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

Gonadal development in Ruditapes philippinarum involves germ cell formation, sex differentiation, vitellogenesis, and maturation, regulated by complex genes-hormone interactions. To elucidate these mechanisms, multi-omics analyses (transcriptomics, proteomics, and steroid targeted metabolomics) and qPCR were applied to gonads across developmental stages in clams. Differential expression analysis revealed 332 testicular and 560 ovarian genes, alongside 174 testicular and 107 ovarian proteins. Integrated omics identified 12 concordantly annotated gene-protein pairs. Expressed levels for 14 genes (8 transcriptome only, 6 multi-omics) demonstrated that Cyclin B2 (CCNB2), Moloney sarcoma oncogene (MOS), and Glutaminase (GLS) peaked during the maturation stage and significantly declined during the partially spent stage (P < 0.05). These three genes were also more highly expressed in the ovary compared to the testis (P < 0.05). Cell Division Cycle 20 (CDC20), Atrial Natriuretic Peptide Receptor Type A (ANPRA), and Rpsr-1 showed highest levels during both maturation and partially spent stage (P < 0.05), with ANPRA predominantly expressed in the testis, and CDC20 and Rpsr-1 in the ovary; Calmodulin (CALM), Glutamine Synthetase (GLUL), Sushi Domain Containing 2 (SUSD2), Rpsr-2, Rpsr-3, Rpsr-4 and CD151 Molecule (Raph Blood Group) (CD151) attained their highest values in the partially spent stage (P < 0.05), where Rpsr-3, Rpsr-4 and CD151 expressed higher in the testis (P < 0.05), and CALM, GLUL, SUSD2 and Rpsr-2 were more pronounced in the ovary (P < 0.05); Phospholipase A2 Group IVA (PLA2G4) exhibited its highest expression during the growing stage, gradually declining as development progressed, which was significantly higher in the testis than in the ovary (P < 0.05). Targeted metabolomics identified 14 steroids in clam gonads, with five steroids-Pregnenolone, 5α-Pregnane-3,20-dione, 17α-Hydroxypregnenolone, 25-Hydroxycholesterol, and 24-dehydro Cholesterol-elevated during the gonadal growing phase (no sex differences). Intriguingly, 17β-estradiol (typically reported in bivalve gonad development) was undetected. Integrated transcriptomic/proteomic analyses confirmed absence of its synthesis pathway, including key enzymes like cytochrome P450 aromatase (CYP19A1). This study established a multi-omics platform to detect the genes-hormone interactions, and evaluate expression dynamics of 14 key regulators across gonadal stages and sexes in R philippinarum, concurrently evaluating steroid hormones roles. These findings provide valuable resources for further research into the comprehensive regulatory mechanisms governing gonad development in clams.