Microbial Ecology

, Volume 78, Issue 2, pp 506–516 | Cite as

Floral and Foliar Source Affect the Bee Nest Microbial Community

  • Jason A. Rothman
  • Corey Andrikopoulos
  • Diana Cox-FosterEmail author
  • Quinn S. McFrederickEmail author
Invertebrate Microbiology


Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.


Solitary bees Microbial communities ITS sequencing 16S rRNA gene sequencing 



The authors would like to thank the UC Riverside Genomics Core facility staff for their Next-Generation Sequencing expertise.

Funding Information

This research was supported by fellowships from the National Aeronautics and Space Administration MIRO Fellowships in Extremely Large Data Sets (Award No: NNX15AP99A) and the United States Department of Agriculture National Institute of Food and Agriculture (NIFA) Predoctoral Fellowship (Award No. 2018-67011-28123) to Jason A. Rothman. Funds were also awarded to Quinn S. McFrederick from the Alfalfa Pollinator Research Initiative and NIFA Hatch (Award No. CA-R-ENT-5109-H).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

248_2018_1300_MOESM1_ESM.docx (2.5 mb)
ESM 1 (DOCX 2585 kb)
248_2018_1300_MOESM2_ESM.xlsx (789 kb)
ESM 2 (XLSX 788 kb)


