male, and the resulting measurements were averaged. The degree of space-use overlap between neighbouring males was calculated by tracing on a single map the polygons of least area for all males on a single reef. Using a digitizing tablet, I then calculated the amount of reef which was shared with one or more males, expressed as a percentage of total area covered by each male. For comparison of behaviours among types of males, I averaged the behaviour scores from each replicate observation of every male observed in 1985. The resulting averages were compared by Mann-Whitney U test (Siegel 1956). Spawning occurred during many of the observation periods, and notes were made of behaviours observed during such periods. To obtain more detailed information on the duration of the spawning period, as well as the frequency and timing of spawning, groups containing tagged females were observed for the entire spawning period. Although six groups contained tagged females, observations were predominantly of two harems which contained the majority of tagged females. During these periods, observers focussed on males, and noted the time and frequency of particular behaviour patterns, and the identity of spawning partners, if known. Spawning observations were continued from the first indication of imminent spawning until 60 minutes after the first successful spawn or until 20 minutes had elapsed since the most recent spawn. Gonadosomatic indices (GSIs) were calculated according to the formula: GSI = 100 * (gonad weight / body weight). Since body weights were not available for all fish, body weights were estimated from the relationship of body weight (BW) to standard length (SL) calculated from a sample of 98 fish: BW = 0.1844 * (SL) + 149.3; r2 = 0.894. GSIs of different groups were compared by t-test following an arc-sin transformation (after Freeman and Tukey 1950 in Zar 1984). 38 C. Results 1. DESCRIPTION OF BEHAVIOURS  La. General maintenance behaviours Lai. Clean: the fish approaches a cleaning station and solicits removal of ectoparasites by cleaner fish or invertebrates (see Randall 1958). Solicitation involves assuming a head-up posture and remaining motionless, usually with the mouth and operculae open. I.a2- Feeding bite: specialized teeth are used to scrape algae from coral and other hard substrate material. I.a3. Feeding bout: several bites directed at the same area of substrate. Different bouts were arbitrarily defined as bites occurring one body length or more apart, or bites separated by more than 3 seconds. I.a4. Swim: using the pectoral fins for propulsion, the individual slowly moves about it's home range, stopping every meter or so to feed, and less frequently to clean or interact with other stoplights. Individuals swim between feeding sites (exposed coral surfaces with algae growing on them), cleaning sites and holes or crevices in the coral, and, except during spawning (see spawning behaviours, below), almost never rise higher than 1 m (3 body lengths) above the substrate. I.a5. Defecate: the fish releases a visible cloud of feces from the anus. During defecation, fish tilt their caudal peduncle and fin upwards. Lb. Social behaviours l.bi. Chase: swimming at high speed towards another individual that is moving away. Males use their pectoral fins for propulsion during mild chases, but during more vigorous chases, the caudal fin is also used, and individuals tilt their bodies to one side at a 45 to 60 degree angle. I distinguished between chases directed at males and those directed at females. I.b2- Bite: physical contact by the mouth or teeth of a fish with the body of another fish. Bites often cause visible wounds on the head or fins of combatants. 39 l.b3- Display: the body is presented laterally towards another individual. Usually performed by Tph males, but occasionally also by females. Colours are usually intensified, and the median fins held erect. This behaviour was occasionally performed while the fish was swimming parallel to another individual, using only the pectoral fins for propulsion. I.b4. Tail Stand: (fig. 4.1a) a Tph fish adopts a posture identical to that adopted during Cleaning (head up). However, Tail Stand usually occurs in the presence of a second Tail-Standing neighbour. Also, this posture was often accompanied by an intensification of colours. I.b5. Patrol: a male swims rapidly along the borders of his territory. The behaviour usually involves brief stops to Feed, Tail-stand, or Clean, and/or to interact with females or neighbouring males. The behaviour is difficult to distinguish from normal swimming movements. To quantify Patrolling, I measured the distance males travel over a fifteen-minute observation period. I.b6- Head-bob: the fish swims parallel to the bottom but in an undulating pattern in the vertical plane. This behaviour was rare, and was only performed by territorial males. I.e. Spawning behaviours l.ci. Solicit: (fig. 4.1b) The female circles slowly at the spawning site, high in the water column (2 to 3 m above the substrate). This behaviour is particularly conspicuous, in that outside the spawning period, females almost never swim more than 1 m above the substrate. Soliciting females tilt their caudal peduncle and caudal fin upwards in a posture resembling that occurring during defecation. 1.C2- Rapid Swim: Using the pectoral fins for propulsion, the male swims around the perimeter of his territory at a more rapid pace, and higher in the water column than during Patrol (1 to 2 m from substrate). Males rarely stop to feed during Rapid Swim, but often Chase females and neighbouring males. 1.C3. Flutter: The male swims steeply upwards near a group of females, using the caudal fin for propulsion. The male often defecated prior to performing Flutter. Figure 4.1 Stoplight behaviour patterns, (a) Tail-stand displays exhibited by males on two adjacent territories, (b) Soliciting female (high in water column). A second female is shown feeding close to the substrate, (c) Male (upper fish) Overswirmuhg a female prior to a Spawning rush, (d) Spawning rush. 41 1.C4- Overswim: (fig. 4.1c). The male approaches a Soliciting female, and orients his body parallel to, but above and slightly ahead of the female. Early Overswim is performed several body-depths above a female, but later the male's abdomen touches the females dorsal area. In this way, the male appears to direct the female in a slowly ascending spiral. I.C5. Head-Flick: A courtship behaviour performed by males while Overswimming females (only while not in contact with the female). The head is jerked to one side (towards the interior of the spiral) several times in succession. 1.C6- Spawning Rush: (fig. 4. Id). Following Overswim, the female swims rapidly upwards, followed by the male. At the culmination of the Spawning Rush, a cloud of gametes is released and the pair swim rapidly towards the substrate. Overswim does not always result in Spawning Rush; often the pair separate during overswim and the preceding sequence is repeated. Spawning Rush is extremely rapid, taking from 1-3 seconds. 1. C7. Streak: A solitary Iph fish rushes up into the water column and ejaculates into the gamete cloud left behind by a spawning pair. This spawning tactic is performed by Iph males (Warner and Robertson 1978). 2. SOCIAL BEHAVIOUR AND THE USE OF SPACE  2. a. Tph males At Glover's reef, many large Tph males defend territories on which they feed, spawn, hide from predators and sleep (i.e. all-purpose territories, Hoffman 1983). However, not all Tph males hold territories. This results in two distinct social types of Tph males; territorial Tph males (TTph males) and bachelor Tph males (BTph males). Territorial males are distributed fairly evenly over patch reefs, and their territories (area: 245.5 +/- 64.1 m2, mean and SE, n=10) are contiguous. The all-purpose territories are defended at all times of day, including both spawning and non-spawning periods. Most territories 42 encompass the home-ranges of 2 to approximately 16 females, although five males on large reefs actively defended territories on which no females were ever observed. This suggests that males do not simply defend females, but defend resources that may be important to females (e.g. food, spawning sites, hiding spaces, etc. - see Robertson and Hoffman 1977, Thresher 1984). This was further suggested by observations of two males on small isolated reefs from which all females had been removed as part of an experiment on the possible social control of sex change. Both males were observed over the following two weeks (10 point-sighting observations), during which only on one occasion was one of the males not found on his home reef. The absent male had returned by the following observation. Use of space by territorial males was documented on patch reefs of various sizes which had from one to 15 territories each (e.g. fig. 4.2a). Overlap among TTph males on a reef inhabited by 10 territorial males (percent of least-area polygon shared with one or more males) was 5.7 +/-1.9 percent (n=10). This low level of overlap indicates exclusive use of space by TTph males. On large reefs, territory boundaries are determined by the presence of other territorial males. This was shown by male-removal experiments on reefs with more than one male. On three reefs documented in 1984, each of two males occupied approximately half the reef. In all three cases, when one male was either wounded or removed, the remaining male subsequently expanded his territory to encompass the entire reef. In 1986, 6 two-male reefs were documented on which one dominant male occupied most of the reef, and a smaller subordinate male occupied a small outlying area. Upon removal of one male, the remaining male expanded his territory to include the entire reef, regardless of previous status. Similarly, 6 territorial males were removed from a large reef inhabited by 15 territorial males. Of these, the territories of 13 males were mapped and are outlined in figure 4.3a. The movements of all remaining males were mapped over the following week (fig. 4.3b). 43 Figure^ Least-area polygons (outlines) of TTph male territories (A) and bachelor male home-ranges (B documented at m m reef in 1985. Shaded area in (A) represents the location of the bachelor male home-ranges depicted in (B). 44 Figure 4,3 (A) Least-area polygons (outlines) of 13 territories documented at w.b. reef in 1984. TTph males are identified by numbers. (B) Least-area polygons (shaded areas) of the same territorial males one week after removal of the six males whose identification numbers are circled in (A). Dotted lines enclose pre-removal territory locations. Fish 17 and 11 were previously identified on a different part of the same reef. The fish labelled "new" was not previously identified. 45 Following removals, territorial males 1,11 and 17 abandoned their old territories to take possession of vacancies. Male 15 abandoned most of his old territory to take over a neighbouring territory. A foreign male ("new") acquired a further vacated territory and males 6, 8, 14 and 16 adjusted or expanded their territory boundaries to include most of the remaining vacancies. One territory that had been experimentally vacated and one abandoned territory remained unoccupied seven days after the original removals. These experiments demonstrate that territory boundaries are partially determined by interactions among neighbouring males. Bachelor males are found in loose groups of 10 to 40 individuals. Use of space by bachelor males was documented at a reef location with TTph males nearby, and at a non-reef, 'channel' location with no nearby TTph males. Bachelor male home-ranges average 148.1 +/-18.1 m2 in area (n=l 1), and are not defended. For example, fig. 4.2b shows least-area polygons for BTph males on M.M. reef during the same period as the TTph males in figure 4.2a. Overlap among individual home ranges of bachelor males (61.2 +/- 7.9 percent, n=l 1) is significantly higher than the home range overlap among territorial males (t-test, P<0.001; data were arc-sin transformed, Zar 1984). However, at all locations there were more bachelors than it was possible to observe, and therefore overlap among bachelor males is likely to be higher than that calculated. One large group of bachelor males was present in the same location three years in a row. Although I did not determine whether the same individuals comprised the group over that period, one individual with particularly striking opercular markings was observed in the group from April to August 1985 and was still there the following season (July 1986). Territorial males actively defend their territories by Displaying to and Chasing neighbouring males. Although bachelor males occasionally Chase or Display to other males, the frequency of these behaviours in bachelors is significantly lower than in territorial males (fig. 4.4; Mann-Whitney U test, Chase: p<0.001; Display: p<0.005). In contrast, bachelor males perform Feeding Bites more frequently than territorial males (fig. 4.4; P<0.001). 46 Displays 0.4 0.2 100 50 Chases Feeding Bites 1 • -21 23 Bachelor Territorial Tph males Tph males Figure 4.4 Comparison of the frequencies (mean + SEM, sample sizes within bars) of Displays, Chases and Feeding Bites exhibited by territorial and bachelor Tph males. 47 Territorial males Patrol the perimeters of their territories, while bachelor males do not exhibit obvious Patrolling behaviour, and move around their home-ranges at a significantly slower average rate than territorial males (fig. 4.5; values calculated during non-spawning periods; t-test, p<0.001). Bachelor males from reef groups differed from those at the channel location primarily in that the latter males migrated at dusk to deeper water over the fore-reef, while the former males did not. During migration, large numbers of fish moved in single file along paths which were consistent day after day. As males moved into deeper water, they joined other groups to form aggregations, some of which included Iph fish. Unfortunately, owing to the depths at which these aggregations occurred, and to the time of day, I was unable to capture any of the Iph fish to determine their sex. However, during daylight hours, the only Iph fish found on the fore-reef were females. Crepuscular migratory behaviour has been described in other parrotfish species (Buckman and Ogden 1973), but its function is unknown. Figure 4.6 compares the size distribution of all territorial Tph males with all bachelor Tph males captured at Glover's reef. Males with territories are clearly larger (>280 mm SL, mean = 317 mm SL), than those without territories (mean = 277 mm SL; Mann-Whitney U test, p<0.001). Groups of particularly small BTph males were often observed on shallow patch reefs that contained no females or TTph males; eight BTph sampled from one such reef ranged in size from 164 to 236 mm SL. These reefs were found in shallow areas with relatively litde current, such as the middle of the lagoon, and behind the reef crest (see Stoddart 1962 for cross sectional views of the lagoon and fringing reef). The substrate in such areas was typified by high levels of sediment, and the coral was overgrown with vegetation and macroscopic algae (Burke 1982). The occurrence of bachelor males at Glover's reef suggests that, whatever resources TTph males defend, habitat containing those resources is in short supply. Alternatively, bachelor males may represent fish which have not fully completed sex change, and are physiologically incapable of territoriality. To test these possibilities, 10 TTph males were removed from a reef also inhabited by approximately 23 bachelor males. Most of the bachelors 48 250, 1 Territorial Bachelor Tph Males Tph Males Figure 4.5 Comparison of distances travelled (mean + SEM, sample sizes within bars) by territorial and bachelor males during 15-nhnute mapping observations. 49 Figure 4.6 Size distribution (10 mm size classes) of all Tph stoplights captured at Glover's Reef, comparing territorial (n=123) and bachelor males (n=100). 50 were individually identified previously during two weeks of mapping studies conducted on ten of them. Three weeks after removing the TTph males, the reef was re-surveyed, and space-use maps were produced for all territorial males present. By that time, seven territories had been taken over by males previously identified as bachelors, and two others were occupied by previously unidentified males. The remaining bachelors remained on their previous home-ranges. This result supports the hypothesis that bachelor males are fully functional males which, given the opportunity to acquire suitable habitat, are capable of defending it. In 1986, TTph males were removed from 18 singleton reefs. The reefs were re-visited every few days to check for transitional or intersex fish. Tph males appeared on 11 of the singleton reefs 18.7 days (average) after the removals. One of the males was previously identified among a group of bachelors approximately 250 m away. I interpret the sudden arrival of Tph males with fully developed testes as evidence that bachelor males occasionally wander from reef to reef in search of vacancies. That these were not recently sex changed females from the experimental reefs is inferred from the suddenness of their appearance, their fully-developed testes, and the complete Tph colouration of most of them, although one was a late Tr male, having nearly complete Tph colouration. These results, together with results from large reefs with numerous bachelors, and small reefs with one TTph male and one subordinate (bachelor) male demonstrate that there is a shortage of "preferred", resource-rich habitat, forcing smaller, competitively inferior males to adopt bachelor status. 2.b. Iph males As noted in chapter three, Iph males are rare in this population. Due to our inability to distinguish Iph males from females, and since we bled and killed any Iph males that we found, no data were collected on their social behaviour, or use of space. Incidental observations of Iph male behaviour during spawning were recorded. On seven occasions, all one to five days after the removal of all TTph males from large reefs, Iph males were observed spawning with 51 females, suggesting that Iph males were already present before the Tph males were removed. The sex of the Iph males was indicated by their size and male-like pair-spawning behaviour, and in four cases was later confirmed histologically. Thus, it appears that Iph males live on or around TTph male territories among the females which they mimic. 2.c. Females Females generally remain within the confines of a single male's territory. Fig. 4.7 shows the least area polygons of replicate space-use maps for females that spawned with males T18 and T13 at W.B. reef in 1985, and outlines the territories of other males in the area. Tagged females that wandered onto neighbouring males' territories were actively chased out by the neighbouring male. Males also occasionally chased their own harem members towards the edges or along the borders of their territories. One female tagged in 1984 was observed twice at locations which did not correspond to her spawning partner's territory. The reason for this is unknown, but may reflect the occasional acts of mating infidelity by females (see Mating System, below). In 1984 and 1985, following experimental removal of all males from a large reef, most of the hundred or so females from all harem groups on the reef, including some of the tagged females in 1985, were found in a single area at spawning time, engaged in pair spawning with Iph males. However, at other times of the day, the females were observed on the home-ranges they had occupied prior to male removals. A similar observation was reported by Dubin (1981) after mass removals of Tph male redband parrotfish (Sparisoma aurofrenatum). Aggressive interactions between Iph fish were observed on several occasions. These included chases, displays, and on one occasion, a mouth-to-mouth display (described in other parrotfish - see Dubin 1981). However, the significance of female-female aggression is not clear. Dominance hierarchies are common in haremic fishes (Robertson 1974, Hoffman 1983, Robertson and Hoffman 1977), including the redband parrotfish, Sparisoma aurofrenatum (Dubin 1981). Unfortunately, in the present study, interactions between tagged females were Figure 4.7 Least-area polygons (shaded areas) depicting the home-ranges of females (identity codes within circles) that spawned with males 18,14,13 and 7 (territories are shown as dark outlines, and TTph male identification numbers are within squares). 