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.

UNLABELLED

Insect gut microbiota play important roles in host health and interactions with the environment. In laboratory and mass-reared insects, gut microbiomes can differ in composition and function compared to wild conspecifics. For fruit flies, such as the Mediterranean fruit fly (medfly; ), these changes can influence male performance and behavior. Overall, understanding factors that influence the ability of bacteria to establish in hosts is important for the establishment of lost or novel microbiota in mass-reared insects. The goal of this study was to evaluate how host age and diet-inoculation method influenced bacterial establishment in laboratory and mass-reared medflies. We used an strain with antibiotic resistance and coupled it with full-length PacBio Kinnex 16S rRNA sequencing to track the establishment of the specific isolates under different adult dietary conditions. We also used two longstanding reared lines of medfly in our study. Our results identified that diet had a strong interaction with age. The target bacterial isolate was detected in medfly when inoculated with liquid diet regardless of age, but those fed a slurry-based diet and a separate water source had less establishment. This was consistent for both fly rearing lines used in the study. 16S rRNA sequencing corroborated the establishment of the specific strain but also revealed some species/strain-level variation of sequences associated with the flies. Additionally, our study illustrates that long-read 16S rRNA sequencing may afford improved characterization of species- and strain-level distribution of Enterobacteriaceae in insects.

IMPORTANCE

Insects form intimate relationships with gut microorganisms that can help facilitate several important roles. The goal of our study was to evaluate factors that influence microbial establishment in lines of the Mediterranean fruit fly (medfly), an important pest species worldwide. Mass-reared insects for the sterile insect technique often possess gut microbiomes that substantially differ from wild flies, which can impact their performance in pest control contexts. Here, we show that liquid-based formulations can be utilized to manipulate the gut microbiota of mass-reared medflies. Furthermore, using near full-length 16S rRNA metabarcoding sequencing, we uncovered strain-level diversity that was not immediately obvious using other approaches. This is a notable finding, as it suggests that full-length 16S rRNA approaches can have marked improvements for some taxa compared to fewer hypervariable regions at approximately the same cost. Our results provide new avenues for exploring and interrogating medfly-microbiome interactions.