私たちは、希釈および農薬製剤の支援のためモリーStedfastに感謝します。また、実験計画の支援のためにプロビット分析ガイダンスのためのトロイ・アンダーソンとザカリーエーデルマンに感謝します。本研究の一部は昆虫財団賞によって、バージニア害虫管理の奨学金によってサポートされていました。

注：このプロトコルは別々のベッドのバグの卵と第一齢を治療するための2つの殺虫剤のアッセイが含まれます。両方のプロトコルは同じ殺虫剤を用いて行ったが、プロトコルが殺虫剤の曝露を確実にするために、容易に試験片を操作するように適合されなければなりませんでした。 1.卵浸漬殺虫剤アッセイ<ol><li>コロニーから交配、よく給餌成人女性のベッドのバグを引き、ろ紙でペトリ皿にメス（5-10）を配置。殺虫剤のアッセイのための十分な卵が存在するであろうことを確実にするために、少なくとも10の反復を持っています。女性は毎日卵を築いているかどうかを確認してください。 <ol><li>彼らは卵を産むたら、ペトリ皿（6センチメートル×5センチ）のうち、女性を取り、毎日新しいろ紙で新しいペトリ皿にそれらを入れて、古いものと新しいペトリ皿上の日付を記録します。 </li><li>アッセイを開始する前に5日齢4個の卵を許可します。胚発生時の殺虫剤の変化に対する感受性は、よO卵の年齢のために制御することが重要です。 Dの光周期、60％RH、および27°C：午前12時12 Lで、すべてのライフステージを開催しています。 </li></ol></li><li>卵が適切に熟成させた後、ソフトチップの鉗子を使用して濾紙から静かにベッドのバグの卵を削除します。静かにピンセットを用いてろ紙に付着した卵の底をこすり、その後、静かに投げベッドのバグの卵を制限するために、卵に多くの圧力を適用せずにつかみます。 </li><li>プラスチック製のペトリ皿の内側に、それぞれのグループに削除されたベッドのバグの卵を置きます。柔軟仕上げ剤シートと外側に各ペトリ皿の底をこすることによりベッドのバグの卵を投げつけから静電気を制限します。すべてを一度にベッドのバグの卵の治療群（1群当たり5-10の卵）。 1グループに複製を考慮し、このアッセイのために複製し、少なくとも5（またはそれ以上）を使用します。 </li><li>イミダクロプリド（0.1％）/β-シフルトリン（0.5％）と制御のための0.21から21μL/ mLの（5異なる濃度の範囲を得るために、殺虫剤の連続希釈液を準備溶剤（水道水）を有する溶液のみ。浸漬アッセイのための十分な容量を持つように各濃度の20ミリリットルを加えます。 </li><li> 35 mLの線で切断した遠心分離管（50mL）を内側カットオフホイールアクセサリとの回転工具を使用し、次いで遠心分離管（直径= 2.54cm）のアルミニウムと同じ回転工具を用いたキャップに穴をカット石のアクセサリーを研削酸化物。切断時にキャップがしっかりとチューブ（ 図1）にねじ込みますように、キャップの端に5センチのままにします。 </li><li>キャップ（〜5cm 2の）に適合するのに十分な大きさの正方形に細かいメッシュをカットします。遠心管のキャップを使用して、遠心管に、任意の凹凸をなくし、しっかりメッシュスクリュー。 </li><li>遠心管の内側にメッシュ上にペイントブラシを使用して、ベッドのバグの卵のグループ（1複製）を配置します。 5秒間の殺虫剤濃度にカット遠心管の内側にベッドのバグのグループを浸し。 </li><li>目のうち、遠心分離管を削除します電子殺虫剤液。実験室の組織に直接遠心管を配置することによって、内部の卵でメッシュを乾燥させます。 </li><li>小さな、細かい毛ペイントブラシを使用して、遠心分離管のうち、卵のグループを削除し、クリーンなフィルターペーパーできれいにペトリ皿に入れます。 </li><li>すべての卵が孵化するのに十分な時間を持っていることを確認するために14日間のレコード卵の死亡率（孵化に失敗）。 </li><li>アボットの式（ - ％コントロールの死亡率/ 100 - 制御死亡率×100％試験の死亡率）を用いた制御処理における死亡率に対する正しいです。 </li><li> LC 50値を計算するために、分析ソフトウェア15に対応する濃度で記録された死亡率を挿入します。 </li></ol>まず齢殺虫剤アッセイ<ol><li>好ましくは1日前に送られ、十分に供給されたベッドのバグ（5成人男性と5成人女性）のグループを分離し、matinのために提供ろ紙でペトリ皿（6センチメートル×5センチ）に入れますグラムと産卵。大人のベッドのバグが合わさると、約7-9日間卵を産むことを許可します。給餌のために、血液16を囲むガラスチューブ内の温水を循環させる人工的な供給システムを使用しています。この研究のために、defibronatedウサギの血液を使用します。 </li><li>ペトリ皿（5センチメートル×6センチ）から成人を削除し、卵をペトリ皿内の第1齢に孵化することができます。ベッドのバグの卵は室温で孵化するのは約7-9日かかります。 </li><li>ペイントブラシを使用して、同じ年齢の無給電最初の齢を収集します。年齢は、毎日最初の齢を除去し、それらが斜線日を記録することによって決定することができます。殺虫剤アッセイまで、新しい、きれいなペトリ皿に入れます。 1齢幼虫は耐性が非常に乾燥剤ではありませんので、殺虫剤のアッセイは、できるだけ早く完了する必要があります。 </li><li>アッセイのために、5つの異なる濃度と制御ソリューの範囲を取得するために、イミダクロプリド/β-シフルトリンの連続希釈液を準備溶剤のみでる。米国のベッドのバグの制御のために標識策定製品はこれらの研究のために使用した、したがって、溶媒が（ラベルに従って）水でした。 </li><li>ピペット個々のろ紙上に対照群に対する各殺虫剤の濃度と水の150μLのアリコート（＃1; 4.2センチメートル直径）。ろ紙の縁部全体と中央付近の液滴を適用することにより、ピペッターを使用して、全体のろ紙の上に殺虫剤を配布します。 </li><li>個々のハードボードパネル（7センチメートル2）の上に個別に処理された濾紙を置きます。 </li><li>しばらくまだ殺虫剤で濡れとペトリ皿の反転底部と第一齢をカバーする個々の濾紙上に最初齢のリリース群（1群あたり10から5）。 </li><li>処理された表面から逃げるの1齢ベッドのバグを防ぐために、ペトリ皿の上に重量を配置します。 </li><li>すべてに24時間曝露後の1齢ベッドのバグのレコード死亡殺虫剤の濃度。 </li><li>アボットの式（ - ％コントロールの死亡率/ 100 - 制御死亡率×100％試験の死亡率）を用いた制御処理における死亡率に対する正しいです。 </li><li> LC 50値を計算するために、分析ソフトウェア15に記録死亡率および濃度を挿入します。 </li></ol>

