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。     

烟粉虱对拟除虫菊酯杀虫剂的抗性机理

通过增效剂生物测定、生化分析以及钠离子通道基因ⅡS4-6 cDNA片段的RT-PCR扩增,探讨了烟粉虱Bemisia tabaci（Gennadius）对拟除虫菊酯杀虫剂的抗性机理。结果表明：对于采自田间的6个烟粉虱抗性品系,磷酸三苯酯（TPP）和胡椒基丁醚（PBO）对氯氰菊酯、溴氰菊酯、氯氟氰菊酯和甲氰菊酯均有显著的增效作用,而DEM对4种拟除虫菊酯杀虫剂均无明显的增效作用。烟粉虱抗性品系的α-NA羧酸酯酶和β-NA羧酸酯酶活性分别是敏感品系的2.16～2.65倍和1.22～1.41倍,抗性品系的谷胱甘肽S转移酶活性与敏感品系没有差异,表明羧酸酯酶和多功能氧化酶在烟粉虱对拟除虫菊酯类杀虫剂的抗性中具有重要的作用,而谷胱甘肽S转移酶与抗性无关。通过RT-PCR克隆了6个烟粉虱田间抗性品系的钠离子通道结构域ⅡS4-6 cDNA片段的序列（420 bp）,发现与敏感品系相比,有2个位点发生突变,分别为L925I突变和I917V突变,L925I突变在所有6个烟粉虱田间抗性种群中均有发生,该位点突变已被证实与拟除虫菊酯类杀虫剂密切相关,表明神经不敏感性可能是烟粉虱对拟除虫菊酯产生抗性的另一个重要因子。     

kdr突变和解毒代谢在B型烟粉虱对高效氯氰菊酯抗性中的作用

本研究明确了kdr突变和解毒代谢在B型烟粉虱Bemisia tabaci对高效氯氰菊酯抗性中的作用。B型烟粉虱NJ品系相对于烟粉虱敏感品系（SUD-S,非B型）对高效氯氰菊酯有266倍的抗性。对NJ品系用高效氯氰菊酯进行群体筛选获得抗性为811倍的NJ-R1品系,对NJ品系进行单对交配筛选获得抗性达2 634倍的NJ-R2品系。在NJ,NJ-R1和NJ-R2品系间,酯酶、多功能氧化酶和谷胱甘肽S-转移酶活性无显著差异,说明在筛选过程中解毒代谢没有发生变化。PASA检测结果表明,NJ-R2品系钠离子通道基因L925I突变（kdr突变）频率为100%,NJ-R1品系为80.6%,NJ品系为55%。由此可见,kdr突变频率的增加是B型烟粉虱种群对高效氯氰菊酯抗性上升的主要原因。在NJ,NJ-R1和NJ-R2品系中,增效醚（PBO）对高效氯氰菊酯的增效作用均为20倍左右,而PBO对SUD-S品系没有任何增效作用。PBO能同时抑制烟粉虱的多功能氧化酶和酯酶,通过与TPP增效作用进行对比表明,在B型烟粉虱中PBO所产生的增效作用主要来源于对酯酶的抑制。因此,B型烟粉虱品系（NJ-R2,NJ-R1和NJ）与非B型SUD-S品系相比存在20倍左右的先天抗性,该先天抗性主要与B型烟粉虱的特有酯酶有关。在B型烟粉虱品系对高效氯氰菊酯的抗性中,抗性水平完全由kdr突变频率高低所决定。     

