Evolution of insecticide resistance in Culex pipiens populations: The Corsican paradox

Culex pipiens mosquitoes from Corsica have been subject to insecticide treatments since 1971, using temephos (an organophosphate). After 17 years, resistance has not developed beyond a 14-fold level. This relatively low resistance is due to the presence of several identified resistance genes, including the insensitive target ( Ace^R ) and overproduced esterases (A1, A4 and B4). The fact that only a low resistance has developed after 17 years of treatment and that this low resistance level is the result of a relatively large number of resistance genes constitute a paradox. To understand this situation and explain why a higher temephos resistance level has not evolved in Corsica as in other parts of the world, it is proposed that the occurrence (through mutation or migration) of efficient resistance genes was a limiting step, and that the only resistance genes available at that time through migration from the surrounding Mediterranean countries had a low cross-resistance to temephos. The local situation of Corsica is discussed in the light of recent data on the world distribution of the known organophosphate resistance genes in this species, and the relative role of mutation and migration in the evolution of insecticide resistance in natural populations.     

不同地区尖音库蚊复合组抗药性的分子特征

通过生物测定、蛋白质电泳和单个蚊虫基因组Southern杂交三种方法对三个不同地区的尖音库蚊复合组蚊虫Culexpipienscomplex的抗性水平、抗性相关基因在种群中的分布及抗性分子特征进行了研究。采自山东高密、北京和云南昆明的尖音库蚊复合组蚊虫 4龄幼虫对敌敌畏、对硫磷 (有机磷类 )和仲丁威 (氨基甲酸酯类 )的抗性水平的测定结果表明 :与北京敏感系相比 ,高密种群对敌敌畏、对硫磷的抗性水平很高 ,北京种群对敌敌畏的抗性水平也很高 (约 30倍 ) ,三个种群对仲丁威的抗性与北京敏感系没有明显的区别。淀粉电泳的结果表明 :高密和北京种群中存在过量产生的非特异性酯酶Estβ11,昆明种群中存在过量产生的非特异性酯酶Estα2和Estβ2 ,单个蚊虫基因组Southern杂交的结果表明 :酯酶Estβ11、Estα2和Estβ2的结构基因发生了不同程度的扩增导致酯酶的过量产生     

Esterase polymorphism and sensitivity to dursban organophosphorus insecticide in Culex pipiens pipiens populations

Esterase polymorphism and Dursban (O,O-dimethyl-2-pyridylphosphorothioate) sensitivity have been investigated in 12 natural populations and three laboratory strains of Culex pipiens pipiens. This mosquito has two esterase loci, Est-1 and Est-2, which were shown to code esterases of the B group (aliesterases) but not cholinesterases. No correlation between Est-1 polymorphism and Dursban sensitivity was found, but the increase of the Est-2(0.64) allele in the populations less sensitive to Dursban was highly significant (r = -0.9850 for 6 df).     

杭州地区尖音库蚊复合组的抗药性动态

用生物测定和淀粉电泳技术对采自杭州市和上海市的尖音库蚊复合组(Culex pipienscomplex)蚊虫的抗性水平及与抗性有关的酯酶进行了研究。抗性水平的测定结果表明:与S-LAB敏感系相比,杭州市种群对DDVP、对硫磷、毒死蜱、邻仲丁基苯基甲基氨基甲酸酯和残杀威的抗性分别是S-LAB敏感品系的3.9,7.6,1.6,1.6,2.5倍。利用淀粉凝胶电泳技术分析了野生种群中抗性羧酸酯酶(EST)等位酶的基因表型及其频率。4个种群中都存在着世界范围内广泛分布的过量产生的非特异性酯酶Estβ1和Estα2/β2,上海种群主要以高活性的酯酶Estβ11为主(98.3%),除了这些基因以外,2004年杭州市下城区又出现了1种新的酯酶基因(50%)。     

