Published October 10, 2025
| Version v1
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Surface swabs outperform most traditional honeybee (Apis mellifera) hive samples for recovery of eDNA and eRNA
Creators
- 1. CSIRO Health and Biosecurity, Canberra, Australia
- 2. CSIRO Centre for Australian National Biodiversity Research, Canberra, Australia
- 3. CSIRO Australian Centre for Disease Preparedness, East Geelong, Australia
- 4. CSIRO Health and Biosecurity, Floreat, Australia
Description
Genetic sampling from European honeybee (Apis mellifera) hives is a promising repository of terrestrial environmental nucleic acids (eNA) from a range of taxonomic sources. eNA samples from honeybee hives can be used to monitor environments via vegetation assessments and plant pathogen detection, while simultaneously assessing honeybee hive health through the identification of honeybee pests and pathogens. However, there is still limited comparison across the different types of hive samples commonly used for biomonitoring. There is also a need for less invasive sampling methods to facilitate adoption of hive eNA surveillance at scale. The aim of this study was to compare the effectiveness of using non-invasive swabbing of hive entrances with common sample types collected from honeybee hives (honey, pollen, and bees) for eNA detection of various taxa. Using a DNA metabarcoding approach for bacteria (16S), animal (COI), fungi (ITS1), and plant (ITS2) identification, and metatranscriptomic sequencing for virus detection, swabbing was found to provide comparable or better detection of taxa compared with more invasive sampling methods. Swabs were most informative for identifying bacterial and fungal communities, while plant detections were similar between swabs and honey samples. Detection of RNA viral diversity was highest in honey samples, however, swabs also showed good recovery of plant and insect viruses. A minimum of two swab replicates was recommended to increase taxa yield for DNA metabarcoding. Our study suggests that swabbing of hive entrances is a highly effective biomonitoring method for non-invasively collecting hive eNA and delivering comprehensive surveillance for animal, plant and microbial communities in the environment.
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References
- Abarenkov K, Nilsson RH, Larsson K-H, Taylor AFS, May TW, Frøslev TG, Pawlowska J, Lindahl B, Põldmaa K, Truong C, Vu D, Hosoya T, Niskanen T, Piirmann T, Ivanov F, Zirk A, Peterson M, Cheeke TE, Ishigami Y, Jansson AT, Jeppesen TS, Kristiansson E, Mikryukov V, Miller JT, Oono R, Ossandon FJ, Paupério J, Saar I, Schigel D, Suija A, Tedersoo L, Kõljalg U (2024) The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: Sequences, taxa and classifications reconsidered. Nucleic Acids Research 52(D1): D791–D797. https://doi.org/10.1093/nar/gkad1039
- Anderson KE, Carroll MJ, Sheehan T, Mott BM, Maes P, Corby‐Harris V (2014) Hive‐stored pollen of honey bees: Many lines of evidence are consistent with pollen preservation, not nutrient conversion. Molecular Ecology 23(23): 469–499. https://doi.org/10.1111/mec.12966
- Ankenbrand MJ, Keller A, Wolf M, Schultz J, Förster F (2015) ITS2 Database V: Twice as Much: Table 1. Molecular Biology and Evolution 32(11): 3030–3032. https://doi.org/10.1093/molbev/msv174
- Apprill A, McNally S, Parsons R, Weber L (2015) Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology 75(2): 129–137. https://doi.org/10.3354/ame01753
- As Y, Eroglu GB (2025) Molecular detection and phylogenetic analysis of five honey bee viruses from wet pollen in Türkiye. Biology Bulletin of the Russian Academy of Sciences 52(3): 89. https://doi.org/10.1134/S1062359024612898
- Balkanska R, Shumkova R, Atsenova N, Salkova D, Dundarova H, Radoslavov G, Hristov P (2023) Molecular detection and phylogenetic analysis of deformed wing virus and sacbrood virus isolated from pollen. Veterinary Sciences 10(2): 140. https://doi.org/10.3390/vetsci10020140
- Banerjee P, Stewart KA, Dey G, Antognazza CM, Sharma RK, Maity JP, Saha S, Doi H, De Vere N, Chan MWY, Lin P-Y, Chao H-C, Chen C-Y (2022) Environmental DNA analysis as an emerging non-destructive method for plant biodiversity monitoring: a review. AoB PLANTS 14: plac031. https://doi.org/10.1093/aobpla/plac031
- Batchelor KL, Bell KL, Campos M, Webber BL (2023) Can honey bees be used to detect rare plants? Taking an eDNA approach to find the last plants in a weed eradication program. Environmental DNA 5(6): 1516–1526. https://doi.org/10.1002/edn3.471
- Biová J, Charrière J-D, Dostálková S, Škrabišová M, Petřivalský M, Bzdil J, Danihlík J (2021) Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR. Insects 12(2): 150. https://doi.org/10.3390/insects12020150
- Boardman L, Marcelino JAP, Valentin RE, Boncristiani H, Standley JM, Ellis JD (2024) Novel eDNA approaches to monitor Western honey bee (Apis mellifera L.) microbial and arthropod communities. Environmental DNA 6(1): e419. https://doi.org/10.1002/edn3.419
- Buchfink B, Reuter K, Drost H-G (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nature Methods 18(4): 366–368. https://doi.org/10.1038/s41592-021-01101-x
- Bushnell B (2014) BBTools software packag. e.
- Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: Architecture and applications. BMC Bioinformatics 10(1): 421. https://doi.org/10.1186/1471-2105-10-421
- Card SD, Pearson MN, Clover GRG (2007) Plant pathogens transmitted by pollen. Australasian Plant Pathology 36(5): 455. https://doi.org/10.1071/AP07050
- Carroll MJ, Brown N, Goodall C, Downs AM, Sheenan TH, Anderson KE (2017) Honey bees preferentially consume freshly-stored pollen. PLoS ONE 12: e0175933. https://doi.org/10.1371/journal.pone.0175933
- Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5: e8613. https://doi.org/10.1371/journal.pone.0008613
- Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34(17): i884–i890. https://doi.org/10.1093/bioinformatics/bty560
- Cirtwill AR, Wirta H (2025) DNA in honey could describe the changes in flower visits and microbe encounters of honey bees over decades. Scientific Reports 15(1): 8807. https://doi.org/10.1038/s41598-025-93315-8
- Coffey MF, Breen J (1997) Seasonal variation in pollen and nectar sources of honey bees in Ireland. Journal of Apicultural Research 36(2): 63–76. https://doi.org/10.1080/00218839.1997.11100932
- Čukanová E, Prodělalová J, Palíková M, Kováčová K, Linhart P, Papežíková I (2023) Can the examination of different types of hive samples be a non-invasive method for detection and quantification of viruses in honey bee (Apis mellifera L. ) colonies? Journal of Veterinary Research (Pulawy) 67(3): 323–331. https://doi.org/10.2478/jvetres-2023-0046
- Danner N, Keller A, Härtel S, Steffan-Dewenter I (2017) Honey bee foraging ecology: Season but not landscape diversity shapes the amount and diversity of collected pollen. PLoS ONE 12: e0183716. https://doi.org/10.1371/journal.pone.0183716
- De Vere N, Jones LE, Gilmore T, Moscrop J, Lowe A, Smith D, Hegarty MJ, Creer S, Ford CR (2017) Using DNA metabarcoding to investigate honey bee foraging reveals limited flower use despite high floral availability. Scientific Reports 7(1): 42838. https://doi.org/10.1038/srep42838
- Doble CJ, Hipperson H, Salzburger W, Horsburgh GJ, Mwita C, Murrell DJ, Day JJ (2020) Testing the performance of environmental DNA metabarcoding for surveying highly diverse tropical fish communities: A case study from Lake Tanganyika. Environmental DNA 2(1): 24–41. https://doi.org/10.1002/edn3.43
- Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19): 2460–2461. https://doi.org/10.1093/bioinformatics/btq461
- Edgar RC (2016) SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. https://doi.org/10.1101/074161
- Eyer M, Neumann P, Dietemann V (2016) A look into the cell: Honey storage in honey bees, Apis mellifera. PLoS ONE 11: e0161059. https://doi.org/10.1371/journal.pone.0161059
- Fetters AM, Ashman T (2023) The pollen virome: A review of pollen‐associated viruses and consequences for plants and their interactions with pollinators. American Journal of Botany 110(6): e16144. https://doi.org/10.1002/ajb2.16144
- Ficetola GF, Pansu J, Bonin A, Coissac E, Giguet‐Covex C, De Barba M, Gielly L, Lopes CM, Boyer F, Pompanon F, Rayé G, Taberlet P (2015) Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data. Molecular Ecology Resources 15(3): 543–556. https://doi.org/10.1111/1755-0998.12338
- Fox J, Weisberg S (2018) An R companion to applied regression. Sage publications. https://doi.org/10.32614/CRAN.package.carData
- Freeland JR (2017) The importance of molecular markers and primer design when characterizing biodiversity from environmental DNA. Genome 60: 358–374. https://doi.org/10.1139/gen-2016-0100
- Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts. Molecular Ecology 2(2): 113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, Di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29(7): 644–652. https://doi.org/10.1038/nbt.1883
