We thank the Bloomington Drosophila Stock Center for the fly stocks; C. Gahr, J. T. Alkema, and S. van Hasselt for their early attempt at developing the pressurized air assay; Jasper Bosman for the advice on cultivating yeast; and Rezza Azanchi and Joel Levine for originally developing the time-lapse monitoring of Drosophila mating behavior. J.A. Gorter was supported by a Neuroscience Research School BCN/NWO Graduate Program grant. This work was supported in part by the Dutch organization for scientific research (NWO) (reference: 821.02.020) to J.C. Billeter.

1. Environmentally Controlled Mating Box
<ol>
	<li>To ensure a controlled and easy to clean test area, setup a stainless-steel kitchen cabinet of 120 cm x 64 cm x 85 cm as illustrated in Figure 1A.
	<ol>
		<li>Drill one hole at the back of the cabinet just below the ceiling and four sets of four holes into the sides, each with a diameter of 2 cm. Drill the first two sets of four holes, on each side of the box at a height of 7 cm from the bottom of the box and with 12.5 cm in between holes. Drill the ot...

<ol>
	<li>Hileman, S. M., Pierroz, D. D., Flier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin+nutrition,+and+reproduction:+timing+is+everything.">Leptin nutrition, and reproduction: timing is everything.</a> J. Clin. Endocrinol. Metab. 85 (2), 804-807 (2000).</li><li>Grosjean, Y., 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=An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila.">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478 (7368), 236-240 (2011).</li><li>Harshman, L. G., Hoffman, A. A., Prout, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+effects+on+remating+in+Drosophila+melanogaster.">Environmental effects on remating in Drosophila melanogaster.</a> Evolution. 42 (2), 312-321 (1988).</li><li>Fricke, C., Bretman, A., 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=Female+nutritional+status+determines+the+magnitude+and+sign+of+responses+to+a+male+ejaculate+signal+in+Drosophila+melanogaster.">Female nutritional status determines the magnitude and sign of responses to a male ejaculate signal in Drosophila melanogaster.</a> J. Evol. Biol. 23 (1), 157-165 (2010).</li><li>Gorter, J. A., Jagadeesh, S., Gahr, C., Boonekamp, J. J., Levine, J. D., Billeter, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nutritional+and+hedonic+value+of+food+modulate+sexual+receptivity+in+Drosophila+melanogaster+females.">The nutritional and hedonic value of food modulate sexual receptivity in Drosophila melanogaster females.</a> Sci. Rep. , 1-10 (2016).</li><li>Wigby, S., 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=Insulin+signalling+regulates+remating+in+female+Drosophila.">Insulin signalling regulates remating in female Drosophila.</a> Proc. Biol. Sci. 278 (1704), 424-431 (2011).</li><li>Ribeiro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dilemmas+of+the+gourmet+fly:+the+molecular+and+neuronal+mechanisms+of+feeding+and+nutrient+decision+making+in+Drosophila.">The dilemmas of the gourmet fly: the molecular and neuronal mechanisms of feeding and nutrient decision making in Drosophila.</a> Front. Neurosci. 7, 1-13 (2013).</li><li>Walker, S. J., Corrales-Carvajal, V. M., Ribeiro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmating+circuitry+modulates+salt+taste+processing+to+increase+reproductive+output+in+Drosophila.">Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila.</a> Curr. Biol. 25 (20), 2621-2630 (2015).</li><li>Hussain, A., &#220;&#231;punar, H. K., Zhang, M., Loschek, L. F., Grunwald Kadow, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptides+modulate+female+chemosensory+processing+upon+mating+in+Drosophila.">Neuropeptides modulate female chemosensory processing upon mating in Drosophila.</a> PLoS Biol. 14 (5), e1002455-e1002428 (2016).</li><li>Avila, F. W., Sirot, L. K., LaFlamme, B. A., Rubinstein, C. D., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+seminal+fluid+proteins:+identification+and+function.">Insect seminal fluid proteins: identification and function.</a> Annu. Rev. Entomol. 56 (1), 21-40 (2011).</li><li>Laturney, M., Billeter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenetics+of+female+reproductive+behaviors+in+Drosophila+melanogaster.">Neurogenetics of female reproductive behaviors in Drosophila melanogaster.</a> BS:ADGEN. 85, 1-108 (2014).</li><li>Liu, H., Kubli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-peptide+is+the+molecular+basis+of+the+sperm+effect+in+Drosophila+melanogaster.">Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. U S A. 100 (17), 9929-9933 (2003).</li><li>Ram, K. R., Wolfner, M. 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. Sci. 276 (1662), 1705-1711 (2009).</li><li>Dukas, R., Jongsma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costs+to+females+and+benefits+to+males+from+forced+copulations+in+fruit+flies.">Costs to females and benefits to males from forced copulations in fruit flies.</a> Anim. Behav. 84 (5), 1177-1182 (2012).</li><li>Yorozu, S., 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=Distinct+sensory+representations+of+wind+and+near-field+sound+in+the+Drosophila+brain.">Distinct sensory representations of wind and near-field sound in the Drosophila brain.</a> Nature. 457 (7235), 201-205 (2009).</li></ol>

The authors have no competing interests to disclose.

