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 other two sets on each side of the box at a height of 35 cm from the bottom. 
		NOTE: The four sets of four holes are used for camera power cables and air pump tubing to enter and exit the cabinet. The hole at the back is used for the power cables of a light board.</li>
		<li>Build a light board with 18 rows of 40 alternating white and red light-emitting diodes (LED) with 2.5 cm space between each light.Mount the white and red LEDs in a circuit with power supply. Connect each LED in series with a resistor of 560 &#8486;, 0.25W and 5% tolerance. 
		NOTE: The emission wavelengths of the red light spanned from 590-661 nm with a sharp peak at 627 nm. The resulting light intensity in the experimental area is approximately 900 lux with both lights on and 90 lux with only the red lights on; this was measured using a smartphone light meter app.</li>
		<li>Attach the light board to the top of the stainless-steel cabinet and pass the power cables through the hole at the back.</li>
		<li>Connect the adapter of the white LEDs to a power control timer to allow switching off the white LED during the dark phase of the experiment. Connect the adapter of the red light, which flies are blind to25, to a regular power supply to keep them on for the whole duration of the experiment.</li>
		<li>Fix one 110 cm and two 54 cm long metal brackets, each with a width of 0.5 cm, to the inner sides of the box at a height 50 cm from the bottom of the box. Place a frosted glass diffusion plate (dimensions of 119.0 cm x 54.5 cm x 0.5 cm) on these brackets.</li>
		<li>Add three layers of filter paper (120 cm x 50 cm) in between the light board and glass plate to diffuse the light and limit glare on the surface of the mating arenas (described in section 4). Pin two filter paper sheets together on their long edge on 120 cm long wooden rods using magnets (on the side), to stick them to the insides of the metal cabinet; perform this action 3 times.</li>
		<li>Attach four fans at the sides of the box to create an air stream that continuously vents the mating box. Attach the first set of 8 cm fans between the light board and glass plate, with the inlet of air at the left side and the exhaust at the right side of the box, to minimize the building-up of heat generated by the light board.</li>
		<li>Attach the second set of 12 cm fans 25 cm above the bottom of the cabinet to create an outward air flow that vents the inside of the cabinet and cools it to a stable 26 &#176;C. Attach the fans at the exhaust side to a suction hose and lead the airstream out of the room to prevent re-cycling of the air in the cabinet.</li>
	</ol>
	</li>
	<li>Set up two stands (approximately 48 cm tall) mounted with two clamps, one at 28 cm and one at 30 cm from the base of the stand. Fix a webcam onto each of the clamps. Connect the 4 cameras to a computer running the monitoring software.</li>
	<li>Place A4 sheets underneath each webcam. Use unprinted white sheets or sheets with pre-numbered grid of 7 by 5 squares with 4 cm axes to accommodate mating arenas (described in section 4). 
	NOTE: An HD webcam camera with 78&#176; wide-angle view and 5-million-pixel resolution can cover an area of 21 cm x 30 cm corresponding to an A4 sheet and monitor between 20 and 35 mating arenas.</li>
</ol>
2. Fly Rearing and Collection
<ol>
	<li>Place 20 male and 20 female wild-type Canton-S flies into fly rearing bottles containing 45 mL rich fly food medium (see section 3) for three to four days. Transfer the same adults three times by first tapping them down and then into fresh bottles.
	<ol>
		<li>Place the bottles in an incubator at 25 &#176;C, and 12 h: 12 h light-dark cycle with lights on at 09:00 (Zeitgeber time (ZT) 0). A new generation will appear about 10 days later.</li>
	</ol>
	</li>
	<li>Anesthetize the resulting newly eclosed flies on carbon dioxide pads for no longer than 5 minutes and collect them into fly food vials using a paint brush.
	<ol>
		<li>Collect virgin (newly eclosed) females and virgin males from the wild-type Canton-S stock bottles into 2.5 cm x 9.5 cm fly rearing vials with 6.5 mL of rich fly food medium.</li>
	</ol>
	</li>
	<li>Age the flies in same sex groups of 20 flies each in fly rearing vials for 5 to 8 days at 25 &#176;C and 12 h: 12 h light-dark cycle and lights on at 09:00 (ZT 0).</li>
	<li>Transfer the flies to fresh fly rearing vials on the day before the experiment.</li>
</ol>
3. Food Medium Preparation
<ol>
	<li>Prepare 1 L of rich fly medium as follows.
	<ol>
		<li>Pour 1 L of tap water in a 2 L glass beaker with a magnetic stir bar and put the beaker on a magnetic hot plate. Keep the stirring off and turn the heating up to 300 &#176;C until boiling temperature is reached. 
