Raja Ben Cheikh

Genes Conferring Resistance to Organophosphorus Insecticides in Culex pipiens (Diptera: Culicidae) From Tunisia

ABSTRACT In Tunisia, the mosquito Culex pipiens shows various organophosphate resistance alleles at Ester and ace-1 loci. The characterization and the distribution pattern of these alleles were studied among 20 populations sampled from north to center of Tunisia. At the Ester locus, Ester4, Ester5, and EsterB12 were present. A new esterase characterized by the same electrophoretic migration as esterase Al was identified: A13, encoded by EsterA13 allele. At the ace-1 locus, the presence of the ace-1R, ace-1D, and F290V mutated alleles was also detected. A large heterogeneity in allelic frequencies at Ester and ace-1 loci was observed among samples, with a high significant genotypic differentiation considering both loci (Fst = 0.077, P <10-5), depicting variations of insecticide treatment intensity between areas. A comparison between populations collected in 1996 and 2005 showed an absence of significant resistance evolution. However, the high frequencies of resistance alleles in 2005 populations suggested that the selection pressures are still important in Tunisia. Strategies for resistance management are discussed in the context of the current knowledge of the Tunisian situation.

Characterization of a Novel High-Activity Esterase in Tunisian Populations of the Mosquito Culex pipiens

Abstract In the mosquito Culex pipiens (L.) (Diptera: Culicidae) esterases contribute to insecticide resistance by their increased activity. These esterases display a heterogeneous geographical distribution, particularly in Tunisia, where they are very diverse. In this study, we extended the characterization of a highly active esterase first detected in 1996: B12. Esterase B12 displayed the fastest electrophoretic mobility of all the previously described highly active esterases. We showed that it was encoded by the EsterB12 allele at the Ester locus, and we isolated a strain, TunB12, homozygous for this allele. TunB12 displayed a low (approximately two- to three-fold) but significant resistance to the organophosphates temephos and chlorpyrifos, and to the pyrethroid permethrin. Only temephos resistance was synergized by S,S,S-tributyl-phosphorotrithioate. Real-time quantitative polymerase chain reaction revealed that the EsterB12 allele was not amplified in TunB12 strain, indicating that B12 high activity could be due to a gene up-regulation mechanism. EsterB12 allele frequencies also were estimated in 20 Tunisian populations collected in 2005. Analyses revealed a large distribution of this allele all over the country. Finally, sequences of EsterB12 were acquired and genetic distance trees were constructed with the resistance Ester alleles already published, providing indications about allele’s origins. The diverse array of highly active esterases in C. pipiens from Tunisia and the possible scenario of the origin of their coding alleles are discussed in the context of their possible evolution.

Evaluation of Chlorpyrifos Resistance and Biochemical Mechanisms of Culex pipiens in Five Localities of Grand Tunis Area, Northeast Tunisia

Five Culex pipiens samples were collected at preimaginal stages from breeding sites in 5 localities of Grand Tunis area, Northeast Tunisia, between March 2002 and November 2005. Larvae were used for bioassays using ethanol solutions of chlorpyrifos and propoxur insecticides. All samples were resistant to chlorpyrifos (RR>1, p 1,000-fold) were recorded in samples # 2, 4, and 5. Our synergist study showed that the increased detoxification by EST (and/or GST) had only a minor role in the chlorpyrifos resistance, although several overproduced esterases, known to be involved in the OPs resistance, were detected in all the resistant field samples. The mortality due to propoxur was significantly correlated with the LC50 of chlorpyrifos and indicated an insensitive AChE.

Evaluation of Fenitrothion against Culex pipiens (Diptera: Culicidae) Larvae in Grand Tunis Area of Tunisia

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.

Resistance of Culex pipiens (Diptera: Culicidae) to Chlorpyrifos Insecticide in Central Tunisia

Field populations collected as larvae in five localities of central Tunisia were used to study the resistance to chlorpyrifos insecticide. The resistance levels exceeded 10,000 folds in samples # 3 (Centre East), and 5 (West Centre) not exceeding 5-fold in samples # 4 (Centre East). Our result showed that detoxification by oxydases, EST and/or GST was responsible, at least in part, in resistance to chlorpyrifos in samples # 1, 3, and 5. Different esterases were detected in all studied samples. Except A2B2 who recorded high frequencies ranged from 22% to 28%, all other detected esterases showed low percentages. Mortality caused by propoxur was very low in samples showed the highest resistance (# 3, and 5) and high in samples showed lowest resistant (# 1, 2, and 4) indicated an insensitive AChE 1. Our results of the resistance of Culex pipiens to chlorpyrifos in central Tunisia are consistent with those found in the literature. These data will help to better plan and program the vector control in Tunisia.

Pirimiphos-Methyl Resistance Status of Field Populations of Culex pipiens(Diptera: Culicidae) From Grand Tunis Area, Northeast Tunisia

Mosquito species are responsible for the transmission of many parasitic diseases and their control leads to the phenomenon of resistance to insecticides. Five Culex pipiens samples were collected from various localities of Grand Tunis area, Northeast Tunisia, between June 2003 and November 2005. All the studied samples were resistant to pirimiphos methyl (organophosphorus insecticide). Our results showed the implication of insensitive acetylcholinesterase and overproduced esterases in the resistance of the Tunisian populations of Culex pipiens. Results were discussed in relation to resistance mechanism.

Study on Resistance of Culex pipiens (Diptera: Culicidae) Populations to Fenitrothion in Northern Tunisia

Four populations of Culex pipiens were collected as larvae in Northern Tunisia to evaluate their resistance status against two insecticides: fenitrothion and propoxur. At LC50, the sample # 1 was susceptible, whereas all the other samples were resistant. The RR50 ranged from 1.08 in sample # 1 to 550 in sample # 3. The A2-B2, A4-B4 (and/or A5- B5), B12 and C1 esterases were found in collected samples and the frequencies ranged from 0.02 to 0.42. Propoxur caused a mortality of 0% in samples # 3 which showed the highest resistance levels to fenitrothion insecticide and 87% in sample # 1 which was susceptible hence the involvement of AChE 1 in the recorded resistance. Our results are essential for the development of such strategies.

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

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.

Evaluation of Fenitrothion Resistance and Biochemical Mechanism in three Populations of Culex pipiens (Diptera: Culicidae) from Southern Tunisia

Evaluation of fenitrothion resistance was realized in three populations of Culex pipiens collected in Southern Tunisia between March 2002 and October 2005. It was not possible to considered bioassays tests to fenitrothion in sample # 3 due to their control-level mortality. The RR50 were 27.1 in sample # 1 and 179 in sample # 2. All the studied samples showed the presence of one or more esterases in their electrophoretic profiles except the sample # 3 which was sensitive to propoxur. The addition of Pb to fenitrothion bioassays indicated the involvement of CYTP450 in the recorded resistance. This result could be explained by the massive use of the permethrine in the control against these insects in southern Tunisia. We also showed that the resistance to fenitrothion was correlated with the propoxur resistance. These results indicate that modifications of the target, AChE1, can be involved in the recorded resistance.