Experimental DNA Demethylation Reduces Expression Plasticity and Thermal Tolerance in Pacific Oysters

Increasing seawater temperatures pose a great threat to marine organisms, especially those settled in fluctuating intertidal areas. DNA methylation, which can be induced by environmental variation, can influence gene expression and mediate phenotypic plasticity. However, the regulatory mechanisms of DNA methylation in gene expression-mediated adaptation to environmental stress have rarely been elucidated. In this study, DNA demethylation experiments were conducted on a typical intertidal species, the Pacific oyster ( Crassostrea gigas ), to determine the direct role of DNA methylation in regulating gene expression and adaptability under thermal stress. The global methylation level and the expression level of DNA methyltransferases ( DNMT1 , DNMT3a ) showed an accordant variation trend under high temperatures, supporting that the genomic methylation status was catalyzed by DNMTs. DNA methylation inhibitor 5-Azacytidine (5-Aza) effectively inhibited DNA methylation level and decreased methylation plasticity at the 6th hour in thermal conditions. In total, 88 genes were identified as candidate DNA methylation-regulated thermal response genes; they exhibited reduced expression plasticity in response to heat stress, possibly caused by the decreased methylation plasticity. Post-heat shock, the thermal tolerance indicated by the survival curve was reduced when oysters were pretreated with 5-Aza, meaning that DNA demethylation negatively affected thermal adaptation in oysters. This study provides direct evidence for the crucial role of DNA methylation in mediating stress adaptation in marine invertebrates and contributes to the theoretical foundations underlying marine resource conservation and aquaculture.