This protocol describes an assay to test mating behavior over 24 h while continuously controlling the environmental cues that a mating couple is hypothesized to use to determine mating frequency. It is possible to increase the mating frequency in response to yeast air delivered through a pressurized air system when the medium contains yeast as well (Figure 2B). Additionally, a similar response in mating frequency can be observed with a simplified food medium containing only agar, peptone, and acetic acid...

Reproductive behaviors typically have high energy costs, especially for females, who produce larger gametes than males and must carefully chose the conditions to raise their developing offspring. Because of the energy cost, it is not surprising that reproduction is connected to nutritional conditions. This is true in most, if not all, animals including mammals, whose puberty can be delayed by malnutrition, and whose sexual drive can be negatively affected by food-restriction1.
The reproduction of the genetic model organism Drosophila melanogaster is also affected by nutritional conditions. Males court at higher level in the presence of food volatiles2, and females are more sexually receptive in the presence of yeast, a major nutrient for egg production and offspring survival3,4,5. This evolutionary conserved reproductive response to food offers the opportunity to study mechanisms that connect environmental food availability to sexual reproduction in a genetically tractable and time-efficient organism. Indeed, work in D. melanogaster has implicated the insulin pathway as an important regulator of the connection between food and mating behavior6. It has also shown that the act of mating itself changes the food preference of females as well as the associated chemosensory neurons7,8,9.

It is clear that food cues affect reproductive behaviors in D. melanogaster. These effects seem to mainly affect females, specifically those who have already mated5. However, to test these acute effects of environmental conditions the assay classically used for female mating behavior might not be very suitable due to the long interruptions between mating episodes. In the classic remating assay, a virgin female first mates with a male, and is immediately isolated and presented with a new male 24 to 48 h later. This classic assay has been used with great success to identify components of the male ejaculate that modify the female behavior and the female response12,13,14,15,16,17,18. The continuous mating assay demonstrated here, is therefore, an addition to classic mating assays that can be used to study the acute effect of environmental conditions on reproductive behaviors.
Using the continuous assay for mating behavior that is explained here, we previously showed that a pair of flies exposed to yeast will remate several times over a 24 h observation period5,19,20,21, while flies not exposed to food will only remate once5. This finding can be puzzling in the light of a large portion of the D. melanogaster literature indicating that females do not remate for several days after an initial mating (reviewed in references10,11). However, this discrepancy can easily be explained by assay conditions, where a female is isolated for one to several days before a new mating opportunity is provided. If the pair does not mate in this hour-long observation period, the female is characterized as not receptive. Moreover, the high mating frequency should not be surprising given that the data from wild-caught flies show that females contain sperm from 4 to 6 males in their storage organs; thus indicating that females naturally remate several times22,23.

Here, we demonstrate the use of this continuous mating assay to unravel how flies gather and combine information about environmental conditions to modulate mating frequency. This assay allows one to test a relatively large number of mating couples for genetic studies and to test the influence of volatile and non-volatile environmental cues. The assay typically runs for 24 h, but can be extended to 48 h, allowing the testing of cycling environmental cues such as the light-dark (LD) cycle. We demonstrate this assay by testing the influence of volatile cues from a yeast culture within a pressurized air system in combination with the availability of non-volatile yeast nutrient in the food substrate.
The pressurized air system continuously pumps volatile cues into a mating arena that contains a food substrate and a test couple (whose mating behavior is monitored). To further determine the specifics through which yeast influences mating, we test a major volatile compound of yeast, namely acetic acid24, in combination with an amino acid content that corresponds to that of yeast in the food substrate, in the form of peptone (amino acids derived from enzymatic digestion of animal proteins). Together these experiments demonstrate how the effect of environmental cues on the mating behavior of D. melanogaster can be tested with this 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

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

Using this continuous assay, mating behavior, and mating frequency in specific, can be determined under experimental environmental conditions. To control environmental conditions, we transformed a stainless-steel kitchen cabinet into a test area, with its own light source and diffusion, which ensures a high abundance of light and a minimum amount of glare from the top of the mating arenas (Figure 1A). The inner test area is completely encased by stainless steel and glass,...

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

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

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

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.

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JoVE Journal

Behavior

9.0K visualizações.  University of Groningen. 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 reprodu...