Upper thermal tolerance of the euryhaline fish Fundulus heteroclitus is lower in freshwater and shaped by environmental salinity-acclimation time interaction.

This study investigates whether salinity affects the upper thermal tolerance (CTmax) of the euryhaline mummichog (Fundulus heteroclitus). Mummichogs were acclimated to seawater (38 ppt, SW) or freshwater (<1 ppt, FW) at 18.5°C (Tacc), then transferred to the opposite salinity. CTmax and physiological profiles were assessed on days 0, 1, 3, 6, and 13 post-transfer. CTmax was consistently lower in FW than in SW, regardless of prior acclimation. Compared to the Tacc controls, CTmax exposure reduced plasma ion levels in FW-transferred fish and increased them in SW-transferred fish. In both salinities, CTmax exposure raised plasma cortisol, glucose, lactate, and hematocrit. Second-order Akaike Information Criterion (AICc) modeling identified the Salinity/Acclimation time interaction as the most parsimonious predictor of CTmax. Furthermore, multivariate analysis reveals that potential underlying mechanisms of thermal shock diverged by environment. In FW, CTmax progressively declined over time, constrained by a direct metabolic tax-the recruitment of high-overhead, residual (ouabain-insensitive) ATPase activity required to maintain ionic stability-alongside a unique stress-erythropoiesis response. Conversely, in SW, thermal performance improved over time and was tightly coupled to osmolality, glucose and cortisol. Additionally, lactate accumulation was a shared response across salinities. We conclude that the synergy of ion-poor environments and rising temperatures creates a unique performance trade-off that imposes a permanent thermal penalty, potentially increasing the climate vulnerability of estuarine populations beyond current ecological forecasts.