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

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

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><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 ...

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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

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

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

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

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

Estimación Laboratorio de red trófica eficiencias de transferencia de los congéneres de PCB a la trucha de lago (<em&gt; Salvelinus namaycush</em&gt;) Desde Su Presa

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

Investigación

Medio ambiente

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

El ensayo de los comportamientos de alimentación depredadores en<em&gt; Pristionchus</em&gt; Y otros nematodos

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

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

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

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

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

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 ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

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

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

La identificación de las feromonas de la lamprea de mar usando fraccionamiento guiado por bioensayo

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

Bioquímica

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

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

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

Protocolo experimental para examinar los perfiles de respuesta conductual en peces larvales: aplicación a la Neuro-estimulantes cafeína

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

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

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

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

Identificación de la actividad hemolítica y fosfolipasa en extractos crudos de anémonas de mar mediante bioensayos sencillos

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

Lethality Bioassay Using Artemia salina L.

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

Lethality Bioassay Using Artemia salina L.

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

Bioensayo de letalidad utilizando Artemia salina L.

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

Química

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

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

Biología

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

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

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

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

Avidez detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

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

Batería de pruebas de comportamiento para evaluar los efectos neurotóxicos en el pez cebra adulto

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

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

Autor destacado: Evaluación de los cambios de comportamiento ambientales e inducidos por drogas en el pez cebra adulto 

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

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

Resumen

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