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.

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.