艾氏剂环氧化酶及细胞色素P－450对小菜蛾抗药性发展的影响

本文对室内长期饲养的小菜蛾（Ｐｌｕｔｅｌｌａ　ｘｙｌｏｓｔｅｌｌａ　Ｌ．）敏感品系和田间采集的抗性种群体内的艾氏剂平氧化酶及细胞色素Ｐ－４５０进行了比较研究。结果证明,艾氏剂环氧化酶在感性和抗性小菜蛾间存在着量及质的差异。抗性种群的艾氏剂环氧化酶的Ｖｍａｘ和Ｋｍ值分别为感性品系的５．４％和６．５倍。抗性种群的细胞色素Ｐ－４５０的含量是感性品系的１．１—１．３倍。艾氏剂环氧化酶在量上及质上的差异及细胞色素Ｐ－４５０含量的提高是导致小菜蛾抗药性发生与发展的重要机制之一。而且质的差异较之量的差异可能起着更为重要的作用．     

棉铃虫的细胞色素P-450单加氧酶系与菊酯类杀虫剂抗药性的关系(英文)

细胞色素Ｐ－４５０单加氧酶系在昆虫抗药性中起重要作用。本文研究了棉铃虫抗性品系与敏感品系间细胞色素Ｐ４５０含量、对－硝基茴香醚Ｏ－脱甲基酶和艾氏剂环氧化酶的差异。结果表明：抗性品系中细胞色素Ｐ－４５０含量、对－硝基茴香醚Ｏ－脱甲基酶活性分别是敏感品系的１．７１倍和２．２１倍,而艾氏剂环氧化酶活性仅为１．３５倍。因此细胞色素Ｐ－４５０含量和对－硝基茴香醚Ｏ－脱甲基酶在棉铃虫抗菊酯品系中起重要作用。进一步讨论了不同抗性品系间抗性机制不同的原因。     

氰戊菊酯和苯巴比妥钠对敏感和抗性棉铃虫中肠多功能氧化酶系的诱导作用

用苯巴比妥钠（2mg/g）和氰戊菊酯（0.2mg/g）拌饲料处理,对敏感品系棉铃虫Helicoverpa armigera中肠的细胞色素P450和细胞色素c还原酶含量均具有明显的诱导作用（两者都使细胞色素P450含量提高了2.24倍,使细胞色素c还原酶的含量分别提高1.33和1.40倍）,但对细胞色素b5诱导作用不显著（仅为对照的1.23和1.15倍）;此外,苯巴比妥钠对敏感棉铃虫中肠的艾氏剂环氧化酶活性和甲氧试卤灵-O-脱甲基酶活性也有显著的诱导作用（分别提高了2.75和2.66倍）,但对7-乙氧香豆素-O-脱乙基酶活性没有诱导作用,而氰戊菊酯对敏感棉铃虫中肠的艾氏剂环氧化酶活性则有2.02倍的诱导作用。同一浓度的苯巴比妥钠和氰戊菊酯使抗性品系棉铃虫中肠的细胞色素P450含量分别提高1.21和1.15倍,使细胞色素c还原酶含量分别提高1.48和1.86倍（差异显著）,但是细胞色素b₅含量没有明显变化（分别为对照的1.15和0.98倍）;此外,氰戊菊酯能使抗性棉铃虫中肠的艾氏剂环氧化酶活性提高1.53倍,但苯巴比妥钠对该酶活性则有明显抑制作用。     

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。     

西花蓟马对吡虫啉抗性机制的研究

为了解大规模应用吡虫啉防治西花蓟马Frankliniella occidentalis(Pergande)的抗性风险,本文研究了吡虫啉与多杀菌素和啶虫咪的交互抗性并比较了多功能氧化酶系(mixed-function oxidases,MFO)的3个组分细胞色素P450、细胞色素b5、甲氧试卤灵O脱甲基酶(methoxyresorufin O-demethylase,MROD)在西花蓟马敏感品系(S)、抗吡虫啉品系(BK)和北京田间品系(BJ)中的含量以及活性。生物测定表明北京田间品系(BJ)对多杀菌素已经产生了较高抗性(RR50=35.38),而对啶虫咪(RR50=3.05)和吡虫啉(RR50=1.82)处于敏感,交互抗性研究结果表明西花蓟马对吡虫啉与多杀菌素(RR50=1.31)和啶虫咪(RR50=3.20)无交互抗性。生化研究表明:西花蓟马抗吡虫啉品系的3个组分含量均显著高于S品系西花蓟马的含量(P<0.05),其中细胞色素P450在是S品系WFT的4.5倍,细胞色素b5含量是S品系WFT的4.23倍,MROD活性是S品系WFT的5.99倍,表明多功能氧化酶系与西花蓟马对吡虫啉的抗药性密切相关。     

