この研究の財政的支援は、国家主要研究開発計画（2016YFC1200601）と中国国立自然科学財団（31390421）によって提供された。

1.白蝶貝の飼育と種識別<ol><li> 27±1℃、湿度70±10％、光14時間：暗期10時間の標準条件下で、カゴGossypium hirsutum （Malvaceae）（cv。Zhe-Mian 1973）をカゴで維持する。 </li><li>個々の成熟白コショウを集め、30μLの溶解緩衝液（10mMトリス、pH8.4,50mM KCl、0.45％[wt / vol] Tween-20,0.2％[wt / vol]ゼラチン、0.45％[vol / vol]ノニデットP40,60μg/ mLプロテイナーゼK）。 </li><li>ホモジネートを65℃で1時間インキュベートし、次に100℃で10分間インキュベートする。 注：65℃でのインキュベーション時間は必要に応じて増やすことができます。 100℃でのインキュベーション時間は、Proteinase Kを十分に不活性化し、DNA損傷を避けるために厳密に制御する必要があります。 </li><li> ミトコンドリアシトクロムオキシダーゼIプライマーに基づいて、whitefly DNA（セクション1.3で得られた）を用いてポリメラーゼ連鎖反応（PCR）を行う。 COI-F：5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3 &#39; COI-R：5&#39;-TAATATGGCAGATTAGTGCATTGGA-3 &#39; <ol><li> 1UのTaq DNAポリメラーゼ、10μlのBuffer、0.2mMのdNTP、0.2mMの各プライマー、2μLのwhitefly DNAを含む25μLの最終容量でPCR反応を行います。 </li><li> 95℃で3分間の変性後、95℃で45秒間（変性）、50℃で45秒間（アニーリング）および72℃で1分間（伸長）の35サイクル、およびさらなる伸長のために72℃でさらに10分間。 </li></ol></li><li> DNAゲル抽出キットを使用してPCR増幅産物を推奨プロトコールで洗浄します。 </li><li>精製したDNAサンプルをDNAサンプルシークエンシングシステムで、製造者の指示に従って配列決定する。 </li><li> Basic Local Alignment Search Tool（BLAST）を使用してシークエンスの潜在種を特定し、他の種の汚染を避けるために配列を分析するes。 </li></ol> 2.内分泌攪乱の同定と局在化<ol><li>白蝶状DNA（ステップ1.3で取得）にPCRを行い、白蝶内の各内毒素の特定の遺伝子を増幅する（PCRレシピはセクション1.4に記載されており、PCR手順およびプライマーは表1に記載されている ）。 </li><li>以前に公表されたプロトコール20 （1％アガロースゲル、ランニングバッファーとしてTris-アセテート-EDTA、電圧5V / cm）に従って増幅した試料をアガロースゲル電気泳動に付して、各細菌の特異的遺伝子を同定する。 </li><li>白い葉の中の細菌種を決定するために、各バンドを回収し、配列決定する（1.5-1.7節参照）。 </li><li>以前に公表されたプロトコール19に従う、内腔の位置を同定するために、ウンシュウの蛍光in situハイブリダイゼーション（FISH）を実施する。 注：必要に応じて蛍光プローブの濃度を上げてください。 </li></o1&gt; 透過型電子顕微鏡（TEM） <ol><li> 2.5％[vol / vol]グルタルアルデヒドを含有するリン酸緩衝液（0.1M、pH7.0）中に白鳥を4時間浸漬して固定する。 </li><li>リン酸緩衝液（0.1M、pH7.0）中でサンプルを3回、毎回15分間洗浄する。 </li><li> 1％[wt / vol] OsO 4を含有するリン酸緩衝液（0.1M、pH7.0）に試料を2時間浸して再度固定する。 </li><li>毎回15分の段階的なエタノール（30％、50％、70％、80％、90％、95％および100％）で試料を脱水する。 </li><li>サンプルを100％アセトンに20分間浸して浸します。 </li><li>サンプルを100％アセトンと100％Spurr樹脂（1：1）との混合物中に室温で1時間置く。 </li><li>試料を100％アセトンと100％Spurr樹脂（1：3）の混合物に室温で3時間移す。 </li><li>サンプルを100％Spurr樹脂（70℃でインキュベート）に9時間以上浸して浸します。 </li><li>白を用いて、マイクロを用いてサンプルを切る私に。 </li><li>絶対ウラニルアセテートに5〜10分間浸漬し、続いて50％アルカリ性クエン酸鉛にさらに5〜10分間浸漬することにより、超薄膜（セクション3.9で得たもの）を染色する。 注：セクション3.1-3.10で利用されている試薬の量は、サンプルによって異なります。サンプルが試薬に浸されていることを確認してください。 </li><li>透過型電子顕微鏡（15,000X）でサンプルを観察し、さらなる使用のために細菌の局在、形状およびサイズを決定する（セクション4.6を参照）。 </li></ol> 4.白蝶菌の解剖および精製<ol><li> 1倍PBS溶液100μLを顕微鏡スライドに加える。綿の葉から3〜4齢のニンフをとり、PBS溶液に浸します。 </li><li> 実体顕微鏡下で細かい昆虫の針を使用し、白蝶身からバクテリアを引き出します。 <ol><li>ゆっくりとニンフの片側の穴を切り、もう一方を少し押してくださいバクテリオミセスを排除するための側面。 </li></ol></li><li> 0.5-10μLピペットで20μLのマイクロローダー（細菌のサイズに合うように先端を切断したもの）をマウントする。個々のバクテリオミウムをPBS溶液からマイクロローダーに抽出する。 </li><li>細菌溶液を洗浄して、細菌溶液をPBS溶液にピペッティングし、細菌を抽出することによって、他の白癬組織の汚染を除去する。 3回繰り返します。 </li><li>直ちに、洗浄した細菌を60μLの1×PBS溶液を含む遠心管にピペットで移す。 </li><li>組み立てたバクテリオロームを5μmのフィルター膜（バクテリアのバクテリアと核の大きさで確認）にシリンジでろ過する。混合液をフィルター膜に完全に移動するために複数回繰り返します。 注：増幅とシークエンシングのより良い結果を得るには、100以上の細菌を集めます。細菌の損失を減らすために1x PBS溶液を使用して最初にフィルター膜をぬらしてください膜に結合する。 </li></ol> Endosymbiontゲノムの増幅<ol><li> いくつかの改変を加えた推奨プロトコールによるDNA増幅キットを用いて、ろ液（主にコナジラミの内胞子を含む）を増幅する。 <ol><li>血液または細胞からのゲノムDNAの増幅の推奨プロトコールを選択し、その後の実験のために緩衝液D2を調製する。 </li><li> 1.5μLのろ液（セクション4.6を参照）を遠心チューブに直接添加し、続いて1.5μLのBuffer D2を加えよく混合します。 </li><li>氷上で10分間インキュベートし、停止液1.5μLを加える。 </li><li> 15μLの反応バッファーと1μLのDNAポリメラーゼを添加し、穏やかに混合する。 </li><li>混合物を30℃で16時間インキュベートし、続いて65℃で3分間インキュベートする。 </li></ol></li><li> bac用に設計された特異的プライマーを使用することにより、増幅された濾液上で直接PCRを実施する（必要に応じて増幅されたサンプルを希釈する）細菌種を確認するために使用した（2.1節参照）。 </li><li>以前に公表された方法21 （PCRレシピはセクション1.4に記載されている）に従ってwhitefly遺伝子ベータアクチンおよびEF1プライマーを使用することによって宿主ゲノムの汚染があるかどうかを確認するためにPCRを行う。 </li></ol> 6.エンドサイトーシスメタゲノム配列決定<ol><li>配列の前に増幅されたろ液を分光光度計と蛍光光度計（製造元の指示に従う）に供して、試料の品質および試料が配列決定の基準を満たすかどうかを試験する。 </li><li>増幅したものを製造者の指示に従ってゲノムシーケンサーに供する。 </li></ol>