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<li>Pritchard, M. J., Hwang, S. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734207/">Severe anemia from bedbugs.</a> Can. Med. Assoc. J. 181, 287-288 (2009).</li>
<li>Goddard, J., de Shazo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Psychological+effects+of+bed+bug+attacks+%28Cimex+lectularius+L%29%5BTitle%5D)+AND+%22Am.+J.+Med%22%5BJournal%5D)">Psychological effects of bed bug attacks (Cimex lectularius L).</a> Am. J. Med. 125, 101-103 (2012).</li>
<li>Harlan, H. J. <a target="_blank" href="https://www.researchgate.net/publication/263133743_Bed_Bugs_101_the_Basics_of_Cimex_lectularius">Bed bugs 101: the basics of Cimex lectularius.</a> Am. Entomol. 52, 99-101 (2006).</li>
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<li>Bell, C. H., Hole, B. D., Evans, P. H. The occurrence of resistance to phosphine in adult and egg stages of strains of Rhyzopertha dominica (F.)(Coleoptera: Bostrichidae). J. Stored Prod. Res. 13, 91-94 (1977).</li>
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<li>Price, L. A., Mills, K. A. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/0022474X88900082">The toxicity of phosphine to the immature stages of resistant and susceptible strains of some common stored product beetles, and implications for their control.</a> J. Stored Prod. Res. 24, 51-59 (1988).</li>
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</ol>

