小菜蛾对杀虫双和杀螟丹抗性的生化遗传分析

在对室内杀虫双和杀螟丹继代选择过程中小菜蛾的抗性发展变化、抗性生化机制、抗性遗传方式和抗性风险评估的基础上,笔者测定了小菜蛾抗杀虫双品系、抗杀螟丹品系和敏感品系及其杂交后代的生物学特性以及蛹的酶活性,通过t值检验酶活性间的差异显著性,从生物统计学和遗传学角度分析酶活性与抗性之间的关系。 1 材料与方法     

Mariner转座子与小菜蛾抗性关系的研究

以抗溴氰菊酯、杀虫双、杀螟丹小菜蛾种群及其敏感品系为研究对象,借助聚合酶链式反应(PCR)、载体筛选、琼脂糖凝胶电泳等实验技术,检测了小菜蛾4个品系的Mariner转座子的存在及其与抗性的关系。结果表明,在抗溴氰菊酯、杀虫双、杀螟丹以及敏感品系的小菜蛾中都存在两种大约500 bp的Mariner转座子片断,初次证明在小菜蛾4个品系中均含有两种Mariner转座子,并发现一种新的转座子基因片断,但是没有发现Mariner转座子与抗性之间存在直接的联系。研究结果将为利用Mariner转座子标签法在小菜蛾中分离定位基因、转化外源基因、转基因昆虫防治害虫等研究提供理论基础。     

小菜蛾溴氰菊酯抗性和敏感种群遗传差异的cDNARDA分析

寻找小菜蛾抗溴氰菊酯品系中差异表达基因,初步分析小菜蛾敏感品系与抗溴氰菊酯品系间的遗传差异.采用改进的cDNA代表性差异分析法,以小菜蛾抗溴氰菊酯品系的cDNA为Driver扩增子、敏感品系的cDNA为Tester扩增子,通过四轮消减杂交,获得1条200bp左右的差异扩增带.将此差异扩增带克隆于pMD18-T载体,随机挑选10个克隆测序.将所得的10个序列与GenBank等数据库作同源比较,发现仅有1个序列与已知泛素基因有较高同源性,但它与小菜蛾抗药性的关系尚未查明.     

小菜蛾对杀虫双的抗性遗传研究

利用室内选育的敏感品系和抗杀虫双品系为亲本,采用剂量对数—死亡机率值回归线(LD-P线)分析法,研究了小菜蛾对杀虫双的抗性遗传方式。结果表明,小菜蛾对杀虫双的抗性为多基因、常染色体遗传,正、反交F_1的显性度(D)值分别为0.39、0.28,即其主效基因为不完全显性。小菜蛾对杀虫双的抗性现实遗传力较低,h~2=0.052,产生抗性的速率较慢,室内选育119代,抗性仅达122.8倍。抗杀虫双品系和遗传杂交后代(F_1、F_2、BC)对拟除虫菊酯类、氨基甲酸酯类、有机磷类的代表杀虫剂溴氰菊酯、灭多威、敌敌畏等的交互抗性测定结果表明,它们对3种杀虫剂无交互抗性;亲本和杂交后代的多功能氧化酶环氧化活性与杀虫双的抗性水平呈正相关性;乙酰胆碱酯酶活性要比敏感品系低;羧酸酯酶活性与敏感品系无明显差异。     

小菜蛾对溴氢菊酯抗药性的随机扩增多态性DNA分析

利用室内选育119代的抗溴氰菊酯小菜蛾Plutella xylostella品系和敏感品系，通过反复回交和自交建立近等基因系。用Operon公司合成的20个引物对近等基因系基因组DNA进行PCR扩增。3个引物在抗性品系中分别产生1条特异扩增带，4个引物在敏感品系中分别产生了1～5条特异扩增带。由于在近等基因系中，除了与抗性相关的基因区外，其它的遗传背景是相同的，因此可以认为这些特异带与小菜蛾对溴氰菊酯的抗性有关。     

小菜蛾对溴氰菊酯抗性选育及其机理

本研究用溴氰菊酯在室内以点滴法处理小菜蛾４龄幼虫，连续继代药剂淘汰选育其抗药性，至Ｆ６５代，抗药性提高到１１６３倍，已形成高抗生品系。其抗性的形成发展趋势为前期相对缓慢，中期较快，后期迅速增长。于１９９２、１９９３、１９９４年分别以氯氰菊酯、敌敌畏、杀虫双、灭虫剂有明显 正交互抗性，对其它非菊酯类杀虫虫没有产生交互抗性。用聚丙烯酰胺凝胶电泳地（ＰＡＧＥ）测定表明，小菜蛾对溴氰菊酯抗性的产生可能与非     

