The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Summary

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

Abstract

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.

Introduction

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 species^3,4 or control pest species^5,6. 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 volatile^7,8 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 pheromones^10,11,12. 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 larvae^{15,16,17,18,19}. Mature males ascend to the spawning grounds, release a multi-component sex pheromone to attract mates, intermittently spawn for approximately a week, and then die^15,20. 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.

Protocol

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

1. 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 °C.
2. Collect the male-conditioned water throughout each night from June to July.
   Note: Sea lampreys naturally die after spawning. If a fish is nearing this point in its life, replace it with a fresh mature male.
3. Extract the conditioned water by solid phase extraction.
   1. Soak the resin in methanol for 4 h before loading it into a column. Load the resin into the column, then pump 10 L of water through the column to eliminate the organic solvent.
   2. Pass the conditioned water from the tank holding the fish via the pumping system to the top of the column. The water passes through a bed of 2 kg of moderately polar polymeric ion-exchange resin (e.g., Amberlite XAD-7 HP resin), contained in a series of four 2.5 L-capacity glass columns. Maintain load speeds between 400 and 600 mL/6 min. Elute the metabolites with 10 L of methanol followed immediately by 5 L of acetone.
      CAUTION: This step uses methanol and acetone. Both are flammable and poisonous.
      Note: The pooled residual can be stored at -80 °C until further processed.
4. Reactivate the column after 1 week of enrichment by washing it with water (approximately 10 L).
5. Remove the organic solvent (the mixture of methanol and water) and harvest the extract by rotatory evaporation for 5 h under reduced pressure (under 300 mbar) at 40 °C. Concentrate the water residue by freeze dryer lyophilization for 48 h at -20 °C until dry.

2. Isolation of Fraction Pools with Chromatography

1. Soak the silica gel (230 - 400 mesh) in the initial mobile phase for 30 min. Transfer the gel with the solvent (suspension) to a glass column. Open the valve of the column to allow the mobile phase to go through. Allow the silica gel to precipitate and form a silica gel bed.
2. Mix the extracts with silica gel (70 - 230 mesh) thoroughly and load them into the liquid chromatography column over the silica gel bed. Elute them with a gradient from 95% CHCl₃ (chloroform)/MeOH (methanol) to 100% MeOH, 2.5 L in total volume. Collect the eluent in individual vials (each 10 ml).
   CAUTION: The chloroform used in this step is a poisonous reagent.
3. Guide the pooling of the eluents into 20 fractions by a thin layer chromatography (TLC) analysis.
   1. Perform the TLC experiment on pre-coated silica gel plates by applying the sample on the starting line and immersing the starting line of the plate in the developing solvents (choose the polarity by the ratio of CHCl₃ to methanol from 100 - 0%) in a sealed glass tank.
   2. Select the ratio of developing solvent to make all the TLC spots with a retention factor (R_f) ranging from 0.3 - 0.8.
   3. After the developing solvent reaches the end line, take the plate out of the tank, and wait 10 min for the solvent to evaporate.
   4. Visualize the spots first under UV light at 254 nm and then stain them by spraying them with an acidic methanol solution of 5% anisaldehyde (20 μL) by a chromatography sprayer and heating them at 85 °C for 3 min.
      Note: The colorful spots on the TLC plate indicate the main component in the fraction.
   5. Combine the eluent to 20 fractions based on the spot color and the R_f similarity of the main component.
4. Concentrate the fraction to a residue by rotatory evaporation under reduced pressure (300 mbar according the property of solvents) at 40 °C for approximately 30 min.

