Name: a method to test the effect of environmental cues on mating behavior in drosophila melanogaster
Uploaded: 2017-07-17T13:00:00
Description: Watch this Scientific Journal Video about A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster at JoVE.com

Vi takker Bloomington Drosophila Stock Center for flybestandene; C. Gahr, JT Alkema og S. van Hasselt for deres tidlige forsøg på at udvikle trykluftanalysen; Jasper Bosman for råd om dyrkning af gær Og Rezza Azanchi og Joel Levine for oprindeligt at udvikle time-lapse overvågningen af Drosophila parringsadfærd. JA Gorter blev støttet af et stipendium fra Neuroscience Research School BCN / NWO Graduate Program. Dette arbejde blev delvist støttet af den nederlandske organisation for videnskabelig forskning (NWO) (reference: 821.02.020) til JC Billeter.

1. Miljøstyret parringsboks <ol><li> For at sikre et kontrolleret og let at rengøre testområde skal du oprette et køkkenskab af rustfrit stål på 120 cm x 64 cm x 85 cm som vist i figur 1A. <ol><li> Bor et hul bag på kabinettet lige under loftet og fire sæt med fire huller i siderne, hver med en diameter på 2 cm. Bor de to første sæt med fire huller på hver side af kassen i en højde på 7 cm fra bunden af ​​kassen og med 12,5 cm mellem hullerne. Bor de to andre sæt på hver side af ka...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+post-mating+response+in+Drosophila+melanogaster+requires+multiple+seminal+fluid+Proteins.">Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid Proteins.</a> PLoS gen. 3 (12), 2428-2438 (2007).</li><li>Yapici, N., Kim, Y. -. J., Ribeiro, C., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor+that+mediates+the+post-mating+switch+in+Drosophila+reproductive+behaviour.">A receptor that mediates the post-mating switch in Drosophila reproductive behaviour.</a> Nature. 451 (7174), 33-37 (2008).</li><li>Yang, C. -. 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=Control+of+the+postmating+behavioral+switch+in+Drosophila+females+by+internal+sensory+neurons.">Control of the postmating behavioral switch in Drosophila females by internal sensory neurons.</a> Neuron. 61 (4), 519-526 (2009).</li><li>H&#228;semeyer, M., Yapici, N., Heberlein, U., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+neurons+in+the+Drosophila+genital+tract+regulate+female+reproductive+behavior.">Sensory neurons in the Drosophila genital tract regulate female reproductive behavior.</a> Neuron. 61 (4), 511-518 (2009).</li><li>Rez&#225;val, C., Pavlou, H. J., Dornan, A. J., Chan, Y. -. B., Kravitz, E. A., Goodwin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuitry+underlying+Drosophila+female+postmating+behavioral+responses.">Neural circuitry underlying Drosophila female postmating behavioral responses.</a> Curr. Biol. , 1-11 (2012).</li><li>Haussmann, I. U., Hemani, Y., Wijesekera, T., Dauwalder, B., Soller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+mediate+the+sex-peptide-regulated+switch+in+female+Drosophila+reproductive+behaviours.">Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours.</a> Proc. Biol. Sci. 280 (1771), 20131938-20131938 (2015).</li><li>Krupp, J. 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=Social+experience+modifies+pheromone+expression+and+mating+behavior+in+male+Drosophila+melanogaster.">Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.</a> Curr. Biol. 18 (18), 1373-1383 (2008).</li><li>Billeter, J. C., Jagadeesh, S., Stepek, N., Azanchi, R., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+females+change+mating+behaviour+and+offspring+production+based+on+social+context.">Drosophila melanogaster females change mating behaviour and offspring production based on social context.</a> Proc. Biol.l Sci. 279 (1737), 2417-2425 (2012).</li><li>Krupp, J. J., Billeter, J. -. C., Wong, A., Choi, C., Nitabach, M. N., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment-dispersing+factor+modulates+pheromone+production+in+clock+cells+that+influence+mating+in+Drosophila.">Pigment-dispersing factor modulates pheromone production in clock cells that influence mating in Drosophila.</a> Neuron. 79 (1), 54-68 (2013).</li><li>Imhof, M., Harr, B., Brem, G., Schl&#246;tterer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mating+in+wild+Drosophila+melanogaster+revisited+by+microsatellite+analysis.">Multiple mating in wild Drosophila melanogaster revisited by microsatellite analysis.</a> Mol. Ecol. , 915-917 (1998).</li><li>Ochando, M. D., Reyes, A., Ayala, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+paternity+in+two+natural+populations+(orchard+and+vineyard)+of+Drosophila.">Multiple paternity in two natural populations (orchard and vineyard) of Drosophila.</a> Proc. Natl. Acad. Sci. U S A. 93 (21), 11769-11773 (1996).</li><li>Becher, P. G., 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=Yeast,+not+fruit+volatiles+mediate+Drosophila+melanogaster+attraction,+oviposition+and+development.">Yeast, not fruit volatiles mediate Drosophila melanogaster attraction, oviposition and development.</a> Func. Ecol. 26 (4), 822-828 (2012).</li><li>Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+visual+transduction.">Drosophila visual transduction.</a> Trends Neurosci. 35 (6), 356-363 (2012).</li><li>Ejima, A., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+for+courtship+suppression+in+Drosophila.">Assay for courtship suppression in Drosophila.</a> Cold Spring Harbor Prot. 2011 (2), 5575 (2011).</li><li>Crickmore, M. A., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+Dopaminergic+and+GABAergic+Neurons+Control+the+Duration+and+Persistence+of+Copulation+in+Drosophila.">Opposing Dopaminergic and GABAergic Neurons Control the Duration and Persistence of Copulation in Drosophila.</a> Cell. 155 (4), 881-893 (2013).</li><li>Bretman, A., Fricke, C., Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+responses+of+male+Drosophila+melanogaster+to+the+level+of+sperm+competition+increase+male+reproductive+fitness.">Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness.</a> Proc. Biol. 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Denne protokol beskriver et test for at teste parringsadfærd i løbet af 24 timer, mens du løbende styrer de miljømæssige signaler, som et parretpar er hypotetiseret til at bruge til at bestemme parringsfrekvensen. Det er muligt at øge parringsfrekvensen som reaktion på gærluft leveret gennem et trykluftsystem, når mediet også indeholder gær ( figur 2B ). Derudover kan et lignende svar i parringsfrekvens observeres med et forenklet fødevaremedium indeholdende kun agar-, pepton- og eddikesyrel...

