Name: A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms (Video) | JoVE Journal
Uploaded: 2015-01-11T14:00:00.000+00:00
Duration: 16 min 3 s
Description: Learn a fish-feeding bioassay to assess antipredatory activity of secondary metabolites from marine organisms. This method uses a model predatory fish and nutritionally comparable food matrix.

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5 minutes

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 chop tissue to pieces or slices that can be inserted into a 50 ml centrifuge tube. Note: Fresh tissue can be used in some cases, but it is often better to cut or chop frozen tissue, which is not subject to squeezing when cut.</li>
	<li>Add tissue pieces to 30 ml of a 1:1 mixture of dichloromethane (DCM) and methanol (MeOH) in a graduated centrifuge tube until a final volume of 40 ml is reached. Be sure to conduct all steps involving the transfer of solvent in a fume hood with adequate ventilation.</li>
	<li>Cap the tube and invert it several times, then agitate repeatedly during a 4 hr extraction period. Note: During this period, water combines with the MeOH and the resulting MeOH:water phase separates from the DCM phase. The tissue is alternately exposed to DCM and MeOH:water as an emulsion as the tubes are agitated.</li>
	<li>Transfer the DCM extract to a round-bottom flask and evaporate to dryness on a rotary evaporator using low heat (&#60;40 &#176;C). Using minimal solvent, transfer the dried extract to a 20 ml scintillation vial. Fit the vial with a rotary evaporator adapter and again evaporate to dryness on a rotary evaporator using low heat (&#60;40 &#176;C). 
	Note: The next step requires the use of a homemade compression instrument that may be assembled by screwing the following items in sequential order onto the end of a threaded rod: (1) nut, (2) washer, and (3) acorn nut. The washer must either be perforated or fitted so that it is less than the internal diameter of a 50 ml centrifuge tube.</li>
	<li>Returning to the graduated centrifuge tube that contains tissue and the MeOH:water extract, squeeze the extraction medium out of the tissue through compression. Transfer the MeOH:water extract to the same round-bottom flask and store chilled (&#60;10 &#176;C).</li>
	<li>Add MeOH to the graduated centrifuge tube until the now dehydrated tissue is submerged for a second extraction of 2 to 6 hr duration, then transfer the new MeOH extract to the chilled round-bottom flask containing the MeOH:water extract. If there is any concern that the tissue has not been fully extracted, repeat the 2 to 6 hr MeOH extraction.</li>
	<li>Dry off the MeOH on a rotary evaporator using low heat (&#60;40 &#176;C). Transfer the remaining aqueous extract from the round-bottom flask to the scintillation vial containing the dried nonpolar extract, using a minimal volume of MeOH to rinse the round-bottom flask.</li>
	<li>Evaporate the aqueous extract to dryness using low heat (&#60;40 &#176;C) on a vacuum concentrator. The scintillation vial now contains the total dry crude organic extract of 10 ml of tissue. Evacuate the head space of the vial with N2 gas to prevent oxidation, seal tightly, and store frozen (-20 &#176;C).</li>
</ol>
2) Food Preparation
<ol>
	<li>Prepare freeze-dried squid mantle powder. 
	Note: Squid mantle provides a source of nutrition that is comparable to that of other benthic invertebrates, and will be used as an ingredient in the substeps of 2.2.
	<ol>
		<li>Thaw frozen rings of squid mantle in warm deionized (DI) water, then puree them in a high-speed blender.</li>
		<li>Pour a thin layer of pureed squid mantle onto a shallow cookie sheet and freeze (-20 &#176;C), then break the sheet of frozen squid puree into small pieces to be lyophilized.</li>
		<li>Lyophilize the frozen squid mantle puree following the operating procedures of the freeze-drier.</li>
		<li>Pulverize the lyophilized pieces of squid mantle puree in a high-speed blender to form a powder.</li>
		<li>In a fume hood, pour the powdered squid mantle into a rotary flour sifter and sift to separate large chunks of tissue from the fine powder.</li>
		<li>Transfer the fine powdered squid mantle to a sealable container. Evacuate the container head space with N2 gas to prevent oxidation and store frozen (-20 &#176;C).</li>
	</ol>
	</li>
	<li>Prepare the food mixture. 
	Note: When running multiple consecutive assays, it is practical to prepare ~100 ml of food mixture, however this recipe may be scaled to smaller volumes if necessary.
	<ol>
		<li>Combine a mixture of 3 g alginic acid and 5 g freeze-dried squid mantle powder with 100 ml of DI water in a 150 ml beaker. Stir vigorously with a microspatula for a few minutes until the powder is fully hydrated and the mixture is homogeneous. 
