白纹伊蚊溴氰菊酯抗性和敏感品系羧酸酯酶性质比较

本文对白纹伊蚊Aedes albopictus溴氰菊酯抗性品系和敏感品系羧酸酯酶的生物化学性质进行了比较。白纹伊蚊抗性品系和敏感品系羧酸酯酶随底物浓度(α-乙酸萘酯或β-乙酸萘酯)的变化比活力变化趋势一致,但抗性品系对这2种底物的比活力均高于敏感品系,抗性品系羧酸酯酶的米氏常数和最大反应速度与敏感品系有显著差异。胆碱酯酶抑制剂测定结果表明,抗性品系羧酸酯酶对敌敌畏和磷酸三苯酯的敏感性高于敏感品系,对残杀威的敏感性低于敏感品系。2个品系羧酸酯酶对脱叶磷的敏感性差异不大。说明羧酸酯酶可能与白纹伊蚊对溴氰菊酯抗性有关。     

有机磷对抗拟除虫菊酯家蝇的毒力

测定敏感、抗溴氰菊酯(Del-R)、抗氯菊酯(2Cl-R)的家蝇品系对有机磷杀虫剂敌敌畏、辛硫磷及马拉硫磷的LD₅₀,α-乙酸萘酯（α-NA）酯酶动力学,酯酶的活性和酯酶的抑制作用。Del-R和2Cl-R的家蝇品系对三种有机磷杀虫剂的抗性倍数为0.966～7.190倍,均为低抗水平。三个家蝇品系的羧酸酯酶活性水平与抑制中浓度存在正相关性,说明羧酸酯酶在抗拟除虫菊酯家蝇对有机磷杀虫剂的抗性中起一定的作用。     

阿维菌素对甘蔗绵蚜解毒酶系活性的影响

采用叶片浸渍法测定了阿维菌素对甘蔗绵蚜的毒力,并用分光光度计法检测了不同浓度药剂处理后活虫体内羧酸酯酶和乙酰胆碱酯酶活性的变化。结果表明,阿维菌素对甘蔗绵蚜的LC50为2.400×10-2mg·mL-1。甘蔗绵蚜体内β-NA羧酸酯酶活性高于α-NA羧酸酯酶,随着阿维菌素处理质量浓度的升高,α-NA羧酸酯酶活性逐渐升高,当质量浓度升高到4.500×10-2mg·mL-1时,活性达到最大,随后开始缓慢下降,而β-NA羧酸酯酶的活性总体呈上升趋势,说明甘蔗绵蚜体内β-NA羧酸酯酶的活性与阿维菌素的质量浓度可能存在一定的相关,β-NA羧酸酯酶活性的升高可能会使甘蔗绵蚜对阿维菌素产生抗性。甘蔗绵蚜体内乙酰胆碱酯酶的活性在阿维菌素处理质量浓度为2.250×10-2mg·mL-1时最高,而在处理质量浓度为4.500×10-2mg·mL-1时则降至最低,后又随阿维菌素处理质量浓度的加大而缓慢上升,乙酰胆碱酯酶的活性与阿维菌素的质量浓度不存在线性相关。     

运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

一个致倦库蚊杀虫剂敏感品系的筛选

化学防治是控制蚊虫传播疾病的主要方法, 抗性监测表明我国蚊虫已对有机磷、 有机氯、 氨基甲酸酯和拟除虫菊酯类杀虫剂产生了不同程度的抗性。蚊虫抗药性的分子机制主要包括靶标抗性和三大解毒酶家族带来的代谢抗性。筛选对杀虫剂敏感的品系是抗性监测和抗性机理研究必不可少的材料。本研究通过从一个致倦库蚊Culex pipiens quinquefasciatus野生种群筛选无乙酰胆碱酯酶G119S突变且具有低活性羧酸酯酶、 P450单加氧酶和谷胱甘肽-S-转移酶的单雌系, 建立了一个对杀虫剂敏感的致倦库蚊品系。该品系的羧酸酯酶活性是敏感品系S-lab的2.5倍, P450单加氧酶和谷胱甘肽-S-转移酶的活性与S-lab相当。生物测定表明, 与S-lab相比, 该品系对有机磷杀虫剂有低于2倍的抗性, 对氨基甲酸酯和拟除虫菊酯类杀虫剂没有抗性, 可以作为相对敏感品系用于抗性监测。     

