Name: a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms
Uploaded: 2015-01-11T14:00:00.000+00:00
Duration: 16 min 3 s
Description: Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

We thank James Maeda and Aaron Cooke for assistance with the filming and editing of this video. Funding was provided by the National Science Foundation (OCE-0550468, 1029515).

注：このプロトコルのステップ3は、脊椎動物動物被験体を伴う。手順は、動物は、可能な限り最も人道的な扱いを受けるように設計されており、ノースカロライナ州ウィルミントンの大学の施設内動物管理使用委員会（IACUC）によって承認されています。 1）組織の抽出<ol><li>これは二次代謝産物の体積濃度が変更されますように絞った、乾燥したアウトまたは過度に湿った水和のその自然の状態ではないです組織を使用してください。 50mlの遠心管に挿入することができる断片またはスライスに組織を切断またはチョップ。注：新鮮な組織は、いくつかのケースで使用することができるが、それは時カットを圧迫を受けない凍結組織を、切断又は切るほうが良い場合が多い。 </li><li> 40ミリリットルの最終体積に達するまで目盛り遠沈管にジクロロメタンの1：1混合物（DCM）およびメタノール（MeOH）で30 mlに1の組織片を追加する。移転すべてのステップを実施するようにしてください適切な換気と換気フード中の溶剤。 </li><li>チューブに蓋をし、それを数回転倒し、4時間の抽出期間中に繰り返し攪拌する。注：この期間中、水はMeOHで結合し、得られたメタノール：水相をDCM相から分離する。組織を交互DCMおよびMeOHにさらされる：チューブなどのエマルジョン等の水が攪拌される。 </li><li>丸底フラスコにDCM抽出物を移し、低熱を使用して、ロータリーエバポレーターで乾燥するまで蒸発させ（&lt;40℃）。最小限の溶媒を用いて20mlのシンチレーションバイアルに乾燥した抽出物を移す。ロータリーエバポレーターアダプタでバイアルを取り付け、再び弱火を使用して、ロータリーエバポレーターで蒸発乾固する（&lt;40℃）。 注：（1）ナット（2）、ワッシャー、および（3）ドングリナット：次のステップでは、ねじロッドの端部上に順番に以下の項目をねじ込むことによって組み立てることができる自家製圧迫器の使用を必要とする。ワッシャーは、穿孔またはいずれかでなければなりませんそれを50mlの遠心分離管の内径よりも小さくなるように嵌め込ま。 </li><li>圧縮により組織から抽出媒体を絞る、水抽出：組織およびMeOHを含んで目​​盛り遠心管に戻る。 MeOHを転送：同じ丸底フラスコに、水抽出物を、冷却保存（&lt;10℃）。 </li><li>水抽出物：今、脱水組織は2から6時間の期間の第二の抽出のために水没されるまで、新たなMeOHをメタノールを含む冷却丸底フラスコに抽出転送した後、卒業した遠心管にメタノールを追加します。組織が完全に抽出されなかったことをいずれかの懸念がある場合は、2〜6時間のメタノール抽出を繰り返す。 </li><li>低熱を使用して、ロータリーエバポレーターでメタノールを乾燥（&lt;40℃）。丸底フラスコをすすぎ、MeOH中の最小体積を用いて、乾燥した非極性抽出物を含有するシンチレーションバイアルに丸底フラスコから残りの水性抽出物を移す。 </li><li> E真空濃縮器で弱火（&lt;40℃）を使用して乾燥するまで水抽出物をvaporate。シンチレーションバイアルは現在、組織の10ミリリットルの総乾燥粗製の有機抽出物を含有する。密栓し、酸化を防ぐためにN 2ガ ​​スをバイアルのヘッドスペースを排気し、ストアは凍結（-20℃）。 </li></ol> 2）食品の準備<ol><li>凍結乾燥したイカマントル粉末を準備します。 注：イカマントルは、他の底生無脊椎動物のものに匹敵する、および2.2のサブ工程の原料として使用される栄養源を提供する。 <ol><li>温かい脱イオン（DI）水中のイカマントルの融解凍結リングは、その後、高速ブレンダーでそれらをピューレ。 </li><li>浅いクッキーシートと凍結にピューレイカマントルの薄層を注ぐ（-20℃）、次いで凍結乾燥される小さな断片に凍結されたイカピューレのシートを破る。 </li><li> FRの操作方法以下の凍結イカマントルピューレを凍結乾燥eezeドライヤー。 </li><li>粉末を形成するために、高速ブレンダー中でイカマントルピューレの凍結乾燥された部分を粉砕する。 </li><li>ヒュームフードでは、回転小麦粉ふるいに粉末状のイカ外套を注ぐと微粉末から組織の大きな塊を分離するためにふるいにかける。 </li><li>密封可能容器に微粉末イカ外套を転送します。酸化を防止し、凍結保存するためのN 2ガ ​​スで容器のヘッドスペースを排気する（-20℃）。 </li></ol></li><li>食品の混合物を準備します。 注：必要に応じて、複数の連続した​​アッセイを実行するとき、それは〜食品の混合物の100ミリリットルを用意することが現実的である、しかしこのレシピは、より小さなボリュームにスケーリングすることができる。 <ol><li> 3グラムアルギン酸150mlのビーカー中で脱イオン水100mlで5gの凍結乾燥したイカマントル粉末の混合物を組み合わせる。粉末が完全に水和され、混合物が均質になるまで数分間ミクロ激しく攪拌する。 注：必要に応じて、食品着色料本STで添加することができるEP：それはむしろ追加することによって、抽出物処理混合物の色にマッチしようとするよりも、両方の処置および対照の混合物が生成されます食品混合物（抽出物処理混合物中の抽出物の天然色素をマスキング）に染料を追加する方が簡単です対照混合物に染料。緑がかった、または茶色がかった食品の色、粗抽出物中の任意の顔料をマスクすることが望ましい場合が多い。 </li><li>卒業シリンジに食品の混合物の正確に10ミリリットルをロードします。このプロセスの間に気泡の混入を避けるように注意してください。 </li><li>冷凍庫からの乾燥原油有機抽出物を20ミリリットルのシンチレーションバイアルを取り外します。その後ミクロとの均一な混合物にエキスをかき混ぜる、メタノールの低下や2を追加します。 </li><li> 20ミリリットルのシンチレーションバイアルに食品マトリックスのロードを10mlシリンジを取り出し、抽出物処理食品混合物を均質化するためにミクロでかき混ぜる。 注：小さい増分（IEで注射器を取り出すために役立つことがあり、2ミリリットルを取り出し、均質化する場合、R。すべての10ミリリットルまでのEPEAT）が均質化されています。 </li></ol></li><li>アッセイペレットを準備します。 <ol><li>シリンジにエキス混合物（約1 ml）を、非常に小さなボリュームをロードし、0.25 MのCaCl 2溶液にシリンジ先端を水没。長い、スパゲッティのようなストランドを形成するために、シリンジの内容物を取り出します。 </li><li>数分後、その後、かみそりの刃でガラスカッティングボード上に4ミリメートル長いペレットにそれをチョップ、硬化ストランドを削除海水をですすぐ。 </li><li>繰り返しますコントロールペレットを作るために組織抽出物を含まずに2.3.1と2.3.2を繰り返します。溶剤添加のために制御する（ステップ2.2.3で扱わ混合物にメタノールを添加することを参照のこと）、溶媒の等量の制御ペレットを扱うようにしてください。陰性対照は、そのアッセイの魚を確認することが望まれる場合、給電思いとどまることができ、2 mgのmlの濃度で安息香酸デナトニウムを追加-1生食液27。 </li></ol></li></ol> 3）嗜好性バイオアッセイ<ol><li>野生捕獲黄相blueheadベラと摂食アッセイを行う、Thalassomaのbifasciatumは、実験室の水槽の不透明な両面区画に3のグループに保管。 </li><li>ゴム球付きガラスピペットを用いて、海水のビーカーから食物ペレットを配信。注：この方法で食べ物を受け取るために魚を訓練するために数日かかる場合があります。食品の配達の前に条件刺激（水槽のガラスにピペットの例えば数回タップ）は、食物ペレットの追加を期待する魚を訓練するために役立つことがあります。 </li><li>スコアリングペレット。容易に魚が消費する場合は、受け入れられたペレットを考えてみましょう。彼らの口腔にそれを取るために1つ以上の魚による3回の試行の最小の後に食べていない場合は、拒否されたペレットを考えて、またはペレットは、そのような試みの後に接近され、無視されている場合。 </li><li>スコアリングのサンプル。注：アッセイ手順は、 図1のフローチャートとして示されている食べたがらない魚のグループ。プロトコルの任意のステップにおいて、制御ペレットはさらに考慮されない。アッセイの一回の実行の2潜在的な結果があります。サンプルは、承認または拒否されるか。 <ol><li>魚のグループが協力的であることを確認するために、制御ペレットで始まる。治療ペレットを提供します。魚が扱わペレットを受け入れると受け入れとして、サンプルスコア。魚が扱わペレットを拒否した場合、魚が給餌を中止しているかどうかを判断するために次の制御ペレットを提供します。魚はその後の制御ペレットを受け入れると拒否されたとして、サンプルスコア。 </li></ol></li><li>レプリケーション。各抽出物のための魚の10の独立したグループでアッセイ手順を繰り返します。 </li></ol> 4）評価の意義<ol><li>フィッシャーの正確確率検定26の修正バージョンで処理されたペレットVSコントロールの消費量の差の有意性を評価します。制御のための限界​​合計と扱わペレットが固定されているように、テストを変更します、ランダムサンプルとしてそれらの両方を処理する。注：7ペレットを食べているときには、p = 0.057を提供します。 7以上のペレットを食べている場合は、したがって、すべての抽出物は、6個以下のペレットが食べられている場合には抑止力とみなされ、口当たりている。 </li><li>抽出物のグループ間の相対的な嗜好性を比較するために、各グループ内で食べられたペレットの平均数を計算します。ペレットの平均数は+標準誤差（SE）≤6を食べている場合ので、反復抽出物のグループが抑止力と考えられている6ペレットしきい値を保管してください。注意：代表的な結果では、グループ割り当ては、種であるので、複製する抽出物は、異なる個体に由来する相対嗜好性は、種間で比較することができる。