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Insecticide resistance has been reported to impact the interactions between mosquitoes and the pathogens they transmit. However, the effect on vector competence for arboviruses still remained to be investigated. We examined the influence of two insecticide resistance mechanisms on vector competence of the mosquito Culex quinquefasciatus for two arboviruses, Rift Valley Fever virus (RVFV) and West Nile virus (WNV). Three Cx. quinquefasciatus lines sharing a common genetic background were used: two insecticide-resistant lines, one homozygous for amplification of the Ester 2 locus (SA2), the other homozygous for the acetylcholinesterase ace-1 G119S mutation (SR) and the insecticide-susceptible reference line Slab. Statistical analyses revealed no significant effect of insecticide-resistant mechanisms on vector competence for RVFV. However, both insecticide resistance mechanisms significantly influenced the outcome of WNV infections by increasing the dissemination of WNV in the mosquito body, therefore leading to an increase in transmission efficiency by resistant mosquitoes. These results showed that insecticide resistance mechanisms enhanced vector competence for WNV and may have a significant impact on transmission dynamics of arboviruses. Our findings highlight the importance of understanding the impacts of insecticide resistance on the vectorial capacity parameters to assess the overall consequence on transmission.

International audience; We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses 1 mg l−1 and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1V or the duplicated ace-1D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

Background Understanding the age-structure of mosquito populations, especially malaria vectors such as Anopheles gambiae, is important for assessing the risk of infectious mosquitoes, and how vector control interventions may impact this risk. The use of near-infrared spectroscopy (NIRS) for age-grading has been demonstrated previously on laboratory and semi-field mosquitoes, but to date has not been utilized on wild-caught mosquitoes whose age is externally validated via parity status or parasite infection stage. In this study, we developed regression and classification models using NIRS on datasets of wild An. gambiae (s.l.) reared from larvae collected from the field in Burkina Faso, and two laboratory strains. We compared the accuracy of these models for predicting the ages of wild-caught mosquitoes that had been scored for their parity status as well as for positivity for Plasmodium sporozoites. Results Regression models utilizing variable selection increased predictive accuracy over the more common full-spectrum partial least squares (PLS) approach for cross-validation of the datasets, validation, and independent test sets. Models produced from datasets that included the greatest range of mosquito samples (i.e. different sampling locations and times) had the highest predictive accuracy on independent testing sets, though overall accuracy on these samples was low. For classification, we found that intramodel accuracy ranged between 73.5–97.0% for grouping of mosquitoes into “early” and “late” age classes, with the highest prediction accuracy found in laboratory colonized mosquitoes. However, this accuracy was decreased on test sets, with the highest classification of an independent set of wild-caught larvae reared to set ages being 69.6%. Conclusions Variation in NIRS data, likely from dietary, genetic, and other factors limits the accuracy of this technique with wild-caught mosquitoes. Alternative algorithms may help improve prediction accuracy, but care should be taken to either maximize variety in models or minimize confounders. Electronic supplementary material The online version of this article (10.1186/s13071-017-2501-1) contains supplementary material, which is available to authorized users.

International audience; While gene copy-number variations play major roles in long-term evolution, their early dynamics remains largely unknown. However, examples of their role in short-term adaptation are accumulating: identical repetitions of a locus (homogeneous duplications) can provide a quantitative advantage, while the association of differing alleles (heterogeneous duplications) allows carrying two functions simultaneously. Such duplications often result from rearrangements of sometimes relatively large chromosome fragments, and even when adaptive, they can be associated with dele-terious side effects that should, however, be reduced by subsequent evolution. Here, we took advantage of the unique model provided by the malaria mosquito Anopheles gambiae s.l. to investigate the early evolution of several duplications, heterogeneous and homogeneous, segregating in natural populations from West Africa. These duplications encompass ~200 kb and 11 genes, including the adaptive insecticide resistance ace-1 locus. Through the survey of several populations from three countries over 3-4 years, we showed that an internal deletion of all coamplified genes except ace-1 is currently spreading in West Africa and introgressing from An. gambiae s.s. to An. coluzzii. Both observations provide evidences of its selection, most likely due to reducing the gene-dosage disturbances caused by the excessive copies of the nona-daptive genes. Our study thus provides a unique example of the early adaptive tra-jectory of duplications and underlines the role of the environmental conditions (insecticide treatment practices and species ecology). It also emphasizes the striking diversity of adaptive responses in these mosquitoes and reveals a worrisome process of resistance/cost trade-off evolution that could impact the control of malaria vectors in Africa.

