Document Type : Original Article


Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran


Resistance to the knockdown effect of pyrethroid insecticides occurs due to mutations at target sites of pyrethroids, meaning the voltage-gated sodium channels gene (VGSC) in the membrane of the neurons. In fleas, this mutation occurs at two sites in the sodium channel in neurons: one is the replacement of leucine with phenylalanine (L1014F) and the other is the replacement of threonine with valine (T929V). In this study, 81 Pulex irritans and 47 Ctenocephalides canis fleas were collected from five provinces in the west and northwest of Iran. Adult fleas were exposed to cypermethrin 0.75%, and the mortality rate was calculated after 1 and 8 hr, and the mutation sites in the VGSC gene were investigated. The lethality of cypermethrin 0.75% for P. irritans was 40.00 - 57.14% after 1 hr and 60.00 - 73.91% after 8 hr. The lethality of this dose for C. canis after 1 and 8 hr of exposure was 33.33 - 41.17% and 66.66 - 80.33%, respectively. The VGSC sequence analysis indicated two mutation sites in the resistant and one mutation site in the susceptible fleas. The VGSC sequence analysis of susceptible P. irritans showed that 5.50% of them were homozygous susceptible and 94.45% were hetero-zygous susceptible. Susceptible C. canis were 5.26% homozygous and 94.73% heterozygous susceptible. All the resistant fleas were homozygous. The development of pyrethroid resistance and high-frequency L1014F mutation in fleas suggest that pyrethroids are likely to be ineffective in controlling fleas. Therefore, monitoring pyrethroid resistance and its underlying mechanisms are necessary for controlling fleas and finding new alternative control methods.


