不同地理种群小菜蛾对甲胺磷的抗药性

采用浸叶法研究了福州建新镇、福建农林大学校园内、闽侯县上街镇和闽侯县甘蔗镇等地田间小菜蛾种群对甲胺磷的抗性水平及抗性机制。结果表明,与敏感小菜蛾相比,上述4个田间小菜蛾种群已对甲胺磷产生了34-82倍的抗性水平。敏感小菜蛾乙酰胆碱酯酶(AChE)的Ki值是田间抗性小菜蛾AChE的Ki值的17.3-39.1倍。不同小菜蛾种群对甲胺磷的抗性水平也存在着差异,小菜蛾种群AChE对甲胺磷的敏感性大小与其对甲胺磷的抗性水平高低显著相关。     

小菜蛾对阿维菌素的抗性遗传方式 和相对适合度研究

就小菜蛾Plutella xylostella对阿维菌素的抗性遗传方式和抗性品系的相对适合度进行了研究。室内选育的阿维菌素抗性品系与同源的敏感品系杂交、F₁代自交、F₁代与亲本回交,结果表明：杂交后的显性度（D）分别为-0.64和-0.52,说明小菜蛾对阿维菌素的抗性是常染色体、不完全隐性遗传;χ²检验证实,可能是多基因控制的抗性遗传。杂交F₁代乙酰胆碱酯酶（AChE）、羧酸酯酶（CarE）和谷胱甘肽转移酶（GST）活性比抗性亲本有所降低,F2代及回交后代三种酶的活性继续降低。种群适合度研究表明,抗性品系相对于敏感品系有0.84的适合度。     

B型烟粉虱田间种群对毒死蜱和敌敌畏抗性的生化机制

通过增效剂生物测定和生化分析,探讨了采自福建省的B型烟粉虱Bemisia tabaci 6个田间种群对毒死蜱和敌敌畏抗性的生化机制。结果表明：与敏感品系SUD-S相比,6个田间种群对毒死蜱和敌敌畏分别具有54.53～78.43倍和6.23～11.25倍的抗性。TPP、PBO和DEM对毒死蜱的增效比分别为3.61～24.94倍、1.14～1.76倍和1.04倍,对敌敌畏的增效比分别为1.67～2.64倍、1.33～1.65倍和1.09倍,表明羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中起着重要作用。烟粉虱抗性种群乙酰胆碱酯酶的K_m值是敏感品系的1.83～4.0倍,V _max值是敏感品系的0.34～0.62倍; 敏感品系乙酰胆碱酯酶的活性在底物浓度大于1.0 mmol/L时受抑制,抗性种群乙酰胆碱酯酶的活性在底物浓度大于16 mmol/L时受抑制;抗性种群乙酰胆碱酯酶对敌敌畏和毒死蜱的敏感度分别比敏感品系低119.92～161.33倍和10.11～14.24倍,表明烟粉虱田间抗性种群乙酰胆碱酯酶可能已发生了变构,由变构引起的乙酰胆碱酯酶不敏感是烟粉虱田间种群对毒死蜱和敌敌畏产生抗性的重要原因。结果提示,乙酰胆碱酯酶不敏感性和羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中均起着重要作用,而乙酰胆碱酯酶不敏感性在对敌敌畏的抗性中起重要的作用,多功能氧化酶和谷胱甘肽S转移酶在烟粉虱对毒死蜱和敌敌畏抗性中所起的作用不大。     

褐飞虱抗吡虫啉品系生物适合度研究

毒力测定结果显示 ,虽然褐飞虱NilaparvatalugensSt l田间品系对吡虫啉还没有表现出明显的抗性 ,但室内筛选品系已经出现了一定水平的抗性 ,抗性品系 1 (R1)和抗性品系 2 (R2 )的抗性倍数分别为 2 5 3 8和 68 92。通过建立抗性品系、田间种群和敏感品系的生命表 ,发现抗性品系适合度显著下降 ,而且R2 品系下降的幅度明显大于R1品系。存在显著变化的因素有低龄若虫存活率、成虫羽化率、交配率、产卵量和孵化率下降 ,雌虫寿命缩短 ;R2 品系还表现为卵历期延长 ,3龄若虫至 5龄若虫存活率下降。R2 品系产卵高峰期迟 ,高峰期日虫量显著低于敏感品系和田间种群。用种群数量趋势指数 (I)来确定抗性品系的相对生物适合度 ,发现与敏感性品系相比 ,R1品系和R2 品系的相对适合度分别为0 60 9和 0 2 45。     

