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      Incidence, Spread and Mechanisms of Pyrethroid Resistance in European Populations of the Cabbage Stem Flea Beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae)

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          Abstract

          Background

          Cabbage stem flea beetle (CSFB), Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major early season pest of oilseed rape throughout Europe. Pyrethroids have been used for controlling this pest by foliar application, but in recent years control failures have occurred, particularly in Germany due to the evolution of knock-down resistance (kdr). The purpose of this study was to investigate the incidence and spread of pyrethroid resistance in CSFB collected in Germany, Denmark and the United Kingdom during 2014. The level of pyrethroid resistance was measured in adult vial tests and linked to the presence of kdr genotypes.

          Results

          Although kdr (L1014F) genotypes are present in all three countries, marked differences in pyrethroid efficacy were found in adult vial tests. Whereas Danish CSFB samples were in general susceptible to recommended label rates, those collected in the UK mostly resist such rates to some extent. Moderately resistant and susceptible samples were found in Germany. Interestingly, some of the resistant samples from the UK did not carry the kdr allele, which is in contrast to German CSFB. Pre-treatment with PBO, prior to exposure to λ-cyhalothrin suggested involvement of metabolic resistance in UK samples.

          Conclusion

          Danish samples were mostly susceptible with very low resistance ratios, while most other samples showed reduced sensitivity in varying degrees. Likewise, there was a clear difference in the presence of the kdr mutation between the three countries. In the UK, the presence of kdr genotypes did not always correlate well with resistant phenotypes. This appears to be primarily conferred by a yet undisclosed, metabolic-based mechanism. Nevertheless our survey disclosed an alarming trend concerning the incidence and spread of CSFB resistance to pyrethroids, which is likely to have negative impacts on oilseed production in affected regions due to the lack of alternative modes of action for resistance management purposes.

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          Most cited references8

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          Allelic discrimination using fluorogenic probes and the 5' nuclease assay.

          K Livak (1999)
          Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.
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            A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum.

            Cytochrome P450-mediated detoxification is one of the most important mechanisms involved in insecticide resistance. However, the molecular basis of this mechanism and the physiological functions of P450s associated with insecticide resistance remain largely unknown. Here, we exploited the functional genomics and reverse genetic approaches to identify and characterize a P450 gene responsible for the majority of deltamethrin resistance observed in the QTC279 strain of Tribolium castaneum. We used recently completed whole-genome sequence of T. castaneum to prepare custom microarrays and identified a P450 gene, CYP6BQ9, which showed more than a 200-fold higher expression in the deltamethrin-resistant QTC279 strain when compared with its expression in the deltamethrin-susceptible Lab-S strain. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6BQ9 and transgenic expression of CYP6BQ9 in Drosophila melanogaster showed that CYP6BQ9 confers deltamethrin resistance. Furthermore, CYP6BQ9 enzyme expressed in baculovirus metabolizes deltamethrin to 4-hydroxy deltamethrin. Strikingly, we also found that unlike many P450 genes involved in insecticide resistance that were reported previously, CYP6BQ9 is predominantly expressed in the brain, a part of the central nervous system (CNS) containing voltage-gated sodium channels targeted by deltamethrin. Taken together, the current studies on the brain-specific insect P450 involved in deltamethrin resistance shed new light on the understanding of the molecular basis and evolution of insecticide resistance.
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              Diversity and Convergence of Sodium Channel Mutations Involved in Resistance to Pyrethroids.

              Pyrethroid insecticides target voltage-gated sodium channels, which are critical for electrical signaling in the nervous system. The intensive use of pyrethroids in controlling arthropod pests and disease vectors has led to many instances of pyrethroid resistance around the globe. In the past two decades, studies have identified a large number of sodium channel mutations that are associated with resistance to pyrethroids. The purpose of this review is to summarize both common and unique sodium channel mutations that have been identified in arthropod pests of importance to agriculture or human health. Identification of these mutations provides valuable molecular markers for resistance monitoring in the field and helped the discovery of the elusive pyrethroid receptor site(s) on the sodium channel.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                30 December 2015
                2015
                : 10
                : 12
                : e0146045
                Affiliations
                [1 ]Department of Agroecology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark
                [2 ]Bayer CropScience AG, Pest Control Biology, Alfred Nobel Str. 50, 40789 Monheim, Germany
                [3 ]Department of Biological Chemistry and Crop Protection, Rothamsted Research, West Common, Harpenden AL5 2JQ, United Kingdom
                Institute of Zoology, Chinese Academy of Sciences, CHINA
                Author notes

                Competing Interests: The involvement of Ralf Nauen from Bayer CropScience did not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: DHH RN SPF MSW MK. Performed the experiments: DHH RN SPF. Analyzed the data: DHH RN SPF MSW MK. Wrote the paper: DHH RN SPF MSW MK.

                Article
                PONE-D-15-44054
                10.1371/journal.pone.0146045
                4696833
                26717570
                8eb2b4b5-c3c0-464a-8e94-ba4a5a189db0
                © 2015 Højland et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 7 October 2015
                : 11 December 2015
                Page count
                Figures: 2, Tables: 3, Pages: 11
                Funding
                AHDB-HGCA part- funded the work done at Rothamsted Research which is an institute of the Biotechnology and Biological Sciences Council of the United Kingdom. This project EvoPPM 0603-00516B was funded by Innovation Fund Denmark and Bayer CropScience. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Research Article
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                All relevant data are within the paper and its Supporting Information files.

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