Experimental and Applied Acarology

, Volume 74, Issue 4, pp 455–461 | Cite as

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of Metarhizium anisopliae on Rhipicephalus microplus tick larvae

  • Anelise Webster
  • Ugo Araújo Souza
  • João Ricardo Martins
  • Guilherme Klafke
  • José Reck
  • Augusto Schrank


Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 106, 107 and 108 conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT50 values obtained were 134.6, 27.2 and 24.8 days for concentrations of 106, 107 and 108 conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT50 values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 106, 107 and 108 conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.


Ixodidae Fungus Biological control LPT LIT Acaripathogenic fungi 



This study was financially supported by Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq).

Supplementary material

10493_2018_235_MOESM1_ESM.tif (60 kb)
Fig. 1 Larval mortality at different concentrations of Metharhizium anisopliae suspension. Panel A, Results of Larval Immersion Test (LIT). Panel B, Results of Larval Packet Test (LPT). The evaluation of tick mortality (counting the number of alive and dead larvae) was done throughout the 21 days of experiment daily. Spore suspension of M. anisopliae at 106 conidia/mL (■), 107 conidia/mL (▲), 108 conidia/mL (X) and control (♦). (TIFF 59 kb)


  1. Arruda W, Lubeck I, Schrank A, Vainstein MH (2005) Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp Appl Acarol 37:231–244. CrossRefPubMedGoogle Scholar
  2. Beys-da-Silva WO, Santi L, Berger M, Guimarães JA, Schrank A, Vainstein MH (2013) Susceptibility of Loxosceles sp. to the arthropodpathogenic fungus Metarhizium anisopliae: potential biocontrol of the brown spider. Trans R Soc Trop Med Hyg 7:59–61. CrossRefGoogle Scholar
  3. Camargo MG, Golo PS, Angelo IC, Perinotto WM, Sá FA, Quinelato S, Bittencourt VR (2012) Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions. Vet Parasitol 188:140–147. CrossRefPubMedGoogle Scholar
  4. Castro-Janer E, Rifran L, Piaggio J, Gil A, Miller RJ, Schumaker TT (2009) In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization. Vet Parasitol 162:120–128. CrossRefPubMedGoogle Scholar
  5. Estrada-Peña A, García Z, Fragoso SH (2006) The distribution and ecological preferences of Boophilus microplus (Acari: Ixodidae) in Mexico. Exp Appl Acarol 38:307–316. CrossRefPubMedGoogle Scholar
  6. Fernandes ÉKK, Angelo IC, Rangel DEN, Bahiense TC, Moraes AML, Roberts DW, Bittencourt VREP (2011) An intensive search for promising fungal biological control agents of ticks, particularly Rhipicephalus microplus. Vet Parasitol 182:307–318CrossRefPubMedGoogle Scholar
  7. Food and Agriculture Organization (FAO) (2004) Resistance management and integrated parasite control in ruminants: Guidelines. Roma: Food and Agriculture Organization, Animal Production and Health Division, 53 pGoogle Scholar
  8. Frazzon APG, Da Silva VI, Masuda A, Schrank A, Vainstein MH (2000) In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet Parasitol 94:117–125CrossRefPubMedGoogle Scholar
  9. Grisi L, Leite RC, Martins JRS, Barros ATM, Andreotti R, Cançado PHD, León AAP, Pereira JB, Villela HS (2014) Reassessment of the potential economic impact of cattle parasites in Brazil. Braz J Vet Parasitol 23:150–156CrossRefGoogle Scholar
  10. Jonsson NN, Miller RJ, Robertson JL (2007) Critical evaluation of the modified adult immersion test with discriminating dose bioassay for Boophilus microplus using American and Australian isolates. Vet Parasitol. 146:307–315. CrossRefPubMedGoogle Scholar
  11. Kaaya GP, Mwangi EN (1995) Control of livestock ticks in Africa: possibilities of biological control using the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae. In: Coons L, Rothshild M (eds) The second international conference on tick-borne pathogens at the host-vector interface: a global perspective, 1995, August 28–September 1, South Africa. Proceedings and Abstracts, vol 1, pp 5–16Google Scholar