著者らは、開示することは何もありません。

A critical step in this assay is to ensure that no eggs that are removed from a surface are damaged prior to the assay. Many insects cement their eggs to a substrate, therefore, a preliminary test may be needed to ensure that removal does not cause mortality. This test can be conducted with several replications of a treated group (bed bugs removed from filter paper) compared to a control group (bed bugs not removed). Similar low rates of mortality (or no mortality) between the treated and control group will ensure that removal did not cause egg mortality. Thus, any mortality recorded for the insecticide assay should be due to the insecticide treatment. The same preliminary experiment can be conducted using first instars to ensure that methods for collecting do not kill the experimental insects. All experiments conducted later should include control replications that do not receive insecticide but are treated with the solvent (for these experiments, tap water was the solvent). 
Preliminary experiments should be conducted with the recommended label rate for formulated products and then serially diluted to determine the correct concentrations to produce a concentration response curve of mortality ranging from 20-80%. Previous insecticide studies on the same insects or compounds may also be insightful when trying to determine the appropriate concentration. Concentrations will need to be modified dependent upon the insect, life stage, and compound used. New solutions should be made each time an experiment is conducted. 
The exposure of first instars to wet residues was necessary to get high mortality. Although this life stage is considered more susceptible to insecticides than older nymphs and adults, high mortality was still difficult to obtain with formulated products. Submerging the first instars into insecticides similar to the eggs was not plausible because of the resultant high mortality, thus this was a limitation to the study. The alternative first instar bioassay was manipulated to maintain first instars on surfaces to force constant contact with the treatment.
Dry residue assays and bottle assays commonly used for insecticide efficacy and resistance studies do not provide methodology for testing against multiple insect life stages17. These assays are often used to determine resistance in insects, and not to test insecticide efficacy. There are limited studies on egg dipping toxicological studies and none that have reported a standard methodology previously that can be repeated for multiple insect species.
This study provides a protocol for testing insecticide efficacy against bed bug eggs and first instars. Efficacy should be evaluated in different life stages, if not all, for a more holistic view of the efficacy of a treatment program. This consideration is important because an insect's susceptibility to insecticides changes with age and development 18. While age is an important factor in insecticide susceptibility, a factor in egg insecticide susceptibility is the eggshell. The eggshell provides a barrier to insecticides and makes insect eggs particularly difficult to control19. 
These toxicological assays provide researchers a way to evaluate insecticide toxicity against various life stages of insects. Insecticidal solutions are prepared to evaluate mortality and to determine LC50 concentrations. While this protocol was used for formulated products, the steps can be manipulated for technical grade compounds and for a variety of insects and life stages to evaluate toxicity.

ベッドのバグは、そのヒト宿主1、2、3、4、5、6の血液損失、皮膚の炎症、不眠症、うつ病、および不安を引き起こす可能性がある重要な都市の害虫です。ベッドのバグを排除し、制御のコストが高く、多くの場合、時間が害虫駆除会社7による家庭への複数の訪問を必要とします。複数の訪問は通常ので、ベッドのバグと、それらを殺すことに関連する困難の不可解な行動が要求されています。具体的には、ベッドのバグの卵は、サイズが小さいためと卵殻による胚の保護を制御することが困難です。 現在、米国の多くの害虫駆除会社のための一般的な方法は、化学殺虫剤を使用して、ベッドのバグのために家を治療することです。それは、よく知られています卵は制御が困難であることを、非常に多くの企業が再治療の8のために2週間の時間枠を実装しています。最初の齢が出現し、うわさによれば、殺虫剤で殺すために容易になりますように、これは、ベッドのバグの卵を孵化するのに十分な時間を可能にします。しかし、ベッドのバグの卵と第一齢に対する液体殺虫剤の有効性を評価する研究で不足があります。 昆虫の卵は、多くの農業害虫に加えて、ベッドのバグ以外の都市害虫に制御するための最も困難なライフステージであることが報告されています。いくつかの研究は、殺虫剤抵抗性を報告しているが、これらの困難の大部分は、卵殻に起因しています。卵の段階での抵抗は、 コナガは 11 コナガ 、10 capitis Triatoma infestansの 9、 ヒトジラミにRhyzoperthaのドミニカ 12を文書化されています<eメートル&gt;ナンキンムシ13、及び複数の格納されている製品の甲虫（ すなわち ノコギリヒラタムシ、モドキTribolium castaneum、Cryptolestes ferrugineusとRhyzoperthaのドミニカ ）。 14卵の段階での抵抗性の発達および未成熟の段階だけでなく、卵殻を通して殺虫剤の浸透に伴う合併症は、これらのライフステージに対する殺虫効力バイオアッセイの必要性を必要とします。 このプロトコルは、一般的なベッドのバグの卵と第一齢段階、 ナンキンムシに殺虫剤の有効性を決定するためのステップバイステップ手順を提供します。これらのプロトコルは両方とも、LC 50値の定量化を可能にする濃度反応アッセイです。濃度 - 応答アッセイは、一般毒性試験に使用され、ただし、このプロトコルは、簡単に、ベッドバグ卵第齢のグループを処理するように適合されています。これらのアッセイは、適合させることができます様々な昆虫の卵や未熟ライフステージのために。

