The emergence of caste-differentiated colonies, which have been defined as ‘superorganisms’, in ants, bees, and wasps represents a major transition in evolution. Lifetime mating commitment by queens, pre-imaginal caste determination and lifetime unmatedness of workers are key features of these animal societies. Workers in superorganismal species like honey bees and many ants have consequently lost, or retain only vestigial spermathecal structures. However, bumble bee workers retain complete spermathecae despite 25-40 million years since their origin of superorganismality, which remains an evolutionary mystery. Here, we show (i) that bumble bee workers retain queen-like reproductive traits, being able to mate and produce colonies, underlain by queen-like gene expression, (ii) the social conditions required for worker mating, and (iii) that these abilities may be selected for by early queen-loss in these annual species. These results challenge the idea of lifetime worker unmatedness in superorganisms, and provide an exciting new tool for the conservation of endangered bumble bee species.

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.

Bumblebees (Bombus spp.) are important pollinators that are widely used for greenhouse crop pollination and are produced annually by commercial breeding facilities. In this study, we used the commercially important bumblebee species Bombus terrestris Linnaeus (Hymenoptera: Apidae: Bombini) to evaluate the effects of artificial box stocking density on the reproductive performance of newly eclosed females under commercial rearing conditions. Newly eclosed gynes and workers were artificially held in separate wooden boxes at different stocking densities (1 to 150 individuals per box for gynes and 1 to 50 individuals per box for workers) before mating tests, ovarian development assessments, or oviposition observations. Higher box stocking density reduced mating success in both gynes and workers, whereas worker ovarian development and oviposition occurred earlier as worker box stocking density increased. At worker stocking densities of 30 individuals per box or higher, workers began to lay eggs on day 6, and oviposition was concentrated between days 6 and 9. These findings show that artificial box stocking density is an important management factor during commercial bumblebee rearing and can be adjusted according to whether the production goal is to maximize female mating success or promote earlier worker-derived male production.

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.

Bumblebees (Bombus spp.) are ecologically indispensable pollinators, and their health directly contributes to the stability of both natural and agricultural ecosystems. Although the gut microbiota of female bumblebee castes has been well characterized, the microbiota of males—despite their essential function in colony reproduction—remains largely unexplored. Moreover, bumblebee males can be produced by both queens and workers, raising fundamental questions about the origins and assembly of their gut microbiota. Here, using the buff-tailed bumblebee Bombus terrestris as a model system and 16S rRNA gene sequencing across five developmental stages (egg, larva, pupa, 1-day-old adult, and 7-day-old adult), we characterized the composition, diversity, and colonization dynamics of the gut microbiota in male offspring produced by queens versus workers. Our results reveal that no consistent bacterial signal was detected in egg and pupal stages, with colonization likely initiating at the larval stage where Lactobacillus is overwhelmingly dominant. The core gut microbiota of queens and workers showed substantial taxonomic overlap with that of their male offspring, suggesting potential maternal contribution. Maternal origin was associated with distinct larval communities: queen-produced male larvae exhibited higher diversity and overlapped more extensively with maternal genera, while worker-produced male larvae displayed an extremely simplified community dominated almost exclusively by Lactobacillus (99.41%). However, these early-life differences gradually decreased after eclosion. By the 1-day-old adult stage, Gilliamella, Snodgrassella, and Candidatus Schmidhempelia emerged as dominant genera, with the latter showing transient enrichment specifically in newly emerged adults. By 7 days of age, the gut microbiota structure became similar across males from different reproductive origins, stabilized by the core adult taxa Gilliamella and Snodgrassella.