Thermal tolerance plasticity of Fundulus heteroclitus is maintained in freshwater and fluctuating temperature conditions.

Understanding how increases in mean temperature and thermal variability impact the physiological flexibility of aquatic species is crucial for predicting the effects of climate change on marine ecosystems. We examined how constant and fluctuating warm temperatures affect thermal tolerance and osmoregulation in Atlantic killifish (Fundulus heteroclitus). Killifish were acclimated for ≥ 4 weeks to control (20 °C), constant (28 °C), and fluctuating (20–28 °C every two days) temperatures in both freshwater (FW) and seawater (SW). Upper thermal tolerance was assessed using critical thermal maximum (CTmax), and plasma osmolality, hematocrit, and glucose levels before and after acute FW→SW and SW→FW transfers. To examine whether long-term acclimation to constant and fluctuating warm temperature is associated with shifts in the steady-state and regulatory responses of gill ionocytes, the gene expression of three branchial ion transporters (ncc2, nkcc1, and cftr1) was measured both before and after an acute salinity transfer. Acclimation to constant and fluctuating warm temperature increased CTmax relative to controls in both FW and SW; however, CTmax was lower in fish acclimated to fluctuating temperature compared to those at constant temperature in FW. Thermal acclimation was linked to an impaired ability to maintain ionic/osmotic homeostasis following an acute transfer from SW→FW, but not from FW→SW. Long-term acclimation to warm temperature in SW was not associated with shifts in either the steady-state or regulatory expression patterns of branchial ion transporters in response to an acute FW transfer. However, upon long-term acclimation to constant and fluctuating warm temperature in FW, we observed an upregulation in the steady-state gene expression of ncc2. Our findings highlight the remarkable plasticity of thermal tolerance in Fundulus heteroclitus under fluctuating temperature and FW conditions. Additionally, we reveal that thermal acclimation is associated with the upregulation of branchial ncc2 gene expression, suggesting a compensatory shift in ionocyte function to enhance ion absorption upon long-term acclimation to warm temperature in FW.