云南烟蚜抗药性机制研究

通过比较云南烟蚜敏感品系和抗性品系的解毒酶(α-乙酸萘酯羧酸酯酶、β-乙酸萘酯羧酸酯酶)和靶标酶(乙酰胆碱酯酶)的活力,研究了烟蚜对有机磷、拟除虫菊酯和氨基甲酸酯类杀虫剂抗性的生化机制,并通过酯酶基因扩增检测和钠离子通道突变检测,研究了其抗性的分子机制。结果表明:α-乙酸萘酯羧酸酯酶活力增强是烟蚜对有机磷类、氨基甲酸酯类杀虫剂及拟除虫菊酯类杀虫剂的抗性机制之一;乙酰胆碱酯酶在烟蚜对有机磷杀虫剂抗性中起重要作用;3个抗性品系烟蚜均没有发生酯酶基因扩增,抗拟除虫菊酯品系烟蚜发生了钠离子通道突变。     

抗有机磷三带喙库蚊的酯酶研究

本文主要利用聚丙烯酰胺凝胶电泳及离体酶学技术研究抗有机磷三带喙库蚊(Culex tritaeniorhynchus Giles)与各种水解酶系的关系.结果表明:三带喙库蚊抗性品系的羧酸酯酶活性明显高于敏感品系.此外,酯酶同功酶谱显示抗性品系有一染色很深的高活性羧酸酯酶带E₄,而胆碱酸酶活性测定结果表明抗性与敏感品系无明显差异.因此本实验证明,该蚊对有机磷杀虫剂之所以产生高度抗性.其主要原因与羧酸酯酶的活力增加有关.这和我们生物测定结果完全一致.     

抑制剂存在下不同种群烟粉虱乙酰胆碱酯酶残余活性频率分布及其与抗药性的关系

为了探讨烟粉虱Bemisia tabaci不同种群个体乙酰胆碱酯酶敏感性差异及其与抗药性的关系, 我们选用室内饲养的烟粉虱SUD S敏感品系和6个田间抗性种群, 采用酶标板酶动力学法测定了各品系 (种群）乙酰胆碱酯酶对抑制剂的敏感性反应以及抑制剂存在时各抗性种群个体乙酰胆碱酯酶残余活性频率分布。结果表明: 在抑制剂浓度为300 μmol/L时, 敏感品系乙酰胆碱酯酶的活性基本上被完全抑制, 可以明显地区分敏感品系与田间抗性种群。在抑制剂浓度为2 000 μmol/L时, 各抗性种群个体乙酰胆碱酯酶残余活性频率分布差异明显, 其中ZZ-R种群和FZ-R种群的乙酰胆碱酯酶残余活性频率分布相似, 大部分个体的乙酰胆碱酯酶残余活性分布在1.00~1.80 mOD/min之间; SM-R种群和ND-R种群的乙酰胆碱酯酶残余活性频率分布也相似, 大部分个体的乙酰胆碱酯酶残余活性分布在0.40~1.00 mOD/min之间; LY-R和NP-R种群大部分个体的乙酰胆碱酯酶残余活性分别分布在1.00~1.60 mOD/min和0.80~1.20 mOD/min之间。各抗性种群乙酰胆碱酯酶高残余活性 (大于1.00 mOD/min）个体频率与对敌敌畏的抗性水平之间具有明显相关性, 相关系数为0.86 (P<0.05）。考虑到乙酰胆碱酯酶对抑制剂作用不敏感是一些昆虫对有机磷和氨基甲酸酯类杀虫剂抗性的重要机制之一, 建议可以将乙酰胆碱酯酶对敌敌畏的敏感性作为烟粉虱抗药性生化检测的一个参考指标。     

