Meta-analysis of gonadal transcriptome provides novel insights into sex change mechanism across protogynous fishes.

Protogyny, being capable of changing from female to male during their lifetime, is prevalent in 20 families of teleosts but is believed to have evolved within specific evolutionary lineages. Therefore, shared regulatory factors governing the sex change process are expected to be conserved across protogynous fishes. However, a comprehensive understanding of this mechanism remains elusive. To identify these factors, we conducted a meta-analysis using gonadal transcriptome data from seven species. We curated data pairs of ovarian tissue and transitional gonad, and employed ratios of expression level as a unified criterion for differential expression, enabling a meta-analysis across species. Our approach revealed that classical sex change-related genes exhibited differential expression levels between the ovary and transitional gonads, consistent with previous reports. These results validate our methodology's robustness. Additionally, we identified novel genes not previously linked to gonadal sex change in fish. Notably, changes in the expression levels of acetoacetyl-CoA synthetase and apolipoprotein Eb, which are involved in cholesterol synthesis and transport, respectively, suggest that the levels of cholesterol, a precursor of steroid hormones crucial for sex change, are decreased upon sex change onset in the gonads. This implies a potential universal influence of cholesterol dynamics on gonadal transformation in protogyny.

Protein expression patterns and metal metabolites in a protogynous hermaphrodite fish, the ricefield eel (Monopterus albus).

BACKGROUND: The ricefield eel Monopterus albus undergoes a natural sex change from female to male during its life cycle, and previous studies have shown the potential mechanisms of this transition at the transcriptional and protein levels. However, the changes in protein levels have not been fully explored, especially in the intersexual stage. RESULTS: In the present study, the protein expression patterns in the gonadal tissues from five different periods, the ovary (OV), early intersexual stage gonad (IE), middle intersexual stage gonad (IM), late intersexual stage gonad (IL), and testis (TE), were determined by untargeted proteomics sequencing. A total of 5125 proteins and 394 differentially expressed proteins (DEPs) were detected in the gonadal tissues. Of the 394 DEPs, there were 136 between the OV and IE groups, 20 between the IM and IE groups, 179 between the IL and IM groups, and 59 between the TE and IL groups. Three candidate proteins, insulin-like growth factor 2 mRNA-binding protein 3 isoform X1 (Igf2bp3), triosephosphate isomerase (Tpi), and Cu-Zn superoxide dismutase isoform X1 [(Cu-Zn) Sod1], were validated by western blotting to verify the reliability of the data. Furthermore, metal metabolite-related proteins were enriched in the IL vs. IM groups and TE vs. IL groups, which had close relationships with sex change, including Cu2+-, Ca2+-, Zn2+- and Fe2+/Fe3+-related proteins. Analysis of the combined transcriptome data revealed consistent protein/mRNA expression trends for two metal metabolite-related proteins/genes [LOC109953912 and calcium Binding Protein 39 Like (cab39l)]. Notably, we detected significantly higher levels of Cu2+ during the sex change process, suggesting that Cu2+ is a male-related metal metabolite that may have an important function in male reproductive development. CONCLUSIONS: In summary, we analyzed the protein profiles of ricefield eel gonadal tissues in five sexual stages (OV, IE, IM, IL, and TE) and verified the plausibility of the data. After preforming the functional enrichment of metal metabolite-related DEPs, we detected the contents of the metal metabolites Zn2+, Cu2+, Ca2+, and Fe2+/Fe3+ at these five stages and screened for (Cu-Zn) Sod1 and Mmp-9 as possible key proteins in the sex reversal process.

Sacculina-Induced Morphological Feminization in the Grapsid Crab Pachygrapsus crassipes.

Rhizocephalan barnacles (Thecostraca: Cirripedia) are parasitic crustaceans that lack appendages, segmentation, and a digestive system in adults, while instead infiltrating their hosts with a nutrient-absorbing system of rootlets. Sacculinids, belonging to the Rhizocephala order, are known for their various parasitization-induced effects on their decapod hosts, such as parasitic castration, reduction in the growth of secondary sexual characteristics, feminization of male crabs, and alteration of host behavior. In this study, we conducted field surveys in Japan at Manazuru Town (Kanagawa) on the Pacific coast, and on Sado Island and Noto Peninsula on the Sea of Japan side, and found that sacculinid-parasite-ratios on the grapsid crab Pachygrapsus crassipes were particularly high on the Sea of Japan coast. Molecular phylogenetic analysis revealed that the Manazuru population forms a single clade with Sacculina yatsui, and both Sado and Noto populations form a single clade with S. confragosa. We further demonstrated that external morphologies of male P. crassipes parasitized by sacculinids were changed to female phenotypes. This host-parasite interaction will be a useful model for understanding molecular mechanisms underlying rhizocephalan-driven morphological and behavioral feminization and castration.

