Abstract
Hibernation is a physiological strategy animals use to survive in hostile environments with extreme temperature challenges and food scarcity. During this state, significant changes occur in metabolism and cellular function, with numerous stress response pathways recalibrated to survive physiological challenges that could otherwise be fatal. Numerous studies were performed to explain the molecular mechanisms of mammalian hibernation, but detailed analyses remain scarce in reptiles. Given the limited understanding of the mechanisms regulating hibernation, we performed a comprehensive analysis of liver gene expression in the Chinese softshell turtle ( Pelodiscus sinensis ) comparing summer active (SA), hibernation (H), and early arousal (EA) states using RNA-sequencing. A total of 435 million high-quality reads were generated, identifying 3,508, 3,607, and 2,993 differentially expressed genes (DEGs) in the SA vs. H, H vs. EA, and EA vs. SA respectively. Gene ontology analysis revealed a shift in metabolic fuel utilization, with the down-regulation of metabolic and cellular processes during hibernation, reflecting a conserved strategy for energy conservation. The transition from hibernation to early arousal was marked by up-regulation of immune-related genes (e.g., CXCL12, ITGA4, PIGR) and endocrine regulators (e.g., CDKN1A, DLL4, IGF1R), facilitating metabolic recovery and cellular protection. Besides, we observed dynamic changes in carbohydrate and lipid metabolism, with down-regulation of hexokinase 2 (HK2) and glucose transporters during hibernation, and up-regulation of lipid metabolism genes (LSS, GPLD1) to support membrane integrity and signaling. Our findings provide insights into the molecular mechanisms underlying hibernation and arousal in ectotherms, with implications for understanding metabolic adaptations, immune regulation, and stress responses in extreme conditions.