Temperature- and genotype-dependent stress response and activation of the hypothalamus-pituitary-interrenal axis during temperature-induced sex reversal in pejerrey Odontesthes bonariensis, a species with genotypic and environmental sex determination.

In the pejerrey Odontesthes bonariensis (Atheriniformes, Atherinopsidae), exposure to high and low temperatures during the critical period of sex determination (CPSD) induce testicular and ovarian differentiation, respectively, regardless of the presence or not of the sex determining gene amhy, which is crucial for testis formation only at intermediate, sexually neutral temperatures. In this study we explored the existence of genotype-specific signaling of Crh (Corticotropin Releasing Hormone) family genes and their associated carrier protein, receptors, and other stress-related genes in response to temperature during the CPSD and the potential involvement of the central nervous system via the hypothalamus-pituitary-interrenal (HPI) axis in the sex determination of this species. The Crh family genes crhb, uts1, ucn3, the receptor crhr1 and the stress-related genes gr1, gr2, nr3c2 were transiently upregulated in the heads of pejerrey larvae during the CPSD by high temperature alone or in combination with other factors. Only crhr2 transcript abundance was not influenced by temperature but independently by time and genotype. In most cases, mRNA abundance was higher in the XX heads compared to that of XY individuals. The mRNAs of some of these genes were localized in the hypothalamus of pejerrey larvae during the CPSD. XX larvae also showed higher whole-body cortisol titers than the XY, downregulation of cyp19a1a and upregulation of the testis-related genes amhy/amha in trunks (gonads) and were 100% masculinized at the high temperature. In contrast, at the low temperature, crhbp and avt were upregulated in the heads, particularly the former in XY larvae. cyp19a1a and amhy/amha were up- and downregulated, respectively, in the gonads, and fish were 100% feminized. Signaling via the HPI axis was observed simultaneously with the first molecular signs of ongoing sex determination/differentiation in the gonads. Overall, the results strongly suggest a temperature-dependent, genotype-specific regulatory action of the brain involving the Crh family of stress-related genes on the process of environmental sex determination of pejerrey.

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10 days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30 days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

Y-specific amh allele, amhy, is the master sex-determining gene in Japanese flounder Paralichthys olivaceus.

Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.

Inclusion of shape parameters increases the accuracy of 3D models for microplastics mass quantification.

As microplastics may bring about adverse effects on living organisms, it is important to establish more precise quantification approaches to better understand their dynamics. One method to determine the concentration of microplastics is to estimate their mass using three-dimensional (3D) models, but its accuracy is not well known. In this study, we evaluated the shape of the particles and verified the accuracy of a 3D model-based mass estimation using samples from a tidal flat facing Tokyo Bay. The particle shape evaluation suggested that the microplastics were flat and irregular in shape; based on these data, we created two types of models to estimate their mass. As a result, an accuracy of mass estimation by our model was higher than other models that consider the slenderness and flatness of particles. The optimization of mass estimation methods based on 3D models may improve the reliability of microplastic evaluation in monitoring studies.

Early warming stress on rainbow trout juveniles impairs male reproduction but contrastingly elicits intergenerational thermotolerance.

The exposure of adult fish to warm or high temperatures is known to impair reproduction, yet the long-term reproductive impacts for treatments at early life are not well clarified. This study aimed to evaluate the effects of warm temperature (WT) during juvenile stage on gonad maturation, gamete quality, and offspring thermotolerance in rainbow trout. While the comparison of basic reproductive parameters in WT females did not reveal any kind of impairment, many WT males showed an atrophied, undeveloped gonad, or a smaller testis with lower milt volume; sperm quality parameters in WT males and deformity rates in the respective progeny were also highly affected. However, despite of such negative effects, many of the remaining progeny presented better rates of survival and growth when exposed to the same conditions as those of parental fish (WT), suggesting that thermal stress in parr stage males elicited intergenerational thermotolerance after a single generation. The present results support that prolonged warming stress during early life stages can adversely affect key reproductive aspects, but contrastingly increase offspring performance at upper thermal ranges. These findings have implications on the capacity of fish to adapt and to cope with global warming.

Where the Ends Meet: An Overview of Sex Determination in Atheriniform Fishes.

