淡色库蚊中抗性相关羧酸酯酶的纯化及其生化性质

在库蚊Culex pipiens品系中,非专一性酯酶活性的升高是对有机磷杀虫剂产生抗性的重要机理之一。应用SDS/PAGE,比较淡色库蚊Culex pipiens pallens抗敌百虫品系(RD)、敏感型品系(S)和抗苄呋菊酯品系(PY)中可溶性总蛋白质带型,显示RD中含有一条特异蛋白带,其它两个品系中未检出。在RD成虫匀浆液总蛋白中含量高达2.1%。分子量测定为66 kD。应用柱层析法分离得到了较纯的纯品。以α-NA为底物测得K_m=64.1 mmol/L,V_max=249.8 mmol/(L·mg·min)。与羧酸酯酶相比较：其K_m值小于已报道的抗性品系及非抗性品系A-酯酶和B-酯酶。V_max值比已报道抗性品系A-酯酶低,比B-酯酶高。较高浓度的敌百虫并不能抑制其酶活,属于A-酯酶。在昆虫体内可能主要通过结合隔离作用(sequestration)提高昆虫对有机磷的耐受性,对有机磷杀虫剂水解作用的可能性也不能排除。     

几种杀虫药剂敏感蚊类酯酶多态性的研究

通过蚊虫酯酶蛋白的淀粉凝胶电泳分析和基因组DNA的限制性酶切片段长度多态性(RFLPs)比较, 对尖音库蚊Culex pipiens、三带喙库蚊Culex tritaeniorhynchus和中华按蚊Anopheles sinensis有机磷杀虫药剂敏感种群的酯酶蛋白和结构基因的多态性进行分析。发现在蛋白质水平上,三带喙库蚊敏感种群(n=54)在酯酶α和β位点分别存在2个和3个等位基因,在DNA水平上有2.9%的个体具有与酯酶β1¹基因1.3 kb Cdna片段同源的1.3 kb单拷贝带存在。发现中华按蚊敏感种群 (n= 50)中具有低活性的非特异性酯酶存在,在蛋白质水平上,酯酶α和β位点各有一个等位基因;在DNA水平上,通过对单个蚊虫基因组DNA的研究未发现有与酯酶β1¹基因同源的酯酶编码基因的存在。对尖音库蚊北京敏感种群(n= 64)的研究发现,在酯酶α和β位点都存在5个等位基因,在DNA水平上,使用一个限制性内切酶(EcoRI),15只蚊虫的样本在酯酶β位点发现了5个等位基因,说明在尖音库蚊北京敏感种群的酯酶β基因周围存在着较大的中性多态性,在有机磷杀虫剂的选择下,这些中性多态性可能会成为基因扩增的潜在因素。     

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。     

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。     

抑制剂存在下不同种群烟粉虱乙酰胆碱酯酶残余活性频率分布及其与抗药性的关系

为了探讨烟粉虱Bemisia tabaci不同种群个体乙酰胆碱酯酶敏感性差异及其与抗药性的关系, 我们选用室内饲养的烟粉虱SUD S敏感品系和6个田间抗性种群, 采用酶标板酶动力学法测定了各品系 (种群）乙酰胆碱酯酶对抑制剂的敏感性反应以及抑制剂存在时各抗性种群个体乙酰胆碱酯酶残余活性频率分布。结果表明: 在抑制剂浓度为300 μmol/L时, 敏感品系乙酰胆碱酯酶的活性基本上被完全抑制, 可以明显地区分敏感品系与田间抗性种群。在抑制剂浓度为2 000 μmol/L时, 各抗性种群个体乙酰胆碱酯酶残余活性频率分布差异明显, 其中ZZ-R种群和FZ-R种群的乙酰胆碱酯酶残余活性频率分布相似, 大部分个体的乙酰胆碱酯酶残余活性分布在1.00~1.80 mOD/min之间; SM-R种群和ND-R种群的乙酰胆碱酯酶残余活性频率分布也相似, 大部分个体的乙酰胆碱酯酶残余活性分布在0.40~1.00 mOD/min之间; LY-R和NP-R种群大部分个体的乙酰胆碱酯酶残余活性分别分布在1.00~1.60 mOD/min和0.80~1.20 mOD/min之间。各抗性种群乙酰胆碱酯酶高残余活性 (大于1.00 mOD/min）个体频率与对敌敌畏的抗性水平之间具有明显相关性, 相关系数为0.86 (P<0.05）。考虑到乙酰胆碱酯酶对抑制剂作用不敏感是一些昆虫对有机磷和氨基甲酸酯类杀虫剂抗性的重要机制之一, 建议可以将乙酰胆碱酯酶对敌敌畏的敏感性作为烟粉虱抗药性生化检测的一个参考指标。     

