The role of the Bemisia tabaci and Trialeurodes vaporariorum cytochrome-P450 clade CYP6DPx in resistance to nicotine and neonicotinoids.

The alkaloid, nicotine, produced by tobacco and other Solanaceae as an anti-herbivore defence chemical is one of the most toxic natural insecticides in nature. However, some insects, such as the whitefly species, Trialeurodes vaporariorum and Bemisia tabaci show strong tolerance to this allelochemical and can utilise tobacco as a host. Here, we used biological, molecular and functional approaches to investigate the role of cytochrome P450 enzymes in nicotine tolerance in T. vaporariorum and B. tabaci. Insecticide bioassays revealed that feeding on tobacco resulted in strong induced tolerance to nicotine in both species. Transcriptome profiling of both species reared on tobacco and bean hosts revealed profound differences in the transcriptional response these host plants. Interrogation of the expression of P450 genes in the host-adapted lines revealed that P450 genes belonging to the CYP6DP subfamily are strongly upregulated in lines reared on tobacco. Functional characterisation of these P450s revealed that CYP6DP1 and CYP6DP2 of T. vaporariorum and CYP6DP3 of B. tabaci confer resistance to nicotine in vivo. These three genes, in addition to the B. tabaci P450 CYP6DP5, were also found to confer resistance to the neonicotinoid imidacloprid. Our data provide new insight into the molecular basis of nicotine resistance in insects and illustrates how divergence in the evolution of P450 genes in this subfamily in whiteflies may have impacted the extent to which different species can tolerate a potent natural insecticide.

Host plant adaptation in the polyphagous whitefly, Trialeurodes vaporariorum, is associated with transcriptional plasticity and altered sensitivity to insecticides.

BACKGROUND: The glasshouse whitefly, Trialeurodes vaporariorum, is a damaging crop pest and an invasive generalist capable of feeding on a broad range of host plants. As such this species has evolved mechanisms to circumvent the wide spectrum of anti-herbivore allelochemicals produced by its host range. T. vaporariorum has also demonstrated a remarkable ability to evolve resistance to many of the synthetic insecticides used for control. RESULTS: To gain insight into the molecular mechanisms that underpin the polyphagy of T. vaporariorum and its resistance to natural and synthetic xenobiotics, we sequenced and assembled a reference genome for this species. Curation of genes putatively involved in the detoxification of natural and synthetic xenobiotics revealed a marked reduction in specific gene families between this species and another generalist whitefly, Bemisia tabaci. Transcriptome profiling of T. vaporariorum upon transfer to a range of different host plants revealed profound differences in the transcriptional response to more or less challenging hosts. Large scale changes in gene expression (> 20% of genes) were observed during adaptation to challenging hosts with a range of genes involved in gene regulation, signalling, and detoxification differentially expressed. Remarkably, these changes in gene expression were associated with significant shifts in the tolerance of host-adapted T. vaporariorum lines to natural and synthetic insecticides. CONCLUSIONS: Our findings provide further insights into the ability of polyphagous insects to extensively reprogram gene expression during host adaptation and illustrate the potential implications of this on their sensitivity to synthetic insecticides.

Frequency and spatial distribution of knock-down resistance (kdr) to pyrethroids in multiple oilseed rape pest species of the genus Ceutorhynchus.

BACKGROUND: The protection of European oilseed rape (OSR) from damaging insects relies on pyrethroid insecticides, but the development of resistance in key coleopteran pests such as the pollen beetle (Brassicogethes aeneus) and the cabbage stem flea beetle (Psylliodes chrysocephala) has resulted in reduced effectiveness of these insecticides. The sodium channel gene mutation L1014F knock-down resistance (kdr) is a contributing factor in resistance to pyrethroids in B. aeneus and P. chrysocephala, but little is known about the status of resistance in weevils of the genus Ceutorhynchus (Coleoptera: Curculonidae). Therefore, the present study investigated pyrethroid susceptibility and the presence of the kdr mutation in four Ceutorhynchus species. RESULTS: The kdr mutation in either its heterozygous or homozygous form was found in all investigated Ceutorhynchus species (C. picitarsis, C. pallidactylus, C. napi and C. obstrictus). Samples where pyrethroids in bioassays still provided control at 100% field rate or below contained kdr at frequencies of ≤12.5%, whilst bioassays using 100% field rate that did not control Ceutorhynchus populations contained homozygous resistant individuals at frequencies of greater than 55%. Field sampling demonstrated that kdr frequencies in populations of C. picitarsis and C. obstrictus collected from across France and Germany ranged from 0 to 100%. CONCLUSION: The present study demonstrated the potential of all four Ceutorhynchus species tested to develop pyrethroid resistance via the L1014F (kdr) mutation. Although kdr frequency varies among species and geographic locations, the risk of loss of pyrethroid insecticide effectiveness is high. Integration of other control tools for resistance management is therefore needed. © 2023 Society of Chemical Industry.

