Aggression Assay: A Method to Study Aggressive Behavior of Zebrafish in Response to a Mirror

1. Conducting the Aggression Assay

1. Conduct the aggression assay in a 19 L rectangular tank measuring 30 cm x 15 cm x 10 cm. Outfit the tank in external opaque partitions surrounding three sides of the aggression tank, leaving the front exposed for viewing, to reduce undesirable outside stimuli. Divide the tank with external markings into four equal rectangular quadrants and fix a mirror permanently (with silicone caulk) within the lower left quadrant, inclined at 22.5° forming a right triangle against the far left side of the tank.
2. Ensure that the temperature of the water in the acclimation tank and aggression tank is within 2 °C of the housing rack. To do so, have fresh, aged water pre-warmed to 27.5-28.5 °C ready to add to the aggression tank. NOTE: It is important to change the water in between each assay to eliminate unwarranted olfactory cues.
3. Transfer, by cupping, a focal fish from the housing rack to a separate acclimation tank where it will acclimate for 10 min.
   NOTE: It is important to reduce stimulus introduced to the fish through improper handling. Transfer individuals as previously described. Specifically, manually and carefully place a small, transparent plastic cup into the tank of interest. Without disrupting the fish as much as transferring by using a net, slowly scoop up the fish into the cup.
4. Ensure that the video camcorder is ready to record by confirming that the aggression assay tank (see step 1.1) is in focus and immediately before transferring the individual into the aggression tank, press record on the device. Transfer focal fish via cupping from acclimation tank to aggression tank.
   NOTE: It is important to record prior to the fish entering the aggression tank to track the exact time of entry.
5. Record the aggression tank for 10 min and 30 sec and then cup the focal fish from the aggression tank back into the acclimation tank.
6. When watching the recording, wait 30 sec after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors.
7. Using the JWatcher scoring system defined for aggressive behaviors, quantify the following behaviors for 10 min: Time spent near mirror, number of mirror approaches, number of darts, number of attempted bites (opening mouth in direction of mirror), and number of lateral displays (dorsal, pectoral, anal, and caudal fin erection).
8.  Score a single aggression assay at least twice, until the assay is scored with high agreement between iterations.