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This protocol is an excerpt from Bantel and Tessier, Taste Preference Assay for Adult Drosophila, J. Vis. Exp. (2016).
1. Starvation
- Prepare fly starvation vials by saturating a cotton ball with 18.2 MΩ water at the bottom of a standard fly vial. Alternatively, similarly saturate a small strip of filter paper with 18.2 MΩ water and place at an angle within the vial.
- Collect flies into sets of ~100 animals on a CO2 pad and then add the flies to a prepared vial.
NOTE: Best results are obtained with animals that are less than 5 days old. However, the exact age of the animals can be controlled as an experimental variable to determine changes in taste preference over time.
- Use a cotton ball or foam stopper to secure the vials closed. Place vials on their side in an environmentally controlled incubator. Maintain the temperature at 25 °C, and the humidity above 70%. Leave vials untouched for 24 hr.
2. Taste Preference Assay
- Prepare all tastants for the assay on the same day as testing.
NOTE: The exact tastants to be used will vary depending on experimental question being asked. The following are example tastants used in this protocol. See section 4 for optimizations.
- Prepare control tastant (1 mM sucrose) by combining 10 µl of 100 mM sucrose solution, 13 µl of red food coloring, and 977 µl of 18.2 MΩ water.
- Prepare experimental tastant (5 mM sucrose) by combining 50 µl of 100 mM sucrose solution, 10 µl of blue food coloring, and 940 µl of 18.2 MΩ water.
- Make assay chambers using a standard 100 mm x 15 mm plastic petri dish prepared in the following manner:
- Place three 10 µl drops of control tastant nearest the edge of the plate at 12 o'clock and another 3 drops at 6 o'clock. Ensure that the spacing between drops is similar.
- Place three 10 µl drops of experimental tastant nearest the edge of the plate at 3 o'clock and another 3 drops at 9 o'clock. Ensure that the spacing between drops is similar.
- Repeat steps 2.2.1 and 2.2.2 for as many replicates as desired.
- Empty 1 vial of ~100 starved flies onto a CO2 pad just long enough to anesthetize all animals (approximately 10 sec). Brush the animals into the middle of a prepared assay chamber and cover with the dish lid.
NOTE: Longer periods of CO2 exposure should be avoided to improve recovery time and limit interference with the feeding behavior. Exposure to ice (~5 min) may be used for anesthetizing to avoid CO2 behavioral effects that may arise from even limited exposure.
- Place the assay chamber in an opaque cardboard box. Be sure to label the outside of the box with the condition and genotype being tested.
- Place the entire setup (assay chamber contained within cardboard box from step 2.4) into a 25 °C incubator with at least 70% humidity for 2 hr.
- Repeat steps 2.3 through 2.5 for all replicates.
- After 2 hours, place the assay chambers, still contained within cardboard boxes, directly into a -20 °C freezer until ready for quantitation.
3. Taste Preference Assay Quantification
- Allow a single assay chamber to warm up to room temperature (approximately 5 min).
- Under a dissection microscope, using a brush or pair of forceps, group animals based on the color of their abdomen: red, blue, purple or clear (Figure 1).
- Record the number of animals in each grouping. Consider clear animals to have not participated in the assay and therefore do not include them in any calculations.
- Calculate the preference index according to one of the following equations:
- If the experimental tastant of interest is added to the red dye, then use (Nred + 0.5Npurple)/(Nred + Nblue + Npurple).
- If the experimental tastant is added to the blue dye, then adjust the equation to (Nblue + 0.5Npurple)/(Nblue + Nred + Npurple).
- Repeat the calculations for all experimental conditions and replicates.
4. Optimization of Taste Preference Assay
- Empirically determine the concentration of food coloring indicators to be used so food coloring does not affect the outcome of the taste assay, as follows:
- Prepare 4 tastants using the same base compound (e.g. 5 mM sucrose) as indicated in step 2.1, but omit the food coloring.
- Add 1.3% red food coloring to one of the tastants. Make the remaining 3 tastants with blue food coloring of varying concentrations in each tube (e.g. 0.6%, 1%, and 1.3%).
- Complete protocol steps 2.2 through 3.4 for each tastant pair: 1.3% red vs. 0.6% blue; 1.3% red vs. 1% blue and 1.3% red vs. 1.3% blue.
- Repeat step 4.1.1-4.1.3 with different percentages of blue food coloring until the preference index averages a value of 0 (Figure 2).
NOTE: As a starting point, 1.3% red food coloring coupled with 1% blue food coloring typically yields good results. If no satisfactory concentration of blue food coloring can be matched to 1.3% dye, then step 4.1.1 through 4.1.3 can be repeated with varying concentrations of red coloring and a constant concentration of blue food coloring.
- Analyze all conditions to be tested with the same optimized food coloring concentrations.
