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.     

High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR

The peach-potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.     

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.     

Sensitivity of the Drosophila para sodium channel to DDT is not lowered by the super-kdr mutation M918T on the IIS4-S5 linker that profoundly reduces sensitivity to permethrin and deltamethrin

DDT inhibits Na channel inactivation and deactivation, promotes Na channel activation and reduces the resting potential of Xenopus oocytes expressing the Drosophila para Na channel. These changes are only marginally influenced by the single mutation M918T (super-kdr) but are reduced approximately 10-fold by either the single mutation L1014F (kdr) or the double mutation L1014F+M918T, both of which confer resistance to the pyrethroids permethrin and deltamethrin. We conclude that DDT binds either to or in the region of L1014 on IIS6 but only weakly to M918 on the IIS4-S5 linker, which is part of a high-affinity binding site for permethrin and deltamethrin.     

Evidence for multiple origins of identical insecticide resistance mutations in the aphid Myzus persicae

The peach-potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage gated sodium channel. Kdr mutations in M. persicae are found in field populations world-wide. In order to investigate whether this situation is due to the mutations arising independently in different populations or by single mutation events that have spread by migration, regions flanking these mutations were sequenced from different geographical areas. The DNA sequences produced, which included a 1 kb intron, were found to be highly conserved. Several different haplotypes were identified containing kdr and super-kdr. Whilst these results could indicate either multiple independent origins of both mutations or recombination following a single origin, given the short timescale of resistance development, multiple independent origins of kdr and super-kdr are the most plausible interpretation.     

Temporal and spatial incidence of alleles conferring knockdown resistance to pyrethroids in the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae), and their association with other insecticide resistance mechanisms

The peach-potato aphid, Myzus persicae (sulzer), is an important arable pest species throughout the world. Extensive use of insecticides has led to the selection of resistance to most chemical classes including organochlorines, organophosphates, carbamates and pyrethroids. Resistance to pyrethroids is often the result of mutations in the para-type sodium channel protein (knockdown resistance or kdr). In M. persicae, knockdown resistance is associated with two amino-acid substitutions, L1014F (kdr) and M918T (super-kdr). In this study, the temporal and spatial distributions of these mutations, diagnosed using an allelic discriminating polymerase chain reaction assay, were investigated alongside other resistance mechanisms (modified acetylcholinesterase (MACE) and elevated carboxylesterases). Samples were collected from the UK, mainland Europe, Zimbabwe and south-eastern Australia. The kdr mutation and elevated carboxylesterases were widely distributed and recorded from nearly every country. MACE and super-kdr were widespread in Europe but absent from Australian samples. The detection of a strongly significant heterozygote excess for both kdr and super-kdr throughout implies strong selection against individuals homozygous for these resistance mutations. The pattern of distribution found in the UK seemed to indicate strong selection against the super-kdr (but not the kdr) mutation in any genotype, in the absence of insecticide pressure. There was a significant association (linkage disequilibrium) between different resistance mechanisms, which was probably promoted by a lack of recombination due to parthenogenesis.     

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.     

Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4-S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.     

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.     

First report of L1014F kdr mutation in Culex quinquefasciatus in Mexico

The L1014F mutation in the voltage‐sodium channel gene has been associated with resistance to DDT and pyrethroids in various arthropod species including mosquitoes. We determined the frequency of the L1014F kdr mutation in 16 field populations of Culex quinquefasciatus from Northeastern Mexico collected between 2008 and 2013. The L1014F was present in all populations analyzed with the lowest frequency (3.33%) corresponding to the population from Monclova collected in 2012, and the highest frequency (63.63%) from the Monterrey population collected in 2012. The presence of a kdr mutation in populations of Cx. quinquefasciatus from northeastern Mexico provides evidence of pyrethroid resistance. This requires a special attention, considering that pyrethroid‐based insecticides are commonly used in vector‐control campaigns, especially against Aedes aegypti (L.).     

Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene

Populations of Plutella xylostella, extending over 3800 km in southern Australia, show no genetic structure as assessed by microsatellite markers; yet outbreaks of pyrethroid resistance occur sporadically in cropping areas. Since mutations in the para voltage-gated sodium channel gene have been implicated in pyrethroid resistance, we looked for DNA sequence variation at this target among Australian moths. We found two resistance mutations previously reported for this species (L1014F and T929I), as well as a novel substitution (F1020S). Of the eight possible haplotypes formed by combinations of these three biallelic polymorphisms, only four were found in Australian populations: the wild-type allele (w), the kdr mutation allele (kdr) with only L1014F, the super-kdr-like combination of L1014F and T929I (skdrl), and the crashdown allele with only F1020S (cdr). Comparison of genotype frequencies among survivors of permethrin assays with those from untreated controls identified three resistant genotypes: skdrl homozygotes, cdr homozygotes and the corresponding heterozygote, cdr/skrdl - the heterozygote being at least as resistant as either homozygote. Spatial heterogeneity of allele frequencies was conspicuous, both across the continent and among local collections, consistent with reported spatial heterogeneity of pyrethroid resistance. Further, high resistance samples were sometimes associated with high frequency of cdr, sometimes high frequency of skdrl, or sometimes with a high combined cdr+skdrl frequency. The skdrl and cdr alleles explain a high proportion of the Australia-wide resistance variation. These data add to evidence that nerve insensitivity by mutations in the para-sodium channel gene is a common pyrethroid resistance mechanism in P. xylostella.     

Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae

How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.     

Novel point mutation in the sodium channel gene of pyrethroid-resistant sea lice Lepeophtheirus salmonis (Crustacea: Copepoda)

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 several different mutation sites have been identified across a range of insect species. To investigate the possibility that a kdr-type mechanism is responsible for pyrethroid resistance in sea lice, a domain II region of the Lepeophtheirus salmonis sodium channel gene was PCR amplified and sequenced. To our knowledge, this is the first published sodium channel sequence from a crustacean. Comparison of sequences from a range of samples, including several individuals from areas in which control failures had been reported, failed to identify any of the mutations within this region that have previously been linked with resistance. Instead, a novel glutamine to arginine mutation, Q945R, in transmembrane segment IIS5 was consistently found in the samples from areas of control failure and may therefore be associated with resistance to pyrethroids in this species.     

The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly sodium channels expressed in Xenopus oocytes

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of approximately 9mV and approximately 5mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel alpha subunit is located at the interface between sodium channel domains I and II.     

Evaluation of resistance to permethrin,cypermethrin and deltamethrin in different populations of Musca domestica (L.), collected from the Iranian dairy cattle farms

The house fly, Musca domestica (Linnaeus) (Diptera: Muscidae), is a major pest for human and livestock health and is also resistant to different insecticides. Herein, six M. domestica populations were collected, five of them from industrial cattle farms and the Koohrang population from a remote area as a susceptible population. The resistance/susceptibility of populations to three pyrethroids was evaluated. High levels of permethrin resistance were observed in all field populations and the resistance ratios (RRs) were estimated to vary from 52- to 129-fold. Resistant populations also exhibited resistance to other pyrethroids (cypermethrin and deltamethrin), with RRs ranging between 45- and 180-fold. According to synergistic (piperonyl butoxide, diethyl maleate and triphenyl phosphate) and enzymatic assays, resistant populations exhibited multiple resistance phenotypes. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (CarEs) were found to be involved in pyrethroid resistance in Isfahan population, P450s and GSTs in Mobarake population and CarEs detoxified pyrethroids in Natanz and Alavijeh populations. As substitution of Leucine (CTT) with Phenylalanine (TTT) at position 1014 of the voltage sensitive sodium channel (VSSC) gene is the most common mutation conferring resistance to pyrethroids in M. domestica, we sequenced a partial fragment of IIS6 and L1014F mutation was detected in all resistant populations. The present study provides valuable information for early detection of pyrethroid resistance and developing resistance management strategies in the house fly populations.     

Distribution of a knockdown resistance mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in western and southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future.     

Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides

Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.