An evolutionarily-unique heterodimeric voltage-gated cation channel found in aphids

We describe the identification in aphids of a unique heterodimeric voltage-gated sodium channel which has an atypical ion selectivity filter and, unusually for insect channels, is highly insensitive to tetrodotoxin. We demonstrate that this channel has most likely arisen by adaptation (gene fission or duplication) of an invertebrate ancestral mono(hetero)meric channel. This is the only identifiable voltage-gated sodium channel homologue in the aphid genome(s), and the channel's novel selectivity filter motif (DENS instead of the usual DEKA found in other eukaryotes) may result in a loss of sodium selectivity, as indicated experimentally in mutagenised Drosophila channels

Insecticide resistance mediated 1 by an exon skipping event

Many genes increase coding capacity by alternate exon usage. The gene encoding the insect nicotinic acetylcholine receptor (nAChR) a6 subunit, target of the bio-insecticide spinosad, is one example of this and expands protein diversity via alternative splicing of mutually exclusive exons. Here, we show that spinosad resistance in the tomato leaf miner, Tuta absoluta is associated with aberrant regulation of splicing of Taa6 resulting in a novel form of insecticide resistance mediated by exon skipping. Sequencing of the a6 subunit cDNA from spinosad selected and unselected strains of T. absoluta revealed all Taa6 transcripts of the selected strain were devoid of exon 3, with comparison of genomic DNA and mRNA revealing this is a result of exon skipping. Exon skipping cosegregated with spinosad resistance in survival bioassays, and functional characterization of this alteration using modified human nAChR a7, a model of insect a6, demonstrated that exon 3 is essential for receptor function and hence spinosad sensitivity. DNA and RNA sequencing analyses suggested that exon skipping did not result from genetic alterations in intronic or exonic cis-regulatory elements, but rather was associated with a single epigenetic modification downstream of exon 3a, and quantitative changes in the expression of trans-acting proteins that have known roles in the regulation of alternative splicing. Our results demonstrate that the intrinsic capacity of the a6 gene to generate transcript diversity via alternative splicing can be readily exploited during the evolution of resistance and identifies exon skipping as a molecular alteration conferring insecticide resistance

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

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedBackground: 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 (Mp alpha 1-5 and Mp beta 1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR beta 1 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.Peer reviewedFinal Published versio

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

© 2014 The Authors. Published by Elsevier Ltd. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence e (http://creativecommons.org/licenses/by-nc-nd/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.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.Peer reviewedFinal Published versio

Vitellogenin gene expression as a biomarker of endocrine disruption in the invertebrate, Mytilus edulis

Vitellogenin levels are often used as a biomarker of endocrine disruption in fish. For invertebrates, there is a general lack of knowledge regarding endocrine regulation and, consequently, there are few direct biomarkers of endocrine disruption in such species. This study focuses on the marine mussel Mytilus edulis, which is often employed in biomonitoring studies. A partial vitellogenin mRNA has been isolated and gene expression quantified using real-time PCR

Characterisation of imidacloprid resistance mechanisms in the brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae)

Effective control of the brown planthopper, Nilaparvata lugens Stal, across rice-growing regions of Asia has been seriously compromised over the last 2 years by the appearance of widespread resistance to the neonicotinoid insecticide, imidacloprid. Sequence analysis of the ligand-binding domain of the nicotinic acetylcholine receptor alpha 1 subunit from two field-collected resistant strains (CHN-2 and IND-11) did not reveal the Y151S point mutation previously implicated in conferring target-site resistance in this species. This result was supported by ligand-binding studies with [(3)H]-imidacloprid that showed no significant change in insecticide binding to isolated membranes from susceptible and resistant strains. In contrast, there was an approximate 5-fold increase in the mixed function oxidase activity for the two resistant strains suggesting that imidacloprid metabolism by increased cytochrome P450 monooxygenase activity is the major mechanism of resistance in these strains. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.Peer reviewe

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis

Mytilus edulis were exposed to 17β-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process. © 2010 Elsevier Ltd. All rights reserved

Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2-16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.Peer reviewedFinal Published versio

Data from: Insecticide resistance mediated by an exon skipping event

Many genes increase coding capacity by alternate exon usage. The gene encoding the insect nicotinic acetylcholine receptor (nAChR) α6 subunit, target of the bio-insecticide spinosad, is one example of this and expands protein diversity via alternative splicing of mutually exclusive exons. Here, we show that spinosad resistance in the tomato leaf miner, Tuta absoluta is associated with aberrant regulation of splicing of Taα6 resulting in a novel form of insecticide resistance mediated by exon skipping. Sequencing of the α6 subunit cDNA from spinosad selected and unselected strains of T. absoluta revealed all Taα6 transcripts of the selected strain were devoid of exon 3, with comparison of genomic DNA and mRNA revealing this is a result of exon skipping. Exon skipping cosegregated with spinosad resistance in survival bioassays, and functional characterization of this alteration using modified human nAChR α7, a model of insect α6, demonstrated that exon 3 is essential for receptor function and hence spinosad sensitivity. DNA and RNA sequencing analyses suggested that exon skipping did not result from genetic alterations in intronic or exonic cis-regulatory elements, but rather was associated with a single epigenetic modification downstream of exon 3a, and quantitative changes in the expression of trans-acting proteins that have known roles in the regulation of alternative splicing. Our results demonstrate that the intrinsic capacity of the α6 gene to generate transcript diversity via alternative splicing can be readily exploited during the evolution of resistance and identifies exon skipping as a molecular alteration conferring insecticide resistance