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。     

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。     

与拟除虫菊酯抗性相关的烟粉虱钠通道基因突变及其检测

通过RT-PCR克隆了烟粉虱Bemisia tabaci (Gennadius) 南京种群(B-生物型)的钠离子通道结构域ⅡS4-6 cDNA片段,证实了与拟除虫菊酯抗性相关的是位于第925位亮氨酸到异亮氨酸的突变(L925I),并建立了L925I突变的PASA检测技术。与SUD-S敏感品系相比,2002年采自南京棉花上的烟粉虱种群对氯氰菊酯具有77倍的抗性,用氯氰菊酯对该种群进行多次筛选后,该种群对氯氰菊酯的抗药性提高到227倍。PASA检测结果表明筛选后的南京种群中100%个体都具有L925I突变(61.1%的个体为L925I突变纯合子,38.9%的个体为杂合子),而未筛选的南京种群只有75%个体具有L925I突变(35%个体为L925I突变纯合子,40%的个体为杂合子,25%的个体为野生型)。该结果表明了烟粉虱钠离子通道L925I突变与对拟除虫菊酯抗性密切相关。还讨论了烟粉虱对拟除虫菊酯抗性的代谢机理。     

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。     

敏感和抗性棉铃虫对溴氰菊酯毒力反应的机理研究

通过生物测定和生物化学方法比较了棉铃虫玎Helicoverpa armigera敏感和抗性种群对溴氰菊酯毒力反应及其3种解毒酶的差异。结果表明,田间抗性种群和室内药剂汰选的抗性种群对溴氰菊酯均有较高的抗性,其抗性倍数分别达到195.8和37 375倍。水解酯酶和多功能氧化酶是导致棉铃虫对溴氰菊酯产生高抗性的重要酶系。特异性抑制剂活体内外抑制作用测试发现,敏感种群和抗性种群均含有较高量的乙酰胆碱酯酶,但两个种群对抑制剂的亲和力反应不同,表明乙酰胆碱酯酶在敏感种群和抗性种群中发生了不同的变化,这种变化可能与棉铃虫对溴氰菊酯的抗性有关。由此推断,棉铃虫对拟除虫菊酯这类中枢神经系统神经毒剂产生抗性,乙酰胆碱酯酶发生变化可能也是一个重要因子。     

广东首次发现Q型烟粉虱种群分布

烟粉虱Bemisia tabaci(Gennadius)是全世界分布的刺吸式传毒昆虫,其中Q型烟粉虱自2003年首次在昆明一品红上发现以来,该种群已在全国10多个省份广泛传播,并有逐步取代B型烟粉虱的趋势。然而,作为较早对外开放的沿海省份,广东省一直未有Q型烟粉虱在田间分布危害的报道。本文利用常规PCR、特异B/Q型烟粉虱引物扩增及DNA测序技术,对近期在广州长湴公园扶桑寄主上采集的烟粉虱种群进行了分子鉴定,并对该种群与国内外其它Q型种群的系统发育关系进行了研究。结果表明,在广州长湴公园扶桑寄主上采集的烟粉虱种群为Q型烟粉虱,在660 bp的mtCOI序列中,该Q型烟粉虱与来自江苏、云南及浙江的Q型烟粉虱种群分别有1²个碱基差异;系统进化关系表明该Q型烟粉虱与江苏、云南的Q型烟粉虱最近,其种群的最初起源应为地中海西岸地区。相比B型烟粉虱,Q烟粉虱被认为具有更强的抗药性和传播植物病毒的能力,Q型烟粉虱在广东的首次发现,表明该烟粉虱种群已在广东野外定殖,其扩散动态、寄主范围及危害程度亟待实时监测。     