  1. Silver KS, Du Y, Nomura Y, et al. Voltage-gated sodium channels as insecticide targets. Adv In Insect Phys 2014; 46: 389-433.
  2. Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000; 26(1): 13-25.
  3. Soderlund DM. Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch Toxicol 2012; 86(2):165-181.
  4. Dong K, Du Y, Rinkevich F, et al. Molecular biology of insect sodium channels and pyrethroid resistance. Insect Biochem Mol Biol 2014; 50: 1-17.
  5. Rinkevich FD, Du Y, Dong K. Diversity and convergence of sodium channel mutations involved in resistance to pyrethroids. Pestic Biochem Phys 2013; 106(3): 93-100.
  6. Enayati AA, Vatandoost H, Ladonni H, et al. Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi. Med Vet Entomol 2003; 17(2): 138-144.
  7. Singh OP, Dykes CL, Sharma G, et al. L1014F-Kdr mutation in Indian Anopheles subpictus (Diptera: Culicidae) arising from two alternative transversions in the voltage-gated sodium channel and a single PIRA-PCR for their detection. J Med Entomol 2015; 52(1): 24-27.
  8. Fabro J, Sterkel M, Capriotti N, et al. Identification of a point mutation associated with pyrethroid resistance in the para-type sodium channel of Triatoma infestans, a vector of Chagas’ disease. Infect Genet Evol 2012; 12(2): 487-491.
  9. Yoon KS, Kwon DH, Strycharz JP, et al. Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera: Cimicidae). J Med Entomol 2008; 45(6): 1092-1101.
  10. Rosario-Cruz R, Guerrero FD, Miller RJ, et al. Molecular survey of pyrethroid resistance mechanisms in Mexican field populations of Rhipicephalus (Boophilus) microplus. Parasitol Res 2009; 105(4): 1145-1153.
  11. Morgan JA, Corley SW, Jackson LA, et al. Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides. Int J Parasitol 2009; 39(7): 775-779.
  12. Kumar R, Nagar G, Sharma AK, et al. Survey of pyrethroids resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: identification of C190A mutation in the domain II of the para-sodium channel gene. Acta Trop 2013; 125(2): 237-245.
  13. Lee SH, Gao JR, Yoon KS, et al. Sodium channel mutations associated with knockdown resistance in the human head louse, Pediculus capitis (De Geer). Pestic Biochem Phys 2003; 75(3): 79-91.
  14. Tomita T, Yaguchi N, Mihara M, et al. Molecular analysis of a para sodium channel gene from pyrethroid-resistant head lice, Pediculus humanus capitis (Anoplura: Pediculidae). J Med Entomol 2003; 40(4): 468-474.
  15. Gellatly KJ, Krim S, Palenchar DJ, et al. Expansion of the knockdown resistance frequency map for human head lice (Phthiraptera: Pediculidae) in the United States using quantitative sequencing. J Med Entomol 2016; 53(3): 653-659.
  16. Liu Z, Valles SM, Dong K. Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides. Insect Biochem Mol Biol 2000; 30(10): 991-997.
  17. Zhu F, Wigginton J, Romero A, et al. Widespread distribution of knockdown resistance mutations in the bed bug, Cimex lectularius (Hemiptera: Cimicidae), populations in the United States. Arch Insect Biochem Physiol 2010; 73(4): 245-257.
  18. Dang K, Toi CS, Lilly DG, et al. Identification of putative kdr mutations in the tropical bed bug, Cimex hemipterus (Hemiptera: Cimicidae). Pest Manag Sci 2015; 71(7): 1015-1020.
  19. Dang K, Toi CS, Lilly DG, et al. Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia. Pest Manag Sci 2015; 71(7): 914-922.
  20. Palenchar DJ, Gellatly KJ, Yoon KS, et al. Quantitative sequencing for the determination of kdr-type resistance allele (V419L, L925I, I936F) frequencies in common bed bug (Hemiptera: Cimicidae) populations collected from Israel. J Med Entomol 2015; 52(5): 1018-1027.
  21. Dang K, Doggett SL, Veera Singham G, et al. Insecticide resistance and resistance mechanisms in bed bugs, Cimex (Hemiptera: Cimicidae). Parasit Vectors 2017; 10(1): 318. doi: 10.1186/s13071-017-2232-3.
  22. Bass C, Schroeder I, Turberg A, et al. Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis. Insect Biochem Mol Biol 2004; 34(12): 1305-1313.
  23. Rust MK, Vetter R, Denholm I, et al. Susceptibility of adult cat fleas (Siphonaptera: Pulicidae) to insecticides and status of insecticide resistance mutations at the Rdl and knockdown resistance loci. Parasitol Res 2015; 114 Suppl 1: S7-S18.
  24. Rust MK. Insecticide resistance in fleas. Insects 2016; 7(1):10. doi: 10.3390/insects7010010.
  25. Bitam I, Dittmar K, Parola P, et al. Fleas and flea-borne diseases. Int J Infect Dis 2010;14: e667-e676.
  26. Beugnet F, Franc M. Insecticide and acaricide molecules and/or combinations to prevent pet infestation by ectoparasites. Trends Parasitol 2012; 28(7): 267-279.
  27. Boyer S, Miarinjara A, Elissa N. Xenopsylla cheopis (Siphonaptera: Pulicidae) susceptibility to Deltamethrin in Madagascar. PLoS One 2014; 9(11): e111998. doi: 10.1371/journal.pone.0111998.
  28. Shyamal B, Ravi Kumar R, Sohan L, et al. Present susceptibility status of rat flea, Xenopsylla cheopis (Siphonaptera: Pulicidae) vector of human plague against organochlorine, organophosphate and synthetic pyrethroids 1. The Nilgiris district, Tamil Nadu, India. J Commun Dis 2008; 40(1): 41-45.
  29. Mazumder S, Sohan L, Reddy MS, et al. Susceptibility status of rodent fleas to different insecticides in plague endemic area Kolar, Karnataka, India. Int J Curr Microbiol Appl Sci 2014; 3(8): 836-841.
  30. Coles TB, Dryden MW. Insecticide/acaricide resistance in fleas and ticks infesting dogs and cats. Parasites Vectors 2014; 7: 8. doi: 10.1186/1756-3305-7-8.
  31. Asmar M, Piazak N, Karimi Y. An illustrated key for flea of Iran [Persian]. Pasteur Institute of Iran, Tehran, 1979.
  32. Martinez‐Torres D, Devonshire AL, Williamson MS. Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects. Pestic Sci1997; 51(3): 265-270.
  33. Sakuma M. Probit analysis of preference data. Appl Entomol Zool 1998; 33(3): 339-347.
  34. Folmer O, Black M, Hoeh W, et al. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994; 3(5): 294-299.
  35. Soderlund DM, Knipple DC. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem Mol Biol 2003; 33(6): 563-577.
  36. Vais H, Williamson MS, Goodson SJ, et al. Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations. J Gen Physiol 2000; 115(3): 305-318.
  37. Ghavami MB, Haghi FP, Alibabaei Z, et al. First report of target site insensitivity to pyrethroids in human flea, Pulex irritans (Siphonaptera: Pulicidae). Pestic Biochem Physiol 2018; 146: 97-105.
  38. Samiei A, Tavassoli M, Mardani K. Molecular analysis of pyrethroid resistance in Cimex hemipterus (Hemiptera: Cimicidae) collected from different parts of Iran. Vet Res Forum 2020; 11(3): 243-248.