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

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.