Population genetics of insecticide resistance in the mosquito Culex pipiens

Thirty years of control of the mosquito Culex pipiens using organophosphate insecticides (OP) has selected for OP-resistance alleles on a world-wide scale. As reviewed here, studies at the levels of gene and population allow identification of the main forces driving this process of adaptation. Three loci are involved in OP-resistance in C. pipiens. For two of these, adaptive mutations were found to be rare events, such that the ubiquitous distribution of certain resistance alleles could only be explained as deriving from a single origin by mutation followed by extensive migration. Population structure analyses confirmed that long-distance migration is frequent. Thus, different resistance alleles could accumulate and compete within populations soon after their origin by mutation. The different selection pressures acting on these alleles, i.e. their selective advantage in the presence of OP and their disadvantage (resistance cost) in absence of OP, were also analysed. Substantial differences in resistance cost among alleles present within the Mediterranean area were discovered. Long-term surveys of Mediterranean populations confirmed the pivotal importance of resistance cost in shaping the evolution of this adaptive polymorphism. Some hypotheses on the functional links between the nature of the initial mutation events and the subsequent evolution of polymorphism are discussed.     

Forty years of erratic insecticide resistance evolution in the mosquito Culex pipiens

One view of adaptation is that it proceeds by the slow and steady accumulation of beneficial mutations with small effects. It is difficult to test this model, since in most cases the genetic basis of adaptation can only be studied a posteriori with traits that have evolved for a long period of time through an unknown sequence of steps. In this paper, we show how ace-1, a gene involved in resistance to organophosphorous insecticide in the mosquito Culex pipiens, has evolved during 40 years of an insecticide control program. Initially, a major resistance allele with strong deleterious side effects spread through the population. Later, a duplication combining a susceptible and a resistance ace-1 allele began to spread but did not replace the original resistance allele, as it is sublethal when homozygous. Last, a second duplication, (also sublethal when homozygous) began to spread because heterozygotes for the two duplications do not exhibit deleterious pleiotropic effects. Double overdominance now maintains these four alleles across treated and nontreated areas. Thus, ace-1 evolution does not proceed via the steady accumulation of beneficial mutations. Instead, resistance evolution has been an erratic combination of mutation, positive selection, and the rearrangement of existing variation leading to complex genetic architecture.     

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.     

Fitness costs of insecticide resistance in natural breeding sites of the mosquito Culex pipiens

Genetic changes conferring adaptation to a new environment may induce a fitness cost in the previous environment. Although this prediction has been verified in laboratory conditions, few studies have tried to document this cost directly in natural populations. Here, we evaluated the pleiotropic effects of insecticide resistance on putative fitness components of the mosquito Culex pipiens. Experiments using different larval densities were performed during the summer in two natural breeding sites. Two loci that possess alleles conferring organophosphate (OP) resistance were considered: ace-1 coding for an acetylcholinesterase (AChE1, the OP target) and Ester, a 'super locus" including two closely linked loci coding for esterases A and B. Resistance ace-1 alleles coding for a modified AChE1 were associated with a longer development time and shorter wing length. The pleiotropic effects of two resistance alleles Ester1 and Ester4 coding for the overproduced esterases A1 and A4-B4, respectively, were more variable. Both A1 and A4-B4 reduced wing length, although only A1 was associated with a longer preimaginal stage. The fluctuating asymmetry (FA) of the wing did not respond to the presence or to the interaction of resistance alleles at the two loci at any of the density levels tested. Conversely, the FA of one wing section decreased when larval density increased. This may be the consequence of selection against less developmentally stable individuals. The results are discussed in relation to the local evolution of insecticide resistance genes.     

Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens

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.     