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International audience; Mosquito vectors’ resistance to insecticides is usually considered a major threat to the recent progresses in malaria control. However, studies measuring the impact of interventions and insecticide resistance reveal inconsistencies when using entomological versus epidemiological indices. First, evaluation tests that do not reflect the susceptibility of mosquitoes when they are infectious may underestimate insecticide efficacy. Moreover, interactions between insecticide resistance and vectorial capacity reveal nonintuitive outcomes of interventions. Therefore, considering ecological interactions between vector, parasite, and environment highlights that the impact of insecticide resistance on the malaria burden is not straightforward and we suggest that vector control still matters despite insecticide resistance.

Insecticide resistance raises concerns for the control of vector-borne diseases. However, its impact on parasite transmission could be diverse when considering the ecological interactions between vector and parasite. Thus we investigated the fitness cost associated with insecticide resistance and Plasmodium falciparum infection as well as their interactive cost on Anopheles gambiae survival and fecundity. In absence of infection, we observed a cost on fecundity associated with insecticide resistance. However, survival was higher for mosquito bearing the kdr mutation and equal for those with the ace-1R mutation compared to their insecticide susceptible counterparts. Interestingly, Plasmodium infection reduced survival only in the insecticide resistant strains but not in the susceptible one and infection was associated with an increase in fecundity independently of the strain considered. This study provides evidence for a survival cost associated with infection by Plasmodium parasite only in mosquito selected for insecticide resistance. This suggests that the selection of insecticide resistance mutation may have disturbed the interaction between parasites and vectors, resulting in increased cost of infection. Considering the fitness cost as well as other ecological aspects of this natural mosquito-parasite combination is important to predict the epidemiological impact of insecticide resistance.

International audience; 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 identies 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 eld 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 identied 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.

BACKROUND: 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. Copyright © 2009 Society of Chemical Industry

Background Ivermectin has been proposed as a novel malaria transmission control tool based on its insecticidal properties and unique route of acquisition through human blood. To maximize ivermectin’s effect and identify potential resistance/tolerance mechanisms, it is important to understand its effect on mosquito physiology and potential to shift mosquito population age-structure. We therefore investigated ivermectin susceptibility and gene expression changes in several age groups of female Anopheles gambiae mosquitoes. Methods The effect of aging on ivermectin susceptibility was analyzed in three age groups (2, 6, and 14-days) of colonized female Anopheles gambiae mosquitoes using standard survivorship assays. Gene expression patterns were then analyzed by transcriptome sequencing on an Illumina HiSeq 2500 platform. RT-qPCR was used to validate transcriptional changes and also to examine expression in a different, colonized strain and in wild mosquitoes, both of which blood fed naturally on an ivermectin-treated person. Results Mosquitoes of different ages and blood meal history died at different frequencies after ingesting ivermectin. Mortality was lowest in 2-day old mosquitoes exposed on their first blood meal and highest in 6-day old mosquitoes exposed on their second blood meal. Twenty-four hours following ivermectin ingestion, 101 and 187 genes were differentially-expressed relative to control blood-fed, in 2 and 6-day groups, respectively. Transcription patterns of select genes were similar in membrane-fed, colonized, and naturally-fed wild vectors. Transcripts from several unexpected functional classes were highly up-regulated, including Niemann-Pick Type C (NPC) genes, peritrophic matrix-associated genes, and immune-response genes, and these exhibited different transcription patterns between age groups, which may explain the observed susceptibility differences. Niemann-Pick Type 2 genes were the most highly up-regulated transcripts after ivermectin ingestion (up to 160 fold) and comparing phylogeny to transcriptional patterns revealed that NPCs have rapidly evolved and separate members respond to either blood meals or to ivermectin. Conclusion We present evidence of increased ivermectin susceptibility in older An. gambiae mosquitoes that had previously bloodfed. Differential expression analysis suggests complex midgut interactions resulting from ivermectin ingestion that likely involve blood meal digestion physiological responses, midgut microflora, and innate immune responses. Thus, the transcription of certain gene families is consistently affected by ivermectin ingestion, and may provide important clues to ivermectin’s broad effects on malaria vectors. These findings contribute to the growing understanding of ivermectin’s potential as a transmission control tool. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2029-8) contains supplementary material, which is available to authorized users.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1–36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5–5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