Atheriniform fishes have recently emerged as attractive models for evolutionary, ecological, and molecular/physiological studies on sex determination. Many species in this group have marked temperature-dependent sex determination (TSD) and yet many species also have a sex determinant gene that provides a strong drive for male differentiation. Thus, in these species the 2 forms of sex determination that were once considered to be mutually exclusive, environmental (ESD) and genotypic (GSD) sex determination, can coexist at environmentally relevant conditions. Here, we review the current knowledge on sex determination in atheriniform fishes with emphasis on the molecular and physiological mechanisms of ESD and GSD, the coexistence and cross-talk between these 2 mechanisms, the possibility of extragonadal transduction of environmental information and/or extragonadal onset of sex determination, and the results of field studies applying novel tools such as otolith increment analysis and molecular markers of genetic sex developed for selected New World and Old World atheriniform species. We also discuss the existence of molecular and histological mechanisms to prevent the discrepant differentiation in parts of the gonads because of ambiguous or conflicting environmental and genetic signals and particularly the possibility that the female is the default state in these species.

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka.

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that ndrg1b is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.

Testicular subcutaneous allografting followed by immunosuppressive treatment promotes maintenance of spermatogonial cells in rainbow trout (Oncorhynchus mykiss).

Germ cell transplantation and testis graft represent promising biotechnologies that can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might remove the transplanted donor cells/tissues and limit the surrogate production of gametes. In this work, we administered emulsion containing-immunosuppressants to verify whether they are capable to prevent immune rejection and promote survival of testis allografts in rainbow trout. In the first part of this study, we demonstrated in vitro that tacrolimus and cyclosporine were able to affect viability, inhibit leucocyte proliferation, and suppress il2 expression in vitro. In in vivo experiments, both doses of tacrolimus (0.5 and 1.5 mg/kg) and the lower dose of cyclosporine (20 mg/kg) significantly inhibited the expression of il2 in head kidney, three days post-injection. A higher dose of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with emulsion containing-tacrolimus. Immunohistochemical, conventional histology, and qRT-PCR (vasa) analysis demonstrated the presence of spermatogonial cells by the fifth week, in animals treated with 0.5 mg/kg of tacrolimus similar as found in autografted group. In the group treated with the highest tacrolimus dose (1.5 mg/kg) and in the non-treated group (without immunosuppressant), no germ cells or their respective markers were detected. il2 expression in head kidney was also suppressed in grafted animals treated with tacrolimus compared to non-treated group. These results suggest that tacrolimus may be a promising immunosuppressant for testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting other activation pathways of the immune system, as performed in human organ transplantation, could be an alternative approach to optimize the immunosuppressive effects in host organisms.

Phenotypic/genotypic sex mismatches and temperature-dependent sex determination in a wild population of an Old World atherinid, the cobaltcap silverside Hypoatherina tsurugae.

Several New World atheriniforms have been recognized as temperature-dependent sex determined (TSD) and yet possess a genotypic sex determinant (amhy) which is primarily functional at mid-range temperatures. In contrast, little is known about the sex determination in Old World atheriniforms, even though such knowledge is crucial to understand the evolution of sex determination mechanisms in fishes and to model the effects of global warming and climate change on their populations. This study examined the effects of water temperature on sex determination of an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae, in which we recently described an amhy homologue. We first assessed the occurrence of phenotypic/genotypic sex mismatches in wild specimens from Tokyo Bay for three years (2014-2016) and used otolith analysis to estimate their birth dates and approximate thermal history during the presumptive period of sex determination. Phenotypic sex ratios became progressively biased towards males (47.3%-78.2%) during the period and were associated with year-to-year increases in the frequency of XX-males (7.3%-52.0%) and decreases in XY/YY-females (14.5%-0%). The breeding season had similar length but was delayed by about 1 month per year between 2014 and 2016, causing larvae to experience higher temperatures during the period of sex determination from year to year. Larval rearing experiments confirmed increased likelihood of feminization and masculinization at low and high temperatures, respectively. The results suggest that cobaltcap silverside has TSD, or more specifically the coexistence of genotypic and environmental sex determinants, and that it affects sex ratios in wild populations.

Crowding stress during the period of sex determination causes masculinization in pejerrey Odontesthes bonariensis, a fish with temperature-dependent sex determination.

The pejerrey is an atherinopsid species from South America that presents a combination of genotypic and environmental (temperature-dependent) sex determination whereby low and high temperatures induce feminization and masculinization, respectively. Masculinization involves a heat-induced stress response leading to increased circulating cortisol and androgens. We tested whether crowding would elicit a similar response as high temperature and affect the sex ratios of pejerrey. Larvae with XX and XY genotypes were reared at 15, 62 and 250 larvae/L in 0.4, 1.6, and 6.4 L containers during a period considered critical for sex determination at 25 °C, a mixed-sex promoting temperature. Fish were analysed at 3-7 weeks for whole-body cortisol and 11-ketotestosterone (11-KT) titer and hydroxy-steroid dehydrogenase (hsd11b2) mRNA transcript abundance, and after completion of gonadal sex differentiation (10-14 weeks) for determination of phenotypic and genotypic sex mismatches. Crowding was associated with depressed growth, higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization compared to intermediate and/or low rearing densities. Perceived crowding (by rearing in containers with mirror-finish, reflecting walls) also caused masculinization. These results suggest the possibility that other environmental factors besides temperature can also affect sex determination in pejerrey and that a stress response leading to increased cortisol and androgen levels, which is potentially perceived by the brain, may be a common feature among different forms of environmental sex determination in this species.

