flies, where a negative slope for one of these regressions would indicate that flies in that treatment group regulated densities across different fruits. For the analyses of the yeast species communities in Experiment 2, we used nonparametric permutational multivariate analysis of variance [27] to test for differences in the yeast community composition among four treatment groups. Analyses using both pairwise Sørensen’s dissimilarities and pairwise Jaccard distances yielded identical qualitative conclusions, so here we report results from the latter. Significance tests were based on 9999 permuta- tions of the community matrix, implemented using the ‘‘adonis’’ function in the ‘‘vegan’’ package of R [28]. We evaluated the multivariate dispersion among communities within each of the four treatment groups using the ‘‘betadisper’’ function with 9999 permutations in the ‘‘vegan’’ package of R [28], [29]. In addition, we analyzed planned contrasts between 1) the yeast communities on fruit exposed to larvae (the larvae and the adults and larvae groups) and the yeast communities on fruit not exposed to larvae (the adults and the no fly groups), and 2) between yeast communities exposed to virgin adult females (the adults and the adults and larvae groups) and the yeast communities not exposed to virgin females (the larvae and the no fly groups). We tested whether yeast community composition and dispersion differed among individual banana fruits by evaluating a reduced dataset confined to the seven bananas that were represented in all four treatment groups. We used individual banana identity as the single explanatory factor in analyses that tested whether banana identity was a significant predictor of yeast community composi- tion (using both pairwise Sørensen’s dissimilarities and pairwise Jaccard distances) and variability (using multivariate dispersion). These analyses did not detect any variation among individual bananas in either yeast community composition (F = 0.86, df = 6,21, P = 0.69) or multivariate dispersion (F = 0.77, df = 6,21, P = 0.61) using nonparametric permutational multivariate analysis of variance. Based on these results, we did not consider banana identity in the analyses of the yeast communities. For statistical analyses that relied on assumptions about normality and heteroscedasticity, we validated these assumptions prior to analysis, and used alternate tests if either assumption was violated (e.g. Welsh’s test when variances were unequal across groups). Unless otherwise indicated, statistical tests were conduct- ed using SPSS version 19. Family-wise errors for multiple tests were controlled used the False Discovery Rate (FDR) procedure [30]. 9. Ethics Information All animal experiments were conducted in conformity with the Guiding principles in the care and use of animals’’ of the Council of the American Physiological Society, and the Guidelines for animal research of the Animal Behavior Society. All experiments comply with the current laws pertaining to studies of Drosophila and yeast in the United States. Results 1. Experiment 1: Effects of Adult Females on Yeast Abundance Adult flies emerged from all 11 pieces of banana that were exposed to mated females and then monitored for two weeks (M14 treatment group: mean = 21.7, S.D. = 12.1 flies per piece), confirming that our experimental protocol was suitable for rearing Drosophila melanogaster, and implying that viable larvae were also present in the mated female, M7 group. Drosophila Effects on Yeast Communities In the absence of flies (no fly group), bananas supported yeast densities ranging from 100 to 108.3 CFU/g. Exposure to either virgin females or to mated females and their offspring increased yeast abundance in the same fruits (virgin females versus no flies: paired t test, t = 5.6, df = 10; mated females versus no flies, t = 5.9, df = 10, both results significant at P,0.001 after correction for multiple tests via FDR). However, the effects of females on yeast density were not uniform across different fruits. Instead, females had the strongest impact on yeast densities in fruits that supported low yeast densities in the absence of flies (Figures 1, 2). This is indicated by negative slopes when the differences in density between the fly-exposed and the no fly pieces were regressed against the densities in the no fly pieces (dvirgin females 2 dno flies) versus dno flies: slope = 20.69, t = 4.78; (dmated females 2 dno flies) versus dno flies: slope = 20.82, t = 9.56, both results significant at P,0.001 after FDR, Figure 1). Looking at these results another way, pieces exposed to flies ended up with a narrow range of yeast densities across fruits that supported a much wider range of densities in the absence of flies (Figure 2). The reduction in the variance of yeast densities across fruits was most pronounced for the pieces exposed to mated females (variance in log (CFU/g): no flies = 7.43, virgin females = 2.16, mated females = 0.63; compar- ison of variance for virgin females versus mated females: Levene’s statistic = 10.06, df = 1,20, P = 0.005). Taken together, these results support the hypothesis that adult female D. melanogaster can ‘vector’ yeasts from fly-conditioned to new fruit substrates. As one would expect if females ‘seeded’ yeasts on fruits, the positive effects of adult females on yeast abundance were strongest for fruits with low densities of yeasts in the absence of flies, resulting in relatively constant yeast densities across female- exposed fruits. Finally, the lower variance in yeast densities in pieces exposed to mated females than in those exposed to virgin females suggested that larvae might help regulate yeast densities across fruits, providing impetus for Experiment 2. 2. Experiment 2: Effects of Larvae, Adult Females and both on Yeast Community Structure and Density Flies had dramatic effects on the yeast communities on bananas. Eight days after they were cut, pieces of banana exposed to virgin adult females (adults), to larvae or to both supported different yeast communities than pieces with no fly exposure (Figure 3). Yeast community composition was affected by adults (F = 2.57, df = 1,37, P = 0.014) and by larvae (F = 5.32, df = 1,37, P = 0.0001), with no significant interaction between them (adult 6 larvae, F = 1.27, df = 1,37, P = 0.255). Processing by larvae dramatically reduced the variability of yeast community composition from one fruit to the next, as evidenced by significant differences in the multivariate dispersion of community composition among the four groups (F = 8.13, df = 3,17, P = 0.0003, Figures 4, S1, S2). Adults did not significantly affect yeast community similarity (average Jaccard distance to centroid with adults present: 0.44, with adults absent: 0.32, F = 1.78, df = 1,39, P = 0.188), but larvae significantly increased the similarity of yeast communities (average Jaccard distance to centroid with larvae present: 0.53, with larvae absent: 0.20, F = 27.97, df = 1,39,P = 0.0001; the latter result significant at P,0.0002 after FDR). Of the pieces processed by larvae, 50% had communities entirely composed of three yeast species, Candida californica, Candida zemplinina and Pichia kluyveri; another 30% had communities entirely composed of two of these species (see Figures 3 and S3). In contrast, of the pieces not exposed to larvae, only 9.5% had yeast communities composed of these three species, and 14% had yeast communities composed of two of them. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e42238