BackgroundKnowledge of microbiota composition, persistence, and transmission as well as the overall function of the bacterial community is important and may be linked to honey bee health. This study aimed to investigate the inter-individual variation in the gut microbiota in honey bee larvae and pupae.ResultsIndividual larvae differed in the composition of major bacterial groups. In the majority of 5th instar bees, Firmicutes showed predominance (70%); however, after larval defecation and during pupation, the abundance decreased to 40%, in favour of Gammaproteobacteria. The 5th instar larvae hosted significantly more (P < 0.001) Firmicutes than black pupae. Power calculations revealed that 11 and 18 replicate-individuals, respectively, were required for the detection of significant differences (P < 0.05) in the Bacteroidetes and Firmicutes abundance between stages, while higher numbers of replicates were required for Actinobacteria (478 replicates) and Gammaproteobacteria (111 replicates).ConclusionsAlthough sample processing and extraction protocols may have had a significant influence, sampling is very important for studying the bee microbiome, and the importance of the number of individuals pooled in samples used for microbiome studies should not be underestimated.