菜蚜茧蜂对甲胺磷和灭多威的抗性机制

采用试管药膜法测定了菜蚜茧蜂 Diaeretiella rapae 对甲胺磷和灭多威的抗性及增效剂的增效作用,并测定了菜蚜茧蜂和菜缢管蚜 Lipaphis erysimi 乙酰胆碱酯酶动力学参数、解毒酶活性及增效剂对菜蚜茧蜂酶活性的体内抑制作用。福州地区菜蚜茧蜂已对甲胺磷和灭多威产生5.6和9.1倍的抗性,胡椒基丁醚、磷酸三苯酯和马来酸二乙酯对两种杀虫剂的抗性品系均有显著增效作用,胡椒基丁醚的作用最为显著。胡椒基丁醚对抗性菜蚜茧蜂杀虫剂敏感性的增效作用远高于对敏感菜蚜茧蜂的影响。3种增效剂对菜蚜茧蜂乙酰胆碱酯酶均无抑制作用;磷酸三苯酯和胡椒基丁醚对羧酸酯酶,马来酸二乙酯对谷胱甘肽S转移酶有显著抑制作用。抗性和敏感菜蚜茧蜂乙酰胆碱酯酶的米氏常数K_m）、最大反应速度（V_max）、羧酸酯酶及谷胱甘肽S转移酶活性值相近,但敏感菜蚜茧蜂乙酰胆碱酯酶的双分子速度常数（K_I）值远高于抗性的。此外,对菜蚜茧蜂和菜缢管蚜乙酰胆碱酯酶、羧酸酯酶和谷胱甘肽S转移酶进行了比较研究。结果表明菜蚜茧蜂对甲胺磷和灭多威的低水平抗性与乙酰胆碱酯酶的不敏感性及解毒酶的解毒代谢有关。     

棉蚜抗吡虫啉品系和敏感品系主要解毒酶活性比较

通过生物测定和生物化学方法比较了棉蚜 Aphis gossypii 对吡虫啉抗性（约为7倍）和敏感品系几种主要解毒酶的活性。结果表明：氧化胡椒基丁醚对两个品系均无明显增效作用。抗性品系中羧酸酯酶和谷胱甘肽S-转移酶的比活力均明显高于敏感品系,抗性品系中羧酸酯酶的K_m值也显著高于敏感品系,说明抗性品系羧酸酯酶与底物的亲和力明显高于敏感品系。上述结果证明羧酸酯酶和谷胱甘肽S-转移酶活力增强在棉蚜对吡虫啉的抗性中起重要作用。     

灰飞虱对噻嗪酮的抗性风险及机理

为研究灰飞虱对噻嗪酮的抗性发展规律及抗性生化机理,采用稻苗喷雾法对灰飞虱种群进行连续筛选获得高抗性品系,估算其现实遗传力并进行田间抗性风险预测;采用稻苗浸渍法测定杀虫剂对灰飞虱的毒力及交互抗性;利用生物化学方法测定不同品系之间的解毒酶活力,探讨灰飞虱对噻嗪酮的抗性生化机理.结果表明:用噻嗪酮对灰飞虱种群连续筛选32代,其抗性倍数达到168.49倍,现实遗传力h~2为0.11.当杀死率为80%~90%时,预计灰飞虱对噻嗪酮的抗性增长10倍,仅需要5~6代.田间实际的现实遗传力要比室内选择种群估计低一些,预计田间抗性提高10倍所需要时间会更长.交互抗性测定结果表明,灰飞虱抗噻嗪酮品系与吡虫啉和噻虫嗪之间有高水平交互抗性,与啶虫脒有低水平交互抗性,与吡蚜酮和毒死蜱无交互抗性.增效作用和解毒酶活力测定结果显示,抗性品系细胞色素P450单加氧酶活力提高最大,酯酶次之,谷胱甘肽-S-转移酶无显著变化.田间使用噻嗪酮防治灰飞虱存在较大抗性风险,可与吡蚜酮和毒死蜱等交替使用以延缓抗性发展;3种解毒酶中,细胞色素P450单加氧酶在灰飞虱对噻嗪酮的抗性发展中起到了重要作用.     

