The for product PKG and the insulin pathway are conserved across many animals, from worms to flies and mammals [36]. PKG has been found to produce behavioural responses to food in flies [14,33], nematodes [37,38], honeybees [39], and ants [40,41]. In particular, PKG interacts with insulin and TGF-beta signaling in worms to regulate quiescence, a state possibly related to satiety [37]. In this study we have focused on the insulin pathway, but potential interactions between for and TGF-beta may be a fruitful area for future study in flies. GEI, foraging, insulin, and energy store allocation Expression of insulin pathway genes such as InR and Pi3k92E (Dp110) is inversely related to the strength of insulin signaling via the foxo transcription factor: foxo is retained in the cytoplasm when signaling is high, but translocates to the nucleus and stimulates transcription of pathway genes when signaling is low [23,24]. Since insulin gene expression is opposite to insulin signaling strength, our finding of greater negative transcriptional effects on insulin pathway genes in rovers (Figure S2) suggests the presence of greater positive insulin signaling in rovers. Since insulin signaling upregulates anabolism and reduces catabolism [19,42], this is consistent with the patterns we find in genes involved in anabolic (fed rover-biased) and catabolic (fed sitter-biased) pro- cesses. And genes which require insulin for expression are higher in fed rovers, while genes repressed by insulin tend to be higher in fed sitters (Figure 7). The finding of genetic interactions between for and genes in the insulin signaling pathway raises questions for future investigation. Insulin signaling in flies can reduce flow through tricarboxylic acid cycle and oxidative phosphorylation and increase flow through the pentose-phosphate shunt, freeing pyruvate and acetyl CoA for lipogenesis and increasing NADPH and precursors for biosynthesis [43]. This is consistent with patterns in the Fed adult rover catabolic groups (Figure 4C, Table S3). Instead of accumulating energy as fat, Fed adult sitters accumulate carbohydrates. Because of the lower density of fat and its higher caloric content, rovers store more energy per unit mass than sitters, a difference which should have implications for life history characteristics such as starvation resistance (see below). Several studies note changes in fat stores in flies with mutations in insulin signaling genes. These include, loss of fat in melted mutants [44], gain of fat in InR, chico and Pi3K (also called Dp110) mutants [45,46] and Pi3K-overexpression in larvae increases accumulation of nutrients in fat [47]. Nuclear foxo reduces fat, phenocopying starvation [44,48], and it reduces head fat body insulin signaling [49]. Thus, there may be multiple different effects of insulin-related genes on fat. Could foxo mediate lower sitter fat levels? Rapamycin treatment (which acts downstream of foxo specifically on the Tor signaling pathway) also produces similar patterns of effects (Figure 7C). Hence, indirect effects of insulin signaling on the Tor pathway could also be involved. In support of this, PDK/Pk61C is the gene in the insulin pathway showing strongest transcriptional regulation in rovers versus both natural and mutant sitters (Figure S2B). PDK phosphorylates ribosomal S6 kinase (S6k), part of Tor regulation of translation [20,21,50]. Indeed, overexpression of Tor has been shown to increase triglycerides in adult male flies [44]. Genes repressed by foxo, rapamycin, or dilp3 ablation are rover- biased in Fed flies, while genes increased by insulin/Tor knockdown are rover-biased in FD flies (Figure 7A–7C); that is, if a gene’s expression is increased by insulin/Tor signaling, it tends to be higher in Fed rovers, while if it’s expression is increased by inhibition of insulin/Tor signaling, it tends to be higher in FD rovers. This is an example of the more general trend illustrated in foraging and Plasticity Figure 5, where the change between Fed and FD flies is larger in rovers than in sitters for behaviours (89%), metabolites (84%), genes (77%), and gene groups (77%). In mammals, a reduced physiological response to food is a sign of insulin resistance; whether this is true of sitters waits further testing. Our data provides direct evidence through genetic crosses and considerable correlational information through patterns of insulin gene expression and meta-analysis that the GEI effects of foraging in Fed and FD flies are mediated at least in part through interaction with the insulin/Tor signaling pathways. Consequences of lipid–carbohydrate allocation differences Whole-body energy stores in fed rovers and sitters differ: how might fatty rovers and starchy sitters differ in life-history? A number of life-history and ecological parameters have been shown to be related to lipid or carbohydrate reserves in flies, including flight capacity, starvation and desiccation resistance. Diptera in general and fruit flies in particular are dependent on glycogen reserves and hemolymph sugars to fuel flight muscles [51,52,53]. Glycogen phosphorylase, which has strong GEI in rovers and sitters, is a rate limiting enzyme for glycogen mobilization to support flight [54] (Figure 4A). Flies selected for postponed ageing show increased flight duration, glycogen reserves and resistance to desiccation, [55], while desiccation-selected flies show higher glycogen levels [56]. Glycogen, desiccation resistance, longevity and stress resistance may form a cluster of correlated traits in flies [57]. Lipid content of adult flies correlates with starvation resistance [58]; among lifespan-selected and other lines, starvation resistance was correlated with lipid content and not glycogen [55]. This correlation extends to sibling species D. simulans [59]. In a cricket species where lipids can be used to support flight, a trade- off between lipid reserves for flight and for egg production has been reported [60,61]. The rover-sitter system, with its dichotomous Y-allocation [62] of energy stores to lipids and carbohydrates, may therefore be useful for studying single-gene influences on traits with costs and benefits associated with energy use and storage including flight capacity, desiccation and starvation resistance. GEI due to foraging and neuromuscular function The patterns of GEI and I between foraging and food (Fed vs. FD) are very consistent for genes whose primary function is in anabolic or catabolic pathways, with I positive in anabolic groups and negative in catabolic groups. However, there are many more genes with significant for GEI. An important set of such genes is involved in nerve and/or muscle function (Table S3). PKG affects synaptic plasticity in mammals [63,64,65,66] and learning and memory in flies [33,34]. The possibility that PKG may cause GEI through its role in regulating ion homeostasis in nerves and muscles deserves further examination. PRKG1, the mammalian homolog of for, regulates calcium and potassium fluxes in smooth muscle relaxation where it is associated with the myosin phosphatase complex, Ca++ATPases, and potassium ion channels [67]. We find a cluster of gene groups with I,0 associated with muscle and actin cytoskeleton, including genes such as wupA (troponin-I) and Prm (paramyosin). These are some of the genes whose expression is most correlated with for in a coexpression analysis across humans, flies, worms, and yeast [68]. Calcium/potassium levels are important in synaptic function and plasticity, and mutations in potassium channel genes affect habituation in the giant-fiber axon escape reflex in flies [69,70]. Habituation of the giant-fiber escape reflex differs in adult rovers and sitters [31]. Rover-sitter differences in PKG are also associated PLoS Genetics | www.plosgenetics.org 8 August 2009 | Volume 5 | Issue 8 | e1000609