<table><tbody><tr><td>Petri dishes</td><td>Fisher Scientific Inc.</td><td>08-757-105</td><td>Plastic</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1001-042</td><td></td></tr><tr><td>Featherweight forceps</td><td>Fisher Scientific Inc.</td><td>4750</td><td></td></tr><tr><td>Temprid SC (imidacloprid/bifenthrin)</td><td>Bayer CropScience</td><td></td><td></td></tr><tr><td>Transport GHP (acetamiprid/bifenthrin)</td><td>FMC Corp.</td><td></td><td></td></tr><tr><td>Centrifuge tube</td><td></td><td>06-443-18</td><td>50 mL; flat-top threaded</td></tr><tr><td>Fine mesh</td><td></td><td>7250C</td><td></td></tr><tr><td>KimWipe</td><td></td><td>06-666A</td><td>11 cm x 21 cm</td></tr><tr><td>Small paint brush</td><td></td><td></td><td>Any small paint brush</td></tr><tr><td>Hardboard panels</td><td></td><td></td><td>Composite wood or tile would be sufficient from a home improvement store</td></tr><tr><td>Metal weights</td><td></td><td></td><td>Any type of metal screw that will hold the weight of the Petri dish down onto a surface</td></tr></tbody></table>

a method for evaluating insecticide efficacy against bed bug, cimex lectularius, eggs and first instars

害虫に対する殺虫剤の標準的な毒性評価は、主に成虫に行われています。評価は用量または殺虫剤の濃度として死亡率増加が増加する用量 - 反応又は濃度 - 応答曲線に基づいています。このような卵や早期齢や幼虫の段階として、試験集団の50％の死亡率をもたらすテストは非成人昆虫のライフステージに対する殺虫剤の毒性を評価するために挑戦することができ、標準的な致死濃度（LC 50）と致死量（LD 50）、 。しかし、この情報は、制御および治療の努力に影響を与えるすべてのベッドのバグのライフステージにおける殺虫剤の有効性を、理解するために不可欠です。このプロトコルは、幼虫最初の齢を治療するためにベッドのバグの卵と接触殺虫アッセイのために修正された標準的な浸漬バイオアッセイを使用しています。これらのアッセイは、さらに、殺虫剤の評価のためのLC 50値を定量化するための濃度-応答曲線を生成します。

