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     

昆虫钠离子通道基因突变及其与杀虫剂抗性关系的研究进展

昆虫电压门控钠离子通道(voltage-gated sodium channel)存在于所有可兴奋细胞的细胞膜上,在动作电位的产生和传导上起重要作用,是有机氯和拟除虫菊酯杀虫剂的靶标位点。在农业和医学害虫控制过程中,由于有机氯和拟除虫菊酯杀虫剂的广泛使用,抗药性问题日益突出。其中,由于钠离子通道基因突变,降低了钠离子通道对有机氯和拟除虫菊酯类杀虫剂的亲和性,从而产生击倒抗性(knock-down resistance, kdr),已成为抗性产生的重要机制之一。本文综述了昆虫钠离子通道的跨膜拓扑结构、功能、进化及其基因的克隆;更重要的是总结了已报道的40多种昆虫40个钠离子通道基因非同义突变,以及钠离子通道基因选择性mRNA剪接和编辑,以及它们与杀虫剂抗性的关系;也评述了钠离子通道基因突变引起蛋白质结构的改变,从而对杀虫剂抗性的影响机制。这些研究对于进一步鉴定与杀虫剂抗性相关的突变及抗性机制,开发有机氯和拟除虫菊酯类杀虫剂抗性分子监测方法具有重要意义。     

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。     

禾谷缢管蚜阳离子通道基因RSC1的克隆、分子特性及诱导表达分析

【目的】SC1通道(sodium channel 1)是昆虫体内一种重要的离子通道,被认为是一种开发新型杀虫剂的神经靶标。本研究拟克隆禾谷缢管蚜Rhopalosiphum padi的SC1通道基因,并初步分析其生理功能及其与SC1类通道、电压门控钠离子通道、电压门控钙离子通道的进化关系。【方法】采用RT-PCR技术,克隆了禾谷缢管蚜SC1基因完整的开放阅读框;利用实时荧光定量PCR技术,分析禾谷缢管蚜成蚜在不同浓度的高效氯氟氰菊酯诱导下SC1基因表达变化。【结果】获得了禾谷缢管蚜SC1基因(命名为RSC1)完整的开放阅读框(Gen Bank登录号为KU640190),其长度6 687bp,编码2 228个氨基酸。RSC1具有SC1通道的结构特征,有一个不同于电压门控钠离子通道和电压门控钙离子通道的特殊DEEA模体(motif)。系统进化分析结果显示,RSC1与电压门控钠离子通道组成一个进化枝,电压门控钙离子通道组成另外一个进化枝,SC1与电压门控钠离子通道在进化上有更近的起源关系。实时荧光定量PCR分析结果表明,LC15,LC35和LC503种剂量的高效氯氟氰菊酯处理6 h后,禾谷缢管蚜RSC1基因表达量相对于清水对照显著下调,表达量分别为对照的0.57,0.82和0.78倍;3种剂量的高效氯氟氰菊酯处理24 h后,禾谷缢管蚜RSC1基因表达量分别为对照的2.19,1.33和1.19倍,其中LC15(0.1484 mg/L)胁迫下RSC1基因的表达量显著上调。【结论】SC1类通道与电压门控钠离子通道在进化起源上有更近的关系。RSC1通道可能是高效氯氟氰菊酯的次级靶标。由于RSC1和其同源基因只存在于节肢动物中,脊椎动物尚未发现该类基因,因此这类通道可能作为开发新型杀虫剂的神经靶标。     

氰氟虫腙对草地贪夜蛾的毒力与田间防效

【目的】本研究旨在明确氰氟虫腙对草地贪夜蛾Spodoptera frugiperda幼虫的毒力水平和田间防治效果,为科学使用氰氟虫腙防治草地贪夜蛾提供参考依据。【方法】采用饲料混毒法在室内测定了氰氟虫腙与4种常用杀虫剂甲维盐、氯虫苯甲酰胺、虱螨脲和茚虫威对草地贪夜蛾3和6龄幼虫的致死中浓度(LC₅₀)及LC₉₀值,以及LC₉₀浓度的这些杀虫剂对3龄幼虫的致死中时(medium lethal time, LT₅₀)值。采用人工喷雾方法测定了玉米田中22%氰氟虫腙悬浮剂(6.6 g/667 m²)、22%氰氟虫腙悬浮剂(17.6 g/667 m²)、5.7%甲维盐水分散剂(1 g/667 m²)和150 g/L茚虫威悬浮剂(2 g/667 m²)对草地贪夜蛾幼虫的防效。【结果】室内生测结果显示,供试的5种杀虫剂中氰氟虫腙对草地贪夜蛾3和6龄幼虫均具有较高的毒力,其LC₅₀值分别为2.64和4.3...     

杀虫药剂的神经毒理学研究进展

大多数杀虫药剂都具有较强的神经毒性,它们对神经系统的作用靶标不同。有机磷类杀虫剂不仅抑制乙酰胆碱酯酶活性和乙酰胆碱受体功能,影响乙酰胆碱的释放,而且还具有非胆碱能毒性,有些有机磷杀虫剂还能引发迟发性神经毒性。新烟碱类杀虫剂作为烟碱型乙酰胆碱受体（nAChR）的激动剂,作用于该类受体的α亚基;它对昆虫的毒性比对哺乳动物的毒性大得多,乃是因为它对昆虫和哺乳动物nAChR的作用位点不同。拟除虫菊酯类杀虫剂主要作用于神经细胞钠通道,引起持续开放,导致传导阻滞;该类杀虫剂也可抑制钙通道。另外,这类杀虫剂还干扰谷氨酸递质和多巴胺神经元递质的释放。拟除虫菊酯类杀虫剂对昆虫的选择毒性很可能是因为昆虫神经元的钠通道结构与哺乳动物的不同。阿维菌素类杀虫剂主要作用于γ-氨基丁酸（GABA）受体,它能促进GABA的释放,增强GABA与GABA受体的结合,使氯离子内流增加,导致突触后膜超级化。由于这类杀虫剂难以穿透脊椎动物的血脑屏障而与中枢神经系统的GABA受体结合,故该类杀虫剂对脊椎动物的毒性远低于对昆虫的毒性。多杀菌素类杀虫剂可与中枢神经系统的nAChR作用,引起Ach长时间释放,此外,这类杀虫剂还可作用于昆虫的GABA受体,改变GABA门控氯通道的功能。     

家蚕para钠通道cDNA片段克隆与序列分析

昆虫神经系统para型钠离子通道是拟除虫菊酯类杀虫剂的主要靶标,已有的研究表明钠离子通道基因发生点突变与昆虫对菊酯类杀虫剂的抗性密切相关。本文通过RT-PCR方法克隆获得了编码家蚕Bombyx mori L.钠离子通道的cDNA片段(GenBank No.EF521818),该片段全长4882bp,部分ORF包含3986bp核苷酸,翻译成1328个氨基酸。蛋白序列分析表明,PCR扩增获得的家蚕钠离子通道cDNA片段所编码的氨基酸与其他昆虫的para型钠离子通道α亚基的氨基酸具有很高的同源相似性,与棉铃虫Heliothis virescens Fabricius、埃及伊蚊Aedes aegypti L.、德国小蠊Blattella germanica L.、果蝇Drosophila melanogaster Meigen和家蝇Musca domestica L.的相似性分别为95%、82%、80%、79%、77%。     

电压门控钠离子通道β亚基的研究

电压门控钠离子通道对Na+的选择性通透是神经元等兴奋性细胞产生动作电位的基础。该通道为跨膜蛋白,主要是由形成孔道的α亚基和一个或几个辅助性的β亚基组成,近年来发现,β亚基对α亚基的调节主要是在调节钠通道的膜上表达和亚细胞定位方面。由于β亚基的突变不仅能够引起动作电位的传导异常,导致神经元功能障碍,引发多种心脏系统疾病,包括恶性心律失常、Brugada综合征、QT间期延长综合征及其他传导性疾病,还能引起亨廷顿病(Huntigton’s diaease,HD)等神经系统疾病。本文就近几年钠离子通道β亚基生理功能的研究及其突变体与疾病的关系等方面作一阐述。     

昆虫钠通道的结构和与击倒抗性有关的基因突变

击倒抗性(kdr)是指昆虫和其他节肢动物由于它们的神经系统对DDT和拟除虫菊酯类杀虫剂的敏感性降低而引起的抗性。电压敏感的钠通道是DDT和拟除虫菊酯类杀虫剂的主要靶标。已知拟除虫菊酯是通过改变位于神经膜上的这类通道而发挥其杀虫效果的,钠通道基因的点突变是产生kdr抗性的主要原因。40年来kdr抗性一直是重要的研究课题,但近10年来在kdr分子生物学方面取得了很大进展。本文主要综述了1996年以来所取得的新进展,着重于钠通道的结构、在14种害虫中与kdr抗性相关的钠通道基因突变及其氨基酸序列的多态性。这些结果有助于对拟除虫菊酯改变钠通道的功能及其机理作进一步探究。     

电压门控钠离子通道疾病的研究进展

细胞膜上的电压门控钠离子通道（Voltage-gated Sodium Channels,VGSCs）是细胞形成动作电位过程中重要的组成构件,由一个大的α亚基和一个或多个不同的β亚基组成,中央是具高度选择性只允许钠离子通过的亲水通道。电压门控钠离子通道在调节细胞膜电位、维持细胞离子稳态、细胞增殖和凋亡等生理过程中发挥着重要作用,因而钠离子通道自身的异变或是相关基因的变异都可能引起一系列身体病变。本文主要介绍了电压门控钠离子通道的结构与功能,阐述了其与癌细胞侵袭转移和神经病理性疼痛的关系,并介绍了几种典型的由钠离子通道基因变异引起的疾病。随着对电压门控钠离子通道及其异常分子机制研究的不断深入,新成果将为生理学、药理学和病理学等领域的研究提供理论基础和新的研究思路,为离子通道疾病的临床预防、诊断与治疗找到新途径。     

Analysis of the action of lidocaine on insect sodium channels

A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNa(v)1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNa(v)1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNa(v)1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNa(v)1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.     

DDT, pyrethrins, pyrethroids and insect sodium channels

The long term use of many insecticides is continually threatened by the ability of insects to evolve resistance mechanisms that render the chemicals ineffective. Such resistance poses a serious threat to insect pest control both in the UK and worldwide. Resistance may result from either an increase in the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts. DDT, the pyrethrins and the synthetic pyrethroids (the latter currently accounting for around 17% of the world insecticide market), act on the voltage-gated sodium channel proteins found in insect nerve cell membranes. The correct functioning of these channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein which prevent the binding of the insecticide and result in the insect developing resistance. Here we review some of the work (done at Rothamsted Research and elsewhere) that has led to the identification of specific residues on the sodium channel that may constitute the DDT and pyrethroid binding sites.     

Insect sodium channels and insecticide resistance

Voltage-gated sodium channels are essential for the generation and propagation of action potentials (i.e., electrical impulses) in excitable cells. Although most of our knowledge about sodium channels is derived from decades of studies of mammalian isoforms, research on insect sodium channels is revealing both common and unique aspects of sodium channel biology. In particular, our understanding of the molecular dynamics and pharmacology of insect sodium channels has advanced greatly in recent years, thanks to successful functional expression of insect sodium channels in Xenopus oocytes and intensive efforts to elucidate the molecular basis of insect resistance to insecticides that target sodium channels. In this review, I discuss recent literature on insect sodium channels with emphases on the prominent role of alternative splicing and RNA editing in the generation of functionally diverse sodium channels in insects and the current understanding of the interactions between insect sodium channels and insecticides.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Mutations in the transmembrane helix S6 of domain IV confer cockroach sodium channel resistance to sodium channel blocker insecticides and local anesthetics

Indoxacarb and metaflumizone are two sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated non-conducting state, a mode of action similar to that of local anesthetics (LAs). Recently, two sodium channel mutations, F1845Y (F⁴ⁱ¹⁵Y) and V1848I (V⁴ⁱ¹⁸I), in the transmembrane segment 6 of domain IV (IVS6), were identified to be associated with indoxacarb resistance in Plutella xylostella. F⁴ⁱ¹⁵ is known to be critical for the action of LAs on mammalian sodium channels. Previously, mutation F⁴ⁱ¹⁵A in a cockroach sodium channel, BgNa_v1-1a, has been shown to reduce the action of lidocaine, a LA, but not the action of SCBIs. In this study, we introduced mutations F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸A/I individually into the cockroach sodium channel, BgNa_v1-1a, and conducted functional analysis of the three mutants in Xenopus oocytes. We found that both the F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations reduced the inhibition of sodium current by indoxacarb, DCJW (an active metabolite of indoxacarb) and metaflumizone. F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations also reduced the use-dependent block of sodium current by lidocaine. In contrast, substitution V⁴ⁱ¹⁸A enhanced the action metaflumizone and lidocaine. These results show that both F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations may contribute to target-site resistance to SCBIs, and provide the first molecular evidence for common amino acid determinants on insect sodium channels involved in action of SCBIs and LA.     

Pyrethroid insecticides: Actions of deltamethrin and related compounds on insect axonal sodium channels

The actions of deltamethrin and eight other pyrethroids were tested on isolated giant axons of the cockroach Periplaneta americana, using microelectrode and oil-gap, single-fibre electrophysiological recording techniques. Deltamethrin at micromolar concentrations induced a slow progressive depolarization of the axon membrane accompanied by a gradual reduction in action potential amplitude. The deltamethrin-induced depolarization was enhanced by an increase in stimulation frequency and was reduced in the presence of the sodium channel blocking agent saxitoxin (1 × 10^?7 M).Other synthetic pyrethroids (biopermethrin and its 1S enantiomer, biotetramethrin, s-bioallethrin, bioresmethrin and its 1S enantiomer, cismethrin and kadethrin) were also studied. In contrast to the findings with deltamethrin all other compounds, apart from the 1S isomers which were inactive, induced prolonged negative (depolarizing) after-potentials. Deltamethrin appears to affect a small fraction of sodium channels which are held in a modified open-state, whereas the pyrethroids which generate large negative after-potentials appear to induce a brief alteration of the open-state sodium channels with a larger number of channels affected. Differences between the actions of pyrethroids on insect axonal sodium channels and whole insects are discussed.