Research Article, Vector Biol J Vol: 2 Issue: 1
Temephos Resistance in Three Populations of Culex pipiensCollected from Three Districts of Southern Tunisia and its Significance for the Resistance Mechanism
*Corresponding Author : Ahmed Tabbabi
Laboratory of Genetics, Faculty of Medicine of Monastir, Monastir University, 5019, Monastir, Tunisia
Tel: +00216 97 085 424
E-mail: [email protected]
Received: June 06, 2017 Accepted: June 23, 2017 Published: June 30, 2017
Citation: Daaboub J, Tabbabi A, Lamari A, Feriani M, Boubaker C, et al. (2017) Temephos Resistance in Three Populations of Culex pipiens Collected from Three Districts of Southern Tunisia and Its Significance for the Resistance Mechanism. Vector Biol J 2:1.doi: 10.4172/2473-4810.1000117
Objectives: The aim of this study was to investigate the resistance to temephos (OP) in three populations of Culex pipiens collected from three districts of Southern Tunisia and its significance for the resistance mechanism.
Methods: Resistance to temephos insecticide was studied on Culex pipiens mosquitoes collected in three localities of Southern Tunisia. Larvae were used for different bioassays and adults were stored in -80°C for esterases identification.
Results: Bioassays revealed the susceptibility of sample # 3. The weakest resistance was recorded in sample # 3 (RR50 = 0.68) and the strongest resistance for the sample # 2 (RR50 = 3.6). At LC95, two samples (# 1, and 3) were susceptible. The use of synergists showed the non-involvement of resistance mechanisms inhibited by DEF and Pb. Our investigation reported that temephos resistance could be explained by the two most common mechanisms of resistance to OP (overproduced esterases and AChE 1 mutation).
Conclusion: Both detoxification mechanisms and target site alteration were involved in the resistance to temephos as reported in our study. These results are very important for the implementation and development of vector control strategies
Keywords: Culex pipiens; Temephos resistance; Overproduced esterases; AChE 1 mutation; Southern Tunisia
The use of insecticides around the world to control mosquitoes is regularly disputed, in particular because of the increasing resistance of these insects to products. This adaptation of mosquitoes threatens the prevention of epidemics in the absence of an alternative to insecticides. It is important to understand that the mosquito does not mutate to resist insecticides! Numerous mutations preexist in the immense populations of mosquitoes. When insecticides are present in the environment, mosquitoes that have mutations favorable to their survival reproduce and pass them on to their offspring, while sensitive mosquitoes die. Mutations that give mosquitoes the ability to resist organophosphates (OPs) are not spawned, but are selected by the environment. More simply, the frequency of mosquitoes carrying these mutations increases in a toxic environment.
Only three loci are responsible for major resistances, Est-2, Est- 3 and ace-1 [1-4]. Est-2 and Est-3 form a super locus (designated by Ester) as they are very close in the genome; these genes encode esterases which trap or metabolise insecticides before they can inhibit acetylcholinesterase synapses. In the case of resistance, these esterases are produced in excess by a process of amplification of the number of copies of the genes which encode them in the genome or an increase in their expression [5-9]. Some resistance alleles have up to 50 copies of Ester, while the sensitive allele contains only one copy . The ace- 1 gene codes for the target of OPs insecticides, acetylcholinesterase 1 (AChE 1). In the case of resistance, this target is mutated, which reduces its affinity for OPs [7,11-13].
Temephos, belongs to the family of organophosphate (OP) insecticides, used in the fight against immature mosquitoes vectors due to its cost-effectiveness and community acceptance . Recent study showed their effectiveness as a larvicide for mosquito control . Many countries used this insecticide in mosquito control. However, its massive use had led to the development of resistance in different countries including Tunisia [16,17].
The aim of this study was to investigate the resistance to temephos (OP) in three populations of Culex pipiens collected from three districts of Southern Tunisia and its significance for the resistance mechanism.
Material and Methods
Between 2002 and 2005, Culex pipiens were sampled in three localities of Southern Tunisia and used for different bioassays tests. S-Lab  was used as a reference population for bioassays. SA2 and SA5 , were used as reference population for biochemical tests.
Insecticides and used synergists
Assays were performed as described by Raymond et al. , using ethanol solutions of temephos (95.5% [AI]), 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.
Bioassay tests for mosquito larvae and data analysis
Bioassays were done on larvae preferably on early fourth stage. Each bioassay test included five different temephos concentrations. Tests were carried out in triplicate with five repetitions of controls without insecticide. The mortality results were read after 24h hours of exposure and were analyzed by using the log-probit program of Raymond , based on Finney .
Starch gel electrophoresis
From each collection, we used sample of adult mosquitoes to study the elevated esterases according to the method of Pasteur . We used two references strains (SA2 and SA5) to identify detected esterases.
