Drosophila Aggression Assay: A Method to Test Behavioral Plasticity in Flies

- To assess aggression in flies, use an enclosed fighting chamber equipped with two entry points, and a divider to keep subjects separated before the trial. Place a food source in the center of the chamber to create conflict for the flies. Then, load previously isolated and marked males through the entry points on either side of the arena. Remove the divider and record the flies' encounters to quantify aggression.

To establish dominance, a male displays stereotypical offensive behaviors that increase in intensity, from approaching and chasing the opponent to lunging, a behavior that indicates aggression, where the attacking fly raises high on its hind legs and snaps down onto the opponent in an attempt to grab it. In contrast, the opponent displays defensive behaviors, like running away in retreat of the attacker. Thus, a loser and a winner emerge from the fight, and a memory of defeat or victory is formed in each opponent.

In the example protocol, we will see how a setup that eliminates animal handling during the aggression assay is used to study memory formation in flies who have lost their first fight, termed "loser" effect.

- Begin behavioral experiments by inserting plastic dividers into each fighting chamber. Remove the cotton plug from the isolation tube and position the tube below an open hole on the side of the apparatus. Allow the fly to enter the fighting chamber by negative geotaxis. Then, close the sliding wall after the fly has entered the chamber. Repeat the procedure to introduce the second fly into the fighting chamber on the other side of the apparatus.

Next, place the apparatus back into the illuminated videotaping position. Begin the video recording and remove the plastic divider to allow the flies to interact. For "loser" effect experiments, record the first fights for 20 minutes to ensure the formation of strong dominance relationships. After 20 minutes, gently replace the plastic divider into the fighting chambers to separate the flies and stop the video.

- To induce a strong "loser" mentality, make sure the flies establish and maintain dominance relationship during the first fights, and then minimize the animals' disturbance when inserting the dividers to separate the flies.

- After a 10-minute period of rest, begin recording and gently remove the plastic divider. Leave the flies to interact for an additional 20 minutes. Stop recording after 20 minutes. To conclude the experiment, remove the flies from the behavioral chambers by negative geotaxis by reversing the procedure described before.