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Issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.-- 9 pages, 3 figures.-- Data accessibility: This article does not contain any additional data ; The hypothesis that epigenetic mechanisms of gene expression regulation have two main roles in vertebrate sex is presented. First, and within a given generation, by contributing to the acquisition and maintenance of (i) the male or female function once during the lifetime in individuals of gonochoristic species; and (ii) the male and female function in the same individual, either at the same time in simultaneous hermaphrodites, or first as one sex and then as the other in sequential hermaphrodites. Second, if environmental conditions change, epigenetic mechanisms may have also a role across generations, by providing the necessary phenotypic plasticity to facilitate the transition: (i) from one sexual system to another, or (ii) from one sex-determining mechanism to another. Furthermore, if the environmental change lasts enough time, epimutations could facilitate assimilation into genetic changes that stabilize the new sexual system or sex-determining mechanism. Examples supporting these assertions are presented, caveats or difficulties and knowledge gaps identified, and possible ways to test this hypothesis suggested ; This study was supported by Spanish Ministry of Science grant no. PID2019-108888RB-I00, with funding from the Spanish government through the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) ; Peer reviewed

13 pages, 4 figures, supplementary data https://doi.org/10.1093/molbev/msz153 ; Domestication of wild animals induces a set of phenotypic characteristics collectively known as the domestication syndrome. However, how this syndrome emerges is still not clear. Recently, the neural crest cell deficit hypothesis proposed that it is generated by a mildly disrupted neural crest cell developmental program, but clear support is lacking due to the difficulties of distinguishing pure domestication effects from preexisting genetic differences between farmed and wild mammals and birds. Here, we use a farmed fish as model to investigate the role of persistent changes in DNA methylation (epimutations) in the process of domestication. We show that early domesticates of sea bass, with no genetic differences with wild counterparts, contain epimutations in tissues with different embryonic origins. About one fifth of epimutations that persist into adulthood are established by the time of gastrulation and affect genes involved in developmental processes that are expressed in embryonic structures, including the neural crest. Some of these genes are differentially expressed in sea bass with lower jaw malformations, a key feature of domestication syndrome. Interestingly, these epimutations significantly overlap with cytosine-to-thymine polymorphisms after 25 years of selective breeding. Furthermore, epimutated genes coincide with genes under positive selection in other domesticates. We argue that the initial stages of domestication include dynamic alterations in DNA methylation of developmental genes that affect the neural crest. Our results indicate a role for epimutations during the beginning of domestication that could be fixed as genetic variants and suggest a conserved molecular process to explain Darwin's domestication syndrome across vertebrates ; Research supported by the Spanish Ministry of Economy and Competitiveness (MINECO) grants "Epifarm" (ref. AGL2013-41047-R) and "Epimark" (ref. AGL2016-78710-R) to F.P. D.A. was supported by a PhD scholarship from the Spanish Government (BES-2011-044860) and an Epimark contract ; Peer Reviewed

13 pages, 5 figures, 3 tables, additional files http://dx.doi.org/10.1186/s12864-017-4345-7 ; [Background]: Understanding the consequences of thermal and chemical variations in aquatic habitats is of importance in a scenario of global change. In ecology, the sex ratio is a major population demographic parameter. So far, research that measured environmental perturbations on fish sex ratios has usually involved a few model species with a strong genetic basis of sex determination, and focused on the study of juvenile or adult gonads. However, the underlying mechanisms at the time of gender commitment are poorly understood. In an effort to elucidate the mechanisms driving sex differentiation, here we used the European sea bass, a fish species where genetics and environment (temperature) contribute equally to sex determination. [Results]: Here, we analyzed the transcriptome of developing gonads experiencing either testis or ovarian differentiation as a result of thermal and/or exogenous estrogen influences. These external insults elicited different responses. Thus, while elevated temperature masculinized genetic females, estrogen exposure was able to override thermal effects and resulted in an all-female population. A total of 383 genes were differentially expressed, with an overall downregulation in the expression of genes involved in both in testicular and ovarian differentiation when fish were exposed to Estradiol-17ß through a shutdown of the first steps of steroidogenesis. However, once the female phenotype was imposed, gonads could continue their normal development, even taking into account that some of the resulting females were fish that otherwise would have developed as males. [Conclusions]: The data on the underlying mechanisms operating at the molecular level presented here contribute to a better understanding of the sex ratio response of fish species subjected to a combination of two of the most common environmental perturbations and can have implications in future conservational policies. ; N.D. was supported first by a scholarship from the Government of Spain (BES-2007-14,273) and then by a contract from the Epigen-Aqua (AGL2010–15939) project granted to F.P. Research was supported by the Spanish Ministry of Economy and Competitiveness grants Consolider Aquagenomics (CDS2007-002) and Epifarm (AGL2013-41047-R) to F.P. ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

[Background] Sex 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. ; [Results] Elevated 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). ; [Conclusions] Taken 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. ; ND was supported first by a scholarship from the Government of Spain (BES-2007-14273) and then by a contract from the Epigen-Aqua Project (AGL2010-15939).

