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

Wij danken de Amerikaanse Geological Survey Hammond Bay biologische Station voor het gebruik van de onderzoeksfaciliteiten van hun en het personeel van Wildlife Service, US Fish en visserij en oceanen Canada voor het verstrekken van zee prikvissen. Dit onderzoek werd gesteund door subsidies van de Visserijcommissie van de grote meren aan Weiming Li en Ke Li.

Alle methoden die hier worden beschreven zijn goedgekeurd door de institutionele Animal Care en gebruik Comité van Michigan State University (AUF # 03/14-054-00 en 02/17-031-00).
1. verzameling en extractie van de Zeeprik geconditioneerd Water
<ol><li>Plaats seksueel volwassen mannelijke zee prikvissen (15-30 dieren) in een tank geleverd met 250 L belucht Lake Huron water onderhouden bij 16-18 ° C.</li>
	<li>Het verzamelen van de man-geconditioneerd water gedurende elke nacht van juni tot ...

<ol>
	<li>Wyatt, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromones and Animal Behavior: Chemical Signals and Signatures. , (2014).</li><li>Zhu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+chemical+ecology:+Olfactory+proteins+from+the+giant+panda+and+their+interactions+with+putative+pheromones+and+bamboo+volatiles.">Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles.</a> Proceedings of the National Academy of Sciences. 114 (46), E9802-E9810 (2017).</li><li>Leal, W. 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A., Bergstedt, R. A., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory-mediated+stream-finding+behavior+of+migratory+adult+sea+lamprey+(Petromyzon+marinus).">Olfactory-mediated stream-finding behavior of migratory adult sea lamprey (Petromyzon marinus).</a> Canadian Journal of Fisheries and Aquatic Sciences. 68, (2011).</li><li>Wagner, C. M., Jones, M. L., Twohey, M. B., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+test+verifies+that+pheromones+can+be+useful+for+sea+lamprey+(Petromyzon+marinus)+control+in+the+Great+Lakes.">A field test verifies that pheromones can be useful for sea lamprey (Petromyzon marinus) control in the Great Lakes.</a> Canadian Journal of Fisheries and Aquatic Sciences. 63 (3), 475-479 (2006).</li><li>Wagner, C. M., Twohey, M. 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B., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sea+lamprey+control:+past,+present,+and+future.">Sea lamprey control: past, present, and future.</a> Great Lakes Fisheries Policy and Management. , 651-704 (2013).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+Novel+Bile+Alcohols+of+Mature+Male+Sea+Lamprey+(Petromyzon+marinus)+Act+as+Chemical+Cues+for+Conspecifics.">Three Novel Bile Alcohols of Mature Male Sea Lamprey (Petromyzon marinus) Act as Chemical Cues for Conspecifics.</a> Journal of Chemical Ecology. 43 (6), 543-549 (2017).</li><li>Hird, S. J., Lau, B. P. -. 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M., Quino&#225;, E., Riguera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assignment+of+absolute+configuration+by+NMR.">The assignment of absolute configuration by NMR.</a> Chemical Reviews. 104 (1), 17-118 (2004).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+Salt-like+Dienones+Having+a+Novel+Skeleton+or+a+Rare+Substitution+Pattern+Function+as+Chemical+Cues+in+Adult+Sea+Lamprey.">Bile Salt-like Dienones Having a Novel Skeleton or a Rare Substitution Pattern Function as Chemical Cues in Adult Sea Lamprey.</a> Organic Letters. , (2017).</li><li>Li, K., Buchinger, T. J., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+natural+products+that+act+as+pheromones+in+fish.">Discovery and characterization of natural products that act as pheromones in fish.</a> Natural Product Reports. , (2018).</li><li>Yambe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Kynurenine,+an+amino+acid+identified+as+a+sex+pheromone+in+the+urine+of+ovulated+female+masu+salmon.">L-Kynurenine, an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon.</a> Proceedings of the National Academy of Sciences. 103 (42), 15370-15374 (2006).</li><li>Rasmussen, L., Lee, T. D., Zhang, A., Roelofs, W. L., Daves, G. 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Vissen leven in een chemische wereld vol van verbindingen nog worden geïdentificeerd. Bioassay-geleide fractionering heeft bewezen essentieel te identificeren en te karakteriseren van biologische actieve moleculen die vele chemische interacties, zoals waargenomen in masu zalm31, Aziatische olifanten32en zee prikvissen33, bemiddelen 34,35. Bioassay-geleide fractionering is een eff...