		NOTE: During the long boiling time in the following steps a proportion of water will evaporate, but together with the added ingredients this protocol results in 1 L of rich fly medium when prepared at room temperature of approximately 22 &#176;C.</li>
		<li>Turn on the stirring to 500 rounds per min (rpm) and add the following ingredients to the boiling water: 10 g agar, 30 g glucose, 15 g sucrose, 15 g cornmeal, 10 g wheat germ, 10 g soy flour, 30 g molasses, 35 g active dry yeast. Wait for the yeast to foam vigorously, then turn down the hot plate temperature to 120 &#176;C.</li>
		<li>After 10 min turn the hot plate down to 30 &#176;C and let the mixture stir until cooled to 48 &#176;C. Monitor temperature by inserting a thermometer directly into the food.</li>
		<li>Dissolve 2 g of p-hydroxy-benzoic acid methyl ester (tegosept,100%) into 10 mL of 96% ethanol. Add this and 5 mL of 1 M propionic acid to the mixture. Stir for 3 min.</li>
		<li>Pour the fly food medium into the arenas (described in section 4) to create a 0.3 cm thick layer at the bottom of the arena.
		<ol>
			<li>Use a 200 mL glass beaker for pouring. When exact quantities are important, use a 10 mL serological pipette.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare fly medium minus yeast exactly as described in step 3.1.1 until 3.1.5, but leave out the yeast in step 3.1.2.</li>
	<li>Prepare medium with agar with or without peptone by mixing 10 g agar and 35 g peptone in 1 L of boiling water and perform steps 3.1.4 to 3.1.5.</li>
</ol>
4. Mating Arena Preparation
<ol>
	<li>Pierce a hole approximately 0.3 cm in diameter on the upper side of a 3.5 cm x 1.0 cm plastic Petri dish using a heated preparation needle (heated to redness in a Bunsen burner). Alternatively, use a soldering iron.</li>
	<li>When preparing food medium with odorous compounds, first pipette 30 &#181;L (1% of the final food medium, e.g. Acetic Acid glacial 100%) of desired compound into the dish for half of the experimental dishes. Leave the other half of the dishes empty for comparison. 
	NOTE: With the set-up described here, a maximum of 140 arenas can be tested at once.</li>
	<li>Using a 10 mL serological pipette, pour 3 mL of food medium at the bottom of the dish on top of the desired compound. Cover it with a cheese cloth to prevent contamination, and leave the medium to solidify for approximately 1 h at room temperature.</li>
	<li>Place a lid on the dishes and tape each shut at two sides. Prepare small paraffin film plugs to cover the holes of the dishes by rolling pieces of paraffin film into 0.2 cm thick rolls and then cut them into 0.5 cm segments.</li>
</ol>
5. Yeast Culture for Odor
<ol>
	<li>Grow dry active yeast on yeast extract peptone dextrose (YPD) agar in a 14.0 cm x 2.06 cm Petri dish. Wear gloves to prevent contamination in this step.
	<ol>
		<li>Prepare YPD agar plates by adding 10 g yeast extract, 20 g peptone, 22 g glucose (0(+)-glucose monohydrate) and 15 g agar (pure) to 1 L of boiling ultrapure water. Layer the bottom of the Petri dish once everything is dissolved and store upside down in the refrigerator at 4 &#176;C, for up to 2 months.</li>
		<li>Sprinkle a few grains of dried yeast on a YPD medium plate, let them dissolve. Then streak the medium plate using a sterile loop. Store the plate in a 30 &#176;C incubator overnight. Afterwards, store the culture in the refrigerator for no longer than 1 week.</li>
	</ol>
	</li>
	<li>Prepare YPD liquid medium in 1 L bottles by adding 10 g yeast extract, 20 g peptone, 22 g glucose (0(+)-glucose monohydrate) and a stir bar to 1 L ultrapure water.
	<ol>
		<li>Autoclave for 25 min at 120 &#176;C and 1 bar pressure. Afterwards, store the bottles at 4 &#176;C for up to 2 months until use.</li>
	</ol>
	</li>
	<li>Fit open bottle caps (4.5 cm) with a 0.32 cm thick silicone septum.
	<ol>
		<li>Cut two small holes in the septum to snugly fit barbed bulkhead fittings. Attach the small polyvinyl Chloride (PVC) tubing (diameters: outer 0.8 cm and inner 0.5 cm) to both outlets that exit the bottle and to only one of the inlets entering the bottle. See figure 1B for illustration.</li>
		<li>Wrap the fitted caps and tubing in aluminum foil and autoclave for 25 min at 120 &#176;C and 1 bar pressure.</li>
	</ol>
	</li>
	<li>Wear gloves to protect against contamination in this step. Dip a sterile 100 &#181;L pipette tip into one of the yeast colonies from the YPD agar plate (described in 5.1) and drop it into the autoclaved YPD liquid medium bottle.
	<ol>