- Hawkins J, De Vere N, Griffith A, Ford CR, Allainguillaume J, Hegarty MJ, Baillie L, Adams-Groom B (2015) Using DNA metabarcoding to identify the floral composition of honey: A new tool for investigating honey bee foraging preferences. PLoS ONE 10: e0134735. https://doi.org/10.1371/journal.pone.0134735
- Hénaff E, Najjar D, Perez M, Flores R, Woebken C, Mason CE, Slavin K (2023) Holobiont Urbanism: Sampling urban beehives reveals cities' metagenomes. Environmental Microbiome 18(1): 23. https://doi.org/10.1186/s40793-023-00467-z
- Kadlečková D, Tachezy R, Erban T, Deboutte W, Nunvář J, Saláková M, Matthijnssens J (2022) The Virome of Healthy Honey Bee Colonies: Ubiquitous Occurrence of Known and New Viruses in Bee Populations. mSystems 7: e00072-22. https://doi.org/10.1128/msystems.00072-22
- Kassambara A (2019) rstatix: Pipe-friendly framework for basic statistical tests. CRAN: Contributed packages. https://doi.org/10.32614/CRAN.package.rstatix
- Koetschan C, Förster F, Keller A, Schleicher T, Ruderisch B, Schwarz R, Müller T, Wolf M, Schultz J (2010) The ITS2 Database III—Sequences and structures for phylogeny. Nucleic Acids Research 38(suppl_1): D275–D279. https://doi.org/10.1093/nar/gkp966
- Koetschan C, Hackl T, Müller T, Wolf M, Förster F, Schultz J (2012) ITS2 Database IV: Interactive taxon sampling for internal transcribed spacer 2 based phylogenies. Molecular Phylogenetics and Evolution 63(3): 585–588. https://doi.org/10.1016/j.ympev.2012.01.026
- Lee E, Vansia R, Phelan J, Lofano A, Smith A, Wang A, Bilodeau GJ, Pernal SF, Guarna MM, Rott M, Griffiths JS (2023) Area Wide Monitoring of Plant and Honey Bee (Apis mellifera) Viruses in Blueberry (Vaccinium corymbosum) Agroecosystems Facilitated by Honey Bee Pollination. Viruses 15(5): 1209. https://doi.org/10.3390/v15051209
- Leray M, Knowlton N, Machida RJ (2022) MIDORI2: A collection of quality controlled, preformatted, and regularly updated reference databases for taxonomic assignment of eukaryotic mitochondrial sequences. Environmental DNA 4(4): 894–907. https://doi.org/10.1002/edn3.303
- Mackay JF, Hewett RE, Smith NT, Waters TL, Scandrett JS (2025) The Foster method: Rapid and non-invasive detection of clinically significant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens. Journal of Apicultural Research 64(2): 693–700. https://doi.org/10.1080/00218839.2024.2306445
- Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. Journal 17(1): 10–12. https://doi.org/10.14806/ej.17.1.200
- McKnight DT, Huerlimann R, Bower DS, Schwarzkopf L, Alford RA, Zenger KR (2019) microDecon: A highly accurate read-subtraction tool for the post-sequencing removal of contamination in metabarcoding studies. Environmental DNA 1(1): 14–25. https://doi.org/10.1002/edn3.11
- McMurdie PJ, Holmes S (2013) phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8: e61217. https://doi.org/10.1371/journal.pone.0061217
- Merget B, Koetschan C, Hackl T, Förster F, Dandekar T, Müller T, Schultz J, Wolf M (2012) The ITS2 Database. Journal of Visualized Experiments 3806(61): e3806. https://doi.org/10.3791/3806
- Milla L, Sniderman K, Lines R, Mousavi‐Derazmahalleh M, Encinas‐Viso F (2021) Pollen DNA metabarcoding identifies regional provenance and high plant diversity in Australian honey. Ecology and Evolution 11(13): 8683–8698. https://doi.org/10.1002/ece3.7679
- Milla L, Schmidt‐Lebuhn A, Bovill J, Encinas‐Viso F (2022) Monitoring of honey bee floral resources with pollen DNA metabarcoding as a complementary tool to vegetation surveys. Ecological Solutions and Evidence 3(1): e12120. https://doi.org/10.1002/2688-8319.12120
- Minamoto T, Miya M, Sado T, Seino S, Doi H, Kondoh M, Nakamura K, Takahara T, Yamamoto S, Yamanaka H, Araki H, Iwasaki W, Kasai A, Masuda R, Uchii K (2021) An illustrated manual for environmental DNA research: Water sampling guidelines and experimental protocols. Environmental DNA 3(1): 8–13. https://doi.org/10.1002/edn3.121
- Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O'hara R, Simpson GL, Solymos P (2019) vegan: Community Ecology Package. R package version 2.5-6.
