Modified Acetylcholinesterase Research Articles

Structuration of multilocus genotypes associated with insecticide resistance of the peach potato aphid, Myzus persicae (Sulzer), in potato fields in southern Belgium.

The peach potato aphid, Myzus persicae, has developed resistance to many insecticides. In Belgium, M. persicae is one of the most common aphids in potato fields and one of the most effective virus vectors. We monitored resistance mutations to pyrethroids, carbamates and neonicotinoids and related these results to microsatellite genotyping to provide information to support the choice of management tactics. Most of the 254 aphids tested (97.6%) displayed at least one mutation conferring resistance to pyrethroids (L1014F, M918L and M918T) and 36.2% additionally carried the modified acetylcholinesterase (MACE) carbamates resistance making them resistant to two insecticide action modes. Ten mutation combinations were detected, two of which were frequent and a strong linkage was found between MACE and M918L mutations. The R81T mutation conferring resistance to neonicotinoids was not detected. Microsatellites highlighted a moderate genetic diversity [69 multilocus genotypes (MLG) detected], severe deviations from Hardy-Weinberg expectations, a highly significant excess of heterozygotes and linkage disequilibrium between all pairs of loci. A structuration of MLGs in association with the mutation combinations was observed. Genetic differentiation was mainly not significant between sampling locations and most MLGs were geographically widespread. These results suggest the likely coexistence of parthenogenesis (obligatory or facultative) and sexual reproduction, and the existence of 'old' parthenogenetic overwintering asexual lineages. The results of this monitoring at a regional scale provide useful information on insecticide resistance, genetic diversity and reproductive modes, and highlight the need to reduce the insecticide selection pressure and to implement mitigating techniques.

Temporal and spatial distribution of insecticide-resistance mutations in the green peach aphid Myzus persicae (Hemiptera: Aphididae) on primary and secondary host plants in central Chile.

The aphid Myzus persicae develops insensitivity to almost all classes of insecticides through target site mutations. The aim of this study was to assess the temporal and spatial distribution of resistant aphids that grow on peach trees and weeds, and establish the role of the secondary hosts as a reservoir of resistant genotypes. The frequency of genotypes (RR, RS, and SS) was affected by the host plant, mainly for knock-down resistance (kdr) and modified acetylcholinesterase (MACE) mutations (P < 0.05). A higher frequency of RS and RR was observed in aphids found on peach trees than on weeds. The frequency of the R allele was significantly higher (P < 0.01) than expected in autumn and on weeds for the kdr and MACE mutations, while the frequency of the S allele was high in all seasons for all insecticide-resistance mutations (P < 0.01). The most abundant multilocus genotype was characterized as being RS for the kdr mutation and SS for both super-kdr and MACE. This study provides farm-scale evidence that weeds are not an important reservoir of the R allele, but they are for the S allele. Hence, the presence of heterozygous genotypes on peach trees is most likely attributable to weeds. © 2017 Society of Chemical Industry.

Genetic variation in target-site resistance to pyrethroids and pirimicarb in Tunisian populations of the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae).

We used molecular assays to diagnose resistance to pyrethroids and pirimicarb in samples of Myzus persicae from field crops or an insect suction trap in Tunisia. Genotypes for resistance loci were related to ones for polymorphic microsatellite loci in order to investigate breeding systems and patterns of genetic diversity, and to inform resistance management tactics. The kdr mutation L1014F conferring pyrethroid resistance was found in all samples. The M918T s-kdr mutation also occurred in most samples, but only in conjunction with kdr. We discovered a previously unreported genotype heterozygous for L1014F but homozygous for M918T. Samples with modified acetylcholinesterase (MACE) conferring resistance to pirimicarb were less common but widespread. 16% of samples contained both the kdr and MACE mutations. Many unique microsatellite genotypes were found, suggesting that M. persicae is holocyclic in Tunisia. There were no consistent associations between resistance and microsatellite markers. This first study of insecticide resistance in M. persicae in North Africa showed genetic variation in insecticide resistance within microsatellite multilocus genotypes (MLGM s) and the same resistance mechanisms to be present in different MLGM s. This contrasts with variation in northern Europe where M. persicae is fully anholocyclic. Implications for selection and control strategies are discussed. © 2016 Society of Chemical Industry.

Survey of Resistance to Four Insecticides and their Associated Mechanisms in Different Genotypes of the Green Peach Aphid (Hemiptera: Aphididae) From Chile