3. Electro-olfactogram (EOG) Recordings to Identify Odorous Fractions/Compounds

1. Pull borosilicate glass capillaries (outer diameter: 1.5 mm; inner diameter: 0.86 mm; length: 100 mm) with a micropipette puller with the heater level set to 65.
2. Score and cut the opening at the tip of the capillary with a diamond-tipped glass cutter and fill it with molten 0.4% agar in 0.9% saline. The opening at the tip of the capillary should be around 10 µm in diameter.
3. Fill the pulled capillary electrodes from steps 3.1 - 3.2 and solid-state electrodes holders pre-fabricated with Ag/AgCl pellets (see Table of Materials) with 3 M KCl using a micropipette.
   Note: Dislodge any air bubbles in the capillary or the electrode holder.
4. Insert the pulled electrodes in the electrode holders.
5. Prepare 100 mL of 10^-5 M L-arginine in charcoal-filtered water from a 10^-2 M L-arginine stock solution in deionized water (stored at 4 °C) in a volumetric flask. Transfer 20 mL of the 10^-5 M L-arginine to a glass vial.
6. For the concentration-response curve, prepare 10 mL of 10-fold dilutions of the fraction pools from step 2.3 in glass vials. Prepare fresh dilutions daily prior to the experiments and use them within a day. Put the glass vials of the working solutions of L-arginine and the fraction pool dilutions in a recirculating water bath to allow the temperature to equilibrate to 8 °C.
7. Anesthetize the lamprey with 3-aminobenzoic acid ethyl ester (MS222; 100 mg/L) and immobilize it with an intramuscular injection of gallamine triethiodide (3 mg/kg of body weight, in 0.9% saline) with a sterile syringe.
   Note: A sufficient depth of anesthesia is determined by observing no gill motion, an inability to maintain an upright posture, and no suction to the sides of the tank using its oral disk. To minimize any microbial contamination before and during surgery, wear sterile gloves and soak the dissection tools in 70% ethanol (v:v) in deionized water for at least 10 min before use.
8. Orient the anesthetized lamprey in a V-shaped stand and wrap it with a wet paper towel to prevent desiccation.
   Note: Do not obstruct the gill openings.
9. Insert a tube of recirculating aerated water containing 50 mg/L of MS222 into the buccal cavity, adjust the flow rate, and ensure that water is exiting via the gill openings to continuously irrigate the gills.
10. Use a sterile scalpel and forceps [under the stereoscopic microscope at a 1.25X magnification (see Table of Materials)] to remove a 5 mm² section of the skin on the surface of the olfactory capsule to expose the olfactory epithelium.
11. Flush the odorant delivery tubing with filtered water and connect it to the valve mounted on a micromanipulator. Place the odorant delivery capillary tube into the olfactory epithelium cavity using the micromanipulator to deliver filtered water to the olfactory epithelium to prevent desiccation when not administering odorants.
12. Mount the recording and reference electrodes on the micromanipulators. Lower the reference electrode onto the external skin near the naris. Using the stereoscopic microscope (1.25X), lower the recording electrode to barely touch the surface of the olfactory epithelium.
13. Transfer the uptake of the odorant delivery tube from the background filtered water to the 10^-5 M L-arginine solution.
14. Turn on the computer, amplifier (set to DC mode), filter, and digitizer. Using the valve driver software, program the procedure to administer a 4 s single pulse of odorant by checking the box of T1, setting T to 4 s, and checking the box of T2.
15. In the data acquisition software (see Table of Materials), set the acquisition mode to a high-speed oscilloscope, click the play icon, and then click Start in the valve driver to trigger the odorant pulse.
    Note: The data acquisition software automatically records the differential EOG response amplitude for 20 s (3 s before, 4 s during the odorant pulse and 13 s afterward). Use the micromanipulator to maneuver the positions of the recording electrode, reference electrode, or odorant delivery tube to increase the signal-to-noise ratio with a maximum response to the L-arginine standard and a minimal response to the blank control (filtered water). Ground the amplifier by switching the AC-DC amplifier switch to GND while moving the electrodes.
16. Start recording the blank control, L-arginine, and then the odorants prepared in step 3.6 from low to high concentrations with a 2 min flush of filtered water between applications.
17. After recording responses to all the odorants to be tested, ground the amplifier and carefully retract the electrodes and odorant delivery capillary tube. Following the approved Institutional Animal Care and Use Committee (IACUC) methods, at the termination of the EOG experiment, euthanize the anesthetized lamprey with an overdose of MS222 (1 g/L).
    Note: Verify successful euthanasia by noting a lack of gill movement and heartbeat for at least 5 min followed by pithing the brain.
18. Analyze and plot the data using analysis software. Determine the threshold of detection of the odorants²² to identify odorous fractions that elicit responses greater than the blank water control. The fraction pools that elicit concentration-dependent olfactory responses that are different than the blank water control are then tested in a behavioral assay (step 4).

