Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostella

BACKGROUND: The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. RESULTS: Frequencies of pyrethroid‐resistant alleles (L1014F, T929I and M918I) and two resistance‐related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real‐time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid‐resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. CONCLUSION: Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field. Copyright © 2010 Society of Chemical Industry     

Over-expression of cytochrome P450 CYP6B7 mRNA and pyrethroid resistance in Australian populations of Helicoverpa armigera (Hübner)

Three cDNA clones for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7 which have 84–87% protein sequence identity have been isolated previously from Helicoverpa armigera, and the CYP6B7 mRNA was found to be over-expressed in a pyrethroid-resistant field population. Subsequent analysis has shown that over-expression is observed in a majority of individuals in all populations tested. Single-pair crosses between resistant and sensitive individuals indicated that the pyrethroid resistance co-segregated with the over-expression of this mRNA. Southern analysis indicated that the over-expression, which was confined to midgut only in many insects, was not related to gene amplification. These observations add weight to the conclusion that CYP6B7 is the cytochrome P450 form involved in pyrethroid resistance, and that over-expression of cytochrome P450 CYP6B7 is a common cause of pyrethroid resistance in H. armigera. The results suggest that specific probes for CYP6B7 protein or mRNA could provide the basis for the development of tools for monitoring pyrethroid resistance due to mixed function oxidase activity in field populations of this insect. © 1998 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     

棉铃虫para同源基因III-IV接头DNA片段序列分析

利用反转录 PCR技术 ,用一对特异性寡核苷酸引物 ,分离获得棉铃虫 para同源基因 III- IV接头约30 0 bp DNA片段 ,发现在 Bao D- R和 Bao D- S品系间存在 4个核苷酸差异 ,但在推导的氨基酸组成上没有差别。对比分析表明 ,分离获得的棉铃虫 III- IV接头氨基酸组成与烟芽夜蛾 hscp片段同一区域有 98.1%的氨基酸相同 ,与德国蜚蠊 CSMA的氨基酸有 93.5%相同 ,与果蝇 para基因有 88.9%的氨基酸相同     

State-dependent modification of voltage-gated sodium channels by pyrethroids

Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor.     

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     

Sodium channel mutations (T929I and F1534S) found in pyrethroid-resistant strains of the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

RNA-seq data analysis of cigarette beetle (Lasioderma serricorne) strains having different sensitivities to pyrethroids identified sodium channel mutations in strains showing pyrethroid resistance: the T929I and F1534S mutations. These results suggest that reduced sensitivity of the sodium channel confers the pyrethroid resistance of L. serricorne. Results also showed that the F1534S mutation mostly occurred concurrently with the T929I mutation. The functional relation between both mutations for pyrethroid resistance is discussed.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

The pyrethroid resistance status and mechanisms in Aedes aegypti from the Guerrero state,Mexico

Dengue is one of the most important vector-borne diseases worldwide and is a public health problem in Mexico. Most programs in dengue endemic countries rely on insecticides for Aedes control. In Mexico, pyrethroid insecticides (mainly permethrin and deltamethrin) have been extensively used over a decade as adulticides and represented a strong selection for insecticide resistance for dengue vectors in several parts of the country. We studied the type, frequency and distribution of insecticide resistance mechanisms in Aedes aegypti from six municipalities in the state of Guerrero selected on the basis of historically intense chemical control and a high risk for dengue transmission. Ae. aegypti eggs were collected from October 2009 to January 2010 using ovitraps. F₁ adults, emerged from these collections, were exposed to permethrin, deltamethrin and DDT in WHO diagnostic tests and showed high resistance levels to both pyrethroids and DDT. This was consistent with the presence of increased metabolic enzyme activities and target site insensitivity due to kdr mutations. Biochemical assays showed elevated esterase and glutathione S-transferase activities in the six municipalities. The V1016I kdr mutation on the IIS6 domain of the sodium channel gene was present in an overall frequency of 0.80. A second mutation, F1534C on the IIIS6 domain of the same gene was also detected, being the first report of this mutation in Guerrero. The multiple resistance mechanisms present in Ae. aegypti from Guerrero state represent a warning for the efficacy of the pyrethroid usage and consequently for the success of the dengue control program.     