11 pages, 5 figures, 2 tables.-- PMID: 18665231 [PubMed].-- Supporting information available: Supplementary Table "Field and laboratory data used for the assessment of the presence of temperature-dependent sex determination in gonochoristic fish" and list of references ; [Background] In gonochoristic vertebrates, sex determination mechanisms can be classified as genotypic (GSD) or temperature-dependent (TSD). Some cases of TSD in fish have been questioned, but the prevalent view is that TSD is very common in this group of animals, with three different response patterns to temperature. ; [Methodology/Principal Findings] We analyzed field and laboratory data for the 59 fish species where TSD has been explicitly or implicitly claimed so far. For each species, we compiled data on the presence or absence of sex chromosomes and determined if the sex ratio response was obtained within temperatures that the species experiences in the wild. If so, we studied whether this response was statistically significant. We found evidence that many cases of observed sex ratio shifts in response to temperature reveal thermal alterations of an otherwise predominately GSD mechanism rather than the presence of TSD. We also show that in those fish species that actually have TSD, sex ratio response to increasing temperatures invariably results in highly male-biased sex ratios, and that even small changes of just 1–2°C can significantly alter the sex ratio from 1:1 (males:females) up to 3:1 in both freshwater and marine species. ; [Conclusions/Significance] We demonstrate that TSD in fish is far less widespread than currently believed, suggesting that TSD is clearly the exception in fish sex determination. Further, species with TSD exhibit only one general sex ratio response pattern to temperature. However, the viability of some fish populations with TSD can be compromised through alterations in their sex ratios as a response to temperature fluctuations of the magnitude predicted by climate change ; Work supported by a Spanish Ministry of Education and Science grant ("Sexratio") to F.P. N.O.A. was supported by a scholarship from the aquaculture network (XRAq) from the Government of Catalonia ; Peer reviewed

12 pages, 4 figures, 1 table, supplementary material https://dx.doi.org/10.1038/s41598-017-10861-6 ; 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 ; DA and ND were supported by Ph.D. scholarships from the Spanish Government (BES-2011-044860 and BES-2007-14273, respectively). Research was supported by the Ministry of Economy and Competitiveness "Epifarm" (AGL2013-41047-R) to FP ; Peer Reviewed

13 pages, 6 figures, 2 tables, supporting information http://dx.doi.org/10.1371/journal.pone.0111304 ; Background: Food supply is a major factor influencing growth rates in animals. This has important implications for both natural and farmed fish populations, since food restriction may difficult reproduction. However, a study on the effects of food supply on the development of juvenile gonads has never been transcriptionally described in fish. Methods and Findings: This study investigated the consequences of growth on gonadal transcriptome of European sea bass in: 1) 4-month-old sexually undifferentiated fish, comparing the gonads of fish with the highest vs. the lowest growth, to explore a possible link between transcriptome and future sex, and 2) testis from 11-month-old juveniles where growth had been manipulated through changes in food supply. The four groups used were: i) sustained fast growth, ii) sustained slow growth, iii) accelerated growth, iv) decelerated growth. The transcriptome of undifferentiated gonads was not drastically affected by initial natural differences in growth. Further, changes in the expression of genes associated with protein turnover were seen, favoring catabolism in slow-growing fish and anabolism in fast-growing fish. Moreover, while fast-growing fish took energy from glucose, as deduced from the pathways affected and the analysis of protein-protein interactions examined, in slow-growing fish lipid metabolism and gluconeogenesis was favored. Interestingly, the highest transcriptomic differences were found when forcing initially fast-growing fish to decelerate their growth, while accelerating growth of initially slow-growing fish resulted in full transcriptomic convergence with sustained fast-growing fish. Conclusions: Food availability during sex differentiation shapes the juvenile testis transcriptome, as evidenced by adaptations to different energy balances. Remarkably, this occurs in absence of major histological changes in the testis. Thus, fish are able to recover transcriptionally their testes if they are provided with enough food supply during sex differentiation; however, an initial fast growth does not represent any advantage in terms of transcriptional fitness if later food becomes scarce. © 2014 Díaz et al. ; Research was supported by the Spanish Ministry of Science projects ''Epigen-Aqua'' (AGL2010-15939) and ''Aquagenomics'' (CDS2007-0002) to FP. ND was supported by a scholarship from the Government of Spain (BES-2007-14273) and then by a contract by the Epigen-Aqua project. LR was supported by an Aquagenomics postdoctoral contract and Epigen-Aqua project ; Peer Reviewed

