Drosophila Effects on Yeast Communities treatment groups; six other bananas provided additional samples for yeast species identifications. The number of pieces analyzed per treatment group were: no flies: 11, adults: 10, larvae: 10, adults and larvae: 10. of the strains isolated from the ‘adults and larvae’ treatment in Experiment 2 were used in subsequent experiments to determine whether the yeasts that dominated fly-conditioned banana were palatable to young larvae (Methods, 7). 3. Preparing ‘Clean’ Larvae Adult flies of isoline 755 [22] were allowed to lay eggs overnight (from 16:00 h, Day 1 to 8:00 h, Day 2) on 5 cm petri dishes filled with Bloomington diet, and covered by a Kimwipe moistened with a solution of inviable brewer’s yeast in distilled water. We collected larvae from these dishes on the morning of Day 4 (24–38 h post- hatch). On 10 occasions throughout the experiment we collected six larvae, placed them individually on a RCBA plate for an hour, then removed them. Twelve hours later, no yeast colonies had grown on any of the 60 plates. These results confirmed that this protocol produced larvae free of the yeast species most relevant to the current study, because we also found that the yeasts consumed and excreted by larvae previously fed fly-conditioned banana, and the three yeast species characteristic of fly-exposed fruits (Pichia kluveri (Bedford ex Kudryavtsev 1960), Candida californica (Anderson & Skinner, Bai, Wu & Robert 2006), and Candida zemplinina (Sipiczki 2003) all grew rapidly and formed large colonies 12 h after being placed on RCBA plates (see Methods 6 and 7). 4. Measurement of Yeast Densities Each piece of banana from Experiment 1 and Experiment 2 was placed in a 7 oz. Whirl-Pak filter bag (Nasco) with 1 ml of saline solution (0.85% NaCl, 0.01%Tween 80) and mixed until homogeneous. Two samples were taken from each banana mixture, and six dilutions per sample (100 to 1025 ) were prepared in a 96 well plate. Ten ml per dilution were pipetted onto a RBCA plate using a track-dilution technique [23]. Plates were incubated at 22–24uC for two days and then yeast colonies at each dilution were counted, and the presence/absence of filamentous fungi was noted. The number of colony forming units (CFUs) of yeast per g of fruit was measured for each sample, and the results for both samples from the same piece were averaged for further analysis. Yeast densities in this study ranged from 100 to more than 1010 CFU/g across fruits, so we used log CFU/g as our index of yeast density in this study. 5. Analyses of Yeast Species Identity Yeasts were plated on RCBA plates (Difco, Sparks, MD, USA, DF1831-17-4), incubated in the dark at 22–24uC for 2–3 days, and then evaluated for colony morphology. One representative of each morphotype was picked from each plate, streaked separately onto potato dextrose agar (PDA, Difco, Sparks, MD, USA, DF0013-07-8) plates until each sample was pure, preserved in a 20% glycerol solution and stored at 280uC. Yeasts were identified by amplifying and sequencing the D1/D2 loop of the large (26S) ribosomal RNA gene, using primers NL1 and NL4 [24], [25]. Purified PCR products were sequenced at the CBS UCDNA Sequencing Facility at UC Davis using ABI BigDye Terminator v3.1 Cycle Sequencing and analyzed on an ABI 3730 Capillary Electrophoresis Genetic Analyzer. Sequences were compared to those in public databases using BLAST nucleotide software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Yeast strains with sequences of 99% or higher sequence identity were considered to belong to the same species. All of the samples in this study were assigned the name of a previously described yeast species. Selected yeast strains were assigned UCDFST identification numbers and deposited in the Phaff Yeast Culture Collection, University of California Davis (www.phaffcollection.org). Several 6. Visualizing Yeast ‘Transplanted’ by Larvae One-day-old larvae (24–36 h post-hatch) from isoline 755 were collected from bananas previously exposed to adults for two days in a fly population cage [21] placed individually on RCBA plates for 0.5 h, and then removed (see also Appendix S2). At this point, no yeast was visible on these RCBA plates. Fourteen hours later, however, the fecal pools left by these larvae were filled with yeast colonies. We measured the number of fecal pools per plate, the length of three randomly selected pools, and the number of CFUs per mm in three linear segments of the larva’s pathway. For each of 38 larvae, we computed the average size of its fecal pools, and the average number of CFUs per mm of travel. On 10 occasions, after measuring fecal pools and yeast trails on the RCBA plate, we placed a new one day old larvae from the population cage on the same plate, and observed its behavior. In all 10 cases, the new larvae began feeding on the yeast in the first larva’s fecal pools as soon as they contacted it (based on observations of feeding movements [26]), indicating that the yeast excreted by young larvae were palatable to other young larvae. 7. The Palatability of Yeast Strains Collected from Bananas Exposed to Adults and Larvae From the yeast strains isolated from the ‘adults and larvae’ treatment in Experiment 2 and then deposited in the Phaff collection, we randomly selected two strains each of Candida californica (strains UCDFST 09–378 and UCDFST 09–541), C. zemplinina (strains UCDFST 09–373 and UCDFST 09–522), and P. kluyveri (strains UCDFST 09–523 and UCDFST 09–554). Pure cultures of each of these strains were streaked onto separate RCBA plates, then a 1–2 mm dot of each strain was lightly touched to the center of a new RCBA plate and allowed to grow for 12 h, at which point the resulting mound of yeast was spread to form a uniform ‘lawn’ approximately 1 cm in diameter. Then, for each replicate, 6 larvae of the same length (61 mm) were selected; each was randomly assigned to a different strain of yeast. A single clean larva was placed on a patch and observed until it began to feed, then observed again for five minutes at the end of an hour. Each larva was then transferred to a fresh RBCA plate for another hour, and removed. Twelve hours later, the number and length of the fecal pools on these plates were recorded (n = 17 replicates). Preliminary analyses indicated no significant differences in the number or length of the fecal pools as a function of strain within species (all p values .0.30), so results from both strains of each species were combined for further analysis. All of the larvae began feeding within 5 min of being placed on the lawn, and all of them had fed to satiation (based on a cessation of feeding and crawling behavior) by the end of an hour on the lawn. Hence, we simply describe these results in the text. 8. Statistical Methods To study the effects of flies on yeast densities, for each banana fruit, we computed the difference between the density for the pieces in each fly-exposed treatment group and the density in the piece not exposed to flies, where d = the yeast density (log CFU/g) in each piece of fruit. For instance, the difference in density between pieces exposed to larvae and pieces not exposed to flies = (dlarvae 2 dno flies). To determine whether flies regulated yeast densities across fruits, we regressed these differences against dno PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e42238