  1. 1.
    Klein A-M, Vaissière BE, Cane JH et al (2007) Importance of pollinators in changing landscapes for world crops. Proc Biol Sci 274:303–313. CrossRefGoogle Scholar
  2. 2.
    Calderone NW (2012) Insect pollinated crops, insect pollinators and us agriculture: trend analysis of aggregate data for the period 1992–2009. PLoS One 7:e37235. CrossRefGoogle Scholar
  3. 3.
    Pitts-Singer TL, Cane JH (2011) The alfalfa leafcutting bee, Megachile rotundata: the world’s most intensively managed solitary bee. Annu Rev Entomol 56:221–237. CrossRefGoogle Scholar
  4. 4.
    Michener CD (2000) The bees of the world. Johns Hopkins University Press, BaltimoreGoogle Scholar
  5. 5.
    Powell JE, Martinson VG, Urban-Mead K, Moran NA (2014) Routes of acquisition of the gut microbiota of the honey bee Apis mellifera. Appl Environ Microbiol 80:7378–7387. CrossRefGoogle Scholar
  6. 6.
    Anderson KE, Rodrigues PAP, Mott BM, Maes P, Corby-Harris V (2015) Ecological succession in the honey bee gut: shift in Lactobacillus strain dominance during early adult development. Microb Ecol 71:1008–1019. CrossRefGoogle Scholar
  7. 7.
    Kwong WK, Medina LA, Koch H, Sing KW, Soh EJY, Ascher JS, Jaffé R, Moran NA (2017) Dynamic microbiome evolution in social bees. Sci Adv 3:e1600513. CrossRefGoogle Scholar
  8. 8.
    Martinson VG, Danforth BN, Minckley RL et al (2011) A simple and distinctive microbiota associated with honey bees and bumble bees. Mol Ecol 20:619–628. CrossRefGoogle Scholar
  9. 9.
    McFrederick QS, Thomas JM, Neff JL et al (2017) Flowers and wild megachilid bees share microbes. Microb Ecol 73:188–200. CrossRefGoogle Scholar
  10. 10.
    McFrederick QS, Cannone JJ, Gutell RR et al (2013) Specificity between lactobacilli and hymenopteran hosts is the exception rather than the rule. Appl Environ Microbiol 79:1803–1812. CrossRefGoogle Scholar
  11. 11.
    McFrederick QS, Vuong HQ, Rothman JA (2018) Lactobacillus micheneri sp. nov., Lactobacillus timberlakei sp. nov. and Lactobacillus quenuiae sp. nov., lactic acid bacteria isolated from wild bees and flowers. Int J Syst Evol Microbiol 68:1879–1884. CrossRefGoogle Scholar
  12. 12.
    Keller A, Grimmer G, Steffan-Dewenter I (2013) Diverse microbiota identified in whole intact nest chambers of the red mason bee Osmia bicornis (Linnaeus 1758). PLoS One 8:e78296. CrossRefGoogle Scholar
  13. 13.
    McFrederick QS, Wcislo WT, Taylor DR et al (2012) Environment or kin: whence do bees obtain acidophilic bacteria? Mol Ecol 21:1754–1768. CrossRefGoogle Scholar
  14. 14.
    Lozo J, Berić T, Terzić-Vidojević A, Stanković S, Fira D, Stanisavljević L (2015) Microbiota associated with pollen, bee bread, larvae and adults of solitary bee Osmia cornuta (Hymenoptera: Megachilidae). Bull Entomol Res 105:470–476. CrossRefGoogle Scholar
  15. 15.
    Rothman JA, Carroll MJ, Meikle WG, Anderson KE, McFrederick QS (2018) Longitudinal effects of supplemental forage on the honey bee (Apis mellifera) microbiota and inter- and intra-colony variability. Microb Ecol 76:814–824. CrossRefGoogle Scholar
  16. 16.
    Jones JC, Fruciano C, Hildebrand F, al Toufalilia H, Balfour NJ, Bork P, Engel P, Ratnieks FLW, Hughes WOH (2018) Gut microbiota composition is associated with environmental landscape in honey bees. Ecol Evol 8:441–451. CrossRefGoogle Scholar
  17. 17.
    Aizenberg-Gershtein Y, Izhaki I, Halpern M (2013) Do honeybees shape the bacterial community composition in floral nectar? PLoS One 8:e67556. CrossRefGoogle Scholar
  18. 18.
    Ushio M, Yamasaki E, Takasu H, Nagano AJ, Fujinaga S, Honjo MN, Ikemoto M, Sakai S, Kudoh H (2015) Microbial communities on flower surfaces act as signatures of pollinator visitation. Sci Rep 5:8695. CrossRefGoogle Scholar
  19. 19.