		<li>Cap this yeast-inoculated YPD liquid medium bottle as well as a YPD medium control bottle (yeast not added) with autoclaved caps mounted with in- and outlet (described in step 5.3). Put both bottles on separate magnetic plates and stir at 100 rpm at room temperature for 24 h before the start of the experiment to allow the yeast culture to grow.</li>
		<li>Connect the inlets of both bottles to separate aquarium pumps to supply air to the yeast culture. 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.</li>
	</ol>
	</li>
</ol>
6. Air Pump Set-up
<ol>
	<li>Attach the large PVC tubing (diameters: outer 1.2 cm and inner 0.9 cm) to a pressurized air supply and lead it through two 1 L glass Erlenmeyer flasks filled with activated charcoal to the 800 mL line to purify the air. Use either pressurized air commonly supplied in labs as air supply, or connect the tubes to an air pump (pressurized air was used here). 
	NOTE: Tubing material should be selected based on the chemical properties of the volatile, and tested to prevent the volatile from sticking to the lining of the tubing (e.g. polytetrafluoroethylene, nylon or stainless steel).</li>
	<li>Make two air splitters from 15 mL tubes and three 1,000 &#181;L pipette tips each.
	<ol>
		<li>Make three holes of ~1 cm diameter. First burn two holes, using a heated preparation needle (heated red in a Bunsen burner), adjacent to each other just below the lid of the 15 mL tube. Then make the third hole by removing the bottom of the tube.</li>
		<li>Glue the 1,000 &#181;L pipette tips into the holes with the narrow end pointing outward. Cut the end of the pipette tips to allow greater air flow.</li>
	</ol>
	</li>
	<li>Connect the large PVC tubing from the outlet of the charcoal-filled Erlenmeyer flask to the pipette tip at the bottom of the 15 mL tube. Add small PVC tubing outlets to the two horizontal pipette tips and lead them towards a control and an experimental bottle.</li>
	<li>To prevent contamination of the YPD medium with microorganisms, attach the small tubing to a sterile syringe filter (0.45 &#181;m pore size) with a plastic push on bulkhead tubing connector going towards the filter, and a screw on plastic bulkhead connector leaving the filter. Then, attach the tubing to the inlet of the YPD culture bottle (see Figure 1B).</li>
	<li>To prevent airborne yeast from traveling from the culture flask into the experimental arena, attach a glass tube (6.5 cm long, outer diameter = 0.5 cm and inner diameter = 0.3 cm) to the outlet of each YPD culture bottle using small tubing. Attach PVC tubing to the other side of the glass tube and lead this towards the lower holes (drilled at each side) of the experiment box.
	<ol>
		<li>Fill the tube with glass fiber and autoclave it before use.</li>
	</ol>
	</li>
	<li>Add another 15 mL tube splitter (described in section 6.2) to the small PVC tubing, at each side of the experimental box, to get two tubes running into the box at both the experimental side (closest to exhaust of fan air stream, right) and the control side (closest to the inlet of the fan air stream, left).</li>
	<li>Prepare 8x 25 mL serological pipettes each with 10 outlets to test 80 mating couples at the same time, i.e. 40 for each air condition.
	<ol>
		<li>Burn 10 holes of ~0.8 cm diameter, 2 cm apart into the pipette.</li>
		<li>Cut the outer part of a 1 mL syringe into one small (2.5 cm) and one big (5 cm) outlet.</li>
		<li>Glue these outlets into the holes with hot glue.</li>
		<li>Wrap a small band of plastic paraffin film around the end of the outlets and attach a 1,000 &#181;L pipette tip. Diameter of the tip opening is 0.1 cm. Use clean tips for each experiment.</li>
	</ol>
	</li>
	<li>Attach two serological pipettes, using a T-splitter with outer diameter &#8805;0.5 cm and short pieces of small PVC tubing, 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).</li>
	<li>Using an air flow monitor, set the air flow so that air velocity at the exit of the 1000 &#181;L pipette tip is 0.5 m/s. This corresponds to an air flow of 0.0017 L/s per tip.</li>
</ol>
7. Monitoring of Mating Behavior
<ol>
	<li>Use a mouth pipette (as described in reference26) to place one experimental female into a small Petri dish (described in section 4) at 15:00 o&#39;clock (ZT 6) and give her 1 h to acclimatize to the mating arena.</li>
	<li>Set up the experimental box (described in section 1) as follows:
	<ol>
		<li>Turn on the lights, i.e. white light LEDs on a 12h :12 h light-dark cycle connected to a timer that switches on the light at 09:00 (ZT0) and continuous red light LEDs to allow monitoring of the flies during the dark phase of the experiment. Turn on the fans to limit heating up of the cabinet by the light source and to ensure excess odors are vented outside the test area.</li>
