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6th International Symposium on Genomics in Aquaculture (GIA), 5-7 May 2021, The environment can influence the epigenome through modifications that alter gene expression and, consequently, can produce the appearance of a new phenotype. DNA methylation and regulatory microRNAs (miRNAs) are two types of epigenetic mechanisms responsible to integrate environmental cues. In fish, epigenetic modifications can follow as result of external inputs, and some can affect sex determination and differentiation. However, the underlying molecular mechanisms remain poorly understood. Currently, the search for epigenetic biomarkers (epimarkers) to identify specific phenotypes is at the forefront of research. The present study aimed to find epimarkers (DNA methylation and miRNAs) in mature fish gonads linked to abnormal thermal conditions that these fish might have suffered during early gonadal development. Zebrafish (Danio rerio) larvae were exposed to either control or high temperature during sex differentiation (18¿32 days post fertilization). DNA methylation in the promoter region of a set of key genes related to sexual development was studied in adult gonads by a targeted sequencing approach (Multiplex Bisulfite Sequencing, MBS). miRNA expression was explored with RNA-seq using Illumina technology. Furthermore, the spatial distribution of a selection of miRNAs was studied in the ovaries and testes by fluorescent in situ hybridization (FISH). Results showed differences in the methylation level of the promoter of some genes between sexes and temperatures, thus allowing to predict, by machine-learning strategies, epimarkers associated with sex and previous thermal exposure. By biocomputational analysis, we identified 24 unique miRNAs targeting 402 RNA transcripts and that responded differentially to heat in the gonads. Some of these miRNAs were mostly located to germ cells. This study identified DNA methylation and miRNAs changes in the zebrafish adult gonads that can be considered permanent epimarkers of past thermal events. This work was supported by MINECO grants AGL2016-787107-R "Epimark" to FP, AGL2015-73864-JIN "Ambisex" to LR and scholarship BES-2014¿069051 to AV. This study identified DNA methylation and miRNAs changes in the zebrafish adult gonads that can be considered permanent epimarkers of past thermal events. This study identified DNA methylation and miRNAs changes in the zebrafish adult gonads that can be considered permanent epimarkers of past thermal events. Currently, the search for epigenetic biomarkers (epimarkers) to identify specific phenotypes at the forefront of research

11th European Zebrafish Meeting, 26-27 October 2020, Sex determination in fish is a labile trait easily influenced by environmental factors, often temperature. A mismatch between genotypic and phenotypic sex usually tends towards genotypic females developing as males referred to as neomales. Neomales are being discovered in wild populations of several fish species. Thus, understanding the genetic basis leading toward neomales is of great interest in a global climate change scenario. Wild zebrafish strains possess a ZZ/ZW chromosomal sex determination system, in contrast to laboratory strains, which lack a Z chromosome and assume a polygenic sex determination mechanism. Whether temperature alters the sex ratio in wild strains resulting in neomales and whether the gonadal transcriptome differs between genetic males (ZZm) and neomales (ZWm) is unknown. We assessed the rate of sex reversal in Nadia and EkkWill wild strains using different families exposed to 28 °C (control) and 34¿36 °C (masculinizing) temperatures during sex. In adults, histomorphometrics and transcriptomic analyses by RNA sequencing provided insights into gonadal morphology and function. Results showed, surprisingly, that both wild strains were at least as susceptible to the masculinizing effects of elevated temperature as the laboratory strain, with abundant spontaneous and temperature-induced neomales, although the two natural strains had different genotype-by-environment interactions. Histologically, Nadia ZWm but not ZZm showed a higher number of spermatozoa after exposure to elevated temperature. Transcriptomic results had a strong family influence. Further, ZWm testes had a transcriptome profile indistinguishable from ZZm testes. Taken together, these results suggest a genetic basis underlying both the production of neomales and their response to temperature with potential functional consequences in reproductive capacity. Identifying the genes involved will improve our understanding of the mechanisms of zebrafish sex determination