New cells added to the preoptic area during sex change in the common clownfish Amphiprion ocellaris.

Sex differences in cell number in the preoptic area of the hypothalamus (POA) are documented across all major vertebrate lineages and contribute to differential regulation of the hypothalamic-pituitary-gonad axis and reproductive behavior between the sexes. Sex-changing fishes provide a unique opportunity to study mechanisms underlying sexual differentiation of the POA. In anemonefish (clownfish), which change sex from male to female, females have approximately twice the number of medium-sized cells in the anterior POA compared to males. This sex difference transitions from male-like to female-like during sex change. However, it is not known how this sex difference in POA cell number is established. This study tests the hypothesis that new cell addition plays a role. We initiated adult male-to-female sex change in 30 anemonefish (Amphiprion ocellaris) and administered BrdU to label new cells added to the POA at regular intervals throughout sex change. Sex-changing fish added more new cells to the anterior POA than non-changing fish, supporting the hypothesis. The observed effects could be accounted for by differences in POA volume, but they are also consistent with a steady trickle of new cells being gradually accumulated in the anterior POA before vitellogenic oocytes develop in the gonads. These results provide insight into the unique characteristics of protandrous sex change in anemonefish relative to other modes of sex change, and support the potential for future research in sex-changing fishes to provide a richer understanding of the mechanisms for sexual differentiation of the brain.

Gonadal and cellular dynamics during protogynous sex change in the harlequin sandsmelt Parapercis pulchella.

Some teleost fishes change their sex, and some of these fishes have specific gonads known as "ovotestes," that is, gonads containing both ovarian and testicular tissues. In this study, we revealed the gonadal transformation process and cell dynamics during the female-to-male sex change in the harlequin sandsmelt, Parapercis pulchella (Pinguipetidae), in which females possess ovotestes. Histological observations revealed that although female ovotestes were composed of oocytes, a few cysts of male germ cells were observed among them. At the initial phase of sex change, male germ cells increased, and spermatogenesis proceeded. After that, oocytes decreased and finally disappeared, and the gonads became functional testes. Immunohistochemistry using antibodies against Pcna (proliferating cell nuclear antigen) as a cell proliferation marker revealed that spermatogonia were Pcna positive, whereas spermatocytes were negative, in female ovotestes. This suggests that spermatogenesis is arrested at the spermatocyte stage. In addition, some somatic cells surrounding oocytes, which were thought to be the female follicle cells, were Pcna positive during sex change, indicating that these cells proliferate during sex change and are reused in male testes after sex change. Also, immunostaining using antibodies against active cleaved-Caspase3a as an apoptosis marker demonstrated that oocytes degenerated through apoptotic cell death at the late transition stage. Together with previous findings in other fishes, these findings suggested that the histological processes in gonads during sex change, such as the order of developmental events, developmental fates of ovarian cavities, and ovotestis structures, are diversified among fish species. In contrast, cellular dynamics of female germ and somatic cells during sex change are common among protogynous species.

Bidirectional sex change and plasticity of gonadal phases in the goby Lubricogobius exiguus.

Of the ca. 500 known hermaphroditic fish species, bidirectional sex change and simultaneous hermaphroditism are currently known in 69 and 57 species, respectively. Both bidirectional sex change and simultaneous hermaphroditism are predicted to evolve when mating opportunities are limited, such as in cases of low-density distribution and low mobility of individuals. However, the plasticity of sex is adaptive in obtaining mating opportunities, especially when there is mate loss. Pair-rearing experiments and histological observations of gonads of the goby Lubricogobius exiguus, which has low-density distributions and low mobility, showed bidirectional sex change. The male-role individuals in pairs had gonads in which only the testis was functional (male-phase), whereas the female-role individuals had two types of gonads: only the ovary was functional (female-phase) or both testis and ovary were functional (simultaneously hermaphroditic phase, SH-phase). In addition, single-rearing experiments showed SH-phase gonads in all individuals, and some cyclic spawning but no self-fertilization occurred. These results revealed that L. exiguus has an unusual sexual pattern among hermaphroditic fishes because they undergo bidirectional sex change and some maintain SH-phase gonads. These findings indicate that the low-density distribution and low mobility of L. exiguus in their natural habitat may have influenced the evolution of this unique sexual pattern.

