Research Article, J Genet Disor Genet Rep Vol: 6 Issue: 2
Evaluation of Fenitrothion against Culex pipiens (Diptera: Culicidae) Larvae in Grand Tunis Area of 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: May 04, 2017 Accepted: May 13, 2017 Published: May 22, 2017
Citation: Tabbabi A, Daaboub J, Lamari A, Ben Cheikh R, Ben Jha I, et al. (2017) Evaluation of Fenitrothion against Culex pipiens (Diptera: Culicidae) Larvae in Grand Tunis Area of Tunisia. J Genet Disor Genet Rep 6:2.doi: 10.4172/2327-5790.1000157
Resistance toward 2 insecticides (fenitrothion and propoxur) was analyzed in 5 samples of Culex pipiens populations collected from various localities of Grand Tunis area, Northeast Tunisia. All studied samples were resistant to fenitrothion and propoxur insecticides. The study of esterase’s activities showed the existence of five overproduced esterases: C1, A1, A2-B2, A4-B4 (and/or A5-B5) and B12. One or several esterases were detected in the studied samples. We also showed that the resistance to fenitrothion was correlated with the propoxur resistance indicating that modifications of the target, the AChE 1, can be involved in the OP resistance. These results should be considered in the current mosquitoes control programs in Tunisia.
Keywords: Culex pipiens; Grand Tunis area; Fenitrothion; Propoxur; Resistance; Esterases; AChE, Control programs
The control of mosquito vectors of human diseases is a global health issue. Their ability to resist to insecticide treatments threatens the prevention of epidemics. This systematic use of insecticides to control mosquito larvae has led to the occurrence of resistance phenomena, which considerably reduces the effectiveness of the treatments . Between 1948 and 1990, the number of insect resistance cases increased steadily: 14 species in 1948, 224 in 1969 and more than 500 in 1990 . In several species of mosquitoes, including Culex pipiens, these resistances have already been demonstrated for conventional insecticides [3,4]. Resistance is studied at several biological levels, using molecular, genetic, cellular, metabolic and ecological techniques among others [5-12]. The detection and monitoring of the resistance developed by natural mosquito populations will be essential to better manage them in the field as long as alternatives to insecticides are not put in place.
The organophosphate insecticides (OPs) and synthetic pyrethroids have been used for a long time to fight against mosquitoes. Fenitrothion belongs to the chemical group of organophosphates which inhibit acetylcholinesterase, an enzyme involved in the regulation of nerve impulses . This insecticide has moderate toxicity to mammals. The aim of this paper was to evaluate the resistance level of fenitrothion against Culex pipiens larvae in Grand Tunis Area of Tunisia.
Materials and Methods
Five populations of Culex pipiens were collected from natural habitats of Grand Tunis area, Northeast Tunisia, between June 2003 and November 2005 (Table 1 and Figure 1). A susceptible strain named S-Lab was used as reference. Two other strains characterized by the presence of esterases A2B2 (SA2 strain) and A5B5 (SA5 strain) were used as positive control in starch electrophoresis.
|Code||Locality||Breeding sites||Date of collection||Mosquito control (used insecticides)||Agricultural pest control|
|1||Sidi Thabet||Ditch||Aug. 2004||Rare (C,P)||Yes|
|2||Sokra||Canal||June 2003||Very frequent (C, Pm, F, P, D)||Yes|
|3||Mannouba||River||June 2005||Occasional (P,D)||Yes|
|4||Ouardia||Ditch||Aug. 2005||Very frequent (C, F, P, D)||None|
|5||Ezzahra||Ditch||Nov 2005||Very frequent (C, F, P, D, T)||None|
C : Chlorpyrifos ; T : Temephos ; Pm : Pirimiphos methyl ; F : Fenitrithion ; P : Permethrin ; D : Deltamethrin
Table 1: Geographic origin of Tunisian populations, breeding site characteristics, and insecticide control.
Assays were performed using two insecticides: fenitrothion (98.5% [AI], brought from laboratory Dr Ehrenstorfer, Germany), and propoxur (99.9% [AI], Bayer AG, Leverkusen , Germany), organophosphates and carbamates compounds, respectively.
Bioassays test and data analysis
Twenty third and/or fourth instar larvaes were used for bioassays. We used five different fenitrothion and propoxur concentrations which affect a mortality of 0% to 100%. Bioassays carried out in triplicates except control where we made five repetitions without insecticide. Reading of the larval mortality was carried out after 24 hours of exposure. Mortality data were analyzed by using the logprobit program of Raymond , based on Finney .
Synergism tests and esterase’s detection
Synergism tests consists to perform the same previous bioassays with the addition of 0.5 ml of the maximum sub-lethal concentration of an esterase inhibitor DEF, S,S,S-tributyl phosphorotrithioate (0.5 μg/ml) to each cup. The same for piperonyl butoxide (PB), an inhibitor of mixed function oxidases. The two synergists were added 4 hours before the start of bioassays. Esterase phenotypes were established by starch electrophoresis (TME 7.4 buffer system) as described by Pasteur et al. [16,17] using adults specimens.
S-Lab and 4 field samples (# 1, 3, 4, and 5) showed a linear dosemortality responses (p<0.05). As indicated in Table 2, all studied samples were resistant at LC50. The RR50 varied from 14.5 in sample # 3 to 887 and sample # 5. Agricultural pest control and used other OPs insecticides were probably responsible for the resistance of populations collected from sites not subject to fenitrothion control.
|Population||Fenitrothion||Fenitrothion +DEF||Fenitrothion +Pb|
|LC50 in µg/l
|LC50 in µg/l
|RSR||LC50 in µg/l
(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 study of the effect of DEF on fenitrothion resistance showed the minor role played by the EST (and/or GST) in sample # 4. In fact, the SR was higher than that found in S-Lab and RR50>1 in the presence of DEF (Table 2). The effect of Pb on fenitrothion resistance showed the same situation in sample # 5. P450 cytochrome mediated monooxygenases inhibited by Pb account for only a part of the fenitrothion resistance for sample # 5 (RR50=112, p<0.05, RSR=7.9).
