Unravelling the neuroendocrine system of nocturnal spawning regulated by circadian clock in the razor clam, Sinonovacula constricta

The razor clam (Sinonovacula constricta), as one of important cultivated molluscs, holds high economic importance. Unlike other bivalve species, S. constricta exclusively spawns at night, so the endogenous regulatory mechanisms driving spawning were preliminarily investigated. To identify crucial genes/proteins that may be involved in regulating nocturnal spawning, ovarian transcriptomes and phosphoproteomes were analysed at four diurnal time points (00:00/ZT16, 06:00/ZT22, 12:00/ZT28, 18:00/ZT34) in S. constricta. Transcriptome analysis revealed 503 and 25 differentially expressed genes (DEGs) when comparing ZT16 vs ZT28 and ZT22 vs ZT34, respectively. DEGs associated with energy and substance metabolism, were predominantly overexpressed in ZT16. Notably, genes related to the circadian clock, the neuro-endocrine system, and reproduction, such as clock, timeless homolog (htim), estradiol 17-beta-dehydrogenase (hsd17b11), 5-hydroxytryptamine receptor (htr), and forkhead box protein O (foxo), exhibited significant changes between the light and dark cycles (P < 0.05). Phosphoproteome analysis identified 177 differentially phosphorylated proteins at ZT16 vs ZT28, which were mainly involved in energy and substance metabolism. The association analysis showed that reproduction related proteins including FoxO were heightened phosphorylation at ZT28. Based on our findings, the circadian clock (clock, htim, etc.), neuro-endocrine system (hsd17bs, htr, gpcr, etc.), and reproduction (foxo and rptor) genes and protein phosphorylation all exhibited circadian rhythm fluctuations, suggesting that they may all be involved in nocturnal spawning of razor clams. These findings establish a foundation for understanding the molecular mechanisms underlying spawning and offer valuable insights into the artificial breeding of molluscs.