The Western honey bee (Apis mellifera) is a vital agricultural pollinator whose populations are threatened by the parasitic mite Varroa destructor and associated pathogens. While the impact of Paenibacillus species on honey bees, particularly Paenibacillus larvae causing American foulbrood, is documented, their effect on the microbiota of Varroa mites remains unclear. This study aimed to investigate the influence of Paenibacillus sp. on the bacterial communities of Varroa mites and adult honey bees. We hypothesized that Paenibacillus sp. would significantly alter the microbiota of Varroa mites but have minimal effect on that of adult honey bees. Utilizing 16S rRNA sequencing data from a previous study, we reanalyzed samples categorized into four groups based on Paenibacillus sp. infection load: highly infected and lowly infected honey bees (A. mellifera) and mites (V. destructor). Infection status was determined by Paenibacillus sp. read counts, with more than three reads indicating high infection. Microbial diversity was assessed using alpha and beta diversity metrics. Co-occurrence networks were constructed to visualize bacterial community assemblies, and network robustness was evaluated through node addition and removal tests. Keystone taxa were identified based on eigenvector centrality and relative abundance. Highly infected Varroa mites exhibited a significant reduction in alpha diversity and a markedly different bacterial community composition compared to lowly infected mites (p < 0.05). Their bacterial co-occurrence networks showed decreased connectivity and robustness, indicating a disruptive effect of Paenibacillus sp. In contrast, adult honey bees displayed no significant differences in alpha diversity or network structure between highly and lowly infected groups (p > 0.05), suggesting a resilient microbiota. Keystone taxa analysis revealed fewer central species in highly infected Varroa mites, potentially impacting network stability. High Paenibacillus sp. infection is associated with significant alterations in the microbiota of Varroa mites, disrupting bacterial communities and potentially affecting mite physiology. The microbiota of adult honey bees appears more robust against Paenibacillus sp. influence. These findings enhance our understanding of the complex interactions within the "honey bee-mite-microorganism" system and may inform future strategies for managing Varroa mite infestations and associated pathogens.

Animal Biotechnology Department Center for Genetic Engineering and Biotechnology Avenue 31 Between 158 and 190 P O Box 6162 Havana 10600 Cuba

ANSES INRAE École Nationale Vétérinaire d'Alfort UMR BIPAR Laboratoire de Santé Animale Maisons Alfort F 94700 France

Czech Academy of Sciences Biology Centre Institute of Entomology České Budějovice 37005 Czech Republic

Direction of Animal Health National Center for Animal and Plant Health Carretera de Tapaste y Autopista Nacional Apartado Postal 10 32700 San José de las Lajas Mayabeque Cuba

EA 7310 Laboratoire de Virologie Université de Corse Corte France

INRAE UR 0045 Laboratoire de Recherches Sur Le Développement de L'Élevage ' Corte France

Pavol Jozef Šafárik University in Košice Faculty of Science Institute of Biology and Ecology Institute of Animal Physiology Košice Slovakia

School of Environmental Sciences University of Guelph 50 Stone Rd E Guelph N1H 2W1 Ontario Canada

University of South Bohemia Faculty of Science České Budějovice Czech Republic

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