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.     

家蝇中乙酰胆碱酯酶、羧酸酯酶和多功能氧化酶活性与抗药性的关系

本文对几种抗药性和敏感性家蝇品系的乙酰胆碱酯酶(AChE)、羧酸酯酶及多功能氧化酶(MFO)进行了测定.结果表明:①抗药性品系和敏感性品系的AChE活力差异不大, 有机磷抗性品系的AChE对对氧磷的不敏感性比敏感性家蝇明显增大.②某些抗药性家蝇的羧酸酯酶活力比敏感性家蝇大.③抗药性家蝇的MFO活力(O-脱甲基和环氧化)比敏感性家蝇均有不同程度的增高.④二氯苯醚菊酯抗性家蝇对有机磷有负交互抗性.     

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。     

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。     

乙酰胆碱酯酶敏感性的变化与家蝇抗药性的关系

结合抗性机理, 对四个家蝇(Musca domestica vicina)品系头部的乙酰胆碱酯酶(AchE)对底物和抑制剂反应的动力学进行了研究.结果表明, AchE敏感性的降低是引起两个有机磷药剂(OP)抗性品系DDVP-R和Trichl-R对OP交互抗性的一个主要因子.Trichl-R对二氯苯醚菊酯无抗性, 而二氯苯醚菊酯抗性品系2Cl—R对所测定的OP有负交互抗性, 并且它的AchE对OP的敏感性较正常品系(NP)AchE的敏感性要高.AchE敏感性的变化, 反应在酶结构上, 主要是酶对抑制剂的亲和性(K_d)的变化, 而磷酸化速率(K₂)影响不大.同时对AchE的底物和抑制剂结合位点及结构变化进行了分析和探讨.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅱ:氧化代谢

氧化代谢的增强是引起家蝇对二氯苯醚菊酯产生抗性的因素之一。抗性家蝇多功能氧化酶的萘羟化活性、对二氯苯醚菊酯的氧化代谢能力和微粒体细胞色素P_(450)含量分别是正常家蝇的2、1.48、1.33倍。正常家蝇和抗性家蝇细胞色素P_(450)在对增效磷(SV_1)和氧化胡椒基丁醚(Pb)的敏感性上也存在着差异。SV_1与多功能氧化酶专一性抑制剂Pb一样,对该酶系催化的萘羟化活性及二氯苯醚菊酯的氧化代谢有明显的抑制作用,这种抑制作用是SV_1在家蝇体内对二氯苯醚菊酯增效的机理之一。SV_1对氧化代谢的抑制与它和微粒体细胞色素P_(450)相互作用形成非活性复合体有关。     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅱ:氧化代谢

氧化代谢的增强是引起家蝇对二氯苯醚菊酯产生抗性的因素之一.抗性家蝇多功能氧化酶的萘羟化活性、对二氯苯醚菊酯的氧化代谢能力和微粒体细胞色素P₄₅₀含量分别是正常家蝇的2、1.48、1.33倍.正常家蝇和抗性家蝇细胞色素P₄₅₀在对增效磷(SV₁)和氧化胡椒基丁醚(Pb)的敏感性上也存在着差异.SV₁与多功能氧化酶专一性抑制剂Pb一样,对该酶系催化的萘羟化活性及二氯苯醚菊酯的氧化代谢有明显的抑制作用,这种抑制作用是SV₁在家蝇体内对二氯苯醚菊酯增效的机理之一.SV₁对氧化代谢的抑制与它和微粒体细胞色素P₄₅₀相互作用形成非活性复合体有关.     

