Analysis of molecular identity and function of putative serotonin receptors in the Giant clam (Tridacna crocea) and the potential role of 5-HT1D-like receptor in reproduction

Abstract

5-hydroxytryptamine (5-HT) is a secondary metabolite that interacts with a corresponding receptor, mediating various processes in organisms. However, the molecular function and characteristics of 5-HT receptor (5-HTR) in giant clam gonads remain unknown. In this study, we cloned full-length complementary DNA (cDNA) of four putative 5-HTRs from Tridacna crocea, designated Tc5-HTR1A-like, Tc5-HTR1D-like, Tc5-HTR2-like and Tc5-HTR4-like genes. SMART analysis revealed that all four Tc5-HTR-like proteins contained a seven-transmembrane structural domain, spanning from GN and ending at Y. Phylogenetic analysis showed that the four Tc5-HTR-like proteins were clustered into four distinct groups. The results of RT-qPCR showed that Tc5-HTR-like genes were widely expressed in all tested tissues, including gonads, suggesting that the function of Tc5-HTR-like genes involves the regulation of reproduction, including gametogenesis and embryonic development. Notably, the Tc5-HTR1D-like gene is highly expressed in oocytes of hermaphrodite and female stages. Estradiol-17β (E2) induced the upregulation of Tc5-HTR1A-like, Tc5-HTR1D-like and Tc5-HTR2-like genes, as well as downregulation of Tc5-HTR4-like gene in the gonad within 72 h. In situ hybridization showed that the Tc5-HTR1D-like gene had a positive signal in oocytes, indicating its role in oogenesis. The knockdown of the Tc5-HTR1D-like gene resulted in a significant reduction in oocyte growth and the number of oocytes. Whole-mount in situ hybridization and RT-qPCR analyses suggested that the spatiotemporal expression pattern of the Tc5-HTR1D-like gene is related to the physiological activities of embryonic development. Our data demonstrate that Tc5-HTR-like genes, especially Tc5-HTR1D-like gene, are essential for regulating gamete growth in T. crocea.