Characterization of emamectin benzoate resistance in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

BACKGROUND: Plutella xylostella (L.) is a destructive pest of cruciferous crops worldwide that has evolved resistance to many insecticides. Here we examined the mode of inheritance, cross-resistance profile, and potential mechanisms of emamectin benzoate resistance in a field-derived strain of P. xylostella from Japan. RESULTS: A field-collected population of P. xylostella, was found to exhibit strong (> 150-fold) resistance to emamectin benzoate in insecticide bioassays when compared with a laboratory susceptible strain. Genetic analysis showed that resistance is inherited as an autosomal, recessive trait, and is conferred by a single or a few closely linked loci. The emamectin benzoate resistant strain also exhibited resistance to abamectin, lepimectin, chlorantraniliprole, lufenuron, spinetoram, indoxacarb, fipronil, dieldrin, endosulfan and lambda-cyhalothrin, demonstrating a remarkable multi-resistance profile. Insecticide bioassays employing inhibitors of detoxification enzymes revealed that piperonyl butoxide (PBO) increased the toxicity of emamectin benzoate in the resistant strain by ten-fold indicating the potential involvement of cytochrome P450 monooxygenases in avermectin resistance. Furthermore, cloning and sequencing of the primary receptor of avermectins, the GluCl channel, revealed the absence of target-site mutations in the resistant strain. CONCLUSIONS: Our data on the mode of inheritance and mechanisms of resistance to emamectin benzoate in a P. xylostella strain from Japan provide a foundation for the development of regional resistance management strategies. However, the high levels of phenotypic resistance in this strain to a diverse range of other insecticide classes available for control illustrate the challenges associated with the sustainable control of this important pest. © 2023 Society of Chemical Industry.

A single point mutation in the Bemisia tabaci cytochrome-P450 CYP6CM1 causes enhanced resistance to neonicotinoids.

The tobacco whitefly, Bemisia tabaci, is a polyphagous crop pest which causes high levels of economic damage across the globe. Insecticides are often required for the effective control of this species, among which the neonicotinoid class have been particularly widely used. Deciphering the mechanisms responsible for resistance to these chemicals is therefore critical to maintain control of B. tabaci and limit the damage it causes. An important mechanism of resistance to neonicotinoids in B. tabaci is the overexpression of the cytochrome P450 gene CYP6CM1 which leads to the enhanced detoxification of several neonicotinoids. In this study we show that qualitative changes in this P450 dramatically alter its metabolic capacity to detoxify neonicotinoids. CYP6CM1 was significantly over-expressed in two strains of B. tabaci which displayed differing levels of resistance to the neonicotinoids imidacloprid and thiamethoxam. Sequencing of the CYP6CM1 coding sequence from these strains revealed four different alleles encoding isoforms carrying several amino acid changes. Expression of these alleles in vitro and in vivo provided compelling evidence that a mutation (A387G), present in two of the CYP6CM1 alleles, results in enhanced resistance to several neonicotinoids. These data demonstrate the importance of both qualitative and quantitative changes in genes encoding detoxification enzymes in the evolution of insecticide resistance and have applied implications for resistance monitoring programs.

Mutation of a nicotinic acetylcholine receptor ß subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

BACKGROUND: Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. RESULTS: Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid) to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR). Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpß1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR ß1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T). CONCLUSION: Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also provides further validation of exisiting models of neonicotinoid binding and selectivity for insect nAChRs.

Insecticide resistance management and industry: the origins and evolution of the Insecticide Resistance Action Committee (IRAC) and the mode of action classification scheme.

