Gilliamella and Snodgrassella, members of core gut microbiota in corbiculate bees, have high species diversity and adaptability to a wide range of hosts. In this study, we performed species taxonomy and phylogenetic analysis for Gilliamella and Snodgrassella strains that we isolated in our laboratory, in combination with published whole-genome. Functional effects of accessory and unique genes were investigated by KEGG category and pathway annotation in pan-genome analysis. Consequently, in Gilliamella, we inferred the importance of carbohydrate metabolism, amino acid metabolism, membrane transport, energy metabolism, and metabolism of cofactors and vitamins in accessory or unique genes. The pathway mentioned above, plus infectious disease, lipid metabolism, nucleotide metabolism as well as replication and repair exert a pivotal role in accessory or unique genes of Snodgrassella. Further analysis revealed the existence of functional differentiation of accessory and unique genes among Apis-derived genomes and Bombus-derived genomes. We also identified eight and four biosynthetic gene clusters in all Gilliamella and Snodgrassella genomes, respectively. Our study provides a good insight to better understand how host heterogeneity influences the bacterial speciation and affects the versatility of the genome of the gut bacteria.

To solve the technical bottleneck caused by the absence of a feasible method for removing the spermatheca in social insects, we developed a microsurgical technique specifically designed for bumblebee females. In this study, the invention of this technique is based on the anatomical characteristics of the sting chamber of bumblebees and uses a bespoke scalpel to precisely remove the spermatheca, which is small in size and deeply embedded within the body. During the removal operation, a small wound was observed and a small amount of hemolymph flowed out. The wound healed very quickly and the survival rate of treated individuals was high. The results showed that there was no significant impact on the critical life activities of queens and workers, including longevity, mating behavior, oviposition capacity, and overwintering survival rate after the spermatheca was removed using this technique. These findings further confirm the feasibility and applicability of the technique and provide strong technical support for exploring the evolutionary dynamics and potential function of the spermatheca in social insects.

Microbiomes vary over time in response to host physiology, inter-microbial interactions, and host-microbiome feedback. Changes in life stage alter host behavior and physiology, exerting strong filters on their microbiomes. Bumble bee queens undergo a critical life stage transition from residing in parental colonies to mating and solitary diapause. Previous work highlights declines in core microbes during this transition, but directionality between the changing microbiome and host physiology remains unclear. To test the temporal relationship between the microbiome and host metabolome during this transition, we compare the gut microbiome and hemolymph metabolome of mated and unmated Bombus terrestris gynes (future queens) at several pre-diapause timepoints. Core microbiota exhibits patterns of decline and recovery, with divergence between mating status at timepoints matching divergence in the metabolome. These patterns exist only for core microbiota members, such as Snodgrassella and Gilliamella , and absent in environmentally associated, such as Apibacter and Xylocopilactobacillus . Abundances of specific gene products correlate significantly with metabolites, and mating status dependent variation in these relationships appear attributable to core microbiota ( Snodgrassella, Gilliamella, Schmidhempelia ) rather than environmentally acquired genera. We conclude that initial declines in core microbiota results from changes in host metabolism, while later divergence reflects increasing environmentally associated colonization and inter-microbial competition feeding back into host metabolome maintenance.