Several studies have outlined that changes in the honeybee gut microbial composition may impair important metabolic functions supporting the honeybees’ life. Gut dysbiosis may be caused by diseases likeNosema ceranaeor by other anthropic, environmental or experimental stressors. The present work contributes to increasing knowledge on the dynamics of the gut microbiome acquisition in caged honeybees, an experimental condition frequently adopted by researchers, with or without infection withN. ceranae, and fed with a bacterial mixture to controlN. ceranaedevelopment. Changes of the gut microbiota were elucidated comparing microbial profile of caged and open-field reared honeybees. The absolute abundance of the major gut microbial taxa was studied with both NGS and qPCR approaches, whereas changes in the functionality were based on RAST annotations and manually curated. In general, all caged honeybees showed important changes in the gut microbiota, with\documentclass[12pt]{minimal}
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				\begin{document}$$\gamma$$\end{document}γ-proteobacteria (Frischella,GilliamellaandSnodgrassella) lacking in all caged experimental groups. Caged honeybees infected withN. ceranaeshowed also a strong colonization of environmental taxa likeCitrobacter,CosenzaeaandMorganella, as well as possibly pathogenic bacteria such asSerratia. The colonization ofSerratiadid not occur in presence of the bacterial mixture. The functionality prediction revealed that environmental bacteria or the supplemented bacterial mixture increased the metabolic potential of the honeybee gut microbiome compared to field and caged controls.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00248-022-02050-4.