Comprehensive analysis of small non-coding RNAs in the liver of Onychostoma macrolepis during overwintering and non-overwintering periods provides insights into the metabolic mechanisms of fish overwintering

During the overwintering period in aquaculture, fish face dual stressors of low temperature and food scarcity, yet the roles of small non-coding RNAs (sncRNAs) in this process remain poorly understood. This study investigated the changes in liver morphology and sncRNA expression levels in Onychostoma macrolepis (O. macrolepis) during the overwintering (January, March, and October) and non-overwintering (June) periods using histopathological examination, high-throughput sequencing, and bioinformatics analysis. Significant changes were observed in liver morphology, sncRNA expression, and tRNA-derived small RNA (tsRNA) splicing patterns between overwintering and non-overwintering periods, with a pronounced bias in tsRNA expression during non-overwintering. Some up-regulated sncRNAs in overwintering, such as miR-30-1 and tiRNA5-Asp-GTC-1, could be involved in inhibiting mitochondrial and ribosome biogenesis, as well as ATPase and pyruvate kinase activity. Compared with the level expression of sncRNAs in overwintering, some sncRNAs were up-regulated in non-overwintering, such as let-7-1 and tiRNA5-Lys-CTT-1, could be regulated genes involved in cell growth and metabolism, enhancing body metabolism, accelerating liver growth, thereby preparing for feeding and reproduction during the non-overwintering period. Functional experiments using fertilized eggs demonstrated that inhibitors of let-7-1 and tiRNA5-Lys-CTT-1 significantly suppressed embryonic development, further demonstrating confirming the accuracy of functional predictions. These results indicate that O. macrolepis can actively regulate relevant sncRNAs in the liver to reduce its own energy metabolism and growth, thereby entering a low-energy metabolic state during overwintering. In conclusion, these findings significantly advance our understanding of the roles of miRNAs and tsRNAs in enabling O. macrolepis to adapt to winter food scarcity and low-temperature stress.