Research reportThe social zebrafish: Behavioral responses to conspecific, heterospecific, and computer animated fish
Introduction
Zebrafish has enjoyed much success in developmental biology for the past three decades and is now utilized as a model organism in the analysis of a number of human diseases [26]. An impressive arsenal of genetic tools has been developed for this species [19]. Given the prolific nature of zebrafish (one may obtain 200–400 eggs from a single female every other day), and the well developed genetic tools, zebrafish has been successfully used in forward genetic studies in which induced random genetic mutations are screened on the basis of the phenotypical change they caused [3]. As a result of the strong genetics and the successful use of zebrafish in screening applications, this species has now been considered for a number of other disciplines. One of these disciplines is behavioral genetics [16]. As we and others have argued before, behavioral analysis can be a comprehensive and unbiased way with which functional changes of the brain may be detected (for review see [15], [13]). Indeed behavioral screening has allowed the identification of zebrafish mutants too, for example, in the area of drug addiction related research [7]. However, behavioral screening tools are rare and zebrafish behavior is relatively understudied [38]. This represents a significant mismatch compared to the sophisticated genetics developed for this species. Briefly, the bottle-neck in forward genetic analysis of brain related mechanisms in zebrafish is the behavioral screening tools. The goal of the current paper is to investigate aspects of social behavior of zebrafish and to explore certain behavioral tests and quantification methods so that future automation and increased throughput of testing may be achieved.
Our rationale to focus on social behavior is two-fold. First, zebrafish is a highly social species that prefers swimming in groups, an aggregation behavior termed shoaling [32] and described in a number of fish species (e.g., [37], or for more recent examples see [45]). Other traditional laboratory organisms including the mouse and the rat, or simpler organisms such as Drosophila or the nematode, do not exhibit the degree of social cohesion and group preference displayed by zebrafish. Thus, in addition to the genetic tools developed for zebrafish, its behavioral characteristics make this species particularly appropriate for the proposed analysis. Second, the mechanisms of social behavior of vertebrates including our own species are not clearly understood and as a result diseases associated with abnormal social behaviors in humans (including social phobias and, e.g., the autism spectrum disorders) have been difficult to treat (e.g., [39], [12], [10], [21]). Given the high nucleotide and amino acid sequence homologies found at the DNA and protein levels between zebrafish and mammals including humans [4] and the similarities of the basic layout of the brain of these species [40], it is not unlikely that the analysis of genetic mechanisms of social behavior of zebrafish will yield results that generalize and translate to other vertebrate species including our own [28], [40].
In the current paper we first investigate how experimental zebrafish respond to live stimulus fish during an encounter in which five fish of each group (experimental and stimulus) are able to freely swim with each other. We compare how experimental zebrafish respond to four different types of stimulus fish: their own conspecifics that look similar to them, their conspecifics whose color is slightly different (a color variant), heterospecific fish that show shoaling tendencies similar to those of zebrafish, and heterospecific species that does not shoal. Greater similarity among group members has been found to reduce predation under natural conditions by minimizing phenotypic oddity [27]. We therefore hypothesize that experimental zebrafish will shoal with stimulus fish most similar to them but not with those whose behavior (non-shoaling vs. shoaling) and/or appearance (color, pattern, and shape) is different.
Previously, preference of zebrafish for certain stimulus fish was investigated in choice paradigms [9]. The results demonstrated that zebrafish were sensitive to certain characteristics of the stimulus fish and the preference was strongly influenced by early experience during development. However, systematic analysis of these cues may not be performed using live stimulus fish. In the second experiment of the current paper, we investigate certain visual characteristics of stimulus fish that experimental zebrafish may prefer or avoid. We utilize a simple software application developed in-house that allowed us to present animated, i.e., moving, images of stimulus fish whose appearance and movement characteristics could be precisely controlled. We explore the effect of modifying the color, the stripe pattern, the body shape, or the location of swimming of the animated stimulus fish and quantify how experimental zebrafish respond to these modifications in comparison to normal unmodified images. We conduct these studies in the hope that in the future our experimental procedures and behavioral quantification methods will help us develop automated and high throughput test paradigms with which the biological mechanisms of social behavior of zebrafish could be studied, a goal that will ultimately lead to better understanding of the genetics of social behavior in other vertebrates including humans.
Section snippets
Animals and housing
Zebrafish (Danio rerio), or zebra danio, were obtained (50% males and females) from a local aquarium store (Big Al's Aquarium Warehouse Services, Mississauga, ON, Canada). At the time of purchase, the fish were 3 cm long and approximately 3 months old. The fish were acclimatized in our zebrafish vivarium for a minimum of 3 months and at their approximate age of 6–8 months (when 4 cm long) were behavioral tested. Zebrafish reach sexual maturity by their age of 3–4 months, live for about 3–5 years,
Experiment 1
The distance among experimental zebrafish was found to be dependent upon the stimulus treatment employed (Fig. 2). Repeated measure ANOVA revealed no significant time interval or time × stimulus treatment interaction effects, but the analysis of the time interval averaged data confirmed a significant stimulus treatment effect (repeated measure ANOVA F(4, 36) = 7.004, p < 0.001). Comparison of the groups (t-test with Holm type-1 error correction) showed that experimental zebrafish had the largest
Discussion
The results presented in this paper confirm that zebrafish are not indifferent to the fish around them. They prefer individuals that exhibit characteristics similar to their own, i.e., they show preference towards their own conspecifics. This feature is not unique to zebrafish as other shoaling fish species have also been found to exhibit preference towards conspecifics ([44], [24]; for adaptive significance of this behavior also see [2]). The preference manifests as mixing with the stimulus
Acknowledgements
We would like to thank R. Khrishnan Nair for his technical help, and N. Miller for software development. Supported by NSERC (#311637 - 06) and NIH/NIAAA (#1R01AA015325-01A2) grants to R.G.
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