Assessing the impact of diet formulation and age on targeted bacterial establishment in laboratory and mass-reared Mediterranean fruit fly using full-length 16S rRNA sequencing

Authors: Mason, Charles; NELSON, ROSALIE - University Of Hawaii; WEAVER, MIKINLEY - Orise Fellow; SIMMONDS, TYLER - Orise Fellow; Geib, Scott; SHIKANO, IKKEI - University Of Hawaii

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2025
Publication Date: 5/15/2025
Citation: Mason, C.J., Nelson, R.C., Weaver, M., Simmonds, T.J., Geib, S.M., Shikano, I. 2025. Assessing the impact of diet formulation and age on targeted bacterial establishment in laboratory and mass-reared Mediterranean fruit fly using full-length 16S rRNA sequencing. Microbiology Spectrum. 13(6). Article e02881-24. https://doi.org/10.1128/spectrum.02881-24.
DOI: https://doi.org/10.1128/spectrum.02881-24

Interpretive Summary: Insect gut microbiomes have important roles in overall host health. In laboratory- and mass-reared insects, gut microbiomes can differ in composition and function compared to wild individuals. For fruit flies such as the Mediterranean fruit fly, these changes can influence male performance and behavior. Poor sexual performance of fruit flies is a major limiting factor in the implementation of sterile insect technique. Our goals were to evaluate factors that influence the re-introduction of beneficial bacteria to the Mediterranean fruit fly. The specific goal of this study was to evaluate how age and diet influenced bacterial establishment in laboratory- and mass-reared medfly. We used an Enterobacter strain with antibiotic resistance that allowed us to track the establishment of the bacteria using high-throughput screens on selective media. We coupled this with full-length 16S rRNA sequencing utilizing PacBio Kinnex HiFi sequencing. We evaluated two age classes and used two different diet formulations for evaluating establishment. Our results identified that diet had a strong interaction with age. Host flies fed a liquid diet with the target bacteria were able to be colonized regardless of age. 16S rRNA sequencing corroborated the establishment of the specific strain, but also revealed some species/strain-level variation of Enterobacter sequences associated with the flies. Our results demonstrate that the host fly microbiome is capable of being manipulated using a liquid diet. Our results also show that there is a utility in full-length 16S rRNA sequence that allows us to uncover greater microbial diversity than with shorter sequence-based methods.

Technical Abstract: Insect gut microbiomes have important roles in overall host health and how hosts function in the environment. In laboratory- and mass-reared insects, gut microbiomes can differ in composition and function compared to wild conspecifics. In the Mediterranean fruit fly (medfly; Ceratitis capitata) as well as other Tephritid species, these changes can influence male performance and behavior. Identifying factors that influence host colonization resistance is important for determining the scalability of bacterial recapitulation in mass-reared fruit flies. The goal of this study was to evaluate how age and diet influenced bacterial establishment in laboratory- and mass-reared medfly. We used an Enterobacter strain with antibiotic resistance and coupled it with full-length 16S rRNA sequencing utilizing PacBio Kinnex library preparation and HiFi sequencing to track the establishment of a specific isolates under different adult dietary conditions. We also used two sources of flies in our study. Our results identified that diet had a strong interaction with age. Host medfly fed a liquid diet with the target bacteria were able to be colonized regardless of age, but those fed a slurry-based diet and separate water source were more resilient. This was consisted for both fly sources used in the study. 16S rRNA sequencing corroborated the establishment of the specific strain, but also revealed some species/strain-level variation of Enterobacter sequences associated with the flies. Our results demonstrate that the medfly microbiome is capable of being manipulated using a liquid diet. Additionally, our study illustrates that long-read full-length16S rRNA sequencing may afford improved characterization of species- and strain-level distribution of Enterobacteriaceae in insects with similar throughput and costs to traditional approached through utilization of Kinnex technology.