Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism was increased as well as the expression of DNA methyltransferases 3a. This study reveals novel aspects on the link of DNA methylation and gene expression patterns.

5-methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. How 5mC contributes to phenotypic variation in invertebrates can be investigated by experimental alteration of methylation patterns. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce global changes into the methylome of mollusk species. Flavanone inhibitor Flv1 was highly efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata generation and in its untreated offspring. Drug treatment triggers significant variation in the morphometric traits in both generations. An epigenotyping by sequencing method corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies one Differential Methylated Region (DMR) out of 8, found both in Flv1-exposed snails and its progeny, demonstrating a multigenerational effect of an induced epimutation. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus associated with reduced gene expression.