We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

Bee Research Institute at Dol Libcice nad Vltavou Czechia

CNR ISPA Agrofood Dept 1 73100 Lecce Italy

Crop Research Institute Prague 6 Czechia

Institute of Organic Chemistry and Biochemistry Prague 6 Czechia

Laboratory of Mass Spectrometry Charles University Prague Faculty of Science Prague 2 Czechia

See more in PubMed

Ho Y. P. & Reddy P. M. Identification of pathogens by mass spectrometry. Clin. Chem. 56, 525–536 (2010). PubMed PMC

Calderaro A. et al.. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification. Sci. Rep. 4, 6803; 10.1038/srep06803 (2014). PubMed DOI PMC

Cornman S. R. et al.. Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera. BMC Genomics 11, 602; 10.1186/1471-2164-11-602 (2010). PubMed DOI PMC

Le Conte Y., Ellis M. & Ritter W. Varroa mites and honey bee health: can Varroa explain part of the colony losses? Apidologie 41, 353–363 (2010).

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

Dainat B., Evans J. D., Chen Y. P., Gauthier L. & Neumann P. Dead or alive: deformed wing virus and Varroa destructor reduce the life span of winter honeybees. Appl. Environ. Microbiol. 78, 981–987 (2012). PubMed PMC

Dainat B., Evans J. D., Chen Y. P., Gauthier L. & Neumann P. Predictive markers of honey bee colony collapse. PLoS ONE 7, e32151; 10.1371/journal.pone.0032151 (2012). PubMed DOI PMC

Dietemann V. et al.. Varroa destructor: research avenues towards sustainable control. J. Apic. Res. 51, 125–132 (2012).

Anderson D. L. & Trueman J. W. H. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp. Appl. Acarol. 24, 165–189 (2000). PubMed

Bowen-Walker P. L. & Gunn A. The effect of the ectoparasitic mite, Varroa destructor on adult worker honeybee (Apis mellifera) emergence weights, water, protein, carbohydrate, and lipid levels. Entomol. Exp. Appl. 101, 207–217 (2001).

Amdam G. V., Hartfelder K., Norberg K., Hagen A. & Omholt S. W. Altered physiology in worker honey bees (Hymenoptera: Apidae) infested with the mite Varroa destructor (Acari: Varroidae): a factor in colony loss during overwintering? J. Econ. Entomol. 97, 741–747 (2004). PubMed

Yang X. L. & Cox-Foster D. L. Impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification. Proc. Natl. Acad. Sci. USA 102, 7470–7475 (2005). PubMed PMC

Erban T., Petrova D., Harant K., Jedelsky P. L. & Titera D. Two-dimensional gel proteome analysis of honeybee, Apis mellifera, worker red-eye pupa hemolymph. Apidologie 45, 53–72 (2014).

vanEngelsdorp D. et al.. Colony Collapse Disorder: a descriptive study. PLoS ONE 4, e6481; 10.1371/journal.pone.0006481 (2009). PubMed DOI PMC

Woltedji D. et al.. Proteome analysis of hemolymph changes during the larval to pupal development stages of honeybee workers (Apis mellifera ligustica). J. Proteome Res. 12, 5189–5198 (2013). PubMed

Bogaerts A., Baggerman G., Vierstraete E., Schoofs L. & Verleyen P. The hemolymph proteome of the honeybee: gel-based or gel-free? Proteomics 9, 3201–3208 (2009). PubMed

Erban T., Jedelsky P. L. & Titera D. Two-dimensional proteomic analysis of honeybee, Apis mellifera, winter worker hemolymph. Apidologie. 44, 404–418 (2013).

Chan Q. W. T., Howes C. G. & Foster L. J. Quantitative comparison of caste differences in honeybee hemolymph. Mol. Cell. Proteomics 5, 2252–2262 (2006). PubMed

Bromenshenk J. J. et al.. Iridovirus and microsporidian linked to honey bee colony decline. PLoS ONE 5, e13181; 10.1371/journal.pone.0013181 (2010). PubMed DOI PMC

Foster L. J. Interpretation of data underlying the link between Colony Collapse Disorder (CCD) and an invertebrate iridescent virus. Mol. Cell. Proteomics 10, M110.006387; 10.1074/mcp.M110.006387 (2011). PubMed DOI PMC

Tokarz R., Firth C., Street C., Cox-Foster D. L. & Lipkin W. I. Lack of evidence for an association between Iridovirus and Colony Collapse Disorder. PLoS ONE 6, e21844; 10.1371/journal.pone.0021844 (2011). PubMed DOI PMC

Tian R. J. et al.. Biological fingerprinting analysis of the interactome of a kinase inhibitor in human plasma by a chemiproteomic approach. J. Chromatogr. A 1134, 134–142 (2006). PubMed

Erban T. Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields. PLoS ONE 6, e22860; 10.1371/journal.pone.0022860 (2011). PubMed DOI PMC

Permyakov E. & Kretsinger R. H. Calcium binding proteins. (John Wiley & Sons, 2011).

