Publications by year
In Press
Singh KS, Hosken DJ, Wedell N, ffrench-Constant R, Bass C, Baxter S, Paszkiewicz K, Sharma MD (In Press). <i>De novo</i> genome assembly of the meadow brown butterfly, <i>Maniola jurtina</i>.
Abstract:
De novo genome assembly of the meadow brown butterfly, Maniola jurtina
AbstractBackgroundMeadow brown butterflies (Maniola jurtina) on the Isles of Scilly represent an ideal model in which to dissect the links between genotype, phenotype and long-term patterns of selection in the wild - a largely unfulfilled but fundamental aim of modern biology. To meet this aim, a clear description of genotype is required.FindingsHere we present the draft genome sequence of M. jurtina to serve as an initial genetic resource for this species. Seven libraries were constructed using DNA from multiple wild caught females and sequenced using Illumina, PacBio RSII and MinION technology. A novel hybrid assembly approach was employed to generate a final assembly with an N50 of 214 kb (longest scaffold 2.9 Mb). The genome encodes a total of 36,294 genes. 90.3% and 88.7% of core BUSCO (Benchmarking Universal Single-Copy Orthologs) Arthropoda and Insecta gene sets were recovered as complete single-copies from this assembly. Comparisons with 17 other Lepidopteran species placed 86.5% of the assembled genes in orthogroups.ConclusionsOur results provide the first high-quality draft genome and annotation of the butterfly M. jurtina.
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Dupeyron M, Singh K, Bass C, Hayward A (In Press). Evolution of Mutator transposable elements across eukaryotic diversity. Mobile DNA
Yang X, Shun D, Wei X, Yang J, Zhao Q, Yin C, Du T, Guo Z, Xia J, Yang Z, et al (In Press). MAPK-directed activation of the whitefly transcription factor CREB leads to P450-mediated imidacloprid resistance. Proceedings of the National Academy of Sciences of USA
2023
Trissi N, Troczka BJ, Ozsanlav-Harris L, Singh KS, Mallott M, Aishwarya V, O'Reilly A, Bass C, Wilding CS (2023). Differential regulation of the Tor gene homolog drives the red/green pigmentation phenotype in the aphid Myzuspersicae.
Insect Biochem Mol Biol,
153Abstract:
Differential regulation of the Tor gene homolog drives the red/green pigmentation phenotype in the aphid Myzuspersicae.
In some aphid species, intraspecific variation in body colour is caused by differential carotenoid content: whilst green aphids contain only yellow carotenoids (β-, γ-, and β,γ-carotenes), red aphids additionally possess red carotenoids (torulene and 3,4-didehydrolycopene). Unusually, within animals who typically obtain carotenoids from their diet, ancestral horizontal gene transfer of carotenoid biosynthetic genes from fungi (followed by gene duplication), have imbued aphids with the intrinsic gene repertoire necessary to biosynthesise carotenoids. In the pea aphid, Acyrthosiphon pisum a lycopene (phytoene) desaturase gene (Tor) underpins the red/green phenotype, with this locus present in heterozygous form in red individuals but absent in green aphids, resulting in them being unable to convert lycopene into the red compounds 3,4-didehydrolycopene and torulene. The green peach aphid, Myzus persicae, separated from the pea aphid for ≈45MY also exists as distinct colour variable morphs, with both red and green individuals present. Here, we examined genomic data for both red and green morphs of M. persicae and identified an enlarged (compared to A. pisum) repertoire of 16 carotenoid biosynthetic genes (11 carotenoid desaturases and five carotenoid cyclase/synthase genes). From these, we identify the homolog of A. pisum Tor (here called carotene desaturase 2 or CDE-2) and show through 3D modelling that this homolog can accommodate the torulene precursor lycopene and, through RNA knockdown feeding experiments, demonstrate that disabling CDE-2 expression in red M. persicae clones results in green-coloured offspring. Unlike in A. pisum, we show that functional CDE-2 is present in the genomes of both red and green aphids. However, expression differences between the two colour morphs (350-700 fold CDE-2 overexpression in red clones), potentially driven by variants identified in upstream putative regulatory elements, underpin this phenotype. Thus, whilst aphids have a common origin of their carotenoid biosynthetic pathway, two aphid species separated for over 40MY have evolved very different drivers of intraspecific colour variation.
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Baril T, Pym A, Bass C, Hayward A (2023). Patterns of transposable element accumulation at resistance loci: a comparative genomic analysis in aphids.
Singh KS, Cordeiro EMG, Hunt BJ, Pandit AA, Soares PL, Correa AS, Zimmer CT, Zucchi MI, Batista C, Dow JAT, et al (2023). The genome sequence of the Neotropical brown stink bug, Euschistus heros provides insights into population structure, demographic history and signatures of adaptation.
Insect Biochem Mol Biol,
152Abstract:
The genome sequence of the Neotropical brown stink bug, Euschistus heros provides insights into population structure, demographic history and signatures of adaptation.
The Neotropical brown stink bug, Euschistus heros, is a major pest of soybean in South America. The importance of E. heros as a pest has grown significantly in recent times due to increases in its abundance and range, and the evolution of insecticide resistance. Recent work has begun to examine the genetic diversity, population structure, and genetic mechanisms of insecticide resistance in E. heros. However, to date, investigation of these topics has been hampered by a lack of genomic resources for this species. Here we address this need by assembling a high-quality draft genome for E. heros. We used a combination of short and long read sequencing to assemble an E. heros genome of 1.4 Gb comprising 906 contigs with a contig N50 of 3.5 MB. We leveraged this new genomic resource, in combination with genotyping by sequencing, to explore genetic diversity in populations of this species in Brazil and identify genetic loci in the genome which are under selection. Our genome-wide analyses, confirm that there are two populations of E. heros co-occurring in different geographical regions in Brazil, and that, in certain regions of the country these populations are hybridizing. We identify several regions of the genome as under selection, including markers associated with putative insecticide resistance genes. Taken together, the new genomic resources generated in this study will accelerate research into fundamental aspects of stinkbug biology and applied aspects relating to the sustainable control of a highly damaging crop pest.
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Zeng B, Liu Y-T, Feng Z-R, Chen F-R, Wu S-F, Bass C, Gao C-F (2023). The overexpression of cytochrome P450 genes confers buprofezin resistance in the brown planthopper, Nilaparvata lugens (Stål).
Pest Manag Sci,
79(1), 125-133.
Abstract:
The overexpression of cytochrome P450 genes confers buprofezin resistance in the brown planthopper, Nilaparvata lugens (Stål).
BACKGROUND: Buprofezin, an insect growth regulator, has been widely used to control brown planthopper (BPH), Nilaparvata lugens, one of the most destructive pests of rice crops in Asia. The intensive use of this compound has resulted in very high levels of resistance to buprofezin in the field, however, the underpinning mechanisms of resistance have not been fully resolved. RESULTS: Insecticide bioassays using the P450 inhibitor piperonyl butoxide significantly synergized the toxicity of buprofezin in two resistant strains of BPH (BPR and YC2017) compared to a susceptible strain (Sus), suggesting P450s play a role in resistance to this compound. Whole transcriptome profiling identified 1110 genes that were upregulated in the BPR strain compared to the Sus strain, including 13 cytochrome P450 genes, eight esterases and one glutathione S-transferase. Subsequently, qPCR validation revealed that four of the P450 genes, CYP6ER1vA, CYP6CW1, CYP4C77, and CYP439A1 were significantly overexpressed in both the BRP and YC2017 strains compared with the Sus strain. Further functional analyses showed that only suppression of CYP6ER1vA, CYP6CW1, and CYP439A1 gene expression by RNA interference significantly increased the toxicity of buprofezin against BPH. However, only transgenic Drosophila melanogaster expressing CYP6ER1vA and CYP439A1 exhibited significant resistance to buprofezin. Finally, the BPR strain was found to exhibit modest but significant levels of resistance to acetamiprid, dinotefuran and pymetrozine. CONCLUSIONS: Our findings provide strong evidence that the overexpression of CYP6ER1vA and CYP439A1 contribute to buprofezin resistance in BPH, and that resistance to this compound is associated with low-level resistance to acetamiprid, dinotefuran and pymetrozine. These results advance understanding of the molecular basis of BPH resistance to buprofezin and will inform the development of management strategies for the control of this highly damaging pest. © 2022 Society of Chemical Industry.
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2022
Oplopoiou M (2022). A genomic approach to investigating avermectin resistance in the diamondback moth, Plutella xylostella L. (Lep: Plutellidae).
Abstract:
A genomic approach to investigating avermectin resistance in the diamondback moth, Plutella xylostella L. (Lep: Plutellidae)
BACKGROUND: Plutella xylostella (L.) causes a severe damage to crop production worldwide. This study aimed to elucidate the mode of inheritance, cross-resistance, mechanisms and synergism of emamectin benzoate resistance in a field-derived multi- resistant Japanese population of P. xylostella. Furthermore, the evolution of avermectin resistance was modelled under insecticide rotation and mixture scenarios to examine which of these two strategies are more effective in slowing resistance development and inform future insecticide resistance management strategies.
RESULTS: a field collected resistant strain of P. xylostella from Japan was established in the laboratory and repeatedly exposed to emamectin benzoate - however, selection did not considerably affect the resistance levels, with the level of emamectin resistance in the Aichi-R non selected (Aichi-R-ns) and selected populations both >230-fold compared with the susceptible reference strain. Genetic analysis showed that resistance in this strain is inherited as an autosomal, incompletely recessive trait, and is conferred by a single or a few closely linked loci. Aichi-R-ns was found to be resistant to emamectin benzoate, abamectin, lepimectin, chlorantraniliprole, lufenuron, spinetoram, indoxacarb, fipronil, dieldrin, endosulfan and lambda-cyhalothrin, demonstrating the strain’s remarkable multi-/cross-resistance profile. Finally, piperonyl butoxide (PBO), a P450 inhibitor, increased the toxicity of emamectin benzoate in the resistant strain by 10-fold, suggesting the involvement of P450s in resistance. Cloning and sequencing of the GluCl channel revealed the absence of target-site mutations and NGS analysis revealed upregulation of P450 genes that may contribute to metabolic resistance.
CONCLUSIONS: No significant effect on resistant levels due to selection in the laboratory may be an indication of a high initial resistance allele frequency. A clear cross-resistant phenotype to abamectin and lepimectin was confirmed in our emamectin benzoate resistant strain. No target-site mutations were detected in the GluCl channel, the primary site of action of avermectins. Synergism studies, NGS analysis and qPCR validation in the near-isogenic resistant strain indicate the potential involvement of cytochrome P450 monooxygenases in avermectin resistance.
Keywords: Plutella xylostella; avermectins; resistance; inheritance; GluCl; cross- resistance; synergism
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Papadimitriou F, Folia M, Ilias A, Papapetrou P, Roditakis E, Bass C, Vontas J, T Margaritopoulos J (2022). Flupyradifurone resistance in Myzus persicae populations from peach and tobacco in Greece.
Pest Manag Sci,
78(1), 304-312.
Abstract:
Flupyradifurone resistance in Myzus persicae populations from peach and tobacco in Greece.
BACKGROUND: Myzus persicae has evolved resistance to various insecticides in Greece. Here we examine the effectiveness of the insecticide flupyradifurone against aphid clones collected from tobacco and peach in Greece during 2017-2020. Furthermore, we monitored the frequency of the neonicotinoid resistance mutation R81T in the sampled clones, and the association between the responses to flupyradifurone and acetamiprid. RESULTS: of 43 clones tested with flupyradifurone, 6.977%, 60.465% and 32.558% showed low (10-14), moderate (19-89) and high (104-1914) resistance factor (RF) values, respectively. Resistance was higher in clones from peach than from tobacco with 42.308% and 17.647% of clones (respectively) failing into the high RF category (median RF values 67.5 and 36.4 for clones from peach and tobacco, respectively). Acetamiprid resistance was detected in clones collected in 2019-2020, in line with our previous study in Greece. The analysis of the whole dataset (54 clones collected during 2017-2020) revealed that all tobacco clones had RF
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Doyle T, Jimenez-Guri E, Hawkes WLS, Massy R, Mantica F, Permanyer J, Cozzuto L, Hermoso Pulido T, Baril T, Hayward A, et al (2022). Genome-wide transcriptomic changes reveal the genetic pathways involved in insect migration.
Mol Ecol,
31(16), 4332-4350.
Abstract:
Genome-wide transcriptomic changes reveal the genetic pathways involved in insect migration.
Insects are capable of extraordinary feats of long-distance movement that have profound impacts on the function of terrestrial ecosystems. The ability to undertake these movements arose multiple times through the evolution of a suite of traits that make up the migratory syndrome, however the underlying genetic pathways involved remain poorly understood. Migratory hoverflies (Diptera: Syrphidae) are an emerging model group for studies of migration. They undertake seasonal movements in huge numbers across large parts of the globe and are important pollinators, biological control agents and decomposers. Here, we assembled a high-quality draft genome of the marmalade hoverfly (Episyrphus balteatus). We leveraged this genomic resource to undertake a genome-wide transcriptomic comparison of actively migrating Episyrphus, captured from a high mountain pass as they flew south to overwinter, with the transcriptomes of summer forms which were non-migratory. We identified 1543 genes with very strong evidence for differential expression. Interrogation of this gene set reveals a remarkable range of roles in metabolism, muscle structure and function, hormonal regulation, immunity, stress resistance, flight and feeding behaviour, longevity, reproductive diapause and sensory perception. These features of the migrant phenotype have arisen by the integration and modification of pathways such as insulin signalling for diapause and longevity, JAK/SAT for immunity, and those leading to octopamine production and fuelling to boost flight capabilities. Our results provide a powerful genomic resource for future research, and paint a comprehensive picture of global expression changes in an actively migrating insect, identifying key genomic components involved in this important life-history strategy.
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Paliwal D, Hamilton AJ, Barrett GA, Alberti F, van Emden H, Monteil CL, Mauchline TH, Nauen R, Wagstaff C, Bass C, et al (2022). Identification of novel aphid-killing bacteria to protect plants.
Microb Biotechnol,
15(4), 1203-1220.
Abstract:
Identification of novel aphid-killing bacteria to protect plants.
Aphids, including the peach-potato aphid, Myzus persicae, are major insect pests of agriculture and horticulture, and aphid control measures are limited. There is therefore an urgent need to develop alternative and more sustainable means of control. Recent studies have shown that environmental microbes have varying abilities to kill insects. We screened a range of environmental bacteria isolates for their abilities to kill target aphid species. Tests demonstrated the killing aptitude of these bacteria against six aphid genera (including Myzus persicae). No single bacterial strain was identified that was consistently toxic to insecticide-resistant aphid clones than susceptible clones, suggesting resistance to chemicals is not strongly correlated with bacterial challenge. Pseudomonas fluorescens PpR24 proved the most toxic to almost all aphid clones whilst exhibiting the ability to survive for over three weeks on three plant species at populations of 5-6 log CFU cm-2 leaf. Application of PpR24 to plants immediately prior to introducing aphids onto the plants led to a 68%, 57% and 69% reduction in aphid populations, after 21 days, on Capsicum annuum, Arabidopsis thaliana and Beta vulgaris respectively. Together, these findings provide new insights into aphid susceptibility to bacterial infection with the aim of utilizing bacteria as effective biocontrol agents.
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Zeng B, Chen F-R, Sun H, Liu Y, Wu S-F, Bass C, Gao C-F (2022). Molecular and functional analysis of chitin synthase genes in Chilo suppressalis (Lepidoptera: Crambidae).
Insect SciAbstract:
Molecular and functional analysis of chitin synthase genes in Chilo suppressalis (Lepidoptera: Crambidae).
The rice stem borer, Chilo suppressalis, has developed a high level of resistance to many of the compounds currently used for control. There is therefore an urgent need to develop novel control methods for C. suppressalis. Insect chitin synthases (CHS) have attracted interest as a potential target for insect pest management. However, to date, CHS have not been characterized in C. suppressalis. Two CHS genes (CsCHS1 and CsCHS2) were identified and cloned from C. suppressalis. Two transcript variants were identified for CsCHS1, CsCHS1a and CsCHS1b. Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument. In contrast, CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut. Knockdown of CsCHS1 by RNA interference significantly inhibited the molting and pupation of C. suppressalis, and knockdown of CsCHS2 significantly affected growth during the larval stage, but had no significant effect on the pupation. Moreover, knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate, larval survivorship, pupation rate, and eclosion rate, but only larval survivorship at the G0 generation was lowered after the knockout of CsCHS2. These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C. suppressalis, and so have potential as insecticidal targets for the control of this highly damaging pest.
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Mavridis K, Papapostolou KM, Riga M, Ilias A, Michaelidou K, Bass C, Van Leeuwen T, Tsagkarakou A, Vontas J (2022). Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.
Pest Manag Sci,
78(1), 263-273.
Abstract:
Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.
BACKGROUND: Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides. RESULTS: a suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI = 0.991-0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g. etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations. CONCLUSION: the highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.
