An integrated transcriptome, proteome and targeted metabolome analysis reveals genes and steroids related to gonadal development in Ruditapes philippinarum

In this study, gonads at different developmental stages of Ruditapes philippinarum were comprehensively analyzed by transcriptomics, proteomics, and steroid targeted metabolomics, and qPCR was employed to analyze the expression levels of 14 genes in clams across different developmental periods. The results showed that 332 and 560 genes were differentially expressed in the testis and ovary, respectively, while 174 and 107 proteins exhibited differential expression in these gonads. Notably, 12 genes (proteins) were annotated in both transcriptome and proteome during the combinative analysis. Expressed levels for 14 genes (8 from transcriptome and 6 from transcriptome-proteome combinative analysis) demonstrated that CCNB2, MOS, and GLS peaked during the maturation stage and significantly declined during the discharge stage. These three genes were also more highly expressed in the ovary compared to the testis. CDC20, ANPRA, and Rpsr-1 showed peak levels during both maturation and discharge phases, with ANPRA predominantly expressed in the testis, and CDC20 and Rpsr-1 in the ovary; CALM, GLUL, SUSD2, Rpsr-2, Rpsr-3, Rpsr-4 and CD151 attained their highest values in the partially spent stage, where Rpsr-3, Rpsr-4 and CD151 were notably higher in the testis, and CALM, GLUL, SUSD2 and Rpsr-2 were more pronounced in the ovary; PLA2G4 exhibited its highest expression during the growing stage, which was significantly higher in the testis than in the ovary, gradually declining as development progressed. Through targeted steroid metabolomics analysis, 14 steroids were identified in clam gonads. Among these, five steroids - Pregnenolone, 5a-Pregnane-3,20-dione, 17a-Hydroxypregnenolone, 25-Hydroxycholesterol, and 24-dehydro Cholesterol - were found in higher levels during the gonadal growing phase, without notable gender-specific differences. These findings indicate that 14 genes and several steroids played important roles in the gonadal development of R. philippinarum, providing a foundation and reference for future studies on regulative regulation and genetic breeding in this species.