Analysis of resistance levels
Linearity of the dose-mortality response was accepted (p>0.05) only for S-Lab and rejected for all samples except # 2. Bioassays revealed the susceptibility of sample # 3 (Table 1). The weakest resistance was recorded in sample # 3 (RR50 = 0.68) and the strongest resistance for the sample # 2 (RR50 = 3.6). At LC95, 2 samples (# 1, and 3) were susceptibles. The use of synergists showed the non-involvement of resistance mechanisms inhibited by DEF and Pb (Table 1).
|Population||Temephos||Temephos +DEF||Temephos +Pb|
|LC50 in μg/l (a)||Slope ± SE||RR50 (a)||LC50 in μg/l (a)||Slope ± SE||RR50 (a)||SR50 (a)||RSR||LC50in μg/l (a)||Slope ± SE||RR50 (a)||SR50 (a)||RSR|
|Slab||1.2 (1.1-1.4)||2.34 ± 0.22||-||0.32 (0.28-0.36)||4.99 ± 0.69||-||3.8 (2.8-5.0)||-||2.2 (1.7-2.8)||1.94 ±0.28||-||0.56 (0.44-0.72)||-|
|1-Tozeur||2.3 (1.4-3.9)||2.73 * ± 0.63||1.9 (1.2-3.0)||-||-||-||-||-||-||-||-||-||-|
|2-Gabès||4.6 (3.8-5.4)||2.35 * ± 0.24||3.6 (2.9-4.5)||2.3 (1.5-3.4)||3.46 ± 0.53||7.2 (4.0-12.8)||1.9 (1.1-3.4)||0.51||6.3 (4.2-9.5)||3.51 ± 1.09||2.9 (1.6-5.1)||0.72 (0.40-1.2)||1.3|
|3- Bordj El Khadra||0.86 (0.62-1.1)||5.07 ± 1.5||0.68 (0.40-1.1)||-||-||-||-||-||-||-||-||-||-|
(a), 95% CI; * The log dose-probit mortality response 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/LC50observed 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 1: Temephos resistance characteristics of Tunisian Culex pipiens in presence and absence of synergists DEF and Pb.
Analysis of resistance mechanisms and genes
We investigated the two most common mechanisms of resistance to OP (overproduced esterases and AChE1 mutation) on field collected populations. The over-produced esterases encoded by the Est-2, and Est-3 loci were present in sample # 1 with frequency of 0.56 and 0.81 in sample # 2. The study of crossresistance temephos/propoxur showed a strong correlation. In fact the mortality due to propxur was 1% for the sample having the strongest resistance to temephos (sample # 2) and 100% for the sample having the lowest resistance (sample # 3) indicated an insensitive AChE.
Previous studies of Ben Cheikh et al.  showed that the temephos resistance levels of the Tunisian Culex pipiens were low (LC50 ranges of 0.0021–0.015lmg/l). This is in agreement with our results showing a low rate of resistance to this insecticide in Southern Tunisia. The resistance of Culex pipiens population collected in South east (sample # 3) may be associated with the use of temephos and other insecticides at different intensities and frequencies of application. In contrast, the susceptibility recorded in far South (sample # 3) could be explained by the absence of vector control in this locality because of lack of human population. Other studies reported LC50 of 0.01 mg/l in this species from India . This value was 0.0000473 mg/l for Culex quinquefasciatus collected in the same country . Recently, Ben Cheikh et al.  showed the contribution of overproduced esterases in the recorded resistance to the temephos insecticide which confirms our starch gel electrophoresis but not synergists results. The involvement of esterases enzymes system in resistance to OPs, carbamates [26,27] and pyrethroids  has been showed in mosquitoes and other insects.
It should be noted that despite many studies reported the temephos resistance in mosquitoes, the involved mechanism are not well characterized. Our investigation reported that temephos resistance could be explained by the AChE 1 insensivity and the increased detoxification by esterases enzymes. This result are in agreement with previous studies showing mutations on the acetylcholinesterase (ace-1) gene have been associated with OP resistance [29-31]. Previous studies on Culex populations showed that mutation in the acetylcholinesterase (ace-1) gene leading to G119S substitution is responsible for insensitivity to OP and carbamate insecticides .
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.
- Nauen R (2007) Insecticide resistance in disease vectors of public health importance. Pest Manag Sci 63: 628-633.
- Braga IA, Valle D (2007) Aedes aegypti: inseticides ,mecanismos. Epidemiologia 279-293.
- Whalon ME, Mota-Sanchez D, Hollingworth RM (2008) Analysis of global pesticide resistance in arthropods. Global Pesticide Resistance in Arthropods. CAB International, Cambridge, MA, United Kingdom.
- Valle D, Belinato TA, Martins AJ (2015) Controle qu´imico de Aedes aegypti. Resistance a inseticides alternativas, in Dengue: Teorias e Praticas. Editora Fiocruz, Rio de Janeiro, Brazil.