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.

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by theace-1gene, distinct and divergent from theace-2gene, which performs this function inDrosophila. This is an unprecedented situation within the Diptera order because bothacegenes derive from an old duplication and are present in most insects and arthropods. Nevertheless,Drosophilapossesses only theace-2gene. 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 forace-1andace-2by 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 thatace-2plays 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 onace-2sequences is constant across the Diptera, irrespective of the presence or absence oface-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover oface-2and the loss oface-1, taking into account our limited knowledge of non-synaptic functions ofacegenes and some specific adaptations of true flies.

Recently there have been calls for the eradication of malaria and the elimination of soil-transmitted helminths (STHs). Malaria and STHs overlap in distribution, and STH infections are associated with increased risk for malaria. Indeed, there is evidence that suggests that STH infection may facilitate malaria transmission. Malaria and STH coinfection may exacerbate anemia, especially in pregnant women, leading to worsened child development and more adverse pregnancy outcomes than these diseases would cause on their own. Ivermectin mass drug administration (MDA) to humans for malaria parasite transmission suppression is being investigated as a potential malaria elimination tool. Adding albendazole to ivermectin MDAs would maximize effects against STHs. A proactive, integrated control platform that targets malaria and STHs would be extremely cost-effective and simultaneously reduce human suffering caused by multiple diseases. This paper outlines the benefits of adding albendazole to ivermectin MDAs for malaria parasite transmission suppression.

Despite its epidemiological importance, the impact of insecticide resistance on vector-parasite interactions and malaria transmission is poorly understood. Here, we explored the impact of Plasmodium infection on the level of insecticide resistance to dichlorodiphenyltrichloroethane (DDT) in field-caught Anopheles gambiae sensu stricto homozygous for the kdr mutation. Results showed that kdr homozygous mosquitoes that fed on infectious blood were more susceptible to DDT than mosquitoes that fed on noninfectious blood during both ookinete development (day 1 after the blood meal) and oocyst maturation (day 7 after the blood meal) but not during sporozoite invasion of the salivary glands. Plasmodium falciparum infection seemed to impose a fitness cost on mosquitoes by reducing the ability of kdr homozygous A. gambiae sensu stricto to survive exposure to DDT. These results suggest an interaction between Plasmodium infection and the insecticide susceptibility of mosquitoes carrying insecticide-resistant alleles. We discuss this finding in relation to vector control efficacy.

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s. , homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles–Plasmodium combination . Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.

