Lactobacillus spp. in honey bees of southwestern Sweden : A feasible gut-analysis via long-read 16S nanopore sequencing

Abstract: Together with other pollinating animals, honeybees maintain an ecological stability and pollinate both wild and cultivated plants, contributing to the preservation of natural ecosystems and overall security of food production. Unfortunately, honeybees across the world are dying at a dramatic rate, which is very dangerous for the biosecurity of this planet. The diversity of Lactobacillus species in the honeybee gut could be a direct indication of honeybee health. Among the objectives of this experiment was to characterize the diversity of Lactobacillus species in the gut of honeybees isolated from four geographically separate hives. This was achieved by extracting the DNA from the honeybee gut, amplifying the entire 16S rRNA marker-gene using the 27F and 1492R universal primers, and performing the full-length 16S nanopore sequencing workflow. Results confirmed the dominance of Lactobacillus in three out of four honeybee gut samples, with more than 70% of all reads mapping to Lactobacillus. The MinION analysis demonstrated, in high reproducibility with previous studies, that the honeybee gut microbiota consists of eight Lactobacillus species: Lactobacillus melliventris, Lactobacillus kimbladii, Lactobacillus mellis, Lactobacillus apis, Lactobacillus helsingborgensis, Lactobacillus kullabergensis, Lactobacillus mellifer, and Lactobacillus bombicola, shown to occur reliably in specific proportions. The findings reported here support the applicability of full-length 16S rRNA nanopore sequencing, and particularly of the portable MinION sequencing device, as an effective and relatively inexpensive method for analyzing Lactobacillus dominant microbiomes, such as the microbiome of the honeybee gut.