with different voltage-dependent K+ currents in larval neuromus- cular junctions, along with differences in neuronal excitability, neurotransmitter release, and synaptic transmission [71]. Rover- sitter differences in neural thermotolerance arise from differences in the regulation of K+ channel activity via a circuit involving PKG, PP2A, and ion channels [72]. Thus our demonstration of rover-sitter differences in gene expression of genes involved in neurotransmitter release, postsynaptic membranes, and calcium- and potassium-channels supports previous studies. It will be important to determine whether foraging interacts epistatically with other genes influencing K+ currents in neurons and muscles. It is also of interest to investigate whether the metabolic effects of allelic variation in for are independent of, or are tied to PKG’s effects on ion homeostasis and neural function. Magnitude of foraging GEI compared to effects of other genes Our study used only a few strains of flies and thus does not speak to the importance of for-mediated effects on the genome in natural populations. However, we are able to consider allelic effects at the for locus relative to the genetic background effects in a principal components analysis which identifies genetic background (BG) and food (Fed vs. FD) as the most important factors, followed by the interaction of BG and food, for genotype main effects, and for interaction effects. Using the Storey-Tibshirani method to estimate the true proportion of differentially expressed genes palt shows that for GEI affects 57% of the highest expression genes. We also found that the effect of the natural sitter background was to intensify gene expression contrasts with rovers but to reduce behavioural contrasts. Thus, an important future step is to quantify the relative importance and roles of for and other genes in a wider variety of natural genetic backgrounds. Our results also speak to evolutionary questions about pleiotropy, epistasis, and plasticity. Pleiotropic genes may affect few traits when redundancy, degeneracy [73], or compensations in gene networks buffer the effects of mutations [74], while mutations in other genes produce large changes [75]. The number of traits influenced by a gene follows a power law, with a few genes having widespread affects [74,76]. It has been proposed that the use of naturally occurring alleles or mild mutations is more relevant to studies of epistasis and network stability than the more common use of knockouts or severe loss of function mutations [73,77,78]. The question of whether some genes can increase phenotypic plasticity and thus whether selection can act to increase or decrease plasticity has been the subject of much debate [2]. In for we have an example of a gene with naturally occurring alleles maintained in a stable polymorphism in the wild [79]. We demonstrate that in adult flies for interacts pervasively with food, producing pleiotropic GEI in behaviours, lipids and carbohydrates, and gene expression. Our quantitative plasticity measure reveals that for6food GEI is often due to rovers having significantly higher food-related plasticity than sitters. We show that for interacts with genes of the insulin signaling pathway to produce some of these effects. The foraging gene may thus provide a suitable context for resolving some of these questions relating to phenotypic plasticity and selection. Methods Strains and rearing The following allelic variants of the chromosome-2 foraging (for) gene were used in this study: the forR (natural rover), fors (natural sitter) variants and fors2 (a sitter mutant strain made on a forR genetic background) [9,13]. All strains share common isogenic third chromosomes from forR and common X chromosomes. foraging and Plasticity Genetic variation on the small fourth and Y chromosomes was not controlled. For tests of epistasis we used three mutants of genes in the insulin signalling pathway (see below). Flies were reared on medium b for behavioural assays, gene array and metabolite experiments (Table S1A) [80]. For additional behavioural assays reported in Table S1B, S1C flies were raised in media described in Table S1A, S1C. Flies were raised in 40 mL plastic vials containing 10 mL food medium in 12/12 h light/dark cycle (lights on 08:00), 2561uC, 7065% relative humidity (standard conditions). Fly rearing and behaviour testing Flies were collected 0–2 days post eclosion, separated under light CO2 anaesthesia, then reared in groups of 25–30 for 4 days. 12–13 males and 12–13 females were used in each rearing group. Adult rearing was done under standard conditions as described above. Flies were transferred to test media the night before behaviour tests. Test media consisted of 10 mL of food medium (Fed), or 10 mL 1% agar for food deprivation (FD) tests in vials. Flies were tested in the morning (9–12 a.m.) after 16–18 hours under Fed or FD conditions. A plexiglass maze was used for the food-leaving assay; the maze is as described [80] and is shown in Figure S1. Each morning mazes were conditioned by passing through one sample of 25 natural sitter flies before testing commenced. Mazes were placed horizontally on a light table with a uniform light intensity of 1000 lumens. Flies were placed in a10675 mm borosilicate glass tube (the ‘‘sugar entry tube’’) containing 0.5 mL 0.25 M sucrose in 1% agar for 15 min prior to test. At start of testing, the sugar tube with flies is placed in the entry port of the maze. Empty glass collection tubes are placed in the 9 exit ports of the maze. After 3 min, flies in collection tubes at exit ports are counted, as are flies remaining in the sugar tube. Food- leaving score is (flies in collection tubes)/(total flies). All treatment conditions were tested on at least 3 different test days. Quantitative complementation crosses To test whether the rover and sitter for alleles interact epistatically with alleles of the three genes involved in the insulin signaling pathway we used a form of quantitative complementation, a method of complementation developed for testing quantitative effects, in this case, gene interactions [27].We asked if one copy of a mutant allele of a gene involved in insulin signaling, in the presence of each of the for alleles, changes food- leaving behaviour. Crosses were made between rover (forR), natural sitter (fors), and mutant sitter (fors2) strains and balanced loss of function mutants in three insulin signaling pathway genes: (a) InR, the insulin receptor (mutant allele: InR93dj-4 [81]); (b) Pi3K92E/ Dp110, the phosphatidylinositol 3-kinase catalytic subunit (Dp110B [25]); and (c) foxo, (foxo21 [22]). Strains carrying mutations in the insulin signaling pathway were: (a) In(3R)GC25, InR93Dj-4/TM3, Sb1 (Bloomington stock 9554) [82]; (b) yw;P[ry+,gH], Dp110B/ TM3,Ser,y+(C) [83]; (c) foxo21/TM6C [22]. All mutants of the insulin signaling pathway were maintained heterozygous with balancer chromosomes which did not carry mutations in these insulin signaling genes and were on a sitter background. Epistasis is identified as a two-way statistical interaction between the variant (rover or sitter) and test genotype (mutant or control) [26] (see below). The balancer heterozygotes control for effects of natural variation in the genetic background that can result in increases or decreases in food leaving behaviour. Metabolite analysis Fourier Transform Ion Cyclotron Resonance Mass Spectrom- etry (FTICR MS) was used to analyze homogenized fly heads PLoS Genetics | www.plosgenetics.org 9 August 2009 | Volume 5 | Issue 8 | e1000609