21 pages, 4 figures, 6 tables, supplementary information https://doi.org/10.1007/s11160-021-09681-9.-- Data availability statement: All the data used in the present study is contained in the online Supplementary information ; The distribution of hermaphroditism in fishes has traditionally been mainly explained by its dependence on biotic factors. However, correlates with major abiotic factors have not been investigated on a quantitative basis and at a global scale. Here, we determined the incidence of hermaphroditism in fish at the family and species level, tested the hypothesis that evolutionary relationships account for the poor presence of hermaphroditism in freshwater species, and tested the association of sexual systems with latitude, habitat type and depth. Functional hermaphroditism is reported in 8 orders, 34 families and 370 species of fishes, all teleosts. Sequential hermaphroditism predominates over simultaneous hermaphroditism at a ratio ~ 5:1 and protogyny (female-to-male sex change) predominates ~ 6:1 over protandry (male-to-female). We found 12 hermaphroditic species that can live in freshwater. However, seven of these species are from four primarily marine families while there are only five species from two mostly freshwater families. Protogynous and bi-directional sex changers have a tighter association with reef-associated tropical and subtropical habitats when compared to protandrous species, which tend to be more plastic in terms of distribution requirements. Finally, simultaneous hermaphrodite species live both in the deep sea and shallow waters in similar proportions. This study can be the basis for further research in specific groups for different purposes, including ecological and evolutionary issues as well as conservation and management of exploited species. Understanding the environmental correlates can help to forecast changes in the distribution or phenology of hermaphrodites in a global change scenario ; We also acknowledge the funding of the Spanish government through the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) and grant PID2019-108888RB-I00. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature ; Peer reviewed

17 pages, 6 figures, 1 table, supplementary material https://doi.org/10.1186/s13072-018-0205-1 ; Abstract Background DNA methylation is one of the main epigenetic mechanisms for the regulation of gene expression in eukaryotes. In the standard model, methylation in gene promoters has received the most attention since it is generally associated with transcriptional silencing. Nevertheless, recent studies in human tissues reveal that methylation of the region downstream of the transcription start site is highly informative of gene expression. Also, in some cell types and specific genes it has been found that methylation of the first intron, a gene feature typically rich in enhancers, is linked with gene expression. However, a genome-wide, tissue-independent, systematic comparative analysis of the relationship between DNA methylation in the first intron and gene expression across vertebrates has not been explored yet. Results The most important findings of this study are: (1) using different tissues from a modern fish, we show a clear genome-wide, tissue-independent quasi-linear inverse relationship between DNA methylation of the first intron and gene expression. (2) This relationship is conserved across vertebrates, since it is also present in the genomes of a model pufferfish, a model frog and different human tissues. Among the gene features, tissues and species interrogated, the first intron's negative correlation with the gene expression was most consistent. (3) We identified more tissue-specific differentially methylated regions (tDMRs) in the first intron than in any other gene feature. These tDMRs have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. (4) Lastly, we identified CpGs in transcription factor binding motifs, enriched in the first intron, the methylation of which tended to increase with the distance from the first exon–first intron boundary, with a concomitant decrease in gene expression. Conclusions Our integrative analysis clearly reveals the important and conserved role of the methylation level of the first intron and its inverse association with gene expression regardless of tissue and species. These findings not only contribute to our basic understanding of the epigenetic regulation of gene expression but also identify the first intron as an informative gene feature regarding the relationship between DNA methylation and gene expression where future studies should be focused ; DA was supported by a Ph.D. scholarship from the Spanish Government (BES-2011-044860). AEC is funded by the RED-BIO project of the Spanish National Bioinformatics Institute (INB) under grant number PT13/0001/0044. The INB is funded by the Spanish National Health Institute Carlos III (ISCIII) and the Spanish Ministry of Economy and Competitiveness (MINECO). Research supported by MINECO grant "Epifarm" (ref. AGL2013-41047-R) to FP. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI) ; Peer Reviewed