53 extremely rare, so investigation of a possible dominance hierarchy was not possible. 3. THE STOPLIGHT MATING SYSTEM 3.a. Location of spawning In anticipation of spawning, females congregate at the edges of patch reefs and Solicit over the sand while the male rapidly swims around the perimeter of his territory. The locations of 143 spawning acts were documented on three territories at W.B. reef in 1985 (fig. 4.8). The same spawning sites are used consistently from day to day, although some harem groups have more than one spawning site. This occasionally made observations of spawning difficult, since the male continually swam between spawning sites during a spawning period, and would sometimes spawn with females at one site before the observer arrived from the other. 3.b. Time of spawning Spawning occurs every day during daylight hours, and was observed as early as 07:28 and as late as 17:50. There is a close correspondence between daily tidal peak and the time of spawning (fig. 4.9). When high tide occurs in the morning, high tide precedes spawning, while the reverse is true when high tide occurs in the late afternoon. Time of spawning progresses later in the day until sunset, when spawning moves back to early morning. On such days, spawning can occur twice; two spawning periods occurred on both June 15 and July 2,1985. On 2/7/85, male T18 spawned between 08:49 and 09:40 and again between 17:36 and 17:50. On the other hand, male T13 spawned between 08:59 and 09:39, but did not spawn during an observation between 17:00 and 18:15. Females can also spawn twice; on the same date, 2 of 3 tagged females that spawned with male T18 in the morning spawned again in the afternoon. Spawning periods last an average of 35.6 +/- 3.0 min. (SE, n=39), ranging from 9 min. at 09:01-09:10 h on 1/7/85, when the harem was observed from 06:50-10:00 h, to 93 min. at 10:52-12:25 h on 21/6/85, when the harem observed from 09:50-12:35 h. On June 23,1985, we observed two harem groups from dawn (05:35) until dusk (18:14) at hourly intervals. On that day, and during 65 other observation periods at W.B. reef in 1985, spawning was observed only 54 Figure 4.8 Location of 143 spawning rushes observed at w.b. reef (stippled area) during mapping studies and spawning observations of territorial males 18,13 and 7 in 1985. Spawning also occurred along the northern and western edges of the reef, but the exact spawning locations were not recorded. 55 ( • ) Time of first spawn (-*-) Predicted time of high tide 20:00 i 1 Jun 3 Jun 8 Jun 13 Jun 18 Jun 23 Jun 28 Jul 3 Jul 8 Date Figure 4.9 Comparison of predicted time of daytime high tide at Glover's Reef with time of first observed spawn at w.b. reef from June 3 to July 10,1985. Tidal predictions based on those for Carrie Bow Caye, approximately 24 km E of Glover's Reef (after Kjerfve et al. 1982). Spawning data unavailable for June 3,4,5 and July 3 and 6. A plus sign (+) indicates the beginning of the first of two spawning periods on a particular date. 56 during the few hours encompassing high tide, and was not observed at other times of the day. Male Identity Time of Observed Spawning T19 11:58 T13 12:02 T15 12:03 T7 12:03 T4 12:11 T5 12:11 Table 4.1 Time of spawning at W.B. reef on July 6,1985. Times denote the time at which spawning was first observed in each harem group. Data are only presented for groups which were visible at the same time from a central location on the reef. Spawning is highly synchronized on a particular reef, usually commencing in all visible harem groups within minutes of each other. For example, the time of first spawning by males visible from a central position on W.B. reef on July 6, 1985 occurred within a 15 minute period (Table 4.1). However, spawning does not appear to be synchronized between reefs, occurring as much as two hours earlier or later on different reefs. On the same date, (6/7/85), spawning was observed on a patch reef approximately 50 m to the southwest of W.B. reef at 13:00 h, and at M.M. reef, 750 m to the west of W.B., at 14:08 h. In general, spawning occurred earlier at reefs to the north and east of W.B. reef, and later to the south and west. The reason for this is unknown; presumably localized topographical differences affect current flow, and the timing of maximum current, which may in turn affect time of spawning. However, the trend for spawning to follow a tidal temporal pattern was consistent from reef to reef. 57 3.c. Frequency of spawning TTph males spawn daily with the same group of females (table 4.2). In addition, the data indicate that females are capable of spawning every day, and sometimes twice a day. On several occasions we arrived at the study site too late to observe the entire spawning period, which may account for some of the gaps in table 4.2. Also, because spawning occurs very rapidly, some females may have spawned while observers were writing notes or attempting to catch up with the rapidly-swimming male. Spawning pair male female (17) June 18 19 20 21 Date 22 23 (24) 25 26 27 July 2 T18 X * * * * * * * * * * ** T18 Y * * * * * * * * * * ** T18 A * * * * * * * * * * * T13 C * * * * * * ** * * * * T13 1 * * * * * * * * * * * T13 2 * * * * * * T13 K * * * * * * * * * * T13 Q * * * * * * * * * T13 T * * * * * * spawning observed between indicated pair on that date ** : two spawns by a female in a single day () : part of the spawning period was missed on that date Table 4.2 Frequency of spawning in two harem groups observed in 1985. 58 3.d. Which individuals spawn? Of the several hundred pair-spawnings observed, the majority (> 95%) involved a TTph male and an Iph female. On a given day, TTph males spawned with nearly all ripe females in their harem group, thus enjoying high mating success. However, four cases of mating infidelity were observed. Three involved tagged females and the fourth involved an untagged female whose infidelity was presumed from the spawning location. Of these four, one female spawned with two males on one day, and three females mated with neighbouring males while their usual mates were engaged in aggressive interactions or in extra-haremic copulation attempts. In contrast, females on singleton reefs are unlikely to wander into other harem groups, as they were never observed more than a few meters from their home reef. As a result, mating fidelity on isolated, singleton patch reefs is probably more strict than on the fore-reef or on larger reefs with several harem groups. Bachelor males apparently do not spawn. They were never observed doing so, despite over 25 h of observation, much of which took place while TTph males were spawning nearby. This was true even in the reef-associated bachelor males which had numerous spawning harem groups nearby. It is possible that channel BTph group-spawn during their crepuscular migrations. Randall and Randall (1963) suggest this might be the case in Sparisoma rubripinne, although they did not observe it directly. However, I did not detect any sign of spawning activity during the migrations I observed. Bachelor males also have slightly smaller gonads relative to their body weight than territorial males (fig. 4.10; p<0.01). This fact supports the contention that bachelors do not spawn. If they group-spawn, BTph would be expected to have larger gonads than pair-spawning TTph males (Choat and Robertson 1974, Robertson and Choat 1975, robertson and Warner 1978). Iph male stoplights have larger testes than TTph or BTph males, both absolutely, and relative to their body weight (fig. 4.10; p<0.001 in both cases). A previous report noted large 1 Males Figure 4.10 Comparison of gonadosomatic indices (GSIs) of females, initial phase males, bachelor and territorial tph males (mean + SEM, sample sizes within bars). 60 GSI in Iph male stoplights in Panama (Robertson and Warner 1978). Large testes in Iph males are believed to be adaptations to sperm competition which may arise as a result of alternative mating strategies such as group spawning (Robertson and Choat 1975, Robertson and Warner 1978). However, group spawning by Iph males has not been observed in this species. I.E. Downs (personal communication cited in Robertson and Warner 1978) apparently observed spawning by pairs of Iph fish, although details are not available. In the present study, Iph pair-spawning was observed following removal of Tph males from large reefs in 1984,1985 and 1987. We successfully speared or netted some of these Iph fish, and determined that the participants in each case were a female and an Iph male. The sequence of behaviours during pair-spawning involving two Iph fish is similar to that involving a Tph male and an Iph female. Although I collected no quantitative data, Iph males do not appear to perform Rapid swim, Patrol or Flutter movements. Iph males Solicit in the same manner as females, but perform male-like Head-Flicking movements and perform Overswim and the final Spawning Rush in what appears to be the normal (upper) Tph male position. On one occasion, three Iph fish participated in the spawning rush, but I could not tell whether all three fish released gametes, nor could I determine the sex of the participants. Iph pair-spawning was never observed while Tph males were present, even though several hundred pair-spawning acts were witnessed. Some Iph male labroids also spawn by 'streaking' (Warner and Robertson 1978, Robertson and Warner 1978), a strategy similar to that employed by satellite males in other teleosts (Jones and King 1952, Gross 1982). This was observed in S. viride during a spawning observation on June 12,1985. At 16:56 h, T13, the territorial male under observation, began a prolonged (109 s) chase of an Iph fish with which the male had not yet spawned. While T13 was thus engaged, the male from a neighbouring territory came onto T13's territory and spawned with a soliciting female at 16:58. As the spawning pair parted and swam to the coral, a small Iph fish swam rapidly to the gamete cloud and released its own milt there. The Iph fish was subsequently chased by T14 who kept the Iph hiding in coral holes at the border of the two 61 territories. Although we were initially unsuccessful at catching the (presumed) Iph male, an Iph male was caught in this location three weeks later. The strategy of streaking may explain the large gonads of Iph males. Since a streaking Iph male is probably engaged in sperm competition with a TTph male, a large testis in the former may increase sperm volume output, and therefore may increase the Iph male's fertility (Choat and Robertson 1975). However, there is no empirical evidence for a relationship between increased milt volume and increases in fertility in teleosts. 4. SOCIAL CONTROL OF SEX CHANGE To investigate possible social control of sex change, I examined the gonads of fish caught on reefs from which TTph males had been removed. These included both small, isolated, single-male patch reefs, and large patch reefs on which numerous TTph males had been removed. For comparison, I examined a large sample of fish that were caught on reefs which had not been disturbed (that is, no stoplight parrotfish had been removed from the reefs). Excluding males, I examined 108 fish captured on reefs from which the resident males had been removed, and 89 fish from previously undisturbed reefs. Eleven intersex fish were found among the former group, and six among the latter (X2 test with Yates' correction for continuity after Zar 1984, p>0.5). These results suggest that male removals do not affect the frequency of sex change by females. It could be argued that additional males on the undisturbed reefs may recently have died or otherwise left. Although I cannot fully discount this argument, all undisturbed reefs had TTph males present. Furthermore, during approximately 130 hours observing 116 territorial males, no TTph males died or vacated their territories by natural means, indicating that the natural mortality rate and rate of territory loss among males is low. Unfortunately, I could not keep long-term data on territory turn-over rates. However, working with the closely-related parrotfish Sparisoma aurofrenatum, Dubin (1981) kept long-term records of territory ownership by Tph males. Dubin (1981) reports that overall, territory turn-over rates were low, although there was a 62 substantial difference between sites; at 'preferred' sites, 142 males held territories for 406 to 568 days, while at less preferred sites, 66 males held territories for 62-94 days. A stronger argument against the conclusion that sex change is not socially induced is that females may have been prevented from changing sex by bachelor males which moved in before sex change could occur. Two intersex fish were found on isolated reefs which had been vacant for 42 and 65 days. However, other isolated singleton reefs were without males for more than 30 days, yet the resident females did not change sex. Furthermore, TTph males and in some cases bachelors were found on the undisturbed reefs on which sex changing females were found. Thus, loss of a male does not result in a one-for-one replacement by sex changing females, and some females change sex even in the presence of Tph males. D. Discussion Stoplight parrotfish at Glover's reef have a complex social and mating system. Large Tph males aggressively exclude all other Tph males from fixed areas ('territories' Brown 1975, Kaufman 1983) which encompass the home ranges of several females. Territories are defended all day, and incorporate feeding areas, mating sites, hiding spaces, and sleeping sites. However, habitat containing the resources which territorial Tph males defend is evidently in short supply. As a result, smaller, competitively inferior Tph males are relegated to less desirable habitat, where they inhabit overlapping, non-defended home ranges in groups of 10 to 40 individuals (bachelor males). Iph males, which are less common than Tph males, take advantage of their similarity to females by inhabiting Tph male territories or territory interstices. Spawning takes place during high tide at mating sites located on Tph male territories. For the most part, spawning is accomplished by a territorial Tph male and the females within his territory. Bachelor Tph males do not appear to spawn. Females spawn nearly every day with the same territorial male, and mating infidelity by females or TTph males, involving extra-haremic copulations with neighbours, is infrequent. Thus, the predominant mating unit appears to be a 63 harem (Robertson and Hoffman 1977), including a single territorial Tph male and a fixed group of females. Iph males spawn by streaking and by pair-spawning with females in the absence of TTph males. However, Iph male pair-spawning was observed only after large numbers of TTph males were removed from their territories, and it is doubtful that such mass mortality of TTph males occurs naturally. For this reason, pair spawning by Iph males is only likely on small, isolated reefs. It is not known whether Iph males are present on such reefs, but one of the 6 Iph males I found came from a two-male reef containing approximately 20 females. In general, the distribution in space or time of females or the resources which females need determines the potential for polygyny (Emlen and Oring 1977, Wittenberger 1981, Robertson and Hoffman 1977, Thresher 1984); when females or resources such as food, mating sites or hiding spaces are patchily distributed, some males may be able to gain access to a large number of females by defending 'patches' from other males (i.e. 'female-defense' or 'resource-defense' polygyny, Emlen and Oring 1977). Robertson (1981) asserts that the tendency of parrotfish to feed at the edges of reefs where spawning sites also occur promotes the defense of all-purpose territories by males, and explains the high frequency of haremic social systems in this group (Choat and Robertson 1975, Robertson and Warner 1978). Dubin (1981) argues that male control of a female's access to favourable spawning sites determines parrotfish social structure, and that food is unimportant. Thus, parrotfish mating systems appear to involve resource-defense polygyny (review for reef fishes by Thresher 1984, see also Baird 1989). Data from stoplight parrotfish agree with the resource-defense hypothesis; in my study area, females are restricted to isolated patches of coral, probably because the risk of predation while travelling over expanses of sand or sea-grass beds is prohibitive. Males defend territories that include either entire small patch reefs, or parts of larger patch reefs. Most territories, which also serve as feeding areas for both males and females, include a reef-edge where the harem group spawns. Finally, some males defend territories that contain no females, and limited evidence from female-removal experiments suggests that males do not abandon their territories 64 after females have been removed. These data are best explained by the hypothesis that males defend resources, rather than females themselves. The results of male removal experiments indicate that sex change in S. viride is not socially controlled; intersex fish are found just as frequently on previously undisturbed reefs as on reefs from which males were removed. Changes in local social conditions, that is, removal of the male from a harem group, have been shown to cause sex change by the largest, most dominant female remaining in the harem in a number of sex changing species, particularly those with strict social and mating systems (Fishelson 1970, Robertson 1972, Hoffman 1983, review in Shapiro 1987). In species with less rigid social and mating systems, such as the saddle-back wrasse (Thalassoma duperrey, Ross et al. 1983), changes in the relative numbers of smaller conspecifics in the immediate vicinity of females stimulate sex change. Shapiro and Lubbock (1980) present a model under which females change sex after a local group recruits sufficient numbers of females from the juvenile population to boost the sex ratio above a threshold value. However, under this model, when a species' sex ratio is biased towards females, as it is in Sparisoma viride (Robertson and Warner 1978), removing males from a population should have a large positive effect on the local ratio of females to males, and should therefore promote sex change. In the present study this did not occur, even after removal of large numbers of males. Investigators should be cautious in extending social mechanisms of proximate control to all sex changing species. Although no species has been shown to change sex after attaining a species-specific size or age (Shapiro 1987), relatively few species have so far been examined with respect to proximate control mechanisms. Socially-induced sex change is only predicted when the costs of imprecise timing of the change are high, and when local social conditions are reliable predictors of an individual's chances of becoming a successful territory holder (Warner 1988, Hoffman et al. 1985). The large number of bachelor males, and, in comparison with most other well-studied protogynous species, the large size and therefore the high degree of mobility of stoplights may combine to make local social conditions an unreliable indicator of the probability that a female will be successful in taking over a territory immediately after changing 65 sex. If so, it is not surprising that sex change does not appear to be under direct social control in this species. 