Exploring the Effects of Rearing Densities on Epigenetic Modifications in the Zebrafish Gonads.

Rearing density directly impacts fish welfare, which, in turn, affects productivity in aquaculture. Previous studies have indicated that high-density rearing during sexual development in fish can induce stress, resulting in a tendency towards male-biased sex ratios in the populations. In recent years, research has defined the relevance of the interactions between the environment and epigenetics playing a key role in the final phenotype. However, the underlying epigenetic mechanisms of individuals exposed to confinement remain elucidated. By using zebrafish (Danio rerio), the DNA methylation promotor region and the gene expression patterns of six genes, namely dnmt1, cyp19a1a, dmrt1, cyp11c1, hsd17b1, and hsd11b2, involved in the DNA maintenance methylation, reproduction, and stress were assessed. Zebrafish larvae were subjected to two high-density conditions (9 and 66 fish/L) during two periods of overlapping sex differentiation of this species (7 to 18 and 18 to 45 days post-fertilization, dpf). Results showed a significant masculinization in the populations of fish subjected to high densities from 18 to 45 dpf. In adulthood, the dnmt1 gene was differentially hypomethylated in ovaries and its expression was significantly downregulated in the testes of fish exposed to high-density. Further, the cyp19a1a gene showed downregulation of gene expression in the ovaries of fish subjected to elevated density, as previously observed in other studies. We proposed dnmt1 as a potential testicular epimarker and the expression of ovarian cyp19a1a as a potential biomarker for predicting stress originated from high densities during the early stages of development. These findings highlight the importance of rearing densities by long-lasting effects in adulthood conveying cautions for stocking protocols in fish hatcheries.

The Model of the Conserved Epigenetic Regulation of Sex.

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.