Research Article - (2017) Volume 6, Issue 2

Study Confirming Resistance to Fenitrothion of Culex pipiens (Diptera: Culicidae) from Central Tunisia

Ahmed Tabbabi1*, Jaber Daaboub1,2, Ali Laamari1, Raja Ben Cheikh1, Ibtissem Ben Jha1 and Hassen Ben Cheikh1
1Laboratory of Genetics, Faculty of Medicine of Monastir, Monastir University, 5019, Monastir, Tunisia
2Department of Hygiene and Environmental Protection, Ministry of Public Health, 1006, Bab Saadoun, Tunis, Tunisia
*Corresponding Author: Ahmed Tabbabi, Laboratory of Genetics, Faculty of Medicine of Monastir, Monastir University, 5019, Monastir, Tunisia, Tel: 73500276, Fax: 73500278 Email:

Abstract

Five natural populations of Culex pipiens were taken as larvae in the central Tunisia to evaluate their resistance level of fenitrothion. Our study showed that all samples were resistant to fenitrothion at LC50. The RR50 ranged from 9.2 in sample # 2 to 59.2 in sample # 5. Starch electrophoresis detected the overproduced esterases in all studied samples. The most frequent esterase A2B2 was detected in samples # 5 with a frequency of 31%. Three other esterases were detected in samples # 1, 2, 3, and 4: A4-B4 and/or A5-B5, A12, and C1. Synergists showed that the involvement of CYTP450 in the resistance to fenitrothion (OP) is not neglected. Cross-resistance of fenitrothion and propoxur was detected indicate the involvement of target site alteration (AChE1) in fenitrothion resistance. It should be noted that study of the polymorphism of AChE 1 will be of great importance.

Keywords: Culex pipiens; Fenitrothion; Propoxur; Resistance; Esterases; AChE1; Central Tunisia

Introduction

In the absence of vaccine and therapeutic treatment, the control of mosquitoes by the use of chemical insecticides remains the preferred weapon. Unfortunately, the intensive and repeated use of the same insecticides (especially pyrethroids and organophosphates) for more than forty years has led to the selection and diffusion of resistance on a global scale [1-4]. The management of these resistances becomes problematic because very few new insecticides are developed for public health. Indeed, almost all the insecticides used against mosquitoes come from the agricultural market, because of the lack of investment by the agrochemical companies in a public health market considered too narrow and not very profitable.

Insecticide resistance is now seen by the World Health Organization (WHO), as a major obstacle to the control of mosquito-borne diseases. Resistance is likely to contribute to the re-emergence of arboviruses because of the inability to maintain effective control of mosquito populations.

The objectives of this work are multiple: to identify areas where insecticide resistance may challenge the control of mosquito vectors and to provide recommendations to the government to improve resistance management and to encourage the deployment of alternative control methods. We tested the fenitrothion insecticide which is an organophosphorous compounds largely used in Culex pipiens control.

Materials and Methods

Mosquitoes

We used eihgt colonies of Culex pipiens in this study: Five natural populations were taken as larvae in the central Tunisia (Table 1 and Figure 1), a sensitive strain called S-Lab to do comparisons with resistant strains, and two resistant strains SA2 and SA5 characterized by the presence of A2B2 and A5B5, respectively. The two last strains were used as reference in starch electrophoresis to identify overproduced esterases of collected populations.

hereditary-genetics-Tunisian-population

Figure 1: Geographic origin of Tunisian population.

Code Locality Breeding sites Date of collection Mosquito control(used insecticides) Agricultural pest control
1 Kalaakebira River July. 2003 Occasional (F,  Pm,  P, D) None
 2 Monastir Ditch Aug. 2003 Rare (C,F) Yes
3 Moknine Water pond Aug. 2003 Very frequent (C) Yes
4 HajebLaayoun River July. 2004 None Yes
 5 Sbiba River Sept. 2004 Rare (Pm, P) Yes

C:Chlorpyrifos; T: Temephos; Pm: Pirimiphosmethyl; F:Fenitrithion; P:Permethrin; D:Deltamethrin

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

Rearing of Culex pipiens in the laboratory

Larvae were directly transferred to the laboratory and putted in plastic basins containing water and rabbit crop which serves as food. Adults were transferred to cages and both sexes were fed on sugar water. Only females then blood fed on birds to be able to lay.

Chemical insecticides

Two insecticides were used for bioassays: the organophosphates fenitrothion (98.5% [AI]), brought from laboratory Dr Ehrenstorfer, Germany) and the carbamate propoxur (99.9% [AI], Bayer AG, Leverkusen, Germany).

