In vivo imaging reveals temperature sensitivity of innate immune cell behavior and tissue regeneration in zebrafish.

Climate change has been linked to increased disease outbreaks in aquatic ectotherms, threatening species persistence. Fish rely primarily on the innate immune system, yet its thermal sensitivity remains poorly understood. We investigated temperature effects on innate immune cell behavior in zebrafish (Danio rerio) larvae by quantifying neutrophil and macrophage recruitment to a wound site and measuring tail regeneration across five ecologically relevant temperatures (22 °C, 28 °C, 31 °C, 33 °C, and 35 °C). Using transgenic zebrafish with fluorescently labelled immune cells, we performed in vivo imaging for 12 h post-tail amputation to track cell recruitment dynamics. Tail regeneration was assessed over three days. Integrating these traits into the thermal performance curve framework enabled comparisons of temperature sensitivities, optima and performance breadth. Neutrophil recruitment rates increased consistently with warming, while macrophage recruitment and tissue regeneration peaked more narrowly around 30-31 °C. This indicates that these processes exhibit distinct thermal sensitivities. By providing real-time, in vivo data on immune cell behavior, this study contributes to improving our understanding of how temperature influences immune responses in fish. Overall, moderate warming (30-31 °C) may enhance the cellular immune response in zebrafish, while higher temperatures disrupt immune resolution, highlighting the need to integrate immune performance into climate change vulnerability assessments of aquatic ectotherms.