棉铃虫对氰戊菊酯-辛硫磷混剂的抗性演化及解毒酶活性变化

用氰戊菊酯-辛硫磷混剂（有效成分1∶10,简称氰-辛混剂）对棉铃虫Helicoverpa armigera室内品系(YS)进行16代的抗性选育,获得棉铃虫对氰-辛混剂的抗性品系（YS-FP）。YS-FP品系与YS品系相比,对氰-辛混剂的抗性为14.7倍,对其中的单剂氰戊菊酯和辛硫磷的抗性分别为2 170倍和3.1倍。随着筛选的进行,氰戊菊酯和辛硫磷之间的共毒系数在F₂代出现短暂的增加,然后逐渐降低,它们之间的互作由增效变为拮抗。交互抗性测定结果表明,YS-FP品系对氯氰菊酯、溴氰菊酯、三氟氯氰菊酯、三唑磷和灭多威产生了明显的交互抗性,对硫丹、多杀菌素和爱玛菌素没有产生交互抗性。YS-FP品系6龄幼虫中肠细胞色素P450氧化酶甲氧基香豆素O-脱甲基活性为YS品系的10倍,3龄幼虫谷胱甘肽S-转移酶和酯酶活性分别是YS品系的1.7倍（CDNB结合作用）和2.4倍（α-NA 酯酶水解作用）。氰-辛混剂的筛选导致了棉铃虫多种解毒酶活性的增加,特别是细胞色素P450氧化酶活性增强最为明显。本研究结果表明氰-辛混剂对棉铃虫的筛选导致了广谱的交互抗性和多种代谢抗性机理,并且两个单剂之间的互作由增效变为拮抗,因此氰 辛混剂在棉铃虫抗性治理中的作用是有限的和暂时的。     

棉铃虫6龄幼虫中肠与脂肪体微粒体P450酶系的比较

报道了棉铃虫6龄幼虫中肠和脂肪体微粒体P450酶系的组成与加单氧酶活性。与脂肪体微粒体相比,中肠策粒体具有更高的细胞色素P450,细胞色素b5和NADPH－细胞色素P450还原酶含量,表现出较高的艾氏剂环氧化酶和对－硝基苯甲醚O－脱甲基酶活性。SDS－PAGE电泳显示,中肠与脂肪体微粒体介于P450分子量范围内（45－60kDa)的蛋白图谱有所不同,反映出中肠和脂肪体微粒体蛋白组成存在差异。通过对－硝基苯甲醚O－脱甲基酶的动力学分析发现脂肪体微粒体的对－硝基苯甲醚O－脱甲基酶对底物有更强的亲和性,表明不同组织来源的P450同功酶存在质的不同。     

小菜蛾对三氟甲吡醚的抗性风险评价与抗性生化机制研究

【目的】明确小菜蛾Plutella xylostella(Linnaeus)对三氟甲吡醚的抗性风险和抗性生化机制,为三氟甲吡醚的合理使用提供科学依据。【方法】采用TabashnikMc Gaughey的阈性状分析方法评估小菜蛾对三氟甲吡醚的抗性风险;采用浸叶法测定增效剂(胡椒基丁醚、磷酸三苯酯和顺丁烯二酸二乙酯)对三氟甲吡醚的增效作用,通过酶动力学方法测定了小菜蛾对三氟甲吡醚抗性和敏感品系的谷胱甘肽-S-转移酶、酯酶和多功能氧化酶的活性。【结果】经过18代次筛选,小菜蛾种群对三氟甲吡醚的抗性水平上升至14.8倍,小菜蛾对三氟甲吡醚的抗性现实遗传力(h~2)为0.1558;当h~2=0.155 8时,在致死率为50%-90%的选择压力下,预计小菜蛾对三氟甲吡醚抗性增加10倍分别需要16.1-7.3代。在小菜蛾三氟甲吡醚抗性品系中,酯酶和多功能氧化酶比活力均显著高于敏感品系,分别为敏感品系的1.34倍和1.45倍;谷胱甘肽-S-转移酶比活力与敏感品系的无显著差异。抗性品系的酯酶抑制剂磷酸三苯酯和多功能氧化酶抑制剂胡椒基丁醚对三氟甲吡醚具有明显增效作用,增效倍数分别为1.21倍和1.43倍;谷胱甘肽-S-转移酶抑制剂顺丁烯二酸二乙酯对三氟甲吡醚没有明显增效作用。【结论】小菜蛾对三氟甲吡醚产生抗性的风险较大,酯酶和多功能氧化酶活性的升高可能是小菜蛾对三氟甲吡醚产生抗性的重要机制。     

