Sublethal thermal preconditioning induces acquired thermotolerance in Acrossocheilus fasciatus via coordinated gill...

Recurrent heatwaves are emerging as a threat to freshwater fishes, yet most studies have focused on the immediate response to a single heat event, leaving the physiological basis of resilience to repeated warming poorly understood. Acrossocheilus fasciatus, a mountain-stream cyprinid, is an ecologically relevant model because field monitoring indicates that summer heatwaves already pose a substantial threat to its wild populations. Here, fish were first exposed to a sublethal thermal preconditioning regime (22 to 30°C, maintained for 72 h), allowed to recover at 22°C for 5 d, and then subjected, together with non-preconditioned controls, to a more severe heat challenge at 34°C for 72 h. Preconditioning significantly increased critical thermal maximum (CTmax) from 33.96 to 34.85°C, reduced mortality during secondary heat stress, and was associated with trends toward higher antioxidant enzyme activities and lower malondialdehyde accumulation. Gill transcriptomics across the control, preconditioned, heat-stressed, and preconditioned heat-stressed groups showed that severe warming induced extensive transcriptional reprogramming, whereas preconditioning left a molecular signature after recovery that reshaped the secondary heat-stress response. By intersecting retained preconditioning-responsive genes with the core heat-responsive program, we identified 393 shared differentially expressed genes that define a mechanistic model of acquired thermotolerance centered on four coordinated modules: complement-mediated damage sensing and clearance, restraint of complement/protease cascade amplification, heme/iron detoxification and acute-phase homeostasis, and epithelial membrane repair and barrier maintenance. Together, these findings show that sublethal thermal preconditioning induces acquired thermotolerance in A. fasciatus and that this phenotype is underpinned by retained gill mucosal reprogramming.