Diel temperature fluctuations alter molecular, biochemical, and whole-animal responses to thermal stress and hypoxia in adult zebrafish.

Aquatic ecosystems naturally experience temperature fluctuations, which are often accompanied by shifts in dissolved oxygen. However, climate change has exacerbated the prevalence and magnitude of these fluctuations, often leaving organisms exposed to sub-optimal conditions. As a result of their inherent relationship, these abiotic factors are thought to share common signalling pathways (cross-talk) and protective mechanisms (cross-tolerance) that confer cross-protection. Much of what we currently understand about the effect of temperature on fish originates from static exposures, despite physiological performance differing in fluctuating environments. Therefore, we aimed to determine whether diel temperature fluctuations influence thermal and hypoxia tolerance, while simultaneously characterizing underpinning molecular and biochemical adjustments. Zebrafish were acclimated to either static control conditions (27 °C) or a thermal flux (23-33 °C), after which they were sampled for brain and liver tissue, or subjected to acute thermal (critical thermal maximum) and hypoxia (time to loss of equilibrium) challenges. We detected that diel thermal variability causes differential expression of microRNAs (miR-301c-5p and let-7d-5p), mRNAs (heat-shock protein genes: hsp70 and hsp90αα), proteins (Hsp70), and metabolic enzyme activity (lactate dehydrogenase: LDH). In addition to these molecular and biochemical changes, fish acclimated to diel temperatures exhibited increased thermal and hypoxia tolerance. Collectively, our results demonstrate that diel thermal variability elicits distinct responses across multiple levels of biological organization, reiterating the importance of incorporating more natural thermal regimes in future studies.