Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI     

The sodium channel gene in Tetranychus cinnabarinus (Boisduval): identification and expression analysis of a mutation associated with pyrethroid resistance

BACKGROUND: The carmine spider mite (CSM), Tetranychus cinnabarinus, is the most harmful mite pest of various crops and vegetable plants. Pyrethroid insecticide fenpropathrin has been used to control insects and mites worldwide, but CSM has developed resistance to this compound. RESULTS: Three synergists together eliminated about 50% resistance against fenpropathrin in the CSM. A point mutation was identified from the sodium channel gene of fenpropathrin‐resistant CSM (FeR) by comparing cDNA sequences between FeR and susceptible (S) sodium channel genes, which caused a phenylalanine (F) to isoleucine (I) change at amino acid 1538 position in IIIS6 of the sodium channel and has been proven to confer strong resistance to pyrethroid in other species. The mRNA expression of the sodium channel gene in the FeR and abamectin‐resistant strain (AbR), which was included as a control, were both relatively lower than in the S. CONCLUSION: These results demonstrate that a mutation (F1538I) is present in the sodium channel gene in FeR of CSM, likely playing an important role in fenpropathrin resistance in T. cinnabarinus, but that decrease in the abundance of sodium channel did not confer this resistance. The F1538I mutation could be used as a molecular marker for detecting kdr resistance in Arachnida populations. Copyright © 2011 Society of Chemical Industry     

Voltage‐sensitive sodium channel mutations S989P + V1016G in Aedes aegypti confer variable resistance to pyrethroids,DDT and oxadiazines

BACKGROUND

Aedes aegypti is a vector of several important human pathogens. Control efforts rely primarily on pyrethroid insecticides for adult mosquito control, especially during disease outbreaks. A. aegypti has developed resistance nearly everywhere it occurs and insecticides are used. An important mechanism of resistance is due to mutations in the voltage‐sensitive sodium channel (Vssc) gene. Two mutations, in particular, S989P + V1016G, commonly occur together in parts of Asia.

RESULTS

We have created a strain (KDR:ROCK) that contains the Vssc mutations S989P + V1016G as the only mechanism of pyrethroid resistance within the genetic background of Rockefeller (ROCK), a susceptible lab strain. We created KDR:ROCK by crossing the pyrethroid‐resistant strain Singapore with ROCK followed by four backcrosses with ROCK and Vssc S989P + V1016G genotype selections. We determined the levels of resistance conferred to 17 structurally diverse pyrethroids, the organochloride DDT, and oxadiazines (VSSC blockers) indoxacarb (proinsecticide) and DCJW (the active metabolite of indoxacarb). Levels of resistance to the pyrethroids were variable, ranging from 21‐ to 107‐fold, but no clear pattern between resistance and chemical structure was observed. Resistance is inherited as an incompletely recessive trait. KDR:ROCK had a > 2000‐fold resistance to DDT, 37.5‐fold cross‐resistance to indoxacarb and 13.4‐fold cross‐resistance to DCJW.

CONCLUSION

Etofenprox (and DDT) should be avoided in areas where Vssc mutations S989P + V1016G exist at high frequencies. We found that pyrethroid structure cannot be used to predict the level of resistance conferred by kdr. These results provide useful information for resistance management and for better understanding pyrethroid interactions with VSSC. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Mutations in the sodium channel associated with pyrethroid resistance in the greenhouse whitefly, Trialeurodes vaporariorum

BACKGROUND: Trialeurodes vaporariorum Westwood is an important pest of protected crops in temperate regions of the world. Resistance to pyrethroid insecticides is long established in this species, but the molecular basis of the mechanism(s) responsible has not previously been disclosed. RESULTS: Mortality rates of three European strains of T. vaporariorum to the pyrethroid bifenthrin were calculated, and each possessed significant resistance (up to 662‐fold) when compared with a susceptible reference strain. Direct sequencing revealed three amino acid substitutions in the para‐type voltage‐gated sodium channel (the pyrethroid and DDT target site) of bifenthrin‐resistant T. vaporariorum at positions previously implicated with pyrethroid or DDT resistance (M918L, L925I and T929I) in other related species. CONCLUSION: This study indicates that resistance to bifenthrin in T. vaporariorum is associated with target‐site insensitivity, and that the specific mutations in the sodium channel causing resistance may differ between localities. Copyright © 2012 Society of Chemical Industry     

