Dietary restriction (DR) extends healthy lifespan in diverse organisms, and reduces fecundity 1,2. DR is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature 3-6. Long life under DR and high fecundity under full feeding would thus be mutually exclusive, through competition for the same, limiting nutrients. We tested this idea, by identifying the nutrients producing the responses of lifespan and fecundity to DR in Drosophila. Adding essential amino acids to a DR diet increased fecundity and decreased lifespan, similar to full feeding, with other nutrients having little or no effect. However, methionine alone increased fecundity as much as full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the DR responses. Lifespan was reduced by amino acids, particularly essential amino acids. Hence an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during DR. Reduced activity of the insulin/Igf signaling pathway extends lifespan in diverse organisms 7, and it protected against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits for lifespan of DR without reduced fecundity, through a suitable balance of nutrients in the diet.

Dietary restriction (DR) extends life span in many organisms, through unknown mechanisms that may or may not be evolutionarily conserved. Because different laboratories use different diets and techniques for implementing DR, the outcomes may not be strictly comparable. This complicates intra- and interspecific comparisons of the mechanisms of DR and is therefore central to the use of model organisms to research this topic. Drosophila melanogaster is an important model for the study of DR, but the nutritional content of its diet is typically poorly defined. We have compared fly diets composed of different yeasts for their effect on life span and fecundity. We found that only one diet was appropriate for DR experiments, indicating that much of the published work on fly "DR" may have included adverse effects of food composition. We propose procedures to ensure that diets are suitable for the study of DR in Drosophila.

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.

BackgroundOutcomes of lifespan studies in model organisms are particularly susceptible to variations in technical procedures. This is especially true of dietary restriction, which is implemented in many different ways among laboratories.Principal FindingsIn this study, we have examined the effect of laboratory stock maintenance, genotype differences and microbial infection on the ability of dietary restriction (DR) to extend life in the fruit fly Drosophila melanogaster. None of these factors block the DR effect.ConclusionsThese data lend support to the idea that nutrient restriction genuinely extends lifespan in flies, and that any mechanistic discoveries made with this model are of potential relevance to the determinants of lifespan in other organisms.

Dietary restriction (DR) is a moderate reduction in food intake, without malnutrition, that extends the healthy life span in many organisms, including Drosophila (1). Although fruit flies have many practical advantages for studying DR, various technical complexities can have large effects on experimental outcomes (1).For DR, it is important that the basic food conditions are optimal. This ensures that increased longevity due to food restriction prolongs a healthy life span rather than returning sick animals to normal health by limiting access to a nutritionally inappropriate diet. We have systematically optimized our conditions for Drosophila DR, eliminated several non-nutritional explanations including water imbalance, and recommend a Brewer's-yeast-based diet (2, 3). Ja et al. (4) report contradictory data. Here, we present additional data in support of our conclusions and point out a flaw in the data concerning the Brewer's yeast diet presented by Ja et al.First, we developed a system that effectively hydrates flies under salt stress (Table 1, experiment 1). Adding 8 g·L −1 NaCl to our standard food [1.0 SYBrewer's (2)] shortened median life span by 24%. Adding to each vial a 200-μl pipette tip filled with water (1% agar) restored normal life span. The rescue was not due to the tip itself because an identical tip filled with dry cotton wool had no effect. Furthermore, addition of a tip containing wet cotton wool also restored normal life span. This was reproducible when the food was made from a different yeast [SYBaker's (2)] with salt added (Table 1, experiment 2). Thus, our technique is effective in delivering water to salt-stressed flies and in rescuing the associated shortening of the life span.Next, we established that the life-span change associated with DR was not rescued by water addition [ Table 1, experiment 3; replicate of our published data (2)].These data demonstrate that DR in Drosophila under our conditions is not due to rescue of hydration stress.These results directly contradict data from an unreplicated experiment by Ja et al. using conditions ostensibly replicating ours, which reported that life-span extension by DR was eliminated by the addition of water, concluding that hydration stress explains DR (figure 2, G-I, in ref. 4). There are two problems with this conclusion. First, Ja et al. used more sugar (100 g·L −1 ) in their concentrated medium (CM) than did we, and this higher concentration causes a significant reduction in egg laying compared with the level we use (50 g·L −1 ) (2), perhaps because of water stress. [Furthermore, fly feeding behavior can be dramatically reduced as sugar concentration increases in this range, which may explain why Ja et al. observed lowered feeding in their CM ( figure 1F in ref. 4) and we do not.] Second, the data in figure 2, G-I, from Ja et al. (4) demonstrate that water addition shortened DR life span to the level of the CM (Fig. 1). Thus the "rescue" of the DR effect by water addition could also be explained by water shortening the life span of DR fli...