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).

NOTA: El paso 3 de este protocolo implica sujetos animales vertebrados. El procedimiento ha sido diseñado para que los animales reciben el tratamiento más humana posible y ha sido aprobado por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de Carolina del Norte en Wilmington. 1) La extracción de tejidos <ol><li> Utilice tejido que se encuentra en su estado natural de hidratación y no exprimido, reseca o demasiado húmedo ya que esto alterará la concentración volumétrica de metabolitos secundarios. Cortar o cortar tejido para piezas o rodajas que se pueden insertar en un tubo de centrífuga de 50 ml. Nota: El tejido fresco se pueden utilizar en algunos casos, pero a menudo es mejor cortar o picar el tejido congelado, que no está sujeto a la compresión cuando se corta. </li><li> Añadir piezas de tejido a 30 ml de una mezcla 1: 1 de diclorometano (DCM) y metanol (MeOH) en un tubo de centrífuga graduado hasta un volumen final de 40 ml se alcanza. Asegúrese de llevar a cabo todas las medidas que impliquen la transferencia dedisolvente en una campana de humos con ventilación adecuada. </li><li> Tapar el tubo e invierta varias veces, luego agitar repetidamente durante un período de extracción de 4 horas. Nota: Durante este período, el agua se combina con el MeOH y el MeOH resultante: fase de agua se separa de la fase de DCM. El tejido se alternativamente expuesto a DCM y MeOH: agua como una emulsión como los tubos se agitan. </li><li> Transferir el extracto de DCM a un matraz de fondo redondo y se evapora a sequedad en un evaporador rotatorio usando calor bajo (&lt;40 ° C). Utilizando disolvente mínima, transferir el extracto seco a un vial de centelleo de 20 ml. Colocar el vial con un adaptador evaporador rotatorio y otra vez evaporar a sequedad en un evaporador rotatorio usando calor bajo (&lt;40 ° C). Nota: El siguiente paso requiere el uso de un instrumento de compresión hecha en casa que puede ser montado atornillando los siguientes elementos en orden secuencial en el extremo de una varilla roscada: (1) tuerca, (2) la arandela, y (3) la tuerca ciega. La arandela o bien debe ser perforada oequipado de modo que sea menor que el diámetro interno de un tubo de centrífuga de 50 ml. </li><li> Volviendo al tubo de centrífuga graduado que contiene el tejido y MeOH: extracto de agua, apriete el medio de extracción de tejido a través de la compresión. Transferir el MeOH: extracto de agua, al mismo matraz de fondo redondo y almacenar refrigerada (&lt;10 ° C). </li><li> Añadir MeOH al tubo de centrífuga graduado hasta que el tejido se sumerge ahora deshidratados para una segunda extracción de 2 a 6 hr duración, a continuación, transferir la nueva MeOH extraer al matraz de fondo redondo refrigerado que contiene el MeOH: extracto de agua. Si hay alguna preocupación de que el tejido no ha sido completamente extraída, repetir la extracción MeOH 2 a 6 hr. </li><li> Seque el MeOH en un evaporador rotatorio usando calor bajo (&lt;40 ° C). Transferir el extracto acuoso restante del matraz de fondo redondo al vial de centelleo que contiene el extracto no polar seco, usando un volumen mínimo de MeOH para enjuagar el matraz de fondo redondo. </li><li> Evaporate el extracto acuoso a sequedad usando calor bajo (&lt;40 ° C) en un concentrador de vacío. El vial de centelleo ahora contiene el extracto orgánico seco total bruta de 10 ml de tejido. Evacuar el espacio superior del vial con gas N2 para evitar la oxidación, sellar herméticamente y almacenar congelado (-20 ° C). </li></ol> 2) Preparación de Alimentos <ol><li> Preparar calamar polvo manto liofilizado. Nota: manto de jibia proporciona una fuente de nutrición que es comparable a la de otros invertebrados bénticos, y será usada como un ingrediente en las subetapas de 2,2. <ol><li> Anillos Descongele del manto de jibia en desionizada caliente (DI), entonces ellos hacen puré en una licuadora de alta velocidad. </li><li> Vierta una fina capa de manto de jibia en puré en una bandeja de horno poco profunda y la congelación (-20 ° C), luego romper la hoja de puré de calamar congelado en trozos pequeños a liofilizar. </li><li> Liofilizar el puré de manto de jibia congelada siguiendo los procedimientos operativos de la freeze de pelo. </li><li> Pulverizar las piezas liofilizadas de puré de manto de jibia en un mezclador de alta velocidad para formar un polvo. </li><li> En una campana de extracción, vierta el manto de jibia en polvo en un tamiz de harina rotativo y tamizar para separar grandes trozos de tejido del polvo fino. </li><li> Transferir el fino manto de jibia en polvo a un recipiente hermético. Evacuar el espacio superior del recipiente con gas N2 para evitar la oxidación y almacenar congelado (-20 ° C). </li></ol></li><li> Preparar la mezcla de alimentos. Nota: cuando se ejecutan múltiples ensayos consecutivos, es práctico para preparar ~ 100 ml de mezcla de comida, sin embargo esta receta puede hacerse a escala en volúmenes más pequeños si es necesario. <ol><li> Combinar una mezcla de 3 g de ácido algínico y 5 g de polvo liofilizado manto de jibia con 100 ml de agua DI en un vaso de precipitados de 150 ml. Se agita vigorosamente con una microespátula durante unos minutos hasta que el polvo está completamente hidratado y la