Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.

Highlights d Interaction between polycomb target genes in ESCs occurs independently of cohesin d Loop extrusion by cohesin disrupts interactions between polycomb target genes d Cohesin removal enhances repression at polycomb target genes with increased interactions

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a "male-like" gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario.A major paradox in developmental biology is the lack of conservation of sex-determining mechanisms even in closely related taxa (1). Thus, in mammals and birds, sex is genetically canalized by a chromosomal sex determination (CSD) system of male (XX/XY in mammals) or female (ZW/ZZ in birds) heterogamety, driven by the action of a master sex-determining gene: sry in mammals and dmrt1 in birds (1). In contrast, fish sex can be very plastic, with the combination of genetic and environmental influences (2). In addition to fish species with CSD, there are species with polygenic sex determination (PSD), in which sex depends on the combined action of several promale and profemale genetic factors, and species with temperature-dependent sex determination (TSD) (3). Understanding how the environment influences sex ratios in vertebrates is of fundamental importance in the study of the evolution of sex-determining mechanisms (4, 5). It is also of practical relevance in evaluating the effects of climate change and chemical pollution (6, 7).The effects of temperature on gene expression during sex differentiation have been investigated in different teleost species, including the African catfish, Clarias gariepinus (8), European sea bass, Dicentrarchus labrax (9, 10), Japanese flounder, Paralichthys olivaceus (11), pejerrey, Odontesthes...

Controlling the sex ratio is essential in finfish farming. A balanced sex ratio is usually good for broodstock management, since it enables to develop appropriate breeding schemes. However, in some species the production of monosex populations is desirable because the existence of sexual dimorphism, primarily in growth or first time of sexual maturation, but also in color or shape, can render one sex more valuable. The knowledge of the genetic architecture of sex determination (SD) is convenient for controlling sex ratio and for the implementation of breeding programs. Unlike mammals and birds, which show highly conserved master genes that control a conserved genetic network responsible for gonad differentiation (GD), a huge diversity of SD mechanisms has been reported in fish. Despite theory predictions, more than one gene is in many cases involved in fish SD and genetic differences have been observed in the GD network. Environmental factors also play a relevant role and epigenetic mechanisms are becoming increasingly recognized for the establishment and maintenance of the GD pathways. Although major genetic factors are frequently involved in fish SD, these observations strongly suggest that SD in this group resembles a complex trait. Accordingly, the application of quantitative genetics combined with genomic tools is desirable to address its study and in fact, when applied, it has frequently demonstrated a multigene trait interacting with environmental factors in model and cultured fish species. This scenario has notable implications for aquaculture and, depending upon the species, from chromosome manipulation or environmental control techniques up to classical selection or marker assisted selection programs, are being applied. In this review, we selected four relevant species or fish groups to illustrate this diversity and hence the technologies that can be used by the industry for the control of sex ratio: turbot and European sea bass, two reference species of the European aquaculture, and salmonids and tilapia, representing the fish for which there are well established breeding programs.

The zebrafish (Danio rerio) has become a well-established experimental model in many research fields but the loss of the primary sex-determining region during the process of domestication renders laboratory strains of zebrafish susceptible to the effects of environmental factors on sex ratios. Further, an essential husbandry aspect -the optimal rearing density to avoid stress-induced masculinization -is not known. We carried out two experiments: the first focusing on the effects of density on survival, growth and sex ratio by rearing zebrafish at different initial densities (9, 19, 37 and 74 fish per litre) for 3 months (6-90 days post-fertilization, dpf ), and the second focusing on the effects of cortisol during the sex differentiation period (15-45 dpf) for zebrafish reared at low density. The results showed an increase in the number of males in groups subjected to the two highest initial rearing densities; we also observed a reduction of survival and growth in a density-dependent manner. Furthermore, zebrafish treated with cortisol during the sex differentiation period showed a complete masculinization of the population; treatment with the cortisol synthesis inhibitor metyrapone negated the effects of exogenous cortisol. Our results indicate that the process of sex differentiation in domesticated zebrafish can be perturbed by elevated stocking density and that this effect is likely to be mediated by an increase in cortisol through the stress response. However, the underlying mechanism needs further study.

The embryonic gonad is the only organ that takes two mutually exclusive differentiating pathways and hence gives rise to two different adult organs: testes or ovaries. The recent application of genomic tools including microarrays, next-generation sequencing approaches, and epigenetics can significantly contribute to decipher the molecular mechanisms involved in the processes of sex determination and sex differentiation. However, in fish, these studies are complicated by the fact that these processes depend, perhaps to a larger extent when compared to other vertebrates, on the interplay of genetic and environmental influences. Here, we review the advances made so far, taking into account different experimental approaches, and illustrate some technical complications deriving from the fact that as development progresses it becomes more and more difficult to distinguish whether changes in gene expression or DNA methylation patterns are the cause or the consequence of such developmental events. Finally, we suggest some avenues for further research in both model fish species and fish species facing specific problems within an aquaculture context.

BackgroundSex in fish is plastic and in several species can be influenced by environmental factors. In sensitive species, elevated temperatures have a masculinizing effect. Previous studies on the effects of temperature on gene expression have been restricted to a few cognate genes, mostly related to testis or ovarian development, and analyzed in gonads once they had completed the process of sex differentiation. However, studies on the effect of temperature at the whole gonadal transcriptomic level are scarce in fish and, in addition, temperature effects at the time of sex differentiation at the transcriptomic level are also unknown. Here, we used the European sea bass, a gonochoristic teleost with a polygenic sex determination system influenced by temperature, and exposed larvae to elevated temperature during the period of early gonad formation. Transcriptomic analysis of the gonads was carried out about three months after the end of temperature exposure, shortly after the beginning of the process of sex differentiation.ResultsElevated temperature doubled the number of males with respect to untreated controls. Transcriptomic analysis of early differentiating female gonads showed how heat caused: 1) an up-regulation of genes related to cholesterol transport (star), the stress response (nr3c1) and testis differentiation (amh, dmrt, etc.), 2) a decrease in the expression of genes related to ovarian differentiation such as cyp19a1a, and 3) an increase in the expression of several genes related to epigenetic regulatory mechanisms (hdac11, dicer1, ehmt2, jarid2a, pcgf2, suz12, mettl22).ConclusionsTaken together, the results of this study contribute to the understanding of how the early environment sets permanent changes that result in long-lasting consequences, in this case in the sexual phenotype. Results also show the usefulness of comparing the effects of heat on the behavior of cognate genes related to sex differentiation as well as that of genes involved in establishing and maintaining cell identity through epigenetic mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1862-0) contains supplementary material, which is available to authorized users.

In natural fish populations, temperature increases can result in shifts in important phenotypic traits. DNA methylation is an epigenetic mechanism mediating phenotypic changes. However, whether temperature increases of the magnitude predicted by the latest global warming models can affect DNA methylation is unknown. Here, we exposed European sea bass to moderate temperature increases in different periods within the first two months of age. We show that increases of even 2 °C in larvae significantly changed global DNA methylation and the expression of ecologically-relevant genes related to DNA methylation, stress response, muscle and organ formation, while 4 °C had no effect on juveniles. Furthermore, DNA methylation changes were more marked in larvae previously acclimated to a different temperature. The expression of most genes was also affected by temperature in the larvae but not in juveniles. In conclusion, this work constitutes the first study of DNA methylation in fish showing that temperature increases of the magnitude predicted by the latest global warming models result in stage-dependent alterations in global DNA methylation and gene expression levels. This study, therefore, provides insights on the possible consequences of climate change in fish mediated by genome-wide epigenetic modifications.