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Mavridis K, Papapostolou KM, Ilias A, Michaelidou K, Stavrakaki M, Roditakis E, Tsagkarakou A, Bass C, Vontas J (2022). Next-generation molecular diagnostics (TaqMan qPCR and ddPCR) for monitoring insecticide resistance in Bemisia tabaci.
PEST MANAGEMENT SCIENCE,
78(11), 4994-5001.
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Pym A, Umina PA, Reidy-Crofts J, Troczka BJ, Matthews A, Gardner J, Hunt BJ, van Rooyen AR, Edwards OR, Bass C, et al (2022). Overexpression of UDP-glucuronosyltransferase and cytochrome P450 enzymes confers resistance to sulfoxaflor in field populations of the aphid, Myzus persicae.
Insect Biochem Mol Biol,
143Abstract:
Overexpression of UDP-glucuronosyltransferase and cytochrome P450 enzymes confers resistance to sulfoxaflor in field populations of the aphid, Myzus persicae.
The green peach aphid, Myzus persicae, is a highly damaging, globally distributed crop pest that has evolved multiple resistance to numerous insecticides. It is thus imperative that insecticides that are not strongly compromised by pre-existing resistance are carefully managed to maximise their effective life span. Sulfoxaflor is a sulfoximine insecticide that retains efficacy against M. persicae clones that exhibit resistance to older insecticides. In the current study we monitored the efficacy of sulfoxaflor against M. persicae populations collected in Western Australia, following reports of control failures in this region. We identified clones with low (4-23-fold across multiple independent bioassay experiments), but significant, levels of resistance to sulfoxaflor compared with a reference susceptible clone. Furthermore, we demonstrate that sulfoxaflor resistance can persist after many months of culturing in the laboratory in the absence of insecticide exposure. Resistance was not conferred by known mechanisms of resistance to neonicotinoid insecticides, that act on the same target-site as sulfoxaflor, i.e. the R81T mutation or overexpresssion of the P450 gene CYP6CY3. Rather, transcriptome profiling of multiple resistant and susceptible clones identified the P450 CYP380C40 and the UDP-glucuronosyltransferase UGT344P2 as highly overexpressed (21-76-fold and 6-33-fold respectively) in the resistant clones. Transgenic expression of these genes demonstrated that they confer, low, but significant, levels of resistance to sulfoxaflor in vivo. Taken together, our data reveal the presence of low-level resistance to sulfoxaflor in M. persicae populations in Australia and uncover two novel mechanisms conferring resistance to this compound. The findings and tools generated in this study provide a platform for the development of strategies that aim to slow, prevent or overcome the evolution of more potent resistance to sulfoxaflor.
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Duarte A, Pym A, Garrood WT, Troczka BJ, Zimmer CT, Davies TGE, Nauen R, O’Reilly AO, Bass C (2022). P450 gene duplication and divergence led to the evolution of dual novel functions and insecticide cross-resistance in the brown planthopper Nilaparvata lugens.
PLOS Genetics,
18(6), e1010279-e1010279.
Abstract:
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.
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Haas J, Hayward A, Buer B, Maiwald F, Nebelsiek B, Glaubitz J, Bass C, Nauen R (2022). Phylogenomic and functional characterization of an evolutionary conserved cytochrome P450-based insecticide detoxification mechanism in bees.
Proc Natl Acad Sci U S A,
119(26).
Abstract:
Phylogenomic and functional characterization of an evolutionary conserved cytochrome P450-based insecticide detoxification mechanism in bees.
The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.
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Umina PA, Bass C, van Rooyen A, Chirgwin E, Arthur AL, Pym A, Mackisack J, Mathews A, Kirkland L (2022). Spirotetramat resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) and its association with the presence of the A2666V mutation.
Pest Manag Sci,
78(11), 4822-4831.
Abstract:
Spirotetramat resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) and its association with the presence of the A2666V mutation.
BACKGROUND: Chemicals are widely used to protect field crops against aphid pests and aphid-borne viral diseases. One such species is Myzus persicae (Sulzer), a global pest that attacks a broad array of agricultural crops and transmits many economically damaging plant viruses. This species has evolved resistance to a large number of insecticide compounds as a result of widespread and repeated chemical use in many parts of the world. In this study, we investigated the evolution of resistance to a new plant protection product, spirotetramat, following reported chemical control failures. RESULTS: Our study provides clear phenotypic and genotypic evidence of spirotetramat resistance in populations of M. persicae from Australia. We show there is cross-resistance to other insecticides within the same chemical group, namely spiromesifen and spirodiclofen. We also demonstrate that resistance is associated with the previously reported mutation, A2226V in the target site of spirotetramat, acetyl-CoA carboxylase. Our genetic analysis found all resistant M. persicae populations belong to the same multi-locus clonal type and carry the A2226V mutation, which appears to be inherited as a dominant trait in this species. CONCLUSION: Our findings provide new insight into the resistance conferred by A2226V and have implications for the control of M. persicae in Australia and worldwide. A diagnostic assay developed in this study should serve as a valuable tool for future resistance monitoring and to support the implementation of pest management strategies. © 2022 the Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Nauen R, Bass C, Feyereisen R, Vontas J (2022). The Role of Cytochrome P450s in Insect Toxicology and Resistance.
Annu Rev Entomol,
67, 105-124.
Abstract:
The Role of Cytochrome P450s in Insect Toxicology and Resistance.
Insect cytochrome P450 monooxygenases (P450s) perform a variety of important physiological functions, but it is their role in the detoxification of xenobiotics, such as natural and synthetic insecticides, that is the topic of this review. Recent advances in insect genomics and postgenomic functional approaches have provided an unprecedented opportunity to understand the evolution of insect P450s and their role in insect toxicology. These approaches have also been harnessed to provide new insights into the genomic alterations that lead to insecticide resistance, the mechanisms by which P450s are regulated, and the functional determinants of P450-mediated insecticide resistance. In parallel, an emerging body of work on the role of P450s in defining the sensitivity of beneficial insects to insecticides has been developed. The knowledge gained from these studies has applications for the management of P450-mediated resistance in insect pests and can be leveraged to safeguard the health of important beneficial insects.
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Bushnell-Crowther E (2022). The genetic basis of adaptive behavioural traits in a global crop pest Myzus persicae (Sulzer).
Abstract:
The genetic basis of adaptive behavioural traits in a global crop pest Myzus persicae (Sulzer)
Myzus persicae is the most economically important aphid pest in temperate regions of the world causing damage to a range of food and commodity crops. This species is highly adaptable and attempts to control M. persicae using chemical insecticides have led to the evolution of resistance – with at least eight genetically distinct mechanisms described to date. In addition to variation in sensitivity to insecticides different genotypes of M. persicae exhibit variation in a range of other traits. These include behavioural traits, such as response to alarm pheromone, predator threat or insecticide presence. Additionally, in certain cases variation in these traits has been associated with genetic alterations that confer insecticide resistance, revealing potential fitness costs associated with resistance. Despite previous studies the extent of variation in behavioural traits within and between different genotypes of M. persicae, the link with insecticide resistance, and their genetic control is poorly understood.
To address this knowledge gap, 110 genetically distinct clonal lines of M. persicae were examined for variation in behavioural responses to three external stimuli with behavioural variation analysed against genomic and transcriptomic variation between clones. Pheromone behavioural assays provided clear evidence that genetically distinct clones of M. persicae vary in their response to (E)-β-Farnesene. Marked variation was observed between high responding clones (>60% total response) and low responding clones (
Abstract.
Hayward A (2022). Understanding insecticide detoxification in the leafcutter bee, Megachile rotundata.
Abstract:
Understanding insecticide detoxification in the leafcutter bee, Megachile rotundata.
Recent research on three managed bee pollinators, namely the Western honeybee (Apis mellifera), buff-tailed bumblebee (Bombus terrestris) and red mason bee (Osmia bicornis), has demonstrated that cytochrome P450 enzymes belonging to the CYP9Q and CYP9BU lineages provide protection to certain insecticides from three different mode of action classes. The alfalfa leafcutter bee (Megachile rotundata) is the world’s most economically important solitary bee species. Nonetheless, it is unclear whether this species has a CYP9Q/BU ortholog that could afford similar levels of protection against insecticides, to those seen in other managed bee pollinators.
To address this question, the M. rotundata CYPome was curated and examined using phylogenetic and syntenic analyses. These investigations revealed that this species lacks a CYP9Q/BU ortholog or closely related P450 enzyme. Topical insecticide bioassays using M. rotundata determined that the species exhibits high sensitivity to all the compounds known to be detoxified by the CYP9Q/BU lineage. For example, M. rotundata is >2,500-fold more sensitive to thiacloprid than A. mellifera. Functional studies, using M. rotundata native microsomes, revealed no significant level of metabolism of any of the insecticides known to be detoxified by the CYP9Q/BU lineages. Radioligand competition assays, on head membrane preparations, showed no significant difference in binding affinity of neonicotinoid and butanolide insecticides at the nicotinic acetylcholine receptor. Taken together these findings indicate that the lack of a CYP9Q/BU ortholog, or closely related P450, in M. rotundata correlates with an inability of microsomal P450s to metabolise certain insecticides in vitro and a high sensitivity to these compounds in vivo.
To understand how wide spread the lack of a CYP9Q/BU ortholog might be, genomic and transcriptomic CYP9 sequences from 75 bee species were examined, using phylogenetic analyses. Five of the six bee families included in the phylogeny had genes that were closely related to, or shared a recent ancestor with CYP9Q/BU lineage (~97%). The Megachilidae showed a lower prevalence of CYP9Q/BU orthologs (50%), most notably in the two species of Megachile, where, based on phylogenetic and syntenic analyses, CYP9DM genes have evolved in place of the CYP9Q/BU lineage. Sequencing of the transcriptomes of three UK and one Canadian Megachile species further substantiated that this entire genus (~1500 species) may have CYP9DM rather than CYP9Q/BU genes. Functional expression of this P450 suggested that, in contrast to several Megachilidae CYP9BU-like enzymes, CYP9DM P450s lack the capacity to bind neonicotinoids.
From the results generated in this thesis it is clear that the use of other managed bee species as a proxy for M. rotundata in ecotoxicological testing is unreliable. This has important implications for regulatory risk assessments. The data also illustrate the utility of using phylogenetic analyses, with targeted functional studies, as a tool to predict the level of sensitivity to insecticides of a bee species.
Abstract.
2021
Haas J, Zaworra M, Glaubitz J, Hertlein G, Kohler M, Lagojda A, Lueke B, Maus C, Almanza M-T, Davies TGE, et al (2021). A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.
Ecotoxicol Environ Saf,
217Abstract:
A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.
Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.
Abstract.
Author URL.
Hu B, Huang H, Hu S, Ren M, Wei Q, Tian X, Esmail Abdalla Elzaki M, Bass C, Su J, Reddy Palli S, et al (2021). Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua.
PLoS Genet,
17(3).
Abstract:
Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua.
The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.
Abstract.
Author URL.
Grant C (2021). Combating insecticide resistance in the tomato leafminer, Tuta absoluta.
Abstract:
Combating insecticide resistance in the tomato leafminer, Tuta absoluta.
BACKGROUND: the tomato leafminer, Tuta absoluta is a damaging pest of tomato crops worldwide. In the UK, T. absoluta is controlled using an integrated pest management (IPM) strategy that includes the pesticides spinosad and chlorantraniliprole, the biocontrol agent Macrolophus pygmaeus and pheromone-based mating disruption. Some growers have reported a loss of efficacy of this technology. There are concerns that T. absoluta may have evolved resistance to these applied chemistries as well as undergone adaptations in its capacity to reproduce asexually. In this thesis I investigate whether pesticide resistance is present in UK populations and identify the molecular mechanisms for this resistance. I will also investigate the capacity T. absoluta to reproduce asexually through parthenogenesis in the absence of males.
RESULTS: I demonstrate that UK populations of T. absoluta are highly resistant to spinosad and identify two novel mechanisms by which resistance has evolved. Analysis of messenger RNA encoding the target site of spinosad, the nicotinic acetylcholine receptor (nAChR) α6 subunit, revealed resistant strains lack exon 4 resulting in a highly truncated protein. In a second resistant strain the deletion of three amino acids is detected in the transmembrane domain of the nAChR - predicted to be the binding site of spinosad. I identify low levels of tolerance to chlorantraniliprole in UK populations and show this resistance can be selected for to produce highly resistant populations. Analysis of the target site of chlorantraniliprole, the ryanodine receptor, identified amino acid substitution G4903V that has been strongly linked to diamide resistance in a range of lepidopteran species including T. absoluta. With regards asexual reproduction, I observed a small but significant increase in the rate of asexual reproduction. This allows persistence of the pest in the presence of the mating disruptor, Isonet T. Marked differences in several other life history traits associated with reproduction were also observed in these populations including increased longevity further allowing T. absoluta’s persistence within the crop.
CONCLUSION: My findings show that the evolution of resistance has rendered spinosad redundant at most sites in the UK. The mechanisms identified are unique to UK populations and so have likely evolved under selection in the UK. Chlorantraniliprole remains effective, however our findings of resistance at low frequency suggest that continued use of this pesticide must be monitored carefully. The low overall occurrence of asexual reproduction observed in this study is unlikely to result in loss of efficacy of mating disruption as reproductive rate remained low. However, the observed changes in longevity and egg laying may allow T. absoluta to persist for longer within the crop, and, together with the increased frequency of parthenogenesis, may reflect selection from the use of Isonet T. Thus, regular monitoring of the reproductive capacity of UK populations should be conducted, along with continual assessment of resistance allele frequencies of pesticides to inform resistance management strategies.
Abstract.
Erdos Z, Chandler D, Bass C, Raymond B (2021). Controlling insecticide resistant clones of the aphid, Myzus persicae, using the entomopathogenic fungus Akanthomyces muscarius: fitness cost of resistance under pathogen challenge.
Pest Manag Sci,
77(11), 5286-5293.
Abstract:
Controlling insecticide resistant clones of the aphid, Myzus persicae, using the entomopathogenic fungus Akanthomyces muscarius: fitness cost of resistance under pathogen challenge.
BACKGROUND: Biological control is a cornerstone of integrated pest management and could also play a key role in managing the evolution of insecticide resistance. Ecological theory predicts that the fitness cost of insecticide resistance can be increased under exposure to invertebrate natural enemies or pathogens, and can therefore increase the value of integrating biological control into pest management. In this study of the peach potato aphid, Myzus persicae, we aimed to identify whether insecticide resistance affected fitness and vulnerability of different aphid clones to the entomopathogenic fungus Akanthomyces muscarius. RESULTS: Insecticide resistant clones were found to be slightly less susceptible to the pathogen than susceptible clones. However, this pattern could also be explained by the influence of length of laboratory culture, which was longer in susceptible clones and was positively correlated with susceptibility to fungi. Furthermore, resistance status did not affect aphid development time or intrinsic rate of increase of aphids. Finally, in a cage trial the application of fungus did not increase the competitive fitness of insecticide resistant clone 'O'. CONCLUSION: We found no fitness cost in reproductive rate or pathogen susceptibility associated with chemical resistance in M. persicae. In contrast, some susceptible clones, particularly those subject to decades of laboratory rearing, showed enhanced susceptibility to a fungal pathogen, but not reduced reproductive fitness, an observation consistent with down-regulation of costly immune functions in culture. Overall, fungal pathogen control is compatible with insecticides and should not increase the selection pressure for resistance of M. persicae to chemical insecticides.
Abstract.
Author URL.
McLeman A (2021). Developing screening tools to identify novel, resistance breaking pesticides.
Abstract:
Developing screening tools to identify novel, resistance breaking pesticides.
Pesticide resistance is estimated to cost the USA $1.4 billion annually. Not only is there a huge economic cost, but the loss of crop yield and higher doses of pesticides needed to control pests damages the ecosystem 1,2. The development of resistance to chemicals is a universal phenomenon and within insect pests more than 440 species are now resistant to one or more pesticidal compound 3,4. As increasing levels of resistance arise and new molecular tools become available the understanding of resistance mechanisms grows and the limitations of pesticides are clarified 5,6. Understanding resistance is vital to counter it 5,7,8.
Still facing high levels of pesticide resistance and the damaging effects of the remaining effective compounds, I here look to identify a novel pesticidal compound to overcome current resistance mechanisms 9. Synthetic compounds made by industrial partner Darr House M.I. were tested for activity against Drosophila melanogaster and Myzus persicae. The first 18 compounds were expected to act on the nicotinic acetylcholine receptor using imidacloprid as a positive control. Four competitively active compounds were found but, following a ban on neonicotinoids in the EU in 2018 and a knock-on lack of interest on the part of major agrochemical companies in novel nAChR compounds, this part of the project was pursued no further 10,11. The next 30 compounds were then tested for activity against the neurotransmitter gamma-aminobutyric acid (GABA) receptor. Here activity was only found against Drosophila not Myzus. Five compounds showed activity against D. melanogaster susceptible strain Canton-S, four then showed activity against metabolic resistant strain Hikone-R with compound 47 being close to resistance breaking.
