Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections

  • Katherine M. Kocan
  • José de la Fuente
  • Raúl Manzano-Roman
  • Victoria Naranjo
  • Wayne L. Hynes
  • Daniel E. SonenshineEmail author


Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.


Defensin Varisin RNA interference Dermacentor variabilis Anaplasma marginale 



This research was partially supported by the Oklahoma Agricultural Experiment Station (project 1669), the Walter R. Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University), Pfizer Animal Health, Kalamazoo, MI, USA, the Junta de Comunidades de Castilla-La Mancha, Spain (project 06036-00 ICS-JCCM), and Ministry of Science and Education (MEC), Spain (project AGL2005-07401). Dr. Raúl Manzano-Roman was funded by Ministerio de Educación y Ciencia, Spain. V. Naranjo was founded by Consejería de Educación, JCCM, Spain. Support, in part, is gratefully acknowledged by a grant from the National Science Foundation, IBN 0212901 (Hynes & Sonenshine) and a grant from the National Research Fund for Tick-borne Diseases (Hynes & Sonenshine).


  1. Boulanger N, Munks RJ, Hamilton JV, Vovelle F, Brun R, Lehane MJ et al (2002) Epithelial innate immunity. A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans. J Biol Chem 277:49921–49926. doi: 10.1074/jbc.M206296200 PubMedCrossRefGoogle Scholar
  2. Ceraul SM, Sonenshine DE, Ratzlaff RE, Hynes WL (2003) An arthropod defensin expressed by the hemocytes of the American dog tick, Dermacentor variabilis (Acari: Ixodidae). Insect Biochem Mol Biol 33:1099–1103. doi: 10.1016/S0965-1748(03) 00122-X PubMedCrossRefGoogle Scholar
  3. Ceraul SM, Dreher-Lesnick S, Gillespie JJ, Sayeedur Rahman M, Azad AF (2007) A new tick defensin isoform and antimicrobial gene expression in response to Rickettsia montanensis challenge. Infect Immun 75:1973–1983. doi: 10.1128/IAI.01815-06 PubMedCrossRefGoogle Scholar
  4. de la Fuente J, Garcia-Garcia JC, Blouin EF, Kocan KM (2001) Major surface protein 1a effects tick infection and transmission of the ehrlichial pathogen Anaplasma marginale. Int J Parasitol 31:1705–1714. doi: 10.1016/S0020-7519(01) 00287-9 CrossRefGoogle Scholar
  5. de la Fuente J, Ayoubi P, Blouin EF, Almazán C, Naranjo V, Kocan KM (2005) Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum. Cell Microbiol 7:549–559. doi: 10.1111/j.1462-5822.2004.00485.x CrossRefGoogle Scholar
  6. de la Fuente J, Almazán C, Blouin EF, Naranjo V, Kocan KM (2006a) Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin. Parasitol Res 100:85–89. doi: 10.1007/s00436-006-0244-6 CrossRefGoogle Scholar
  7. de la Fuente J, Almazán C, Blas-Machado U, Naranjo V, Mangold AJ, Blouin EF et al (2006b) The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction. Vaccine 24:4082–4095. doi: 10.1016/j.vaccine.2006.02.046 CrossRefGoogle Scholar
  8. de la Fuente J, Blouin EF, Manzano-Roman R, Naranjo V, Almazán C, Pérez de la Latra JM et al (2007a) Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale. Genomics 90:712–722. doi: 10.1016/j.ygeno.2007.08.009 CrossRefGoogle Scholar
  9. de la Fuente J, Manzano-Roman R, Blouin EF, Naranjo V, Kocan KM (2007b) Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells. BMC Infect Dis 7:110. doi: 10.1186/1471-2334-7-110 CrossRefGoogle Scholar
  10. de la Fuente J, Kocan KM, Almazán C, Blouin EF (2007c) RNA interference for the study and genetic manipulation of ticks. Trends Parasitol 23:427–433. doi: 10.1016/ CrossRefGoogle Scholar
  11. Dreher-Lesnick SM, Mulenga A, Simser JA, Azad AF (2006) Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis. Insect Mol Biol 15:445–453. doi: 10.1111/j.1365-2583.2006.00657.x PubMedCrossRefGoogle Scholar
  12. Dumler JS, Barbet AC, Bekker CPJ, Dasch GA, Palmer GH, Ray SC et al (2001) Reorganization of the genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 51:2145–2165PubMedGoogle Scholar
