Name: functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica
Uploaded: 2018-08-23T14:30:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+resistance+in+house+flies+from+the+United+States:+Resistance+levels+and+frequency+of+pyrethroid+resistance+alleles.">Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles.</a> Pesticide Biochemistry and Physiology. 107 (3), 377-384 (2013).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole+transcriptomal+linkage+analysis+of+gene+co-regulation+in+insecticide+resistant+house+flies,+Musca+domestica.">A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica.</a> BMC Genomics. 14, 803 (2013).</li><li>Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+resistance+in+mosquitoes:+impact,+mechanisms,+and+research+directions.">Insecticide resistance in mosquitoes: impact, mechanisms, and research directions.</a> Annual Review of Entomology. 60, 537-559 (2015).</li><li>Grigoraki, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+and+genetic+study+reveal+amplified+carboxylesterase+genes+implicated+in+temephos+resistance,+in+the+Asian+tiger+mosquito+Aedes+albopictus.">Transcriptome profiling and genetic study reveal amplified carboxylesterase genes implicated in temephos resistance, in the Asian tiger mosquito Aedes albopictus.</a> e0003771. 9, e0003771 (2015).</li><li>Grigoraki, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylesterase+gene+amplifications+associated+with+insecticide+resistance+in+Aedes+albopictus:+Geographical+distribution+and+evolutionary+origin.">Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.</a> PLOS Neglected Tropical Diseases. 11, e0005533 (2017).</li><li>Wheelock, C., Shan, G., Ottea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+carboxylesterases+and+their+role+in+the+metabolism+of+insecticides.">Overview of carboxylesterases and their role in the metabolism of insecticides.</a> Journal of Pesticide Science. 30, 75-83 (2005).</li><li>Feng, X., Li, M., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylesterase+genes+in+pyrethroid+resistant+house+flies,+Musca+domestica.">Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica.</a> Insect Biochemistry and Molecular Biology. 92, 30-39 (2018).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.</a> J Immunol Methods. 65 (1-2), 55-63 (1983).</li><li>Jarvis, D. 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最近数十年で表現し、大量の蛋白質の生化学的, 機能的定量法と体外酵素の特性をこのように分離異種発現システムに広く使用されています。日には、大腸菌、 ・組成分析、 Sacccharomyces 酵母、ハスモンヨトウ frugiperdaなどいくつかの別のモデル システムは組換え蛋白質の表現との選択に適応されている、興味の蛋白質22,<sup class="xref...

