Author Summary Normal individual differences in the foraging (for) gene of the fruit fly Drosophila melanogaster result in two behavioral types called rover and sitter. Larval rovers show a greater behavioral response to changes in their food environment than sitters. The for gene makes an enzyme called PKG, which is found in the head of the fly, as well as in most other organisms, including humans. Here, we demonstrate that adult rovers and sitters differ in their metabolic response to changes in their food environment. We measure metabolites in rovers and sitters and show that rovers store energy predominantly as lipids, whereas sitters store it as carbohydrates. We also examine expression levels of genes in rover and sitter heads when the flies are well-fed or food-deprived. We find that for affects levels of gene products involved in carbohydrate and fat metabolism and insulin signaling. We confirm an interaction between for and insulin signaling genes by using genetic mutants to measure their combined effects on fly food-leaving behavior. Our results show that natural variation in this single gene can affect plasticity of large numbers of traits. For behaviour, metabolite and gene expression measures we calculate I, the difference between rover and sitter plasticity, as I = (fed rovers – FD rovers)2(fed sitters – FD sitters); that is, I is the difference between rover response to food and sitter response (for cases when two sitter strains are used, see Statistical Methods). I can be measured for behavioural, metabolic, and gene expression traits, and gives useful information about GEI. I is proportional to the Analysis of Variance (ANOVA) interaction term used to calculate the significance of GEI. Thus, the ‘‘direction of GEI’’, refers to the sign of I. Behaviour The food-leaving assay measures the proportion of flies that traverse a maze after leaving a vial containing sucrose in agar (see Methods and Figure S1). This ‘‘food-leaving’’ behaviour shows significant GEI interaction (Figure 1). To determine how general this pattern of GEI is we repeated these experiments rearing flies on a variety of different food media and in all cases we found significant and similar patterns in GEI (Table S1 for descriptions of food media and statistical tables). Specifically, rover scores increase more from FD to Fed environments than sitter scores. The direction of the GEI is positive (I.0) in all cases. Metabolites We examined Fed and FD forR and fors2 heads to determine compounds most strongly associated with the for response to feeding state, using Fourier Transform Ion Cyclotron Resonance Mass Spectroscopy (FTICR MS, Methods) to detect 750 putative metabolites. We found a significant influence of for (forR or fors2) and feeding state on compounds with the molecular weight (MW) and chemical properties of triacylglycerols (TAG) and polysac- charides (PS) (Figure 2A and 2B; Table S2A, S2B). There was a significant main effect of for in carbohydrates, and significant GEI in both carbohydrates and lipids, but in opposite directions (I.0 for lipids, I,0 for carbohydrates). The largest differences were found in smaller MW compounds. Incorporating MW into ANOVA of TAG compounds gave p(for6food) = 1.3?10213 and p(for6MW) = 1.9?1025; PS compounds had p(for6food6MW) = 0.03 (Table S2B). (Note that the term food in the ANOVA describes the feeding state of Fed vs. FD and the term for refers to foraging and Plasticity Figure 1. foraging gene by food interaction in behaviour. The interaction between for and food [Fed vs. food deprived (FD)] is significant (p = 0.021) and positive in sign (rovers increase more than sitters; I.0) (for medium composition and ANOVA results see Table S1A, S1B, and Methods). The food-leaving score (arcsine-transformed proportion of flies leaving a known food source and traversing a maze; see Methods for assay details) is plotted61 standard error of the mean. doi:10.1371/journal.pgen.1000609.g001 the strains forR or fors2). Thus, GEI interactions are found for metabolites but their direction I depends on metabolite type. Rovers have a larger drop in lipids than sitters in the change from Fed to FD, while sitters have a greater drop in carbohydrates than rovers. Whole-fly spectrophotometric measures of total carbohydrates, lipids, and proteins (Methods) showed that adult rovers had almost twice as much energy stored in whole-body lipid and about half the energy stored in carbohydrates compared to adult sitters, whereas protein levels normalized to dry weight were not significantly different between genotypes (Figure 3 gives full statistics). Thus, for genotype strongly affects energy storage strategies. A main effect of genotype in fed flies is consistent with Figure 2. Rovers and sitters use energy stores differently. Change in the heads of flies in (A) triacylglycerols (TAG) and (B) polysaccharides (PS) between Fed and food deprived (FD) states is higher in rovers than in mutant sitters (positive GEI interaction I.0) for TAG but lower for PS (negative GxE interaction I,0; both interactions significant, Table S2A has ANOVA details). Total signal/noise ratio levels determined using FTICR shown on vertical axis (Methods). FTICR measurements were done on rovers (forR) and mutant sitters (fors2). doi:10.1371/journal.pgen.1000609.g002 PLoS Genetics | www.plosgenetics.org 2 August 2009 | Volume 5 | Issue 8 | e1000609