Name: soil sampling and isolation of entomopathogenic nematodes (steinernematidae, heterorhabditidae)
Uploaded: 2014-07-11T15:00:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae at JoVE.com

これらのプロトコルの多くの改善に貢献し、サム·ギュキム、キャサリンPlichtaビクトリアミランダ·トンプソン：著者は、在庫ラボの過去のメンバーに感謝したいと思います。我々は、SP株式に（補助金のIOS-0840932およびIOS-0724978）国立科学財団の支援を認める。

1。土壌サンプルの採取<ol><li>各サンプリングサイトは4m 2 - 2の最小面積をカバーすることを検討してください。 </li><li>少なくとも15cmの深さ（ 図4）での土壌サンプルを収集します。 </li><li>この領域内に少なくとも5のランダムサンプルを取る。 </li><li>サンプルあたり3サブサンプルを取る。研究の目的は、サブサンプルを組み合わせて、または別のそれらを保つのどちらかによって異なります。 </li><li>ビニール袋に各サンプルを置きます。サンプル（ 図4）の漏れを回避するために二重の袋詰めを考えてみましょう。 </li><li>防水マーカーでラベルサンプル。各サンプリングサイトから以下の情報が含まれる：（ローカリティ/エリア/サイト名やGPSなどの座標情報）サイト情報を、日付、生息地、関連する植生、温度、標高など 注：必要に応じて、その後の識別のための試料を用いて収集した昆虫や他の無脊椎動物の存在を収集し、および/または記録します。彼らは潜在的な漢方を表すことができアルEPNホスト。 </li><li>水で徹底的に洗浄および/または70％エタノールまたは0.5％の漂白剤溶液でそれらを消毒することにより、サンプル間のきれいな収集ツール。 </li><li>研究室への輸送中に - （15°C 8）クーラー内のサンプルを保管してください。 </li><li>土壌の組成、質感、水分、導電性又は他の所望の土壌パラメータに関する情報を得るために分析用の土壌サンプルの一部を取る。 </li></ol> 。土壌サンプルからの2線虫の分離：昆虫苦しませるテクニック<ol><li>ごみを取り除き（ すなわち 、岩などの木材や樹皮、葉の部分。）腐生の微生物による汚染を避けるために、サンプルを用いて収集した。 </li><li>土を湿らせて、線虫の移動を容易にするために水を加える。 NOTE：ゆっくりと土壌の水分レベルを増加させるためにスプレーボトルを使用する。 </li><li>蓋付きの清潔なプラス​​チック容器に湿った土壌の約200〜250ミリリットルを配置します。 </li><li>昆虫の餌を追​​加。各サンプル（ 図5）の土壌表面に5から10昆虫を検討してください。 </li><li>蓋付きの容器をカバーし、上下逆にコンテナを回す。 </li><li> （ - 25°Cの通常22）暗闇の中で、RTでコンテナを維持する。 </li><li> 3日、死んだ虫を削除 - すべての2の容器を確認してください。 注：土壌サンプルをさらに苦しませるために、各コンテナに追加の健全な昆虫を追加します。 </li><li>容器から死んだ虫を取り除く。注：濃い紫色の死体に赤レンガをheterorhabditidsによって寄生されたのに対し、茶色や黄土色に着色した死体は、通常、steine​​rnematidsによって寄生されています。 </li><li>滅菌水に死体を洗浄します。 </li><li> （下記の手順を参照）、線虫の子孫の回収のために変更されたホワイトトラップに死体を置く。 </li></ol>感染した死体から3センチュウ回復：変更されたホワイト罠<ol><li> 50（または60）ミリメートル直径の上に置きます。大きなお皿（100ミリメートル）の内部ペトリ皿。 </li><li> 1罪を設定小さい皿の内側GLE円形の濾紙（ワットマン＃1）。 </li><li>必ず死体を作るより小さな皿のフィルター紙の上に置いて、死体は汚染を避けるために、お互いに手を触れないでください。 </li><li> CAで外側（大きい）ペトリ皿を埋める。滅菌蒸留水20ml。死体を保持している皿に水を追加しないでください。 </li><li>蓋（ 図6）を持つ大規模なシャーレとその内容をカバーしています。 </li><li>それに応じてお料理にラベルを付けます。線虫の名前（種/分離株）、感染日付（日付感染が設定された）とトラップの日付（これはトラップが設定されている日付です）：以下の情報を追加します。 </li><li>感染幼虫期（IJS）の出現が起こるまで、室温でトラップしておいてください。線虫種および/または考慮さ菌株によって25日 - このプロセスは、10の間に取ることができます。 </li><li>トラップの大きなお皿を取り出し、ビーカーに線虫で水を注ぐことによってIJSで収穫水。 </li><li>線虫は、Bの底にデカントすることができますエーカー。このプロセスには数分かかる場合があります。 </li><li>必ず線虫は、ビーカーの底に残っていること、丁寧に水を注ぐ。 </li><li>より多くの水を追加し、線虫をデカントできるようにすることで、線虫を洗浄します。水がきれいになるまで3回 - このステップでは、2を繰り返すことができます。 </li><li>組織培養フラスコ（250ml）に線虫サスペンションを配置します。 3000線虫/ mlに - 1000懸濁液の濃度を保つ。 </li><li> 20℃ - 低温室で、10間のインキュベーター内で線虫懸濁液と店のフラスコ 注：EPNの保存期間が可変であるように、定期的に保存されたフラスコを確認してください。 heterorhabditidsは、より定期的な検診が必要な場合があり、一方、継代培養を必要とせずに、12ヶ月 - 通常steine​​rnematids 6のために保存することができます。 </li></ol>