66 CHAPTER FIVE Hormonal Control of Sex and Colour Change A. Introduction Protogynous sex change involves remarkable and seemingly irreversible changes in gonadal anatomy and function, as well as in colouration and behaviour. The physiological mechanism that controls these changes has long been recognized for its potential importance in contributing to a complete understanding of the mechanisms of sex differentiation in vertebrates (Stoll 1955, Atz 1964, Chan 1970, Reinboth 1962a, 1988, Adkins-Regan 1986). In higher vertebrates, the translation of genotypic sex to phenotypic sex is thought to be accomplished by endocrine events during a critical period early in development (Jost 1965, review in Wilson et al. 1981). Although the exact mechanisms involved are not clear (e.g. Bogart 1987), it is generally agreed that hormones secreted by the developing gonad have a profound influence on further differentiation of gonadal and somatic tissues, including the brain and behaviour (Wilson et al. 1981, MacLusky and Naftolin 1981, Forest 1983, Schumacher et al. 1987). The mechanism of sex differentiation in teleosts is poorly understood (Yamamoto 1962, 1969, Harrington 1974, Hunter and Donaldson 1983). Conceptually, models of sex differentiation have been dominated by the view that hormonal factors play key roles in the process (Essenberg 1926, Witschi and Crown 1937, Turner 1946, D'Ancona 1950, Yamamoto 1962,1969, Hunter and Donaldson 1983, Reinboth 1988). It is well established that sex steroids can have profound effects on sex differentiation of juveniles of gonochoristic teleosts. In particular, when administered to larval individuals, testosterone and its synthetic derivatives, as well as 11-ketotestosterone - the major teleost androgen (Billard et al. 1982, Fostier et al. 1983), and 17p-estradiol - the major teleost estrogen (Ng and Idler 1983) can cause sterilization, masculinization, or feminization, (or none of the above), depending on the choice of species and steroid, and the dose and duration of treatment (Schreck 1974, Hunter and Donaldson 1983, 67 Piferrer and Donaldson 1987). Although it is not known whether these effects represent pharmacological or biological actions, they at least suggest that steroid hormones may be involved in sex differentiation. Few investigators have examined the influence of exogenous hormones on sex change in hermaphroditic teleosts. Androgens aclministered to females of protogynous species usually (Stoll 1955, Reinboth 1962a, 1970,1972, Fishelson 1975, Okada 1962) but not always (Reinboth 1962b, 1967, Tang et al. 1974a) induce what appears to be normal, but precocious sex change. On the other hand, estrogens have little effect in either protogynous or protandric species, except in causing general damage to tissues (Reinboth 1970, Okada 1964, Chan and Yeung 1983). Unfortunately, studies of this sort do not provide a clear picture of the endocrine events which occur over the course of sex change; in nearly all cases, the androgens tested have been synthetic steroids of unknown biological relevance (Shapiro 1979, Chan and Yeung 1983). Efforts to determine the steroid-hormonal profile of sex changing species have largely been restricted to in vitro investigations of the metabolites produced by gonadal tissues following incubation with radio-active steroid precursors. Such studies, however, have revealed few, if any, consistent trends. Furthermore, the relevance of information on steroid metabolic pathways in the gonads to actual blood levels of hormones is not clear (reviews by Reinboth 1979, Chan and Yeung 1983). Measurement of important reproductive hormones circulating in blood (in vivo) has recently become possible with the application of steroid radioimmunoassay techniques to teleosts (e.g. Scott et al. 1980a,b, Kime and Manning 1982, Fostier et al. 1981, Lamba et al. 1983, Cochran 1987, Kobayashi 1988). If the mechanism of sex change involves the endocrine system, then measurement of hormones in blood sampled before, during and after sex change, coupled with hormone treatment should provide important evidence for such a mechanism. In this chapter I present the first evidence that sex change in a protogynous marine species involves characteristic changes in the concentration of three teleost sex steroids (11-ketotestosterone, '11KT', testosterone, 'T' , and 17p-estradiol, 'E2') measured by radioimmunoassay of blood 68 plasma sampled before, during or after sex change. Since these hormones have not previously been measured in plasma from parrotfish, I also present the results of validation studies for radioimmunoassays for 11KT, T, and E 2 in plasma from stoplight parrotfish. B. Materials and Methods 1. RADIOIMMUNOASSAY PROCEDURES AND ASSAY VALIDATION  La. Chemicals Fresh Diethyl-Ether (BDH, AnalaR grade) and n-Heptane (BDH assured grade) were used in the extraction procedures, but were not re-distilled prior to assay. Assay buffer (a fresh batch was prepared for each assay run) was a standard phosphate buffer (11.5 g Na2HP04-2H20,2.63 g NaH2PC»4 in 2 L distilled water, 0.05M pH 7.1), with sodium azide added as preservative (0.065 g/L; BDH) and gelatin (1.0 g/L; BDH) added to limit non-specific binding. Antibodies to testosterone and 17p-estradiol were purchased from Miles Scientific Inc., who provide a list of cross-reacting steroids. Anti-11-ketotestosterone was generously donated by Dr. D.R. Idler, Memorial University, St. John's Newfoundland. Cross-reactivity between the antibodies and other steroids known to be present in fish plasma was minimal (cross-reactivities determined by the antibody suppliers). Of particular concern was cross-reactivity between anti-11-ketotestosterone and testosterone, and vice-versa, since non-specificity of these types of antibodies has been problematic in previous studies (see Kime and Hyder 1983, Scott et al. 1980a). Antibodies against testosterone cross-react with 11-ketotestosterone by 1.5% and with 1 lp-hydroxytestosterone by 2.5 % (Miles Scientific). Dr. Idler reported (personal communication) that anti-11-ketotestosterone does not cross-react significantly with 1 lp-hydroxytestosterone, llp-hydroxyandrostenedione or adrenosterone, (nor with estrone, Cortisol, corticosterone or cortisone). I further determined that cross-reaction of anti-11-ketotestosterone with testosterone is 0.06% at the level of 50% binding. Bound and free steroids were separated with a dextran-coated charcoal solution (1.0 g activated charcoal [Sigma] and 0.1 g dextran T-70 [Pharmacia] in 100 ml buffer). Radioactive 69 steroids were purchased from Amersham as [l,2,6,7]-3H-Testosterone, [2,4,6,7]-3H-17p-estradiol, and [l,2]-3H-ll-ketotestosterone. Radioactivity was counted in a commercially available liquid-scintillation cocktail for use with aqueous solutions (Scintiverse II, Fisher). Lb. Extraction of steroids from plasma In preliminary experiments, plasma samples measured without prior extraction did not dilute in parallel with serial dilutions of standard steroid. Thus, diethyl-ether/heptane (4:1 v/v) solvent extraction was used to separate steroids from plasma. Plasma aliquots were thawed over ice, and 100-200 ^ 1 was transferred to a glass test-tube (16x125 mm). Controls made up of distilled water alone were also used to test for a 'blank' effect in the assay (i.e. to control for possible contaminants which can cause overestimates of steroid levels, Abraham 1975). Approximately 1000 cpm of labelled steroid (in 100 1^ diluent) were added to the samples, which were then vortexed and left at room temperature for 30 minutes to allow the label to equilibrate with endogenous steroids. A similar quantity of labelled steroid was added directly to counting vials as "100% recovery" tubes. Four ml diethyl ether and 1 ml n-heptane were added, vortexed for 15 seconds, left to separate 1 min., re-vortexed, and finally left to separate for 5 minutes before being placed in an ultra deep-freeze (-70°C). Once the aqueous layer was well frozen, the organic layer was poured into clean glass test-tubes. Tubes containing the frozen aqueous layer were rinsed with a further 1 ml of solvent, and this was again decanted. Tubes containing the solvent were placed in a warm water bath (37°C), and a light stream of compressed air was blown over them to speed evaporation of the solvent. Dried extracts were re-constituted in assay buffer (1:10, v/v), vortexed, capped, and left at 4°C overnight to redissolve. A 500 ^ 1 aliquot was then withdrawn and added to scintillation vials for recovery determinations. 70 I.e. Radioimmunoassay procedure The procedures used for RIA are similar to those described previously for use in other teleost species (Scott et al. 1980b, Van Der Kraak et al. 1984). Doubling dilutions of standard steroid were made up from a set of deep frozen, concentrated standards. The final standard concentrations ranged from 1000 to 7.8 pg/ml for T, 1000 to 15.6 pg/ml for 11KT, and 500 to 1.9 pg/ml for E2. Polystyrene test tubes (12x75 mm) were pre-marked with sample or standard identification numbers, and 200 1^ of standard or unknown were added, each being assayed in duplicate. Previously deep-frozen antibodies were made up in assay buffer immediately prior to assay, and 200 1^ of the antibody solution was added to each assay tube. Final antibody dilutions were: 1:120,000 for anti- 11KT; 1:60 for anti-T; and 1:100 for anti-E2. The antibody-cold hormone complex was mixed, and then 200 1^ of label containing 1200-1400 cpm for 3H-11KT and 1800-2000 cpm for 3H-T and 3H-E2 were added to all standards and unknowns. Control tubes, also made up in duplicate, consisted of the following: 'total counts', containing label and 600 1^ diluent but no antibody, no cold hormone, and no charcoal; '100% binding', containing label, antibody, charcoal, and 200 H l diluent, but no cold hormone; and 'non-specific binding', containing label, charcoal, plasma extract and 200 H l diluent but no antibody. The assay mixture was left overnight at 4°C. Subsequently, 200 1^ of an ice-cold solution of dextran-coated charcoal was added to each tube to separate bound from free steroids. Tubes were left in an ice bath for 12 minutes, and were then centrifuged at 4°C in a pre-cooled centrifuge at 4000 rpm for 12 minutes. The supernatant containing bound steroids was decanted into polypropylene scintillation vials (minivials, Fisher). Three ml of counting fluid were added, the tubes mixed, placed in a scintillation counter (Beckmann LS9000), and counted for 10 minutes or until 104 counts had accumulated. The counting program included automatic correction for quench based on comparison with an internal, high activity radioactive standard. 71 1 .d. Determination of unknown values Average cpm values for each replicate standard were converted to percent bound scores according to the formula: % Bound = (sample counts - non-specific binding counts) / total counts. Standard curves were generated for each assay run by plotting percent bound against the concentration of cold hormone for each standard on semi-log graph paper, and drawing a smooth line through the points. Unknown concentrations were calculated using the percentage binding for each unknown and interpolating from the standard curve. Replicate values for each unknown were averaged. I.e. Assay validation To determine the accuracy of the assays, pooled samples with high levels of endogenous steroid or samples with added standard steroid were measured after serial dilution with diluent and compared with serial dilutions of standard steroid. The standard and unknown dilution curves were plotted on semi-log graph paper to determine parallelism. To further test the assay's accuracy, known amounts of standard steroid at several concentrations were added to plasma with low endogenous hormone levels and the samples were assayed using the normal procedure. A least-squares linear regression was performed on the concentration of steroid added versus that measured, and the correlation coefficients was determined for each assay. Precision (or intra-assay variation) of each RIA was tested by determining the coefficient of variation for several replicates of the same sample measured in a single assay run. CV's less than 20% were considered acceptable (Cekan 1975). Replicability (inter-assay variation) was determined by assaying several samples twice in separate assay runs. Least-squares linear regressions were performed, and correlation coefficients were determined, comparing expected versus measured values in each case. Interassay variation was monitored by running samples with known hormone values in each run, and by comparing the percent of total radioactivity bound by antibodies in the absence of cold hormone. Substantial deviations in this parameter indicated that the assay was not performing 72 optimally. In such cases, results from unknowns were ignored, and steps were taken to restore the assay to optimal performance before re-assaying the samples. 2. ANALYSIS Each hormone was analyzed separately for each sample, and means and standard error measurements calculated for groups of samples classified according to sex or colour phase. Owing to the propensity of some groups to have unusually low variances (due to uniformly low or undetectable levels of a particular steroid among individuals), non-parametric methods of statistical analysis were employed. This involved non-parametric ANOVA, followed by non-parametric, multiple comparison tests (Zar 1984). 3. HORMONE ADMINISTRATION As initial RIA studies suggested that 11KT may play a causal role in sex change, I investigated the effects on gonadal histology and colouration of aclministering 11KT to functional females. Adult females were captured, sexed by cannulation, and treated with either a single intramuscular injection of 11KT (experimentals; 5 ng/g bw) or the vehicle alone (controls; 0.9% saline with 2 drops of Tween-80 per 250 ml). In a preliminary experiment, females were treated with a potent synthetic androgen (methyl-testosterone, MT; 200 ng/g bw), released onto their home reefs and re-captured 10-14 days later. However, the rate of re-capture using this method was low (5 of 13 treated fish were found, and of these, only three were caught). Therefore, in 1987,1 lKT-treated and control fish were housed in a triangular cage (3 m/ side) made of plastic-coated chain-link fence, and held down with coral boulders on nylon "herring web" netting. Coral boulders and coconut palm fronds were added to the interior and sides of the cage to provide cover for the subjects. Several females received abrasions while trying to escape and died within 48 hr of being placed within the cage. Several more disappeared without trace, and others were killed by large predators. Five 1 lKT-treated and five control-treated females survived to be included in the experiment. Fish were observed daily for evidence of 73 colour change. Two 11KT-treated fish were killed on days 5 and 9, two on day 10 and one on day 16. Three control-treated fish were killed on day 10, and two on day 15. Gonads from all subjects were preserved in Bouin's fluid for histological analysis. C. Results 1. A S S A Y VALIDATION Following solvent extraction of steroids from plasma, all samples diluted in parallel with the standard curve. Recovery determinations after solvent extraction gave consistent results, although estradiol was extracted with somewhat lower efficiency than T or 11KT. Mean extraction efficiency for 11KT and T was 100.2 +/- 0.40%, n=105 and 98.2 +/- 0.42%, n=96, respectively, while that for E2 was 88.05 +/- 0.58%, n=63. Following this initial determination, extraction efficiency was assumed to be constant and was measured only occasionally to make sure that it remained so. The low extraction efficiency of E2 was taken into account in calculating a sample's final E2 concentration. Sensitivity, the lowest dose of standard steroid which falls on the linear portion of the standard curve, was approximately 4 pg/ml for E2, 8 pg/ml for T and 16 pg/ml for 11KT. Validation studies showed the RIAs for 11KT, T and E2 to be accurate, precise, and reliable (table 5.1). Linear regressions were not significantly different from Y=X (p > 0.05; see Cekan 1975, Abraham 1975 for steroid RIA validation procedures). The level of 11KT in plasma from females was below the detection limit for the assay (<80 pg/ml plasma). Possibly, components of female plasma mask endogenous 11KT, causing the low measurements. However, recovery of standard 11KT added to female plasma was quantitative (table 5.1). In 1987, values of T and 11KT in plasma from six males gave results which were more than 4 standard deviations above the mean values obtained from other males. The reason for such high levels in these males is unknown; therefore, they were considered outlying samples (Zar 1984, Cekan 1975), and were discarded. 74 Precision Steroid CV level (pg/ml) n 11KT 12.3% 400 7 10.1% 1400 8 T 10.4% 350 5 9.4% 1280 5 E2 5.1% 430 5 11.7% 1340 5 Accuracy2 Steroid Regression (pg/ml) correl. coeff. n 11KT Y=( 1.022 * X) -15.9 r = 0.951 23 T Y=(0.975 * X) + 56.4 r = 0.991 14 E2 Y=(1.016 * X) + 60.5 r = 0.998 9 Replicability3 Steroid Regression (pg/ml) correl. coeff. n 11KT Y=(1.012 * X) - 90.2 r = 0.918 21 T Y=(1.102 *X)-81.7 r = 0.990 15 E2 Y=(0.989 * X) - 77.0 r = 0.979 12 Table 5.1 Validation of radioimmunoassays for 11KT, T and E2 measured in parrotfish plasma. 1 - A pool of plasma with either low or high endogenous levels measured n times in a single assay run. CV = (SD/mean)*100% 2 - A sample with low steroid levels to which standard steroid was added at several dosages. Regression is the amount added (X) versus the amount that endogenous steroid levels increased (Y). 3 - Samples that were assayed twice in separate RIAs. Regression is the amount measured in the first RIA (X) versus the amount measured in the second (Y). 2. HORMONAL CORRELATES OF SEX AND COLOUR CHANGE  2.a. Sex change I classified fish according to the histological condition of their gonads. Seventeen fish had both ovarian and testicular germinal elements in their gonads, and blood samples from them 75 were classified as 'intersex'. Fish whose gonads contained either testicular or ovarian germinal elements alone were classified as male or female, respectively, and blood samples from them were classified accordingly. Kruskal-Wallace non-parametric ANOVA revealed a significant difference in plasma steroid levels among groups (p<0.001 for 11KT, T and E2). Levels of 11-ketotestosterone were below the level of detectability in females (< 80 pg/ml plasma), but were dramatically higher in intersexes and males (fig. 5.1a; p<0.001). Conversely, 17p-estradiol levels were much higher in females than in intersex fish and males (fig. 5.1c; p<0.001). Thus, sex change involves a striking increase in plasma levels of 11KT, and an equally striking decrease in E2. On the other hand, females and intersex fish had similar, moderate levels of testosterone (fig. 5. lb, p>0.05), indicating that T is unlikely to be important in sex change. Then again, the high levels of testosterone in males compared with females (p<0.001) suggests that testosterone may be involved in the maintenance of male characteristics once sex change has been accomplished. 2.b. Colour phase change In Chapter Three, I showed that although some individuals change sex and colouration at the same time, others maintain Iph colouration after sex change. This suggests that sex change and colour change are asynchronous, and that colour change may be controlled by a mechanism that is independent of the mechanism controlling sex change. To examine the hormonal profile of fish undergoing colour phase change, I determined the plasma hormone levels of males (gonadal sex determined by histological examination) that were classified at the time of capture as either initial colour phase (Iph), transitional colour phase (Tr), or terminal colour phase (Tph). Non-parametric ANOVA revealed significant differences among groups (p<0.01 for 11KT, T andE2). Five of six Iph males had undetectable levels of 11-ketotestosterone, while transitional and Tph males had higher levels (fig. 5.2a; p<0.001). Thus, the high levels of 11KT found during sex change apparently subside to low levels in Iph males, but peak a second time during colour phase change. Iph males had elevated levels of 17p-estradiol compared with either 76 11-Ketotestosterone Female Intersex Male Figure 5.1 Plasma levels (mean + SEM, sample sizes within bars) of (a) 1 lketotestosterone (upper panel), (b) testosterone (middle panel) and (c) estradiol (lower panel) in females, intersex fish and males. Within each panel, bars with different superscripts are statistically different (see text for level of significance). 77 11-Ketotestosterone Initial Transitional Terminal Phase Phase Figure 5.2 Plasma levels (mean + SEM, sample sizes within bars) of (a) llketotestosterone (upper panel), (b) testosterone (middle panel) and (c) estradiol (lower panel) in functional males with Iph, transitional (Tr) or Tph colouration. Within each panel, bars with different superscripts are statistically different (see text for level of significance). 78 transitional or Tph males (fig. 5.2c; p<0.005, and p<0.002, respectively), although in absolute terms, levels of E2 in all males were low compared with the levels in mature females (c/fig. 5.1c). Finally, levels of testosterone in males with transitional colouration were similar to those in Iph males (p>0.05), but were significantly lower than those in Tph males (fig. 5.2b; p<0.02). As during sex change, therefore, testosterone does not seem to play a significant role in promoting colour phase change. 