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.