Proteome and Transcriptome Analysis of Gonads Reveals Intersex in Gigantidas haimaensis.

Sex has proven to be one of the most intriguing areas of research across evolution, development, and ecology. Intersex or sex change occurs frequently in molluscs. The deep-sea mussel Gigantidas haimaensis often dominates within Haima cold seep ecosystems, but details of their reproduction remain unknown. Herein, we conducted a combined proteomic and transcriptomic analysis of G. haimaensis gonads to provide a systematic understanding of sexual development in deep-sea bivalves. A total of 2,452 out of 42,238 genes (5.81%) and 288 out of 7,089 proteins (4.06%) were significantly differentially expressed between ovaries and testes with a false discovery rate (FDR) <0.05. Candidate genes involved in sexual development were identified; among 12 differentially expressed genes between sexes, four ovary-biased genes (ß-catenin, fem-1, forkhead box L2 and membrane progestin receptor α) were expressed significantly higher in males than females. Combining histological characteristics, we speculate that the males maybe intersex undergoing sex change, and implied that these genes may be involved in the process of male testis converting into female gonads in G. haimaensis. The results suggest that this adaptation may be based on local environmental factors, sedentary lifestyles, and patchy distribution, and sex change may facilitate adaptation to a changing environment and expansion of the population. The findings provide a valuable genetic resource to better understand the mechanisms of sex change and survival strategies in deep-sea bivalves.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex-ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex-reversing stimuli more frequently, which may lead to higher sex-reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR-based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L-1 17α-ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L-1 EE2 and a glyphosate-based herbicide with 3 µg L-1 or 3 mg L-1  glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

Stable and persistent male-like behavior during male-to-female sex change in the common clownfish Amphiprion ocellaris.

Many fish species exhibit natural sex change as part of their life, providing unique opportunities to study sexually-differentiated social behaviors and their plasticity. Past research has shown that behavioral sex change in the female-to-male (protogynous) direction occurs rapidly and well before gonadal sex change. However, little is known about the timecourse of behavioral sex change in male-to-female (protandrous) sex-changing species, limiting our ability to compare patterns of behavioral sex change across species and identify conserved or divergent underlying mechanisms. Using the protandrous sex changing anemonefish Amphiprion ocellaris, we assessed behavior (aggression and parental care) and hormones (estradiol and 11-ketotestosterone) in fish over six months of sex change, and compared those fish against their non-changing partners as well as control males and females. Contrary to expectations, we found that sex-changing fish displayed behavior that was persistently male-like, and that their behavior did not become progressively female-like as sex change progressed. Hormones shifted to an intermediate profile between males and females and remained stable until gonads changed. These results support a new perspective that the timecourse for protandrous sex change in anemonefish is completely distinct from other well-established models, such that behavioral sex change does not occur until after gonadal sex change is complete, and that sex-changing fish have a stable and unique behavioral and hormonal phenotype that is distinct from a male-typical or female-typical phenotype. The results also identify aspects of sex change that may fundamentally differ between protandrous and protogynous modes, motivating further research into these remarkable examples of phenotypic plasticity.

Cyt-C Mediated Mitochondrial Pathway Plays an Important Role in Oocyte Apoptosis in Ricefield Eel (Monopterus albus).

