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.     

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.     

New methods for the detection of insecticide resistant Myzus persicae in the U.K. suction trap network

1  Myzus persicae is a highly polyphagous pest of U.K. agriculture. It presents particular control difficulties because it has developed resistance to several insecticide classes.
2 For almost 20 years, M. persicae collected in the U.K. suction trap network have been analysed for insecticide resistance and the data disseminated to growers via a resistance bulletin. These data are generated by the biochemical analysis of individuals for two major resistance phenotypes: (i) elevated carboxylesterase and (ii) modified acetylcholinesterase (MACE).
3 The development of new polymerase chain reaction (PCR)-based technologies using fluorescently labelled probes has allowed other resistance mechanisms, such as knockdown resistance to pyrethroids (kdr/super-kdr), to be detected and has greatly increased the speed and accuracy of resistance monitoring. Unfortunately, these newer PCR-based assays are incompatible with the older biochemical assays.
4 The present study describes the development and testing of new compatible methods for detecting elevated carboxylesterases and MACE for use on M. persicae caught in the field or suction traps.
5 These new tests have significant advantages over present methodologies by allowing individual aphids to be tested for three resistance mechanisms quickly and accurately on a single platform.     

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.     

Dynamics and mechanisms of permethrin resistance in a field population of the horn fly, Haematobia irritans irritans

A study was conducted at the Pressler ranch, near Kerrville, Texas, USA between 2002 and 2006 to determine the dynamics and mechanisms of resistance to permethrin in a field population of the horn fly, Haematobia irritans irritans (L.). Changes of resistance to pyrethroid insecticide associated with use of a pour-on formulation of cyfluthrin in 2002 and use of diazinon ear tags in subsequent years were studied using a filter paper bioassay technique and a polymerase chain reaction assay that detects two sodium channel mutations, kdr and super-kdr resistance alleles. A maximum of 294-fold resistance to permethrin was observed in the summer of 2002. A significant decrease in the resistance level was observed in spring 2003, and resistance continued to decline after animals were treated with diazinon ear tags. In response to pyrethroid treatments, the allelic kdr and super-kdr frequency increased from 56.3% to 93.8% and from 7.5% to 43.8%, respectively in 2002, and decreased significantly in 2003 when the pyrethroid insecticide was no longer used to treat animals. Females were found to have a higher allelic super-kdr frequency than males in 2002, while no difference was detected between males and females in the allelic kdr frequency. There was a significant positive correlation between frequencies of the sodium channel mutations and levels of permethrin resistance, suggesting that the sodium channel mutations, kdr and super-kdr , are the major mechanisms of resistance to pyrethroids in this horn fly population. Results of synergist bioassays also indicated possible contributions of two metabolic detoxification mechanisms, the mixed function oxidases (MFO) and glutathione S-transferases (GST). Compared to a horn fly infestation of an untreated herd, treatments with the pyrethroid pour-on formulation failed to control horn flies at the Pressler ranch in 2002. Sustained control of horn flies was achieved with the use of diazinon ear tags in 2003 and subsequent years.     

Clonal turnover of MACE-carrying peach-potato aphids (Myzus persicae (Sulzer), Homoptera: Aphididae) colonizing Scotland

Peach-potato aphids, Myzus persicae (Sulzer), collected in Scotland in the years 1995 and 2002-2004 were characterized using four microsatellite loci and three insecticide resistance mechanisms. From 868 samples, 14 multilocus genotypes were defined (designated clones A-N). Five of these (denoted A, B, H, M and N) carried modified acetylcholinesterase (MACE) resistance, the most recent resistance mechanism to have evolved in M. persicae. The current paper shows that the continued presence of MACE aphids is due to turnover, as clones A and B were replaced in field samples by clones H, M and N in later seasons. Thus, insecticide-resistant populations in Scotland can be attributed to multiple waves of rapid clone colonisations and not to the continued presence of stable resistant clones or mutation or sexual recombination in local populations. The MACE clones carried varying levels of the other insecticide resistance mechanisms, kdr and esterase. The presence of these mechanisms could alter the clones success in the field depending on insecticide spraying (positive selection) and resistance fitness costs (negative selection).     

The attack of the clones: tracking the movement of insecticide-resistant peach-potato aphids Myzus persicae (Hemiptera: Aphididae)

Myzus persicae (Sulzer) collected in Scotland were characterized for four microsatellite loci, intergenic spacer fingerprints and the resistance mechanisms modified acetylcholinesterase (MACE), overproduced carboxylesterase and knockdown resistance (kdr). Microsatellite polymorphisms were used to define a limited number of clones that were either fully susceptible to insecticides or possessed characteristic combinations of resistance mechanisms. Within these clones, intergenic spacer fingerprints could either be very consistent or variable, with the latter indicating ongoing evolution within lineages, most likely derived from the same zygote. Two clones (termed A and B) possessed all three resistance mechanisms and predominated at sites treated with insecticides. Their appearance on seed potatoes and oilseed rape in Scotland in 2001 coincided with extensive insecticide use and severe control failures. Clones C, I and J, with no or fewer resistance mechanisms, were found in samples from 1995 and were dominant at untreated sites in 2001. A comparison of Scottish collections with those from other UK and non-UK sites provides insight into the likely origins, distribution and dynamics of M. persicae clones in a region where asexual (anholocyclic) reproduction predominates, but is vulnerable to migration by novel genotypes from areas of Europe where sexual (holocyclic) reproduction occurs.     

