二斑叶螨对甲氰菊酯的抗性选育及解毒酶活力变化

为了明确二斑叶螨Tetranychus urticae Koch对甲氰菊酯产生抗性的机理,在室内模拟田间药剂的选择压力, 用甲氰菊酯对二斑叶螨敏感品系（S）进行逐代汰选,选育至38代时, 获得了抗性倍数( resistance ratio, RR)为247.35的抗甲氰菊酯品系（Fe-R）。对S和Fe-R解毒酶活性的分析表明,Fe-R₃₈体内羧酸酯酶（carboxylesterase, CarE）、酸性磷酸酯酶(acid phosphatase, ACP)、 碱性磷酸酯酶(alkaline phosphatase, ALP)、谷胱甘肽-S-转移酶(glutathione s-transferase, GSTs)和多功能氧化酶(mixed function oxidase, MFO)较S体内相应酶的活力显著升高（P< 0.05）,其相对比值（R/S）分别为1.822,13.941,3.789,4.262和17.386。此外,筛选至第9,19,25,32代时,除Fe-R₂₅和Fe-R₃₂的MFO活性与S相比有显著性差异（P< 0.05）外,其余解毒酶（CarE,ACP,ALP,GSTs）的活性与S相比均无显著性差异(P>0.05)。筛选至第38代时, 5种解毒酶的活力与S相比均差异显著(P<0.05)。结果说明二斑叶螨Fe-R随着筛选代数的增加（第25代后）,MFO活性的上升可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

阿维菌素对棉铃虫的抗性选育及其对解毒酶活性的影响

为了了解棉铃虫对阿维菌素的抗药性机理,用阿维菌素对棉铃虫(Heliothis armigera)进行了抗性选育,并对选育的27代棉铃虫解毒酶的活性进行了检测.选育从F0至F21代,抗性缓慢波动上升,达到选育前的121.19倍;F0至F27代抗性迅速增长,达到选育前的821.73倍,抗性发展趋势呈现S型曲线.随着选育代数的增加,对乙酰胆碱酯酶(AChE)没有明显的影响;羧酸酯酶(CarE)活性,F27是F0的2.52倍(α-NA)和2.83倍(β- NA),从20开始,活性在较高水平上波动;谷胱甘肽转移酶(GST)活性,F27是F0的2.12倍,且从F16开始,活性在较高水平上波动.选育的抗性品系,增效醚对阿维菌素增效6.77倍.上述结果说明棉铃虫对abamectin的抗性机制也涉及MFO、GSTs、CarE等多个酶系统,但与乙酰胆碱酯酶(AChE)无关.     

截形叶螨抗哒螨灵品系和敏感品系体内解毒酶活性的变化

【目的】通过对截形叶螨 Tetranychus truncatus Ehara抗哒螨灵品系体内解毒酶活性分析和增效剂与哒螨灵混用的增效作用测定,明确截形叶螨对哒螨灵的抗性动态及抗性机理,获得抗性治理的途径。【方法】采用室内生测法培育截形叶螨抗哒螨灵品系,微量滴度酶标板测定抗性和敏感品系体内解毒酶比活性、米氏常数（K_m）及最大反应速度（V_max）,再用增效醚（PBO）、顺丁烯二酸二乙酯（DEM）和磷酸三甲苯酯（TPP）进行增效作用测定。【结果】室内筛选的截形叶螨对哒螨灵产生了抗性,筛选至49代,抗性倍数达到955.25;PBO,TPP和DEM对哒螨灵药剂有不同程度的增效作用,相对增效系数分别为95.97%, 85.14%和97.37%;抗哒螨灵品系体内的羧酸酯酶（CarE）、谷胱甘肽转移酶（GSTs）、多功能氧化酶（MFO）活性较敏感品系显著性提高（P＜0.05）,酸性磷酸酯酶（ACP）和碱性磷酸酯酶（ALP）的活性与敏感品系差异不大(P>0.05);抗性品系中的CarE,GSTs和MFO 3种解毒酶的米氏常数（K_m）下降,最大反应速度（V_max）高于敏感品系。【结论】截形叶螨对哒螨灵产生抗性可能与其体内CarE,GSTs和MFO 3种解毒酶与底物的亲和力提高和代谢能力增强有关;3种增效剂(PBO, TPP和 DEM)与哒螨灵混用能提高对截形叶螨的毒杀效果。     