  13. Fogaca AC, Lorenzini DM, Kaku LM, Esteves E, Bulet P, Daffre S (2004) Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile. Dev Comp Immunol 28:191–200. doi: 10.1016/j.dci.2003.08.001 PubMedCrossRefGoogle Scholar
  14. Ganz T, Lherer RI (1994) Defensins. Curr Opin Immunol 6:584–589. doi: 10.1016/0952-7915(94) 90145-7 PubMedCrossRefGoogle Scholar
  15. Gillespie JP, Kanost MR, Trenczek T (1997) Biological mediators of insect immunity. Annu Rev Entomol 42:611–643. doi: 10.1146/annurev.ento.42.1.611 PubMedCrossRefGoogle Scholar
  16. Hoffmann JA, Hetru C (1992) Insect defensins: inducible antibacterial peptides. Immunol Today 13:411–415. doi: 10.1016/0167-5699(92) 90092-L PubMedCrossRefGoogle Scholar
  17. Hynes WL, Ceraul SM, Todd SM, Seguin KC, Sonenshine DE (2005) A defensin-like gene expressed in the black-legged tick, Ixodes scapularis. Med Vet Entomol 19:339–344. doi: 10.1111/j.1365-2915.2005.00579.x PubMedCrossRefGoogle Scholar
  18. Hynes WL, Stokes MM, Hensley SM, Todd SM, Sonenshine DE (2008) Using RNA interference to determine the role of varisin in the innate immune system of the hard tick Dermacentor variabilis (Acari: Ixodidae). Exp Appl Acarol 45. doi: 10.1007/s10493-008-9158-6
  19. Johns R, Sonenshine DE, Hynes WL (2000) Tick immunity to microbial infections: anti-bacterial peptides or proteins in the hemolymph of the hard tick Dermacentor variabilis (Acari: Ixodidae). Acarology Proc 10th Internatl Congr, Canberra, Australia, pp 638–644Google Scholar
  20. Johns R, Sonenshine DE, Hynes WL (2001a) Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis. Insect Biochem Mol Biol 31:857–865. doi: 10.1016/S0965-1748(01)00031-5 PubMedCrossRefGoogle Scholar
  21. Johns R, Ohnishi J, Broadwater A, Sonenshine DE, deSilva A, Hynes WL (2001b) Contrasts in tick immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis (L.) versus immunocompetence in Dermacentor variabilis. J Med Entomol 38:99–107PubMedCrossRefGoogle Scholar
  22. Kocan KM (1986) Development of Anaplasma marginale in ixodid ticks: coordinated development of a rickettsial organism and its tick host. In: Sauer J, Hair JA (eds) Morphology, physiology and behavioral ecology of ticks. Ellis Horwood Ltd, Chichester, pp 472–505Google Scholar
  23. Kocan KM, Hair JA, Ewing SA (1980) Ultrastructure of Anaplasma marginale Theiler in Dermacentor andersoni Stiles and Dermacentor variabilis (Say). Am J Vet Res 41:1966–1976PubMedGoogle Scholar
  24. Kocan KM, Goff WL, Stiller D, Claypool PL, Edwards W, Ewing SA et al (1992a) Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred successively from infected to susceptible cattle. J Med Ent 29:657–668Google Scholar
  25. Kocan KM, Stiller D, Goff WL, Claypool PL, Edwards W, Ewing SA et al (1992b) Development of Anaplasma marginale in male Dermacentor andersoni transferred from infected to susceptible cattle. Am J Vet Res 53:499–507PubMedGoogle Scholar
  26. Kocan KM, de la Fuente J, Blouin EF, Garcia-Garcia JC (2004) Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia. Parasitology 129:S285–S300. doi: 10.1017/S0031182003004700 PubMedCrossRefGoogle Scholar
  27. Kocan KM, Yoshioka J, Sonenshine DE, de la Fuente J, Ceraul SM, Blouin EF et al (2005) Capillary tube feeding system for studying tick/pathogen interactions of Dermacentor variabilis (Acari: Ixodidae) and Anaplasma marginale (Rickettisales: Anaplasmataceae). J Med Entomol 42:864–874. doi: 10.1603/0022-2585(2005) 042[0864:CTFSFS]2.0.CO;2 PubMedCrossRefGoogle Scholar
  28. Lai R, Lomas LO, Jonczy J, Turner PC, Rees HH (2004) Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum. Biochem J 379:681–685. doi: 10.1042/BJ20031429 PubMedCrossRefGoogle Scholar