		Note: If desired, food coloring may be added at this step: it is easier to add dye to the food mixture that will generate both treated and control mixtures (masking the natural pigment of the extract in the extract-treated mixture) rather than trying to match the color of the extract-treated mixture by adding dye to the control mixture. A greenish or brownish food color is often desirable to mask any pigments in the crude extract.</li>
		<li>Load exactly 10 ml of food mixture into a graduated syringe. Take care to avoid the inclusion of air bubbles during this process.</li>
		<li>Remove the 20 ml scintillation vial with dry crude organic extract from the freezer. Add a drop or two of MeOH, then stir the extract into a homogeneous mixture with a microspatula.</li>
		<li>Eject the loaded 10 ml syringe of food matrix into the 20 ml scintillation vial and stir with a microspatula to homogenize the extract-treated food mixture. 
		Note: It may help to eject the syringe in smaller increments (i.e., eject 2 ml and homogenize, then repeat until all 10 ml have been homogenized).</li>
	</ol>
	</li>
	<li>Prepare the assay pellets.
	<ol>
		<li>Load a very small volume of the extract mixture (~1 ml) into a syringe, and submerge the syringe tip in a solution of 0.25 M CaCl2. Eject the contents of the syringe to form a long, spaghetti-like strand.</li>
		<li>After a few minutes, remove the hardened strand, chop it into 4 mm long pellets on a glass cutting board with a razor blade, then rinse in seawater.</li>
		<li>Repeat steps 2.3.1 and 2.3.2 without including tissue extract to make control pellets. Be sure to treat control pellets with an equivalent volume of solvent (see addition of MeOH to treated mixture in step 2.2.3) to control for solvent addition. If a negative control is desired to confirm that assay fish can be deterred from feeding, add denatonium benzoate at a concentration of 2 mg ml-1 to the raw food mixture27.</li>
	</ol>
	</li>
</ol>
3) Palatability Bioassays
<ol>
	<li>Perform feeding assays with wild-caught yellow-phase bluehead wrasse, Thalassoma bifasciatum, kept in groups of three in opaque-sided compartments of laboratory aquaria.</li>
	<li>Deliver food pellets from a beaker of seawater using a glass pipette with a rubber bulb. Note: It may take a few days to train fish to receive food in this manner. A conditioning stimulus (e.g. a few taps of the pipette on the aquarium glass) that precedes the delivery of food may be helpful to train the fish to expect the addition of food pellets.</li>
	<li>Scoring pellets. Consider a pellet accepted if readily consumed by the fish. Consider a pellet rejected if not eaten after a minimum of three attempts by one or more fish to take it into their mouth cavity, or if the pellet is approached and ignored after one such attempt.</li>
	<li>Scoring samples. Note: The assay procedure is depicted as a flowchart in Figure 1. Groups of fish that refuse to eat control pellets at any step in the protocol are not considered further. There are two potential outcomes of a single run of the assay: the sample is either accepted or rejected.
	<ol>
		<li>Begin with a control pellet to confirm that the group of fish is cooperative. Offer a treated pellet. If the fish accept the treated pellet, score the sample as accepted. If the fish reject the treated pellet, offer a subsequent control pellet to determine whether the fish have ceased feeding. If the fish accept the subsequent control pellet, score the sample as rejected.</li>
	</ol>
	</li>
	<li>Replication. Repeat the assay procedure with ten independent groups of fish for each extract.</li>
</ol>
4) Evaluating Significance
<ol>
	<li>Evaluate the significance of differences in the consumption of control vs treated pellets with a modified version of Fisher&#8217;s exact test26. Modify the test so that the marginal totals for control and treated pellets are fixed, treating them both as random samples. Note: This provides p = 0.057 when 7 pellets are eaten; hence, any extract is considered deterrent if 6 or fewer pellets are eaten, and palatable if 7 or more pellets are eaten.</li>
	<li>To compare the relative palatability among groups of extracts, calculate a mean number of pellets eaten within each group. Keep the threshold at 6 pellets so a group of replicate extracts are considered deterrent if the mean number of pellets eaten + standard error (SE) &#8804;6. Note: In the representative results, the group assignment is species, so replicate extracts come from distinct individuals and the relative palatability may be compared among species.</li>
</ol>

