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

## TL;DR

Old honeybee combs accumulate harmful microbes and pesticides over generations, threatening colony health and suggesting the need for regular comb replacement.

## Contribution

This study combines microbiome and metabolomic analyses to reveal how multi-generational cocoon accumulation in honeybee combs increases health risks.

## Key findings

- Older cocoons contain higher levels of harmful bacteria, fungi, and pesticide residues.
- Microbial diversity shifts significantly with multiple generations of cocoon accumulation.
- Pesticides and pathogens show strong positive correlations, compounding health risks.

## Abstract

Honeybees reuse the same comb cells many times, leaving behind silk-like cocoon layers. Over generations, these layers build up inside old combs. The accumulation of cocoons within brood cells of old combs is a key factor causing a series of negative impacts on bee colonies. By studying both the microbes and chemicals present in cocoons from new and old combs, we discovered that older cocoons contain more harmful bacteria and fungi, as well as higher levels of pesticide residues. These pollutants and pathogens appear to work together, increasing health risks to bee brood. Our findings show that regularly replacing old combs can help protect honeybee colonies from hidden threats. This practice supports more sustainable beekeeping, which in turn benefits agricultural productivity and environmental health.

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 Apis mellifera 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 (p < 0.05). In the fungal community, the Shannon and Pielou_e indices significantly increased, while the Simpson and Faith_pd indices significantly declined (p < 0.05). Potential pathogens such as Melissococcus and the mycotoxin-producing fungus Wallemia 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.