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

Overview

Aggression is an innate behavior and previous fighting experience affects fighting strategy and outcome of later contests in flies. This video describes an assay used to study Drosophila's aggressive behavior. The example protocol features the assay performed in a setup that eliminates fly handling and therefore reduces stress for the animals, which would otherwise impact the subjects' performance.

Protocol

This protocol is an excerpt from Trannoy et al., A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster, J. Vis. Exp. (2015).

1. Behavioral Experiment

1. Perform the behavioral experiments with 6-7-day-old male flies.
2. Insert plastic dividers into each fighting chamber.
3. Remove the cotton plug from the isolation tube and position the tube below an open hole on the side of the apparatus.
4. Let the fly enter into the fighting chamber by negative geotaxis, usually within a few seconds.
5. Close the sliding wall after the fly has left the tube and entered the chamber.
6. Repeat the procedure to introduce the second fly into the fight chamber on the other side of the apparatus.
7. Place the apparatus back in the illuminated videotaping position.
8. Begin video recording and remove the plastic divider to allow flies to interact.
   1. For Aggression Experiments:
      1. In these experiments, record fights for at least 20 min to ensure the formation of strong dominance relationships.
      2. After the desired time, stop video recording.
      3. Remove flies from the fighting chambers by reversing the procedure described above.
      4. Thoroughly wash the behavioral apparatus with mild detergent and water after each use and rinse.
   2. For "Loser" Effect Experiments:
      NOTE: In these experiments, first fights are considered as the conditioning phase and second fights as the test phase. The period of rest between fights can differ but 10 min allows formation of a strong "loser" effect.
      1. Follow the procedure described above to perform a first fight.
      2. CRITICAL STEP: To induce the formation of a "loser" mentality, ensure that first fights are at least 20 min long in order to establish strong dominance relationships. After 20 min, re-insert the plastic divider into fighting chambers to separate the flies.
      3. After a 10 min period of rest, gently remove the plastic divider to allow flies to interact for second fights.
      4. At the end of the second 20 min fight, stop the video recording.
      5. Remove flies from behavioral chambers as above.
      6. Once again, thoroughly clean the behavioral apparatus.

Materials

Name	Company	Catalog Number	Comments
Behavioral chambers	Mike machine		Fabricated in-house. Directly contact the company for more information.
Borosilicate glass vials	VWR International	47729-576	16 x 10 mm
Cotton	Fisherbrand	22-456-881	Any brand can be used
Srew cap tube	VWR International	10011-394	15 mm diameter, 10 mm height
Plastic slices	Electron Microscopy Sciences	70329-40	22 x 40 x 0.25 mm Thickness
Light source (bulb)	VWR International	500003-418	Any brand can be used
Timer	VWR International	62344-641	Any brand can be used
Binocular	Nikon	SMZ-745	Any brand can be used
Camera (SONY Handycam HDR-CX330)	B&H	SOHDRCX330B	Any brand can be used