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。     

The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance

The primary routes of insecticide resistance in all insects are alterations in the insecticide target sites or changes in the rate at which the insecticide is detoxified. Three enzyme systems, glutathione S-transferases, esterases and monooxygenases, are involved in the detoxification of the four major insecticide classes. These enzymes act by rapidly metabolizing the insecticide to non-toxic products, or by rapidly binding and very slowly turning over the insecticide (sequestration). In Culex mosquitoes, the most common organophosphate insecticide resistance mechanism is caused by co-amplification of two esterases. The amplified esterases are differentially regulated, with three times more Est beta 2(1) being produced than Est alpha 2(1). Cis-acting regulatory sequences associated with these esterases are under investigation. All the amplified esterases in different Culex species act through sequestration. The rates at which they bind with insecticides are more rapid than those for their non-amplified counterparts in the insecticide-susceptible insects. In contrast, esterase-based organophosphate resistance in Anopheles is invariably based on changes in substrate specificities and increased turnover rates of a small subset of insecticides. The up-regulation of both glutathione S-transferases and monooxygenases in resistant mosquitoes is due to the effects of a single major gene in each case. The products of these major genes up-regulate a broad range of enzymes. The diversity of glutathione S-transferases produced by Anopheles mosquitoes is increased by the splicing of different 5' ends of genes, with a single 3' end, within one class of this enzyme family. The trans-acting regulatory factors responsible for the up-regulation of both the monooxygenase and glutathione S-transferases still need to be identified, but the recent development of molecular tools for positional cloning in Anopheles gambiae now makes this possible.     

Molecular and kinetic evidence for allelic variants of esterase Estβ1 in the mosquito Culex quinquefasciatus

Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.     

Characterization of amplified esterase Estβ12 associated with organophosphate resistance in a multi-resistant population of the mosquitoCulex quinquefasciatus from Cuba

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estα2 ^1\ Estβ2 ¹ esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estβ1s are much rarer. Individuals with the Estβ1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estα2 ¹\ Estβ2 ¹ amplicons. To test the hypothesis that this is because Estβ1 is less able to bind insecticide than the common amplified esterases, Estβ1² was purified from the multi-resistant Habana strain of Culex quinquefasciatus , from Cuba. In its native form Estβ1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estβ1² with several OP insecticides were similar to those for the commonly elevated esterases Estα2¹ and Estβ2¹, and much higher than for the electrophoretically identical non-elevated Estβ1³ and Estα3. Hence the apparent selective advantage of the Estα2 ¹\ Estβ2 ¹ amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.     

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.     

不同地域有机磷杀虫药剂抗性库蚊复合组酯酶B1扩增的研究

在库蚊Culex pipiens品系中,非专一性酯酶的过量产生是对有机磷杀虫药剂抗性的普遍机理。酯酶基因位于紧密连锁的A和B座位上。现已知所有酯酶B的过量产生都是基因扩增的结果。为了确定不同国家库蚊品系的酯酶B1的过量产生是否都是相同DNA单基因型扩增的结果,我们构建了酯酶B1结构基因扩增区的限制性内切酶酶切图谱,分析了限制性酶切片段长度多态性(RFLP)。研究发现不同地理位置的酯酶B1库蚊,如法属圭亚那,委内瑞拉、波多黎各岛、美国加利福尼亚和中国北京,都有着相同的单基因扩增,但在扩增水平上有较大的差异。我们认为无论在美洲或亚洲,凡是酯酶B1扩增的库蚊都为同一个起源,之后经迁移而传播到各地;同时发现酯酶B1扩增的库蚊与酯酶A2一B2扩增的库蚊相比,其迁移有一定的局限性;并且酯酶B1扩增的库蚊仅仅限于美国、加勒比和中国的一些地区,而酯酶A2-B2扩增的库蚊则广泛地分布于美国、加勒比、亚洲、非洲、太平洋各岛及欧洲等地。     

