Subarctic Drosophila species cold-acclimated in laboratory maintain full capacity for oxygen consumption in their flight muscle mitochondria.

Mitochondria play a central role in energy metabolism and cellular survival, yet their seasonal adaptations in insects enduring prolonged dormancy remain poorly understood. We investigated whether winter-like lab acclimation alters mitochondrial density/abundance and respiratory capacity in the flight muscles of three Drosophila species from subarctic climatic zone and D. melanogaster. Flies were acclimated to either warm (20 °C and long day) or cold (3 °C and short day) conditions, and mitochondrial traits were assessed using microCT, microscopy, molecular proxies of mtDNA abundance, and high-resolution respirometry. Contrary to expectations, cold acclimation induced little to no change in flight muscle structure, mitochondrial density and abundance across all species. Similarly, mitochondrial oxygen consumption capacity measured at 20 °C remained largely unchanged between warm- and cold-acclimated phenotypes, despite pronounced interspecific differences. The only consistent physiological trend was a modest increase in the relative contribution of glycerol-3-phosphate pathway to respiration in cold-acclimated flies. These findings suggest that subarctic Drosophila species enter seasonal dormancy with mitochondria that retain the capacity for normal rates of oxygen consumption. Our study provides a foundation for future work examining mitochondrial performance under ecologically relevant winter (low-temperature) conditions and their resistance to severe cold-induced stress.