4. Two-choice Maze Behavioral Bioassay to Identify Behaviorally Active Fractions/Compounds

1. Acclimate the sexually mature female sea lamprey in the release cage in the maze (see Supplementary Figure 1) for 5 min. Maintain the flow rate of the river water in the maze
   Note: The maze is built from varnished marine grade wood and measures 6.5 m in length and 1.2 m in width with a divider measuring 2.7 m in length to separate the maze into two channels at the upstream end. Water is temporarily diverted into the maze. The water depth should be maintained at 0.19 m and the velocity should be maintained at 0.07 m/s ± 0.01.
2. Release the sea lamprey and record the cumulative amount of time the lamprey spends in the experimental and control channel each containing river water for 10 min.
   Note: If the sea lamprey fails to enter the experimental and control channel for at least 10 s during this 10 min period, end the trial, as this is an indication of inactivity or strong side bias.
3. Apply the test stimulus (i.e., a putative pheromone at 10^-12 M dissolved in 50% methanol/deionized water) to the randomly assigned experimental channel and the vehicle (50% methanol/deionized water) to the control channel using peristaltic pumps at constant rates of 200 mL/min for 5 min.
   Note: The detection threshold concentration of the test stimulus as determined with the electro-olfactogram recordings should be used as the initial concentration for the behavioral test.
4. Apply the test stimulus and the vehicle for an additional 10 min and record the cumulative amount of time the lamprey spends in the experimental and control channel.
5. Flush the maze with water for 10 min before the start of the next trial. Repeat steps 4.1 - 4.4 with at least 7 lampreys if sufficient test stimulus is available.
6. Calculate an index of preference²² for each trial and evaluate the significance using a Wilcoxon signed-rank test.
   Note: The index results in a single number that can be either positive or negative. A positive value of the index of preference indicates attraction, whereas a negative value of the index of preference indications repulsion. If the index of preference is significantly different from zero, the fraction is deemed active.
   
   Here,
   B_c = the time spent by the test lamprey in the control channel before the odorant application,
   B_e = the time spent in the experimental channel before the odorant application,
   A_c = the time spent in the control channel after the odorant application, and
   A_e = the time spent in the experimental channel after the odorant application.

5. Chromatographic Isolation of Pure Compounds from Active Fractions

1. Repeat steps 2.1 - 2.4 with the fraction pools that induce olfactory responses (step 3) and elicit behavioral responses (step 4).
2. Further purify the active fractions into compounds with size-exclusion chromatography using a Sephadex LH-20 column.
   1. Prepare the sample in 0.5 mL in the initial mobile phase [CHCl₃-MeOH (1:1) or MeOH 100%], load into the corresponding column a CHCl₃-MeOH (1:1) column and then a MeOH (100%) column, and elute them to yield the compounds.

6. Structure Elucidation of a Pure Compound with Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR)

1. Dissolve and dilute the purified compound in the initial mobile phase (usually, methanol to water, 1:1, v:v) of high-performance liquid chromatography (HPLC) to form a 10 μL solution of approximately 1 μg/mL.
   1. Transfer the sample solution to HPLC vials and set them into the autosampler of HPLC. Inject the sample (10 μL) into the electrospray ionization mass spectrometry (ESIMS) and record the high-resolution electrospray ionization mass spectrometry (HRESIMS) spectra using a chromatography mass spectrometer²³.
2. Predict the molecular formula according to the database in the mass spectrometer software (see Table of Materials)²⁴.
   1. Open the chromatogram of the injected sample and obtain its mass spectrum by selecting the chromatographic peak.
   2. Input the measured mass to ion (m/z) value (4 decimals) in Elemental composition under the Tool module. Set the tolerance parameter to PPM < 5.
   3. Adjust the symbol parameters to fit the element composition of the measured molecule. The software produces a prediction of molecular formula based on the single mass analysis.
3. Select the deuterated solvents (600 μL, CH₃OH-d₄ or DMSO-d₆) according to the protocol²⁵ to dissolve the samples.
   1. Completely dissolve the sample in the selected deuterated solvents to form a solution with a concentration ranging approximately from 0.1 to 10 mg/mL. Transfer the sample solution into an NMR tube to record the 1D (¹H, ¹³C) and 2D NMR [¹H-¹H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC)] spectra on a 900 MHz NMR spectrometer²⁶.
4. Place the NMR tube with the sample inside the spinner turbine. Use the depth gauge to ensure the sample height is in the middle of the measuring window. Open the NMR software (see Table of Materials) and click the Lift button to change the sample in the magnet.
   Note: Hold a hand over the top of the magnet to feel the gas coming from the top of the magnet. At the same time, a whistling sound should be audible.
5. Gently place the sample on the air cushion on top of the magnet and push the Lift button again to descend the sample into the NMR magnet.
   Note: Listen for a “click” noise to indicate the sample is in the right position.
6. Create a new dataset and load the default parameters recommended by the NMR instrument (see Table of Materials).
   1. Type “lock” to invoke the automatic locking procedure and select the solvent at the prompt page.
   2. For fine-tuning, in the Manipulate panel, adjust the button in the Unlock model, adjust the cursor on the manipulate panel, and lock the magnet again.
   3. On the prompt Atma page, adjust the parameter in the manipulate panel to the tuning procedure, which may take a few minutes. Wait until the tuning is completed to proceed.
   4. Start the automatic shimming routine by typing “topshim” in the prompt. Wait until the shimming is completed to proceed.
   5. Type “rga” to set automatic receiving gain adjustments, then type “d1” to set the delay between pulses, then type “zg” to start the acquisition, and wait until the acquisition finishes.
   6. Type “ef” or “efp” to process the data and then type “apk” for automatic phasing. Process the data on the NMR processing software.
7. Elucidate the chemical structure by an interpretation of the NMR data analysis.
8. Count the carbon signals in the ¹³C NMR spectrum. Select the matched molecular formula from the predicted result in high-resolution mass spectrometry (HR-MS).
9. Integrate the proton signals in the ¹H NMR spectrum and assign the connectivity of the carbon- and proton-based on the signals in the HSQC spectrum.
10. Assign the connectivity of the carbon skeleton based on the correlations in the ¹H-¹H COSY and HMBC spectrum.
11. Tentatively assign the chemical structure based on the structure rationale²⁷. Search the tentative structure in the chemical structure databases. Compare the tentative structure with the analogs in references.
12. Assign the relative configuration with the Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum.
    Note: Since the identity properties of enantiomers displayed on spectrometry and spectroscopic methods are identical, the absolute configuration of some compounds, especially those with 2’-alcohol and 2’-NH₂, has to be determined with a derivatization reaction. After the derivatization reaction, the differences indicated on the spectra facilitate the unequivocal assignment of the absolute configurations of most compounds²⁸.

