Whole-body homogenates restore disrupted microbiota composition in a model insect better than feces or no restoration treatment

Antibiotic treatment can disrupt gut microbiota and pose challenges and opportunities for the establishment or restoration of healthy microbial communities. Using the fruit fly, Drosophila melanogaster, as an experimental model, we evaluated the impact of two types of microbial transplants--fly feces and whole-body fly homogenates--on host microbiota composition, following, or independent of, tetracycline-induced community disruption. Using 16S rRNA sequencing, we compared community beta diversity between treatments. We show that antibiotic treatment significantly altered microbiota composition and community structure relative to untreated controls. Flies inoculated with whole body homogenates of age-matched, antibiotic-free flies had a more similar microbial community composition to the untreated communities than flies exposed to fly feces or to flies that received no restoration treatment. We also found that the presence of Wolbachia was associated with variation in microbiota composition and specific locomotor functions. These findings show that whole-body homogenates are a superior method for microbiota restoration in Drosophila melanogaster and contribute to a growing body of research on microbial community restoration following disturbance. IMPORTANCEGut microbes play a critical role in animal biology, influencing digestion, immunity, development, and behavior. Disruptions to the gut microbiota--whether from antibiotics, disease, or other interventions--can have lasting effects, and restoring these communities remains an important challenge across biological and biomedical research. Model organisms like fruit flies (Drosophila melanogaster) provide a powerful system for testing microbial restoration methods because their gut communities are relatively simple and easy to manipulate. In this study, we compared various strategies for repopulating the microbiota of flies following antibiotic treatment. We found that flies fed whole-body homogenates of untreated flies more closely resembled the microbiota of untreated flies than flies exposed to fly feces or to flies that received no restoration treatment. These findings contribute to broader efforts to understand and develop methods for microbiota recovery following disturbance and suggest applications across animal systems.