  29. Lehane MJ, Wu D, Lehane SM (1997) Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans. Proc Natl Acad Sci U S A 94:11502–11507. doi: 10.1073/pnas.94.21.11502 CrossRefGoogle Scholar
  30. Ma Y, Creanga A, Lum L, Beachy PA (2006) Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443:359–363. doi: 10.1038/nature05179 PubMedCrossRefGoogle Scholar
  31. Manzano-Roman R, Almazán C, Naranjo V, Blouin EF, Kocan KM, de la Fuente J (2007) Expression of perilipin in human promyelocytic cells in response to Anaplasma phagocytophilum infection results in modified lipid metabolism. J Med Microbiol (in press)Google Scholar
  32. Nakajima Y, van Naters-Yasui AV, Taylor D, Yamakawa M (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31:747–751. doi: 10.1016/S0965-1748(01) 00066-2 PubMedCrossRefGoogle Scholar
  33. Nakajima Y, van Naters-Yasui A, Taylor D, Yamakawa M (2002) Antibacterial peptide defensin is involved in midgut immunity of the soft tick, Ornithodoros moubata. Insect Mol Biol 11:611–618. doi: 10.1046/j.1365-2583.2002.00372.x PubMedCrossRefGoogle Scholar
  34. Richardson KC, Jarret L, Finke FH (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313–323PubMedGoogle Scholar
  35. Rudenko N, Golovchenko M, Edwards MJ, Grubhoffer L (2005) Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection. J Med Entomol 42:36–41. doi: 10.1603/0022-2585(2005) 042[0036:DEOIRT]2.0.CO;2 PubMedCrossRefGoogle Scholar
  36. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC et al (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci U S A 101:1892–1897. doi: 10.1073/pnas.0308698100 PubMedCrossRefGoogle Scholar
  37. Siedz CA, Holko M, de Veer MJ, Silverman RH, Williams BR (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5:834–839. doi: 10.1038/ncb1038 CrossRefGoogle Scholar
  38. Simser JA, Macaluso KR, Mulenga A, Azad AF (2004) Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis, and an embryonic cell line of the Rocky Mountain wood tick, D andersoni. Insect Biochem Mol Biol 34:1253–1246. doi: 10.1016/j.ibmb.2004.07.003 CrossRefGoogle Scholar
  39. Sonenshine DE (1993) Biology of ticks, vol 2. Oxford University Press, New YorkGoogle Scholar
  40. Sonenshine DE, Hynes WL, Ceraul SM, Mitchell RD, Benzine T (2005) Host blood proteins and peptides in the midgut of the tick Dermacentor variabilis contribute to bacterial control. Exp Appl Acarol 36:207–223. doi: 10.1007/s10493-005-2564-0 PubMedCrossRefGoogle Scholar
  41. Sukumaran B, Narasimhan S, Anderson JF, DePonte K, Marcantonio N et al (2006) An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands. J Exp Med 203:1507–1517. doi: 10.1084/jem.20060208 PubMedCrossRefGoogle Scholar
  42. Todd SM, Sonenshine DE, Hynes WL (2007) Tissue and life-stage distribution of a defensin gene in the Lone Star Tick, Amblyomma americanum. Med Vet Entomol 21:141–147. doi: 10.1111/j.1365-2915.2007.00682.x PubMedCrossRefGoogle Scholar
  43. Tsuji N, Battsetseg B, Boldbaatar D, Miyoshi T, Xuan X, Oliver JH Jr et al (2007) A babesial vector tick defensin against Babesia parasites. Infect Immun 75:3633–3640. doi: 10.1128/IAI.00256-07 PubMedCrossRefGoogle Scholar
  44. Zhou J, Ueda M, Umemiya R, Battsetseg B, Boldbaatar D, Xuan X et al (2006) A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. Insect Biochem Mol Biol 36:527–535. doi: 10.1016/j.ibmb.2006.03.003 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Katherine M. Kocan
    • 1
  • José de la Fuente
    • 1
    • 2
  • Raúl Manzano-Roman
    • 1
  • Victoria Naranjo
    • 2
  • Wayne L. Hynes
    • 3
  • Daniel E. Sonenshine
    • 3
    Email author
  1. 1.Department of Veterinary Pathobiology, Center for Veterinary Health SciencesOklahoma State UniversityStillwaterUSA
  2. 2.Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM)Ciudad RealSpain
  3. 3.Department of Biological SciencesOld Dominion UniversityNorfolkUSA

Personalised recommendations