While synthetic compounds are popular, natural sources are not only a source of inspiration for synthetic products but natural products used for pest control have advantages of being environmentally friendly and constantly evolving with their pests. I tested 9 botanical sources for insecticidal and repellent activity against D. melanogaster and the Peach potato aphid M. persicae. Extracts from samples were taken using a methanol extraction technique. Rosemary extract results suggest potential lethal effects on Drosophila but development of this product would be required to concentrate the lethal effects above 40%. All extracts: basil, chilli, garlic, lemongrass, nasturtium leaves, flowers and seeds and rosemary showed repellent activity against Myzus except dill extract which had no effect. An increase in nymph droppings was seen for Myzus treated with basil suggesting possible problems for use of this compound as aphid control.
To address the problem of cost and identification of novel active pesticides a Fly-Tox panel was developed using D. melanogaster as a model screening tool containing metabolic P450 resistance genes from multiple economically important pests and pollinator species. In this thesis four lines were developed containing Cyp6cm1, Cyp6bq23, Cyp6bq9 and Cyp337b3 but conferral of resistance was unsuccessful. Alternative lines from the published Fly-Tox panel were used to test the use of the screening tool with novel insecticides from chapter 2 and 3; one nAChR and one GABA targeting compound. These novel compounds were compared against positive controls; imidacloprid and fipronil, and showed a successful test run of a section of the screening tool.
No resistance breaking bee-safe compounds were identified in this thesis but there was a successful trial of the Fly-Tox screening tool of transgenic Drosophila showing the value of this new resource in pesticidal discovery science.
There were also findings of broad metabolic capabilities of the gene Cyp6er1, known to metabolise neonicotinoids, but also found to be active against suspected GABA targeting novel compound 47.
Abstract.
Yang X, Wei X, Yang J, Du T, Yin C, Fu B, Huang M, Liang J, Gong P, Liu S, et al (2021). Epitranscriptomic regulation of insecticide resistance.
Sci Adv,
7(19).
Abstract:
Epitranscriptomic regulation of insecticide resistance.
N 6-methyladenosine (m6A) is the most prevalent messenger RNA modification in eukaryotes and an important posttranscriptional regulator of gene expression. However, the biological roles of m6A in most insects remain largely unknown. Here, we show that m6A regulates a cytochrome P450 gene (CYP4C64) in the global whitefly pest, Bemisia tabaci, leading to insecticide resistance. Investigation of the regulation of CYP4C64, which confers resistance to the insecticide thiamethoxam, revealed a mutation in the 5' untranslated region of this gene in resistant B. tabaci strains that introduces a predicted m6A site. We provide several lines of evidence that mRNA methylation of the adenine at this position, in combination with modified expression of m6A writers, acts to increase expression of CYP4C64 and resistance. Collectively, these results provide an example of the epitranscriptomic regulation of the xenobiotic response in insects and implicate the m6A regulatory axis in the development of insecticide resistance.
Abstract.
Author URL.
Singh KS, De-Kayne R, Omufwoko KS, Martins DJ, Bass C, ffrench-Constant R, Martin SH (2021). Genome assembly of Danaus chrysippus and comparison with the Monarch Danaus plexippus.
Singh KS, De-Kayne R, Omufwoko KS, Martins DJ, Bass C, ffrench-Constant R, Martin SH (2021). Genome assembly of Danaus chrysippus and comparison with the Monarch Danaus plexippus. G3: Genes, Genomes, Genetics, 12(3).
Singh KS, Cordeiro EMG, Troczka BJ, Pym A, Mackisack J, Mathers TC, Duarte A, Legeai F, Robin S, Bielza P, et al (2021). Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.
Commun Biol,
4(1).
Abstract:
Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.
The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host-plant associations, uncovering the widespread co-option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.
Abstract.
Author URL.
Troczka BJ, Singh KS, Zimmer CT, Vontas J, Nauen R, Hayward A, Bass C (2021). Molecular innovations underlying resistance to nicotine and neonicotinoids in the aphid Myzus persicae.
Pest Manag Sci,
77(12), 5311-5320.
Abstract:
Molecular innovations underlying resistance to nicotine and neonicotinoids in the aphid Myzus persicae.
The green peach aphid, Myzus persicae, is a globally distributed highly damaging crop pest. This species has demonstrated an exceptional ability to evolve resistance to both synthetic insecticides used for control, and natural insecticides produced by certain plants as a chemical defense against insect attack. Here we review work characterizing the evolution of resistance in M. persicae to the natural insecticide nicotine and the structurally related class of synthetic neonicotinoid insecticides. We outline how research on this topic has provided insights into long-standing questions of both evolutionary and applied importance. These include questions pertaining to the origins of novel traits, the number and nature of mutational events or 'adaptive steps' underlying the evolution of new phenotypes, and whether host plant adaptations can be co-opted to confer resistance to synthetic insecticides. Finally, research on the molecular mechanisms underlying insecticide resistance in M. persicae has generated several outstanding questions on the genetic architecture of resistance to both natural and synthetic xenobiotics, and we conclude by identifying key knowledge gaps for future research. © 2021 the Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Abstract.
Author URL.
Grant C, Jacobson R, Bass C (2021). Parthenogenesis in UK field populations of the tomato leaf miner, Tuta absoluta, exposed to the mating disruptor Isonet T.
Pest Manag Sci,
77(7), 3445-3449.
Abstract:
Parthenogenesis in UK field populations of the tomato leaf miner, Tuta absoluta, exposed to the mating disruptor Isonet T.
BACKGROUND: the tomato leafminer, Tuta absoluta is a damaging pest of tomato crops worldwide. In the UK T. absoluta is controlled using an integrated pest management (IPM) strategy that includes pheromone-based mating disruption. However, some growers have reported a loss of efficacy of this technology, and there are concerns that T. absoluta may evolve resistance via changes in its capacity to reproduce asexually. In this study we investigated the reproductive capacity of virgin populations of T. absoluta collected from a UK glasshouse before (EVH2016) and after (EVH2019) the introduction of the mating disrupter Isonet T. RESULTS: in line with earlier reports, we demonstrate that UK populations of T. absoluta can reproduce parthenogenetically, and observed a small but significant increase in the rate of parthenogenesis associated with the use of Isonet T. Marked differences in several other life history traits associated with reproduction were also observed between the two virgin populations, with the EVH2019 strain producing fewer eggs, a delayed onset of egg laying and increased lifespan. CONCLUSION: the low rate of parthenogenetic reproduction seen in this study is unlikely to result in loss of efficacy of mating disruption. However, the observed changes in longevity and egg laying may allow T. absoluta to persist for longer within the crop, and, together with the increased rate of parthenogenesis, may reflect selection from the use of Isonet T. Thus, regular monitoring of the reproductive capacity of UK populations should be conducted, and mating disruption used only as part of IPM to avoid the emergence of resistance. © 2021 the Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Abstract.
Author URL.
Panini M, Chiesa O, Troczka BJ, Mallott M, Manicardi GC, Cassanelli S, Cominelli F, Hayward A, Mazzoni E, Bass C, et al (2021). Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae.
Proc Natl Acad Sci U S A,
118(23).
Abstract:
Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae.
The evolution of resistance to insecticides threatens the sustainable control of many of the world's most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.
Abstract.
Author URL.
2020
Reid RJ, Troczka BJ, Kor L, Randall E, Williamson MS, Field LM, Nauen R, Bass C, Davies TGE (2020). Assessing the acute toxicity of insecticides to the buff-tailed bumblebee (Bombus terrestris audax).
Pesticide Biochemistry and Physiology,
166Abstract:
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.
Abstract.
Singh KS, Hosken DJ, Wedell N, Ffrench-Constant R, Bass C, Baxter S, Paszkiewicz K, Sharma MD (2020). De Novo Genome Assembly of the Meadow Brown Butterfly, Maniola jurtina.
G3 (Bethesda),
10(5), 1477-1484.
Abstract:
De Novo Genome Assembly of the Meadow Brown Butterfly, Maniola jurtina.
Meadow brown butterflies (Maniola jurtina) on the Isles of Scilly represent an ideal model in which to dissect the links between genotype, phenotype and long-term patterns of selection in the wild - a largely unfulfilled but fundamental aim of modern biology. To meet this aim, a clear description of genotype is required. Here we present the draft genome sequence of M. jurtina to serve as a founding genetic resource for this species. Seven libraries were constructed using pooled DNA from five wild caught spotted females and sequenced using Illumina, PacBio RSII and MinION technology. A novel hybrid assembly approach was employed to generate a final assembly with an N50 of 214 kb (longest scaffold 2.9 Mb). The sequence assembly described here predicts a gene count of 36,294 and includes variants and gene duplicates from five genotypes. Core BUSCO (Benchmarking Universal Single-Copy Orthologs) gene sets of Arthropoda and Insecta recovered 90.5% and 88.7% complete and single-copy genes respectively. Comparisons with 17 other Lepidopteran species placed 86.5% of the assembled genes in orthogroups. Our results provide the first high-quality draft genome and annotation of the butterfly M. jurtina.
Abstract.
Author URL.
McLeman A, Troczka BJ, Homem RA, Duarte A, Zimmer C, Garrood WT, Pym A, Beadle K, Reid RJ, Douris V, et al (2020). Fly-Tox: a panel of transgenic flies expressing pest and pollinator cytochrome P450s.
Pesticide Biochemistry and Physiology,
169Abstract:
Fly-Tox: a panel of transgenic flies expressing pest and pollinator cytochrome P450s
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.
Abstract.
Lueke B, Douris V, Hopkinson JE, Maiwald F, Hertlein G, Papapostolou KM, Bielza P, Tsagkarakou A, Van Leeuwen T, Bass C, et al (2020). Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci.
Pesticide Biochemistry and Physiology,
166Abstract:
Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci
Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.
Abstract.
Dupeyron M, Baril T, Bass C, Hayward A (2020). Phylogenetic analysis of the Tc1/mariner superfamily reveals the unexplored diversity of pogo-like elements.
Mob DNA,
11Abstract:
Phylogenetic analysis of the Tc1/mariner superfamily reveals the unexplored diversity of pogo-like elements.
BACKGROUND: Tc1/mariner transposons are widespread DNA transposable elements (TEs) that have made important contributions to the evolution of host genomic complexity in metazoans. However, the evolution and diversity of the Tc1/mariner superfamily remains poorly understood. Following recent developments in genome sequencing and the availability of a wealth of new genomes, Tc1/mariner TEs have been identified in many new taxa across the eukaryotic tree of life. To date, the majority of studies focussing on Tc1/mariner elements have considered only a single host lineage or just a small number of host lineages. Thus, much remains to be learnt about the evolution of Tc1/mariner TEs by performing analyses that consider elements that originate from across host diversity. RESULTS: We mined the non-redundant database of NCBI using BLASTp searches, with transposase sequences from a diverse set of reference Tc1/mariner elements as queries. A total of 5158 Tc1/mariner elements were retrieved and used to reconstruct evolutionary relationships within the superfamily. The resulting phylogeny is well resolved and includes several new groups of Tc1/mariner elements. In particular, we identify a new family of plant-genome restricted Tc1/mariner elements, which we call PlantMar. We also show that the pogo family is much larger and more diverse than previously appreciated, and we review evidence for a potential revision of its status to become a separate superfamily. CONCLUSIONS: Our study provides an overview of Tc1-mariner phylogeny and summarises the impressive diversity of Tc1-mariner TEs among sequenced eukaryotes. Tc1/mariner TEs are successful in a wide range of eukaryotes, especially unikonts (the taxonomic supergroup containing Amoebozoa, Opisthokonta, Breviatea, and Apusomonadida). In particular, ecdysozoa, and especially arthropods, emerge as important hosts for Tc1/mariner elements (except the PlantMar family). Meanwhile, the pogo family, which is by far the largest Tc1/mariner family, also includes many elements from fungal and chordate genomes. Moreover, there is evidence of the repeated exaptation of pogo elements in vertebrates, including humans, in addition to the well-known example of CENP-B. Collectively, our findings provide a considerable advancement in understanding of Tc1/mariner elements, and more generally they suggest that much work remains to improve understanding of the diversity and evolution of DNA TEs.
Abstract.
Author URL.
Pym A (2020). The biological and molecular factors influencing control of Trialeurodes vaporariorum and Bemisia tabaci on different host plants.
Abstract:
The biological and molecular factors influencing control of Trialeurodes vaporariorum and Bemisia tabaci on different host plants
The greenhouse whitefly Trialeurodes vaporariorum and the tobacco whitefly Bemisia tabaci are global crop pests which cause damage to a wide variety of plants encompassing fruits, vegetables and ornamentals. Their sap-sucking nature combined with their aptitude to act as vector for plant viruses means that control of these organisms is vital for crop management, generally involving the use of synthetic insecticides. Previous work has observed fluctuation in tolerance to insecticides within populations of whiteflies feeding on different host plants.
To investigate this, lines of whiteflies were established on five different host plants; pumpkin (Cucurbita pepo), cucumber (Cucumis sativus), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) and French bean (Phaseolus vulgaris). Bioassays on these populations revealed large differences in tolerance to insecticides across multiple chemical classes. The general trend observed saw the lines of whiteflies on the nightshade hosts, tobacco and tomato, developing an increased tolerance to pesticides. RNA sequencing from the same whitefly lines revealed large scale changes in gene expression, again particularly in the nightshade-reared lines. Many of the over-expressed genes belonged to detoxification enzyme super-families, which have previously been implicated in insecticide resistance.
Sequencing of the first T. vaporariorum genome using 10x genomics discovered multiple novel genes encoding detoxification enzymes. Comparisons between the two whitefly species T. vaporariorum and B. tabaci revealed a 1.5-fold increase in detoxification enzymes present in B. tabaci which could partly explain why this species is considered a greater global pest.
Four T. vaporariorum cytochrome-P450s have previously been associated with resistance to insecticides; CYP6CM2, 3 and 4 with neonicotinoids and CYP4G61 with the juvenile hormone analog pyriproxyfen. To identify whether they were able to confer resistance, the three genes were inserted into D. melanogaster lines and bioassayed using the GAL4/UAS system to test their potential to convey resistance to insecticides. No link between the CYP6CM2-4 genes and resistance to neonicotinoids was discovered although an increased tolerance to the natural plant compound nicotine was observed. Similarly, CYP4G61 did not appear to confer resistance to pyriproxyfen in flies, a result confirmed by further microsomal analysis.
Abstract.
Singh KS, Troczka BJ, Duarte A, Balabanidou V, Trissi N, Carabajal Paladino LZ, Nguyen P, Zimmer CT, Papapostolou KM, Randall E, et al (2020). The genetic architecture of a host shift: an adaptive walk protected an aphid and its endosymbiont from plant chemical defenses.
Science Advances,
6(19).
Abstract:
The genetic architecture of a host shift: an adaptive walk protected an aphid and its endosymbiont from plant chemical defenses
A complex series of mutational events protected a mutualistic symbiosis during the shift of an insect to a toxic host plant.
Abstract.
Douris V, Denecke S, Van Leeuwen T, Bass C, Nauen R, Vontas J (2020). Using CRISPR/Cas9 genome modification to understand the genetic basis of insecticide resistance: Drosophila and beyond.
Pesticide Biochemistry and Physiology,
167Abstract:
Using CRISPR/Cas9 genome modification to understand the genetic basis of insecticide resistance: Drosophila and beyond
Chemical insecticides are a major tool for the control of many of the world's most damaging arthropod pests. However, their intensive application is often associated with the emergence of resistance, sometimes with serious implications for sustainable pest control. To mitigate failure of insecticide-based control tools, the mechanisms by which insects have evolved resistance must be elucidated. This includes both identification and functional characterization of putative resistance genes and/or mutations. Research on this topic has been greatly facilitated by using powerful genetic model insects like Drosophila melanogaster, and more recently by advances in genome modification technology, notably CRISPR/Cas9. Here, we present the advances that have been made through the application of genome modification technology in insecticide resistance research. The majority of the work conducted in the field to date has made use of genetic tools and resources available in D. melanogaster. This has greatly enhanced our understanding of resistance mechanisms, especially those mediated by insensitivity of the pesticide target-site. We discuss this progress for a series of different insecticide targets, but also report a number of unsuccessful or inconclusive attempts that highlight some inherent limitations of using Drosophila to characterize resistance mechanisms identified in arthropod pests. We also discuss an experimental framework that may circumvent current limitations while retaining the genetic versatility and robustness that Drosophila has to offer. Finally, we describe examples of direct CRISPR/Cas9 use in non-model pest species, an approach that will likely find much wider application in the near future.
Abstract.
ffrench-Constant R, Martin S, Bass C, Singh K, Traut W, Gordon I, Smith D, Martins D (2020). Whole-chromosome hitchhiking driven by a male-killing endosymbiont. PLoS Biology
Hu B, Ren M, Fan J, Huang S, Wang X, Elzaki MEA, Bass C, Palli SR, Su J (2020). Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua.