Bretana N. A. et al.. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses. PLoS ONE 7, e40694; 10.1371/journal.pone.0040694 (2012). PubMed DOI PMC

Lanzi G. et al.. Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L). J. Virol. 80, 4998–5009 (2006). PubMed PMC

Wang H. et al.. Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera). PLoS ONE 8, e74508; 10.1371/journal.pone.0074508 (2013). PubMed DOI PMC

Govan V. A., Leat N., Allsopp M. & Davison S. Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses. Virology 277, 457–463 (2000). PubMed

de Miranda J. R. et al.. Complete nucleotide sequence of Kashmir bee virus and comparison with acute bee paralysis virus. J. Gen. Virol. 85, 2263–2270 (2004). PubMed

McMenamin A. J. & Genersch E. Honey bee colony losses and associated viruses. Curr. Opin. Insect. Sci. 8, 121–129 (2015). PubMed

Kevan P. G., Hannan M. A., Ostiguy N. & Guzman-Novoa E. A summary of the Varroa-virus disease complex in honey bees. Am. Bee J. 146, 694–697 (2006).

de Miranda J. R., Gauthier L., Ribiere M. & Chen Y. P. Honey bee viruses and their effect on bee and colony health. In: eds. Sammataro D. & Yoder J. A. Honey bee colony health: challenges and sustainable solutions. (CRC Press, 2012). p. 71–102.

Ravoet J. et al.. Comprehensive bee pathogen screening in Belgium reveals Crithidia mellificae as a new contributory factor to winter mortality. PLoS ONE 8, e72443; 10.1371/journal.pone.0072443 (2013). PubMed DOI PMC

Ravoet J. et al.. Widespread occurrence of honey bee pathogens in solitary bees. J. Invertebr. Pathol. 122, 55–58 (2014). PubMed

Katsuma S. et al.. Novel Macula-like virus identified in Bombyx mori cultured cells. J. Virol. 79, 5577–5584 (2005). PubMed PMC

Iwanaga M. et al.. Infection study of Bombyx mori macula-like virus (BmMLV) using a BmMLV-negative cell line and an infectious cDNA clone. J. Virol. Methods 179, 316–324 (2012). PubMed

Fujiyuki T. et al.. Novel insect picorna-like virus identified in the brains of aggressive worker honeybees. J. Virol. 78, 1093–1100 (2004). PubMed PMC

Ongus J. R. et al.. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J. Gen. Virol. 85, 3747–3755 (2004). PubMed

de Miranda J. R. & Genersch E. Deformed wing virus. J. Invertebr. Pathol. 103, S48–S61 (2010). PubMed

Ryabov E. V. et al.. A virulent strain of deformed wing virus (DWV) of honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission. PLoS Pathogens 10, e1004230; 10.1371/journal.ppat.1004230 (2014). PubMed DOI PMC

Martin S., Hogarth A., van Breda J. & Perrett J. A scientific note on Varroa jacobsoni Oudemans and the collapse of Apis mellifera L. colonies in the United Kingdom. Apidologie 29, 369–370 (1998).

Zhang Q. S. et al.. Detection and localisation of picorna-like virus particles in tissues of Varroa destructor, an ectoparasite of the honey bee, Apis mellifera. J. Invertebr. Pathol. 96, 97–105 (2007). PubMed

Yue C. & Genersch E. RT-PCR analysis of deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor). J. Gen. Virol. 86, 3419–3424 (2005). PubMed

Tentcheva D. et al.. Polymerase Chain Reaction detection of deformed wing virus (DWV) in Apis mellifera and Varroa destructor. Apidologie 35, 431–439 (2004).

Tentcheva D. et al.. Comparative analysis of deformed wing virus (DWV) RNA in Apis mellifera and Varroa destructor. Apidologie 37, 41–50 (2006).

