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

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>
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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. 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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. 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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.

a-method-to-test-effect-environmental-cues-on-mating-behavior

Research

JoVE Journal

Behavior

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Measuring Sensitivity to Viewpoint Change with and without Stereoscopic Cues

The speed and accuracy of object recognition is compromised by a change in viewpoint; demonstrating that human observers are sensitive to this transformation. Here we discuss a novel method for simulating the appearance of an object that has undergone a rotation-in-depth, and include an exposition of the differences between perspective and orthographic projections. Next we describe a method by which human sensitivity to rotation-in-depth can be measured. Finally we discuss an apparatus for creating a vivid percept of a 3-dimensional rotation-in-depth; the Wheatstone Eight Mirror Stereoscope. By doing so, we reveal a means by which to evaluate the role of stereoscopic cues in the discrimination of viewpoint rotated shapes and objects.

We discuss a novel method forviewpoint-rotation of visual stimuli, and demonstrate using a mirror stereoscopethe three-dimensional percept of rotation-in-depth. The technique can be used to investigate the role of stereoscopic cues in encoding viewpoint-rotated figures.

The speed and accuracy of object recognition is compromised by a change in viewpoint; demonstrating that human observers are sensitive to this ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen distribution. It is also possible that aberrant behavior of oxygen-sensing neurons could induce hypoxia-like behavior in the presence of normal oxygen levels. In D. melanogaster, development at low oxygen levels results in inhibition of growth and sluggish behavior during the larval phases. However, these established manifestations of oxygen deficit overlap considerably with the phenotypes of many mutations that regulate growth, stress responses or locomotion. As result, there is currently no assay available to identify i) cellular hypoxia induced by a mutation or ii) hypoxia-like behavior when induced by abnormal neuronal behavior.
We have recently identified two distinctive behaviors in D. melanogaster larvae that occur at normal oxygen levels in response to internal detection of hypoxia. First, at all stages, such larvae avoid burrowing into food, often straying far away from a food source. Second, tunneling into a soft substratum, which normally occurs during the wandering third instar stage is completely abolished if larvae are hypoxic. The assay described here is designed to detect and quantitate these behaviors and thus to provide a way to detect hypoxia induced by internal damage rather than low external oxygen. Assay plates with an agar substratum and a central plug of yeast paste are used to support animals through larval life. The positions and state of the larvae are tracked daily as they proceed from first to third instar. The extent of tunneling into the agar substratum during wandering phase is quantitated after pupation using NIH ImageJ. The assay will be of value in determining when hypoxia is a component of a mutant phenotype and thus provide insight into possible sites of action of the gene in question.

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

The protocol describes a simple assay to identify Drosophila melanogaster larvae that are experiencing hypoxia under normal atmospheric oxygen levels. This protocol allows hypoxic larvae to be distinguished from other mutants that show overlapping phenotypes such as sluggishness or slow growth.

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents&#39; proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The ...

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.