3. EFFECTS OF ANDROGENS ON FEMALES The RIA studies indicated that 11KT rises abruptly in plasma concentration during sex change, suggesting that 11KT may play an important causal role. On the other hand, increased 11KT may be a result of sex change, rather than a cause. However, in a preliminary experiment, treatment of females with methyl-testosterone, a soluble synthetic androgen, induced colour phase change and rapid proliferation of spermatogenic tissue in the three females examined histologically (fig. 5.3d). Encouraged by these findings, I investigated the possibility that 11KT plays a causal role in sex and/or colour phase change by comparing the effects of 11KT or a control treatment on the gonadal histology and external colouration of females. Gonads from three 11KT-treated females had crypts of spermatocytes or spermatids by days 10, 10 and 16, respectively (e.g. fig. 5.3a and b), while no control-treated females showed evidence of sex change by days 10 or 15 (fig. 5.3c). Histologically, 1 lKT-induced sex change appears to be identical with the early stages of 'natural' sex change; in both situations, spermatogenic crypts form among degenerating and resorbing oocytes along the ovarian lamellae (see Chapter Three). Two 1 lKT-treated females that were sacrificed on days 5 and 9 had not begun spermatogenesis, but did show oocyte atresia and resorption. However, atretic oocytes are not in themselves sufficient evidence for sex change (Sadovy and Shapiro 1987), since functional females also undergo atresia as a normal part of the reproductive cycle (Chapter Three of present study, Khoo 1975, Chan et al. 1967). 79 Figure 5.3 (a) and (b): Cross-sections of gonads from females treated with 11KT, showing tailed spermatozoa (sz) and a crypt of primary spermatocytes (sc). a and b: 598X. (c) Cross-section of the gonad from a female treated with saline/tween-80, showing vitellogenic oocytes (arrows) and some degenerating oocytes (do), (d) Cross-section of the gonad from a female treated with methyl-testosterone, showing numerous spermatogenic crypts (sc, spermatocytes; st, spermatids), degenerating oocytes (do) and yellow-bodies (yb). c and d: 239 X. 80 Four of five females treated with 11KT, including one that did not show spermatogenesis, had transitional colouration as early as day seven of treatment. On the other hand, no control-treated fish had transitional colouration. The 1 lKT-treated female that was sacrificed on day 9 did not show evidence of sex or colour change, and did not have detectable levels of 11KT in its plasma, suggesting that the treatment may have failed to deliver sufficient drug to this fish's circulatory system. 11-ketotestosterone was detectable in all other experimental fish (median 631, range 105 - 1332 pg/ml plasma), but in none of the controls (<80 pg/ml). None of the 1 lKT-treated fish that changed colour, nor any of the three MT-treated fish had detectable levels of estradiol (<80 pg/ml plasma; 855 pg/ml in the fish that failed to change sex or colour), while all five controls had moderate E2 levels (median 575, range 236 - 1064 pg/ml plasma). These results suggest that the atresia seen in androgen treated fish may result from inhibition of aromatase activity. That 11KT did not simply induce an overall depression of steroidogenesis is suggested by the fact that levels of testosterone were not significantly different between treatment groups; the median level of T was 480 pg/ml in experimentals (range 133 -1011), and was 824 pg/ml (range 333 - 2614) in controls (Mann-Whitney U test, p>0.05). D. Discussion Overall, the range of plasma levels for all three sex steroids are similar to the values found in the few other perciform teleosts in which plasma levels of sex steroids have been measured (Kime and Hyder 1983, Kadmon et al. 1985, Malison et al. 1987, Thomas et al. 1987, Yeung and Chan 1987, Shih and Yu 1988). Sex differences in 11KT and E2 in parrotfish are consistent with the reported biological functions of these steroids in fishes; 11KT, which is a potent teleost androgen (Billard et al. 1982, Fostier et al. 1983,1987, Lofts 1986, Adkins-Regan 1986), is found at higher levels in males than in females, and 17p-estradiol, which plays a key role in vitellogenesis (Lazier et al. 1987, Ng and Idler 1983), is found at higher concentrations in females than in males. 81 The presence of detectable levels of 11-ketotestosterone in the earliest intersexual stages, and a corresponding absence in the preceding female phase, suggests that 11KT is a natural androgen in Sparisoma viride, perhaps playing a role in stimulating the onset of sex change. Experimental induction of sex change in functional females by administering 11KT confirms this. Together, these results lend strong support to the hypothesis that sex change is under the control of hormones, and in particular, 11KT. This is the first report that a naturally-occurring androgen (11KT) promotes protogynous sex change and increases in concentration in the earliest stages of sex change detectable by light microscopy. Evidently, the increased 11KT in intersex fish subsides to low levels in Iph males. This is surprising, since Iph males have been observed to spawn and have larger testes than terminal phase males. Thus, although 11KT may play a role in the onset of sex change, this hormone is not necessary for the maintenance of spermatogenesis or the initial phase male pattern of reproductive behaviour. On the other hand, levels of 11KT increase again during colour change. This, plus the fact that 11KT-treated fish developed transitional colouration, suggests an additional role for 11KT in promoting the onset of colour phase change. Winn and Bardach (1960) reported that the synthetic androgen methyl-testosterone induced transitional colouration in initial phase stoplight parrotfish (unfortunately, as the latter investigators did not examine the gonads of treated fish, it is unknown whether sex change also occurred). The present findings extend the results of Winn and Bardach (1960) to include a similar effect of the natural androgen 11-ketotestosterone in inducing colour change in stoplight parrotfish. Interestingly, Reinboth and Becker (1984) found that gonads from primary (non sex-changing) initial phase males of Coris julis (Labridae) had an extremely low level of llp-hydroxylase activity {i.e. low capacity to produce 11KT or llp-hydroxytestosterone [110HT] from radio-labelled testosterone), while gonads from secondary terminal colour phase males produced high levels of 11-oxygenated androgens. This suggests that 11-oxygenated androgens (11KT and/or 1 lOHT) may play a role in promoting Tph male characteristics 82 (colouration, behaviour and relative testis size) in both S. viride and C. julis, and perhaps in labroid fishes generally. Testosterone does not appear to be involved in the initiation of either sex or colour change; although the mean level of T in intersex fish is intermediate between that of females and males, the difference is not significant in either case. Nor was there as difference in levels of T between Iph and transitionally coloured males. The function of testosterone in adult teleosts is a matter of controversy. Depending on the species, testosterone is reportedly found at higher concentrations in males than in females (e.g. Kime and Hyder 1983), at similar concentrations in males and females (e.g. Wingfield and Grimm 1977), and in some species, testosterone concentrations can reach higher levels in females than in males (e.g. Scott et al. 1980a). Scott et al. (1980a,b) suggest that the presence of testosterone in plasma may be incidental to its function as a precursor of 11-ketotestosterone in males, and of 17p-estradiol in females. On the other hand, testosterone has been shown to have androgenic effects by promoting spermatogenesis, secondary sex characteristics and/or male behaviour patterns in several species, although its potency is much less than that of 11KT (Stoll 1955, Hunter and Donaldson 1983, Liley and Stacey 1983, Norris 1986, Fostier et al. 1987). In the present investigation, the high levels of testosterone in males compared with females, and in Tph males compared with transitional colour phase males suggests that high levels of testosterone are associated with Tph male status, and may play a role in maintaining Tph male colouration and behaviour. Both sex-changing females and females injected with 11KT show a dramatic decrease in plasma levels of E2- Estradiol plays a major role in teleost reproduction, particularly in vitellogenesis and oocyte maturation (Ng and Idler 1983, Fostier et al. 1983). Thus, the decrease in plasma levels of E2 may be related to the cessation of vitellogenesis and the onset of oocyte resorption which occur early in sex change (Chan and Yeung 1983, Reinboth 1970). Experimental studies, perhaps using simultaneous administration of E2 and 11KT may confirm this. 83 It remains to be seen why initial phase males do not change colour phase during sex change when 11KT levels increase. Perhaps suppression of E2 synthesis is necessary for colour change. This is suggested by the elevated levels of E2 in Iph males compared with males with transitional or Tph colouration. Furthermore, 11KT treated fish, which changed colour phase, had significantly depressed levels of E2 compared with controls which did not change colour. However, this evidence is circumstantial; direct evidence for a role of E2 in the inhibition of colour change awaits experimental studies which I could not perform. Previously, attempts to measure hormones in blood taken from hermaphroditic species have been rare. This is primarily for logistical reasons, in that most well-known sex changing species are small, marine species from which field collection of sufficiently large blood samples is difficult. In 1976, Idler et al. (1976) compared the levels of 11-ketotestosterone and llp-hydroxytestosterone (HOHT) in blood samples from two gonochoristic species with a limited number of samples from four species of hermaphroditic teleosts. There was a predominance of 11-ketotestosterone in gonochoristic species, while 11-OHT was predominant in the hermaphrodites. Although Idler and co-workers attributed this result to a basic physiological difference between hermaphrodites and gonochorists, this conclusion has recently been questioned; the difference in steroid hormonal profile may result from temperature-dependent differences in testosterone metabolism (Kime and Hyder 1983, Yeung and Chan 1987). More recently, the advent of highly sensitive and accurate radioimmunoassay techniques has facilitated the measurement of steroids in relatively small volumes of plasma. Yeung and Chan (1987) used radioimmunoassays to measure the profile of sex steroids in plasma from the protogynous freshwater species, Monopterus albus. Interpretation of the results from that investigation is difficult, because M. albus is a seasonally spawning species, and changes in plasma steroid levels are confounded by changes associated with the spawning cycle (Yeung and Chan 1987). The clearest result was that in the postspawned/inactive phase, androstenedione increased in early and mid intersex fish compared with females, late intersex fish and males. 84 This finding led the latter authors to conclude that androstenedione may play a role in sex change. Unfortunately, although the same research group previously found that injections of a variety of androgens failed to induce sex change in M. albus, androstenedione was not tested (Tang et al. 1974a). On the other hand, injections of a mammalian gonadotropin preparation induce sex change in females of this species (Tang et al. 1974b), suggesting that sex change in M. albus may be controlled by gonadotropin directly (Chan and Yeung 1983). If so, differences in androstenedione concentrations between intersex fish and females may be a result, rather than a cause of sex change in M. albus. Reports that androgens can induce sex change when administered to individuals of protogynous species have been criticized because investigators often failed to confirm the sex of subjects prior to testing {e.g. Chan and Yeung 1983). Furthermore, no reports have yet shown that the effective steroid is also a major circulating hormone; indeed, as most studies have employed synthetic steroids (with the exception of Tang et al. 1974a), it is not always clear whether positive results represent biological or pharmacological effects (Shapiro 1979). The present investigation is the first to show that in a protogynous species, a naturally occurring androgen can induce precocious sex and colour change in functional adult females. This result is quite different from the commonly-reported effects of sex steroids on sex differentiation in juvenile gonochorists; in studies of the latter type, gonadal sex can be manipulated only during a species-specific critical period of sensitivity to the hormones during the larval stages (Donaldson and Benfey 1987, Hunter and Donaldson 1983). The wide range of sizes over which sex change naturally occurs in S. viride suggests that either the period of sensitivity to hormonal factors has, in an evolutionary sense, moved to a much later stage in ontogeny, or that individuals are sensitive to these hormones throughout life. 85 CHAPTER SIX Hormones and Social Status in Tph Males A. Introduction Small Tph male stoplights appear to be excluded from high-quality habitat by larger territorial males. The small males apparentiy adopt a bachelor male strategy, in which they do not defend territories. Presumably, in doing so they avoid the high energetic costs and increased risk of physical injury due to territorial defense and fighting when there would be few benefits to territoriality (Warner 1984b; present study, Chapter Seven). However, bachelor males are capable of opportunistically assuming territorial behaviour when suitable habitat becomes available, such as when a large male dies. The proximate mechanism that regulates territorial behaviour in stoplight males may involve the endocrine system; in fishes, the endocrine system is known to be responsive to changes in the external environment via the brain-pituitary-gonad axis (e.g. Lam 1983, Kyle et al. 1985, Scott et al. 1984, Liley et al. 1986a, b, Liley et al. 1987). Androgens are known to exert finely-tuned control over the expression of reproductive aggression in birds (e.g. Searcy and Wingfield 1980, Wingfield et al. 1987), reptiles (Moore and Marler 1987), rodents (Sachser and Prove 1984) and ungulates (Bouissou 1983). Furthermore, there is good evidence in Anabantids, Cichlids and Gasterosteids that castration decreases aggressive behaviour and androgen treatment to castrates or intact fish restores or increases aggressive behaviour, at least in reproductive contexts, suggesting that androgens may play a causal role in controlling reproductive aggression in teleosts (review by Villars 1983, Munro and Pitcher 1983; see also Munro and Pitcher 1985, Hannes et al. 1984, Hannes 1984). In this chapter I explore the possibility that androgens may be involved in controlling territorial behaviour in Tph male stoplights. First I determine the steroid hormonal 'profiles' of bachelor and territorial Tph males. Second, I determine whether a causal relationship exists 86 between behavioural status and hormonal condition by allowing bachelor males access to vacated territories and observing the effect on steroid hormone concentrations. Finally, I examine the effects of male-male interactions on androgen levels by subjecting males to experimental territorial intrusion. B. Materials and Methods 1. Hormonal Correlates of Social Status Blood samples were taken from Tph males of known social status (i.e. from bachelor or territorial males). Using radioimmunoassays, I measured the levels of sex steroids in these plasma samples. Since the levels of 17p-estradiol were consistently low in all Tph males, in this chapter I only consider 11-ketotestosterone and testosterone. Bachelor males were found in two distinct situations; reef associated bachelors were found on reefs that contained harem groups, while channel bachelors were found well away from females and territorial males. RIA results from blood samples taken from reef and channel bachelors were grouped separately. 2. Effect of Access to Vacant Territories To investigate the effect of acquiring a territory on the plasma androgen levels in bachelor males, territory vacancies were created. To do so, known territorial males were removed from reefs. In 1985, males that took over vacant territory locations were captured and bled either one or three weeks later. Hormone levels in these 'new territorial Tph males' (new TTph males) were compared with the levels in bachelors and in the established TTph males that had been removed. Because the sample sizes were low, I repeated the experiment in 1986. However, the procedure was modified slightly, in that all new TTph males were sampled one week after territory acquisition, and their hormone levels were compared with those of established territorial Tph males from a separate reef 70 m away. I present the results from each year separately. 87 3. Simulated Territorial Intrusion Since the previous experiment showed that bachelor males exhibit increased androgen levels following territory acquisition, I investigated whether the increase was due to aggressive interactions with neighbouring males or to the acquisition of, or increased proximity to territory resources. To test the former hypothesis, males on singleton reefs were subjected to simulated territorial intrusion (STI). To test the 'access to territory resources' hypothesis, bachelor males were allowed access to vacant territories on singleton reefs without neighbours with which to interact. (a) STI For STI, I captured Tph males from non-study reefs to serve as intruders and transported them to study reefs in a 301 container of sea water. At the study site, an intruder male was transferred into a cage that was hung over the side of the boat. The cage consisted of a cylinder (diameter 80 cm, length 80 cm) constructed from plastic-coated chicken wire. A door (25 x 20 cm) at one end of the cylinder was held in place and closed with tying wire. The cage was subsequently moved onto the reef and placed on the substrate in a prominent position. The 'intrusion' was carried out for a thirty-minute period in the morning and again in the afternoon, and was repeated over seven days. Intrusions were occasionally missed because of bad weather or the lack of a stimulus fish, but each male received STI on at least 6 days. Controls received an empty cage, but were otherwise treated identically. Both control and experimental males were captured and bled immediately after the final stimulation. The behaviour of control and STI-treated males was documented during the first 15 minutes of stimulus periods (at least three observations per male); replicate observations on each male were averaged. The following behaviours were recorded: the time to first physical contact with the cage, the total time spent by the territorial male outside a distance of five body lengths from the cage, the frequency of Feeding Bites, Feeding Bouts, and agonistic behaviours directed at the caged intruder. The latter group of behaviours included: Tail-Stand Displays; Bites, in which the male contacted the cage with his mouth or teeth; Vigorous Attacks, in which a 88 vigorous thrust of the caudal fin propelled the territorial male towards the cage, and resulted in a Bite; and Circles, in which the male moved >180° around the circumference of the cage but within 5 body lengths of it. (b) Access to singleton reefs Following the STI experiment, I revisited all reefs from which control or experimental males had been removed, and captured and bled any 'new singleton' males that arrived there. Plasma levels of 11KT and T in these males were compared with the levels in the controls from the STI experiment and with the bachelor males described in part 1 above. 