Deciphering the genetic basis of sex differentiation in silver-lipped pearl oyster (Pinctada maxima) by the integrative transcriptome analysis

The silver-lipped pearl oyster (Pinctada maxima) is the largest pearl-producing oyster and known for producing high-value large lustrous pearls. It exhibits the feature of sequential hermaphroditism and the production of pearls displays sex dimorphism. Thus, deciphering the molecular mechanisms of sex determination/differentiation is crucial for the development of pearl oyster breeding industry. In this study, integrative transcriptomic analyses were conducted of P. maxima using isoform and RNA sequencing (Iso-Seq and RNA-Seq). A total of 4,581 differential expression genes (DEGs) were identified between ovary and testis, gene co-expression network analysis identified four related modules, including three sex-specific modules and one sexually shared module. Through integrative analysis of hub genes and DEGs, our study revealed multiple genes key for gonad development in P. maxima, of which FOXL2, NANOS1, and β-catenin were important for ovary development, and DMRT, SOX30, FEM1, and FOXJ1 were crucial for testis development. Interestingly, genes of sexually shared module were significantly enriched in spliceosome pathway, and alternative splicing analysis revealed the abundant and widespread distribution of alternative splicing in P. maxima gonadal tissues. More active alternative splicing process in testis were observed than in ovary. Nearly half (47.83%, 375) of the identified genes undergoing differential alternative splicing (DASGs) were genes undergoing different transcript usage (DTUGs), and only 24% DTUGs were DEGs. The genes related to sex differentiation, like DMRT, β-catenin and U2AF2, were found to have sex-specific and/or sex-biased isoforms. Our study provides a deep insight into the molecular basis of sex differentiation in P. maxima, contributing to a comprehensive understanding of the reproductive regulation network in pearl oysters.