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To investigate whether alterations in mitochondrial metabolism affect longevity in  Drosophila melanogaster,  we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of  Indy,  which encodes a  Drosophila  Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these  Indy  mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the  Indy  mutations themselves. Moreover, neither  Indy  mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the  Indy  mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original  Indy  line with long-lived males is infected by the cytoplasmic symbiont  Wolbachia , and the longevity of  Indy  males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for  Indy  mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the  Indy  orthologues  nac-2  and  nac-3  in the nematode  Caenorhabditis elegans .

No Influence of Indy on Lifespan in Drosophila after Correction for Genetic and Cytoplasmic Background Effects