In other words, we used the 3 independent studies to tell us which genes may be transcriptionally regulated by insulin signaling. We then used our data to ask, for the same genes, what the rover-sitter difference in expression is under the two food conditions. Our hypothesis is that rovers have higher insulin signaling when Fed than sitters, but not necessarily when FD. Hence we predict that genes requiring insulin signaling for their expression should have higher expression levels in Fed rovers than in Fed sitters, but that this difference may not exist in FD rovers and sitters. Similarly, if genes are shown in the independent study to have lower expression when insulin signaling is high (or equivalently, higher expression when insulin expression is reduced), then we predict those genes should have lower expression in Fed rovers than in Fed sitters. In the first study, Buch et al. [28] ablated dilp3 secreting cells in adults and used microarrays to compare ablation lines which had reduced insulin signaling to that of controls. Figure 7A shows a summary of the four rover-sitter comparisons for this study. Bars on the right labelled ‘‘expression down’’ are for genes whose expression was reduced by dilp3 ablation (i.e. insulin signaling increases expression of these) and bars on the left (‘‘expression up’’) are for genes whose expression was increased by dilp3 ablation (genes repressed by insulin signaling). Genes with expression reduced by dilp3 ablation (Figure 7A, left) show a negative GEI interaction sign I (rovers higher when food deprived, mutant sitters higher when fed). Conversely genes increased by dilp3 ablation (Figure 7A, right) show a positive GEI interaction sign I (no difference when food deprived, rovers higher when fed), in accordance with our predictions. Figure 7B gives the four comparisons for genes whose expression was changed by foxo overexpression [29]; Figure 7C is for genes changed by rapamycin treatment [30]. In each case the pattern is similar to dilp3 ablation: genes with expression increased by a manipulation equivalent to lowering insulin/Tor signaling (genes reduced by insulin) show the negative I GEI interaction, while genes whose expression is reduced by the manipulation (genes increased by insulin) show positive I. Full statistics are given in Table S6. This table also shows that the pattern in I is more significant when natural sitters are used in the analysis than when only mutant sitters are used, so the trends shown in Figure 7 apply to both mutant and natural sitters. In summary, the patterns of GEI interaction strength I in rover- sitter gene expression of genes affected by three different manipulations of insulin/Tor signaling in three independent studies [28,29,30] are consistent with our hypothesis in each study. Genes requiring insulin signaling for expression show positive rover-sitter I and genes inhibited by insulin signaling show negative rover-sitter I. Discussion The foraging gene in Drosophila which encodes PKG is known for its importance as a natural variant affecting behavioural and neural plasticity [11,14,31,32,33,34,35]. We now demonstrate that it also affects metabolic, gene expression, and behavioural plasticity in adult flies. Specifically, rovers show a greater response to changes in their food environment than either mutant or natural sitters for the majority of behavioural, metabolite, and gene expression traits studied here. The pattern of such changes is matched by the pattern of expression of positive regulators of insulin signaling. Combining for alleles with mutants of positive insulin signaling regulators makes rover responses sitter-like, but does not change sitter responses. Collectively these findings suggest that the effect on metabolic, gene expression, and behavioural plasticity of foraging works in part through the insulin signaling pathway. foraging and Plasticity Figure 7. Meta-analysis of 3 manipulations of the insulin/Tor signaling identifies rover-biased genes. Three published studies decreased insulin/Tor effects via (A) ablation of dilp3 expressing cells [28], (B) overexpression of constitutively active foxo [29], or (C) rapamycin [30]. We used data from these papers to identify sets of genes in each study whose expression went up or down in response to the particular insulin/Tor manipulation (Table S6 gives full statistics and methods). For each gene set (expression up or down), we plot average log2 fold change between rovers and mutant sitters in our study on the vertical axis, one bar for FD flies and one for Fed flies. When gene expression is reduced by insulin signaling (e.g. increases due to dilp3/ foxo/rapamycin ablation), food deprived rovers have significantly higher mean expression than sitters (far left in each panel). When gene expression is increased by insulin signaling (e.g. decreases due to dilp3/ foxo/rapamycin ablation), fed rover expression is higher than sitters (far right each panel). This was true for gene sets used from the ablation of dilp3 expressing cells publication [28] (A), the overexpression of foxo publication [29] (B), and the rapamycin treatment paper [30] (C). This results in significant negative interactions (Table S6) for genes repressed by insulin signaling, and significant positive interactions for genes increased by insulin. Error bars = 1 s.e.m. Blue bars, mean rover expression is higher than sitter, red bars, mean sitter expression is higher than rover. doi:10.1371/journal.pgen.1000609.g007 PLoS Genetics | www.plosgenetics.org 7 August 2009 | Volume 5 | Issue 8 | e1000609