JOURNAL OF HAZARDOUS MATERIALS,
398 Author URL.
2019
Mallott M, Hamm S, Troczka BJ, Randall E, Pym A, Grant C, Baxter S, Vogel H, Shelton AM, Field LM, et al (2019). A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella.
Insect Biochem Mol Biol,
115Abstract:
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.
Abstract.
Author URL.
Abeku TA, Helinski MEH, Kirby MJ, Kefyalew T, Awano T, Batisso E, Tesfaye G, Ssekitooleko J, Nicholas S, Erdmanis L, et al (2019). Correction to: Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.
Malar J,
18(1).
Abstract:
Correction to: Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.
Please be advised that one of the author names is incorrectly spelled in the published article: 'Irene Kyomuhagi' should be 'Irene Kyomuhangi'.
Abstract.
Author URL.
Singh KS, Troczka BJ, Beadle K, Field LM, Davies TGE, Williamson MS, Nauen R, Bass C (2019). Extension of Partial Gene Transcripts by Iterative Mapping of RNA-Seq Raw Reads.
IEEE/ACM Transactions on Computational Biology and Bioinformatics,
16(3), 1036-1041.
Abstract:
Extension of Partial Gene Transcripts by Iterative Mapping of RNA-Seq Raw Reads
Many non-model organisms lack reference genomes and the sequencing and de novo assembly of an organisms transcriptome is an affordable means by which to characterize the coding component of its genome. Despite the advances that have made this possible, assembling a transcriptome without a known reference usually results in a collection of full-length and partial gene transcripts. The downstream analysis of genes represented as partial transcripts then often requires further experimental work in the laboratory in order to obtain full- length sequences. We have explored whether partial transcripts, encoding genes of interest present in de novo assembled transcriptomes of a model and non-model insect species, could be further extended by iterative mapping against the raw transcriptome sequencing reads. Partial sequences encoding cytochrome P450s and carboxyl/cholinesterase were used in this analysis, because they are large multigene families and exhibit significant variation in expression. We present an effective method to improve the contiguity of partial transcripts in silico that, in the absence of a reference genome, may be a quick and cost-effective alternative to their extension by laboratory experimentation. Our approach resulted in the successful extension of incompletely assembled transcripts, often to full length. We experimentally validated these results in silico and using real-time PCR and sequencing.
Abstract.
Beadle K, Singh KS, Troczka BJ, Randall E, Zaworra M, Zimmer CT, Hayward A, Reid R, Kor L, Kohler M, et al (2019). Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis.
PLoS Genet,
15(2).
Abstract:
Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis.
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.
Abstract.
Author URL.
Pym A, Singh KS, Nordgren Ã…, Davies TGE, Zimmer CT, Elias J, Slater R, Bass C (2019). Host plant adaptation in the polyphagous whitefly, Trialeurodes vaporariorum, is associated with transcriptional plasticity and altered sensitivity to insecticides.
BMC Genomics,
20(1).
Abstract:
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.
Abstract.
Author URL.
Troczka BJ, Homem RA, Reid R, Beadle K, Kohler M, Zaworra M, Field LM, Williamson MS, Nauen R, Bass C, et al (2019). Identification and functional characterisation of a novel N-cyanoamidine neonicotinoid metabolising cytochrome P450, CYP9Q6, from the buff-tailed bumblebee Bombus terrestris.
Insect Biochem Mol Biol,
111Abstract:
Identification and functional characterisation of a novel N-cyanoamidine neonicotinoid metabolising cytochrome P450, CYP9Q6, from the buff-tailed bumblebee Bombus terrestris.
Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides.
Abstract.
Author URL.
Carabajal Paladino LZ, ProvaznÃková I, Berger M, Bass C, Aratchige NS, López SN, Marec F, Nguyen P (2019). Sex Chromosome Turnover in Moths of the Diverse Superfamily Gelechioidea.
Genome Biol Evol,
11(4), 1307-1319.
Abstract:
Sex Chromosome Turnover in Moths of the Diverse Superfamily Gelechioidea.
Sex chromosomes play a central role in genetics of speciation and their turnover was suggested to promote divergence. In vertebrates, sex chromosome-autosome fusions resulting in neo-sex chromosomes occur frequently in male heterogametic taxa (XX/XY), but are rare in groups with female heterogamety (WZ/ZZ). We examined sex chromosomes of seven pests of the diverse lepidopteran superfamily Gelechioidea and confirmed the presence of neo-sex chromosomes in their karyotypes. Two synteny blocks, which correspond to autosomes 7 (LG7) and 27 (LG27) in the ancestral lepidopteran karyotype exemplified by the linkage map of Biston betularia (Geometridae), were identified as sex-linked in the tomato leafminer, Tuta absoluta (Gelechiidae). Testing for sex-linkage performed in other species revealed that while LG7 fused to sex chromosomes in a common ancestor of all Gelechioidea, the second fusion between the resulting neo-sex chromosome and the other autosome is confined to the tribe Gnoreschemini (Gelechiinae). Our data accentuate an emerging pattern of high incidence of neo-sex chromosomes in Lepidoptera, the largest clade with WZ/ZZ sex chromosome system, which suggest that the paucity of neo-sex chromosomes is not an intrinsic feature of female heterogamety. Furthermore, LG7 contains one of the major clusters of UDP-glucosyltransferases, which are involved in the detoxification of plant secondary metabolites. Sex chromosome evolution in Gelechioidea thus supports an earlier hypothesis postulating that lepidopteran sex chromosome-autosome fusions can be driven by selection for association of Z-linked preference or host-independent isolation genes with larval performance and thus can contribute to ecological specialization and speciation of moths.
Abstract.
Author URL.
Grant C, Jacobson R, Ilias A, Berger M, Vasakis E, Bielza P, Zimmer CT, Williamson MS, Ffrench-Constant RH, Vontas J, et al (2019). The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.
Pest Manag Sci,
75(8), 2079-2085.
Abstract:
The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.
BACKGROUND: the tomato leafminer, Tuta absoluta, is an economically important pest of tomatoes in Europe, Africa, Asia and South America. In the UK this species is controlled using an integrated pest management (IPM) programme which incorporates the insecticides spinosad and chlorantraniliprole. In response to UK grower concerns of loss of efficacy of these compounds at certain sites, insecticide bioassays were performed on five populations collected from four commercial glasshouses and potential mechanisms of resistance investigated. RESULTS: We observed high levels of resistance to spinosad in four of the strains, and in two of these tolerance to chlorantraniliprole. Selection of one of these strains with chlorantraniliprole rapidly resulted in a line exhibiting potent resistance to this compound. Sequencing of messenger RNA encoding the nicotinic acetylcholine receptor (nAChR) α6 subunit, target of spinosad, revealed Taα6 transcripts in the spinosad-resistant strains that lack exon 4 and encode a highly truncated protein, or contain a triplet deletion in the predicted first transmembrane domain resulting in the loss of a highly conserved amino acid. Sequencing of the ryanodine receptor gene, encoding the target of diamide insecticides, of the chlorantraniliprole-selected line revealed an amino acid substitution (G4903V) that has been previously linked to diamide resistance in populations of T. absoluta in the Mediterranean and South America. CONCLUSION: Taken together our results reveal emerging resistance in UK populations of T. absoluta to two of the most important insecticides used as part of IPM, with significant implications for the control of this species in the UK. © 2019 Society of Chemical Industry.
Abstract.
Author URL.
Hawkins NJ, Bass C, Dixon A, Neve P (2019). The evolutionary origins of pesticide resistance.
Biol Rev Camb Philos Soc,
94(1), 135-155.
Abstract:
The evolutionary origins of pesticide resistance.
Durable crop protection is an essential component of current and future food security. However, the effectiveness of pesticides is threatened by the evolution of resistant pathogens, weeds and insect pests. Pesticides are mostly novel synthetic compounds, and yet target species are often able to evolve resistance soon after a new compound is introduced. Therefore, pesticide resistance provides an interesting case of rapid evolution under strong selective pressures, which can be used to address fundamental questions concerning the evolutionary origins of adaptations to novel conditions. We ask: (i) whether this adaptive potential originates mainly from de novo mutations or from standing variation; (ii) which pre-existing traits could form the basis of resistance adaptations; and (iii) whether recurrence of resistance mechanisms among species results from interbreeding and horizontal gene transfer or from independent parallel evolution. We compare and contrast the three major pesticide groups: insecticides, herbicides and fungicides. Whilst resistance to these three agrochemical classes is to some extent united by the common evolutionary forces at play, there are also important differences. Fungicide resistance appears to evolve, in most cases, by de novo point mutations in the target-site encoding genes; herbicide resistance often evolves through selection of polygenic metabolic resistance from standing variation; and insecticide resistance evolves through a combination of standing variation and de novo mutations in the target site or major metabolic resistance genes. This has practical implications for resistance risk assessment and management, and lessons learnt from pesticide resistance should be applied in the deployment of novel, non-chemical pest-control methods.
Abstract.
Author URL.
Hayward A, Beadle K, Singh KS, Exeler N, Zaworra M, Almanza M-T, Nikolakis A, Garside C, Glaubitz J, Bass C, et al (2019). The leafcutter bee, Megachile rotundata, is more sensitive to N-cyanoamidine neonicotinoid and butenolide insecticides than other managed bees. Nature Ecology & Evolution, 3(11), 1521-1524.
Martin SH, Singh KS, Gordon IJ, Omufwoko KS, Collins S, Warren IA, Munby H, Brattström O, Traut W, Martins DJ, et al (2019). Whole-chromosome hitchhiking driven by a male-killing endosymbiont.
2018
Dermauw W, Pym A, Bass C, Van Leeuwen T, Feyereisen R (2018). Does host plant adaptation lead to pesticide resistance in generalist herbivores?.
Current Opinion in Insect Science,
26, 25-33.
Abstract:
Does host plant adaptation lead to pesticide resistance in generalist herbivores?
Most herbivorous arthropods feed on one or a few closely related plant species; however, certain insect and mite species have a greatly expanded host range. Several of these generalists also show a remarkable propensity to evolve resistance to chemical pesticides. In this review, we ask if the evolution of mechanisms to tolerate the diversity of plant secondary metabolites that generalist herbivores encounter, has pre-adapted them to resist synthetic pesticides. Critical examination of the evidence suggests that a generalist life-style per se is not a predictor of rapid resistance evolution to pesticides. Rather the prevalence of pesticide resistance in generalist herbivores probably reflects their economic importance as pests and thus the strong selection imposed by intensive pesticide use.
Abstract.
Bass C, Jones CM (2018). Editorial overview: Pests and resistance: Resistance to pesticides in arthropod crop pests and disease vectors: mechanisms, models and tools. Current Opinion in Insect Science, 27, iv-vii.
Jones CM, Lim KS, Chapman JW, Bass C (2018). Genome-Wide Characterization of DNA Methylation in an Invasive Lepidopteran Pest, the Cotton Bollworm Helicoverpa armigera.
G3 (Bethesda),
8(3), 779-787.
Abstract:
Genome-Wide Characterization of DNA Methylation in an Invasive Lepidopteran Pest, the Cotton Bollworm Helicoverpa armigera.
The genes and genomes of insect pests are shaped by the wide array of selective forces encountered in their environments. While the molecular adaptations that evolve are beginning to be understood at the genomic and transcriptomic level, they have been less well characterized at an epigenetic level. Here, we present a genome-wide map of DNA methylation at single-nucleotide resolution for the cotton bollworm moth, Helicoverpa armigera, a globally invasive pest of agriculture. We show that methylation is almost identical in the larvae and adults of H. armigera and that, through whole-genome bisulfite sequencing (WGBS), at the most ∼0.9% of CpG sites in this species are methylated. We find that DNA methylation occurs primarily in exons, is positively correlated with gene expression, and that methylated genes are enriched for cellular "housekeeping" roles. H. armigera has an exceptional capacity for long-range migration. To explore the role of methylation in influencing the migratory phenotype of H. armigera, we performed targeted bisulfite sequencing on selected loci from 16 genes that were differentially expressed between adult moths exhibiting distinct flight performance in behavioral assays. While most CpG sites in these genes were not methylated between flight phenotypes, we identified hypermethylation in a demethylase (KDM4) that targets lysine-specific histone modifications, which are strongly associated with transcription and methylation. The H. armigera methylome provides new insights into the role of DNA methylation in a noctuid moth and is a valuable resource for further research into the epigenetic control of adaptive traits in this important pest.
Abstract.
Author URL.
Zimmer C, Garrood W, Singh K, Randall E, Bettina L, Gutbrod O, Matthiesen S, Kohler M, Nauen R, Davies TGE, et al (2018). Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper. Current Biology, 28, 268-274.
Bass C, Field LM (2018). Neonicotinoids.
Current Biology,
28(14), R772-R773.
Abstract:
Neonicotinoids
Neonicotinoids are a class of pesticides widely used in agriculture. In this Quick Guide, Bass and Field explain their mechanism of action and discuss the evidence supporting a potential role in pollinator declines.
Abstract.
Manjon C, Troczka B, Zaworra M, Beadle K, Randall E, Hertlein G, Singh K, Zimmer C, Homem R, Lueke B, et al (2018). Unravelling the molecular determinants of bee sensitivity to neonicotinoid insecticides. Current Biology, 28, 1-7.
2017
Field LM, Bass C, Davies TGE, Williamson MS, Zhou JJ (2017). Aphid genomics and its contribution to understanding aphids as crop pests. In (Ed) Aphids as crop pests, 37-49.
Panini M, Tozzi F, Zimmer CT, Bass C, Field L, Borzatta V, Mazzoni E, Moores G (2017). Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).
Pest Manag Sci,
73(9), 1873-1882.
Abstract:
Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).
BACKGROUND: Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase-based mechanism has been attributed to pyrethroid resistance and over-expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. RESULTS: in vitro interactions between putative synergists and CYP6CM1, B and Q-type esterases were investigated, and structure-activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B-type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q-esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. CONCLUSIONS: Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industry.
Abstract.
Author URL.
ffrench-Constant RH, Bass C (2017). Does resistance really carry a fitness cost?.
Current Opinion in Insect Science,
21, 39-46.
Abstract:
Does resistance really carry a fitness cost?
Insecticide resistance mutations are widely assumed to carry fitness costs. However studies to measure such costs are rarely performed on genetically related strains and are often only done in the laboratory. Theory also suggests that once evolved the cost of resistance can be offset by the evolution of fitness modifiers. But for insecticide resistance only one such example is well documented. Here we critically examine the literature on fitness costs in the absence of pesticide and ask if our knowledge of molecular biology has helped us predict the costs associated with different resistance mechanisms. We find that resistance alleles can arise from pre-existing polymorphisms and resistance associated variation can also be maintained by sexual antagonism. We describe novel mechanisms whereby both resistant and susceptible alleles can be maintained in permanent heterozygosis and discuss the likely consequences for fitness both in the presence and absence of pesticide. Taken together these findings suggest that we cannot assume that resistance always appears de novo and that our assumptions about the associated fitness costs need to be informed by a deeper understanding of the underlying molecular biology.
Abstract.
Garrood WT, Zimmer CT, Gutbrod O, Lüke B, Williamson MS, Bass C, Nauen R, Emyr Davies TG (2017). Influence of the RDL A301S mutation in the brown planthopper Nilaparvata lugens on the activity of phenylpyrazole insecticides.
Pesticide Biochemistry and Physiology,
142, 1-8.
Abstract:
Influence of the RDL A301S mutation in the brown planthopper Nilaparvata lugens on the activity of phenylpyrazole insecticides
We discovered the A301S mutation in the RDL GABA-gated chloride channel of fiprole resistant rice brown planthopper, Nilaparvata lugens populations by DNA sequencing and SNP calling via RNASeq. Ethiprole selection of two field N. lugens populations resulted in strong resistance to both ethiprole and fipronil and resulted in fixation of the A301S mutation, as well as the emergence of another mutation, Q359E in one of the selected strains. To analyse the roles of these mutations in resistance to phenylpyrazoles, three Rdl constructs: wild type, A301S and A301S + Q359E were expressed in Xenopus laevis oocytes and assessed for their sensitivity to ethiprole and fipronil using two-electrode voltage-clamp electrophysiology. Neither of the mutant Rdl subtypes significantly reduced the antagonistic action of fipronil, however there was a significant reduction in response to ethiprole in the two mutated subtypes compared with the wild type. Bioassays with a Drosophila melanogaster strain carrying the A301S mutation showed strong resistance to ethiprole but not fipronil compared to a strain without this mutation, thus further supporting a causal role for the A301S mutation in resistance to ethiprole. Homology modelling of the N. lugens RDL channel did not suggest implications of Q359E for fiprole binding in contrast to A301S located in transmembrane domain M2 forming the channel pore. Synergist bioassays provided no evidence of a role for cytochrome P450s in N. lugens resistance to fipronil and the molecular basis of resistance to this compound remains unknown. In summary this study provides strong evidence that target-site resistance underlies widespread ethiprole resistance in N. lugens populations.