Reproduktiv adfærd har typisk høje energikostnader, især for kvinder, der producerer større gameter end mænd og skal omhyggeligt vælge vilkårene for at hæve deres udviklende afkom. På grund af energikostnaden er det ikke overraskende, at reproduktion er forbundet med ernæringsmæssige forhold. Dette gælder i de fleste, om ikke alle, dyr, herunder pattedyr, hvis pubertet kan forsinkes af underernæring, og hvis seksuelle drev kan blive negativt påvirket af fødevarebegrænsning 1 . Reproduktionen af ​​den genetiske modelorganisme Drosophila melanogaster påvirkes også af næringsbetingelser. Haner domstol på højere niveau i nærværelse af fødevarer flygtige 2 , og kvinder er mere seksuelt modtagelige i nærværelse af gær, et vigtigt næringsstof til ægproduktion og afkom overlevelse 3 , 4 , 5 . Det herEvolutionært konserveret reproduktivt respons på mad giver mulighed for at studere mekanismer, der forbinder miljøtilgængeligheden til seksuel reproduktion i en genetisk tålelig og tidseffektiv organisme. Faktisk har arbejdet i D. melanogaster impliceret insulinvejen som en vigtig regulator for forbindelsen mellem mad og parringsadfærd 6 . Det har også vist, at handlingen med parring selv ændrer kvindens madpræference såvel som de tilhørende kemosensoriske neuroner 7 , 8 , 9 . Det er klart, at fødevaresignaler påvirker reproduktive adfærd i D. melanogaster . Disse virkninger synes primært at påvirke kvinder, især dem, der allerede har parret 5 . For at teste disse akutte virkninger af miljømæssige forhold kan analysen klassisk anvendt til kvindelig parringsadfærd dogIkke meget passende på grund af de lange afbrydelser mellem parringspisoder. I den klassiske remitterende analyse mødes en jomfru kvinde først med en mand, og isoleres straks og præsenteres med en ny mand 24 til 48 timer senere. Dette klassiske assay er blevet anvendt med stor succes til at identificere komponenter af det mandlige ejakulat, der ændrer den kvindelige opførsel og kvindespørgsmålet 12 , 13 , 14 , 15 , 16 , 17 , 18 . Den kontinuerlige parringsanalyse demonstreret her er derfor en tilføjelse til klassiske parringsanalyser, der kan anvendes til at studere den akutte virkning af miljømæssige forhold på reproduktionsadfærd. Ved hjælp af det kontinuerlige assay for parringsadfærd, der forklares her, viste vi tidligere, at et par fluer udsat for gær vil remate sEveral gange over en 24 timers observationsperiode 5 , 19 , 20 , 21 , mens fluer ikke udsættes for mad, vil kun remate en gang 5 . Denne opdagelse kan være uoverskuelig i lyset af en stor del af D. melanogaster litteraturen, der tyder på, at kvinder ikke remitterer i flere dage efter en indledende parring (gennemgået i henvisninger 10 , 11 ). Denne uoverensstemmelse kan dog let forklares ved analysevilkår, hvor en kvinde er isoleret i en til flere dage, før der gives en ny parring mulighed. Hvis parret ikke parrer i denne times lange observationsperiode, er kvinden karakteriseret som ikke modtagelig. Desuden bør den høje parringsfrekvens ikke være overraskende, da dataene fra vildefangne ​​fluer viser, at kvinder indeholder sæd fra 4 til 6 mænd i deres opbevaringsorganer; Således iDicere, at kvinder normalt afholder flere gange 22 , 23 . Her demonstrerer vi brugen af ​​denne kontinuerlige parringsanalyse for at ophæve, hvordan fluer samler og kombinerer information om miljøforhold for at modulere parringsfrekvens. Dette assay gør det muligt