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+coral+reef+fish+learn+to+avoid+unpalatable+prey+using+visual+cues.">Do coral reef fish learn to avoid unpalatable prey using visual cues.</a> Animal Behaviour. 85, 339-347 (2013).</li><li>Pawlik, J. R., Fenical, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+re-evaluation+of+the+ichthyodeterrent+role+of+prostaglandins+in+the+Caribbean+gorgonian+coral,+Plexaura+homomalla.">A re-evaluation of the ichthyodeterrent role of prostaglandins in the Caribbean gorgonian coral, Plexaura homomalla.</a> Marine Ecology Progress Series. 52, 95-98 (1989).</li><li>Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+properties+of+secondary+metabolites+from+the+Caribbean+gorgonian+coral+Erythropodium+caribaeorum.">Defensive properties of secondary metabolites from the Caribbean gorgonian coral Erythropodium caribaeorum.</a> Marine Ecology Progress Series. 75, 1-8 (1991).</li><li>Pawlik, J. R., Fenical, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+Pterogorgia+anceps,+a+Caribbean+gorgonian+coral.">Chemical defense of Pterogorgia anceps, a Caribbean gorgonian coral.</a> Marine Ecology Progress Series. 87, 183-188 (1992).</li><li>Chanas, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+skeleton+of+a+sponge+provide+a+defense+against+predatory+reef+fish.">Does the skeleton of a sponge provide a defense against predatory reef fish.</a> Oecologia. 107 (2), 225-231 (1996).</li><li>Chanas, B., Pawlik, J. R., Lindel, T., Fenical, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+the+Caribbean+sponge+Agelas+clathrodes+(Schmidt).">Chemical defense of the Caribbean sponge Agelas clathrodes (Schmidt).</a> Journal of Experimental Marine Biology and Ecology. 208 (1-2), 185-196 (1997).</li><li>Wilson, D. M., Puyana, M., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+the+Caribbean+reef+sponge+Axinella+corrugata+against+predatory+fishes.">Chemical defense of the Caribbean reef sponge Axinella corrugata against predatory fishes.</a> Journal of Chemical Ecology. 25 (12), 2811-2823 (1999).</li><li>Chanas, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+predatory+reef+fish+II.+Spicules,+tissue+toughness,+and+nutritional+quality.">Defenses of Caribbean sponges against predatory reef fish II. Spicules, tissue toughness, and nutritional quality.</a> Marine Ecology Progress Series. 