First Report of Biochemical Mechanisms of Insecticide Resistance in the Field Population of Culex pipiens (Diptera: Culicidae) From Sari, Mazandaran, North of Iran

  • Seyed Hassan Nikookar 1Department of Medical Entomology and Vector Control, School of Public Health and Health Sciences Research Center, Addiction Institute, Mazandaran University of Medical Sciences, Sari, Iran
  • Mahmoud Fazeli-Dinan 2Department of Medical Entomology and Vector Control, School of Public Health and Health Sciences Research Center, Mazandaran University of Medical Science, Sari, Iran
  • Seyyed Payman Ziapour Department of Parasitology, Zoonosis Research Center, Pasteur Institute of Iran, Amol, Iran
  • Fatemeh Ghorbani 4Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Yaser Salim-Abadi 5Department of Health Services and Health Promotion, School of Health, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
  • Hassan Vatandoost Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran
  • Ahmad Ali Hanafi-Bojd Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Ahmadali Enayati Department of Medical Entomology and Vector Control, School of Public Health and Health Sciences Research Center, Addiction Institute, Mazandaran University of Medical Sciences, Sari, Iran
Keywords: Culex pipiens, Insecticide resistanc, Enzyme, Iran


Background: Culex pipiens play an important role in transmission of infectious diseases. Vector control by chemi­cal pesticides, leads inevitably to resistance development. Understanding the underlying resistance mechanisms can help improve the control programmes and insecticide resistance management.Methods: The total contents of cytochrome p450s and the activities of glutathione S-transferases, alpha- and beta-esterases and inhibition rates of acetylcholine esterase (by propoxur) were measured in the field population of Cx. pipiens collected from Sari County, North of Iran, in 2016 and the results were compared with those of the laborato­ry susceptible strain according to the biochemical assay methods of WHO for adult mosquitoes. Independent sample t-test was used to compare the mean values of enzyme activities/contents between filed and laboratory susceptible popula­tions.Results: The enzyme ratio of cytochrome p450s, alpha- and beta-esterases in the field population was 2.07, 3.72 and 1.36 respectively when compared with the results of the laboratory population. Although not statistically significant, the mean GSTs activities in the field population was marginally less than the laboratory population (ER=0.92). Ace­tylcholinesterase was insensitive to propoxur in 62.82% of the individuals of the tested field population. There was a significant difference (P< 0.05) between all values of the activities/contents of the enzyme in the field population except for GSTs compared with the laboratory susceptible strain. The highest enzyme activity was related to alpha esterase.Conclusion: The present study showed a range of metabolic mechanisms, comprising p450s and esterases combined with target site insensitivity of AChE, contributing to organophosphate, carbamate and pyrethroid resistance in the field population of Cx. pipiens.


1. Weitzel T, Jawień P, Rydzanicz K, Lonc E, Becker N (2015) Culex pipiens sl and Culex torrentium (Culicidae) in Wrocław area (Poland): occurrence and breeding site preferences of mosquito vectors. Para¬si¬tol Res. 114: 289–295
2. Turell MJ (2012) Members of the Culex pipiens complex as vectors of viruses. J Am Mosq Control Assoc. 28: 123–126.
3. de Wispelaere M, Despres P, Choumet V (2017) European Aedes albopictus and Culex pipiens Are Competent Vectors for Japanese Encephalitis Virus. PLoS Negl Trop Dis. 11: e0005294.
4. Bravo-Barriga D, Parreira R, Almeida AP, Calado M, Blanco-Ciudad J, Serrano-Aguilera FJ, Pérez-Martín JE, Sánchez-Peinado J, Pinto J, Reina D, Frontera E (2016) Culex pipiens as a potential vector for transmission of Dirofilaria immitis and other unclassified Filarioidea in South¬west Spain.Vet Parasitol. 223: 173–180.
5. Ventim R, Ramos JA, Osorio H, Lopes RJ, Perez-Tris J, Mendes L (2012) Avian ma¬laria infections in western European mos¬quitoes. Parasitol Res. 111: 637–645.
6. Shi L, Hu H, Ma K, Zhou D, Yu J, Zhong D, Fang F, Chang X, Hu Sh, Zou F (2015) Development of resistance to pyrethroid in Culex pipiens pallens population un¬der different insecticide selection pres¬sures. PloS Negl Trop Dis. 9: e0003928.