亚致死质量浓度联苯肼酯对截形叶螨解毒酶系的影响

【目的】明确联苯肼酯的亚致死质量浓度对截形叶螨Tetranychus truncatus解毒酶系的影响,从而为进一步揭示截形叶螨的代谢抗性及联苯肼酯的科学使用提供理论依据和指导。【方法】采用叶片浸渍法测定了联苯肼酯对截形叶螨雌成螨的LC50,应用毒力回归方程计算截形叶螨雌成螨死亡率为10%和30%时联苯肼酯的亚致死质量浓度LC10和LC30,并处理截形叶螨雌成螨,以甲醇水溶液作为对照,测定并分析其体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)、多功能氧化酶(MFO)的比活力及酶动力学常数(米氏常数Km和最大反应速率Vmax)的变化。【结果】比活力与对照相比,LC10、LC30处理后,截形叶螨雌成螨体内CarE、GSTs和MFO的比活力均显著升高(P0.05),且LC30处理组CarE、GSTs(除24 h外)比活力显著高于LC10(P0.05)。酶动力学常数:体内CarE的Km显著减小或无明显变化(P0.05),Vmax显著增大(P0.05),即CarE与底物亲和力增大,反应速率加快;而GSTs和MFO的Km显著增大或无明显变化(P0.05),Vmax均显著减小(P0.05),说明GSTs和MFO与底物亲和力低,反应速率降低。【结论】联苯肼酯对截形叶螨雌成螨体内的解毒酶系有诱导作用,CarE在截形叶螨对该杀螨剂的代谢中起主导作用。     

朱砂叶螨不同抗性品系酯酶同工酶研究

在室内模拟田间药剂的选择压力,用3种不同药剂及其组合(轮用和混用)对朱砂叶螨Tetranychus cinnabarinus 进行抗药性选育.经过40余代的筛选,朱砂叶螨对甲氰菊酯、阿维菌素和哒螨·阿维混剂分别产生了68.5、8.7和6.7倍的抗性;甲氰菊酯和阿维菌素混用和轮用分别对甲氰菊酯产生了5.6倍和28.7倍抗性.朱砂叶螨各品系酯酶同工酶电泳结果和同工酶谱谱带密度扫描表明,与敏感品系相比,各抗性筛选品系的酯酶活性均有不同程度增加;阿维菌素抗性品系活性最强,酯酶带迁移距离明显远于其他抗性品系,表明该品系酯酶体系中存在变构酯酶.     

二斑叶螨多重抗性品系最优内参基因的筛选及CYP392A亚家族基因的表达分析

【目的】筛选出二斑叶螨Tetranychus urticae Koch抗甲氰菊酯、阿维菌素及螺螨酯混剂的实时定量PCR最优内参基因。【方法】选取5.8S rRNA, α-tubulin, TBP, β-actin, ELFn, RPL13a, GAPDH和SDHA 8个候选内参基因,以GeNorm, BestKeeper和Normfinder 3个软件分析这8个基因在二斑叶螨多重抗性品系中的表达稳定性, 并以筛选的内参基因分析二斑叶螨P450酶系CYP392A亚家族基因的表达水平。【结果】经GeNorm, BestKeeper和Normfinder 3个软件综合评价确定ELFn基因为二斑叶螨敏感品系(susceptible strain, SS)和多重抗性品系(multi-pesticide resistant strain, Mp-R)各发育阶段的最优内参基因。以ELFn为内参基因对二斑叶螨CYP392A亚家族16个基因表达量进行分析,结果表明：经多重抗性选育40代后,Mp-R品系卵期CYP392A1表达量显著上调;CYP392A16基因在各发育阶段表达量极显著高于SS品系相应发育阶段;其他基因表达量在敏感品系和抗性品系之间差异不显著。【结论】筛选出了SS和Mp R品系中各发育阶段最佳内参基因为EFLn;Mp-R品系CYP392A亚家族16个基因的表达量在幼螨和若螨阶段低于卵与成螨阶段,其中CYP392A16基因在二斑叶螨多重抗性的形成中起主要作用。该结果为二斑叶螨多重抗性研究奠定了一定基础。     

