Varroa destructor mite is a major threat to honeybee (Apis mellifera) populations, contributing to colony losses through parasitism and pathogen transmission. While extensive research has focused on Varroa biology and its role as a virus vector, its microbiome remains poorly understood, particularly regarding geographic variation. Here, we investigated the microbial diversity, composition, and functional potential of Varroa mite microbiota collected from two neighboring countries, Czechia and Slovakia. Using high-throughput sequencing and network analysis, we assessed alpha and beta diversity metrics, microbial co-occurrence patterns, and predicted metabolic functions. Our results revealed significant differences in microbial diversity between the two regions, with some bacterial taxa appearing more prevalent in specific populations. Network analysis suggested potential variation in the structural stability of microbial communities in Varroa mites, raising the possibility that geographic factors may influence microbial interactions. Functional profiling indicated region-associated differences in predicted metabolic pathways, possibly linked to certain bacterial taxa. While these findings provide new insights into the Varroa microbiome and its potential ecological role, the interpretation of geographic influence remains a subject of ongoing investigation to better understand its scope and underlying mechanisms. A deeper understanding of these microbial dynamics may contribute to the development of novel strategies for Varroa mite management and the conservation of honeybee health.

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

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

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

Faculty of Science Pavol Jozef Šafárik University Košice Slovakia

Faculty of Science University of South Bohemia České Budějovice Czech Republic

School of Environmental Sciences University of Guelph Guelph ON Canada

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Oksanen J, G Simpson, FG Blanchet, R Kindt, P Legendre, P Minchin, RB O’Hara, P Solymos, MHH Stevens, E Szoecs, et al. 2022 Community ecology package ‘vegan.’

Almeida EL, Ribiere C, Frei W, Kenny D, Coffey MF, O’Toole PW (2023) Geographical and seasonal analysis of the honeybee microbiome. Microb Ecol 85:765–778 PubMed

Fernandes AD, JM Macklaim, TG Linn, G Reid, GB Gloor 2013 ANOVA-like differential expression (ALDEx) analysis for mixed population RNA-Seq. Parkinson J, editor. PLoS One 8:e67019.

Nadal-Jimenez P, S Siozios, CL Frost, R Court, E Chrostek, GC Drew, JD Evans, DJ Hawthorne, JB Burritt, GDD Hurst 2022 Arsenophonus apicola sp. nov., isolated from the honeybee Apis mellifera. Int J Syst Evol Microbiol 72:005469.

Pakwan C, M Kaltenpoth, B Weiss, P Chantawannakul, G Jun, T Disayathanoowat 2018 Bacterial communities associated with the ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae of the honey bee (Apis mellifera). FEMS Microbiol Ecol 94.

Barberán A, Bates ST, Casamayor EO, Fierer N (2012) Using network analysis to explore co-occurrence patterns in soil microbial communities. ISME J 6:343–351 PubMed

Bastian M, Heymann S, Jacomy M (2009) Gephi: An open source software for exploring and manipulating networks. Proc Int AAAI Conf Weblogs Soc Media 3:361–362

Bleau N, Bouslama S, Giovenazzo P, Derome N (2020) Dynamics of the honeybee (Apis mellifera) gut microbiota throughout the overwintering period in Canada. Microorganisms 8:1146 PubMed PMC

Bokulich NA, Kaehler BD, Rideout JR, Dillon M, Bolyen E, Knight R, Huttley GA, Gregory Caporaso J (2018) Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6:1–17

Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME2. Nat Biotechnol 37:852–857 PubMed PMC

Bray JR, Curtis JT (1957) An ordination of the upland forest communities of southern wisconsin. Ecol Monogr 27:325–349

Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583 PubMed PMC

Caspi R, Billington R, Fulcher CA, Keseler IM, Kothari A, Krummenacker M, Latendresse M, Midford PE, Ong Q, Ong WK et al (2018) The MetaCyc database of metabolic pathways and enzymes. Nucleic Acids Res 46:D633–D639 PubMed

Chandra S, Šlapeta J (2020) Biotic factors influence microbiota of nymph ticks from vegetation in Sydney. Australia Pathogens 9:566 PubMed

Chazdon RL, Chao A, Colwell RK, Lin S-Y, Norden N, Letcher SG, Clark DB, Finegan B, Arroyo JP (2011) A novel statistical method for classifying habitat generalists and specialists. Ecol 92:1332–1343

Allaire JJ, P Ellis, C Gandrud, K Kuo, BW Lewis, J Owen, K Russell, J Rogers, C Sese, C Yetman 2025 D3 JavaScript Network Graphs from R Package “networkD3.”

DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072 PubMed PMC

Douglas GM, Maffei VJ, Zaneveld JR, Yurgel SN, Brown JR, Taylor CM, Huttenhower C, Langille MGI (2020) PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 38:685–688 PubMed PMC

Engel P, Moran NA (2013) The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev 37:699–735 PubMed

Freitas S, D Yang, S Kumar, H Tong, DH Chau 2021 Evaluating graph vulnerability and robustness using tiger. Proc Int Conf Inf Knowle Manag:4495–4503.

Faith DP (1992) Conservation evaluation and phylogenetic diversity. Biol Conserv 61:1–10

Faust K, Raes J (2012) Microbial interactions: From networks to models. Nat Rev Microbiol 10:538–550 PubMed

Hubert J, Bicianova M, Ledvinka O, Kamler M, Lester PJ, Nesvorna M, Kopecky J, Erban T (2017) Changes in the bacteriome of honey bees associated with the parasite Varroa destructor, and pathogens Nosema and Lotmaria passim. Microb Ecol 73:685–698 PubMed

Hubert J, Erban T, Kamler M, Kopecky J, Nesvorna M, Hejdankova S, Titera D, Tyl J, Zurek L (2015) Bacteria detected in the honeybee parasitic mite Varroa destructor collected from beehive winter debris. J Appl Microbiol 119:640–654 PubMed

Hubert J, Kamler M, Nesvorna M, Ledvinka O, Kopecky J, Erban T (2016) Comparison of Varroa destructor and worker honeybee microbiota within hives indicates shared bacteria. Microb Ecol 72:448–459 PubMed

Friedman J, EJ Alm 2012 Inferring correlation networks from genomic survey data. Von Mering C, editor. PLoS Comput Biol 8:e1002687.

Jones L, Brennan GL, Lowe A, Creer S, Ford CR, De Vere N (2021) Shifts in honeybee foraging reveal historical changes in floral resources. Commun Biol 4:37 PubMed PMC

Kakumanu ML, Reeves AM, Anderson TD, Rodrigues RR, Williams MA (2016) Honey bee gut microbiome is altered by in-hive pesticide exposures. Front Microbiol 7:1255 PubMed PMC

Kanehisa M (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28:27–30 PubMed PMC

Keller A, McFrederick QS, Dharampal P, Steffan S, Danforth BN, Leonhardt SD (2021) (More than) Hitchhikers through the network: The shared microbiome of bees and flowers. Curr Opin Insect Sci 44:8–15 PubMed

Kwong WK, Moran NA (2016) Gut microbial communities of social bees. Nat Rev Microbiol 14:374–384 PubMed PMC

Li C, M Tang, X Li, X Zhou 2022 Community dynamics in structure and function of honey bee gut bacteria in response to winter dietary shift. Martiny JBH, editor. mBio 13:e01131–22.

Liu H-H, Chen L, Shao H-B, Gao S, Hong X-Y, Bing X-L (2024) Environmental factors and the symbiont Cardinium influence the bacterial microbiome of spider mites across the landscape. Microb Ecol 87:1

Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550 PubMed PMC

Maes PW, Floyd AS, Mott BM, Anderson KE (2021) Overwintering honey bee colonies: Effect of worker age and climate on the hindgut microbiota. Insects 12:224 PubMed PMC

Moran NA (2015) Genomics of the honey bee microbiome. Curr Opin Insect Sci 10:22–28 PubMed PMC

Moro CV, Thioulouse J, Chauve C, Zenner L (2011) Diversity, geographic distribution, and habitat-specific variations of microbiota in natural populations of the chicken mite, Dermanyssus gallinae. J Med Entomol 48:788–796 PubMed

Peschel S, CL Müller, E Von Mutius, A-L Boulesteix, M Depner 2021 NetCoMi: Network construction and comparison for microbiome data in R. Brief Bioinform 22:bbaa290.