7. Gao Q, Xiong C, Su F, Cao H, Zhou J, Jiang Q (2016) Structure, Spatial and Tem¬poral Distribution of the Culex pipiens Complex in Shanghai, China. Int J En-vi¬ron Res Public Health. 13: 1150.
8. Vinogradova E, Shaikevich E, Ivanitsky A (2007) A study of the distribution of the Culex pipiens complex (Insecta: Dip-tera: Culicidae) mosquitoes in the European part of Russia by molecular methods of iden¬tification. Comp Cytogenet. 1: 129–138.
9. Hesson JC, Schäfer M, Lundström JO (2016) First report on human-biting Culex pipiens in Sweden. Parasit Vectors. 9: 632.
10. Chaulk AC, Carson KP, Whitney HG, Fon¬seca DM, Chapman TW (2016) The ar¬rival of the northern house mosquito Culex pipiens (Diptera: Culicidae) on New-foundland's Avalon Peninsula. J Med Entomol. 53: 1364–1369.
11. Zaim M (1987) The distribution and larval habitat characteristics of Iranian Culic¬inae. Journal of the American Mosquito Control Association. 3: 568–573.
12. Moosa-Kazemi S, Vatandoost H, Ni-kookar H, Fathian M (2009) Culicinae (Diptera: culicidae) mosquitoes in Chabahar Coun¬ty, Sistan and Baluchistan Province, south¬eastern iran. Iran J Arthropod Borne Dis. 3: 29–35.
13. Dehghan H, Sadraei J, Moosa-Kazemi SH, Abolghasemi E, Solimani H, Jaffari No¬doshan A, Najafi MH (2016) A pictorial key for Culex pipiens Complex (Dip¬tera: Culicidae) In Iran. J Arthropod Borne Dis. 10: 291–302.
14. Nikookar S, Fazeli-Dinan M, Azari-Ha-midi¬an S, Mousavinasab S, Arabi M, Zia¬pour S, Shojaee J, Enayati AA (2017) Species composition and abundance of mosquito larvae in relation with their hab¬itat characteristics in Mazandaran Prov¬ince, northern Iran. Bull Entomol Res. 107: 598–610.
15. Nikookar SH, Azari-Hamidian S, Fazeli-Dinan M, Nasab SNM, Aarabi M, Ziapour SP, Ziapour P, Enayati AA (2016) Species composition, co-occurrence, asso¬ciation and affinity indices of mosquito larvae (Diptera: Culicidae) in Mazanda¬ran Province, northern Iran. Acta Trop. 157: 20–29.
16. Nikookar SH, Moosa-Kazemi SH, Yaghoobi-Ershadi MR, Vatandoost H, Oshaghi MA, Ataei A, Anjamrooz M (2015) Fauna and larval habitat characteristics of mos¬qui¬toes in Neka County, northern Iran. J Ar¬thropod Borne Dis. 9: 253–266.
17. Nikookar SH, Moosa-Kazemi SH, Oshaghi MA, Vatandoost H, Yaghoobi-Ershadi MR, Enayati AA, Motevali-Haghi F, Zia¬pour SP, Fazeli-Dinan M (2015) Biodi¬ver¬sity of culicid mosquitoes in rural Neka township of Mazandaran Province, north¬ern Iran. J Vector Borne Dis. 52: 63–72.
18. Nikookar SH, Moosa-Kazemi SH, Oshaghi M, Yaghoobi-Ershadi M, Vatandoost H, Kianinasab A (2010) Species compo¬si¬tion and diversity of mosquitoes in neka County, Mazandaran Province, northern iran. J Arthropod-Borne Dis. 4: 26–34.
19. McCarroll L, Hemingway J (2002) Can in¬secticide resistance status affect parasite transmission in mosquitoes? Insect Bi¬ochem Mol Biol. 32: 1345–1351.
20. Labbe P, Berticat C, Berthomieu A, Unal S, Bernard C, Weill M, Lenormand T (2007) Forty years of erratic insecticide resistance evolution in the mosquito Cu¬lex pipiens. PLoS Genet. 3(11): e205.
21. Akıner MM, Ekşi E (2015) Evaluation of insecticide resistance and biochemical mech¬anisms of Culex pipiens L. in four localities of east and middle mediter¬ra-nean basin in Turkey. Int J Mosq Res. 2: 39–44.
