Water-independent effects of dietary restriction in Drosophila

Abstract

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. Table 1. Providing a water source to flies rescues the life-shortening effect caused by addition of salt (NaCl) to food, but not life-span alterations due to dietary restriction 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 flies, rather than increasing the life span of those on CM. Fig. 1. Life-span differences due to DR and water addition. Ja et al. (4) reported significant extension of life span when the concentration of nutrients in a Brewer's yeast diet were diluted (DR vs. CM) and that this difference was eliminated by the addition ... These data presented from our laboratory, both here and elsewhere (2, 3), robustly demonstrate that hydration stress does not explain DR under the conditions that we use. Furthermore, we have discovered that DR in Drosophila is mediated by an amino acid imbalance in the food that is not modified by water addition (3).