for en at teste et relativt stort antal parringspar til genetiske undersøgelser og for at teste indflydelsen af ​​flygtige og ikke-flygtige miljøindikatorer. Assayet løber typisk i 24 timer, men kan forlænges til 48 timer, hvilket muliggør afprøvning af cykliske miljøindikatorer, såsom den lysmørke (LD) cyklus. Vi demonstrerer denne analyse ved at teste indflydelsen af ​​flygtige signaler fra en gærkultur i et trykluftsystem i kombination med tilgængeligheden af ​​ikke-flygtigt gærnæringsstof i fødevaresubstratet. Trykluftsystemet pumper kontinuerligt flygtige signaler ind i en parringsarena, der indeholderEt fødevaresubstrat og et testpar (hvis parringsadfærd overvåges). For yderligere at bestemme de særlige forhold, hvorigennem gær påvirker parring, tester vi en større flygtig forbindelse af gær, nemlig eddikesyre 24 , i kombination med et aminosyreindhold, der svarer til gærets i fødevaresubstratet i form af pepton (amino Syrer afledt af enzymatisk fordøjelse af animalske proteiner). Sammen viser disse eksperimenter, hvordan effekten af ​​miljøindikatorer på D. melanogasters parringsadfærd kan testes med dette assay.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel kitchen cabinet</td>
			<td>Horecaworld</td>
			<td>7412.0105</td>
			<td></td>
		</tr>
		<tr>
			<td>White LEDs</td>
			<td>Lucky Light</td>
			<td>ll-583wc2c-001</td>
			<td>Cold white, 20 mAmp and 2 V</td>
		</tr>
		<tr>
			<td>Red LEDs</td>
			<td>Lucky Ligt</td>
			<td>ll-583vc2c-v1-4da</td>
			<td>Wavelength between 625 nm, 20 mAmp and 6 V</td>
		</tr>
		<tr>
			<td>Resistor</td>
			<td>Royal Ohm</td>
			<td>CFR0W4J0561A50</td>
			<td>560 ohm, 0.25 W, 250 V and 5 % tolerance</td>
		</tr>
		<tr>
			<td>Smartphone light meter app</td>
			<td>Patrick Giudicelli</td>
			<td></td>
			<td>Light/Lux Meter FREE, version 1.1.1</td>
		</tr>
		<tr>
			<td>Power timer</td>
			<td>Alecto</td>
			<td>TS-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal brackets</td>
			<td></td>
			<td></td>
			<td>Sharp angle 5 by 5 mm,&#160; 2 x 5450 and 1 x 1100 mm long</td>
		</tr>
		<tr>
			<td>Frosted glass plate</td>
			<td></td>
			<td></td>
			<td>1190 x 545 x 5 mm</td>
		</tr>
		<tr>
			<td>Filter paper sheets</td>
			<td>LEE filters</td>
			<td>220</td>
			<td>White frost</td>
		</tr>
		<tr>
			<td>Small fan</td>
			<td>Nanoxia Deep silence</td>
			<td>4260285292828</td>
			<td>80 mm Ultra-Quiet PC Fan, 1200 RPM</td>
		</tr>
		<tr>
			<td>Big fan</td>
			<td>Nanoxia Deep silence</td>
			<td>4260285292910</td>
			<td>120 mm Ultra-Quiet PC Fan, 650-1500 RPM</td>
		</tr>
		<tr>
			<td>Webcam camera</td>
			<td>Logitech</td>
			<td>950270</td>
			<td>B910 HD WEBCAM OEM, Angle: 78-degree, resolution: 5-million-pixel&#160;&#160;</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>DeskShare</td>
			<td></td>
			<td>Security monitor pro</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fly rearing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly rearing bottles</td>
			<td>Flystuff</td>
			<td>32-130</td>
			<td>6oz Drosophila stock bottle</td>
		</tr>
		<tr>
			<td>Flypad</td>
			<td>Flystuff</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild-type flies</td>
			<td></td>
			<td></td>
			<td>Canton-S</td>
		</tr>
		<tr>
			<td>Fly rearing vials</td>
			<td>Dominique Dutscher</td>
			<td>789008</td>
			<td>Drosophila tubes narrow 25x95 mm</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Sanyo</td>
