The Molecular Mechanism of Clock in Thermal Adaptation of Two Congeneric Oyster Species

Clock genes regulate physiological and metabolic processes by responding to changes in environmental light and temperature, and genetic variations in these genes may facilitate environmental adaptation, offering opportunities for resilience to climate change. However, the genetic and molecular mechanisms remain unclear in marine organisms. In this study, we investigated the role of a key clock gene, the circadian locomotor output cycles kaput (<i>Clock</i>), in thermal adaptation using DNA affinity purification sequencing (DAP-Seq) and RNA interference (RNAi)-based transcriptome analysis. In cold-adapted <i>Crassostrea gigas</i> and warm-adapted <i>Crassostrea angulata</i>, <i>Clock</i> was subject to environmental selection and exhibited contrasting expression patterns. The transcriptome analysis revealed 2054 differentially expressed genes (DEGs) following the knockdown of the <i>Clock</i> expression, while DAP-Seq identified 150,807 genes regulated by <i>Clock</i>, including 5273 genes located in promoter regions. The combined analyses identified 201 overlapping genes between the two datasets, of which 98 were annotated in public databases. These 98 genes displayed distinct expression patterns in <i>C. gigas</i> and <i>C. angulata</i> under heat stress, which were potentially regulated by <i>Clock</i>, indicating its role in a molecular regulatory network that responds to heat stress. Notably, a heat-shock protein 70 family gene (<i>Hsp12b</i>) and a tripartite motif-containing protein (<i>Trim3</i>) were significantly upregulated in <i>C. angulata</i> but showed no significant changes in <i>C. gigas</i>, further highlighting their critical roles in thermal adaptation. This study preliminarily constructs a thermal regulatory network involving <i>Clock</i>, providing insights into the molecular mechanisms of clock genes in thermal adaptation.