Physical interactions facilitate sex change in the protogynous orange-spotted grouper,Epinephelus coioides

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange-spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11-ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change.     

Socially controlled sex-change in the half-moon grouper,Epinephelus rivulatus,at Ningaloo Reef,Western Australia

The cues controlling sex-change have been elucidated for various species of hermaphroditic fishes that inhabit coral reefs, but not for the epinepheline serranids. A male removal experiment conducted on an assemblage of the half-moon grouper, Epinephelus rivulatus, demonstrated that protogynous sex-change in this species is socially controlled, possibly by the suppressive dominance of males and a threshold sex ratio. The experiment showed that a reproductively ripe female can change sex and become a male with ripening testis within 3 weeks. However, this process can be delayed, slowed, or stopped by the presence of other males in the area.     

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring,sex hormone shifts and gene profiles

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42′ 02.6″ N, 114° 32′ 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17β-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.     

The influence of social factors on juvenile sexual differentiation in a diandric,protogynous grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The influence of social factors on juvenile sexual differentiation was explored in a diandric, protogynous grouper Epinephelus coioides. Experimental social units were established as singles, pairs and quartets using sexually undifferentiated juveniles at an age of 22 weeks post-hatching (WPH); all gonads were examined histologically at 130 WPH, the age of first sexual maturation. The percentage of primary males was about 39% at the end of the experiment, higher than the <5% obtained under mariculture conditions or from wild stocks for similar age or body size. This study demonstrated for the first time that social factors can significantly influence juvenile sexual differentiation in E. coioides.     

Size, maturation and the social control of sex reversal in the coral reef fish Anthais squamipinnis

A major hypothesis to explain the causal initiation of protogynous sex reversal is that females change sex upon reaching a critical size. A study of the coral reef fish Anthias squamipinnis shows that the size hypothesis does not hold. Females from two neighbouring, but spatially discrete and probably genetically homogeneous populations on Aldabra Island changed sex at distinctly different sizes. Previous laboratory and field studies in which sex reversal has followed the removal of a male from social groups have been uncontrolled and thus permit the interpretation that sex reversal is caused by non-specific social disruption or by causes other than male removal. In this study, a male was removed from each of eleven single-male and five multi-male social groups in the laboratory ( N = 8 male removals) and in the field ( N = 19 male removals). In each group, the result was that one female changed sex. Laboratory controls made it unlikely that sex reversal was induced by non-specific disruption and field observations showed that sex reversals resulted from male removals and were not coincidental, ongoing events. Previous statements that sex change is controlled by the presence or absence of a male, by inhibition of a female's tendency to change sex, or by aggression or dominance are shown, by an analysis of the complexity of issues, to be premature. Gonadal histology on 130 specimens confirmed that this species is a monandric, protogynous hermaphrodite and provided details of gonadal transformation.     

Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper,Epinephelus coioides

Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.     

Field evidence for bi-directional sex change in the polygynous gobiid fish Trimma okinawae

The social condition of bi-directional sex change in the gobiid fish Trimma okinawae was investigated at Akamizu Beach, Kagoshima, Japan. Social groups of T. okinawae usually consisted of a large male and one or more smaller females. The number of females in the group was positively correlated with male body size and groups were usually separated from each other by 1–3 m. In total, 22 instances of female-to-male sex change and three instances of male-to-female sex change were observed during the 16 months that social groups were monitored. Two individuals changed sex twice: female to male and back to female. Female-to-male sex change occurred when the male disappeared from a group. Either the largest remaining female changed sex to male or a large female from another group immigrated and changed sex to male. Larger individuals appear to benefit from becoming male because they can monopolize the breeding opportunities with several females, as reported in other protogynous fishes. Sex change from male-to-female only occurred when a solitary male joined another group as a subordinate. Mortality rates are high in these small fish, therefore joining another group and reproducing as a female is likely to increase the reproductive value of a solitary male.     

