are compared, but again as for RNS, the difference between rovers and natural sitters is larger than the difference between rovers and mutant sitters, suggesting that the genetic background of natural sitters may intensify this difference. Genetic test of interaction of foraging and insulin genes The finding that rovers show larger responses to food, and the known role of insulin signaling in the response to food, suggests that rovers might also show a larger impact of changes to insulin signaling. We therefore tested whether for interacts with the insulin signaling pathway by means of quantitative complementation crosses for epistasis between mutant insulin pathway genes and alleles of for [25,26,27]. We crossed each of the three for genotypes to loss of function mutants of the fly insulin receptor InR, phosphatidylinositol-3-kinase Pi3K92E (or Dp110), and foxo (Methods). InR and Dp110 are positive regulators of insulin signaling; foxo is a negative regulator. Based on our gene expression data, we hypothesized that rovers had higher insulin signaling than sitters, so we expected crosses of rovers with loss of function insulin mutants to be more sitter-like than their controls. We tested food-deprived adults of the resulting 18 trans heterozygote genotypes and compared food-leaving scores of for;mutant to the for;Balancer which controlled for genetic back- ground effects (see Methods). Recall that food-deprived homozy- gous rovers show low levels of food leaving behaviour (Figure 1) while sitters have higher levels. As expected, the control for;Balancer flies show the previously found lower level of behavioural response for rovers compared to sitters (Figure 6A and 6B, solid lines), indicating no direct effects of, or interactions with, the balancer chromosome background. There is, however, a significant epistatic interaction in the for;InR and for;dp110 flies (Figure 6A and 6B dashed lines; Table S5). Rovers crossed to these insulin pathway mutants become more sitter-like. In contrast, the interaction with negative regulator foxo is not significant (Table S5). Mutants of foraging and Plasticity positive regulators of insulin signaling make rover food-leaving behaviour more like sitters (reduces RNS), while a mutant of negative regulator foxo trended towards making rovers less like sitters (increases RNS). This suggests that there is a significant (epistatic) interaction between for and the two positive regulators of the insulin signaling pathway tested here. This is consistent with rovers experiencing greater shifts in insulin signaling effects between Fed and FD states than sitters. There are also differences between natural and mutant sitters in the interaction with InR (Table S5) suggesting that the difference in genetic backgrounds between these strains may also affect this interaction. For this behavioural measure, natural sitters are intermediate between rovers and mutant sitters, a difference from the trend found in gene expression overall (via RNS, Figure 5) or in regulators of insulin signaling (Figure S2). Thus the effect of the background difference between natural sitters and the other strains varies between gene expression and behavioural measures. Meta-analysis of insulin and foraging effects on gene expression We performed a bioinformatic meta-analysis comparing our array results to those from three published microarray studies which manipulated insulin/Tor signaling [28,29,30]. This pro- vides additional evidence for transcriptional parallels between for and insulin. We use these studies to identify sets of genes which were up- or down-regulated by the manipulation of insulin/Tor signaling, and which had high enough expression levels in our data for reliable comparison. To ensure independence of the three analyses we used sets of genes which did not overlap between studies (see Table S6 for gene selection criteria). For each up- or down-regulated set of genes identified from a study we calculated the mean log2 fold change between rovers and mutant sitters when Fed or FD. This gave four comparisons per study (up/down regulated in study 1, 2 or 36Fed/FD in our data). Figure 6. Insulin pathway genes interact with foraging alleles in expression and in food-leaving behaviour. Flies carrying different for alleles crossed to mutants of the positive regulators (A) InR and (B) Dp110 (dashed curves) show almost none of the normal food-deprived rover-sitter food-leaving difference (compare to Figure 1 and solid balancer curves in this figure). In these quantitative complementation crosses, the food leaving behaviour of FD for;mutant transheterozygotes is compared to the FD for;Balancer transheterozygote controls. The difference in food leaving between the control balancer and mutant cross depends significantly on foraging allele, demonstrating interaction between the mutant gene and for. p(Interaction) = 0.012 (InR), p = 0.046 (Dp110). Data is arcsine transformed means61 standard error for trials on n days (n = 11 for InR and Dp110). Behaviour assays were performed on FD flies as described in Figure 1 and Methods. Full ANOVA statistics are in Table S5. doi:10.1371/journal.pgen.1000609.g006 PLoS Genetics | www.plosgenetics.org 6 August 2009 | Volume 5 | Issue 8 | e1000609