拟除虫菊酯抗性家蝇的交互抗药性研究

拟除虫菊酯的广泛使用使家蝇普遍产生了抗药性,为有效的控制家蝇的危害,需要了解家蝇对轮换或新杀虫剂的交互抗性状况。作者用点滴法测定了两个实验室汰选的拟除虫菊酯抗性家蝇品系对几种杀虫剂的交互抗性,结果表明:二氯苯醚菊酯和溴氰菊酯之间存在较高程度的交互抗药性;拟除虫菊酯抗性较高的家蝇对作用机制不同的新农药(多杀菌素、氟虫腈)表现较低程度的交互抗性。     

拟除虫菊酯对家蝇Ca—ATPase和Ca—Mg—ATPase的抑制作用

通过对家蝇神经系统的Ca-ATPase、Ca-Mg-ATPase性质的研究,表明Ca-ATPase、Ca-Mg-AT-Pase反应的适宜pH值分别为7.0-8.5和6.5。适温皆为35-40℃;底物（ATP）最适浓度均为0.5mmol/L。比较测定了家蝇三个品系中两种ATPase的活性及拟除虫菊酯对该酶的抑制作用,实验证明,敏感与Del-R、2Cl-R品系间Ca-ATPase、Ca-Mg-ATPase活力无明显的差异。溴氰菊酯、氯菊酯可部分地抑制敏感品系家蝇Ca-ATPase活性,而对拟除虫菊酯抗性品系Ca-ATPase无抑制作用,从而证明,Del-R、2Cl-B品系Ca-ATPase对拟除虫菊酯的敏感性已明显降低,这可能能是击倒抗性机制之一。实验还表明,拟除虫菊酯对Ca-Mg-ATPase基本上无抑制作用,这说明在家蝇中,Ca-Mg-ATPase并不是拟除虫菊酯的一个靶标位点。     

拟除虫菊酯的结构与害虫抗药性的关系

以家蝇Musca domestica vicina L.为试虫,用汰选方法研究了家蝇对六种不同拟除虫菊酯的抗性发展.结果表明,家蝇对不同化学结构和不同光学异构体组分的拟除虫菊酯抗性差别很大.对溴氧菊酯、顺式氯氰菊酯抗性发展很快,氯氰菊酯次之,氰戊菊酯、氰戍菊酯A和氰戊菊酯A_σ抗性发展较慢.用抗溴氰菊酯家蝇品系和点滴法测定了十种拟除虫菊酯和七种有机磷杀虫剂的毒力,讨论了它们之间的交互抗性和结构与抗性的关系.溴氰菊酯(抗性比值24.00)、氯氰嫡酯(抗性比值20.11)、顺式氯氰菊酯(抗性比值38.10)和二氯苯醚菊酯(抗性比值11.04),结构相近的交互抗性比较严重.氰戊菊酯(抗性比值4.64)、氰戊菊酯A(抗性比值5.97)、戊菊酯(抗性比值4.49)和氟氰菊酯(抗性比值4.12)化学结构中醇部分与溴氰菊酯相同、酸部分不同,它们与溴氰菊酯交互抗性水平较低.联苯菊酯(抗性比值1.98)化学结构中酯和醇部分都与溴氰菊酯不同,其交互抗性水平较低,杀螟松等七种有机磷杀虫剂,除敌敌畏与溴氰菊酯有轻微交互坑性外,其它均无交互抗性.     

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。     

钠离子通道与蜜蜂狄斯瓦螨对氟胺氰菊酯的抗性机理

狄斯瓦螨Varroadestructor是全世界蜜蜂最严重的寄生虫 ,目前 ,它对主要防治药物———拟除虫菊酯类的氟胺氰菊酯已产生明显抗性 ,严重影响其防治效果。近年来神经生理学研究结果证实 :电压门控的钠离子通道是拟除虫菊酯作用的位点。钠通道结构的改变 ,是拟除虫菊酯类杀虫剂毒理的主要基础 ,也是产生抗药性的基础。该文介绍了近年来国内外研究电压门控钠离子通道、拟除虫菊酯对钠通道的作用、钠通道与拟除虫菊酯的抗性和狄斯瓦螨对氟胺氰菊酯抗性机理研究的新进展     

家蝇对DDT和拟除虫菊酯的重要抗性机制——中枢神经系统的不敏感性

本文以电生理技术研究了四个品系的家蝇Musca domestica vicina Macq.中枢神经系统(CNS)对DDT、二氯苯醚菊酯和澳氰菊酯的敏感性,结果表明:三种抗性家蝇,DDT高抗品系(DDT-R)、二氯苯醚菊酯高抗品系(2C1-R)和溴氰菊酯高抗品系(Dec-R)的中枢神经系统(CNS)对三种杀虫剂的敏感性与敏感家蝇相比均明显降低,而且,GNS的不敏感性随杀虫剂LD₅₀的升高有逐渐上升的趋势.我们认为,CNS不敏感性是家蝇对DDT相拟除虫菊酯产生抗性的一个重要机制,也是产生交互抗性的一个重要原因.     

