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。     

甜菜夜蛾抗药性研究现状

就有关甜菜夜蛾Spodoptera exigua(Hbner)对常用杀虫剂的抗药性现状作综述。甜菜夜蛾对拟除虫菊酯类、有机磷类、氨基甲酸酯类杀虫剂已产生了较高水平的抗药性,其中山东泰安抗性种群对氯氟氰菊酯的抗性高达2445.5倍;对多杀菌素等生物杀虫剂产生了中低水平的抗性;对昆虫生长调节剂如虫酰肼的敏感性也有所降低,但昆虫生长调节剂依然是比较理想的防治药剂。对交互抗性及抗性治理也作了阐述。     

钠离子通道疾病及其抑制剂生物学功能研究进展

电压门控型钠离子通道(Voltage-gated sodium channel,VGSC)广泛分布于兴奋性细胞,是电信号扩大和传导的主要介质,在神经细胞以及心肌细胞兴奋传导等方面发挥重要作用。钠离子通道结构和功能的异常会改变细胞的兴奋性,从而导致多种疾病的发生,如神经性疼痛、癫痫,以及心律失常等。目前临床上多采用钠离子通道抑制剂治疗上述疾病。近些年,研究人员陆续从动物的毒液中分离纯化出具有调控钠离子通道功能的神经毒素。这些神经毒素多为化合物或小分子多肽。现已有医药研发公司将这些天然的神经毒素进行定向设计改造成钠离子通道靶向药物用于临床疾病的治疗。此外,来源于七鳃鳗Lampetra japonica口腔腺的富含半胱氨酸分泌蛋白(Cysteine-rich buccal gland protein,CRBGP)也首次被证明能够抑制海马神经元和背根神经元的钠离子电流。以下针对钠离子通道疾病及其抑制剂生物学功能的最新研究进展进行分析归纳。     

Sequence and transcript expression of the super-kdr locus of the horn fly,Haematobia irritans

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.     

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。     

Mutations in the Bemisia tabaci para sodium channel gene associated with resistance to a pyrethroid plus organophosphate mixture

The voltage-gated sodium channel is the primary target site of pyrethroid insecticides. In some insects, super knockdown resistance (super-kdr) to pyrethroids is caused by point mutations in the linker fragment between transmembrane segments 4 and 5 of the para-type sodium channel protein domain II (IIS4-5). Here, we identify two mutations in the IIS4-5 linker of the para-type sodium channel of the whitefly, Bemisia tabaci: methionine to valine at position 918 (M918V) and leucine to isoleucine at position 925 (L925I). Although each mutation was isolated independently from strains >100-fold resistant to a pyrethroid (fenpropathrin) plus organophosphate (acephate) mixture, only L925I was associated with resistance in strains derived from the field in 2000 and 2001. The L925I mutation occurred in all individuals from nine different field collections that survived exposure to a discriminating concentration of fenpropathrin plus acephate. Linkage analysis of hemizygous male progeny of unmated heterozygous F1 females (L925I×wild-type) shows that the observed resistance is tightly linked to the voltage-gated sodium channel locus. The results provide a molecular tool for better understanding, monitoring and managing pyrethroid resistance in B. tabaci.     

Equivalent intensity but differential dominance of sodium channel blocker insecticide resistance conferred by F1845Y and V1848I mutations of the voltage-gated sodium channel in Plutella xylostella

Two point mutations (F1845Y and V1848I) in the voltage-gated sodium channel gene of Plutella xylostella are involved in the target-site resistance to sodium channel blocker insecticides (SCBIs). The contribution of the individual mutations to the SCBI resistance and the associated inheritance modes is as yet unclear. Through 2 rounds of single-pair crossing and marker-assisted selection, 2 P. xylostella strains (1845Y and 1848I) bearing homozygous F1845Y or V1848I mutant alleles were successfully established from a field-collected population, and the contribution of each mutation to SCBI resistance, as well as associated inheritance patterns, was determined. When compared with the susceptible SZPS strain, each of the mutations individually conferred equally high-level resistance to indoxacarb (378 and 313 fold) and metaflumizone (734 and 674 fold), respectively. However, dominance levels of resistance to SCBIs were significantly different between the 2 resistant strains. Resistance of the 1845Y strain to indoxacarb and metaflumizone was inherited as an autosomal and incompletely dominant trait (D values ranged from 0.43 to 0.76). In contrast, that of the 1848I strain followed an autosomal but incompletely recessive to semidominant mode (D values: −0.24 to 0.09). Our findings enriched the current understanding of inheritance and mechanisms of SCBI resistance in P. xylostella, and will help develop resistance management programs for P. xylostella and other economic pests.     

CRISPR/Cas9 mediated G4946E substitution in the ryanodine receptor of Spodoptera exigua confers high levels of resistance to diamide insecticides

Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyR^G4946E substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyR^G4946V mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyR^G4946E mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyR^G4946E mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyR^G4946E mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyR^G4946E mutation.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.     

昆虫击倒抗性基因突变对钠通道功能的影响

该文综述了昆虫钠通道基因的表达与功能特性、击倒抗性突变的功能和这些突变对钠通道门控的影响,以及钠通道基因突变与抗性表现型之间的因果关系;还讨论了这些突变增强击倒抗性的分子机理。     

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth,Plutella xylostella

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field‐evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750‐fold more resistant to indoxacarb and 70‐fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNa_v protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field‐collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34‐ to 870‐fold) and metaflumizone (1‐ to 70‐fold). The two mutations were never found to co‐exist in the same allele of PxNa_v, suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.     

与拟除虫菊酯抗性相关的烟粉虱钠通道基因突变及其检测

通过RT-PCR克隆了烟粉虱Bemisia tabaci (Gennadius) 南京种群(B-生物型)的钠离子通道结构域ⅡS4-6 cDNA片段,证实了与拟除虫菊酯抗性相关的是位于第925位亮氨酸到异亮氨酸的突变(L925I),并建立了L925I突变的PASA检测技术。与SUD-S敏感品系相比,2002年采自南京棉花上的烟粉虱种群对氯氰菊酯具有77倍的抗性,用氯氰菊酯对该种群进行多次筛选后,该种群对氯氰菊酯的抗药性提高到227倍。PASA检测结果表明筛选后的南京种群中100%个体都具有L925I突变(61.1%的个体为L925I突变纯合子,38.9%的个体为杂合子),而未筛选的南京种群只有75%个体具有L925I突变(35%个体为L925I突变纯合子,40%的个体为杂合子,25%的个体为野生型)。该结果表明了烟粉虱钠离子通道L925I突变与对拟除虫菊酯抗性密切相关。还讨论了烟粉虱对拟除虫菊酯抗性的代谢机理。     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene

Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth-instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S-transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock-down resistant (kdr)-type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage-gated sodium channel gene was obtained by RT-PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.     

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field‐collected population of Spodoptera exigua. The field‐collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH‐S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH‐S strain by crossing WF with WH‐S, followed by three rounds of backcrossing with WH‐S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH‐S strain. Compared with WH‐S, the near‐isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.