Full-Length 16S rRNA Gene Sequencing Resolution Reveals Variable Compositional Features of Medfly Gut Microbiomes

Abstract Insects have interconnected relationships with gut microorganisms which can have impacts on behavior and survival. Understanding the foundational ecological principles of insect-associated microorganisms can reveal how insects utilize microbes to cope under challenging conditions. Surveys of insect microbiomes often utilize 16S rRNA gene (16S) sequencing of metagenomic DNA, with target amplicon regions and lengths typically selected based on the limitations of short-read sequencing technology read lengths. Targeting partial 16S amplicons may obfuscate ecological signal, and methods which are agnostic to amplicon length allow targeting of the entire 16S gene and may aid in overcoming this hurdle. In this study, we illustrate the advantage of full-length 16S sequencing utilizing PacBio HiFi sequencing in comparison to V4 which provides new insight into the gut microbiome community composition of an invasive insect. We evaluated the gut microbiome of mass-reared medfly males (Mediterranean fruit fly, Ceratitis capitata) that were collected across a nine-month sampling period. Full-length 16S PCR products were prepared into a Kinnex 16S library, sequenced on a PacBio Revio system, and the resulting HiFi sequences were processed into amplicon sequence variants (ASVs). Substantial differences in bacterial ASV compositions were observed across medfly cohorts using full-length amplicons, which were subdued using V4 regions. Species-level gut microbiome variation were supported with genomes assembled from shotgun metagenomic sequencing. Our results highlight how highly accurate long-read sequencing of full-length 16S amplicons can uncover ecological interactions between host and gut microbiomes and serve as a bridge between short-fragment amplicons and shotgun metagenomics.