This method can help us answer key questions about flies'response to temperature changes, such as differences between genotypes, the interaction with other sensory cues, or the function of different temperature receptors. The main advantage of this technique is that it allows for multiple fast and precise temperature changes controlling time and space and in an automated fashion. Begin by placing 20 male and 20 female flies in a rearing bottle containing 45 milliliters of fly food medium, and placing the bottle inside a 25 degree celsius incubator under 12 hour light, 12 hour dark cycles.
After 10 days, anesthetize the newly eclosed flies on carbon dioxide pads for a maximum of four minutes. And use a paintbrush to collect virgin flies in 2.5 by 9.5 centimeter fly rearing vials containing 6.5 milliliters of fresh fly food medium, separated by sex into groups of 20 flies per vial. Then return the vials to the incubator for five to seven days.
To set up a temperature-controlled arena, turn on the arena and open the temperature phases script in the control computer. Verify that the temperature sequence is properly set and check that the duration of each experimental phase is set to 60 seconds. Under the start experimental block section, confirm these settings:the desired number of phases, iterative on/off setup of the indicative red light emitting diodes, two degrees celsius temperature increase per phase, and 16 degrees celsius as the starting temperature.
Next, run the temperature phases script. The software will initialize for five seconds then stop. For temperature behavioral experiments, place a strand of white conductive tape on the top of the copper tiles of the arena, ensuring that all the edges are covered.
Place a heated aluminum ring around the copper tiles and use a clean tissue to wipe the glass cover. Place the cover on the top of the aluminum ring, leaving a gap through which a fly can be blown in and tap an acclimated fly vial two times to force the flies to the bottom of the vial. Using a mouth aspirator, trap one fly and close the vial before putting it back into the incubator.
Blow the fly into the arena through the gap between the glass cover and the aluminum ring and immediately push the glass clover to close the gap. Place a frame of lights around the arena to ensure symmetric illumination and start recording with the video recording program. Then press the space bar of the control computer to begin running the experimental phases.
To track the videos, open the fly steps tracking software and open the configuration file inside the fly tracker folder. Set the location of the videos in video folder, and the names of the videos in video files. Specify the borders of the fly arena in arena settings based on the x/y pixel coordinates of multiple points at the edge of the arena.
Specific the location of the inactive red LEDs in LED settings, based on the x/y pixel coordinates of the location of the center of the LEDs. To check the location of the borders of the fly arena, set debug to true in the arena settings, click save, and run the script in the terminal. A screen capture of the video will appear with a blue square formed by the coordinates inputted in arena settings.
Then change debug in arena settings to false. Click save, and run the screen in the terminal again to begin the tracking process. In a typical temperature behavioral experiment, single flies exposed to 16, 20, or 24 degrees celsius exhibit a higher locomotion at the beginning of the experiment, than after five minutes.
The temperature-controlled arena can also be used to compare fly behavioral responses from different genetic backgrounds to dynamic temperature changes. For example, in this experiment, the speed of all of the tested species increased according to their own response curves as the temperature increased, until reaching a point of maximum performance, after which their speeds decayed and the flies perished. When individual flies were exposed to 40 degrees celsius in the middle and one side tile, with the other side tile at a comfortable 22 degrees celsius the the Wild-type flies quickly stopped moving along the arena and remain in the comfortable location.
In contrast, the classic memory mutant Dunce flies keep exploring the arena and spend less time than controls in the comfortable location. Further, testing combinations of temperature and location are also useful in understanding the function of different temperature receptors during dynamic temperature changes. As illustrated in this experiment in which individual d melanogaster mutants were exposed to increasing temperatures while a shifting comfortable location at 22 degrees celsius was also provided.
While performing these procedure, remember to perform the steps in order and quickly so you can capture as much of flies behavior as possible. Next to this procedure other methods like negative geotaxis or heat choke assays can be used to answer additional questions such as where do flies move normally or have a normal heat resistance. The implications of this technique extend to us understanding neurological disorders or mutations in which temperature perception or pain play a role.
Generally, individuals new to this method will struggle because each step is simple by itself but they're required to be perfectly coordinated so the technique needs to be practiced.
