Name: harvesting venom toxins from assassin bugs and other heteropteran insects
Uploaded: 2018-04-21T13:00:00.000+00:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

我々 は金融を認めるオーストラリア国立保健・医学研究評議会 (主要研究員、オーストラリアの研究評議会 (助成金 DP130103813 と G.F.K.、EABU に DECRA 交わり DE160101142 に LP140100832) からサポートG.F.K. に APP1044414)、クイーンズランド大学 (A.A.W. に員) など。

このプロトコルは、レスポンシブル ・ ケアと教育・研究における動物の使用(PPL 4.20.11) と同様に、国立健康医学研究議会ののケアのためのオーストラリアのコードとの使用でクイーンズランド大学のポリシーに準拠します。科学的な目的のための動物(8th版 2013)。
注意: すべき envenomated は暗殺者のバグを処理するとき注意してください。守備の毒を吐く種を処理するとき、目を保護するために注意してください。全体で世話をする実験動物を傷つけないように。これには、輪ゴムなどの拘束圧の監視、および、テングが壊れていないことが含まれます。
注: 必要に応じて、0.5-2 の CO2の露出によって動物を anaesthetize 分や毒で収穫前の 4-10 ° C に冷却を目指す 1-3 安全な移送と拘束を容易にします。Anaesthetization は必須ではないが、アジャイルまたは強力な標本の安全制限を容易にする可能性があります。しかし、動物は毒を収穫できるように目がさめている必要があります。プロテアーゼ阻害薬を追加するかどうかを決定するときダウン ストリーム アプリケーションを覚えておいてください。
1. 電気刺激による毒の毒素の収穫
<ol><li>ライブ標本から毒素を収穫するを取得します。</li>
	<li>正極・負極の事前に用意されたプラスチック ピンセットは、どちらかの先端にマウント。電化のピンセットを electrostimulator または電圧の調整が可能な定電圧源に接続します。<ol>
<li>小さな (~ 10 mm) と大 (〜 25 mm) サシガメ、15 と 25 V のピーク電圧をそれぞれ使用します。</li>
		<li>巨大な水のバグなど大きな heteropterans は、最大 40 V を使用します。</li>
	</ol>
</li>
	<li>胸部にゴムバンドを使用して、プラットフォームにそれらのストラップがライブのバグを抑制します。</li>
	<li>適切な収集のヒントに口吻の先端を配置します。サシガメ、P200 ピペット チップを使用します。巨大な水のバグ、P200 先端開口部のサイズを増やすために下肢をカットします。<ol>
<li>優しく清潔なピンセットの閉じたペアでテングを持ち上げ、テングの終わり上コレクション先端の開口を押してください。</li>
		<li>必要に応じて、収集に口吻を配置する前に取り込み 〜 5 μ L の純水がヒントします。これは収穫された毒が薄くなることが毒、チップ内部の残りの損失を低減します。</li>
	</ol>
</li>
	<li>電気刺激を適用します。2.5 M NaCl/50% グリセロールなどの導電性ゲルの刺激電極を浸しなさい。胸部に電極を適用します。Belostomatids、頭の背側後面に 2 つの電極を適用します。</li>
	<li>Autodegradation を防ぐために毒を格納します。毒を押し出し後、-60 ° C または-20 ° C でチューブやプロテアーゼ阻害剤のカクテルを含むチューブにすばやく転送します。</li>
	<li>1.5 と 1.6 の手順を繰り返して十分な毒を取得または今後さらに毒はありません。</li>
</ol>2. ハラスメントによる毒の毒素の収穫
<ol><li>毒を収穫するため動物を準備し、1.1 と 1.3 1.4 のサブセクションで説明するようコレクション前庭で口吻の先端を配置します。</li>
	<li>毒が自然に押し出されて、2.3 の手順に進みます。ない場合は、優しく触って脚、腹部と触角にピンセットで毒を生成まで動物を苦しめます。</li>
	<li>すぐに必要な場合、管含むプロテアーゼ抑制剤のカクテル、-60 ° C または-20 ° C の管へ毒を転送します。</li>