Evidence that estrogen regulates the sex change of honeycomb grouper (Epinephelus merra), a protogynous hermaphrodite fish

Circulating estradiol-17beta (E2) levels decrease precipitously during female to male (protogynous) sex change in fish. Whether this drop in E2 levels is a cause or consequence of sex change is still largely unknown. The present study treated adult female honeycomb groupers (Epinephelus merra) with aromatase inhibitor (AI, Fadrozole), either alone or in combination with E2, to investigate the role of estrogen in protogynous sex change. Control fish had ovaries undergoing active vitellogenesis; the gonads of AI-treated fish had already developed into testes, which produced sperm capable of fertilization. In contrast, co-treatment of fish with E2 completely blocked AI-induced sex reversal. AI treatment significantly reduced circulating levels of E2, whereas the addition of E2 to AI prevented the loss. The plasma androgen (testosterone and 11-ketotestosterone) levels were increased in the AI-treated fish, while the levels in the E2-supplemented fish were low compared to controls. Present results show that E2 plays an important role in maintaining female sex of hermaphrodite fishes, and that the inhibition of E2 synthesis causes oocyte degeneration leading to testicular differentiation in the ovary.     

Bidirectional sex-change behavior and physiological aspects in the Gorgeous goby Lythrypnus pulchellus (Gobiidae)

The Gorgeous goby Lythrypnus pulchellus shows extreme sexual plasticity with the bidirectional sex-change ability socially controlled in adults. Therefore, this study describes how the hierarchical status affects hormone synthesis through newborn hormone waste products in water and tests the influence of body size and social dominance establishment in sex reversal duration and direction. The associated changes in behavior and hormone levels are described under laboratory conditions in male–male and female–female pairs of similar and different body sizes, recording the changes until spawning. The status establishment occurred in a relatively shorter time period in male and female pairs of different sizes (1–3 days) compared to those of similar size (3–5 days), but the earlier one did not significantly affect the overall time of sex change (verified by pair spawning). The changes in gonads, hormones, and papilla occurred in sex-changer individuals, but the first one was observed in behavior. Courtship started at 3–5 days in male pairs and from 2 h to 1 day in female pairs of both groups of different and similar sizes. Hormones did not gradually move in the new sexual phenotype direction during the sex-change time course. Nonetheless, estradiol regulated sex change and 11-ketotestosterone enabled bidirectional sex change and was modulated by agonistic interactions. Cortisol is associated with status and gonadal sex change. In general, similar mechanisms underlie sex change in both directions with a temporal change sequence in phases. These results shed new light on sex-change mechanisms. Further studies should be performed to determine whether these localized changes exist in the steroid hormone synthesis along the brain–pituitary gonad axis during social and bidirectional sex changes in L. pulchellus.     

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.     

Spawning dynamics of orange roughy,Hoplostethus atlanticus,in mid-slope waters of New Zealand

Synopsis The social and reproductive biology of the sand tilefish,Malacanthus plumieri (Malacanthidae), was studied at Glover's Reef, Belize, where this species occurs in colonies over sand-rubble flats. Individuals each occupy a home burrow refuge and a surrounding home range. Home range overlap among adjacent fish of the same sex is low, and individuals defend exclusive use of much of their home range against all conspecifics except mates (i.e., territoriality). Areas defended by males overlap the territories of up to 6 females; and male territory area is positively related to the number of female residents. Males maintain dominance over females within their territories by aggression, including intervention into some female disputes. Females spawn pelagically-dispersed eggs as frequently as every day. Each female spawns near her burrow, almost exclusively with the male whose defended area encompasses her territory (harem polygyny). Tilefish colonies therefore consist of a mosaic of female territories over which adjacent male territories are superimposed. Histological evidence and observation of behavioral sex change in one female revealed thatM. plumieri is capable of protogynous sex reversal. Females did not change sex in response to removal of one male. Occurrence of small transitional fish indicates that the onset of sex change is controlled by factors other than size-related social hierarchies within harems or colonies.     

Sex change of primary males in a diandric labrid Halichoeres trimaculatus: coexistence of protandry and protogyny within a species

In the threespot wrasse Halichoeres trimaculatus , sex change of primary males was observed in the field and confirmed by aquarium experiments. In other words, protandry and protogyny coexisted within this species. Moreover, male-to-female-to-male sex change and female-to-male-to-female sex change were observed in aquarium experiments; i.e . reversed sex change occurred in both protandrous and protogynous hermaphrodites. These results suggest that only the direction of sex differentiation before maturation may differ between the two sexual types that have been regarded as a primary male and a protogynous hermaphrodite.     