家蝇对DDT和拟除虫菊酯的重要抗性机制——中枢神经系统的不敏感性

本文以电生理技术研究了四个品系的家蝇Musca domestica vicina Macq.中枢神经系统(CNS)对DDT、二氯苯醚菊酯和澳氰菊酯的敏感性,结果表明:三种抗性家蝇,DDT高抗品系(DDT-R)、二氯苯醚菊酯高抗品系(2C1-R)和溴氰菊酯高抗品系(Dec-R)的中枢神经系统(CNS)对三种杀虫剂的敏感性与敏感家蝇相比均明显降低,而且,GNS的不敏感性随杀虫剂LD₅₀的升高有逐渐上升的趋势.我们认为,CNS不敏感性是家蝇对DDT相拟除虫菊酯产生抗性的一个重要机制,也是产生交互抗性的一个重要原因.     

家蝇对拟除虫菊酯农药的抗性机制

本文对二氯苯醚菊酯和溴氰菊酯分别选择的高抗性家蝇(Musca domestica vicina)品系2Cl-R及Dec-R的抗性机制进行了研究.应用生物测定、增效剂,体内试验的表皮穿透作用、离体条件的解毒酶系活性的增加以及家蝇头部ATP酶活力的研究结果表明,两种拟除虫菊酯高抗性家蝇品系的表皮穿透性均比正常品系NP为慢,特别是Dec-R品系极慢.酯酶和多功能氧化酶及其末端的细胞色素P-450的活性在两个抗性品系中都比NP品系有不同程度的增高,但2Cl-R品系以氧化酶为主,而Dec-R品系似以酯酶占优势.Dec-R品系的Na⁺—K⁺-ATP酶活力低于NP品系的46%,而2Cl-R品系与NP品系相等.Mg²⁺-ATP酶活性在两个抗性品系中均高于正常品系.Mg²⁺-ATP酶可能也是拟除虫菊酯的一个重要靶标部位.     

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。     

家蝇抗药性的测定及抗性的培育

<正> 家蝇Musca domestica L.是传布疾病的主要媒介昆虫之一。由于它生活周期短、繁殖能力强,分布范围广、用药次数多,已成为国际性的抗药性最严重的害虫。 据了解,国外某些地区的家蝇几乎对所有的杀虫剂都有抗性,即使最近使用的拟除虫菊酯、保幼激素及灭幼脲TH60-40亦不例外。我国使用杀虫剂的种类     

Antigenic Crossreactivity between Bacterial and Plant Cytochrome P-450 Monoxygenases

Although cytochrome P-450 monoxygenases mediate critical reactions in plant microsomes, characterization of their activities has been difficult due to their inherent instability and the lack of a crossreacting P-450 antibody. We have surveyed the effects of protein stabilizing agents on t-cinnamic acid hydroxylase (t-CAH), a prominent microsomal P-450, and on total P-450 monoxygenase content. Trans-cinnamic acid is the most effective protecting agent for t-CAH activity. Leupeptin, a broad spectrum protease inhibitor, stabilizes t-CAH activity and increases the apparent P-450 content more than serine protease inhibitors such as phenylmethylsulfonyl fluoride. The combination of t-cinnamic acid and protease inhibitors increase the level of detectable t-CAH activity 4- to 14-fold over the levels detected by previously published procedures. In order to estimate the molecular weights and diversity of the plant P-450 monoxygenases in wounded pea epicotyls, we have prepared two polyclonal antibodies against the Pseudomonas putida camphor hydroxylase (P-450_cam). One of the heterologous antibodies cross-reacts with constitutive microsomal polypeptides between 52 and 54 kilodaltons and several pea (Pisum sativum L.) mitochondrial proteins between 47 and 48 kilodaltons. The other polyclonal antibody cross-reacts strongly with two wound-induced polypeptides (65 and 47 kilodaltons) and weakly with one constitutive polypeptide (58 kilodaltons). We conclude that at least two subclasses of plant P-450 monoxygenases share common epitopes with the bacterial P-450 enzyme.     

Screening of bacterial cytochrome P450s responsible for regiospecific hydroxylation of (iso)flavonoids

Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from Streptomyces avermitilis MA4680, Bacillus and Nocardia farcinica IFM10152 strains. As electron transfer redox partners with the P450s in Escherichia coli system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from S. avermitilis MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using S. avermitilis MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4',5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4',5,7-dihydroxyisoflavone) to produce 3',4',5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3',4',5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS-MS and GC-MS and 1H NMR.     

Dap1/PGRMC1 binds and regulates cytochrome P450 enzymes

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.     