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利害の対立が宣言されていません。

このデモでは、一般的に、EPNとその共生細菌を研究するために使用されている多くの技術を説明する2つのプレゼンテーションの最初のものです。これらの技術は、実験室でのEPN培養の成功確立のための重要なステップを表し、また、研究のためのモデル生物として、EPNを採用し、他のバイオアッセイのための基礎を形成する。 EPNの様々な土壌調査では、その豊かさと分...

多くの線虫、昆虫に関連付けられ、それらの宿主の相互作用が有益から有害な範囲である。ここに、我々はまた、昆虫病原性線虫（あるいはEPN）として知られている昆虫の病原体である線虫のグループに焦点を当てる。 SteinernematidaeとHeterorhabditidae 11,13：二つの線虫の家族が線虫のこのグループに属している。その病原性効果は、実際には通性嫌気性の腸内細菌との相互作用によって付与される（ ラブダス細菌がheterorhabditids関連付けるsteinernematidsとのPhotorhabdus細菌に関連付ける）2。細菌は土壌中に住んでいる唯一の自由生活線虫ステージによって別の昆虫宿主（またdauerの少年、感染少年やIJとして知られている）、3段目の感染性若年性からベクトル化されています。かつて昆虫の中に、線虫は、大規模な敗血症による昆虫宿主を殺す昆虫の血リンパに細菌を放出する。また、細菌昆虫の組織を分解し、それらが成熟し増殖することができ、線虫 &#39;食料源となる。通常、成体線虫の1つまたは2つの世代の昆虫死体内に生成される。より以前の栄養の関係よりも、やり方を専門とし、彼らが寄生する他の昆虫を待ちます土に虫の死骸から外に移動するので、その腸内にこれらの細胞を育てる中で、いくつかの細菌細胞との最後の大人の世代の再アソシエートの子孫6,11,14（ 図1）。 彼らは、農業害虫3,4,5である昆虫の広い範囲に寄生することができるように1927年に発見されているので、EPNは化学農薬への貴重な選択肢と考えられてきた。しかし、過去20年間、これらの線虫とその共生細菌は、宿主微生物の生物生態や進化の側面を研究するための可鍛性のモデル系として認識されているinteractioNS 14。 このプレゼンテーションの目的は、方法及び最も一般的に土壌のサンプリングとEPNの単離のために使用される技術を示すことである。さまざまなツールが利用可能で、含む土壌試料収集するために使用することができます。土壌の芯抜き、こて、ポストホールディガー、オーガー、サンプリングチューブ、ハンドシャベル、とりわけ（ 図2）。 研究の目的に応じて、2つのサンプリング戦略を考慮することができる：a）層状と、b）ランダムサンプリング（ 図3）。層化サンプリングは、通常、所与の期間にわたって特定の又はdemarked領域における集中的な研究の一部として使用される。一般に、トランセクトはdemarkedされ、土壌サンプルを、一定期間（ 図3A）15上トランセクトに規定の間隔で採取する。広範な領域に着目すると、ランダムサンプリングが一般的である。このサンプリング戦略は、EPNのfの多様性を研究するために使用されているROM広い地域または地域（ 図3B）12。多くの要因は、標高、土壌のテクスチャや生息地の多様な範囲（ 例えば 、耕地、森林、牧草地、公園、海岸、水辺エリアなど）を含めた研究の焦点に応じて考えることができる。 私たちは、もともと寝具、Akhurst 1で説明した土壌サンプルからEPNを回復するための簡単かつ選択的な代替手段を表した昆虫苦しませる手法を示している。これは、線虫（IJステージ）昆虫宿主に引き付けられ、それに寄生されることを前提としている選択的な手順である。この技術はまた、真菌、および細菌8,10のような他の昆虫の病原体の単離のために考慮することができる。 我々はまた、感染した昆虫のホスト7,14から線虫の子孫を検索するために使用される修正されたホワイトトラップ技術を実証する。これが満たさ簡単です劣化昆虫死体からの不要な物を「クリーンな」線虫の子孫を取得する利点を提供していますHOD。 最後に、この記事で説明して示した技術が考案および/または、私たちの研究室だけでなく、様々な同僚や協力者によって記載されているもので行われるものに対応する。