Abstract.
Abeku TA, Helinski MEH, Kirby MJ, Ssekitooleko J, Bass C, Kyomuhangi I, Okia M, Magumba G, Meek SR (2017). Insecticide resistance patterns in Uganda and the effect of indoor residual spraying with bendiocarb on kdr L1014S frequencies in Anopheles gambiae s.s.
Malar J,
16(1).
Abstract:
Insecticide resistance patterns in Uganda and the effect of indoor residual spraying with bendiocarb on kdr L1014S frequencies in Anopheles gambiae s.s.
BACKGROUND: Resistance of malaria vectors to pyrethroid insecticides has been attributed to selection pressure from long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and the use of chemicals in agriculture. The use of different classes of insecticides in combination or by rotation has been recommended for resistance management. The aim of this study was to understand the role of IRS with a carbamate insecticide in management of pyrethroid resistance. METHODS: Anopheles mosquitoes were collected from multiple sites in nine districts of Uganda (up to five sites per district). Three districts had been sprayed with bendiocarb. Phenotypic resistance was determined using standard susceptibility tests. Molecular assays were used to determine the frequency of resistance mutations. The kdr L1014S homozygote frequency in Anopheles gambiae s.s. was used as the outcome measure to test the effects of various factors using a logistic regression model. Bendiocarb coverage, annual rainfall, altitude, mosquito collection method, LLIN use, LLINs distributed in the previous 5 years, household use of agricultural pesticides, and malaria prevalence in children 2-9 years old were entered as explanatory variables. RESULTS: Tests with pyrethroid insecticides showed resistance and suspected resistance levels in all districts except Apac (a sprayed district). Bendiocarb resistance was not detected in sprayed sites, but was confirmed in one unsprayed site (Soroti). Anopheles gambiae s.s. collected from areas sprayed with bendiocarb had significantly less kdr homozygosity than those collected from unsprayed areas. Mosquitoes collected indoors as adults had significantly higher frequency of kdr homozygotes than mosquitoes collected as larvae, possibly indicating selective sampling of resistant adults, presumably due to exposure to insecticides inside houses that would disproportionately affect susceptible mosquitoes. The effect of LLIN use on kdr homozygosity was significantly modified by annual rainfall. In areas receiving high rainfall, LLIN use was associated with increased kdr homozygosity and this association weakened as rainfall decreased, indicating more frequency of exposure to pyrethroids in relatively wet areas with high vector density. CONCLUSION: This study suggests that using a carbamate insecticide for IRS in areas with high levels of pyrethroid resistance may reduce kdr frequencies in An. gambiae s.s.
Abstract.
Author URL.
Haddi K, Berger M, Bielza P, Rapisarda C, Williamson MS, Moores G, Bass C (2017). Mutation in the ace-1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance.
Journal of Applied Entomology,
141(8), 612-619.
Abstract:
Mutation in the ace-1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance
The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a major invasive pest that has spread throughout many countries in the Mediterranean basin and parts of Asia over the last decade. The control of T. absoluta has relied heavily on the use of chemical insecticides, a strategy that has led to the evolution of resistance. In this study, biological and molecular methods were used to determine the susceptibility of five strains of T. absoluta to the organophosphate chlorpyrifos and to investigate the molecular mechanisms underlying resistance to this class of insecticides. High levels of resistance to chlorpyrifos were observed in all five strains tested. Cloning and sequencing of the gene encoding the organophosphate target site, ace-1, of T. absoluta revealed the presence of an alanine to serine substitution at a position that has been previously linked with organophosphate resistance across a range of different insect and mite species. The presence of this mutation at high frequency in T. absoluta populations originating from various countries further supports the suggestion that the rapid expansion of this species is, in part, mediated by the resistance of this pest to chemical insecticides.
Abstract.
Roditakis E, Steinbach D, Moritz G, Vasakis E, Stavrakaki M, Ilias A, GarcÃa-Vidal L, MartÃnez-Aguirre MDR, Bielza P, Morou E, et al (2017). Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).
Insect Biochem Mol Biol,
80, 11-20.
Abstract:
Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).
Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.
Abstract.
Author URL.
Panini M, Tozzi F, Bass C, Zimmer CT, Field L, Borzatta V, Mazzoni E, Moores G (2017). The interactions of piperonyl butoxide and analogues with the metabolic enzymes FE4 and CYP6CY3 of the green peach aphid Myzus persicae (Hemiptera: Aphididae).
Pest Manag Sci,
73(2), 371-379.
Abstract:
The interactions of piperonyl butoxide and analogues with the metabolic enzymes FE4 and CYP6CY3 of the green peach aphid Myzus persicae (Hemiptera: Aphididae).
BACKGROUND: Piperonyl butoxide (PBO) is a well-known insecticide synergist capable of interacting with phase 1 metabolic enzymes, specifically esterases and cytochrome P450s. In this study, structure-activity relationship analyses were used to characterise the interaction of around 30 analogues of PBO with the esterase FE4 and the P450 CYP6CY3 from insecticide-resistant Myzus persicae (Sulzer), in order to predict the synthesis of more potent inhibitors. RESULTS: Enzyme inhibition studies were performed against esterase and oxidase activities and, together with in silico modelling, key activity determinants of the analogues were identified and optimised. Novel analogues were then designed and synthesised, some of which showed greater inhibition against both enzymatic systems: specifically, dihydrobenzofuran moieties containing an alkynyl side chain and a butyl side chain against FE4, and benzodioxole derivatives with a propyl/butyl side chain and an alkynyl ether moiety for CYP6CY3. CONCLUSIONS: in vitro assays identified potential candidate synergists with high inhibitory potency. The in vivo confirmation of such results will allow consideration for a possible use in agriculture. © 2016 Society of Chemical Industry.
Abstract.
Author URL.
Zimmer CT, Panini M, Singh KS, Randall EL, Field LM, Roditakis E, Mazzoni E, Bass C (2017). Use of the synergist piperonyl butoxide can slow the development of alpha-cypermethrin resistance in the whitefly Bemisia tabaci.
Insect Mol Biol,
26(2), 152-163.
Abstract:
Use of the synergist piperonyl butoxide can slow the development of alpha-cypermethrin resistance in the whitefly Bemisia tabaci.
The development of insecticide resistance in insect pests of crops is a growing threat to sustainable food production, and strategies that slow the development of resistance are therefore urgently required. The insecticide synergist piperonyl butoxide (PBO) inhibits certain insect detoxification systems and so may delay the evolution of metabolic resistance. In the current study we characterized resistance development in the silverleaf whitefly, Bemisia tabaci, after selection with either a neonicotinoid (thiacloprid) or pyrethroid (alpha-cypermethrin) insecticide alone or in combination with PBO. Resistance development was significantly suppressed (> 60%) in the line selected with alpha-cypermethrin + PBO compared to the line selected with alpha-cypermethrin alone. RNA sequencing (RNAseq) analyses revealed an increase in frequency of a knock-down resistance mutation but no differentially expressed genes were identified that could explain the sensitivity shift. No significant difference was observed in the level of resistance between the thiacloprid and thiacloprid + PBO selected lines, and RNA sequencing (RNAseq) analyses revealed that the cytochrome P450 monooxygenase CYP6CM1, known to metabolize neonicotinoids, was significantly upregulated (>10-fold) in both lines. The findings of this study demonstrate that PBO used in combination with certain insecticides can suppress the development of resistance in a laboratory setting; however, the mechanism by which PBO supresses resistance development remains unclear.
Abstract.
Author URL.
2016
Zimmer CT, Garrood WT, Puinean AM, Eckel-Zimmer M, Williamson MS, Davies TGE, Bass C (2016). A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.
Insect Biochem Mol Biol,
73, 62-69.
Abstract:
A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.
Spinosad, 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.
Abstract.
Author URL.
Garrood WT, Zimmer CT, Gorman KJ, Nauen R, Bass C, Davies TGE (2016). Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia.
Pest Management Science,
72(1), 140-149.
Abstract:
Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia
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.
Abstract.
Alptekin S, Bass C, Nicholls C, Paine MJI, Clark SJ, Field L, Moores GD (2016). Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes.
Insect Mol Biol,
25(2), 171-180.
Abstract:
Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes.
Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group. Although previous work has suggested that this results from enhanced metabolism of the former by detoxification enzymes, the specific enzyme(s) involved remain to be characterized. In this work, a pretreatment of honey bees with a sublethal dose of thiacloprid resulted in induced insensitivity to the same compound immediately following thiacloprid feeding. A longer pretreatment time resulted in no, or increased, sensitivity. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were over-expressed significantly in insecticide-treated bees compared with untreated controls. These included five P450s, CYP6BE1, CYP305D1, CYP6AS5, CYP315A1, CYP301A1, and a carboxyl/cholinesterase (CCE) CCE8. Four of these P450s were functionally expressed in Escherichia coli and their ability to metabolize thiacloprid examined by liquid chromatography-mass spectrometry (LC-MS) analysis.
Abstract.
Author URL.
Berger M, Puinean AM, Randall E, Zimmer CT, Silva WM, Bielza P, Field LM, Hughes D, Mellor I, Hassani-Pak K, et al (2016). Insecticide resistance mediated by an exon skipping event.
Mol Ecol,
25(22), 5692-5704.
Abstract:
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.
Abstract.
Author URL.
Ffrench-Constant RH, Williamson MS, Davies TGE, Bass C (2016). Ion channels as insecticide targets.
J Neurogenet,
30(3-4), 163-177.
Abstract:
Ion channels as insecticide targets.
Ion 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.
Abstract.
Author URL.
Bass C, Jones CM (2016). Mosquitoes boost body armor to resist insecticide attack.
Proc Natl Acad Sci U S A,
113(33), 9145-9147.
Author URL.
Silva WM, Berger M, Bass C, Williamson M, Moura DMN, Ribeiro LMS, Siqueira HAA (2016). Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).
Pesticide Biochemistry and Physiology,
131, 1-8.
Abstract:
Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)
The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in field populations.
Abstract.
2015
Jones CM, Papanicolaou A, Mironidis GK, Vontas J, Yang Y, Lim KS, Oakeshott JG, Bass C, Chapman JW (2015). Genomewide transcriptional signatures of migratory flight activity in a globally invasive insect pest.
MOLECULAR ECOLOGY,
24(19), 4901-4911.
Author URL.
Troczka BJ, Williams AJ, Bass C, Williamson MS, Field LM, Davies TGE (2015). Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae.
Gene,
556(2), 106-112.
Abstract:
Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae.
The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel.
Abstract.
Author URL.
Abeku TA, Helinski MEH, Kirby MJ, Kefyalew T, Awano T, Batisso E, Tesfaye G, Ssekitooleko J, Nicholas S, Erdmanis L, et al (2015). Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.
Malar J,
14Abstract:
Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.
BACKGROUND: Scale-up of malaria interventions seems to have contributed to a decline in the disease but other factors may also have had some role. Understanding changes in transmission and determinant factors will help to adapt control strategies accordingly. METHODS: Four sites in Ethiopia and Uganda were set up to monitor epidemiological changes and effectiveness of interventions over time. Here, results of a survey during the peak transmission season of 2012 are reported, which will be used as baseline for subsequent surveys and may support adaptation of control strategies. Data on malariometric and entomological variables, socio-economic status (SES) and control coverage were collected. RESULTS: Malaria prevalence varied from 1.4 % in Guba (Ethiopia) to 9.9 % in Butemba (Uganda). The most dominant species was Plasmodium vivax in Ethiopia and Plasmodium falciparum in Uganda. The majority of human-vector contact occurred indoors in Uganda, ranging from 83 % (Anopheles funestus sensu lato) to 93 % (Anopheles gambiae s.l.), which is an important factor for the effectiveness of insecticide-treated nets (ITNs) or indoor residual spraying (IRS). High kdr-L1014S (resistance genotype) frequency was observed in A. gambiae sensu stricto in Uganda. Too few mosquitoes were collected in Ethiopia, so it was not possible to assess vector habits and insecticide resistance levels. ITN ownership did not vary by SES and 56-98 % and 68-78 % of households owned at least one ITN in Ethiopia and Uganda, respectively. In Uganda, 7 % of nets were purchased by households, but the nets were untreated. In three of the four sites, 69-76 % of people with access to ITNs used them. IRS coverage ranged from 84 to 96 % in the three sprayed sites. Half of febrile children in Uganda and three-quarters in Ethiopia for whom treatment was sought received diagnostic tests. High levels of child undernutrition were detected in both countries carrying important implications on child development. In Uganda, 7-8 % of pregnant women took the recommended minimum three doses of intermittent preventive treatment. CONCLUSION: Malaria epidemiology seems to be changing compared to earlier published data, and it is essential to have more data to understand how much of the changes are attributable to interventions and other factors. Regular monitoring will help to better interpret changes, identify determinants, modify strategies and improve targeting to address transmission heterogeneity.
Abstract.
Author URL.
Zhang Y, Wang X, Yang B, Hu Y, Huang L, Bass C, Liu Z (2015). Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.
J Neurochem,
135(4), 686-694.
Abstract:
Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.
Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance.
Abstract.
Author URL.
Silva WM, Berger M, Bass C, Balbino VQ, Amaral MHP, Campos MR, Siqueira HAA (2015). Status of pyrethroid resistance and mechanisms in Brazilian populations of Tuta absoluta.
Pestic Biochem Physiol,
122, 8-14.
Abstract:
Status of pyrethroid resistance and mechanisms in Brazilian populations of Tuta absoluta.
The tomato leafminer, Tuta absoluta, is a major pest of tomato crops worldwide. This study surveyed the resistance of T. absoluta populations from four regions in Brazil to pyrethroid insecticides, the frequencies of L1014F, T929I and M918T Na channel mutations, and the role of detoxification metabolism in the resistance. Resistance ratios varied from 1- to 11-times among populations and insecticides, but control failure likelihood assays showed that all pyrethroids assessed exhibited no efficacy at all (and thus, 98-100% control failure likelihood) against all T. absoluta populations. The activity of glutathione S-transferase and cytochrome P450-mediated N-demethylation in biochemical assays was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes may play a role in resistance. TaqMan assays were used to screen for the presence of knockdown resistance (kdr) mutations and revealed that the L1014F kdr mutation was fixed in all populations and associated with two super-kdr mutations, M918T and particularly T929I, at high frequency. Altogether, results suggest that control failures are because of mutations in the domain II of the sodium channel, as a prevailing mechanism of resistance to pyrethroids in populations of T. absoluta in Brazil. But, enhanced cytochrome P450-dependent monooxygenases and GST activities also play an important role in the resistance of some populations, which reinforce that pyrethroids must not be used overall to control T. absoluta.
Abstract.
Author URL.
Bass C, Denholm I, Williamson MS, Nauen R (2015). The global status of insect resistance to neonicotinoid insecticides.
Pestic Biochem Physiol,
121, 78-87.
Abstract:
The global status of insect resistance to neonicotinoid insecticides.
The first neonicotinoid insecticide, imidacloprid, was launched in 1991. Today this class of insecticides comprises at least seven major compounds with a market share of more than 25% of total global insecticide sales. Neonicotinoid insecticides are highly selective agonists of insect nicotinic acetylcholine receptors and provide farmers with invaluable, highly effective tools against some of the world's most destructive crop pests. These include sucking pests such as aphids, whiteflies, and planthoppers, and also some coleopteran, dipteran and lepidopteran species. Although many insect species are still successfully controlled by neonicotinoids, their popularity has imposed a mounting selection pressure for resistance, and in several species resistance has now reached levels that compromise the efficacy of these insecticides. Research to understand the molecular basis of neonicotinoid resistance has revealed both target-site and metabolic mechanisms conferring resistance. For target-site resistance, field-evolved mutations have only been characterized in two aphid species. Metabolic resistance appears much more common, with the enhanced expression of one or more cytochrome P450s frequently reported in resistant strains. Despite the current scale of resistance, neonicotinoids remain a major component of many pest control programmes, and resistance management strategies, based on mode of action rotation, are of crucial importance in preventing resistance becoming more widespread. In this review we summarize the current status of neonicotinoid resistance, the biochemical and molecular mechanisms involved, and the implications for resistance management.
Abstract.
Author URL.
2014
Zimmer CT, Maiwald F, Schorn C, Bass C, Ott M-C, Nauen R (2014). A de novo transcriptome of European pollen beetle populations and its analysis, with special reference to insecticide action and resistance.
Insect Mol Biol,
23(4), 511-526.
Abstract:
A de novo transcriptome of European pollen beetle populations and its analysis, with special reference to insecticide action and resistance.
