Research Article, J Genet Disor Genet Rep Vol: 6 Issue: 2

Pirimiphos-methyl Resistance in Culex pipiens (Diptera: Culicidae) in Southern Tunisia

Tabbabi Ahmed^1*, Daaboub Jaber^1,2, Laamari Ali¹, Ben Cheikh Raja¹ and Ben Cheikh Hassen¹

¹Laboratory of Genetics, Faculty of Medicine of Monastir, Monastir University, Monastir-5019, Tunisia

²Department of Hygiene and Environmental Protection, Ministry of Public Health, Bab Saadoun, Tunis-1006, Tunisia

*Corresponding Author : Tabbabi A, Laboratory of Genetics
Faculty of Medicine of Monastir, Monastir University, Monastir- 5019, Tunisia
Tel: +216 97 085 424
E-mail: [email protected]

Received: April 17, 2017 Accepted: May 02, 2017 Published: May 08, 2017

Citation: Tabbabi A, Daaboub J, Laamari A, Ben Cheikh R, Ben Cheikh H (2017) Pirimiphos-methyl Resistance in Culex pipiens (Diptera: Culicidae) in Southern Tunisia. J Genet Disor Genet Rep 6:2.doi: 10.4172/2327-5790.1000155

Abstract

Keywords: Culex pipiens; Primiphos methyl; Resistance; Insensitive AChE; Esterases; Southern Tunisia

Introduction

Culex pipiens is showing locally invasive or interfering with everyday life, tourism and economic activities [1-3]. Many control methods have been tested. Many of these actions have had unintended negative or collateral consequences, and none has shown lasting results. This mosquito is favored by the fact that it is rapidly reproduced in large numbers and is highly adaptive. In particular, it has demonstrated the ability to adapt to many insecticides, which are sometimes only temporarily or partially effective [4-9], while affecting non-target species that were precisely predators of the mosquito.

The majority of synthetic insecticides are compounds that inactivate a vital target of the nervous system, resulting in death of the insect. Unfortunately, the populations of treated insects evolve very rapidly towards resistance [10,11]. In order to combat resistance, the use of insecticidal molecules directed against another target may prove effective for a more or less short time depending on the frequency of the occurrence of new adaptive mutations.

After the massive use of DDT (organochlorine), resistance was very quickly selected in the 1960s. Two other families of insecticides were then used: organophosphates (OP) and carbamates (CX). These molecules inhibit AChE, the enzyme responsible for the hydrolysis of acetylcholine in cholinergic synapses. This inhibition prolongs the nerve impulse, which leads rapidly to the death of the insect by tetany. In the mosquito Culex pipiens, two main mechanisms of resistance to these insecticides have been demonstrated: The first corresponds to an increased detoxification of OPs by carboxylesterases that trap or degrade them [12]. In the case of resistance, these esterases are overproduced by a process of amplification or overexpression of the genes that encode them [13,14]. The second mechanism corresponds to a modification of the target, AChE, which reduces its affinity for insecticides and leads to its insensitivity [15,16].

Pirimiphos-methyl is an organophosphorus insecticide used in a wide range of vectors control and is being considered by WHO for addition to potable water in containers as a mosquito larvicide treatment. Recently, results have shown that pyrimiphos-methyl alone has little effect on larvae of Aedes aegypti whereas its efficacy on adults is particularly good [17]. The aim of this study is to investigate the pirimiphos-methyl resistance of three populations of Culex pipiens in southern Tunisia.

Materials and Methods

Mosquitoes

Three Culex pipiens samples were collected at preimaginal stages from breeding sites in 3 localities of southern Tunisia between March 2002 and October 2005 (Table 1 and Figure 1). Comparisons of different bioassays were made with a reference strain called S-Lab [18]. SA2 and SA5 are two resistant strains homozygous for Ester2 and Ester5, respectively [19]. They were used for correct identification of detected esterases.