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A. 89 (7), 2699-2702 (1992).</li><li>Nirgianaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+infections+of+the+whitefly+Bemisia+tabaci.">Wolbachia infections of the whitefly Bemisia tabaci.</a> Curr. Microbiol. 47 (2), 93-101 (2003).</li><li>Everett, K. D., Thao, M. L., Horn, M., Dyszynski, G. E., Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chlamydiae+in+whiteflies+and+scale+insects:+endosymbionts+'Candidatus+Fritschea+bemisiae'+strain+Falk+and+'Candidatus+Fritschea+eriococci'+strain+Elm.">Novel chlamydiae in whiteflies and scale insects: endosymbionts 'Candidatus Fritschea bemisiae' strain Falk and 'Candidatus Fritschea eriococci' strain Elm.</a> Int. J. Syst. Evol. Micr. 55, 1581-1587 (2005).</li></ol>

著者は何も開示することはない。

Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

節足動物1では 、相対的な宿主と密接な共生関係を形成する細菌が広がっている1 。内分泌攪乱物質は、栄養代謝、繁殖、環境ストレスへの応答2,3、4などのような宿主の局面にほとんど全ての発育段階5において影響を及ぼすことが実証されている5 。しかし、協会の基盤となる仕組みはまだほとんど知られていません。ゲノミクスは、細菌の潜在的な機能と役割を研究する際に重要かつ重要です。いくつかの基本的な情報、 すなわち 、分類学的状態、機能遺伝子、代謝経路、分泌系は、共生している共生生物の潜在的役割についての光を放出するゲノム配列から推測することができる。ハイスループット配列決定の開発により、膨大な数の細菌ゲノムが配列決定されている様々な機能を明らかに6 。 内寄生虫は、アブラムシ7 、ナンキンムシ8 、オオバコ9 、褐斑病10および蝉11などの半翅類において極めて重要である。例えば、アブラムシのBuchneraは、必須の共生生物として、アブラムシのゲノム12由来の遺伝子とともに、必須のアミノ酸生合成に関与することが示されている。さらに、 ブフネラ（Buchnera）の転写調節もまた明らかにされている13 。 オオバコでは、 Carsonellaが配列決定され、今までに発見された最小の細菌ゲノムにランクされている14 。これらの内在性の特徴はすべて、ゲノム配列に基づいて推測されます。これらの内在性物質はインビトロで培養することができないので、seqのために適切な細菌を単離するためにいくつかのアプローチが適用されている覚醒する。アブラムシでは、遠心分離および濾過により内腔菌を抽出し、さらなるゲノムおよび転写分析に供する5 。褐斑病菌では、昆虫ゲノム10全体とともに内生菌が配列決定される10 。 Whitefly B. tabaciは、35種以上の形態学的に区別不能な種（潜在種）を含む種複合体であり、そのうち2種の侵略種が世界中に侵入し、農業生産に多大な害をもたらした15 。注目すべきことに、 B. tabaci種内の内在性物質は、害虫16の発生において重要であることを示している。今日まで、8種の内在性物質が、 ハリネリチア 、 リケッチア 、 アルセノフォヌス 、 カルディニウム 、em&gt; Wolbachia、 FritschaおよびHemipteriphilusは、17,18を定義した。 以前に記載された半体とは異なり、コナジラミB. tabaciはわずか1mmの非常に小さな昆虫である。ほとんどの内在性物質は、バクテリア細胞19 （B細胞において細菌をさらに形成する共生物質を含む特殊細胞）に限定される。さらに、これらの内在性物質はインビトロで培養することができない。バチルス・タバチ（ B. tabaci）から内寄生虫を得るための唯一の方法は、細菌を解剖することである。しかし、解剖が困難である。第一に、脆弱な細菌は、常に分離するのは難しい白蝶の他の組織とつながります。第二に、白蝶の小さなサイズは、十分な細菌の分離を制限する。第3に、内在性細菌は細菌の中に集まり、単一の細菌種を獲得することを非常に複雑にする。

<table><tbody><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA polymerase</td><td>Takara</td><td>R001A</td><td>including rTaq, 10&amp;times;Buffer and dNTP</td></tr><tr><td>Gel DNA extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DNA sample sequencing system</td><td>ABI</td><td>ABI-3730XL</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>EM UC7</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7650 TEM</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>20 &amp;mu;L microloader</td><td>Eppendorf</td><td>F2771951</td><td></td></tr><tr><td>Filter holder</td><td>Millipore</td><td>SX0001300</td><td></td></tr><tr><td>Filter membrane filter</td><td>Millipore</td><td>SMWP001300</td><td>5.0 &amp;mu;m SMWP</td></tr><tr><td>REPLI-g UltraFast Mini Kit</td><td>Qiagen</td><td>150033</td><td>DNA amlification kit</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Genome Sequencer</td><td>Illumina</td><td>Hiseq 2000</td><td></td></tr></tbody></table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