小菜蛾对杀虫双、杀螟丹抗性品系选育及其抗性消长规律的研究(英语)

用改进的蛭石萝卜苗法饲养小菜蛾,以点滴法处理对杀虫双、杀螟丹两种药剂淘汰选育的小菜蛾抗性品系和不以药剂处理的敏感品系的各项生物学特性与同期田间种群比较,各品系间差异不显著,均没有生长发育和繁殖上的不利情况.5年来药剂淘汰选育,杀虫双品系至F_(85)代其抗性增长178倍;杀螟丹品系F_(80)代其抗性增长87倍,均已形成高抗品系.两个抗性品系经停止药剂淘汰选育后5代,抗性倍数分别从167倍降至57倍和74倍降至16倍,说明这两个抗性品系的抗性是不稳定的,在没有药剂选择压力的情况下,开始几代抗性下降较快,当下降到一定水平后抗性便比较稳定,似乎很难恢复原有的敏感性.     

湖南不同地区小菜蛾对药剂敏感性比较

采用浸叶法在室内测定了湖南长沙和怀化地区小菜蛾Plutella xylostella(L.)田间种群对10种药剂的敏感性。结果表明:湖南长沙和怀化地区田间小菜蛾除对丁醚脲和BT制剂仍处于敏感(抗性倍数<3)状态外,对其它8种药剂产生了不同程度的抗药性,其中以长沙地区小菜蛾田间种群对高效氯氰菊酯抗性倍数最高(抗性倍数为33.58)。长沙和怀化种群对药剂的相对毒力倍数以巴丹最低(1.20)而以多杀菌素最高(2.59)。     

艾氏剂环氧化酶及细胞色素P-450对小菜蛾抗药性发展的影响

本文对室内长期饲养的小菜蛾(Plutella xylostella L.)敏感品系和田间采集的抗性种群体内的艾氏剂环氧化酶及细胞色素P-450进行了比较研究。结果证明,艾氏剂环氧化酶在感性和抗性小菜蛾间存在着量及质的差异。 抗性种群的艾氏剂环氧化酶的Vmax和Km值分别为感性品系的5.4倍和6.5倍。抗性种群的细胞色素P-450的含量是感性品系的1.1-1.3倍。艾氏剂环氧化酶在量上及质上的差异及细胞色素P-450含量的提高是导致小菜蛾抗药性发生与发展的重要机制之一。而且质的差异较之量的差异可能起着更为重要的作用,     

小菜蛾对三氟甲吡醚的抗性风险评价与抗性生化机制研究

【目的】明确小菜蛾Plutella xylostella(Linnaeus)对三氟甲吡醚的抗性风险和抗性生化机制,为三氟甲吡醚的合理使用提供科学依据。【方法】采用TabashnikMc Gaughey的阈性状分析方法评估小菜蛾对三氟甲吡醚的抗性风险;采用浸叶法测定增效剂(胡椒基丁醚、磷酸三苯酯和顺丁烯二酸二乙酯)对三氟甲吡醚的增效作用,通过酶动力学方法测定了小菜蛾对三氟甲吡醚抗性和敏感品系的谷胱甘肽-S-转移酶、酯酶和多功能氧化酶的活性。【结果】经过18代次筛选,小菜蛾种群对三氟甲吡醚的抗性水平上升至14.8倍,小菜蛾对三氟甲吡醚的抗性现实遗传力(h~2)为0.1558;当h~2=0.155 8时,在致死率为50%-90%的选择压力下,预计小菜蛾对三氟甲吡醚抗性增加10倍分别需要16.1-7.3代。在小菜蛾三氟甲吡醚抗性品系中,酯酶和多功能氧化酶比活力均显著高于敏感品系,分别为敏感品系的1.34倍和1.45倍;谷胱甘肽-S-转移酶比活力与敏感品系的无显著差异。抗性品系的酯酶抑制剂磷酸三苯酯和多功能氧化酶抑制剂胡椒基丁醚对三氟甲吡醚具有明显增效作用,增效倍数分别为1.21倍和1.43倍;谷胱甘肽-S-转移酶抑制剂顺丁烯二酸二乙酯对三氟甲吡醚没有明显增效作用。【结论】小菜蛾对三氟甲吡醚产生抗性的风险较大,酯酶和多功能氧化酶活性的升高可能是小菜蛾对三氟甲吡醚产生抗性的重要机制。     

