The Timeless (Tim) Gene Participates in Circadian Rhythm Regulation of the Ridgetail White Prawn, Exopalaemon carinicauda.

We previously found that period (per) may exert a positive regulatory effect on ovarian development under the modulation of circadian rhythms. This study further explores the function of the core clock gene timeless. Thus, we cloned the tim gene (Ec-tim) from the ridgetail white prawn, Exopalaemon carinicauda. The full-length Ec-tim cDNA sequence is 6102 bp in length and encodes a protein of 1543 amino acids with a conserved TIM domain. Quantitative real-time PCR (qPCR) analysis revealed that Ec-tim displays prominent eyestalk-specific expression in both adult and sexually mature individuals. Under different light-dark (L:D) cycles, Ec-tim exhibited regular oscillations under various L:D ratios within a 24 h period, and even under constant darkness (0 L:24 D) (p < 0.05), suggesting that tim is an endogenous clock gene. After RNAi-mediated silencing of Ec-tim, the transcript levels of key circadian clock genes, including per and cryptochrome 1 (cry1), were significantly downregulated relative to the control group (p < 0.01). In contrast, the mRNA level of the ecdysone receptor gene (EcR) was markedly upregulated (p < 0.01). These findings suggest that tim may regulate the expression of per and cry1 to form a trimeric complex, synergistically modulating circadian rhythms to further affect ovarian development in shrimp. Collectively, this study provides valuable references for further research on circadian regulation and reproductive development in crustaceans.