- Parada AE, Needham DM, Fuhrman JA (2016) Every base matters: Assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology 18(5): 1403–1414. https://doi.org/10.1111/1462-2920.13023
- Pawlowski J, Apothéloz-Perret-Gentil L, Mächler E, Altermatt F (2020) Environmental DNA applications for biomonitoring and bioassessment in aquatic ecosystems. https://doi.org/10.5167/UZH-187800
- Pereira KDS, Meeus I, Smagghe G (2019) Honey bee-collected pollen is a potential source of Ascosphaera apis infection in managed bumble bees. Scientific Reports 9(1): 4241. https://doi.org/10.1038/s41598-019-40804-2
- Piggott MP, Banks SC, Broadhurst BT, Fulton CJ, Lintermans M (2021) Comparison of traditional and environmental DNA survey methods for detecting rare and abundant freshwater fish. Aquatic Conservation 31(1): 173–184. https://doi.org/10.1002/aqc.3474
- Prosser SWJ, Hebert PDN (2017) Rapid identification of the botanical and entomological sources of honey using DNA metabarcoding. Food Chemistry 214: 183–191. https://doi.org/10.1016/j.foodchem.2016.07.077
- Ribani A, Utzeri VJ, Taurisano V, Fontanesi L (2020) Honey as a source of environmental dna for the detection and monitoring of honey bee pathogens and parasites. Veterinary Sciences 7(3): 113. https://doi.org/10.3390/vetsci7030113
- Ribani A, Taurisano V, Utzeri VJ, Fontanesi L (2022) Honey environmental DNA can be used to detect and monitor honey bee pests: Development of methods useful to identify aethina tumida and galleria mellonella infestations. Veterinary Sciences 9(5): 213. https://doi.org/10.3390/vetsci9050213
- Ribière C, Hegarty C, Stephenson H, Whelan P, O'Toole PW (2019) Gut and whole-body microbiota of the honey bee separate thriving and non-thriving hives. Microbial Ecology 78(1): 195–205. https://doi.org/10.1007/s00248-018-1287-9
- Roberts JMK, Anderson DL, Durr PA (2018a) Metagenomic analysis of varroa-free australian honey bees (Apis mellifera) shows a diverse Picornavirales virome. The Journal of General Virology 99(6): 818–826. https://doi.org/10.1099/jgv.0.001073
- Roberts JMK, Ireland KB, Tay WT, Paini D (2018b) Honey bee‐assisted surveillance for early plant virus detection. The Annals of Applied Biology 173(3): 285–293. https://doi.org/10.1111/aab.12461
- Roberts JMK, Jooste AEC, Pretorius L-S, Geering ADW (2023) Surveillance for avocado sunblotch viroid utilizing the european honey bee (Apis mellifera). Phytopathology 113(3): 559–566. https://doi.org/10.1094/PHYTO-08-22-0295-R
- Roberts JMK, Hall RJ, Shams F, Encinas‐Viso F, Bravo F, Soroka J, Milla L, Snape N, Martoni F, Walford A, Gleeson D, Trujillo‐González A (2025) Environmental DNA methods for detection of Varroa destructor in honey bee (Apis mellifera) hives. Environmental DNA 7(3): e70109. https://doi.org/10.1002/edn3.70109
- Roessink I, Van Der Steen JJM (2021) Beebread consumption by honey bees is fast: Results of a six-week field study. Journal of Apicultural Research 60(5): 659–664. https://doi.org/10.1080/00218839.2021.1915612
- Scriver M, Zaiko A, Pochon X, Von Ammon U (2023) Harnessing decay rates for coastal marine biosecurity applications: A review of environmental DNA and RNA fate. Environmental DNA 5(5): 960–972. https://doi.org/10.1002/edn3.405
- Selig C, Wolf M, Muller T, Dandekar T, Schultz J (2007) The ITS2 Database II: Homology modelling RNA structure for molecular systematics. Nucleic Acids Research 36(Database): D377–D380. https://doi.org/10.1093/nar/gkm827