  12. Kaaya GP, Samish M, Hedimbi M, Gindin G, Glazer I (2011) Control of tick populations by spraying Metarhizium anisopliae conidia on cattle under field conditions. Exp Appl Acarol 55:273–281CrossRefPubMedGoogle Scholar
  13. Leemon DM, Jonsson NN (2008) Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus. J Invertebr Pathol 97:40–49. CrossRefPubMedGoogle Scholar
  14. Luz C, D’Alessandro WB, Rodrigues J, Fernandes ÉK (2016) Efficacy of water- and oil-in-water-formulated Metarhizium anisopliae in Rhipicephalus sanguineus eggs. Parasitol Res 115:143–149. CrossRefPubMedGoogle Scholar
  15. Martins JR, Furlong J (2001) Avermectin resistance of the cattle tick Boophilus microplus in Brazil. Vet Rec 149:64PubMedGoogle Scholar
  16. Onofre SB, Miniuk CM, Barros NM, Azevedo JL (2001) Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus. Am J Vet Res 62:1478–1480CrossRefPubMedGoogle Scholar
  17. Pal S, St Leger RJ, Wu LP (2007) Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster. J Biol Chem 282:8969–8977. CrossRefPubMedGoogle Scholar
  18. Perinotto WMS, Angelo IC, Golo PS, Camargo MG, Quinelato S, Santi L, Vainstein MH, Beys-da-Silva WO, Salles CMC, Bittencourt VREP (2014) Metarhizium anisopliae (Deuteromycetes: Moniliaceae). Pr1 activity: biochemical marker of fungal virulence in Rhipicephalus microplus (Acari: Ixodidae). Biocontrol Sci Technol 24:123–132. CrossRefGoogle Scholar
  19. Quinelato S, Golo PS, Perinotto WM, Sá FA, Camargo MG, Angelo IC, Moraes AM, Bittencourt VR (2012) Virulence potential of Metarhizium anisopliae s.l. isolates on Rhipicephalus (Boophilus) microplus larvae. Vet Parasitol 190(3–4):556–565.
  20. Reck J, Klafke GM, Webster A, Dall’Agnol B, Scheffer R, Souza UA, Corassini VB, Vargas R, Silveira J, Martins JR (2014) First report of fluazuron resistance in Rhipicephalus microplus: a field tick population resistant to six classes of acaricides. Vet Parasitol 201:128–136. CrossRefPubMedGoogle Scholar
  21. Robertson JL, Preisler HK, Russel RM (2003) PoloPlus Probit and Logit Analysis-user’s guide. Berkeley: LeOra Software, 36 pGoogle Scholar
  22. Robertson LJ, Russell RM, Presler HK, Savin NE (2007) Bioassays with arthropods. CRC Press, Boca Raton, p 199Google Scholar
  23. Sabatini GA, Kemp DH, Hughes S, Nari A, Hansen J (2001) Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus. Vet Parasitol 95:53–62CrossRefPubMedGoogle Scholar
  24. Santi L, Silva LAD, Beys-da-Silva WO, Corrêa APF, Rangel DEN, Carlini CR, Schrank A, Vainstein MH (2011) Virulence of the entomopathogenic fungus Metarhizium anisopliae using soybean oil formulation for control of the cotton stainer bug Dysdercus peruvianus. World J Microbiol Biotechnol 27:2297–2303. CrossRefGoogle Scholar
  25. Schrank A, Vainstein MH (2010) Metarhizium anisopliae enzymes and toxins. Toxicon 56:1267–1274. CrossRefPubMedGoogle Scholar
  26. Shaw RD (1966) Culture of an organophosphorus-resistant strain of Boophilus microplus (Can.) and an assessment of its resistance spectrum. Bull Entomol Res 56(3):389–405CrossRefPubMedGoogle Scholar
  27. Stone BF, Haydock KP (1962) A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.) Bull Entomol Res 53:563–578CrossRefGoogle Scholar
  28. Vega FE, Kaaya HF (2012) Insect pathology. Academic Press, CambridgeGoogle Scholar
  29. Wang C, St Leger RJ (2007) The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants. Eukaryot Cell 6:808–816. CrossRefPubMedCentralPubMedGoogle Scholar
  30. Webster A, Reck J, Santi L, Souza UA, Dall’Agnol B, Klafke GM, Beys-da-Silva WO, Martins JR, Schrank A (2015) Integrated control of an acaricide-resistant strain of the cattle tick Rhipicephalus microplus by applying Metarhizium anisopliae associated with cypermethrin and chlorpyriphos under field conditions. Vet Parasitol 207(3–4):302–308CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF)Eldorado do SulBrazil
  2. 2.Centro de BiotecnologiaUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil

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