Insecticide resistance is a long-standing problem affecting the efficacy and utility of crop protection compounds. Insecticide resistance also impacts the ability and willingness of companies around the world to invest in new crop protection compounds and traits. The Insecticide Resistance Action Committee (IRAC) was formed in 1984 to provide a coordinated response by the crop protection industry to the problem of insecticide resistance. Since its inception, participation in IRAC has grown from a few agrochemical companies in Europe and the US to a much larger group of companies with global representation and an active presence (IRAC Country Groups) involving an even wider array of companies in more than 20 countries. The focus of IRAC has also evolved from that of defining and documenting cases of insecticide resistance to a pro-active role in addressing insecticide resistance management (IRM) providing an array of informational and educational tools (videos, posters, pamphlets) on insect pests, bioassay methods, insecticide mode of action and resistance management, all publicly available through its website (https://irac-online.org/). A key tool developed by IRAC is the Insecticide Mode of Action (MoA) Classification Scheme, which has evolved from a relatively simple acaricide classification started in 1998 to the far broader scheme that now includes biologics as well as insecticides and acaricides. A separate MoA Classification Scheme has also been recently developed for nematicides. The IRAC MoA Classification Scheme coupled with expanding use of MoA labeling on insecticide and acaricide product labels provides a straightforward means to implement IRM. An overview of the history of IRAC along with some of its notable accomplishments and future directions are reviewed. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella, is a damaging pest of cruciferous crops, and has evolved resistance to many of the insecticides used for control, including members of the diamide class. Previous work on the molecular basis of resistance to diamides has documented mutations in the target-site, the ryanodine receptor, in resistant populations of P. xylostella worldwide. In contrast the role of metabolic resistance to this insecticide class is significantly less clear. Here we show that overexpression of a flavin-dependent monooxgenase (FMO) confers resistance to the diamide chlorantraniliprole in P. xylostella. Transcriptome profiling of diamide resistant strains, with and without target-site resistance, revealed constitutive over-expression of several transcripts encoding detoxification enzymes compared to susceptible strains. Two of these, CYP6BG1, and PxFMO2 were particularly highly overexpressed (33,000 and 14,700-fold, respectively) in a resistant strain (HAW) lacking target-site resistance. After 17 generations without diamide selection the resistance of the HAW strain fell by 52-fold and the expression of PxFMO2 by > 1300-fold, however, the expression of CYP6BG1 declined by only 3-fold. Generation of transgenic Drosophila melanogaster expressing these genes demonstrated that PxFMO2, but not CYP6BG1, confers resistance in vivo. Overexpression of PxFMO2 in the HAW strain is associated with mutations, including a putative transposable element insertion, in the promoter of this gene. These enhance the expression of a reporter gene when expressed in a lepidopteran cell line suggesting they are, at least in part, responsible for the overexpression of PxFMO2 in the resistant strain. Our results provide new evidence that insect FMOs can be recruited to provide resistance to synthetic insecticides.

Use of an individual-based simulation model to explore and evaluate potential insecticide resistance management strategies.

BACKGROUND: Tools with the potential to predict risks of insecticide resistance and aid the evaluation and design of resistance management tactics are of value to all sectors of the pest management community. Here we describe use of a versatile individual-based model of resistance evolution to simulate how strategies employing single and multiple insecticides influence resistance development in the pollen beetle, Meligethes aeneus. RESULTS: Under repeated exposure to a single insecticide, resistance evolved faster to a pyrethroid (lambda-cyhalothrin) than to a pyridine azomethane (pymetrozine), due to difference in initial efficacy. A mixture of these compounds delayed resistance compared to use of single products. The effectiveness of rotations depended on the sequence in which compounds were applied in response to pest density thresholds. Effectiveness of a mixture strategy declined with reductions in grower compliance. At least 50% compliance was needed to cause some delay in resistance development. CONCLUSION: No single strategy meets all requirements for managing resistance. It is important to evaluate factors that prevail under particular pest management scenarios. The model used here provides operators with a valuable means for evaluating and extending sound resistance management advice, as well as understanding needs and opportunities offered by new control techniques. © 2016 Society of Chemical Industry.