Apoptosis plays a key role in the effective removal of excessive and defective germ cells, which is essential for sequential hermaphroditism and sex change in vertebrates. The ricefield eel, Monopterus albus is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. Previous studies have demonstrated that apoptosis is involved in sex change in M. albus. However, the apoptotic signaling pathway is unclear. In the current study, we explored the underlying mechanism of apoptosis during gonadal development and focused on the role of the mitochondrial apoptosis signaling pathway in sex change in M. albus. Flow cytometry was performed to detect apoptosis in gonads at five sexual stages and ovary tissues exposed to hydrogen peroxide (H2O2) in vitro. Then the expression patterns of key genes and proteins in the mitochondrial pathway, death receptor pathway and endoplasmic reticulum (ER) pathway were examined. The results showed that the apoptosis rate was significantly increased in the early intersexual stage and then decreased with the natural sex change from female to male. Quantitative real-time PCR revealed that bax, tnfr1, and calpain were mainly expressed in the five stages. ELISA demonstrated that the relative content of cytochrome-c (cyt-c) in the mitochondrial pathway was significantly higher than that of caspase8 and caspase12, with a peak in the early intersexual stage, while the levels of caspase8 and caspase12 peaked in the late intersexual stage. Interestingly, the Pearson's coefficient between cyt-c and the apoptosis rate was 0.705, which suggests that these factors are closely related during the gonadal development of M. albus. Furthermore, the cyt-c signal was found to be increased in the intersexual stage by immunohistochemistry. After incubation with H2O2, the mRNA expression of mitochondrial pathway molecules such as bax, apaf-1, and caspase3 increased in ovary tissues. In conclusion, the present results suggest that the mitochondrial apoptotic pathway may play a more important role than the other apoptotic pathways in sex change in M. albus.

Age-based life history of three groupers in the southern Gulf of Mexico.

The three most important grouper fishery species in southern Gulf of Mexico, Epinephelus morio, Mycteroperca bonaci and Mycteroperca microlepis, do not have life history parameters data reported such as growth, age and natural mortality essential to management. Individuals from the three species were captured in the Campeche Bank (CB) at different times from 1988 to 2001. Sagittae were thin-sectioned to count annuli: n = 1081 for E. morio, n = 920 for M. bonaci and n = 251 for M. microlepis. Otoliths from the three species exhibited successive opaque and translucent bands deposited with an annual frequency, occurring between January and April for E. morio and M. bonaci, and between January and March for M. microlepis. Estimated age in the three grouper species ranged from 0+ to 20 years. The most frequent ages were 7-10 years for E. morio and 8-12 years for M. bonaci and M. microlepis. The von Bertallanfy Growth Function model parameters (L∞ , K and t0 ) were consistent with reported growth patterns for Epinephelidae family species, as established by growth performance Ф' (near 3). The CB groupers appear to reach maximum ages over 13 years with K values between 0.09 and 0.12 year-1 , indicating all three studied species displayed relatively slow growth rate and medium longevity. Lifespan values ranged from 13 to 33 years for E. morio, from 18 to 30 years for M. bonaci and from 17 to 33 years for M. microlepis. A50 was 6.1 years for E. morio and 5.5 years for both M. bonaci and M. microlepis. Age at which all females were mature was 8 years for M. bonaci and 10 years for E. morio and M. microlepis. P50 was 9.3 years for E. morio, 13.6 years for M. bonaci and 11.6 years for M. microlepis. Age at which 100% of individuals were male was 13 years for E. morio, 19 years for M. bonaci and 15 years for M. microlepis. Values of M at age exhibited an exponential decrease with age for all three species, but overall values were 0.13 year-1 for E. morio, 0.14 year-1 for M. bonaci and 0.17 year-1 for M. microlepis. For the three species size at age varied by geographical distribution area, but particularly for E. morio the discrepancy in average size between published studies may be the consequence of the fishing effort exerted on the stock and/or the lack of regulation for the fishery prior to 2001; after this stock biomass fell below the reference limit point and was declared overexploited. Resource managers could use the information provided in this study as a reference, representing pre-overexploitation values, to evaluate the current status of these populations and ensure sustainable management actions for the CB grouper fisheries.

A Comparative Study of Urinary Complication Rates before and after the Incorporation of a Urethral Lengthening Technique during Masculinizing Genital Gender Affirmation Surgery.