X Annual Chromatin and Epigenetics symposium, Organized by the Chromatin and Epigenetics section of the Catalan Society of Biology (SCB) and the Barcelona Chromatin Club (BCC), 6 March 2020, Barcelona, In many organisms, sex can be determined by a combination of genetic and environmental influences. Recently accumulated evidence shows that changes in environmental conditions can affect population sex ratios through epigenetic regulation of key genes involved in gonadal development. Further, altered sex ratios can persist in the offspring even when the environmental cue is no longer present (a multigenerational effect) and that epigenetic changes tend to be paternally transmitted. However, evidence of transgenerational effects (i.e., beyond the first non-exposed generation) in subsequent generations is scarce and a matter of debate. Here, we used a laboratory strain of zebrafish, where sex determination is dependent on genetic and environmental influences, to study possible multi-and transgenerational effects of exposure to abnormally elevated temperature during the critical period of sex differentiation. To this end, we used eight different families of which we selected five to capture sufficient biological variability in sex ratio at control (28°C) and sex ratio response to elevated (35°C) temperature in the parental (P) generation. Sex ratios were determined at the non-exposed F1 and F2 offspring generations from parents kept at 28°C (control) and35°C (treated). All offspring were raised at 28°C. Global DNA methylation was also assessed in males of the P, F1 and F2 generations derived from exposed and non-exposed fathers. Results showed a consistent increase in the number of males in the P generation in all five families but only a persisting male-skewed sex ratio in the 35°C-derived, unexposed offspring of the F1 generation in two families, denoting family-dependent multigenerational effects. However, no transgenerational effects were observed in any of the families. Likewise, DNA methylation was significantly decreased only in the testis of the 35°C-derived males in the F1 generation but not of the F2 generation and, surprisingly, neither in the 35°C-exposed males of the P generation. Taken together, our results show great interfamily variation, not only in sex ratio response to elevated temperature, but also on its multigenerational effects, denoting a strong influence of genetics. Alterations in the testicular epigenome in F1 males calls for attention to possible, previously-unnoticed effects of temperature in the unexposed offspring of heat-exposed parents in a global warning scenario

Memoria de tesis doctoral presentada por Alejandro Valdivieso Muñoz para obtener el título de Doctor en Genética, Microbiología y Estadística por la Universitat de Barcelona (UB), realizada bajo la dirección del Dr. Francesc Piferrer y de la Dra. Laia Ribas del Institut de Ciències del Mar (ICM-CSIC).-- 318 pages, annexes, In many organisms, sex is determined by a combination of genetic and environmental influences. Epigenetic regulatory mechanisms such as DNA methylation integrate both types of influences. However, understanding how genetic and environmental changes shape the sexual phenotype and the role of epigenetics in this process is far from clear. To address these questions that are of major interest in reproductive and evolutionary biology, in this study we used two zebrafish (Danio rerio) wild strains, with an intact sex-determining loci (sar4), where sex determination is thought to follow a monofactorial system with female heterogamety (ZZ/ZW). We also used the laboratory AB strain, a consolidated model for many research areas, with loss of sar4during the process of domestication, and with polygenic sex determination. Genetic variation was accounted for by using different families of each strain, which were exposed to biotic (rearing density) and abiotic (temperature) environmental perturbations during critical stages of sex differentiation. Elevated rearing density resulted in lower survival and growth, increased masculinization and delayed gonad maturation. Transcriptomic analysis of the adult gonads showed that masculinization was achieved by upregulation of male–related genes and downregulation of female–related genes and, importantly, the participation of the cortisol-mediated stress response. By comparing the gonadal transcriptomes of females resistant to heat-and crowding-induced stress, a common set of differentially expressed genes were identified, constituting novel biomarkers to aid in the identification of hidden effects environmental perturbations. Temperature was able to alter DNA methylation levels of the regulatory regions of sex–and stress–related genes in a clear sex-related fashion. By using machine-learning procedures, we identified specific methylation profiles of some CpG sites in the promoter regions of key genes (cyp19a1a, amh and foxl2a) involved in sex differentiation and in the response to the environment. In wild strains, we discovered elevated rates of spontaneous sex reversal at control temperature, identified novel sexual genotypes and showed genotype-dependent rates of sex reversal under elevated temperature, with possible consequences in sperm production. Contrary to expectations, the presence of sar4in wild strains did not confer higher resistance to temperature when compared to the situation in the AB strain. Finally, effects of elevated temperature on sex ratio and/or DNA methylation in the gonads were inherited, at least in males, in the F1 but only in a family-dependent manner while effects were never detected in the F2. In summary, we developed novel DNA methylation-based biomarkers capable of predicting phenotypic sex and whether fish had been previously exposed to abnormal environmental conditions, paving the way for similar developments in other species. Taken together, these results contribute to our understanding of the role of DNA methylation in shaping the sexual phenotype and can aid towards obtaining a better picture of how environmental changes may affect natural populations in a global warning scenario