Bioassays and data analysis

Assays were realized according to Raymond et al. [20], using ethanol solutions of pirimiphos methyl (99% [AI]), brought from laboratory Dr Ehrenstorfer, Germany, and propoxur (99.9% [AI], Bayer AG, Leverkusen , Germany). The effect on OPs resistance of 2 synergists, the DEF (98% [AI], Chem Service, England), and the Pb (94% [AI], Laboratory Dr Ehrenstorfer, Germany) , was studied. Data were analysed by probit analysis [21] using a BASIC program [22].

Over-produced esterases

Esterases were characterized on homogenates of adult thorax and abdomen using the method of Pasteur et al. (1981,1988) [23,24].

Results and Discussion

S-Lab was the only strain that has linear dose-mortality response. With the exception of susceptibility to propoxur, the susceptibility of the third population (sample # 3) of Culex pipiens to pirimiphosmethyl was not considered due to control-level mortality. Both other studied samples were resistant to pirimiphos methyl. This result can be explained by the frequent control of these areas against this mosquito (frequent monitoring by the pirimiphos methyl, Table 1) because of the alerted by inhabitants about the mosquito bites. The RR₅₀ was 6.9 in sample # 1 and 27 in sample # 2 (Table 2).

Table 1: Geographic origin of Tunisian populations, breeding site characteristics, and insecticide control.

Table 2: Pirimiphosmethyl resistance characteristics of Tunisian Culex pipiens in presence and absence of synergists DEF and Pb.

The tolerance to pirimiphos methyl in S-Lab and the two field samples decreased significantly with the addition of the DEF (SR₅₀>1, p<0.05) (Table 2). No synergistic action was recorded by the DEF in samples # 1 and 2. Therefore, esterases (and/or GST) had no effect on the pirimiphos methyl resistance in both samples. These results confirm those found by Tabbabi et al. [9] which showed the noninvolvement of esterases (and/or GST) in the resistance of Culex pipiens strain subjected to temephos (organophosphorus) selection pressure. The tolerance to pirimiphos-methyl decreased significantly with the addition of the Pb in S-Lab (SR₅₀=7.24, p<0.05) and in both samples, and the SR was not significantly higher than that recorded in S-Lab (Table 2). Therefore, the CYP450 had no effect on the pirimiphos methyl resistance in both populations. Other previous studies on Culex pipiens from Tunisia [4], Culex quinquefasciatus from Venezuela [25] and USA [26], and Musca domestica [27] showed the minor role of oxydases in resistance to chlorpyrifos insecticide (organophosphorus).

Mortality caused by propoxur was 1% in samples # 2, 46% in samples # 1 and 100% in sample # 3. A significantly correlation was observed between the mortality due to propoxur and the LC₅₀ of pirimiphos methyl (P<0.01) showed an insensitive AChE. These results are in agreement with previous studies [9,28,29] which showed the major role of insensitive AChE in resistance to OPs insecticides. It was not possible to carry out toxicological tests on population # 3 because of control-level mortality but as it was even possible to keep samples and to study its AChE and esterases activities. The latter were very sensitive to propoxur. This can be explained by the absence of control actions in this region (occasionally by the permethrin).

With the exception of the sample # 3, all the studied samples showed the presence of one or more esterases in their electrophoretic profiles. The frequency of 0.03 and 0.56 were those of A4-B4 (and / or A5-B5) esterases found in samples # 1 and 2, respectively. The frequency of 0.03 was those of The B12 esterases observed in samples # 2. The frequency of C1 esterases was 0.28 in sample # 1. The A2B2 esterases were observed just in sample # 2 with a frequency of 0.11. Our results confirms the previous studies which showed the major implication of esterases in the resistance to OPs insecticides [4,25,27,30-36].

Acknowledgements

This work was kindly supported by the Ministry of Higher Education and Scientific Research of Tunisia by funds allocated to the Research Unit (Génétique 02/UR/08-03) and by DHMPE of the Minister of Public Health of Tunisia. We are very grateful to S. Ouanes, for technical assistance, A. Ben Haj Ayed and I. Mkada for help in bioassays, S. Saïdi, Tunisian hygienist technicians for help in mosquito collecting, and M. Nedhif and M. Rebhi for their kind interest and help.