The green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) is amajor pest of agriculture worldwide that has proved to be particularly adept at evolving insecticide resistance. Several mechanisms that confer resistance to many insecticide types have been described in M. persicae. We measured the resistance status of nine multilocus genotypes (MLGs) of this aphid species collected in Chile. MLGs were identified using microsatellite markers, and these MLG clonal populations were measured for the presence of modified acetylcholinesterase (MACE), kdr and super kdr mutations, and enhanced carboxyl esterase activity. Toxicological bioassays were used to estimate aphid LC50 when treated with metamidophos (organophosphate), pirimicarb (dimethyl carbamate), cyfluthrin (pyrethroid), and imidacloprid (neonicotinoid). Two MLGs presented >20-fold resistance to pirimicarb, which was associated with the MACE mutation in the heterozygous condition. The kdr mutation was found in only four MLGs in the heterozygous condition and they showed resistance ratios (RR) to cyfluthrin of less than sevenfold. The super kdr mutation was not detected. Enhanced carboxyl esterase activity was predominantly found in the susceptible (S) to first level of resistance (R1) with RR to metamidophos less than eight-fold. Finally, RR to imidacloprid was also less than eight-fold in all MLGs tested. A few MLGs with resistance to pirimicarb were found, while susceptibility to cyfluthrin, metamidophos and imidacloprid was still predominant. A significant positive correlation between imidacloprid tolerance with pirimicarb resistance was detected, as well as between imidacloprid and metamidophos tolerance. With the increase in the use of neonicotinoid insecticides, better rotation of insecticides with different modes of action will be necessary to prevent further development of M. persicae insecticide resistance in Chile.

Monitoring for MACE resistance in the tobacco-adapted form of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in the eastern United States

Organophosphate and carbamate insecticides exert their neurotoxic effects by inhibiting acetylcholinesterase (AChE), thereby, prolonging the action of acetylcholine at cholinergic synapses, resulting in neuronal hyperexcitation. Mutations at the AChE target site confer modified acetylcholinesterase (MACE) phenotypes. Target-site insensitivity of AChE was characterized in field-collected, tobacco-adapted forms of the green peach aphid, Myzus persicae (Sulzer), from nine different states in the eastern United States from 2004 to 2007. The specific activity of the AChE among the 65 aphid colonies screened by Ellman's assay ranged from 0.017–0.259 U/min/mg protein. Eight colonies, with a wide range of specific activities were chosen to study the inhibition of AChE in the presence of two carbamate insecticides, methomyl and pirimicarb. IC 50 values for methomyl ranged from 0.35 to 2.4 μM, while six out of eight colonies had lower values that ranged from 0.16 to 0.30 μM for pirimicarb. Two colonies that were inhibited by methomyl had very high IC 50 values for pirimicarb, 40.4 and 98.6 μM respectively. The target-site insensitivity in these two colonies that are resistant to pirimicarb could be due to an ace2 gene mutation. This is the first instance where MACE phenotypes in M. persicae from the United States were studied and confirmed. The results indicate that the possible insensitivity due to MACE resistance in some colonies may render selected carbamate insecticides ineffective. Concerns of MACE resistance in managing the tobacco-adapted form of the green peach aphid on tobacco in the United States are discussed.

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

Abstract1 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.

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).

Insecticide resistance and genetic composition of Myzus persicae (Hemiptera: Aphididae) on field potatoes in New Zealand

The prevalence of insecticide resistance mechanisms in populations of Myzus persicae on potatoes in New Zealand was determined in five parthenogenetic aphid lineages raised from individuals collected from Pukekohe in the North Island, and 66 from Canterbury and one from the West Coast in the South Island during mid-summer (January) to early autumn (March) 2005. For each lineage, the genotypes, determined using four microsatellite loci, were compared with the presence or absence of resistance mechanisms assessed using a combination of biochemical tests, polymerase chain reaction-based allelic discrimination and diagnostic dose bioassays. The 72 lineages comprised 23 genotypes (defined using microsatellite markers), with 60% containing one or more mechanisms that confer resistance to between one and three of any of four insecticide classes. In all, 38% contained high or extreme elevated carboxylesterases (E-Carb-R 2 and -R 3) that confer resistance primarily to organophosphates, and some resistance to carbamates and pyrethroids; 19% contained modified acetylcholinesterase (MACE) that gives strong resistance to the di-methyl carbamates, pirimicarb and triazamate; 54% contained a mutation in a voltage-gated sodium channel gene, called knockdown resistance (kdr), that confers resistance to pyrethroids; and 36% contained a second mutation of the gene (super-kdr) that gives enhanced resistance to pyrethroids. In all, 10% of the lineages also showed low-level resistance to neonicotinoids, as shown in imidacloprid diagnostic dose bioassays. Most of the microsatellite genotypes appeared to disclose cycles of sexual reproduction, the clones of which were susceptible to all classes of insecticide chemistry, except two that had acquired MACE possibly by breeding with an invasive MACE genotype or an individual carrying a locally derived MACE mutation. However, lineages carrying multiple resistance mechanisms belonged to two resistant genotypes that formed widely distributed clones. Behavioural bioassays on selected lineages from New Zealand and Europe showed that M. persicae clones from both regions carried E-Carb R 3, or kdr/s-kdr, and showed consistently low responses to the aphid alarm pheromone ( E)- β-farnesene. Methods are discussed for exploiting such putative fitness costs of resistance traits in M. persicae aphids to minimise the development of resistance and restore efficacy to insecticide programmes.