江苏Q型烟粉虱抗性监测及其靶标基因克隆

应用成虫浸叶生测法研究了江苏省扬州、无锡和东台3个地区Q型烟粉虱Bemisia tabaci(Gennadius)田间种群对5种杀虫剂的抗药性。结果表明,与相对敏感种群相比,Q型烟粉虱田间种群已对氯氰菊酯和吡虫啉产生了低到中等水平抗性,对阿维菌素仍然表现敏感。进一步通过RCR扩增获得了长度分别为287bp和184bp的烟粉虱乙酰胆碱酯酶ace1和para-同源钠离子通道基因片断。序列分析表明,江苏Q型烟粉虱存在与有机磷抗性相关的乙酰胆碱酯酶F331W突变和与拟除虫菊酯抗性相关的钠离子通道L925I和T929V突变。     

寄主植物对B型烟粉虱(Bemisia tabaci)几种主要解毒酶活性的影响

研究测定了棉花、一品红、茄子和番茄4个B型烟粉虱(Bemisia tabaci)种群岐睳A羧酸酯酶、猹睳A羧酸酯酶、乙酰胆碱酯酶和谷胱甘肽S-转移酶活性.结果表明:岐睳A羧酸酯酶活性与猹睳A羧酸酯酶活性比值均大于1,说明B型烟粉虱水解岐睳A的能力高于对猹睳A的水解能力;B型烟粉虱不同寄主种群岐睳A羧酸酯酶、猹睳A羧酸酯酶活性个体分布频率均存在一定差异.B型烟粉虱番茄种群羧酸酯酶活性最高(93.06 mOD/(mg protein·min)),是棉花种群的1.49倍.B型烟粉虱茄子种群乙酰胆碱酯酶活性明显高于其他寄主种群,达极显著差异水平(p＜0.01);茄子种群乙酰胆碱酯酶活性分布频率在＞4U/mg protein区间段的分布高达85%,与其他3个寄主种群分布明显不同.B型烟粉虱茄子种群谷胱甘肽S-转移酶活性最高,与其他3个寄主种群间的差异达极显著水平(p＜0.01).上述结果表明,B型烟粉虱主要解毒酶活性在不同的寄主植物上具有一定的生理可塑性.研究有利于揭示该害虫寄主范围广和寄主适应性强的生理生态学基础.     

运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

白纹伊蚊溴氰菊酯抗性和敏感品系羧酸酯酶性质比较

本文对白纹伊蚊Aedes albopictus溴氰菊酯抗性品系和敏感品系羧酸酯酶的生物化学性质进行了比较。白纹伊蚊抗性品系和敏感品系羧酸酯酶随底物浓度(α-乙酸萘酯或β-乙酸萘酯)的变化比活力变化趋势一致,但抗性品系对这2种底物的比活力均高于敏感品系,抗性品系羧酸酯酶的米氏常数和最大反应速度与敏感品系有显著差异。胆碱酯酶抑制剂测定结果表明,抗性品系羧酸酯酶对敌敌畏和磷酸三苯酯的敏感性高于敏感品系,对残杀威的敏感性低于敏感品系。2个品系羧酸酯酶对脱叶磷的敏感性差异不大。说明羧酸酯酶可能与白纹伊蚊对溴氰菊酯抗性有关。     

烟粉虱田间种群的抗药性

采用成虫浸叶生测法分别测定了福建省福州、漳州、龙岩、三明、南平、宁德等地烟粉虱田间种群对13种杀虫剂的抗性.结果表明,福建省6个烟粉虱田间种群对氯氟氰菊酯、甲氰菊酯、氯氰菊酯、溴氰菊酯、乙酰甲胺磷、毒死蜱产生了高水平抗性,抗性倍数分别达838.38～2460.52、244.64～834.29、116.02～266.35、81.75～124.18、425.18～875.56和54.53～78.43倍,对乐果产生了中等水平抗性,抗性倍数达14.16～17.66倍,对敌敌畏产生了中等偏低水平的抗性,抗性倍数达6.23～11.25倍,对灭多威的抗性水平较低,抗性倍数仅为4.07～5.66倍.漳州种群已对烟碱类杀虫剂产生了中等水平抗性,对吡虫啉、啶虫脒和噻虫嗪的抗性倍数分别达23.08、10.32和24.60倍,而其它地区烟粉虱种群对吡虫啉、啶虫脒和噻虫嗪的抗性水平较低,甚至不明显,抗性倍数分别仅为1.31～3.28、1.82～7.23和1.39～5.45倍.福建省各地区烟粉虱种群对阿维菌素尚未产生明显的抗药性.     

