Insecticide resistance to organophosphates in Culex pipiens complex from Lebanon
© Osta et al.; licensee BioMed Central Ltd. 2012
Received: 28 March 2012
Accepted: 3 July 2012
Published: 3 July 2012
Analysis of Culex pipiens mosquitoes collected from a single site in Lebanon in 2005, revealed an alarming frequency of ace-1 alleles conferring resistance to organophosphate insecticides. Following this, in 2006 the majority of municipalities switched to pyrethroids after a long history of organophosphate usage in the country; however, since then no studies have assessed the impact of changing insecticide class on the frequency of resistant ace-1 alleles in C. pipiens.
C. pipiens mosquitoes were captured indoors from 25 villages across the country and subjected to established methods for the analysis of gene amplification at the Ester locus and target site mutations in ace-1 gene that confer resistance to organophosphates.
We conducted the first large-scale screen for resistance to organosphosphates in C. pipiens mosquitoes collected from Lebanon. The frequency of carboxylesterase (Ester) and ace-1 alleles conferring resistance to organophosphates were assessed among C. pipiens mosquitoes collected from 25 different villages across the country between December 2008 and December 2009. Established enzymatic assay and PCR-based molecular tests, both diagnostic of the major target site mutations in ace-1 revealed the absence of the F290V mutation among sampled mosquitoes and significant reduction in the frequency of G119S mutation compared to that previously reported for mosquitoes collected from Beirut in 2005. We also identified a new duplicated ace-1 allele, named ace-1 D13 , exhibiting a resistant phenotype by associating a susceptible and a resistant copy of ace-1 in a mosquito line sampled from Beirut in 2005. Fisher’s exact test on ace-1 frequencies in the new sample sites, showed that some populations exhibited a significant excess of heterozygotes, suggesting that the duplicated allele is still present. Starch gel electrophoresis indicated that resistance at the Ester locus was mainly attributed to the Ester 2 allele, which exhibits a broad geographical distribution.
Our analysis suggests that the frequency of resistant ace-1 alleles in mosquito populations can be downshifted, and in certain cases (F290V mutation) even eliminated, by switching to a different class of insecticides, possibly because of the fitness cost associated with these alleles.
An important global strategy to contain mosquito-borne diseases is vector control using chemical insecticides. However, the strong dependence on insecticides for mosquito control worldwide and the use of such chemicals in agriculture has led to the physiological resistance of important mosquito vectors in recent years, including Anopheles gambiae[1, 2], Aedes aegypti[3–5], and Culex pipiens[6, 7]. Hence, monitoring insecticide resistance in mosquito populations is crucial in order to ensure the sustainability of vector control programs .
Lebanon is a temperate country where two potentially important mosquito vectors of disease are prevalent, C. pipiens, which transmits filarial worms, West Nile (WNV) and several encephalitis viruses [9, 10], and Aedes albopictus the vector for Chikungunya (CHIKV)  and dengue viruses (DENV) . Ae.aegypti, the primary vector of DENV is not present in the country. Despite the prevalence of potential vectors of disease, arboviral diseases are absent from Lebanon with the exception of some cases of WNV infections , while WNV has been responsible for numerous and iterative outbreaks in Israel , a country at the southern border of Lebanon. In the past, however, both DENV  and WNV  were highly prevalent in the country and a Dengue epidemic affected thousands of individuals in Beirut between the years 1945 and 1946 .
