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).

NOTE: Step 3 of this protocol involves vertebrate animal subjects. The procedure has been designed so that animals receive the most humane treatment possible and has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina Wilmington.
1) Tissue Extraction
<ol>
	<li>Use tissue that is in its natural state of hydration and not squeezed, dried-out or overly wet as this will alter the volumetric concentration of secondary metabolites. Cut or ch...

<ol>
	<li>Paul, V. J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecological roles of marine natural products. , (1992).</li><li>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=Marine+invertebrate+chemical+defenses.">Marine invertebrate chemical defenses.</a> Chemical Reviews. 93 (5), 1911 (1993).</li><li>Hay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+chemical+ecology:+what&#39;s+known+and+what&#39;s+next.">Marine chemical ecology: what&#39;s known and what&#39;s next.</a> Journal of Experimental Marine Biology and Ecology. 44 (5), 476-476 (1996).</li><li>McClintock, J. B., Baker, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Marine Chemical Ecology. , (2001).</li><li>Amsler, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Algal Chemical Ecology. , (2008).</li><li>Hay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+chemical+ecology:+Chemical+signals+and+cues+structure+marine+populations,+communities,+and+ecosystems.">Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems.</a> Annual Review of Marine Science. 1, 193-212 (2009).</li><li>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=The+chemical+ecology+of+sponges+on+Caribbean+reefs:+Natural+products+shape+natural+systems.">The chemical ecology of sponges on Caribbean reefs: Natural products shape natural systems.</a> BioScience. 61 (11), 888 (2011).</li><li>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=Antipredatory+Defensive+Roles+of+Natural+Products+from+Marine+Invertebrates.">Antipredatory Defensive Roles of Natural Products from Marine Invertebrates.</a> Handbook of Marine Natural Products. , 677-710 (2012).</li><li>Pawlik, J. R., Amsler, C. D., Ritson-Williams, R., McClintock, J. B., Baker, B. J., Paul, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+Chemical+Ecology:+A+Science+Born+of+Scuba.+.">Marine Chemical Ecology: A Science Born of Scuba. .</a> Research and Discoveries: The Revolution of Science through Scuba. 39, 53-69 (2013).</li><li>Randall, J. E., Hartman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sponge-feeding+fishes+of+the+West+Indies.">Sponge-feeding fishes of the West Indies.</a> Marine Biology. 1, 216-225 (1968).</li><li>Bakus, G. J., Green, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+in+sponges+and+holothurians+&#8212;+geographic+pattern.">Toxicity in sponges and holothurians &#8212; geographic pattern.</a> Science. 185, 951-953 (1974).</li><li>Pawlik, J. R., Chanas, B., Toonen, R. J., Fenical, W. <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.+1.+Chemical+deterrency.">Defenses of Caribbean sponges against predatory reef fish. 1. Chemical deterrency.</a> Marine Ecology Progress Series. 127, 183-194 (1995).</li><li>Schulte, B. A., Bakus, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predation+deterrence+in+marine+sponges+&#8212;+laboratory+versus+field+studies.">Predation deterrence in marine sponges &#8212; laboratory versus field studies.</a> Bulletin of Marine Science. 50, 205-211 (1992).</li><li>Jackson, J. B. C., Buss, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathy+and+spatial+competition+among+coral+reef+invertebrates.">Allelopathy and spatial competition among coral reef invertebrates.</a> Proceedings of the National Academy of Sciences. 72, 5160-5163 (1975).</li><li>Bakus, G. J. <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+mechanisms+on+the+great+barrier+reef.">Chemical defense mechanisms on the great barrier reef.</a> Australia. Science. 211, 497-499 (1981).</li><li>Gemballa, S., Schermutzki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+haplosclerid+sponges+preferred:+a+field+study+on+the+diet+of+the+dotted+sea+slug+Peltodoris+atromaculata+(doridoidea:+nudibranchia).">Cytotoxic haplosclerid sponges preferred: a field study on the diet of the dotted sea slug Peltodoris atromaculata (doridoidea: nudibranchia).</a> Marine Biology. 144, 1213-1222 (2004).