Direct RNA Sequencing Reveals Sex-Biased Transcriptomic and Epitranscriptomic Regulation in Procambarus clarkii

The red swamp crayfish (<i>Procambarus clarkii</i>) 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 <i>P. clarkii</i>, 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 <i>Dmrt7</i>, <i>FR</i>, <i>Fruitless</i>, <i>IAGBP</i>, <i>RDH</i>, and <i>Vtg</i>. Collectively, these findings provide the first comprehensive epitranscriptomic landscape of <i>P. clarkii</i> gonads, underscoring the pivotal role of post-transcriptional regulation in sex determination and offering valuable insights for mono-sex breeding strategies in aquaculture.