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<li>Kubanek, J., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+antifeedant+triterpene+glycosides+from+the+Caribbean+sponge+Erylus+Formosus%5BTitle%5D)+AND+%22Natural+Product+Letters%22%5BJournal%5D)">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/pubmed?term=((Does+the+odor+from+sponges+of+the+genus+Ircinia+protect+them+from+fish+predators%5BTitle%5D)+AND+%22Journal+of+Chemical+Ecology%22%5BJournal%5D)">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/pubmed?term=((Defenses+of+Caribbean+sponges+against+invertebrate+predators.+I.+Assays+with+hermit+crabs%5BTitle%5D)+AND+%22Marine+Ecology+Progress+Series%22%5BJournal%5D)">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/pubmed?term=((Defense+of+Caribbean+sponges+against+invertebrate+predators.+II.+Assays+with+sea+stars%5BTitle%5D)+AND+%22Marine+Ecology+Progress+Series%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+anti-predatory+defenses+of+Red+Sea+and+Caribbean+sponges.+I.+Chemical+defense%5BTitle%5D)+AND+%22Marine+Ecology+Progress+Series%22%5BJournal%5D)">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/pubmed?term=((Testing+for+defensive+synergy+in+Caribbean+sponges%3A+Bad+taste+or+glass+spicules%5BTitle%5D)+AND+%22Journal+of+Experimental+Marine+Biology+and+Ecology%22%5BJournal%5D)">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><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

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...

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 ...