細菌共生生物は、宿主と親密な関係を形成し、ほとんどの場合宿主に利点を与える。ゲノム情報は、宿主における細菌共生生物の機能および進化を研究するために重要である。ほとんどの共生物質はインビトロで培養できないため、ゲノム配列決定のために適切な量の細菌を単離する方法は非常に重要です。コナジラミBemisia tabaciでは、いくつかの内寄生虫が同定されており、複数のアプローチによる有害生物の発生および生殖において重要であると予測されている。しかし、協会を支える仕組みはほとんど知られていません。この障害は部分的に、白癬菌の内在性物質（ほとんどが細菌細胞に抑制されている）が宿主細胞から分離するのが困難であるという事実に由来する。ここでは、主に解剖学的検査によって白癬B. tabaciからの内在性物質の同定、抽出および精製のための段階的プロトコールを報告するオンにして濾過する。この方法によって調製された内毒素試料は、依然として異なる内毒素種の混合物であるが、その後のゲノム配列決定およびバチルス・タバチにおける内在性の可能な役割の分析に適している。この方法は、他の昆虫から内生菌を単離するためにも使用することができる。

ここでは、 B. tabaci複合体の中東アジアマイナー1（MEAM1）種を例として取り上げた。コナジラミを飼育するためのコットンおよびコナジラミのいくつかの発生段階を、 図1に示す。コットン植物、成虫コナジラミ、およびコナジラミの第1、 第 2 および第 4齢の幼虫（第3齢の幼虫は第 4齢の幼虫と同様に見える）。第...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

ここでは、白斑Bemisia tabaciからの内胚葉を切開および濾過により単離するためのプロトコールを提示する。増幅後、DNAサンプルは、その後のシークエンシングおよび内寄生虫とコナジラミ間の共生の研究に適している。

白蝶貝からの内生菌の抽出方法<em&gt;ベミシア・タバチ</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

methods-for-extraction-endosymbionts-from-whitefly-bemisia

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

9.8K ビュー.  Zhejiang University. ここでは、白斑Bemisia tabaciからの内胚葉を切開および濾過により単離するためのプロトコールを提示する。増幅後、DNAサンプルは、その後のシークエンシングおよび内寄生虫とコナジラミ間の共生の研究に適している。 

ビデオ: Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

フィールドを使用してでコナジラミ、タバココナジラミ、モデル システムとして綿の付着の昆虫のための生命表の開発のための方法論

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

環境学

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

遺伝学

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

細胞遺伝学的手法を用いたタバココナジラミの卵の受精率を決定します。

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

タバココナジラミの迅速同定のループ介在増幅法 (LAMP) アッセイ

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

蛍光<em&gt;その場での</emウイルスと植物及び昆虫組織における共生細菌の局在化のための&gt;ハイブリダイゼーション（FISH）

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

免疫・感染学

Trichogramma dendrolimiによる人工宿主寄生と毒液抽出

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

著者スポットライト:Trichogramma parasitoid Waspsからの効率的な毒抽出法 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

生化学

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

コナジラミを使用した送信植物ウイルス

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

白蝶貝からの内生菌の抽出方法

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フィールドを使用してでコナジラミ、タバココナジラミ、モデルシステムとして綿の付着の昆虫のための生命表の開発のための方法論

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

細胞遺伝学的手法を用いたタバココナジラミの卵の受精率を決定します。

タバココナジラミの迅速同定のループ介在増幅法 (LAMP) アッセイ

蛍光

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

ウイルス感染とキイロショウジョウバエのホストウイルスの相互作用の解析

コナジラミを使用した送信植物ウイルス

白蝶貝からの内生菌の抽出方法

要約

さらに動画を探す

この動画の章

フィールドを使用してでコナジラミ、タバココナジラミ、モデル システムとして綿の付着の昆虫のための生命表の開発のための方法論

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

細胞遺伝学的手法を用いたタバココナジラミの卵の受精率を決定します。

タバココナジラミの迅速同定のループ介在増幅法 (LAMP) アッセイ

蛍光

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

人工宿主を用いた寄生バチ Trichogramma dendrolimi からの毒液抽出

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

コナジラミを使用した送信植物ウイルス

フィールドを使用してでコナジラミ、タバココナジラミ、モデルシステムとして綿の付着の昆虫のための生命表の開発のための方法論

ウイルス感染とキイロショウジョウバエのホストウイルスの相互作用の解析