Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

不同抗性水平棉铃虫地理种群的基因交流对抗性演化的影响

采自我国长江流域棉区的棉铃虫Helicoverpa armigera(Hubner)四川简阳种群、湖北武穴种群、湖南衡阳种群、江苏南京种群和浙江杭州种群对辛硫磷、三氟氯氰菊酯和硫丹的抗性水平分别为8.05～38.80、2.86～290.00和5.87～19.83倍。抗性较高的简阳种群和较低的杭州种群正、反交F₁代对上述3种农药的抗性为13.16和16.69倍、10.43和19.00倍及12.42和10.21倍,抗性遗传的显性系数为-0.2220、-0.2147和0.0700,表明对辛硫磷、三氟氯氰菊酯抗性呈不完全隐性方式遗传,而硫丹抗性则为半隐性遗传。P1自交F2代对辛硫磷、三氟氯氰菊酯和硫丹的抗性水平分别为17.66、7.57和9.03倍,F₁代对杭州种群回交F₂代的抗性倍数分别为8.79、3.Oo和6.61。其结果,高抗区和低抗区棉铃虫种群的基因流动将导致对三种农药抗性水平明显下降。     

SUBSTRATE SPECIFICITY AND DEVELOPMENTAL CHANGES OF ACETYLCHOLINESTERASE IN COTTON BOLLWORM*

Abstract The substrate specificity and developmental changes of acetylcholinesterase (AChE) of cotton bollworm, Helicoverpa armigera Hübner, were investigated during 1991 to 1994. The insects were collected from Handan suburbs of Hebei Province and Guan County of Shandong Province. The results show that the specific activity and Michaelis constants (k_m) of AChE toward acetylthiocholine (ATCH) and acetyl-β-methyl-thiocholine (MeTCh) regularly varied with the developmental stage of cotton bollworm. The two peaks of the specific activity were observed respectively in the third instar and sixth instar of larvae. The specific activity of AChE in pupae was the lowest and that in heads of four-days moth was the highest in various developmental stages of cotton bollworms. The tendency of K_m and maximum velocity (V_max) was identical with the change of specific activity in the AChEs of cotton bollworm. The activation energy (Ea) of AChE toward MeTCh in pupae and adults was 3. 9–4. 3 times as much as that of, larvae in cotton bollworms from Handan of Hebei Province. It suggests that the spending energies of AChE for hydrolysing substrate are different in larva, pupa and adult. The optimum conditions of AChE reaction with ATCh in larvae were 50–100 mg of tissue weights for the amount of enzyme, 10–20 minutes for the reaction time, 35°C for the reaction temperature and 8. 0 for the reaction pH value.     

棉铃虫乙酰胆碱酯酶(AChE)的体躯分布及部分纯化

该文研究了棉铃虫Helicoverpa armigera乙酰胆碱酯酶(AChE)的体躯分布及部分纯化技术。在幼虫和成虫体内均以头部AChE活性最高;AChE在成虫和幼虫头、胸、腹部的活性分布分别为78.2%、3.9%、17.9%和90.3%、6.5%和3.2%。通过用Sepharose 4B和Sephadex G-200纯化后,AChE的最高纯化倍数为3.66和17.74倍。样品和洗脱液中含有Triton X-100时,纯化倍数明显高于无Triton X-100的处理组。     

棉铃虫乙酰胆碱酯酶的底物专一性和发育期变化(英)

通过对河北省邯郸地区和山东省冠县的棉铃虫(Helicoverpa armigera H(?)bner)乙酰胆碱酯酶(AChE)研究,结果表明,棉铃虫乙酰胆碱酯酶对乙酰硫代胆碱(ATCh)和乙酰-β-甲基硫代胆碱(MeTCh)的比活力以及米氏常数(K_(?))值随其发育阶段呈有规律的变化,AChE比活力在幼虫期呈现两个高峰,一个在三龄,另一个在化蛹前;在蛹期AChE比活力没有明显的变化,而且比活力值比较低;到成虫期第4天有一个明显的高峰,比活力值高于其它任何虫态。K_(?)和最大反应速度(V_(max))的变化趋势基本上与比活力一致。棉铃虫AChE的K_(?)和比活力随其生长发育阶段的周期性变化对于指导用有机磷和氨基甲酸酯类杀虫药剂的化学防治具有重要的意义。不同地点采集到的棉铃虫AChE对AICh和MeTCh水解的活化能有所不同,邯郸地区的棉铃虫AChE水解MeTCh的活化能,蛹和成虫是幼虫期的3.9-4.3倍,而冠县棉铃虫水解MeTCh的活化能则变化不大。AChE水解ATCh的活化能,邯郸地区棉铃虫的AChE不同虫态间变化不大,冠县棉铃虫AChE,幼虫和成虫期约是蛹期的4倍。这说明不同生长发育时期,棉铃虫AChE对底物的水解所消耗的能量是不同的。棉铃虫幼虫AChE的最适反应条件是酶量以重量计为50-100mg,反应时间为10-20min,反应温度为35℃,反应体系的pH值为8.0。     

