Mitochondria have been suggested to be paramount for temperature adaptation in insects. Considering the large range of environments colonized by this taxon, we hypothesized that species surviving large temperature changes would be those with the most flexible mitochondria. We thus investigated the responses of mitochondrial oxidative phosphorylation (OXPHOS) to temperature in three flying insects: the honeybee (Apis mellifera carnica), the fruit fly (Drosophila melanogaster) and the Colorado potato beetle (Leptinotarsa decemlineata). Specifically, we measured oxygen consumption in permeabilized flight muscles of these species at 6, 12, 18, 24, 30, 36, 42 and 45°C, sequentially using complex I substrates, proline, succinate, and glycerol-3-phosphate (G3P). Complex I respiration rates (CI-OXPHOS) were very sensitive to temperature in honeybees and fruit flies with high oxygen consumption at mid-range temperatures but a sharp decline at high temperatures. Proline oxidation triggers a major increase in respiration only in potato beetles, following the same pattern as CI-OXPHOS for honeybees and fruit flies. Moreover, both succinate and G3P oxidation allowed an important increase in respiration at high temperatures in honeybees and fruit flies (and to a lesser extent in potato beetles). However, when reaching 45°C, this G3P-induced respiration rate dropped dramatically in fruit flies. These results demonstrate that mitochondrial functions are more resilient to high temperatures in honeybees compared to fruit flies. They also indicate an important but species-specific mitochondrial flexibility for substrate oxidation to sustain high oxygen consumption levels at high temperatures and suggest previously unknown adaptive mechanisms of flying insects’ mitochondria to temperature.

Many factors negatively impact domesticated honeybee (Apis mellifera) health causing a global decrease in their population year after year with major losses occurring during winter, and the cause remains thus far unknown. Here, we monitored for 12 months North American colonies of honeybees enduring important temperature variations throughout the year, to assess the metabolism and immune system of honeybees of summer and winter individuals. Our results show that in flight muscle, mitochondrial respiration via complex I during winter is drastically reduced compared to summer. However, the capacity for succinate and glycerol-3-phosphate (G3P) oxidation by mitochondria is increased during winter, resulting in higher mitochondrial oxygen consumption when complex I substrates, succinate and G3P were assessed altogether. Pyruvate kinase, lactate dehydrogenase, aspartate aminotransferase, citrate synthase and malate dehydrogenase tend to have reduced activity levels in winter unlike hexokinase, NADH dehydrogenase and pyruvate dehydrogenase. Transcript abundance of highly important immunity proteins like Vitellogenin and Defensin-1 were also increased in winter bees, and a stronger phagocytic response as well as a better hemocyte viability was observed during winter. Thus, a reorganization of substrate utilization favoring succinate and G3P while negatively affecting complex I of the ETS is occurring during winter. We suggest that this might be due to complex I transitioning to a dormant conformation through post-translational modification. Winter bees also have an increased response for antibacterial elimination in honeybees. Overall, this study highlights previously unknown cellular mechanisms between summer and winter honeybees that further our knowledge about this important species.