Feromonen zijn specifieke chemische signalen vrijgegeven door personen die hen helpen bij het lokaliseren van voedselbronnen, opsporen van roofdieren en bemiddelen van sociale interacties van soortgenoten1. Feromoon communicatie bij insecten geweest goed bestudeerde2; echter, de identificatie van chemische en biologische functie van aquatische gewervelde feromonen zijn niet bestudeerd zo uitgebreid. Kennis van de identiteit en de functie van de feromonen vrijgegeven kunnen worden toegepast om het herstel van bedreigde soorten3,4 of besturingselement pest soorten5,6. De toepassing van deze technieken vereist de isolatie en de karakterisering van de componenten van bioactieve feromoon.
Feromoon identificatie is een tak van de Scheikunde natuurproduct. Vooruitgang in feromoon onderzoek gedeeltelijk beperkt vanwege de aard van de feromoon moleculen zichzelf gebleven. Feromonen zijn vaak instabiel en uitgebracht in kleine hoeveelheden, en slechts een paar bemonsteringstechnieken bestaan om minieme hoeveelheden van vluchtige7,,8 of9van de in water oplosbare verbindingen te detecteren. Benaderingen voor het identificeren van feromonen omvatten 1) een gerichte screening van bekende verbindingen, metabolomica 2) en 3) bioassay-geleide fractionering. Een gerichte screening van bekende stoffen getest verkrijgbare metabole bijproducten van fysiologische processen in hypothetische om te functioneren als feromonen. Deze aanpak beperkt omdat onderzoekers alleen bekende en beschikbare verbindingen kunnen testen. Echter, het heeft geleid tot de succesvolle identificatie van geslachtshormonen in goudvis die functie als feromonen10,11,12. Metabolomica is een tweede feromoon identificatie benadering die potentiële klein molecuul stofwisselingsproducten in een biologisch systeem13onderscheidt. Een vergelijking van de metabolische profielen van twee groepen (dat wil zeggen, een actieve versus een inactieve uittreksel) de identificatie van een potentiële metabole profiel mogelijk maakt van die de metaboliet is gezuiverd is, wat de structuur is opgehelderd, en de topicale is bevestigd14. Additieve of synergetische effecten van complexe formuleringen van bepaalde mengsels zijn meer kans om te worden gedetecteerd met metabolomica omdat metabolieten worden beschouwd samen in plaats van als een reeks van breuken13. Echter de uitvoeringvan metabolomica is afhankelijk van de beschikbaarheid van synthetische verwijzingen omdat de resulterende gegevens doen niet de opheldering van nieuwe structuren vergemakkelijken.
Bioassay-geleide fractionering is een geïntegreerde, iteratieve aanpak dat twee velden omvat: Scheikunde en biologie. Deze aanpak maakt gebruik van de resultaten van fysiologische en gedragsmatige bioassays te begeleiden van de isolatie en identificatie van de verbinding van een actieve feromoon. Een ruwe extract is gefractioneerde door een chemische eigenschap (d.w.z.moleculaire grootte, polariteit, etc.) en getest met electro-olfactogram (EOG) opnames en/of in een bioassay. De bioactieve componenten worden gescreend uit herhaalt u deze stap voor fractionering en EOGs en/of bioassays. De structuren van zuivere actieve verbindingen zijn toegelicht door spectrometrische en spectroscopische methoden, waarmee het moleculaire gewicht en structurele informatie tot een sjabloon van de stof te worden gesynthetiseerd. Bioassay-geleide fractionering kan opleveren divers metabolieten en potentieel nieuwe feromonen met unieke chemische skeletten die zijn waarschijnlijk niet kan worden voorspeld uit de biosynthetic trajecten.
Hier beschrijven we de bioassay-geleide fractionering-protocol dat wordt gebruikt om te isoleren en karakteriseren de topicale van mannelijke Zeeprik seks feromoon verbindingen. De Zeeprik (Petromyzon marinus) is een ideale gewervelde model aan de studie van feromoon communicatie omdat deze vissen sterk afhankelijk zijn van de olfactorische detectie van chemische signalen te bemiddelen hun anadrome levensgeschiedenis bestaat uit drie verschillende fasen: larven, jeugdige en volwassene. Zeeprik larven ingraven in het sediment van zoetwater stromen, een drastische metamorfose ondergaan en transformeren in jonge exemplaren die naar een meer of Oceaan migreren, waar ze talloze vis parasitize. Na het loskoppelen van de host-vis, migreren de volwassenen terug in de paaitijd stromen, geleid door de grote afstanden trekkende feromonen vrijgegeven door stream-ingezeten larven15,16,17,18,19 . Volwassen mannetjes opstijgen naar de paaigronden, release een multi-component seks feromoon voor het aantrekken van stuurlieden, met tussenpozen paaien voor ongeveer een week en dan sterven15,20. De identificatie van de Zeeprik feromonen is belangrijk omdat een modulatie van het feromoon communicatiesysteem onder de is controle van de invasieve zee prikvissen in de Laurentian GREATLAKES21overwogen opties.