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel composed of 5 protein subunits arranged around a central cation selective pore. Several classes of natural and synthetic insecticides mediate their effect through interacting at nAChRs. This review examines the basic pharmacology of the neonicotinoids and related chemistry, with an emphasis on sap-feeding insects from the order Hemiptera, the principle pest target for such insecticides. Although the receptor subunit stoichiometry for endogenous invertebrate nAChRs is unknown, there is clear evidence for the existence of distinct neonicotinoid binding sites in native insect preparations, which reflects the predicted wide repertoire of nAChRs and differing pharmacology within this insecticide class. The spinosyns are principally used to control chewing pests such as Lepidoptera, whilst nereistoxin analogues are used on pests of rice and vegetables through contact and systemic action, the pharmacology of both these insecticides is unique and different to that of the neonicotinoids.

Target-site resistance to neonicotinoids.

Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

An individual-based model of the evolution of pesticide resistance in heterogeneous environments: control of Meligethes aeneus population in oilseed rape crops.

Preventing a pest population from damaging an agricultural crop and, at the same time, preventing the development of pesticide resistance is a major challenge in crop protection. Understanding how farming practices and environmental factors interact with pest characteristics to influence the spread of resistance is a difficult and complex task. It is extremely challenging to investigate such interactions experimentally at realistic spatial and temporal scales. Mathematical modelling and computer simulation have, therefore, been used to analyse resistance evolution and to evaluate potential resistance management tactics. Of the many modelling approaches available, individual-based modelling of a pest population offers most flexibility to include and analyse numerous factors and their interactions. Here, a pollen beetle (Meligethes aeneus) population was modelled as an aggregate of individual insects inhabiting a spatially heterogeneous landscape. The development of the pest and host crop (oilseed rape) was driven by climatic variables. The agricultural land of the landscape was managed by farmers applying a specific rotation and crop protection strategy. The evolution of a single resistance allele to the pyrethroid lambda cyhalothrin was analysed for different combinations of crop management practices and for a recessive, intermediate and dominant resistance allele. While the spread of a recessive resistance allele was severely constrained, intermediate or dominant resistance alleles showed a similar response to the management regime imposed. Calendar treatments applied irrespective of pest density accelerated the development of resistance compared to ones applied in response to prescribed pest density thresholds. A greater proportion of spring-sown oilseed rape was also found to increase the speed of resistance as it increased the period of insecticide exposure. Our study demonstrates the flexibility and power of an individual-based model to simulate how farming practices affect pest population dynamics, and the consequent impact of different control strategies on the risk and speed of resistance development.

Behavioural avoidance and enhanced dispersal in neonicotinoid-resistant Myzus persicae (Sulzer).

BACKGROUND: The peach potato aphid Myzus persicae is a major agricultural pest capable of transmitting over 100 plant viruses to a wide range of crops. Control relies largely upon treatment with neonicotinoid insecticides such as thiamethoxam (TMX). In 2009, a strain denoted FRC, which exhibits between 255- and 1679-fold resistance to current neonicotinoids previously linked to metabolic and target site resistance, was discovered in France. Dispersal behaviour may potentially further enhance the resistance of this strain. This study investigated this possibility and is the first to compare the dispersal behaviour of aphid clones of the same species with differing levels of neonicotinoid resistance. RESULTS: Comparing the dispersal behaviour of the FRC strain with that of a clone of lower neonicotinoid resistance (5191A), and a susceptible clone (US1L) highlighted several differences. Most importantly, the FRC strain exhibited an increased ability to locate untreated areas when presented with an environment consisting of both TMX-treated and untreated plant tissue. CONCLUSION: The altered dispersal behaviour of the FRC may partially account for the high level of neonicotinoid resistance exhibited by this strain in the field. Since the dispersal of aphid vectors is key to the transmission of viruses across crop fields this has implications for current crop protection practice.

A mutation (L1014F) in the voltage-gated sodium channel of the grain aphid, Sitobion avenae, is associated with resistance to pyrethroid insecticides.