			<td>MIR-154</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic hot plate</td>
			<td>Heidolph</td>
			<td>505-20000-00</td>
			<td>MR Hei-Standard</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Caldic Ingredients B.V.</td>
			<td>010001.26.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Gezond&#38;wel</td>
			<td>1019155</td>
			<td>Dextrose/Druivensuiker</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Van Gilse</td>
			<td></td>
			<td>Granulated sugar</td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>Flystuff</td>
			<td>62-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat germ</td>
			<td>Gezond&#38;wel</td>
			<td>1017683</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy flour</td>
			<td>Flystuff</td>
			<td>62-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Molasses</td>
			<td>Flystuff</td>
			<td>62-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Active dry yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Flystuff</td>
			<td>20-258</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Peptone (bacto)</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td>1000631000</td>
			<td>Glacial, 100%</td>
		</tr>
		<tr>
			<td>Small petridish</td>
			<td>Greiner bio-one</td>
			<td>627102</td>
			<td>35 x 10 mm with vents</td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Bemis NA</td>
			<td></td>
			<td>Parafilm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast and pressurised air set-up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big petridish</td>
			<td>Gosselin</td>
			<td>BP140-01</td>
			<td>140 x 20.6 mm</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Millipore corporation</td>
			<td></td>
			<td>MiliQ</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar (pure)</td>
			<td>BD</td>
			<td>214530</td>
			<td>bacto</td>
		</tr>
		<tr>
			<td>Glucose (0(+)-glucose monohydrate)&#160;</td>
			<td>Merck</td>
			<td>18270000004</td>
			<td></td>
		</tr>
		<tr>
			<td>Open caps</td>
			<td>Schott</td>
			<td>29 240 28&#160;</td>
			<td>GL45</td>
		</tr>
		<tr>
			<td>Silicone septum</td>
			<td>VWR</td>
			<td>548-0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Barbed bulkhead fittings</td>
			<td>Nalgene</td>
			<td>6149-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Large PVC tubing</td>
			<td></td>
			<td></td>
			<td>diameter: outer 1.2 cm and inner 0.9 cm</td>
		</tr>
		<tr>
			<td>Small PVC tubing</td>
			<td></td>
			<td></td>
			<td>diameters: outer 0.8 cm and inner 0.5 cm</td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Falcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aquarium pump</td>
			<td>Sera precision</td>
			<td></td>
			<td>Sera air 110 plus, AC 220-240 V, 50/60 Hz, 3 W and pressure &#62;100 mbar</td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Superfish</td>
			<td>A8040400</td>
			<td>Norit activated carbon</td>
		</tr>
		<tr>
			<td>Disposible filter unit</td>
			<td>Whatman</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>VWR</td>
			<td>612-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot glue</td>
			<td>Pattex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Whatman</td>
			<td></td>
			<td>FP 30/pore size 0.45 mm CA-S</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistics software</td>
			<td>R</td>
			<td></td>
			<td>lme4 package</td>
		</tr>
	</tbody>
</table>