湖南宁乡烟区烟蚜的抗性监测及其相关酶的活性测定

为明确湖南宁乡烟区烟蚜对杀虫剂的抗性现状,测定烟蚜田间种群对吡虫啉、啶虫脒、氯氟氰菊酯、辛硫磷及灭多威5种杀虫剂的敏感性。结果表明,烟蚜田间种群对烟碱类的啶虫脒和吡虫啉表现出中等抗性,与敏感品系相比,抗性倍数分别为25. 20和21. 03;对菊酯类的氯氟氰菊酯表现为低抗水平,抗性倍数为5. 19;对辛硫磷和灭多威均表现为敏感性降低,抗性倍数分别为2. 38和2. 86。本研究又测定了烟蚜田间与室内敏感种群相关酶活力的变化,结果表明,田间种群体内解毒酶、保护酶与消化酶活力均显著高于室内敏感种群。试验结果可为延缓田间烟蚜抗性及烟蚜的综合治理提供理论依据。     

东南沿海地区小菜蛾对Bt δ-内毒素和Bt制剂的抗性检测

采用浸叶法测定了2003年秋季、2004年春季采自广东惠州、福建福州、浙江杭州和江苏南京的小菜蛾Plutella xylostella田间种群对Cry1Aa、Cry1Ab、Cry1Ac和Cry2Aa以及Bt制剂kurstaki亚种 (Bacillus thuringiensis subsp. kurstaki, Btk）的抗性水平。与敏感品系PHI-S相比,广东惠州田间小菜蛾种群的抗性水平最高,其对Cry1Ab和Cry1Ac的抗性分别达到了168和120倍,均为高抗水平; 对Btk制剂的抗性有47倍,达到了中抗水平;对Cry1Aa和Cry2Aa具有低水平抗性 (分别为5.8和5.6倍)。福建福州、浙江杭州和江苏南京田间小菜蛾种群抗性水平相近,对Cry1Ab和Cry1Ac具有低至中等水平抗性 (8～28倍),对Btk制剂具有低水平抗性 (3.5～7倍),对Cry1Aa和Cry2Aa还没有产生明显抗性。因此,在我国东南沿海地区要注意Btk制剂与Bt其他亚种制剂或其他生物杀虫剂轮换使用,以减小Bt制剂对小菜蛾的选择压力,延缓小菜蛾对Bt抗性的发展。     

Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid

The Asian citrus psyllid, Diaphorina citri Kuwayama, is a worldwide pest of citrus, which vectors the putative causal pathogen of huanglongbing. Current management practices warrant continuous monitoring of field populations for insecticide resistance. Baseline activities of acetylcholinesterase (AChE), general esterase, and glutathione S-transferase as well as sensitivity of AChE to selected organophosphate and carbamate insecticides were established for a susceptible laboratory strain (Lab) and compared with several field populations of D. citri from Florida. The specific activity of AChE in various D. citri populations ranged from 0.77 to 1.29 microM min(-1) mg of protein(-1); the Lab strain was characterized by the highest activity. Although reduced AChE sensitivity was observed in the Lab strain compared with field populations, overlap of 95% confidence intervals of I50 values (concentration required for 50% AChE activity inhibition) suggests no significant difference in AChE sensitivity among all populations tested for a given insecticide. There was no significant evidence of target site insensitivity in field populations that were exposed to the selected organophosphate and carbamate insecticides tested. The specific activity of general esterase and glutathione S-transferase was lowest in the Lab strain and was generally comparable to that of the field populations evaluated. The current data provide a mode-of-action specific baseline for future monitoring of resistance to organophosphate and carbamate insecticides in populations of D. citri.     