Induction of female-to-male sex change in the honeycomb grouper (Epinephelus merra) by 11-ketotestosterone treatments

The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol-17beta (E2) levels alone triggers female-to-male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to-male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to-male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.     

Reproductive styles of shallow-water groupers (Pisces: Serranidae) in the eastern Gulf of Mexico and the consequences of fishing spawning aggregations

Synopsis Seasonal and spatial aspects of spawning for three commercially important grouper species in the northeastern Gulf of Mexico are detailed. These species — all of which are protogynous hermaphrodites - spawn in deep water (> 25 m for red grouper,> 40 m for gag and scamp), making it difficult to observe spawning behaviors without ROV or submersible support. They respond to intense fishing pressure in ways that are directly related to their respective reproductive styles. Species that aggregate appear to be more susceptible to such pressures than those that do not, as evidenced by marked skewing of sex ratios in favor of females. Gag, Mycteroperca microlepis, have suffered a drop in the proportion of males from 17% to 1% in the last 20 years; scamp, Mycteroperca phenax, have dropped from 36% to 18%; and red grouper, Epinephelus morio, which do not aggregate, have shown little change in the sex ratio over the past 25–30 years.     

Status-dependent behavioural sex change in a polygynous coral-reef fish, Halichoeres melanurus

To examine how a change in an individual's social status could influence its behavioural sex, we conducted male "removal-and-return" experiments in the polygynous wrasse, Halichoeres melanurus. This coral-reef fish is a protogynous hermaphrodite: the largest female (LF) living in a male's territory typically completes functional sex change within 2–3 weeks after the male's disappearance. In this experiment we removed males from their territories just prior to spawning time, about 1 h before sunset. In 12 of 30 trials, the resident LF spawned in the male role with smaller females, 21–98 min after male removal. Previous research suggests the LF should readily adopt male sexual behaviour to retain smaller females as future mates. However, the LFs of smaller body size were less likely to immediately perform male-role behaviour. This could be related to females' preference for larger mates: smaller LFs would be less likely to be chosen by other females, even if they could complete sex change and defend a territory. When a male was returned immediately after an occurrence of female–female spawning, the LF subsequently spawned in the female role with the returned male (6 of 12 trials). It could be adaptive for the LFs to accept a larger male as a mate rather than to fight against it. Thus, behavioural sex is reversible in H. melanurus, changing rapidly with social status. Electronic Publication     

Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.     

Sex inversion of sexually immature honeycomb grouper (Epinephelus merra) by aromatase inhibitor

Previous studies have shown that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that the treatments with aromatase inhibitor (AI) cause estrogen depletion and complete sex inversion of pre-spawning females into functional males. In the present study, we examined whether AI causes sex inversion of sexually immature females. Female honeycomb groupers were implanted with various doses of Fadrozole (0, 100, 500 and 1000 microg/fish) in the non-breeding season, and resultant changes in the gonadal structures and the plasma levels of sex steroid hormones (estradiol-17 beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) were examined three months after implantation. Vehicle-implanted groups did not change sex, while 100 and 500 microg AI-implanted groups had turned into transitionals with intersex gonad. In contrast, the highest dose receiving group exhibited both transitional and male phases. Transitional phase gonad had atretic oocytes and spermatogenic germ cells at the late stages of spermatogenesis, while male phase testis contained spermatozoa accumulated in the seminiferous tubules. All males released sperm upon slight pressure on the abdomen. In the AI-implanted fish, plasma levels of E2 decreased in a dose-dependent manner, while the levels of 11-KT were high in the highest dose receiving group. Present results suggest that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that treatments with AI potentially inhibits endogenous E2 production in vivo, causing oocyte degeneration and subsequently the sex inversion from female to male. The Fadrozole could be an important tool for manipulating the sex of hermaphrodite fishes.     

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex‐linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex‐reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress‐induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North‐Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female‐to‐male sex‐reversed adults had similar body mass as normal males. We recorded no events of male‐to‐female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human‐induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex‐reversed individuals surviving to adulthood may participate in breeding.