Bioassays and data analysis

Third and fourth instar larvae were used to do bioassays according standard protocol of the World Health Organization (WHO). [5]. Brieftly, five insecticide concentrations were used for each assay and five replicates for each concentration. Lethal concentrations (LCs) and all related data were calculated via probit analysis [6]. The sensitive strain S-Lab was used as reference to do analysis.

Mechanisms involved in the resistance to fenitrothion

We used two synergists to identify the role of esterases, GST, and CYTP450 in the recorded resistance. The only exception compared to previous assays was to add 0.5 ml of the maximum sub-lethal concentration of an esterase inhibitor, S, S, S-tributylphosphorotrithioate, (0.5 μg/ml) to each cup with 0.5 ml of insecticide and piperonyl butoxide (pb), an inhibitor of mixed function oxidases. The addition should be done 4 hours before the start of the bioassays. Esterase phenotypes were established by starch electrophoresis (TME 7.4 buffer system) as described by Pasteur et al. [7,8] using adults specimens.

Results

Fenitrothion resistance

The S-Lab was the only strain with accepted linearity of the dosemortality response (p<0.05). As indicate in Table 2, all samples were resistant to fenitrothion at LC50. The RR50 ranged from 9.2 in sample # 2 to 59.2 in sample # 5.

Population Fenitrothion Fenitrothion +DEF Fenitrothion +Pb
LC50 in µg/l
(a)
Slope
± SE
RR50
(a)
LC50 in µg/l
(a)
Slope
± SE
RR50
(a)
SR50
(a)
RSR LC50  in µg/l
(a)
Slope
± SE
RR50
(a)
SR50
(a)
RSR
S-Lab 3.3
(1.7-6.3)
3.19
± 0.94
- 1.3
(1.0-1.6)
2.43
± 0.26
- 2.5
(1.2-5.2)
- 2.8
(0.18-44)
1.44
± 0.93
- 1.1
(0.34-3.9)
-
 1-kalaa Kebira 51
(26-98)
1.52
± 0.3
15.2
(7.18-32.3)
17
(8.5-36)
1.07
± 0.19
13.3
(8.8-19.9)
2.8
(1.8-4.5)
1.1 14
(12-17)
1.6
± 0.11
5.0
(2.0-12.4)
3.5
(2.4-5.1)
3.0
2-Monastir 30
(25-37)
1.27
± 0.08
9.2
(4.8-17.4)
21
(12-35)
1.04
± 0.12
16.1
(11.5-22.5)
1.4
(1.1-1.8)
0.57 24
(19-30)
1.23*
± 0.08
8.3
(3.2-21.0)
1.2
(1.1-1.5)
1.1
3-Moknine 115
(45-291)
0.93
± 0.18
34.4
(16.8-70.4)
38
(15-97)
0.81
± 0.15
28.8
(19.4-42.9)
2.9
(1.9-4.6)
1.2 105
(45-249)
0.63
± 0.08
36.4
(14.7-90.3)
1.0
(0.74-1.6)
0.94
4-Hajeb laayoun 103
(63-169)
1.56
± 0.25
30.9
(15.2-62.5)
17
(9.6-31)
1.32
± 0.29
13.1
(8.8-19.5)
5.9
(3.9-8.9)
2.3 12
(9.1-17)
1.76
± 0.26
4.2
(1.3-13.0)
8.4
(5.6-12.8)
7.3
5-Sbiba 198
(103-377)
2.54
± 0.64
59.2
(24.3-144)
247
(111-548)
1.82
± 0.43
185
(101-337)
0.80
(0.36-1.7)
0.32 61
(46-81)
1.89
± 0.19
21.3
(8.5-53.6)
3.2
(1.7-5.8)
2.8

(a), 95% CI;  * The log dose-probit mortality responses is parallel to that of  S-Lab. RR50, resistance ratio at LC50 (RR50=LC50 of the population considered/LC50 of Slab); SR50, synergism ratio (LC50 observed in absence of synergist/LC50 observed in presence of synergist). RR and SR considered significant (P<0.05) if their 95%CI did not include the value 1. RSR, relative synergism ratio (RR for insecticide alone/RR for insecticide plus synergist).

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

The tolerance to fenitrothion insecticide decreased in S-Lab (SR50=2.5, p<0.05) and 4 among 5 field samples when DEF were added to bioassays (Table 2) despite SR of all samples were not significantly higher than that recorded in S-Lab. That’s why no detoxification role in resistance has been given to EST (and/or GST). The fenitrothion resistance in S-Lab did not change after addition of PB synergist (SR50 = 1.16, p<0.05). As showed in Table 2, only the SR50 of sample # 4 was significantly higher than that recorded in S-Lab. That’s why detoxification caused by CYTP450 played a minor role in recorded resistance in sample # 4 (RR50=4.2, p<0.05, RSR=7.3).

