previously fed these yeasts were comparable in size to those of larvae of the same age collected from a natural food substrate (fly- conditioned banana). We also found that viable adults emerged from eggs laid on bananas prepared using our protocol, implying that the yeast communities that developed on fly-exposed banana were adequate to support larval growth and development. Indeed, the traditional use of unsterilized pieces of banana to culture and collect Drosophila melanogaster (e.g. [39], [40]), in itself implies that the yeasts which develop on fly-conditioned banana are suitable for larval growth and development. In the future, it would be useful to confirm that a mixed yeast diet of C. candida, C. zemplinina and P. kluyveri on sterile banana supports larval growth and survival, and if so, compare the growth, survival or fecundity of flies raised on this diet to that of flies raised on banana with other yeast communities. Our results may also help shed light on the functional significance of larval mobility in D. melanogaster. In nature, most larvae are highly mobile (‘rovers’), although a sizeable minority are more sedentary (‘sitters’) [41]. Thus far, studies of the fitness consequences of larval mobility have been conducted in the laboratory using artificial food substrates, typically at densities in which larvae compete with one another for food (e.g. [42], [43]). However, our results suggest that highly mobile larvae might have positive effects on their own future food supplies, especially when larvae occur at low densities in a suitable new substrate, as was the case in our study. In nature, low larval densities at a suitable new substrate are most likely to occur when a single ‘pioneering’ fertile female first locates and begins to oviposit on a newly available patch of fruit, a situation in which the larvae within a fruit are full or half sibs. If larvae ‘transplant’ palatable yeasts across suitable substrates or improve conditions for yeast growth by churning fruit substrates as they travel, then highly mobile larvae may improve future foraging conditions for themselves and perhaps also for their siblings, by encouraging a high density of palatable yeasts in their natal patch. Although studies of interactions between Drosophila larvae and yeasts on natural substrates are rare, there are hints that the larvae of flies other than D. melanogaster might affect the yeast communities in natural substrates in ways comparable to those reported here. As was noted in the Introduction, Starmer and Fogleman found that D. mojavensis larvae encouraged yeast-yeast interactions that theory suggests might increase the qualitative stability of yeast communities in the necrotic agria (Stenocereus gummosus) cactus tissue in which that species breeds [12]. More recently, Morais et al. [17] studied D. serido and the yeasts in rots of another cactus (Pilosocereus arrabidae), and found higher yeast species diversity in rots in which adult flies were captured while feeding than in rots that contained developing larvae. These results are consistent with the hypothesis that the larvae of other drosophilids might reduce the alpha and beta diversity of yeast communities, although they are of course also consistent with alternate hypotheses (e.g. adult flies might feed in patches with higher yeast diversity than those in which they lay their eggs). More generally, our results demonstrate that larvae Drosophila melanogaster engage in ‘niche construction’ [5], [44], since, as a result of their activities (food consumption, burrowing, excretory patterns, etc.) larvae modify the micro-biotic environment in which they themselves will live and develop. In addition, larvae may also satisfy the criteria for ‘ecosystem engineers’, since they physically modified the substrate in which they lived in ways likely to affect other organisms living in that same substrate [45], [46]. For instance, larvae burrowing behavior may have contributed to the differences between the communities of yeast and filamentous Drosophila Effects on Yeast Communities fungi we observed in larval-exposed fruits versus control fruits; confirmation of this hypothesis would merit further study. Finally, we can ask about the nature of the relationship between Drosophila and the strains of yeast that were favored on fly-exposed bananas. In particular, we can ask whether the interactions of flies and yeast on banana substrates satisfy the criterion for a special type of harvest mutualism [3], [47], in which one organism vectors and encourages the growth and survival of another organism, which it then consumes. A more general term for this type of mutualism is ‘farming’. Agriculture in both animals and humans involves three actions by farmers: 1) sowing: the transfer of propagules into areas in which they can grow, 2) cultivating: processes that favor the growth or survival of edible species (the crop), and 3) harvesting: consumption of the crop [48], [49]. The simplest forms of agriculture occur when farmers sow or cultivate wild, free-living food items, which they then consume [48-50]. In rare cases, more advanced forms of agriculture may evolve, in which farmers and their crops become highly dependent on one another, in which crops change in ways that benefit the farmers (domestication) and/ or in which farmers adjust their behavior in ways that benefit their crops. Not surprisingly, to date, most studies of animal farming have focused on examples of advanced forms of agriculture (e.g. fungal farming by ants, termites and beetles [49], [51], [52], algal farming by damselfish [53] or bacterial farming by amoebae [54]. Here we ask whether Drosophila melanogaster might be engaging in a simple form of agriculture, analogous to the forms of proto- farming that must have preceded the evolution of advanced agriculture in animals and humans. Proto-farming occurs when farmers consume, sow and/or cultivate wild species, leading to a situation in which the growth rate, survival or density of an edible species at a given locality is higher in the presence than in the absence of the farmer. In this situation, a farmed species need not be the most nutritious wild food item that is available to the farmer. This is because a crop is a species that is capable of growing, surviving and reproducing under the conditions created by the farmers, and free-living species whose traits facilitate sowing, cultivation and harvesting are not necessarily the highest quality food items that occur in the surrounding environment [55], [56]. As was noted earlier, to date there is evidence that Drosophila both consume (harvest) and vector (sow) yeast onto new substrates. The results of the current study indicate that Drosophila melanogaster also encouraged the development of yeast communities that were composed of three species of yeast (C. californica, C. zemplinina and P. kluvyeri) on banana. If further studies show that a diet comprised of these yeasts on banana supports larval growth and development, then the fly-yeast-banana system would satisfy the basic criteria for animal proto-agriculture. However, it is also clear that relationships between D. melanogaster and these yeast species are by no means obligatory: Candida californica, C. zemplinina and P. kluvyeri have been collected from substrates that in nature support a wide range of Drosophila species and other saprophytic arthropods (see Appendix S3), and conversely, D. melanogaster thrives on a wide range of species of fruits and yeasts [1], [2], [57]. Nor is it known whether the facilitating effects of D. melanogaster larvae on these yeasts in banana substrates are simply by-products of larval physiology and behavior (e.g. burrowing behavior, excretory products and patterns, inefficient digestion that results in the excretion of viable yeast cells), or whether particular aspects of larval behavior, physiology or morphology have been altered by selection in ways that enhance the growth of these or other strains of dietary yeasts in the fruit substrates in which larvae live and develop. Conversely, PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e42238