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.     

Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.     

Electrophoretic analysis of esterase isozymes in organophosphate-resistant mosquitoes (Culex pipiens)

Esterase isozymes from individual mosquitoes, Culex pipiens, were analyzed by thin layer agar gel electrophoresis. Changes in esterase isozyme patterns during the developmental stages were studied. In most strains, only one band was found in eggs. During larval development many bands were gradually formed, and the most anodal bands were lost upon pupation. These larva-specific esterases may be controlled by three or more co-dominant alleles. In the adult, many bands were present that were probably controlled by at least seven loci. Est-2 may be controlled by co-dominant alleles; a recessive silent allele was also found.Esterase isozyme patterns of organophosphorus insecticide-resistant and susceptible strains were compared. Resistant strains had very active esterase bands but different patterns were found in malathion and temephos-resistant strains. The strong bands presumably hydrolyse the insecticides to which the strains are resistant, or their PO analogues. Their substrate specificity was studied with various esters.     

Recombination between two amplified esterase alleles in Culex pipiens

Esterase gene amplification at the Ester superlocus provides organophosphate resistance in the mosquito Culex pipiens (L.). In this study we explored the possibility of recombination between two amplified esterase alleles, thus generating a composite amplified allele. To do that, females heterozygous for two distinct amplified alleles (Ester(2) and Ester(4)) were crossed with males homozygous for a third resistance allele (Ester(8)). Among analyzed offspring, one recombinant composite allele (Ester(2-4)) was detected, providing a rate of recombination of approximately 0.2%. This is the first report of a recombination between two distinct amplified esterase alleles. This phenomenon renders the predictability of allele evolution considerably more complex than was previously thought.     

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     

Chromosomal organization of the amplified esterase B1 gene in organophosphate-resistant Culex pipiens quinquefasciatus Say (Diptera, Culicidae)

In Culex pipiens quinquefasciatus from California, high resistance to organophosphorus insecticides is due to an increased production of the detoxifying esterase B1 resulting from a 250-fold amplification of the structural gene. The chromosomal organization of this amplified gene was studied by in situ hybridization techniques. Esterase B1 gene copies were found to be clustered on a single chromosome, tentatively identified as chromosome II.     

Cytoplasmic factors in the gonads of Culex pipiens complex mosquitoes

Electron microscopic examination of the gonads of six species of the $\text{Culex pipiens}$ complex revealed rickettsia-like, intracellular symbionts in $\text{C.pipiens}$, $\text{C.molestus}$, $\text{C.pallens}$ and $\text{C.fatigans}$.¹ In the two endemic Australian members, $\text{C.a}$$\text{C.globocoxitus}$, no rickettsia-like symbionts were found, but virus-like particles were seen in the cytoplasm of the ovaries and certain associated tissues.     

Characterization of a novel high-activity esterase in Tunisian populations of the mosquito Culex pipiens

AIn 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 Ester(B12) 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 Ester(B12) allele was not amplified in TunB12 strain, indicating that B12 high activity could be due to a gene up-regulation mechanism. Ester(B12) 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 Ester(B12) 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.     

Identification of two distinct amplifications of the esterase B locus inCulex pipiens (L.) mosquitoes from mediterranean countries

Two new highly active esterases were detected by starch electrophoretic studies inCulex pipiens mosquitoes from the area of Montpellier (France) and from Cyprus. We demonstrate here that both the French and the Cyprus esterases B are overproduced due to amplification of the coding gene. The production of the esterase B is approximately 50- and 500-fold higher in mosquitoes from France and Cyprus, respectively, than in susceptible insects, whereas the number of gene copies is about 25 and 250. Differences of about 7- and 95-fold were also found in the degree of chlorpyrifos resistance. RFLP comparison of the amplified region containing the esterase B gene revealed large differences between French and Cyprus mosquitoes. It thus appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified. We therefore named the haplotypes in mosquitoes from France and Cyprus B4 and B5, respectively. The estimated genetic distance between these two haplotypes is not smaller than those observed in all pair comparisons of other known esterase B haplotypes. These results are discussed in the context of amplification phenomena.This work was supported in part by Grant 89.1540 from the Ministère de l'Education Nationale and by the CNRS/Programme Environnement.     

库蚊野生种群扩增等位基因重组现象分析

羧酸酯酶 (carboxylesterases)的过量产生是库蚊Culexpipiens对有机磷杀虫剂 (OP)产生抗性的主要机制。由est 3和est 2组成的酯酶超级基因座 (estersuper locus)的基因扩增是引起酯酶基因扩增的主要遗传学基础。通过淀粉电泳研究了采自广州、佛山、郑州的库蚊野生蚊虫种群 ,发现在这些种群中存在着扩增等位基因重组现象。该现象可能是蚊虫受到杀虫药剂的选择压力、等位基因多样性和等位基因型频率的影响。这将提供一个研究抗性进化的自然模型。     

Biochemical genetics of phosphohydrolase polymorphism in Culex pipiens complex.

1. Single 4th-instar larvae were used in the investigation of alkaline phosphohydrolase (APH) variability in Culex pipiens quinequefasciatus. The genetic basis of isozyme variability was determined from genetic crosses performed with isogenic and hybrid strains of mosquitoes. 2. Isozyme electromorphs presented four enzyme activity zones, three monomorphic and one polymorphic, correspondent with four APH gene loci (aph1, aph2, aph3 and aph4). 3. All isozymes migrated anodically, with aph4 isozymes migrating most rapidly. 4. Enzyme polymorphism was evident only at aph4 locus, with three allozymes present. 5. aph4 allozymes are conditioned by multiple, co-dominant alleles transmitted in a Mendelian manner. 6. Differential frequencies and selection for aph4 alleles and genotypes are suggested by data from genetic crosses.     

我国尖音库蚊复合组蚊虫的杂交及其与Wolbachia感染的关系

为了了解我国尖音库蚊复合组蚊虫间杂交卵的不孵化现象和明确该现象与共生微生物Wolbachia感染的关系,对该复合组实验室种群4个亚种进行了笼内杂交和抗生素处理后的杂交。试验表明: 在复合组蚊虫中骚扰库蚊Culex pipiens molestus与淡色库蚊Cx. Pipiens pallens、致倦库蚊Cx. Ipiens quinquefasciatus与尖音库蚊Cx. Pipiens pipiens之间存在有单向胞质不融合现象,骚扰库蚊的雄虫与尖音库蚊、致倦库蚊和淡色库蚊的雌虫杂交卵的孵化率分别为0.06%、0.46%和0.19%;该胞质不融合现象可以通过抗生素处理而消除,处理后骚扰库蚊雄虫与其余3个亚种雌虫F₃杂交卵的孵化率均有提高,分别为89.49%（t=3.90×10^-28＜t_0.01=2.704）、23.39%（t=9.15×10^-7＜t_0.01=2.660_）和22.27%（t=5.08×10^-4＜t_0.01=2.750）,并可因抗生素处理而产生新的不融合类型。