The pollen beetle Meligethes aeneus is the most important coleopteran pest in European oilseed rape cultivation, annually infesting millions of hectares and responsible for substantial yield losses if not kept under economic damage thresholds. This species is primarily controlled with insecticides but has recently developed high levels of resistance to the pyrethroid class. The aim of the present study was to provide a transcriptomic resource to investigate mechanisms of resistance. cDNA was sequenced on both Roche (Indianapolis, IN, USA) and Illumina (LGC Genomics, Berlin, Germany) platforms, resulting in a total of ∼53 m reads which assembled into 43 396 expressed sequence tags (ESTs). Manual annotation revealed good coverage of genes encoding insecticide target sites and detoxification enzymes. A total of 77 nonredundant cytochrome P450 genes were identified. Mapping of Illumina RNAseq sequences (from susceptible and pyrethroid-resistant strains) against the reference transcriptome identified a cytochrome P450 (CYP6BQ23) as highly overexpressed in pyrethroid resistance strains. Single-nucleotide polymorphism analysis confirmed the presence of a target-site resistance mutation (L1014F) in the voltage-gated sodium channel of one resistant strain. Our results provide new insights into the important genes associated with pyrethroid resistance in M. aeneus. Furthermore, a comprehensive EST resource is provided for future studies on insecticide modes of action and resistance mechanisms in pollen beetle.
Abstract.
Author URL.
Dabiré RK, Namountougou M, Diabaté A, Soma DD, Bado J, Toé HK, Bass C, Combary P (2014). Distribution and frequency of kdr mutations within Anopheles gambiae s.l. populations and first report of the ace.1 G119S mutation in Anopheles arabiensis from Burkina Faso (West Africa).
PLoS One,
9(7).
Abstract:
Distribution and frequency of kdr mutations within Anopheles gambiae s.l. populations and first report of the ace.1 G119S mutation in Anopheles arabiensis from Burkina Faso (West Africa).
An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G119S mutation in An. arabiensis populations collected at 8 sites [corrected]. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.
Abstract.
Author URL.
Zimmer CT, Bass C, Williamson MS, Kaussmann M, Wölfel K, Gutbrod O, Nauen R (2014). Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus.
Insect Biochem Mol Biol,
45, 18-29.
Abstract:
Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus.
The pollen beetle (Meligethes aeneus F.) is widespread throughout much of Europe where it is a major coleopteran pest of oilseed rape (Brassica napus). The reliance on synthetic insecticides for control, particularly the pyrethroid class, has led to the development of populations with high levels of resistance. Resistance to pyrethroids is now widespread throughout Europe and is thought to be mediated by enhanced detoxification by cytochrome P450Å› and/or mutation of the pyrethroid target-site, the voltage-gated sodium channel. However, in the case of cytochrome P450 mediated detoxification, the specific enzyme(s) involved has (have) not yet been identified. In this study a degenerate PCR approach was used to identify ten partial P450 gene sequences from pollen beetle. Quantitative PCR was then used to examine the level of expression of these genes in a range of pollen beetle populations that showed differing levels of resistance to pyrethroids in bioassays. The study revealed a single P450 gene, CYP6BQ23, which is significantly and highly overexpressed (up to ∼900-fold) in adults and larvae of pyrethroid resistant strains compared to susceptible strains. CYP6BQ23 overexpression is significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Functional recombinant expression of full length CYP6BQ23 along with cytochrome P450 reductase in an insect (Sf9) cell line showed that it is able to efficiently metabolise deltamethrin to 4-hydroxy deltamethrin. Furthermore we demonstrated by detection of 4-hydroxy tau-fluvalinate using ESI-TOF MS/MS that functionally expressed CYP6BQ23 also metabolizes tau-fluvalinate. A protein model was generated and subsequent docking simulations revealed the predicted substrate-binding mode of both deltamethrin and tau-fluvalinate to CYP6BQ23. Taken together these results strongly suggest that the overexpression of CYP6BQ23 is the primary mechanism conferring pyrethroid resistance in pollen beetle populations throughout much of Europe.
Abstract.
Author URL.
Bass C, Puinean AM, Zimmer CT, Denholm I, Field LM, Foster SP, Gutbrod O, Nauen R, Slater R, Williamson MS, et al (2014). The evolution of insecticide resistance in the peach potato aphid, Myzus persicae.
Insect Biochemistry and Molecular Biology,
51(1), 41-51.
Abstract:
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. © 2014 the Authors.
Abstract.
2013
Vezenegho SB, Chiphwanya J, Hunt RH, Coetzee M, Bass C, Koekemoer LL (2013). Characterization of the Anopheles funestus group, including Anopheles funestus-like, from Northern Malawi.
Transactions of the Royal Society of Tropical Medicine and Hygiene,
107(12), 753-762.
Abstract:
Characterization of the Anopheles funestus group, including Anopheles funestus-like, from Northern Malawi
Background: Limited information is available on malaria vector composition, feeding habits and malaria transmission in northern Malawi. Evidence of mosquito species diversity in this area was established in 2009, when Anopheles funestus-like, a new member of the An. funestus group was described. Additional biological information is needed to identify this species and to understand its role in malaria transmission. Methods: Anopheline mosquitoes were collected in northern Malawi and analyzed for Plasmodium species infection, blood meal source and susceptibility to insecticides. A new hydrolysis probe assay was designed to identify An. funestus-like. Results: Anopheles funestus and An. rivulorum predominated in the indoor collections. Most An. funestus-like were collected indoors, mainly fed on animals and were uninfected with P. falciparum. Anopheles funestus showed insecticide resistance to deltamethrin and bendiocarb. A high-precision hydrolysis probe assay was successfully developed to identify An. funestus-like. Discussion: Four species in the An. funestus group were collected in Karonga. Resistance to deltamethrin and bendiocarb was observed in An. funestus and further investigation is needed on the insecticide resistance mechanisms. Anopheles funestus-like, while collected indoors, is mainly zoophilic and most likely not a malaria vector. © the Author 2013. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved.
Abstract.
Puinean AM, Elias J, Slater R, Warren A, Field LM, Williamson MS, Bass C (2013). 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.
Pest Manag Sci,
69(2), 195-199.
Abstract:
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 β1 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.
Abstract.
Author URL.
Namountougou M, Diabaté A, Etang J, Bass C, Sawadogo SP, Gnankinié O, Baldet T, Martin T, Chandre F, Simard F, et al (2013). First report of the L1014S kdr mutation in wild populations of Anopheles gambiae M and S molecular forms in Burkina Faso (West Africa).
Acta Tropica,
125(2), 123-127.
Abstract:
First report of the L1014S kdr mutation in wild populations of Anopheles gambiae M and S molecular forms in Burkina Faso (West Africa)
We investigated the occurrence of the L1014F and L1014S kdr mutations in malaria vector populations in Burkina Faso (West Africa). A cross-sectional survey was conducted at 10 sites all located in cotton cultivation areas which are assumed to be the major insecticide resistance selection foci in Burkina Faso. The hot ligation method was used to detect the two kdr mutations in field collected Anopheles gambiae s.l. samples. For the first time in Burkina Faso the L1014S mutation was identified in both M and S forms of An. gambiae s.s. populations collected from the site of Koupela in the central-eastern region at low frequency. Furthermore, the L1014S mutation was also found in one specimen of An. arabiensis collected from the Dano site. The data generated in this study provides additional evidence of the spread of the L1014S mutation into An. gambiae s.l. populations in West Africa. It is now important to evaluate the role of the L1014S mutation in the pyrethroid resistance phenotype and assess its potential impact on the efficacy of pyrethroid-based control measures in West Africa where several resistance mutations now coexist. © 2012.
Abstract.
Bass C, Zimmer CT, Riveron JM, Wilding CS, Wondji CS, Kaussmann M, Field LM, Williamson MS, Nauen R (2013). Gene amplification and microsatellite polymorphism underlie a recent insect host shift.
Proceedings of the National Academy of Sciences of the United States of America,
110(48), 19460-19465.
Abstract:
Gene amplification and microsatellite polymorphism underlie a recent insect host shift
Host plant shifts of herbivorous insects may be a first step toward sympatric speciation and can create new pests of agriculturally important crops; however, the molecular mechanisms that mediate this process are poorly understood. Certain races of the polyphagous aphid Myzus persicae have recently adapted to feed on tobacco (Myzus persicae nicotianae) and show a reduced sensitivity to the plant alkaloid nicotine and cross-resistance to neonicotinoids a class of synthetic insecticides widely used for control. Here we show constitutive overexpression of a cytochrome P450 (CYP6CY3) allows tobacco-adapted races of M. persicae to efficiently detoxify nicotine and has preadapted them to resist neonicotinoid insecticides. CYP6CY3, is highly overexpressed in M. persicae nicotianae clones from three continents compared with M. persicae s.s. and expression level is significantly correlated with tolerance to nicotine. CYP6CY3 is highly efficient (compared with the primary human nicotine-metabolizing P450) at metabolizing nicotine and neonicotinoids to less toxic metabolites in vitro and generation of transgenic Drosophila expressing CYP6CY3 demonstrate that it confers resistance to both compounds in vivo. Overexpression of CYP6CY3 results from the expansion of a dinucleotide microsatellite in the promoter region and a recent gene amplification, with some aphid clones carrying up to 100 copies. We conclude that the mutations leading to overexpression of CYP6CY3 were a prerequisite for the host shift of M. persicae to tobacco and that gene amplification and microsatellite polymorphism are evolutionary drivers in insect host adaptation.
Abstract.
Carvalho RA, Omoto C, Field LM, Williamson MS, Bass C (2013). Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.
PLoS One,
8(4).
Abstract:
Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.
The 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.
Abstract.
Author URL.
Yang N, Xie W, Jones CM, Bass C, Jiao X, Yang X, Liu B, Li R, Zhang Y (2013). Transcriptome profiling of the whitefly Bemisia tabaci reveals stage-specific gene expression signatures for thiamethoxam resistance.
Insect Molecular Biology,
22(5), 485-496.
Abstract:
Transcriptome profiling of the whitefly Bemisia tabaci reveals stage-specific gene expression signatures for thiamethoxam resistance
Bemisia tabaci has developed high levels of resistance to many insecticides including the neonicotinoids and there is strong evidence that for some compounds resistance is stage-specific. To investigate the molecular basis of B. tabaci resistance to the neonicotinoid thiamethoxam we used a custom whitefly microarray to compare gene expression in the egg, nymph and adult stages of a thiamethoxam-resistant strain (TH-R) with a susceptible strain (TH-S). Gene ontology and bioinformatic analyses revealed that in all life stages many of the differentially expressed transcripts encoded enzymes involved in metabolic processes and/or metabolism of xenobiotics. Several of these are candidate resistance genes and include the cytochrome P450 CYP6CM1, which has been shown to confer resistance to several neonicotinoids previously, a P450 belonging to the Cytochrome P450s 4 family and a glutathione S-transferase (GST) belonging to the sigma class. Finally several ATP-binding cassette transporters of the ABCG subfamily were highly over-expressed in the adult stage of the TH-R strain and may play a role in resistance by active efflux. Here, we evaluated both common and stage-specific gene expression signatures and identified several candidate resistance genes that may underlie B. tabaci resistance to thiamethoxam. © 2013 the Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.
Abstract.
2012
Carvalho R, Yang Y, Field LM, Gorman K, Moores G, Williamson MS, Bass C (2012). Chlorpyrifos resistance is associated with mutation and amplification of the acetylcholinesterase-1 gene in the tomato red spider mite, Tetranychus evansi.
Pesticide Biochemistry and Physiology,
104(2), 143-149.
Abstract:
Chlorpyrifos resistance is associated with mutation and amplification of the acetylcholinesterase-1 gene in the tomato red spider mite, Tetranychus evansi
The tomato red spider mite, Tetranychus evansi is an invasive pest of many African countries where it causes significant damage to a range of solanaceous crops. In Malawi the control of T. evansi relies heavily on the use of chemical pesticides and this species has evolved resistance to members of the pyrethroid and organophosphate (OP) classes. In this study the molecular mechanisms underlying resistance to the organophosphate chlorpyrifos were investigated in two resistant strains of T. evansi from Malawi and France. Cloning and sequencing of the gene encoding the OP target (. ace-1) revealed an amino acid substitution at just one of the positions (331) previously implicated in OP resistance across a range of different insect and mite species. The amino acid residue usually found at this position in susceptible insects and mites is a phenylalanine (F) but was a tyrosine (Y) in all sequenced clones of the France strain and a tyrosine or tryptophan (W) in sequenced clones of the Malawi strain. Additionally we found evidence that the ace-1 locus is amplified in the resistant strains, with direct measurement of gene copy number by quantitative PCR showing there are around 8-10 copies of the gene in both the France and the Malawi strain. Sequencing of clones of ace-1 from the Malawi strain indicated that individual mites have fewer copies of the W331 allele than the Y331 allele. The enhanced copy number of the ace-1 gene in T. evansi and copy number variation of the two alleles seen in the Malawi strain may be a mechanism to compensate for fitness costs associated with the mutant alleles as has been proposed for T. urticae. © 2012.
Abstract.
Bass C, Hebsgaard MB, Hughes J (2012). Genomic resources for the brown planthopper, Nilaparvata lugens: Transcriptome pyrosequencing and microarray design.
Insect Science,
19(1), 1-12.
Abstract:
Genomic resources for the brown planthopper, Nilaparvata lugens: Transcriptome pyrosequencing and microarray design
The brown planthopper, Nilaparvata lugens is a pest of cultivated rice throughout Asia and is controlled using insecticides and/or resistant rice varieties. This species has developed resistance to many classes of insecticide and biotypes have developed that are virulent against formerly resistant rice cultivars. Insects use a suite of detoxification enzymes, including cytochrome P450s, glutathione S-transferases and carboxyl/cholinesterases to defend themselves against plant secondary metabolites and pesticides. Pyrosequencing on the Roche 454-FLX platform was used to produce a substantial expressed sequence tag (EST) dataset to complement the existing Sanger sequenced ESTs in GenBank. A total of 78959 reads were combined with the 37392 publically available Sanger ESTs; these assembled into 8 911 contigs and 10620 singletons. Analysis of the distribution of tentative unique genes (TUGs) with the gene ontology for biological processes and molecular functions suggests that the 454 and Sanger EST assembly is broadly representative of the N. lugens transcriptome. The brown planthopper transcriptome was found to contain 31 TUGs encoding P450s, nine encoding glutathione S-transferases and 26 encoding carboxyl/cholinesterases and many of these are putatively involved in the detoxification of xenobiotics. The Agilent eArray platform was used to construct an oligonucleotide microarray populated with probes for ∼19000 unigene sequences, including all those known to encode detoxification enzymes. The genomic resources developed in this study will be useful to the community studying this crop pest and will help elucidate the molecular mechanism underlying insecticide resistance and planthopper adaptation to resistant rice cultivars. © 2011 the Authors Journal compilation © Institute of Zoology, Chinese Academy of Sciences.
Abstract.
Haddi K, Berger M, Bielza P, Cifuentes D, Field LM, Gorman K, Rapisarda C, Williamson MS, Bass C (2012). Identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (Tuta absoluta).
Insect Biochemistry and Molecular Biology,
42(7), 506-513.
Abstract:
Identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (Tuta absoluta)
The tomato leaf miner. Tuta absoluta (Lepidoptera) is a significant pest of tomatoes that has undergone a rapid expansion in its range during the past six years and is now present across Europe, North Africa and parts of Asia. One of the main means of controlling this pest is through the use of chemical insecticides. In the current study insecticide bioassays were used to determine the susceptibility of five. T. absoluta strains established from field collections from Europe and Brazil to pyrethroids. High levels of resistance to λ cyhalothrin and tau fluvalinate were observed in all five strains tested. To investigate whether pyrethroid resistance was mediated by mutation of the. para-type sodium channel in. T. absoluta the IIS4-IIS6 region of the. para gene, which contains many of the mutation sites previously shown to confer knock down (kdr)-type resistance to pyrethroids across a range of different arthropod species, was cloned and sequenced. This revealed that three kdr/super-kdr-type mutations (M918T, T929I and L1014F), were present at high frequencies within all five resistant strains at known resistance 'hot-spots'. This is the first description of these mutations together in any insect population. High-throughput DNA-based diagnostic assays were developed and used to assess the prevalence of these mutations in 27 field strains from 12 countries. Overall mutant allele frequencies were high (L1014F 0.98, M918T 0.35, T929I 0.60) and remarkably no individual was observed that did not carry kdr in combination with either M918T or T929I. The presence of these mutations at high frequency in. T. absoluta populations across much of its range suggests pyrethroids are likely to be ineffective for control and supports the idea that the rapid expansion of this species over the last six years may be in part mediated by the resistance of this pest to chemical insecticides. © 2012.
Abstract.
Karatolos N, Williamson MS, Denholm I, Gorman K, ffrench-Constant RH, Bass C (2012). Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly trialeurodes vaporariorum.
PLoS ONE,
7(2).
Abstract:
Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly trialeurodes vaporariorum
Background: the juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood) which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s) are poorly understood. Results: Bioassays against eggs of a German (TV8) population of T. vaporariorum revealed a moderate level (21-fold) of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel) displaying a much higher resistance ratio (>4000-fold). The enzyme inhibitor piperonyl butoxide (PBO) suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s). Quantitative PCR highlighted a single P450 gene (CYP4G61) that was highly over-expressed (81.7-fold) in TV8pyrsel. Conclusion: Over-expression of a single cytochrome P450 gene (CYP4G61) has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen. © 2012 Karatolos et al.