    Engel P, Kwong WK, McFrederick QS, Anderson KE, Barribeau SM, Chandler JA, Cornman RS, Dainat J, de Miranda JR, Doublet V, Emery O, Evans JD, Farinelli L, Flenniken ML, Granberg F, Grasis JA, Gauthier L, Hayer J, Koch H, Kocher S, Martinson VG, Moran N, Munoz-Torres M, Newton I, Paxton RJ, Powell E, Sadd BM, Schmid-Hempel P, Schmid-Hempel R, Song SJ, Schwarz RS, vanEngelsdorp D, Dainat B (2016) The bee microbiome: impact on bee health and model for evolution and ecology of host-microbe interactions. MBio 7:e02164–e02115. Google Scholar
  20. 20.
    Tehel A, Brown MJF, Paxton RJ (2016) Impact of managed honey bee viruses on wild bees. Curr Opin Virol 19:16–22. CrossRefGoogle Scholar
  21. 21.
    Graystock P, Blane EJ, McFrederick QS et al (2016) Do managed bees drive parasite spread and emergence in wild bees? Int J Parasitol Parasites Wildl 5:64–75. CrossRefGoogle Scholar
  22. 22.
    Singh R, Levitt AL, Rajotte EG, Holmes EC, Ostiguy N, vanEngelsdorp D, Lipkin WI, dePamphilis CW, Toth AL, Cox-Foster DL (2010) RNA viruses in hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PLoS One 5:e14357. CrossRefGoogle Scholar
  23. 23.
    Foley K, Fazio G, Jensen AB, Hughes WOH (2012) Nutritional limitation and resistance to opportunistic Aspergillus parasites in honey bee larvae. doi:
  24. 24.
    Hedtke SM, Blitzer EJ, Montgomery GA, Danforth BN (2015) Introduction of non-native pollinators can lead to trans-continental movement of bee-associated fungi. PLoS One 10:e0130560. CrossRefGoogle Scholar
  25. 25.
    Boomsma JJ, Jensen AB, Meyling NV, Eilenberg J (2014) Evolutionary interaction networks of insect pathogenic fungi. Annu Rev Entomol 59:467–485. CrossRefGoogle Scholar
  26. 26.
    Evison SE, Jensen AB (2018) The biology and prevalence of fungal diseases in managed and wild bees. Curr Opin Insect Sci 26:105–113. CrossRefGoogle Scholar
  27. 27.
    Antonovics J (2005) Plant venereal diseases: insights from a messy metaphor. New Phytol 165:71–80. CrossRefGoogle Scholar
  28. 28.
    Li JL, Cornman RS, Evans JD, Pettis JS, Zhao Y, Murphy C, Peng WJ, Wu J, Hamilton M, Boncristiani HF, Zhou L, Hammond J, Chen YP (2014) Systemic spread and propagation of a plant-pathogenic virus in European honeybees, Apis mellifera. MBio 5:e00898–e00813. Google Scholar
  29. 29.
    Card SD, Pearson MN, Clover GRG (2007) Plant pathogens transmitted by pollen. Australas Plant Pathol 36:455. CrossRefGoogle Scholar
  30. 30.
    Herrera CM, Pozo MI, Medrano M (2013) Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity. Ecology 94:273–279. CrossRefGoogle Scholar
  31. 31.
    Junker RR, Romeike T, Keller A, Langen D (2014) Density-dependent negative responses by bumblebees to bacteria isolated from flowers. Apidologie 45:467–477. CrossRefGoogle Scholar
  32. 32.
    Horne M (1995) Leaf area and toughness: effects on nesting material preferences of Megachile rotundata (Hymenoptera: Megachilidae). Ann Entomol Soc Am 88:868–875. CrossRefGoogle Scholar
  33. 33.
    Frankie G, Thorp R, Hernandez J et al (2009) Native bees are a rich natural resource in urban California gardens. Calif Agric 63:113–120CrossRefGoogle Scholar
  34. 34.
    Conrad A, Biehler D, Nobis T, Richter H, Engels I, Biehler K, Frank U (2013) Broad spectrum antibacterial activity of a mixture of isothiocyanates from nasturtium (Tropaeoli majoris herba) and horseradish (Armoraciae rusticanae radix). Drug Res (Stuttg) 63:65–68. CrossRefGoogle Scholar
  35. 35.
    Pulverer G (1969) Allyl isothiocyanate: a new broad-spectrum antibiotic from nasturtium. Ger Med Mon 14:27–30Google Scholar
  36. 36.
    Pitts-Singer TL, Bosch J (2010) Nest establishment, pollination efficiency, and reproductive success of Megachile rotundata (Hymenoptera: Megachilidae) in relation to resource availability in field enclosures. Environ Entomol 39:149–158. CrossRefGoogle Scholar
  37. 37.
    Engel P, James RR, Koga R, Kwong WK, McFrederick QS, Moran NA (2013) Standard methods for research on Apis mellifera gut symbionts. J Apic Res 52:1–24. CrossRefGoogle Scholar
  38. 38.
    Pennington MJ, Rothman JA, Jones MB, McFrederick QS, Gan J, Trumble JT (2017) Effects of contaminants of emerging concern on Megaselia scalaris (Lowe, Diptera: Phoridae) and its microbial community. Sci Rep 7:8165. CrossRefGoogle Scholar