云南烟蚜抗药性机制研究

通过比较云南烟蚜敏感品系和抗性品系的解毒酶(α-乙酸萘酯羧酸酯酶、β-乙酸萘酯羧酸酯酶)和靶标酶(乙酰胆碱酯酶)的活力,研究了烟蚜对有机磷、拟除虫菊酯和氨基甲酸酯类杀虫剂抗性的生化机制,并通过酯酶基因扩增检测和钠离子通道突变检测,研究了其抗性的分子机制。结果表明:α-乙酸萘酯羧酸酯酶活力增强是烟蚜对有机磷类、氨基甲酸酯类杀虫剂及拟除虫菊酯类杀虫剂的抗性机制之一;乙酰胆碱酯酶在烟蚜对有机磷杀虫剂抗性中起重要作用;3个抗性品系烟蚜均没有发生酯酶基因扩增,抗拟除虫菊酯品系烟蚜发生了钠离子通道突变。     

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。     

新疆棉铃虫对溴氰菊酯和硫丹 抗性的生化机理研究

通过生物测定和生化分析研究了新疆棉铃虫Helicoverpa armigera (Hübner)敏感种群和室内筛选获得的抗性种群对硫丹和溴氰菊酯的反应及其α-乙酸萘酯酶和乙酰胆碱酯酶的动态活性反应。结果表明,筛选后新疆棉铃虫对硫丹和溴氰菊酯产生的抗性倍数分别为13倍和66倍。两个抗性种群的α-乙酸萘酯酶和乙酰胆碱酯酶比活力均高于敏感种群。相应杀虫剂预处理后,α-乙酸萘酯酶酶活力受到抑制。抗性种群的α-乙酸萘酯酶对底物的亲和力高于敏感种群,但Vmax低于敏感种群。抗性种群的乙酰胆碱酯酶对底物的亲和力显著低于敏感种群,Vmax比敏感种群高。聚丙烯酰胺凝胶电泳显示,两个抗性种群都有一条特异性酶带,其迁移率相近,且均可被甲基对氧磷抑制。因此推测,α-乙酸萘酯酶参与了新疆棉铃虫对硫丹和溴氰菊酯的抗性,具有代谢和阻断作用;乙酰胆碱酯酶对抗性的产生也起到了重要作用。     

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.     

福建省烟粉虱田间种群抗药性发展及其影响因素

采用成虫浸叶生测法对福建省不同地区烟粉虱田间种群的抗药性发展进行监测.结果表明： 福建各地烟粉虱田间种群对氯氟氰菊酯、甲氰菊酯、毒死蜱仍保持较高水平抗性,对灭多威的抗性水平较低,对阿维菌素未产生明显的抗药性;烟粉虱田间种群对烟碱类杀虫剂的抗性发展极为迅速,其中漳州种群对吡虫啉、噻虫嗪已由2005年的中等水平抗性（抗性倍数分别为23和25倍）发展为2009年的高水平抗性（抗性倍数分别为103和228倍）,其他地区种群对吡虫啉、噻虫嗪也由2005年的低水平抗性（抗性倍数分别为1.5～3.3倍和1.7～5.5倍）发展为2009年的中等水平抗性（抗性倍数分别为23～33倍和29～49倍）.采用mtDNA CO I分子标记技术对福建省不同地区和寄主植物上采集的8个烟粉虱种群的生物型进行鉴定发现,采自漳州变叶木上的烟粉虱种群为本地土著种(未知生物型),其他7个采自大田蔬菜作物的烟粉虱种群均为B型.寄主植物和温度对烟粉虱种群的药剂敏感性影响有限,吡虫啉等烟碱类杀虫剂高强度使用是B型烟粉虱田间种群对该类型药剂迅速形成抗性的重要原因.     