不同地理种群尖音库蚊复组抗性动态和遗传多样性

通过生物测定、蛋白质电泳和等位酶分析等方法对5个不同地区的尖音库蚊复组蚊虫Culex pipiens complex的抗性水平、种群中非特异性酯酶基因表型分布和种群遗传多样性进行了研究。不同地理种群的抗性检测结果表明：5个种群分别对敌敌畏、对硫磷、氯菊酯和溴氰菊酯的抗性较高,对残杀威、巴沙和胺菊酯的抗性较低;朝阳种群对敌敌畏抗性最高（55.7倍）,武汉种群次之;佛山种群对氯菊酯和溴氰菊酯的抗性比率高达123倍和23.9倍。酯酶电泳结果显示：5个种群间酯酶多态性存在差异,广州和佛山两个库蚊种群酯酶表型多态性最高,有B1,A2-B2,A8-B8,A9-B9,B10和A11-B11等6种酯酶表型,提示高活性酯酶是主要的抗性机制。群体遗传学研究表明：每位点平均等位基因数（A）为2.76,平均多态位点百分率（P）为64.45％,平均预期杂合度（He）为0.1943,种群间遗传分化系数（Fst）值为0.10,平均基因流(Nm)＝2.57,说明5个种群有较丰富的遗传多样性,种群内遗传多样性高于种群之间。据此推测,种群间可以通过迁徙等方式进行基因交流,使得遗传结构、抗性水平朝一致性方向变化。本研究对我国尖音库蚊复组蚊虫的综合治理有一定指导意义。     

淡色库蚊 (Culex pipiens pallens Coquillett)的抗性酯酶表现型及其在杀虫剂压力下的变化趋势(英文)

对来自杭州、临海和金华三个城市的淡色库蚊种群进行了抗性酯酶表现型频率分布的测定和分析 ,同时对三个种群进行双硫磷、敌百虫、毒死蜱和马拉硫磷等四种有机磷杀虫剂的抗性品系筛选 ,逐代测定和分析各种群在不同杀虫剂压力下 ,其抗性酯酶表现型频率分布的变化。结果表明 ,三个自然种群中都存在酯酶B1 、B2 纯合表现型及酯酶B1 /B2 杂合表现型 ,其中酯酶B1 纯合表现型占优势。各自然种群中的抗性酯酶表现型频率分布有差异。经过杀虫剂的逐代筛选 ,各种群相对于某一杀虫剂的压力 ,表现出选择较为单一的抗性酯酶表现型的趋势 :双硫磷有利于酯酶B1 纯合表现型的选择 ,敌百虫和毒死蜱有利于酯酶B2 纯合表现型的选择 ,马拉硫磷则似乎有利于酯酶B1 纯合表现型和酯酶B1 /B2 杂合表现型的选择 ,但酯酶B1 纯合表现型相对于B1 /B2 杂合表现型来说 ,对马拉硫磷抗性更强一些。根据研究结果 ,就蚊虫的抗性酯酶基因对不同杀虫剂的选择优势及相应的蚊媒控制策略进行了讨论。     