a method to test the effect of environmental cues on mating behavior in drosophila melanogaster

En persons seksuelle drev påvirkes af genotype, erfaring og miljøforhold. Hvordan disse faktorer interagerer med at modulere seksuel adfærd forbliver dårligt forstået. I Drosophila melanogaster påvirker miljømæssige signaler, såsom fødevaretilgængelighed, parringsaktivitet, der tilbyder et traktabelt system til at undersøge mekanismerne, der modulerer seksuel adfærd. I D. melanogaster registreres miljømærker ofte via kemosensoriske gustatoriske og olfaktoriske systemer. Her præsenterer vi en metode til at teste virkningen af ​​miljømæssige kemiske tegn på parringsadfærd. Assayet består af en lille parringsarena indeholdende madmedium og parringpar. Parringsfrekvensen for hvert par overvåges kontinuerligt i 24 timer. Her præsenterer vi anvendeligheden af ​​dette assay for at teste miljøforbindelser fra en ekstern kilde gennem et trykluftsystem samt manipulering af miljøkomponenterne direkte i parringsarenaen. Den uSe et trykluftsystem er især nyttigt til at teste virkningen af ​​meget flygtige forbindelser, mens manipulering af komponenter direkte i parringsarenaen kan være af værdi for at fastslå en forbindelses tilstedeværelse. Dette assay kan tilpasses til at svare på spørgsmål om indflydelse af genetiske og miljømæssige tegn på parringsadfærd og fecundity samt andre reproduktive adfærd hos mænd og kvinder.