Cross-resistance fenitrothion/Propoxur

Mortality caused by propoxur were 72%, 41%, 11%, 79% and 12% for samples # 1, 2, 3, 4 and 5, respectively. A strong correlation were recorded between mortality due to propoxur and LC50 of fenitrothion [Spearman rank correlation, (r) = -0.69 (P<0.01)].

Esterase’s activities

Starch electrophoresis detected the overproduced esterases in all studied samples. The most frequent esterase A2B2 was detected in samples # 5 with a frequency of 31%. Three other esterases were detected in samples # 1, 2, 3, and 4: A4-B4 and/or A5-B5, A12 and C1.

Discussion

The status of fenitrothion resistance in Culex pipiens was studied in central Tunisia to have data on levels of resistance of this species to this insecticide. Our study showed different levels of resistance to fenitrothion in the five population of Culex pipiens collected in central Tunisia despite almost all the insecticides used against mosquitoes come from the agricultural market (Table 1). Cross-resistance of public health and agriculture insecticides could explain the recorded resistance in all studied populations. It should be noted that fenitrothion resistance levels recorded in other areas of the wolrd is lower than recorded in Tunisia [9,10].

Our synergist study showed the non-involvement of EST (and/or GST) in fenitrothion resistance, although starch electrophorsis showed several overproduced esterases in all studied samples. These enzymes are probably involved in recorded resistance. Indeed, the action of the synergist employed in the toxicological tests (DEF) does not always result in the inhibition of esterases and GSTs. Many previous studies confirmed the association between resistance to OPs insecticides and overproduced esterases and/or GST [11-15]. Similar results have been reported in Malaysian Culex quinquefasciatus [16] and Malaysian Aedes aegypti [17].

The involvement of CYTP450 in the resistance to fenitrothion (OP) could explain its involvement in the resistance to chlorpyrifos (OP) on Culex pipiens from Tunisia [11]. However, previous studies showed the strong correlation between oxydases and pyrethroid resistance in Malaysian Culex quinquefasciatus [18], Aedes albopictus [19,20], and Aedes aegypti [21].

A strong correlation was recorded between mortality due to propoxur and LC50 of fenitrothion (OP) indicating the involvement of AChE 1 in the recorded resistance. Similar studies were recorded by Labbé et al. [22] in several mosquito species.

The involvement of both metabolic mechanisms and target site alteration in multiple insecticide resistance has been reported in Culex quinquefasciatus from many parts of the world [23-25]. Furthermore, reduced insecticide penetration has been recorded in Culex quinquefasciatus [26].

Conclusion

In conclusion, it would be interesting to develop a spatial analysis using geographic information systems (GIS) to correlate the presence of different insecticides, treatments and tolerance of populations to insecticides throughout the country.