4. Analysis Differences between mean hormone levels among several groups were analyzed by one-way ANOVA followed by Tukey multiple comparisons test (Zar 1984). When comparing several groups against a control, Dunnet's test was employed, and when only two groups were being compared, Student's t-test was used (Zar 1984). The behaviour of control and experimental groups in the simulated territorial intrusion experiment was compared using a non-parametric test for independent groups (Mann-Whitney U test; Siegel 1956). C . Results 1. HORMONAL CORRELATES OF TERRITORIAL AND BACHELOR STATUS Plasma levels of testosterone and 11-ketotestosterone differed significantly among BTph and TTph males (T: p<0.001; 11KT: p<0.001). Testosterone was significantly higher in territorial terminal phase males compared with bachelor terminal phase males from either channel or reef groups (fig. 6.1a; p<0.001 and p<0.01, respectively). Furthermore, T was higher in bachelors that were situated near territorial males compared with channel bachelors inhabiting a rubble and sea-grass bed several hundred meters away from territorial males (p<0.001). Similarly, 11KT was significantly higher in territorial males compared with bachelors from 89 3000 2000 05 E CO J5 Q . T3 O CD CO 1000 1500 p> 1000 500 Territorial Tph Reef Bachelor Channel Bachelor Figure 6.1 Plasma levels (mean + SEM, sample sizes within bars) of (a) testosterone (upper panel) and (b) 11-ketotestosterone (lower panel) in territorial Tph males and bachelor males from "reef and "channel" groups. 90 either reef or channel groups (fig. 6.1b; p<0.001 and p<0.01, respectively), and was higher in reef bachelors compared with channel bachelors p<0.01). 2. EFFECT OF REMOVING TERRITORIAL MALES The above correlation does not indicate whether high or low androgen titers are a cause or an effect of territoriality or bachelorhood; it may be, for instance, that low endogenous androgen levels render some males incapable of territorial behaviour. However, the fact that bachelor males take over experimentally vacated territories suggests that bachelor males are capable of territoriality, despite initially low androgen levels. To investigate this, I examined the effects of allowing males access to artificially vacated territories on endogenous levels of testosterone and 11-ketotestosterone. In 1985, one week after taking over or expanding a territory, plasma levels of both T (fig. 6.2a) and 11KT (fig. 6.2b) in new territorials were dramatically higher than those in undisturbed territorial males (p<0.001 for testosterone and 11-ketotestosterone) or in reef bachelor males (cf figures 6.1a and 6.1b; p<0.001 in both cases). The same result occurred in 1986; both T and 11KT were again markedly increased in new TTph males compared with undisturbed TTph males (fig. 6.3, p<0.01 for both HKTandT). However, by three weeks after taking over a territory, levels of T and 11KT in new TTph males were not significantly different from the levels in undisturbed territorial males (fig. 6.2a and 6.2b; p>0.05), but were still significantly higher than the levels in reef bachelors that failed to take over a territory (fig. 6.2a and 6.2b; p<0.01 for both steroids). These results confirm the hypothesis that the expression of territorial behaviour and high plasma levels of androgen are directly related, and further demonstrate that androgen levels are highest during the first week after territory acquisition, falling to lower levels by three weeks after the male had taken over a vacated territory. 91 6000 O ) 2000 Q_ 1000 Undisturbed NewTTph NewTTph Reef teach. TTph (1 week) (3 weeks) (3 weeks) Figure 6.2 Plasma levels (mean + SEM, sample sizes within bars) of (a) testosterone (upper panel) and (b) 11-ketotestosterone (lower panel) in undisturbed territorial males, new territorial males sampled one or three weeks after taking over a territory, and in bachelor males sampled three weeks after nearby territorial males were removed. Data are from 1985. 92 3 0 0 0 r CTJ E CO a? ^ 2 0 0 0 E I I Testosterone 11-Ketotestosterone a wamm o 55 U) Q . 1 0 0 0 0 8 Controls 1 5 NewTTph Figure 6.3 Plasma levels (mean + SEM, sample sizes within bars) of testosterone and 11-ketotestosterone in established territorial males and in new territorial Tph males sampled one week after taking over a vacant territory in 1986. 93 3. EFFECT OF SIMULATED TERRITORIAL INTRUSION AND ACCESS TO SINGLETON REEFS Patterns of androgen secretion similar to the pattern observed after territory acquisition in male stoplights have recently been reported in wild populations of passerine birds (e.g. Silverin and Wingfield 1982, Wingfield 1984a, 1985). In these species, free-living males undergo spring-time increases in plasma testosterone levels that are over and above the increases inducible by changes in day length in the laboratory. These peaks in testosterone production occur during periods of intense social interaction, and are a response to behavioural challenges from other males for the territory or its resources ('the challenge hypothesis' - Wingfield 1984b, 1985, Wingfield etal. 1987). Could a mechanism similar to that found in passerine birds produce the changes in hormone concentrations observed in stoplight parrotfish after territory acquisition? According to the challenge hypothesis, increased levels of T and 11KT one week after territory takeover may result from interactions with neighbouring males. Presumably, the lower androgen levels found three weeks after territory takeover indicate that by that time, dominance-relationships between neighbouring TTph males have been established, and boundary disputes have become less frequent. Alternatively, the increased hormone concentrations may be a response to females, a high-quality food supply, or other 'desirable' resources on the territory. I tested these alternative hypotheses by maintaining territory resources at a constant level while manipulating the level of territorial interactions through the use of STI, and subsequently by maintaining territorial interactions at a minimum while allowing males access to vacated singleton territories. If territorial interactions cause increased androgen levels in new TTph males, then males subjected to STI without increased access to territory resources should have higher androgen levels than controls. On the other hand, if access to territory resources also stimulates increased androgen levels, males that acquire territories on isolated reefs should show androgen levels similar to those taking over territories on larger, more densely populated reefs. 94 Measure STI Controls Sample size 9 10 Time to first contact 78.3(47-244) >900 (900) with cage (seconds) Time >5 body lengths 36.5 (0-67.5) 899.6 (891-900) from cage (seconds) Vigorous Attacks 5.3 (3.3-24.5) 0 (0) (n/15 min.) Bites 33.8 (1.5-83) 0 (0) (n/15 min.) Circles 29.0 (9-63) 0 (0) (n/15 min.) Tail-Stand Displays 3.3(0-31) 0(0) (n/15 min.) Feeding Bites 0 (0) 51.1 (28-68) (n/15 min.) Feeding Bouts 0 (0) 17.3 (12-35) (n/15 min.) Table 6.1 Effects of Simulated Territorial Intrusion on the Behaviour of Tph Males on Isolated Singleton Reefs. Data are medians with ranges in parentheses •. Males subjected to simulated territorial intrusion responded rapidly and vigorously with agonistic behaviours directed at the 'intruding' males (table 6.1). As expected, experimental males exhibited a much greater frequency of Bites, Chases, Vigorous Attacks and Tail Stand displays directed at the 'intruder' than control males (p<0.001 in all cases). Experimental males spent a considerable portion (95.94%) of the 15-minute observation period within 5 body lengths of the cage, while control males spent very little time there (0.04%; p<0.01). Experimentals also took a shorter time than controls to come into contact with the cage after it was placed on the reef (p<0.001); in fact, while empty cages were never physically contacted by control males, cages containing intruders suffered extensive damage from the aggressive activities of 95 experimental males. Finally, experimental males did not feed during periods of intrusion, while control males fed consistently (p<0.001 for Feeding Bites and Feeding Bouts). Plasma androgen levels were significantly higher in males subjected to simulated territorial intrusion compared with males receiving empty cages (fig. 6.4; p<0.01 for T and 11KT). This lends clear support to the hypothesis that increased levels of 11-ketotestosterone and testosterone during territory takeover are due to interactions with neighbouring males while territory boundaries are being established (i.e. the challenge hypothesis, Wingfield 1984b). In contrast, levels of both 11-ketotestosterone and testosterone in males that took over isolated reefs were similar to the levels in control males (p>0.05 for T and 11KT). This result does not support the hypothesis that access to territory resources or females stimulate the high levels of androgens during the initial period after territory takeover. When compared with the levels in bachelor males (see figure 6.1), testosterone and 11-ketotestosterone were significantly elevated in new singleton-TTph males (c/fig. 6.4; T: p<0.01; 11KT: p<0.05). This implies that access to vacant territories on singleton reefs does stimulate androgen levels in bachelor males, although not to the same extent as during the first week after territory acquisition on larger reefs with numerous Tph males (cf figures 6.2 and 6.3). It should be noted that the recent history of the new singleton-TTph males was not known. However, while it is possible that the new singleton TTph males were recendy sex changed fish that were somehow incapable of increasing their androgen levels, this seems doubtful. None of the males had transitional colouration, and all had fully developed testes; these males were probably ex-bachelors or perhaps territorial males that moved onto the study reefs from elsewhere. 96 4000 CO E co CL 3000 o D) Q -2000 100O Testosterone Y/A 11-Ketotestosterone 8 Control STI-treated New Singleton (empty cage) (caged intruder) TTph Figure 6.4 Plasma levels (mean + SEM, sample sizes within bars) of testosterone and 11-ketotestosterone in singleton males exposed to simulated territorial intrusion (STI), or to empty cages (controls), and in males that took over vacant singleton reefs (new sTTph). 97 D. Discussion The results presented in this chapter demonstrate a relationship between high plasma androgen concentrations and the expression of territorial behaviour in Tph male stoplight parrotfish; territorial males have higher levels of T and 11KT compared with bachelor males. The increases in agonistic and spawning behaviours associated with becoming territorial are therefore correlated with increased plasma androgen levels. Since the higher levels of testosterone and 11-ketotestosterone in territorial males can be induced in bachelor males by allowing them access to vacant territories, it appears that high androgen levels are a result of the difference in social status, although, as I suggest below, high androgen levels may also play a role in maintaining territorial status. In males that took over vacant territories, androgen concentrations initially increased to levels over and above those found in established territorial males, although by three weeks after territory acquisition, concentrations of T and 11KT had fallen to levels not significantly different from those in established TTph males. Simulated territorial intrusion also promoted increased androgen levels in established territorial males even though these males did not gain further access to territorial resources or gain increased proximity to females. Thus, the sharp increase in androgen levels during the initial period of territory establishment appears to be due to behavioural challenges from neighbouring males as they establish territory boundaries. Presumably, the fact that levels of T and 11KT fall to lower levels by three weeks after territory upheaval reflects the fact that by that time, territory boundaries have stabilized. According to the challenge hypothesis, the higher levels of androgens in plasma from established territorial stoplights (compared with bachelors) may be a response to aggressive interactions among territorial males. The behavioural differences between bachelor males and territorial males generally support this view. Furthermore, higher levels of T and 11KT in reef-associated bachelors compared with channel bachelors may be related to increased aggression experienced by reef bachelors as a result of their proximity to territorial males. 98 Interestingly, however, there was no significant difference in testosterone or 11-ketotestosterone concentrations between established TTph males on a large patch reef with numerous males on contiguous territories and established territorial males on singleton reefs (compare fig. 6.3 with fig. 6.4; p>0.05). In addition, new singleton-TTph males exhibited a significant increase in plasma levels of T and 11KT when compared with bachelor males. These results were surprising, since the challenge hypothesis predicts that males in high-density areas should experience higher levels of territorial intrusion, and should therefore have higher androgen concentrations than males in low density situations. The maintenance of moderate androgen levels in males on isolated, singleton reefs suggests that factors other than male-male interactions may also stimulate increased production of T and 11KT. One possibility is that increased proximity to reproductively active females may stimulate the secretion of androgens; hormonal responses by males to the presence of active females has been demonstrated in birds and mammals (Moore 1983, review by Harding 1981), and has also been shown in teleosts (Kyle et al. 1985, Scott et al. 1984, Liley et al. 1986a, b, Stacey et al. 1987, Dulka et al. 1987, Liley et al. 1987). However, other factors associated with territory takeover, such as the presence of a high-quality food supply, spawning sites, and so on, have not been examined and these may also be stimulatory. Higher androgen levels in territorial compared with non-territorial males has previously been described in impalas (Bramley and Neaves 1972). Illius et al. (1976) have suggested that since proximity to females promotes increased testosterone levels (at least in some male mammals), and since testosterone is believed to promote aggression in ungulates, the proximity to females enjoyed by haremic, territorial males should further enable them to maintain their status (see Bouissou 1983). Work on birds strongly suggests that increased androgen levels during territory establishment cause increased territorial aggression and territorial vigilance, thus promoting the chances of successful establishment (Silverin 1980, Wingfield 1984b, Wingfield et al. 1987). As mentioned in the introduction, there is strong evidence that androgens play a causal role in the control of reproductive aggression in teleosts (Villars 1983, Liley and Stacey 99 1983). Thus, I suggest that the increased androgen concentrations in established territorial stoplights is necessary to maintain the higher level of aggressiveness required by their status. Furthermore, I suggest that increases above the 'baseline' androgen levels that occur during prolonged territorial disputes may stimulate increased aggressiveness and territory vigilance, thereby maximizing the chances of successful territory defense. In summary, these findings demonstrate that when a territory becomes vacant, bachelor males, whose androgen levels are normally low, are capable of taking over the territory and becoming territorial. Bachelors that do so exhibit a moderate increase in plasma levels of T and 11KT. On large reefs with numerous contiguous territories, the initial period of territory establishment or territory expansion is accompanied by an increased level of androgen secretion that produces circulating concentrations of T and 11KT substantially above those found in established TTph males. The sudden rise in androgen levels appears to be a response to aggressive challenges from neighbouring males, and when territory boundaries have stabilized, androgen levels subside to lower levels. In contrast, on small, isolated reefs, interactions with neighbouring males appear to be rare, and as a result, the initial period of territory establishment involves only minor increases in androgen levels. 100 CHAPTER SEVEN General Discussion The major objective of this thesis was to investigate the behavioural endocrinology of a protogynous sex changing teleost, Sparisoma viride. Knowledge about the endocrine events underlying sex change may provide important insights into the poorly understood endocrine-behaviour relationship in teleosts generally (reviewed in Liley 1980, Liley and Stacey 1983, Moore 1986, Liley et al. 1987). In this chapter I will summarize the important findings from my investigations, and will discuss some of the more important and interesting issues raised by the findings. 1. The Adaptive Significance and Optimal Timing of Sex Change The stoplight parrotfish is a protogynous hermaphrodite (Reinboth 1968, Robertson and Warner 1978, present investigation - Chapter Three). However, my data show that this species exhibits a complex pattern of sex change; some individuals change sex before, while others do so after functioning as females. The issue is further confused by the presence of two extremely different colour phases (Iph and Tph). Thus, some individuals change sex and colour simultaneously (Iph female to Tph male), while others do not, functioning for a time as Iph males before eventually becoming Tph males. Several evolutionary hypotheses have been proposed to account for the occurrence of protogyny in fishes (see Atz 1964, Smith 1967, Ghiselin 1969, Warner etal. 1975). The most widely accepted explanation is Ghiselin's (1969) size-advantage model (Warner 1988). This model argues that protogyny may evolve when large males are able to mate with a disproportionate number of females at the expense of small males. In such circumstances, a fitness advantage may accrue to individuals that function as females when small, and change sex into males when they grow large enough to compete for the highly successful dominant or territorial male status (Ghiselin 1969, Warner et al. 1975). 