The red swamp crayfish (Procambarus clarkii) is a globally important freshwater crustacean that exhibits pronounced sexual dimorphism, with males growing faster than females. However, the molecular mechanisms underlying sex differentiation in crustaceans remain poorly understood. In this study, Oxford Nanopore-based Direct RNA Sequencing (DRS) was employed to analyze the gonadal transcriptomes of male and female P. clarkii, identifying 20,001 previously unannotated genes and revealing extensive sex-specific differences in transcript structure, alternative splicing, and RNA modifications. Ovarian transcripts had shorter poly(A) tails and more frequent alternative splicing, while male gonads showed greater enrichment of m⁶A and psU modifications in the 3′ UTRs. qPCR validation confirmed the sex-biased expression of key candidate genes, including Dmrt7, FR, Fruitless, IAGBP, RDH, and Vtg. Collectively, these findings provide the first comprehensive epitranscriptomic landscape of P. clarkii gonads, underscoring the pivotal role of post-transcriptional regulation in sex determination and offering valuable insights for mono-sex breeding strategies in aquaculture.

Crayfish with cyan soft-carapace and red hard-carapace differ considerably in market value. To investigate the causes underlying this difference in shell color, we selected 120 healthy Procambarus clarkii of similar size (9–10 cm), equally divided by shell color and sex. Muscle quality, pigment deposition, and DGAT2 mRNA expression were compared between the two shell-color types. The results showed that: (1) Muscle hardness was significantly higher in red hard-carapace crayfish than in cyan soft-carapace crayfish (p < 0.05), whereas no significant difference in muscle elasticity was observed among groups (p > 0.05). (2) A significant interaction between shell color and sex influenced crude fat and ash content (p < 0.05). Crude fat content was higher in red hard-carapace crayfish, and ash content was highest in male red hard-carapace crayfish, significantly exceeding that in male cyan soft-carapace crayfish (p < 0.05). (3) Moisture and crude protein content were not significantly affected by the interaction between shell color and sex (p > 0.05), with no notable differences across groups. (4) The contents of astaxanthin, lutein, and β-carotene in the shell were significantly influenced by the interaction between sex and shell color (p < 0.05). Astaxanthin was lowest in female cyan soft-carapace crayfish, and lutein was highest in male red hard-carapace crayfish, and β-carotene showed no significant differences. (5) DGAT2 mRNA expression was highest in hepatic tissue across all groups (p < 0.05). In gonads, expression was higher in ovaries of cyan soft-carapace crayfish and in testes of red hard-carapace crayfish (p < 0.05). (6) DGAT2 mRNA expression in the inner membrane, muscle, and intestine was significantly affected by the interaction between shell color and sex (p < 0.05), with specific expression patterns varying among tissues and groups. These findings indicate that shell color and sex interact to influence nutritional composition, carotenoid profile, and gene expression, providing insights into the mechanisms of body color formation and its physiological correlates.