1st EPIgenetics in MARine biology congress (EPIMAR) and French Groupement de Recherche Épigenétique en Ecologie et Evolution (GDR 3E) 1st WebConference Epigenetics in Ecology and Evolution, 6-9 October 2020, Epigenetic regulatory mechanisms can contribute to integrate genomic and environmental information to determine sex. In fish, these processes are poorly understood. Some evidence shows that environmental changes can affect sex ratios through epigenetic changes in genes involved in gonadal development. Altered sex ratios can potentially persist in subsequent generations even when the environmental cue is no longer present, but evidence is also scarce. To elucidate both questions, we exposed zebrafish (Danio rerio) larvae of several independent families to high temperature during sex differentiation (18¿32 days post fertilization). We used a targeted sequencing approach to analyze DNA methylation profiles of genes related to sex development and the stress response in adult gonads. Results showed sex-related differences in DNA methylation levels of steroidogenic enzymes (e.g., cyp19a1a, hsd17b1 and hsd11b2) and transcription and growth factors (e.g., dmrt1 and amh). In testes, elevated temperature increased methylation levels of cyp19a1a, cyp11a1 and amh, while it decreased methylation of dmrt1. To study transgenerational effects, five families previously exposed to temperature (parental, P), were raised until to the first (F1) and second (F2) generation. Family sex ratios and global DNA methylation in the testis were assessed. As expected, the P generation was masculinized by temperature. Family-dependent multigenerational effects (to the F1) were observed but no transgenerational effects (to the F2) were observed in any family. Global DNA methylation was significantly decreased in the testis of the 35°C-derived males in the F1 generation but not in the F2 generation neither in the P generation. These alterations in the testicular epigenome in F1 males suggests possible, previously unnoticed, effects of temperature in the unexposed offspring of heat-exposed parents. This novel finding is relevant in a global warning scenario and calls for similar studies in other species

10 Congress of Iberian Association for Comparative Endocrinology : 10º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC) : 10º Congresso da Associação Ibérica de Endocrinologia Comparativa (AIEC), 13-15 July 2017, Vigo, Spain.-- 1 page, Fish exhibit different types of reproduction in which gonads show sexual plasticity. Fish sex determining mechanisms and sex differentiation processes can be modulated by the influence of the environment where they live. Thus, external factors such as temperature or rearing density are known to influence the final sex ratios in some fish populations. However, the molecular mechanisms in which a bipotential gonad differentiates towards an ovary or a testis are not well understood. Thus, functional genomic strategies can help researchers in this field to decipher the complexity of molecular processes in which the final sex of an organism is determined. Here, we report our work carried out in two fish species with important commercial interests, the European sea bass (Dicentrarchus labrax) and the turbot (Scophthalmus maximus), and the work developed in a fish model species, the zebrafish (Danio rerio). In this presentation, we summarize the results of different types of experiments in our laboratory using these fish species in which we identify different transcriptomic profiles based on gene expression and molecular pathway analysis along gonadal development. We focus not only in the analysis of canonical genes related to the endocrinology of testis and ovarian differentiation, but also consider genes related to the stress response and to epigenetic regulatory mechanisms. Next, we compare the molecular common and species-specific patterns found among the sex differentiation processes in these fish species, describing the influences of temperature during early periods of development. Further, we show how temperature and density factors are able to modulate the final sex ratio in zebrafish demonstrating that sex ratio response to elevated temperature is family-specific. Heat-exposed zebrafish revealed new transcriptomic profiles in which some heat-treated females displaying normal ovarian morphology in fact exhibited a ¿male-like¿ gonadal transcriptome, a novel observation in fish physiology and vertebrate biology. Together, these studies contribute to our understanding on how changes in environmental conditions during early life influence sex ratios and thus fish population structure

