1887

Abstract

Meliponiculture – the management of stingless bee colonies – is an expanding activity in Brazil with economic, social and environmental potential. However, unlike in apiculture, the pathogens that impact on meliponiculture remain largely unknown. In southern Brazil, every year at the end of the summer, managed colonies of the stingless bee Melipona quadrifasciata manifest a syndrome that eventually leads to collapse. Here we characterize the M. quadrifasciata virome using high-throughput sequencing, with the aim of identifying potentially pathogenic viruses, and test whether they are related to the syndrome outbreaks. Two paired viromes are explored, one from healthy bees and another from unhealthy ones. Each virome is built from metagenomes assembled from sequencing reads derived either from RNA or DNA. A total of 40 621 reads map to viral contigs of the unhealthy bees’ metagenomes, whereas only 11 reads map to contigs identified as viruses of healthy bees. The viruses showing the largest copy numbers in the virome of unhealthy bees belong to the family Dicistroviridae – common pathogenic honeybee viruses – as well as Parvoviridae and Circoviridae, which have never been reported as being pathogenic in insects. Our analyses indicate that they represent seven novel viruses associated with stingless bees. PCR-based detection of these viruses in individual bees (healthy or unhealthy) from three different localities revealed a statistically significant association between viral infection and symptom manifestation in one meliponary. We conclude that although viral infections may contribute to colony collapses in the annual syndrome in some meliponaries, viruses spread opportunistically during the outbreak, perhaps due to colony weakness.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001273
2019-06-06
2019-08-22
Loading full text...

Full text loading...

