63 research outputs found
Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia
This is the final version of the article. Available from Wiley via the DOI in this record.BACKGROUND: We report on the status of imidacloprid and ethiprole resistance in Nilaparvata lugens Stål collected from across South and East Asia over the period 2005-2012. RESULTS: A resistance survey found that field populations had developed up to 220-fold resistance to imidacloprid and 223-fold resistance to ethiprole, and that many of the strains collected showed high levels of resistance to both insecticides. We also found that the cytochrome P450 CYP6ER1 was significantly overexpressed in 12 imidacloprid-resistant populations tested when compared with a laboratory susceptible strain, with fold changes ranging from ten- to 90-fold. In contrast, another cytochrome P450 CYP6AY1, also implicated in imidacloprid resistance, was underexpressed in ten of the populations and only significantly overexpressed (3.5-fold) in a single population from India compared with the same susceptible strain. Further selection of two of the imidacloprid-resistant field strains correlated with an approximate threefold increase in expression of CYP6ER1. CONCLUSIONS: We conclude that overexpression of CYP6ER1 is associated with field-evolved resistance to imidacloprid in brown planthopper populations in five countries in South and East Asia.This work was funded by Bayer CropScience. Rothamsted Research receives grant-aided support from the Biotechnology and Biosciences Research Council of the United Kingdom
Host plant adaptation in the polyphagous whitefly, Trialeurodes vaporariorum, is associated with transcriptional plasticity and altered sensitivity to insecticides
This is the final version. Available from BMC via the DOI in this record. 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.ER
Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper
This is the final version of the article. Available from Elsevier via the DOI in this record.Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3–5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6–8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait.This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement n°646625), the Biotechnology and Biological Sciences Research Council of the UK (BB/G023352/1), and Bayer Crop Science
P450 gene duplication and divergence led to the evolution of dual novel functions and insecticide cross-resistance in the brown planthopper Nilaparvata lugens
This is the final version. Available on open access from Public Library of Science via the DOI in this recordData Availability: All relevant data are within the manuscript and its Supporting Information files.The sustainable control of many highly damaging insect crop pests and disease vectors is threatened by the evolution of insecticide resistance. As a consequence, strategies have been developed that aim to prevent or delay resistance development by rotating or mixing insecticides with different modes of action (MoA). However, these approaches can be compromised by the emergence of mechanisms that confer cross-resistance to insecticides with different MoA. Despite the applied importance of cross-resistance, its evolutionary underpinnings remain poorly understood. Here we reveal how a single gene evolved the capacity to detoxify two structurally unrelated insecticides with different MoA. Using transgenic approaches we demonstrate that a specific variant of the cytochrome P450 CYP6ER1, previously shown to confer resistance to the neonicotinoid imidacloprid in the brown planthopper, N. lugens, also confers cross-resistance to the phenylpyrazole ethiprole. CYP6ER1 is duplicated in resistant strains, and we show that while the acquisition of mutations in two encoded substrate recognition sites (SRS) of one of the parologs led to resistance to imidacloprid, a different set of mutations, outside of known SRS, are primarily responsible for resistance to ethiprole. Epistatic interactions between these mutations and their genetic background suggest that the evolution of dual resistance from the same gene copy involved functional trade-offs in respect to CYP6ER1 catalytic activity for ethiprole versus imidacloprid. Surprisingly, the mutations leading to ethiprole and imidacloprid resistance do not confer the ability to detoxify the insecticide fipronil, another phenylpyrazole with close structural similarity to ethiprole. Taken together, these findings reveal how gene duplication and divergence can lead to the evolution of multiple novel functions from a single gene. From an applied perspective they also demonstrate how cross-resistance to structurally unrelated insecticides can evolve, and illustrate the difficulty in predicting cross-resistance profiles mediated by metabolic mechanisms.European Union Horizon 2020Medical Research Council (MRC
Assessing the acute toxicity of insecticides to the buff-tailed bumblebee (Bombus terrestris audax)