Insecticide resistance in Chinese populations of the Culex pipiens complex through esterase overproduction

In most parts of China, mosquitoes have been subjected to organophosphate (OP) insecticide treatments since the mid-1960s, and resistance gene monitoring in the Culex pipiens complex (Diptera: Culicidae) started in only a few locations from the end of the 1980s. Many resistant alleles at the Ester locus have been found in field populations, including those commonly found around the world ( Ester ^B1 and Ester ² ), and those endemic to China ( Ester ^B6 , Ester ^B7 , Ester ⁸ , and Ester ⁹ ). This situation is atypical, and may represent a complex situation for the evolution of insecticide resistance genes in China. To increase our understanding of the Chinese situation and our ability to manage resistance in the C. pipiens complex, a large study was performed. Twenty field populations were sampled from Beijing to Guangzhou. Bioassays with five insecticides (dichlorvos, parathion, chlorpyrifos, 2-sec-butylphenyl methyl carbamate, and propoxur) disclosed resistance levels variable according to the geographic origin, and up to 85-fold for dichlorvos. Six overproduced esterases were identified, including two that have not been previously described. Most of them were found in all samples, although at variable frequencies, suggesting variable selection or a transient situation, e.g., each one was recently restricted to a particular geographic area. The results are discussed in the context of recent alterations to insecticide campaigns, and of the evolution of resistance genes in Chinese C. pipiens populations.     

A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus

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.     

Controlling Culex pipiens: antagonists are more efficient than a neonicotinoid insecticide

Species vulnerability to pesticides depends on physiological sensitivity, the potential to recover, and the ecological context. We assessed the vulnerability of the mosquito Culex pipiens to a repeated treatment with thiacloprid in outdoor microcosms with and without antagonists (competitive and predatory invertebrates). Microcosms were treated repeatedly (three times) with thiacloprid at a concentration of 0.1, 1, or 10 µg/liter. In microcosms without antagonists, the abundance of Cx. pipiens larvae decreased moderately after the second and the third exposures to 10 µg/liter thiacloprid. In microcosms with antagonists, the abundance of Cx. pipiens larvae declined to approximately zero in the control group and the low concentration treatments during the five weeks of observation. By contrast, the abundance of Cx. pipiens larvae temporarily increased at 10 µg/liter thiacloprid after the second and third contamination. We explained this positive effect on the development of Cx. pipiens because of the decrease in competition due to the elimination of sensitive antagonists combined with the high recovery potential of Cx. pipiens. Based on these results, natural antagonists must be supported for the sustainable control of mosquitoes.     

Multiple incompatibilities within populations of Culex pipiens L. in southern France

Strains of Culex pipiens derived from natural populations collected in southern France were crossed to determine their ability to give fertile offspring. Uni- and bi-directional incompatibilities occurred between these strains and each of them had its own crossing properties. Compatibility or incompatibility of a cross seemed to be related to the geographic distance separating the parental colonies, but not to their ecological origin (hypogeous or epigeous). Our results showed evidence for the variation in time of crossing properties of a strain.     

Detection and widespread distribution of sodium channel alleles characteristic of insecticide resistance in Culex pipiens complex mosquitoes in China

Culex pipiens complex mosquitoes are widely distributed throughout China and are known to be important disease vectors. Two pyrethroid resistance associated mutations have been identified in Cx. pipiens complex (Diptera: Culicidae), but there is little information on the diversity and distribution of kdr alleles in pyrethroid resistance in Cx. pipiens complex mosquitoes in China. In the present study, we report on a modified three tube allele-specific (AS)-PCR method for detecting the 1014F and 1014S alleles. The new technique was applied to identify the distribution of the two alleles in natural Cx. pipiens complex populations in China. The results confirmed that the new method is both sensitive and specific. The 1014F allele was found in all 14 of the field populations tested (frequency ranged from 6.8 to 76.2%) and the 1014S allele was found in almost two-thirds (frequency from 2.4 to 28.6%), indicating that the genotypes known to be associated with pyrethroid resistance are widespread in China. The resistance-associated alleles were more common in southern Chinese sampling sites than in northern sites. The coexistence of the two resistant mutations in individual mosquitoes was also observed in five of the field populations. Two alternative mutations within the L1014 codon were identified in Culex pipiens molestus Forskal, 1775, including a non-synonymous mutation resulting in a 1014C substitution.