composition, after yeast propagules have reached natural sub- strates. For instance, Starmer and Fogelman [12] seeded pairs of yeasts from the agria cactus yeast community onto sterile agria cactus tissue, and then estimated the carrying capacity of each yeast species based on fitted growth curves. They found that in the absence of larvae, all yeast-yeast effects on carrying capacity were positive, but when larvae were present various negative and positive-negative interactions appeared. They interpreted these results as suggesting that yeast communities with larvae might be more qualitatively stable than those lacking larvae. Others have suggested that larvae digging behavior might affect the develop- ment of microbial communities that favor larval growth and development [19], or that larvae may disperse yeast cells through substrates as a result of their foraging activities [2]. However, to our knowledge, no one has studied the effects of Drosophila adults or larvae on the density or composition of yeast communities in natural fruit substrates. In principle, there are at least two reasons why we might expect Drosophila to manage the yeast communities in natural substrates in ways that are favorable for larval growth and development. First, the breeding habitats of Drosophila (e.g. rotting fruits, flowers, mushrooms, soft rots, leaf litter, tree bark, exudates and fluxes) are also home to other animals, including other flies, beetles, nematodes and mites [1]. As a result, adult flies often oviposit on substrates that already harbor yeasts, bacteria, filamentous fungi (i.e., molds) and other microbes left by previous visitors, and other animals could easily ‘contaminate’ an oviposition site with other microbes after a female Drosophila had laid her eggs there. Hence, vectoring alone may not guarantee that a newly colonized breeding site will eventually support high densities of yeasts that support offspring development. Second, many Drosophila breed in isolated patches of substrate, to which their offspring will be confined until they initiate pupation, a process that typically requires a week or more. In this situation, larval growth and development depends upon the yeast communities that develop and are maintained within their natal patch over an extended period of time. Moreover, high densities of palatable yeasts are most likely to be important towards the end of the larval development period (third instar), when larval food intake rates are highest [20]. Hence, when a small number of Drosophila larvae first emerge in newly available natural substrates, one might expect them to engage in activities early in the larval period that encourage (or at least, do not discourage) high densities of nutritious yeasts in their natal patch a week or so later, when their food requirements will be highest. Here, we investigate the effects of adult and larval Drosophila melanogaster (Meigen, 1830) on the yeast communities that are present at the end of the larval period on pieces of cultivated banana fruits (Musa acuminata (Colla, 1820)). In order to mimic a natural situation in which a small number of adult flies first arrive and begin to oviposit on structurally intact, non-sterile fruits, we cut matched pieces from the same banana fruits (including skin), and compared the yeast communities in pieces exposed to D. melanogaster adult females, to larvae, and to both adults and larvae to the yeast communities that developed in matches pieces from the same fruits that were not exposed to flies. This protocol allowed us to control for variation among fruits in their microbial communities at the onset of the study, and for differences among fruits in any characteristics that might affect the microbial communities that subsequently developed in them. In addition, we investigated the effects of larvae on the filamentous fungi in fruit, and showed that young larvae are able to ‘transplant’ viable, palatable yeast in their fecal pools as they travel around a substrate. Drosophila Effects on Yeast Communities Materials and Methods 1. Experiment 1: Effects of Adult Females on Yeast Abundance The main goal of this experiment was to test the hypothesis that adult D. melanogaster ‘vector’ yeasts to fruit, by comparing yeast abundance on pieces of banana fruit exposed to virgin adult females previously fed conditioned banana to the yeast abundance on pieces of the same fruits not exposed to flies. We also included two additional treatment groups with mated females to 1) confirm that our experimental protocol generated viable larvae, and 2) evaluate the potential effects of larvae on yeast abundance. On the afternoon of Day 1, newly emerged females and adult males from isoline 141 ([21], Appendix S1) were placed in vials with Bloomington diet, a medium which contains inactive brewer’s yeast (Saccharomyces cerevisiae), but no viable yeast. Twenty females and 14 males were added to each vial; these were the ‘mated females’ used in two treatments: M7 and M14. In the afternoon of Day 3, newly emerged virgin females from isoline 141 were collected and held in vials (20 females per vial) with Bloomington diet; these were the virgin females used in the ‘virgin female’ treatment. On the morning of Day 4, two screened 800 ml jars were each provisioned with 5 g of fly-conditioned banana that had previously been exposed to flies in a population cage for 3–4 days [21]. The mated females were transferred into one jar; the virgin females were placed in the other. On the morning of Day 5, four pieces of banana 15.0 (6 20.1) g each were cut from the center of the same banana and placed individually into 100–ml beakers covered with sterile mesh tops. Five virgin females were transferred into the ‘virgin female’ beaker, five mated females each were transferred into the M7 and M14 beakers; no flies were transferred into the fourth beaker (‘no flies’). The beakers were maintained at 90–100% humidity and 25uC throughout the experiment. All females were removed from the beakers 24 hours later, on the morning of Day 6. Yeast density was measured on Day 12 (7 days after cutting) for the no fly, virgin female and M7 treatment groups (see Methods, 4). The M14 beaker was monitored for 7 more days, at which time all of the flies that emerged from that piece were counted. This experiment was repeated using 11 different banana fruits. 2. Experiment 2: Effects of Larvae, Adults and Both on Yeast Species Diversity and Abundance Methods for this experiment were the same as those described above for Experiment 1, with the following exceptions. Four 15 g slices of the same banana were each placed in a 100 ml beaker, and then randomly assigned to the following treatment groups: ‘adults’, ‘larvae’, ‘adults and larvae’, and ‘no flies’. Virgin adult females were prepared as described for the virgin female treatment in Experiment 1. Clean one-day old larvae were prepared as described in Methods, 3. Twenty larvae were gently transferred onto the banana pieces in the ‘larvae’ and the ‘adults and larvae’ beakers, five adult females were then added to the ‘adults; and the ‘adults and larvae’ beakers; no flies were added to the ‘no flies’ beaker. All adult females were removed 24 h later. Eight days after the fruit was cut, all four pieces were analyzed for yeast abundance (Methods, 4). At this point, most of the larvae had not yet eclosed, so it was not possible to measure their size or age as adults. Yeast abundance was measured for a total of 36 bananas. For a subset of the pieces in this experiment, the yeast colonies from the highest dilutions on the track-dilution plates used to quantify yeast abundance were categorized based on morphology, and then each morphotype was identified to species (Methods, 5). For seven bananas we were able to identify the yeasts for all four PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e42238