A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

Although the gut microbial communities in adult bumblebees and their associated functionalities are widely studied, descriptive data on the larval gut microbiota are still limited. The gut microbiota of a fully sampled wild Bombus pascuorum nest has been characterized, using the multiplexed Illumina MiSeq 16S ribosomal RNA amplicon sequencing technique. The nesters and foragers inhabiting the same nest showed the typical core bacterial sequences and only marginal differences in their characterized gut microbiota. The gut microbial communities within the adult and larval specimens differed strongly, as the typical core gut bacteria in the adult bumblebees are absent in the larval bumblebees. The bacterial communities within the larval gut are dominated by bacterial phylotypes of Enterobacteriaceae and Lactobacillaceae, supplemented with genera belonging to Corynebacteriales and Bacillales. The function of this larval gut microbiota, being different from the adult, remains to be determined.

The interaction between the insect host and its microbiota plays a central role in insect health and is mostly studied in relation to the digestive system. Nonetheless, there are numerous microorganisms occupying multiple habitats in and on insects. We studied microbial communities in the gut and fat body of bumblebees (Bombus terrestris) using the V4 region of the 16S rRNA gene on the Illumina MiSeq platform. In one of the two study locations, the fat body microbial composition was marked by the dominant presence of Arsenophonus sp. and Phyllobacterium sp. Bumblebees infected with Apicystis bombi, a eukaryotic parasite multiplying in the fat body, had a significant higher relative abundance of Arsenophonus sp. compared with the non-infected individuals. In general, the infection of A. bombi correlated with a more interlinked microbial association network, as we observed an increase of significant associations between the relative abundance of bacteria present in the gut and fat body of infected bumblebees. The causality within this potential prokaryotic-eukaryotic relation is important when assessing the health impact on bees.