22. Liu N (2015) Insecticide resistance in mos¬quitoes: impact, mechanisms, and research directions. Annu Rev Entomol. 60: 537–559.
23. Scott JG, Yoshimizu MH, Kasai S. Pyre-throid resistance in Culex pipiens mos-quitoes (2015) Pestic Biochem Physiol. 120: 68–76.
24. Zhang H, Meng F, Qiao C, Cui F (2012) Identification of resistant carboxylester¬ase alleles in Culex pipiens complex via PCR-RFLP. Parasit Vectors. 5: 209.
25. Zaim M, Mousavi SB, Baghestani MA, Aitio A (2017) An assessment of agricultural pesticide use in Iran, 2012–2014. J En-viron Health Sci Eng. 15: 1–8.
26. Naseri-Karimi N, Vatandoost H, Bagheri M, Chavshin AR (2015) Susceptibility status of Culex pipiens against deltame-thrin and DDT, Urmia County, West Azerbaijan Province, northwestern Iran. Asian Pac J Trop Dis. 5: S77–S79.
27. Ataie A, Moosa-Kazemi SH, Vatandoost H, Yaghoobi-Ershadi MR, Bakhshi H, Anjomruz M (2015) Assessing the sus-cep¬tibility status of mosquitoes (Diptera: Culicidae) in a dirofilariasis focus, north¬western Iran. J Arthropod Borne Dis. 9 (1): 7–21.
28. Fathian M, Vatandoost H, Moosa-Kazemi SH, Raeisi A, Yaghoobi-Ershadi MR, Oshaghi MA, Sedaghat MM (2015) Sus¬ceptibility of Culicidae mosquitoes to some insecticides recommended by WHO in a malaria endemic area of southeastern Iran. J Arthropod Borne Dis. 9(1): 22–34.
29. Salim-Abadi Y, Oshaghi MA, Enayati AA, Abai MR, Vatandoost H, Eshraghian MR, Mirhendi H, Hanafi-Bojd AA, Gorouhi MA, Rafi F (2016) High Insecticides Re¬sistance in Culex pipiens (Diptera: Cu¬licidae) from Tehran, Capital of Iran. J Arthropod Borne Dis. 10(4): 483–492.
30. Ghorbani F, Vatandoost H, Hanafi-Bojd AA, Abai MR, Nikookar H, Enayati AA (2018) High resistance of vector of West Nile virus, Culex pipiens Linnaeus (Dip¬tera: Culicidae) to different insecticides recommended by WHO in northern Iran. J Arthropod Borne Dis. 12: 24–30.
31. Brown FV, Logan RAE, Wilding CS (2019) Carbamate resistance in a UK pop¬ula-tion of the halophilic mosquito Ochlerotatus detritus implicates selection by agricultural usage of insecticide. Int J Pest Manag. 65: 284–292.
32. Safi NHZ, Ahmadi AA, Nahzat S, Zia-pour SP, Nikookar SH, Fazeli-Dinan M, Enayati AA, Hemingway J (2017) Evidence of metabolic mechanisms playing a role in multiple insecticides resistance in Anoph¬eles stephensi populations from Afghan¬istan. Malar J. 16: 100.
33. Ziapour SP, Kheiri S, Fazeli-Dinan M, Sahraei-Rostami F, Mohammadpour RA, Aarabi M, Nikookar SH, Sarafrazi M, Asgarian F, Enayati A (2017) Pyrethroid resistance in Iranian field populations of Rhipicephalus (Boophilus) annulatus. Pes¬tic Biochem Physiol. 136: 70–79
34. Enayati AA, Vatandoost H, Ladonni H, Town¬son H, Hemingway J (2003) Molec¬ular evidence for a kdr‐like pyrethroid re¬sistance mechanism in the malaria vec¬tor mosquito Anopheles stephensi. Med Vet Entomol. 17: 138–144.
35. Soltani A, Vatandoost H, Oshaghi MA, Ma¬leki-Rav¬asan N, Enayati AA, Asgar-ian F (2015) Resistance mechanisms of Anopheles stephensi (Diptera: Cu-licidae) to Temephos. J Arthropod Borne Dis. 9: 71–83.
36. Marcombe S, Mathieu RB, Pocquet N, Riaz MA, Poupardin R, Sélior S, Darriet F, Reynaud S, Yébakima A, Corbel V (2012) Insecticide resistance in the dengue vec¬tor Aedes aegypti from Martinique: dis¬tri¬bu¬tion, mechanisms and relations with en¬vi¬ronmental factors. PLoS One. 7: e30989.