施用噻虫嗪可降低B型烟粉虱群体的遗传多样性

用4个具有多态性的微卫星位点（BEM15、BEM25、BEM31和BT-b53）分析了施用化学农药噻虫嗪 （thiamethoxam）对B型烟粉虱Bemisia tabaci（Gennadius）群体遗传结构的影响。结果表明：噻虫嗪汰选18代的B型烟粉虱处理种群（抗性种群）抗药性上升到对照种群（敏感种群）的19.08倍,同时其微卫星位点不同等位基因频率分布发生了变化,等位基因数（N_a）平均值由2.750降到2.250,杂合度（h）平均值由0.459降到0.299（降低了34.8%）,多态信息含量（PIC）平均值由0.393降到0.250,有效等位基因数（N_e）平均值由2.051降到1.488;抗性种群与敏感种群的标准遗传距离为0.15。结果提示,化学农药的施用可能是B型烟粉虱群体遗传结构的重要影响因素之一。     

Assessment of insecticide resistance in five insect pests attacking field and vegetable crops in Nicaragua

Field populations of Hypothenemus hampei (Ferrari), Plutella xylostella (L.), Spodoptera exigua (Hübner), Helicoverpa zea (Boddie) and Bemisia tabaci (Gennadius) were tested for resistance to several insecticides commonly used in Nicariagua. Assays were conducted to estimate the LD50s or LC50s and the corresponding resistance ratios. A diagnostic concentration was used to discriminate between susceptible and resistant strains of H. hampei. The tests with >6,000 H. hampei adults collected from six different sites indicate the absence of resistance to endosulfan. Resistance to cypermethrin, deltamethrin, chlorfluazuron, thiocyclam, and methamidophos was documented in six field populations of P. xylostella. High levels of resistance to cypermethrin and deltamethrin, but moderate levels of resistance to chlorpyriphos and methomyl, were also documented in two field populations of S. exigua. Moderate levels of resistance to cypermethrin, deltamethrin and chlorpyriphos were also documented in three field populations of H. zea. Moderate to high levels of resistance to bifenthrin, methamidophos and endosulfan were documented in four field populations of B. tabaci. The presence of significant correlations between LD50s or LC50s suggests the occurrence of cross-resistance or simultaneous selection for resistance by different insecticides with different modes of action. Our data could not differentiate between these two possibilities. Because insecticides will continue being used in Nicaragua, a resistance management program is urgently needed. The implementation of integrated pest management tactics must be accompanied by specific regulations for pesticide registration. In the future, pesticide registration regulations in Nicaragua should include periodic resistance monitoring. The mechanisms to cover the costs of resistance monitoring and resistance management should also be established.     

北京和湖南烟粉虱生物型及其抗药性监测

烟粉虱是蔬菜等园艺作物上为害严重、最难以防治的害虫之一,生产上防治该虫主要还是依靠化学药剂。本研究鉴定了北京、湖南地区设施蔬菜上烟粉虱的生物型,并监测了其对6种不同杀虫剂的抗药性。结果表明,北京、湖南地区蔬菜烟粉虱生物型分别是Q型和B型。两地烟粉虱对阿维菌素尚处于敏感状态;对烯啶虫胺及其他烟碱类杀虫剂噻虫嗪和吡虫啉的抗药性达中抗到高抗水平,湖南地区抗性水平比北京地区更高,抗性倍数最高达到71.58倍;传统杀虫剂毒死蜱和联苯菊酯对湖南、北京两地烟粉虱的毒杀活性差。     

Assessment of cross-resistance potential to neonicotinoid insecticides in Bemisia tabaci (Hemiptera: Aleyrodidae)

