抗性品系棉蚜乙酰胆碱酯酶和羧酸酯酶的变异

用浸叶法测定了采自我国不同地区(泰安、莱阳、南京、北京和安阳)的棉蚜品系Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ对久效磷、甲胺磷、抗蚜威和灭多威等杀虫剂的抗性水平,各棉蚜品系对杀虫剂的抗性依次为Ⅴ>Ⅳ>Ⅲ,Ⅱ>Ⅰ。进一步研究表明,Ⅴ和Ⅳ品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降,Ⅱ品系次之,Ⅲ和Ⅰ品系接近于敏感品系。Ⅴ和Ⅳ品系乙酰胆碱酯酶的Km值显著下降,表明酶发生了质的变化。不同棉蚜抗性品系的酯酶（全酯酶和羧酸酯酶）活性均显著升高,其中Ⅲ品系的酯酶活力为Ⅱ品系的2倍。Ⅴ品系羧酸酯酶Km值达2460.4 μmol/L,而Ⅳ品系仅为84.4 μmol/L,该两个品系羧酸酯酶发生了质的变化。研究结果表明,不同抗性程度的棉蚜品系均存在代谢抗性和靶标抗性。低抗水平的棉蚜品系,以代谢抗性为主,靶标抗性为辅;中抗水平的棉蚜品系,抑或由于解毒代谢酶的活性显著增强,也可能由于靶标的敏感性显著下降;而高抗水平的棉蚜品系,依赖于代谢抗性和靶标抗性的联合作用。     

我国麦蚜抗药性及研究现状

麦蚜是危害我国小麦Triticum aestivum L.生产的主要害虫,具有分布广、数量大、繁殖力强以及远距离迁飞等特点,不仅直接吸食小麦汁液,还传播多种植物病毒,每年造成小麦减产10%~30%。目前对麦蚜的防控主要以化学防治为主,但由于化学杀虫剂长期或不合理的使用,多地麦蚜对常用杀虫剂产生了不同程度的抗性。本文从麦蚜抗药性测定方法、抗性水平及交互抗性、代谢和靶标抗性机制、以及麦蚜抗药性综合治理等方面进行了综述,以期为麦蚜的防治及杀虫剂的持续合理使用提供理论参考和依据。     

昆虫抗药性分子机制研究的新进展

昆虫抗性机制的研究对于抗性监测、治理及新农药的研制具有重要意义。在过去几十年中,人们对与昆虫杀虫剂抗性有关的昆虫行为、生理代谢活动以及作用靶标等进行了广泛的研究。已经证实,昆虫的抗药性与行为改变、生理功能改变、解毒功能增强以及靶标不敏感性有关。近年来,随着分子生物学以及昆虫基因组学的发展,昆虫抗药性的分子机理有了突破性进展,已发现并克隆了一些靶标基因,与抗药性相关的基因突变也得到广泛验证。本文综述了昆虫的抗药性机理在分子生物学上的研究最新进展,重点阐述了与昆虫抗性相关基因的扩增、表达及基因结构的改变等相关内容。     

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。     

棉蚜啶虫脒抗性种群交互抗性和增效剂增效作用的研究

【目的】明确棉蚜Aphis gossypii Glover啶虫脒抗性品系与其它杀虫剂的交互抗性现状以及增效剂的增效作用,为延缓和治理棉蚜对啶虫脒的抗性提供依据。【方法】采用单头反选育和群体汰选的方式,获得了棉蚜啶虫脒敏感和抗性品系;采用叶片药膜法测定了13种杀虫剂对啶虫脒的交互抗性以及增效剂对啶虫脒的增效作用。【结果】经过室内棉蚜敏感和抗性品系的筛选,获得了相对抗性倍数为82.33倍的棉蚜啶虫脒抗性品系。棉蚜啶虫脒抗性品系的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为:吡蚜酮,甲基阿维菌素;交互抗性倍数在5~10倍的药剂为:噻虫嗪,联苯菊酯,毒死蜱,马拉硫磷,丙溴磷,辛硫磷;交互抗性倍数在10~15倍的药剂为:硫丹,阿维菌素,高效氯氰菊酯,三唑磷,氧化乐果;交互抗性倍数大于1 5倍的药剂为:吡虫啉。增效剂实验表明,TPP和PBO在啶虫脒敏感品系中增效作用不明显,但在抗性品系中增效作用显著。在啶虫脒抗性品系中的增效比为1.77、1.61,在啶虫脒敏感品系中的增效比为1.02、1.03。DEM在啶虫脒抗性、敏感品系中的增效作用均不明显,增效比为1.04、1.02。TPP和PBO对啶虫脒有很好的增效作用。以室内棉蚜敏感品系(LC_(50)为0.180 mg/L)为基础,对新疆各主要棉区的棉蚜种群进行了啶虫脒药剂的抗性调查,结果表明新疆各主要棉区棉蚜对啶虫脒的相对抗性倍数为6.1~22.0倍。【结论】由此说明新疆主要棉区棉蚜对啶虫脒具有一定的抗性风险,生产中可以利用无交互抗性的吡蚜酮和甲基阿维菌素来治理抗性棉蚜种群。     

