Validating thermal refuge-seeking behaviour in zebrafish (Danio rerio) during acute thermal challenge

Ectotherms, such as fish, are highly dependent on the stability of their environment to regulate body temperature, performance, and metabolism. Increasing temperatures cause behavioural changes in fish which can be observed and used as indices for determining upper thermal limits. The thermal agitation temperature (T_ag) is a recent, and ecologically significant, sublethal index for the upper thermal limit. Previous studies have described thermal agitation as the endpoint, prior to the critical thermal maximum (CT_max), where fish start exhibiting apparent refuge-seeking and thermal avoidance behaviour. It is an assumption that fish are seeking thermal refuge at T_ag, but evidence for this is lacking. Therefore, this study aimed to validate this assumption by using zebrafish (Danio rerio) and providing them with thermal refuge while increasing their environment's temperature past T_ag. The behavioural responses of D. rerio were observed and their spatial movements were tracked using the animal-tracking software, AnimalTA. The analysis from this study indicated that refuge is sought out prior to T_ag and distance between shoal members increases after T_ag, indicating D. rerio may trade-off the protective value of a shoal to search for thermal refuge. Our study demonstrates that with refuge available, D. rerio can surpass T_ag until refuge itself exceeds T_ag, validating that agitation is refuge-seeking behaviour, but a mechanism of last resort. This insight improves our understanding of fish responses to thermal stress and emphasizes the value of using T_ag as a sublethal metric alongside CT_max in thermal tolerance studies, with potential applications in ecology and conservation contexts.