The different percentages of mortalities caused by propoxur 46%, 21%, 48%, 18%, and 0% were recorded in the resistant samples # 1, 2, 3, 4, and 5, respectively. All recorded percentages were correlated with the resistance rates of fenitrothion (P<0.01).
The study of esterase’s activities showed the existence of five esterases: C1, A1, A2-B2, A4-B4 (and/or A5-B5) and B12. One or several esterases were detected in the studied samples with different percentages ranged from 0.02 to 0.47. The involvement of these enzymes in the recorded resistance confirmed partially the previous results of the synergist used in bioassays.
Several authors studied and evaluated the resistance rate of many mosquitos’ species in the world like Rong et al.  who carried out the level of fenitrothion resistance and other insecticides on An. gambia from Benin. Authors in this study showed the high level of resistance to fenitrothion of Culex pipiens populations collected from Tunisia. This level is higher than existed in other countries of the world: It was 11.2-fold in Cx. pipiens quinquefasciatus from Brazil , and 4.1-fold in Aedes aegypti from Cuba . The fenitrothion resistance levels of Culex pipiens were low in Senegal [21,22].
The study of the effect of DEF on fenitrothion resistance showed the minor role played by the EST (and/or GST) in one among five used samples. These results were not in agreement with those found by starch electrophoresis where five esterases were detected to be involved in the resistance of all field samples. Several studies confirmed the correlation between OPs insecticides and overproduced esterases and GST in Culex pipiens and other insect species [8,10,23-25]. CYP450 inhibited by Pb account for only a part of the fenitrothion resistance for one among 5 tested samples. These results were in agreement with those found by Ben Cheikh et al.  obtained on Culex pipiens from Tunisia resistant to chlorpyrifos (OP).
We showed also a strong correlation between fenitrothion resistances of all resistant samples with the propoxur resistance. The AChE 1 is probably involved in the recorded resistance. The resistance allele, Ace-1R, is present worldwide and causes OPs resistance in several mosquito species [3,9,26].
Our field work presents a certain weakness due to a lack of physicochemical characterization of the mosquito breeding sites. A hypothesis about a possible relationship between chemical disturbances of water and the potential for resistance of larvae to insecticides would be of great importance.
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.
- Brogdon WG, McAllister JC (1998) Insecticide resistance and vector control. Emergence of infectious diseases 4: 605-613.
- Mouches C, Pauplin Y, Agarwal M, Lemieux L, Herzog M, et al. (1990) Characterization Of Amplification Core And Esterase-B1 Gene Responsible For Insecticide Resistance In Culex. Proceedings Of The National Academy Of Sciences Of The United States Of America 87: 2574-2578.
- 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.
- 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.
- Scott JG (1989) Cross-resistance to the biological insecticide abamectin in pyrethroidresistant house flies. Pesticide Biochemistry and physiology34: 27-31.
- Liu N, Scott JG (1997) Phenobarbital induction of CYP6D1 is due to a trans acting factor on autosome 2 in house flies, Musca domestica. Insect Molecular Biology 6: 77-81.
- Cheikh BH, 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.
- 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.
- 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.
- 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.
- 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 Biochemphysiol 79: 84-93.
- Alout H, Berthomieu A, Hadjivassilis A, Weill M (2007) A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus. Insect Biochem Mol Biol 37: 41-47.
- Aldridge WN (1950) Some properties of specific cholinesterase with particular reference to the mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E605) and analogues. Biochemistry Journal 46: 451-460.
- Raymond M, Prato G, Ratsira D (1993) PROBIT. Analysis of mortality assays displaying quantal response, CNRS-UM II. License L93019. Avenix, George d’Orques, France.
- Finney DJ (1971) Probit Analysis. Cambridge University Press
- 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
- Pasteur N, Pasteur G, Bonhomme F, Britton- Davidian J (1988) Practical Isozyme Genetics. Ellis Horwood
- Rong LS, Ann AT, Ahmad NW, Lim LH, Azirun MS (2012) Insecticide susceptibility status of field-collected Aedes (Stegomyia) aegypti (L.) at a dengue endemic site in Shah Alam, Selangor, Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 43:34.
- Bracco JE, Barata JMS, Marinotti O (1999) Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from Sào Paulo, Brazil. Mem Inst Oswaldo Cruz94: 115-120.
- Rodriguez MM, Bisset J, Fernandez DMD, Lauzan L, Soca A (2001) Detection of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Cuba and Venezuela. J Med Entomol 38: 623-628.
- Diop A (2008) Rapports annuels du Projet OMS/GATES sur le controle et la biologie des vecteurs du paludisme. Programme National de Lutte contre le Paludisme, Ministere de la Sante et de la Prevention, Senegal.
- Diop A (2010) Rapports annuels du Projet OMS/GATES sur le controˆle et la biologie des vecteurs du paludisme. Programme National de Lutte contre le Paludisme, Ministere de la Sante et de la Prevention, Senegal.
- Liu H, Xu Q, Zhang L, Liu N (2005) Chlorpyrifos resistance in mosquito Culex quinquefasciatus. J Med Entomol 42: 815-820
- 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 Plutella xylostella. Insect Biochem Mol Biol 28: 651-658.
- 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 Musca domestica. Insect Biochem Mol Biol 31: 1145-1153.
- Labbe P, Berthomieu A, Berticat C, Alout H, Raymond M, et al. (2007) Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens. Mol Biol Evol 24: 1056-1067.