德国小蠊抗药性机理

德国小蠊(Blattella germanica)是一种重要的室内卫生害虫,化学防治是目前主要的防治手段,但抗药性已逐渐成为制约德国小蠊防治的关键因素。德国小蠊抗药性的产生存在多种机理,本文从行为抗性、表皮穿透性降低、代谢抗性、靶标抗性及微生物降解等方面对德国小蠊抗药性产生机理进行了综述,重点阐述了近年来德国小蠊抗药性机理最新的研究进展。     

简易快速检测棉蚜对有机磷抗性的生化方法──滤纸酯酶反应法

棉蚜AphisgossypiiGlover是危害棉苗的主要害虫之一。有关棉蚜的抗药性研究已做了很多工作,先后报道了棉蚜对内吸磷、对硫磷、乐果等有机磷农药的抗药性[1－3]。近几年来,由于频繁过量连续使用农药,导致棉蚜的抗药性水平迅速上升。例如山东高密地区近几年使用有机磷农药防治棉蚜,造成抗性逐年递增。而对抗药性的监测目前主要是使用生物测定法和酶活性分析法。前者使用虫量大,点滴操作要求严格,不易掌握;后者操作条件要求苛刻,且需一定的仪器设备等条件,不易在基层单位推广。孙耘芹[2],郑炳宗[4]指出棉蚜对有机磷杀虫剂的抗性主要…     

亚洲稻区灰飞虱抗药性研究进展

灰飞虱Laodelphax striatellus(Fallen)是亚洲稻区的一种重要传毒昆虫。20世纪60年代以来,灰飞虱先后对有机氯、有机磷以及氨基甲酸酯类杀虫剂产生了不同程度的抗药性,其抗性机理主要涉及解毒代谢增强和靶标敏感性下降。随着吡虫啉等新型药剂使用面积的扩大,加强抗性监测,制定科学合理的抗性治理策略势在必行。     

抗吡虫啉棉蚜种群对啶虫脒和噻虫胺的交互抗性及相关酶学机理

【目的】通过对乙酰胆碱受体β1亚基突变后的抗吡虫啉棉蚜Aphis gossypii (Glover)种群的继续筛选, 明确该种群的抗性发展规律以及对其他新烟碱类杀虫剂啶虫脒和噻虫胺的交互抗性及相关酶学机理。【方法】采用浸渍法连续对抗吡虫啉棉蚜进行室内筛选、 测定噻虫胺和啶虫脒对抗吡虫啉棉蚜种群的毒力; 选择LC₂₀剂量吡虫啉、 啶虫脒和噻虫胺处理抗性棉蚜, 采用生化分析法测定其体内羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶的活性变化, 并观察其生物学特性的变化。【结果】本研究对抗性棉蚜突变种群用吡虫啉继续筛选至75代, 抗性倍数达到72.6倍, RF75停止用药筛选12代（RF₇₅₊₁₂）, 抗性仍达72.0倍。且RF₇₅₊₁₂对噻虫胺和啶虫脒的交互抗性可分别达11.9倍和20.1倍。噻虫胺对抗吡虫啉棉蚜的蜜露分泌和体重的抑制作用均大于吡虫啉和啶虫脒。噻虫胺对RF₇₅₊₁₂的羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶均具有明显的抑制作用, 而啶虫脒的抑制作用较小。【结论】结果表明乙酰胆碱受体基因突变棉蚜种群对吡虫啉的抗性水平不仅升高, 且停止用药后其抗性可稳定遗传; 第二代新烟碱类的噻虫胺在抗吡虫啉棉蚜靶标突变种群的治理中具有较大的应用价值。     

害虫抗药性进化的遗传起源与分子机制

根据生物进化理论深入理解害虫抗药性进化的遗传起源,并根据解释基因新功能进化的基因重复理论,推测认为基因重复为抗性基因变异提供了原材料。最后,根据现有抗性报道的例子将抗性突变的分子机制进行归类,并发现在多样化的抗性突变中存在一定的规律性,如靶标位点的点突变导致抗性的机制是靶标抗性机制的主要形式,基因扩增或基因过表达导致的代谢酶活性增加是代谢抗性的重要机制,这种规律性与变异的适合度密切相关。     

PCR detection of pirimicarb resistance in Australian field isolates of Aphis gossypii Glover (Aphididae: Hemiptera)