127 (1), 195-211 (1995).</li><li>Albrizio, S., Ciminiello, P., Fattorusso, E., Magno, S., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphitoxin,+a+new+high+molecular+weight+antifeedant+pyridinium+salt+from+the+Caribbean+sponge+Amphimedon+compressa.">Amphitoxin, a new high molecular weight antifeedant pyridinium salt from the Caribbean sponge Amphimedon compressa.</a> Journal of Natural Products. 58 (5), 647-652 (1995).</li><li>Assmann, M., Lichte, E., Pawlik, J. R., K&#246;ck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Marine Ecology Progress Series. 207, 255-262 (2000).</li><li>Kubanek, J., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antifeedant+triterpene+glycosides+from+the+Caribbean+sponge+Erylus+Formosus.">New antifeedant triterpene glycosides from the Caribbean sponge Erylus Formosus.</a> Natural Product Letters. 15 (4), 275-285 (2001).</li><li>Pawlik, J. R., McFall, G., Zea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+odor+from+sponges+of+the+genus+Ircinia+protect+them+from+fish+predators.">Does the odor from sponges of the genus Ircinia protect them from fish predators.</a> Journal of Chemical Ecology. 28 (6), 1103-1115 (2002).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+invertebrate+predators.+I.+Assays+with+hermit+crabs.">Defenses of Caribbean sponges against invertebrate predators. I. Assays with hermit crabs.</a> Marine Ecology Progress Series. 195, 125-132 (2000).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+of+Caribbean+sponges+against+invertebrate+predators.+II.+Assays+with+sea+stars.">Defense of Caribbean sponges against invertebrate predators. II. Assays with sea stars.</a> Marine Ecology Progress Series. 195, 133-144 (2000).</li><li>Burns, E., Ifrach, I., Carmeli, S., Pawlik, J. R., Ilan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-predatory+defenses+of+Red+Sea+and+Caribbean+sponges.+I.+Chemical+defense.">Comparison of anti-predatory defenses of Red Sea and Caribbean sponges. I. Chemical defense.</a> Marine Ecology Progress Series. 252, 105-114 (2003).</li><li>Jones, A. C., Blum, J. E., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+for+defensive+synergy+in+Caribbean+sponges:+Bad+taste+or+glass+spicules.">Testing for defensive synergy in Caribbean sponges: Bad taste or glass spicules.</a> Journal of Experimental Marine Biology and Ecology. 322 (1), 67 (2005).</li></ol>

