Project description:This study investigated how gut-derived bacterial supplementation influences the honey bee gut microbiota, metabolism, and proteome under three experimental settings: (i) controlled laboratory conditions (C versus B), (ii) semi-controlled laboratory conditions with or without social interaction with older nestmates (COB versus BOB), and (iii) field conditions (CON versus TRT). A bacterial mixture containing Lactobacillus helsingborgensis, L. apis, Bifidobacterium choladohabitans, and B. polysaccharolyticum was administered. Gut samples were collected after 10 days and analyzed for microbiome (16s rRNA sequencing), metabolome (1H NMR), and proteome profiles (LFQ-proteomics, laboratory experiment only). Proteomic profiling revealed distinct group-specific host responses. Control bees (C and COB) upregulated ribosomal proteins linked to protein synthesis and cellular stress. Supplemented bees in group B showed strong induction of major royal jelly proteins (MRJP1 and MRJP5), associated with neural modulation and colony behavior. In BOB, elevated expression of mitochondrial enzymes suggested improved energy metabolism in the presence of social context. Bacterial supplementation induces reproducible, condition-dependent changes across microbiota, metabolic, and proteomic layers. These findings provide mechanistic insight into how microbial interventions reshape gut function and support honey bee health under realistic environmental conditions.