Mosquito control in Lebanon depended heavily on organophosphate (OPs) usage before the year 2006. The most commonly used OPs included, dichlorvos, malathion, diazinon and chlorpyriphos. However, after that date the use of OPs dwindled; dichlorvos, malathion and diazinon were eventually discontinued, while chlorpyriphos has remained in use in a limited number of villages. On the other hand, there has been a significant shift towards the use of pyrethroids in most villages, according to the feedback obtained from several municipalities and major insecticide distributors across the country. The most commonly used pyrethroids are alpha-cypermethrin, deltamethrin and tetramethrin. In Lebanon, insecticides are used almost exclusively to control adult mosquito populations by spraying along the roads in villages and around houses, while no strategies exist to identify and treat larval habitats. Resistance to OPs can be metabolic or due to target site modifications. The former is characterized by the amplification of esterases A and B that sequester these insecticides , preventing them from reaching their target, the ace-1 gene-encoded acetylcholinesterase. Target-site modifications are due to three distinct mutations in ace-1, resulting in three substitutions, G119S found in several mosquito species, F290V found only in Culex pipiens, and F331W found only in Culex tritaeniorhynchus, (numbered according to Torpedo californica ace), which independently render the enzyme less sensitive to OP insecticides.
Data from C. pipiens mosquitoes sampled in 2005 from Beirut indicated a high frequency of both G119S and F209V mutations . Here, we conducted a large-scale one-year survey, between December 2008 and December 2009, to measure the impact of switching to pyrethroids on the residual OP resistance in C. pipiens mosquito populations. The study involved analysis of gene amplification at the Ester locus and target site mutations in ace-1 gene in mosquitoes captured indoors across the country.
Collection sites and mosquito strains
Starch gel electrophoresis
The frequency of Ester 2 (A2-B2) locus among the sampled mosquitoes was determined by Starch gel electrophoresis using TEM 7.4 buffer systems and revealed according to Pasteur et al.. The reference strain SA2  was used as a control for esterase amplification at Ester 2 .
Detection of the G119S and F290V mutations in ace-1
The G119S mutation was detected using a diagnostic PCR test followed by RFLP, as previously described . Briefly, legs of individual mosquitoes were ground in extraction buffer (0.1 M Tris–HCl, pH 8.0, 0.01 M EDTA, 1.4 M NaCl, 2% cetyltrimethyl ammonium bromide), DNA was then extracted with chloroform, precipitated in isopropanol, and resuspended in sterile water. A 374 bp amplicon was amplified from exon 3 of ace-1 gene using the primers CpEx3dir, 5’-CGACTCGGACCCACTCGT-3’, and CpEx3rev, 5’-GACTTGCGACACGGTACTGCA-3’, and the following PCR conditions: 30 cycles, 95 °C for 5 min, 95 °C for 40 sec, 60 °C for 1 min and 72 °C for 50 sec. Since the G119S mutation in exon 3 creates an AluI restriction site , the PCR product was digested with AluI (this generates two fragments of 272 and 102 bp) and products were analyzed on 1.5% agarose gel. Detection of the F290V mutation was performed using the PASA diagnostic test as previously described  except that the PCR conditions were modified as follows: 30 cycles, 95 °C for 5 min, 95 °C for 40s, 61 °C for 1 min and 72 °C for 50s.
Témoin-dichlorvos-propoxur- (TDP) test
The TDP test was performed exactly as described by Alout et al. to identify all possible phenotypes at the ace- 1 locus: phenotype [V], mosquitoes containing only the F290V mutated enzyme; [RR], mosquitoes containing only the G119S mutated enzyme, [SS] those containing susceptible enzyme; [VS], [VR], [RS] and [VRS], mosquitoes containing two or three (VRS) enzyme forms.
Detection of ace-1 gene duplications
Due to the similarity of duplicated allele sequences with non-duplicated ace-1 alleles, it is not possible to directly detect the presence of the duplicated allele in natural populations [6, 27]. However, an excess of heterozygote phenotypes at the ace-1 locus have been shown to suggest robustly the presence of a duplicated allele in a population [28, 29]. Thus, excess of heterozygotes (as indicated by a negative Fis, i.e. a significant departure from the expected frequencies under Hardy-Weinberg assumptions) was tested for each sampled population using the Genepop software (Fisher's exact tests, ).
