Background: The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentallyacquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes residewithin infected flies. Tsetseâs bacterial microbiota impacts many aspects of the flyâs physiology. However, littleis known about the structure of tsetseâs midgut-associated bacterial communities as they relate to geographicallydistinct fly habitats in east Africa and their contributions to parasite infection outcomes. We utilized culture dependentand independent methods to characterize the taxonomic structure and density of bacterial communities that residewithin the midgut of tsetse flies collected at geographically distinct locations in Kenya and Uganda.Results: Using culture dependent methods, we isolated 34 strains of bacteria from four different tsetse species(G. pallidipes, G. brevipalpis, G. fuscipes and G. fuscipleuris) captured at three distinct locations in Kenya. Toincrease the depth of this study, we deep sequenced midguts from individual uninfected and trypanosomeinfected G. pallidipes captured at two distinct locations in Kenya and one in Uganda. We found that tsetseâsobligate endosymbiont, Wigglesworthia, was the most abundant bacterium present in the midgut of G. pallidipes, andthe density of this bacterium remained largely consistent regardless of whether or not its tsetse host was infected withtrypanosomes. These fly populations also housed the commensal symbiont Sodalis, which was found at significantlyhigher densities in trypanosome infected compared to uninfected flies. Finally, midguts of field-captured G. pallidipeswere colonized with distinct, low density communities of environmentally acquired microbes that differed intaxonomic structure depending on parasite infection status and the geographic location from which the flieswere collected.Conclusions: The results of this study will enhance our understanding of the tripartite relationship betweentsetse, its microbiota and trypanosome vector competence. This information may be useful for developingnovel disease control strategies or enhancing the efficacy of those already in use.Keywords: Tsetse fly, Glossina, Symbiont, Wigglesworthia, Sodalis, Microbiota, African trypanosome, Metagenomics