Genetic Analysis of Metalaxyl Insensitivity Loci in Phytophthora infestans Using Linked DNA Markers

ABSTRACT Previous studies indicated that incompletely dominant loci determine insensitivity by oomycetes to phenylamide fungicides such as metalaxyl. To compare the bases of insensitivity in different strains of the late blight pathogen, Phytophthora infestans, crosses were performed between sensitive isolates and isolates from Mexico, the Netherlands, and the United Kingdom that displayed varying levels of insensitivity. Segregation analyses indicated that metalaxyl insensitivity was determined primarily by one locus in each isolate, and that two of the isolates were heterozygous and the other homozygous for the insensitive allele. Metalaxyl insensitivity was also affected by the segregation of additional loci of minor effect. DNA markers linked to insensitivity were obtained by bulked segregant analysis using random amplified polymorphic DNA (RAPD) markers and the Dutch and Mexican crosses. By studying the linkage relationships between these markers and the insensitivity in each cross by RAPD or restriction fragment length polymorphism analysis, it appeared that the same chromosomal locus conferred insensitivity in the Mexican and Dutch isolates. However, a gene at a different chromosomal position was responsible for insensitivity in the British isolate.     

Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction

BACKGROUND: Pediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin‐ and pyrethroid‐based pediculicides is due to knockdown (kdr)‐type point mutations in the voltage‐sensitive sodium channel α‐subunit gene. Early detection of resistance is crucial for the selection of effective management strategies. RESULTS: Kdr allele frequencies of lice from 14 countries were determined using the serial invasive signal amplification reaction. Lice collected from Uruguay, the United Kingdom and Australia had kdr allele frequencies of 100%, while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining seven countries investigated, including seven US populations, two Argentinian populations and populations from Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11 to 97%. CONCLUSION: The newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr‐type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin‐ and pyrethroid‐based pediculicides, the T917I mutation appears to be a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined. Copyright © 2010 Society of Chemical Industry     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

基于转录组测序解析草地贪夜蛾对溴氰虫酰胺的解毒代谢分子机制

为阐明草地贪夜蛾Spodoptera frugiperda对溴氰虫酰胺的解毒代谢分子机制,通过LC₅₀的溴氰虫酰胺诱导草地贪夜蛾3龄幼虫后,利用酶活测定和转录组测序鉴定解毒代谢相关基因,并采用实时荧光定量PCR技术对细胞色素P450单加氧酶(cytochrome P450 monooxygenase,P450)基因进行验证分析。结果表明,经LC₅₀的溴氰虫酰胺处理后,草地贪夜蛾3龄幼虫体内3种解毒代谢酶活性较对照均有所升高,但仅P450活性较对照显著升高,而谷胱甘肽S-转移酶和羧酸酯酶与对照无显著差异。经LC₅₀的溴氰虫酰胺处理后草地贪夜蛾3龄幼虫转录组中共筛选到1 408个差异表达基因,其中上调表达的基因有935个,下调表达的基因有473个。药物代谢-细胞色素P450通路、药物代谢-其他酶通路及细胞色素P450对异生物质的代谢通路中有超过20个基因存在差异表达。在草地贪夜蛾转录组中筛选鉴定到121个P450基因,其中,属于CYP2、CYP3、CYP4以及Mito家簇的基因分别有9、45、58和9个,而经LC_5...     

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     

Actions of Tefluthrin on Rat Na(v)1.7 Voltage-Gated Sodium Channels Expressed in Xenopus Oocytes

In rats expression of the Na_v1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Na_v1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 μM modified of Na_v1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked “late current” at the end of a 40 ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to twofold by the application of a train of up to 100 5 ms depolarizing prepulses. These effects of tefluthrin on Na_v1.7 channels were qualitatively similar to its effects on rat Na_v1.2, Na_v1.3 and Na_v1.6 channels assayed previously under identical conditions. However, Na_v1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Na_v1.3 and Na_v1.6 channels. It is likely that Na_v1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Na_v1.3, Na_v1.6 and Na_v1.8) and two pyrethroid-resistant isoforms (rat Na_v1.2 and Na_v1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.     

