Ryanodine receptors: next generation of insecticide targets

Ryanodine receptors (RyRs) are calcium channels located on the endo(sarco)plasmic reticulum of muscle cells and neurons. They are the largest ion channels known made up of four monomers, each 565kDa in size. Mammals have 3 different RyR isoforms, encoded by different genes, while insects express only one isoform of the receptor, which is only 46% similar (at the amino acid level) to its mammalian counterpart(s). RyRs function to regulate the release of luminal Ca2+ stores into the cell cytoplasm and play a key role in muscle excitation-contraction coupling (ECC). The plant alkaloid ryanodine, from which the receptor derives its name, has been investigated extensively as a potential pest control agent, but to date no commercial products have been identified. Recently two synthetic insecticides selectively targeting pest RyRs were introduced to the market. These compounds belong to the novel group of insecticides called diamides. In this study two insect ryanodine receptors were isolated sequenced and cloned into suitable expression vectors from economically important pests M. persicae and P. xylostella to identify protein site of interaction for the novel compounds. Both proteins were expressed in HEK 293 cells and Sf9 cells and analysed for evidence of function using ryanodine binding assays and calcium release imaging. In the case of M. persicae RyR the expression level was not sufficient to obtain any functional data. However the expression of P. xylostella RyR showed evidence of function in both HEK and Sf9 cells. Functional studies showed that expressed P. xylostella RyR can bind [3H] ryanodine and respond to various caffeine concentrations; the protein was also sensitive to both diamide compounds. DNA sequencing of RyR from field evolved diamide resistant strains of P. xylostella identified a mutation causing amino acid change G4946E. Functional analysis of modified RyR construct in Sf9 cells showed significantly reduced sensitivity to to both diamide compounds while retaining caffeine and ryanodine sensitivity comparable to the expressed WT form

Diamide resistance: 10 years of lessons from Lepidopteran pests

Diamide insecticides selectively acting on insect ryanodine receptors (RyR) were launched to the market more than 10 years ago, particularly targeted for the control of lepidopteran pest species in diverse agronomic and horticultural cropping systems. They are now globally registered in many countries and provide reliable control levels in most settings. However, their frequent application, due to alternative mode of action chemistries often not providing sufficient levels of control, has resulted in the selection of diamide resistance in some of the world’s most destructive lepidopteran species, including populations of diamondback moth, tomato leafminer, rice stem borer and more recently beet armyworm. High levels of diamide resistance, compromising diamide efficacy at recommended field rates, has been shown to be conferred by RyR target-site mutations affecting diamide binding. The present work reviews the global status of diamide insecticide resistance in lepidopteran pests, with special reference to RyR target-site alterations. Furthermore, we discuss principles enabling the prediction of the impact and spread of diamide resistance, based on population genetics and associated fitness costs as influenced by the known target-site mutations recently described. In this context, we reiterate calls by the Insecticide Resistance Action Committee to implement effective diamide insecticide resistance management by following a three-step strategy of resistance identification, tracking and prediction according to the protocols discussed in this article

Assessing the acute toxicity of insecticides to the buff-tailed bumblebee (Bombus terrestris audax)

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 LD50 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

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human−Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains

An analysis of variability in genome organisation of intracellular calcium release channels across insect orders

Using publicly available genomic data, combined with RT-PCR validation, we explore structural genomic variation for two major ion channels across insect classes. We have manually curated ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R) ORFs and their corresponding genomic structures from 26 different insects covering major insect orders. We found that, despite high protein identity for both RyRs (> 75%) and IP3Rs (~67%), the overall complexity of the gene structure varies greatly between different insect orders with the simplest genes (fewest introns) found in Diptera and the most complex in Lepidoptera. Analysis of intron conservation patterns indicated that the majority of conserved introns are found close to the 5′ end of the channels and in RyR around the highly conserved mutually exclusive splice site. Of the two channels the IP3Rs appear to have a less well conserved organisation with a greater overall number of unique introns seen between insect orders. We experimentally validated two of the manually curated ORFs for IP3Rs and confirmed an atypical (3799aa) IP3R receptor in Myzus persicae, which is approximately 1000 amino acids larger than previously reported for IP3Rs

Rapid selection for resistance to diamide insecticides in Plutella xylostella via specific amino acid polymorphisms in the ryanodine receptor

Diamide insecticides, such as flubendiamide and chlorantraniliprole, are a new class of insecticide with a novel mode of action, selectively activating the insect ryanodine receptor (RyR). They are particularly active against lepidopteran pests of cruciferous vegetable crops, including the diamondback moth, Plutella xylostella. However, within a relatively short period following their commercialisation, a comparatively large number of control failures have been reported in the field. In this review we summarise the current body of knowledge regarding the molecular mechanisms of diamide resistance in P. xylostella. Resistant phenotypes collected from different countries can often be linked to specific target site mutation(s) in the ryanodine receptors' transmembrane domain. Metabolic mechanisms of resistance have also been proposed. Rapid resistance development is probably a consequence of over-reliance on this one class of chemistry for diamondback moth control

Diamide insecticide resistance in transgenic Drosophila and Sf9-cells expressing a full-length diamondback moth ryanodine receptor carrying an I4790M mutation

BACKGROUNDResistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus.RESULTSI4790M when introduced into P. xylostella RyR expressed in an insect-derived Sf9 cell line was found to mediate just a ten-fold reduction in chlorantraniliprole efficacy (compared to 104- and 146-fold reductions for the G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥150-fold reduction. I4790M-mediated resistance to flubendiamide was estimated to be >24-fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4-fold resistance to chlorantraniliprole and 22-fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Although the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a noncompetitive environment, it does, however, have potentially major impacts on mobility at both the larval and adult stages.CONCLUSIONSI4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect

P450 gene duplication and divergence led to the evolution of dual novel functions and insecticide cross-resistance in the brown planthopper Nilaparvata lugens

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

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

This is the final version. Available on open access from Elsevier via the DOI in this recordData availability: Sequence data has been deposited with the NCBI Short Read Archive as BioProject PRJNA548670.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.Syngenta LtdBiotechnology and Biological Sciences Research Council (BBSRC

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