Separating good from bad – a methodological assessment of the critical temperature that separates stressful and permissive temperatures in ectotherms

Andreas Havbro Faber , Frederik Due Møller, Michael Ørsted, Bodil Kirstine Ehlers, Johannes Overgaard

Context and aim: Estimating the thermal limits of ectothermic organisms is critical for predicting their responses to climate change. A key physiological threshold in this context is the critical temperature (Tc), which separates the permissive temperature range, where organisms maintain homeostasis and complete their life cycle, from the stressful range, where thermal stress causes physiological disruption and eventually mortality. We aimed to evaluate experimental methods for estimating Tc in ectotherms and assess their practical utility. - Methods: We evaluated four experimental methods to estimate Tc under heat stress in two model ectotherms, the aquatic plant Lemna gibba and the insect Drosophila suzukii. Two methods identify Tc as the end-points of either the classic thermal performance curve, or the thermal death time curve, whereas the other two exploit the antagonistic injury-repair processes above and below Tc. - Results: For both species, three of the four methods that vary in exposure duration and temperature intensity all successfully produced consistent Tc estimates, while one failed to identify Tc. When comparing practical aspects of different methodologies, we find that an assay combining high constant temperature assays interrupted by lower temperatures offer a fast and practical method for estimating Tc. Collectively, Tc coincided with the temperature at which performance declined sharply and mortality increased, supporting its interpretation as a biologically meaningful upper thermal limit for ectotherms. - Conclusions: Tc represents a standardized physiological threshold reflecting the physiological transition from sustained homeostasis to the accumulation of acute thermal damage. Its consistent estimation across methods highlights its utility for comparing thermal limits across studies and species. We propose that Tc should be adopted as a standardized parameter for quantifying upper thermal limits and assessing organismal vulnerability in a warming world.