Functional Genomics and Enzymatic Diversity of Gut Bacteria in Apis mellifera: A Multi-Approach Study from India

The gut microbiota of the western honey bee Apis mellifera plays a vital role in host nutrition, digestion, immunity, and overall colony health. Although the functional and enzymatic capabilities of bee-associated microbes are increasingly recognized, studies integrating culture-dependent screening with metagenomic functional profiling remain scarce. This study characterizes the gut bacterial communities of forager and hive bees from the Indian subcontinent using cultivation, 16S rRNA gene sequencing, enzyme assays, and metagenomic analysis. A total of 165 isolates were obtained, yielding 85 unique strains deposited in GenBank. Metagenomic assembly generated 7.78 million non-redundant genes, including 11,050 KEGG-annotated and 2.43 million CAZy-annotated genes. Forager bees showed pronounced enrichment of carbohydrate-processing pathways such as glycolysis/gluconeogenesis (22.9%), galactose metabolism (4.42%), starch and sucrose metabolism, and ABC transporters (9.80%), consistent with their nectar- and pollen-rich diet. Culture-based biochemical assays revealed substantial enzymatic diversity among isolates belonging to Bacillus, Enterobacter, Serratia, Cedecea, Clostridium, Lysinibacillus, and Aneurinibacillus. High invertase activities were recorded in Xanthomonas sp. HAmf44 (2.509 U/mg), Clostridium argentinense HAmf20 (2.470 U/mg), Lysinibacillus fusiformis HAmh15 (2.509 U/mg), and Bacillus paralicheniformis HAmh05 (2.333 U/mg). Strong lipolytic activities were observed in Cedecea davisae HAmf19 (6.062 U/mg), Pseudomonas aeruginosa HAmh21 (5.927 U/mg), and Enterobacter cloacae HAmf26 (3.349 U/mg). Significant variation among isolates (p = 0.001) underscored the functional diversity of the gut microbiota. Integrating KEGG orthologs with species abundance revealed that dominant taxa-including Gilliamella, Snodgrassella, Lactobacillus, and Bifidobacterium-drive key metabolic pathways. Overall, this study provides the first combined enzymatic and metagenomic assessment of A. mellifera gut microbiota from India and identifies high-performing strains with probiotic potential to enhance honey bee nutrition and colony productivity.