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).

REMARQUE: L&#39;étape 3 de ce protocole implique des sujets sur des animaux vertébrés. La procédure a été conçu afin que les animaux bénéficient d&#39;un traitement le plus humain possible et a été approuvé par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) à l&#39;Université de Caroline du Nord Wilmington. 1) Extraction de tissus <ol><li> Utilisez tissu qui est dans son état naturel d&#39;hydratation et pas pressé, desséché ou trop humide que ceci modifie la concentration volumétrique de métabolites secondaires. Couper ou couper le tissu en morceaux ou en tranches qui peuvent être insérés dans un tube de centrifugeuse de 50 ml. Remarque: tissu frais peut être utilisé dans certains cas, mais il est souvent préférable de couper ou hacher tissu congelé, qui ne est pas soumis à presser quand on les coupe. </li><li> Ajouter les morceaux de tissu de 30 ml d&#39;un mélange 1: 1 de dichlorométhane (DCM) et du methanol (MeOH) dans un tube à centrifuger gradué jusqu&#39;à un volume final de 40 ml est atteinte. Assurez-vous de procéder à toutes les étapes impliquant le transfert desolvant dans une hotte avec une ventilation adéquate. </li><li> Boucher le tube et inverser plusieurs fois, puis agiter à plusieurs reprises pendant une période d&#39;extraction de 4 heures. Remarque: Au cours de cette période, l&#39;eau se combine avec le MeOH et le MeOH en résulte: la phase aqueuse se sépare de la phase DCM. Le tissu est exposé alternativement à DCM et du MeOH: eau, une émulsion telle que les tubes sont agités. </li><li> Transférer l&#39;extrait DCM à un ballon à fond rond et évaporer à sec sur un évaporateur rotatif, à feu doux (&lt;40 ° C). Utilisation minimale de solvant, transférer l&#39;extrait séché à une 20 ml flacon à scintillation. Monter le flacon avec un adaptateur évaporateur rotatif et à nouveau évaporer à sec sur un évaporateur rotatif, à feu doux (&lt;40 ° C). Note: La prochaine étape nécessite l&#39;utilisation d&#39;un instrument de compression maison qui peut être assemblé par vissage les éléments suivants dans l&#39;ordre séquentiel sur l&#39;extrémité d&#39;une tige filetée: (1) écrou, (2) la rondelle, et (3) écrou borgne. La rondelle doit soit être perforé oumonté de sorte qu&#39;il soit inférieur au diamètre interne d&#39;un tube de centrifugation de 50 ml. </li><li> En revenant au tube à centrifuger gradué qui contient le tissu et le MeOH: extrait de l&#39;eau, presser le milieu d&#39;extraction hors du tissu lors de la compression. Transférer le MeOH: extrait de l&#39;eau à la même ballon à fond rond et stocker refroidie (&lt;10 ° C). </li><li> Ajouter MeOH dans le tube à centrifuger gradué jusqu&#39;à ce que le tissu est immergé maintenant déshydraté pour une seconde extraction de durée de 2 à 6 h, puis transférer le nouveau MeOH extrait dans le ballon à fond rond contenant le réfrigérées MeOH: extrait de l&#39;eau. Se il ya une inquiétude que le tissu n&#39;a pas été complètement extraite, répéter l&#39;extraction de MeOH 2-6 h. </li><li> Séchez le MeOH sur un évaporateur rotatif, à feu doux (&lt;40 ° C). Transférer l&#39;extrait aqueux restant dans le ballon à fond rond dans le flacon de scintillation contenant l&#39;extrait séché non polaire, en utilisant un volume minimal de MeOH pour rincer le ballon à fond rond. </li><li> Evaporate l&#39;extrait aqueux à sec utilisant la chaleur faible (&lt;40 ° C) sur un concentrateur sous vide. Le flacon de scintillation contient maintenant l&#39;extrait sec organique brut total de 10 ml de tissu. Évacuer l&#39;espace du flacon avec du gaz N 2 pour éviter l&#39;oxydation de la tête, fermer hermétiquement et congelez (-20 ° C). </li></ol> 