Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

Summary

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.

Abstract

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

Introduction

There is a growing body of literature investigating the associations between distinct behaviors within individual animals from a given population. These associations are termed behavioral syndromes, and the measurements typically include boldness, aggression, exploratory behavior, and sociability^1-5. Behavioral syndromes are valuable for both direct and indirect reasons. Directly, knowledge of behavioral syndromes can provide a more complete view of evolutionary theory, population structure, and population dynamics³. Indirectly, knowledge regarding behavioral associations may inform fields that quantify behavior such as pharmacology⁶ ,....

Protocol

The following methodologies for the housing, care, and study of zebrafish have been approved by the Saint Joseph's University IACUC.

1. Zebrafish Housing and Care

1. Obtain the subject zebrafish from a local supplier, or from wild-caught populations. Please note that housing the zebrafish is subject to IACUC guidelines and additional permits are required for housing wild-caught populations.
2. Identify and separate zebrafish according to sex. Alternatively, skip this step or replace with a different method of separation based on the study hypothesis.
   NOTE: Sex characteristics are difficult to determine in zebrafis....

Results

Depending on the nature of the study, and specific protocol employed, several distinct results are possible in a behavioral syndromes experiment. The following tables and figures, where indicated, are adapted from our previous study published in the journal Behavioural Processes¹⁴ and the journal Zebrafish¹⁷. When the proposal (as described above) is carried out in its entirety, two sets of results, 'within assay correlations' and 'between assay correlations,' are ex.......

Discussion

The protocol will determine if there are consistent associations in boldness, aggression, and shoaling behaviors in zebrafish. If there are consistent associations in a given population between any of these behaviors, then a behavioral syndrome is present. By studying a population's natural behavioral syndrome, researchers can have a more complete understanding of its behavioral dynamic, population structure, and possibly evolutionary history³. Furthermore, manipulating the environment that affects these b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zebrafish Rack System	Aquaneering Inc	Cat. # ZS550
Pet Valu Tropical Fish Food, 224.0 g	Pet Valu	Cat. # 31700
Premium Grade Brine Shrimp Eggs, 16 oz	Brine Shrimp Direct
1.5 L Trapezoidal Tank	Pentair Aquatic Ecosystems	Cat. # itsts-a
19 L rectangular tank	That Fish Place	211932
76 L rectangular tank	That Fish Place	212180
Hitachi KP-D20A CCD Camera	Prescott's, Inc.
Nikon AF Nikkor 35-105 mm f/305~4.5s MACRO lens	Nikon Corporation
ArtMinds Square Mirror, Value Pack 3" x 3"	Michaels	Cat. # 10334162
Jwatcher
SPSS Statistics Base	IBM
R	The R Foundation