A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

The overall goal of this method is to introduce the formation of loser and winner effects in drosophila, melanin, gaster by eliminating fly handling from the behavioral procedure. Most animal behavioral studies that are carried out in a laboratory setting involved handling of the animals. Handling of the animals in turn usually induces stress in the animals and stress can interfere with the behavioral studies that you're trying to perform to try to eliminate the stressful handling of animals.

We developed a new chamber for our aggression studies in fruit flies that eliminates completely the handling of the animals prior, during, or after the experiments. Using these new chambers, all of the behavioral parameters that we've measured have turned out to be better. Using this method, we minimize the behavioral viability among individuals.

We decrease the time necessary to generate clear dominance relationship, and we were able to induce strong loser and winner mentalities. These chambers are easy to use and can be multipurpose to study different fruit fly behaviors. To begin the experiment, raise and maintain fly stocks in a 12 hour light dark cycle incubator at 25 degrees Celsius and 50%relative humidity.

Next, prepare isolation vials by heating fresh fly food until it is melted. Using a pasti pipette transfer approximately 1.5 milliliters of melted fly food into single empty glass test tubes and allow the food to solidify with a fine paintbrush. Gently remove a late stage male pupa from the stock vial and place it on the side of a test tube containing freshly prepared food.

Close the isolation vials with cotton store the isolation vials in the incubator. After isolation, the flies are ready to be painted. Anesthetize the fly with carbon dioxide, then with a fine tooth pick.

Place a small dot of acrylic paint on the thorax and avoid painting the head wings, abdomen, or legs. Let the paint dry for approximately 10 seconds and gently transfer the flies back to their original isolation vials. Return the isolation vials to the incubator.

Continue with apparatus preparation by heating fresh fly food until it is melted. Use a posterior pipette to transfer 0.8 milliliters of food into the lid of the screw cap vial, and allow the food to cool. Once the food is cool.

Apply a small drop of fresh yeast paste to the center of the food cup surface. Wash the apparatus and individual fighting chambers with water before each use and dry them with a paper towel. Next, apply a drop of putty on the food cup and place it into the center of each fighting chamber.

Cover the fighting chambers with the apparatus lid. Place the covered apparatus under a light source and install a video camera above the structure. 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 geo axis. 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. So to induce a strong loser mentality, make sure the flies establish and maintain a dominance relationship during the first fight, and then minimize the animal's 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 geo axis by reversing the procedure described before the new method was used to observe first fights.

On average, the first encounter is observed after two minutes. The first lunge occurs after four minutes, and flies establish a dominance relationship after eight minutes. During the remaining 12 minutes, the dominance relationship is reinforced.

As winners lunge more and more and losers retreat more and more. The new approach was used to quantify the loser effect the percentage of losers that lose second fights during second fights. A significant effect is observed when previous losers were paired with familiar winners or with unfamiliar winners for their second fights.

While attempting this procedure, it's important to remember to perform aggression asay using six to seven days old male flies that were rising in social isolation. So once mastered these chambers allow easy introduction, monitoring, interactions, and separation of the flies without any handling at all, and that induces a strong loser effect. The use of dividers to separate the flies after the first fight allows variation in the experimental protocol.

We can keep the winners and pair them with different opponent on the second fight to induce a winner effect With small adjustments, the same chambers can be used to study courtship, locomotion, and other social behaviors of the fruit fly. After watching this video, you should have a good understanding of how to perform aggression assays and how to induce looser effect without manipulation of the flies before and during the experiments.