Integrated Transcriptome and Methylome Analyses Reveal Sex-Specific Molecular Responses to Chronic Heat Stress in Tongue Sole (Cynoglossus semilaevis)

Chronic heat stress poses a major challenge to marine aquaculture, yet its sex-associated molecular basis remains poorly understood in Chinese tongue sole (Cynoglossus semilaevis). Juveniles were exposed to a control temperature (24 °C) or elevated temperature (30 °C) for two months, and liver samples from low-temperature females (LF), high-temperature females (HF), low-temperature males (LM), and high-temperature males (HM) were analyzed by RNA sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS). Principal component analysis indicated a strong temperature-associated separation, while sex-related separation under heat stress was smaller and was mainly observed along the second principal component. In high-temperature relative to low-temperature comparisons, females showed a broader set of differentially expressed genes (DEGs; LF vs. HF, 1968) than males (LM vs. HM, 506). Differential methylation analyses indicated that cytosine-guanine (CG) methylation was the predominant heat-associated epigenetic signal. Integrative analysis identified 624 overlapping genes between DEGs and CG-associated differentially methylated genes (CG-DMGs) in females and 177 in males, suggesting broader methylation-associated transcriptional remodeling in females. Functional enrichment associated the female overlap genes with immune response, inflammatory signaling, lipid metabolism, and detoxification, whereas male overlap genes were more closely associated with proteostasis, autophagy, and DNA replication/repair. Correlation analyses suggested modest methylation–expression coupling and highlighted candidate W-linked genes, including H2AZ2 and ANKRD13A. Overall, these results should be regarded as a preliminary baseline for understanding sex-associated molecular responses to chronic heat stress in tongue sole and as a source of candidate genes and pathways for future validation and heat-resilience breeding.