Aphis gossypii Glover (cotton aphid) is a major secondary pest of Australian cotton that readily develops resistance to the carbamate insecticide pirimicarb (Pirimor®) and to organophosphates generally. To test the pirimicarb resistance status of Australian strains of A . gossypii , a polymerase chain reaction (PCR) assay followed by restriction enzyme assay (REA) was designed to identify the Ace I polymorphism S431F known to be responsible for resistance. The method was tested against reference and 33 field strains collected over two consecutive seasons. Both methods confirmed pirimicarb resistance in two field strains, one from each cotton season, giving credence to the molecular technique described. The PCR assay proved specific for the Ace I gene. This PCR REA assay has the potential to replace bioassay for the routine pirimicarb resistance monitoring in A . gossypii. With the molecular assay providing results in 48 h, compared with 4–8 weeks for bioassay, such an assay could be used before insecticide control.     

辽宁棉区棉蚜抗药性监测与治理抗性研究

应用FAO推荐的微量毛细管点滴法,测定了棉蚜对六种常用杀虫剂的LD50,并与敏感蚜比较。得出抗药性倍数。棉蚜对有机磷杀虫剂的抗药性,增加缓慢,从1960年至今增加了500～700倍。而对于拟菊酯类杀虫剂的抗性增加较快,仅3～4年就达到667。7～9937．4倍。每年增加1.5～2倍。本项研究还应用棉蚜的防治指标和综合治理措施,对抗性棉蚜进行了治理抗性的试验研究。     

华北棉区主要害虫抗药性监测与治理技术示范

【目的】华北地区转基因Bt棉大面积种植后,刺吸式口器害虫已成为棉花主要害虫。本研究旨在监测明确棉花主要害虫对田间常用杀虫剂抗性水平变化,以指导田间合理用药。【方法】2013-2015年分别采用叶片药膜法和点滴法系统监测了河北省邱县、山东省滨州市、河南省西华县棉铃虫Helicoverpa armigera、棉蚜Aphis gossypii、绿盲蝽Apolygus lucorum对常用杀虫药剂的抗性动态变化情况。【结果】棉铃虫对辛硫磷、高效氯氟氰菊酯抗性呈上升趋势,抗性倍数都在20倍以上,对甲氨基阿维菌素苯甲酸盐从敏感状态转为中等水平抗性,抗性倍数在10倍以上。棉蚜对杀虫剂抗性水平都比较高,特别是对氧化乐果、高效氯氰菊酯、吡虫啉都已产生了高水平抗性,抗性倍数都在100倍以上。绿盲蝽对吡虫啉从敏感状态转为中等水平抗性,抗性倍数在10倍以上,对马拉硫磷、灭多威等药剂抗性倍数还维持在10倍以下,对高效氯氟氰菊酯只监测到滨州种群产生了中等水平的抗性,抗性倍数达到了95倍。在明确棉花害虫抗药性水平的基础上,对山东滨州棉花害虫实施了以轮换用药为主的抗性治理示范,示范区比农户自防对照区减少3次用药,增加棉花产量7.53%,节本增收109.16元,取得了较好的示范效果和经济效益。【结论】当前华北棉区主要害虫抗性水平上升,急需开展以轮换用药为主的抗性治理措施。     

Efficacy of new insecticides for management of Helicoverpa spp. (Lepidoptera: Noctuidae) in Australian grain crops

Abstract  Insecticide resistance in Helicoverpa armigera (Hübner) has led to the reduced efficacy of some older insecticide groups (pyrethroids and carbamates) and serious crop losses. Eight small-plot experiments were conducted to evaluate new insecticides for the management of H. armigera in grain crops. Several products showed efficacy equivalent to or better than the commercial standard, thiodicarb. Indoxacarb and spinosad at rates 50% or less of the registered rates for cotton were consistently superior to other tested products across the range of crops treated and provided residual protection for up to 14 d. The insect growth regulator compound, methoxyfenozide, was slower acting than other products tested, but demonstrated potential for H. armigera management. Pyridalyl performed well and warrants further evaluation in grain crops. We discuss the positioning of new compounds in an Insecticide Resistance Management Strategy (IRMS) in relation to a farming system that incorporates both grain and cotton crops. Use guidelines are recommended for indoxacarb, the first new compound to be registered in selected grain crops and cotton in Australia. These guidelines include restricted-use periods and limits on the number of applications per crop. It is anticipated that additional new compounds will be registered in grain crops, leading to the reduced selection pressure on the limited number of efficacious products. Coordinated insecticide use across farming systems and compatibility with developing integrated pest management programs should be fundamental considerations for the future IRMS.     