武汉地区小菜蛾对溴氰菊酯的抗性回复及交互抗性

与小菜蛾敏感品系相比,武汉市张家湾小菜蛾田间种群的F₁、F₄和F₇,代对溴氰菊酯的抗性指数分别为362.79、30.78和10.91倍,抗性回复显著。用浸叶法测定Bt、抑太保和杀虫双制剂对两个小菜蛾种群的毒力,证明小菜蛾对这3类药剂与溴氰菊酯间无交互抗性。研究结果可为小菜蛾抗药性治理方案的制订提供依据。     

小菜蛾对杀虫药剂敏感品系的选育

通过单配选育的小菜蛾敏感品系对敌敌畏、灭多威、溴氰菊酯和杀虫双的敏感水平达到了选育目标,对4个药剂的敏感度LD_(50)值分别为0.0786、0.0356、0.0222和1.8949μg/头,并在生物学特性上与亲本无差异。该敏感品系可作为小菜蛾抗药性研究和新农药筛选的重要材料。     

小菜蛾对阿维菌素的抗性遗传分析及交互抗性研究

利用室内选育的抗阿维菌素小菜蛾品系和敏感品系分析了小菜蛾对阿维菌素的抗性遗传。结果表明,小菜蛾对阿维菌素的抗性为常染色体、不完全隐性遗传,而且可能是由多基因控制的抗性遗传。阿维菌素抗性品系对4 种杀虫剂的抗性谱测定结果表明,对马拉硫磷、溴氰菊酯、灭多威和农梦特无交互抗性。     

小菜蛾对溴氰菊酯的抗性消退与抗性恢复动态

利用选育的抗溴氰菊酯小菜蛾 Plutella xylostella L. 品系第100代为虫源,在不接触任何杀虫剂条件下继代繁殖50代,观察了小菜蛾对溴氰菊酯的抗性消退动态;同时对消退过程中的小菜蛾施加溴氰菊酯选择压力和利用消退过程中的小菜蛾与抗性品系、非抗性品系杂交,观察了小菜蛾对溴氰菊酯的抗性恢复动态.结果表明:①小菜蛾对溴氰菊酯的抗性消退表现为1～15代较慢,15～25代较快,25代后又缓慢的趋势,抗性消退也使氯菊酯、氯氰菊酯交互抗性水平下降;②溴氰菊酯抗性消退过程中,当连续受到一定程度的溴氰菊酯选择压力时,抗性会在几代之内很快恢复;③与其它品系杂交的后代受到一定剂量溴氰菊酯连续处理后,3～4代就能达到或接近抗溴氰菊酯品系第100代的抗性水平,对氯菊酯、氯氰菊酯的交互抗性水平也随之迅速上升.     

Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L) fumigated with phosphine

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.     

杀虫剂室内选育亚洲玉米螟抗药性及交互抗性的研究

用4种杀虫剂及1种混合剂以点滴法分别逐代处理敏感性亚洲玉米螟五龄幼虫,以选育其抗药性。至30代,该虫对速灭杀丁及呋喃丹产生_的抗性分别达40.3及30.2倍,而后经5—6代正常饲养,其抗性虽有所衰退,但仍明显,可认为已成为抗性品系;而用甲基对硫磷、甲基对硫磷+速灭杀丁混合剂及辛硫磷(24代)处理,其抗性仅分别达1.8、3.4及4.0倍,由于获得抗性不明显,将此三种试虫称之选育品系。抗性品系对多种药剂存在着明显的交互抗性;而选育品系对其选育剂的抗性虽不明显,但它却对另外的有些药剂产生了抗性。     

小菜蛾抗多杀菌素和敏感品系耐低温能力的比较

通过杂交筛选得到小菜蛾对多杀菌素敏感和高抗的纯合子品系，对二个品系在适温和经低温处理后的生长发育特性进行了比较，以探明其抗性是否伴有适合度的变化以及适合度变化与温度的关系。结果发现:高抗品系的卵经6℃低温处理14 d，孵化出的幼虫仅有7.7％正常化蛹，显著低于敏感品系61.4％的正常化蛹率；经6℃低温处理28 d，高抗和敏感品系的卵孵化率分别为27％和75％，二者差异显著；高抗品系的蛹经 5℃低温处理7～14 d后，蛹的 发育历期显著延长，其子代卵的存活力也显著下降。在适温25℃下，抗性和敏感品系之间多项生物学特性均无显著差异或差异很小。这些结果表明，小菜蛾对多杀菌素的抗性伴随有耐低温能力的适合度代价。     