Results and discussion
Prevalence of Ester 2 in sampled mosquitoes
Frequency of Ester 2 phenotype in the mosquito populations sampled
[Ester 2 ]
Geographical distribution of AChE1 G119S substitution and evidence for ace-1 duplication
The analysis of insecticide resistance in a small sample of C. pipiens mosquitoes collected from Beirut in 2005 revealed an alarming frequency of the G119S resistant AChE1 distributed as follows : 41.4% [RS], 37.9% [RR], 8.6% [VS] and 12.1% [VRS]. None of the mosquitoes sampled exhibited the [SS] phenotype (susceptible enzyme), suggesting that in 2005 there was indeed a strong selective pressure on the ace-1 locus due to the past heavy usage of OPs. Here, we screened a mosquito line, that was selected with OP for several generations from this 2005 Beirut sample, for the presence of a duplicated ace-1 allele as previously reported . The duplicated ace-1 allele (or haplotype) originated from duplication of the ace-1 gene (termed ace-1 D or D) which associates a resistant and a susceptible copy (termed D(R) and D(S), respectively), resulting in "permanent heterozygotes" . This screen revealed the presence of a new duplicated allele associating the D7S susceptible copy already found in Algeria  and a new DR resistant copy (Figure 2), hence exhibiting an overall resistant phenotype. This new allele (as of the complete haplotype) should thereafter be named ace-1 D13 , according to previous nomenclature  (ace-1 D13 susceptible and resistant copies are available in GenBank under references JX007790 and JX007791, respectively. All other duplicated alleles can be found in GenBank under references JX007766 to JX007789). Also, considering only the G119S mutation, the ace-1 allele frequencies in the 2005 sample reported above  show a significant departure from the Hardy-Weinberg equilibrium (Genepop, , Fisher’s exact-test for an excess of heterozygotes; Fis = −0.34, p-value =0.018). This provides additional support to the presence of at least one ace-1 duplicated allele in the 2005 Beirut sample, as previously reported in other locations [27, 35].
Frequency of ace-1 alleles based on diagnostic PCR analysis
Interestingly, the F290V substitution that was previously detected at low frequency only in five Mediterranean countries including Lebanon , was not detected in the present study using both the PASA (Table 2) and TDP tests, despite the large samples collected across the country. A plausible explanation for the disappearance of the F290V substitution from natural populations is the increased resort to pyrethroids, which do not select for this mutation. Moreover, even in municipalities where chlorpyriphos is still used, the resistance conferred by the F290V mutation to this insecticide is 150-fold weaker than that conferred by G119S, according to Alout et al.. Thus, high fitness cost in addition to weak insecticide selection of the F290V AChE1 may explain the loss of this mutation from C. pipiens in Lebanon. The reason why F290V was prevalent at low frequencies in samples collected from Beirut in 2005 may be due to the insecticide dichlorvos which was commonly used at that time together with chlorpyriphos. F290V was shown to confer approximately 10 fold higher resistance to dichlorvos compared to the G119S mutation .
In summary, this study shows that the frequencies of resistant ace-1 alleles carrying the G119S and F290V substitutions were dramatically reduced in C. pipiens mosquitoes collected between 2008 and 2009 when compared to those recorded in 2005. This is probably due to increased dependence on pyrethroid insecticides in recent years that do not select for these mutations. We propose that using a rotation system, whereby the use of different classes of insecticides is alternated on a yearly basis, should maintain resistance ace-1 alleles at a low frequency in C. pipiens mosquitoes. Nevertheless, cross-resistance between pyrethroids and OP through elevated esterases should be considered and evaluated.
We thank all friends who helped in mosquito collections. We also thank the different municipalities, Boecker International Sal and Debbane Group for providing information regarding insecticide usage in Lebanon. MAO and KK were funded by AUB-URB and Lebanese CNRS, MW was funded by CNRS institutional grants. All sequence data were obtained on the Environmental Genomic Platform of the SFR Montpellier-Environnement-Biodiversité.
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