1. Introduction
Euryhaline organisms, living in transitory habitats such as coastal lagoons or estuaries, are generally characterized by a high phenotypic plasticity. Prolonged or repeated exposure to an environmental stressor leads to changes in phenotypic responses and modification in physiological performances. Salinity strongly fluctuates in transitory habitats. Fish living in these habitats must have efficient strategies and mechanisms that operate at the gene, transcript and protein levels allowing them to respond through acclimation and to adapt. Epigenetic mechanisms like DNA methylation, histone modifications and non-coding RNA play an essential role in promoting phenotypic variation through the modulation of gene expression patterns (Bird et al. 2002). In fish, DNA methylation is the most studied epigenetic modification process in which methyl groups are transferred to the cytosines of DNA by specific DNA methyltransferases. This process potentially regulates gene expression without affecting the DNA sequence (Jones 2012). In vertebrate genomes, DNA is methylated at a high rate with about 60-80% of the cytosine-phosphate guanine (CpG) dinucleotides methylated (Feng et al., 2010). However, a subset of <10% of CpGs form clusters termed CpG islands, that are often associated with genes and known to cover part of their promoter region and at least a part of one exon (Larsen et al. 1992). CpG islands are unmethylated regions, which facilitate active gene transcription. DNA methylation plays a significant role in many biological functions through the regulation of gene expression (Suzuki and Bird, 2008). According to the methylated context considered, however, DNA methylation can have a different role in the regulation of gene expression with either an activation, inhibition, or, will remain without any functional effect (Jones 2012). DNA methylation at promoter level has often been associated with gene silencing in vertebrates (Newell-Price et al. 2000). However, recent studies suggest that DNA methylation dynamics and its regulatory role in gene expression is much more complex and depends on the cell type and genomic context (Smith et al. 2020). Promoter DNA hypermethylation has for example also been associated with high transcriptional activity by several authors (Smith et al. 2020; De Larco et al. 2003). The role of DNA methylation at gene body level was less investigated. It could be involved in transcription elongation, alternative splicing or controlling alternative promoter usage (Suzuki and Bird, 2008; Maunakea et al. 2010; Jones 2012). First introns of human genes are considered as enriched in CpG islands and are thus likely involved in transcriptional regulation (Li et al. 2012). In mammals, Brenet et al. (2011) have established a negative correlation between DNA methylation and gene expression in the first exon, and this correlation was stronger than between promoter DNA methylation and gene expression. Anastasiadi et al. (2018) have shown an inverse correlation between DNA methylation in the first intron and gene expression in European sea bass muscles and testes. The functional role of DNA methylation in different genomic contexts is therefore worth considering and requires further investigations.
Environmentally-induced changes in DNA methylation play an important role in mediating phenotypic responses that provide a substrate for selection (Flores et al. 2013). Epigenetic and genetic components are known to be important for acclimation and adaptation to salinity. In euryhaline fish that switch between salinity-contrasted habitats, epigenetic mechanisms are expected to be the major mechanism of regulation since it allows for rapid and reversible acclimation. In fish, the effect of salinity on DNA methylation has mainly been investigated in stickleback Gasterosteus aculeatus (Artemov et al. 2017; Metzger et Schulte 2018; Heckwolf et al. 2020) and recently in yellow croaker (Larimichthys crocea ) (Yang et al., 2023).Using whole genome bisulfite sequencing in a marine population of stickleback, a majority of hypomethylated cytosines have been shown at a salinity of 21 ppt relative to 2 ppt, suggesting that salinity affects DNA methylation rate. Additionally, genes known to be involved in ion transport in fish were identified with changes in mRNA expression and DNA methylation (Metzger and Schulte 2018). Using a comparative approach between stickleback populations along a natural salinity cline, and gills as a target tissue, differential methylated CpG sites were associated with osmoregulatory processes, notably ion transport and channel activity as well as water homeostasis (Heckwolf et al. 2020). In the gills of brown trout (Salmo trutta ) fed with salt-enriched diets, short-term DNA methylation changes were shown using methylation-sensitive amplified polymorphism (MSAP) (Morán et al. 2013). Studies in marine euryhaline fish species at the genome-wide scale with single base-pair resolution methods are still lacking in order to investigate the role of DNA methylation dynamics in salinity acclimation (Metzger et Schulte 2016).
The European sea bass Dicentrarchus labrax is a main aquaculture species in the Mediterranean area, and has recently become an important model for genetic and epigenetic studies. In this species, environmentally-induced DNA methylation has been investigated in response to temperature (Navarro-Martín et al. 2011; Anastasiadi et al. 2017). D. labrax lives in coastal waters and enters estuaries and coastal lagoons that serve as feeding grounds (Pickett et al. 2004. Dufour et al. 2009). D. labrax have also been observed in freshwater streams that are connected to the coastal lagoons or estuaries. These transitory habitats are characterized by unpredictable salinity fluctuations with salinities ranging from 0 to over 60 ppt in Mediterranean lagoons. The influence of salinity on D. labrax DNA methylation dynamics is still unknown. Also, the functional role of DNA methylation changes on the transcription of genes and modulation of stress response remains poorly investigated.
In this study, we provide a high-resolution analysis of DNA methylation in European sea bass using whole-genome bisulfite sequencing (WGBS) in order to address the question if a 2-week freshwater transfer affects DNA methylation patterns in the gill tissue of D. labraxjuveniles. DNA methylation was analyzed in different genomic regions (promoters vs gene bodies). RNAseq was performed to explore differentially expressed genes following freshwater exposure. To determine if DNA methylation has a functional role in salinity acclimation, we investigated the correlations between gene expression and DNA methylation levels. Salinity-responsive genes identified by RNAseq exhibiting differential DNA methylation patterns were highlighted in order to identify genes or gene families whose expression could be modulated by DNA methylation dynamics.