		<li>Connect webcam cameras to a computer and start them with the monitoring software for picture monitoring.</li>
		<li>For each camera, set the focus, brightness, and zoom in the monitoring software.
		<ol>
			<li>Right-click on the camera screen, open &#34;camera properties,&#34; and unclick &#34;automatic focus.&#34; Adjust the &#34;focus&#34; to clarify of the grid or any written words on the paper sheet. If necessary, change the &#34;brightness&#34; and &#34;zoom.&#34;</li>
		</ol>
		</li>
		<li>Set the program of the monitoring software to capture 1 picture every 2 min. Right-click on each camera screen and choose &#34;edit camera&#34; and then the &#34;Actions&#34; option. Click to start the actions &#34;At regular intervals&#34; and change the time to &#34;2 minutes.&#34; Choose &#34;Take Photo&#34; for select actions to perform and finally click on &#34;ok.&#34;</li>
		<li>Right-click on each camera screen and select &#34;start monitoring.&#34;</li>
	</ol>
	</li>
	<li>After 1 h (at ZT 7), transfer a wildtype male to the Petri dish using the mouth pipette, place the dish on the A4 paper sheetsunder the webcam cameras, and click &#34;start monitoring&#34; for 24 h. 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.</li>
	<li>To analyze the mating behavior of the couple, do the following.
	<ol>
		<li>Select and open all pictures in an image viewing software and page through them in chronological order.</li>
		<li>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 that it is placed under the webcam camera. Record the time stamp from the pictures.</li>
		<li>Mark the start time of each copulation in the same row into the spreadsheet. Count a mating as an incident when the male has mounted the female and the couple remains moderately stationary and in the same posture for at least five consecutive frames (10 min). 
		NOTE: This criterion is based on the reported length of copulation, ranging from 12 to 27 min in D. melanogaster, and the observation that copulations of 10 min and over are fertile27,28.</li>
		<li>Count the number of copulations for each row in the spreadsheet to determine the mating frequency. Alternatively, subtract the start time of the experiment from the time of first mating for each row as a measure of mating latency, or subtract the time of the first mating from the time of the second mating as a measure of remating latency.
		<ol>
			<li>To calculate the remating latency, make sure to define the dates of the first and the second mating as consecutive days in the spreadsheet software.</li>
		</ol>
		</li>
		<li>Analyze the data with mixed effects models, assuming normal distribution of the data and including the date of the experiment as a random factor, using a statistical software (see the table of materials) to determine the statistical significance of the independent variables&#8211;food medium, air type, and interaction&#8211;as previously described5.</li>
		<li>Select the best explaining model by performing the backwards elimination of non-significant independent variables using log-likelihood ratio tests and the associated Akaike information. After running the model, visually inspect the data residuals to confirm normality. Confirm the homogeneity of variances using the Levene&#39;s test. In the case of unequal homogeneity, square-root transform the data.</li>
	</ol>
	</li>
</ol>

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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.

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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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Yeast and pressurised air set-up&lt;/strong&gt;</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 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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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analysis&lt;/strong&gt;</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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Behavior

9.0K Views.  University of Groningen. We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

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

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

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

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Chapters in this video

Methods to Assay Drosophila Behavior

A Single-fly Assay for Foraging Behavior in Drosophila

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Conditions Affecting Social Space in Drosophila melanogaster

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Drosophila Courtship Conditioning As a Measure of Learning and Memory