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Hematophagous Stomoxys (stable) fly populations in dairy barns are sustained by a constant availability of cattle hosts and manure, which serve as major reservoirs of both zoonotic and opportunistic bacterial pathogens. However, the composition of the Stomoxys fly microbiota, the mechanisms by which flies acquire their microbiome, and the ability of potentially pathogenic bacteria to colonize and persist in fly hosts remain to be investigated. Here, we longitudinally collected fly and manure samples from two connected dairy facilities. High throughput 16S rRNA gene amplicon sequencing was then used to characterize and compare bacterial communities present on or within flies and in manure collected from the same facility, while culture-dependent methods were used to verify the viability of clinically relevant bacteria. Bacterial alpha diversity was overall higher in manure samples as compared to fly samples, with manure-associated bacterial communities being dominated by members of the Bacteroidales, Eubacteriales, and Oscillospirales. In contrast, flies harbored relatively low-complexity communities dominated by members of the Enterobacterales, Staphylococcales, and Lactobacillales. Clinically relevant bacterial strains, including Escherichia spp. and other taxa associated with mastitic cows housed in the same facilities, were detected in paired fly and manure samples but exhibited dramatically elevated abundances in fly samples as compared to manure samples. Viable colonies of Escherichia, Klebsiella, and Staphylococcus spp. were also readily isolated from fly samples, confirming that flies harbor culturable mastitis-associated bacteria. This study identifies biting flies as bona fide carriers of opportunistically pathogenic bacterial taxa on dairy farms. IMPORTANCE: Disease prevention on dairy farms has significant implications for cattle health, food security, and zoonosis. Of particular importance is the control of bovine mastitis, which can be caused by diverse bacteria, including Klebsiella, Escherichia coli, Streptococcus, and Staphylococcus spp. Despite being one of the most significant and costly cattle diseases worldwide, the epidemiology of bovine mastitis is not well understood. This study provides parallel culture-independent and culture-dependent evidence to support the carriage of opportunistically pathogenic bacteria by Stomoxys flies on dairy farms. We further show that the fly microbiota is enriched in clinically relevant taxa-the vast majority of which can be traced to the manure habitats in which flies breed. Altogether, our results identify biting flies as underrecognized carriers of bacterial taxa associated with environmental bovine mastitis and other opportunistic infections in vertebrates and offer important insights into mechanisms of microbial acquisition by these and other medically important insects.

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Vector-borne diseases constitute a major global public health threat. The most significant arthropod disease vectors are predominantly comprised of members of the insect order Diptera (true flies), which have long been the focus of research into host-pathogen dynamics. Recent studies have revealed the underappreciated diversity and function of dipteran-associated gut microbial communities, with important implications for dipteran physiology, ecology, and pathogen transmission. However, the effective parameterization of these aspects into epidemiological models will require a comprehensive study of microbe-dipteran interactions across vectors and related species. Here, we synthesize recent research into microbial communities associated with major families of dipteran vectors and highlight the importance of development and expansion of experimentally tractable models across Diptera towards understanding the functional roles of the gut microbiota in modulating disease transmission. We then posit why further study of these and other dipteran insects is not only essential to a comprehensive understanding of how to integrate vector-microbiota interactions into existing epidemiological frameworks, but our understanding of the ecology and evolution of animal-microbe symbiosis more broadly.

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Interactions between species shape the formation and function of microbial communities. In the gut microbiota of animals, cross-feeding of metabolites between microbes can enhance colonization and influence host physiology. We examined a mutually beneficial interaction between two bacteria isolated from the gut microbiota of Drosophila, i.e., Acetobacter fabarum and Lactobacillus brevis After developing an in vitro coculture assay, we utilized a genetic screen to identify A. fabarum genes required for enhanced growth with L. brevis The screen, and subsequent genetic analyses, showed that the gene encoding pyruvate phosphate dikinase (ppdK) is required for A. fabarum to benefit fully from coculture. By testing strains with mutations in a range of metabolic genes, we provide evidence that A. fabarum can utilize multiple fermentation products of L. brevis Mutualism between the bacteria in vivo affects gnotobiotic Drosophila melanogaster; flies associated with A. fabarum and L. brevis showed >1,000-fold increases in bacterial cell density and significantly lower triglyceride storage than monocolonized flies. Mutation of ppdK decreased A. fabarum density in flies cocolonized with L. brevis, consistent with the model in which Acetobacter employs gluconeogenesis to assimilate Lactobacillus fermentation products as a source of carbon in vivo We propose that cross-feeding between these groups is a common feature of microbiota in DrosophilaIMPORTANCE The digestive tracts of animals are home to a community of microorganisms, the gut microbiota, which affects the growth, development, and health of the host. Interactions among microbes in this inner ecosystem can influence which species colonize the gut and can lead to changes in host physiology. We investigated a mutually beneficial interaction between two bacterial species from the gut microbiota of fruit flies. By coculturing the bacteria in vitro, we were able to identify a metabolic gene required for the bacteria to grow better together than they do separately. Our data suggest that one species consumes the waste products of the other, leading to greater productivity of the microbial community and modifying the nutrients available to the host. This study provides a starting point for investigating how these and other bacteria mutually benefit by sharing metabolites and for determining the impact of mutualism on host health.

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A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter, so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB, show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits.

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