The overall goal of this procedure is to assess the anti predatory activity of secondary metabolites from the tissues of marine organisms. Hello, I'm Dr.Joseph Pollock at the University of North Carolina Wilmington, and I'm joined by graduate student Michael Marty, who has been the team leader in putting together this video on performing bioassays to test for chemical defenses in the tissues of marine organisms. We began developing the methods you are about to see in the late 1980s.At that time, natural products chemists were assigning ecological roles to secondary metabolites without any experimental evidence. Meanwhile, ecologists were extrapolating defensive roles from toxicity assays that had no ecological relevance. Here we Present an approach designed to assess the anti predatory activity of the secondary metabolites from tissues of marine organisms.This approach satisfies four important criteria for feeding bioassays. One, we use an appropriate generalist predator. Two, we exhaustively extract organic metabolites of all polarities from the tissues.Three, we put those metabolites into an experimental food at the same volumetric concentration as found in the organism. And four, our experimental design and statistical approach provides a meaningful metric to indicate relative distasteful. In these feeding experiments, we use the blue head thoma by fossum, a model predatory fish appropriate for assays using tissues from Caribbean marine invertebrates because this fish species is common on Caribbean coral reefs and is known to sample a wide assortment of benthic invertebrates.Now Michael will describe the protocol in detail. First, the tissue must be extracted. Add a one-to-one mixture of dichloride, methane and methanol to a graduated centrifuge tube to a final volume of 30 milliliters.Then cut chunks of tissue from your target organism. This can be done with fresh or frozen tissue. We work with sponges, so this film demonstration will feature the orange elephant ear sponge, alus, plet roadies.You may need to cut very small pieces of tissue to get the volume right. Using volumetric displacement, you can extract exactly 10 milliliters of tissue, cap the tube, and invert it, then agitate for four hours during this period, water combines with the methanol and the resulting methanol water phase separates from the di chloro methane phase. The tissue is alternately exposed to each extraction medium as the tubes are agitated.After this extraction period, two distinct phases will be apparent. The polar phase of methanol and water will sit on top of the nonpolar phase of chloro methane using a passer pipette transfer. The di chloro methane extract from the graduated centrifuge tube to a round bottom flask, the graduated centrifuge tube should be stored in the refrigerator.For the next few steps, attach the round bottom flask to a rotary evaporator for all steps on the rotary evaporator. Make sure that the temperature of the water bath remains below 40 degrees Celsius. Following basic operating procedures for the rotary evaporator, dry the di chloro methane in the round bottom flask resuspend the dried non-polar extract using a very small amount of di chloro methane.Some agitation or swirling may be helpful to remove dried material from the sides of the round bottom flask. Then transfer the chloro methane extract from the round bottom flask to a 20 milliliter scintillation vial. Instead of placing the cap on the scintillation vial, attach a rotary evaporator adapter to the scintillation vial and again, evaporate to dryness On a rotary evaporator, The scintillation Vial with the dried non-polar extract can be stored in the refrigerator during the next few steps.Returning to the graduated centrifuge tube that contains tissue and the methanol water extract, we will use a homemade instrument that squeezes the extraction medium out of the tissue through compression, compressed the tissue. Then transfer the methanol water extract to the round bottom flask and store chilled in the refrigerator. Add methanol to the graduated centrifuge tube until the tissue is submerged for a second extraction of two to six hour duration.Once the second methanol extraction is complete, remove the chilled round bottom flask from the refrigerator, compress the contents of the graduated centrifuge tube and transfer this new methanol extract to the original round bottom flask, which contains the methanol water extract from earlier. At this point, if there is any concern that the tissue has not been fully extracted, the two to six hour methanol extraction step may be repeated. Attach the round bottom flask to a rotary evaporator and dry off the methanol as before.Remember to keep the temperature of the water bath below 40 degrees Celsius. The round bottom flask will contain an aqueous extract that must be combined with the dried non-polar extract in the 20 milliliter scintillation vial. Therefore, transfer the contents of the round bottom flask to the 20 milliliter scintillation vial.If necessary, rinse the round bottom flask with a small amount of methanol to collect all of the remaining extract. The scintillation vial now contains the dried non-polar extract and the polar extract. However, the polar extract is suspended in a small volume of liquid.Using a vacuum concentrator on low heat evaporate the aqueous extract to dryness. The scintillation vial now contains a dry, crude organic extract of 10 milliliters of tissue, blow down the vial with dye nitrogen gas and store in the freezer at negative 20 degrees Celsius. To test the palatability of these tissue extracts, they must first be reconstituted in a food matrix that is nutritionally comparable to the target organism.Rings of squid mantle are an appropriate protein source for bioassays of invertebrate tissues and can be readily prepared into a lyophilized powder. For this bioassay to begin place frozen rings of squid mantle in warm deionized water. Once thawed, decant some of the water and pour the rings into a blender.Blend thoroughly on high until the squid mantle becomes a viscous liquid. Add Water if the material is too thick to blend. Pour a thin layer of liquified squid mantle onto a shallow cookie sheet and spread it evenly.Place the cookie sheet in a freezer at negative 20 degrees Celsius. The sheet of frozen squid must be broken into smaller pieces to facilitate the process of lyophilization. Smaller pieces are better because they will dry out faster.In the freeze dryer. Place the broken pieces of frozen squid in the chamber and follow the operating procedures for the freeze dryer. Once lyophilized, the pieces of squid must be pulverized.This may be accomplished with a blender. Place the dried pieces of squid into the blender and run on high until the material appears as a uniform Powder. The next step should be Conducted in a fume hood to reduce inhalation of particulate squid dust.Pour the freeze dried squid powder into a rotary flower sifter. Hold a container below the sifter to catch the powder passing through the sieve. Rotate the crank to sift the freeze dried