Experimental & Applied Acarology

, Volume 36, Issue 3, pp 207–223 | Cite as

Host Blood Proteins and Peptides in the Midgut of the Tick Dermacentor variabilis Contributeto Bacterial Control

  • Daniel E. Sonenshine
  • Wayne L. Hynes
  • Shane M. Ceraul
  • Robert Mitchell
  • Tiffany Benzine


Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C18 Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit α-, β-7, γ-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit α- and β-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut’s response to microbial challenge differs from that of the hemolymph. In summary, the midgut’s antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins.


Antimicrobial peptides Hemoglobin fragments Midgut Ticks 


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  1. Agyei, A.D., Runham, N.W., Blackstock, N. 1991Histochemical localization of acid phosphatase and non-specific esterase in the midguts of two species of tick, Boophilus microplusRhipicephalus appendiculatusas determined by light microscopyParasitol. Res.77629634PubMedGoogle Scholar
  2. Beerntsen, B., James, A.A., Christensen, B.M. 2000Genetics of mosquito vector competenceMicrobiol. Mol. Biol. Rev.64115137PubMedGoogle Scholar
  3. Boulanger, N., Brun, R., Ehret-Sabatier, L., Kunz, C., Bulet, P. 2002Immunopeptides in the defense reactions of Glossina morsitans to bacterial and Trypanosoma brucei brucei infectionsInsect Biochem. Mol. Biol.32369375PubMedGoogle Scholar
  4. Ceraul, S.M., Sonenshine, D.E., Hynes, W.L. 2002Investigations into the resistance of the tick, Dermacentor variabilis (Say) (Acari: Ixodidae) following challenge with the bacteriumEscherichi coli (Enterobacteriales: Enterobacteriaceae)J. Med. Entomol.39376383PubMedGoogle Scholar
  5. Ceraul, S.M., Sonenshine, D.E., Ratzlaff, R.E., Hynes, W.L. 2003An arthropod defensin expressed by the hemocytes of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)Insect Biochem. Mol. Biol.3310991103PubMedGoogle Scholar
  6. Cohen, S.L., Chait, B.T. 1997Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelsAnal. Biochem.247257267CrossRefPubMedGoogle Scholar
  7. Coons, L.B., Rosell-Davis, R., Tarnowski, B.I. 1986