Generation of a white-albino phenotype from cobalt blue and yellow-albino rainbow trout (Oncorhynchus mykiss): Inheritance pattern and chromatophores analysis.

Albinism is the most common color variation described in fish and is characterized by a white or yellow phenotype according to the species. In rainbow trout Oncorhynchus mykiss, aside from yellow-albino phenotypes, cobalt blue variants with autosomal, recessive inheritance have also been reported. In this study, we investigated the inheritance pattern and chromatophores distribution/abundance of cobalt blue trouts obtained from a local fish farm. Based on crosses with wild-type and dominant yellow-albino lines, we could infer that cobalt blue are dominant over wild-type and co-dominant in relation to yellow-albino phenotype, resulting in a fourth phenotype: the white-albino. Analysis of chromatophores revealed that cobalt blue trouts present melanophores, as the wild-type, and a reduced number of xanthophores. As regards to the white-albino phenotype, they were not only devoid of melanophores but also presented a reduced number of xanthophores. Cobalt blue and white-albino trouts also presented reduced body weight and a smaller pituitary gland compared to wild-type and yellow-albino phenotypes. The transcription levels of tshb and trh were up regulated in cobalt blue compared to wild type, suggesting the involvement of thyroid hormone in the expression of blue color. These phenotypes represent useful models for research on body pigmentation in salmonids and on the mechanisms behind endocrine control of color patterning.

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae).

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self-sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south-east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark-based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishes.

Differential Expression of Genes Related to Sexual Determination Can Modify the Reproductive Cycle of Astyanax scabripinnis (Characiformes: Characidae) in B Chromosome Carrier Individuals.

The species complex Astyanax scabripinnis is one of the most studied with respect to origin, distribution, and frequency of B chromosomes, and is considered a model organism for evolutionary studies. Research using population inferences about the occurrence and frequency of the B chromosome shows seasonal variation between sexes, which is associated with the presence of this supernumerary element. We hypothesized that the B chromosome could influence the sex ratio of these animals. Based on this assumption, the present work aimed to investigate if differences exist among levels of gene expression with qRT-PCR of the amh (associated with testicular differentiation) and foxl2a (associated with ovarian differentiation) genes between B-carrier and non-B-carrier individuals. The results showed that for the amh gene, the difference in expression between animals with B chromosomes was not accentuated compared to that in animals without this chromosome. Expression of foxl2a in B-carrier females, however, was reduced by 73.56% compared to females that lacked the B chromosome. Males had no difference in expression of the amh and foxl2a genes between carriers and non-carriers of the B chromosome. Results indicate that the presence of B chromosomes is correlated with the differential expression of sex-associated genes. An analysis of these results integrated with data from other studies on the reproductive cycle in the same species reveals that this difference in expression may be expanding the reproductive cycle of the species.

The Duplicated Y-specific amhy Gene Is Conserved and Linked to Maleness in Silversides of the Genus Odontesthes.

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.

Environmental regulation of sex determination in fishes: Insights from Atheriniformes.

Sex determination is the first step toward the establishment of phenotypic sex in most vertebrates. Aquatic poikilotherms such as teleost fishes exhibit a high diversity of sex-determination mechanisms and gonadal phenotypes that are remarkably plastic and responsive to a variety of environmental factors (e.g., water temperature, pH, salinity, photoperiod, population density). This chapter reviews current knowledge of genotypic and environmental sex determination systems in fishes with special reference to Atheriniformes-one of the best-characterized taxa in this field-and offers perspectives to guide and stimulate further research.

Spatiotemporal Correlations between amh and cyp19a1a Transcript Expression and Apoptosis during Gonadal Sex Differentiation of Pejerrey, Odontesthes bonariensis.

