Genome-wide DNA Methylation Difference Between Sexes in Juveniles and Epigenetic Regulation of Sexual Development in Intersex Crabs Eriocheir sinensis.

Epigenetic regulation of sexual development is widely documented in model species, yet data for commercial invertebrates remain limited. Here, we used whole-genome bisulfite sequencing (WGBS) to profile genome-wide DNA methylation and identify differentially methylated regions (DMRs) between the female and male juveniles at stage III in the Chinese mitten crab Eriocheir sinensis. Global methylated cytosines accounted for approximately 1.44% and 1.45% of all cytosines in the female and male genomes, respectively. Among the CpG, CHG, and CHH contexts, CpG showed the highest mean methylation levels, at 8.15% in females and 8.08% in males. A total of 21,038 nominally significant DMRs were identified between sexes, with 11,557 (55%) hypermethylated DMRs in males. Notably, the methylation level of the ZW chromosome was the highest compared to the ZZ chromosome and the autosomes, suggesting sex-biased epigenetic modification within the ZW/ZZ sex determination system. On autosomes, the hypermethylated DMRs in females were mainly detected in the gene body regions of Sex lethal, Male-specific lethal 3, Feminization-1c, and Feminization-1a. On the ZW chromosome, 233 DMRs were hypermethylated in females, whereas 161 were hypermethylated in males. Furthermore, in the intersex crabs (ZW) bearing male first gonopods, the promoter of LOC113817324 exhibited intersex-specific hypermethylation in the ovary, whereas intronic methylation of the Serine/threonine-protein kinase (ATR) remained low, mirroring the pattern observed in normal male (ZZ) testes. These findings reveal sex- and intersex-associated DNA methylation patterns in E. sinensis and provide candidate epigenetic loci for further investigation of sexual development.