</ol>3.「吐き」種の毒から嫌がらせで毒の毒素の収穫
<ol><li>Anaesthetize、または、部分的に anaesthetize、任意の時期尚早守備吐きようにエンクロージャから取り外す前に昆虫。</li>
	<li>毒吐く動作を引き起こします。含まれており、標準 90 x 16 mm ペトリ皿の深い蓋を使用して虫の位置を変更します。防ぐため飛行する昆虫の上 1-4 cm のやや後方の蓋を保持します。ほとんどの昆虫はしばしば立て続けに数回を吐き出すでしょう。すべての毒を皿の下側に収集します。</li>
	<li>ペトリ皿の下側に毒を収集するには、10 μ L の純水で洗浄します。すばやく-60 ° C または-20 ° C でチューブやプロテアーゼ阻害剤のカクテルを含む管にそれを転送します。</li>
</ol>4. 腺解剖によって毒の毒素の収穫
<ol><li>動物を犠牲に。大きく anaesthetize またはを使用して動物を殺す > CO2を 5 分露出。動物のハウジング筐体の空気穴に直接パイプを純粋な CO2 。</li>
	<li>ピン昆虫解剖トレイ。暗殺者のバグの腹側表面 (4.3) を解剖します。巨大な水のバグの背側表面 (4.4) を解剖します。</li>
	<li>腹側郭清<ol>
<li>毒液腺に穴をあけることがなく昆虫を押しながら後部の腹部に 3 本のピンを挿入します。</li>
		<li>小型のメスを使用して腹部の腹側表面に短い正中切開をカットします。外骨格のみをカットし、内部構造を損傷しないように注意しながら、頭を前方正中切開を拡張するのにミニチュアはさみを使用します。</li>
		<li>内部構造を公開するには、するには、正中切開から昆虫の側に伸びる複数の水平カットを確認します。内部構造を明らかにする腹側の外骨格の各フラップ、ピンバックします。</li>
		<li>大きなサシガメの中間腹部、前腹部、最初と 2 番目の足の間と第 1 戦の前に 4 つの横切開を作る。</li>
	</ol>
</li>
	<li>背郭清<ol>
<li>基地の近くの翼を削除します。毒液腺に穴をあけることがなく昆虫を押しながら後部の腹部に 3 本のピンを挿入します。</li>
		<li>ミニチュアはさみやメス、外骨格のみをカットし、内部構造を損傷しないように注意しながら使用して腹部に頭から正中切開をカットします。</li>
		<li>昆虫の 2 つの半分を離れて強制的に。公開されている内部の空洞を残して昆虫の長さに沿って横方向にいくつかのピンを配置します。</li>
		<li>ピンセットを使用して飛行筋肉を削除します。</li>
	</ol>
</li>
	<li>郭清トレイを洪水します。簡単に可視化バグが内部構造をフロートし、するを許可する浸水までは、PBS を追加します。</li>
	<li>マイクロ剪刀、ピンセットを使用すると、気管や神経組織結合を慎重に取り外します。毒液腺のように細長い、消化管の各側面に沿って延びる透過する構造体です。<ol>
<li>前葉および後葉門部で会った 2 つのダクトとその特徴的な外観、主な腺を識別します。</li>
		<li>必要な場合は、肺門からダクトをトレースして付属腺を識別します。門部から発せられる 2 つのダクトを切断することによって主要な腺を無料します。</li>
	</ol>
</li>
	<li>必要な腺ルーメンを収穫します。30 μ L の PBS または PBS を含有した氷の腺を遠心機に転送プラスのプロテアーゼ抑制剤のカクテル。鋭いピンで腺を槍します。<ol>
<li>10 s および腺ルーメンを空にして (1 分、5,000 × g、4 ° C) 遠心分離機の渦。ピンセットを使用して腺組織を削除します。</li>
	</ol>
</li>
	<li>毒素抽出物を明らかにします。遠心分離機 (5 分、17,000 × g、4 ° C) 任意の固体粒子、上澄みを保持とペレットを破棄を削除します。Autoproteolytic の劣化を防ぐため-60 ° C または-20 ° C で保存します。</li>
</ol>

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サシガメ毒を収穫の最も重要なステップは、研究の目的に応じて適切な方法を選択します。かめむし類の毒を収穫のために提示する 3 つの方法のそれぞれが長所と短所下流のアプリケーションに応じて。
テング (プロトコル 1-3) から毒を追放するバグを誘発する腺組織で毒の汚染を回避します。さらに、これらのメソッドは非殺傷、バグズ ・ ライフのコースで何度も繰?...