棉蚜抗氧化乐果品系的羧酸酯酶基因突变

用氧化乐果对室内敏感品系棉蚜Aphis gossypii (Glover)进行抗性选育,经24代筛选,抗性指数达到124.7倍。以α-乙酸萘酯（α-NA）为底物,比较了氧化乐果敏感和抗性品系棉蚜羧酸酯酶的比活力,发现抗性品系羧酸酯酶比活力明显小于敏感品系。对这两个品系的羧酸酯酶基因进行了克隆,通过对抗性和敏感品系羧酸酯酶基因核苷酸序列及推导的氨基酸序列比较,发现抗性品系有4个氨基酸残基发生了替代 (His¹⁰⁴→Arg, Ala¹²⁸→Val, Thr³³³→Asp, Lys⁴⁸⁴→Arg)。对其蛋白质三维结构分析推测只有His¹⁰⁴→Arg的替代是位于其活性中心。棉蚜氧化乐果敏感和抗性品系羧酸酯酶基因cDNA全长的GenBank登录号分别为AY485216和AY485214。     

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。     

小菜蛾芳基酰胺酶表达及其与呋喃虫酰肼抗性的关系

以对硝基乙酰苯胺为底物,采用紫外分光光度计比色法,研究了小菜蛾Plutella xylostella(L.)体内芳基酰胺酶酶促反应的最适反应条件、各发育阶段和不同组织中芳基酰胺酶的活性及亚细胞分布及其与小菜蛾对呋喃虫酰肼抗性的关系。结果表明,小菜蛾芳基酰胺酶最适反应条件为35℃,pH7.5,底物浓度为1.2×10-3mol/L,反应时间30min。利用非变性聚丙烯凝胶电泳活性染色及SDS-PAGE法测得小菜蛾芳基酰胺酶的分子量为61.1ku。小菜蛾4龄幼虫的芳基酰胺酶活性最高。不同亚细胞层中,芳基酰胺酶活力大小依次为线粒体层>微粒体层>细胞核和细胞碎片层>可溶性部分。芳基酰胺酶主要分布在小菜蛾4龄幼虫的腹部,其余依次为表皮、头部和胸部。小菜蛾呋喃虫酰肼抗性品系芳基酰胺酶的活性是敏感品系的3.89倍,表明该酶参与了小菜蛾对呋喃虫酰肼的抗性形成。     

Phosphine resistance in the rust red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): inheritance, gene interactions and fitness costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.     

The rph2 gene is responsible for high level resistance to phosphine in independent field strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SR(QLD)), New South Wales (SR(NSW)) and South Australia (SR(SA)), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SR(QLD) and SR(NSW). We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SR(SA) and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.     

对硫磷真菌降解酶的分离纯化和性质

黑曲霉AspergillusnigerY－8在液体培养基中30℃培养5d,其菌体用超声波破碎后,经硫酸铵沉淀、DEAE-纤维素层析、Sephadex－100凝胶过滤,得到凝胶电泳均一的对硫磷降解酶,其比活为6.94,提纯倍数为13.6倍,收率为17．4％。酶作用的最适温度是50℃,最适pH为7.5,在40℃以下和pH6.0～9.0之间稳定,此酶为单亚基蛋白,凝胶过滤法测得分子量为42000,含糖14.6％,SDS、Hg2+、Ag+、Fe3+对酶有强烈的抑制作用,金属螫合剂EDTA对酶活无影响。此酶对甲基对硫磷、敌敌畏、亚胺硫磷也有较好的降解作用,当以对硫磷为底物时,Km为0.43mmol／L,Vmax为1.24μmol·mg-1·min-1。     

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae (L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.     

