The overall goal of this assay is to measure and manipulate the male Drosophila mating drive in order to examine motivation in a reductionist system. This method can help answer key questions in the study of motivation like what genes and neurons control mating drive potentially revealing the logic behind the motivational circuitry. The main advantage of this method is that it provides a high throughput assay for measuring mating drive that can be used in concert with the powerful genetic tools available in the fly.
At least three days before a 2-D satiety assay prepare female flies from a bottle of white 11 18 over heat shock hid fly stock. Once 20 to 50%of the pupae have eclosed and there are at least five males to propagate the stock, flip flies in to a new bottle. Next heat shock the original bottle in a 37 degree Celsius hot water bath for one hour to sufficiently activate heat shock hid to kill male larvae and pupae.
After the heat shock only females will eclose from these bottles. To prepare the arena for experiments partially assemble the 2-D satiety arena and tighten it with thumb nuts and hex screws. Next place fresh fly food in to a clean, microwave safe bottle and add just enough water to cover the bottom surface.
Microwave the food on high for 30 to 45 seconds. Visually check the progress and stir the food every 15 seconds until it is melted. Once the food is melted use a blunted 1000 microliter pipette tip to slowly transfer about 1 milliliter of melted food in to each chamber.
Allow the food to re-solidify at four degrees Celsius for 10 minutes before completely assembling the arena. The completed arena can be used for experiments. Store the leftover food at four degrees Celsius for reuse.
Before starting the 2-D satiety assay, set the incubator to 23 degrees Celsius for standard room temperature experiments and make sure the humidity is greater than 30%Leave a cup of water in the incubator if the incubator does not have humidity control. Place the 2-D satiety assay arena in the incubator for 30 minutes to equilibrate the temperature and prevent condensation in the chambers during the course of the experiment. Keep the rotating doors in the open position.
Next use a fly aspirator to aspirate 15 to 20 virgin females in to each chamber of the arena. After the female flies are transferred, aspirate one male in to each chamber. Then place the loaded arena in to incubator under a standard consumer camcorder and film the flies for 4.5 hours.
After filming use CO2 or cold temperature to anesthetize the flies before removing them from the arena. Score videos by noting when each male fly starts and ends each mating. Use the provided code to generate an ethogram.
After logging all mating times, sum the number of matings for each fly. Aspirate one male in to each of the 32 chambers in a courtship arena. Then aspirate one female in to each chamber.
Film the flies for twenty minutes using a standard consumer camcorder. Score the courtship index to quantify courtship over a five minute window. If a male fly does not start courtship in the first fifteen minutes, the courtship index is zero.
Naive wild type flies should show a courtship index greater than 0.9 Whereas a fully satiated fly should have a courtship index below 0.2. After performing a 2-D satiety assay, aspirate the male flies from the chambers in to food vials. Isolate the male flies from the females at 23 degrees Celsius for the desired number of days.
Then perform a 2-D satiety assay with the recovered males and score their matings over the course of just one hour. To characterize the Drosophila mating drive, three day old wild type Canton-S males were tested in a 2-D satiety assay. Over 4.5 hours males mate an average of 4.8 times.
78%of matings initiate in the first two hours. Matings become less frequent as the assay progresses. This decline in mating behavior is also seen when the males are tested in courtship assays with new females and is recovered after the males have been isolated from females for three days.
At the end of 2-D satiety assays dopaminergic stimulation increased both courtship and copulation. The reversal effects are not observed with parental controlled genotypes. Once mastered, this assay can be prepared in 30 minutes and scored in an hour and a half.
This assay can be performed in concert with genetic and neuronal manipulations such as RNAi knockdown and optogenetics to determine which genes and neurons control mating drive.
