Acta Parasitologica

, Volume 58, Issue 1, pp 64–69 | Cite as

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

  • Aneta Strachecka
  • Grzegorz Borsuk
  • Krzysztof Olszewski
  • Jerzy Paleolog
  • Zbigniew Lipiński
Original Paper

Abstract

The aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method — as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.

Keywords

Proteolytic system proteins inhibitor activities acaracides Varroa destructor 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anson M. 1938. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology, 22, 79–84.PubMedCrossRefGoogle Scholar
  2. Bania J., Polanowski A. 1999. Bioinsecticides and insect defense mechanisms. Postępy Biochemii, 45, 143–150.PubMedGoogle Scholar
  3. Bode W., Fernandez-Catalan C., Nagase H., Maskos K. 1999. Endoproteinase — protein inhibitor interaction. Acta Pathologica, Microbiologica et Immunologica Scandinavica, 107, 3–10. DOI: 10.1111/j.1699-0463.1999.tb01520.x.CrossRefGoogle Scholar
  4. Colin M., Tchamitchian M., Bonmatin J., Pasquale S. 2001. Presence of chitinase in adult Varroa destructor, an ectoparasitic mite of Apis mellifera. Experimental and Applied Acarology, 25, 947–955. DOI: 10.1023/A:1020657906024.CrossRefGoogle Scholar
  5. Evans J.D., Aronstein K., Chen Y.P., Hetru C., Imler J.L., Jiang H., Kanost M., Thompson G.J., Zou Z., Hultmark D. 2006. Immune pathways and defence mechanisms in honey bee Apis mellifera. Insect Molecular Biology, 15, 645–656. DOI: 10.1111/j.1365-2583.2006.00682.x.PubMedCrossRefGoogle Scholar
  6. Felicioli R., Garzelli B., Vaccari L., Melfi D., Balestreri E. 1997. Activity staining of protein inhibitors of proteases on gelatincontaining polyacrylamide gel electrophoresis. Analytical Biochemistry, 244, 176–179. DOI: 10.1006/abio.1996.9917.PubMedCrossRefGoogle Scholar
  7. Frączek R., Żółtowska K., Lipiński Z. 2009. The activity of nineteen hydrolases in extracts from Varroa destructor and in hemolymph of Apis mellifera ceranica workers bees. Journal of Apicultural Science, 53, 43–51.Google Scholar
  8. Garedew A., Schmolz E., Lamprecht I. 2004. The energy and nutritional demand of the parasitic life of the mite Varroa destructor. Apidologie, 35, 419–430. DOI: 10.1051/apido:2004032.CrossRefGoogle Scholar
  9. Koch W., Ritter W. 1991. Experimental Examinations Concerning the Problem of Deformed Emerging Bees After Infestation with Varroa jacobsoni. Zentralblatt fur Veterinarmedizin B, 38, 337–344.Google Scholar
  10. Kralj J., Fuchs S. 2006. Parasitic Varroa destructor mites influence flight duration and homing ability of infested Apis mellifera foragers. Apidologie, 37, 577–587. DOI: 10.1051/apido:2006040.CrossRefGoogle Scholar
  11. Laemmli U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  12. Lee T., Lin Y. 1995. Trypsin inhibitor and trypsin — like protease activity in air — or submergence — grown rice (Oryza sativa L.) coleoptiles. Plant Science, 106, 43–54. DOI: 10.1016/0168- 9452(95)04058-3.CrossRefGoogle Scholar
  13. Lima P.R.M., Brochetto-Braga M.R., Chaud-Netto J. 2000. Proteolytic activity of Africanized honeybee (Apis mellifera: hymenoptera, apidae) venom. Journal of Venomous Animals and Toxins, 6, 104–113. DOI: 10.1590/S0104-79302000000100004.CrossRefGoogle Scholar
  14. Malone L.A., Todd J.H., Burgess E., Christeller J.T. 2004. Development of hypopharyngeal glands in adult honey bees fed with a Bt toxin, a biotin-binding protein and a protease inhibitor. Apidologie, 35, 655–664. DOI: 10.1051/apido:2004063.CrossRefGoogle Scholar