An insensitive acetylcholinesterase confers resistance to methomyl in the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae)

Two forms of acetylcholinesterase were identified in field populations of the beet armyworm, Spodoptera exigua (Hübner), collected from cotton in San Joaquin Valley, CA. Strains (BESS and BKRR) homogeneous for each variant were isolated and their relative susceptibilities to methomyl, chlorpyrifos, and chlorpyrifos-oxon assessed by topical application bioassay. In comparisons with a laboratory susceptible strain (DOW), BKRR and BESS expressed 68-fold and sevenfold resistance, respectively, to the carbamate methomyl. Neither strain was cross-resistant to chlorpyrifos or its oxygen analog (chlorpyrifos-oxon). In biochemical studies, the BKRR AChE enzyme was approximately 30-fold and sixfold more insensitive to methomyl and chlorpyrifos-oxon, respectively, compared with the DOW enzyme. The correlation between the toxicological and biochemical studies provides strong evidence that target-site insensitivity is an important mechanism of resistance to methomyl. The lack of significant cross-resistance to chlorpyrifos suggests that the insensitive AChE in these field populations was selected by methomyl alone and not by the organophosphate.     

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。     

MOLECULAR MECHANISMS OF TARGET RESISTANCE IN THE ORGANOPHOSPHATE- AND PYRETHROID-RE-SISTANT HELICOVERPA ARMIGERA (HUBNER)

Abstract  The molecular mechanisms of target resistance, i. e. acetyicholinesterase (AChE) and sodium channel insensitivity, in the organophosphate(0P)- and pyrethroid(Py)-resistant (R) Helicoverpa armigera were investigated. The activity and V_max of AChE from R strain were 1.09– and 1.23-fold of the susceptible(S) strain respectively, but the K_M value of AChE in R strain was only 0.67-fold of S stain. The K_i values of AChE from the R strain were 0.44 for DDVP and 0.50 for malathion respectively, as compared with those of the S strain. These data showed that the AChE from R strain might be qualitatively altered. The knockdown resistance ( kdr ) of the resistant H. armigera was also identified by PCR technique. The fragments of IIS6, linker of 11–111 and a part of IIS5-IIs6 in the sodium channel were cloned and sequenced, and then compared to amino acid sequences from the R and S strains. and other insect species. There was no difference to be found at amino acid level in the above fragments cloned. It was suggested that the Rdr was not involved in the resistance of R strain.     

Mutation in ace1 associated with an insecticide resistant population of Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.     

棉铃虫抗药性的生理生化机制研究

本文报道了棉铃虫Helicoverpa armigera田间抗性种群对杀虫剂抗药性的生理生化机制。抗性种群(HJ-R)5龄幼虫羧酸酯酶、谷胱甘肽转移酶、多功能氧化酶活力均明显高于相对敏感种群(HD-S)。两种群乙酰胆碱酯酶对杀虫剂敏感性没有显著差异。HJ-R种群的腹神经索对氰戊菊酯表现了2-3倍的神经不敏感性。HJ-R种群对氨基甲酸酯类杀虫剂的抗性主要是由代谢机制引起,其中多功能氧化酶可能起主导作用;对菊酯的抗性是由多功能氧化酶、酯酶、以及神经不敏感性几个因子综合作用的结果。     

