Name: the identification of sea lamprey pheromones using bioassay-guided fractionation
Uploaded: 2018-07-17T15:00:00
Description: Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

We thank the U.S. Geological Survey Hammond Bay Biological Station for the use of their research facilities and the staff of U.S. Fish and Wildlife Service and Fisheries and Oceans Canada for providing sea lampreys. This research was supported by grants from the Great Lakes Fishery Commission to Weiming Li and Ke Li.

All methods described here have been approved by the Institutional Animal Care and Use Committee of Michigan State University (AUF# 03/14-054-00 and 02/17-031-00).
1. Collection and Extraction of Sea Lamprey Conditioned Water
<ol>
	<li>Place sexually mature male sea lampreys (15 - 30 animals) in a tank supplied with 250 L of aerated Lake Huron water maintained at 16 - 18&#160;&#176;C.</li>
	<li>Collect the male-conditioned water throughout each night from June to July. 
	Note: Sea lampr...

<ol>
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Fish live in a chemical world full of compounds yet to be identified. Bioassay-guided fractionation has proven essential to identify and characterize bioactive molecules that mediate many chemical interactions, such as those observed in masu salmon31, Asian elephants32, and sea lampreys33,34,35. Bioassay-guided fractionation is an effective approach to accurately trace and pinpoint...

Pheromones are specific chemical signals released by individuals that aid them in locating food sources, detecting predators, and mediating social interactions of conspecifics1. Pheromone communication in insects has been well studied2; however, the chemical identification and biological function of aquatic vertebrate pheromones have not been studied as extensively. Knowledge of the identity and function of the pheromones released can be applied to facilitate the recovery of threatened species3,4 or control pest species5,6. The application of these techniques necessitates the isolation and characterization of the bioactive pheromone components.
Pheromone identification is a branch of natural product chemistry. Progress in pheromone research has been partially limited due to the nature of the pheromone molecules themselves. Pheromones are often unstable and released in small quantities, and only a few sampling techniques exist to detect minute amounts of volatile7,8 or water-soluble compounds9. Approaches to identify pheromones include 1) a targeted screening of known compounds, 2) metabolomics, and 3) bioassay-guided fractionation. A targeted screening of known compounds tests commercially available metabolic by-products of physiological processes hypothesized to function as pheromones. This approach is limiting because researchers can only test known and available compounds. However, it has resulted in the successful identification of sex hormones in goldfish that function as pheromones10,11,12. Metabolomics is a second pheromone identification approach that distinguishes potential small molecule metabolic products within a biological system13. A comparison of the metabolic profiles of two groups (i.e., an active versus an inactive extract) enables the identification of a potential metabolic profile from which the metabolite is purified, the structure is elucidated, and the bioactivity is confirmed14. Additive or synergistic effects of complex formulations of specific mixtures are more likely to be detected with metabolomics because metabolites are considered together rather than as a series of fractions13. Yet, the implementation of metabolomics relies on the availability of synthetic references because the resulting data do not facilitate the elucidation of novel structures.
Bioassay-guided fractionation is an integrated, iterative approach that spans two fields: chemistry and biology. This approach uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. A crude extract is fractionated by a chemical property (i.e., molecular size, polarity, etc.) and tested with electro-olfactogram (EOG) recordings and/or in a bioassay. The bioactive components are screened out by repeating these steps of fractionation and EOGs and/or bioassays. The structures of pure active compounds are elucidated by spectrometric and spectroscopic methods, which provide the molecular weight and structural information to produce a template of the compound to be synthesized. Bioassay-guided fractionation can yield diverse metabolites and potentially novel pheromones with unique chemical skeletons that are unlikely to be predicted from the biosynthetic pathways.
Here, we describe the bioassay-guided fractionation protocol used to isolate and characterize the bioactivity of male sea lamprey sex pheromone compounds. The sea lamprey (Petromyzon marinus) is an ideal vertebrate model to study pheromone communication because these fish rely heavily on the olfactory detection of chemical cues to mediate their anadromous life history comprised of three distinct stages: larvae, juvenile, and adult. Sea lamprey larvae burrow into the sediment of freshwater streams, undergo a drastic metamorphosis, and transform into juveniles that migrate to a lake or ocean where they parasitize large host fish. After detaching from the host fish, the adults migrate back into spawning streams, guided by the migratory pheromones released by stream-resident larvae15,16,17,18,19. Mature males ascend to the spawning grounds, release a multi-component sex pheromone to attract mates, intermittently spawn for approximately a week, and then die15,20. The identification of sea lamprey pheromones is important because a modulation of the pheromone communication system is among the options considered to control the invasive sea lampreys in the Laurentian Great Lakes21.