Background Mass drug administration (MDA) of ivermectin to humans for control and elimination of filarial parasites can kill biting malaria vectors and lead to Plasmodium transmission reduction. This study examines the degree and duration of mosquitocidal effects resulting from single MDAs conducted in three different West African countries, and the subsequent reductions in parity and Plasmodium sporozoite rates. Methods Indoor-resting, blood-fed and outdoor host-seeking Anopheles spp. were captured on days surrounding MDAs from 2008–2013 in Senegalese, Liberian and Burkinabé villages. Mortality was assessed on a portion of the indoor collection, and parity status was determined on host-seeking mosquitoes. The effect of MDA was then analysed against the time relative to the MDA, the distributed drugs and environmental variables. Results Anopheles gambiae survivorship was reduced by 33.9% for one week following MDA and parity rates were significantly reduced for more than two weeks after the MDAs. Sporozoite rates were significantly reduced by >77% for two weeks following the MDAs in treatment villages despite occurring in the middle of intense transmission seasons. These observed effects were consistent across three different West African transmission dynamics. Conclusions These data provide a comprehensive and crucial evidence base for the significant reduction in malaria transmission following single ivermectin MDAs across diverse field sites. Despite the limited duration of transmission reduction, these results support the hypothesis that repeated MDAs with optimal timing could help sustainably control malaria as well as filarial transmission. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-417) contains supplementary material, which is available to authorized users.

International audience; Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management. Citation: Alout H, Labbé P, Berthomieu A, Djogbénou L, Leonetti J-P, et al. (2012) Novel AChE Inhibitors for Sustainable Insecticide Resistance Management. PLoS ONE 7(10): e47125.

International audience; 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. (C) 2010 Elsevier Ltd. All rights reserved.

Resistance to insecticides was monitored on Culex pipiens quinquefasciatus mosquitoes collected in twelve localities of La Reunion, a geographically isolated island of the Indian Ocean. This mosquito is of medical concern in the region as a known vector for filariasis and a potential vector for West Nile and Rift Valley Fever viruses. Our bioassays indicated the presence of resistance to all tested insecticides, i.e. organochlorides, organophosphates and pyrethroids. A molecular investigation revealed a higher frequency of resistance genes in the coastal areas compared to elevated rural sites, probably reflecting the different nature of insecticide pressures together with the genetic cost of resistance alleles. A simple molecular test was developed to detect Rdl R allele, encoding a γ-aminobutyric acid (GABA) receptor resistant to dieldrin. Unexpectedly high Rdl R frequencies were recorded over the whole island, despite this insecticide having been banned for over 15 years. This resistant allele was also detected for the first time in two samples of Aedes albopictus , a species recently involved in severe Chikungunya epidemics on the island. Rdl R selection in these two mosquito species discloses current insecticide pressures in urban areas, from unknown origins, that should be taken into account to develop vector control strategies.

International audience; 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.

BackgroundThe role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s.Methods and resultsTo establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression.ConclusionsThese results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa.

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.

International audience; 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 AChE I 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 AChE I and show that resistance is associated with a single Phe-to-Val substitution of residue 290, which also lines the active site. Comparison of AChEI 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. (c) 2006 Elsevier Ltd. All rights reserved.

The widespread insecticide resistance raises concerns for vector control implementation and sustainability particularly for the control of the main vector of human malaria, Anopheles gambiae sensu stricto. However, the extent to which insecticide resistance mechanisms interfere with the development of the malignant malaria parasite in its vector and their impact on overall malaria transmission remains unknown. We explore the impact of insecticide resistance on the outcome of Plasmodium falciparum infection in its natural vector using three An. gambiae strains sharing a common genetic background, one susceptible to insecticides and two resistant, one homozygous for the ace-1(R) mutation and one for the kdr mutation. Experimental infections of the three strains were conducted in parallel with field isolates of P. falciparum from Burkina Faso (West Africa) by direct membrane feeding assays. Both insecticide resistant mutations influence the outcome of malaria infection by increasing the prevalence of infection. In contrast, the kdr resistant allele is associated with reduced parasite burden in infected individuals at the oocyst stage, when compared to the susceptible strain, while the ace-1 (R) resistant allele showing no such association. Thus insecticide resistance, which is particularly problematic for malaria control efforts, impacts vector competence towards P. falciparum and probably parasite transmission through increased sporozoite prevalence in kdr resistant mosquitoes. These results are of great concern for the epidemiology of malaria considering the widespread pyrethroid resistance currently observed in Sub-Saharan Africa and the efforts deployed to control the disease.