To explore the function of the period gene (Ec-per) in Exopalaemon carinicauda, we cloned the gene of 4611 bp with a 5′UTR of 201 bp, a 3′UTR of 813 bp, and an ORF of 3597 bp encoding 1198 amino acids. The predicted protein includes two PAS and one PERIOD domain. qPCR analysis revealed that Ec-per was expressed across all tissues tested at different developmental stages and during both embryonic and larval stages. Moreover, Ec-per oscillated rhythmically under different conditions of light-to-dark (L:D) ratios, including continuous darkness (0 L:24 D), where changes in the photoperiod influenced amplitude and phase shifts. The knockdown of Ec-per mRNA significantly reduced the expression of the circadian-related genes timeless (tim) and cryptochrome 1 (cry1) (p < 0.05). This suggests that Ec-per is an endogenous clock gene that may participate in molecular feedback loops and synergistically regulate the circadian rhythms through interacting with tim and cry1. RNA interference of Ec-per also markedly downregulated ecdysone receptor mRNA (p < 0.05), suggesting a positive role in the ovarian development of E. carinicauda. In situ hybridization further demonstrated that Ec-per is involved in oocyte proliferation and the accumulation of exogenous nutrients. This study provides new insights for promoting ovarian development and artificial breeding in crustaceans through optimized light-cycle management.

Litopenaeus vannamei, commonly known as the Pacific white shrimp, is one of the most economically significant species in global aquaculture, valued for its rapid growth and adaptability. However, the mechanisms regulating its growth, especially under high-density farming and environmental stress, remain poorly understood. Previous study predicted that LvSlc12A2 was involved in growth regulation. To further reveal the function of this gene in the growth regulation of the whiteleg shrimp, in this study, we explore its function using RT-qPCR, RNA interference, overexpression, and tissue in situ hybridization. RT-qPCR results showed that LvSlc12A2 was highly expressed in gills (about 62%), followed by the hepatopancreas, with the lowest expression in muscle (0.08%, compared to the gills). Myostatin (LvMstn) was mainly expressed in the heart, and molt-inhibiting hormone (LvMIH) in the ventral nerve. In situ hybridization of gill tissues using the mRNA of the gene as a probe revealed strong LvSlc12A2 signals in the gill stratum and epithelial cells. Overexpression of LvSlc12A2, significantly decreased the osmotic gene aquaporin (LvAqp), while knockdown increased its expression. Additionally, levels of growth-related inhibitory genes LvMstn and LvMIH increased significantly after LvSlc12A2 overexpression and were downregulated after its knockdown, suggesting LvSlc12A2 negatively regulates growth, possibly in synergy with LvMstn and LvMIH. These findings indicate LvSlc12A2 influences growth both by negative regulation and by modulating osmotic balance in gill tissues.

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.