The authors declare that they have no competing financial interests.

明細書に記載の手順は、海洋生物にantipredatory化学防御を評価するための比較的単純な、生態学的に関連する研究室プロトコルを提供する。ここでは、メソッドのこのセットによって満たされる重要な基準を確認してください。 （1）適切な捕食者を。この送りアッセイはblueheadベラ、Thalassoma bifasciatum、カリブ海全域サンゴ礁上で最も豊富な魚の1を採用して?...

化学生態学は、化学者と生態学者のコラボレーションにより開発された。地上波化学生態学のsubdisciplineがしばらくの周りされているが、海洋化学生態学のはわずか数十年古いですが、海洋生物1-8の進化生態学と社会構造に重要な洞察を提供してきた。スキューバダイビングとNMR分光法の緊急の技術を利用して、有機化学者は急速に1970年代と1980年代9底生海洋無脊椎動物と藻類由来の新規の代謝産物を記述する出版物を多数生成した。二次代謝産物は、経験的証拠なしに新しい化合物に生態学的に重要な特性を帰さこれらの出版物の多くは、いくつかの目的を果たす必要があることを仮定する。約同時に、生態学者は、以前あちこちに知られているスキューバダイビングの出現を利用することと底生動物や植物の分布と存在量を記述したそのような浚渫などの比較的効果サンプリング方法をメートル。これらの研究者の仮定が固着し、ソフトボディの何かが化学的に捕食者10によって消費を避けるために擁護しなければならないということでした。種の存在量にそうでなければ説明的な作業だったものに経験主義を導入するための努力では、いくつかの生態毒性アッセイ11からの化学防御を外挿する開始しました。最も毒性アッセイは半アッセイ生物を死滅させるために責任の抽出物の乾燥質量濃度の後続の決定に、脊椎動物の組織の粗有機抽出物の水性懸濁液に、魚全体、または他の生物の曝露を含んでいた。しかし、毒性アッセイは、潜在的な捕食者は、自然条件の下で獲物知覚している様子をエミュレートしていない、とその後の研究では、毒性や嗜好性12-13の間には何の関係は認められません。それは、権威ある雑誌に出版物はほとんど、あるいは全くecologicaを持つ技術を使用していることは驚くべきことであるLの関連性14-15、これらの研究はまだ広く引用されている今日。これは、毒性データに基づいて、研究は16〜18を発表され続けることに注意することは、さらに憂慮すべきである。明細書に記載のバイオアッセイ法は、antipredatory化学防御を評価するために、海洋化学生態学者のための生態学的に関連したアプローチを提供するために、1980年代後半に開発されました。方法は、より生態学的に意味のある毒性データよりも嗜好性データを提供する、栄養的に同等の食品マトリックス中の天然の濃度で標的生物からの粗有機抽出物をサンプリングするために、モデル捕食者を必要とする。 海洋生物の組織のantipredatory活性を評価する一般的なアプローチは、4つの重要な基準は、（1）適切なジェネラリスト捕食者は、摂食アッセイにおいて使用されなければならない、（2）すべての極性の有機代謝産物は徹底的の組織から抽出されなければならない生物体を標的とする、（3）代謝産物をbしなければならないeは、それらを抽出した生物に見られるのと同じ体積濃度で栄養的に適切な実験食品に混入、及び（4）実験デザインと統計的アプローチは、相対的なdistastefulnessを示すために意味のある測定基準を提供する必要があります。 以下に概説する手順は、カリブ海の海洋無脊椎動物でantipredatory化学防御を評価するために特別に設計されています。この種はカリブ海のサンゴ礁に一般的であり、底生無脊椎動物19の幅広い品揃えをサンプリングすることが知られているので、私たちはモデル捕食魚として、blueheadベラ、Thalassoma bifasciatumを採用。標的生物由来の組織は、まず、抽出された食品の混合物と混合し、最終的にTのグループに提供されbifasciatumは、それらが抽出処理された食品を拒否するかどうかを観察した。この方法を用いたアッセイのデータは、海洋生物12,20-21、Lの防御的な化学に重要な洞察を提供してきたIFE履歴トレードオフ22-24、およびコミュニティ生態学25-26。

