Christoph T. Zimmer

Gene amplification and microsatellite polymorphism underlie a recent insect host shift

Significance Insect host shifts may lead to sympatric speciation and can create new crop pests, however identifying the genetic changes involved has proved elusive. We studied a subspecies of the aphid Myzus persicae that has recently host shifted to tobacco and are resistant to the plant alkaloid nicotine. We found these races overexpress a cytochrome P450 enzyme (CYP6CY3) that allows them to detoxify nicotine and also certain synthetic insecticides. Overexpression of CYP6CY3 is caused by gene amplification (up to 100 copies) and expansion of a dinucleotide microsatellite in the promoter. Our findings provide insights into the molecular drivers of insect host shifts. 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.

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 P450ś 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.

Field‐evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia

Abstract 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.

Pyrethroid resistance and thiacloprid baseline susceptibility of European populations of Meligethes aeneus (Coleoptera: Nitidulidae) collected in winter oilseed rape.

BACKGROUND Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae), is a major pest in European winter oilseed rape. Recently, control failures with pyrethroid insecticides commonly used to control this pest have been reported in many European countries. For resistance management purposes, the neonicotinoid insecticide thiacloprid was widely introduced as a new mode of action for pollen beetle control. RESULTS A number of pollen beetle populations collected in Germany, France, Austria, Great Britain, Sweden, Denmark, Finland, Poland, Czech Republic and Ukraine were tested for pyrethroid resistance using lambda-cyhalothrin-coated glass vials (adult vial test). Most of the populations tested exhibited substantial levels of resistance to lambda-cyhalothrin, and resistance ratios ranged from < 10 to > 2000. A similar resistance monitoring bioassay for the neonicotinoid insecticide thiacloprid was developed and validated by assessing baseline susceptibility data for 88 European pollen beetle populations. A variation of less than fivefold in response to thiacloprid was detected. The thiacloprid adult vial bioassay is based on glass vials coated with an oil-dispersion-based formulation of thiacloprid, resulting in a much better bioavailability compared with technical material. Analytical measurements revealed a > 56 and 28 day stability of thiacloprid and lambda-cyhalothrin in coated glass vials at room temperature, respectively. No cross-resistance between thiacloprid and lambda-cyhalothrin based on log-dose probit-mortality data was detected. CONCLUSION Pyrethroid resistance in many European populations of M. aeneus was confirmed, whereas all populations are susceptible to thiacloprid when tested in a newly designed and validated monitoring bioassay based on glass vials coated with oil-dispersion-formulated thiacloprid. Based on the homogeneous results, it is concluded that thiacloprid could be an important chemical tool for pollen beetle resistance management strategies in European winter oilseed rape.

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.

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.

Baseline susceptibility and insecticide resistance monitoring in European populations of Meligethes aeneus and Ceutorhynchus assimilis collected in winter oilseed rape

Pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and cabbage seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), are important pests in the production of European winter oilseed rape, Brassica napus L. (Brassicaceae), which is grown on several million hectares in Europe. Insecticide treatments are common to control both pests once they exceed economic damage thresholds; however, not many chemical classes are available for their control in European oilseed rape. Particularly pollen beetles recently developed high levels of pyrethroid resistance impairing field control at recommended rates in many countries, whereas no resistance is yet reported to another important insecticide, thiacloprid. The major objective of this study was to investigate the spatio‐temporal susceptibility status of pollen beetle against the recently introduced insecticide thiacloprid. From 2009 to 2012 more than 630 populations of pollen beetle collected in 13 countries were monitored for resistance to thiacloprid by using an adult vial test. No shifting to lower susceptibility of pollen beetle to thiacloprid has been observed between 2009 and 2012. Furthermore, we were able to show that pollen beetle larvae are extremely susceptible to thiacloprid, whereas within strains larvae are significantly more resistant than adults to pyrethroids such as lambda‐cyhalothrin. Dose‐response data for thiacloprid against cabbage seed weevil populations collected in 2011 in Germany, Sweden, and Ukraine showed a 10‐fold higher intrinsic sensitivity compared to pollen beetle, and showed only a low variation in response. In addition, we also tested 17 cabbage seed weevil populations collected in five countries against lambda‐cyhalothrin with low variation in response (three‐fold), suggesting full baseline susceptibility and no resistance to pyrethroids. The implications of the data presented for resistance management in coleopteran pests in winter oilseed rape are discussed.

Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae).

Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90-100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting pyrethroid treatments by expressing a target-site mutation, underpins the importance of diversity in available chemistry for resistance management tactics based on mode of action rotation in order to guarantee sustainable winter oilseed rape cultivation in Europe.

The interactions between 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.