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.     

Seasonal changes of methamidophos susceptibility and biochemical properties in Plutella xylostella (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae (Hymenoptera: Braconidae)

Methamidophos resistance and acetylcholinesterase (AChE) insensitivity to methamidophos, dichlorvos, and carbofuran were determined in the field populations of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) collected from the corresponding hosts between October 1998 and December 2003 in Fuzhou and Minhou, Fijian, China. Resistance levels to methamidophos and AChE insensitivity to the three insecticides in the two species of insects were high during autumn and spring and low during summer. Resistance to methamidophos was 15.3- and 12.6-fold higher in resistant F0 parents of P. xylostella and C. plutellae than in their susceptible F11 progeny, respectively. The bimolecular rate constant (k(i)) values of AChE to methamidophos, dichlorvos, and carbofuran were 4.6-, 6.3-, and 7.7-fold higher in F11 progeny of P. xylostella, and 3.7-, 4.5-, and 3.7-fold higher in F11 progeny of C. plutellae than those in their F0 parents, respectively. Compared with susceptible F11 progeny, the resistance ratios for methamidophos were 4.2-29.8 and 3.8-13.1 in 21 field populations of P. xylostella and C. plutellae, respectively. The k(i) values of AChE to methamidophos, dichlorvos, and carbofuran were 2.0-21.6-, 3.6-9.5-, and 2.6-9.2-fold higher in F11 progeny of P. xylostella, and 1.8-7.6-, 1.9-4.6-, and 2.2-7.6-fold higher in F11 progeny of C. plutellae than those in 21 field populations, respectively. Significant correlative variations of methamidophos resistance as well as significant correlative variations of k(i) values of AChE to insecticides between the two species of insects also were found in space and time. The k(i) values of AChE to insecticides in C. plutellae were far higher than those in P. xylostella. There were no obvious differences in the Km and Vmax of AChE between F0 parents and F11 progeny of P. xylostella and C. plutellae, respectively. But carboxylesterase activity was 1.6-fold higher in F0 parents of C. plutellae than in F11 progeny, and glutathione S-transferase activity was 1.5-fold higher in F0 parents of P. xylostella than in F11 progeny. The results suggested that the AChE insensitivity to insecticides might play the most important role in methamidophos resistance in the two species of insects. From these results, a spatial and temporal correlative evolution of methamidophos resistance and insensitive AChE was found to exist between P. xylostella and C. plutellae.     

河北转Bt基因棉田棉铃虫对杀虫剂的抗性及相关酶活性的变化

为明确河北省推广种植植转Bt基因抗虫棉（简称Bt棉）后, 棉铃虫Helicoverpa armigera (Hübner)对常用杀虫剂的抗药性水平及其生化机理, 2011-2012年采用点滴法对保定南郊、 沧州南皮、 邢台巨鹿3个地区的田间种群以及敏感种群进行了室内毒力测定, 并采用生化分析法对4个种群相关的羧酸酯酶（carboxylesterase, CarE）、 谷胱甘肽S 转移酶(glutathione S-transferases, GSTs)和乙酰胆碱酯酶(acetylcholinesterase, AChE)的活性进行了研究。结果表明: 3个田间种群对高效氯氰菊酯和氰戊菊酯处于中至高抗水平, 抗性倍数为20.02~73.70倍; 对灭多威处于低至中抗水平, 抗性倍数为6.27~11.84倍; 对高效氯氟氰菊酯(抗性倍数: 1.07~4.20倍), 辛硫磷、 毒死蜱和马拉硫磷(抗性倍数: 1.00~2.69倍), 以及氯虫苯甲酰胺（抗性倍数: 2.00~3.67倍）均处于敏感水平。3个田间种群的CarE, GSTs和AChE活性分别是敏感种群的1.06~1.23, 1.20~1.63和1.15~1.23倍, 这可能与其对高效氯氰菊酯、 氰戊菊酯和灭多威产生的抗性有关。     