柑橘全爪螨两个田间种群抗性监测及羧酸酯酶生化特性研究

采用叶碟浸渍法测定了重庆北碚和万州地区柑橘全爪螨Panonychus citri(McGregor)田间种群对阿维菌素、毒死蜱、甲氰菊酯和哒螨灵的抗性水平。结果表明,同室内敏感品系相比,北碚种群对毒死蜱、甲氰菊酯和哒螨灵的相对抗性水平分别达到3倍、3倍和22倍;万州种群对阿维菌素、毒死蜱、甲氰菊酯和哒螨灵的相对抗性水平分别达到2倍、35倍、10倍和2倍。柑橘全爪螨2个地理种群的羧酸酯酶CarE的生化特性研究发现,CarE酶活的增高和毒死蜱的抗性存在一定的相关性。毒死蜱对不同地理种群柑橘全爪螨CarE的抑制效果不同,对抗性倍数较高的万州种群抑制效果最差。     

不同地理区域桔小实蝇解毒酶系活性及其与抗药性水平关系

昆虫体内的解毒酶活性是反映其抗药性水平的主要生理指标，本文比较了桔小实蝇Bactrocera dorsalis的几种解毒酶活性和抗药性水平关系。测定了9个地理品系和相对敏感品系的桔小实蝇成虫的三种解毒酶，即多功能氧化酶(Mixed function oxidase, MFO)、羧酸酯酶（Carboxylesterase, CarE）和谷胱甘肽Ｓ-转移酶(Glutathione S-transferase, GST)的活性,利用药膜法测定其对敌百虫(Trichlorphon)、高效氯氰菊酯(β-cypermethrin)和阿维菌素(Avermectin)的抗性水平，比较了各地理品系的桔小实蝇这些酶活性和对不同杀虫剂的抗性水平的关系，并作通径分析。结果表明：广东广州地区桔小实蝇体内的MFO-O-脱甲基活性最高，为相对敏感品系酶活性的1.4782倍；而广东茂名地区品系酶活性最低，只有0.8649倍。广东惠州地区桔小实蝇体内CarE活性最高，为相对敏感品系酶活性1.8147倍；而广西南宁地区的桔小实蝇体内酶活性最低，为敏感品系的0.9636倍。广东茂名地区桔小实蝇GST活性最高，是相对敏感品系2.2557倍；而广东广州地区桔小实蝇GST活性最低，只有相对敏感品系的1.1622倍。抗性水平表明：各地理品系对敌百虫的抗性水平为相对敏感品系的2.1552倍至100.2271倍之间，对高效氯氰菊酯的抗性水平在1.0065到26.0026倍之间，对阿维菌素的抗性水平在2.3353倍至29.0688倍之间。相关性分析表明：桔小实蝇体内的GST活性和对敌百虫抗性水平的相关系数为0.41，存在显著性正相关；CarE活性与桔小实蝇对高效氯氰菊酯抗性水平存在极显著正相关，相关系数为0.50。通径分析结果表明：GST活性对敌百虫的抗药性水平的直接通径系数为0.4414，对敌百虫的抗性上升起到正向作用；MFO-O-脱甲基活性、CarE活性和GST活性对高效氯氰菊酯抗性水平的直接通径系数分别为0.3311，0.4946和0.1775，均起到正向作用；GST活性与阿维菌素的抗性水平的直接通径系数很小，为0.0668。结果显示了桔小实蝇的解毒酶与抗药性水平关系密切，在抗性发展中起到了促进作用。     