2) Préparation des aliments <ol><li> Préparer calmars poudre de manteau lyophilisée. Remarque: manteau de calmar est une source d&#39;alimentation qui est comparable à celle d&#39;autres invertébrés benthiques, et sera utilisé comme ingrédient dans les sous-étapes de 2,2. <ol><li> Anneaux Décongeler du manteau calmars dans déminéralisée chaude (DI) de l&#39;eau, puis les réduire en purée au mélangeur à haute vitesse. </li><li> Versez une fine couche de purée calmars manteau sur une plaque à biscuits peu profonde et le gel (-20 ° C), puis casser la feuille de purée calmars congelés en petits morceaux pour être lyophilisée. </li><li> Lyophiliser le manteau calmars congelés purée en suivant les procédures de fonctionnement de la freeze-cheveux. </li><li> Pulvériser les pièces lyophilisés de calmars manteau purée dans un mélangeur à haute vitesse pour former une poudre. </li><li> Dans une hotte, versez le manteau de calmar en poudre dans une farine tamis rotatif et tamiser pour séparer de grands morceaux de tissu de la poudre fine. </li><li> Transférer le manteau calmars fine poudre dans un récipient hermétique. Évacuer l&#39;espace de tête de récipient avec du gaz N 2 pour éviter l&#39;oxydation et les conserver congelés (-20 ° C). </li></ol></li><li> Préparer le mélange de nourriture. Remarque: Lorsque vous exécutez plusieurs essais consécutifs, il est pratique pour préparer ~ 100 ml de mélange de nourriture, cependant cette recette peut être adapté à de plus petits volumes si nécessaire. <ol><li> Moissonneuse un mélange de 3 g d&#39;acide alginique et 5 g de poudre lyophilisée du manteau calmar avec 100 ml d&#39;eau désionisée dans un bêcher de 150 ml. Remuer vigoureusement avec une microspatule pendant quelques minutes jusqu&#39;à ce que la poudre est entièrement hydraté et le mélange est homogène. Remarque: Si vous le souhaitez, colorant alimentaire peut être ajoutée à ce stPE: il est plus facile d&#39;ajouter un colorant au mélange de la nourriture qui va générer les deux mélanges traités et témoins (masquant le pigment naturel de l&#39;extrait dans le mélange de l&#39;extrait traité) plutôt que d&#39;essayer de faire correspondre la couleur du mélange d&#39;extraction traité par addition de colorant au mélange de commande. Un colorant alimentaire verdâtre ou brunâtre est souvent souhaitable pour masquer des pigments dans l&#39;extrait brut. </li><li> Chargez exactement 10 ml de mélange de la nourriture dans une seringue graduée. Prenez soin d&#39;éviter l&#39;inclusion de bulles d&#39;air lors de ce processus. </li><li> Retirez le flacon à scintillation de 20 ml avec de l&#39;extrait organique brut sec du congélateur. Ajouter une ou deux gouttes de MeOH, puis incorporer l&#39;extrait dans un mélange homogène avec une microspatule. </li><li> Éjecter le chargé seringue de 10 ml de la matrice alimentaire dans le flacon à scintillation de 20 ml et remuez avec une microspatule pour homogénéiser le mélange alimentaire extrait traité. Remarque: Il peut être utile pour éjecter la seringue par incréments plus petits (ce est à dire, éjecter 2 ml et homogénéiser, alors r.épéter jusqu&#39;à ce que tous les 10 ml ont été homogénéisé). </li></ol></li><li> Préparer les granulés de dosage. <ol><li> Chargez un très petit volume du mélange d&#39;extrait (~ 1 ml) dans une seringue, et plonger la pointe de la seringue dans une solution de 0,25 M CaCl 2. Ejecter le contenu de la seringue pour former un long brin de spaghetti-like. </li><li> Après quelques minutes, retirer le brin durci, couper en 4 mm de long pastilles sur une planche à découper en verre avec une lame de rasoir, puis rincer à l&#39;eau de mer. </li><li> Répétez les étapes 2.3.1 et 2.3.2 sans