海南普通大蓟马对拟除虫菊酯药剂抗性监测及钠通道突变检测

普通大蓟马Megalurothrips usitatus在海南省对豇豆造成严重危害且抗药性逐渐增强。本研究测定了2019年至2021年海南省普通大蓟马对氯菊酯和甲氰菊酯的抗性。结果表明,海口、乐东和三亚3个地理种群对甲氰菊酯处于极高水平抗性,对氯菊酯处于高水平抗性,且抗性逐年增强。对普通大蓟马钠离子通道序列分析发现存在M283R突变,该突变位于钠离子通道同源结构域Ⅰ。突变频率检测显示,2019年至2021年连续3年海口种群该突变位点的突变频率分别为1/30、1/30、3/30,有升高趋势。本研究发现海南省普通大蓟马从2019年到2021年对拟除虫菊酯类药剂的抗药性呈逐年上升趋势。     

Pyrethroid resistance in the tomato red spider mite, Tetranychus evansi, is associated with mutation of the para-type sodium channel

BACKGROUND: The tomato red spider mite, Tetranychus evansi (Baker and Pritchard), is a serious pest of solanaceous crops in many African countries. In this study an investigation has been conducted to establish whether mutation of the para‐type sodium channel underlies pyrethroid resistance in T. evansi strains collected in Southern Malawi. RESULTS: Two T. evansi strains from Malawi showed tolerance to the organophosphate chlorpyrifos and resistance (20–40‐fold) to the pyrethroid bifenthrin, but were susceptible to two contemporary acaricides (abamectin and fenpyroximate) in insecticide bioassays. Cloning of a 3.1 kb fragment (domains IIS5 to IVS5) of the T. evansi para gene from pyrethroid‐resistant and pyrethroid‐susceptible strains revealed a single non‐synonymous mutation in the resistant strains that results in an amino acid substitution (M918T) within the domain II region of the channel. Although novel to mites, this mutation confers high levels of resistance to pyrethroids in several insect species where it has always been associated with another mutation (L1014F). This is the first report of the M918T mutation in the absence of L1014F in any arthropod species. Diagnostic tools were developed that allow sensitive detection of this mutation in individual mites. CONCLUSION: This is the first study of pyrethroid resistance in T. evansi and provides contemporary information for resistance management of this pest in Southern Malawi. Copyright © 2011 Society of Chemical Industry     

A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance

Sodium channel mutations were investigated through nucleotide sequencing of three cDNA fragments amplified from permethrin resistant and susceptible Aedes aegypti from northern Thailand. There was a novel nucleotide substitution (T → G) at the second position of codon 1552 resulting in the replacement of Phenylalanine by Cysteine in segment 6 domain III. This amino acid was indicated by another study to involve an aromatic-aromatic contact between the sodium channel protein and the first aromatic ring of the pyrethroid alcohol moiety. Reciprocal crosses between the homozygous parental susceptible and resistant strains indicated that resistance was autosomal and incompletely recessive, and highly associated with the homozygous mutation. The bioassay of the F₂ progeny, formed by backcrossing the F₁ with the resistant parental strain, did not show a clear plateau curve across the range of doses, suggesting that resistance to permethrin was controlled by more than one gene locus. Other possible resistance mechanisms involved are discussed.     

Use of biochemical and DNA diagnostics for characterising multiple mechanisms of insecticide resistance in the peach-potato aphid,Myzus persicae (Sulzer)

The peach-potato aphid Myzus persicae (Sulzer) can resist a range of insecticides by over-producing detoxifying esterase and having mutant-insensitive forms of the target proteins, acetylcholinesterase (AChE), and the sodium channel. Using a combination of bioassays, biochemical and DNA diagnostics, it is now possible to diagnose all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. A survey of 58 samples of wide geographic origin showed that all 46 resistant clones had amplified esterase genes (E4 or FE4) conferring broad-spectrum resistance to pyrethroids, organophosphates and carbamates. These occurred in combination with insensitive AChE (11 clones), conferring resistance to pirimicarb and triazamate, and/or mutant sodium channel genes (25 clones), conferring knockdown (kdr) resistance to pyrethroids and DDT. Amplified esterase genes were in linkage disequilibrium with both insensitive AChE and the kdr mutation, reflecting tight physical linkage, heavy selection favouring aphids with multiple mechanisms, and/or the prominence of parthenogenesis in many M. persicae populations. An ability to monitor individual mechanisms with contrasting cross-resistance profiles has important implications for the development of resistance management recommendations. ©1997 SCI     

A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.