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. Ben Cheikh H, Marrakchi M, Pasteur N (1995) Mise en évidence d’une très forte résistance au chlorpyrifos et à la perméthrine chez les moustiques Culex pipiens de Tunisie. Arch. Institut Pasteur Tunis 72 : 7-12.
  2. Weill M, Fort P, Berthomieu A, Dubois MP, Pasteur N (2002) A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila. Proc Roy SocLond (Biol. Sci) 269: 2007-2016.
  3. Russel RJ, Claudianos C, Campbell PM, Horne I, Sutherland TD, et al. (2004) Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides. Pestic. Biochem. physiol 79: 84-93.
  4. Alout H, Berthomieu A, Hadjivassilis A, Weill M (2007) A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culexpipiens mosquitoes from Cyprus. Insect Biochem. Mol. Biol 37: 41-47.
  5. WHO (1963) Insecticide resistance and vector control: 13th Report of the WHO Expert Committee on Insecticides. WHO Tech Rep Ser 265.
  6. 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.
  7. Pasteur N, Iseki A, Georghiou GP (1981) Genetic and biochemical studies of the highly active esterasesA′and B associated with organophosphate resistance in mosquitoes of the Culexpipiens complex. Biochemical Genetics 19: 909–919.
  8. Pasteur N, Pasteur G, Bonhomme F, Britton-Davidian J (1988) Practical isozyme genetics. Ellis Horwood, Chichester, UK.
  9. Bracco JE, Barata JMS, Marinotti O (1999) Evaluation of insecticide resistance and biochemical mechanisms in a population of Culexquinquefasciatus (Diptera: Culicidae) from Sào Paulo, Brazil. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 94: 115-120.
  10. Sathantriphop S, Paeporm P, Supaphathom K (2006) Detection of insecticide resistance status in Culexquinquefasciatus and Aedesaegypti to four majorgroups of insecticides. TropBiomed 23: 97–101.
  11. Ben Cheikh H, Haouas-Ben Ali Z, Marquine M, Pasteur N (1998) Resistance to organophosphorus and pyrethroid insecticides in Culexpipiens (Diptera: Culicidae) from Tunisia. J MedEntomol 35: 251-260.
  12. Liu H, Xu Q, Zhang L, Liu N (2005) Chlorpyrifos resistance in mosquito Culexquinquefasciatus. J. Med. Entomol 42: 815-820.
  13. Huang HS, Hu NT, Yao YE, Wu CY, Chiang SW, et al. (1998) Molecular cloning and heterologous expression of a glutathione-s-tranferase involved in insecticide resistance from the diamondback moth Plutellaxylostella. Insect Biochem Mol. Biol 28: 651-658.
  14. Weill 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 Muscadomestica. Insect Biochem. Mol. Biol 31: 1145-1153.
  15. Hemingway J, Hawkes NJ, McCarroll L, Ranson H (2004) The molecular basis of insecticide resistance in mosquitoes. Insect Biochemistry and Molecular Biology 34: 653-665
  16. Lee HL (1990) A rapid and simple biochemical method for the detection of insecticide resistance due to elevated esterase activity in mosquito larvae of Cx. quinquefasciatus. Trop. Biomed 7: 21–28.
  17. Chen CD, Nazni WA, Lee HL, Seleena B, Sofian-Azirun M (2008) Biochemical detection of temephos resistance in Aedes (Stegomyia) aegypti (Linnaeus) from dengue-endemic areas of Selangor state, Malaysia. Proc ASEAN Congr Trop. Med. Parasitol 3: 6–20.
  18. Wan-Norafikah O, Nazni WA, Lee HL, Zainol-Ariffin P, Sofian-Azirun M (2013b) Development of permethrin resistance in Culexquinquefasciatus Say in Kuala Lumpur, Malaysia. Saudi J BiolSci 20: 241–250.
  19. Wan-Norafikah O, Nazni WA, Lee HL, Zainol-Ariffin P, Sofian-Azirun M (2013a) Susceptibility of AedesalbopictusSkuse (Diptera: Culicidae) to permethrin in Kuala Lumpiur, Malaysia. Asian Biomed 7: 51–62.
  20. Wan-Norafikah O, Nazni WA, Lee HL, Chen CD, Wan-Norjuliana WM (2008) Detection of permethrin resistance in AedesalbopictusSkuse, collected from Titiwangsa Zone, Kuala Lumpur, Malaysia. Proc ASEAN Congr Trop. Med. Parasitol 3: 69–77.
  21. Wan-Norafikah O, Nazni WA, Lee HL, Zainol-Ariffin P, Sofian-Azirun M (2010) Permethrin resistance in Aedesaegypti (Linnaeus) collected from Kuala Lumpur, Malaysia. J. Asia-Pacific Entomol 13: 175–182.
  22. Labbé P, Berthomieu A, Berticat C, Alout H, Raymond M, et al. (2007) Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culexpipiens. Mol. Biol. Evol 24: 1056-1067.
  23. Sarkar M, Bhattacharyya IK, Borkotoki A, Goswami D, Rabha B (2009a) Insecticide resistance and detoxifying enzyme activity in the principal bancroftianfilariasis vector, Culexquinquefasciatus, in northeastern India. Med. Vet. Entomol 23: 122–131.
  24. Sarkar M, Borkotoki A, Baruah I, Bhattacharyya IK, Srivastava RB (2009b) Molecular analysis of knock down resistance (kdr) mutation and distribution of kdr genotypes in a wild population of Culexquinquefasciatus from India. Trop. Med. Int. Health. 14: 1097–1104.
  25. Corbel V, N’Guessan R, Brengues C, Chandre F, Djogbenou S(2007) Multiple insecticide resistance mechanisms in Anopheles gambiae and Culexquinquefasciatus from Benin, West Africa. Acta Trop 101: 207–216
  26. Stone BF, Brown AWA (1969) Mechanisms of resistance to fenthion in Culexpipiensfatigans.Wied Bull (WHO) 40: 401–408.
Citation: Tabbabi A, Daaboub J, Laamari A, Cheikh RB, Cheikh HB, et al. (2017) Study Confirming Resistance to Fenitrothion of Culex pipiens (Diptera: Culicidae) from Central Tunisia. Hereditary Genet 6:176.

Copyright: © 2017 Tabbabi A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.