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).

OBS: Steg 3 i detta protokoll omfattar ryggradsdjur ämnen. Förfarandet har utformats så att djuren får den mest human behandling möjligt och har godkänts av Institutional Animal Care och användning kommittén (IACUC) vid University of North Carolina Wilmington. 1) Vävnads Extraktion <ol><li> Använd vävnad som är i sitt naturliga tillstånd av hydrering och inte pressas, torkade ut eller alltför våt som detta kommer att förändra volymetriska koncentrationen av sekundära meta...

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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=Evidence+of+a+resource+trade-off+between+growth+and+chemical+defenses+among+Caribbean+coral+reef+sponges.">Evidence of a resource trade-off between growth and chemical defenses among Caribbean coral reef sponges.</a> Marine Ecology Progress Series. 406, 71-78 (2010).</li><li>Leong, 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=Comparison+of+reproductive+patterns+among+7+Caribbean+sponge+species+does+not+reveal+a+resource+trade-off+with+chemical+defenses.">Comparison of reproductive patterns among 7 Caribbean sponge species does not reveal a resource trade-off with chemical defenses.</a> Journal of Experimental Marine Biology and Ecology. 401 (1-2), 80-84 (2011).</li><li>Pawlik, J. R., Loh, T. -. L., McMurray, S. E., Finelli, C. 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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=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. 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Det förfarande som beskrivs häri tillhandahåller en relativt enkel, ekologiskt relevanta laboratorieprotokoll för bedömning antipredatory kemiskt försvar hos marina organismer. Här granskar vi de viktiga kriterier som är uppfyllda av denna uppsättning metoder: (1) Lämplig rovdjur. Detta utfodring analys sysselsätter Thalassoma bifasciatum, Thalassoma bifasciatum, en av de mest förekommande fiskar på korallrev i hela Karibien. Den bluehead är en generalist köt...

Kemisk ekologi utvecklas genom samarbete mellan kemister och ekologer. Medan subdiscipline av marksänd kemisk ekologi har funnits under en längre tid, är det av marin kemisk ekologi bara några decennier gammal men har gett viktiga insikter i evolutionär ekologi och samhällsstruktur av marina organismer 1-8. Med utnyttjande av de framväxande teknologier sportdykning och NMR-spektroskopi, organiska kemister snabbt genererat ett stort antal publikationer som beskriver nya metaboliter från bentiska marina ryggradslösa djur och alger under 1970- och 1980-talen 9. Förutsatt att sekundära metaboliter måste tjäna något syfte, många av dessa publikationer skrivs ekologiskt viktiga egenskaper till nya föreningar utan empiriska bevis. Vid ungefär samma tid var ekologer också dra nytta av tillkomsten av dykning och beskriva de distributioner och bestånd av bottendjur och växter tidigare kända from relativt ineffektiva provtagningsmetoder såsom muddring. Antagandet av dessa forskare var att allt fastsittande och mjuk arbetsföra måste kemiskt varas att undvika konsumtion av rovdjur 10. I ett försök att introducera empiri vad var annars beskrivande arbete med arter abundances började några ekologer extrapolera kemiskt försvar från toxicitetsanalyser 11. De flesta toxicitetsanalyser involverade exponeringen av hel fisk eller andra organismer till vattensuspensioner av råa organiska extrakt av ryggradslösa vävnader, med efterföljande bestämning av de torra koncentrationer av extrakt som är ansvariga för att döda hälften av analys organismer. Men toxicitets analyserna inte efterlikna det sätt på vilket potentiella rovdjur uppfattar offer under naturliga förhållanden, och senare studier har funnit något samband mellan toxicitet och smaklighet 12-13. Det är förvånande att publikationer i ansedda tidskrifter som används tekniker som har liten eller ingen ecological relevans 14-15 och att dessa studier fortfarande allmänt citeras idag. Det är ännu mer alarmerande att notera att studier baserade på toxicitetsdata fortsättningsvis att publiceras 16-18. Bioassay beskrivs häri utvecklades i slutet av 1980-talet för att ge ett ekologiskt relevant strategi för marina kemiska ekologer att bedöma antipredatory kemiskt försvar. Metoden kräver en modell rovdjur att prova en rå organisk utdrag ur målorganismen vid en naturlig koncentration i en näringsmässigt jämförbar livsmedelsmatris, som ger smak uppgifter som är mer ekologiskt meningsfulla än toxicitetsdata. Den allmänna metod för bedömning av antipredatory aktiviteten i vävnaderna av marina organismer omfattar fyra viktiga kriterier: (1) en lämplig generalist rovdjur måste användas i foderanalyser, (2) organiska metaboliter av alla polariteter måste uttömmande heras från vävnaden av rikta organism, (3) de metaboliter skall be blandas i en näringsmässigt lämplig experimentell mat vid samma volymetriska koncentrationen som finns i organismen från vilken de extraherade, och (4) den experimentella designen och statistisk metod måste ge en meningsfull metrisk att indikera relativa distastefulness. Proceduren som beskrivs nedan är speciellt utformad för att utvärdera antipredatory kemiskt försvar i Karibien marina ryggradslösa djur. Vi använder den Thalassoma bifasciatum, Thalassoma bifasciatum, som modell rovfiskar eftersom denna art är vanligt med Caribbean korallrev och är känd för att prova ett brett sortiment av bottendjur 19. Vävnad från målorganismen först utvinns, kombineras sedan med en livsmedelsblandning, och slutligen erbjuds till grupper av T. bifasciatum att observera om de avvisar extraktbehandlade livsmedel. Analys data med hjälp av denna metod har gett viktiga insikter i den defensiva kemi marina organismer 12,20-21, life historia avvägningar 22-24 och samhällsekologi 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.

Här rapporterar vi resultaten av denna bioanalys för sex arter av vanliga karibiska svampar (Figur 2). Dessa data ursprungligen publicerades 1995 av et al. Pawlik 12 och demonstrera kraften i denna strategi för att kartlägga skillnader i kemiska försvarsstrategier bland samarbete före taxa. Resultaten redovisas som en genomsnittlig antal livsmedel pellets ätit + standardfel (SE) för varje art. Nästan inga pellets åts i analyser med råa organiska extrakt från Agelas cl...

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

Denna bioassay använder en modell rovfisk att bedöma förekomsten av utfodring-avskräckande metaboliter från organiska extrakt av vävnader av marina organismer vid naturliga koncentrationer med hjälp av en näringsmässigt jämförbar livsmedelsmatris.

En fisk-matning Laboratorie Bioassay att bedöma Antipredatory aktivitet av sekundära metaboliter från vävnader av marina organismer

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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