Objectives Masculinizing genital gender affirmation surgery (MgGAS) consists of operative procedures designed to help the transition of transmen in their journey toward male gender role. Phalloplasty and urethral lengthening remain the most challenging of these surgeries, as the female urethra (4 cm long) must be lengthened to male dimensions (15-29 cm) with anastomosis at two sites, the native urethra/pars fixa urethra and the pars fixa urethra-penile urethra. As a result, there is a high incidence of urinary complications such as strictures and fistulae. Authors incorporated a urethral lengthening technique to reduce urinary complications in MgGAS. They compare the rates of urinary complications rates in cohorts before and after the introduction of this technique. Materials and Methods Authors have been performing phalloplasty since past 27 years, utilizing mainly free radial artery forearm flap (fRAFFp 431 cases) and pedicled anterolateral thigh flap (pALTp 120 cases). A retrospective review and comparison of urinary complications were performed before and after the introduction of their new technique since March 2017. Results There was a statistically significant reduction in the incidence of stricture with and without fistulae (25.94% with conventional and 4.17% with urethral lengthening technique p = 0.001) and fistulae alone (12.81% with conventional and 2.78% with urethral lengthening technique p = 0.011) in fRAFFp cases. In pALTp cases, the respective reductions were 43.08 to 17.07%, p = 0.006 (significant), and 13.85 to 4.88%, p = 0.197 (not statistically significant). Conclusion Over years, the rates of urinary complications in MgGAS have remained constant, varying from 25 to 58% for strictures and 17 to 75% for fistulae as noted by many authors. Authors noted that in most of their cases, strictures occurred at distal pars fixa urethra (DPFU)-penile urethra anastomosis and incorporated a urethral lengthening technique, which lengthens the DPFU by 3 to 5 cm at this anastomotic site, thus significantly reducing the anastomotic tension and the rate of urinary complications.

Phenotypic plasticity rather than genotype drives reproductive choices in Hydra populations.

Facultative clonality is associated with complex life cycles where sexual and asexual forms can be exposed to contrasting selection pressures. Facultatively clonal animals often have distinct developmental capabilities that depend on reproductive mode (e.g., negligible senescence and exceptional regeneration ability in asexual individuals, which are lacking in sexual individuals). Understanding how these differences in life history strategies evolved is hampered by limited knowledge of the population structure underlying sexual and asexual forms in nature. Here we studied genetic differentiation of coexisting sexual and asexual Hydra oligactis polyps, a freshwater cnidarian where reproductive mode-dependent life history patterns are observed. We collected asexual and sexual polyps from 13 Central European water bodies and used restriction-site associated DNA sequencing to infer population structure. We detected high relatedness among populations and signs that hydras might spread with resting eggs through zoochory. We found no genetic structure with respect to mode of reproduction (asexual vs. sexual). On the other hand, clear evidence was found for phenotypic plasticity in mode of reproduction, as polyps inferred to be clones differed in reproductive mode. Moreover, we detected two cases of apparent sex change (males and females found within the same clonal lineages) in this species with supposedly stable sexes. Our study describes population genetic structure in Hydra for the first time, highlights the role of phenotypic plasticity in generating patterns of life history variation, and contributes to understanding the evolution of reproductive mode-dependent life history variation in coexisting asexual and sexual forms.

Impact of bisphenol-A and synthetic estradiol on brain, behavior, gonads and sex hormones in a sexually labile coral reef fish.

Endocrine disrupting chemicals, such as bisphenol A (BPA) and ethinylestradiol (EE2), are detected in the marine environment from plastic waste and wastewater effluent. However, their impact on reproduction in sexually labile coral reef fish is unknown. The objective of this study was to determine impacts of environmentally relevant concentrations of BPA and EE2 on behavior, brain gene expression, gonadal histology, sex hormone profile, and plasma vitellogenin (Vtg) levels in the anemonefish, Amphiprion ocellaris. A. ocellaris display post-maturational sex change from male to female in nature. Sexually immature, male fish were paired together and fed twice daily with normal food (control), food containing BPA (100 µg/kg), or EE2 (0.02 µg/kg) (n = 9 pairs/group). Aggression toward an intruder male was measured at 1, 3, and 6 months. Blood was collected at 3 and 6 months to measure estradiol (E2), 11-ketotestosterone (11-KT), and Vtg. At the end of the study, fish were euthanized to assess gonad morphology and to measure expression of known sexually dimorphic genes in the brain. Relative to control, BPA decreased aggression, altered brain transcript levels, increased non-vitellogenic and vitellogenic eggs in the gonad, reduced 11-KT, and increased plasma Vtg. In two BPA-treated pairs, both individuals had vitellogenic eggs, which does not naturally occur. EE2 reduced 11-KT in subordinate individuals and altered expression of one transcript in the brain toward the female profile. Results suggest BPA, and to a lesser extent EE2, pollution in coral reef ecosystems could interfere with normal reproductive physiology and behavior of the iconic sexually labile anemonefish.