inclure extrait de tissu pour fabriquer des pastilles de contrôle. Assurez-vous de traiter les pastilles de contrôle avec un volume équivalent de solvant (voir addition de MeOH au mélange traité à l&#39;étape 2.2.3) pour contrôler les plus solvant. Si un contrôle négatif désire confirmer que les poissons d&#39;essai peut être dissuadée de l&#39;alimentation, le benzoate de dénatonium ajouter à une concentration de 2 mg ml -1 au mélange de la nourriture crue 27. </li></ol></li></ol> 3) appétenceBioassays <ol><li> Effectuer des essais d&#39;alimentation avec du jaune phase labre bluehead capturés dans la nature, Thalassoma bifasciatum, gardé par groupes de trois dans les compartiments opaque-verso de laboratoire aquariums. </li><li> Délivrer boulettes de nourriture à partir d&#39;un bêcher d&#39;eau de mer en utilisant une pipette en verre avec une poire en caoutchouc. Remarque: Il peut prendre quelques jours pour former poisson pour recevoir de la nourriture de cette manière. Un stimulus conditionné (par exemple à quelques robinets de la pipette sur la vitre de l&#39;aquarium) qui précède la livraison de nourriture peut être utile de former le poisson de se attendre à l&#39;ajout de boulettes de nourriture. </li><li> Marquant pellets. Envisager une pastille accepté si facilement consommé par les poissons. Envisager une pastille rejetée si pas mangé après un minimum de trois tentatives par un ou plusieurs poissons à prendre dans leur cavité buccale, ou si le culot est approché et a ignoré après une telle tentative. </li><li> Échantillons notation. Remarque: La procédure de test est dépeint comme un organigramme de la figure 1 Groupes de poissons qui refusent de manger.pastilles de contrôle à ne importe quelle étape dans le protocole ne sont pas considérées. Il ya deux résultats potentiels d&#39;un seul passage de l&#39;essai: l&#39;échantillon est accepté ou rejeté. <ol><li> Commencez avec une pastille de contrôle pour confirmer que le groupe de poissons est coopérative. Offrez une pastille traitée. Si les poissons acceptent le culot traité, marquer l&#39;échantillon comme acceptée. Si les poissons rejettent le culot traité, offrir une pastille de contrôle a posteriori afin de déterminer si les poissons ont cessé l&#39;alimentation. Si les poissons acceptent la pastille de contrôle a posteriori, marquer l&#39;échantillon comme rejetée. </li></ol></li><li> Replication. Répétez la procédure de test avec dix groupes indépendants de poissons pour chaque extrait. </li></ol> 4) Importance évaluation <ol><li> Évaluer l&#39;importance des différences dans la consommation du contrôle vs granulés traités avec une version modifiée du test exact de Fisher 26. Modifier le test de sorte que les totaux marginaux pour le contrôle et granulés traités sont fixés, Les traiter deux échantillons aléatoires. Remarque: Cette offre p = 0,057 lorsque sept pastilles sont consommés; donc, tout extrait est considéré comme dissuasif si les six ou moins pellets sont consommés, et agréable au goût si 7 ou plusieurs pastilles sont consommés. </li><li> Pour comparer l&#39;appétence rapport entre les groupes d&#39;extraits, calculer un nombre moyen de pellets consommés au sein de chaque groupe. Gardez le seuil à 6 pastilles ainsi un groupe d&#39;extraits répétées sont considérées comme dissuasif si le nombre moyen de pellets mangé + erreur standard (SE) ≤6. NOTE: Dans les résultats représentatifs, l&#39;affectation de groupe est l&#39;espèce, de sorte répliquant extraits proviennent d&#39;individus distincts et la palatabilité rapport peuvent être comparés entre les espèces. </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=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.

