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Widespread resistance to pyrethroids threatens malaria control in Africa. Consequently, several countries switched to carbamates and organophophates insecticides for indoor residual spraying. However, a mutation in the ace-1 gene conferring resistance to these compounds (ace-1(R) allele), is already present. Furthermore, a duplicated allele (ace-1(D)) recently appeared; characterizing its selective advantage is mandatory to evaluate the threat. Our data revealed that a unique duplication event, pairing a susceptible and a resistant copy of the ace-1 gene spread through West Africa. Further investigations revealed that, while ace-1(D) confers less resistance than ace-1(R), the high fitness cost associated with ace-1(R) is almost completely suppressed by the duplication for all traits studied. ace-1 duplication thus represents a permanent heterozygote phenotype, selected, and thus spreading, due to the mosaic nature of mosquito control. It provides malaria mosquito with a new evolutionary path that could hamper resistance management.

Background: Malaria is endemic in sub-Saharan Africa with considerable burden for human health. Major insecticide resistance mechanisms such as kdr(R) and ace-1(R) alleles constitute a hindrance to malaria vector control programs. Anopheles gambiae bearing both kdr and ace-1 resistant alleles are increasingly recorded in wild populations. In order to maintain the efficacy of vector control strategies, the characterization of concomitant kdr and ace-1 resistance, and their pleiotropic effects on malaria vector phenotype on insecticide efficacy are important., Methods: Larval and adult bioassays were performed with different insecticide classes used in public health following WHO standard guidelines on four laboratory Anopheles gambiae strains, sharing the same genetic background but harboring distinct resistance status: KISUMU with no resistance allele; ACERKIS with ace-1(R) allele; KISKDR with kdr(R) allele and ACERKDRKIS with both resistance alleles' ace-1(R) and kdr(R) ., Results: Larval bioassays indicate that the homozygote resistant strain harboring both alleles (ACERKDRKIS) displayed slightly but significantly higher resistance level to various insecticides like carbamates (bendiocarb, p < 0.001; propoxur, p = 0.02) and organophosphates (chlorpyriphos-methyl, p = 0.002; fenitrothion, p < 0.001) when compared to ACERKIS strain. However, no differences were recorded between ACERKDRKIS and KISKDR resistance level against permethrin (Pyrethroid, p = 0.7) and DDT (Organochlorine, p = 0.24). For adult bioassays, the percentages of mosquitoes knocked down were significantly lower for ACERKDRKIS than for KISKDR with permethrin (p = 0.003) but not with deltamethrin. The percentage of mortality from adult bioassays was similar between ACERKDRKIS and ACERKIS with carbamates and organophosphates, or between ACERKDRKIS and KISKDR with pyrethroid and DDT. Concerning acetylcholinesterase enzyme, ACERKDRKIS strain showed similarAChE1 activity than that of ACERKIS., Conclusion: The presence of both kdr(R) and ace-1(R) alleles seems to increase the resistance levels to both carbamate and organophosphate insecticides and at operational level, may represent an important threat to malaria vector control programs in West Africa.

The status of genes conferring resistance to organophosphate and carbamate insecticides has been examined in Culex pipiens pipiens mosquitoes sampled in Algeria. Presence of overproduced esterases was sporadic, but acetylcholinesterase-1 resistant alleles were observed in almost all samples. We focused our study on the AChE1 G119S substitution characterized in almost all samples, mostly at the heterozygous state. A genetic test revealed the presence of ace-1 duplication associating a susceptible and a resistant ace-1 copy. Molecular characterization showed a high occurrence of ace-1 duplication with six distinct duplicated alleles out of four samples. The inferred frequency of duplicated allele suggests that it is replacing the single resistant G119S allele. Finally, we discuss the mechanism at the origin of these duplicated haplotypes and their consequences on the management of insecticide resistance., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised., Results: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain., Conclusions: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR-RLFP assay developed in this study.