四种杀虫剂对两种书虱羧酸酯酶和乙酰胆碱酯酶的抑制作用

选用有机磷类杀虫剂(敌敌畏、毒死蜱、对氧磷)和氨基甲酸酯类杀虫剂(丁硫克百威),通过生物测定(药膜法)和生化测定(比色法)比较了嗜卷书虱和嗜虫书虱对所选药剂的敏感差异性。根据LC50可知嗜虫书虱对所选药剂比嗜卷书虱敏感。离体酶活性分析结果显示嗜卷书虱和嗜虫书虱的羧酸酯酶只对敌敌畏敏感,且嗜卷书虱比嗜虫书虱更敏感;4种药剂对乙酰胆碱酯酶均有强烈的抑制作用,同样是嗜卷书虱比嗜虫书虱敏感。乙酰胆碱酯酶的动力学研究结果和离体酶活性测定相一致。聚丙烯酰胺凝胶电泳分析显示,4种杀虫剂离体条件下对2种书虱的酯酶同工酶的抑制能力有明显差异,其中敌敌畏的抑制力最强;但对不同同功酶的抑制趋势(对大分子的抑制似乎较强)是一致的。酶的敏感性分析结果与生测结果比较表明,2种书虱的耐药力差异与其乙酰胆碱酯酶和酯酶对药剂的敏感性无关。如要弄清耐药力机制,需做进一步研究。     

Cross-resistance and biochemical mechanisms of abamectin resistance in the B-type Bemisia tabaci

Abstract:  To understand the risk of resistance and the possible mechanisms of resistance to abamectin in B-type Bemisia tabaci (Gennadius) better, a resistant strain of B. tabaci was selected in the laboratory and cross-resistance pattern and resistance mechanisms to abamectin were investigated. The NJ-Abm strain of B. tabaci was derived from a field population (NJ) collected in Nanjing, China in 2002 with 18 generations of selection with abamectin in the laboratory. Compared with the unselected NJ strain, the selected NJ-Abm strain developed 14.5-fold resistance to abamectin and showed significant cross-resistance to emamectin benzoate (4.4-fold) and imidacloprid (3.4-fold), but no cross-resistance to fipronil. The oxidase inhibitor piperonyl butoxide (PBO) and glutathione S -transferase inhibitor diethyl maleate (DEM) produced significant synergism on abamectin in the NJ-Abm strain (with synergistic ratios of 3.9- and 4.1-fold respectively); however, the esterase inhibitor triphenyl phosphate (TPP) did not act synergistically with abamectin. Biochemical analysis confirmed that P450 monooxygenase activity and glutathione S -transferase activity of the NJ-Abm strain were elevated to 2.1- and 2.0-fold, respectively, compared with that of the NJ strain. This indicated that enhanced metabolism mediated by P450 monooxygenase and glutathione S -transferase is likely to be involved in abamectin resistance and cross-resistance to imidacloprid and emamectin benzoate in the NJ-Abm strain.     

Comparison of chlorpyrifos resistance in Culex pipiens pipiens (Diptera: Culicidae) collected from Northern and Southern Tunisia

In this study, we investigated resistance to the organophosphates chlorpyrifos in Tunisian populations of Culex pipiens pipiens. Three field populations were collected from Northern and central Tunisia between 2003 and 2005 and used for the bioassays tests. Our results registered moderate and high levels of resistance to chlorpyrifos which ranged from 33.8 to 111. The chlorpyrifos resistant populations were highly resistant to propoxur indicated an insensitive acetylcholinesterase 1 (AChE 1). The highest frequency of AChE 1 resistant phenotypes (64%) was recorded in the most resistant population (sample # 1). Bioassays conducted in the presence of synergists showed that not esterases were involved as the resistance mechanism to chlorpyrifos. However, CYP450 was partly involved in the resistance of the most resistant sample (# 1). Starch electrophoresis showed that three esterases were present in studied samples: A2‐B2, A4‐B4 and B12. Results are discussed in relation to the selection pressure caused by insecticide treatments.