Mutualisms are among the most ubiquitous interactions in nature yet the general rules governing them have remained elusive. The natural mutualism between fruit flies and yeast, two common model organisms in molecular biology, is a particularly useful relationship for investigating these parameters. Drosophila feed on yeasts throughout their entire lifecycle and nonmotile yeasts depend on Drosophila to vector them to new, sugar-rich substrates. In the laboratory, Drosophila melanogaster can discriminate between and prefer different strains of Saccharomyces cerevisiae. However, in nature, Drosophila are associated with a variety of yeast species in addition to S. cerevisiae. The efforts detailed in this thesis are focused on characterizing the natural associations between wild Drosophila and yeast and testing the relevance of the fine-scale specificity between flies and yeast observed in the laboratory under more natural contexts.

First, I present a detailed dissection of the associations between Drosophila and natural yeasts over two harvest seasons in organic wineries. Using targeted, amplicon sequencing methods, I found that the fungal communities vectored by Drosophila in wineries are distinct between winery microhabitats. However, the structure in these fungal communities is not a direct result of Drosophila behavior. Instead, a diversity of yeast species, even those that are not commonly associated with flies, are adequate partners in the fly-yeast mutualism at a wide range of relevant temperatures. While many yeast species can stimulate oviposition in Drosophila, ovipositional responses vary depending on the volatile profile emitted by a particular yeast species. Using gas chromatography-mass spectrometry and synthetic compounds, I found that a minimal blend of isoamyl acetate, acetic acid, 2-phenylethyl ester, and ethanol is sufficient for stimulating oviposition. However, the strength of the oviposition response is dependent on the volatile composition of the overall blend.

Flies initially locate yeast through olfaction and a large portion of D. melanogaster odorant receptors are dedicated to detecting yeast metabolites. Kelly Schiabor, a former graduate student in the lab, demonstrated a correlation between two allelic variants of the odorant receptor 22 (Or22) in D. melanogaster and sensitivity to the volatiles produced by yeast grown under different nitrogen conditions. In collaboration with Carolyn Elya, another graduate student in the lab, I tested the hypothesis that the chimeric allele of Or22 confers heightened sensitivity to yeast grown on sugar-rich but nitrogen-limited substrates (YVN), a nutrient composition much like a natural, fruity substrate. Through extensive genotyping of natural populations, bidirectional crosses between chimeric and non-chimeric lines, and functional allele replacement of a non-chimeric Or22 allele with a chimeric allele, we found that Or22 alone cannot account for behavioral sensitivity to YVN. Even so, the signs of selection at the Or22 locus across wild D. melanogaster populations suggest that this receptor confers some kind of adaptive function in wild flies.

Because wineries are not purely natural ecosystems, I next characterized the fly-yeast mutualism in Hawaiian Drosophila. The Hawaiian Drosophila are a diverse species group that has radiated across the Hawaiian Islands and exhibit very specific host plant adaptations. It has been hypothesized that the microbial communities on host plants, rather than host plants themselves, mediate host plant discrimination in Hawaiian Drosophila. I characterized the fungal communities associated with three closely related species of Hawaiian Drosophila and their respective host plants but found no clear association between the fungi associated with flies and host plants. However, the yeast species isolated from host plants and flies produce distinct volatile profiles, suggesting that different yeast species would affect the overall volatile bouquet of a plant substrate. Unfortunately, direct behavioral assays were not possible because the Hawaiian Drosophila-yeast system is limited by sample size and the ability to rear healthy fly populations in the laboratory. Nonetheless, the role of fungi in Hawaiian Drosophila host plant discrimination remains an ecologically significant question and an open area of investigation given the appropriate resources.

Initially, the range of suitable yeast species in the fly-yeast mutualism was a surprising observation of these studies. However, this flexibility is clearly beneficial to the fitness of both flies and yeast from an evolutionary standpoint, as both organisms exist in dynamic environments. Even though their mutualism is nonspecific, both flies and yeast have continually coevolved conserved mechanisms and fine-tuned behaviors for ensuring a close association with each other.