foraging and Plasticity whether fors2 or fors are used in comparison to forR. The rover-natural sitter RNS comparison is more rover-biased (has more cases with rovers changing more than sitters) than the rover-mutant sitter comparison, but the correlation between the two is good (for genes, r = 0.83, p,10215), so we show the conservative, common genetic background, rover-sitter mutant distributions in Figure 5. The direction of for-dependent response to feeding state (GEI I) and plasticity (RNS) are different measures, as shown in Table S3. Most groups with significantly negative GEI I have RNS.0. Of the traits with RNS,0, most are mitochondrial groups involved in fatty acid beta oxidation or oxidative phosphorylation complexes I–V. Among metabolites (Table S2A, Table S4) only the group containing PS polysaccharides has significant RNS,0. So, sitters are more plastic than rovers (have a higher magnitude of change in response to food) for a small subset of traits having to do with sugars (among metabolites) or mitochondrial catabolic pathways (functional groups), but rovers are more plastic for the majority of gene and metabolite groups. Hypothesis: plasticity differences and the insulin pathway We hypothesized that the for-dependent metabolic plasticity might be mediated by the insulin signaling pathway. This is because rovers exhibit a higher plasticity in response to feeding state and the insulin signaling pathway is a key regulator of the response to food [18]. In the cell, binding of DILPS (Drosophila insulin-like peptides) to the insulin receptor (InR) triggers a signaling cascade with major effects on gene expression [19]. Protein translation is increased via phosphorylation of key members of the TOR pathway by the kinases Akt1 (dPKB) and Pk61C (dPDK) [20,21]. Negative homeostatic control of insulin signaling occurs on the transcrip- tional level – when signaling is high, foxo is phosphorylated by Akt1 and sequestered in the cytoplasm, but when signaling drops foxo translocates to the nucleus where it stimulates transcription of genes such as InR and the negative regulator of translation Thor (d4EBP) [22,23,24]. At the transcript level, then, many insulin pathway genes have an inverse relationship to the level of insulin signaling. Our results show that transcription of positive regulators decreased more in fed rovers than sitters, resulting in a negative GEI interaction coefficient I for the group of positive regulators as a whole (Figure S2). This normal inverse relationship between transcription and insulin signaling is more evident in rovers than sitters. As with RNS, this is true whether mutant or natural sitters Figure 5. foraging GEI is due to plasticity differences: rovers respond more to food than sitters. In each histogram, the horizontal axis is the measure RNS (relative nutrient sensitivity; Methods) of which genotype has larger response to food. Blue bars, rovers respond more (RNS.0); red bars, mutant sitters respond more. (A) Behavioural plasticity: RNS measured using 9 different food media (Table S1C). RNS.0 for 8 of 9 (89%) and RNS = 20.004 for the ninth. Student t for RNS?0, t = 2.99, df = 8, p = 0.009. (B) Metabolite plasticity: RNS for compounds with a significant response to food. 84% of these had RNS.0. Chi-square contingency test x2 = 65.3, df = 1, p = 6.3610216. (C) Gene expression plasticity: RNS for 1,000 genes with significant food response. Of these, 77% had RNS.0 (x2 = 305.3,df = 1, p,2.2610216). (D) Functional group plasticity. RNS for 300 Gene Ontology groups with significant food response. In 77% of these RNS.0 (x2 = 88.6,df = 1, p,2.2610216). Average mutant sitter change on food deprivation is about K of rover. For simplicity, only rover versus mutant sitter RNS values are shown. This is conservative; rover versus natural sitter gene and gene group RNS distributions were more biased in favour of rovers than the rover vs. mutant sitter (Kolmogorov-Smirnov test for genes, D = 0.236, p,2.2610216; for gene groups D = 0.233, p = 0.000022). doi:10.1371/journal.pgen.1000609.g005 PLoS Genetics | www.plosgenetics.org 5 August 2009 | Volume 5 | Issue 8 | e1000609