</li><li>Voogd, N. J., Cleary, D. F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+species+traits+to+environmental+variables+in+Indonesian+coral+reef+sponge+assemblages.">Relating species traits to environmental variables in Indonesian coral reef sponge assemblages.</a> Marine and Freshwater Research. 58, 240-249 (2007).</li><li>Mollo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+promoting+marine+invasions:+a+chemolecological+approach.">Factors promoting marine invasions: a chemolecological approach.</a> Proceedings of the National Academy of Sciences. 105, 4582-4586 (2008).</li><li>Randall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+habits+of+reef+fishes+of+the+West+Indies.">Food habits of reef fishes of the West Indies.</a> Studies in Tropical Oceanography. 5, 665-847 (1967).</li><li>O'Neal, 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=A+reappraisal+of+the+chemical+and+physical+defenses+of+Caribbean+gorgonian+corals+against+predatory+fishes.">A reappraisal of the chemical and physical defenses of Caribbean gorgonian corals against predatory fishes.</a> Marine Ecology Progress Series. 240, 117-126 (2002).</li><li>Hines, D. 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=Assessing+the+antipredatory+defensive+strategies+of+Caribbean+non-scleractinian+zoantharians+(Cnidaria):+is+the+sting+the+only+thing.">Assessing the antipredatory defensive strategies of Caribbean non-scleractinian zoantharians (Cnidaria): is the sting the only thing.</a> Marine Biology. 159 (2), 389-398 (2012).</li><li>Walters, K. D., 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=Is+there+a+trade-off+between+wound-healing+and+chemical+defenses+among+Caribbean+reef+sponges.">Is there a trade-off between wound-healing and chemical defenses among Caribbean reef sponges.</a> Integrative and Comparative Biology. 45 (2), 352-358 (2005).</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=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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sponge+Communities+on+Caribbean+Coral+Reefs+Are+Structured+by+Factors+That+Are+Top-Down,+Not+Bottom-Up.">Sponge Communities on Caribbean Coral Reefs Are Structured by Factors That Are Top-Down, Not Bottom-Up.</a> PLoS ONE. 8 (5), e62573 (2013).</li><li>Loh, T. -. L., 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=Chemical+defenses+and+resource+trade-offs+structure+sponge+communities+on+Caribbean+coral+reefs.">Chemical defenses and resource trade-offs structure sponge communities on Caribbean coral reefs.</a> Proceedings of the National Academy of Science. 111, 4151-4156 (2014).</li><li>Miller, A. M., 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=Do+coral+reef+fish+learn+to+avoid+unpalatable+prey+using+visual+cues.">Do coral reef fish learn to avoid unpalatable prey using visual cues.</a> Animal Behaviour. 85, 339-347 (2013).</li><li>Pawlik, J. R., Fenical, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+re-evaluation+of+the+ichthyodeterrent+role+of+prostaglandins+in+the+Caribbean+gorgonian+coral,+Plexaura+homomalla.">A re-evaluation of the ichthyodeterrent role of prostaglandins in the Caribbean gorgonian coral, Plexaura homomalla.</a> Marine Ecology Progress Series. 52, 95-98 (1989).</li><li>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=Defensive+properties+of+secondary+metabolites+from+the+Caribbean+gorgonian+coral+Erythropodium+caribaeorum.">Defensive properties of secondary metabolites from the Caribbean gorgonian coral Erythropodium caribaeorum.</a> Marine Ecology Progress Series. 75, 1-8 (1991).</li><li>Pawlik, J. R., Fenical, W. <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+Pterogorgia+anceps,+a+Caribbean+gorgonian+coral.">Chemical defense of Pterogorgia anceps, a Caribbean gorgonian coral.</a> Marine Ecology Progress Series. 87, 183-188 (1992).</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=Does+the+skeleton+of+a+sponge+provide+a+defense+against+predatory+reef+fish.">Does the skeleton of a sponge provide a defense against predatory reef fish.</a> Oecologia. 107 (2), 225-231 (1996).</li><li>Chanas, B., Pawlik, J. R., Lindel, T., Fenical, W. <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+sponge+Agelas+clathrodes+(Schmidt).">Chemical defense of the Caribbean sponge Agelas clathrodes (Schmidt).</a> Journal of Experimental Marine Biology and Ecology. 208 (1-2), 185-196 (1997).</li><li>Wilson, D. M., Puyana, M., 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=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.

