This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.
The overall goal of this high throughput assay is to provide a simple, efficient, and scalable platform to identify genes and neurons that control a simple choice behavior in Drosophila. The main advantage of this method is actually you can infectioning as an eggening choice, and a single animal in a highly suitable manner. To begin the experiment, place eight females and six males into a narrow food vial filled with standard fly media.
Then use the female progenies grown from this vial for the egg-laying experiments. Two to three days after female eclosion, make fresh yeast paste by mixing six grams of active yeast with 10 milliliters of 0.5%propionic acid. Use a spatula to apply the yeast paste onto the side wall of the food vials.
Next, place 30 previously collected eclosed females and 20 males into the vials. After four days, check the surface of the food medium. If the surface is wet from the larvae actively burrowing in the food, then the female flies are ready for egg-laying experiments.
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 milliliter sucrose substrate by placing 750 microliters of two molar sucrose solution into the tube. Keep a bottle of melted 1%agrose 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. The preference index, or PI, was calculated as the number of eggs laid by female flies when given the choice between a plain substrate and a sucrose containing substrate.
Females robustly preferred the plain substrate for egg-laying. Following this procedure, other methods like radio recording are opportunities genetics can be performed in order to answer other questions like how the flies behave prior depositing each egg.