Potential role of DNA methylation of cyp19a1a promoter during sex change in protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen has a pivotal role in early female differentiation and further ovarian development. Aromatase (Cyp19a) is responsible for the conversion of androgens to estrogens in vertebrates. In teleosts, cyp19a1a and it paralog cyp19a1b are mainly expressed in the ovary and hypothalamus, respectively. Decreased plasma estrogen levels and lower cyp19a1a expression are associated with the initiation of female-to-male sex change in protogynous grouper. However, an 17α-methyltestosterone (MT)-induced the sex change from a female to a precocious male is a transient phase, and a reversible sex change (induced male-to-female) occurs after chemical withdrawal. Thus, we used this characteristic to study the epigenetic modification of cyp19a1a promoter in orange-spotted grouper. CpG-rich region with a CpG island is located on the putative regulatory region of distal cyp19a1a promoter. Our results showed that cyp19a1a promoter exhibited tissue-specific methylation status. Low methylation levels of distal cyp19a1a promoter and hypomethylated (0-40%) clones of cyp19a1a promoter region were widely observed in the ovary but not shown in testis and other tissues. In femaleness, higher numbers of hypomethylated clones of cyp19a1a promoter region were observed in the vitellogenic oocyte stage compared to the primary oocyte stage. Furthermore, decreased numbers of hypomethylated clones of cyp19a1a promoter region were associated with the maleness during the female-to-male sex change. DNA methylation inhibitor (5-aza-2'-deoxycytidine) delayed the spermatogenesis process (according to germ cell stage and numbers: by decrease of sperm and increase of spermatocytes) but did not influence the reversed sex change in MT-induced bi-directional sex change. These results suggest that epigenetic modification of cyp19a1a promoter may play an important role during the sex change in orange-spotted grouper.

Age, growth and reproductive life-history characteristics infer a high population productivity for the sustainably fished protogynous hermaphroditic yellowspotted rockcod (Epinephelus areolatus) in north-western Australia.

The yellowspotted rockcod, Epinephelus areolatus, is a small-sized grouper that is widely distributed throughout the Indo-Pacific, where it forms a valuable component of the harvest derived from multispecies fisheries along continental and insular shelves. Samples of E. areolatus were collected from 2012 to 2018 from commercial catches and research surveys in the Kimberley, Pilbara and Gascoyne regions of north-western Australia to improve the understanding of the life history, inherent vulnerability and stock status of this species. Histological analysis of gonads (n = 1889) determined that E. areolatus was a monandric protogynous hermaphrodite. Non-functional spermatogenic crypts were dispersed within the ovaries of 23% of mature functioning females; nonetheless, these crypts were not observed during the immature female phase. The length and age at which 50% of females matured were 266 mm total length (LT ) and 2.7 years, respectively. The spawning period was protracted over 10-12 months of the year with biannual peaks at the start of spring and autumn (i.e., September and March) when the photoperiod was at its mid-range (i.e., 12.1 h). Estimates of the lengths and ages at which 50% of E. areolatus change sex from female to male were very similar (i.e., <5% difference) between the Kimberley and Pilbara regions, i.e., L 50 sc of 364 and 349 mm LT and A 50 sc of 7.9 and 7.3 years, respectively. A maximum age of 19 years was observed in all three regions, but there was significant regional variation in growth. These variations in growth were not correlated with latitude; instead a parabolic relationship was evident, where the smallest mean length-at-age and fastest growth rates (k) occurred in the mid-latitudes of the Pilbara region. In the Kimberley and Pilbara regions, individuals were not fully selected by commercial fish traps until 5-6 years of age, hence, several years after reaching maturity. These life-history characteristics infer a high population productivity, which underpins the sustainable harvest of this species, despite comprising the largest catches of all epinephelids in the multispecies tropical fisheries across north-western Australia.

Development of reproductive potential in protogynous coral reef fishes within Philippine no-take marine reserves.