  39. 39.
    Pennington MJ, Rothman JA, Jones MB, McFrederick QS, Gan J, Trumble JT (2018) Effects of contaminants of emerging concern on Myzus persicae (Sulzer, Hemiptera: Aphididae) biology and on their host plant, Capsicum annuum. Environ Monit Assess 190:125. CrossRefGoogle Scholar
  40. 40.
    McFrederick QS, Rehan SM (2016) Characterization of pollen and bacterial community composition in brood provisions of a small carpenter bee. Mol Ecol 25:2302–2311. CrossRefGoogle Scholar
  41. 41.
    Pennington MJ, Rothman JA, Dudley SL, Jones MB, McFrederick QS, Gan J, Trumble JT (2017) Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant. Proc Natl Acad Sci 114:E9923–E9931. CrossRefGoogle Scholar
  42. 42.
    Smith DP, Peay KG (2014) Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS One 9:e90234. CrossRefGoogle Scholar
  43. 43.
    McFrederick QS, Rehan SM (2018) Wild bee pollen usage and microbial communities co-vary across landscapes. Microb Ecol:1–10.
  44. 44.
    Kembel SW, O’Connor TK, Arnold HK et al (2014) Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest. Proc Natl Acad Sci U S A 111:13715–13720. CrossRefGoogle Scholar
  45. 45.
    Hanshew AS, Mason CJ, Raffa KF, Currie CR (2013) Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities. J Microbiol Methods 95:149–155. CrossRefGoogle Scholar
  46. 46.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. CrossRefGoogle Scholar
  47. 47.
    Callahan BJ, McMurdie PJ, Rosen MJ et al (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. CrossRefGoogle Scholar
  48. 48.
    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 6:90. CrossRefGoogle Scholar
  49. 49.
    Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. CrossRefGoogle Scholar
  50. 50.
    Abarenkov K, Henrik Nilsson R, Larsson K-H, Alexander IJ, Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Ursing BM, Vrålstad T, Liimatainen K, Peintner U, Kõljalg U (2010) The UNITE database for molecular identification of fungi—recent updates and future perspectives. New Phytol 186:281–285. CrossRefGoogle Scholar
  51. 51.
    Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87. CrossRefGoogle Scholar
  52. 52.
    Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. CrossRefGoogle Scholar
  53. 53.
    Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490CrossRefGoogle Scholar
  54. 54.
    Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, Collman RG, Bushman FD, Li H (2012) Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics 28:2106–2113. CrossRefGoogle Scholar
  55. 55.
    R Core Team (2018) R: A language and environment for statistical computing.Google Scholar
  56. 56.
    Oksanen J, Blanchet FG, Friendly M, et al (2017) vegan: Community Ecology PackageGoogle Scholar
  57. 57.
    Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. CrossRefGoogle Scholar
  58. 58.
    Wickham H (2009) ggplot2: elegant graphics for data analysisGoogle Scholar
  59. 59.
    Faust K, Raes J (2016) CoNet app: inference of biological association networks using Cytoscape. F1000Research 5:1519. CrossRefGoogle Scholar
  60. 60.
    Anderson MJ, Walsh DCI (2013) PERMANOVA, ANOSIM, and the Mantel test in the face of heterogeneous dispersions: what null hypothesis are you testing? Ecol Monogr 83:557–574. CrossRefGoogle Scholar
  61. 61.
    Thomma BPHJ (2003) Alternaria spp.: from general saprophyte to specific parasite. Mol Plant Pathol 4:225–236. CrossRefGoogle Scholar
  62. 62.
    McFrederick QS, Mueller UG, James RR (2014) Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut. Proc R Soc B Biol Sci 281:20132653. CrossRefGoogle Scholar
  63. 63.
    Takamatsu S, Matsuda S, Grigaliunaite B (2013) Comprehensive phylogenetic analysis of the genus Golovinomyces (Ascomycota: Erysiphales) reveals close evolutionary relationships with its host plants. Mycologia 105:1135–1152. CrossRefGoogle Scholar
