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 border="0" cellpadding="0" cellspacing="0">
	<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

9.3K 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

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

水化学と海洋微生物学へのフィールドガイド - 海における幽玄選手の解明

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

環境学

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

水 - パルス振幅変調（WATER-PAM）蛍光測定を用いて細菌/植物プランクトンの共培養を評価するために小音量バイオアッセイ

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

光合成細菌の増殖を探検する鉄（II）リッチ先カンブリア時代マリン湧昇システムの実験室でのシミュレーション

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

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 ...

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 ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

水中ステレオ ビデオのツールを使用して魚の密度を定量化する新方式の開発

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

海洋モデルとしてフジツボタテジマフジツボ improvisus培養と遺伝子発現

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Cohoused 水生動物の長期のビデオ追跡: ノルウェー オマール海老 (アカザエビ norvegicus) の毎日の歩行活動の事例

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

マイクロフォーカスX線CT(マイクロCT)アクチニアエクイナ(クニダリア)、ハーモトホーsp.(アネリダ)、およびゼノトゥルベラジャポニカ(キセナコエロモルファ)のイメージング

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...