The overall goal of this procedure is to provide a simple Y maze assay to test olfaction in Drosophila melon Gaster. This is accomplished by first starving flies for 16 to 18 hours, so they're motivated to explore new environments with volatile stimuli. The second step of the procedure is to assemble the Y maze setup containing the odor sources on filter paper, such as acetic acid in solvent versus solvent alone.
The third step is to quickly and gently introduce the flies into the loading vial. After several hours, the final step is to score the flies location in the maze. Ultimately, the results can show a response toward odorant from the data obtained with the Y maze.
We first as the idea for this method when we decided to have a simple and inexpensive method to test the olfactory response of phyla. An advantage of this technique over other option is that it allows the flies to make a decision whether too much stress Demonstrating the procedure will be Megan Simon, a PhD student from my laboratory For this protocol have ready an ISO inized fly line with stable, robust behavioral phenotypes. Any such stock will serve as a reference control for the olfactory test, for each genetic strain to be used for the experiment back, cross the strain to the reference strain for five generations.
Pair single experimental strain females to a few isogenic males for each back cross. For olfactory experiments, maintain the drosophila on standard corn flour medium with an antibiotic under 1212 light cycle. And at 25 degrees Celsius.
The experiments should always be executed at 25 degrees Celsius and under light in the far red spectra, not visible to drosophila. It must also be possible to fully ventilate the room between experiments before conducting the assay the following morning. At the end of the day, transfer cohorts of 10 to 20 experimental lines to glass vials with moisture provided from a wet towel.
The flies must be tested in 16 to 18 hours, or they could die due to de or starvation the next day. Assemble the Y maze. Start by joining the Y shaped connector to two glass vials using foam connectors with trimmed pipette tips, which make the conduits their tapering shape makes these one-way passages.
The third branch of the Y is attached to a loading tube made from the large portion of a pipette tip and allows passage from the loading vial to the choice point of the maze. Beyond each tapering tunnel is a glass vial, one of which must be loaded with an odorant load. These as 40 microliter boluses soaked into 28 millimeters squares of tissue paper.
Which of the two contains the odorant should alternate randomly to conduct the test first.Cool. The flies on ice briefly, so the flies are slowed down. This should be under far red light from LED bulbs, and at 25 degrees Celsius, gently drop the cohort of flies into the loading vial and load the Y maze with the test flies.
Do not use gas anesthesia. It is really critical to transfer the flies to the loading vial both quickly and gently. It is actually the most difficult step of this experiment because flies have to wake up rapidly before the diffusion of others in the Y mass.
The flies should be allowed to explore the maze for 24 hours, so a maximum number of flies move to one or the other glass vial. The number of flies present in the different tubes serves to calculate the olfactory index. Standard statistical methods should be applied to this value gathered from the behavior of up to 10 flies.
If 10 fly cohorts are not providing consistent results, the trials can be run with 20 fly cohorts. To clean up the maze components, soak them dismantled in RBS 35 MD overnight. Follow this with rinses in tap water and a final rinse in DI water before air drying.
Standard responses to an odorant. Using the Y maze include attraction, indifference, or avoidance. For example, cantons flies strongly avoid 10%acetic acid, whereas they do not avoid 10%pheno acetic acid.
While attempting this procedure, it's important to remember that this wave asay represent an artificial environment for the flies. Following this procedure as a method like teammates fulfilled arena or internal assays can be performed in order to confirm the olfactory responses of flies and the different conditions. After watching this video, you should have a good understanding of how to simply test olfactory responses of filano gaste using the SW asay.
