Optimized High-Resolution Respirometry Defines the Bioenergetic Signature of Drosophila melanogaster Thoracic Muscle

Drosophila melanogaster provides a robust model system for investigating mitochondrial metabolism in muscle tissue, particularly due to availability of fly strains that recapitulate genetic mitochondrial disorders, muscle atrophy, and related pathological conditions. However, most experimental approaches are adaptations of protocols originally developed for mammalian muscles, primarily rodents. Here, we systematically applied and refined variations of these mammalian-based protocols to female adult thoracic muscles of D. melanogaster, with particular emphasis on tissue permeabilization, respiratory substrates, respiratory chain inhibitors, and oxidative phosphorylation uncouplers. Our findings appointed pyruvate as the principal respiratory substrate and suggested succinate as a major anaplerotic contributor to the tricarboxylic acid cycle during NADH dehydrogenase-supported respiration. In addition, Drosophila thoracic muscle exhibits a maximal respiratory flux strongly dependent on ADP levels, with oxidative phosphorylation operating near the maximal capacity of the electron transfer system. We compared Drosophila thoracic muscle with Drosophila larvae fillet muscle, as well as murine and human skeletal muscle, revealing species- and tissue-specific differences in mitochondrial metabolism. Finally, we validated our refined protocol in a PINK1 RNAi Drosophila model, extending previous evidence of mitochondrial dysfunction associated with PINK1 deficiency in flies.