10 th European Zebrafish Meeting (EZM), 3-7 July 2017, Budapest, Hungary

Plant and Animal Genomics (PAG) Asia, 29-31 May 2017, Seoul, South Korea, One way to increase the production of farmed fish is through exploitation of the fact that in many species one sex, usually females, grows faster than the other. Thus, for the production of monosex stocks, it is necessary first to understand the process of sex differentiation in fish in order to achieve its control. Here, using a tropical species (zebrafish, Danio rerio), a temperate water species (European sea bass, Dicentrarchus labrax) and a cold water species (turbot, Scophthalmus maximus), we investigated common patterns of gene expression during sex differentiation and in response to heat. The genes and signaling pathways involved in ovarian and testicular differentiation are fairly conserved albeit with species-specific differences in their interaction and/or temporal expression. Likewise, exposure to heat has only moderate effects on males and the testis transcriptome. In contrast, elevated temperature can result in genetic females developing functional testis (neomales) with a normal male transcriptome and capable of producing viable sperm. This represents a mismatch between genotype and phenotype. In species with a simple chromosomal system of sex determination like the turbot (ZW/ZZ), neomales can be used to generate novel genotypes (WW, superfemales) to obtain the desired all-female stocks. Recently, we discovered that some heat-exposed zebrafish females have apparently normal ovaries but with a male transcriptome (pseudofemales). In this case, the mismatch is between form and function. However, the fate of pseudofemales, including whether they can produce viable gametes and of what sex is, at present, unknown, Supported by MINECO AGL2016-78710-R grant to FP.

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)

18th International Congress of Comparative Endocrinology (ICCE18) held jointly with 4th Biennial Conference of the North American Society for Comparative Endocrinology (NASCE) and 9th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology (ISAREN), 4-9 June 2017, Banff National Park, Alberta, Canada, Temperature is the main abiotic factor that affects multiple biological functions at different organization levels by changing the rates of physiological processes and chemical reactions. This is especially relevant for poikilothermic animals such as fish and of particular interest in processes like the sexual development. Sex in fishis very plastic since fish exhibit several types of reproduction, including gonochorism, various forms of hermaphroditism, and unisexuality. Further, sex determination can range from genetic sex determination (GSD)to environmental sex determination (ESD). Temperature-dependent sex determination (TSD) has been identified in several species but now is recognized that even species with GSD may have populations with sex ratio response to temperature which, under certain environmental conditions, can produce skewed sex ratios. Here, we used data from many species to show type of reproduction-related differences in thermal preferences and, using a cold water species (turbot, Scophthalmus maximus), a temperate water species (sea bass, Dicentrarchus labrax) and a tropical species (zebrafish, Danio rerio), we investigated common patterns of gene expression in response to heat. We focus not only in the analysis of genes and signaling pathways related to the endocrinology of testis and ovarian differentiation, but also consider genes related to the stress response and to epigenetic regulatory mechanisms. Further, we compared effects of temperature during early development at both the time of sex differentiation and in juveniles and adults. We describe the appearance of new phenotypes (neomales and pseudofemales) as a result of elevated temperature, suggest the existence of transgenerational transmission of epigenetic marks related to sexual development and discuss the possible consequences for natural populations in a global warming scenario, Supported by: MINECO AGL2013-41047-R and 2016-78710-R grants to FP. DA and AV were the recipients of MINECO FPI scholarships