<table><tbody><tr><td>Dichloromethane</td><td>Fisher Scientific</td><td>D37-20</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A41220</td><td></td></tr><tr><td>Anhydrous Calcium Chloride</td><td>Fisher Scientific</td><td>C614-500</td><td></td></tr><tr><td>Cryocool Heat Transfer Fluid</td><td>Fisher Scientific</td><td>20-548-146</td><td>For vacuum concentrator</td></tr><tr><td>Alginic Acid Sodium Salt High Viscosity</td><td>MP Biomedicals</td><td>154723</td><td></td></tr><tr><td>Squid mantle rings</td><td>N/A</td><td>N/A</td><td>Can be purchased at grocery store</td></tr><tr><td>Denatonium benzoate</td><td>Aldrich</td><td>D5765</td><td></td></tr><tr><td>50 ml graduated centrifuge tube</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>20 ml scintillation vial</td><td>Fisher Scientific</td><td>03-337-7</td><td></td></tr><tr><td>Disposable Pasteur pipets</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>Rubber bulbs for Pasteur pipets</td><td>Fisher Scientific</td><td>03-448-24</td><td></td></tr><tr><td>Red bulbs for pellet delivery</td><td>Fisher Scientific</td><td>03-448-27</td><td></td></tr><tr><td>250 ml round-bottom flask</td><td>Fisher Scientific</td><td>10-067E</td><td></td></tr><tr><td>Scintillation vial adapter for rotavap</td><td>Fisher Scientific</td><td>K747130-1324</td><td></td></tr><tr><td>Weightboats</td><td>Fisher Scientific</td><td>02-202B</td><td></td></tr><tr><td>Microspatula</td><td>Fisher Scientific</td><td>21-401-10</td><td></td></tr><tr><td>5 ml graduated syringe</td><td>Fisher Scientific</td><td>14-817-53</td><td></td></tr><tr><td>10 ml graduated syringe</td><td>Fisher Scientific</td><td>14-817-54</td><td></td></tr><tr><td>Razor blade</td><td>Fisher Scientific</td><td>S17302</td><td></td></tr></tbody></table>

a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

ここでは、一般的なカリブ海のスポンジの6種（ 図2）については、このバイオアッセイの結果を報告する。これらのデータは、最初Pawlik らによって1995年に出版された。12と共起する分類群の間で化学防衛戦略の違いを調査するために、このアプローチの力を証明している。結果は、それぞれの種のために、標準誤差（SE）+食べ食物ペレットの平均数として?...

Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

このバイオアッセイは、栄養的に同等の食品マトリックスを使用して、天然の濃度での海洋生物の組織の有機抽出物からの給電抑止代謝産物の存在を評価するためのモデル捕食魚を用いる。

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

a-fish-feeding-laboratory-bioassay-to-assess-antipredatory-activity

Research

JoVE Journal

Environment

9.4K ビュー.  University of North Carolina Wilmington. このバイオアッセイは、栄養的に同等の食品マトリックスを使用して、天然の濃度での海洋生物の組織の有機抽出物からの給電抑止代謝産物の存在を評価するためのモデル捕食魚を用いる。 

ビデオ: A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

湖トラウトのPCB同族体の正味栄養の導入効率の実験室推定（<em&gt;イワナnamaycush</em&gt;）獲物から

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

環境学

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 ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

海洋細菌の生態毒性学的方法<em&gt; Vibrio anguillarum</em&gt;環境汚染物質の急性毒性を評価する

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

バイオアッセイ誘導分別を使用して海ヤツメウナギ フェロモンの同定

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

生化学

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

アルテミアサリナLを用いた致死性バイオアッセイ。

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

化学

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

生物学

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

成体ゼブラフィッシュの神経毒性影響を評価するための行動試験バッテリー

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

著者スポットライト:成体ゼブラフィッシュにおける環境および薬物誘発性行動変化の評価 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

要約

さらに動画を探す

この動画の章

湖トラウトのPCB同族体の正味栄養の導入効率の実験室推定（

で捕食摂食行動を分析します

海洋細菌の生態毒性学的方法

バイオアッセイ誘導分別を使用して海ヤツメウナギフェロモンの同定

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

アルテミアサリナLを用いた致死性バイオアッセイ。

甲殻類との長期致死毒性試験アルテミアfranciscana

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

単一水槽内の一連の行動試験を用いた成獣ダニオ・レリオの神経毒性評価

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

要約

さらに動画を探す

この動画の章

湖トラウトのPCB同族体の正味栄養の導入効率の実験室推定（

で捕食摂食行動を分析します

海洋細菌の生態毒性学的方法

バイオアッセイ誘導分別を使用して海ヤツメウナギ フェロモンの同定

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

アルテミアサリナLを用いた致死性バイオアッセイ。

甲殻類との長期致死毒性試験アルテミアfranciscana

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

単一水槽内の一連の行動試験を用いた成獣ダ ニオ・レリオ の神経毒性評価

バイオアッセイ誘導分別を使用して海ヤツメウナギフェロモンの同定

単一水槽内の一連の行動試験を用いた成獣ダニオ・レリオの神経毒性評価