References

  1. Villas-Bôas J. Manual Tecnológico: Mel de Abelhas sem Ferrão Instituto Sociedade, População e Natureza; 2012; p96
    [Google Scholar]
  2. de Melo GAR. Catálogo Moure. 2013;http://www.moure.cria.org.br/catalogue 11 May 2018
  3. Jaffé R, Pope N, Carvalho AT, Maia UM, Blochtein B et al. Bees for development: Brazilian survey reveals how to optimize stingless beekeeping. PLoS One 2015;10:e0121157 [CrossRef]
    [Google Scholar]
  4. Freitas BM, Imperatriz-Fonseca VL, Medina LM, Kleinert AdeMP, Galetto L et al. Diversity, threats and conservation of native bees in the Neotropics. Apidologie 2009;40:332–346 [CrossRef]
    [Google Scholar]
  5. Blochtein B, Marques BH. Himenópteros In Livro Vermelho Da Fauna Ameaçada de Extinção No Rio Grande Do Sul Porto Alegre: EDIPUCRS; 2003; pp95–109
    [Google Scholar]
  6. Fundação Zoobotânica do Rio Grande do 2014; Avaliação do Estado de Conservação de Espécies Fauna. https://secweb.procergs.com.br/livlof/?id_modulo=1&id_uf=23&ano=2012 15 May 2018
  7. Brosi BJ, Delaplane KS, Boots M, De Roode JC. Ecological and evolutionary approaches to managing honeybee disease. Nat Ecol Evol 2017;1:1250–1262 [CrossRef]
    [Google Scholar]
  8. Díaz S, de Souza Urbano S, Caesar L, Blochtein B, Sattler A et al. Report on the microbiota of Melipona quadrifasciata affected by a recurrent disease. J Invertebr Pathol 2017;143:35–39 [CrossRef]
    [Google Scholar]
  9. Kwong WK, Moran NA. Gut microbial communities of social bees. Nat Rev Microbiol 2016;14:374–384 [CrossRef]
    [Google Scholar]
  10. Message D, Teixeira É, Jong D. Situação da sanidade das abelhas no Brasil In Polinizadores no Brasil: contribuição e perspectivas para a biodiversidade, uso sustentável, conservação e serviços ambientais São Paulo: Edusp; 2012; pp237–256
    [Google Scholar]
  11. Teixeira EW, Chen Y, Message D, Pettis J, Evans JD. Virus infections in Brazilian honey bees. J Invertebr Pathol 2008;99:117–119 [CrossRef]
    [Google Scholar]
  12. Teixeira EW, Chen YP, Message D, Boncristiani HF, Pettis JS et al. Israeli acute paralysis virus in Africanized honey bees in southeastern Brazilian Apiaries. J Apic Res 2012;51:282–284 [CrossRef]
    [Google Scholar]
  13. Bailey L, Ball BV. Honey Bee Pathology London, UK: Academic Press; 1991; p208
    [Google Scholar]
  14. Chen YP, Higgins JA, Feldlaufer MF. Quantitative real-time reverse transcription-PCR analysis of deformed wing virus infection in the honeybee (Apis mellifera L.). Appl Environ Microbiol 2005;71:436–441 [CrossRef]
    [Google Scholar]
  15. Brettell LE, Mordecai GJ, Schroeder DC, Jones IM, da Silva JR et al. A comparison of Deformed wing virus in deformed and asymptomatic honey bees. Insects 2017;8:28 [CrossRef]
    [Google Scholar]
  16. Benaets K, Van Geystelen A, Cardoen D, De Smet L, de Graaf DC et al. Covert deformed wing virus infections have long-term deleterious effects on honeybee foraging and survival. Proc Biol Sci 2017;284:20162149 [CrossRef]
    [Google Scholar]
  17. de Miranda JR, Cordoni G, Budge G. The acute bee paralysis virus-Kashmir bee virus-Israeli acute paralysis virus complex. J Invertebr Pathol 2010;103:S30–S47 [CrossRef]
    [Google Scholar]
  18. Bailey L, Woods RD. Two more small RNA viruses from honey bees and further observations on sacbrood and acute bee-paralysis viruses. J Gen Virol 1977;37:175–182 [CrossRef]
    [Google Scholar]
  19. Bonning BC, Miller WA. Dicistroviruses. Annu Rev Entomol 2010;55:129–150 [CrossRef]
    [Google Scholar]
  20. Christian PD, Scotti PD. Picorna-like viruses of insects In The Insect Viruses New York: Plenum Press; 1998; pp301–.336
    [Google Scholar]
  21. Bailey L, Gibbs AJ, Woods RD. Two viruses from adult honey bees (Apis mellifera Linnaeus). Virology 1963;21:390–395 [CrossRef]
    [Google Scholar]