The procedure described herein provides a relatively simple, ecologically relevant laboratory protocol for assessing antipredatory chemical defenses in marine organisms. Here we review the important criteria that are satisfied by this set of methods:
(1) Appropriate predator. This feeding assay employs the bluehead wrasse, Thalassoma bifasciatum, one of the most abundant fishes on coral reefs throughout the Caribbean. The bluehead is a generalist carnivore known to sample a w...

Chemical ecology developed through the collaboration of chemists and ecologists. While the subdiscipline of terrestrial chemical ecology has been around for some time, that of marine chemical ecology is only a few decades old but has provided important insights into the evolutionary ecology and community structure of marine organisms1-8. Taking advantage of the emergent technologies of SCUBA diving and NMR spectroscopy, organic chemists rapidly generated a great number of publications describing novel metabolites from benthic marine invertebrates and algae in the 1970s and 1980s9. Assuming that secondary metabolites must serve some purpose, many of these publications ascribed ecologically important properties to new compounds without empirical evidence. At about the same time, ecologists were also taking advantage of the advent of SCUBA diving and describing the distributions and abundances of benthic animals and plants previously known from relatively ineffective sampling methods such as dredging. The assumption of these researchers was that anything sessile and soft-bodied must be chemically defended to avoid consumption by predators10. In an effort to introduce empiricism to what was otherwise descriptive work on species abundances, some ecologists began extrapolating chemical defenses from toxicity assays11. Most toxicity assays involved the exposure of whole fish or other organisms to aqueous suspensions of crude organic extracts of invertebrate tissues, with subsequent determination of the dry mass concentrations of extracts responsible for killing half the assay organisms. However, toxicity assays do not emulate the manner in which potential predators perceive prey under natural conditions, and subsequent studies have found no relationship between toxicity and palatability12-13. It is surprising that publications in prestigious journals used techniques having little or no ecological relevance14-15 and that these studies are still widely cited today. It is even more alarming to note that studies based on toxicity data continue to be published16-18. The bioassay method described herein was developed in the late 1980s to provide an ecologically relevant approach for marine chemical ecologists to assess antipredatory chemical defenses. The method requires a model predator to sample a crude organic extract from the target organism at a natural concentration in a nutritionally comparable food matrix, providing palatability data that are more ecologically meaningful than toxicity data.
The general approach to assessing the antipredatory activity of the tissues of marine organisms includes four important criteria: (1) an appropriate generalist predator must be used in feeding assays, (2) organic metabolites of all polarities must be exhaustively extracted from the tissue of the target organism, (3) the metabolites must be mixed into a nutritionally appropriate experimental food at the same volumetric concentration as found in the organism from which they were extracted, and (4) the experimental design and statistical approach must provide a meaningful metric to indicate relative distastefulness.
The procedure outlined below is designed specifically to assess antipredatory chemical defenses in Caribbean marine invertebrates. We employ the bluehead wrasse, Thalassoma bifasciatum, as a model predatory fish because this species is common on Caribbean coral reefs and is known to sample a wide assortment of benthic invertebrates19. Tissue from the target organism is first extracted, then combined with a food mixture, and finally offered to groups of T. bifasciatum to observe whether they reject the extract-treated foods. Assay data using this method have provided important insights into the defensive chemistry of marine organisms12,20-21, life history trade-offs22-24, and community ecology25-26.

<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>
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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.

Here we report results of this bioassay for six species of common Caribbean sponges (Figure 2). These data were initially published in 1995 by Pawlik et al.12 and demonstrate the power of this approach to survey differences in chemical defense strategies among co-occurring taxa. Results were reported as a mean number of food pellets eaten + standard error (SE) for each species. Almost no pellets were eaten in assays with crude organic extracts from Agelas clathrodes, Amph...

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A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

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

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9.3K Views.  University of North Carolina Wilmington. This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.