Comparative analysis of gut bacteria-associated enzymatic differentiation in honeybees Apis cerana and Apis mellifera

The present study elucidates the enzymatic diversity, functional specialization, and biotechnological potential of culturable gut bacterial communities in Apis mellifera and Apis cerana , two key pollinator species. Gut samples were analyzed using 16 S rRNA gene sequencing to characterize bacterial diversity, followed by qualitative and quantitative screening of culturable isolates for cellulolytic, hemicellulolytic, and lipolytic activities. A total of 34 bacterial isolates were recovered from distinct gut compartments, of which 31 showed significant enzymatic activity ( p < 0.05). Phylogenetic analysis revealed predominant affiliation with Bacillota and Pseudomonadota , including Apilactobacillus , Fructobacillus , Ralstonia , Enterobacter , Bombella , Citrobacter , and Burkholderia . Among these, Apilactobacillus (1.874 ± 0.12 U/mg) and Enterobacter (1.643 ± 0.11 U/mg) exhibited the highest cellulolytic activity. Ralstonia showed the strongest hemicellulolytic potential (1.674 ± 0.24 U/mg), while Bombella demonstrated the highest lipolytic activity (3.541 ± 0.22 U/mg). Enzyme activity was significantly higher in midgut isolates (45%) compared to hindgut (29%) and foregut (26%) isolates ( p < 0.01), indicating compartment-specific specialization. Hierarchical clustering and PCA revealed distinct functional structuring between the species, with A. cerana showing a cohesive enzymatic network and A. mellifera exhibiting greater metabolic heterogeneity. These findings highlight bee-microbiota co-evolution and the industrial potential of bee-derived enzymes for biomass conversion and biowaste valorization.