BACKGROUND: The grain aphid, Sitobion avenae Fabricius (Hemiptera: Aphididae), is an important pest of cereal crops. Pesticides are the main method for control but carry the risk of selecting for resistance. In response to reports of reduced efficacy of pyrethroid sprays applied to S. avenae, field samples were collected and screened for mutations in the voltage-gated sodium channel, the primary target site for pyrethroids. Aphid mobility and mortality to lambda-cyhalothrin were measured in coated glass vial bioassays. RESULTS: A single amino acid substitution (L1014F) was identified in the domain IIS6 segment of the sodium channel from the S. avenae samples exhibiting reduced pyrethroid efficacy. Bioassays on aphids heterozygous for the kdr mutation (SR) or homozygous for the wild-type allele (SS) showed that those carrying the mutation had significantly lower susceptibility to lambda-cyhalothrin. CONCLUSION: The L1014F (kdr) mutation, known to confer pyrethroid resistance in many insect pests, has been identified for the first time in S. avenae. Clonal lines heterozygous for the mutation showed 35-40-fold resistance to lambda-cyhalothrin in laboratory bioassays, consistent with the reported effect of this mutation on pyrethroid sensitivity in other aphid species.

The evolution of insecticide resistance in the peach potato aphid, Myzus persicae.

The peach potato aphid, Myzus persicae is a globally distributed crop pest with a host range of over 400 species including many economically important crop plants. The intensive use of insecticides to control this species over many years has led to populations that are now resistant to several classes of insecticide. Work spanning over 40 years has shown that M. persicae has a remarkable ability to evolve mechanisms that avoid or overcome the toxic effect of insecticides with at least seven independent mechanisms of resistance described in this species to date. The array of novel resistance mechanisms, including several 'first examples', that have evolved in this species represents an important case study for the evolution of insecticide resistance and also rapid adaptive change in insects more generally. In this review we summarise the biochemical and molecular mechanisms underlying resistance in M. persicae and the insights study of this topic has provided on how resistance evolves, the selectivity of insecticides, and the link between resistance and host plant adaptation.

Development of a high-throughput real-time PCR assay for the detection of the R81T mutation in the nicotinic acetylcholine receptor of neonicotinoid-resistant Myzus persicae.

BACKGROUND: Myzus persicae is a globally important aphid pest that is mainly controlled through the application of chemical insecticides. Recently, a clone of M. persicae exhibiting control-compromising levels of resistance to neonicotinoid insecticides was described. The resistance of this clone was associated with reduced affinity of imidacloprid for the target site (the nicotinic acetylcholine receptor) as a result of mutation of a key amino acid residue (R81T) in the loop D region of a nAChR ß1 subunit. The potent levels of resistance conferred by this mechanism are cause for considerable concern, and the frequency and distribution of the mutation in worldwide populations of M. persicae require careful monitoring. In this study, a high-throughput assay has been developed that allows detection of the mutation in individual aphids. RESULTS: A real-time TaqMan assay to detect the R81T substitution was developed that proved to be sensitive and specific in tests of analytical sensitivity and in a blind genotyping trial of DNA extracted from individual aphids comprising the three possible genotypes. The assay was then used to examine the frequency of the R81T mutation in aphids collected and stored in ethanol from peach orchards in southern France. The R81T frequency varied from 33 to 100% in seven populations from the department of Gard, France. CONCLUSIONS: This study describes a rapid and sensitive assay that very effectively detects the R81T mutation in individual aphids. The results also have practical significance for the control of M. persicae in southern France and provide contemporary data to inform current resistance management strategies.

Investigating the mode of action of sulfoxaflor: a fourth-generation neonicotinoid.

BACKGROUND: The precise mode of action of sulfoxaflor, a new nicotinic acetylcholine receptor-modulating insecticide, is unclear. A detailed understanding of the mode of action, especially in relation to the neonicotinoids, is essential for recommending effective pest management practices. RESULTS: Radiolabel binding experiments using a tritiated analogue of sulfoxaflor ([(3) H]-methyl-SFX) performed on membranes from Myzus persicae demonstrate that sulfoxaflor interacts specifically with the high-affinity imidacloprid binding site present in a subpopulation of the total nAChR pool. In competition studies, imidacloprid-like neonicotinoids displace [(3) H]-methyl-SFX at pM concentrations. The effects of sulfoxaflor on the exposed aphid nervous system in situ are analogous to those of imidacloprid and nitenpyram, and finally the high-affinity sulfoxaflor binding site is absent in a Myzus persicae strain (clone FRC) possessing a single amino acid point mutation (R81T) in the ß-nAChR, a region critical for neonicotinoid interaction. CONCLUSION: The nicotinic acetylcholine receptor pharmacological profile of sulfoxaflor in aphids is consistent with that of imidacloprid. Additionally, the insecticidal activity of sulfoxaflor and the current commercialised neonicotinoids is affected by the point mutation in FRC Myzus persicae. Therefore, it is suggested that sulfoxalfor be considered a neonicotinoid, and that this be taken into account when recommending insecticide rotation partnering for effective resistance management programmes.