Efficacy of the pyrethroid alpha-cypermethrin against Bactrocera oleae populations from Greece, and improved diagnostic for an iAChE mutation

BACKGROUND: The most important pest of olive orchards worldwide is the olive fruit fly Bactrocera oleae (Gmelin). Its control in Greece has been based on organophosphates (OPs), but their intense use has led to the development of resistance. A test previously developed to monitor the trait may not be as robust as originally thought. The pyrethroid alpha-cypermethrin has recently been registered for bait sprays, as an alternative to OPs. RESULTS: The susceptibility of 20 B. oleae populations to alpha-cypermethrin was examined. Variation was observed in their response, with LD(50) ranging from 0.14 to 3.28 ng insect(-1) and resistance factors from 2.3 to 54.7. Resistance mechanisms were investigated. Cytochrome P450 monoxygenase activities showed an association with resistance. Sequences in the domain IIS4-IIS6 of the B. oleae para-type sodium channel were also analysed, but no resistance-associated mutations were identified. Finally, a novel diagnostic assay able to reliably monitor the frequency of the iAChE G488S resistance mutation was developed. CONCLUSION: This is the first attempt to evaluate the efficacy of alpha-cypermethrin against B. oleae from Greece. Data showed that it can be used effectively, but also highlighted the importance of continuous monitoring. The IIS4-IIS6 sodium channel region is the default area in which to look for resistance mutations if target-site resistance to pyrethroids arises. The application of the novel iAChE molecular diagnostic may facilitate the introduction of pyrethroids alongside OPs currently in use.     

Pyrethroids, knockdown resistance and sodium channels

Knockdown resistance to DDT and the pyrethrins was first described in 1951 in the housefly (Musca domestica L.). This trait, which confers reduced neuronal sensitivity to these insecticides, was subsequently shown to confer cross-resistance to all synthetic pyrethroid insecticides that have been examined to date. As a consequence, the worldwide commercial development of pyrethroids as a major insecticide class over the past three decades has required constant awareness that pyrethroid overuse has the potential to reselect this powerful resistance mechanism in populations that previously were resistant to DDT. Demonstration of tight genetic linkage between knockdown resistance and the housefly gene encoding voltage-sensitive sodium channels spurred efforts to identify gene mutations associated with knockdown resistance and understand how these mutations confer a reduction in the sensitivity of the pyrethroid target site. This paper summarizes progress in understanding pyrethroid resistance at the molecular level, with particular emphasis on studies in the housefly.     

Genetics of Pyrethroid Resistance in a Strain (ALHF) of House Flies (Diptera: Muscidae)

Five house fly lines were derived from crosses of the pyrethroid-resistant ALHF (wildtype) and the susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each line was homozygous for one mutant-type marker from aabys. The level of resistance to permethrin was measured for each line to determine the genetic linkage of pyrethroid resistance in ALHF. Permethrin resistance in ALHF was 6600-fold compared with that in aabys. Resistance in flies bearing a mutant-type marker on autosome 4 was similar to that in ALHF. Flies with mutant-type markers on autosomes 1 and 2 had relatively lower resistance than ALHF; flies with mutant-type markers on autosomes 3 and 5 had much lower levels of resistance. These results demonstrated that factors on autosomes 3 and 5 play very important roles in pyrethroid resistance, whereas factors on autosomes 1 and 2 may have relatively small roles in resistance. Piperonyl butoxide (PBO) increased toxicity of permethrin in strains with mutant-type markers on autosomes 3 and 4 similar to that in ALHF. Slightly decreased synergism ratios in strains with autosomes 1 and 2 mutant-type markers compared with ALHF indicated that factors on autosomes 1 and 2 might make a small contribution in P450 monooxygenase-mediated resistance. However, when the autosome 5 mutant-type marker was present, PBO did not substantially decrease resistance, suggesting that the factor(s) on autosome 5 plays the most important role in P450 monooxygenase-mediated resistance. The resistance ratios of permethrin + PBO in strains with mutant-type markers on autosomes 1, 2, and 5 were significantly lower than those in ALHF, suggesting that factors on autosomes 1, 2, and 5 might be involved in pyrethroid resistance mechanisms other than P450-mediated detoxication. Injection did not change levels of resistance in the house flies tested, revealing that decreased rate of cuticular penetration (pen) probably does not play an important role in pyrethroid resistance in ALHF. The interaction and regulation of different mechanisms and/or factors involved in pyrethroid resistance in house flies are discussed.