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
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).

OPMERKING: Stap 3 van dit protocol gaat gewervelde dier onderwerpen. De procedure is zo ontworpen dat de dieren krijgen de meest humane behandeling mogelijk en is goedgekeurd door de Institutional Animal Care en gebruik Comite (IACUC) aan de Universiteit van North Carolina Wilmington goedgekeurd. 1) Tissue Extraction <ol><li> Gebruik weefsel dat is in zijn natuurlijke staat van hydratatie en niet geperst, uitgedroogde of overdreven nat als dit de volumetrische concentratie van secundaire m...

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De hier beschreven procedure voorziet in een relatief eenvoudige, ecologisch relevante laboratorium protocol voor de beoordeling van antipredatory chemische afweer in mariene organismen. Hier bespreken we de belangrijkste criteria die worden voldaan door deze set van methoden: (1) Passende roofdier. Deze voeden test maakt het bluehead lipvis, Thalassoma bifasciatum, een van de meest voorkomende vissen op de koraalriffen in het hele Caribische gebied. De bluehead is een gene...

Chemische ecologie ontwikkeld door de samenwerking van chemici en ecologen. Terwijl de subdiscipline van terrestrische chemische ecologie is al enige tijd, dat van mariene chemische ecologie is slechts een paar decennia oud, maar heeft belangrijke inzichten opgeleverd in de evolutionaire ecologie en de gemeenschap structuur van mariene organismen 1-8. Te profiteren van de opkomende technologieën van duiken en NMR-spectroscopie, organisch chemici snel geleid tot een groot aantal publicaties beschrijven roman metabolieten van bentische ongewervelde zeedieren en algen in de jaren 1970 en 1980 9. Aannemende dat secundaire metabolieten bepaald doel, veel van deze publicaties toegeschreven ecologisch belangrijke eigenschappen om nieuwe verbindingen zonder empirisch bewijs moet dienen. Rond dezelfde tijd werden ecologen ook te profiteren van de komst van het duiken en het beschrijven van de uitkeringen en abundanties van bodemdieren en planten eerder weer bekendben relatief ineffectief bemonsteringsmethoden zoals baggeren. De aanname van deze onderzoekers was dat alles sessiele en zacht lichaam chemisch moet worden verdedigd om het verbruik door roofdieren 10 te vermijden. In een poging om de empirie te introduceren aan wat anders beschrijvende werk aan soorten op, sommige ecologen begon extrapoleren chemische afweer van toxiciteit assays 11. De meeste toxiciteit testen betrokken de blootstelling van hele vis of andere organismen op waterige suspensies van ruwe organische extracten van ongewervelde weefsels, met daaropvolgend de bepaling van het drooggewicht concentraties van extracten die verantwoordelijk zijn voor het doden van de helft van de test organismen. Echter, toxiciteitstests niet emuleren de wijze waarop potentiële roofdieren waarnemen prooien onder natuurlijke omstandigheden en latere studies hebben geen relatie tussen toxiciteit en smakelijkheid 12-13 gevonden. Het is verrassend dat publicaties in prestigieuze tijdschriften gebruikte technieken die weinig of geen ecological relevantie 14-15 en dat deze studies worden nog steeds veel geciteerd vandaag. Het is zelfs nog alarmerender om op te merken dat de studies op basis van gegevens over de toxiciteit blijven 16-18 worden gepubliceerd. De biologische analysemethode hierin beschreven werd ontwikkeld in de late jaren 1980 tot een ecologisch relevante benadering voor mariene chemische ecologen verstrekken aan antipredatory chemische afweer te beoordelen. De methode vereist een model roofdier om een ​​ruwe organische extract uit het doelorganisme proeven op een natuurlijke concentratie in een qua voedingswaarde vergelijkbaar voedsel matrix, het verstrekken van smakelijkheid gegevens die meer ecologisch zinvolle dan toxiciteitsgegevens. De algemene benadering van de beoordeling van de antipredatory activiteit van de weefsels van mariene organismen omvat vier belangrijke criteria: (1) een passende generalist roofdier moet worden gebruikt voor het voederen assays, (2) organische metabolieten van alle polariteiten moet uitputtend worden gewonnen uit het weefsel van de doelorganisme, (3) de metabolieten moet be gemengd in een voedingsoogpunt geschikte experimentele eten op dezelfde volumetrische concentratie in het organisme waaruit ze werden geëxtraheerd, en (4) het experimenteel ontwerp en statistische benadering moet zorgen voor een betekenisvolle uitdrukking relatieve distastefulness geven. De hieronder beschreven procedure is speciaal ontworpen om antipredatory chemische afweer in Caribisch ongewervelde zeedieren te beoordelen. We maken gebruik van de bluehead lipvis, Thalassoma bifasciatum, als model roofvissen, omdat deze soort komt veel voor op Caribische koraalriffen en staat bekend om een breed assortiment van benthische ongewervelden 19 proeven. Weefsel van het doelorganisme wordt eerst geëxtraheerd, vervolgens met een mengsel van voedsel, en uiteindelijk aangeboden aan groepen T. bifasciatum om te zien of ze verwerpen de-extract behandelde voedingsmiddelen. Assay gegevens met behulp van deze methode hebben belangrijke inzichten opgeleverd in het defensief chemie van mariene organismen 12,20-21, life geschiedenis trade-offs 22-24, en de gemeenschap ecologie 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.

Hier rapporteren we de resultaten van de bioassay zes soorten van gemeenschappelijk Caribbean sponsen (figuur 2). Deze gegevens werden in eerste instantie gepubliceerd in 1995 door Pawlik et al. 12 en tonen de kracht van deze benadering van verschillen in chemische verdediging strategieën tussen co-voorkomende taxa overzien. De resultaten werden gerapporteerd als een gemiddeld aantal van voedsel pellets gegeten + standaardfout (SE) voor elke soort. Bijna geen pellets werden gegeten ...

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

Dit bioassay maakt gebruik van een model roofvissen aan de aanwezigheid van het voeden-afschrikmiddel metabolieten van organische extracten van de weefsels van mariene organismen in de natuurlijke concentraties met behulp van een qua voedingswaarde vergelijkbaar voedsel matrix beoordelen.

Een vis van borstvoeding Laboratory Bioassay de Antipredatory activiteit van secundaire metabolieten te beoordelen vanuit de weefsels van mariene organismen

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

Research

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