101 In the stoplight parrotfish, there are apparently considerable fitness benefits to attaining large male status; territorial males spawn daily with as many as 16 females, while females spawn only once, and occasionally twice, each day. Furthermore, the majority of small males gain little or no reproductive success, since among males without territories, only Iph males have been observed to spawn, and then only in rare circumstances. Thus, as predicted by the size-advantage model, the distribution of reproductive success among males is highly skewed towards large individuals. The size-advantage model has also been used to predict the optimal age or size at which individuals should change sex. In order to maximize lifetime reproductive success, protogynous individuals should change sex only when they are large enough to acquire dominant or territorial male status (Warner et al. 1975, Hoffman et al. 1985, Warner 1988). Since the majority of territorial male stoplights at Glover's reef are larger than 28 cm SL, and all TTph males are larger than 26 cm, females should change sex at 26-28 cm SL if they are to optimize their lifetime reproductive success. The size-advantage model predicts that females that change sex before they reach the optimal size may incur costs associated with decreased daily mating success compared with individuals that change sex later and, due to their larger size, immediately gain a territory (Hoffman etal. 1985, Aldenhoven 1986). Male stoplights that are smaller in standard length than the smallest territorial male are found over the entire size range of mature females, although they appear to derive from at least two distinct ontogenies: secondary gonochorism and true protogyny. Also, all but two of seventeen intersex fish were smaller than 26 cm SL. Thus, stoplight females appear to become males well before their expectations of acquiring a territory warrant sex change. In doing so, they may suffer an immediate decrease in daily mating success which, for some individuals, can remain low for a considerable period. Why do stoplights become bachelor Tph males when they clearly have the alternative of remaining females or being Iph males? Presumably, there is an advantage to early sex change. One possibility is that bachelor males have higher daily reproductive success than females or Iph 102 males. However, bachelor Tph males do not spawn; not only have they never been observed doing so, but bachelor males also have smaller testes, both absolutely and relative to their body size than territorial or Iph males. This is a strong indication that bachelor males do not spawn by the common labroid interference-spawning tactics of streaking or group-spawning (Robertson and Choat 1975, Warner and Robertson 1978). It has been proposed that 'early' sex change, resulting in the production of non-spawning, bachelor males, may be an adaptive strategy employed by some individuals of protogynous angelfishes {Centropyge spp.; Moyer and Zaiser 1984, Aldenhoven 1986). Although bachelor males carry an initial cost of little or no reproduction following sex change, Aldenhoven (1986) maintains that bachelor males are more successful at acquiring vacant territories than females that must first change sex. In stoplight parrotfish, females may successfully acquire a territory immediately after sex change only in rare circumstances, such as when an isolated reef is left vacant for long periods. Bachelor Tph males, on the other hand, were highly successful at taking over vacant territories, often within hours of a vacancy being created. Thus, in agreement with Aldenhoven, it is reasonable to suggest that in stoplight parrotfish, bachelor males enjoy increased probability of successfully taking over vacant territories compared with females that must first change sex and colour. Bachelors may have a similar advantage over Iph males that presumably must change colour phase to be a successful territorial male. By abandoning reproduction, bachelor males may also gain an advantage of increased growth compared with females (Hoffman et al. 1985). Increased growth may in-turn promote the bachelor's chances of reaching the body size necessary for successful territory defence. On the other hand, individuals that change sex early lose the benefit of guaranteed mating success as females. Aldenhoven's (1986) model predicts that there is no advantage to becoming a bachelor when larger females are present in the harem, since size is extremely important in success at winning territories (Warner et al. 1975, Hoffman et al. 1985). Similarly, early sex change would not be adaptive when there are so many bachelors that the chances of an individual bachelor successfully acquiring a territory diminish. That is, the bachelor strategy 103 may be density-dependent (Maynard-Smith 1976, Austad 1984). In these respects, S. viride may be quite different from C. bicolor, female stoplights change sex in the presence of larger males and females, and, judging from the size of some bachelor groups, the 'critical' density of' bachelors above which individuals stay female rather than becoming bachelors appears to be extremely high, or non-existent. One reason for these discrepancies may be that while there is no inter-reef migration in C. bicolor (Aldenhoven 1986), stoplight bachelor males can and do migrate between reefs. A consequence of the potential for migration is that a female stoplight parrotfish may be unable to effectively assess her chances of taking over a harem in the event that a territorial male dies, since a bachelor from elsewhere can move in and take over quite rapidly. Instead, females should change sex early and become bachelor males themselves. Indeed, the potential for inter-reef migration may be a reason that social control of sex change has not evolved in this species. The point at which females change sex may be conditional upon growth rate, mortality, food supply, absolute size or age, or other factors that are relevant to the prospects of acquiring a territory at some time in the future, (e.g. Warner et al. 1975) but not necessarily on the presence or relative size of other stoplights in the local vicinity (cf. Fishelson 1970, Robertson 1972, Ross et al. 1983, Shapiro and Lubbock 1980). 2. Iph Males: An Alternative Life History As noted above, information available on the size, sex and colour distribution of stoplights suggests that S. viride follows at least two distinct life-history trajectories (figure 3.9); some individuals change sex as immature females, becoming Iph males for a period before eventually becoming Tph males, while others change sex and colour as mature females, becoming Tph males. The alternative reproductive behaviours in stoplights are irreversible, since once they have changed sex, Iph males cannot revert to being a female, but whether the differences in ontogeny result from genetic differences or different phenotypic responses to 104 environmental factors by genetically equipotent individuals is not known, and similarly whether the alternative tactics confer equal reproductive success is not yet known. In coho salmon {Oncorhynchus kisutch) and blue-gill sunfish (Lepomis macrocheirus), it has been shown that irreversible alternative life-history tactics can be maintained in a population by frequency-dependent selection (Dominey 1984, Gross 1985, Gross and Charnov 1980). Thus, when individuals employing one tactic become more common, selection favours individuals employing the other, less common tactic. Overall, individual fitness is approximately equal in individuals adopting the different tactics. The result is an equilibrium which is evolutionarily stable (ESS - Maynard-Smith 1976, reviews by Austad 1984, Dominey 1984). Not all alternatives confer equal reproductive success, however (Dominey 1984). For example, poor conditions during the juvenile or larval period may restrict the size of some individuals, with the result that their expected lifetime reproductive success through territoriality (for example) is vanishingly small. Such individuals may adopt an alternative strategy (such as sneaking) which, although it confers lower overall benefits than large males can acquire through territoriality, nevertheless confers greater reproductive benefits on small individuals than they would otherwise acquire by attempting to be territorial (Austad 1984). This strategy has been labelled "making the best of a bad job" (e.g. Austad 1984). Then again, alternative tactics may be maintained in a population when individuals employing either strategy experience differential success in different situations (Dominey 1984, Austad 1984). In the diandric blue-headed wrasse (Thalassoma bifasciatum), sex changing protogynous hermaphrodites co-exist in an apparently stable equilibrium with gonochoristic males (Warner et al. 1975). Both gonochores and hermaphrodites eventually become highly successful Tph males, but primary males gain reproductive success while small through Iph interference spawning tactics, while small hermaphrodites are females. Warner and Hoffman (1980) argue that primary males are maintained in the population as a result of differential reproductive success of males employing alternative male mating strategies at different densities; at high densities (i.e. on large reefs), central mating territories are abandoned by large 105 males because of the high costs of defending them from groups of Iph males. This results in a high level of mating success among primary Iph males and decreased selection for protogyny. At lower densities, Iph males are effectively precluded from spawning by the aggressive activities of large primary and secondary Tph males, and protogyny is favoured. Sex changers and gonochores are believed to contribute offspring to the population in proportions that reflect the underlying distribution of high and low density populations on large and small reefs, respectively (Warner and Hoffman 1980). Could Iph male stoplights be equivalent to primary males in other labroid species (as previously suggested by Robertson and Warner 1978) and be maintained in the population by habitat-dependent reproductive success of Iph males? At Glovers Reef, Iph males spawn by streaking and occasional pair-spawning. However, they were successful in only limited circumstances; streaking was only observed once while a TTph male was involved in a prolonged agonistic interaction and a neighbouring TTph male performed an extra-haremic copulation. Similarly, Iph male pair-spawning was observed only after large numbers of TTph males were removed. Three factors may enhance the reproductive success of Iph males. First, the presence of small, isolated patch reefs may allow Iph males on them to pair-spawn with females when the resident Tph male dies. If the Tph male is not promptly replaced by a BTph male, Iph males may enjoy extended periods of pair-spawning and gain high levels of reproductive success. For instance, a pair-spawning Iph male that spawns with 10 females every day for 65 days (the maximum time a reef was observed without a Tph male) can accomplish as much reproductive success as a female that pair-spawns for nearly two years (however, as noted in Chapter Four, it is not clear whether there are Iph males on the smallest reefs, although one was found on a small reef with two TTph males and about 20 females). Second, on larger reefs, closely packed territories with numerous females among which an Iph male can hide, as well as certain types of substrate, such as large boulders with numerous crevices and other hiding spaces, could enhance the anonymity of Iph males, thereby allowing them increased success at streaking. Third, again 106 through increased anonymity within harems, the success of streaking by Iph males may also increase as the number of females increases (Robertson and Choat 1975, Warner et al. 1975, Warner and Robertson 1978). Because they enhance the reproductive success of small males, all three factors may lead to decreased selection for protogyny, and therefore, a stable equilibrium between secondarily gonochoristic Iph males and protogynous females appears possible. The low frequency of Iph males within the overall population may thus be a reflection of the underlying distribution of habitats in which the above factors promote Iph male success. In comparison with territorial Tph males, Iph males are small, both in Belize and in Panama, as are functional males that have transitional colouration which I presume were previously Iph males. This, plus the small size of some BTph males, suggests that there may be constraints on Iph male reproductive success, and that when these constraints are high, Iph males abandon the Iph male tactic and change colour phase. One such constraint may be the number of other Iph males; as Iph males become more abundant, the daily reproductive success rate of an individual Iph male will likely decrease, and vigilance by TTph males may increase. Furthermore, a low proportion of females on a given reef may also lower the potential reproductive success of Iph males through pair-spawning. Thus, density-dependent mechanisms may determine the local value of being an Iph male. When conditions are such that Iph male reproductive success is low, individuals may abandon the Iph male tactic, change colour phase, and make the best of a bad situation by becoming small BTph males and awaiting the opportunity to take over a territory. Similarly, Iph males of T. bifasciatum abandon reproduction altogether when they find themselves on small reefs where the chances of spawning through Iph tactics are low. Such individuals grow 1.5 times faster than females (Warner 1984b). These males were shown to be "making the best of a bad job", since they were not fully compensated for lost reproduction, and since they resumed reproductive activity when transplanted onto larger reefs (Warner 1984b). Such a tactic may have some benefits of its own; Hoffman et al. (1985) have shown that, because they forego reproduction, fish that change sex early may experience increased growth or survival compared with functioning females, and therefore may increase 107 their chances of ultimately reaching territorial male status. While I was unable to measure either growth or survival in stoplights, the hypothesis that Iph males in poor habitat abandon reproductive activity in favour of increased growth seems reasonable. Females that produce and spawn clutches of eggs every day almost certainly devote less energy to growth and are more likely to be preyed upon than individuals that do not mate, such as BTph males (see Warner 1984b). 3. The Hormonal Control of Reproductive Behaviour It is well known that hormones can exert two major kinds of effects on behavioural as well as on morphological sex characteristics: (1) activational effects, in which the effects on previously organized behaviours or structures are reversible, and are a response to the prevailing endocrine state of the animal; (2) organizational effects, in which the presence of a hormone during a species-specific critical period permanently channels the development of the organism along a particular ontogenetic pathway (e.g. female or male; Phoenix et al. 1959); reviews for higher vertebrates in Wilson et al. 1981, Davidson 1973, Hutchison 1978, Leshner 1978, MacLusky and Naftolin 1981; for lower vertebrates see Hunter and Donaldson 1983, Adkins-Regan 1986, Moore 1986, Liley and Stacey 1983, Liley et al. 1987). In teleosts, induction of male behaviour patterns by androgen administration to adult females has been reported for several gonochoristic species. However, in the few species in which behaviour has been specifically examined, masculinization resulting from this treatment paradigm appears to be incomplete; more complete masculinization is attained only by treatement in the early post-hatching stages (Billy 1982, Liley and Stacey 1983, Adkins-Regan 1986). Thus, as in higher vertebrates, sexual dimorphism in reproductive behaviour of teleosts is not simply a reflection of sexual differences in adult gonadal hormone secretion (i.e. it is not simply an activational phenomenon). Rather, there appears to be hormone-induced sexual dimorphism in the underlying substrate for reproductive behaviour (an organizational effect; Adkins-Regan 1986). Given the proclivity of commercial aquaculture operations for using early hormone treatments to produce monosex cultures of fish (Donaldson and Benfey 1987), 108 permanent effects of hormones on the development of behaviour may have practical significance. However, the demonstration of permanent, organizing effects of hormone treatment does not prove that the hormone normally performs this function during development. Direct evidence for an organizational effect requires.demonstration that the organizing hormone is present during the critical developmental period, and that castration or anti-hormone treatment (withdrawing the hormone) in the early developmental period prevents, and hormonal replacement therapy facilitates behavioural organization (e.g. in mammals, Hart 1968). Unfortunately, this has not been accomplished in teleosts, owing largely to the small size of differentiating embryos. In this respect, the phenomenon of sequential hermaphroditism provides a natural form of the classical castration and replacement therapy experiment in animals undergoing sexual (re)differentiation. In Chapter Five, I showed that biologically relevant steroids undergo dramatic changes in plasma concentration over the course of protogynous sex change in stoplight parrotfish. The results of treatment with 11KT suggest that this steroid plays a direct role in controlling the onset of gonadal and colourational sex change. Correlational data further suggests that 11KT plays a similar role in behavioural sex change. However, it is not clear whether this is an activational or an organizational effect. Although high levels of 11-ketotestosterone are correlated with the presence of some aspects of male reproductive behaviour, such as territorial aggression, Patrolling and Rapid Swimming, female behaviours are associated with high levels of E2. Thus, the behaviour patterns exhibited by an individual may simply reflect the current state of the gonad; the animal behaves as female or male depending on the current circulating hormone levels (activational effects). In stoplights, circulating levels of gonadal hormones provide reliable indicators about the state of the gonad and external colouration, and therefore the appropriateness of female or male behaviour patterns. One would expect the evolution of behavioural responsiveness to such cues. 109 However, Iph males are capable of spermatogenesis and pair-spawning despite the absence of RIA-detectable quantities of 11KT, although Iph males are not territorial. Furthermore, females have high levels of testosterone, yet do not behave as males. These data indicate that females are insensitive to the androgenic effects of relatively high levels of testosterone, while Iph males are highly sensitive to androgens. I suggest that one action of 11-ketotestosterone in parrotfish may be to sensitize an individual to the presence of circulating androgens (an organizing effect). This hypothesis explains not only the lack of male characteristics in functional females, but also the obvious male characteristics of Iph males despite their low levels of 11KT. The sensitizing action of 11-ketotestosterone could operate by a mechanism similar to the mechanism in female rodents of estrogen-induced sensitivity to the presence of progesterone (see Davidson 1973, Leshner 1978), in which estrogen induces the production of receptors to progesterone in specific target organs (O'Malley and Schrader 1976, Etgen 1984). In mammals, the behavioural organizing effects of androgens are both masculinizing and defeminizing and these effects appear to be independent (Leshner 1978, MacLusky and Naftolin 1981). In parrotfish, 11-ketotestosterone appears to have a masculinizing effect, at least on gonadal and colourational sex, and following sex change, female sexual behaviour diminishes. I have suggested that decreased production of E2 may play a role in the cessation of vitellogenesis that occurs during sex change, and in the induction of Tph colour patterns, although direct testing of either hypothesis was not possible. Decreased E2 in intersex fish and males may also play a role in defeminizing patterns of reproductive behaviour; comparing females with males, there is a positive correlation between levels of E2 and the expression of female behaviour patterns. Furthermore, one intersex fish with reduced levels of estradiol (#326,107 pg E2/ml plasma) did not spawn during 3.5 h of observation while nearby stoplights spawned. On the other hand, defeminization is also correlated with increased levels of 11KT. Thus, it is not yet possible to determine whether defeminization of reproductive behaviour is due to decreased E2, or increased 11KT, or to changes in non-steroidal factors that I did not examine (see Stacey 110 1981, Liley and stacey 1983). It would perhaps be illuminating to simultaneously treat females with 11KT and E2 to determine if such treatment maintains female behaviour patterns, vitellogenesis and/or Iph colouration while instigating spermatogenesis and male behaviour patterns. If so, this would indicate a role for reduced E2 in defeminization. Unfortunately, owing to the logistical problems of direct experimentation, I was unable to pursue this further. It is not clear what 11KT does to instigate sex change. Presumably, increased 11KT levels represent one step in a series of physiological events that result in the production of a male from a functional female. There has been considerable speculation that sex-specific cell-surface antigens (Sxs, formerly H-Y antigen; Wiberg 1987) may play a causal role in gonadal differentiation in gonochoristic vertebrates (e.g. Wachtel et al. 1975, Ohno et al. 1979) and even in promoting sex change in protogynous teleosts (Reinboth 1987, 1988, Shapiro 1979,1987, Chan and Yeung 1983). However, such speculation may have been premature; sex-specific antigens are inducible in female Cons julis by testosterone injection (e.g. Reinboth et al. 1987). Furthermore, there is evidence from mammalian studies that testis organization can take place in the absence of Sxs antigen (McLaren et al. 1984), although in such cases, spermatogenesis appears to be incomplete (Bourgoyne et al. 1986). Thus, Sxs antigens may play a role in facilitating or promoting spermatogenesisonce sex change has begun, presumably being expressed as a result of increased 11KT concentrations (see Adkins-Regan 1986), but the antigens are not involved in the induction of sex change. Clearly, more work is required to fully elucidate the role of hormones in controlling all aspects of protogynous sex change. I measured only three of numerous possible steroids, any of which could play a role in sex change. Non-steroid hormones also play a role in sexual differentiation in mammals (e.g., mullerian inhibiting substance; Wilson et al. 1981), although these substances have not been identified in teleosts. Furthermore, the factor responsible for increased 11KT levels in intersex fish remains undetermined. Increased 11KT may be promoted by changes in.gonadotropin production from the pituitary, although sex differentiation in higher vertebrates is initiated independendy of gonadotropin production (Wilson et al. 1981). I l l Nevertheless, the present results should provide a basis for further, more detailed investigations of the intriguing phenomenon of sex change. Details of the hormonal correlates of sex change have been published for only a few species (Cardwell and Liley 1987, Yeung and Chan 1987). Comparisons across species should reveal relationships between hormones and behavioural, colourational and gonadal sex change that, together, may lead to a broader understanding of the evolutionary significance of the hormonal control mechanisms found in those species and in vertebrates generally. 