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

- To test a female fly's preference for egg-laying, or oviposition sites, provide an individual female fly with a choice between two different substrates on which to deposit her eggs within a test chamber. The fly will compare the available options and choose the sites she prefers, usually the substrate best suited for her offspring.

Ensure the fly is primed to lay many eggs, so there are robust egg numbers in each trial. At the end of the experiment, remove the fly, and count the eggs on each substrate to determine the fly's oviposition preference.

In the example protocol, we will use a high-throughput assay to test egg-laying preference for plain or sucrose-containing agarose substrates.

- Continue with the experiment by obtaining previously constructed acrylic egg-laying chambers. Insert plastic sheets into the top, loading a piece of the chamber to serve as the bottom surface.

Next, anesthetize the females on a carbon dioxide pad, and load them individually into each egg-laying arena. Allow the flies 30 minutes to recover from the carbon dioxide and to acclimate to the new environment. While the flies are adjusting, fill a tube with agarose up to the 10 milliliter mark. Then, prepare a 150 millimolar sucrose substrate by placing 750 microliters of 2 molar sucrose solution into the tube. Keep a bottle of melted 1% agarose in a water bath.

Prepare the plain substrate in the same manner, but replace the sucrose solution with distilled water. Next, obtain the substrate piece of the chamber and pipette 1,000 microliters of the agarose substrate into each trough. Set the agarose aside to solidify for 30 minutes.

Once the agarose substrates and flies are ready, assemble all three pieces of the egg-laying chamber and then take out the plastic sheets. Place the chambers in fly incubators overnight.

Anesthetize females by injecting carbon dioxide into the chamber. Disassemble the chamber and discard the anesthetized flies. Finally, take pictures of the eggs for record-keeping.