Identifying the presence of neonicotinoidresistant peach-potato aphid (Myzus persicae) in the peach-growing regions of southern France and northern Spain.

BACKGROUND: The neonicotinoid class of insecticides is a key component of pest management strategies used by stone fruit producers in Europe. Neonicotinoids are currently one of the most important tools for control of the peach-potato aphid (Myzus persicae). Overreliance on neonicotinoids has led to the development of resistance through a combination of metabolic and target-site resistance mechanisms in individual aphids. A resistance monitoring project was conducted by Syngenta in 2010 to determine the resistance status of M. persicae populations collected from France and Spain, and to determine the frequency of the target-site mutation in those populations. RESULTS: Resistance monitoring suggests that resistance to neonicotinoids is relatively widespread in populations of M. persicae collected from peach orchards in the Languedoc-Roussillon, Provence-Alpes-Cote d'Azur and Rhone-Alpes regions of France, and resistance can be associated with the frequency of the target-site mutation (R81T). The R81T mutation in its heterozygous form is also present in Spanish populations and is associated with neonicotinoid resistance. CONCLUSION: The widespread nature of neonicotinoid resistance in southern France and the potential for resistance development in northern Spain highlight the need for a coordinated management strategy employing insecticides with different modes of action to reduce the selection pressure with neonicotinoids.

Rapid, multiplexed microfluidic phage display.

The development of a method for high-throughput, automated proteomic screening could impact areas ranging from fundamental molecular interactions to the discovery of novel disease markers and therapeutic targets. Surface display techniques allow for efficient handling of large molecular libraries in small volumes. In particular, phage display has emerged as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Yet, the process becomes cumbersome and time-consuming when multiple targets are involved. Here we demonstrate for the first time a microfluidic chip capable of identifying high affinity phage-displayed peptides for multiple targets in just a single round and without the need for bacterial infection. The chip is shown to be able to yield well-established control consensus sequences while simultaneously identifying new sequences for clinically important targets. Indeed, the confined parameters of the device allow not only for highly controlled assay conditions but also introduce a significant time-reduction to the phage display process. We anticipate that this easily-fabricated, disposable device has the potential to impact areas ranging from fundamental studies of protein, peptide, and molecular interactions, to applications such as fully automated proteomic screening.

Susceptibility of standard clones and European field populations of the green peach aphid, Myzus persicae, and the cotton aphid, Aphis gossypii (Hemiptera: Aphididae), to the novel anthranilic diamide insecticide cyantraniliprole.

BACKGROUND: Parthenogenetic clones of the green peach aphid, Myzus persicae (Sulzer), and the cotton aphid, Aphis gossypii Glover, were tested with the anthranilic diamide insecticide cyantraniliprole (i.e. DuPont(™) Cyazypyr(™) ) in systemic-uptake bioassays to investigate potential for cross-resistance conferred by mechanisms of insecticide resistance to organophosphates, carbamates and pyrethroids and, in the case of M. persicae, reduced sensitivity to neonicotinoids. These data were compared with the response of field samples of M. persicae and A. gossypii collected from around Europe. RESULTS: Cyantraniliprole was not cross-resisted by any of the known insecticide resistance mechanisms present in M. persicae or A. gossypii. The compound was equally active against resistant and susceptible aphid strains. The responses of the M. persicae field samples were very consistent with a maximum response ratio of 2.9 compared with a standard laboratory clone. The responses of the A. gossypii field samples were more variable, although a majority of the responses were not statistically different. CONCLUSION: Cyantraniliprole is currently the only anthranilic diamide (IRAC MoA 28) insecticide targeting aphid species such as M. persicae and A. gossypii. There is no evidence to suggest that the performance of this compound is affected by commonly occurring mechanisms that confer resistance to other insecticide chemistries. Cyantraniliprole is therefore a valuable tool for managing insecticide resistance in these globally important pests.