- Rooker S, Guillemaud T, Berge J, Pasteur N, Raymond M (1996) Co-amplification of esterase A and B genes as a single unit in Culex pipiens mosquitoes. Heredity 77: 555-561.
- Hemingway J, Hawkes N, Prapanthadara L, Jayawardenal KG, Ranson H (1998) The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance. Philos Trans R Soc Lond B Biol Sci 353: 1695-1699.
- Hemingway J, Hawkes N, McCarroll I, Ranson H (2004) The molecular basis of insecticide resistance in mosquitoes. Insect Biochem. Mol Biol 34: 653-665.
- 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.
- Ranson H, Claudianos C, Ortelli F, Abgrall C, Hemingway J, et al.(2002) Evolution of supergene families associated with insecticide resistance .Science (Washington DC) 298: 179-181.
- Weill M, Berticat C, Raymond M, Chevillon C (2000) Quantitative PCR to estimate the number of amplified esterase genes in insecticide resistant mosquitoes. Anal Biochem 285: 267-270.
- Weill M, Lutfalla G, Mogensen K, Chandre F, Berthomieu A, et al. (2003) Comparative genomics: Insecticide resistance in mosquito vectors. Nature 423: 136-137.
- 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.
- 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.
- World Health Organization (2009) Dengue guidelines for diagnosis, treatment, prevention and control: New edition. Geneva, Switzerland.
- Mukhopadhyay AK, Patnaik SK, Babu PS (2006) Susceptibility status of some culicine mosquitoes to insecticides in Rajahmundry town of Andhra Pradesh, India. J Vector Borne Dis 43: 39-41.
- Ben Cheikh H, Haouas-Ben Ali Z, Marquine M, Pasteur N (1998) Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia. J Med Entomol 35: 251-260
- Ben Cheikh R, Berticat C, Ben Cheikh H, Berthomieu A, Weille M (2008) Characterization of a novel high-activity esterase in Tunisian populations of the mosquito Culex pipiens. J Econ Entomol 101: 484-491.
- Georghiou GP, Metcalf RL, Gidden FE (1966) Carbamate-resistance in mosquitos. Selection of Culex pipiens fatigans Wiedemann (=C. quinquefasciatus Say) for resistance to Baygon. Bull World Health Organ 35: 691-708.
- 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.
- Raymond M, Fournier D, Bride JM, Cuany A, Bergé J, et al. (1986) Identification of resistance mechanisms in Culex pipiens (Diptera: Culicidae) from southern France: insensitive acetylchlinesterase and detoxifying oxidases. J Econ Entomol 79: 1452-1458
- Raymond M, Prato G, Ratsira D (1993) PROBIT. Analysis of mortality assays displaying quantal response. Praxeme (Licence No. L93019), Saint Georges d’Orques, France.
- Finney DJ (1971) Probit analysis. Cambridge University Press, Cambridge. England.
- 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.
- Badawy MEI, Taktak NEM, Awad OM, Elfiki SA, Abou El-Ela NE (2015) Larvicidal activity of temephos released from new chitosan/alginate/gelatin capsules against Culex pipiens. Int J Mosq Res 2: 45–55.
- Dorta DM, Vasuki V, Rajavel A (1993) Evaluation of organophosphorus and synthetic pyrethroid insecticides against six vector mosquito species. Rev Saude Publica 27: 391-397.
- Paeporn P, Komalamisra N, Deesin V, Rongsriyam Y, Eshita Y, et al. (2003) Temephos resistance in two forms of Aedes aegypti and its significance for the resistance mechanism. Southeast Asian J Trop Med Public Health 34: 786-792.
- Marcombe S, Mathieu RB, Pocquet N, Riaz MA, Poupardin R (2012) Insecticide resistance in the dengue vector Aedes aegypti from Martinique: Distribution, mechanisms and relations with environmental factors. PLoS One 7: 30989.
- Ganesh KN, Urmila J, Vijayan VA (2003) Pyrethroid susceptibility & enzyme activity in two malaria vectors, Anopheles stephensi (Liston) &. A. culicifacies (Giles) from Mysore, India. Indian J Med Res 117: 30-38.
- Nabeshima T, Mori A, Kozaki T, Iwata Y, Hidoh O, et al. (2004) An amino acid substitution attributable to insecticide-insensitivity of acetylcholinesterase in a Japanese encephalitis vector mosquito, Culex tritaeniorhynchus. Biochem Biophys Res Commun 313: 794-801.
- Weill M, Malcolm C, Chandre F, Mogensen K, Berthomieu A, et al. (2004) The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors. Insect Mol Biol 13: 1-7.
- Alout H, Labbe P, Berthomieu A, Pasteur N, Weill M (2009) Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations. Insect Biochem Mol Biol 39: 884-891.