  15. Mathieu L., Faucon J.P. 2000. Changes in the response time for Varroa jacobsoni exposed to amitraz. Journal of Apicultural Research, 39, 155–158.Google Scholar
  16. Milani N. 1995. The resistance of Varroa jacobsoni Oud. to pyrethroids a laboratory assay. Apidologie, 26, 415–429. DOI:10.1051/apido:19950507.CrossRefGoogle Scholar
  17. Mira A. 2000. Exuviae eating: a nitrogen meal? Journal of Insect Physiology, 46, 605–610. DOI: 10.1016/S0022-1910(99)00 146-8.PubMedCrossRefGoogle Scholar
  18. Roberts T., Hutson D. 1999. Tau-fluvalinate. Metabolic Pathways of Agrochemicals. Part Two: Insecticides and Fungicides, 670–677.Google Scholar
  19. Romero-Vera C., Otero-Colina G. 2002. Effect of single and successive infestation of Varroa destructor and Acarapis woodi on the longevity of worker honey bees Apis mellifera. American Bee Journal, 142, 54–57.Google Scholar
  20. Salvy M., Martin C., Bagneres A., Provost E., Roux M., Le Conte Y., Clement J. 2001. Modifications of the cuticular hydrocarbon profile of Apis mellifera worker bees in the presence of the ectoparasitic mite Varroa jacobsoni in brood cells. Parasitology, 122, 145–159.PubMedCrossRefGoogle Scholar
  21. SAS Institute. 2002–2003. SAS/STAT User’s Guide release 9.13, Cary, NC, Statistical Analysis System Institute, license 86636.Google Scholar
  22. Schacterle G., Pollack R. 1973. Simplified method for quantitative assay of small amounts of protein in biological material. Analytical Biochemistry, 51, 654–655.PubMedCrossRefGoogle Scholar
  23. Schneider P., Drescher W. 1987. The influence of Varroa jacobsoni Oud. on weight; development on weight and hypopharyngeal glands; and longevity of Apis mellifera L. Apidologie, 18, 101–110.CrossRefGoogle Scholar
  24. Schoofs L., Salzet M. 2002. Trypsin and chymotrypsin inhibitors in insects and gut leeches. Current Pharmaceutical Design, 8, 125–133. DOI: 1381-6128/02S35.00.00.CrossRefGoogle Scholar
  25. Strachecka A. 2010. Proteazy i inhibitory proteaz na powierzchni ciaŁa pszczoŁy miodnej (Apis mellifera) jako element odporności nieswoistej. Rozprawa doktorska (Proteases and protease inhibitors on the body surface of the honey bee (Apis mellifera) as an element of non-specific resistance). UMCS in Lublin, 24–25.Google Scholar
  26. Strachecka A., Gryzińska M., Krauze M., Grzywnowicz K. 2011. Profile of the Body Surface Proteolytic System in Apis mellifera Queens. Czech Journal of Animal Science, 56, 15–22.Google Scholar
  27. Strachecka A., Paleolog J., Grzywnowicz K. 2008. The surface proteolytic activity in Apis mellifera. Journal of Apicultural Science, 52, 49–56.Google Scholar
  28. Tewarson N., Engles W. 1982. Undigested uptake of non-host proteins by Varroa jacobsoni. Journal of Apicultural Research, 21, 222–225.Google Scholar
  29. Terra W., Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology, 109B, 1–62, DOI: 10.1016/0305-0491 (94)90141-4.Google Scholar
  30. Watkins M. 1997. Resistance and its relevance to beekeeping. Bee World, 78, 15–22.Google Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2013

Authors and Affiliations

  • Aneta Strachecka
    • 1
  • Grzegorz Borsuk
    • 1
  • Krzysztof Olszewski
    • 1
  • Jerzy Paleolog
    • 1
  • Zbigniew Lipiński
    • 2
  1. 1.Department of Biological Basis of Animal Production, Faculty of Biology and Animal BreedingUniversity of Life Sciences in LublinLublinPoland
  2. 2.Institute of Animal Reproduction and Food Research of Polish Academy of SciencesOlsztynPoland

Personalised recommendations