棉铃虫田间种群抗药性的生态学干扰和生化机制研究

从生态遗传学角度和抗性机制两个方面研究了作物布局和解毒酶系对棉铃虫高效氯氰菊酯抗药性的影响,通过田间采样,测定了姜堰,如皋,兴化3个地区的棉铃虫对高效氯氰菊酯的抗药性。结果表明,作物布局对棉铃虫菊酯抗性的发展具有明显的影响,寄主相对单一的棉花连片种植区（兴化）棉田棉铃虫对高效氯氰菊酯的抗性水平最高,是棉花春玉米混栽区（如皋）棉田棉铃虫抗药性的3．5倍,在邻近春玉米种植区的稻棉区（姜堰）,棉田棉铃虫的抗药性水平,是4－5km外玉米田棉铃虫的4．8倍,研究同时表明,四代棉铃虫的抗药性比3代棉铃虫的抗药性下降了2．3倍,棉铃虫离体解毒酶和乙酰胆碱酯酶活性测定表明,棉铃虫的羧酸酯酶,谷胱甘肽S－转移酶和乙酰胆碱酯酶活性与棉铃虫对高效氯氰菊酯的抗药性水平有显著的相关性,对姜堰南部棉田和如振戴庄棉田棉铃虫的活体增效试验表明,多功能氧化酶是抗性棉铃虫对高效氯氰菊酯重要的解毒酶系,增效醚可分别增效22．79倍和12．33倍,羧酸酯酶对抗性棉铃虫的解毒代谢也有部分贡献,磷酸三苯酯可分别增效4．41倍和3．70倍。     

棉铃虫对拟除虫菊酯杀虫剂敏感性的修复及其乙酰胆碱酯酶的变化

利用生物测定和生物化学方法研究了抗药性棉铃虫Helicoverpa armigera(Hubner)对拟除虫菊酯敏感性修复及其体内乙酰胆碱酯酶的变化,结果表明：当去除药剂选择压后,棉铃虫对拟除虫菊酯的抗性为不稳定。随着停止用药的时间加长,敏感性增加。从田间收集的棉铃虫在室内饲养后第1代对拟除虫菊酯的抗性下降速度较迅速。特异性酶抑制剂对敏感和抗性棉铃虫种群的乙酰胆碱酯酶亲和力试验结果证实,乙酰胆碱酯酶在抗性棉铃虫对杀虫剂反应发生变化的过程中发生改变,这种酶的改变可能对棉铃虫抗药性的不稳定性有重要影响。     

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。     

棉铃虫对茚虫威的抗性机理:PBO,DEF和DEM的增效作用及解毒酶活性

【目的】本研究旨在明确不同棉铃虫Helicoverpa armigera种群对茚虫威的抗性水平及抗性机理,以科学有效防治这一害虫,避免其对茚虫威的抗性快速发展。【方法】采用浸叶法测定了棉铃虫不同种群,即相对敏感种群(CP)、汰选种群(TP)、沂水种群(YP)(采自山东沂水)和邯郸种群(HP)(采自河北邯郸)3龄幼虫对茚虫威抗性水平及增效醚(PBO)、脱叶磷(DEF)、顺丁烯二酸二乙酯(DEM)对茚虫威的增效作用;并测定了不同种群棉铃虫3龄幼虫体内多功能氧化酶(MFO)、羧酸酯酶(CarE)和谷胱甘肽-S-转移酶(GST)3种解毒酶及乙酰胆碱酯酶(ACh E)活性。【结果】CP种群对茚虫威敏感,TP,YP及HP种群对茚虫威的抗性倍数(resistence ratio,RR)分别为4.36,8.06和15.34倍,分别表现为敏感性降低、低水平抗性和中等水平抗性。在TP种群中,当棉铃虫3龄幼虫取食3种增效剂处理的叶片时,在0,6和12 h处理时间内增效作用随时间而升高,且PBO和DEF对茚虫威的增效作用优于DEM。增效剂PBO,DEF和DEM饲喂棉铃虫3龄幼虫12 h后对HP种群的增效倍数分别为3.86,2.52和4.57倍,对CP种群增效作用分别为1.11,0.52和0.91倍。酶活力对比研究发现,HP种群的棉铃虫MFO,CarE和GST活性显著高于CP种群和TP种群,YP种群的棉铃虫MFO和CarE活性显著高于CP种群,CarE活性显著低于HP种群,但是ACh E活性在棉铃虫种群间差异不显著。【结论】结果提示,当达到中等抗性水平时,MFO,CarE和GST活性显著升高与棉铃虫对茚虫威的抗性有关,而代谢抑制剂PBO,DEF和DEM对茚虫威有明显的增效作用。     