殺虫剤抵抗性は現在家はえ制御世界中1,2の主要な問題であります。殺虫剤抵抗性のメカニズムを容易にこの問題のより良い理解を決定するため、新しい戦略を効果的に防止したり、抵抗の開発3の拡散を最小限に努力。カルボキシルエステラーゼ、主要な解毒酵素の一つとして、多くの隔離と様々 な昆虫4,5,6の殺虫剤の代謝における役割のため注目を集めています。私たちの以前の研究は家のハエで複数カルボキシルエステラーゼを識別しているの発現量恒常耐性 ALHF 株にまで規制がありませんがまたペルメトリン治療7 への応答で高い誘導のレベルをすることができます。.ただし、殺虫剤の代謝におけるこれらのカルボキシルエステラーゼ遺伝子の機能的な特徴は、探検するために残ります。
初期の 1980 年代8の最初のレポート以来高蛋白質生産効率性真核生物のタンパク質のプロセッシング機能9バキュロ ウイルスを介する遺伝子発現システムに広く採用されています。このバイナリ システムは 2 つの本質的な要素で構成される: 構築された組換えバキュロ ウイルスの宿主細胞と遺伝子組換えバキュロ ウイルスによる感染細胞による興味のあるタンパク質の大量発現に外国の遺伝子を提供します。過去十年にわたってバキュロ ウイルス媒介電池式システムは、組換えタンパク質、細胞質酵素から昆虫の膜結合型蛋白質に至るまでの数千を生成する広く使用されています、哺乳類細胞の10。私たちの以前の研究は正常にこのシステム11Sf9 昆虫細胞での複数の CYP450 酵素を表明しています。本研究で我々 は Sf9 昆虫細胞に感染するカルボキシルエステラーゼ組換えバキュロ ウイルスを構築、異なるペルメトリン治療し、大規模な表現カルボキシルエステラーゼ タンパク質の in vitroの免疫寛容を検討機能探査。以前研究12,13で採択された「昆虫ホモジュネートからの複数のカルボキシルエステラーゼ アイソザイムの混合物を調査して、代わりこのバキュロ ウイルス媒介昆虫細胞発現系により特定の式とターゲットタンパク質の生化学的および構造特性より良い評価のための分離。
テトラゾリウム塩を用いた測定 (MTT) は、高スループットの比色定量法を開発し、細胞生存率を測定するために最適化されました。このアッセイは、生細胞のみがこの分析できる有機溶剤14に溶解した後、暗い紫色着色された塩の沈殿物に黄色の MTT の試薬を代謝できるメカニズムに基づいています 15。トリパン ブルー色素排除とチミジン滴定分析16,17などの時間のかかる方法がより正確なのいくつかは近年開発されています。ただし、MTT の細胞に基づく試金はまだ現在、細胞生存率を迅速に検出する最も迅速かつ簡単に操作の方法として認識されます。ここでは、MTT の試金を使用殺虫剤の治療に対して免疫寛容を探索します。強くカルボキシルエステラーゼ組換えバキュロ ウイルスに感染している場合に、セルの強化されたトレランスは、順番で殺虫剤抵抗性への関与を示唆している殺虫剤、カルボキシルエステラーゼの代謝の役割をサポートします。
さらに、体外代謝アッセイは、この研究も行われました。カルボキシルエステラーゼの加水分解の活動を反映するように α ラウリン酢酸 (α-NA) と β-ナフチル酢酸 (β-NA) などの一般的な基材を使用して一般的なカルボキシルエステラーゼの試金と比較して、体外代謝研究は正確な方法とみなされる殺虫剤18に向かってカルボキシルエステラーゼの活動を直接測定します。このメソッドは正常に様々 な昆虫の殺虫剤抵抗11,19,20協会における複数チトクロム p450 の特性評価に採用されています。ただし、このメソッドがまだ適用されていないカルボキシルエステラーゼの研究。バキュロ ウイルスによる発現システムによって生成するカルボキシルエステラーゼ タンパク質の可用性、我々 の関与の強力な証拠を提供することができます。 さらにペルメトリンに向かってカルボキシルエステラーゼの体外代謝研究を実行できます。ピレスロイド耐性を家の中でカルボキシルエステラーゼのハエします。

<table><tbody><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>New England Biolabs inc.</td><td>M0491L</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen by life technology</td><td>K240020</td><td>S.O.C medium and universal M13 sequence primers were included in this kit.</td></tr><tr><td>PureLink HiPure Plasmid Miniprep Kit</td><td>Invitrogen by life technology</td><td>K210002</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme mix for BaculoDirect&lt;sup&gt;TM&lt;/sup&gt; Kits</td><td>Invitrogen by life technology</td><td>11791-023</td><td></td></tr><tr><td>BaculoDirect C-Term Linear DNA Transfection Kit</td><td>Invitrogen by life technology</td><td>12562-019</td><td>Cellfectin transfection reagent and ganciclovir were included in this kit</td></tr><tr><td>pENTR-CAT plasmid</td><td>Invitrogen by life technology</td><td></td><td>Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL</td></tr><tr><td>Heat inactivated Fetal Bovine Serum, Certified</td><td>Gibco by Life Technologies</td><td>10082-139</td><td></td></tr><tr><td>Sf9 cells in Sf-900 III SFM</td><td>Gibco by Life Technologies</td><td>12659017</td><td></td></tr><tr><td>Insect Cell-PE LB Insect Cell Protein Extraction &amp;amp; Lysis Buffer</td><td>G Biosciences by A Geno Technology Inc</td><td>786-411</td><td></td></tr><tr><td>Sf-900 III SFM (1&amp;times;) Serum Free Medium Complete</td><td>Gibco by Life Technologies</td><td>12658-019</td><td></td></tr><tr><td>Grace&#039;s Insect Medium, unsupplemented</td><td>Gibco by Life Technologies</td><td>11595030</td><td></td></tr><tr><td>Permethrin (isomers) analytical standard</td><td>SUPELCO by Solutions Within&lt;sup&gt;TM&lt;/sup&gt;</td><td>442748</td><td></td></tr><tr><td>Methanol (analytical graded)</td><td>Sigma-Aldrich</td><td>67-56-1</td><td></td></tr><tr><td>Acetonitrile (analytical graded)</td><td>Sigma-Aldrich</td><td>75-05-8</td><td></td></tr><tr><td>GHP Acrodisc 25 mm Syringe Filters with 0.45 &amp;mu;m GHP Membrane (HPLC Certified)</td><td>Pall Life Sciences</td><td>21890388</td><td></td></tr><tr><td>Alliance Waters 2695 HPLC System</td><td>Waters</td><td></td><td></td></tr><tr><td>T100 Thermal Cycle</td><td>Bio-Rad Laboratories Inc.</td><td>1861096</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophotometers</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica

カルボキシルエステラーゼを介した代謝は、さまざまな昆虫の殺虫剤抵抗性に大きな役割を果たすと考えられます。いくつかのカルボキシルエステラーゼ遺伝子は、探検するために殺虫剤耐性での役割が残っているに対し、抵抗性イエバエ フライひずみの調整を発見されました。ここでは、我々 はカルボキシルエステラーゼの機能解析のためのプロトコルを設計しました。3 つの例の実験を紹介する: (1) 式とバキュロ ウイルス媒介昆虫ハスモンヨトウ frugiperda (Sf9) セル式システムによるカルボキシルエステラーゼ タンパク質の分離(2) の細胞ベースの MTT (3-[4, 5-dimethykthiazol-2-イル]-2, 5-diphenyltetrazolium ブロマイド) 異なるペルメトリン治療昆虫細胞の耐性を測定する細胞毒性アッセイ(3)の in vitro代謝の研究ペルメトリンに向かってカルボキシルエステラーゼの代謝機能を調査します。MdαE7 抵抗性イエバエから複製されたカルボキシルエステラーゼ遺伝子系統 ALHF を飛ぶし、Sf9 細胞感染の組換えバキュロ ウイルスを作成するために使用します。異なるペルメトリン治療に対してセル目は、MTT アッセイを測定しました。パーメスリン抵抗性制御グループ (猫組換えバキュロ ウイルス感染細胞と GFP 遺伝子組換えバキュロ ウイルス感染細胞) の比較実験グループ (MdαE7 組換えバキュロ ウイルス感染細胞) の高められた細胞トレランス治療は、それにより化学損傷から細胞を保護する殺虫剤の代謝で MdαE7 の機能を提案しました。それに加えて、カルボキシルエステラーゼ蛋白質は昆虫 Sf9 細胞で発現され分離体外代謝研究を行うこと。かなりの in vitro代謝効率 MdαE7 のペルメトリン、殺虫剤の代謝でカルボキシルエステラーゼの関与を示す直接の方が示唆し、飛ぶため殺虫剤抵抗性の家を授与します。

異なるペルメトリン トリートメント (MTT の試金) に向かって細胞生存率
MdαE7 組換えバキュロ ウイルス感染 Sf9 細胞 (実験群) と猫組換えバキュロ ウイルス感染 (感染するバキュロ ウイルス キット提供) 細胞 (群) のペルメトリンの細胞毒性を調べた。拡張セル許容範囲が MdαE7 強く発現細胞を用いたペルメトリンには、殺虫?...

Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

ここでは、プロトコルを提案家フライ カルボキシルエステラーゼ タンパク質の in vitroバキュロ ウイルス媒介昆虫細胞発現系を生成し、後で機能的、それにより代謝ペルメトリンの役割を特徴づけるピレスロイドを授与セルベースの MTT アッセイと体外代謝研究を行うことにより抵抗。

殺虫剤抵抗性イエバエ、イエバエのカルボキシルエステラーゼの機能特性

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

functional-characterization-carboxylesterases-insecticide-resistant

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

7.4K ビュー.  Auburn University. ここでは、プロトコルを提案家フライ カルボキシルエステラーゼ タンパク質の in vitroバキュロ ウイルス媒介昆虫細胞発現系を生成し、後で機能的、それにより代謝ペルメトリンの役割を特徴づけるピレスロイドを授与セルベースの MTT アッセイと体外代謝研究を行うことにより抵抗。