9 pages, 6 figures, 1 table, supplementary information http://jeb.biologists.org/content/220/6/1056?panels_ajax_tab_tab=jnl_template_cob_tab_supp&panels_ajax_tab_trigger=supplemental ; 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 ; This study was supported by Ministerio de Economía y Competitividad grants from Spanish government ('EpiFarm': AGL2013-41047-R) to F.P. L.R. was supported by an Epifarm contract; A.V. and N.D. were supported by Ministerio de Economía y Competitividad PhD scholarships (BES-2014-069051 and BES-2007-14273, respectively) ; Peer Reviewed

[Background] The role of epigenetic modifications such as DNA methylation during vertebrate sexual development is far from being clear. Using the zebrafish model, we tested the effects of one of the most common DNA methyltransferase (dnmt) inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), which is approved for the treatment of acute myeloid leukaemia and is under active investigation for the treatment of solid tumours. Several dose–response experiments were carried out during two periods, including not only the very first days of development (0–6 days post-fertilization, dpf), as done in previous studies, but also, and as a novelty, the period of gonadal development (10–30 dpf). ; [Results] Early treatment with 5-aza-dC altered embryonic development, delayed hatching and increased teratology and mortality, as expected. The most striking result, however, was an increase in the number of females, suggesting that alterations induced by 5-aza-dC treatment can affect sexual development as well. Results were confirmed when treatment coincided with gonadal development. In addition, we also found that the adult gonadal transcriptome of 5-aza-dC-exposed females included significant changes in the expression of key reproduction-related genes (e.g. cyp11a1, esr2b and figla), and that several pro-female-related pathways such as the Fanconi anaemia or the Wnt signalling pathways were downregulated. Furthermore, an overall inhibition of genes implicated in epigenetic regulatory mechanisms (e.g. dnmt1, dicer, cbx4) was also observed. ; [Conclusions] Taken together, our results indicate that treatment with a DNA methylation inhibitor can also alter the sexual development in zebrafish, with permanent alterations of the adult gonadal transcriptome, at least in females. Our results show the importance of DNA methylation for proper control of sexual development, open new avenues for the potential control of sex ratios in fish (aquaculture, population control) and call attention to possibly hidden long-term effects of dnmt therapy when used, for example, in the treatment of prepuberal children affected by some types of cancer ; Work supported by Ministry of Economy and Competitiveness (MEC) grants from Spanish government ("EpiFarm": AGL2013-41047-R) and Aquagenomics (CDS2007-0002) to FP LR was supported by an Epifarm contract ; Peer reviewed

12 pages, 5 figures, 3 tables, supplemental material https://doi.org/10.1038/s41598-020-60376-w.-- The datasets supporting this article have been uploaded as part of the supplementary material ; The Sparids are an ideal group of fishes in which to study the evolution of sexual systems since they exhibit a great sexual diversity, from gonochorism (separate sexes) to protandrous (male-first) and protogynous (female-first) sequential hermaphroditism (sex change). According to the size-advantage model (SAM), selection should favour sex change when the second sex achieves greater reproductive success at a larger body size than the first sex. Using phylogenetic comparative methods and a sample of 68 sparid species, we show that protogyny and protandry evolve from gonochorism but evolutionary transitions between these two forms of sequential hermaphroditism are unlikely to happen. Using male gonadosomatic index (GSI) as a measure of investment in gametes and proxy for sperm competition, we find that, while gonochoristic and protogynous species support the predictions of SAM, protandrous species do not, as they exhibit higher GSI values than expected even after considering mating systems and spawning modes. We suggest that small males of protandrous species have to invest disproportionally more in sperm production than predicted not only when spawning in aggregations, with high levels of sperm competition, but also when spawning in pairs due to the need to fertilize highly fecund females, much larger than themselves. We propose that this compensatory mechanism, together with Bateman's principles in sequential hermaphrodites, should be formally incorporated in the SAM ; This project was supported by Spanish Government grant AGL2016-78710-R to FP and the Santander Universities Travel Award Grant 2016/2017 to CB and SP that facilitated the collaboration among the three institutions involved in this study. SP was supported by an AGL2016-78710-R contract