References

1. Vinogradova EB (2000) Culex pipiens Mosquitoes: Taxonomy, Distribution, Ecology, Physiology, Genetics, Applied Importance, and Control. Pensoft Publishers, Sofia, pp: 250.
2. Abdel-Hamid YM, Soliman MI, Allam KM (2009) Spatial distribution and abundance of culicine mosquitoes in relation to the risk of Filariasis transmission in El Sharqiya Governorate, Egypt. Egypt Acad J biology Sci 1: 39-48.
3. . Abdel-Hamid YM, Soliman MI, Allam KM (2011) Mosquitoes (Diptera: Culicidae) in relation to the risk of disease transmission in El Ismailia governorate, Egypt. J Egypt Soc Parasitol 41: 109-118.
4. Cheikh BH, Ali-Houas BZ, Marquine M, Pasteur N (1998) Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia (North Africa). J Med Entomol 35: 251-260.
5. Cheikh BR, Berticat C, Berthomieu A, Pasteur N, Cheikh BH, et al. (2008) Characterization of a Novel High-Activity Esterase in Tunisian Populations of the Mosquito Culex pipiens. J Econ Entomo: 484-491.
6. Cheikh BR, Berticat C, Berthomieu A, Pasteur N, Cheikh BH, et al. (2009) Genes conferring resistance to organophosphorus insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia. J Med Entomo 46: 523-530.
7. Daaboub J, Cheikh BR, Lamari A, Jha BI, Feriani M, et al. (2008) Resistance to pyrethroid insecticides in Culex pipiens pipiens (Diptera: Culicidae) from Tunisia. Acta Trop 107: 30-36.
8. Tabbabi A, Daaboub J, Laamari A, Cheikh BH (2016) New Esterases Amplification Involved in Organophosphate Resistance in Culex Pipiens Mosquitoes from Tunisia. The Journal of Middle East and North Africa Sciences 2: 1-2.
9. Tabbabi A, Laamari A, Daaboub J, Jha BI, Cheikh BH (2017) Cross-Resistance to Pyrethroid and Organophosphorus Insecticides Induced by Selection with Temephos in the Potential Mosquito Vector of West Nile Virus (Culex Pipiens) from Tunisia. The Journal of Middle East and North Africa Sciences 3: 25-29.
10. Kioulos I, Kampouraki A, Morou E, Skavdis G, Vontas J (2013) Insecticide resistance status in the major West Nile virus vector Culex pipiens from Greece. Pest Manag Sci Doi 70: 623-627.
11. El Lalami OA, El-Akhal F, El Amri N, Maniar S, Faraj C (2014) Etat de la résistance du moustique Culex pipiens vis-à-vis du téméphos au centre du Maroc. Bull Soc Pathol Exot 107: 194-198.
12. Raymond M, Berticat C, Weill M, Pasteur N, Chevillon C (2001) Insecticide resistance in the mosquito Culex pipiens: what we have learned about adaptation? Genetica 112-113: 287-296.
13. Guillemaud T, Rooker S, Pasteur N, Raymond M (1996) Testing the unique amplification event and the worldwide migration hypothesis of insecticide resistance genes with sequence data. Heredity 77: 535-543.
14. Guillemaud T, Makate N, Raymond M, Hirst B, Callaghan A (1997) Esterase gene amplification in Culex pipiens. Insect Mol Biol 6: 319-327.
15. aylor M, Feyereisen R (1996) Molecular biology and evolution of resistance to toxicants. Molecular Biology and Evolution 13: 719-734.
16. Weill M, Fort P, Berthomieu A, Dubois MP, Pasteur N, et al. (2002) A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and in non-homologous to the ace gene in Drosophila. Proc R Soc Lond B 269: 2007-2016.
17. Chung YK, Lam-Phua SG, Chua YT, Yatiman R (2001) Evaluation of biological and chemical insecticide mixture against Aedes aegypti larvae and adults by thermal fogging in Singapore. Medical and Veterinary Entomology 15: 321-327.