不同种群棉铃虫三龄幼虫酯酶活性频率分布与抗药性的关系

选用室内饲养的棉铃虫Helicoverpa armigera偃师和湖北2个敏感品系、对辛硫磷高抗的PCP20品系、对氰戊菊酯高抗的YG45品系及1999或2000年采自山东阳谷、河北邯郸和河南安阳的田间高抗种群,江苏徐州、湖北武汉的田间中等抗性水平种群和新疆沙湾的田间敏感种群,采用酶标板酶动力学法测定了各品系（种群）的3龄幼虫个体酯酶活性频率分布和平均酯酶活性。结果表明,偃师敏感品系、湖北敏感品系和新疆沙湾田间敏感种群的酯酶活性个体频率分布相似,三个品系（种群）的平均酯酶活性相近,分别为991、1 138、1 055 mOD·min^-1·larva^-1。室内选育的PCP20抗性品系、YG45抗性品系及山东阳谷、河北邯郸 、河南安阳田间高抗种群的高酯酶活性（活性在1 800 mOD·min^-1·larva^-1以上）个体频率明显高于三个敏感品系（种群）,平均酯酶活性在1 510～2 482 mOD·min^-1·larva^-1之间。江苏徐州、湖北武汉的田间中抗水平种群高酯酶活性个体频率及平均酯酶活性都介于敏感和高抗品系（种群）之间,平均酯酶活性为1 258～1.404 mOD·min^-1·larva^-1。棉铃虫各品系（种群）平均酯酶活性与对拟除虫菊酯类杀虫剂抗性个体频率的相关性要比对有机磷类的高,相关系数分别为0.82和0.42。分析各品系（种群）高酯酶活性个体频率与棉铃虫对拟除虫菊酯类、有机磷类杀虫剂抗性个体频率的相关性,得到相似的结果。考虑到酯酶并不是棉铃虫对拟除虫菊酯抗性的主要机理,建议酯酶活性可作为棉铃虫抗药性生化检测的一个参考指标。本文还讨论了酯酶与棉铃虫对拟除虫菊酯类杀虫剂及有机磷类杀虫剂抗性的关系。     

云南彝族人群CYP2E1基因多态性与酒依赖的关联性研究

目的：探讨云南彝族人群中的酒精依赖患者和云南彝族人群中健康人在CYP2E1基因的一个SNP（Rs3813867）的等位基因和基因型频率的不同,试图找出酒依赖的危险基因,比较它与其他人群之间在CYP2E1PstI位（rs3813867）基因多态性的不同。方法：对110个酒精依赖者和330名健康的志愿者不喝酒（对照组）的CYP2E1PstI位的多态性,等位基因频率和基因型频率进行测定。采用PCR—RFLP方法进行基因分型。结果：CYP2 E1 Psfl位的多态性,等位基因频率和基因型频率是相似的在酒精依赖者和对照组（72．7％vs72．1％,C1／C1）,（25．5％vs25．8％,C1／C2）,（1．8％vs2．1％为C1／C2）和（85．5％vs85％c1的）,（14．5％VSl5％为c2）。结论：CYP2E1的基因型和等位基因分布在酒精依赖组和对照组之间没有显着性差异（P〉0．05）,在这两个民族在AD组和对照组基因型分布有差异（P〈0．001）。     

快速检测蚊虫乙酰胆碱酯酶活性的方法

本研究采用微板法,根据乙酰硫代胆碱-二硫双硝基苯甲酸法的原理,检测了敏感品系和抗残杀威抗性品系淡色库故CulexpipienspallensCoguillett）单个蚊虫的乙酰胆碱酯酶（AchE）的活性。给果表明：敏感品系和抗性品系蚊虫的AchE活性有较大差别,敏感品系蚊虫的AchE可被一定量的杀虫剂抑制,而抗性品系的AchE则不被抑制,因此该方法能够区分敏感蚊虫和抗性蚊虫,用于抗性测定,具有简单易行,检测快速等优点。     