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。     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅰ:水解代谢

水解代谢在家蝇对二氯苯醚菊酯抗性中起重要作用。正常家蝇和抗性家蝇酯酶在对SV₁、SV₂等抑制剂的敏感性和电泳性质上存在着差异。抗性家蝇酯酶水解二氯苯醚菊酯的活性较高,水解乙酸-α-萘酯的活性相对比正常家蝇要低。SV₁及其在体内的代谢产物SV₂在离体和活体情况下对家蝇酯酶都有明显的抑制作用。SV₁和SV₂抑制相同的酯酶电泳条带,但SV₁的抑制作用相对小一些。SV_t对酯酶的抑制是它在家蝇体内对二氯苯醚菊酯增效的机理之一。     

辛硫磷和溴氰菊酯混剂对家蝇抗性发展的影响

以家蝇(Musca domestica vicina Macquart)为试虫,用辛硫磷溴氰菊酯单剂及不同配比的混剂进行汰选试验。所有混剂选育的家蝇抗性发展都很缓慢,而单剂抗性发展都很快。增效试验表明,辛硫磷与溴氰菊酯混配有明显增效作用,特别是对抗性品系。生化分析结果表明,对澳氰菊酯的抗性发展与酯酶的酶活升高有关。对辛硫磷抗性发展与多功能氧化酶的酶活升高和乙酰胆碱酯酶敏感性降低有关。混剂选育的家蝇其对单剂的敏感性的变化及酶系的变化,随着混剂的配比而变化。     

辛硫磷和溴氰菊酯混剂对家蝇抗性发展的影响

以家蝇（ＭｕｓｃａｄｏｍｅｓｔｉｃａｖｉｃｉｎａＭａｃｑｕａｒｔ）为试虫,用辛硫磷溴氰菊酯单剂及不同配比的混剂进行汰选试验。所有混剂选育的家蝇抗性发展都很缓慢,而单剂抗性发展都很快。增效试验表明,辛硫磷与溴氰菊酯混配有明显增效作用,特别是对抗性品系。生化分析结果表明,对溴氰菊酯的抗性发展与酯酶的酶活升高有关。对辛硫磷抗性发展与多功能氧化酶的酶活升高和乙酰胆碱酯酶敏感性降低有关。混剂选育的家蝇其对单剂的敏感性的变化及酶系的变化,随着混剂的配比而变化     

抗溴氰菊酯家蝇在不同用药方式下的敏感性变化及其机制

以具有极高抗水平的抗溴氰菊酯家蝇Musca domestica vicina Macquart DR0品系为试虫,模拟田间几种常见的用药方式(混用、轮用、使用增效剂),在室内进行平行汰选,并以不用药和继续用原药汰选的为比较,研究试虫在这几种用药方式下的敏感性变化及其变化机制。抗性家蝇用辛溴混剂、辛硫磷以及溴氰菊酯＋SV₁汰选后,在F₁₆(F₁₇)代以前,对溴氰菊酯及汰选药剂的抗性发展相对都比较缓慢;F₁₆(F₁₇)代以后,用溴氰菊酯＋SV₁汰选的家蝇对溴氰菊酯的敏感性迅速下降,抗性发展很快。家蝇对溴氰菊酯的敏感性变化与药剂中溴氰菊酯的选择压有关。生化分析结果表明,在不同用药方式汰选下,家蝇体内酯酶、多功能氧化酶、谷胱甘肽-S-转移酶、乙酰胆碱酯酶的酶活或特性发生了不同的变化。     

不同杀虫剂选育对家蝇抗药性水平及kdr基因频率的影响

采用杀虫剂（溴氰菊酯和甲基嘧啶磷）筛选及不接触药物自然衰退的方法,研究了家蝇Musca domestica氯氟氰菊酯高抗品系(Cyh-R)对杀虫剂的抗性变化,探讨蝇类抗药性治理的方法。用点滴法测定氯氟氰菊酯对不同家蝇品系的毒力,比较抗药性的变化,结合特异性等位基因PCR扩增（PASA）技术检测了不同家蝇品系的kdr基因频率,探讨kdr基因频率与抗性水平之间的关系。结果表明:甲基嘧啶磷筛选后,氯氟氰菊酯对第2~8代Cyh-R品系的LD₅₀呈递减趋势,从F₀的2.8434 μg/蝇降为F₈的0.4404 μg/蝇,但第8~18代Cyh-R品系的LD₅₀呈逐代递增趋势;溴氰菊酯筛选后,氯氟氰菊酯对Cyh-R品系第2~16代的LD₅₀呈上升趋势,从F₀的2.8434 μg/蝇升至F₁₆的24.3249 μg/蝇;表明了施用有机磷杀虫剂可降低其对氯氟氰菊酯的抗药性,而施用拟除虫菊酯药剂则有助于其对氯氟氰菊酯抗药性的增长;不使用任何杀虫剂也能降低其对氯氟氰菊酯的抗药性,但下降速率低于甲基嘧啶磷。PASA技术检测表明Cyh-R品系的kdr抗性基因频率为88.8%,不经过任何药剂筛选其kdr抗性基因频率下降程度最大,达到69.7%;甲基嘧啶磷筛选后其结果降为78.8%,而经溴氰菊酯筛选后kdr抗性基因频率则明显升高,达到98.9%。通过对kdr抗性基因频率和抗性水平进行相关和回归分析表明kdr抗性基因频率与家蝇对氯氟氰菊酯的LD₅₀呈对数关系,即LD₅₀值高的品系其kdr抗性基因频率相应的也较高。建议在家蝇防治中考虑轮换用药。     

溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响

以敏感品系家蝇和溴氰菊酯抗性品系家蝇(Musca domestica L.)为材料,研究和比较了神经毒剂溴氰菊酯对其脑突触体蛋白磷酸化作用的影响。结果表明,浓度为10^-5 mol/L溴氰菊酯抑制了敏感品系家蝇脑突触体蛋白磷酸化作用,而对抗性品系家蝇脑突触体蛋白磷酸化作用无明显影响。若反应体系中加入2.5×10^-6 mol/L的cAMP显著激活了敏感品系家蝇脑突触体蛋白磷酸化水平,但是当0.6 mmol/Lca²⁺或0.6 mmol/L Ca²⁺加10^-5 mol/L钙调蛋白时明显增强了抗性品系家蝇脑突触体蛋白磷酸化水平,甚至超过了其对敏感品系的作用水平。此外,还发现不同浓度的溴氰菊酯可抑制突触膜上的Na/K-ATP酶和Ca-ATP酶活力,浓度越高抑制作用也越大,并且敏感品系家蝇对溴氰菊酯的敏感度要高于抗性品系。     

Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.     

Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus, in northeastern India

The insecticide resistance status of Culex quinquefasciatus Say (Diptera: Culicidae) to DDT and deltamethrin across army cantonments and neighbouring villages in northeastern India was investigated. In India, DDT is still the insecticide of choice for public health programmes. In military stations, pyrethroids, especially deltamethrins, are used for insecticide‐treated nets (ITNs). Recent information on the levels of resistance to DDT and deltamethrin in mosquito populations of northeastern India is scare. Continued monitoring of insecticide resistance status, identification of the underlying mechanisms of resistance in local mosquito populations and the establishment of a baseline data bank of this information are of prime importance. Insecticide susceptibility assays were performed on wild‐caught adult female Cx. quinquefasciatus mosquitoes to the discriminating doses recommended by the World Health Organisation (WHO) to DDT (4%) and deltamethrin (0.05%). Across all study sites, mortality as a result of DDT varied from 11.9 to 50.0%, as compared with 91.2% in the susceptible laboratory strain (S‐Lab), indicating that Cx. quinquefasciatus is resistant to DDT. The species was found to be 100% susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knock‐down times (KDT) in response to deltamethrin varied significantly between study sites (P < 0.01) from 8.3 to 17.8 min for KDT₅₀ and 37.4 to 69.5 min for KDT₉₀. All populations exceeded the threshold level of alpha‐esterase, beta‐esterase and glutathion S‐transferase (GST) established for the S‐Lab susceptible strain, and all populations had 100% elevated esterase and GST activity, except Missamari and Solmara. Beta‐esterase activity in Field Unit II (96.9%) was less than in any of the other populations. Benganajuli had the highest activity level for all the enzymes tested. There was a significant correlation between all enzyme activity levels and insecticide resistance phenotype by populations (P < 0.05). The results presented here provide the first report and baseline information of the insecticide resistance status of Cx. quinquefasciatus in northeastern India, and associated information about biochemical mechanisms that are essential for monitoring the development of insecticide resistance in the area.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.