Inheritance of resistance to pyriproxyfen in Bemisia tabaci (Hemiptera: Aleyrodidae) males and females (B biotype)

We evaluated effects of the insect growth regulator pyriproxyfen on Bemisia tabaci (Gennadius) (B biotype) (Hemiptera: Aleyrodidae) males and females in laboratory bioassays. Insects were treated with pyriproxyfen as either eggs or nymphs. In all tests, the LC50 for a laboratory-selected resistant strain was at least 620 times greater than for an unselected susceptible strain. When insects were treated as eggs, survival did not differ between males and females of either strain. When insects were treated as nymphs, survival did not differ between susceptible males and susceptible females, but resistant males had higher mortality than resistant females. The dominance of resistance decreased as pyriproxyfen concentration increased. Resistance was partially or completely dominant at the lowest concentration tested and completely recessive at the highest concentration tested. Hybrid female progeny from reciprocal crosses between the susceptible and resistant strains responded alike in bioassays; thus, maternal effects were not evident. Rapid evolution of resistance to pyriproxyfen could occur if individuals in field populations had resistance with traits similar to those of the laboratory-selected strain examined here.     

Microplate adaptation of Gomori's assay for quantitative determination of general esterase activity in single insects

Esterase activity is monitored in mosquitoes and other arthropod species because high levels of these enzymes can be associated with pesticide resistance. In the 1950s, G. Gomori devised a colorimetric method to detect esterase activity based on their capacity to hydrolyze aryl-esters. We modified this method for use in microtiter plates. Mosquito homogenates (Culex quinquefasciatus Say and C. pipiens L.) from strains susceptible and resistant to insecticides were allowed to hydrolyze alpha-naphthyl acetate in the presence of Triton X-100 and a specific acetylcholinesterase inhibitor. The alpha-naphthol product was detected colorimetrically by a diazo-coupling reaction with Fast Garnet GBC salt. Triton X-100 improved the extraction of esterases and maintained the azo compound in solution. The linear range of the method was 2-20 nmoles of alpha-naphthol; this high sensitivity permitted accurate determinations in 1/30 portions of single adult mosquitoes from the strain with the lowest esterase activity. To avoid variations due to changes in temperature and duration of assay, results were normalized to equivalent enzyme activity units obtained in a spectrophotometer at 25 degrees C. Depending on the number of homogenate dilutions required, performance of the assay in microplates allowed the simultaneous analysis of 20-80 samples. Female mosquitoes showed higher enzyme activity than males when expressed in nmoles/min per mosquito, but differences were reduced when results were expressed as specific activity (nmoles/min per mg protein). A mosquito strain resistant to organophosphates due to the presence of high levels of esterases showed about 200 times more esterase activity than a susceptible strain or a strain resistant due to insensitive acetylcholinesterase.     

植物次生物质和杀虫剂对分月扇舟蛾谷胱甘肽S-转移酶的抑制作用

本论文采用分光光度计法研究了植物次生物质和杀虫剂对分月扇舟蛾Closteraanastomosis(L.)谷胱甘肽S-转移酶(GSTs)的体外抑制作用。结果表明各植物次生物质和杀虫剂对分月扇舟蛾GSTs活性的体外抑制作用存在差异。当植物次生物质和杀虫剂终浓度为8.34×10-5mol/L时,槲皮素和单宁酸对分月扇舟蛾GSTs活性抑制作用最强,分别为64.41%和58.61%;三唑磷、毒死蜱、辛硫磷、氟铃脲、氟虫腈和哒螨灵都有较强的抑制作用,对GSTs活性抑制率超过20%;氧化乐果、水胺硫磷、丙溴磷、马拉硫磷、灭多威、联苯菊酯、高效氯氰菊酯、甲氰菊酯、高效氯氟氰菊酯、吡虫啉和啶虫脒对分月扇舟蛾GSTs有中等抑制作用;其他杀虫剂对分月扇舟蛾GSTs有较弱(或没有)抑制作用。此外,在一定的浓度范围内,槲皮素、单宁酸和辛硫磷对分月扇舟蛾GSTs活性的抑制作用存在明显的剂量效应关系。槲皮素、单宁酸和辛硫磷对分月扇舟蛾GSTs的抑制中浓度分别为2.19×10-5,2.62×10-5,1.49×10-4mol/L。因此,此研究明确了槲皮素和单宁酸作为防治分月扇舟蛾的新型防治剂或增效剂具有很好的潜力。