Abstract.
Dabiré RK, Namountougou M, Sawadogo SP, Yaro LB, Toé HK, Ouari A, Gouagna LC, Simard F, Chandre F, Baldet T, et al (2012). Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: Bionomics, infection rate and susceptibility to insecticides.
Parasites and Vectors,
5(1).
Abstract:
Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: Bionomics, infection rate and susceptibility to insecticides
Background: Historical studies have indicated that An. gambiae s.s. is the predominant malaria vector species in Bobo-Dioulasso the second biggest city of Burkina Faso (West Africa). However, over the last decade, An. arabiensis appears to be replacing An. gambiae s.s. as the most prevalent malaria vector in this urban setting. To investigate this species transition in more detail the present study aims to provide an update on the malaria vector composition in Bobo-Dioulasso, and also the Plasmodium infection rates and susceptibility to insecticides of the local An. gambiae s.l. population. Methods: an entomological survey was carried out from May to December 2008 in Dioulassoba and Kodeni (central and peripheral districts respectively), which are representative of the main ecological features of the city. Sampling consisted of the collection of larval stages from water bodies, and adults by monthly indoor residual spraying (IRS) using aerosol insecticides. Insecticide susceptibility tests were performed using the WHO filter paper protocol on adults emerged from larvae. PCR was used to determine vector species and to identify resistance mechanisms (kdr and ace-1R). The Plasmodium infection rate was estimated by ELISA performed on female mosquitoes collected indoors by IRS. Results: An. arabiensis was found to be the major malaria vector in Bobo-Dioulasso, comprising 50 to 100% of the vector population. The sporozoite infection rate for An. arabiensis was higher than An. gambiae s.s. at both Dioulassoba and Kodeni. An. gambiae s.l. was resistant to DDT and cross-resistant to pyrethroids at the two sites with higher levels of resistance observed in An. gambiae s.s. than An. arabiensis. Resistance to 0.1% bendiocarb was observed in the An. gambiae s.s. S form but not the M form or in An. arabiensis. The L1014F kdr mutation was detected in the two molecular forms of An. gambiae s.s. at varying frequencies (0.45 to 0.92), but was not detected in An. arabiensis, suggesting that other mechanisms are involved in DDT resistance in this species. The ace-1R mutation was only detected in the S molecular form and was observed at the two sites at similar frequency (0.3). Conclusions: over the last ten years, An. arabiensis has become the major malaria vector in Bobo-Dioulasso city where it was formerly present only at low frequency. However, the ecological determinant that enhances the settlement of this species into urban and peri-urban areas of Bobo-Dioulasso remains to be clarified. The impact of the changing An. gambiae s.l. population in this region for vector control including resistance management strategies is discussed. © 2012 Dabire et al.; licensee BioMed Central Ltd.
Abstract.
Troczka B, Zimmer CT, Elias J, Schorn C, Bass C, Davies TGE, Field LM, Williamson MS, Slater R, Nauen R, et al (2012). Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor.
Insect Biochem Mol Biol,
42(11), 873-880.
Abstract:
Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor.
Diamide insecticides such as chlorantraniliprole and flubendiamide are a new class of insecticide that selectively target insect ryanodine receptors (RyR), a distinct class of homo-tetrameric calcium release channels which play a pivotal role in calcium homeostasis in numerous cell types. Resistance to these insecticides has recently been reported in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a global lepidopteran pest of cruciferous crops. In the present study a region of the gene encoding the proposed diamide binding site of the RyR from P. xylostella collected from the Philippines and Thailand and found to be over 200-fold resistant to both chlorantraniliprole and flubendiamide compared to susceptible strains, were amplified by RT-PCR and sequenced. Comparison of the sequence with those from several susceptible reference strains revealed non-synonymous mutations in each of the resistant strains that in both cases lead to a glycine to glutamic acid substitution (G4946E) in the protein. The independent evolution of the same amino acid substitution within a highly conserved region of the proposed diamide binding site in two geographically separated resistant strains of P. xylostella strongly suggests a causal association with diamide resistance. Furthermore we designed a pyrosequencing-based diagnostic assay for resistance monitoring purposes that can be used to detect the G4946E mutation in field-collected samples of diamondback moth. The implications of the reported findings for resistance management strategies are discussed.
Abstract.
Author URL.
Nauen R, Zimmer CT, Andrews M, Slater R, Bass C, Ekbom B, Gustafsson G, Hansen LM, Kristensen M, Zebitz CPW, et al (2012). Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.
Pesticide Biochemistry and Physiology,
103(3), 173-180.
Abstract:
Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.
Pollen beetle. Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle. para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed. kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using. tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-. kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed. © 2012 Elsevier Inc.
Abstract.
2011
Hofinger BJ, Russell JR, Bass CG, Baldwin T, Dos Reis M, Hedley PE, Li Y, MacAulay M, Waugh R, Hammond-Kosack KE, et al (2011). An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations.
Molecular Ecology,
20(17), 3653-3668.
Abstract:
An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations
In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats. © 2011 Blackwell Publishing Ltd.
Abstract.
Brown NA, Bass C, Baldwin TK, Chen H, Massot F, Carion PWC, Urban M, van de Meene AML, Hammond-Kosack KE (2011). Characterisation of the Fusarium graminearum-Wheat Floral Interaction.
J Pathog,
2011Abstract:
Characterisation of the Fusarium graminearum-Wheat Floral Interaction.
Fusarium Ear Blight is a destructive fungal disease of cereals including wheat and can contaminate the crop with various trichothecene mycotoxins. This investigation has produced a new β-glucuronidase (GUS) reporter strain that facilitates the quick and easy assessment of plant infection. The constitutively expressed gpdA:GUS strain of Fusarium graminearum was used to quantify the overall colonisation pattern. Histochemical and biochemical approaches confirmed, in susceptible wheat ear infections, the presence of a substantial phase of symptomless fungal growth. Separate analyses demonstrated that there was a reduction in the quantity of physiologically active hyphae as the wheat ear infection proceeded. A simplified linear system of rachis infection was then utilised to evaluate the expression of several TRI genes by RT-qPCR. Fungal gene expression at the advancing front of symptomless infection was compared with the origin of infection in the rachis. This revealed that TRI gene expression was maximal at the advancing front and supports the hypothesis that the mycotoxin deoxynivalenol plays a role in inhibiting plant defences in advance of the invading intercellular hyphae. This study has also demonstrated that there are transcriptional differences between the various phases of fungal infection and that these differences are maintained as the infection proceeds.
Abstract.
Author URL.
Bass C, Field LM (2011). Gene amplification and insecticide resistance.
Pest Manag Sci,
67(8), 886-890.
Abstract:
Gene amplification and insecticide resistance.
Pesticide resistance in arthropods has been shown to evolve by two main mechanisms, the enhanced production of metabolic enzymes, which bind to and/or detoxify the pesticide, and mutation of the target protein, which makes it less sensitive to the pesticide. One route that leads to enhanced metabolism is the duplication or amplification of the structural gene(s) encoding the detoxifying enzyme, and this has now been described for the three main families (esterases, glutathione S-transferases and cytochrome P450 monooxygenases) implicated in resistance. More recently, a direct or indirect role for gene duplication or amplification has been described for target-site resistance in several arthropod species. This mini-review summarises the involvement of gene duplication/amplification in the insecticide/acaricide resistance of insect and mite pests and highlights recent developments in this area in relation to P450-mediated and target-site resistance.
Abstract.
Author URL.
Bass C, Puinean AM, Andrews M, Cutler P, Daniels M, Elias J, Paul VL, Crossthwaite AJ, Denholm I, Field LM, et al (2011). Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.
BMC Neuroscience,
12Abstract:
Mutation of a nicotinic acetylcholine receptor β 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 Mpβ1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 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. © 2011 Bass et al; licensee BioMed Central Ltd.
Abstract.
Bass C, Carvalho RA, Oliphant L, Puinean AM, Field LM, Nauen R, Williamson MS, Moores G, Gorman K (2011). Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.
Insect Molecular Biology,
20(6), 763-773.
Abstract:
Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens
The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. Insect Molecular Biology © 2011 the Royal Entomological Society.
Abstract.
Araújo RA, Williamson MS, Bass C, Field LM, Duce IR (2011). Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel.
Insect Molecular BiologyAbstract:
Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel
The maize weevil, Sitophilus zeamais, is the most important pest affecting stored grain in Brazil and its control relies heavily on the use of insecticides. The intensive use of compounds such as the pyrethroids has led to the emergence of resistance, and previous studies have suggested that resistance to both pyrethroids and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) may result from reduced sensitivity of the insecticide target, the voltage-gated sodium channel. To identify the molecular mechanisms underlying pyrethroid resistance in S. zeamais, the domain II region of the voltage-gated sodium channel (para-orthologue) gene was amplified by PCR and sequenced from susceptible and resistant laboratory S. zeamais strains that were selected with a discriminating dose of DDT. A single point mutation, T929I, was found in the para gene of the resistant S. zeamais populations and its presence in individual weevils was strongly associated with survival after DDT exposure. This is the first identification of a target-site resistance mutation in S. zeamais and unusually it is a super-kdr type mutation occurring in the absence of the more common kdr (L1014F) substitution. A high-throughput assay based on TaqMan single nucleotide polymorphism genotyping was developed for sensitive detection of the mutation and used to screen field-collected strains of S. zeamais. This showed that the mutation is present at low frequency in field populations and is a useful tool for informing control strategies. © 2011 the Authors. Insect Molecular Biology © 2011 the Royal Entomological Society.
Abstract.
Araújo RA, Williamson MS, Bass C, Field LM, Duce IR (2011). Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel.
Insect Molecular Biology,
20(4), 437-445.
Abstract:
Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel
The maize weevil, Sitophilus zeamais, is the most important pest affecting stored grain in Brazil and its control relies heavily on the use of insecticides. The intensive use of compounds such as the pyrethroids has led to the emergence of resistance, and previous studies have suggested that resistance to both pyrethroids and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) may result from reduced sensitivity of the insecticide target, the voltage-gated sodium channel. To identify the molecular mechanisms underlying pyrethroid resistance in S. zeamais, the domain II region of the voltage-gated sodium channel (para-orthologue) gene was amplified by PCR and sequenced from susceptible and resistant laboratory S. zeamais strains that were selected with a discriminating dose of DDT. A single point mutation, T929I, was found in the para gene of the resistant S. zeamais populations and its presence in individual weevils was strongly associated with survival after DDT exposure. This is the first identification of a target-site resistance mutation in S. zeamais and unusually it is a super-kdr type mutation occurring in the absence of the more common kdr (L1014F) substitution. A high-throughput assay based on TaqMan single nucleotide polymorphism genotyping was developed for sensitive detection of the mutation and used to screen field-collected strains of S. zeamais. This showed that the mutation is present at low frequency in field populations and is a useful tool for informing control strategies. © 2011 the Royal Entomological Society.
Abstract.
Nyoni BN, Gorman K, Mzilahowa T, Williamson MS, Navajas M, Field LM, Bass C (2011). Pyrethroid resistance in the tomato red spider mite, Tetranychus evansi, is associated with mutation of the para-type sodium channel.
Pest Management Science,
67(8), 891-897.
Abstract:
Pyrethroid resistance in the tomato red spider mite, Tetranychus evansi, is associated with mutation of the para-type sodium channel
BACKGROUND: the tomato red spider mite, Tetranychus evansi (Baker and Pritchard), is a serious pest of solanaceous crops in many African countries. In this study an investigation has been conducted to establish whether mutation of the para-type sodium channel underlies pyrethroid resistance in T. evansi strains collected in Southern Malawi. RESULTS: Two T. evansi strains from Malawi showed tolerance to the organophosphate chlorpyrifos and resistance (20-40-fold) to the pyrethroid bifenthrin, but were susceptible to two contemporary acaricides (abamectin and fenpyroximate) in insecticide bioassays. Cloning of a 3.1 kb fragment (domains IIS5 to IVS5) of the T. evansi para gene from pyrethroid-resistant and pyrethroid-susceptible strains revealed a single non-synonymous mutation in the resistant strains that results in an amino acid substitution (M918T) within the domain II region of the channel. Although novel to mites, this mutation confers high levels of resistance to pyrethroids in several insect species where it has always been associated with another mutation (L1014F). This is the first report of the M918T mutation in the absence of L1014F in any arthropod species. Diagnostic tools were developed that allow sensitive detection of this mutation in individual mites. CONCLUSION: This is the first study of pyrethroid resistance in T. evansi and provides contemporary information for resistance management of this pest in Southern Malawi. © 2011 Society of Chemical Industry.
Abstract.
Karatolos N, Pauchet Y, Wilkinson P, Chauhan R, Denholm I, Gorman K, Nelson DR, Bass C, ffrench-Constant RH, Williamson MS, et al (2011). Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.
BMC Genomics,
12Abstract:
Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes
Background: 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. © 2011 Karatolos et al; licensee BioMed Central Ltd.
Abstract.
2010
Puinean AM, Foster SP, Oliphant L, Denholm I, Field LM, Millar NS, Williamson MS, Bass C (2010). Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.
PLoS Genetics,
6(6), 1-11.
Abstract:
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. © 2010 Puinean et al.
Abstract.
de Carvalho RA, Limia CEG, Bass C, de Azeredo-Espin AML (2010). Changes in the frequency of the G137D and W251S mutations in the carboxylesterase E3 gene of Cochliomyia hominivorax (Diptera: Calliphoridae) populations from Uruguay.
Veterinary Parasitology,
170(3-4), 297-301.
Abstract:
Changes in the frequency of the G137D and W251S mutations in the carboxylesterase E3 gene of Cochliomyia hominivorax (Diptera: Calliphoridae) populations from Uruguay
The New World Screwworm (NWS) fly Cochliomyia hominivorax is one of most important myiasis-causing flies in the Neotropics. It is responsible for severe losses to the livestock industry through both mortality and the loss of productivity of infested animals. In Uruguay, NWS represents a significant problem. To date this pest has been controlled by the application of chemical insecticides, mainly the pyrethroid and organophosphate (OP) classes. However, the intensive use of these compounds over many years has led to the evolution of resistance which has the potential to compromise the effectiveness of current control strategies. One mechanism by which resistance has occurred in this and related dipteran species is through two mutations (G137D and W251S) in the carboxylesterase E3 enzyme that have enhanced ability to hydrolyze certain insecticides. In this study changes in the frequency of these mutations in C. hominivorax was investigated in three different Uruguayan regions in 2003 and 2009. All three regions analyzed showed a reduction in the frequency of the G137D mutation and a significant increase in frequency of the W251S mutation, and this may be related to the current intense use of dimethyl-OP and pyrethroid insecticides. The findings of this study provide current information on the frequency of these resistance-associated mutations in NWS in Uruguay and may help select appropriate chemicals for NWS control as part of potential pest management strategies. © 2010 Elsevier B.V.
Abstract.
Bass C, Nikou D, Vontas J, Williamson MS, Field LM (2010). Development of high-throughput real-time PCR assays for the identification of insensitive acetylcholinesterase (ace-1<sup>R</sup>) in Anopheles gambiae.
Pesticide Biochemistry and Physiology,
96(2), 80-85.
Abstract:
Development of high-throughput real-time PCR assays for the identification of insensitive acetylcholinesterase (ace-1R) in Anopheles gambiae
Resistance to the organophosphate and carbamate insecticides through insensitivity of the target site enzyme, acetylcholinesterase has recently been reported in Anopheles gambiae populations in West Africa. To date, screening for the mutation (G119S of the ace-1 gene) conferring this insensitivity has employed a simple PCR-RFLP diagnostic. However, this has the disadvantage of requiring digestion of the amplified fragment and subsequent gel electrophoresis of the products. To overcome this, and thus increase throughput and reduce costs, we have developed two assays based on real-time PCR (TaqMan and melt-curve) that represent true 'closed-tube' approaches. The two new platforms were compared to PCR-RFLP to genotype over 280 samples. The two new methods compared favourably with PCR-RFLP with the TaqMan assay delivering the greatest specificity and sensitivity of the three approaches. This assay is also cheaper to run than PCR-RFLP and results are obtained in a single step. © 2009 Elsevier Inc. All rights reserved.
Abstract.
Bass C, Nikou D, Vontas J, Donnelly MJ, Williamson MS, Field LM (2010). The Vector Population Monitoring Tool (VPMT): High-Throughput DNA-Based Diagnostics for the Monitoring of Mosquito Vector Populations.
Malar Res Treat,
2010Abstract:
The Vector Population Monitoring Tool (VPMT): High-Throughput DNA-Based Diagnostics for the Monitoring of Mosquito Vector Populations.
