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.