No oxygen limitation of upper thermal tolerance in zebrafish regardless of acclimation temperature

Understanding the physiological mechanisms that limit the upper thermal tolerance of ectothermic animals during acute warming, particularly the role of tissue oxygen supply, is a key area of interest. Studies manipulating water oxygen levels show mixed results, suggesting that the effect of oxygen availability on upper thermal limits is species and context-dependent. Given the importance of thermal acclimation in modulating acute thermal limits, we investigated whether thermal acclimation impacts the oxygen dependence of Critical Thermal Maximum (CT_max) in adult zebrafish (Danio rerio). We conducted a series of three experiments. First, we measured routine and maximum metabolic rates and CT_max of 20°C-acclimated fish across four oxygen levels: 50, 100, 150, and 250 % air saturation to investigate the relationship between water oxygen level and aerobic scope. Second, we acclimated zebrafish to 20, 28, and 34°C for 13-17 days, and tested their CT_max under acute oxygen exposure (30, 100, 200 % air sat). Third, we expanded our sample size for fish acclimated to 14° and 20°C and assessed CT_max across the different oxygen levels. Thermal acclimation strongly influenced CT_max, with colder acclimation lowering CT_max (-6.2°C at 14°C, -2.7°C at 20°C) and warmer acclimation increasing it (+1.5°C at 34°C) relative to the optimal temperature (28°C). Hyperoxia did not increase CT_max at any acclimation temperature, despite significantly expanding their aerobic scope at 20°C. While hypoxia at 50 % air saturation did not reduce aerobic scope or CT_max, severe hypoxia (30 % air saturation) reduced CT_max across all acclimation temperatures. Our findings indicate that upper thermal limits in adult zebrafish are oxygen-independent across a broad range of oxygen levels, regardless of thermal history. Our study suggests that multiple temperature-sensitive physiological functions, rather than only the oxygen limitation mechanism, determines the acute upper thermal tolerance of fish.