The overall goal of this procedure is to quantify spontaneous locomotor activity of fruit flies. This is accomplished by first collecting and aging experimental flies. The second step is to set up population monitors in a temperature controlled incubator and download the appropriate software.
Next, the flies are transferred to glass files and then placed inside population monitors where the activity monitors are run. The final step is to analyze the data. Ultimately, population monitors are used to show differences in spontaneous physical activity of flies.
The main advantage of this technique over existing methods, such as negative geo axis, is that it allows for simple, reliable, and objective continuous recording of spontaneous physical activity of multiple populations of flies. This method can help us to determine how different manipulations affect flies. Spontaneous physical activity demonstrating the procedure will be Suzanne Kowski, a lab assistant from a laboratory To begin prepare food according to the steps listed in the accompanying text protocol and let it cool down with constant mixing on a hot plate.
Aliquot five milliliters of food into each narrow glass vial, making sure the amount of food is below the lowest ring of the population. Monitor after the food cools down to room temperature. Cover the vials with sponge plugs and keep them at four degrees Celsius for up to two weeks.
Warm up the vials to room temperature before use. Next grow Canton S flies in plastic vials with standard laboratory food. Keep the vials in a humidified temperature controlled chamber at 25 degrees Celsius on a 12 hour light dark cycle.
Clear adult flies from the vials in the morning. Then separate newly enclosed flies by sex on a carbon dioxide pad within eight hours after welo to ensure that the female flies are virgins. Prepare 10 vials of parental flies by placing five to 10 day old virgin male and female.
Flies in a standard food vial with several grains of yeast. Keep the flies in an incubator at 25 degrees Celsius with a 12 hour light dark cycle for two days. Pass the flies to a new plastic vial every other day and keep the vials with the resulting eggs in an incubator at 25 degrees Celsius.
Then clear and discard the flies that he closed during the first day and return the vials to the incubator to collect a more synchronized population of flies on day two. Next, on a carbon dioxide pad, quickly collect 25 male and 25 female flies per vial with a metal spoon within 24 hours. Write down the day of ELO on the vial.
Assemble at least five Replicate vials each. For experimental and control groups. Keep the vials in temperature controlled environmental chambers at 25 degrees Celsius with a 12 hour light dark cycle.
Pass the flies to a new plastic vial every other day for 10 days. Using a funnel place the population monitors in a temperature controlled incubator. Next, connect each monitor with a four wire telephone cable to the power supply interface unit or PSIU via a five-way splitter.
Then connect the telephone cable to the PSIU. Connect the PSIU to a 100 to 240 volt power outlet, and plug the power supply output connector into one of the PSIU mating jax. Then download the USB software used by the PSIU to synthesize a data link between the computer program and the PSIU and activity monitors.
Start the program. Then under preferences. Choose the serial port PSIU for Macintosh.
Then select the reading interval. Next, select the monitors by their unique manufacturer number. Select the monitor range that corresponds to the numbers given to the monitors.
Make sure that all the monitors are properly connected by looking for the green light. A red light indicates that the connection is lost. A black box indicates that the system is off or that the USB connection has not been properly made.
Separate male and female flies of the same age. On a carbon dioxide pad, place 10 male and 10 female flies into separate room temperature. Glass vi.
Use at least three vials for each experimental and control line, and for each sex of flies, keep the vials on their sides until the flies recover from carbon dioxide to ensure they do not get stuck in the food. Leave them for two hours at room temperature to recover from the carbon oxide. Then place the vials inside the population.
Monitors housed in the incubators. Pass the flies after three or four days to new vials to avoid food drying. At the end of the experiment, scan the data to eliminate duplicate readings and ensure the recordings are complete.
Using file scan 110 x, select the interval for data collection and discard the data collected within the first 24 hours at the selected time period. The program will send the current total count for each monitor to the computer and start counting again from zero. Next, choose an experimental name and copy the files from the computer data folder to save the data.
Begin data analysis by carefully copying the data collected in the text files into columns of Excel spreadsheets. Then calculate the total activity within a desired period of time for each monitor that represents the sum of activity collected at three different heights of infrared beams. Finally determine the average locomotor activity and the standard deviation between the three monitors that represent three biological replicates using the two tailed student T-test.
Spontaneous locomotor activity in drosophila depends on the sex of the fly and on the light dark cycle. As demonstrated by this graph of the average activity of male and female flies, the arrow marks the transition from light to dark. Where a decrease in activity is seen, the total activity of 20 day old male flies increases when the flies are given half the amount of standard food on a low calorie diet compared to the decreased activity of flies on a high calorie diet.
After watching this video, you should have a good understanding of how to determine the spontaneous physical activity of drosophila. This method is helpful to determine how different genetics or environmental manipulations affect the activity of flies.