7. EOG and Bioassay to confirm pure compounds are odorous and behaviorally active

1. Repeat steps 3.1 - 3.5.
2. For the concentration-response curve, prepare 10 mL of 10-fold dilutions of the pure compounds from 10^-6 M - 10^-13 M of a 10^-3 M pheromone stock solution in 50% methanol/water stored at -20 °C based on the molecular weight determined in step 6.2. Put the glass vials with the working solutions of L-arginine and the pure compounds in a recirculating water bath to allow the temperature to equilibrate to 8 °C.
3. Repeat steps 3.7 - 3.18.
4. Repeat steps 4.1 - 4.2.
5. Apply the pure compound at the EOG detection threshold concentration to the randomly assigned experimental channel and the vehicle (50% methanol/deionized water) to the control channel using a peristaltic pump at constant rates of 200 mL/min for 5 min.
6. Repeat steps 4.4 - 4.6.

Results

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 ...

Discussion

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 lampreys^33,34,35. Bioassay-guided fractionation is an effective approach to accurately trace and pinpoint...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Premium standard wall borosilicate capillaries with filament	Warner Instruments	G150F-4	recording and reference electrode (OD 1.5 mm, ID 0.86 mm)
Pipette puller instrument	Narishige	PC-10	pulls electrodes for EOGs
Diamond-tipped glass cutter	Generic		cut tip of electrodes for EOG
Borosilicate glass capillaries	World Precision Instruments	1B150-4	odorant delivery tube for EOG
Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm	Warner Instruments	ESP-M15N	recording electrode holder
Reference electrode holder E Series with handle with  Ag/AgCl pellet  for glass capillary OD 1.5 mm	Warner Instruments	E45P-F15NH	reference electrode holder
1 mm pin	Warner Instruments	WC1-10	to bridge reference and recording electrode holders
2 mm pin	Warner Instruments	WC2-5	to bridge reference and recording electrode holders
Agar	Sigma	A1296	molten agar to fill electrodes
Potassium chloride (KCl)	Sigma	P9333	3M KCl to fill electrodes and electrode holders
Micropipette microfil	World Precision Instruments	MF28G-5	to fill electrodes and electrode holders
L-Arginine	Sigma	A5006	positive control odorant for EOG
Methanol	Sigma	34860
Water bath	Custom made	N/A	holds odorants for EOG
3-aminobenzoic acid ethyl ester (MS222)	Syndel USA	Tricaine1G	EOG anesthetic
Gallamine triethiodide	Sigma	G8134-5G	EOG paralytic
1 mL syringe	BD Biosciences	301025	to administer paralytic
Subcutaneous needle 26G 5/8	BD Biosciences	305115	to administer paralytic
Roller clamp	World Precision Instruments	14043-20	adjust flow rate of anesthic into lamprey's mouth
Sodium chloride (NaCl)	J.T. Baker	3624-05	for preparation of 0.9% saline
V-shaped plastic stand as specimen stage	Custom made	N/A	holds lamprey during EOG
Plastic trough	Custom made	N/A	holds V-shaped plastic stand during EOG
Scalpel Blades - #11	Fine Science Tools	10011-00	for EOG dissection
Scalpel Handle - #3	Fine Science Tools	10003-12	for EOG dissection
Straight ultra fine forceps	Fine Science Tools	11252-00	for EOG dissection, Dumont #5SF Forceps
