Comparison of Inoculation Routes for Honeybee Gut Microbiota Assembly Under Laboratory Conditions

The honeybee (Apis mellifera) gut microbiota serves as a model for host–microbe studies, as newly emerged workers provide a low-complexity, nearly germ-free system. Although multiple inoculation strategies are commonly applied under laboratory conditions, their comparative effects on microbial colonization remain incompletely understood. Here, we systematically compared four commonly used inoculation strategies, including pollen mixture feeding, oral feeding, dunk inoculation, and comb-exposed inoculation, under controlled laboratory conditions using an identical microbial source. We assessed gut microbiota establishment using spike-in 16S rRNA gene sequencing, enabling the quantification of both relative and absolute bacterial abundances, together with analyses of community composition, alpha diversity, and beta diversity. Our results showed that method-dependent differences were observed. Pollen mixture feeding and oral feeding produced highly comparable outcomes across the measured parameters, suggesting that dietary incorporation and direct oral delivery are similarly effective for establishing core gut communities (p > 0.05). Dunk inoculation also supported core bacterial colonization, although differences in beta diversity indicated some variation in community structure (p < 0.05). Comb-exposed inoculation showed the most pronounced differences, with significantly reduced bacterial abundance, lower alpha diversity, and decreased colonization of specific core taxa including Bifidobacterium and Snodgrassella alvi (p < 0.05). Importantly, despite these differences, none of the inoculation methods resulted in atypical communities dominated by noncore taxa, indicating that the core gut microbiota remained broadly conserved across treatments. Collectively, our findings indicate that although inoculation delivery methods differed fundamentally in inoculum volume, microbial concentration, and the mechanisms by which microbes were introduced into the bee gut, these distinct strategies can introduce measurable differences, while the established gut communities remain consistently dominated by core bacterial taxa. These results provide a basis for selecting appropriate inoculation strategies in experimental studies of honeybee microbiota.