二斑叶螨对阿维菌素、哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化

在室内模拟田间药剂的选择压力,用阿维菌素、哒螨灵和甲氰菊酯对二斑叶螨Tetranychuc urticae逐代处理,以选育其抗性种群。选育至12代,对阿维菌素抗性增长到6.72倍,对哒螨灵抗性增长到12.1倍,对甲氰菊酯抗性增长到19.9倍。酶抑制剂和离体酶活性的测定结果表明,阿维菌素抗性种群的多功能氧化酶和谷胱甘肽S-转移酶的活性均有所提高;二斑叶螨对哒螨灵的抗性可能与多功能氧化酶、羧酸酯酶的活性增强有关;而羧酸酯酶、多功能氧化酶和谷胱甘肽S-转移酶活性的增强可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

阿维菌素对小菜蛾的抗性选育及其对解毒酶活性的影响

用阿维菌素对小菜蛾Plutella xylostella (L.)进行了抗性选育,并对选育过程中小菜蛾解毒酶的活性进行了研究。选育从F₀至F₂₁代,抗性缓慢波动上升,达到选育前的122.91倍;F₂₁至F₂₇代,抗性迅速增长,达到选育前的812.73倍,抗性发展趋势呈现S型曲线。随着选育代数的增加,对乙酰胆碱酯酶(AChE)没有明显的影响;羧酸酯酶(CarE)活性,F₂₇是F₀的1.5倍,从F₂₂开始,活性在较高水平上波动;谷胱甘肽转移酶(GST)活性F₂₇是F₀的2.2倍,且从F₁₈开始,活性在较高水平上波动。选育的抗性品系,增效醚对阿维菌素增效6.34倍。     

朱砂叶螨抗药性监测

本文采用药膜法建立了朱砂叶螨Tetranychus cinnabarinus（Boisduval）对5种杀螨剂的敏感基线,并对6个不同地理种群的朱砂叶螨进行了抗药性监测,结果表明：5种药剂杀螨活性由高到低分别为阿维菌素〉丁氟螨酯〉氧化乐果〉炔螨特〉甲氰菊酯,其对朱砂叶螨雌成螨的LC50值分别为0.08、2.19、67.89、201.19和605.27mg/L;朱砂叶螨各地理种群已对甲氰菊酯和炔螨特产生了低、中水平的抗性,其抗性倍数分别介于2.93～16.22与4.85～14.35之间,其中云南种群对这2种杀螨剂抗性最高,对氧化乐果与丁氟螨酯处于敏感性降低阶段,其抗性倍数分别介于2.35～4.26与1.56～2.11之间,对阿维菌素还未产生明显抗性;对阿维菌素和甲氰菊酯的增效剂生物测定结果表明,三类解毒酶系（多功能氧化酶、谷胱甘肽S-转移酶和酯酶）都不同程度地参与了朱砂叶螨抗药性的形成。     

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。     

Thiamethoxam Resistance in the House Fly,Musca domestica L.: Current Status,Resistance Selection,Cross-Resistance Potential and Possible Biochemical Mechanisms

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC₅₀ ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.     

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。     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Effect of rotational use of insecticides on pyrethroids resistance in Helicoverpa armigera (Lep.: Noctuidae)

Abstract:  To investigate fluctuation in susceptibility to insecticides in the field, natural populations of Helicoverpa armigera were collected from the same field in the region of Multan, Pakistan in 2002 and 2003. The populations were examined against pyrethroids (viz. cypermethrin, esfenvalerate and fenpropathrin) and new insecticides (viz. spinosad, abamectin and indoxacarb). In 2002, the resistance ratio (RR) of cypermethrin and esfenvalerate was significantly higher than fenpropahrin compared with susceptible population. The susceptibility to cypermethrin, esfenvalerate and fenpropathrin increased significantly in 2003; however, the RR for cypermethrin was about half the RR (38) of esfenvalerate (101) and fenpropathrin (89). The toxicity of spinosad, abamectin and indoxacarb was identical in both years. In the field experiments, abamectin was more effective than other compounds tested whereas fenpropathrin, cypermethrin and esfenvalerate had similar toxicity. These results might have important implications in resistance management and suggest that the rotational use of spinosad, abamectin and indoxacarb could help to avoid the development of multiple resistant in H. armigera .