<table><tbody><tr><td>&lt;em&gt;Galleria mellonella&lt;/em&gt;</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>For insect baiting technique</td></tr><tr><td>Filter paper, 55 mm Grade 1 Cellulose</td><td>Whatman/VWR</td><td>28450-048</td><td>Infection chamber and White trap assembly</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td>Infection chamber and White trap assembly</td></tr><tr><td>100 x 15 mm Petri dishes</td><td>VWR</td><td>25384-088</td><td>Infection chamber and White trap assembly</td></tr><tr><td>ZIPloc plastic bags</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Mechanical pipettor P1000, P200 ml</td><td>VWR</td><td>89130-562; 89130-566&amp;nbsp;</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Pippetor tips 1000ml, 200 ml</td><td>VWR</td><td>16466-004 ; 53510</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Bleach</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>Sodium hypochlorite</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>70% Ethyl alochol</td><td>AAPER</td><td>17212945</td><td>Soil sampling , disinfecting</td></tr><tr><td>Shovels</td><td>Ace Hardware or local hardware store</td><td>N/A</td><td>Soil sampling</td></tr><tr><td>Tubular soil sampler</td><td>Accuproducts</td><td></td><td>Soil sampling</td></tr><tr><td>Soil probe, long handle</td><td>Nupla&amp;nbsp;</td><td>PRB4T 69401 Classic</td><td>Soil sampling</td></tr><tr><td>Measuring tape</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Flagging tape, various colors</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Tissue culture flasks</td><td>VWR</td><td>Falcon, 29185-304&amp;nbsp;</td><td>White trap, collection of EPN</td></tr><tr><td>Wash bottles, polyethylene, wide mouth</td><td>VWR</td><td>16650-107</td><td>White trap, collection of EPN</td></tr></tbody></table>

soil sampling and isolation of entomopathogenic nematodes (steinernematidae, heterorhabditidae)

昆虫病原性線虫（別名EPN）は昆虫の広い範囲に寄生する土壌に生息する線虫の基を表す。 Steine​​rnematidaeとHeterorhabditidae：これらの線虫は二つの家族に属しています。今まで、70以上の種がSteine​​rnematidaeに記載されており、Heterorhabditidae約20種が存在する。線虫は、腸内細菌と共生のパートナーシップを持っており、彼らは一緒に昆虫種の広い範囲を殺す強力な殺虫複合体として機能します。 ここに、私たちは土壌から、EPNを収集するために考慮される最も一般的な技術に焦点を当てています。このプレゼンテーションの後半では、昆虫苦しませる技術、土壌サンプルからEPNを単離するために広く使用されているアプローチ、および感染した昆虫から、これらの線虫の回復のために使用されている修正されたホワイトトラップ技術に焦点を当てています。これらの方法および技術は、EPNカルトの成功確立のための重要なステップであるURES研究室でも、他の生物学の分野での研究のためのモデル生物として、これらの線虫を考え、他のバイオアッセイのための基礎を形成する。このプレゼンテーションに示す手法が実施され、/またはSPストック研究室のメンバーだけでなく、様々な著者が記載されているものが設計したものに相当する。

土壌サンプルの採取 EPNの3段目の感染少年は、土壌（ 図1）に存在するこれらの線虫のライフサイクルの中で唯一の自由生活のステージです。したがって、土壌サンプルを採取することは、これらの線虫から回復するための非常に効率的な方法である。 図4は、サンプルを採取する位置の深さを示す土壌断面を示している。サンプルの水分を維持?...