かめむし類の毒は、強力生理活性物質1です。たとえば、キスのバグ (クロタマゴバチ)、ベッドのバグ (Cimicidae) などのカメムシの吸血の毒/唾液分泌を容易に止血2を破壊して給餌。これらの毒の毒素は、凝固、血小板凝集と血管収縮、痛みなど複数の経路をターゲットし、経路をかゆみ。他のほとんどのかめむし類種から毒は吸血するのではなく、捕食を容易にするために合わせられます。その毒は、麻痺、死および無脊椎動物3,4に注入する組織の液状化を引き起こします。脊椎動物に注入される毒液抜本的な効果はあります。例えば、脊椎動物にサシガメHolotrichius innesiから毒の注入により痛み、筋麻痺、出血;このバグによってマウス envenomated は呼吸麻痺5のためすぐに死にます。
トランスクリプトームおよびプロテオーム研究は、いくつかのかめむし類の毒のタンパク質組成を明らかにしました。捕食性の種の毒は、プロテアーゼ他酵素とペプチド及びタンパク質の未知構造と機能6,7、8に富んでいます。バグ キス毒は、血液凝固、血小板凝集や血管収縮2,9メンバーに深く影響を与える triabin タンパク質ファミリーが豊富です。ただし、どの毒素の根底にある毒のほとんどの生物活性は知られていません。たとえば、キス バグブラジルサシガメ ジャガイモの毒は、鎮痛剤、ナトリウム チャンネル10を阻害すると報告されているが、解明する責任のコンポーネントが残っています。同様に、どのコンポーネントのサシガメ毒麻痺や痛みの原因は知られていません。特定毒生物活性と構造と、新しい毒の毒素の機能を特徴づける担当の毒素を識別するための前提条件は、毒を得ています。
電気5,6,7,8,11,12,13守備の挑発が毒を heteropterans から入手します。応答4,8、機械的に圧迫、胸郭12,14,15,16毒液腺8,17 を解剖 ,18,19,20,21,22、および23ムスカリン性アセチルコリン受容体のアゴニストのアプリケーション。2 つの独立したルーメン、前方メイン腺 (AMG) と後部主な腺 (PMG) 主な腺から成っているかめむし類の毒腺の形態は複雑に潜在的な長所と短所の任意のメソッドを判断として付属腺 (AG) を関連付けられています。これらの異なる腺コンパートメント獲物の捕獲や防衛、口腔外消化8,17を含むさまざまな生物学的機能特化する可能性があります異なるタンパク質の分泌物を生成します。Peiratine と ectrichodiine の暗殺者バグで AMG は獲物の捕獲および口腔外消化17PMG に関連付けられています。しかし、harpactorine の AMG8防御毒を分泌する仮説があるに対し、 Pristhesancus plagipennis PMG のバグは獲物の捕獲や消化を特化されています。AG は、サシガメ8小さな分泌機能を持つ、巨大な水のバグ23プロテアーゼ ストレージの主要なサイトに記載されています。明らかに、さらに作業は様々 なカメムシ サブグループ間で各腺区画の機能を明らかにする最も毒の毒素の機能を決定する必要があります。本報告ではこの目標に向かって heteropterans から毒の毒素を収穫するためのプロトコルについて述べる。