  22. Maori E, Lavi S, Mozes-Koch R, Gantman Y, Peretz Y et al. Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination. J Gen Virol 2007;88:3428–3438 [CrossRef]
    [Google Scholar]
  23. Boncristiani HF, Evans JD, Chen Y, Pettis J, Murphy C et al. In vitro infection of pupae with Israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (Apis mellifera). PLoS One 2013;8:e73429 [CrossRef]
    [Google Scholar]
  24. McMahon DP, Fürst MA, Caspar J, Theodorou P, Brown MJF et al. A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees. J Anim Ecol 2015;84:615–624 [CrossRef]
    [Google Scholar]
  25. Wilfert L, Long G, Leggett HC, Schmid-Hempel P, Butlin R et al. Deformed wing virus is a recent global epidemic in honeybees driven by Varroa mites. Science 2016;351:594–597 [CrossRef]
    [Google Scholar]
  26. Tehel A, Brown MJ, Paxton RJ. Impact of managed honey bee viruses on wild bees. Curr Opin Virol 2016;19:16–22 [CrossRef]
    [Google Scholar]
  27. Ueira-Vieira C, Almeida LO, de Almeida FC, Amaral IMR, Brandeburgo MAM et al. Scientific note on the first molecular detection of the acute bee paralysis virus in Brazilian stingless bees. Apidologie 2015;46:628–630 [CrossRef]
    [Google Scholar]
  28. Brutscher LM, McMenamin AJ, Flenniken ML. The buzz about honey bee viruses. PLoS Pathog 2016;12:e1005757 [CrossRef]
    [Google Scholar]
  29. Nazzi F, Le Conte Y. Ecology of Varroa destructor, the major ectoparasite of the Western honey bee, Apis mellifera. Annu Rev Entomol 2016;61:417–432 [CrossRef]
    [Google Scholar]
  30. Mordecai GJ, Wilfert L, Martin SJ, Jones IM, Schroeder DC. Diversity in a honey bee pathogen: first report of a third master variant of the deformed wing virus quasispecies. ISME J 2016
    [Google Scholar]
  31. Ryabov EV, Childers AK, Chen Y, Madella S, Nessa A et al. Recent spread of Varroa destructor virus-1, a honey bee pathogen, in the United States. Sci Rep 2017;7: [CrossRef]
    [Google Scholar]
  32. Singh R, Levitt AL, Rajotte EG, Holmes EC, Ostiguy N et al. RNA viruses in hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PLoS One 2010;5:e14357 [CrossRef]
    [Google Scholar]
  33. Gisder S, Genersch E. Special issue: honey bee viruses. Viruses 2015;7:5603–5608 [CrossRef]
    [Google Scholar]
  34. Schoonvaere K, Smagghe G, Francis F, de Graaf DC. Study of the metatranscriptome of eight social and solitary wild bee species reveals novel viruses and bee parasites. Front Microbiol 2018;9: [CrossRef]
    [Google Scholar]
  35. Galbraith DA, Fuller ZL, Ray AM, Brockmann A, Frazier M et al. Investigating the viral ecology of global bee communities with high-throughput metagenomics. Sci Rep 2018;8: [CrossRef]
    [Google Scholar]
  36. Remnant EJ, Shi M, Buchmann G, Blacquière T, Holmes EC et al. A diverse range of novel RNA viruses in geographically distinct honey bee populations. J Virol 2017;91: [CrossRef]
    [Google Scholar]
  37. Tentcheva D, Gauthier L, Zappulla N, Dainat B, Cousserans F et al. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl Environ Microbiol 2004;70:7185–7191 [CrossRef]
    [Google Scholar]
  38. Bull JC, Ryabov EV, Prince G, Mead A, Zhang C et al. A strong immune response in young adult honeybees masks their increased susceptibility to infection compared to older bees. PLoS Pathog 2012;8:e1003083 [CrossRef]
    [Google Scholar]
  39. DeGrandi-Hoffman G, Chen Y. Nutrition, immunity and viral infections in honey bees. Curr Opin Insect Sci 2015;10:170–176 [CrossRef]
    [Google Scholar]
  40. Dolezal AG, Hendrix SD, Scavo NA, Carrillo-Tripp J, Harris MA et al. Honey bee viruses in wild bees: viral prevalence, loads, and experimental inoculation. Plos One 2016;11:e0166190 [CrossRef]
    [Google Scholar]
  41. Daughenbaugh KF, Martin M, Brutscher LM, Cavigli I, Garcia E et al. Honey bee infecting lake Sinai viruses. Viruses 2015;7:3285–3309 [CrossRef]
    [Google Scholar]