EPICONCEPT Workshop 2016, Cross-species Epigenetics, Gametogenesis and Embryogenesis, 18-19 May 2016, Velingrad, Bulgaria.-- 1 page, The zebrafish (Danio rerio) laboratory strain is slightly different from the wild strain due to the many crossings carried out during many generations. This caused the loss of the telomeric region of chromosome 4, which harbored the putative sex gene. Hence the sex determination system in laboratory strains of zebrafish behaves as a polygenic system. This provides a unique system where to test and understand the effect of environmental factors on sex determination and differentiation in fish. It is well known that environmental factors can be stressors that alter the homeostatic state of the fish, and even if these stressors are applied during the earlier stages of development of zebrafish they can modulate the final sex. In this study, zebrafish of the AB strain was exposed to either elevated temperature or rearing at high density during their differentiation period, to determine how these two factors alter population sex ratios. In addition, we were interested to determine the possible existence of transgenerational effects temperature. Here we exposed one zebrafish generation either to normal (control; 28°C) or elevated (34–36°C) temperature and report the effect on sex ratios in the subsequent unexposed (F1 and F2) generations, searching for transgenerational epigenetic inheritance, and trying to establish whether the effect is paternally or maternally transmitted. We also report the effects of density on sex ratios, establishing the threshold in terms of rearing density in which the effects on sex ratio become apparent, Supported by MINECO grant AGL2013-41047-R “Epifarm” to FP

EPICONCEPT Conference, Epigenetics and Periconception Environment, 26-29 September 2016, Giardini Naxos, Italy.-- 1 page, The European sea bass (Dicentrarchus labrax) and the turbot (Scophthalmus maximus) are two of the most important species for marine aquaculture in Europe, with a production in 2014 of 157,000 and 72,000 tonnes, respectively. Both species are gonochoristic and while the sea bass has a polygenic sex determination system the turbot has a chromosomal system of the ZW/ZZ type. For different reasons, in both of them there is much interest in knowing about sex determination and differentiation in order to produce monosex all-female stocks. Regarding the sea bass, under farming conditions most fish develop as males, which grow less than females and mature earlier, one third of them precociously during the first year. On the other hand, the turbot exhibits the largest sexual growth dimorphism recorded in farmed fish in Europe. Thus, understanding sex determination and differentiation in these species is essential to bring sex under our control. In this talk, we will first discuss essential aspects of sex determination and differentiation in fish, with particular emphasis on the fishes sex ratio response to temperature. Next, we will discuss our research both in sea bass and turbot. In the sea bass, we will explain how by integrating different lines of evidence we ended up with a change of paradigm as regards to the temperature-resistant fish. We will also show effects of temperature on sex differentiation and gonad gene expression. In the turbot, we will explain the use of a genetic marker to produce WW “superfemales”, which are now being used in the industry. Finally, we will discuss our work on epigenetics and how we try to integrate environmental and genetic information for sea bass broodstock management and juvenile production

EPICONCEPT Workshop 2015 Periconception Environment, Epigenetics and Periconception Environment, 26-29 April 2015, Dubrovnik, Croatia.-- 1 page, There are species of fish in which sex determination is genetic, whereas in others is based on an interaction between genetic and environmental factors. Several studies have shown that environmental factors such as temperature and rearing density are able to affect adult se ratios, especially if animals have been exposed during early development. Furthermore, it has been described that temperature is capable of modulating the methylation levels of many genes involved in sex differentiation. Thus, there is evidence of the involvement of epigenetic regulatory mechanisms in the integration of environmental effects to modulate sex ratios in fish. Here, we used laboratory strains of zebrafish, in which sex determination mimics a polygenic system due to the loss of the major component of its genetic sex determination system due to the process of domestication. We used these fish to explore both the involvement of epigenetic mechanisms and transgenerational effects in the long-term modulation of gene expression patterns due to early exposure to either elevated temperature or rearing density. The studied endpoints, which were affected by these treatments, included not only population sex ratios but also genes involved in the stress response (i.e., cortisol synthesis and signalling), DNA methylation (i.e., different dnmts) and gonadal sex differentiation (i.e., different transcription factors and steroidogenic enzymes). Together, these studies contribute to our understanding of the importance of epigenetic regulatory elements during early environment in the establishment of the adult phenotype, Supported by MINECO grant AGL2013-41047-R “Epifarm” to FP

