Final Report Summary - ALTERNATIVE OXIDASE (Investigating the roles of the mitochondrial alternative oxidase of Ciona intestinalis) The respiratory chain alternative enzymes constitute additional pathways to the mitochondrial oxidative phosphorylation (OXPHOS) system, in which oxygen consumption is uncoupled from ATP production. In contrast to the majority of eukaryotic organisms, including other invertebrate animals, vertebrates and arthropods lack any such alternative pathway. However, the expression of alternative oxidases and NADH dehydrogenases from tunicates and fungi in mammalian cultured cells and in the fruitfly Drosophila melanogaster has proven to be benign and to counteract deleterious effects of defective OXPHOS systems, inspiring therapeutic possibilities for mitochondrial and related diseases. The observed benefits are in paradox with the fact that genes for alternative enzymes were lost independently, early in the evolution of both Vertebrata and Arthropoda. This project had two main scientific objectives: 1) to investigate the physiological importance of the mitochondrial alternative oxidase (AOX) of Ciona intestinalis, and the effects caused by the disturbance of its expression for general and mitochondrial metabolism in marine invertebrates; and 2) to investigate the roles of C. intestinalis AOX expression in transgenic lines of Drosophila melanogaster and its possible detrimental effects on mitochondrial metabolism and aerobic performance for ‘higher’ metazoans. The ultimate goal was to identify possible cellular pathways that the AOX is involved in, what regulates its functions, and how the enzyme can influence organism metabolism, providing insights into the evolution of the respiratory chain alternative pathways in animals and how these can be used in putative therapies for mitochondrial disorders. The first objective encountered many unforeseen issues, so the role of AOX in Ciona intestinalis remains elusive, although we have learned much about the biology of this organism. On the other hand, the second objective progressed substantially towards the understanding of how nutrition, temperature and tissue-specificity affects the development and male competitiveness of AOX-expressing flies. We identified that carbohydrate-limited diets reduced the rate of adult eclosion for AOX-expressing flies to only 10-35%. The concentration of yeast extract, the main source of amino acids in the diet, has also been tested, but showed no significant effect. Total cellular ATP, lactate and triglyceride levels, and the NAD+/NADH ratio were significantly higher in larva developing in standard high-sugar food, as expected, but no difference was detected between AOX-expressing and nonexpressing control flies. At 25°C, on standard diet, AOX-expressing flies have no drastic alterations. However, at 29°C, our data shows that AOX-expressing flies cultured in standard high-sugar, zero sugar and high fat diets have problems during adult eclosion. There appears to be a correlation between low levels of sugar/high levels of fat and low percentage of adult eclosion for AOX-expressing flies. In sperm competition assays, which tested the ability of the sperm of AOX-expressing males to compete against wild-type males, all lines expressing AOX under the tubulin promoter were defective. Sperm competition in Drosophila occurs via a dislodgement/substitution mechanism, in which the second male dislodges the sperm of the first male from the female’s spermateca and replaces it with its own gametes. For AOX-expressing males, we detected small seminal vesicles and an accumulation of material on the distal end of the testes, which appears to be mature spermatozoids that never leave the testes to be stored in the seminal vesicles, making these males less competitive. In combination, the results obtained during the development of this project show for the first time that AOX expression in flies can affect fitness, by disturbing development under dietary and temperature stresses and decreasing the competitiveness of males. Future work on the putative use of AOX in gene therapies for mitochondrial disorders will have to take this data into careful consideration to minimize side effects in patients.