  42. Potts SG, Biesmeijer JC, Kremen C, Neumann P, Schweiger O et al. Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol 2010;25:345–353 [CrossRef]
    [Google Scholar]
  43. Brown MJF, Paxton RJ. The conservation of bees: a global perspective. Apidologie 2009;40:410–416 [CrossRef]
    [Google Scholar]
  44. Dainat B, Evans JD, Chen YP, Gauthier L, Neumann P. Predictive markers of honey bee colony collapse. PLoS One 2012;7:e32151 [CrossRef]
    [Google Scholar]
  45. Berthoud H, Imdorf A, Haueter M, Radloff S, Neumann P. Virus infections and winter losses of honey bee colonies (Apis mellifera). J Apic Res 2010;49:60–65 [CrossRef]
    [Google Scholar]
  46. Highfield AC, El Nagar A, Mackinder LCM, Noël LM, Hall MJ et al. Deformed wing virus implicated in overwintering honeybee colony losses. Appl Environ Microbiol 2009;75:7212–7220 [CrossRef]
    [Google Scholar]
  47. vanEngelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E et al. Colony collapse disorder: a descriptive study. PLoS One 2009;4:e6481 [CrossRef]
    [Google Scholar]
  48. Cox-foster DL, Conlan S, Holmes EC, Palacios G, Evans JD et al. A metagenomic survey of collapse disorder. Science 2007;318:283–287
    [Google Scholar]
  49. Cornman RS, Tarpy DR, Chen Y, Jeffreys L, Lopez D et al. Pathogen webs in collapsing honey bee colonies. PLoS One 2012;7:e43562 [CrossRef]
    [Google Scholar]
  50. Goulson D, Nicholls E, Botías C, Rotheray EL. Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science 2015;347:1255957 [CrossRef]
    [Google Scholar]
  51. Heard TA. The role of stingless bees in crop pollination. Annu Rev Entomol 1999;44:183–206 [CrossRef]
    [Google Scholar]
  52. de Souza FS, Kevill JL, Correia-Oliveira ME, de Carvalho CAL, Martin SJ. Occurrence of deformed wing virus variants in the stingless bee melipona subnitida and honey bee Apis mellifera populations in Brazil. J Gen Virol 2019;100:289–294 [CrossRef]
    [Google Scholar]
  53. Xu P, Liu Y, Graham RI, Wilson K, Wu K. Densovirus is a mutualistic symbiont of a global crop Pest (Helicoverpa armigera) and protects against a baculovirus and Bt Biopesticide. PLoS Pathog 2014;10:e1004490 [CrossRef]
    [Google Scholar]
  54. Dhar AK, Robles-Sikisaka R, Saksmerprome V, Lakshman DK. Biology, genome organization, and evolution of parvoviruses in marine shrimp. Adv Virus Res 2014;89:85-139 [CrossRef]
    [Google Scholar]
  55. Szelei J, Woodring J, Goettel MS, Duke G, Jousset FX et al. Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics. J Invertebr Pathol 2011;106:394–399 [CrossRef]
    [Google Scholar]
  56. Smits SL, Zijlstra EE, van Hellemond JJ, Schapendonk CME, Bodewes R et al. Novel cyclovirus in human cerebrospinal fluid, Malawi, 2010–2011. Emerg Infect Dis 2013;19: [CrossRef]
    [Google Scholar]
  57. Tan le V, van Doorn HR, Nghia HD, Chau TT, Tu le TP et al. Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections. MBio 2013;4:e00231–13 [CrossRef]
    [Google Scholar]
  58. Phan TG, Luchsinger V, Avendaño LF, Deng X, Delwart E. Cyclovirus in nasopharyngeal aspirates of Chilean children with respiratory infections. J Gen Virol 2014;95:922–927 [CrossRef]
    [Google Scholar]
  59. Hilgert-Moreira SB, Nascher CA, Callegari-Jacques SM, Blochtein B. Pollen resources and trophic niche breadth of Apis mellifera and Melipona obscurior (Hymenoptera, Apidae) in a subtropical climate in the Atlantic rain forest of southern Brazil. Apidologie 2014;45:129–141 [CrossRef]
    [Google Scholar]
  60. Ramalho M, Kleinert-Giovannini A, Imperatriz-Fonseca VL. Important bee plants for stingless bees (Melipona and Trigonini) and Africanized honeybees (Apis mellifera) in neotropical habitats: a review. Apidologie 1990;21:469–488 [CrossRef]
    [Google Scholar]
