Sex is one of the most plastic phenotypes in the evolutionary process. Some species exhibit diverse and plastic patterns of sex determination, even retaining the capacity for sex plasticity in adulthood [1,2]. Alterations in epigenetic modifications triggered by environmental factors are one of the reasons for the occurrence of sexual plasticity. In half-smooth tongue sole (Cynoglossus semilaevis), temperature regulates sex determination-related genes through DNA methylation, leading to the occurrence of masculinization in fish with a female genotype [3]. Environmental stimuli trigger elevated methylation of aromatase genes in bluehead wrasse (Thalassoma bifasciatum), which in turn inhibits the maintenance of female gonads and ultimately causes the reversal of female-to-male [1]. During sex reversal, alterations in DNA methylation are regulated by DNA methylation-related enzyme genes [1]. In addition to DNA methylation modifications, histone demethylation modifications are also capable of triggering sex reversal in temperature-dependent sex determination in red-eared slider turtles (Trachemys scripta elegans) [4]. Therefore, epigenetic regulatory mechanisms possess a critical role in sexual plasticity.

Mollusca are the second largest phylum in the animal kingdom following arthropods, with complex and diverse patterns of sex determination. Bivalves exhibit a prevalent capacity for sexual plasticity, with the Pacific oyster (Crassostrea gigas) being a typical representative of sex reversal due to its relatively pronounced ability for sexual reversal. The Pacific oyster, a world-wide cultured species of significant economic value, lives a sessile life in the intertidal zone, and its complex and variable habitat enhanced its capacity for phenotypic plasticity [5]. The Pacific oyster belongs to a sequential hermaphroditic species, and its sex can be reversed at some point in its life [6,7]. The primary sex ratio (female-to-male) of Pacific oysters is approximately 66 %, with an elevated percentage of females as the age increases [6]. Under warm and adequate food conditions, the sex ratio tends to be biased toward females and vice versa [8]. Therefore, the Pacific oyster provides good material for studying the epigenetic regulatory mechanism of sex reversal in Mollusca.

Sexual plasticity and its evolutionary advantages are evident, especially for sessile oysters. The time window for sex reversal in adult oysters occurs in the resting stage [9,10], during which undifferentiated germ cells or progenitor cells exist in the gonads [11]. These cells will differentiate into male or female gonads under the drive of sex-determining related genes [[12], [13], [14]]. At present, several genes (DMl, Dsx, SoxH, SoxE, Foxl2, β-Catenin, Foxl2os, etc.) with potential involvement in the sex determination signaling pathway were obtained in the Pacific oyster. However, unlike vertebrates, widely expressed genes rather than genes of the sex determination pathway are the primary targets of differential methylation between male and female gonads in the Pacific oyster [15]. For instance, conserved sex-specific genes such as Foxl2 and Dmrt1, which are heavily methylated in vertebrates, are almost unmethylated in oysters [5,15]. However, the mechanisms by which environmental factors trigger sex determination in Pacific oysters, leading to sex reversal, and the potential molecular regulatory mechanisms involved remain unresolved.

In this study, single-base resolution maps of DNA methylation were generated for the gonads of the same individual of the Pacific oyster pre- and post- sex reversal and for the gonads of sex non-reversed individuals. Comparative analysis of gonadal transcriptome and methylome pre- and post-sex reversal was used to explore the epigenetic regulatory mechanisms of sex determination in the Pacific oyster. In addition, the regulatory mechanisms of sex reversal in the Pacific oyster were investigated by comparing the transcriptome and methylome of sexually reversed individuals with those of non-sexually reversed individuals. The results reveal the epigenetic mechanisms underlying the maintenance of gonadal plasticity and organ structural reversal in oysters, providing new insights into the epigenetic regulation of sex determination and sex reversal in invertebrates.