In the European sea bass (Dicentrarchus labrax) variations in offspring sex ratios exist when reared in normal conditions of temperature for early development (13-16ºC). Evidence shows that when reared at higher temperatures (>17ºC) a proportion of the fish which would develop as females under normal conditions are masculinized. Previous research has shown that the DNA methylation of aromatase, a key gene for sexual development, is affected by temperature. Epigenetics integrates genomic and environmental information to beget the final phenotype. Hence, the changes in DNA methylation in response to high temperature can affect the expression of genes relevant for sexual development. Previously, we studied the gonadal DNA methylation profiles on a panel of seven genes related to sexual development in sea bass exposed at low (LT) and high temperatures (HT). We created four families from four sires and one dam (F0), we exposed larvae to LT and HT and examined the DNA methylation by a targeted sequencing approach in juvenile gonads (F1, one year). The genes most affected by both genetics and environment were cyp19a1a and dmrt1, with opposite sex-specific methylation and temperature responses. In the current study, we aim to include a larger proportion of genes in the analysis. We created eight families by crossing an external female with eight males among F1 of the first experiment that were exposed at different temperatures; two LT, three HT and 3 HT neomales (NM). Based on phenotypic and genotypic data of the offspring (sire type, body weight, body length and sex tendency estimated from a genomic animal model using 57K SNP genotypes) we selected fish representative of each group: by sex, temperature and sire type. In total, we have produced 130 libraries (110 Gb of sequencing data) by Reduced Representation Bisulfite sequencing technique to analyze the DNA methylome of grandparents’ gametes (F0, n=2), sires and dam gametes (F1, n=9) and offspring juvenile gonads (F2, n=119). With this approach and the large dataset produced we aim to understand what is the nature of epigenetic inheritance by the comparison of DNA methylation profiles of sperm of the exposed sires (P) and F1 gonads to isolate imprinted regions that escape genomic reprogramming. We will search for differences in DNA methylation of P sperm and F1 gonads between LT males, HT males and HT neomales to understand what is the contribution of the genotype and of the environment to the DNA methylation profiles, and to elucidate whether an additive effect of elevated temperature across generations exists. The comparison of the different methylome profiles will reveal the relationships between grandparent, parent-to-offspring, and the conserved epigenetic effect of temperature regardless of the family. Preliminary results show that there is a total of 4,891 and 3,992 differentially methylated cytosines in females and males, respectively, between progeny exposed at LT and HT from sires exposed at LT (F1). Next, we are going to study how are these results among the progeny of sires exposed at HT (both males and neomales). Research funded by the European Union's 7th Framework Programme under Grant Agreement 652831 (AQUAEXCEL2020, Transnational Access project AE040073) grant and by the Ministry of Economy and Competitiveness ‘Epimark’ (AGL2016-78710-R) grant to FP

6th International Symposium on Genomics in Aquaculture (GIA), 5-7 May 2021