Hypothermia-exposure-induced oxidative stress dysregulates cell fate and perturbs cellular homeostasis and function, thereby disturbing fish health. To evaluate the impact of hypothermia on the freshwater drum (Aplodinotus grunniens), an 8-day experiment was conducted at 25 °C (control group, Con), 18 °C (LT18), and 10 °C (LT10) for 0 h, 8 h, 1 d, 2 d, and 8 d. Antioxidant and non-specific immune parameters reveal hypothermia induced oxidative stress and immunosuppression. Liver ultrastructure alterations indicate hypothermia induced mitochondrial enlargement, nucleoli aggregation, and lipid droplet accumulation under hypothermia exposure. With the analysis of the transcriptome, differentially expressed genes (DEGs) induced by hypothermia were mainly involved in metabolism, immunity and inflammation, programmed cell death, and disease. Furthermore, the inflammatory response and apoptosis were evoked by hypothermia exposure in different immune organs. Interactively, apoptosis and inflammation in immune organs were correlated with antioxidation and immunity suppression induced by hypothermia exposure. In conclusion, these results suggest hypothermia-induced inflammation and apoptosis, which might be the adaptive mechanism of antioxidation and immunity in the freshwater drum. These findings contribute to helping us better understand how freshwater drum adjust to hypothermia stress.

Aplodinotus grunniens, known as freshwater drum, is a kind of eurythermal freshwater fish that is widely distributed in North America. In 2019, our research group reached a milestone on its artificial breeding and cultivation and have investigated its physiological adaption to the environment, providing a breakthrough and prospects for aquaculture. However, its adaptability and metabolic homeostasis to hypothermia is not fully understood. In this experiment, cold stress was conducted at 18 °C (LT18) and 10 °C (LT10) with 25 °C as control (Con) for 8 days to explore the effects of short-term hypothermia on the physiology and metabolism of freshwater drum. From the results, the level of free essential amino acids in LT18 and LT10 decreased significantly after 2 days cold stress compared with Con. Furthermore, plasma total triglyceride (TG) content and lipase (LPS) activity were decreased at LT10 for 2d. With RNA-seq in the liver, metabolic-related signaling, especially amino acid synthesis and lipid metabolism, was inhibited by hypothermia. Specifically, the PPAR signaling pathway is correlated with the inhibition of lipid and amino acid metabolism induced by hypothermia. These data confirmed that PPAR signaling maintains lipid and amino acid metabolic homeostasis during cold stress. These results give a theoretical foundation for hypothermia resistance in the area of metabolic homeostasis for freshwater drum.

Our previous study demonstrated that low temperature could induce hepatic inflammation and suppress the immune and oxidation resistance of freshwater drum. However, the metabolism, especially the glucose and lipid metabolism involved, is poorly studied. To further explore the chronic hypothermia response of freshwater drum, an 8-day hypothermia experiment was conducted at 10 °C to investigate the effect of chronic hypothermia on glucose and lipid metabolism via biochemical and physiological indexes, and metabolic enzyme activities, miRNAs and mRNA-miRNA integrate analysis in the liver. Plasma and hepatic biochemical parameters reveal chronic hypothermia-promoted energy expenditure. Metabolic enzyme levels uncover that glycolysis was enhanced but lipid metabolism was suppressed. Differentially expressed miRNAs induced by hypothermia were mainly involved in glucose and lipid metabolism, programmed cell death, disease, and cancerization. Specifically, KEGG enrichment indicates that AMPK signaling was dysregulated. mRNA-miRNA integrated analysis manifests miR-1 and AMPK, which were actively co-related in the regulatory network. Furthermore, transcriptional expression of key genes demonstrates hypothermia-activated AMPK signaling by miR-1 and subsequently inhibited the downstream glucogenic and glycogenic gene expression and gene expression of fatty acid synthesis. However, glycogenesis was alleviated to the control level while fatty acid synthesis was still suppressed at 8 d. Meanwhile, the gene expressions of glycolysis and fatty acid oxidation were augmented under hypothermia. In conclusion, these results suggest that miR-1/AMPK is an important target for chronic hypothermia control. It provides a theoretical basis for hypothermia resistance on freshwater drum.