Multi-omics joint analysis reveals the genome-wide DNA methylation landscape of Manila clam (Ruditapes philippinarum) and the epigenetic regulation mechanism of immune response induced by pathogens

DNA methylation is an important epigenetic modification in invertebrate immunity, but its role in bivalves under pathogen infection remains unclear. Here, we combined whole-genome bisulfite sequencing (WGBS) and RNA-seq to investigate the immune response of Ruditapes philippinarum challenged by Vibrio anguillarum. Methylation in R. philippinarum occurred mainly in the CpG context, with an average genome-wide level of 9.99%. After infection, the resistant group (VaR) showed significantly higher methylation levels than the control (Con) and susceptible (VaS) groups. We identified 5,355 differentially methylated regions (DMRs) and 2,827 differentially methylated genes (DMGs), which were enriched in immune and metabolic pathways including Notch, mTOR, FoxO, fatty acid metabolism, and glutathione metabolism. Integration with transcriptome data revealed 794 gene body and 177 promoter methylation-related DEGs, with overlapping genes enriched in pathways such as AGE-RAGE, ubiquitin-mediated proteolysis, phagosome, Notch, Wnt, FoxO, and mTOR. Key immune-related DMGs included MRC1, Tnfsf10, Pik3cb, TRAF3, STAT5B, AP-1, and notch1a in gene body region, and CASP6, MIF, and nfkb2 in promoters. BSP sequencing confirmed that MIF promoter methylation was significantly lower in VaR than in Con and VaS, and negatively correlated with gene expression. Functional assays showed that demethylation reduced survival under infection, indicating that DNA methylation promotes pathogen resistance. Overall, promoter methylation repressed immune gene expression, whereas gene body methylation exerted both activating and inhibitory effects. This study provides the first genome-wide methylation map of R. philippinarum, revealing dynamic epigenetic regulation under pathogen stress and offering new insights into invertebrate immune mechanisms. The dataset includes raw BSP sequencing data, qPCR, enzyme activity, and survival measurements, allowing other researchers to reproduce analyses, interpret methylation-gene expression relationships, and explore immune regulatory mechanisms in invertebrates.