Ion channels as insecticide targets.

Published onlineJournal ArticleIon channels remain the primary target of most of the small molecule insecticides. This review examines how the subunit composition of heterologously expressed receptors determines their insecticide-specific pharmacology and how the pharmacology of expressed receptors differs from those found in the insect nervous system. We find that the insecticide-specific pharmacology of some receptors, like that containing subunits of the Rdl encoded GABA receptor, can be reconstituted with very few of the naturally occurring subunits expressed. In contrast, workers have struggled even to express functional insect nicotinic acetylcholine receptors (nAChRs), and work has therefore often relied upon the expression of vertebrate receptor subunits in their place. We also examine the extent to which insecticide-resistance-associated mutations, such as those in the para encoded voltage-gated sodium channel, can reveal details of insecticide-binding sites and mode of action. In particular, we examine whether mutations are present in the insecticide-binding site and/or at sites that allosterically affect the drug preferred conformation of the receptor. We also discuss the ryanodine receptor as a target for the recently developed diamides. Finally, we examine the lethality of the genes encoding these receptor subunits and discuss how this might determine the degree of conservation of the resistance-associated mutations found

Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tThe fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide evidence that both target-site and metabolic mechanisms underlie the resistance of S. frugiperda to pyrethroids and organophosphates.BBSRCNational Council for Scientific and Technological Development of Brazi

Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay

Background: The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. Methods: A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. Arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. Results: The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract DNA. Conclusion: This study describes a rapid and sensitive assay that very effectively identifies the two main malaria vectors of the An. gambiae species complex from the non-vector sibling species. The method is based on TaqMan SNP genotyping and can be used to screen single legs from dried specimens. In regions where An. merus/melas/ bwambae, vectors with restricted distributions, are not present it can be used alone to discriminate vector from non-vector or in combination with the Walker TaqMan assay to distinguish An. arabiensis and An. gambiae s.s

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Published onlineComparative StudyEvaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques. METHODS: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol. RESULTS: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples. CONCLUSION: This study describes a new high-throughput TaqMan assay that very effectively detects the four Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.BBSRCInnovative Vector Control Consortiu

A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.

This is the final version of the article. Available from the publisher via the DOI in this record.Open Access funded by Biotechnology and Biological Sciences Research CouncilSpinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.This work was, in part, funded by a research grant (BB/G023352/1) from the Biotechnology and Biological Sciences Research Council of the UK to CB

Slightly generalized Generalized Contagion: Unifying simple models of biological and social spreading

We motivate and explore the basic features of generalized contagion, a model mechanism that unifies fundamental models of biological and social contagion. Generalized contagion builds on the elementary observation that spreading and contagion of all kinds involve some form of system memory. We discuss the three main classes of systems that generalized contagion affords, resembling: simple biological contagion; critical mass contagion of social phenomena; and an intermediate, and explosive, vanishing critical mass contagion. We also present a simple explanation of the global spreading condition in the context of a small seed of infected individuals.Comment: 8 pages, 5 figures; chapter to appear in "Spreading Dynamics in Social Systems"; Eds. Sune Lehmann and Yong-Yeol Ahn, Springer Natur

Superrigid subgroups and syndetic hulls in solvable Lie groups

This is an expository paper. It is not difficult to see that every group homomorphism from the additive group Z of integers to the additive group R of real numbers extends to a homomorphism from R to R. We discuss other examples of discrete subgroups D of connected Lie groups G, such that the homomorphisms defined on D can ("virtually") be extended to homomorphisms defined on all of G. For the case where G is solvable, we give a simple proof that D has this property if it is Zariski dense. The key ingredient is a result on the existence of syndetic hulls.Comment: 17 pages. This is the final version that will appear in the volume "Rigidity in Dynamics and Geometry," edited by M. Burger and A. Iozzi (Springer, 2002

Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. RESULTS: Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication). BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. CONCLUSION: Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.CASE PhD studentship BBSRCBayer CropScienceRothamsted Researc

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

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