This method can help answer key questions in the field of exercise biology, such as how does exercise response differ for animals of different ages and what genetic pathways control exercise response. The main advantage of this technique is that it allows researchers, for the first time, to measure activity in flies performing rotationally-induced exercise in real time. Demonstrating the procedure will be Louis Patrick Watanabe, a graduate student from my laboratory.
Prior to the planned experiment, set up crosses of male and female flies so that each controlled progeny can be collected for the experiment. To begin, load eight glass specimen tubes with 10 anesthetized test flies per vial. A standard experimental group consists of 10 vials of 10 flies of each sex.
To load the vials into the REQS, insert the vial into a slot of the activity monitor from the front. Then, put the second cap on the vial and center the vial, moving the O-ring as needed. House the REQS in an incubator for controlled environmental conditions.
Allow the animals to recover from the anesthesia for an hour in this incubator before proceeding. For assays longer than eight hours, vials containing food that is very securely attached to one end of the tube may be used. Following the acclimatization period, power up the device to start the rotations.
Adjust the speed to the desired speed, such as four RPM. No tube should make contact with the bottom surface if they are properly positioned. For a typical assay, simply initiate the software and record the activity for the desired time, such as for two hours when using vials without food.
Upon initiation, the software automatically saves the data to a text file. In the software, a green connection light indicates the software is collecting data. The recording frequency can be adjusted under the Preferences tab.
Typically, five minute intervals are used. During the assay, do not open the incubator, as the animals are very sensitive to any disturbance and are likely to respond with increased activity. At the end of the assay, terminate the data collection by closing the software, then turn off the REQS and inspect the tubes.
Take note of any deaths that occurred during the exercise regime. Generally, four RPM for two hours is not lethal to healthy strains. Finally, unload the vials by anesthetizing the flies with carbon dioxide.
The data file can be analyzed with any statistical package. If there are time points where an activity score of zero is recorded for all positions, then there was a cut out in the connection during these time points. If cut outs occur, stabilize the connections leading to the rotating phone jack using tape.
Four genetically distinct drosophila lines were assayed with the REQS using the described protocol. Each group was composed of 100 flies split into 10 vials. The average activity was calculated in five minute bins over the two hour assay, conducted at four RPM.
The mean exercise activity between all four genotypes was significantly different, however, no significant difference was found between virgin male and virgin female flies, so their data is presented together in this analysis. After its development, this technique paved the way for researchers in the field of exercise genetics to explore various exercise responses in drosophila melanogaster and have studied the impact of exercise on animal behavior and physiology. Following this procedure, other methods, such as body composition analysis, stress tests, and fitness tests can be used to answer additional questions regarding the impacts of exercise treatments on the animals.