La procédure décrite ici fournit un protocole de laboratoire relativement simple, écologiquement pertinent pour évaluer défenses chimiques antiprédatrices dans les organismes marins. Ici nous passons en revue les critères importants qui sont satisfaits par cet ensemble de méthodes: (1) prédateur appropriée. Ce test alimentation utilise le labre bluehead, Thalassoma bifasciatum, l&#39;un des poissons les plus abondantes sur les récifs coralliens dans les Caraïbes...

l&#39;écologie chimique développé grâce à la collaboration de chimistes et écologistes. Alors que le sous-discipline de l&#39;écologie terrestre chimique a été autour depuis un certain temps, celle de l&#39;écologie marine chimique est âgé de seulement quelques décennies, mais a fourni des informations importantes sur la structure des organismes marins 1-8 l&#39;écologie et de la communauté de l&#39;évolution. Profitant des technologies émergentes de plongée sous-marine et la spectroscopie RMN, chimistes organiques générés rapidement un grand nombre de publications décrivant de nouveaux métabolites invertébrés et les algues marines benthiques dans les années 1970 et 1980 9. En supposant que les métabolites secondaires doivent servir à quelque chose, beaucoup de ces publications attribuées propriétés écologiquement importantes à de nouveaux composés, sans preuves empiriques. À peu près au même moment, les écologistes ont également été profitent de l&#39;avènement de la plongée sous-marine et en décrivant les distributions et l&#39;abondance d&#39;animaux et de plantes benthiques déjà connus from méthodes d&#39;échantillonnage relativement inefficaces comme le dragage. L&#39;hypothèse de ces chercheurs était que tout sessiles et corps mou doivent être défendus chimiquement pour éviter la consommation par les prédateurs 10. Dans un effort pour introduire l&#39;empirisme à ce qui était contraire travail descriptif sur l&#39;abondance des espèces, certains écologistes ont commencé à extrapoler défenses chimiques à partir de tests de toxicité 11. La plupart des tests de toxicité en cause l&#39;exposition des poissons entiers ou autres organismes à des suspensions aqueuses d&#39;extraits organiques bruts de tissus d&#39;invertébrés, avec la détermination ultérieure des concentrations d&#39;extraits secs de masse responsables de la mort de la moitié des organismes d&#39;essai. Cependant, des essais de toxicité ne imitent la manière dont les prédateurs potentiels perçoivent proies dans des conditions naturelles, et des études ultérieures ne ont trouvé aucune relation entre la toxicité et la palatabilité 12-13. Il est surprenant que les publications dans des revues prestigieuses ont utilisé des techniques ayant peu ou pas ecological pertinence 14-15 et que ces études sont toujours largement cités aujourd&#39;hui. Il est encore plus alarmant de constater que les études basées sur des données de toxicité continuent d&#39;être publiés 16-18. La méthode d&#39;analyse biologique décrit ici a été développé à la fin des années 1980 pour fournir une approche écologiquement pertinentes pour les écologistes chimiques marines pour évaluer défenses chimiques antiprédatrices. La méthode nécessite un prédateur de modèle pour déguster un extrait organique brut de l&#39;organisme cible à une concentration naturelle dans une matrice alimentaire comparables sur le plan nutritionnel, fournissant des données d&#39;appétence qui sont écologiquement plus significative que les données de toxicité. L&#39;approche générale pour évaluer l&#39;activité antiprédatrices des tissus d&#39;organismes marins comprend quatre critères importants: (1) un prédateur généraliste approprié doit être utilisé dans des essais d&#39;alimentation, (2) métabolites organiques de toutes les polarités doivent être exhaustive extraites du tissu de la cibler organisme, (3) les métabolites doivent be mélangé dans un aliment nutritionnellement expérimental approprié à la même concentration volumétrique que l&#39;on trouve dans l&#39;organisme à partir duquel ils ont été extraits, et (4) la conception expérimentale et approche statistique doivent fournir un paramètre significatif pour indiquer distastefulness relative. La procédure décrite ci-dessous est conçu spécifiquement pour évaluer défenses chimiques antiprédatrices chez les invertébrés marins des Caraïbes. Nous employons le labre bluehead, Thalassoma bifasciatum, comme un poisson prédateur modèle, car cette espèce est commune sur les récifs coralliens des Caraïbes et est connu pour déguster un large assortiment des invertébrés benthiques 19. Le tissu de l&#39;organisme cible est d&#39;abord extrait, puis combiné avec un mélange de nourriture, et enfin offert à des groupes de T. bifasciatum d&#39;observer se ils rejettent les aliments extrait traité. Données test en utilisant cette méthode ont fourni des renseignements importants sur la chimie de défense des organismes marins 12,20-21, ll&#39;histoire de l&#39;IFE compromis 22-24, 25-26 et l&#39;écologie des communautés.

