Mitochondrial responses to thermal stress: ROS dynamics and metabolic shifts in Drosophila

Temperature critically impacts ectotherm metabolism, notably mitochondrial respiration, enzyme activity, and ATP production. However, the effect of temperature on reactive oxygen species (ROS) production remains poorly understood in these organisms. Here, we investigated the thermal sensitivity of H₂O₂ production by isolated mitochondria from Drosophila melanogaster. We measured H₂O₂ emission rates at six temperatures (18-45 °C) during: (i) oxidative phosphorylation (OXPHOS) fueled by NADH-linked substrates feeding electrons into complex I (CI), as well as by FADH₂-linked substrates such as proline, succinate, and glycerol-3-phosphate (G3P); and (ii) during non-phosphorylating conditions with FADH₂-linked substrates as well as using defined substrate/inhibitor combinations such as pyruvate, malate and rotenone (P/M-driven), as well as supported by proline, succinate, and G3P when inhibitors are present. We calculated relative H₂O₂ emission rates and compared them with previously measured enzyme activities and oxygen consumption rates. Our results show marked thermal sensitivity of H₂O₂ emission during OXPHOS and when P/M-driven. At elevated temperatures, increased ROS production by NADH-linked substrates during OXPHOS coincided with a decline in CI-induced oxygen consumption capacity and pyruvate dehydrogenase (PDH) activity, indicating a dysfunction in NADH-producing and -consuming systems. In contrast, substrates feeding electrons into the Q pool via FADH₂ oxidation support respiration at high temperature decoupled from ROS production, suggesting a metabolic strategy to sustain respiration while limiting oxidative stress. These findings highlight that mitochondrial thermal sensitivity involves a complex regulation of ROS metabolism. Our study provides new insights into mitochondrial ROS dynamics and their implications for upper thermal tolerance in insects.