棉铃虫不同发育阶段微粒体P450酶系组成和活性的比较

比较了棉铃虫Helicoverpa armigera 6龄幼虫、蛹、成虫微粒体P450单加氧酶系组成及其活性。P450含量在6龄幼虫中肠>（脂肪体=蛹）>成虫,NADPH-细胞色素还原酶在幼虫中肠>幼虫脂肪体>蛹>成虫;6龄幼虫脂肪体微粒体与蛹脂肪体微粒体P450含量相近,但NADPH-细胞色素还原酶活性前者是后者的4.2倍;成虫微粒体的细胞色素P450和NADPH细胞色素P450还原酶含量很低,几乎未检测出。用对-硝基苯甲醚和艾氏剂为底物测定P450酶系活性表明,与6龄幼虫相比,蛹和成虫具有极低的单加氧酶活性,其O-脱甲基酶活性未检出,艾氏剂环氧化酶活性比幼虫低2～3个数量级。     

小菜蛾对定虫隆的抗性生化机制初探

以定虫隆汰选的抗性指数为23.78倍抗性种群(CH-R),并以上海田间种群(SH-R,对定虫隆的抗性指数为9.16倍)作为参比种群,研究小菜蛾Plutella xylostlla(L.)对定虫隆的抗性机制,结果表明：CH—R种群多功能氧化酶O-脱甲基活力比相对敏感种群(SZ-S)提高1倍,而SH-R种群多功能氧化酶O-脱甲基活力虽有所提高,但与SZ-S种群相比差异并不显著;CH-R和SH-R种群羧酸酯酶比活力均明显高于Sz-S种群,分别为SZ-S种群的4.61和2.18倍,且CH-R种群的Km仅为SZ-S种群的1/20;酸性磷酸酯酶和碱性磷酸酯酶比活力种群间无明显差异;CH-R种群的几丁质酶活力降低48%,酚氧化酶活力降低60%。说明小菜蛾对定虫隆的抗性机制具有多因子性,多功能氧化酶解毒代谢能力的提高可能是主导抗性机制之一,羧酸酯酶、几丁质酶和酚氧化酶也参与了小菜蛾对定虫隆的抗性。     

CYTOCHROME P450 MONOOXYGENASES ASSOCIATED WITH PYRETHROID RESISTANCE IN HELICOVERPA ARMIGERA (HÜBNER)

Abstract  The relationship between the cytochrome P-450 (cytP⁴⁵⁰ monooxygenase and insecticide resistance mechanisms in Helicoverpa armigera was studied. The level of cytP450 and activity of p -ni-troanisole ( p -NA) O-demethylase in resistant strain were 1. 71 times and 2. 21 times respectively higher as compared with those of the susceptible strain. Aldrin epoxidase activity showed 1. 35 fold strain difference. Therefore aldrin epoxidase might not be important for H. armigera resistance mechanism, the level of cytP450 and activity of p -NA O-demethylase might play a vital role in H. armigera resistance mechanisms. The reason of different resistance mechanisms among different H. armigera resistant strains is discussed.     

Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

Genetics of cytochrome P450 in two insecticide-resistant strains of the housefly,Musca domestica L.

A susceptible strain of Musca domestica containing visible mutant markers on chromosomes II, III, and V was crossed with multiresistant R-Fc and R-diazinon strains. F₁ flies were backcrossed to the mutant parent, resultant progenies were isolated according to phenotype, and substrains were established. The level of resistance to diazinon, aldrin epoxidase activity, and cytochrome P450 difference spectra of microsomes from each substrain were measured. Titers of cytochrome P450, measured as CO spectra, as well as type I, type II, and type III cytochrome P450 substrate difference spectra were compared in microsomal preparations obtained from phenotypes containing vatious resistant chromosome combinations. In both resistant strains, high levels of cytochrome P450 were controlled by a gene(s) on chromosome II. In R-diazinon, qualitative spectral changes were also controlled by chromosome II, whereas in R-Fc both chromosomes II and V contributed to qualitative changes in cytochrome P450. Both quantitative and qualitative characteristics were intermediate in heterozygous flies, suggesting incomplete dominance for their inheritance. Findings are discussed in relation to known genetics of microsomal resistance to insecticides.     

