This method can help answer key questions in the chemical ecology field, such as the identification of pheromones. The main advantage of this technique is that it links novel compounds to pheromone function. To begin this procedure, place 15-30 sexually mature male sea lampreys in a tank supplied with 250 liters of aerated Lake Huron water, maintained at 16-18 degrees Celsius.
To extract the conditioned water by solid-phase extraction, pass it from the tank via the pumping system to the top of the column. The water passes through a bed of two kilograms of moderately polar, polymeric ion-exchange resin contained in a series of four one-liter capacity glass columns. Next, remove the organic solvent and harvest the extract by rotary evaporation for five hours under reduced pressure at 40 degrees Celsius.
To isolate the fraction pools, soak the silica gel in the initial mobile phase for 30 minutes. Transfer the gel with the solvent to a glass column and open its valve to allow the mobile phase to go through. Next, mix the extracts with silica gel thoroughly and load them into the liquid chromatography column over the silica gel bed.
Elute them with a gradient from 95%chloroform-to-methanol to 100%methanol, resulting in a total of 2.5 liters, then collect the eluent in individual 10-milliliter vials. Subsequently, 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 solvent in a sealed glass tank. After the developing solvent reaches the end line, take the plate out of the tank and wait 10 minutes for the solvent to evaporate.
Visualize the spots, first under UV light at 254 nanometers, then stain the plate by spraying it with an acidic methanol solution of 5%anisaldehyde using a chromatography sprayer and heating it at 85 degrees Celsius for three minutes. Afterwards, concentrate the fraction to a residue by rotary evaporation under reduced pressure at 40 degrees Celsius for approximately 30 minutes. To perform EOG recording, fill the poled capillary electrodes and the solid state electrode holders, pre-fabricated with silver-silver chloride pellets, with three-molar potassium chloride using a micropipette.
Then, insert the poled electrodes in the electrode holders. Next, prepare 100 milliliters of 10 to the minus five-molar L-arginine in charcoal-filtered water from a 10 to the minus two-molar L-arginine stock solution in deionized water in a volumetric flask. For the concentration response curve, prepare 10 milliliters of 10-fold dilutions of the fraction pools.
Now, orient an anesthetized lamprey in a V-shaped stand and wrap it with a wet paper towel to prevent desiccation. Insert a tube of recirculating, aerated water containing 50 milligrams per liter 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.
Flush the odorant delivery tubing with filtered water and connect it to the valve mounted on a micromanipulator. Next, place the odorant delivery capillary tube into the olfactory epithelium cavity to deliver filtered water to the olfactory epithelium to prevent desiccation when not administering odorants. Then, mount the recording and reference electrodes on the micromanipulators.
Lower the reference electrode onto the external skin near the nares. Using the stereoscopic microscope, lower the recording electrode to barely touch the surface of the olfactory epithelium. Subsequently, transfer the uptake of the odorant delivery tube from the background filtered water to the 10 to the minus five-molar L-arginine solution.
Turn on the computer, amplifier, filter, and digitizer. Using the valve driver software, program the procedure to administer a four-second single pulse of odorant by checking the box of T1, setting T to four seconds, and checking the box of T2.In the data acquisition software, 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.
Start recording the blank control, L-arginine, and then the odorants from low to high concentrations with a two-minute flush of filtered water between applications. To identify behaviorally active fractions, acclimate the sexually mature female sea lamprey in the release cage in the maze for five minutes. 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 minutes.
Next, apply the test stimulus to the randomly assigned experimental channel in the vehicle to the control channel using peristaltic pumps at constant rates of 200 milliliter per minute for five minutes, then apply the test stimulus in the vehicle for an additional 10 minutes and record the cumulative amount of time the lamprey spends in the experimental and control channels. In this procedure, further purify the active fractions into compounds with size-exclusion chromatography using a sephadex LH-20 column. Prepare the sample in 0.5 milliliters of the initial mobile phase, loading it into the chloroform-to-methanol column and then the methanol column, and elute the active fractions to yield the compound.
Dissolve and dilute the purified compound in the initial mobile phase of HPLC to form a 10-microliter solution of approximately one microgram per milliliter. Next, transfer the sample solution to the HPLC vials and set them into the auto-sampler of HPLC. Inject the sample into the electrospray ionization mass spectrometry and record the high-resolution electrospray ionization mass spectrometry spectra using a chromatography mass spectrometer.
Completely dissolve the sample in the selected deuterated solvents to form a solution with a concentration ranging approximately from 0.1 to 10 milligrams per milliliter. Transfer the sample solution into an NMR tube to record the 1D and 2D NMR spectra on a 900-megahertz NMR spectrometer. Shown here is a 600-megahertz NMR spectrometer.
Five novel putative sea lamprey pheromones were identified and their structures were elucidated with spectrometric and spectroscopic methods. These putative pheromones were stimulatory to the adult sea lamprey olfactory epithelium and had low detection thresholds in electro-olfactogram recordings. In the two-choice maze behavioral assays, ovulated female sea lampreys were attracted to Petromyzone A, Petromyzene A, and Petromyzene B, and repulsed from Petromyzone C.While attempting this interdisciplinary procedure, it's important to remember to coordinate the chemical and biological experiments to carry out this iterative protocol.
Following this procedure, other methods like X-ray crystallography and derivatization reactions can be performed in order to answer additional questions like the stereochemistry of the compounds. After its development, this technique paved the way for researchers in the field of natural products and neuroethology to explore the plausible biosynthesis and biological function of the pheromones in aquatic organisms like sea lamprey. Don't forget that working with chloroform can be extremely hazardous and precautions, such as working in the chemical hood, wearing gloves, and safety goggles, should always be taken while handling this reagent.