我国棉花主产区棉蚜对吡虫啉的抗性监测及抗性机理

【目的】由于生长周期短、繁殖率高,棉蚜Aphis gossypii容易对杀虫剂产生抗药性。本研究旨在明确我国棉花主产区棉蚜对吡虫啉的抗性水平及抗性机理。【方法】采用浸叶法测定了北京海淀,河北廊坊和邯郸,山东德州,河南许昌,以及新疆奎屯和阿克苏地区棉蚜对吡虫啉的抗性水平;测定了不同种群棉蚜3种解毒酶（多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶）及乙酰胆碱酯酶的活性;并对靶标基因烟碱型乙酰胆碱受体（nAChR）β1亚基基因进行了突变检测。【结果】北京海淀、河南许昌和河北邯郸的棉蚜对吡虫啉敏感;河北廊坊、新疆阿克苏、山东德州及新疆奎屯地区的棉蚜对吡虫啉的抗性倍数(resistance ratio, RR)分别为22.6, 26.3,53.5和61.1倍,为中等水平抗性。酶活力对比研究发现,阿克苏和奎屯地区的棉蚜多功能氧化酶的比活力分别是敏感种群(北京种群)的1.7和1.8倍,羧酸酯酶的比活力分别是敏感种群的1.6和1.7倍,谷胱甘肽S-转移酶的比活力均是敏感种群的1.5倍,但是乙酰胆碱酯酶比活力在棉蚜种群间差异不显著。靶标基因突变检测表明,河北廊坊、新疆阿克苏、山东德州及新疆奎屯棉蚜种群nAChR β1亚基均存在与吡虫啉抗性相关的精氨酸到苏氨酸（R81T）突变。【结论】结果提示,多功能氧化酶、羧酸酯酶和谷胱甘肽S-转移酶活力升高以及nAChR β1亚基R81T突变与棉蚜对吡虫啉的抗性形成相关。     

天津地区家蝇抗药性水平及与两种解毒酶的关系

用点滴法对采自天津市7个郊区县的7个不同生境野外家蝇种群的抗药性进行了测定并与实验室内的相对敏感种群进行比较;对不同地区家蝇种群进行了羧酸酯酶（carboxylesterase, CarE）和谷胱甘肽S-转移酶（glutathione S-transferase, GSTs）活性检测。结果表明,不同家蝇种群对DDVP、高效氯氰菊酯和残杀威的抗性倍数不同,对DDVP抗性最高为18.563倍,最低为1.885倍;对高效氯氰菊酯最高为14.071倍,最低为1.071倍;对残杀威抗性倍数最高为7.499倍,最低为1.071倍。从CarE平均比活力看,室外家蝇种群CarE的比活力均高于室内相对敏感种群的比活力,CarE活性的分布在敏感种群和不同地区野外种群间具有明显的重叠现象。天津不同地区F₂代家蝇GSTs的比活力抗性种群普遍高于室内相对敏感种群,大约在2～4倍之间,不同地区间也具有比较大的差异,而抗药性高的种群GSTs活性也高,GSTs活性变化与不同种群抗性差异是相符的。     

贵州从江侗族威宁彝族、荔波瑶族谷胱甘肽S-转移酶M l、Tl 和Pl 的基因多态性研究

目的：研究贵州从江侗族、威宁彝族、荔波瑶族的GSTs基因多态性。方法：在隔离自然人群中,采用多重等住基因特异聚合酶链反应方法分析GSTM1和GSTT1基因多态性,同时采用PCR-RFLP的方法和TaqMan-MGB探针基因分型方法分析GSTP1（A1578G）基因多态性。结果：贵州从江侗族、成宁彝族、荔波瑶族的GSTM1和GSTT1纯合缺失基因型频率分别为59．6％～71．2％、39．4％～72.5％。其GSTP1（A1578G）基因型频率分别为：野生型（AA）为63.3％～75％、杂合子（AG）为23.2％～35．8％、纯合突变型（GG）为0～1.9％。等位基因频率：A为81．2％～86．6％,G为13．4％～18.8％。结论：贵州从江侗族、威宁彝族、荔波瑶族的GSTM1纯合缺失基因型频率在民族间差异无统计学意义,GSTP1（A1578G）基因型频率和等住基因频率在民族间差异无统计学意义,且其等位基因频率均符合Hardy-Weinberg平衡,但其GSTT1纯合缺失基因型频率在民族间差异有统计学意义（P〈0．05）。     

