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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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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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><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.

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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Universidad San Sebastian

Research

JoVE Journal

Environment

9.4K 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.

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

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Biochemistry

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Chemistry

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Neuroscience

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

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

Summary

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Chapters in this video

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Lethality Bioassay Using Artemia salina L.

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System