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.     

Relationship between the para-homologous sodium channel point mutation (g --> c at nucleotide 2979) and knockdown resistance in the German cockroach using multiplex polymerase chain reaction to discern genotype

Extensive use of pyrethroid insecticides for urban pest control has led to widespread pyrethroid resistance in the German cockroach. A mutation at nucleotide position 2979 (G to C, causing a leucine to phenylalanine change) in the S6 transmembrane segment of domain II of the para-homologous voltage-gated sodium channel has been previously identified in knockdown-resistant cockroaches and demonstrated by site-directed mutagenesis to reduce channel sensitivity to pyrethroids. In a recent survey, 83% of pyrethroid-resistant German cockroach populations were found to possess this mutation. A German cockroach strain with a low incidence of the L993F mutation was subjected to selection pressure with cypermethrin and subsequently evaluated over several generations for the knockdown resistance phenotype. Correspondingly, we determined the genotype of individual cockroaches of each population at the 2979 position of the para-homologous gene. Genotype was discerned by development of a polymerase chain reaction method that employed a mismatched primer-template set. A direct relationship was observed between mean knockdown time and the presence of the kdr mutation. Furthermore, individuals homozygous for the kdr mutation exhibited a significantly higher mean knockdown time than heterozygotes or wildtype cockroaches. This is the first report demonstrating the progressive expression of the kdr allele in response to insecticide selection pressure.     

Analogous pleiotropic effects of insecticide resistance genotypes in peach-potato aphids and houseflies

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach-potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.     

Incidence of insecticide resistance alleles in sexually-reproducing populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae) from southern France

Intensive chemical treatments have led to the development of a number of insecticide resistance mechanisms in the peach-potato aphid Myzus persicae (Sulzer). Some of these mechanisms are known to be associated with negative pleiotropic effects (resistance costs). Molecular and biochemical methods were used to determine the genotypes or phenotypes associated with four insecticide resistance mechanisms in single aphids from sexually-reproducing populations in southern France. The mechanisms considered were E4 and FE4 carboxylesterase overproduction, modified acetycholinesterase, and kdr and rdl resistance-associated mutations. A new method for determining individual kdr genotypes is presented. Almost all resistant individuals overproduced FE4 carboxylesterase, whereas modified acetylcholinesterase was rare. Both the kdr and rdl resistance mutations were present at high frequencies in French sexually-reproducing populations. The frequencies of insecticide resistance genes were compared before and after sexual reproduction in one peach orchard at Avignon to evaluate the potential impact of selection on the persistence of resistance alleles in the over-wintering phase. The frequencies of the kdr and rdl mutations varied significantly between autumn and spring sampling periods. The frequency of the kdr mutation increased, probably due to pyrethroid treatments at the end of the winter. Conversely, the frequency of the rdl mutation decreased significantly during winter, probably because of a fitness cost associated with this mutation.     

Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin

Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para(74) and para(DN7) fly lines showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para(74) lies within the S6 transmembrane region and the amino acid substitution associated with para(DN7) lies within the S4-S5 linker region. These sites are analogous to the mutations in domain II underlying knockdown resistance (kdr) and super-kdr, naturally occurring forms of pyrethroid resistance found in houseflies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para(74) embryos placed in primary culture for three days to two weeks. The mutant para(74) sodium currents were kinetically similar to wild type currents, in activation, inactivation and time to peak. The only observed difference between para(74) and wild-type neurons was in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect on para(74) mutant sodium channels.     

Genetic diversity and insecticide resistance of Myzus persicae (Hemiptera: Aphididae) populations from tobacco in Chile: evidence for the existence of a single predominant clone

The tobacco-feeding race of Myzus persicae (Sulzer), formerly known as M. nicotianae Blackman, was introduced into Chile during the last decade. In order to evaluate the genetic diversity and insecticide resistance status of Chilean tobacco aphid populations, a field survey was conducted in 35 tobacco fields covering a 300 km latitudinal survey. The populations sampled were characterized using microsatellite markers and morphometric multivariate analysis. Insecticide resistance levels were assessed through a microplate esterase assay and the mutation status of the kdr gene. All samples collected corresponded to the same anholocyclic aphid genotype, and showed morphological variation within the range expected for the tobacco-feeding race of M. persicae. Esterase activity showed the level and variability expected for an R1 clone lacking mutations in the sodium channels (susceptible kdr), thus corresponding to a type slightly resistant to organophosphate and carbamate, and susceptible to pyrethroid insecticides.     

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.     

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.     

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.     

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.