<table><tbody><tr><td>Electostimulator</td><td>Grass Technologies</td><td>S48 Square Pulse Stimulator</td><td>Electrostimulator allowing pulsed electrostimulation</td></tr><tr><td>Featherlight tweezers</td><td>Australian Entomological Supplies</td><td>E122B</td><td>For handling live venomous insects</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>4693124001</td><td>For preventing autoproteolytic digestion of venom</td></tr><tr><td>Dissection equipment</td><td>Australian Entomological Supplies</td><td>E152Micro</td><td>For fine dissections</td></tr><tr><td>Insect pins</td><td>Australian Entomological Supplies</td><td>E162</td><td>For fine dissections</td></tr></tbody></table>

harvesting venom toxins from assassin bugs and other heteropteran insects

かめむし類昆虫サシガメ (サシガメ科) や巨大な水のバグ (タガメ) などの捕食性と毒の先祖から子孫し、現存する heteropterans の大半は、この栄養戦略を保持します。いくつかの heteropterans は、脊椎動物の血を主食に移行した (キスのバグ、クロタマゴバチ; などとベッドのバグ、Cimicidae) 餌植物 (ほとんどの Pentatomomorpha) に戻っている他の中。ただし、唾液キスのバグが吸血、少し容易にするために使用を除いてかめむし類の毒クモ、サソリやヘビの毒に比べてについて知られています。
かめむし類の毒の毒素の解析への 1 つの障害は、構造と両方は形態学的に複雑な複数の生物学的役割 (防衛、獲物のキャプチャ、および口腔外消化) を実行毒/口唇腺の機能です。この記事ではカメムシ毒を収集するを用いて正常に 3 つの方法をについて説明します。まず、便利な方法は頻繁に注入されるときに致命的な毒を収集する捕食動物、腺組織で汚染がなくなると電気刺激を示します。第二に、動物の穏やかな嫌がらせが毒テングや heteropterans のいくつかのグループで吐く毒から押し出しを生成するのに十分であることを示します。第三に、我々 は毒液腺を取得する麻酔動物の解剖によって毒の毒素を収穫する方法を説明します。この方法は他の方法を補完するものではなく電気刺激と嫌がらせが効果的なイチイから毒素の収穫を可能性があります。これらのプロトコルは、研究者かめむし類の構造機能解析と応用医学と農業の昆虫から毒素を収穫する有効になります。

P. plagipennis harpactorine と reduviine Platymeris ラダマンテュスなどのいくつかのかめむし類種は確実に電気刺激 (表 1) への応答では毒の大量 (5-20 μ L) をもたらします。一般に、ほとんどの peiratine、reduviine、および harpactorine のバグは、このメソッドへの応答で毒を生成します。Stenopodaine バグの間では、電気刺激はOncocephalus sp. Thodelmus s...

Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

ただし、異翅亜目の多くの昆虫 (昆虫綱: 半翅目) は毒、毒組成とその毒の毒素の機能はほとんど知られてないです。このプロトコルでは、さらに特性評価、電気刺激、嫌がらせや腺の解剖を使用してのかめむし類の毒を収穫する方法について説明します。

暗殺者のバグやその他のカメムシの昆虫から毒の毒素を収穫

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

harvesting-venom-toxins-from-assassin-bugs-other-heteropteran

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

13.0K ビュー.  The University of Queensland. ただし、異翅亜目の多くの昆虫 (昆虫綱: 半翅目) は毒、毒組成とその毒の毒素の機能はほとんど知られてないです。このプロトコルでは、さらに特性評価、電気刺激、嫌がらせや腺の解剖を使用してのかめむし類の毒を収穫する方法について説明します。

ビデオ: Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

環境学

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属や微細藻類の生産性に関するその他の無機汚染物質の定量

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

で捕食摂食行動を分析します<em&gt; Pristionchus</em&gt;およびその他の線虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

プラスチックペレットからの有機塩素系農薬の抽出と塑性解析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

大気エアロゾルからの抽出および界面活性剤の特性評価

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

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

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

ブラックウィドウスパイダーシルクを生産腺の顕微解剖

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

生物学

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

暗殺者のバグやその他のカメムシの昆虫から毒の毒素を収穫

要約

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Helminth Collection and Identification from Wildlife

重金属や微細藻類の生産性に関するその他の無機汚染物質の定量

で捕食摂食行動を分析します

プラスチックペレットからの有機塩素系農薬の抽出と塑性解析

大気エアロゾルからの抽出および界面活性剤の特性評価

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

ブラックウィドウスパイダーシルクを生産腺の顕微解剖

プロテオームとトランスクリプトーム解析のための蜘蛛から毒の抽出と毒液腺Microdissections