Microbiome–Metabolome Analyses Reveal Compound Risks from Multiple-Generation Cocoon Accumulation in Honeybee Combs

The accumulation of cocoons within brood cells of old combs is a key factor causing a series of negative impacts on bee colonies. Previous studies did not sufficiently address this dynamic nature as the core microenvironment for preimaginal bee development. During this accumulation, the enrichment of potentially harmful microorganisms and chemical substances may pose a latent threat to colony health. This study combined microbiome and metabolomics analyses to systematically investigate the potential colony health risks posed by multi-generational accumulation of cocoons in <i>Apis mellifera</i> combs. The results demonstrated that with the growing number of brood rearing generations, the microbial diversity within the cocoons underwent significant shifts. For the bacterial community within multiple-generation cocoons, the Simpson index exhibited a significant increase, whereas indices including Sobs, Ace, and Chao showed significant decreases (<i>p</i> < 0.05). In the fungal community, the Shannon and Pielou_e indices significantly increased, while the Simpson and Faith_pd indices significantly declined (<i>p</i> < 0.05). Potential pathogens such as <i>Melissococcus</i> and the mycotoxin-producing fungus <i>Wallemia</i> became significantly enriched, reaching alarming relative abundances of 42.70% and 13.52%, respectively, in the multiple-generation cocoons. Metabolomic analysis further revealed the enrichment of 685 differential metabolites, including persistent exogenous pesticides such as cyanazine and pymetrozine, etc. Correlation analysis uncovered a significant positive relationship (r > 0.8) between these pesticide residues and pathogen abundance, indicating interactions between pollutants and pathogens that may exacerbate risks. This study reveals the aggravation of microecological imbalance and chemical pollution load within the cocoons of old combs and therefore provides strong scientific support for risk assessment of comb age in colony health management and offers practical guidance for the sustainable development of beekeeping.