有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析

致倦库蚊Culex qinquefasciatus是丝虫病的主要传染媒介。通过生物测定、单个蚊虫酯酶α2和β2基因拷贝数分析和酯酶β基因序列比较, 分析了抗性水平、抗性相关基因在种群中的分布及其基因拷贝数等的抗性分子特征。应用快速PCR仪（realtime quantitatIve PCRs）直接检测库蚊中酯酶基因和mRNA拷贝数。结果显示：上海致倦库蚊对对硫磷的抗性LC₅₀为8.12, 酯酶活性升高是上海致倦库蚊种群对有机磷杀虫药剂产生抗性的主要机理。编码致倦库蚊酯酶β的氨基酸序列同编码尖音库蚊酯酶B1的氨基酸序列相比同源性为98%;同致倦库蚊酯酶B2氨基酸序列相比同源性为100%,同环蹶库蚊酯酶B3氨基酸序列相比同源性为90%, 上海致倦库蚊中酯酶α和β基因均扩增。有机磷抗性的上海和PellRR蚊虫种群中单个蚊虫酯酶α2 和β2定量基因拷贝数均不同,其同一蚊虫个体的酯酶α2 比酯酶β2基因的拷贝数高,但没有明显的规律性,酯酶结构基因的扩增是上海致倦库蚊种群对有机磷杀虫药剂抗性的主要机理,估计在野外种群的杂合个体中存在多种调控机制。     

The molecular basis of two contrasting metabolic mechanisms of insecticide resistance

The esterase-based insecticide resistance mechanisms characterised to date predominantly involve elevation of activity through gene amplification allowing increased levels of insecticide sequestration, or point mutations within the esterase structural genes which change their substrate specificity. The amplified esterases are subject to various types of gene regulation in different insect species. In contrast, elevation of glutathione S-transferase activity involves upregulation of multiple enzymes belonging to one or more glutathione S-transferase classes or more rarely upregulation of a single enzyme. There is no evidence of insecticide resistance associated with gene amplification in this enzyme class. The biochemical and molecular basis of these two metabolically-based insecticide resistance mechanisms is reviewed.     

Variation in general esterase activity within a population of Haematobia irritans (Diptera: Muscidae).

Control of the horn fly, Hematobia irritans (L.), is generally dependent on chemical insecticides. However, the biology and behavior of the horn fly favors rapid development of insecticide resistance. To prolong the effectiveness of the insecticide option, information is required regarding the mechanisms of insecticide resistance. Metabolic hydrolysis of insecticides by esterases is a detoxification mechanism in many insect species. Measurement of general esterase activity within populations of horn flies may provide a diagnostic tool for resistance management. In this study we evaluated the amount of variation in general esterase activity within female and male horn fly samples from a population that had not been exposed to insecticides for 8 yr. We found considerable variation in general esterase activity within samples of each sex, with females demonstrating the greater variation. The observed variation is thought to be the result of age-structure dynamics within the population. The amount of inherent variation makes it difficult to detect small mean differences between populations, thus limiting the utility of general esterase assays. Thus, effective diagnosis of esterase-mediated resistance mechanisms can only be achieved by the identification of specific detoxification esterases and the design of assays, either biochemical or molecular, for their detection and measurement.     

在无杀虫剂压力下淡色库蚊的抗性稳定性研究(英文)

本文对淡色库蚊（Ｃｕｌｅｘ ｐｉｐｉｅｎｓ ｐａｌｌｅｎｓ Ｃｏｑｕｉｌｌｅｔｔ）的毒死蜱抗性品系、抗一敏杂交品系及抗性酯酶Ｂ_２纯合品系在无杀虫剂压力选择下的抗性水平变化进行了研究。结果表明,在毒死蜱抗性品系中,抗性水平逐代下降,ＬＣ_（５０）从Ｆ_０代的０．２０９９ｍｇ／Ｌ快速降至Ｆ_８代的０．０２６２ｍｇ／Ｌ,然后继续缓慢地降至Ｆ_（１６）代的０．０２０７ｍｇ／Ｌ。从毒死蜱抗性品系中的这种抗性水平下降,可以推断该品系中并不是纯的高抗性酯酶基因扩增个体,在传代中抗性个体的比例由于其生物学方面的不适应性而逐步减少。本研究中的抗性品系与敏感品系的杂交群体的抗性变化趋势也证实了这种推理。在纯的抗性酯酶Ｂ_２基因扩增品系中,抗性水平则在后代中基本上保持稳定,并且所有的个体抗性水平和酯酶活力水平更趋集中。本文进一步对蚊虫抗性的演替进行了讨论。     

Genetics of insecticide resistance in mosquito vectors of disease

Early studies on the genetics of insecticide resistance showed that single major semi-dominant genes were generally involved, and biochemical studies defined a limited number of enzymes and structural nerve proteins that were encoded by these genes. Recent advances in resistance detection now allow the measurement of genotype frequencies for some of these resistance mechanisms. Molecular studies are in progress for most of the major resistance genes, and the amplified esterase B(1) gene in Culex quinquefasciatus has been cloned. Changes in resistance gene expression occur, with increasing age of the adult insect, by way of specific mechanisms, and amplified esterase-based resistance genes that are not expressed can occur in aphids. Here Janet Hemingway assesses current knowledge of the genetics of insecticide resistance in mosquitoes.     