18. Georghiou GP, Meltcalf RL, Gidden FE (1966) Carbamate resistance in mosquitoes. Selection of Culex pipiens fatigans Wied for resistance to Baygon. Bull WHO 35: 691-708.
19. Berticat C, Boquien G, Raymond M, Chevillon C (2002) Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes. Genet Res 83: 189-196.
20. Raymond M, Fournier D, Bride JM, Cuany A, Bergé JB, et al. (1986) Identification of resistance mechanisms in Culex pipiens (Diptera: Culicidae) from southern France: insensitive acetylcholinesterase and detoxifying oxidases. J Econ Entomol 79: 1452.
21. Finney DJ (1971) Probit Analysis. Cambridge University Press.
22. Raymond M, Foumier D, Bergé JB, Cuany A, Bride JM, et al. (1985) Single-mosquito test to determine genotypes with an acetylcholinesterase insensitive to inhibition to propoxur insecticide. J Am Mosq Control Assoc l: 425-427.
23. Pasteur N, Iseki A, Georghiou GP (1981) Genetic and biochemical studies of the highly active esterases A′and B associated with organophosphate resistance in mosquitoes of the Culex pipiens complex. Biochemical Genetics 19: 909-919.
24. Pasteur N, Pasteur G, Bonhomme F, Britton- Davidian J (1988) Practical Isozyme Genetics. Ellis Horwood.
25. Bisset, JA, Rodriguez MM, Diaz C, Soca A (1999) Characterization of resistance to organophosphate insecticides, carbamates, and pyrethroids in Culex quinquefasciatus from the state of Miranda, Venezuela. Rev Cubana Med Trop 51: 89-94.
26. Liu H, Xu Q, Zhang L, Liu N (2005) Chlorpyrifos resistance in mosquito Culex quinquefasciatus. J Med Entomol 42: 815-820.
27. Liu N, Yue X (2000) Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae). J Econ Entomol 93: 1269-1275.
28. Pasteur N, Marquine M, Cheikh BH, Bernard C, Bourguet D (1999) A new mechanism conferring unprecedented high resistance to chlorpyrifos in Culex pipiens (Diptera : Culicidae). J Med Entomol 36: 794-802.
29. Weill M, Lutfalla G, Mogensen K, Chandre F, Berthomieu A, et al. (2003) Comparative genomics: Insecticide resistance in mosquito vectors. Nature (London) 423: 136-137.
30. Whyard S, Downe AFR, Walker VK (1994) Isolation of an esterase conferring insecticide resistance in the mosquito Culex tarsalis . Insect Biochem Mol Biol 24: 819-827.
31. Tomita T, Kono Y, Shimada T (1996) Chromosomal localization of amplified esterase genes in insecticide resistant Culex mosquitoes. Insect Biochem Mol Biol 26: 853-857.
32. Hemingway J, Karunaratne SH (1998) Mosquito carboxylesterases: A review of the molecular biology and biochemistry of a major insecticide resistance in mechanism. Med Vet Entomol 12: 1-12.
33. Hemingway J, Hawkes N, Prapanthadara L, Jayawardenal KG, Ranson H (1998) The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance. Phil trans R Soc Lond B Biol Sci 353: 1695-1699.
34. Chevillon C, Raymond M, Guillemaud T, Lenormand T, Pasteur N (1999) Population genetics of insecticide resistance in the mosquito Culex pipiens. Biol J Ann Soc 68: 147-157.
35. Raymond M, Chevillon C, Guillemaud T, Lenormand T, Pasteur N (1998) An overview of the evolution of overproduced esterases in the mosquito Culex pipiens. Phil Trans R Soc Lond B 353: 1707-1711.
36. Wei SH, Clark AG, Syvanen M (2001) Identification and cloning of a key insecticide-metabolizing glutathione -S- transferase (MdGST-6A) from a hyper insecticide-resistant strain of the house fly Musca domestica. Insect Biochem. Mol Bio 31: 1145-1153.