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

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Abstract
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. Human life expectancy is increasing in many populations. Research on aging has gained great attention recently by discoveries of mutations that slow down aging in relatively short-lived models. Studies carried out in yeast, worms, and flies have revealed evolutionarily conserved mechanisms of aging, which are therefore likely to be relevant to mammals, including humans. Therefore, they can provide an important stepping stone for more time-consuming and expensive experiments on mammals. Lifespan studies can be complicated by interactions of genes under study with the environment and with other genes. These effects can be substantially larger than the effects of some mutations with a bona fide effect on lifespan. Here, the authors studied aging in fruit flies using previously described long-lived mutants in the gene Indy, as positive controls for other experiments. Surprisingly, they discovered that Indy mutations do not increase lifespan when the genetic background effects are removed. Similarly, knockdown of genes with a similar function in worms do not increase lifespan in this study. The work presented provides an illustration of how genetic background, and possibly the presence of endosymbionts, can confound studies of the genetics of aging and lead to the spurious appearance of single gene effects on aging where none in fact exist.