Supporting Information Figure S1 Behaviour testing apparatus. A plastic maze originally used for geotaxis experiments [80] is placed horizontal on a light table adjusted to produce 1,000 lumens illumination. Darker areas on photograph edges are due to camera contrast adjustment; actual illumination is even over maze surface. The entry tube contains agar with 0.25 M sucrose (Methods). 24–26 flies are placed in this tube 15 minutes before entry to the maze. 9 empty (no agar or sugar) collection tubes block exit points from maze. At time 0 the entry tube is placed in the maze entry. Numbers of flies in collection tubes is counted every minute until termination of run at 3 minutes. Experiments were conducted in a darkened room maintained at 25 C and humidified to 60%RH. Found at: doi:10.1371/journal.pgen.1000609.s001 (2.47 MB TIF) Figure S2 Positive regulators of insulin signaling - rovers change expression more than sitters. (A) Group mean expression. The average log2 expression of a group of positive regulators of insulin signaling (InR, chico/dIRS, Pi3K92E/dPIK3CB/dp110, Pi3K21B/ dp60, Pk61C/dPDK, Akt1) is shown for rovers (blue), mutant sitters (red), and natural sitters (pink) in two food environments. Average expression shows strong negative I or GxE interaction - that is, rovers show the downregulation expected [23,24] in Fed flies much more than sitters (for6Food F1,97 = 15.52, p = 0.00015, group ANOVA). Natural sitters have a different genetic background (BG) from rovers and mutant sitters. The effect of the BG is to strengthen the negative GEI (I = 20.33, rover vs sitter mutant; I = 20.48, R vs s; BG6food F1,97 = 9.42, p = 0.0029). RNS (plasticity of response) is positive - rovers change more than sitters. Error bars are 61 s.e.m. (B) Range of Individual gene expression between Fed and food deprived (FD) heads. Expression of positive regulators of insulin signaling tends to be higher in the food-deprived state due to foxo-mediated upregulation of tran- scription [23,24]. The vertical axis shows log2 fold change between Fed and FD flies. Found at: doi:10.1371/journal.pgen.1000609.s002 (1.36 MB TIF) Figure S3 qRTPCR results. qRTPCR was done with extracts from heads of forR (blue) and fors2 (red) flies using actin (Act57B) as a reference gene for triplicate samples from fed and food deprived (FD) flies. Data are normalized to fed fors2 levels and shown as log2 values. (A,B) Trehalase (Treh) PCR and array; (C,D) phospho- enolpyruvate carboxykinase (GTP) (CG10924). Pearson’s correla- tion between array and qPCR values: Treh t = 105.9, df = 1, References 1. Via S, Gomulkiewicz R, Dejong G, Scheiner SM, Schlichting CD, et al. (1995) Adaptive phenotypic plasticity - consensus and controversy. Trends Ecol Evol 10: 212–217. 2. 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