棉铃虫对氰戊菊酯等杀虫剂抗性的选育及其生化机理

从用药水平低的棉田采集棉铃虫Helicover pa armigera(Hubner),在室内恒温条件下以人工饲料饲养,用氰戊菊酯等4种杀虫剂和1种混合剂经点滴法分别逐代处理棉铃虫幼虫,以选育其抗药性。用氰戊菊酯选择15代,抗性达311倍;而用灭多威、甲基对硫磷和辛硫磷分别选择13代、14代及13代,抗性仅分别达10.8倍、3.5倍及5.2倍,抗性发展较慢;甲基对硫磷与辛硫磷的混合剂选择12代,抗性也只有4.8倍。氰戊菊酯和溴氰菊酯、三氟氯氰菊酯间存在明显的交互抗性。用生测法测定酶抑制剂和生化法测定酶活性的结果表明,棉铃虫对氰戊菊酯的抗性主要与幼虫体内多功能氧化酶和羧酸酯酶的活性提高有关。     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

西花蓟马的抗药性及其治理策略

西花蓟马Frankliniella occidentalis（Pergande）是世界性的园艺作物上的重要害虫,几乎对每种类型的杀虫剂均产生了抗药性,包括有机磷、氨基甲酸酯、拟除虫菊酯类杀虫剂和多杀菌素等。本文对国外西花蓟马的抗药性发展现状和抗性机制进行了总结,并提出了抗性治理策略,即科学合理使用杀虫剂,结合栽培防治、物理防治、生物防治和寄主植物抗性等方式降低杀虫剂对西花蓟马的选择压,从而达到抗性治理的目的。     

棉铃虫对Bt杀虫剂的抗性遗传方式

随着转基因棉花种植面积的日益增加,棉铃虫Helicoverpa armigera（Hübner)对Bt的抗性已经成为一个不容忽视的问题。发展转多价基因作物是当前缓解害虫对Bt抗性的最有效措施。本研究以经室内多年筛选的、抗性倍数达2 000多倍的Bt杀虫剂（含多种蛋白）抗性品系为材料,通过生物测定和不同的杂交试验,测定棉铃虫对Bt杀虫剂的抗性遗传方式,以期为Bt生物农药的抗性治理提供一定的依据,同时为制定棉铃虫对转多基因作物的抗性治理策略提供一定的参考。对敏感亲本和抗性亲本杂交产生的F1代的研究结果表明,杂交品系的抗性倍数分别为22.2倍和24.6倍;抗性显性度D值均小于0,分别为-0.20和-0.17,抗性为常染色体不完全隐性遗传。对4种回交后代和2种自交后代F2的研究结果表明,实际死亡率与期望死亡率差异较大,说明抗性是由单基因多个位点或多基因控制。     

Genetic diversity of the cotton aphid Aphis gossypii in the unstable environment of a cotton growing area

1 Spatial and temporal habitat heterogeneity represented by annual crops is a major factor influencing population dynamics of phytophagous insect pests such as the cotton aphid Aphis gossypii Glover. We studied the effects of instability of the cotton agroecosystem resulting from the temporary availability of the plant resource and the repeated use of insecticides on the genetic variability of the cotton aphids.
2 Samples of A. gossypii were collected in cotton plots, treated or not with insecticides and from vegetable crops (Malvaceae, Cucurbitaceae and Solanaceae) within the cotton growing area of northern Cameroon. The genetic structure of the samples was assessed using eight microsatellite markers. Insecticide resistance was estimated through the detection of two mutations in the ace -1 gene that are associated with insensitivity of acetylcholinesterase to carbamate and organophosphate insecticides.
3 The results obtained show that both host plants and insecticides act in genetic structuring of A. gossypii . Ninety-three percent of aphids collected on cotton were characterized by the same microsatellite multilocus genotype, Burk1 , which also displays the insecticide resistant alleles.
4 During the dry season, the cotton crop season after, the genotype Burk1 was principally found on two other malvaceous cultivated plants, rosella and okra, acting as suitable reservoir plants. The ability of the cotton aphid to move among asynchronous suitable habitats in response to changes in resource availability enables the pest to exploit unstable cropping systems. An understanding of the cotton aphid life system may aid to improve strategies for integrated resistance management.     

Screening of broad spectrum natural pesticides against conserved target arginine kinase in cotton pests by molecular modeling

Cotton is an economically important crop and its production is challenged by the diversity of pests and related insecticide resistance. Identification of the conserved target across the cotton pest will help to design broad spectrum insecticide. In this study, we have identified conserved sequences by Expressed Sequence Tag profiling from three cotton pests namely Aphis gossypii, Helicoverpa armigera, and Spodoptera exigua. One target protein arginine kinase having a key role in insect physiology and energy metabolism was studied further using homology modeling, virtual screening, molecular docking, and molecular dynamics simulation to identify potential biopesticide compounds from the Zinc natural database. We have identified four compounds having excellent inhibitor potential against the identified broad spectrum target which are highly specific to invertebrates.