This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.
The overall goal of this protocol is to standardized the methodology used to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Directly, this method can answer questions in evolutionary theory, population structure and population dynamics. When combined with interdisciplinary techniques it can be applied to fields such as pharmacology, toxicology, behavioral genetics and endocrinology.
The main advantage of this technique is that the boldness, aggression, shoaling combination encompasses many well studied, easily quantifiable behaviors in Zebrafish. And each assay can be modified to answer alternative experimental questions. Begin by randomly separating the Zebrafish into the appropriate ratio of focal fish to stimulus fish.
Store all fish in Zebrafish housing racks under a strict 12 to 12 hour light/dark cycle. Feed flake food to all focal and stimulus fish everyday. And only feed brine shrimp once a week on non-research days.
Next, set up three assay tanks in the laboratory. One for aggression, boldness and shoaling in locations that minimize the possibility for adverse external stimuli. And allow enough room for a video camera to capture the entire tank.
Set up the video camera, so that it has enough clarity to view the tank labels, as well as to be to distinguish subtle behaviors of the focal fish. Keep the camera maintained in the same position with a full view of the tank during all behavioral assay recordings, in order to minimize unintended differences in behavioral scoring as a result of differential recordings. Next, define a labeling scheme to track the date, fish number and assay number for all behavioral assays.
Label the outside of the tank with tape in view of the camera. Record this information in a lab notebook for quick reference and future quality control. Finally, set up an acclimation tank that will be used between all assays, as well as directly after the individuals are removed from the housing rack.
Begin by obtaining a 19 liter rectangular tank, measuring 30 by 15 by 10 centimeters. Outfit the tank with a 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.
Use silicone caulk to fix a mirror within the left lower quadrant, inclined at 22.5 degrees, forming a right triangle against the far left side of the tank. Have fresh, aged water, pre-warmed to 27.5 to 28.5 degrees celsius, ready to add to the aggression tank in order to ensure that the temperature of the water in the acclimation tank and aggression tank are within two degrees celsius of the housing rack. Cup a focal fish from the housing rack and transfer it into a separate acclimation tank where it will acclimate for 10 minutes.
Confirm that the video camera is focused on the aggression assay tank and press record. Then, immediately transfer the focal fish via cupping from acclimation tank to the aggression tank. Record the aggression tank for 10 minutes and 30 seconds.
Then, cup the focal fish from the aggression tank back into the acclimation tank. When watching the recording after the assay is complete, wait 30 seconds after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors. Finally, score a single aggression assay at least twice until the assay is scored with high agreement between iterations.
To set up the boldness assay, first obtain a small 1.5 liter trapezoidal tank, measuring 15 centimeters tall with a 26.5 centimeter top and 22.5 centimeter bottom, and a 6 centimeter width. Wrap opaque paper around three sides of the tank, and leave the front exposed for viewing. Place a single thin piece of tape external to the tank to demarcate the top from the bottom portion of the tank.
Next, ensure that temperature of the water in the boldness tank is within two degrees celsius of the housing rack. Transfer the focal fish from the housing rack to the acclimation tank and let it acclimate for 10 minutes. Ensure that the video camera is focused on the boldness assay and press record on the device.
Immediately transfer the focal fish from the acclimation tank to the boldness tank and record for eight minutes and 30 seconds. When watching the recording after the assay is complete, allow for an additional 30 second acclimation period after the fish is introduced to the tank before scoring. Finally, score a single boldness assay at least twice, until the assay is scored with high agreement between iterations.
To set up the shoaling assay, obtain a 76 liter tank that has three compartments divided by two glass dividers sealed with silicone caulk. Externally, use tape to mark two preference zones that are six point three five centimeters from each of the glass dividers that designate each stimulus zone. Apply external opaque partitions around three sides of the shoaling tank and leave the front exposed for viewing.
Next, select stimulus shoals to acclimate for at least 12 hours before testing and have them remain in the shoaling tank throughout the course of the experiment. Transfer the focal fish from the housing rack into a separate acclimation tank via cupping where it will acclimate for 10 minutes. Then transfer, by cupping, the focal fish from the acclimation tank to the central compartment of the shoaling tank.
Allow for a 10 minute acclimation period before a 10 minute live scoring observation period, in which shoaling fish are defined as being within the preference zone in the chamber housing the target shoal. Next, quantify the time spend in each preference zone, as well as the time spent in the central volume. Finally, after the assays are completed, transfer the fish into a separate terminal tank that will serve as the final destination tank for all used focal individuals.
This protocol looked at consistent associations in aggression, boldness and shoaling behaviors of Zebrafish to determine if a behavioral syndrome was present. Here, component one represents aggression behaviors, while component two is associated with boldness behaviors. Since aggression and boldness behaviors are not represented by the same component, it can be concluded that there is an absence of boldness aggression behavioral syndrome in Zebrafish.
Once mastered, the entire protocol of one focal fish can be done in 70 minutes. While attempting this procedure, it's important to remember to familiarize yourself and your team with each target behavior in order to minimize observer bias. Scoring the same video recording multiple times will also increase confidence in your results.
Following this procedure, other methods in pharmacological, genetic or physiological fields can be performed in order to answer additional questions about what internal and external factors affect behavioral syndromes. After watching this video, you should have a good understanding of how to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Thanks for watching and good luck with your experiments!