淡色库蚊酯酶等位基因及其在自然种群中的频率分布

酯酶基因扩增所产生的酯酶活性升高是库蚊Culex pipiens对有机磷杀虫剂抗性的主要机理之一。采用分子杂交技术和限制性酶切片段长度多态性(RFLP)分析,已鉴定出多种酯酶等位基因类型。该文通过酯酶基因特异性片段的PCR扩增及扩增片段的酶切片段分析,对淡色库蚊Culex pipiens pallens四种有机磷抗性品系的酯酶等位基因进行分型,并测定分析自然种群中不同酶型的频率分布。研究结果表明：PCR分型方法具有快速、准确的特点。不同的有机磷杀虫剂对酯酶等位基因具有明显的选择作用。双硫磷品系为B₁型;毒死蜱和敌百虫品系为B₂型;马拉硫磷品系为B₁型和B₁/B₂杂合型。不同地区采集的种群表现出不同的酶型频率分布。该文就杀虫剂对酯酶等位基因选择作用及自然种群的酶型频率分布进行了讨论。     

Molecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.     

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%)。     

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.     

STABILITY OF ORGANOPHOSPHATE RESISTANCE IN CULEX PIPIENS PALLENS COQUILLETT UNDER RELAXATION OF INSECITICDE SELECTION*

Abstract The stability of resistance in the organophosphate resistant strain of Culex pipiens pallens with amplification of single gene coding for esterase B₂ was determined under relaxation of insecticide selection. Insecticide resistance and amplification of esterase were both lost when strains were not homozygous for the presence of amplified gene, probably due to biological disadvantage of the esterase gene. LC₅₀ and LC₉₅ of chlorpyrifos for this strain were 0. 2099 mg/L and 0.9036 mg/L in the F₀ generation respectively, but 0. 0207 mg/L and 2. 8027 mg/L respectively in the F₁₆ generation. In the homozygous strain, insecticide resistance was still stable and amplification of esterase remained after 16 generations under relaxation of insecticide selection, which indicated that copy of esterase gene was not lost in the offspring of this strain. The evolution of insecticide resistance in mosquitoes is discussed.     

不同地区致倦库蚊种群相关酯酶 基因的特征分析

分别从广州、沙市、武汉近郊采集到致倦库蚊Culex pipiens quinquefasciatus,对单只蚊虫进行淀粉凝胶电泳和Southern杂交方法的分析结果表明：3个实验种群中均分布有与抗性有关的高活性酯酶β1¹,酯酶α2/β2分布于广州实验种群中;广泛分布于地中海地区尖音库蚊Culex pipiens种群中的酯酶α4/β4、α5/β5在以上3个种群中均不存在。但是,在3个实验种群中均发现存在有一对新的高活性酯酶α8/β8,其电泳迁移率和限制性酶切片段均与目前已报道的几种高活性酯酶不同。含这两对新酯酶的蚊虫将应进一步从种群中纯化,纯合蚊虫新品系做分子特征的研究。     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

French mosquito populations invaded by A2-B2 esterases causing insecticide resistance

In the mosquito Culex pipiens L., the two associated esterases A2 and B2 are responsible for resistance to organophosphorus (OP) insecticides in many countries of Africa, Asia and North America. We report here their presence and geographic spread in French populations based on the analysis of 168 samples collected from 1984 to 1990. First detected in 1986 in one sample from southern France, these esterases were progressively found in new geographic locations, so that in 1990 their distribution covered at least four contiguous French regions. RFLP analysis of the amplified B2 gene indicates that A2-B2 arrived in France most likely through migration. This genetic invasion is discussed in the light of the recent occurrence of A2-B2 in the mosquito genome, and of the consequences of this new resistance factor in natural populations already possessing other insecticide resistance genes.     

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.