- Shirazi S, Meyer RS, Shapiro B (2021) Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding. Ecology and Evolution 11(22): 15766–15779. https://doi.org/10.1002/ece3.8239
- Smith DP, Peay KG (2014) Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS ONE 9: e90234. https://doi.org/10.1371/journal.pone.0090234
- Soares S, Rodrigues F, Delerue-Matos C (2023) Towards DNA-Based Methods Analysis for Honey: An Update. Molecules 28(5): 2106. https://doi.org/10.3390/molecules28052106
- Tremblay ÉD, Duceppe M, Thurston GB, Gagnon M, Côté M, Bilodeau GJ (2019) High‐resolution biomonitoring of plant pathogens and plant species using metabarcoding of pollen pellet contents collected from a honey bee hive. Environmental DNA 1(2): 155–175. https://doi.org/10.1002/edn3.17
- Trujillo-González A, Edmunds RC, Becker JA, Hutson KS (2019) Parasite detection in the ornamental fish trade using environmental DNA. Scientific Reports 9(1): 5173. https://doi.org/10.1038/s41598-019-41517-2
- Utzeri VJ, Schiavo G, Ribani A, Tinarelli S, Bertolini F, Bovo S, Fontanesi L (2018) Entomological signatures in honey: An environmental DNA metabarcoding approach can disclose information on plant-sucking insects in agricultural and forest landscapes. Scientific Reports 8(1): 9996. https://doi.org/10.1038/s41598-018-27933-w
- Valentini A, Taberlet P, Miaud C, Civade R, Herder J, Thomsen PF, Bellemain E, Besnard A, Coissac E, Boyer F, Gaboriaud C, Jean P, Poulet N, Roset N, Copp GH, Geniez P, Pont D, Argillier C, Baudoin J, Peroux T, Crivelli AJ, Olivier A, Acqueberge M, Le Brun M, Møller PR, Willerslev E, Dejean T (2016) Next‐generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Molecular Ecology 25(4): 929–942. https://doi.org/10.1111/mec.13428
- Vamos E, Elbrecht V, Leese F (2017) Short COI markers for freshwater macroinvertebrate metabarcoding. Metabarcoding and Metagenomics 1: e14625. https://doi.org/10.3897/mbmg.1.14625
- Van Der Heyde M, Bunce M, Nevill P (2022) Key factors to consider in the use of environmental DNA metabarcoding to monitor terrestrial ecological restoration. The Science of the Total Environment 848: 157617. https://doi.org/10.1016/j.scitotenv.2022.157617
- Veilleux HD, Misutka MD, Glover CN (2021) Environmental DNA and environmental RNA: Current and prospective applications for biological monitoring. The Science of the Total Environment 782: 146891. https://doi.org/10.1016/j.scitotenv.2021.146891
- White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
- Wickham H (2016) ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag, New York [ISBN 978-3-319-24277-4].
- Wirta H, Abrego N, Miller K, Roslin T, Vesterinen E (2021) DNA traces the origin of honey by identifying plants, bacteria and fungi. Scientific Reports 11(1): 4798. https://doi.org/10.1038/s41598-021-84174-0
- Wood DE, Lu J, Langmead B (2019) Improved metagenomic analysis with Kraken 2. Genome Biology 20(1): 257. https://doi.org/10.1186/s13059-019-1891-0
- Xiong F, Shu L, Zeng H, Gan X, He S, Peng Z (2022) Methodology for fish biodiversity monitoring with environmental DNA metabarcoding: The primers, databases and bioinformatic pipelines. Water Biology and Security 1(1): 100007. https://doi.org/10.1016/j.watbs.2022.100007
- Yates MC, Derry AM, Cristescu ME (2021) Environmental RNA: A revolution in ecological resolution? Trends in Ecology & Evolution 36(7): 601–609. https://doi.org/10.1016/j.tree.2021.03.001