Sex-biased transcriptome dynamics during embryogenesis of the Eriocheir sinensis

The embryonic stages of Eriocheir sinensis are critical for establishing early sex-differentiation signals, yet the molecular mechanisms remain unclear, partly due to the long-standing lack of effective techniques for sex identification in embryos. In this study, sex was distinguished using a sex-specific molecular marker available exclusively in our research group. Transcriptome sequencing combined with weighted gene co-expression network analysis (WGCNA) was then employed to compare sex-biased gene expression across three embryonic stages (blastula, gastrula, and egg-nauplius). Results showed a "development-dominated, sex-emerging" transcriptional pattern. WGCNA identified 19 co-expression modules in which the gene expression was mainly different among developmental stages. Only a clear sex bias was showed at the blastula stage with green and light-yellow modules. Combined with differentially expressed genes (DEGs), the germline and reproduction related genes (VASA, WNT7, FTZ-F1, and GATA4) were upregulated at the male blastula. Notably, the marked sex regulators (Sxl, tra-2c, fem-1b, runt1a, FoxL2, and Sox-14) showed stage-dependent expression and peaked at the gastrula stage, as supported by both DEG analysis and Mfuzz trend clustering. This gastrula-peaked pattern was accompanied by enrichment of spliceosome and nucleocytoplasmic transport pathways in both sexes. It is suggested that sex differences during embryo genesis may partly be shaped at the RNA/splicing level. These findings reveal the connection between developmental and sex-regulatory programs in E. sinensis embryo stages, providing new molecular insights into crustacean sex differentiation.