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Premium standard wall borosilicate capillaries with filament&#160;</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">G150F-4</td>
			<td style="width:436px;">recording and reference electrode (OD 1.5 mm, ID 0.86 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller instrument&#160;</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">PC-10</td>
			<td>pulls electrodes for EOGs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond-tipped glass cutter</td>
			<td style="width:199px;">Generic</td>
			<td style="width:160px;"></td>
			<td>cut tip of electrodes for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillaries</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">1B150-4</td>
			<td>odorant delivery tube for EOG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">ESP-M15N</td>
			<td>recording electrode holder</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:340px;">Reference electrode holder E Series with handle with&#160; Ag/AgCl pellet&#160; for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">E45P-F15NH</td>
			<td>reference electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC1-10</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">2 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC2-5</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Agar</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A1296</td>
			<td>molten agar to fill electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Potassium chloride (KCl)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">P9333</td>
			<td>3M KCl to fill electrodes and electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micropipette microfil</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">MF28G-5</td>
			<td>to fill electrodes and electrode holders&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">L-Arginine</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A5006</td>
			<td>positive control odorant for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Water bath</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds odorants for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-aminobenzoic acid ethyl ester (MS222)</td>
			<td style="width:199px;">Syndel USA</td>
			<td>Tricaine1G</td>
			<td>EOG anesthetic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gallamine triethiodide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">G8134-5G</td>
			<td>EOG paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mL syringe</td>
			<td style="width:199px;">BD Biosciences</td>
			<td>301025</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Subcutaneous needle 26G 5/8</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:160px;">305115</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Roller clamp</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">14043-20</td>
			<td>adjust flow rate of anesthic into lamprey&#39;s mouth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Sodium chloride (NaCl)</td>
			<td style="width:199px;">J.T. Baker</td>
			<td style="width:160px;">3624-05</td>
			<td>for preparation of 0.9% saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">V-shaped plastic stand as specimen stage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds lamprey during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic trough</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds V-shaped plastic stand during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Blades - #11</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10011-00</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Handle - #3</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10003-12</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">11252-00</td>
			<td>for EOG dissection, Dumont #5SF Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Curved ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>11370-42</td>
			<td>for EOG dissection, Moria MC40B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight pring Scissors</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Stereomicroscope</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">Discovery V8</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Illuminator light</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">CL 1500 ECO</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic tubing</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to connect re-circulating EOG setup and water baths</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Odorant delivery tubing&#160;</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">In line filter and gasket set</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">TCFA1201035A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micromanipulators</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">MM-3</td>
			<td>to position electrodes and odorant delivery capillary tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic holding devices</td>
			<td style="width:199px;">Kanetec</td>
			<td style="width:160px;">MB-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Valve driver</td>
			<td style="width:199px;">Arduino</td>
			<td style="width:160px;">custom made</td>
			<td>to control the opening of the valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromagnetic valve</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">LFAA1201618H</td>
			<td>valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog AC/DC amplifier</td>
			<td>Digitimer Ltd.</td>
			<td style="width:160px;">NL106</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog DC pre-amplifier with headstage</td>
			<td>Digitimer Ltd.</td>
			<td>NL102G</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Low-pass 60 Hz filter</td>
			<td>Digitimer Ltd.</td>
			<td>NL125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Digitizer</td>
			<td>Molecular Devices LLC</td>
			<td style="width:160px;">Axon Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Dell computer (OptiPlex 745) running Axoscope data acquistion software</td>
			<td style="width:199px;">Molecular Devices LLC</td>
			<td>AxoScope version 10.4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Faraday cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>Electromagnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Two-choice maze</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>waterproofed marine grade plywood covered with plastic liner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Trash pump</td>
			<td style="width:199px;">Honda</td>
			<td>WT30XK4A</td>
			<td>fills maze with water from nearby river</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Peristaltic pump with tubing</td>
			<td style="width:199px;">Cole Parmer</td>
			<td style="width:160px;">Masterflex 07557-00</td>
			<td>to adminster odorants in maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Inverter Generator&#160;</td>
			<td style="width:199px;">Honda</td>
			<td style="width:160px;">EU1000i</td>
			<td>powers perstaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Release cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>used to acclimate lamprey in the maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Mesh</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>used to contain the dimensions of the maze and minimize water turbulance with mesh rollers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Buckets (5 gallon)</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to mix odorants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Flow meter</td>
			<td style="width:199px;">Marsh-McBirney</td>
			<td>Flo-Mate 2000</td>
			<td>to measure discharge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XAD 7 HP resin</td>
			<td style="width:199px;">Dow chemical</td>
			<td style="width:160px;">37380-43-1</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:199px;">Yamato</td>
			<td style="width:160px;">BM 200</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Freeze dryer</td>
			<td style="width:199px;">Labconco</td>
			<td style="width:160px;">CentriVap&#160; Concentrator</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">chloroform</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">CX1050</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 70-230 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 230-400 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Pre-coated silica gel TLC plates</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">99571</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anisaldehyde</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A88107</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex LH-20</td>
			<td style="width:199px;">GE Healthcare</td>
			<td style="width:160px;">17-0090-01</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite XAD 7 HP resin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">XAD7HP</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4, 2.5L capacity glass columns</td>
			<td>Ace Glass Inc.</td>
			<td>5820</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TQ-S TOF LC Mass spectrometer (or equivalent)</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binary HPLC pump</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">1525</td>
			<td>for isolation of fraction pools/compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent NMR spectrometer, 900MHz (or equivalent)</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotovap drying system</td>
			<td style="width:199px;">Buchi</td>
			<td style="width:160px;">RII</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV lamp (254 nm)</td>
			<td style="width:199px;">Spectronics Co.</td>
			<td style="width:160px;">ENF-240C</td>
			<td>for thin layer chromatography&#160;</td>
		</tr>
	</tbody>
</table>

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.

A diagram summarizing the steps described in the protocol of the bioassay-guided fractionation is shown in Figure 1. The protocol involves steps to isolate and characterize the structure, the olfactory potency, and the behavioral activity of 5 putative sea lamprey pheromones (Figure 2). Using the mass spectrometric and NMR data (Figure 3 and Figure 4), the structures of ...

Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

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 ...

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