Gisder S., Aumeier P. & Genersch E. Deformed wing virus: replication and viral load in mites (Varroa destructor). J. Gen. Virol. 90, 463–467 (2009). PubMed

Moore J. et al.. Recombinants between deformed wing virus and Varroa destructor virus-1 may prevail in Varroa destructor-infested honeybee colonies. J. Gen. Virol. 92, 156–161 (2011). PubMed

Bakonyi T., Farkas R., Szendroi A., Dobos-Kovacs M. & Rusvai M. Detection of acute bee paralysis virus by RT-PCR in honey bee and Varroa destructor field samples: rapid screening of representative Hungarian apiaries. Apidologie 33, 63–74 (2002).

Di Prisco G. et al.. Varroa destructor is an effective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera. J. Gen. Virol. 92, 151–155 (2011). PubMed

Chen Y. P., Pettis J. S., Evans J. D., Kramer M. & Feldlaufer M. F. Transmission of Kashmir bee virus by the ectoparasitic mite Varroa destructor. Apidologie 35, 441–448 (2004).

Shen M., Yang X., Cox-Foster D. & Cui L. The role of Varroa mites in infections of Kashmir bee virus (KBV) and deformed wing virus (DWV) in honey bees. Virology 342, 141–149 (2005). PubMed

Tentcheva D. et al.. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl. Environ. Microbiol. 70, 7185–7191 (2004). PubMed PMC

Camazine S. & Liu T. P. A putative iridovirus from the honey bee mite, Varroa jacobsoni Oudemans. J. Invertebr. Pathol. 71, 177–178 (1998). PubMed

Kleespies R. G., Radtke J. & Bienefeld K. Virus-like particles found in the ectoparasitic bee mite Varroa jacobsoni Oudemans. J. Invertebr. Pathol. 75, 87–90 (2000). PubMed

Voet D. & Voet J. G. Biochemistry. 3rd edition. (John Wiley & Sons, 2004). PubMed

Santillan-Galicia M. T., Carzaniga R., Ball B. V. & Alderson P. G. Immunolocalization of deformed wing virus particles within the mite Varroa destructor. J. Gen. Virol. 89, 1685–1689 (2008). PubMed

Cicero J. M. & Sammataro D. The salivary glands of adult female Varroa destructor (Acari: Varroidae), an ectoparasite of the honey bee, Apis mellifera (Hymenoptera: Apidae). Int. J. Acarol. 36, 377–386 (2010).

Bowen-Walker P. L., Martin S. J. & Gunn A. The transmission of deformed wing virus between honeybees (Apis mellifera L.) by the ectoparasitic mite Varroa jacobsoni Oud. J. Invertebr. Pathol. 73, 101–106 (1999). PubMed

Genersch E. & Aubert M. Emerging and re-emerging viruses of the honey bee (Apis mellifera L). Vet. Res. 41, 54; 10.1051/vetres/2010027 (2010). PubMed DOI PMC

Martin S. J. et al.. Global honey bee viral landscape altered by a parasitic mite. Science 336, 1304–1306 (2012). PubMed

Allen M. F., Ball B. V., White R. F. & Antoniw J. F. The detection of acute paralysis virus in Varroa jacobsoni by the use of a simple indirect ELISA. J. Apic. Res. 25, 100–105 (1986).

Ball B. V. & Allen M. F. The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni. Ann. Appl. Biol. 113, 237–244 (1988).

Nguyen B. K. et al.. Effects of honey bee virus prevalence, Varroa destructor load and queen condition on honey bee colony survival over the winter in Belgium. J. Apic. Res. 50, 195–202 (2011).

Shibuya N. & Nakashima N. Characterization of the 5′ internal ribosome entry site of Plautia stali intestine virus. J. Gen. Virol. 87, 3679–3686 (2006). PubMed

Bonning B. C. & Miller W. A. Dicistroviruses. Annu. Rev. Entomol. 55, 129–150 (2010). PubMed

Asgari S. & Johnson K. N. Insect virology. (Caister Academic Press, 2010).

Azzami K., Ritter W., Tautz J. & Beier H. Infection of honey bees with acute bee paralysis virus does not trigger humoral or cellular immune responses. Arch. Virol. 157, 689–702 (2012). PubMed PMC

Rossmann M. G. et al.. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317, 145–153 (1985). PubMed

Oberste M. S., Maher K., Kilpatrick D. R. & Pallansch M. A. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J. Virol. 73, 1941–1948 (1999). PubMed PMC

Mateu M. G. et al.. A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C. J. Gen. Virol. 71, 629–637 (1990). PubMed

Lochridge V. P. & Hardy M. E. A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization. J. Virol. 81, 12316–12322 (2007). PubMed PMC