mezcla es homogénea. Nota: Si lo desea, colorante de alimentos se puede añadir en este ptep: es más fácil añadir colorante a la mezcla de la comida que va a generar ambas mezclas tratados y de control (enmascarar el pigmento natural del extracto en la mezcla de extracto de tratados) en lugar de tratar de hacer coincidir el color de la mezcla de extracto de tratados mediante la adición teñir a la mezcla de control. Un color verdoso o pardusco alimentos es a menudo deseable para enmascarar cualquier pigmentos en el extracto crudo. </li><li> Cargar exactamente 10 ml de mezcla de alimentos en una jeringa graduada. Tenga cuidado para evitar la inclusión de burbujas de aire durante este proceso. </li><li> Retire el vial de centelleo de 20 ml con extracto orgánico crudo seco del congelador. Añadir una o dos gotas de MeOH, luego añada el extracto en una mezcla homogénea con un microespátula. </li><li> Expulsar el cargado de la jeringa 10 ml de la matriz de los alimentos en el vial de centelleo de 20 ml y se agita con una microespátula para homogeneizar la mezcla de alimentos tratados con el extracto. Nota: Puede ser útil para expulsar la jeringa en incrementos más pequeños (es decir, expulsar 2 ml y homogeneizar, entonces r.EPEAT hasta que todos los 10 ml se han homogeneizado). </li></ol></li><li> Preparar las pastillas de ensayo. <ol><li> Cargar un volumen muy pequeño de la mezcla de extracto (~ 1 ml) en una jeringa, y sumergir la punta de la jeringa en una solución de 0,25 M de CaCl2. Expulsar el contenido de la jeringa para formar una hebra de espagueti largo. </li><li> Después de unos minutos, retire el hilo endurecido, cortar en 4 mm gránulos largos en una tabla para cortar vidrio con una hoja de afeitar, luego enjuague con agua de mar. </li><li> Repita los pasos 2.3.1 y 2.3.2 sin incluir extracto de tejido para hacer bolitas de control. Asegúrese de tratar bolitas de control con un volumen equivalente de disolvente (ver adición de MeOH a la mezcla tratada en la etapa 2.2.3) para el control de la adición de disolvente. Si se desea un control negativo para confirmar que los peces ensayo puede ser disuadido de alimentación, añadir benzoato de denatonio a una concentración de 2 mg ml -1 a la mezcla de alimentos crudos 27. </li></ol></li></ol> 3) La palatabilidadLos bioensayos <ol><li> Realizar ensayos de alimentación con el pez cabeza azul silvestre amarilla fase, Thalassoma bifasciatum, mantenidos en grupos de tres en compartimentos opacos lados de acuarios de laboratorio. </li><li> Entregar bolitas de comida de un vaso de agua de mar con una pipeta de vidrio con una pera de goma. Nota: Puede tomar algunos días para entrenar a los peces a recibir alimentos de esta manera. Un estímulo acondicionado (por ejemplo, un par de toques de la pipeta sobre el cristal del acuario) que precede a la entrega de los alimentos puede ser útil para entrenar a los peces a esperar la adición de bolitas de comida. </li><li> La puntuación pellets. Considere un pellet aceptada si se consume fácilmente por los peces. Considere un pellet rechazada si no se consumen después de un mínimo de tres intentos de uno o más peces para tomar en su cavidad bucal, o si el sedimento se acercó e hizo caso omiso después de uno de esos intentos. </li><li> Muestras de puntuación. Nota: El procedimiento de ensayo es representado como un diagrama de flujo en la Figura 1 Grupos de peces que se niegan a comer.Bolitas de control en cualquier paso en el protocolo no se consideran aún más. Hay dos posibles resultados de una única prueba de la prueba: la muestra se acepta o se rechaza. <ol><li> Comienza con una bolita de control para confirmar que el grupo de peces es cooperativa. Ofrecer un pellet tratada. Si los peces aceptan la pastilla tratada, la puntuación de la muestra aceptada. Si los peces rechazan el sedimento tratado, ofrecer una pastilla de control posterior para determinar si los peces han dejado de alimentación. Si los peces aceptan la pastilla de control posterior, la puntuación de la muestra ha sido rechazada. </li></ol></li><li> Replicación. Repita el procedimiento de ensayo con diez grupos independientes de pescado para cada extracto. </li></ol> 4) Importancia Evaluación <ol><li> Evaluar la importancia de las diferencias en el consumo de control frente gránulos tratados con una versión modificada de la prueba exacta de Fisher 26. Modificar la prueba por lo que los totales marginales para el control y pellets tratados son fijos, Tratándolos ambas muestras aleatorias. Nota: Esto proporciona p = 0,057 cuando se comen 7 pelotillas; por lo tanto, cualquier extracto se considera elemento de disuasión si se consumen 6 o menos bolitas, y agradable al paladar si se comen 7 o más pastillas. </li><li> Para comparar la palatabilidad relativa entre grupos de extractos, calcular una media del número de gránulos comidos dentro de cada grupo. Mantenga el umbral en 6 gránulos así que un grupo de extractos replicados se entienden disuasorio si el número medio de gránulos comido + error estándar (SE) ≤6. NOTA: En los resultados representativos, la asignación de grupo es la especie, por lo que replican extractos provienen de individuos distintos y la palatabilidad relativa se pueden comparar entre las especies. </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.