37. Morou E, Dowd AJ, Rajatileka S, Steven A, Hemingway J, Ranson H, Paine M, Vontas J (2010) A simple colorimetric as¬say for specific detection of glutathione-s transferase activity associated with DDT resistance in mosquitoes. PLoS Negl Trop Dis. 4: e808.
38. Gunasekaran K, Muthukumaravel S, Sahu S, Vijayakumar T, Jambulingam P (2011) Glutathione S Transferase activity in In¬dian vectors of malaria: a defense mech¬anism against DDT. J Med Entomol. 48: 561–569.
39. Enayati AA, Ladonni H (2006) Biochemi-cal assay baseline data of permethrin re¬sistance in Anopheles stephensi (Dip-tera: Culicidae) from Iran. Pakistan J Biol Sci. 9: 1265–1270.
40. Brooke B, Kloke G, Hunt R, Koekemoer L, Tem E, Taylor M, Small G, Hem¬ing¬way J, Coetzee M (2001) Bioassay and biochemical analyses of insecticide re-sistance in southern African Anopheles funestus (Diptera: Culicidae). Bull Ento¬mol Res. 91(4): 265–272.
41. Fonseca-González I, Quiñones ML, McAl¬lister J, Brogdon WG (2009) Mixed-func¬tion oxidases and esterases associated with cross-r esistance between DDT and lambda-cyhalothrin in Anopheles dar¬lingi Root 1926 populations from Colombia. Mem Inst Oswaldo Cruz. 104: 18–26.
42. Achaleke J, Martin T, Ghogomu RT, Vais¬sayre M, Brévault T (2009) Ester-ase‐ mediated resistance to pyrethroids in field populations of Helicoverpa armigera (Lep¬idoptera: Noctuidae) from Central Africa. Pest Manag Sci. 65: 1147–1154.
43. Zayed ABB, Szumlas DE, Hanafi HA, Fryauff DJ, Mostafa AA, Allam KM, Brogdon WG (2006) Use of bioassay and microplate assay to detect and measure insecticide resistance in field popu¬la¬tions of Culex pipiens from filariasis endemic areas of Egypt. J Am Mosq Control Assoc. 22(3): 473–482.
44. Che-Mendoza A, Penilla RP, Rodríguez DA (2009) Insecticide resistance and gluta¬thi¬one S-transferases in mosquitoes: a review. Afr J Biotechnol. 8: 1386–1397.
45. Enayati AA, Asgarian F, Amouei A, Sharif M, Mortazavi H, Boujhmehrani H, Hem¬ingway J (2010) Pyrethroid insecticide re¬sistance in Rhipicephalus bursa (Acari, Ixodidae). Pestic Biochem Physiol. 97: 243–248.
46. Bourguet D, Capela R, Raymond M (1996) An insensitive acetylcholinesterase in Cu¬lex pipiens (Diptera: Culicidae) from Por¬tugal. J Econ Entomol. 89: 1060–1066.
47. Pethuan S, Jirakanjanakit N, Saengtharatip S, Chareonviriyaphap T, Kaewpa D, Rongnoparut P (2007) Biochemical stud¬ies of insecticide resistance in Aedes (Steg¬omyia) aegypti and Aedes (Stegomyia) al¬bopictus (Diptera: Culicidae) in Thailand. Trop Biomed. 24: 7–15.
48. Emtithal AE-S, Thanaa AE-B (2012) Ef-ficacy of some insecticides on field pop-ulations of Culex pipiens (Linnaeus) from Egypt. J Basic Appl Zool. 65: 62–73.
49. Tabbabi A, Daaboub J, Laamari A, Cheikh R, Cheikh H (2017) Pirimiphos-Methyl resistance status of field populations of Culex pipiens (Diptera: Culicidae) from Grand Tunis area, northeast Tunisia. He¬reditary Genet. 6(1): 175.
50. Alout H, Labbe P, Berthomieu A, Ma-koundou P, Fort P, Pasteur N, Weill M (2016) High chlorpyrifos resistance in Culex pipiens mosquitoes: strong syn¬er-gy between resistance genes. Heredity (Edinb). 116: 224–231.