Two amino acid substitutions in acetylcholinesterase 1 (AChE1), G119S and F290V, are responsible for resistance to organophosphate and carbamate insecticides in Culex pipiens mosquitoes. These mutations generate very different levels of insensitivity to insecticide inhibitors. We described here a biochemical method that rapidly identifies AChE1 variants (susceptible, G119S and F290V, named S, R and V, respectively) present in individual mosquitoes. We investigated the frequency of AChE1 phenotypes in 41 field samples collected around the Mediterranean Sea. F290V substitution was found only in 15 samples and at low frequency, whereas G119S was highly spread in all samples. However, seven V distinct alleles were identified whereas only one R allele was present. The [V] enzymatic phenotype was never observed alone, and the V allele was always found associated with the susceptible and/or G119S AChE1 ([VS], [VR] or [VRS] phenotypes). Furthermore, we showed the presence of duplicated alleles, associating a susceptible and a V copy of the ace-1 gene, in most individuals analyzed for its presence. Evolutionary forces driving the large number of F290V ace-1 alleles and their low frequency in Mediterranean countries are discussed., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: Insecticide resistance is a rapid and recent evolutionary phenomenon with serious economic and public health implications. In the mosquito Anopheles gambiae s.s., main vector of malaria, resistance to organophosphates and carbamates is mainly due to a single amino-acid substitution in acetylcholinesterase 1 (AChE1). This mutation entails a large fitness cost. However, a resistant duplicated allele of the gene encoding AChE1 (ace-1), potentially associated to a lower fitness cost, recently appeared in An. gambiae., Methods: Using molecular phenotype data collected from natural populations from West Africa, the frequency of this duplicated allele was investigated by statistical inference. This method is based on the departure from Hardy-Weinberg phenotypic frequency equilibrium caused by the presence of this new allele., Results: The duplicated allele, Ag-ace-1(D), reaches a frequency up to 0.65 in Ivory Coast and Burkina Faso, and is potentially present in Benin. A previous study showed that Ag-ace-1(D), present in both M and S molecular forms in different West Africa countries, was generated by a single genetic event. This single origin and its present distribution suggest that this new allele is currently spreading., Conclusion: The spread of this less costly resistance allele could represent a major threat to public health, as it may impede An. gambiae control strategies, and thus increases the risk of malaria outbreaks.

In natural populations of mosquitoes, high level of resistance to carbamates (CX) and organophosphates (OP) is provided by insensitive acetylcholinesterase (AChE1). Different alleles conferring resistance have been identified at the ace1 locus. They differ from the wild-type by only one amino-acid substitution. The comparison of the biochemical characteristics of mutated recombinant proteins and AChE1 in resistant mosquito extracts confirmed the role of each substitution in insensitivity. Selection of these different resistant alleles in field populations depends likely on the insecticides used locally. Theoretical modelling studies are initiated to develop novel strategies of mosquito control.

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.

Characterization of insecticide resistance provides data on the evolutionary processes involved in the adaptation of insects to environmental changes. Studying the dominance status and resistance level represents a great interest, in terms of understanding resistance evolution in the field to eventually adapt vector control. Resistance and dominance levels conferred by the G119S mutation of acetylcholinesterase (ace-1R) of the mosquito Anopheles gambiae s.s. (Diptera: Culicidae) were studied for various insecticides belonging to different classes, using strains sharing the same genetic background. Our survey shows that the homozygote resistant strain AcerKis displayed a very high resistance level to various carbamates (range 3,000- to 5,000-fold) compared with that of various organophosphates (range 12- to 30-fold). Furthermore, the dominance status varied between semi-recessivity with fenitrothion and chlorpyrifos methyl insecticides to semidominance with temephos, carbosulfan, and propoxur. These results indicate that this resistance mechanism could spread rapidly in the field and then compromise the use of organophosphate and carbamate compounds in public health. This study underlines the necessity to monitor the ace-1R mutation in natural populations before planning and implementing malaria control programs based on the use of these insecticides.

Insecticide resistance owing to insensitive acetylcholinesterase (AChE)1 has been reported in several mosquito species, and only two mutations in the ace-1 gene have been implicated in resistance: 119S and 331W substitutions. We analyzed the AChE1 resistance status of Culex vishnui (Theobald) and Culex tritaeniorhynchus Giles sampled in various regions of China. These two species displayed distinct mutations leading to AChE1 insensitivity; the 119S substitution in resistant C. vishnui mosquitoes and the 331W substitution in resistant C. tritaeniorhynchus. A biochemical test was validated to detect the 331W mutation in field samples. The comparison of the recombinant G119S and 331W mutant proteins produced in vitro with the AChE1 extracted from resistant mosquitoes indicated that the AChE1 insensitivity observed could be specifically attributed to these substitutions. Comparison of their biochemical characteristics indicated that the resistance conferred by these mutations depends on the insecticide used, regardless of its class. This resistance seemed to be fixed in the Cx. tritaeniorhynchus populations sampled in a 2000-km transect, suggesting a very high level of insecticide application or a low fitness cost associated with this 331W mutation.