El procedimiento descrito en este documento proporciona un protocolo relativamente simple, de laboratorio ecológicamente relevante para la evaluación de las defensas químicas antidepredación en los organismos marinos. En este artículo revisamos los criterios importantes que son satisfechas por este conjunto de métodos: (1) predador apropiado. Este ensayo de alimentación emplea el pez cabeza azul, Thalassoma bifasciatum, uno de los peces más abundantes en los arrecif...

Ecología Química desarrolla a través de la colaboración de los químicos y los ecologistas. Mientras que la especialidad de la ecología química terrestre ha estado alrededor por algún tiempo, el de la ecología química marina es sólo unas pocas décadas, pero ha aportado importantes conocimientos sobre la estructura de la ecología y la comunidad evolutiva de los organismos marinos 1-8. Aprovechando las tecnologías emergentes de buceo y la espectroscopia de RMN, los químicos orgánicos generados rápidamente un gran número de publicaciones que describen nuevos metabolitos de invertebrados y algas marinas bentónicas en los años 1970 y 1980 9. Suponiendo que los metabolitos secundarios deben servir algún propósito, muchas de estas publicaciones atribuidas ecológicamente propiedades importantes a nuevos compuestos sin evidencia empírica. Casi al mismo tiempo, los ecologistas también se están aprovechando de la llegada de buceo y la descripción de la distribución y abundancia de animales bentónicos y plantas conocidas previamente froestoy métodos de muestreo relativamente ineficaces como el dragado. La asunción de estos investigadores era que nada sésiles y de cuerpo blando deben ser defendidos químicamente para evitar el consumo por los depredadores 10. En un esfuerzo por introducir el empirismo a lo que era el trabajo de otra manera descriptiva sobre la abundancia de especies, algunos ecologistas comenzaron extrapolando defensas químicas a partir de ensayos de toxicidad 11. La mayoría de los ensayos de toxicidad implicaron la exposición del pescado entero u otros organismos a las suspensiones acuosas de extractos orgánicos crudos de tejidos de invertebrados, con la posterior determinación de las concentraciones en masa seca de los extractos responsables de matar a la mitad de los organismos de ensayo. Sin embargo, los ensayos de toxicidad no emulan la manera en que los depredadores potenciales perciben presa en condiciones naturales, y estudios posteriores no han encontrado relación entre la toxicidad y la palatabilidad 12-13. Es sorprendente que las publicaciones en revistas de prestigio utilizan técnicas que tienen poca o ninguna ecological relevancia 14-15 y que estos estudios son aún ampliamente citados hoy. Es aún más alarmante observar que los estudios basados ​​en datos de toxicidad se siguen publicando 16-18. El método de bioensayo descrito en este documento se elaboró ​​a finales de 1980 para proporcionar un enfoque ecológicamente relevante para los ecologistas químicos marinos para evaluar las defensas químicas antidepredación. El método requiere un modelo depredador para muestrear un extracto orgánico crudo desde el organismo diana a una concentración natural en una matriz alimentaria nutricionalmente comparable, proporcionando datos palatabilidad que son ecológicamente más significativa que los datos de toxicidad. El enfoque general a la evaluación de la actividad antidepredación de los tejidos de organismos marinos incluye cuatro criterios importantes: (1) un depredador generalista apropiado debe ser utilizado en ensayos de alimentación, (2) los metabolitos orgánicos de todas las polaridades deben ser exhaustiva extraído del tejido de la orientar organismo, (3) los metabolitos deben be mezclado en un alimento experimental nutricionalmente adecuada a la misma concentración volumétrica tal como se encuentra en el organismo a partir del cual fueron extraídos, y (4) el diseño experimental y enfoque estadístico deben proporcionar una métrica significativa para indicar mal sabor relativa. El procedimiento descrito a continuación está diseñado específicamente para evaluar las defensas químicas antidepredación en los invertebrados marinos del Caribe. Empleamos el pez cabeza azul, Thalassoma bifasciatum, como un pez depredador modelo porque esta especie es común en los arrecifes de coral del Caribe y se sabe que degustar una amplia variedad de invertebrados bentónicos 19. Tejido del organismo diana se extrae primero, a continuación, se combina con una mezcla de alimentos, y, finalmente, se ofreció a los grupos de T. bifasciatum para observar si rechazan los alimentos tratados con extracto. Los datos de ensayo que utilizan este método han aportado importantes conocimientos sobre la química defensiva de los organismos marinos 12,20-21, lhistoria ife compensaciones 22-24 y ecología de comunidades 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.