卵はイミダクロプリド/β-シフルトリンの5の異なる濃度（μL/ mL）に浸漬しました。最初の齢は、イミダクロプリド/β-シフルトリン5つの異なる濃度（ 図2）の処理された表面上に置きました。 ハーラン、リッチモンド、およびエピックセンター（ 表1）：私たちはC. lectulariusの三つの異なる集団を使用します。ハーラン株はピレスロイド系殺虫剤の影響を受けやすいと博士ハロルドハーランから2005年に買収されました。リッチモンドとエピックセンター株は両方デルタメトリンに耐性です。リッチモンド株はOH、エピックセンター株はシンシナティで2008年に採取した2008年にリッチモンド、バージニア州の高齢者グループホームから収集しました。 高いLC 50値は、より高い濃度の50％を死滅させるために必要であったことを示しリッチモンドとエピックセンター株からの試験集団の卵。卵と同様に、リッチモンドとエピックセンター集団からの最初の齢は、試験集団の50％を殺すために殺虫剤の高い濃度を必要としました。異なる文字が続くLC 50値は大きく異なっています。 <img alt="図1" class="xfigimg" src="/files/ftp_upload/55092/55092fig1.jpg" /> 修正された遠心管の1.写真を図。メッシュの正方形片がキャップにねじ込むことができるように穴が、キャップに切断されています。別の殺虫剤を使用する場合のメッシュ片を廃棄し、交換することができます。ベッドのバグの卵は、ペイントブラシを使用してメッシュ上に直接置き、次いで浸漬アッセイ用殺虫剤の溶液に沈めています。 <a href="http://ecsource.jove.com/files/ftp_upload/55092/55092fig1large.jpg" target="_blank">このfigurの拡大版をご覧になるにはこちらをクリックしてください。</a>電子。 <img alt="図2" class="xfigimg" src="/files/ftp_upload/55092/55092fig2.jpg" /> 用量応答グラフの2表現図。イミダクロプリド/β - シフルトリンのハーラン歪みベッドバグ卵のパーセント反応（死亡率）はy軸上にプロットされ、ベッドバグがさらされたことをログ濃度をx軸上に表されています。投与量は増加し、死亡率は、試験した最高濃度で100％に達したとして、卵の死亡率が増加しました。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>ライフステージ/ひずみ</td><td colspan="2"> n個 </td><td> LC 50（95％CI） </td><td>スロープ±SE </td><td> X 2（DF） </td></tr><tr><td>卵</td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td>ハーラン</td><td colspan="2"> 250 </td><td> 0.41a（<html> 0.276から0.548） </td><td> 1.86±0.24 </td><td> 33.42（23） </td></tr><tr><td>リッチモンド</td><td colspan="2"> 320 </td><td> 1.23b（0.59から2.10） </td><td> 1.13±0.14 </td><td> 82.57（30） </td></tr><tr><td>エピックセンター</td><td colspan="2"> 400 </td><td> 2.10b（1.049から4.587） </td><td> 0.95±0.10 </td><td> 149.91（38） </td></tr><tr><td>まず齢</td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td>ハーラン</td><td colspan="2"> 150 </td><td> 0.04a（0.030から0.063） </td><td> 2.16±0.34 </td><td> 38.17（28） </td></tr><tr><td>リッチモンド</td><td colspan="2"> 195 </td><td> 4.81b（1.94から10.26） </td><td> 0.66±0.12 </td><td> 45.87（37） </td></tr><tr><td>エピックセンター</td><td colspan="2"> 190 </td><td> 19.72b（8.18から184.48） </td><td> 0.75±0.17 </td><td> 45.39（36） </td></tr></tbody></table>キャンベルおよびMiller 2015年13から変更されたベッドのバグの卵と1齢LC 50値の &quot;&gt; 表1の比較。

Watch this Scientific Journal Video about A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars at JoVE.com

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

殺虫剤の評価は、多くの場合、成虫ではなく、未熟な段階を標的としています。ここでは、最初の幼虫ベッドのバグのステージと比較して、ベッドのバグの卵に対する殺虫剤を評価するためのプロトコルを提示します。他の昆虫は、非成人期段階で殺虫剤の有効性を評価するためにこれらのプロトコルを調整することができます。

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 ...

a-method-for-evaluating-insecticide-efficacy-against-bed-bug-cimex

Nanyang Technological University

Research

JoVE Journal

Environment

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

11.3K ビュー.  University of Florida. 殺虫剤の評価は、多くの場合、成虫ではなく、未熟な段階を標的としています。ここでは、最初の幼虫ベッドのバグのステージと比較して、ベッドのバグの卵に対する殺虫剤を評価するためのプロトコルを提示します。他の昆虫は、非成人期段階で殺虫剤の有効性を評価するためにこれらのプロトコルを調整することができます。 

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

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

ブナスケール昆虫への抵抗のためのアメリカのブナを（画面ための技術<em&gt;クリプトコッカスfagisuga</em&gt;リンド。）

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

環境学

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

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

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

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

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 ...

生化学

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

コーヒーベリーボーラーハイポテネマスハンペイに対する対照剤および殺虫剤をテストするための方法論

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

孤独なミツバチの幼虫に対する農薬の効果の評価

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

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

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

情報化学物質にトコジラミの嗅覚ニューロンの応答を検出するために単一感覚子録音を使用しました

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

神経科学

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. ...

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 ...

トコジラミに対する殺虫剤の有効性の評価方法、

要約

さらに動画を探す

ブナスケール昆虫への抵抗のためのアメリカのブナを（画面ための技術

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

コーヒーベリーボーラーハイポテネマスハンペイに対する対照剤および殺虫剤をテストするための方法論

孤独なミツバチの幼虫に対する農薬の効果の評価

モニタリングの殺虫剤抵抗性のためのバイオアッセイ

情報化学物質にトコジラミの嗅覚ニューロンの応答を検出するために単一感覚子録音を使用しました

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

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