Investigating shoal formation in the zebrafish

2.3.1 The zebrafish

The zebrafish(Brachydanio rerio)(Figure XIV.1.) originally lives in the East-Indies, however it became long ago a well known, commonly kept species among the aquarists. It is small (body length is about 6 cm), easy to breed (if it is kept in ideal conditions, they can spawn in every 10 days, and they lay 50-100 eggs at a time); peaceful with other fish and it is even pretty to look at. Besides the aquaria of the enthusiasts, the zebrafish became a favorite subject of a multitude of scientific research as well. Just as the other members of the Danio fishes, zebrafish live in groups in their entire life, basically right after they start to swim first time in their lives.

Figure XIV.1: A group of adult zebrafish

2.3.2 Zebrafish in the biological research

It may seem surprising that a fish can become such a widely used subject of the biological laboratories as some of the rodents, or the fruit fly. However, the zebrafish is a popular research subject around the world, and especially the geneticists use it for testing the effect of mutagens, or various environmental factors that affect gene expression.

Among the vertebrates the zebrafish was among the first few species, of which the full genome was sequenced.

The zebrafish is an ideal subject for investigating the early ontogeny and ontogenic deviations, as the larvae of this species are completely transparent, thus the development of the inner organs are well visible. Another relevant research field where the zebrafish is among the leading subjects is the study oflateralization(Halpern et al., 2003).

In a broad sense lateralization means such processes of the neural system, which usually are expressed also on the level of behavior, and can be characterized with a well defined left-right asymmetry.

2.3.3 Testing social behaviour of the zebrafish in the laboratory

When talking about social behaviour in the zebrafish, scientists usually narrow their scope of interest to the shoal forming, in other words social attraction and the regulation of maintaining group cohesion in this species. This phenomenon is primarily not important anymore from the point of view of zebrafish-ethology. Instead, the dynamics of zebrafish shoals (social attraction to conspecifics) offers an easy-to-test model for investigating such factors that can affect even human behavior. Not surprisingly, zebrafish are often used in human-related genetic, physiological and neurological research. Zebrafish have relatively simple nervous system, their genetic setup is well known, and additionally there are no strict rules from the aspect of animal welfare when lethal or seriously invasive experiments are conducted on fish. Recently such testing procedures were invented, which unify the be-nefits of easy manipulation of fish social behavior with the automated data collection and analysis. Here we provide basic details of this test apparatus.

Factors affecting the shoal formation in the zebrafish (Brachy-daniorerio)

• The subjects (one or more zebrafish) are placed into a small aquarium. The aquarium contains 15 cm deep water, nothing else.

• To the opposite sides of the aquarium two flat screen computer monitors are placed. These serve as channels of stimulus presentation for the fish in the aquarium (see Figure XIV.2).

• Fish are presented with visual presentations from a computer. These presentations show usually 2D images of other fish, which move back and forth horizontally (like as they would ‘swim’).

• The most interesting aspect of the fish’ behavior in this type of device is whether they form a tighter shoal as a reaction to particular presentations on the monitors. For describing the group’s behavior, a few key variables are collected. If the presentation is showing such fish images, which attract the subjects, they swim closer to that monitor (supposedly for joining to the projected ‘fish’, forming a larger shoal). Consequently, the distribution of the zebrafish in the test aquarium will be unequal, as the subjects are drawn to one of the monitors. Another important parameter to describe shoal formation is the inter-fish distance within the group. Obviously, when the subjects form a tighter group, the distance between them will be shorter. Independently of their actual location in the aquarium, the zebrafish swim closer to each other if they are presented with a fear-eliciting object (like a predator image) from above, or on some of the monitors.

Figure XIV.2: Schematic picture of the testing apparatus for following group forming behaviour of the zebrafish (based on Gerlai et al.). One or more zebrafish are placed into a small aquarium, which is equipped with two flat screen computer monitors along its opposite walls. Through the monitors researchers show different ‘fish presentations’ to the subjects. On the graph at the bottom, the results of such an experiment are shown, when images of zebrafish were shown on one of the monitors (black stripe along the x-axis). The subject swam closer to the presentation, and it is detected through the shortened distance between the fish and the wall in front of the monitor.

2.3.4 Factors affecting the group formation of zebrafish

Researchers of several laboratories worked on the details of the mechanism and ontogeny of group behaviour in the zebrafish. They described several neurophysiologic factors that underlay the social behavior of these fish. It turned out that ontogeny of group behavior takes a different course between particular inbred lines of zebrafish.

In a series of applied studies such methods were developed, which enabled the researchers to employ zebrafish’

group behaviour as indicator of the adverse effects of alcohol on humans.