Watch this Scientific Journal Video about Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae at JoVE.com

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

昆虫病原性線虫、昆虫の広い範囲に寄生する土壌に生息する回虫です。それぞれ、土壌サンプル、感染昆虫死体から線虫の回復のために、昆虫苦しませると変更されたホワイトトラップ：我々は2つ​​の技術を使用した土壌からこれらの線虫を分離するために使用されるサンプリング方法を示しています。

土壌のサンプリングと昆虫病原線虫の分離（Steine​​rnematidae、Heterorhabditidae）

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

soil-sampling-isolation-entomopathogenic-nematodes-steinernematidae

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

24.9K ビュー.  University of Arizona. 昆虫病原性線虫、昆虫の広い範囲に寄生する土壌に生息する回虫です。それぞれ、土壌サンプル、感染昆虫死体から線虫の回復のために、昆虫苦しませると変更されたホワイトトラップ：我々は2つ​​の技術を使用した土壌からこれらの線虫を分離するために使用されるサンプリング方法を示しています。 

ビデオ: Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

連続の設計および動作<sup&gt; 13</sup&gt; Cと<sup&gt; 15</sup均一または差動、代謝および構造·&gt; N標識化会議所、工場同位体ラベル化

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

環境学

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

統合フィールドLysimetryと間隙水土壌中の化学物質の移動性の評価のためのサンプリングと設立植生

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

開発<em&gt; Metarhizium anisopliae</em&gt; Mycoinsecticideとして：分離から畑作まで

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont&#8211;free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock&#8217;s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

in vivoおよび昆虫病原線虫の体外飼育には（SteinernematidaeとHeterorhabditidae）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

生物工学

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

ベアマン・ファネルによる野生の線虫の迅速な分離

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

遺伝学

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

昆虫病原性線虫の培養と遺伝子操作

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

免疫・感染学

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壌サンプルからの病原性真菌の単離と選択と昆虫害虫に対する真菌性毒性の評価

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

生物学

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

土壌試料からの昆虫病原性菌類の分離方法の比較

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

自己 カエノールハブディティス 線虫の生態学的調査を行うための高度にスケーラブルなアプローチ

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

モデル線虫の天然微生物叢の分離と特性評価

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

土壌のサンプリングと昆虫病原線虫の分離（Steinernematidae、Heterorhabditidae）

要約

さらに動画を探す

この動画の章

連続の設計および動作

統合フィールドLysimetryと間隙水土壌中の化学物質の移動性の評価のためのサンプリングと設立植生

開発

in vivoおよび昆虫病原線虫の体外飼育には（SteinernematidaeとHeterorhabditidae）

ベアマン・ファネルによる野生の線虫の迅速な分離

昆虫病原性線虫の培養と遺伝子操作

土壌サンプルからの病原性真菌の単離と選択と昆虫害虫に対する真菌性毒性の評価

土壌試料からの昆虫病原性菌類の分離方法の比較

自己カエノールハブディティス線虫の生態学的調査を行うための高度にスケーラブルなアプローチ

モデル線虫の天然微生物叢の分離と特性評価

土壌のサンプリングと昆虫病原線虫の分離（Steine​​rnematidae、Heterorhabditidae）

要約

さらに動画を探す

この動画の章

連続の設計および動作

統合フィールドLysimetryと間隙水土壌中の化学物質の移動性の評価のためのサンプリングと設立植生

開発

in vivoおよび昆虫病原線虫の体外飼育には（SteinernematidaeとHeterorhabditidae）

ベアマン・ファネルによる野生の線虫の迅速な分離

昆虫病原性線虫の培養と遺伝子操作

土壌サンプルからの病原性真菌の単離と選択と昆虫害虫に対する真菌性毒性の評価

土壌試料からの昆虫病原性菌類の分離方法の比較

自己 カエノールハブディティス 線虫の生態学的調査を行うための高度にスケーラブルなアプローチ

モデル線虫の天然微生物叢の分離と特性評価

土壌のサンプリングと昆虫病原線虫の分離（Steinernematidae、Heterorhabditidae）

自己カエノールハブディティス線虫の生態学的調査を行うための高度にスケーラブルなアプローチ