Drosophila Egg-Laying Preference Assay: A Method to Test Decision-Making in Flies

This protocol is an excerpt from Gou et al., High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster, J. Vis. Exp. (2016).

1. Chamber Construction, Assembly, Assay Setup

1. Have a machine shop build acrylic egg-laying chambers (Figure 1A-D). The engineering drawing of different pieces were shown in Supplementary Figure 1-3. High resolution pictures can also be found here (http://www.rebeccayang.org/pdf/chamber%20design.pdf).
2. Insert plastic sheets into loading (top) piece of the chamber as shown in Figure 1C. This serves as the bottom surface while loading flies into individual egg-laying arenas.
3. Anesthetize females on a CO₂ pad and load them individually into each egg-laying arena. Allow ~30 min for flies to recover from the CO₂ and to become acclimated to the new environment.
4. Prepare the agarose substrates.
   1. For convenience, keep a premade bottle of melted 1% agarose in a 55 °C water bath.
   2. Add the desired amount of stock sucrose solution (2 M) into a 50 ml conical tube and mixing it with appropriate amount of agarose. For example, to prepare the 150 mM sucrose substrate, place 750 µl of 2 M sucrose solution into the tube and then fill the tube with agarose to the 10 ml mark.
   3. Prepare the plain substrate in the same manner but add distilled water instead of sucrose solution.
      NOTE: The final concentration of agarose in this protocol is slightly less than 1%. In our experience, the exact concentration of agarose does not matter so long as it is controlled to be within ~0.9 – 1.1% and that the two substrates are of the same agarose concentration.
5. Take the substrate (bottom) piece of the chamber and pipette 1,000 µl of agarose substrate into each trough as seen in Figure 1D.
6. Allow agarose to solidify for ~30 min.
7. Once the agarose substrates and flies are ready, assemble all three pieces of the egg-laying chamber and then take out the plastic sheets.
8. Place the chambers in fly incubators.
   NOTE: Length of egg-laying experiments can vary depending on experimental needs. We typically run the experiment O/N (14 – 16 hr). Also, no significant impact of circadian timing on egg-laying preferences was observed.
9. Anesthetize females by injecting CO₂ into the chamber. Disassemble the chamber, discard the anesthetized flies into fly morgue (i.e., an empty coffee can filled with some corn oil). Take pictures of the results for recordkeeping (see Figure 2).
10. Count the number of eggs manually and calculate preference indices for analysis. Calculate preference index as (N_a – N_b)/(N_a + N_b) where N_a and N_b represent number of eggs on site a vs. site b, respectively.