Sex determination in pejerrey is genetically prescribed by the Y chromosome-linked anti-müllerian hormone amhy but is also strongly influenced by water temperature during the critical period of sex determination. Its gonadal differentiation is characterized by a cephalocaudal and left-to-right histological gradient in both sexes that presumably helps prevent discrepant intersex development in different regions of the gonads in response to ambiguous thermal and genetic stimuli, but the relation of this gradient to molecular processes of sex differentiation is unknown. In this study, we investigated the spatiotemporal expression patterns of amh, gonadal aromatase (cyp19a1a), and apoptosis in relation to the histological gradient in ovaries and testes at an intermediate, sexually neutral temperature. The location and timing of expression of amh, cyp19a1a, and apoptosis seemed to be highly coordinated with the time of gonadal sex differentiation and the histological gradient of gonadal sex differentiation. Apoptosis occurred predominantly in the anterior region of the right gonads and is surmised to be a process to delay differentiation in this area compared to the left gonad, possibly as a means to ensure uniform development in both gonads. Aromatase expression early during development was noted even in putative XY males, supporting the notion of primacy of female development in pejerrey gonads. Thus, apoptosis may be particularly important to prevent discrepant gonadal differentiation in XY individuals where genetic pro-male (amhy), pro-female (cyp19a1a), and thermal stimuli may antagonize.

Sex determination in Neotropical fish: Implications ranging from aquaculture technology to ecological assessment.

The high biodiversity of fish in the Neotropical region contrasts with scarce or biased studies on the mechanisms involved in the sex determination in members of this fauna. In this review, we attempted to compile the information available on determination, differentiation, and manipulation of sex for Neotropical species, with special focus on silversides and other two speciose groups, known as characins (Characiformes) and catfishes (Siluriformes). Currently, there is plenty of information available on chromosomal sex determination systems, which includes both male and female heterogamety with many variations, and sex chromosomes evolution at the macro chromosomal level. However, there is hitherto a blank in information at micro, gene/molecule levels and in research related to the effects of environmental cues on sex determination; most of reported studies are limited to silversides and guppies. In view of such a high diversity, it is critically necessary to establish key model species for relevant Neotropical fish taxa and also multi-disciplinary research groups in order to uncover the main patterns and trends that dictate the mechanisms of sex determination and gonadal differentiation in this icthyofauna. By increasing our knowledge on sex determination/differentiation with the identification of sex chromosome-linked markers or sex-determining genes, characterization of the onset timing of morphological gonadal differentiation, and determination of the environmental-hormonal labile period of gonadal sex determination in reference species, it will be possible to use those information as guidelines for application in other related groups. Overall, the strategic advance in this research field will be crucial for the development of biotechnological tools for aquaculture industry and for conservation of fish fauna from the Neotropical Region.

Differential Expression of dmrt1 in Astyanax scabripinnis (Teleostei, Characidade) Is Correlated with B Chromosome Occurrence.

Astyanax is an abundant fish genus in South America. Some species of this group are characterized by the presence of B chromosomes and absence of morphologically differentiated sex chromosomes. In this study, we used quantitative real-time polymerase chain reaction to characterize mRNA expression of dmrt1 in Astyanax scabripinnis gonads. Maturing gonads of males with the B chromosome overexpressed dmrt1. Our findings suggest that B chromosomes may have an adaptive role in A. scabripinnis sex determination and maintenance.

Optimization of protocols for microinjection-based delivery of cryoprotective agents into Japanese whiting Sillago japonica embryos.

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas » artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.

Demonstration of viability and fertility and development of a molecular tool to identify YY supermales in a fish with both genotypic and environmental sex determination.

The pejerrey possesses a genotypic sex determination system driven by the amhy gene and yet shows marked temperature-dependent sex determination. Sex-reversed XY females have been found in a naturally breeding population established in Lake Kasumigaura, Japan. These females could mate with normal XY males and generate YY "supermale" individuals that, if viable and fertile, would sire only genotypic male offspring. This study was conducted to verify the viability, gender, and fertility of YY pejerrey and to develop a molecular method for their identification. Production of YY fish was attempted by crossing a thermally sex-reversed XY female and an XY male, and rearing the progeny until sexual maturation. To identify the presumable YY individuals, we first conducted a PCR analysis using amhy-specific primers to screen only amhy-positive (XY and YY) fish. This screening showed that 60.6% of the progeny was amhy-positive, which suggested the presence of YY fish. We then conducted a second screening by qPCR in order to identify the individuals with two amhy copies in their genome. This screening revealed 13 individuals, all males, with values twice higher than the other 30 amhy-positive fishes, suggesting they have a YY complement. This assumption as well as the viability, fertility, and "supermale" nature of these individuals was confirmed in progeny tests with XX females that yielded 100% amhy-positive offspring. These results demonstrate that qPCR can obviate progeny test as a means to identify the genotypic sex and therefore may be useful for the survey of all three possible genotypes in wild populations.