  61. Melo MA. Efeito de Apis mellifera Linnaeus, 1758 (Hymenoptera, Apidae) sobre a utilização de fontes de pólen por Melipona quadrifasciata Lepeletier, 1836 (Hymenoptera, Apidae) na região de Viçosa, MG. 2004; p. 70http://www.locus.ufv.br/bitstream/handle/123456789/9970/texto completo.pdf?sequence=1&isAllowed=y 10 Dec 2018
  62. Mazzei M, Carrozza ML, Luisi E, Forzan M, Giusti M et al. Infectivity of DWV associated to flower pollen: experimental evidence of a horizontal transmission route. PLoS One 2014;9:e113448 [CrossRef]
    [Google Scholar]
  63. Nazzi F, Brown SP, Annoscia D, Del Piccolo F, Di Prisco G et al. Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies. PLoS Pathog 2012;8:e1002735 [CrossRef]
    [Google Scholar]
  64. Di Prisco G, Cavaliere V, Annoscia D, Varricchio P, Caprio E et al. Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees. Proc Natl Acad Sci 2013;110:18466–18471 [CrossRef]
    [Google Scholar]
  65. Tritschler M, Vollmann JJ, Yañez O, Chejanovsky N, Crailsheim K et al. Protein nutrition governs within-host race of honey bee pathogens. Sci Rep 2017;7: [CrossRef]
    [Google Scholar]
  66. Evans JD, Spivak M. Socialized medicine: individual and communal disease barriers in honey bees. J Invertebr Pathol 2010
    [Google Scholar]
  67. Meunier J. Social immunity and the evolution of group living in insects. Philos Trans R Soc Lond B Biol Sci 2015;370:20140102 [CrossRef]
    [Google Scholar]
  68. Jones B, Shipley E, Arnold KE. Social immunity in honeybees-Density dependence, diet, and body mass trade-offs. Ecol Evol 2018;8:4852–4859 [CrossRef]
    [Google Scholar]
  69. de Sales Lima FE, Cibulski SP, Witt AA, Franco AC, Roehe PM. Genomic characterization of two novel polyomaviruses in Brazilian insectivorous bats. Arch Virol 2015;160:1831–1836 [CrossRef]
    [Google Scholar]
  70. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning : A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory; 1989; p1626
    [Google Scholar]
  71. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19:455–477 [CrossRef]
    [Google Scholar]
  72. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 2011;29:644–652 [CrossRef]
    [Google Scholar]
  73. MacManes MD. On the optimal trimming of high-throughput mRNA sequence data. Front Genet 2014;5: [CrossRef]
    [Google Scholar]
  74. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. blast+: architecture and applications. BMC Bioinformatics 2009;10:421 [CrossRef]
    [Google Scholar]
  75. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–2079 [CrossRef]
    [Google Scholar]
  76. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359 [CrossRef]
    [Google Scholar]
  77. Wickham H. ggplot2: Elegant Graphics for Data Analysis New York: Springer-Verlag; 2016
    [Google Scholar]
  78. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014;30:2068–2069 [CrossRef]
    [Google Scholar]
  79. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012;28:1647–1649 [CrossRef]
    [Google Scholar]
  80. Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 2011;27:1164–1165 [CrossRef]
    [Google Scholar]
  81. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014;30:1312–1313 [CrossRef]
    [Google Scholar]
  82. Rambaut A. FigTree v.1.4.2. 2014;http://tree.bio.ed.ac.uk/software/figtree/ 10 March 2018
  83. Abramson JH. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiol Perspect Innov 2011;8:1 [CrossRef]
    [Google Scholar]
  84. Erguler K. Barnard: Barnard’s unconditional test. 2014;https://CRAN.R-project.org/package=Barnard 19 October 2018
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001273
Loading
/content/journal/jgv/10.1099/jgv.0.001273
Loading

Data & Media loading...

Supplementary material 1

PDF

Supplementary material 1

EXCEL

Supplementary material 1

EXCEL

Supplementary material 1

EXCEL

Supplementary material 1

EXCEL

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error