酶标板法监测棉蚜乙酰胆碱酯酶对杀虫剂的不敏感性

用酶标板法测定了不同浓度有机磷和氨基甲酸酯杀虫剂在反应不同时间内对棉蚜Aphis gossypii乙酰胆碱酯酶(AChE)的抑制作用。结果表明有机磷杀虫剂甲基1605,辛硫磷,久效磷,氧乐果和乙酰甲胺磷对棉蚜AChE均无明显的抑制作用。当用0.01 mol/L灭多威与酶及底物反应1 h对北京棉蚜(敏感)种群AChE的抑制率可达82.4%,与反应2 h 89.4%仅差7%。因此以0.01 mol/L灭多威反应1 h测定棉蚜AChE对它的敏感性是合理的。通过测定北京地区寄主为鼠李和棉花以及山东高密寄主为棉花的棉蚜种群中个体AChE活性的分布和灭多威对其抑制的分布,表明3个棉蚜种群中AChE个体频率的分布差异不大,而灭多威对三个种群个体AChE的抑制率小于30%的个体分别为2.4%、16%和29%。抑制率大于70%的个体分别为72%、33%和1%,与生物测定结果一致。因此,用酶标板法测定棉蚜个体AChE对氨基甲酸酯的不敏感性频率可作为棉蚜对氨基甲酸酯抗性的监测技术,为棉蚜化学防治提供依据。     

有机磷和拟除虫菊酯抗性棉铃虫靶标抗性的分子机理(英文)

对有机磷和拟除虫菊酯抗性 (R)棉铃虫靶标抗性的分子机理 ,即乙酰胆碱酯酶 (AChE)和钠通道敏感度降低进行了研究。根据AChE的动力学常数表明 ,R品系AChE的活性和Vmax值分别是S品系的 1 0 9和 1 2 3倍 ,但R品系的AChE的Km 值仅是S品系的 0 6 7倍。R品系AChE对DDVP和马拉硫磷的Ki值分别是S品系的 0 4 4和 0 55。这表明AChE发生了质的变化。还应用PCR技术对抗性棉铃虫的击倒抗性 (kdr)进行了鉴定 ,克隆了钠通道的IIS6序列、IIS5和IIS6连接片段以及II和III连接片段 ,测序后比较了R和S品系以及其它昆虫的同源性 ,结果在氨基酸水平未发现有任何差异 ,这表明该抗性棉铃虫品系不涉及kdr。     

棉铃虫AChE不同分子型对抑制剂的敏感度及其与抗药性的关系(英文)

在分离到的棉铃虫AChE五种不同的分子型中 ,2 .1s和 8.7sAChE抗性品系对毒扁豆碱的敏感度明显低于敏感品系 ,成虫头部I50 值分别相差 1 86.3和 84.8倍 ,幼虫I50 值分别相差 1 0 1 0 倍和 1 0 5 倍。幼虫 5.3sAChE对毒扁豆碱的敏感度抗性品系和敏感品系相差达 1 2 3倍 ,而成虫则没有差异。研究结果表明 2 .1s、5.3s和 8.7sAChE敏感度降低可能是造成棉铃虫对有机磷和氨基甲酸酯类杀虫药剂产生抗性的主要原因     

Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations

Two amino acid substitutions in acetylcholinesterase 1 (AChE1), G119S and F290V, are responsible for resistance to organophosphate and carbamate insecticides in Culex pipiens mosquitoes. These mutations generate very different levels of insensitivity to insecticide inhibitors. We described here a biochemical method that rapidly identifies AChE1 variants (susceptible, G119S and F290V, named S, R and V, respectively) present in individual mosquitoes. We investigated the frequency of AChE1 phenotypes in 41 field samples collected around the Mediterranean Sea. F290V substitution was found only in 15 samples and at low frequency, whereas G119S was highly spread in all samples. However, seven V distinct alleles were identified whereas only one R allele was present. The [V] enzymatic phenotype was never observed alone, and the V allele was always found associated with the susceptible and/or G119S AChE1 ([VS], [VR] or [VRS] phenotypes). Furthermore, we showed the presence of duplicated alleles, associating a susceptible and a V copy of the ace-1 gene, in most individuals analyzed for its presence. Evolutionary forces driving the large number of F290V ace-1 alleles and their low frequency in Mediterranean countries are discussed.