To begin, obtain the flies prepared for habituation assay. Place one milliliter of the preferred odorant diluted with paraffin liquid light in a 1.5 milliliter microfuge tube. Vortex the tube for 10 minutes and cover it with evenly perforated plastic wrap.
Using a wire, suspend the odorant tubes in separate media bottles containing flies. Cover the bottle well with cotton and wrap it with craft paper to prevent a fusion of the odor and vapor. Label the control and odor-containing bottles as naive and odor-exposed respectively.
Maintain these induction bottles for three days in an incubator. For the Y-maze assay, attach the Y-maze to the adapter, fix the bottom of the Y-maze to the entry vial, and then attach to climbing chambers to the two arms of the Y-maze. Using odor-free silicone tubes, connect the tapering end of each climbing chamber with the reagent bottle containing the odorant.
With the vacuum pump, drive the odorant from the gas bottles to the two arms of the Y and let it saturate for 15 minutes. Gently introduce the star flies of each vial into the entry vial of the Y-maze setup. After brief acclimatization, connect the entry vial with the Y-maze adapter to begin the test.
Let the flies climb the Y-maze arms and get trapped in the two collection chambers for over one minute. Upon completion, tap back the flies to the bottom of the entry vial and switch the position of the arms of the Y-maze to avoid side bias. Record the number of flies climbing each of the two arms of Y-maze within the time duration.
The row knockdown flies did not get habituated after a three day odorant exposure as compared to the wild-type flies.