Empirical evidence for increases in the reproductive potential (egg output per unit area) of coral reef fish in no-take marine reserves (NTMRs) is sparse. Here, we inferred the development of reproductive potential in two species of protogynous reef fishes, Chlorurus bleekeri (Labridae: Scarinae) and Cephalopholis argus (Epinephelidae), inside and outside of Philippine NTMRs. We estimated key reproductive parameters and applied these to species-specific density and length data from 17 NTMRs (durations of protection 0-11 years) and paired fished sites (controls) in a space-for-time substitution approach. For C. argus, we also used density and length data collected almost annually over 29 years from a NTMR and an adjacent control at Apo Island. The results suggest that C. bleekeri can develop 6.0 times greater reproductive potential in NTMRs than controls after 11 years of protection, equivalent to approximately 582,000 more eggs produced 500 m-2 inside NTMRs. Enhancement of reproductive potential in C. argus was not evident after 11 years in the space-for-time substitution. At Apo Island NTMR, reproductive potential of C. argus increased approximately 6-fold over 29 years but NTMR/control ratios in reproductive potential decreased through time (from 3.2 to 2.4), probably due to spillover of C. argus from the NTMR to the control. C. argus was estimated to produce approximately 113,000 more eggs 500 m-2 inside Apo Island NTMR at the 29th year of protection. Ratios of reproductive potential between NTMR and controls in C. bleekeri and C. argus were often greater than corresponding ratios in density or biomass. The study underscores the importance of species-specific reproductive life history traits that drive variation in the development of larval fish subsidies that originate from NTMRs.

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.

Correlated Evolution of Sex Allocation and Mating System in Wrasses and Parrotfishes.

In accordance with predictions of the size-advantage model, comparative evidence confirms that protogynous sex change is lost when mating behavior is characterized by weak size advantage. However, we lack comparative evidence supporting the adaptive significance of sex change. Specifically, it remains unclear whether increasing male size advantage induces transitions to protogynous sex change across species, as it can within species. We show that in wrasses and parrotfishes (Labridae) the evolution of protogynous sex change is correlated with polygynous mating and that the degree of male size advantage expressed by polygynous species influences transitions between different types of protogynous sex change. Phylogenetic reconstructions reveal strikingly similar patterns of sex allocation and mating system evolution with comparable lability. Despite the plasticity of sex-determination mechanisms in labrids, transitions trend toward monandry (all males derived from sex-changed females), with all observed losses of protogyny accounted for by shifts in the timing of sex change to prematuration. Likewise, transitions in mating system trend from the ancestral condition of lek-like polygyny toward greater male size advantage, characteristic of haremic polygyny. The results of our comparative analyses are among the first to confirm the adaptive significance of sex change as described by the size-advantage model.

Influence of protogynous sex change on recovery of fish populations within marine protected areas.

Marine protected areas (MPAs) are increasingly implemented as a conservation tool worldwide. In many cases, they are managed adaptively: the abundance of target species is monitored, and observations are compared to some model-based expectation for the trajectory of population recovery to ensure that the MPA is achieving its goals. Most previous analyses of the transient (short-term) response of populations to the cessation of fishing inside MPAs have dealt only with gonochore (fixed-sex) species. However, many important fishery species are protogynous hermaphrodites (female-to-male sex-changing). Because size-selective harvest will predominantly target males in these species, harvesting not only reduces abundance but also skews the sex ratio toward females. Thus the response to MPA implementation will involve changes in both survival and sex ratio, and ultimately reproductive output. We used an age-structured model of a generic sex-changing fish population to compare transient population dynamics after MPA implementation to those of an otherwise similar gonochore population and examine how different features of sex-changing life history affect those dynamics. We examined both demographically open (most larval recruitment comes from outside the MPA) and demographically closed (most larval recruitment is locally produced) dynamics. Under both scenarios, population recovery of protogynous species takes longer when fishing was more intense pre-MPA (as in gonochores), but also depends heavily on the mating function, the degree to which the sex ratio affects reproduction. If few males are needed and reproduction is not affected by a highly female-biased sex ratio, then population recovery is much faster; if males are a limiting resource, then increases in abundance after MPA implementation are much slower than for gonochores. Unfortunately, the mating function is largely unknown for fishes. In general, we expect that most protogynous species with haremic mating systems will be in the first category (few males needed), though there is at least one example of a fish species (though not a sex-changing species) for which males are limiting. Thus a better understanding of the importance of male fish to population dynamics is needed for the adaptive management of MPAs.