DNA sequencing approaches for gut microbiome analyses of highbush blueberry-pollinating honey bees (Apis mellifera)

The honey bee gut microbiome, known to impact bee health and immunity, consists of five to eight core members that mainly reside in the hindgut. Targeted 16S rRNA amplicon sequencing has been the standard approach to characterize this microbial community. However, shotgun metagenomic sequencing is an untargeted approach that may provide broader insight into the less dominant members of the gut microbiome. Here, we compared the resolution of 16S rRNA amplicon and shotgun metagenomic sequencing to characterize the gut microbiome of honey bees pollinating highbush blueberries from whole bee, abdomen, dissected whole gut, and hindgut samples. We investigated the relative representation of gut bacteria reads in the different samples to determine which was most informative for the characterization of the gut microbiome. We also evaluated the impact of sequencing depth and different bioinformatics databases on the taxonomic profiling of the honey bee gut microbiome. Species rarefaction analysis showed that sequencing samples at a depth of 40 million reads (i.e. deep sequencing) revealed species at a greater rate than samples sequenced at a depth of less than 40 million reads (i.e. shallow sequencing). Based on these results, we used metagenomic sequencing data from whole gut samples to study gut microbiome profiles over time in highbush blueberry-pollinating honey bees. The work presented here will guide methodological approaches for bee gut microbiome studies tailored to research questions and diagnostic testing related to bee health.