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。     

不同杀虫剂选育对家蝇抗药性水平及kdr基因频率的影响

采用杀虫剂（溴氰菊酯和甲基嘧啶磷）筛选及不接触药物自然衰退的方法,研究了家蝇Musca domestica氯氟氰菊酯高抗品系(Cyh-R)对杀虫剂的抗性变化,探讨蝇类抗药性治理的方法。用点滴法测定氯氟氰菊酯对不同家蝇品系的毒力,比较抗药性的变化,结合特异性等位基因PCR扩增（PASA）技术检测了不同家蝇品系的kdr基因频率,探讨kdr基因频率与抗性水平之间的关系。结果表明:甲基嘧啶磷筛选后,氯氟氰菊酯对第2~8代Cyh-R品系的LD₅₀呈递减趋势,从F₀的2.8434 μg/蝇降为F₈的0.4404 μg/蝇,但第8~18代Cyh-R品系的LD₅₀呈逐代递增趋势;溴氰菊酯筛选后,氯氟氰菊酯对Cyh-R品系第2~16代的LD₅₀呈上升趋势,从F₀的2.8434 μg/蝇升至F₁₆的24.3249 μg/蝇;表明了施用有机磷杀虫剂可降低其对氯氟氰菊酯的抗药性,而施用拟除虫菊酯药剂则有助于其对氯氟氰菊酯抗药性的增长;不使用任何杀虫剂也能降低其对氯氟氰菊酯的抗药性,但下降速率低于甲基嘧啶磷。PASA技术检测表明Cyh-R品系的kdr抗性基因频率为88.8%,不经过任何药剂筛选其kdr抗性基因频率下降程度最大,达到69.7%;甲基嘧啶磷筛选后其结果降为78.8%,而经溴氰菊酯筛选后kdr抗性基因频率则明显升高,达到98.9%。通过对kdr抗性基因频率和抗性水平进行相关和回归分析表明kdr抗性基因频率与家蝇对氯氟氰菊酯的LD₅₀呈对数关系,即LD₅₀值高的品系其kdr抗性基因频率相应的也较高。建议在家蝇防治中考虑轮换用药。     

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。     

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。     

ESTERASE PHENOTYPES IN NATURAL POPULATIONS OF CULEX PIPIENS PALLENS (DIPTERA: CULICIDAE) AND THEIR VARIATIONS UNDER INSECTICIDE SELECTION*

Abstract Three populations of Culex pipiens pallens Coquillett, from Hangzhou, Linhai and Jinhua, were determined for their probability of resistant esterase phenotypes. Further, each population was selected with dipterex, chlorpyrifos, temephos and malathion at a given dose of 60%‐70% mortality and was determined for the variation of resistant esterase phenotypes in its offspring. The results indicated that esterase B₁ phenotype was dominant in three populations although different natural populations have a different probability distribution of esterase B phenotypes. Insecticide selection showed that various esterase loci seemed to have different selective advantages under different insecticide pressures. The esterase B₁ phenotype was selected under temephos, and the esterase B₂ phenotype was selected under dipterex and chlorpyrifos. Except for the esterase B₁ phenotype, the heterozygote of B₁/B₂ phenotypes was detected under malathion pressure. These results are discussed in relation to the selective advantages of esterase genes to OP pressure and mosquito control strategies.     

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean.     

Esterase B1 activity variation within and among insecticide resistant, susceptible and heterozygous strains of Culex quinquefasciatus (Diptera: Culicidae)

Amplification of the esterase B1 gene of Culex quinquefasciatus Say results in high titers of an esterase enzyme that confers resistance to organophosphate insecticides. Esterase activity of individuals was measured in samples from an organophosphate resistant strain (Tem-R), a susceptible strain (S-Lb), and their reciprocal F1 progeny. Within-strain variation, as measured by coefficients of variation, was fairly consistent between sexes within strains and among strains (average, 12%). On average, individuals from the Tem-R strain had about 120 times the esterase activity of individuals from the S-Lab strain. The mean esterase activities of the F1 strains were significantly higher than the average of the Tem-R and S-Lab strain mean esterase activities, suggesting enhanced expression of the amplified esterase B1 genes in F1 individuals. Reciprocal F1 strains did not differ significantly in esterase activity or resistance, indicating that maternal effects do not influence either of these measures in these strains. The levels of esterase activity of the strains are discussed in relation to their resistance.     

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.     

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.     

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.