<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-geleide fractionering is een iteratieve aanpak die gebruikmaakt van de resultaten van fysiologische en gedragsmatige bioassays te begeleiden van de isolatie en identificatie van de verbinding van een actieve feromoon. Deze methode heeft geleid tot de succesvolle karakterisering van de chemische signalen die functie als feromonen in een groot aantal diersoorten. Zee prikvissen vertrouwen op olfaction te detecteren van de feromonen die gedrags- of fysiologische reacties bemiddelen. Wij gebruiken deze kennis van de biologie van de vis te poneren functies van vermeende feromonen en begeleiden van de isolatie en identificatie van actieve feromoon onderdelen. Chromatografie wordt gebruikt om te extraheren, concentreren en scheiden stoffen uit de geconditioneerde water. Electro-olfactogram (EOG) opnames zijn uitgevoerd om te bepalen welke fracties olfactorische reacties uitlokken. Twee-keuze doolhof gedrags testen worden vervolgens gebruikt om te bepalen als een van de geurige breuken ook gedragsgestoorde actief zijn en een voorkeur veroorzaken. Spectrometrische en spectroscopische methoden bieden het moleculaire gewicht en de structurele informatie om te helpen met de opheldering van de structuur. De topicale van de zuivere verbindingen is bevestigd met EOG en gedrags vitrotests. De gedrags reacties waargenomen in de doolhof moeten uiteindelijk worden gevalideerd in een instelling van het veld om te bevestigen hun functie in de instelling van een natuurlijke stream. Deze bioassays spelen een dubbelrol te 1) begeleiden het proces en 2) bevestigen en de topicale van geïsoleerde onderdelen nader te definiëren. Wij rapporteren hier, de representatieve resultaten van een Zeeprik feromoon identificatie die het nut van de bioassay-geleide fractionering aanpak illustreren. De identificatie van de Zeeprik feromonen is vooral belangrijk omdat een modulatie van haar feromoon communicatiesysteem onder de is controle van de invasieve Zeeprik in de Laurentian grote meren overwogen opties. Deze methode kan gemakkelijk worden aangepast om het karakteriseren van de chemische communicatie in een breed scala van taxa en licht werpen op waterbasis chemische ecologie.

Een samenvatting van de stappen die worden beschreven in het protocol van de bioassay-geleide fractionering diagram is afgebeeld in Figuur 1. Het protocol bestaat uit stappen om te isoleren en karakteriseren van de structuur, de olfactorische potentie en de gedragsmatige activiteit van 5 putatief Zeeprik feromonen (Figuur 2). Met behulp van de massa-spectrometrische en NMR-gegevens (Figuur 3 en <stro...

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

Hier presenteren we een protocol om te isoleren en karakteriseren van de structuur, olfactorische potentie en gedragsmatige reactie van vermeende feromoon verbindingen van zee prikvissen.

De identificatie van de Zeeprik feromonen met behulp van de Bioassay-geleide fractionering

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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