The typical between-fish distance in adult zebrafish is about five-six times of their body length. This distance is not affected significantly by the size of the aquarium, in other words a group of adult zebrafish will be approximately the same size in a very large or in a smaller tank. Juvenile fish behave differently. Young zebrafish ‘use the space’

if they can, in a large aquarium they scatter to a bigger extent than the adults. The between-fish distance settles to the usual five-six times body length when the fish reach half year of age (Engeszer et al., 2007).

Among the environmental factors that probably affect group cohesion the effect of food and predators were tested.

If food was scattered in the test aquarium, zebrafish loosened the group (the between-fish distance increased).

Factors affecting the shoal formation in the zebrafish (Brachy-daniorerio)

When a hawk-like silhouette was ‘flown’ over the aquarium, the fish reacted initially with a quick dispersal. After about a half minute though, the group pulled together, with a shorter between-fish distance than prior to the pred-ator presentation (Suboski et al., 1990).

What are those factors (key stimuli), which initiatesocial attraction⁴in the zebrafish? A logical hypothesis would be that the conspicuous horizontal stripes of this species play a role in recognition of conspecifics. In an experiment where the subjects were shown presentations of zebrafish-sized fish images, wearing horizontal or vertical stripes, it was found that the arrangement of the stripes does not affect social attraction in the zebrafish (see Figure 3).

When researchers manipulated the colour of the fish stimuli, an interesting phenomenon was found: zebrafish were attracted stronger to the conspecific images that showed yellowish coloration instead of the natural colour pattern.

This result can be regarded as an effect of asupernormal stimulus. In these tests always adult female fish are used, that are especially attracted to the golden bands appearing on the male zebrafish. Therefore the yellow fish images may attract the female subjects through sex-specific channels. These results can be summarized that pattern and colour are not very important for zebrafish as elicitors of social attraction. However, when other experiments used artificially altered shapes of zebrafish images (elongated or shortened), this caused diminishing social attraction in the subjects (Saverino & Gerlai, 2008). This gave us a proof that the contour (and perhaps the size) of the other fish is important to elicit social attraction in the zebrafish.

Figure XIV.3: Different types of fish-stimuli that were used as presentations for zebrafish to elicit social attraction (based on Gerlai et al.). A: natural looking zebrafish; B: shortened image of a zebrafish; C: elongated image of a zebrafish; D and E: yellow and red colour variants; F: fish without stripes; G: fish with vertical stripes.

The connection between particularneuro-transmittersand the ontogeny of shoal formation was discovered with neurophysiologic experiments. For this purposes the brains of fish from different age classes had to be removed and the concentration of the neuro-transmitters had to be measured. The concentration of the dopamine and dopamine-like transmittersshowed similarly growing curves as the willingness to form a shoal in zebrafish with the age (Buske & Gerlai, 2012). When fish were treated with a chemical that blocks the dopamine D1 receptors in the brain, fish stopped to form a shoal (while their vision and moving ability, along other important behaviors, remained unaffected (Scerbina et al., 2012)). There are also other results that support the connection between the dopaminergic system and social behaviour. In an inbred strain of zebrafish (named as ‘AB’ strain) the willingness to form a group grows steadily along the ontogeny. At the same time in the ‘TU’ strain there is a sudden increase

4See also Chapter 10 (Huddling)

Factors affecting the shoal formation in the zebrafish (Brachy-daniorerio)

of group forming between the ages day 25 and 50. By measuring the dopamine concentration in the brains of the fish, the results showed a linear increase in the ‘AB’ strain, while in the ‘TU’ strain the dopamine level has a steep increase along the ontogeny (Scerbina et al., 2012).

Our last example is about how zebrafish became the model for testing the effect of alcohol on social behavior.

Embryos (while still in the egg) were treated with different physiological densities of alcohol-water solutions (0.25%-1.00%). It turned out that the membrane of fish eggs provides considerable protection against the alcohol diffusion. When the treated fish hatched, they were raised and tested as adults in the above described testing appar-atus. When they were presented with images of conspecifics, it was found that even the lowest density of alcohol solution weakened the social attraction towards the zebrafish presentations. Fish that were treated with the highest density of alcohol (1.00%) were absolutely not attracted to the images of conspecifics. With proper control exper-iments it was also shown that the effect of alcohol on the deterioration of social attraction was not caused by side-effects on the fish’ visual sense or their motoric functions (Gerlai et al., 2006; Gerlai et al., 2008).

3. MATERIALS

3.1 Subjects

The experiments will be conducted on adult female zebrafish. In Experiment 1 a single fish is placed to the testing aquarium, and in Experiment 2 four fish will serve as subjects.