Insecticide resistance status of Myzus persicae (Hemiptera: Aphididae) populations from peach and tobacco in mainland Greece

The susceptibility of 88 and 38 field samples of Myzus persicae (Sulzer) to imidacloprid and deltamethrin respectively was examined using the FAO dip test bioassay. The field samples were collected from tobacco and peach from various regions of Greece in the period from 2004 to 2006. In addition, 497, 349 and 370 clones originating from peach and tobacco were screened for the three known resistance mechanisms, elevated esterases, modified acetylcholinesterase (MACE) and knockdown resistance (kdr) respectively, using biochemical and DNA diagnostics. Most of the samples assayed with imidacloprid showed low resistance factors (RFs)-39% below 5 and 21% between 5 and 10. However, 9% of the samples (all from tobacco) showed relatively high RF values (24-73). Differences were found between crops, with higher RF values recorded in samples from tobacco than in those from peach. Bioassays with deltamethrin revealed the development of strong resistance in the populations examined. The RFs were mostly higher than 23, and in 29% of the samples they were extremely high (152-436). Finally, the three known resistance mechanisms were found in high frequencies in the populations examined, although some differences between crops and years were detected. The implications of the study for management schemes against M. persicae are discussed.

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.

Variation of Musca domestica L. acetylcholinesterase in Danish housefly populations

Anti-cholinesterase resistance is in many cases caused by modified acetylcholinesterase (MACE). A comparison was made of toxicological data and AChE activity gathered from 21 field populations and nine laboratory strains of houseflies, Musca domestica L., to elucidate the best way of generating data to provide advice for management strategies and gathering information for resistance risk assessment on the organophosphates azamethiphos and dimethoate and the carbamate methomyl, which have been the primary insecticides used against adult houseflies in Denmark. Cluster analysis was performed and > 2000 houseflies were assigned to one of three phenotypes based on total acetylcholinesterase activity as well as inhibition by azamethiphos, methomyl or omethoate. A cluster, i.e. a phenotype, with high total AChE activity and high sensitivity to azamethiphos and less sensitivity to inhibition by methomyl and omethoate was shown to be linked to methomyl resistance. It was not possible to define any clusters that could be linked to azamethiphos or dimethoate resistance. The five mutations V180L, G262A, G262V, F327Y and G365A causing anticholinesterase resistance in houseflies were all identified in the Danish housefly strains. The data are very heterogeneous, and a correlation of molecular genetic background and resistance of phenotypes is not obvious with the available data.

Use of the RFLP‐PCR diagnostic test for characterizing MACE and kdr insecticide resistance in the peach potato aphid Myzus persicae

The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids.

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.

Association of MACE-based insecticide resistance in Myzus persicae with reproductive rate, response to alarm pheromone and vulnerability to attack by Aphidius colemani.

Reproductive success and response to alarm pheromone, both potentially important components of fitness, were assessed using clones of Myzus persicae (Sulzer) to establish associations with insecticide resistance conferred by insensitive modified acetylcholinesterase (MACE). Both traits showed significant trends that were apparently related to this mechanism. MACE forms appeared to reproduce at slower rates than non-MACE forms expressing moderate (R1) levels of another resistance mechanism based on elevated carboxylesterase. However, MACE forms were more responsive to alarm pheromone than their non-MACE counterparts. The potential implications for parasitoid performance were tested using two clones showing clear differences in alarm response. The level of parasitism of M persicae by the parasitoid Aphidius colemani (Viereck) was significantly lower in MACE forms on pepper crops compared to non-MACE forms. In addition, the distribution of MACE and non-MACE forms differed on the pepper plants, with more MACE forms being found on the growing points. The presence of the parasitoid A colemani did not alter this change in distribution.

Variation in response to neonicotinoid insecticides in peach-potato aphids, Myzus persicae (Hemiptera: Aphididae).

Laboratory bioassays applying the neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram against clones of the peach-potato aphid Myzus persicae (Sulzer) demonstrated that these compounds effectively circumvent the known carboxylesterase, modified acetylcholinesterase (MACE) and knock-down (kdr) insecticide resistance mechanisms in this species. However, some clones showed cross-tolerance (up to 18-fold) of these compounds relative to susceptible standards. A survey assessing the frequency of neonicotinoid tolerance in M persicae in the UK, based on samples collected from the field and glasshouses between 1997 and 2000, showed that such tolerance is still rare. Experiments on neonicotinoid-susceptible and -tolerant populations of M persicae under simulated field conditions in the laboratory showed that, although the latter were well controlled by imidacloprid applied at recommended application rates, they were more likely to survive and reproduce when this compound was applied at lower concentrations. Such conditions are probably periodically present in imidacloprid-treated field and glasshouse crops. Selection favouring tolerant forms of M persicae could lead to increases in their frequency and the evolution of more potent resistance to neonicotinoids.

Intensification of insecticide resistance in UK field populations of the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) in 1996

AbstractThe well-established carboxylesterase-based resistance to insecticides in Myzus persicae Sulzer has recently been accentuated by the appearance of aphids with a modified acetylcholinesterase (MACE) insensitive to pirimicarb and the novel aphicide, triazamate. This target site resistance mechanism was found in M. persicae from crops in the UK for the first time in 1996, together with especially large proportions of aphids with R2 and R3 levels of carboxylesterases, a combination that was associated with serious insecticide failures. This paper describes the incidence of both mechanisms and discusses the implications for future recommendations for aphid control in the UK.