Inoculation route shapes the assembly of honey bee gut microbiota under laboratory conditions

Honey bees are key pollinators for ecosystems and agriculture. Numerous studies have demonstrated the role of the gut microbiota in their health, contributing to nutrition, detoxification, defense against pathogens, among other functions. This microbiota is composed of a core set of species, including Snodgrassella alvi, Gilliamella apicola, Lactobacillus Firm 4, and Bombilactobacillus. In this context, our objective was to evaluate different bacterial inoculation routes to reconstitute the typical honey bee gut microbiota under laboratory conditions. Individuals close to emergence were obtained from a healthy colony and incubated in the laboratory. Immediately after emergence, different inoculation routes were tested: administration of gut contents from nurse bees, administration of bacterial cultures, and contact with adult bees. As a control, one group of bees did not receive any treatment. In addition, a group of pupae that completed metamorphosis and emerged in the laboratory was included. After five days, the gut was dissected and the microbiota was analyzed using 16S rRNA gene sequencing and qPCR. Bees emerging from pupae and control bees showed a low bacterial load. Bees that received bacterial cultures developed a stable but poorly diverse microbiota lacking some core members. In contrast, bees that received gut contents developed a stable microbiota similar to that of field bees. This assay allowed the identification of the most effective method to reconstitute the gut microbiota, providing a useful technique for future studies under controlled conditions in which this microbiota is a key factor.