Integrated transcriptomic and metabolomic analyses reveal the differential response mechanisms of Gymnocypris eckloni gills to different intensities of acute heat stress.

Temperature represents one of the most critical environmental factors constraining the aquaculture of cold-water fish species. As the primary respiratory organ directly interfacing with the external environment, the gill is among the tissues most sensitive to thermal stress. To elucidate the physiological and molecular mechanisms underlying the thermal response of the plateau cold-water fish Gymnocypris eckloni, this study aimed to investigate the effects of acute heat stress of varying intensities on gill tissue damage and its intrinsic response mechanisms. After acclimation at 16 °C (control group, CO), G. eckloni were subjected to a gradual temperature increase at a rate of 1 °C h-1 to 22 °C (moderate heat group, MH) and 26 °C (high heat group, HH), respectively, and maintained for 12 h. Gill tissues were subsequently collected for histopathological examination, biochemical assays, transcriptomic, and metabolomic analyses to systematically assess the impacts of acute thermal stress on gill morphology, antioxidant defense, immune response, ion regulation, and the expression profiles of related genes and metabolites. Histological observations revealed that heat stress induced apparent gill lesions, including lamellar bending, epithelial edema, protrusion, and detachment, with more severe damage in the HH group. Biochemical analysis demonstrated that high temperature markedly suppressed total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity, increased malondialdehyde (MDA) content, and activated Na+/K+-ATPase activity. Meanwhile, complement component 3 (C3) levels decreased, whereas tumor necrosis factor-α (TNF-α) concentration increased. Integrated multi-omics analysis indicated that moderate heat stress primarily activated adaptive immune and antioxidant pathways, such as the IL-17 signaling and glutathione metabolism pathways. In contrast, high heat stress induced enrichment of the endoplasmic reticulum (ER) protein processing pathway, altered mitochondrial lipid metabolism characterized by acylcarnitine accumulation, and dysregulation of the arginine metabolism pathway. Collectively, acute heat stress induced pronounced structural damage and oxidative stress in the gills of G. eckloni, disturbing immune and osmoregulatory homeostasis. The fish exhibited distinct responses to thermal stress of different intensities: under moderate heat, adaptive compensatory mechanisms involving metabolic resource mobilization and immune modulation were activated, whereas high heat exceeded the physiological compensation capacity, leading to organelle-associated stress responses and metabolic imbalance, thereby shifting from adaptive regulation toward cellular stress and injury. The arginine metabolism pathway may represent a pivotal mechanism mediating the acute thermal stress response in the gills of G. eckloni.