13 pages, 4 figures, 2 tables ; Epigenetics integrates genomic and environmental information to produce a given phenotype. Here, the model of Conserved Epigenetic Regulation of Sex (CERS) is discussed. This model is based on our knowledge on genes involved in sexual development and on epigenetic regulation of gene expression activation and silencing. This model was recently postulated to be applied to the sexual development of fish, and it states that epigenetic and gene expression patterns are more associated with the development of a particular gonadal phenotype, e.g., testis differentiation, rather than with the intrinsic or extrinsic causes that lead to the development of this phenotype. This requires the existence of genes with different epigenetic modifications, for example, changes in DNA methylation levels associated with the development of a particular sex. Focusing on DNA methylation, the identification of CpGs, the methylation of which is linked to sex, constitutes the basis for the identification of Essential Epigenetic Marks (EEM). EEMs are defined as the number and identity of informative epigenetic marks that are strictly necessary, albeit perhaps not sufficient, to bring about a specific, measurable, phenotype of interest. Here, we provide a summary of the genes where DNA methylation has been investigated so far, focusing on fish. We found that cyp19a1a and dmrt1, two key genes for ovary and testis development, respectively, consistently show an inverse relationship between their DNA methylation and expression levels, thus following CERS predictions. However, in foxl2a, a pro-female gene, and amh, a pro-male gene, such relationship is not clear. The available data of other genes related to sexual development such as sox9, gsdf, and amhr2 are also discussed. Next, we discuss the use of CERS to make testable predictions of how sex is epigenetically regulated and to better understand sexual development, as well as the use of EEMs as tools for the diagnosis and prognosis of sex. We argue that CERS can aid in focusing research on the epigenetic regulation of sexual development not only in fish but also in vertebrates in general, particularly in reptiles with temperature sex-determination, and can be the basis for possible practical applications including sex control in aquaculture and also in conservation biology ; This study was supported by the Spanish Ministry of Science grants AGL2016–787107-R "Epimark" to FP and AGL2015-73864-JIN "Ambisex" to LR. DA was supported by an Epimark contract, AV and NS-B were supported by Spanish government scholarships (BES-2014-069051 and BES-2017-079744, respectively); LR and JM-P were supported by Ambisex contracts ; Peer Reviewed

This article is part of the Research Topic Genomics in aquaculture to better understand species biology and accelerate genetic progress.-- 13 pages, 3 figures ; 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 ; This research work was supported by the Spanish Government (Consolider Ingenio Aquagenomics: CSD2007-00002 project) and Spanish Ministerio de Ciencia e Innovación (AGL2009-13273 and AGL2010-15939) projects to Paulino Martínez and Francesc Piferrer ; Peer Reviewed

20 pages, 10 figures, 7 additional files ; [Background] Controlling sex ratios is essential for the aquaculture industry, especially in those species with sex dimorphism for relevant productive traits, hence the importance of knowing how the sexual phenotype is established in fish. Turbot, a very important fish for the aquaculture industry in Europe, shows one of the largest sexual growth dimorphisms amongst marine cultured species, being all-female stocks a desirable goal for the industry. Although important knowledge has been achieved on the genetic basis of sex determination (SD) in this species, the master SD gene remains unknown and precise information on gene expression at the critical stage of sex differentiation is lacking. In the present work, we examined the expression profiles of 29 relevant genes related to sex differentiation, from 60 up to 135 days post fertilization (dpf), when gonads are differentiating. We also considered the influence of three temperature regimes on sex differentiation. ; [Results] The first sex-related differences in molecular markers could be observed at 90 days post fertilization (dpf) and so we have called that time the onset of sex differentiation. Three genes were the first to show differential expression between males and females and also allowed us to sex turbot accurately at the onset of sex differentiation (90 dpf): cyp19a1a, amh and vasa. The expression of genes related to primordial germ cells (vasa, gsdf, tdrd1) started to increase between 75–90 dpf and vasa and tdrd1 later presented higher expression in females (90-105 dpf). Two genes placed on the SD region of turbot (sox2, fxr1) did not show any expression pattern suggestive of a sex determining function. We also detected changes in the expression levels of several genes (ctnnb1, cyp11a, dmrt2 or sox6) depending on culture temperature. ; [Conclusion] Our results enabled us to identify the first sex-associated genetic cues (cyp19a1a, vasa and amh) at the initial stages of gonad development in turbot (90 dpf) and to accurately sex turbot at this age, establishing the correspondence between gene expression profiles and histological sex. Furthermore, we profiled several genes involved in sex differentiation and found specific temperature effects on their expression. ; The study was supported by a project from the Spanish Ministerio de Ciencia e Innovación (AGL2010-22326-C02-01 and CDS2007-0002). Diego Robledo was supported by a FPU fellowship from the Ministerio de Educación Cultura y Deporte of the Spanish Government. Laia Ribas was supported by Aquagenomics and EpigenAqua postdoctoral contract.