ビデオ: Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

免疫・感染学

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

トコジラミに対する殺虫剤の有効性の評価方法、<em&gt;ナンキンムシ</em&gt;、卵とファースト齢

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

環境学

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

コナガからのリアノジン受容体の N 末端ドメインの結晶構造

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

生化学

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

CRISPR/Cas9ゲノム編集 ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

行動学

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

モニタリングの殺虫剤抵抗性のためのバイオアッセイ

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

生物学

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

蚊を殺虫化合物小説の毒性試験のプロトコル

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

蚊やショウジョウバエに対する殺虫剤毒性を定量するための局所適用バイオアッセイ

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed worldwide and cause a severe threat to public health. To date, considering the fast-emerging insecticide resistance in the mosquito, the development of new botanical insecticides to control and reduce Ae. albopictus is urgent and crucial. Therefore, to investigate the decoction effect of the plant C. abrotanoides L. on mosquito larvae killing, we detected the mortality of larvae after treatment with different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) of decoction within a series of time points (12 h, 24 h, 36 h, and 48 h). We found that 24 h with 180 mg/mL C. abrotanoides L. decoction treatment killed 92.35% of mosquitoes relative to the control treatment. Meanwhile, 36 h with 120 mg/mL could also kill more than 90% of mosquitoes. Furthermore, Carassius auratus populations were exposed to 120 mg/mL C. abrotanoides L. decoction for 36 h and were not dead. The mortality evaluation indicated that this concentration is not a harmful level of ecological environmental pollution. This study provides a possible plant candidate that could be used for designing plant-derived insecticides. Additionally, these methods can be altered and applied to other mosquito species.

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

Using the Carpesium abrotanoides L. plant for mosquito larvae control can effectively reduce the Aedes albopictus mosquito population and provide a foundation for designing plant-derived insecticides.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

オリエンタルショウジョウバエバクトセラ背側のCRISPR/Cas9変異誘発のためのプロトコル

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

フィーディングアッセイによる Helicoverpa armigera に対する植物化学的影響の評価

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

著者スポットライト:害虫管理のための給餌アッセイの開発と課題 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigeraに対するファイトケミカルの殺虫効果を評価するための摂食アッセイシステムの開発

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

殺虫剤抵抗性イエバエ、イエバエのカルボキシルエステラーゼの機能特性

要約

さらに動画を探す

この動画の章

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

トコジラミに対する殺虫剤の有効性の評価方法、

コナガからのリアノジン受容体の N 末端ドメインの結晶構造

CRISPR/Cas9ゲノム編集ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

モニタリングの殺虫剤抵抗性のためのバイオアッセイ

蚊を殺虫化合物小説の毒性試験のプロトコル

蚊やショウジョウバエに対する殺虫剤毒性を定量するための局所適用バイオアッセイ

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

オリエンタルショウジョウバエバクトセラ背側のCRISPR/Cas9変異誘発のためのプロトコル

Helicoverpa armigeraに対するファイトケミカルの殺虫効果を評価するための摂食アッセイシステムの開発

殺虫剤抵抗性イエバエ、イエバエのカルボキシルエステラーゼの機能特性

要約

さらに動画を探す

この動画の章

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

トコジラミに対する殺虫剤の有効性の評価方法、

コナガからのリアノジン受容体の N 末端ドメインの結晶構造

CRISPR/Cas9ゲノム編集 ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

モニタリングの殺虫剤抵抗性のためのバイオアッセイ

蚊を殺虫化合物小説の毒性試験のプロトコル

蚊やショウジョウバエに対する殺虫剤毒性を定量するための局所適用バイオアッセイ

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

オリエンタルショウジョウバエバクトセラ背側のCRISPR/Cas9変異誘発のためのプロトコル

Helicoverpa armigeraに対するファイトケミカルの殺虫効果を評価するための摂食アッセイシステムの開発

CRISPR/Cas9ゲノム編集ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定