    Bloodmeal digestion ticks

    Sauer, J.R.Hair, J.A. eds. Morphology, Physiology and Behavioral Biology of TicksEllis HorwoodChichesterUK248279
    Google Scholar
  8. Fogaca, A.C., da Silva, P.I.,Jr., Miranda, M.T., Bianchi, A.G., Miranda, A., Ribolla, P.E., Daffre, S. 1999Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplusJ. Biol. Chem.2742533025334PubMedGoogle Scholar
  9. Gillespie, J.P., Kanost, M.R., Trenczek, T. 1997Biological mediators of insect immunityAnnu. Rev. Entomol.42611643PubMedGoogle Scholar
  10. Gough, J.M., Kemp, D.H. 1995Acid phosphatase in midgut digestive cells in partially fed females of the cattle tick, Boophilus microplusJ. Parasitol.81341349PubMedGoogle Scholar
  11. Grunclova, L., Fouguier, H., Hypga, V., Kopacek, P. 2003Lysozyme from the gut of the soft tick, Ornithodoros moubatathe sequencephylogeny and post-feeding regulationDevelop. Comp. Immunol.27661660CrossRefGoogle Scholar
  12. Hiemstra, P.S., den Barselaar, M.T., Nibbering, P.H., van Furth, R. 1999Ubiquicidin, a novel murine microbicidal protein present in the cytosolic fraction of macrophagesJ. Leukoc. Biol.66426428Google Scholar
  13. Johns, R., Sonenshine, D.E., Hynes, W.L. 2000Responses of the tick, Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales)J. Med. Entomol.37265270PubMedGoogle Scholar
  14. Johns, R., Sonenshine, D.E., Hynes, W.L. 2001aIdentification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilisInsect Biochem. Mol. Biol.31857865CrossRefGoogle Scholar
  15. Johns, R., Ohnishi, J., Broadwater, A., Sonenshine, D.E., deSilva, A., Hynes, W.L. 2001bContrasts in tick immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis (L.) versus immunocompetence in Dermacentor variabilisJ. Med. Entomol.3899107Google Scholar
  16. Kopacek, P., Vogt, R., Jindrak, L., Weise, C., Safarik, I. 1999Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubataInsect Biochem. Mol. Biol.29195205Google Scholar
  17. Kovar, V., Kopacek, P., Grubhoffer, L. 2000Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubataInsect Biochem. Mol. Biol.30195205PubMedGoogle Scholar
  18. Lebendiker, M. 2004Native acidic gel protocolAlexander Silberman Institute of Life SciencesHebrew University, IsraelWeb Site: http://www.Is.huji.ac.il/∼ ∼purification/Protocols/PAGE_Acidic.html.Google Scholar
  19. Lowenberger, C.A., Kamal, S., Chiles, J., Paskewitz, S., Bulet, P., Hoffman, J.A., Christensen, B. 1999Mosquito–Plasmodium interactions in response to immune activation of the vectorExp. Parasitol.915969PubMedGoogle Scholar
  20. Macaluso, K.R., Sonenshine, D.E., Ceraul, C.M., Azad, A.F. 2001Infection and transovarial transmission of rickettsiae in Dermacentor variabilis ticks acquired by artificial feedingVector Borne Zoonotic Dis.1L4553CrossRefGoogle Scholar
  21. Macaluso, K.R., Sonenshine, D.E., Ceraul, S.M., Azad, A.F. 2002Rickettsial infection in Dermacentor variabilis (Acari: Ixodidae) inhibits transovarial transmission of a second RickettsiaJ. Med. Entomol.39809813PubMedGoogle Scholar
  22. Mulenga, A., Misao, O., Sugimoto, C. 2003Three serine proteinases from midguts of the hard tick Phipicephalus appendiculatus; cDNA cloning and preliminary characterizationExp. Appl. Acarol.29151164PubMedGoogle Scholar
  23. Nakajima, Y., van Naters-Yasui, A., Taylor, D., Yamakawa, M. 2002Antibacterial peptide defensin is involved in midgut immunity of the soft tick, Ornithodoros moubataInsect Mol. Biol.11611618PubMedGoogle Scholar
  24. Nakajima, Y., Ogihara, K., Taylor, D., Yamakawa, M. 2003Antibacterial hemoglobin fragments from the midgut of the soft tick, Ornithodoros moubata (Acari: Argasidae)J. Med. Entomol.407881PubMedGoogle Scholar
  25. Parish, C.A., Jiang, H., Tokiwa, Y., Berova, N., Nakanishi, K., McCabe, D., Zuckerman, W., Xi, M.M., Gabay, J.E. 2001Broad spectrum antimicrobial activity of hemoglobinBioorg. Med. Chem.9377382PubMedGoogle Scholar
  26. Rao, J., Herr, J.C., Reddi, P., Wolkowitz, M.J., Bush, L.A., Sherman, N.E., Black, M., Flickinger, C.J. 2003Cloning and characterization of a novel sperm associated iso-antigen (E-3) with defensin/lectin-like motifs exclusively expressed in rat epididymidisBiol. Reprod.68290301PubMedGoogle Scholar
  27. Rechav, Y., Zyzak, M., Fielden, L.J., Childs, J.E. 1999Comparison of methods for introducing and producing artificial infection of ixodid ticks (Acari: Ixodidae) with Ehrlichia chaffeensisJ. Med. Entomol.36414419PubMedGoogle Scholar
  28. Richman, A.M., Dimopoulos, G., Deelye, D., Kafatos, F.C. 1997Plasmodium activates the innate response of Anopheles gambiae mosquitoesEMBO J.1661146119PubMedGoogle Scholar
  29. Ribeiro, J.M. 1988The midgut hemolysin of Ixodes dammini (Acari: Ixodidae)J. Parasitol.74532537PubMedGoogle Scholar
  30. Sonenshine, D.E. 1991Biology of Ticks, Vol. 1Oxford University PressNew YorkGoogle Scholar
  31. Sonenshine, D.E. 1993Biology of Ticks, Vol. 2Oxford University PressNew YorkGoogle Scholar
  32. Sonenshine, D.E., Homsher, P.J., VandeBerg, J.S., Dawson, D. 1981Fine structure of the foveal glands and foveae dorsales of the American dog tick, Dermacentor variabilis (Say)J. Parasitol.67627646PubMedGoogle Scholar
  33. Sonenshine, D.E., Ceraul, S.M., Hynes, W.L., Macaluso, K.R., Azad, A.F. 2002Expression of defensin-like peptides in tick hemolymph and midgut in response to challenge with Borrelia burgdorferiBacillus subtilisExp. Appl. Acarol.28127134PubMedGoogle Scholar
  34. Stojilkovic, I., Evavold, B.D., Kumar, V. 2001Antimicrobial properties of porphyrinsExpert Opin. Investig. Drugs10309320PubMedGoogle Scholar
  35. Vaughan, J.A., Azad, A.F. 1993Patterns of erythrocyte digestion by bloodsucking insects: constraints on vector competenceJ. Med. Entomol.30214216PubMedGoogle Scholar
  36. Vizioli, J., Richman, A.M., Uttenweiler, J.S., Blass, C., Bulet, P. 2001The defensin peptide of the malaria vector mosquitoAnopheles gambiae: antimicrobial activities and expression in adult mosquitoesInsect Biochem. Mol. Biol.31241248PubMedGoogle Scholar
  37. Zhu, K., Dillwith, J.W., Bowman, A.S., Sauer, J.R. 1997Identification of hemolytic activity in saliva of the lone star tick (Acari: Ixodidae)J. Med. Entomol.34160166PubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Daniel E. Sonenshine
    • 1
  • Wayne L. Hynes
    • 1
  • Shane M. Ceraul
    • 1
  • Robert Mitchell
    • 1
  • Tiffany Benzine
    • 1
  1. 1.Department of Biological SciencesOld Dominion UniversityNorfolkUSA

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