Ved anvendelse af denne kontinuerlige analyse, parringsadfærd og parringsfrekvens i specifikke kan bestemmes under eksperimentelle miljøforhold. For at styre miljøforholdene forvandlede vi et køkkenskab af rustfrit stål til et testområde med egen lyskilde og diffusion, hvilket sikrer en høj overflod af lys og et minimum af blænding fra toppen af ​​parringsarenaerne ( Figur 1A ) . Det indre testområde er helt indkapslet af rustfrit stål og glas, hvilket gør...

Watch this Scientific Journal Video about A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster at JoVE.com

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

Vi demonstrerer en analyse for at analysere de miljømæssige og genetiske signaler, der påvirker parringsadfærd i frugtfly Drosophila melanogaster .

En metode til at teste effekten af ​​miljøvinkler på parringsadfærd i<em&gt; Drosophila melanogaster</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

The overall goal of this behavioral assay, is to quantify the effect of environmental queues on reproductive behaviors, such as courtship and mating. This method can help answer key questions in the field of behavioral biology and eurogenetics. Such as the inference of genetic and environmental factors on reproductive behaviors and the mechanisms that moderate those behaviors.This technique offers speed and flexibility in testing a wide range of environmental queues that affect reproduction. And that with a very good number of replicates. Demonstrating the procedure will be Jenke Gorter a grad student from my lab.Begin the experiment by preparing one liter of rich fly medium. Pour one liter of tap water in a two liter glass beaker with magnetic stir bar. And put the beaker on a magnetic hot plate.Keep the stirring off and turn the heat up to 300 degrees Celsius, until the boiling temperature is reached. Turn on the stirring to 500 rpm and add the ingredients to the boiling water. Wait for the yeast to foam vigorously.Then turn down the hot plate temperature to 120 degrees Celsius. After 10 minutes turn the hot plate down to 30 degrees Celsius. And let the mixture stir, until it cools to 48 degrees Celsius.Monitor the temperature by inserting a thermometer directly into the food. Dissolve two grams of Tegosept into 10 milliliters of 96%ethanol. Combine the mixture and five milliliters of one molar propionic acid to the beaker.Stir for three minutes. Heat a needle to redness in a bunsen burner. And pierce a hole 0.3 centimeters in diameter on the upper side of a 3.5 centimeter by 1.0 centimeter plastic Petri dish.When preparing food medium, with odorous compounds, first pipette 30 microliters of the desired compound into the dish for 1/2 of the experimental dishes. Leave the other 1/2 of the dishes empty for comparison. Using a five milliliter serological pipette pour three milliliters of food medium, covering the bottom of the dish.Cover it with a cheesecloth to prevent contamination and allow the medium to solidify for one hour, at room temperature. Next, place a lid on the dishes and tape the lids shut on two sides. Prepare small paraffin film plugs to cover the holes of the dishes by rolling pieces of paraffin film into zero point two centimeter thick rolls.And then cut them into zero point five centimeters segments. Cut two small holes in the septum to snugly fit barbed, bulkhead fittings. Fit open four point five centimeter bottle caps with a zero point three two centimeter thick Silcone septum.Attach the small PVC tubing to both outlets that exit the bottle and to only one of the inlets entering the bottle. Next, attach a glass tube to the outlet of each YPD culture bottle, using small tubing. Fill the tube with glass fiber.And Autoclave it before use. Wrap the fitted caps and tubing in aluminum foil. And Autoclave them for 25 minutes at 120 degrees Celsius and one bar pressure.Dip a sterile 100 microliter pipette tip into one of the yeast colonies from the YPD Agar plate and drop it into the Autoclaved YPD liquid medium bottle. Cap this yeast and oculated YPD liquid medium bottle, as well as, a YPD medium control bottle, without yeast, with Autoclaved caps, mounted within an outlet. To prevent contamination of the YPD medium with microorganisms, attach the small tubing to a zero point four five micrometer sterile syringe filter, with a plastic push on the bulkhead tubing connector, going toward the filter.And a screw on plastic bulkhead connector leaving the filter. Then attach the tubing to the inlet of the YPD culture bottle. Place the bottles on separate magnetic plates.And stir the solution at 100 rpm, at room temperature, for 24 hours before the start of the experiment. Connect the inlets of both bottles to a tube connected to pressurized air. Make sure to connect the outlet of the experimental yeast bottle to a tube venting the yeast smell out of the experimental room, to prevent interference with the experiment.Attach PVC tubing to the outlet side of the glass tube, and lead this toward the lower holes of the experiment box. Using a T-splitter, with outer diameter greater than or equal to zero point five centimeters, and short pieces of small PVC tubing, attach two serological pipettes to each of the two outlets at both sides. Tape the pipettes flat on the white paper sheet under the cameras, in the steel box.Use a mouth pipette to place one experimental female into a small Petri dish at ZT six. And allow the fly to acclimatize to the mating arena for one hour. After one hour, at ZT seven, transfer a wild type male to the Petri dish.And click Start Monitoring for 24 hours. For the air pump experiment, place the dish in such a way, that a pipette outlet is connected to the entrance hole of the mating arena. To analyze the mating behavior of the couple, select an open all pictures, in an image viewing software.And page through them in chronological order. Write down the date, experiment number, dish number, and start time, into a spreadsheet within the same row. Take the start time of each arena from the moment it is placed under the web cam camera.Record the time stamp from the pictures. Mark the start time of each copulation in the same row, into the spread sheet. Count a mating as an incident, when the male has mounted the female and the couple remains moderately stationery and in the same posture, for at least five consecutive frames.Analysis of yeast air, separated for food medium, shows that a mating couple does not respond to yeast odors when there is no yeast present in the food medium. But they do increase their mating frequency in yeast air when yeast is also added to the food medium. Female receptivity increases upon the presence of Acetic Acid.But only when Peptone is present in the medium, demonstrating that the flies need to simultaneously sense Amino Acids and Acetic Acid, to increase their mating frequency. Once mastered, this technique can be set up in six hours. And the behavioral analysis can be performed in five hours, if done properly.

Research

JoVE Journal

Behavior

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

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