Identification and characterization of a laterally transferred alternative oxidase (AOX) in a terrestrial insect, the dipteran Pseudolycoriella hygida

Alternative oxidase (AOX) (EC 1.10.3.11) is a terminal oxidase in the mitochondrial inner membrane that branches the canonical electron transport system (ETS). AOX is ubiquitous in plants, frequently found in fungi and protists and presents a more sporadic distribution in metazoans. More recently, AOX has gained attention due to its potential application in gene therapy for treatment of mitochondrial diseases. Here we characterized the AOX in the basal Dipteran, Pseudolycoriella hygida using a combination of genomic analyses, molecular, functional and in vivo survival assays. AOX is a single copy gene that encodes three developmental stage specific protein isoforms. AOX localizes to the mitochondria in adult thoracic muscles, which present cyanide-resistant respiration that is sensitive to the AOX inhibitor salicylhydroxamic acid (SHAM). Both the cyanide-resistant respiration and AOX levels gradually increase during aging, but are not influenced by thermal stress. Thoracic mitochondria respire using substrates derived from several metabolic routes, such as pyruvate, proline, acylcarnitine, NADH and glycerol-3P, and present values of oxidative phosphorylation capacity ((P-L)/E = 0.70) and coupling (P/L = 4.35; L/E = 0.21). Adult flies exhibit a high survival resistance for SHAM-sensitive complex III inhibition. Together, our results demonstrate the presence of a functional AOX in a terrestrial arthropod and provide insights regarding AOX function in animals and evolution of respiratory systems in metazoans. Psl. hygida emerges as a natural and valuable model for comprehensive AOX research at the whole-organism level which complements models expressing the heterologous enzyme.