Aquí mostramos los resultados de este bioensayo para seis especies de esponjas del Caribe comunes (Figura 2). Estos datos fueron publicados inicialmente en 1995 por Pawlik et al. 12 y demuestran el poder de este enfoque para estudiar las diferencias en las estrategias de defensa química entre concurrentes taxones. Los resultados se presentaron como una media del número de bolitas de comida comido + error estándar (SE) para cada especie. Casi no hay sedimentos se comen en ensayos ...

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

Este bioensayo emplea un pez depredador modelo para evaluar la presencia de metabolitos de alimentación-disuasión a partir de extractos orgánicos de los tejidos de organismos marinos naturales en concentraciones utilizando una matriz de comida comparable nutricionalmente.

Un pez de amamantar Laboratorio Bioensayo para evaluar la actividad antidepredación de metabolitos secundarios de los tejidos de organismos marinos

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

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

Research

JoVE Journal

Environment

9.4K vistas.  University of North Carolina Wilmington.  Este bioensayo emplea un pez depredador modelo para evaluar la presencia de metabolitos de alimentación-disuasión a partir de extractos orgánicos de los tejidos de organismos marinos naturales en concentraciones utilizando una matriz de comida comparable nutricionalmente. 

Video: 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.

Desentrañar los jugadores invisibles en el Océano - Guía de Campo a la química del agua y Microbiología Marina

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

Investigación

Medio ambiente

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 ensayo biológico Volumen pequeños para evaluar bacteriana / fitoplancton Co-cultura Usando AGUA-Pulse-Amplitud Modulada (AGUA-PAM) Fluorometría

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.

Automatizado Gel Tamaño Selección para mejorar la calidad de las bibliotecas de secuenciación de próxima generación Preparado a partir de muestras de agua del medio ambiente

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

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP, un método mejorado para InEx<em&gt; In Situ</em&gt; Medición de las partículas y compuestos disueltos procesada por Alimentadores Active Suspension

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

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.

Simulación de un laboratorio de hierro (II) ricos sistema de afloramiento Precámbrico marina para explorar el crecimiento de bacterias fotosintéticas

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.

Método ecotoxicológico con bacterias marinas<em&gt; Vibrio anguillarum</em&gt; Evaluar la Toxicidad Aguda de los Contaminantes Ambientales

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.

Extracción de plaguicidas organoclorados de pellets de plástico y análisis de tipos de plástico

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

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

Adherencia de bacterias a las superficies de midieron en el laboratorio de la planta

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

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.

Desarrollo de nuevos métodos para cuantificar la densidad de peces utilizando herramientas de estéreo y vídeo subacuáticos

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.

El percebe Balanus improvisus como modelo Marina - cultivo y expresión del gen

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

Un pez de amamantar Laboratorio Bioensayo para evaluar la actividad antidepredación de metabolitos secundarios de los tejidos de organismos marinos

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