Sex-biased alternative splicing in the Zhikong scallop (Chlamys farreri): from transcript isoforms to protein structure

Alternative splicing (AS) is a key post-transcriptional process that enhances transcriptome and proteome diversity, yet its role in bivalve reproduction remains unclear. Here, we employed a multi-faceted approach to investigate sex-specific AS patterns in the gonads of the Zhikong scallop ( Chlamys farreri ). The full-length gonadal transcriptomes were constructed using PacBio Iso-Seq and Illumina RNA-Seq, and identified 447,560 and 423,600 splicing junctions in the ovary and testis. Splicing junctions were significantly enriched in protein-coding genes compared to non-coding genes (average: 16.8 vs 1.6 in the ovary; 18.2 vs 1.7 in the testis), while non-coding genes featured longer introns than coding genes (average: 48,387 bp vs 10,034 bp in the ovary; 48,284 bp vs 9,294 bp in the testis), suggesting a potential link between intron length and splicing frequency. A total of 2,543 differential alternative splicing (DAS) events between sexes was detected, with skipped exon (SE) constituting the predominant AS type. Notably, ovary-biased splicing favored shorter isoforms (exclusion), whereas testis-biased splicing preferred longer isoforms (inclusion). Splicing alterations in approximately 97.8% of DAS genes led to transcript-level expression changes. Frameshift analysis indicated that most mutually exclusive exon (MXE) and SE events maintained open reading frame integrity, whereas approximately 50% of other AS types introduced premature termination codons (PTCs), potentially triggering nonsense-mediated decay (NMD). Key DAS events and corresponding transcript isoforms were validated by RT-qPCR, and dual FISH confirmed germ cell-specific localization of sex-biased isoforms of A1 cf and Jpt1 . Structural modeling indicated that sex-specific exon skipping altered adjacent α-helices or random coils without disrupting core domains, likely performing nuanced adjustments to protein conformation, stability, and interaction networks via intrinsically disordered regions. These results reveal a widespread landscape of sex-biased AS in scallop gonads and provide a foundational resource for future functional studies on splicing-mediated regulation of bivalve reproduction.