Bacterial supplementation shapes honey bee gut microbiota and host metabolism under controlled and field conditions

Gut bacteria are essential to honey bee (Apis mellifera) health, supporting digestion, immunity, and resilience against stressors. Probiotic-based strategies have been proposed to enhance core gut symbionts, yet the underlying mechanisms and influences of environmental context on these effects are not fully understood. This study examined the influence of gut-derived bacterial supplementation on the honey bee gut microbiome, proteome, and metabolome across three conditions: (i) controlled cages, (ii) semi-controlled cages allowing contact with nestmates, and (iii) field conditions. Treatment groups received a bacterial supplement containing Lactobacillus helsingborgensis, Lactobacillus apis, Bifidobacterium choladohabitans, and Bifidobacterium polysaccharolyticum, while control groups received only a sucrose solution. Gut samples from 10-day-old bees were analyzed. Supplemented bees showed strong gut colonization by Bifidobacterium and Lactobacillus, especially under controlled and semi-controlled conditions. In the field, L. helsingborgensis remained significantly enriched in the treatment group, demonstrating short-term ecological resilience in the natural hive environment. Proteomic changes in supplemented bees included an increased abundance of major royal jelly protein precursors and mitochondrial-associated proteins, together with reduced abundance of several ribosomal and translation-related proteins involved in peptide biosynthesis and cellular protein quality control. Metabolomic analysis revealed reproducible shifts across all three conditions. Treatment groups showed higher concentrations of microbial fermentation products (acetate, succinate) and potential neuromodulatory metabolites (ornithine, γ-aminobutyrate), while sucrose, N-acetylglucosamine, and uridine were constantly lower compared to controls. These findings highlight reproducible, context-dependent effects of bacterial supplementation on honey bee gut physiology and provide a framework for interpreting microbiome-based interventions in pollinator health.IMPORTANCEHoney bees are essential pollinators whose health is influenced by their gut microbiome. Probiotic applications aimed at improving gut health have been proposed, yet outcomes remain inconsistent and vary across settings. Results from laboratory experiments often differ from those observed under field conditions, making it difficult to understand the complex dynamics of eusocial insect colonies. Here, we evaluate honey bee gut-derived bacterial supplementation across controlled, semi-controlled, and field settings using bacterial profiling, proteomic, and metabolomic analyses. We demonstrate that bacterial-supplemented groups consistently reshape gut community composition and modulate host physiological processes, but in a context-dependent manner. These results provide a unified understanding of how microbial interventions function at colony and individual levels, guiding the rational design of probiotic strategies to support honey bee health under realistic conditions.