4. Behavioural Endocrinology in the Field Investigations of the behavioural-endocrinology of animals in natural or semi-natural conditions have shown that the artifical environment of the laboratory can have a profound effect on the results from such studies (e.g. Wallen and Winston 1984, Wingfield 1984a,b; Liley etal. 1986a). While laboratory studies provide vital information about some of the effects of hormones on behaviour, and on the influence of behavioural experiences on the endocrine system, the highly controlled environment of the laboratory makes it nearly impossible for the investigator to uncover important factors and relationships not thought of in the design of experiments. Thus, studies of hormone-behaviour relationships conducted on free-living animals provide a body of knowledge that is complementary to that obtained from studies in the laboratory. Recent investigations of the relationship between circulating androgen levels and territorial behaviour in free-living birds show that small changes in the pattern of hormone secretion can have drastic effects on behaviour (Silverin and Wingfield 1982, Wingfield 1984b,c), to the extent that such changes can influence the mating system (Wingfield 1984a). Perhaps even more striking is the simplicity of this relationship; such simplicity may allow rapid evolutionary transition from one mating system to another (for instance, from monogamy to polygyny, ibid.), should such a change become adaptive, possibly involving altered reaction-rates of only a few enzymes in the steroid metabolic pathway. 112 The present field investigation illustrates this point. Large Tph male stoplight parrotfish accrue considerable fitness benefits from territoriality; they spawn daily with a group of females with whom the males enjoy almost exclusive access. However, territorial behaviour is not without costs (Cade 1984, Cox and LeBoeuf 1977, Clutton-Brock et al. 1979, Wingfield 1985, Silverin and Wingfield 1982). If these costs are high, selection should favour the evolution of mechanisms that limit high levels of territorial aggression to periods when it is adaptive; such a mechanism seems to operate in male stoplights. Bachelor Tph male stoplights are relegated by large territorial males to low quality habitat. Bachelor males do not spawn, and, probably since there would be few benefits to doing so, do not defend home ranges. When given access to a vacant territory, however, bachelor males rapidly and opportunistically assume territorial behaviour patterns. Some aspect of the vacant territory apparendy stimulates increased circulating androgen levels. Based on laboratory studies of other teleost species (Villars 1983, Liley and Stacey 1983), I have suggested that this increase serves to maintain the higher level of sexual and aggressive responsiveness demanded by the change in status. Wingfield (1985) suggests that the relationship between testosterone secretion and periods of territorial instability in birds is adaptive in that it limits periods of increased testosterone production, and therefore intense aggressive behaviour, to a minimum. The latter author argues that territorial behaviours are energetically costly and may attract the attention of predators. The fact that the initial increase in testosterone and 11-ketotestosterone in stoplights subsides within three weeks suggests that there may be constraints on the maintenance of high androgen levels. As in passerine birds, territorial behaviours in stoplights are also likely to be energetically demanding. In addition, it appears that time spent in territorial aggression may detract from investment in other crucial activities, such as parental care (Silverin 1980) or feeding; territorial stoplight males feed less frequently than bachelor males, probably because territorials must spend more time in territory maintenance activities. Furthermore, aggressive interactions between Tph males often resulted in visible wounds and torn fins in the combatants; 113 thus, fighting could have a direct effect on mortality, time spent in control of the harem, and even attractiveness to females. The relationship between hormones and behaviour is believed to be a dynamic, two-way relationship. Not only do hormones affect the development and ongoing expression of behaviour, but behavioural experiences, acting through the CNS, can also modify the rates of production or release of hormones, and therefore, can further modify behavioural responsiveness (Leshner 1978, O'Connel et al. 1981, Harding 1983, Wingfield et al. 1987, Liley et al. 1986a, b, 1987). Increasingly, investigators are also becoming aware that the behaviour-endocrine relationship represents an adaptive response to specific selective constraints (see Silver and Cooper 1983, Crews and Moore 1986, Wingfield etal. 1987, Liley et al. 1987). Thus, the endocrine system provides a mechanism for modifying behaviour in order to optimally track an ongoing, or changing situation. This mechanism allows adaptive responses to situations that could not be predicted accurately by 'hard-wired', genetic control mechanisms. Despite this stimulating view of behavioural endocrinology, the list of species that have been examined is still exceptionally small. The benign nature of the environment inhabited by coral reef fishes (i.e. clear, shallow, warm water) provides investigators with an opportunity to observe behaviour directly in circumstances that would be unavailable in species inhabiting temperate regions. Furthermore, coral reef fishes represent a cluster of species with various phylogenetic histories, and representing a broad spectrum of ecology and social organisation. Undoubtedly, the wide range of reproductive behaviour exhibited by these fishes involves numerous 'solutions' to the problem of adaptively controlling behaviour. The current challenge to behavioural endocrinology is to uncover, explore and compare these solutions in as wide a range of species as possible, and in time to expose the factors that determine which behaviour-endocrine control mechanisms are employed in a given species. 114 CHAPTER EIGHT References Cited Abraham, G.E. 1975. Radioimmunoassay of steroids in biological materials. Acta Endocrinol. Suppl. 183:7-42. Adkins-Regan, E. 1986. Hormones and sexual differentiation. In: D.O. Norris and R.E. Jones (eds.). Hormones and reproduction in fishes, amphibians, and reptiles. 1-29. Plenum Press, New York. Ahsan, S.N. 1966. Cyclic changes in the testicular activity of the lake chub, Couesius plumbeus (Agassiz). Can. J. Zool. 44:149-159. Aldenhoven, J.M. 1986. Different reproductive strategies in a sex-changing coral reef fish Centropyge bicolor (Pomacanthidae). Aust. J. Mar. Freshwater Res. 37:353-360. Atz, J.W. 1964. Intersexuality in fishes. In: Intersexuality in Vertebrates Including Man (C.N. Armstrong and A.J. Marshall, eds.). pp. 145-232. Academic Press, New York. Austad, S.N. 1984. A classification of alternative reproductive behaviors and methods for field-testing ESS models. Am. Zool. 24:309-319. Baird, T.A. The adaptive significance of coloniality and harem polygyny in the sand tilefish, Malacanthus plumieri. Ph.D. Thesis. University of British Columbia, Vancouver, Canada. Barlow, G.H. 1975. On the sociobiology of four Puerto Rican parrotfishes (Scaridae). Mar. biol. 33:281-293. Billard, R., A. Fostier, C. Weil and B. Breton. 1982. Endocrine control of spermatogenesis in teleosts. Can. J. Fish. Aquat. Sci. 39:65-79. Billy, A.J. and N.R. Liley. 1985. The effects of early and late androgen treatments and induced sexual reversals on the behavior of Sarotherodon mossambicus (Pisces: Cichlidae). Horm. Behav. 19:311-330. Bogart, M.H. 1987. Sex determination: a hypothesis based on steroid ratios. J. Theor. Biol. 128:349-357. Bouissou, M.F. 1983. Androgens, aggressive behaviour and social relationships in higher mammals. Horm. Res. 18:43-61. Bourgoyne, P.S., E.R. Levy, and A. McLaren. 1986. Spermatogenic failure in male mice lacking H-Y antigen. Nature (London). 320:170-172. Bohlke, J.E. and C.C.G. Chaplin. 1968. Fishes of the Bahamas and Adjacent Tropical Waters. 771 pp. Livingston Press, Wynnewood. 115 Boschung, H.TJr., J.D. Williams, D.W. Gotshall, D.K. Caldwell and M.C. Caldwell. 1983. The Audobon Society Field Guide to North American Fishes, Whales and Dolphins. A.A. Knopf Inc., New York. Bramley, P.S. and W.B. Neaves. 1972. The relationship between social status and reproductive activity in male impala (Acpyceros melampus). J. Reprod. Fertil. 31:77-81. Breder, C.M.Jr., and D.E. Rosen. 1966. Modes of Reproduction in Fishes. Natural History Press, New York. Brown, J.L. 1975. The Evolution of Behavior. Norton, New York. Buckman, N.S. and J.C. Ogden. 1973. Territorial behaviour of the striped parrotfish Scarm croicensh (Bloch)(Scaridae). Ecology_54:1377-1382. Burke, R.R. 1982. Reconnaissance study of the geomorphology and benthic communities of the outer barrier reef platfor, Belize. In: Rutzler, K and I.G. Macintyre, (eds.). The Atlantic Barrier Reef Ecosystem at Carrie Bow Cay, Belize, I. structure and communities. Smithsonian Institution Press, Washington. Cade, W.H. 1984. Genetic variation underlying sexual behavior and reproduction. Am. Zool. 24:355-356. Cardwell, J.R. and N.R. Liley. 1987. Hormonal correlates of sex change and colour phase change in the stoplight parrotfish {Sparisoma viride, Scaridae). In:D.R. Idler, L.W. Crim and J.M. Walsh (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987.p. 129. Cekan, Z. 1975. Assessment of reliability of steroid radioimmunoassays. J. Steroid Biochem. 6:271-275. Chan, S.T.H. 1970. Natural sex reversal in vertebrates. Philos. Trans. R. Soc. London, Ser. B. 259:59-71. Chan, S.T.H. and W.S.B. Yeung. 1983. Sex control and sex reversal in fish under natural conditions, pp. 171-222. In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. LXb. Academic Press, New York. Chan, S.T.H., A. Wright and J.G. Phillips. 1967. The atretic structures in the gonads of the ricefield eel (Monopterus albus) during natural sex-reversal. J. Zool., Lond 153:527-539. Charnov, E.L. 1982. The Theory of Sex Allocation. Princeton Univ. Press. Princeton, NJ. Choat, J.H. and D.R. Robertson. 1975. Protogynous hermaphroditism in fishes of the family Scaridae. In: Intersexuality in the Animal Kingdom. (R. Reinboth, ed.).pp. 263-283. Springer-Verlag, Berlin. Clavijo, I. 1982. Distribution, reproductive biology, and the social structure of the redband parrotfish Sparisom aurofrenatum. Ph.D. Thesis, University of Puerto Rico, Mayaguez. Clutton-Brock, T.H., S.D. Albon, R.M. Gibson and F.E. Guiness. 1979. The logical stag: Adaptive aspects of fighting in red deer (Cervus elaphus L.). Anim. Behav. 27:211-225. 116 Cochran, R.C. 1987. Serum androgens during the annual reproductive cycle of the male mummichog, Fundulus heteroclitus. Gen. Comp. Endocrinol. 65:141-148. Cox, C.R. and BJ. LeBouef. 1977. Female incitation of male competition: a mechanism in sexual selection. Amer. Natur. 111:317-335. Crews, D. and M.C. Moore. 1986. Evolution of mechanisms controlling mating behavior. Science 231:121-125. Crews, D., B. Camazine, M. Diamond, R. Mason, R.R. Tokarz, and W.R. Garstka. 1984. Hormonal independence of courtship behavior in the male garter snake. Horm. Behav. 18:29-41. Culling, C.F.A. 1963. Handbook of Histopathological Techniques. 2nd e d. Butterworth and Co. Ltd., London. 553pp. D'Ancona, U. Ermaphroditismo ed intersessualita nei Teleostei. Experientia. 5:381-389. Davidson, J.M. 1972. Hormones and reproductive behavior. In: Levine, S. (ed.) Hormones and Behavior. Academic Press, New York. Davis, T.L.O. 1977. Reproductive biology of the freshwater catfish Tandanus tandanus Mitchell, in the Gwydir river, Australia. I: structure of the gonads. Aust. J. Mar. Freshwater Res. 28:159-169. DeVlaming, V. 1984. Oocyte development patterns and hormonal involvements among teleosts. In: Rankin, J.C, T.J. Pitcher and R. Duggan (eds.) Control Processes in Fish Physiology, pp. 176-199. CroomHelm, London. Dominey, W. 1984. Alternative mating tactics and evolutionarily stable strategy. Am. Zool. 24:385-396. Donaldson, E.M and T.J. Benfey. 1987. Current status of induced sex manipulation. In: Idler, D.R., L.W. Grim and J.M. Walsh, (eds). Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7, 1987. pp.108-119. Dubin, R.E. 1981. Social behavior and ecology of some Caribbean parrotfish (Scaridae). Ph.D. Thesis. University of Alberta, Edmonton, Canada. Dulka, J.G., P.W. Sorensen and N.E. Stacey. 1987. Socially-stimulated gonadotropin release in male goldfish: differential circadian sensitivities to a steroid pheromone and spawning stimuli. In: Idler, D.R., L.W. Crim and J.M. Walsh. Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. p 160. Emlen, S.T. and L.W. Oring, 1977. Ecology, sexual selection and the evolution of mating systems. Science 197:215-223. Essenberg, J.W. 1926. Complete sex reversal in viviparous teleosts. Biol. Bull. 51:98-109. Etgen, A.M. 1984. Progestin receptors and the activation of female reproductive behavior: A critical review. Horm. Behav. 18:411-430. Fishelson, L. 1970. Protogynous sex reversal in the fish Anthias squamipinnis (Teleostei, Anthiidae) regulated by presence or absence of male fish. Nature (London). 277:90-91. 117 Fishelson, L. 1975. Ecology and physiology of sex reversal in Anthias squamipinnis (Teleostei, Anthiidae). In: Intersexuality in the Animal Kingdom. (R. Reinboth, ed.). pp.284-294. Springer-Verlag, Berlin. Forest, M.G. 1983. Role of androgens in fetal and pubertal development. Horm. Res. 18:69-83. Fostier, A., R. Billard, B. Breton, M. Legendre, and S. Marlot. 1981. Plasma 11-oxotestosterone and gonadotropin during the beginning of spermiation in rainbow trout (Salmo gairdneri, R.) Gen Comp. Endocrinol. 46:428-434. Fostier, A., B. Jalabert, R. Billard, B. Breton and Y. Zohar. 1983. The gonadal steroids. In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. IXa. pp. 277-372. Academic Press, New York. Fostier, A., F. Le Gac and M. Loir. 1987. Steroids in male reproduction. In: Idler, D.R., L.W. Crim and J.M. Walsh. Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. pp.239-245. Fricke, H.W. 1975. Control of different mating systems in a coral reef fish by one environmental factor. Anim. Behav. 28:591-596. Ghiselin, M.T. 1969. The evolution of hermaphroditism among animals. Quart. Rev. Biol. 44:189-208. Gross, M.R. 1982. Sneakers, satellites, and parentals: Polymorphic mating strategies in North American sunfishes. Z. Tierpsychol. 60:1-26. Gross, M.R. 1985. Disruptive selection for alternative life histories in salmon. Nature (London). 313:47-48. Gross, M.R. and E.L. Charnov. 1980. Alternative male life histories in bluegill sunfish. Proc. Natl. Acad. Sci. 77:6937-6940. Hannes, R.P. 1984. Androgen and corticoid levels in blood and body extracts of high- and low-ranking swordtail males (Xiphophorus helleri) before and after social isolation. Z. Tierpsychol. 66: 70-76. Hannes, R.P., D. Franck and F. Liemann. 1984. Effects of rank-order fights on whole-body and blood concentrations of androgens and corticosteroids in the male swordtail (Xiphophorus helleri). Z. Tierpsychol. 65: 53-65. Harding, C F . 1983. Hormonal influences on avian aggressive behavior. In: Svare, B.B. (ed.). Hormones and Aggressive Behavior. Plenum Press, New York. pp. 435-467. Harrington, R.W. Jr. 1974. Sex determination and differentiation in fishes. In: Control of Sex in Fishes. (Schreck, C.B., ed.), VPI-SG-74-01, pp. 4-12. Virginia Polytechnical Institute, Blacksburg. Hart, B.L. 1968. Neonatal castration: influences on neural organization of sexual reflexes in male rats. Science. 160:1135-1136. Henderson, N.E. 1962. the annual cycle in the testis of the eastern brook trout, Salvelinus fontinalis (Mitchill). Can. J. Zool. 40:631-641. 118 Hunter, G.A. and E.M. Donaldson. 1983. Hormonal sex control and its application to fish culture. In: Hoar, W.S., DJ. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. LXb. pp. 223-303. Academic Press, New York. Hutchison, R.E. 1978. Hormonal differentiation of sexual behaviour in Japanese quail. Horm. Behav. 11:363-387. Hoffman, S.G. 1983. Sex-related foraging behavior in sequentially hermaphroditic hogfishes (Bodianus spp.). Ecology 64:798-808. Hoffman, S.G, M.P. Schildauer and R.R. Warner. 1985. The costs of changing sex and the ontogeny of males under contest competition for mates. Evolution. 39:915-927. Hurk, R. van den, and J. Peute. 1979. Cyclic changes in the ovary of the rainbow trout, Salmo gairdneri, with special reference to the sites of steroidogenesis. Cell Tissue Res. 199:289-306. Idler, D.R., R. Reinboth, J.M. Walsh, and B. Truscott. 1976. A comparison of 11-hydroxytestosterone and 11-ketotestosterone in blood of ambisexual and gonochoristic teleosts. Gen. Comp. Endocrinol. 30:517-521. Idler, D.R., L.W. Crim and J.M. Walsh. 1987. (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7, 1987. Illius, A.W., N.B. Haynes, and G.E. Lamming. 1976. Effects of ewe proximity on peripheral plasma testosterone levels and behaviour in the ram. J. Reprod. Fertil. 48:25-32. Johannes, R.E. 1978. Reproductive strategies of coastal marine fishes in the tropics. Environ. Biol. Fishes. 3:65-84. Jones, J.W. and G.M. King 1952. The spawning of the male salmon parr (Salmo salar Linn juv.). J. Zool. London. 122:615-619. Jost, A. 1965. Gonadal hormones in the sex differentiation of the mammalian fetus. In: Organogenesis (R.L. de Haan andH. Ursprung, eds.). pp. 611-628. Holt, New York. Kadmon, G., Z. Yaron and H. Gordin. 1984. Sequence of gonadal events and oestradiol levels in Spams aurata (L.) under two photoperiod regimes. J. Fish Biol. 26:609-620. Kaufmann, 1981. On the definitions and functions of dominance and territoriality. Ann. Rev. Biol. 58:1-20. Khoo, K.H. 1974. Steroidogenesis and the role of steroids in the endocrine control of oogenesis and vitellogenesis in the goldfish, Carassius auratus. Ph.D. Thesis, University of British Columbia, Vancouver, Canada. Khoo, K.H. 1975. The corpeus luteum of goldfish (Carassius auratus L.) and its functions. Can. J. Zool. 53:1306-1323. Khoo, K.H. 1979. The histochemistry and endocrine control of vitellogenesis in goldfish ovaries. Can. J. Zool. 57:617-626. 119 Kime, D.E. and N.J. Manning. 1982. Seasonal patterns of free and conjugated androgens in the brown trout Salmo trutta. Gen. Comp. Endocrinol. 48: 222-231. Kime, D.E. and M. Hyder. 1983. The effect of temperature and gonadotropin on testicular steroidogenesis in Sarotherodon (Tilapia) mossambicus in vitro. Gen. Comp. Endocrinol. 50:105-115. Kjerfve, B., K. Rutzler, and G.H. Kierspe. 1982. Tides at Carrie Bow Cay, Belize. Smiths. Contrib. Mar. Sci. 42:47-51. Kobayashi, M., K. Aida and I. Hanyu. 1988. Hormone changes during the ovulatory cycle in goldfish. Gen. Comp. Endocrinol. 69:301-307. Kyle, A. L., N.E. Stacey, R.E. Peter and R. Billard. 1985. Elevations in gonadotrophin concentrations and milt volumes as a result of spawning behavior in the goldfish. Gen. Comp. Endocrinol. 57:10-22. Lam, T.J. 1983. Environmental influences on gonadal activity. In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. LXb. pp. 65-116. Academic Press, New York. Lamba, V.J., S.V. Goswami, and B.I. Sundararaj. 1983. Circannual and circadian variations in plasma levels of steroids (Cortisol, Estradiol-17p, estrone, and testosterone) correlated with the annual gonadal cycle in the catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 50:205-225. Lazier, C.B., M. Mann, and T.P. Mommsen. 