Laboratory bioassays were carried out with four neonicotinoid insecticides on multiple strains of Bemisia tabaci (Gennadius) to evaluate resistance and cross-resistance patterns. Three imidacloprid-resistant strains and field populations from three different locations in the southwestern USA were compared in systemic uptake bioassays with acetamiprid, dinotefuran, imidacloprid and thiamethoxam. An imidacloprid-resistant strain (IM-R) with 120-fold resistance originally collected from Imperial Valley, California, did not show cross-resistance to acetamiprid, dinotefuran or thiamethoxam. The Guatemala-resistant strain (GU-R) that was also highly resistant to imidacloprid (RR=109-fold) showed low levels of cross-resistance when bioassayed with acetamiprid and thiamethoxam. However, dinotefuran was more toxic than either imidacloprid or thiamethoxam to both IM-R and GU-R strains as indicated by low LC50s. By contrast, a Q-biotype Spanish-resistant strain (SQ-R) of B. tabaci highly resistant to imidacloprid demonstrated high cross-resistance to the two related neonicotinoids. Field populations from Imperial Valley (California), Maricopa and Yuma (Arizona), showed variable susceptibility to imidacloprid (LC50s ranging from 3.39 to 115 microg ml(-1)) but did not exhibit cross-resistance to the three neonicotinoids suggesting that all three compounds would be effective in managing whiteflies. Yuma populations were the most susceptible to imidacloprid. Dinotefuran was the most toxic of the four neonicotinoids against field populations. Although differences in binding at the target site and metabolic pathways may influence the variability in cross-resistance patterns among whitefly populations, comparison of whitefly responses from various geographic regions to the four neonicotinoids indicates the importance of ecological and operational factors on development of cross-resistance to the neonicotinoids.     

Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)

The two most damaging biotypes of Bemisia tabaci, B and Q, have both evolved strong resistance to the neonicotinoid insecticide imidacloprid. The major mechanism in all samples investigated so far appeared to be enhanced detoxification by cytochrome P450s monooxygenases (P450s). In this study, a polymerase chain reaction (PCR) technology using degenerate primers based on conserved P450 helix I and heme-binding regions was employed to identify P450 cDNA sequences in B. tabaci that might be involved in imidacloprid resistance. Eleven distinct P450 cDNA sequences were isolated and classified as members of the CYP4 or CYP6 families. The mRNA expression levels of all 11 genes were compared by real-time quantitative RT-PCR across nine B and Q field-derived strains of B. tabaci showing strong resistance, moderate resistance or susceptibility to imidacloprid. We found that constitutive over-expression (up to approximately 17-fold) of a single P450 gene, CYP6CM1, was tightly related to imidacloprid resistance in both the B and Q biotypes. Next, we identified three single-nucleotide polymorphic (SNP) markers in the intron region of CYP6CM1 that discriminate between the resistant and susceptible Q-biotype CYP6CM1 alleles (r-Q and s-Q, respectively), and used a heterogeneous strain to test for association between r-Q and resistance. While survivors of a low imidacloprid dose carried both the r-Q and s-Q alleles, approximately 95% of the survivors of a high imidacloprid dose carried only the r-Q allele. Together with previous evidence, the results reported here identify enhanced activity of P450s as the major mechanism of imidacloprid resistance in B. tabaci, and the CYP6CM1 gene as a leading target for DNA-based screening for resistance to imidacloprid and possibly other neonicotinoids in field populations.     

韭菜迟眼蕈蚊对新烟碱类杀虫剂的抗药性及抗性机制初探

【目的】为明确韭菜迟眼蕈蚊Bradysia odoriphaga对新烟碱类杀虫剂的抗性水平及其抗性机制。【方法】通过测定不同地区韭菜迟眼蕈蚊对3种新烟碱类杀虫剂吡虫啉、噻虫嗪和噻虫胺的敏感度,及通过增效剂实验和酶活性测定,初步探索抗性产生机制,为韭菜迟眼蕈蚊抗性治理提供依据。【结果与结论】4个不同的韭菜迟眼蕈蚊田间种群对3种新烟碱类杀虫剂均产生了不同水平的抗性。其中,唐山种群对3种新烟碱杀虫剂均产生了较高的抗性。研究发现,唐山种群的7-乙氧基香豆素-O-脱乙基酶(ECOD)比活力为(3.89±0.31)pmol/(mg·pro·min),显著高于敏感品系。增效剂PBO对唐山种群的吡虫啉毒力的增效比为2.64,高于对敏感品系的增效比1.08。因此,P450s酶活性的升高与韭菜迟眼蕈蚊对吡虫啉的抗性有关。