  64. 64.
    Bates ST, Reddy GSN, Garcia-Pichel F (2006) Exophiala crusticola anam. nov. (affinity Herpotrichiellaceae), a novel black yeast from biological soil crust in the Western United States. Int J Syst Evol Microbiol 56:2697–2702. CrossRefGoogle Scholar
  65. 65.
    Chen C, Verkley GJM, Sun G, Groenewald JZ (2016) Redefining common endophytes and plant pathogens in Neofabraea, Pezicula, and related genera. Fungal Biol-UK 120:1291–1322. CrossRefGoogle Scholar
  66. 66.
    Martínez-Espinoza AD, García-Pedrajas MD, Gold SE (2002) The Ustilaginales as plant pests and model systems. Fungal Genet Biol 35:1–20. CrossRefGoogle Scholar
  67. 67.
    Woudenberg JHC, Hanse B, van Leeuwen GCM, Groenewald JZ, Crous PW (2017) Stemphylium revisited. Stud Mycol 87:77–103. CrossRefGoogle Scholar
  68. 68.
    Žabka M, Drastichová K, Jegorov A, Soukupová J, Nedbal L (2006) Direct evidence of plant-pathogenic activity of fungal metabolites of Trichothecium roseum on apple. Mycopathologia 162:65–68. CrossRefGoogle Scholar
  69. 69.
    Rosa CA, Lachance MA, Silva JOC et al (2003) Yeast communities associated with stingless bees. FEMS Yeast Res 4:271–275. CrossRefGoogle Scholar
  70. 70.
    DeGrandi-Hoffman G, Corby-Harris V, DeJong EW et al (2016) Honey bee gut microbial communities are robust to the fungicide Pristine® consumed in pollen. Apidologie 48:1–13. Google Scholar
  71. 71.
    Walterson AM, Stavrinides J (2015) Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiol Rev 39:968–984. CrossRefGoogle Scholar
  72. 72.
    Loncaric I, Heigl H, Licek E, Moosbeckhofer R, Busse HJ, Rosengarten R (2009) Typing of Pantoea agglomerans isolated from colonies of honey bees (Apis mellifera) and culturability of selected strains from honey. Apidologie 40:40–54. CrossRefGoogle Scholar
  73. 73.
    Anderson KE, Carroll MJ, Sheehan TH, Lanan MC, 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. Mol Ecol 23:5904–5917. CrossRefGoogle Scholar
  74. 74.
    Halpern M, Fridman S, Atamna-Ismaeel N, Izhaki I (2013) Rosenbergiella nectarea gen. nov., sp. nov., in the family Enterobacteriaceae, isolated from floral nectar. Int J Syst Evol Microbiol 63:4259–4265. CrossRefGoogle Scholar
  75. 75.
    Peeters N, Guidot A, Vailleau F, Valls M (2013) Ralstonia solanacearum , a widespread bacterial plant pathogen in the post-genomic era. Mol Plant Pathol 14:651–662. CrossRefGoogle Scholar
  76. 76.
    McArt SH, Koch H, Irwin RE, Adler LS (2014) Arranging the bouquet of disease: floral traits and the transmission of plant and animal pathogens. Ecol Lett 17:624–636. CrossRefGoogle Scholar
  77. 77.
    Richardson LL, Adler LS, Leonard AS, Andicoechea J, Regan KH, Anthony WE, Manson JS, Irwin RE (2015) Secondary metabolites in floral nectar reduce parasite infections in bumblebees. Proc Biol Sci 282:20142471. CrossRefGoogle Scholar
  78. 78.
    Stange R, Schneider B, Albrecht U, Mueller V, Schnitker J, Michalsen A (2017) Results of a randomized, prospective, double-dummy, double-blind trial to compare efficacy and safety of a herbal combination containing Tropaeoli majoris herba and Armoraciae rusticanae radix with co-trimoxazole in patients with acute and uncomplicated cystitis. Res Rep Urol 9:43–50. Google Scholar
  79. 79.
    Shykoff JA, Bucheli E (1995) Pollinator visitation patterns, floral rewards and the probability of transmission of Microbotryum violaceum, a veneral disease of plants. J Ecol 83:189. CrossRefGoogle Scholar
  80. 80.
    Batra LR, Batra SWT (1985) Floral mimicry induced by mummy-berry fungus exploits host’s pollinators as vectors. Science 228:1011–1013. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduate Program in MicrobiologyUniversity of CaliforniaRiversideUSA
  2. 2.Department of EntomologyUniversity of CaliforniaRiversideUSA
  3. 3.Department of BiologyUtah State UniversityLoganUSA
  4. 4.USDA-ARS Pollinating Insect-Biology, Management, and Systematics ResearchLoganUSA

Personalised recommendations