Global warming is inducing frequent and severe heatwaves, especially in the Mediterranean area, and changing the neutral range of ambient temperature of farmed fish. Evidence of maladaptation is increasing with every increment of warming , al though current research with a selected fast growing fish strain of gilthead sea bream (Sparus aurata ) reports increased thermal tolerance and growth rates at the IATS aquaculture infrastructure during the extreme climate period from 2022-2023. We wanted to deepen our understanding of the epigenetic mechanisms of this fish population which enabled them to overcome the record- breaking temperatures which generated a water surface temperature of 30 ºC during two consecutive days . More precisely we were interested in exploring tissue- and seasonal-specific DNA methylation changes that can today be identified at a genomic-scale with third-generation sequencing. T he specific aim of this study was t o map seasonal changes of the epigenome from two metabolically active tissues (liver, white skeletal muscle) , and explore whether this is a key driver of different tissue-specific metabolic capabilities of a gilthead sea bream strain.
Fish were fed a commercial-based diet formulation at the IATS flow-through system under natural day-length and temperature conditions . Liver and skeletal muscle tissue portions were taken at two different sampling points (summer (S), July-2022 ; winter (W), February-2023; 5-6 fish/tissue/season). Sequencing libraries were generated with Oxford Nanopore Technologies (ONT) PCR-cDNA Barcoding Kit (SQK-PCB111.24) and sequenced on a PromethION 24 (R10.4.1 chemistry flow cells) . FAST5 files were modification-aware basecalled using Guppy and CSIC reference genome. R esulting files were interrogated for methylation (5mC) using ModKit. Differentially modified (DM) 5mC were calculated with the MethylKit package over those CpG sites with a minimum mapping coverage of 10 reads in at least 5 individuals of each experimental group , and considered significant at a q < 0.05. A total of 99.5 million reads were basecalled with a median Phred score of 16, and an average N50 of 16 kb. Methylation calling rendered ~2.9M 5mC passing the prevalence filters. From them, a total of ~735k DM5mC were found between all the pairwise comparisons, denoting a less- variable epigenome with changing season (3-5k DM5mC) than tissue in both summer and winter (219-407k DM5mC) . In any case, CpG sites followed the same distribution pattern regar dless of the comparison, being predominant in introns (~51%), followed by promoters (~17%), transposable elements (~15%), exons (~11%) and CpG islands (~6%). T he crossing of epigenetic marks and host transcriptome expression remains to be analysed to translate on a functional basis the changing tis sue and seasonal epigenetic landscape . Altogether, this work will offer novel genome-scale methylation marks that would contribute to improve selective breeding and environmental priming for producing tailored climate resilient gilthead sea bream in a challenging scenario.
This study was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/024).