46份野生葡萄株系对霜霉病的抗性鉴定及其相关基因的表达

为明确不同野生葡萄株系对霜霉病的抗性差异,以18个种的46份野生葡萄株系为试材,采用叶盘法鉴定其对霜霉病的抗性,并利用实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)技术对部分关键基因进行定量分析,探讨其在不同抗病株系中的表达模式差异。结果表明,46份野生葡萄株系的病情指数为0～34.72,其中17份为感病株系,病情指数在25.93～34.72;21份为抗病株系,病情指数在5.32～24.35;5份圆叶葡萄株系均表现为免疫,病情指数为0.00;云南-元谋2、云南-2和木扎岭-3为高抗株系,病情指数分别为1.81、4.40和1.62。当被霜霉病菌侵染后,抗病株系和感病株系中的PAL、PR1、TLP和NPR1基因的诱导表达模式不同;与感病株系相比,抗病株系中的TLP、PR1和NPR1基因有强烈的诱导表达,PAL基因在感病株系比在抗病株系中表达量高。在免疫株系普莱德和高抗株系云南-元谋2中,NPR1与其它3个基因的表达模式差异最大;TLP在抗病株系蘡薁-林县中与其它3个基因的表达模式差异最大;在感病株系秋-嵩县中,NPR1与TLP表达模式相近,PAL和PR1表达模式相近。研究表明,在中国野生葡萄种质中,云南-元谋2、云南-2和木扎岭-3对霜霉病有良好的抗性,可作为抗病育种的原始材料;抗性基因可能在抗病株系中发挥着重要作用。     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Fipronil resistance mechanisms in the rice stem borer,Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Thiamethoxam acts as a target‐site synergist of spinosad in resistant strains of Frankliniella occidentalis

BACKGROUND: Previous studies have suggested that the resistance mechanism towards spinosad in Frankliniella occidentalis (Pergande) is an altered target site. Like the neonicotinoids, the spinosyns act on nicotinic acetylcholine receptors (nAChRs) in insects, but at a distinct site. The changes in nAChRs related to spinosad resistance in thrips might involve interaction with neonicotinoids. In this study, the efficacy of spinosad and neonicotinoids, alone and in combination, was evaluated in susceptible and spinosad‐resistant thrips strains. RESULTS: The neonicotinoids tested were imidacloprid, thiacloprid, acetamiprid, thiamethoxam and clothianidin. No cross‐resistance was shown between spinosad and any of the neonicotinoids. However, an increased toxicity was observed when a mixture of spinosad with thiamethoxam or clothianidin was tested. No synergism was found in the susceptible strains. The more spinosad‐resistant the thrips strain, the stronger was the synergism. CONCLUSION: Data suggest that spinosad and thiamethoxam may interact at the nAChRs in spinosad‐resistant thrips, facilitating enhanced insecticidal action. Copyright © 2012 Society of Chemical Industry     

Pyrethroid synergists suppress esterase-mediated resistance in Indian strains of the cotton bollworm, Helicoverpa armigera (Hübner)

The role of esterase in pyrethroid resistance was studied in the final larval instar of different strains of the cotton bollworm, Helicoverpa armigera. The resistant strains viz., Nagpur strain and the Delhi strain were found to have elevated midgut esterase activity in comparison to the susceptible strain. Nagpur strain and Delhi strain have 2.24 and 1.73-fold higher esterase activity, respectively, than that of the susceptible strain. The Native PAGE displayed important differences in the midgut esterase isozyme pattern between the susceptible and the pyrethroid-resistant strains. Out of the 10 esterase isozyme observed, susceptible strain lacked three bands, E2, E6 and E10 that were found in the resistant strains. The potency of the synergists piperonyl butoxide (PBO) and dihydrodillapiole (DDA) as esterase inhibitor were also studied both in vitro and in vivo. The in vitro results clearly show that both PBO and DDA inhibited esterase activity in the two resistant strains, while there was almost no esterase inhibition in the homogenate of the susceptible strain. The in vivo inhibition studies (topical application of PBO and DDA followed by biochemical analysis) illustrated that PBO- and DDA-esterase binding is rather slow and non permanent process. Esterase inhibition did not occur immediately after the synergist treatment but at 4 and 8 h post treatment in case of PBO and DDA, respectively. Native PAGE revealed that the in vivo esterase inhibition caused by both PBO and DDA was due to the binding of the synergist with the E6 isozyme which was not present in the susceptible strain.