51. Bkhache M, Tmimi FZ, Charafeddine O, Faraj C, Failloux AB, Sarih Mh (2016) First report of L1014F-kdr mutation in Culex pipiens complex from Morocco. Parasit Vectors. 9: 644.
52. Xu W, Liu S, Zhang Y, Gao J, Yang M, Liu X, Tao L (2017) Cypermethrin re-sistance conferred by increased target insensitivity and metabolic detox¬ifica-tion in Culex pipiens pallens Coq. Pestic Bi¬ochem Physiol. 142: 77–82.
53. Johnson BJ, Fonseca DM (2016) Insec¬ti-cide resistance alleles in wetland and res¬idential populations of the West Nile virus vector Culex pipiens in New Jersey. Pest Manag Sci. 72: 481–488.
54. Bagheri M, Terenius O, Oshaghi MA, Mota-zakker M, Asgari S, Dabiri F, Vatan-doost H, Mohammadi Bavani M, Chavshin AR (2015) West Nile virus in mosquitoes of Iranian wetlands. Vector Borne Zoon¬o¬tic Dis. 15: 750–754.
55. Hemingway J (1998) Field and Labor¬a¬to-ry Manual for the Mechanistic Detection of Insecticide Resistance in Insects. World Health Organization, Geneva.
56. Penilla PR, Rodriguez AD, Hemingway J, Torres JL, Arredondo-Jimenez Ji, Ro-dri¬guez MH (1998) Resistance manage-ment strategies in malaria vector mosquito con¬trol. Baseline data for a large‐scale field trial against Anopheles albimanus in Mex¬ico. Med Vet Entomol. 12: 217–233.
57. Reid MC, McKenzie FE (2016) The con-tribution of agricultural insecticide use to increasing insecticide resistance in Afri¬can malaria vectors. Malar J. 15: 107.
58. Low VL, Chen CD, Lee HL, Tan TK, Chen CF, Leong CS, Lim YAL, Lim PE, Norma-Rashid Y, Sofian-Azirun M (2013) Enzymatic characterization of insecti-cide resistance mechanisms in field popula¬tions of Malaysian Culex quinquefascia¬tus Say (Diptera: Culicidae). PloS one. 8: e79928.
59. Shen B, Dong HQ, Tian H-S, Ma L, Li XL, Wu GL, Zhu CL (2003) Cyto-chrome p450 genes expressed in the deltame¬thrin-susceptible and-resistant strains of Culex pipiens pallens. Pestic Biochem Physiol. 75: 19–26.
60. Wan-Norafikah O, Nazni WA, Lee HL, Zainol-Ariffin P, Sofian-Azirun M (2010) Permethrin resistance in Aedes aegypti (Linnaeus) collected from Kuala Lum¬pur, Malaysia. J Asia Pac Entomol. 13: 175–182.
61. Wan-Norafikah O, Nazni WA, Lee HL, Zainol-Ariffin P, Sofian-Azirun M (2013) Susceptibility of Aedes albopictus Skuse (Diptera: Culicidae) to permethrin in Kua¬la Lumpiur, Malaysia. Asian Biomed. 7: 51–62.
62. Vatandoost H, Zaim M, Hanafi-Bojd AA, Mousavi B, Nikpour F (2015) Supply of chemical pesticides in agricultural in Iran (2015–2016). institute for environmental research, Tehran university of Medical Sciences.
63. Yadouleton AWM, Asidi A, Djouaka RF, Braïma J, Agossou CD, Akogbeto MC (2009) Development of vegetable farm-ing: a cause of the emergence of insecti¬cide resistance in populations of Anoph¬eles gambiae in urban areas of Benin. Malar J. 8: 103.
64. Sarkar M, Bhattacharyya I, Borkotoki A, Goswami D, Rabha B, Baruah I, Sri-vasta¬va R (2009) Insecticide resistance and de¬toxifying enzyme activity in the prin¬cipal bancroftian filariasis vector, Culex quinquefasciatus, in northeastern India. Med Vet Entomol. 23: 122–131.
How to Cite
Nikookar SH, Fazeli-Dinan M, Ziapour SP, Ghorbani F, Salim-Abadi Y, Vatandoost H, Hanafi-Bojd AA, Enayati A. First Report of Biochemical Mechanisms of Insecticide Resistance in the Field Population of Culex pipiens (Diptera: Culicidae) From Sari, Mazandaran, North of Iran. J Arthropod Borne Dis. 13(4):378-390.
Original Article