Regular monitoring of mosquito vector populations is an integral component of most vector control programmes. Contemporary data on mosquito species composition, infection status, and resistance to insecticides are a prerequisite for effective intervention. For this purpose we, with funding from the Innovative Vector Control Consortium (IVCC), have developed a suite of high-throughput assays based on a single "closed-tube" platform that collectively comprise the "Vector Population Monitoring Tool" (VPMT). The VPMT can be used to screen mosquito disease vector populations for a number of traits including Anopheles gambiae s.l. and Anopheles funestus species identification, detection of infection with Plasmodium parasites, and identification of insecticide resistance mechanisms. In this paper we focus on the Anopheles-specific assays that comprise the VPMT and include details of a new assay for resistance todieldrin Rdl detection. The application of these tools, general and specific guidelines on their use based on field testing in Africa, and plans for further development are discussed.
Abstract.
Author URL.
2009
Vezenegho SB, Bass C, Puinean M, Williamson MS, Field LM, Coetzee M, Koekemoer LL (2009). Development of multiplex real-time PCR assays for identification of members of the Anopheles funestus species group.
Malaria Journal,
8(1).
Abstract:
Development of multiplex real-time PCR assays for identification of members of the Anopheles funestus species group
Background. The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR). One disadvantage of AS-PCR is the requirement for post-PCR processing by gel electrophoresis of PCR products. In this study, three new high-throughput 'closed-tube' assays were developed and compared with the previously described AS-PCR technique. Methods. Protocols for three fluorescence-based assays based on Melt Curve Analysis (MCA), High Resolution Melt (HRM) and TaqMan SNP genotyping were developed to detect and discriminate Anopheles parensis, Anopheles leesoni, Anopheles vaneedeni, Anopheles rivulorum and An. funestus s.s. The sensitivity and specificity of these assays were compared with the widely used AS-PCR in a blind trial using DNA extracted from wild-caught mosquitoes. Results. The TaqMan assay proved to be the most sensitive and specific of the three new assays. The MCA and HRM assays initially gave promising results, but were more sensitive to both DNA quality and quantity and consequently showed a higher rate of incorrect identifications. Conclusion. The TaqMan assay proved to be the most robust of the three protocols tested in this study. This assay very effectively identified all five members of the An. funestus group using fluorescently-labeled probes with distinct emission and excitation spectra allowing their independent detection in a single reaction. This method is at least as sensitive and specific as the gold standard AS-PCR approach and because it has no requirement for post-PCR processing is simpler and more rapid to run. The one disadvantage of the TaqMan assay is the cost of this assay, both in terms of initial capital outlay and running cost per sample, which is higher than AS-PCR. However, the cost of both the real-time PCR machine and fluorescently labelled probes required is falling and in the future the cost of this assay is likely to become closer to that of standard PCR. © 2009 Vezenegho et al; licensee BioMed Central Ltd.
Abstract.
Dabiré KR, Diabaté A, Namontougou M, Djogbenou L, Kengne P, Simard F, Bass C, Baldet T (2009). Distribution of insensitive acetylcholinesterase (ace-1<sup>R</sup>) in Anopheles gambiae s.l. populations from Burkina Faso (West Africa).
Tropical Medicine and International Health,
14(4), 396-403.
Abstract:
Distribution of insensitive acetylcholinesterase (ace-1R) in Anopheles gambiae s.l. populations from Burkina Faso (West Africa)
Objective: to investigate through countrywide sampling at 20 localities across the three different agro-climatic zones of Burkina Faso, the distribution of the acetylcholinesterase insensitive mutation ace-1R, which confers resistance to organophosphates (OP) and carbamates (CM) insecticides in An. gambiae s.l. Methods: Adult mosquitoes were collected by indoor aerosol spraying from August to October 2006. Specimens were identified to species by polymerase chain reaction (PCR) assay and characterized for the ace-1 R mutation using a PCR-restriction fragment length polymorphism diagnostic. Results: Collected mosquitoes were a mixture of An. gambiae s.s. and An. arabiensis across the Sudan (98.3%vs. 1.7%), Sudan-sahelian (78.6%vs. 21.4%) and the Sahel (91.5%vs. 8.5%) ecotypes. The An. gambiae S-form predominated in the Sudan sites from the West (69%vs. 31% for the M form) but was not found in the Sahel (100% M form). The ace-1R mutation was dispersed throughout the Sudan and Sudan-sahelian localities at moderate frequency (
Abstract.
Dabiré KR, Diabaté A, Namountougou M, Toé KH, Ouari A, Kengne P, Bass C, Baldet T (2009). Distribution of pyrethroid and DDT resistance and the L1014F kdr mutation in Anopheles gambiae s.l. from Burkina Faso (West Africa).
Transactions of the Royal Society of Tropical Medicine and Hygiene,
103(11), 1113-1120.
Abstract:
Distribution of pyrethroid and DDT resistance and the L1014F kdr mutation in Anopheles gambiae s.l. from Burkina Faso (West Africa)
This study reports on the distribution of pyrethroid and DDT resistance and the L1014F knockdown resistance (kdr) mutation in Anopheles gambiae s.l. populations from 21 localities in three different climatic zones of Burkina Faso from August to October 2006. The susceptibility of these populations was assessed by bioassay using DDT (4%), permethrin (1%) and deltamethrin (0.05%). Anopheles gambiae were resistant to both permethrin and DDT in the Sudanian regions but were susceptible in the central and sahelian areas and susceptible to deltamethrin at all sites except Orodara, although mortality values in some populations were close to the resistance threshold. The kdr frequency varied from 0.4 to 0.97 in populations from the Sudanian region and was lower in populations from the Sudano-sahelian and sahelian areas (0.047 to 0.54). Compared to the last survey of kdr in An. gambiae populations conducted in 2000, the kdr frequency did not differ in the S form but had increased in the M form (0.6), with an extended distribution into the Sudano-sahelian region. The frequency of kdr was also found to have increased in An. arabiensis populations (0.28), where it was formerly reported in only a single specimen. These results have practical significance for malaria vector control programs. © 2009 Royal Society of Tropical Medicine and Hygiene.
Abstract.
Ramphul U, Boase T, Bass C, Okedi LM, Donnelly MJ, Müller P (2009). Insecticide resistance and its association with target-site mutations in natural populations of Anopheles gambiae from eastern Uganda.
Transactions of the Royal Society of Tropical Medicine and Hygiene,
103(11), 1121-1126.
Abstract:
Insecticide resistance and its association with target-site mutations in natural populations of Anopheles gambiae from eastern Uganda
Insecticide resistance in Anopheles gambiae threatens the success of malaria vector control programmes in sub-Saharan Africa. In order to manage insecticide resistance successfully, it is essential to assess continuously the target mosquito population. Here, we collected baseline information on the distribution and prevalence of insecticide resistance and its association with target-site mutations in eastern Uganda. Anopheles gambiae s.l. adults were raised from wild-caught larvae sampled from two ecologically distinct breeding sites and exposed to WHO discriminating concentrations of DDT, permethrin, deltamethrin, bendiocarb and malathion. Survival rates to DDT were as high as 85.4%, alongside significant resistance levels to permethrin (38.5%), reduced susceptibility to deltamethrin, but full susceptibility to bendiocarb and malathion. Using molecular diagnostics, susceptible and resistant specimens were further tested for the presence of knockdown resistance (kdr) and acetylcholinesterase 1 resistance (ace-1R) alleles. While ace-1R and kdr L1014F ('kdr west') alleles were absent, the kdr L1014S ('kdr east') allele was present in both populations. In A. gambiae s.s. L1014S was closely associated with DDT and, to a lesser degree, with permethrin resistance. Intriguingly, the association between DDT resistance and the presence of L1014S is consistent with a co-dominant effect, with heterozygous individuals showing an intermediate phenotype. © 2009 Royal Society of Tropical Medicine and Hygiene.
Abstract.
2008
Ridl FC, Bass C, Torrez M, Govender D, Ramdeen V, Yellot L, Edu AE, Schwabe C, Mohloai P, Maharaj R, et al (2008). A pre-intervention study of malaria vector abundance in Rio Muni, Equatorial Guinea: Their role in malaria transmission and the incidence of insecticide resistance alleles.
Malaria Journal,
7Abstract:
A pre-intervention study of malaria vector abundance in Rio Muni, Equatorial Guinea: Their role in malaria transmission and the incidence of insecticide resistance alleles
Background. Following the success of the malaria control intervention on the island of Bioko, malaria control by the use of indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLITN) was extended to Rio Muni, on the mainland part of Equatorial Guinea. This manuscript reports on the malaria vectors present and the incidence of insecticide resistant alleles prior to the onset of the programme. Methods. Anopheles mosquitoes were captured daily using window traps at 30 sentinel sites in Rio Muni, from December 2006 to July 2007. The mosquitoes were identified to species and their sporozoite rates, knockdown resistance (kdr) and acetylcholinesterase (AChE) sensitivity measured, to define the role of vector species in malaria transmission and their potential susceptibility to insecticides. Results. A total of 6,162 Anopheles mosquitoes were collected of which 4,808 were morphologically identified as Anopheles gambiae s.l. 120 Anopheles funestus, 1,069 Anopheles moucheti, and 165 Anopheles nili s.l. Both M and S molecular forms of Anopheles gambiae s.s. and Anopheles melas were identified. Anopheles ovengensis and Anopheles carnevalei were the only two members of the An. nili group to be identified. Using the species-specific sporozoite rates and the average number of mosquitoes per night, the number of infective mosquitoes per trap per 100 nights for each species complex was calculated as a measure of transmission risk. Both kdr-w and kdr-e alleles were present in the S-form of An. gambiae s.s. (59% and 19% respectively) and at much lower frequencies in the M-form (9.7% and 1.8% respectively). The kdr-w and kdr-e alleles co-occurred in 103 S-form and 1 M-form specimens. No insensitive AChE was detected. Conclusion. Anopheles gambiae s.s, a member of the Anopheles gambiae complex was shown to be the major vector in Rio Muni with the other three groups playing a relatively minor role in transmission. The demonstration of a high frequency of kdr alleles in mosquito populations before the onset of a malaria control programme shows that continuous entomological surveillance including resistance monitoring will be of critical importance to ensure the chosen insecticide remains effective. © 2008 Ridl et al; licensee BioMed Central Ltd.
Abstract.
Bass C, Williamson MS, Field LM (2008). Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex.
Acta Trop,
107(1), 50-53.
Abstract:
Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex.
Two high-throughput assays for the identification of members of the Anopheles gambiae sensu lato species complex have recently been reported. These methods, are based on TaqMan single nucleotide polymorphism (SNP) genotyping that enables rapid scoring of mosquito DNA samples in real-time PCR reactions. Unfortunately, both assays are restricted in the number of species that they can identify and a combination of the two assays may be required to identify all possible species in certain regions. To overcome this limitation, and thereby further increase throughput while reducing costs, we have developed a new multiplex real-time PCR assay for identifying members of the An. gambiae complex. The new method uses three probes labelled with fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of the two main malaria vectors from the non-vector sibling species, and can be used on single mosquito legs from silica-dried specimens. A genotyping trial of over 450 specimens collected from 13 countries in sub-Saharan Africa showed the multiplex assay to be highly specific and sensitive and it compared well against the two previously reported TaqMan assays and standard allele-specific PCR.
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Bass C, Nikou D, Blagborough AM, Vontas J, Sinden RE, Williamson MS, Field LM (2008). PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.
Malaria Journal,
7Abstract:
PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods
Background. 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. © 2008 Bass et al; licensee BioMed Central Ltd.
Abstract.
Mzilahowa T, Ball AJ, Bass C, Morgan JC, Nyoni B, Steen K, Donnelly MJ, Wilding CS (2008). Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: Evidence for larval selection.
Medical and Veterinary Entomology,
22(3), 258-263.
Abstract:
Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: Evidence for larval selection
Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l. (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT50 of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT50 did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis. They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages. © 2008 the Authors.
Abstract.
2007
Bass C, Nikou D, Donnelly MJ, Williamson MS, Ranson H, Ball A, Vontas J, Field LM (2007). Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods.
Malaria Journal,
6Abstract:
Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods
Background. Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods. Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe - Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results. The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores. Conclusion. The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput. © 2007 Bass et al; licensee BioMed Central Ltd.
Abstract.
Bass C, Williamson MS, Wilding CS, Donnelly MJ, Field LM (2007). Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay.
Malar J,
6Abstract:
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 (
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2006
Bass C, Lansdell SJ, Millar NS, Schroeder I, Turberg A, Field LM, Williamson MS (2006). Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).
Insect Biochem Mol Biol,
36(1), 86-96.
Abstract:
Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).
As part of a program to monitor the susceptibility of cat flea populations to the insecticide imidacloprid we have examined the cat flea nicotinic acetylcholine receptor, the target site protein of the neonicotinoid group of insecticides. Seven nAChR subunits (six alpha-type and one beta-type) were identified in cat flea using a degenerate PCR-based strategy. Five of these were expressed in vitro by creating chimeras containing the N-terminal ligand-binding domain of the cat flea subunits and the C-terminal region of the Drosophila Dalpha2 (SAD) subunit. Two of the five chimeric subunits, Cfalpha1/Dalpha2 and Cfalpha3/Dalpha2, when co-expressed with rat beta2 in Drosophila S2 cells, showed high-affinity binding of both epibatidine (Kd=1.6+/-0.6 and 0.13+/-0.06nM, respectively), and imidacloprid (Ki=142+/-34 and 28.7+/-2.4nM, respectively). It is likely therefore that Cfalpha1 and Cfalpha3 contribute to nAChR populations in vivo that are sensitive to imidacloprid. The identification of cat flea nAChR subunits that have a high affinity for imidacloprid presents candidate genes in which to look for resistance-associated mutations if target-site resistance to imidacloprid arises in domestic pet flea populations.
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Bass C, Hendley R, Adams MJ, Hammond-Kosack KE, Kanyuka K (2006). The Sbm1 locus conferring resistance to Soil-borne cereal mosaic virus maps to a gene-rich region on 5DL in wheat.
Genome,
49(9), 1140-1148.
Abstract:
The Sbm1 locus conferring resistance to Soil-borne cereal mosaic virus maps to a gene-rich region on 5DL in wheat.
A mosaic disease caused by Soil-borne cereal mosaic virus (SBCMV) is becoming increasingly important, particularly in winter wheat in Europe. As there are currently no effective cultural practices or practical environmentally friendly chemicals for disease control, host plant resistance is an important objective in breeding programs. However, development of resistant cultivars is slow owing to difficulties in germplasm screening for resistance. Therefore, there is a need to identify molecular markers linked to SBCMV-resistance gene(s), so that quick and accurate laboratory-based marker-assisted selection rather than prolonged field-based screens for resistance can be used in developing resistant cultivars. We previously demonstrated that resistance to SBCMV in Triticum aestivum 'Cadenza' is controlled by a single locus. In this work, we used AFLP and microsatellite technology to map this resistance locus, with the proposed name Sbm1, to the distal end of chromosome 5DL. Interestingly, several expressed disease-resistance gene analogues also map to this gene-rich region on 5DL. Closely linked (approximately 17 cM interval) markers, BARC110 and WMC765, RRES01 and BARC144, that flank Sbm1 will be very useful in breeding for selection of germplasm carrying Sbm1.
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2005
Hill N, Moor G, Cameron MM, Butlin A, Preston S, Williamson MS, Bass C (2005). Single blind, randomised, comparative study of the Bug Buster kit and over the counter pediculicide treatments against head lice in the United Kingdom.
British Medical Journal,
331(7513), 384-386.
Abstract:
Single blind, randomised, comparative study of the Bug Buster kit and over the counter pediculicide treatments against head lice in the United Kingdom
Objective: to compare the effectiveness of the Bug Buster kit regimen with a single treatment of over the counter pediculicides for eliminating head lice. Design: Single blind, multicentre, randomised, comparative clinical study. Setting: Four counties in England and one county in Scotland. Participants: 133 young people aged 2-15 years with head louse infestation; 56 were allocated to the Bug Buster kit and 70 to pediculicide treatment. Interventions: Home use of proprietary pediculicides (organophosphate or pyrethroid) or the Bug Buster kit. Main outcome measure: Presence of head lice 2-4 days after end of treatment: day 5 for the pediculicides and day 15 for the Bug Buster kit. Results: the cure rate using the Bug Buster kit was significantly greater than that for the pediculicides (57% v 13%; relative risk 4.4, 95% confidence interval 2.3 to 8.5). Number needed to treat for the Bug Buster kit compared with the pediculicides was 2.26. Conclusion: the Bug Buster kit was the most effective over the counter treatment for head louse infestation in the community when compared with pediculicides.
Abstract.
2004
Bass C, Schroeder I, Turberg A, M Field L, Williamson MS (2004). Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis.
Insect Biochem Mol Biol,
34(12), 1305-1313.
Abstract:
Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis.
Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4-S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4-IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the 'common' kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.
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Bass C, Schroeder I, Turberg A, Field LM, Williamson MS (2004). Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).
Pest Manag Sci,
60(12), 1157-1162.
Abstract:
Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).
In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations.
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