The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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,

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Protokół

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 ....

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Wyniki

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 .......

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Dyskusje

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.......

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Materiały

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