Jornada Anual de la Secció d’Aqüicultura de la Societat Catalana de Biologia, Avenços en Recerca en Aqüicultura, 12 de Juny de 2015, Barcelona.-- 2 pages, Fish exhibit all types of reproduction systems known in vertebrates in which sex determination and sex differentiation establish the final gender. Several environmental factors such as density, temperature or toxicity have been described to affect these two processes in fish, altering the final sex ratio. It is knownthat fish subjected to stress release higher cortisol plasma levels. Cortisol can, in turn, masculinize fish. In previous experiments using zebrafish (Danio rerio) in our laboratory, we have observed masculinization in fish subjected to either high density or cortisolt reated diets. Here, we aimed to understand the molecular mechanisms by which fish are masculinized by density and cortisol. For that purpose, we assessed gene expression in juvenile ovaries and testes by RTPCR of four canonical reproductionrelated genes, five genes involved in the glucocorticoid pathway and one epigenetic gene in fish subjected to different densities and cortisol treatments, during 6-90 and 10-45 days post fertilization, respectively. Results showed a significant downregulation of glucocorticoid receptor (nr3c1) in ovaries and testes and also downregulation of DNA methyltransferase 1 (dnmt1) and gonadal aromatase (cyp19a1a) in ovaries of fish subjected to high density. On the other hand, no differences in any of the studied genes were found in testes of fish fed with cortisol treated diets. In conclusion, this study shows that high density induced masculinization, involving changes in the expression of key sex differentiation genes, notably the downregulation of aromatase

XIV Jornada de Biologia de la Reproducció, 2 July 2015, Bellaterra

Trabajo final presentado por Alejandro Valdivieso Muñoz para el Máster en Genética y Genómica de la Universitat de Barcelona (UB), realizado bajo la dirección de la Dra. Laia Ribas y del Dr. Francesc Piferrer del Institut de Ciències del Mar (ICM-CSIC).-- 42 pages, 10 figures, supplements, Zebrafish is a suitable model organism for studying vertebrate biology in many fields. One of the fields that zebrafish is becoming a powerful tool for its usefulness as a fish model is in aquaculture industry. In aquaculture, the fish is farmed in limited spaces in order to reduce costs, growing the fish at high densities in the facilities. It has been shown that in some fish species high density affects the reproduction and as a result of this effect the sex ratio is altered. Sex determination in zebrafish is ruled by genetic factors and by environmental factors (GSD+TE) following a polygenic sex determination system (PSD) while sex differentiation is defined as gonochoristic of undifferentiated type. It has been reported that environmental factors i.e., temperature affect sex differentiation inducing masculinization in zebrafish. Previous studies in the laboratory showed that density appears to be an environmental factor when zebrafish were highly stocked in the tanks. To better understand the effect of density on sex determination and differentiation in zebrafish we proposed two experiments: experiment 1 where zebrafish were established at different densities during their development (0–90 dpf) and experiment 2 where zebrafish were established at low density, and were feed with treated food during their gonadal development (15–45 dpf). The results were obtained by gene expression studies, transcriptomic ovarian tissues analysis, whole-body cortisol and studies related to survival rates, growth and effects of exogenous compounds. We observed that cortisol was involved and had a relevant role in the process of sex differentiation in zebrafish. In conclusion, our results suggested that density is an environmental factor that directly affects sex ratios in zebrafish inducing masculinization and altering the transcriptome