Nguyen PN, Rehan SM (2023) Environmental effects on bee microbiota. Microb Ecol 86:1487–1498 PubMed

Pielou EC (1966) The measurement of diversity in different types of biological collections. J Theor Biol 13:131–144

Piou V, Schurr F, Dubois E, Vétillard A (2022) Transmission of deformed wing virus between Varroa destructor foundresses, mite offspring and infested honey bees. Parasit Vectors 15:333 PubMed PMC

Qi S, Al Naggar Y, Li J, Liu Z, Xue X, Wu L, El-Seedi HR, Wang K (2022) Acaricide flumethrin-induced sublethal risks in honeybees are associated with gut symbiotic bacterium Gilliamella apicola through microbe-host metabolic interactions. Chemosphere 307:136030 PubMed

Ramsey SD, Ochoa R, Bauchan G, Gulbronson C, Mowery JD, Cohen A, Lim D, Joklik J, Cicero JM, Ellis JD et al (2019) Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph. Proc Natl Acad Sci USA 116:1792–1801 PubMed PMC

Rering CC, Beck JJ, Hall GW, McCartney MM, Vannette RL (2018) Nectar-inhabiting microorganisms influence nectar volatile composition and attractiveness to a generalist pollinator. New Phytol 220:750–759 PubMed

Rosenkranz P, Aumeier P, Ziegelmann B (2010) Biology and control of Varroa destructor. J Invertebr Pathol 103:S96–S119 PubMed

Röttjers L, Vandeputte D, Raes J, Faust K (2021) Null-model-based network comparison reveals core associations. ISME Commun 1:36 PubMed PMC

Schwarz RS, Teixeira ÉW, Tauber JP, Birke JM, Martins MF, Fonseca I, Evans JD (2014) Honey bee colonies act as reservoirs for two Spiroplasma facultative symbionts and incur complex, multiyear infection dynamics. MicrobiologyOpen 3:341–355 PubMed PMC

Seeley TD, Visscher PK (1985) Survival of honeybees in cold climates: The critical timing of colony growth and reproduction. Ecol Entomol 10:81–88

Shannon CE (1948) A mathematical theory of communication. Bell Syst Tech J 27:379–423

Skičková Š, Kratou M, Svobodová K, Maitre A, Abuin-Denis L, Wu-Chuang A, Obregón D, Said MB, Majláthová V, Krejčí A et al (2024a) Functional redundancy and niche specialization in honeybee and Varroa microbiomes. Int Microbiol 28:795–810 PubMed

Skičková Š, Svobodová K, Maitre A, Wu-Chuang A, Abuin-Denis L, Piloto-Sardiñas E, Obregon D, Majláth I, Majláthová V, Krejčí A et al (2024b) Differential impact of Paenibacillus infection on the microbiota of Varroa destructor and Apis mellifera. Heliyon 10:e39384 PubMed PMC

Taylor M, Mediannikov O, Raoult D, Greub G (2012) Endosymbiotic bacteria associated with nematodes, ticks and amoebae. FEMS Immunol Med Microbiol 64:21–31 PubMed

Van Treuren W, Ponnusamy L, Brinkerhoff RJ, Gonzalez A, Parobek CM, Juliano JJ, Andreadis TG, Falco RC, Ziegler LB, Hathaway N et al (2015) Variation in the microbiota of Ixodes ticks with regard to geography, species, and sex. Goodrich-Blair H, editor. Appl Environ Microbiol 81:6200–6209 PubMed PMC

Vannette RL, Fukami T (2018) Contrasting effects of yeasts and bacteria on floral nectar traits. Ann Bot 121:1343–1349 PubMed PMC

Wu J, Liu F, Sun J, Wei Q, Kang W, Wang F, Zhang C, Zhao M, Xu S, Han B (2024) Toxic effects of acaricide fenazaquin on development, hemolymph metabolome, and gut microbiome of honeybee (Apis mellifera) larvae. Chemosphere 358:142207 PubMed

Ge Y, Z Jing, Q Diao, J-Z He, Y-J Liu 2021 Host species and geography differentiate honeybee gut bacterial communities by changing the relative contribution of community assembly processes. Bailey MJ, editor. mBio 12:e00751–21.

Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer K-H, Whitman WB, Euzéby J, Amann R, Rosselló-Móra R (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12:635–645 PubMed

Anaconda Documentation 2020 Anaconda Software Distribution.

Lhomme S 2015 Analyse spatiale de la structure des réseaux techniques dans un contexte de risques. Cybergeo: European Journal of Geography. [accessed 2024 Jan 23]. http://journals.openedition.org/cybergeo/26763

R Core Team 2023 A language and environment for statistical computing. R foundation for statistical computing.

RStudio Team 2020 RStudio: Integrated development for R.

Vaidyanathan R, Y Xie, J Allaire, J Cheng, C Sievert, K Russell 2023 Htmlwidgets: HTML widgets for R.