Analysis of sample types and sequencing methods for optimal characterization of the honey bee (Apis mellifera) gut microbiome

The honey bee (Apis mellifera) gut microbiome has been characterized using 16S rRNA amplicon and shotgun metagenomic sequencing. It mainly consists of a conserved microbial community that directly impacts bee health and immunity. Our study aimed to identify the most informative sample and sequencing approach to characterize the honey bee gut microbiome. Whole bee, abdomen, gut and hindgut from individual bees, or from a pool of ten bees, were analyzed using 16S rRNA amplicon and metagenomic shallow and deep sequencing. At the genus level, samples from the individual or pooled bees resulted in similar findings for all sequencing approaches. Kraken 2 analyses accurately identified members of the honey bee microbiome. However, metagenomic sequencing was found to be advantageous at the species level. Species rarefaction analysis revealed that deep-sequenced samples discovered species at a greater rate than shallow-sequenced samples, which indicated that taxa abundance information is gained through increased sequencing depth. While data obtained from whole bee and abdomen were informative, the gut and hindgut yielded a higher proportion of microbial reads compared to host reads. Therefore, sequencing gut or hindgut samples at a greater depth is an ideal approach for bee microbiome analyses to address questions related to bee health.