Gene duplication is thought to be the main potential source of material for the evolution of new gene functions. Several models have been proposed for the evolution of new functions through duplication, most based on ancient events (Myr). We provide molecular evidence for the occurrence of several (at least 3) independent duplications of the ace-1 locus in the mosquito Culex pipiens, selected in response to insecticide pressure that probably occurred very recently (<40 years ago). This locus encodes the main target of several insecticides, the acetylcholinesterase. The duplications described consist of 2 alleles of ace-1, 1 susceptible and 1 resistant to insecticide, located on the same chromosome. These events were detected in different parts of the world and probably resulted from distinct mechanisms. We propose that duplications were selected because they reduce the fitness cost associated with the resistant ace-1 allele through the generation of persistent, advantageous heterozygosis. The rate of duplication of ace-1 in C. pipiens is probably underestimated, but seems to be rather high.

In insects, selection of insecticide-insensitive acetylcholinesterase (AChE) is a very common resistance mechanism. Mosquitoes possess both AChE1 and AChE2 enzymes and insensitivity is conferred by single amino-acid changes located near the active site of the synaptic AChE1. Only two positions have been reported so far to be involved in resistance, suggesting a very high structural constraint of the AChE1 enzyme. In particular, the G119S substitution was selected in several mosquitoes' species and is now largely spread worldwide. Yet, a different type of AChE1 insensitivity was described 10 years ago in a Culex pipiens population collected in Cyprus in 1987 and fixed thereafter as the ACE-R strain. We report here the complete amino-acid sequence of the ACE-R AChE1 and show that resistance is associated with a single Phe-to-Val substitution of residue 290, which also lines the active site. Comparison of AChE1 activities of the recombinant F290V protein and ACE-R mosquito extracts confirmed the causal role of the substitution in insensitivity. Biochemical characteristics of the mutated protein indicated that the resistance level varies with the insecticide used. A molecular diagnosis test was designed to detect this mutation and was used to show that it is still present in Cyprus Island.

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies.

Organophosphate/carbamate target resistance has emerged in Culex pipiens L. (Diptera: Culicidae), the vector of Wuchereria bancrofti and West Nile virus (family Flaviviridae, genus Flavivirus) in China. The insensitive acetylcholinesterase was detected in only one of 20 samples collected on a north-to-south transect. According to previous findings, a unique mutation, G119S in the ace-1 gene, explained this high insensitivity. Phylogenetic analysis indicates that the mutation G119S recently detected in China results from an independent mutation event. The G119S mutation thus occurred at least three times independently within the Cx. pipiens complex, once in the temperate (Cx. p. pipiens) and twice in the tropical form (Cx. p. quinquefasciatus). Bioassays performed with a purified G119S strain indicated that this substitution was associated with high levels of resistance to chlorpyrifos, fenitrothion, malathion, and parathion, but low levels of resistance to dichlorvos, trichlorfon, and fenthion. Hence, it is possible that in China, dichlorvos, trichlorfon, and fenthion will still achieve effective control even in the presence of the G119S mutation.

High insecticide resistance resulting from insensitive acetylcholinesterase (AChE) has emerged in mosquitoes. A single mutation (G119S of the ace-1 gene) explains this high resistance in Culex pipiens and in Anopheles gambiae. In order to provide better documentation of the ace-1 gene and the effect of the G119S mutation, we present a three-dimension structure model of AChE, showing that this unique substitution is localized in the oxyanion hole, explaining the insecticide insensitivity and its interference with the enzyme catalytic functions. As the G119S creates a restriction site, a simple PCR test was devised to detect its presence in both A. gambiae and C. pipiens, two mosquito species belonging to different subfamilies (Culicinae and Anophelinae). It is possibile that this mutation also explains the high resistance found in other mosquitoes, and the present results indicate that the PCR test detects the G119S mutation in the malaria vector A. albimanus. The G119S has thus occurred independently at least four times in mosquitoes and this PCR test is probably of broad applicability within the Culicidae family.

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.