神经递质释放与家蝇对拟除虫菊酯抗性关系研究

通过生物测定比较溴氰菊酯、氯菊酯和DDT对Dec-R,2C1-R,DDT-R和敏感(SP)家蝇Musca domestica vicina的毒力,表明三个抗性品系对溴氰菊酯、氯菊酯和DDT均有很高的抗性,抗性倍数分别达120 912,6 032和112.2倍,并对上述三种杀虫剂有明显的交互抗性和抗击倒效应。杂交试验表明Dec-R对溴氰菊酯的抗性是一个隐性基因,电生理试验表明抗性家蝇中枢神经系统(CNS)对药剂敏感度的降低是其产生抗性和交互抗性的重要机制。研究结果表明Dec-R和2CLR家蝇品系中存在有击倒抗性因子(Kdr)。当用1×10^-7mol/L溴氰菊酯对SP家蝇脑突触体在提高K+浓度去极化后,可加强3H-胆碱的释放,而在Dec-R品系中,溴氰菊酯浓度提高到1×10^-4m0l/L也未能加强³H-胆碱的释放,表明溴氰菊酯与神经递质的释放和钠通道亲和性的降低是抗性的主要机制。     

击倒抗性和钠离子通道

综述了击倒抗性与钠离子通道关系的研究进展。毒理学和电生理学的研究表明,在许多拟除虫菊酯类杀虫剂抗性昆虫中存在击倒抗性。分子遗传学研究进一步发现,击倒抗性与钠离子通道位点连锁。最近的研究表明,昆虫神经系统对拟除虫菊酯类杀虫剂敏感性下降的击倒抗性机制是钠离子通道结构基因突变。但仍有一些问题,如突变的保守性和分布,需要进一步研究、阐明。     

不同光学异构体组成的氯氰菊酯对敏感及抗性家蝇的神经电生理学研究

采用敏感家蝇(Musca domestica vicina L.)及由5种不同光学异构体组成的氯氰菊酯选育的抗性家蝇中胸足离体标本,观测五种药剂对足感觉神经纤维冲动发放的影响。各种异构体组成的氯氰菊酯均可引起感觉神经纤维发放的增加,然后逐渐降低,直至完全阻断。以阻断时间和加药剂量为参数,求出神经敏感度。结果表明：五种药剂作用于敏感家蝇的神经敏感度与室内生物测定的LD₅₀值(μg/头)无相关性,而与供试药剂中反α体与顺α体的比例有关,反α体所占比例越多的药物,神经敏感度越高。抗性家蝇的神经敏感度与敏感 家蝇相比大幅度下降,可以认为神经敏感度降低是家蝇对氯氰菊酯产生抗性的主要机制。抗性家蝇中,氯氰菊酯品系(RC₁)的抗生水平最高,但它的神经敏感度较其它抗性品系也高,由此推测,RC₁,的抗性机制与其它晶系有所不同。     

Biochemical alterations elicited in rat liver microsomes by oxidation and reduction products of chloroform metabolism

The feasibility of an oxygen-independent mechanism of chloroform bioactivation was indicated by the covalent binding to lipid and protein occurring in anaerobic incubations of CHCl3 and microsomes in the presence of NADPH. Under these conditions, the loss of cytochrome P-450 and the inhibition of related monoxygenases were also observed. The chloroform anoxic biotransformation was negligible in uninduced microsomes and seemed to be catalyzed mainly by phenobarbital-inducible P-450 isozymes. Biotransformation could also be supported by NADH as the source of reducing equivalents. Anaerobic metabolism of chloroform led to decreased levels of the main PB-induced P-450 isozymes even at low CHCl3 concentration and did not affect benzo[a]pyrene hydroxylase activity. These effects were not decreased by thiolic compounds. The oxidation products of chloroform caused a general impairment of the monoxygenase system, probably related to the formation of protein aggregates with very high molecular weight. In the presence of physiological concentrations of GSH, the targets of aerobically-produced metabolites were lipids and, to a smaller extent, P-450. At low CHCl3 concentrations and/or in the presence of GSH the most changes to microsomal structures seemed to be produced by the reductively-formed intermediates.     

Cytochrome P-450 and hydroxylating activity of microsomal preparations from whole houseflies

1. A new microsomal preparation, obtained from whole houseflies is described in terms of its cytochrome P-450 content and its hydroxylating activity. 2. Microsomes prepared from whole-fly brei, obtained with the aid of a mortar (a procedure that avoids the destruction of sarcosomes), contain 0.265nmol of cytochrome P-450 and hydroxylate naphthalene at a rate of 28.5nmol/mg of microsomal protein in 30min at 30 degrees C. This corresponds to 104nmol of naphthalene hydroxylated/nmol of cytochrome P-450. This is the highest rate ever reported for housefly and rat liver microsomal preparations. 3. Microsomal fractions prepared by procedures that do not retain the integrity of sarcosomes show the presence in the CO-difference spectrum of a 428nm peak. This cytochrome is associated with sarcosomal microsomes and it may be involved in the inhibition of insect microsomal mixed-function oxidases, although other factors cannot be discarded at present. 4. The inability to show cytochrome P-450 in microsomal fractions isolated from whole houseflies by other procedures may be at least partially due to a masking effect brought about by contamination with the sarcosomal cytochrome.     

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.