Cheng S.-F., Huang Y.-P., Chen L.-H., Hsu Y.-H. & Tsai C.-H. Chloroplast phosphoglycerate kinase is involved in the targeting of Bamboo mosaic virus to chloroplasts in Nicotiana benthamiana plants. Plant Physiol. 163, 1598–1608 (2013). PubMed PMC

Hyodo K., Kaido M. & Okuno T. Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes. Front. Plant Sci. 5, 321; 10.3389/fpls.2014.00321 (2014). PubMed DOI PMC

Smith G. C. M. & Jackson S. P. The DNA-dependent protein kinase. Genes Dev. 13, 916–934 (1999). PubMed

Cooper A. et al.. HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration. Nature 498, 376–379 (2013). PubMed

Ritter W. Varroatosis, a new disease of the bee Apis mellifera. Anim. Res. Dev. 14, 17–35 (1981).

Glinski Z. & Jarosz J. Alterations in haemolymph proteins of drone honey bee larvae parasitized by Varroa jacobsoni. Apidologie 15, 329–338 (1984).

Danty E. et al.. Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera. Insect Biochem. Mol. Biol. 28, 387–397 (1998). PubMed

Fujita T. et al.. Proteomic analysis of the royal jelly and characterization of the functions of its derivation glands in the honeybee. J. Proteome Res. 12, 404–411 (2013). PubMed

Li R. et al.. Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland. BMC Genomics 14, 766; 10.1186/1471-2164-14-766 (2013). PubMed DOI PMC

Matysiak J., Hajduk J., Pietrzak L., Schmelzer C. E. H. & Kokot Z. J. Shotgun proteome analysis of honeybee venom using targeted enrichment strategies. Toxicon 90, 255–264 (2014). PubMed

Fraczek R., Zoltowska K. & Lipinski Z. The activity of nineteen hydrolases in extracts from Varroa destructor and in hemolymph of Apis mellifera carnica worker bees. J. Apic. Sci. 53, 43–51 (2009).

Fraczek R., Zoltowska K., Lipinski Z. & Dmitryjuk M. Proteolytic activity in the extracts and in the excretory/secretory products from Varroa destructor parasitic mite of honeybee. Int. J. Acarol. 38, 101–109 (2012).

Lopienska-Biernat E., Dmitryjuk M., Zaobidna E., Lipinski Z. & Zoltowska K. The body composition and enzymes of carbohydrate metabolism of Varroa destructor. J. Apic. Sci. 57, 93–100 (2013).

Dmitryjuk M., Zoltowska K., Fraczek R. & Lipinski Z. Esterases of Varroa destructor (Acari: Varroidae), parasitic mite of the honeybee. Exp. Appl. Acarol. 62, 499–510 (2014). PubMed

Zioni N., Soroker V. & Chejanovsky N. Replication of Varroa destructor virus 1 (VDV-1) and a Varroa destructor virus 1–deformed wing virus recombinant (VDV-1–DWV) in the head of the honey bee. Virology 417, 106–112 (2011). PubMed

Ren J. Y., Cone A., Willmot R. & Jones I. M. Assembly of recombinant Israeli acute paralysis virus capsids. PLoS ONE 9, e105943; 10.1371/journal.pone.0105943 (2014). PubMed DOI PMC

Cargile B. J., Bundy J. L., & Stephenson J. L. Jr. Potential for false positive identifications from large databases through tandem mass spectrometry. J. Proteome Res. 3, 1082–1085 (2004). PubMed

Searle B. C. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics 10, 1265–1269 (2010). PubMed

Erban T. & Stara J. Methodology for glutathione S-transferase purification and localization in two-dimensional gel electrophoresis performed on the pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae). J. Asia Pacific Entomol. 17, 369–373 (2014).

Altschul S. F., Gish W., Miller W., Myers E. W. & Lipman D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990). PubMed

Waterhouse A. M., Procter J. B., Martin D. M. A., Clamp M. & Barton G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009). PubMed PMC

Blom N., Gammeltoft S. & Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, 1351–1362 (1999). PubMed

Varroa destructor parasitism has a greater effect on proteome changes than the deformed wing virus and activates TGF-β signaling pathways

Changes in the Bacteriome of Honey Bees Associated with the Parasite Varroa destructor, and Pathogens Nosema and Lotmaria passim

Comparison of Varroa destructor and Worker Honeybee Microbiota Within Hives Indicates Shared Bacteria

Comparison of tau-fluvalinate, acrinathrin, and amitraz effects on susceptible and resistant populations of Varroa destructor in a vial test

Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases