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。     

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。     

Characterization of the M918T sodium channel gene mutation associated with strong resistance to pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer)

Recent advances in the characterisation of insect sodium channel gene sequences have identified a small number of point mutations within the channel protein that are implicated in conferring target-site resistance to pyrethroid insecticides (so-called knockdown resistance or kdr). The L1014F (leucine-to-phenylalanine) mutation located in the centre of segment 6 of the domain II region (IIS6) of the sodium channel (the so-called kdr trait) has been detected in the peach-potato aphid, Myzus persicae (Sulzer), and is considered to be the primary cause of pyrethroid resistance in this species. Here we report on the characterisation of a second mutation, M918T (methione-to-threonine), within the nearby IIS4-S5 intracellular linker (the so-called super-kdr trait) in a field clone also possessing L1014F, with both mutations present in heterozygous form. The resistance phenotype of M. persicae clones possessing various combinations of L1014F and M918T to a wide range of pyrethroids (both Type I and II) was assessed in leaf-dip bioassays and to lambda-cyhalothrin applied at up to ten times the recommended field rate as foliar sprays to aphids feeding on whole plants. Bioassay results demonstrated that presence of both mutations was associated with extreme resistance to all the pyrethroids tested relative to aphids lacking the mutations. Furthermore, this resistance well exceeded that shown by aphids that were homozygous for L1014F but lacking M918T. However, pre-treatment with piperonyl butoxide in the leaf-dip bioassays failed to suppress pyrethroid resistance in aphids carrying one or both of the mutations. The relevance of these findings for monitoring and managing pyrethroid resistance in M. persicae populations in the field is discussed.     

Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.     

Levels of cross-resistance to pyrethroids conferred by the Vssc knockdown resistance allele 410L+1016I+1534C in Aedes aegypti

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.     

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     

Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides

The gene para in Drosophila melanogaster encodes an α subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of target-site resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed.     

Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides

The gene para in Drosophila melanogaster encodes an α subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of target-site resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed. Received: 9 May 1997 / Accepted: 21 July 1997     

Inheritance of L1014F and M918T sodium channel mutations associated with pyrethroid resistance in Myzus persicae

Two amino acid substitutions (L1014F and M918T) in the voltage-gated sodium channel confer target-site resistance to pyrethroid insecticides in the peach potato aphid, Myzus persicae. Pyrethroid-resistant and -susceptible M. persicae clones with various combinations of these mutations were crossed under laboratory conditions, and the genotypes of aphid progeny were analysed by direct DNA sequencing of the IIS4-S6 region of the sodium channel gene. Segregation patterns showed that in aphids heterozygous for both L1014F and M918T, both mutations were present in the same resistance allele. Despite these mutations appearing largely recessive in other pest species, such aphids exhibited strong resistance to pyrethroids in leaf-dip bioassays. These results have important implications for the spread and management of pyrethroid resistance in field populations.