江苏沿江棉区棉铃虫世代增殖规律及主害代预测技术

通过聚类分析 ,可将沿江棉区历年棉铃虫世代间的增殖状况划分为 4类 ,相应的主害代第 4代棉铃虫的发生类型依次属于迟发适生型、早发暴发型、基数 -天气制约型和基数制约型。分析各类成因发现 ,3代基数是影响 4代发生量的主要因子。运用逐步回归分析 ,组建了 4代棉铃虫发生期和发生量的预测模型     

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。     

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.     

棉铃虫抗药性基因频率的测定(英文)

本文介绍了用单个棉铃虫测定杀虫药剂抗性基因频率的灵敏方法。该技术是基于比较三个相等量单个棉铃虫的乙酰胆碱酯酶的活性,每个棉铃虫要用微量滴定板同时做3个试验（也可用Enppen－dorf管）（No．1－3）：1）不加抑制剂（No．1）;2）不加酶源（No．2）;3）加一定浓度的残杀威以抑制编码敏感AceS等位基因的乙酰胆碱酯酶,但不能抑制编码抗性AceR等位基因的乙酰胆碱酯酶。如果No．1和No．3的强度一样强,说明残杀威对乙酰胆碱酯酶的活性没有影响,那么基因型应是纯合抗性（AceRR,No3＝No．1）。No．2和地．3的强度一样弱时,说明残杀威完全抑制了乙酰碱酯酶的活性,那么基因型应是纯合敏感（AceSS,No．3＝No．2）。No．3的强度介于No．1（强）和No．2（弱）之间显示出残杀威部分地抑制了乙酰胆碱酯酶的活性;基因型是杂合型（AceSR,No．2＜No.3＜No．1）。作者用该方法测定了1995,1996两年从河北省邯郸和固安县棉田采集的棉铃虫,在邯郸棉铃虫抗药性高发地区,其抗性基因频率AceRR分别是68．3％和65．3％;但在棉铃虫抗药性低发区的固安县其抗性基因频率分别是23．9％和29．4％。     

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。     

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。     

中国棉铃虫P450家族的CYP6B2基因与抗药性关系

运用ＲＴ－ＰＣＲ技术从棉铃虫五龄幼虫扩增到一个６２７ｂｐ的ＤＮＡ片段,测序表明这一片段为ＣＹＰ６Ｂ２基因的功能保守区,以这一片段为探针对棉铃虫除除虫菊酯抗性（Ｒ）及敏感（Ｓ）品系的总ＲＮＡ进行点杂交。ＲＮＡ点杂交表明未经氰戊菊酯诱导的情况下Ｒ品系的ＣＹＰ６Ｂ２基因的ｍＲＮＡ水平明显高于Ｓ品系,即为转录水平的调控,而经氰戊菊酯诱导后杂交表明Ｓ品系明显０被氰戊菊酯诱导,但Ｒ品系不被诱导。由此推测,棉铃     

RESISTANCE ALLEVIATION IN THE LARVAL COTTON BOLLWORM TO FENVALERATE AFTER PRE-TREATMENT WITH BACILLUS THURINGIENSIS

Abstract  Resistance alleviation and mechanism in the 2nd instar larvae of one susceptible and two resistant strains of the cotton bollworm, Helicoverpa armigera to fenvalerate were studied at 24 h after the larvae were treated with Bacillus thuringiensis Berliner ( B.t. ) preparation. The results showed that responses of larvae with B. t pretreatment to fenvalerate were much more sensitive than those without B. t pretreatment. Compared with the susceptible strain (YZ-S), net ratios between LD₅₀s with and without B. t pretreatment in the two resistant strains increased approximate 62. 6% and 80. 9%, respectively) inhibition of specific inhibitors to esterases displayed that efficacy of malaoxon and paraoxon methyl to acetyl-cholinesterase increased from 66.1% to 99. 8% in vitro and in vivo ; dynamic factors of acetylcholinesterase were significantly changed, in which K _m and V _max values decreased from 45. 58% to 68. 62% and K _i values of malaoxon and paraoxon methyl to acethylcholinesterase increased approximate 60%. It suggested that the sensitivity of the 2nd instar larvae of resistant H. armigera to fenvalerate might increase after B. t pretreatment for 24 h, and change of acetylcholinesterase was an important factor during resistance alleviation by B. t pretreatment. The research showed that it is practicable to bring the coordinated use of B. t and chemical insecticides in IPM system.     