<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.

Nous rapportons ici les résultats de cet essai biologique pour six espèces d&#39;éponges commun des Caraïbes (figure 2). Ces données ont été initialement publiés en 1995 par Pawlik et al. 12 et démontrent la puissance de cette approche pour étudier les différences dans les stratégies de défense chimiques entre taxons concomitants. Les résultats ont été rapportés comme un nombre moyen de pastilles d&#39;aliments consommés + erreur standard (SE) pour chaque espèce. P...

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

Ce bio-essai emploie un poisson prédateur de modèle pour évaluer la présence de métabolites alimentation-dissuasion à partir d&#39;extraits organiques des tissus d&#39;organismes marins à des concentrations naturelles en utilisant une matrice d&#39;aliments comparable nutritionnel.

Un poisson-alimentation Laboratoire d&#39;essais biologiques pour évaluer l&#39;activité antiprédatrices des métabolites secondaires dans les tissus d&#39;organismes marins

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

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9.3K vues.  University of North Carolina Wilmington.  Ce bio-essai emploie un poisson prédateur de modèle pour évaluer la présence de métabolites alimentation-dissuasion à partir d'extraits organiques des tissus d'organismes marins à des concentrations naturelles en utilisant une matrice d'aliments comparable nutritionnel. 

Vidéo: 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.

Démêler les joueurs invisibles dans l&#39;océan - un guide de terrain à la chimie de l&#39;eau et de microbiologie marine

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

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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.

Un essai biologique de petit volume pour évaluer bactérienne / phytoplancton co-culture avec de l&#39;eau-Pulse-modulé en amplitude (EAU-PAM) Fluorimétrie

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.

Sélection de la taille Gel automatisé pour améliorer la qualité des bibliothèques de séquençage de prochaine génération Préparé à partir d&#39;échantillons d&#39;eau de l&#39;environnement

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.

Laboratoire de simulation d&#39;un fer (II) riche système Upwelling précambrien Marine à explorer la croissance des bactéries photosynthétiques

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.

Méthode écotoxicologique avec des bactéries marines<em&gt; Vibrio anguillarum</em&gt; Évaluer la toxicité aiguë des contaminants environnementaux

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.

Extraction de pesticides organochlorés à partir de granulés en plastique et analyse de type plastique

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.

Développement de nouvelles méthodes de quantification de la densité des poissons à l’aide d’outils de stéréo-vidéo sous-marine

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.

balane Balanus improvisus comme modèle Marine - culture et l’Expression des gènes

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.

Suivi à long terme de vidéo d’animaux aquatiques Cohoused : une étude de cas de l’activité locomotrice quotidienne de la langoustine (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 ...

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.

Microfocus X-ray CT (microCT) Imagerie d'Actinia equina (Cnidaria), Harmothoe sp. (Annelida), et 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. ...