SINGLE-HELICOVERPA ARMIGERA TEST TO DETERMINE INSECTICIDE RESISTANCE GENOTYPE FREQUENCY*

Abstract A sensitive technique for identifying insecticide resistance genotype frequency of the Ace gene existing in natural populations of Helicoverpa armigera in China is described. The technique is based on the comparison of acetylcholinesterase (AChE) activity in 3 equal aliquots, which are taken from the homogenate of a single H. urnzigera: 1) in absence of inhibitor (well No. 1); 2) in absence of enzyme precursor (well No. 2); and 3) in presence of a concentration of propoxur inhibiting the AChE coded by the Ace^Sallele but not by the Ace^Rallele (well No. 3). An intensity of same strength in wells No. 1 and No. 3 indicates that propoxur has no effect on AChE activity, and the genotype should be Ace^RR(when well No. 3 = well No. 1). A same weak intensity in well No. 2 and No. 3 (and a strong intensity in well No. l), indicates that propoxur completely inhibited AChE activity; and the genotype should be Ace^Ss(when well No. 3 = well No. 2). Well No. 3 displaying an intermediate intensity between well No. 1 (strong intensity) and well No. 2 (weak intensity) indicates that AChE activity was partially inhibited by the propoxur concentration and genotype should be Ace^Rs(when well No. 2<well No. 3<well No. 1). The genotype frequency was determined in 1995 and 1996. the Ace^SSis 76. 1%, 70. 6% for low resistance populations respectively whereas the Ace^RRis 68. 3%, 65. 3% for high resistance population respectively.     

Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii).

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5'-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09:A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5'-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.     

Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover

Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.     

Polymorphism in the acetylcholinesterase gene of Musca domestica L. field populations in Turkey

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphates (OPs) and carbamates (CBs) in insects. Ace mutations have been identified in OP and CB resistant strains of Musca domestica. In this study, the Ace gene was partially amplified and sequenced at amino acid positions 260, 342, and 407 to determine the frequencies of these mutations in housefly samples collected from farms and garbage disposal sites of 16 provinces in the Aegean and Mediterranean regions of Turkey. In addition, the percent remaining AChE activities in these samples were assayed by using three OPs (malaoxon, paraoxon, and dichlorvos) and one CB (carbaryl) compound as inhibitors. In all the analyzed samples, 13 different combinations at the three amino acid positions were identified and the L/V260-A/G342-F/Y407 combination was found in the highest frequency. No susceptible individual was detected. The highest mean percent remaining AChE activities were detected in the individuals having the L260-A/G342-F/Y407 genotype when malaoxon and paraoxon were used as inhibitors and in the individuals with the L260-A342-F/Y407 combination when dichlorvos and carbaryl were used as inhibitors. The obtained data were heterogeneous and there was no exact correlation between the molecular genetic background and the resistance phenotypes of the flies. The findings of this study at the molecular and biochemical levels indicate the presence of significant control problems in the field.     

Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from São Paulo, Brazil

To establish an insecticidal resistance surveillance program, Culex quinquefasciatus mosquitoes from S?o Paulo, Brazil, were colonized (PIN95 strain) and analyzed for levels of resistance. The PIN95 strain showed low levels of resistance to organophosphates [malathion (3.3-fold), fenitrothion (11.2-fold)] and a carbamate [propoxur (3.0-fold)]. We also observed an increase of 7.4 and 9.9 in alpha and beta esterase activities, respectively, when compared with the reference IAL strain. An alteration in the sensitivity of acetylcholinesterase to insecticide inhibition was also found in the PIN95 mosquitoes. The resistant allele (Ace.1R), however, was found at low frequencies (0.12) and does not play an important role in the described insecticide resistance. One year later, Cx. quinquefasciatus mosquitoes were collected (PIN96 strain) at the same site and compared to the PIN95 strain. The esterase activity patterns observed for the PIN96 strain were similar to those of the PIN95 mosquitoes. However the occurrence of the Ace.1R allele was statistically higher in the PIN96 strain. The results show that esterase-based insecticide resistance was established in the PIN95 Cx. quinquefasciatus population and that an acethylcholinesterase based resistant mechanism has been selected for. A continuous monitoring of this phenomenon is fundamental for rational mosquito control and insecticide application programs.     

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.