不同地区小菜蛾种群羧酸酯酶的毒理学性质研究

在1995～1997年对湖北武汉、河北张家口地区小菜蛾Plutella xylostella(L.)种群的抗药性进行了研究。结果表明对阿维菌素的抗性和台湾敏感种群相比,武汉种群抗性为4.3倍,张家口种群抗性为1.8倍;对马拉硫磷的抗性武汉和张家口种群分别为2.2和2.9倍;对氟铃脲的抗性分别为3.2和0.5倍;对溴氰菊酯的抗性分别为2.4和1.7倍。对羧酸酯酶(Care)的研究结果表明,三个种群幼虫CarE对a-乙酸萘酯或β-乙酸萘酯(a或β-NA)水解活性差异显著,但成虫Care活性没有明显差异。武汉和张家口种群幼虫CarE对a-NA和β-NA的亲和力没有明显差异,但是武汉种群幼虫Care对底物的亲和力高于张家口种群。敏感品系Care对a—NA的亲和力明显高于对β-NA,相差约3倍。不同类型的抑制剂对小菜蛾幼虫CarE的抑制能力不同。增效磷和对氧磷对敏感品系CarE水解a-NA具有明显的抑制作用,分别比对武汉种群Care的抑制作用大4.577倍(SVl)和2.576倍(对氧磷)。     

棉铃虫对Bt生物农药早期抗性及与转Bt基因棉抗虫性的关系

用饲料感染法建立了棉铃虫Helicoverpa rmigera(Hubmer)敏感品系(SUS1)对Bt生物农药的敏感毒力基线和区分剂量,1995年测定了五省六县棉铃虫初孵幼虫对Bt生物农药的敏感性,结果表明：山东阳谷、河北邯郸、河南新乡、安徽萧县及江苏丰县棉铃虫已产生早期抗性,抗性个体百分率为5%～10%,与敏感品系相比,LC₅₀值稍有增加,但斜率b值明显变小;而江苏东台棉铃虫仍属敏感。这是国内外首次诊测到棉铃虫对Bt生物农药抗性。用棉叶喂饲法测定比较了转Bt基因棉花品系对不同种群棉铃虫的抗虫性效果,结果表明：用早期抗性的阳谷和新乡棉铃虫初孵幼虫接虫5d后平均死亡率较敏感品系下降16%～29%,说明棉铃虫对Bt农药与转Bt生物基因棉花品系间存在交互抗性。还讨论了Bt农药的抗性治理对策。     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.     

Differences in the cytochrome P-450S from resistant and susceptible house flies

A comparison of difference spectra formed with microsomal cytochrome P-450 from a susceptible and a resistant strain of house flies shows both quantitative and qualitative differences. The differences are similar to those observed for the same spectra between normal and phenobarbital- or 3-methylcholanthrene-treated mammals. The relationship of these findings to resistance, synergism, and cytochrome P-450 in the house fly is discussed.     

甜菜夜蛾抗高效氯氟氰菊酯近等基因系的构建

将甜菜夜蛾Spodoptera exigua抗性种群雄成虫与敏感种群雌成虫杂交,杂交后代再自交后,用高效氯氟氰菊酯杀死敏感和部分杂合个体的剂量(50 μg/mL)处理4龄幼虫,对1天后存活的幼虫再用250 μg/mL汰选,将存活幼虫发育成的雄成虫与敏感种群的雌成虫回交,再经自交和选育,如此循环6次,获得了甜菜夜蛾抗高效氯氟氰菊酯近等基因系。对该基因系构建过程中各代幼虫的抗性水平和相关生物学特性进行了监测和比较,结果表明获得的抗高效氯氟氰菊酯近等基因系抗性种群的抗药性仍保持较高水平;酯酶同工酶的电泳分析表明抗高效氯氟氰菊酯近等基因系抗性种群与敏感种群图谱相近,而与亲本抗性种群之间存在明显差异。