This is the final version. Available from Elsevier / Academic Press via the DOI in this record. The buff-tailed bumblebee, Bombus terrestris audax is an important pollinator within both landscape ecosystems and agricultural crops. During their lifetime bumblebees are regularly challenged by various environmental stressors including insecticides. Historically the honey bee (Apis mellifera spp.) has been used as an ‘indicator’ species for ‘standard’ ecotoxicological testing, but it has been suggested that it is not always a good proxy for other eusocial or solitary bees. To investigate this, the susceptibility of B. terrestris to selected pesticides within the neonicotinoid, pyrethroid and organophosphate classes was examined using acute insecticide bioassays. Acute oral and topical LD values for B. terrestris against these insecticides were broadly consistent with published results for A. mellifera. For the neonicotinoids, imidacloprid was highly toxic, but thiacloprid and acetamiprid were practically non-toxic. For pyrethroids, deltamethrin was highly toxic, but tau-fluvalinate only slightly toxic. For the organophosphates, chlorpyrifos was highly toxic, but coumaphos practically non-toxic. Bioassays using insecticides with common synergists enhanced the sensitivity of B. terrestris to several insecticides, suggesting detoxification enzymes may provide a level of protection against these compounds. The sensitivity of B. terrestris to compounds within three different insecticide classes is similar to that reported for honey bees, with marked variation in sensitivity to different insecticides within the same insecticide class observed in both species. This finding highlights the need to consider each compound within an insecticide class in isolation rather than extrapolating between different insecticides in the same class or sharing the same mode of action.European Union Horizon 2020Biotechnology and Biological Sciences Research Council (BBSRC
Fly-Tox: A panel of transgenic flies expressing pest and pollinator cytochrome P450s
This is the final version. Available from the publisher via the DOI in this record.There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions.European Research Council (ERC)European Union's Horizon 2020 research and innovation programmeBiotechnology and Biological Sciences Research Council (BBSRC
Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis
This is the final version. Available from the publisher via the DOI in this record.The Osmia bicornis whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MPJT00000000. The RNAseq data generated in this study has been deposited in the Sequence Read Archive (SRA) under accession SRP065762. Accession numbers of the bee P450 genes manually curated in this study are shown in S5 Table. All other relevant data are within the paper and its Supporting Information files.The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.Biotechnology and Biological Science Research Council (BBSRC)Bayer AGEuropean Research Council (ERC
Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na+ channel of Anopheles culicifacies from Orissa, India
<p>Abstract</p> <p>Background</p> <p>Knockdown resistance in insects resulting from mutation(s) in the voltage gated Na<sup>+ </sup>channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common <it>kdr </it>mutation in insects, was reported in <it>Anopheles culicifacies</it>-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an <it>An. culicifacies </it>population from Malkangiri district of Orissa, India.</p> <p>Methods</p> <p><it>Anopheles culicifacies sensu lato (s.l.) </it>samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The <it>An. culicifacies </it>population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.</p> <p>Results</p> <p>DNA sequencing of <it>An. culicifacies </it>individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.</p> <p>Conclusions</p> <p>This is the first report of the presence of L1014S (homologous to the <it>kdr-e </it>in <it>An. gambiae</it>) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of <it>An. culicifacies </it>in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.</p
Unravelling the molecular determinants of bee sensitivity to neonicotinoid insecticides
This is the final version of the article. Available from the publisher via the DOI in this record.The impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e., N-nitroguanidine compounds such as imidacloprid and thiamethoxam, are as intrinsically toxic to bees as to the insect pests they target. However, this is not the case for all neonicotinoids, with honeybees orders of magnitude less sensitive to N-cyanoamidine compounds such as thiacloprid [2]. Although previous work has suggested that this is due to rapid metabolism of these compounds [2, 3, 4, 5], the specific gene(s) or enzyme(s) involved remain unknown. Here, we show that the sensitivity of the two most economically important bee species to neonicotinoids is determined by cytochrome P450s of the CYP9Q subfamily. Radioligand binding and inhibitor assays showed that variation in honeybee sensitivity to N-nitroguanidine and N-cyanoamidine neonicotinoids does not reside in differences in their affinity for the receptor but rather in divergent metabolism by P450s. Functional expression of the entire CYP3 clade of P450s from honeybees identified a single P450, CYP9Q3, that metabolizes thiacloprid with high efficiency but has little activity against imidacloprid. We demonstrate that bumble bees also exhibit profound differences in their sensitivity to different neonicotinoids, and we identify CYP9Q4 as a functional ortholog of honeybee CYP9Q3 and a key metabolic determinant of neonicotinoid sensitivity in this species. Our results demonstrate that bee pollinators are equipped with biochemical defense systems that define their sensitivity to insecticides and this knowledge can be leveraged to safeguard bee health.his study received funding from Bayer AG. C.B. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 646625 ). C.B. and K.B. received funding from Biotechnology and Biological Sciences Research Council (BBSRC, award number 15076182 ). The work at Rothamsted forms part of the Smart Crop Protection (SCP) strategic programme ( BBS/OS/CP/000001 ) funded through the Biotechnology and Biological Sciences Research Council’s Industrial Strategy Challenge Fund
Voltage-gated sodium channels as targets for pyrethroid insecticides
The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity
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