Curved ultra fine forceps	Fine Science Tools	11370-42	for EOG dissection, Moria MC40B
Straight pring Scissors	Fine Science Tools	15003-08	for EOG dissection
Stereomicroscope	Zeiss	Discovery V8	for EOG dissection
Illuminator light	Zeiss	CL 1500 ECO	for EOG dissection
Plastic tubing	Generic		to connect re-circulating EOG setup and water baths
Odorant delivery tubing	Custom made	N/A
In line filter and gasket set	Lee Company	TCFA1201035A
Micromanipulators	Narishige	MM-3	to position electrodes and odorant delivery capillary tube
Magnetic holding devices	Kanetec	MB-K
Valve driver	Arduino	custom made	to control the opening of the valve for odor stimulation
Electromagnetic valve	Lee Company	LFAA1201618H	valve for odor stimulation
NeuroLog AC/DC amplifier	Digitimer Ltd.	NL106	to increase the amplitude of the elictrical signal
NeuroLog DC pre-amplifier with headstage	Digitimer Ltd.	NL102G	to increase the amplitude of the elictrical signal
Low-pass 60 Hz filter	Digitimer Ltd.	NL125
Digitizer	Molecular Devices LLC	Axon Digidata 1440A
Dell computer (OptiPlex 745) running Axoscope data acquistion software	Molecular Devices LLC	AxoScope version 10.4
Faraday cage	Custom made	N/A	Electromagnetic noise shielding
Two-choice maze	Custom made	N/A	waterproofed marine grade plywood covered with plastic liner
Trash pump	Honda	WT30XK4A	fills maze with water from nearby river
Peristaltic pump with tubing	Cole Parmer	Masterflex 07557-00	to adminster odorants in maze
Inverter Generator	Honda	EU1000i	powers perstaltic pump
Release cage	Custom made	N/A	used to acclimate lamprey in the maze
Mesh	Generic		used to contain the dimensions of the maze and minimize water turbulance with mesh rollers
Buckets (5 gallon)	Generic		to mix odorants
Flow meter	Marsh-McBirney	Flo-Mate 2000	to measure discharge
XAD 7 HP resin	Dow chemical	37380-43-1	for extraction of conditioned water
Methanol	Sigma	34860	for extraction of conditioned water
Water bath	Yamato	BM 200	for extraction of conditioned water
Freeze dryer	Labconco	CentriVap Concentrator	for extraction of conditioned water
chloroform	Sigma	CX1050	for isolation of fraction pools
Silica gel 70-230 mesh	Sigma	112926-00-8	for isolation of fraction pools
Silica gel 230-400 mesh	Sigma	112926-00-8	for isolation of fraction pools
Pre-coated silica gel TLC plates	Sigma	99571	for isolation of fraction pools
anisaldehyde	Sigma	A88107	for isolation of fraction pools
Sephadex LH-20	GE Healthcare	17-0090-01	for isolation of fraction pools
Amberlite XAD 7 HP resin	Sigma	XAD7HP	for extraction of conditioned water
4, 2.5L capacity glass columns	Ace Glass Inc.	5820	for extraction of conditioned water
Acetone	Sigma	650501	for extraction of conditioned water
TQ-S TOF LC Mass spectrometer (or equivalent)	Waters Co.	N/A	for structure elucidation
Binary HPLC pump	Waters Co.	1525	for isolation of fraction pools/compounds
Agilent NMR spectrometer, 900MHz (or equivalent)	Agilent	N/A	for structure elucidation
Rotovap drying system	Buchi	RII	for extraction of conditioned water
UV lamp (254 nm)	Spectronics Co.	ENF-240C	for thin layer chromatography