squid material.Large strands of connective tissue that were not adequately blended in previous steps will be retained in the sifter yielding a finer powder using a funnel if necessary, transfer the freeze dried squid powder to a sealable container, blow down with dye nitrogen gas and store frozen at negative 20 degrees Celsius. Having prepared the freeze dried squid mantle powder, we're now ready to make a batch of the assay food mixture. When running multiple consecutive assays in the same day, it is practical to make a large batch of the food mixture.Here we demonstrate the preparation process for a mixture of 100 milliliters volume, using a clean whey boat mass out three grams of alginic acid and five grams of freeze dried squid mantle powder. Place these dry ingredients into a 150 milliliter beaker before adding water. Gently stir the dry ingredients with a micros spatula to help mix them together.Add 100 milliliters of deionized water and stir vigorously. It may take a few minutes to fully hydrate the powder. Be sure that you have a homogeneous mixture before moving past this step.If you choose to dye the food matrix one consistent color, add the color dye at this step. Load a graduated syringe with exactly 10 milliliters of food mixture. At this step, volumetric precision is paramount, so take extreme care to avoid the inclusion of air bubbles.Remove the 20 milliliter scintillation vial with dry crude organic extract from the freezer and add a drop or two of methanol. Do not add too much methanol. This will prevent the food matrix from setting up.Using a micros spatula and minimal solvent resuspend the extract into a homogeneous mixture. Be sure to scrape the sides of the vial to ensure that the entire extract is resuspended. Eject the syringe with 10 milliliters of food matrix into the 20 milliliter scintillation vial with resuspended homogenized extract.Stir this mixture with the micros spatula until it is homogeneous. Load a very small volume of the extract mixture. One milliliter is plenty into a syringe.Submerge the syringe tip in a solution of 0.25 molar calcium chloride and eject the contents of the syringe to form a long spaghetti like strand. The strand will harden in this solution. After a couple of minutes, remove the hardened strand and lay it out on a glass cutting board.Use a razor blade to chop the strand into four millimeter long pellets. Collect the extract treated pellets and rinse them in sea water. To prepare control pellets.Follow this same procedure without the addition of any tissue extract. The addition of food coloring may be necessary to match the color of control and treated pellets. To prepare a negative control that fish will certainly refuse to eat.Add deton and benzoate powder to the raw food mixture at a concentration of two milligrams per milliliter as described by Miller and Pollock. 2013, Feeding assays are run With wild caught yellow phase blue head RAs thala by fum fish are kept in groups of three in opaque sided compartments of laboratory aquarium. An appropriate utensil for the delivery of food pellets is a glass pipette with a rubber bulb.A pellet is considered accepted if readily consumed by the fish. A pellet is considered rejected, if not eaten after a minimum of three attempts by one or more fish to take it into their mouth cavity. Or if the pellet is approached and ignored after one such attempt.Uncooperative groups of fish that refuse to eat control pellets must not be considered in assays. This flowchart demonstrates the assay procedure in all cases begin with a control pellet to confirm that the group of fish is cooperative. Offer a treated pellet.If the fish accept the treated pellet, this sample is scored as accepted. If the fish reject the treated pellet, a subsequent control must be offered to confirm whether the fish have ceased feeding. If the fish accept the subsequent control pellet, the sample is scored as rejected.Each sample must be run with 10 independent groups of fish. Here is an example run of this feeding assay. First, a control pellet is offered the fish accept the control pellet.Second, a treated pellet is offered. In this case, the fish reject the treated pellet. Therefore, a subsequent control pellet must be offered.The fish accept the subsequent control and the sample may be scored as rejected. Each sample must be run with 10 independent groups of fish. The significance of differences in the consumption of control versus treated pellets should be evaluated with a modified version of fisher's exact test as presented by low and Pollock 2014.The test is modified so that the marginal totals for control and treated pellets are fixed, treating them both as random samples. This provides a P value equal to 0.057 when seven pellets are eaten. Hence, any extract is considered deterrent if six or fewer pellets are eaten and palatable.If seven or more pellets are eaten. When comparing palatability among groups of extracts, the mean number of pellets eaten should be used as will be shown in the representative results. Here we report the results of this bioassay for six species of common Caribbean sponges.These data were initially published by Pollock et al 1995, and demonstrate the power of this approach to elucidate differences in chemical defense strategies among co-occurring taxa. Each category on the Y axis represents extracts from a single species of sponge. Several individuals of each species were sampled, extracted, and tested, denoted by the number in parentheses following the species name.Results are reported as a mean number of food pellets eaten, plus standard error for each species. Almost no pellets were eaten in assays with crude organic extract from alus, pleth, pheon, compressor, and lycine called foris. In contrast, pellets made with extract from Cali spongy of Vais.Geo Osa and Michel lavis were readily consumed in the assay. Fewer than six pellets were eaten for the first three species, so they may be considered significantly deterrent. In contrast, the second three species were not significantly different from the controls and they may be considered palatable.The methods just demonstrated provide a powerful approach to studying anti predatory chemical defenses in tandem with manipulative field experiments and surveys. The results of these bioassays have helped my research group understand the factors that controlled the distribution and abundance of marine invertebrates on Caribbean coral reefs. Importantly, This method satisfies the need for ecological relevance in chemical ecology beyond chemical ecology.The results of these bioassays may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

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Learn a fish-feeding bioassay to assess antipredatory activity of secondary metabolites from marine organisms. This method uses a model predatory fish and nutritionally comparable food matrix.

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms (Video) | JoVE Journal

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

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