杀虫药剂和植物次生性物质对棉铃虫羧酸酯酶的诱导作用

棉铃虫Helzcoverpa armigera中肠羧酸酯酶(CarEs)比活力随幼虫发育阶段呈有规律的变化。5日龄以前CarEs比活力增长比较缓慢,5日龄以后Cares比活力快速增长,8日龄开始CarEs比活力下降,直到预蛹期,CarEs比活力又开始上升。用聚丙烯酰胺凝胶电泳分离到的9条同功酶带中,E2、E4、E5、E6、E7和E9对β-乙酸萘酯(β-NA)的水解活性高于对a—乙酸萘酯(a-NA),E1、E3、和E8对上述两种底物的水解活性正好相反,对a-NA活性高于对β-NA。低剂量的对硫磷、马拉硫磷、倍硫磷、增效磷(SVl)、灭多威和溴氰菊酯对棉铃虫中肠CarEs比活力和对底物的亲和力具有明显的影响。药剂处理后48 h,Cares的比活力明显降低,对底物的亲和力则与药剂的种类有关。马拉硫磷处理后48h,在观察到的6条主要同功酶带中,E3的活性明显降低,其次是E6、E2和E1;而E5的活性则明显升高,斟的活性也有所升高。用含0.01%的芸香苷、2-十三烷酮和槲皮素的人工饲料饲养棉铃虫经2代或7代后,CarEs比活力均明显升高。     

江苏棉区第5代棉铃虫种群动态的模拟及预测

用澳大利亚热带害虫研究中心开发的通用制模软件Dymex为外壳,以棉铃虫Helicoverpa armigera种群生命表为基础,构建了棉铃虫种群动态模型,以逐日历史气温统计值为驱动变量进行了第4、5代棉铃虫种群动态的模拟。结果表明,除早发高温的年份外,其它年份的绝大多数第5代棉铃虫在江苏棉区11月底前不能发育至蛹而无法安全越冬,故不能成为翌年第1代的有效虫源。根据模拟结果可预测第4代越冬蛹量及第5代卵的发生期、发生量及其越冬蛹数量并制成了简便易用的预测检索表。     

棉铃虫种群适合度研究

本试验比较了我国各棉区棉铃虫Helicoverpa armzgcra(Hubner)种群幼虫期和蛹期发育速度与温度的关系,并模拟了长江流域种群和华北种群在北京地区的发育动态。研究结果表明在20℃～30℃温区内不同地理种群之间的发育速度无明显的感温分化现象,但在自然条件下长江流域种群和华北地区种群的蛹滞育动态则有显著的区别,如湖南衡阳种群和四川简阳种群50%蛹滞育的时间较北京种群晚30d和22d。对各地理种群发育动态、北京地区气候特点和滞育蛹越冬试验结果的综合分析表明,北京地区应为棉铃虫温带生态型生态区北界,棉铃虫亚热带生态型在华北气候环境下不能完成年生活史。     

苏云金杆菌预处理棉铃虫二龄幼虫对氰戊菊酯抗性缓解及机理研究(英文)

本文研究了Ｂ．ｔ预处理棉铃虫二龄幼虫对氰戊菊酯抗性缓解及其机理。结果表明,Ｂ．ｔ预处理后的幼虫对氰戊菊酯的敏感性提高。与敏感性种群相比较,在Ｂ．ｔ预处理和未处理两个抗性种群的幼虫对氰戊菊酯的ＬＤ_５０之间纯比值分别大约增加了６２．６％和８０．９％。特异性酶抑制剂对乙酰胆碱酯酶的抑制作用显示,在活体内,马拉氧磷和甲基对氧磷对乙酰胆碱酯酶抑制效率提高了６６．１％和９９．８％;在活体外抑制效率（Ｉ_５０） ６０％和６７．５％及５５．６５％和８６．４％。同时,乙酰胆碱酯酶动力学各因子变化显著。在Ｂ．ｔ预处理的抗性棉铃虫幼虫中,Ｋｍ值从４５．５８％减少到６８．６２％;Ｋｉ值分别增加了２１．２％和４０．９％及３８．２％和６１．５％。这些结果证实Ｂ．ｔ预处理２４ｈ抗性棉铃虫二龄幼虫对氰戊菊酯的敏感性提高,乙酸胆碱酯酶的变化是重要原因之一。这一研究为在ＩＰＭ系统中将微生物杀虫剂和化学杀虫剂协调使用缓解棉铃虫对拟除虫菊酯类杀虫剂的抗性提供理论基础。