1987. Estrogen receptors and regulation of vitellogenesis. In:D.R. Idler, L.W. Crim and J.M. Walsh (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7, 1987.pp. 178-182. Leshner, A.I. 1978. An Introduction to Behavioral Endocrinology. Oxford University Press, New York. Liley, N.R. 1969. Hormones and reproductive behaviour. In: Hoar, W.S., and D.J. Randall, (eds.). Fish Physiology, vol. Ul, pp. 73-116. Academic Press, New York. Liley, N.R. 1980. Patterns of hormonal control in the reproductive behavior of fish, and their relevance to fish management and culture programs. In: Bardach, J.E., J.J. Magnuson, R.C. May and J.M. Reinhart, (eds.). Fish Behavior and its Use in the Capture and Culture of Fishes. ICLARM Conf. Proc. 5. pp. 210-246. Int. Cent. Living Aquat. Res. Manage., Manila, Philippines. Liley, N.R. and N.E. Stacey 1983. Hormones, pheromones and reproductive behavior. In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. LXb. pp. 1-63. Academic Press, New York. Liley, N.R., A. Fostier, B. Breton andE.S.P. Tan. 1986a. Endocrine changes associated with spawning behavior and social stimuli in a wild population of rainbow trout (Salmo gairdneri) 1. Males. Gen. Comp. Endocrinol. 62:145-156. Liley, N.R., A. Fostier, B. Breton and E.S.P. Tan. 1986b. Endocrine changes associated with spawning behavior and social stimuli in a wild population of rainbow trout (Salmo gairdneri) 2. Females. Gen. Comp. Endocrinol. 62:145-156. 120 Liley, N.R., J.R. Cardwell and Y. Rouger. 1987. Current status of hormones and sexual behaviour in fish. In.D.R. Idler, L.W. Crim and J.M. Walsh (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7, 1987.pp. 142-149. Linville, J.E., L.H. Hanson and S.A. Sower. Endocrine events associated with spawning behavior in the sea lamprey (Petromyzon marinus). Horm. behav. 21:105-117. Lofts, B. Testicular function. In: D.O. Norris and R.E. Jones (eds.). Hormones and Reproduction in Fishes, Amphibians, and Reptiles. 283-325. Plenum Press, New York. MacLusky, N.J. and F. Naftolin. 1981. Sexual differentiation of the central nervous system. Science. 211:1294-1302. Malison, J.A., T.B. Kayes, B.C. Wentworth and C H . Amundson. 1987. Control of sexually related dimorphic growth by gonadal steroids in yellow perch (Perca flavescens). In: Idler, D.R., L.W. Crim, and J.M. Walsh (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. p. 206. Maynard-Smith, J. 1976. Evolution and the theory of games. Amer. Sci. 54:8-14. McLaren, A., E. Simpson, K. Tomonari, P. Chandler and H. Hogg. 1984. Male sexual differentiation in mice lacking H-Y antigen. Nature (London). 312:552-555. Moe, M.A. Jr. 1969. Biology of the red grouper Epinephelus morio (Valenciennes) from the eastern Gulf of Mexico. Fla. Dept. Nat. Resour. Mar. Res. Lab. Prof. Ser. 1-95. Moore, F.L. 1986. Regulation of reproductive behavior. In: D.O. Norris and R.E. Jones (eds.). Hormones and Reproduction in Fishes, Amphibians, and Reptiles. 505-522. Plenum Press, New York. Moore, M . C 1984. Changes in territorial defense produced by changes in circulating levels of testosterone: a possible hormonal basis for mate-guarding behaviour in white-crowned sparrows. Behaviour 88:215-226. Moore, M.C. 1987. Castration affects territorial and sexual behaviour of free-living male lizards, Scleroporus jarrovi. Anim. Behav. 35: 1193-1199. Moore, M.C. and C.A. Marler. 1987. Effects of testosterone manipulations on nonbreeding season territorial aggression in free-living male lizards, Scleroporus jarrovi. Gen. Comp. Endocrinol. 65:225-232. Moyer, J.T. and M.J. Zaiser. 1984. Early sex change: a possible mating strategy of Centropyge Angelfishes (Pisces: Pomacanthidae). J. Ethology. 2:63-67. Munro, A.D. and T.J. Pitcher. 1983. Hormones and agonistic behaviour in teleosts. In: J.C. Rankin, T.J. Pitcher, and R.T. Duggan (eds.), Control Processes in Fish Physiology, pp. 155-175, Croom Helm, London. Munro, A.D. and T.J. Pitcher. 1985. Steroid hormones and agonistic behaviour in a cichlid teleost, Aequidens pulcher. Horm. and Behav. 19:353-371. Nagahama, Y. 1983. The functional morphology of teleost gonads. In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. IXa. pp. 223-275. Academic Press, New York. 121 Ng, T.B. and D.R. Idler. 1983. Yolk formation and differentiation in teleost fishes .In: Hoar, W.S., D.J. Randall, and E.M. Donaldson (eds). Fish Physiology, vol. IXa. pp. 373-404. Academic Press, New York. Norris, D.O. 1986. Regulation of male gonaducts and sex accessory structures. In: D.O. Norris and R.E. Jones (eds.). Hormones and Reproduction in Fishes, Amphibians, and Reptiles. 327-354. Plenum Press, New York. O'Connell, M.E., C. Reboulleau, J.J. Feder, and R. Silver. 1981. Social interactions and androgen levels in birds. I. Female characteristics associated with increased plasma androgen levels in the male ring dove (Streptopelia risoria). Gen. Comp. Endocrinol. 44: 454-463. Ogden, J.C. and N.S. Buckman. 1973. Movements, foraging groups, and diurnal migrations of the striped parrotfish, Scarus croicencis Bloch (Scaridae). Ecology. 64:589-596. Ohno, S., Y. Nagai, S. Ciccarese and H. Iwata. 1979. Testis-organizing H-Y antigen and the primary sex-determining mechanism of mammals. Recent Prog. Horm. Res. 35: 449-476. Okada, Y.K. 1962. Sex reversal in the Japanese wrasse, Halichoeres poecilopterus. Proc. Jap. Acad. 38-508-513. Okada, Y.K. 1964. Effects of androgen and estrogen o sex reversal in the wrasse, Halichoeres poecilopterus. Proc. Jap. Acad. 40: 541-544. O'Malley, B.W. and W.T. Schrader. 1976. The receptors of steroid hormones. Sci. Am. 234:32-43. Phoenix, C.H., R.W. Goy, A.A. Gerall and W.C. Young. 1959. Organizing effects of prenatally administered testosterone propionate in the tissues mediating mating behavior in the female guinea pig. Endocrinology 65: 369-387. Piferrer, F. and E.M. Donaldson. 1987. Influence of estrogen, aromatizable and non-aromatizable androgen during ontogenesis on sex differentiation in coho salmon (Oncorhynchus kisutch). In: Idler, D.R., L.W. Crim and J.M. Walsh, (eds). Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. p.135. Pollard, D.A. 1972. The biology of a landlocked form of the normally catadromous salmoniiform fish Galaxias maculatus (Jenyns).IU: Structure of the gonads. Aust. J. Mar. Freshwater Res. 23:17-38. Policansky, D. 1982. Sex change in plants and animals. Ann. Rev. Ecol. System. 13:471-495. Policansky, D. 1987. Evolution, sex, and sex allocation. Bioscience. 37:466-475. Randall, J.E. 1958. A review of the labrid fish genus Labroides, with descriptions of two new species and notes on ecology. Pac. Sci. 12: 327-347. Randall, J.E. 1967. Food habits of reef fishes of the West Indies. Proc. Int. Conf. Trop. Oceanog., Stud. Trop. Oceanog. 5:655-847. Randall, J.E. and H.A. Randall. 1963. the spawning and early development of the atlantic parrotfish, Sparisoma rubripinne, with notes on other scarid and labrid fishes. Zoologica: N.Y. Zool. Soc. 48:49-59. 122 Reinboth, R. 1962a. Morphologische und funktionelle zweigeschlechtilichkeit bei marinen teleostiern (Serranidae, Sparidae, Centracanthidae, Labridae). Zool. Jb. (Physiol.) 69:405-480. Reinboth, R. 1962b. The effects of testosterone on female Coris julis (L.), a wrasse with spontaneous sex-inversion. Gen. Comp. Endocrinol. 2:abstr.39. Reinboth, R. 1967. Biandric teleost species. Gen. comp. Endocrinol. 9:abstr.l46. Reinboth, R. 1968. Protogynie bei papageifischen (Scaridae). Z. Naturf. 23b:852-855. Reinboth, R. 1970. Intersexuality in fishes. In: Hormones and the environment. Mem. Soc. Endocrinol. 18:516-543. Reinboth, R. 1972. Hormonal control of the teleost ovary. Am. Zool. 12:307-324. Reinboth, R. 1975. Spontaneous and hormone-induced sex-inversion in wrasses (Labridae). Pubbl. Staz. Zool. Napoli 39. suppl. 550-573. Reinboth, R. 1979. On steroidogenic pathways in ambisexual fishes. Proc. Indian Natl. Sci. Acad., B45:421-428. Reinboth, R. 1982. The problem of sexual bipotentiality as exemplified by teleosts. Reprod. Nutr. Dev. 22:397-403. Reinboth, R. 1987. Natural sex inversion. In: Idler, D.R., L.W. Crim and J.M. Walsh, (eds). Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. pp. 124-127. Reinboth, R. 1988. Physiological problems of teleost ambisexuality. Env. Biol. Fishes. 22:249-259. Reinboth, R. and B. Becker. 1984. In vitro studies on steroid metabolism by gonadal tissues from ambisexual teleosts. I. Conversion of [14C]Testosterone by males and females of the protogynous wrasse Coris julis L. Gen. Comp. Endocrinol. 55:245-250. Reinboth, R., A. Mayerova, C. Ebensperger, and U. Wolf. 1987. The occurrence of serological H-Y antigen (Sxs antigen) in the diandric protogynous wrasse, Coris julis (L.) (Labridae, Teleostei). Differentiation 34:13-17. Richter, C.J.J. and H.J.Th. Goos. 1982. (eds). Proc. Second Int. Symp. Reprod. Physiol. Fish. Pudoc, Wageningen, Netherlands. Robertson, D.R. 1972. Social control of sex reversal in a coral reef fish. Science 177:1007-1009. Robertson, D.R. 1974. A study of the ethology and reproductive biology of the labrid fish, Labroides dimidiatus at Heron Island, Great Barrier Reef. Ph.D. Thesis, University of Queensland, St. Lucia, Qld. Robertson, D.R. 1981. The social and mating systems of two labrid fishes, Halichoeres maculipinna and H. garnoti, off the Caribbean coast of Panama. Mar. Biol. 64:327-340. 123 Robertson, D.R. and J.H. Choat. 1974. Protogynous hermpaphroditism and social systems in labrid fish. Proc. Second. Int. Symp. Coral Reefs. 1:217-225. Robertson, D.R. and S.G. Hoffman. 1977. The roles of female mate choice and predation in the mating system of some tropical labroid fishes. Z. Tierpsychyol. 45:298-320. Robertson, D.R., R. Reinboth, and R.W. Bruce. 1982. Gonochorism, protogynous sex-change and spawning in three sparisomatinine parrotfishes from the western Indian Ocean. Bull. Mar. Sci. 32:868-879. Robertson, D.R. and R.R. Warner. 1978. Sexual patterns in the labroid fishes of the western Caribbean, II: The parrotfishes (Scaridae). Smiths. Contrib. Zool. 254:27-54. Roede, M.J. 1972. Color as related to size, sex and behaviour in seven Caribbean labrid fish species (genera Thalassoma, Halichoeres, Hemipteronotus). Stud. Fauna Curacao 24:1-264. Ross, R.M. 1984. Catheterization: a non-harmful method of sex identification for sexually monomorphic fishes. Prog. Fish Cult. 46:151-152. Ross, R.M., G.S. Losey, and M. Diamond. 1983. Sex change in a coral-reef fish: Dependence of stimulation and inhibition on relative size. Science 221:574-575. Sachser, N. and E. Prove. 1986. Social status and plasma-testosterone-titers in male guinea pigs (Cavia apereaf. porcellus). Ethology. 71:103-114. Sadovy, Y. and D.Y. Shapiro. 1987. Criteria for the diagnosis of hermaphroditism in fishes. Copeia. 1987(1): 136-156. Searcy, W.A. and J.C. Wingfield. 1980. The effects of androgen and anti-androgen on dominance and aggressiveness in male red-winged blackbirds. Horm. Behav. 14: 126-135. Schreck, C.B. 1974. Hormonal treatment and sex manipulation in fishes. In: Control of Sex in Fishes. (Schreck, C.B., ed.), VPI-SG-74-01, pp. 84-106. Virginia Polytechnical Institute, Blacksburg. Schumacher, M., J.J. Legros and J. Balthazart. 1987. Steroid hormones, behavior and sexual dimorphism in animals and men: the nature-nurture controversy. Endocrinology. 90:129-156. Scott, A.P., V.J. Bye, and S.M. Baynes. 1980a. Seasonal variations in sex steroids of females rainbow trout (Salmo gairdneri Richardson). J. Fish Biol. 17:587-592. Scott, A.P., V.J. Bye, S.M. Baynes, and J.R.C. Springate. 1980b. Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 17,495-505. Scott, A.P., D.S. MacKenzie and N.E. Stacey. 1984. Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. U. Steroid hormones. Gen. Comp. Endocrinol. 56:349-359. Searcy, W.A. and J.C. Wingfield. 1980. The effects of androgen and antiandrogen on dominance and aggressiveness in male red-winged blackbirds. Horm. and Behav. 14: 126-135. 124 Shapiro, D.Y. 1979. Social behavior, group structure, and the control of sex reversal in hermaphroditic fish. Adv. Study Behav. 10:43-102. Shapiro, D.Y. 1981. The sequence of coloration changes during sex reversal in the tropical marine fish Anthias squamipinnis (Peters). Bull. Mar. Sci. 31:383-398. Shapiro, D.Y. 1987. Differentiation and evolution of sex change in fishes. Bioscience. 37:490-497. Shapiro, D.Y. and R. Lubbock. 1980. Group sex ratio and sex reversal. J. Theor. Biol. 82:411-426. Shih, S.H. and PJ. Yu. Seasonal changes of estradiol and effects of steroid hormones on plasma 17n-estradiol and androgen in the premature grouper, Plectropomus leopardus (abstr. #27). In: First Int. Symp. on Fish Endocrinol., Edmonton, Alberta, Canada. June 12-17 1988. p.38. Siegel, S. 1956. Nonparametric statistics for the behavioral sciences. McGraw-Hill, New York. 312 pp. Silver, R. and M. Cooper. 1983. Avian behavioural endocrinology. Bioscience. 33:567-572. Silverin, B. 1980. Effects of long-acting testosterone treatment on free-living pied flycatchers, Ficedula hypoleuca, during the breeding period. Anim. Behav. 28: 906-912. Silverin, B. and J.C. Wingfield. 1982. Patterns of breeding behaviour and plasma levels of hormones in a free-living population of pied flycatchers, Ficedula hypoleuca. J. Zool. Lond. 198: 117-129. Smith, C L . 1967. Contributions to a theory of hermaphroditism. J. Theor. Biol. 17:76-90. Stacey, N.E. 1977. The regulation of spawning behavior in the female goldfish, Carassius auratus. Ph.D. Thesis. University of British Columbia, Vancouver, Canada. Stacey, N.E. 1981. Hormonal regulation of female sexual behavior in teleosts. Am."Zool. 21:305-316. Stacey, N.E., D.S. MacKenzie, T.A. Marchant, A.L. Kyle and R.E. Peter. 1984. Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. 1. Gonadotropin, growth hormone, and thyroid hormones. Gen. Comp. Endocrinol. 56:333-348. Stacey, N.E., P.W. Sorensen, J.G. Dulka, G.J. Van Der Kraak, and T.J. Hara. 1987. Teleost sex pheromones: recent studies on identity and function. In: Idler, D.R., L.W. Crim and J.M. Walsh, (eds). Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987. pp.150-153. Stoddart, D.R. 1962. Three Caribbean atolls: Turneffe Islands, Lighthouse Reef, and Glover's Reef, British Honduras. Atoll Res. Bull. 87:1-151. Stoll, L.M. 1955. Hormonal control of the sexually dimorphic pigmentation of Thalassoma bifasciatum. Zoologica 40:125-131. 125 Tang, F., S.T.H. Chan and B. Lofts.1974a. Effect of steroid hormones on the process of natural sex reversal in the ricefield eel, Monopterus albus (Zuiew). Gen. Comp. Endocrinol. 24:227-241. Tang, F., S.T.H. Chan and B. Lofts. 1974b. Effect of mammalian luteinizing hormone in the natural sex reversal of the ricefield eel, Monopterus albus (Zuiew). Gen. Comp. Endocrinol. 24:242-248.. Thomas, P., NJ. Brown, and J.M. Trant. 1987, Plasma levels of gonadal steroids during the reproductive cycle of females spotted seatrout Cynoscion nebulosus. In: Idler, D.R., L.W. Crim and J.M. Walsh, (eds.) Proc. Third Int. Symp. on Reprod. Physiol, of Fish. St. John's, Nfld. Aug. 2-7,1987.p 219. Thresher, R.E. 1979. Social behaviour and ecology of two sympatric wrasses (Labridae: Halichoeres sp.) off the coast of Florida. Mar. Biol. 53:161-172. Thresher, R.E. 1984. Patterns of Reproduction in Coral Reef Fishes. TFH publications, Neptune City. 399 pp. Tokarz, R.R. 1987. Effects of the antiandrogens cyproterone acetate and flutamide on male reproductive behavior in a lizard (Anolis sagrei). Horm. and Behav. 21:1-16. Tomlinson, N. 1966. The advantages of hermaphroditism and parthenogenesis. J. Theor. Biol. 11:54-58. Tribble, G.W. 1982. Social organization, patterns of sexuality, and behaviour of the wrasse Coris dorsomaculata at Miyaka-jima, Japan. Environ. Biol. Fish. 7:29-38. Trivers, R.L. 1972. Parental investment and sexual selection. In: Campbell, B.(ed). Sexual Selection and the Descent of Man. 1871-1971. 136-179. Aldine, Chicago. Turner, C L . 1946. Retention of response to ethynyl testosterone in females of Gambusia affinis. J. Exp. Zool. 102:357-369. Van Der Kraak, G., H.M. Dye, and E.M. Donaldson. 1984. Effects of LH-RH and des-Glyio[D-Ala6]LH-RH-ethylamide on plasma sex steroid profiles adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 55:36-45. Victor, B. 1987. The mating system of the Caribbean straight-tailed razorfish Xyrichtys martinicencis. Bull. Mar. Sci. 40:152-160. Villars, T.A. 1983. Hormones and aggressive behavior in Teleost fishes. In: Svare, B.B. (ed.), Hormones and aggressive behavior. Plenum Press, New York. pp. 407-433. Wachtel, S.S., G.C. Koo and E.A Boyse. 1975. Evolutionary conservation of HY (male) antigen. Nature (London). 254:270-272. Wallen, K. and L.A. Winston. 1984. Social complexity and hormonal influences on sexual behaviour in rhesus monkeys (Macaca mulatto). Physiol. Behav. 32:629-637. Warner, R.R. 1975a. The adaptive significance of sequential hermaphroditism in animals. Am. Nat. 109:61-82. 126 Warner, R.R. 1975b. The reproductive biology of the protogynous hermaphrodite Pimelometopon pulchrum (Pisces: Labridae). Fish. Bull. 73:262-283. Warner, R.R. 1982. Mating systems, sex change and sexual demography in the rainbow wrasse, Thalassoma lucasanum. Copeia. 1982(3):653-661. Warner, R.R. 1984a. Mating behavior and hermaphroditism in coral reef fishes. Am. Scient. 72:128-136. Warner, R.R. 1984b. Deferred reproduction as a response to sexual selection in a coral reef fish: a test of the life historical consequences. Evolution. 38:148-162. Warner, R.R. 1988. Sex change in fishes; hypotheses, evidence and objectives. Environ. Biol. Fish. 22:81-90. Warner, R.R. and I.F. Downs. 1977. Comparative life histories: Growth vs reproduction in normal males and sex-changing hermaphrodites of the striped parrotfish, Scarus croicencis. Proc. Third Int. Coral Reef Symp. Rosenstiel School of Mar. Atm. Sci, Univ. of Miami. Warner, R.R. and S.G. Hoffman. 1980. Local population size as a determinant of mating system and sexual composition in two tropical marine fishes (Thalassoma spp.). Evolution 34:508-518. Warner, R.R. and D.R. Robertson. 1978.Sexual patterns in the labroid fishes of the western Caribbean, I: The wrasses (Labridae). Smiths. Contrib. Zool. 254:1-27. Warner, R.R., D.R. Robertson, and E.G. Leigh, Jr. 1975. Sex change and sexual selection. Science. 190:633-638. Wiberg, U.H. 1987. Facts and considerations about sex-specific antigens. Human Genetics 76:207-219. Wilson, J.D., F.W. George, and J.E. Griffin. 1981. The hormonal control of sexual development. Science. 211:1278-1284. Wingfield, J.C. 1984a. Environmental and endocrine control of reproduction in the song sparrow, Melospiza melodia. I: temporal organization of the breeding cycle. Gen. Comp. Endocrinol. 56:406-416. Wingfield, J.C. 1984b. Environmental and endocrine control of reproduction in the song sparrow, Melospiza melodia. U: agonistic interactions as environmental information stimulating secretion of testosterone. Gen. Comp. Endocrinol. 56:417-424. Wingfield, J.C. 1984c. Androgens and mating systems: testosterone-induced polygyny in normally monogamous birds. The Auk. 101:665-671. Wingfield, J.C. 1985. Short term changes in plasma levels of hormones during establishment and defense of a breeding territory in male song sparrows, Melospiza melodia. Horm. Behav. 19,174-187. Wingfield, J.C. and A.S. Grimm. 1977. Seasonal changes in plasma Cortisol, testosterone and oestradiol-17p in the plaice, Pleuronectes platessa L. Gen. Comp. Endocrinol. 31:1-11. 127 Wingfield, J.C. and B. Silverin. 1986. Effects of corticosterone on territorial behaviour of free living male song sparrows Melospiza melodia. Horm. and Behav. 20: 405-417. Wingfield, J.C, G.F. Ball, A.M. Dufty Jr., R.E. Hegner, and M. Ramenofsky. Testosterone and aggression in birds. Am. Sci. 75:602-608. Winn, H.E. and J.E. Bardach. 1957. Behavior, sexual Achromatism and species of parrotfishes. Science. 125:885-886. Winn, H.E. and J.E. Bardach. 1960. Some aspects of the comparative biology of parrotfishes at Bermuda. Zoologica: N.Y. Zool. Soc. 45:29-35. Witschi, E. and W.N. Crown. 1937. Hormones and sex determination in fishes and frogs. Anat. Rec. Suppl. 70(1):205. Wittenberger, J.F. 1981. Animal Social Behavior. Duxbury Press, Boston. Wydoski, R. and L. Emory. 1983. Tagging and marking. In: Nielsen, L.A. and D.L. Johnson, (eds.). Fisheries Techniques. Am. Fish. Soc, Southern Printing Company, Blacksburg. 215-237. Yamamoto, T. 1962. Hormonic factors affecting gonadal sex differentiation in fish. Gen. Comp. Endocrinol. Suppl. 1:341-345. Yamamoto, T. 1969. Sex differentiation. In: Hoar, W.S. and D.J. Randall (eds). Fish Physiology, vol. III. pp.117-175. Academic Press, New York. Yeung, W.S.B. and S.T.H. Chan. 1987. The plasma sex steroid profiles in the freshwater, sex-reversing teleost fish Monopterus albus (Ziuew). Gen. Comp. Endocrinol. 65:233-242. Zar, J.H. 1984. Biostatistical Analysis. Prentice Hall, London. 620 pp. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098250/manifest

Comment

Related Items