Social Interaction and Distribution of Female Zebrafish (Danio rerio) in a Large Aquarium

The zebrafish (Danio rerio) is native to freshwater streams and rice paddies in the Ganges River of East India, Bangladesh, and Burma. Although this species is widely used as a genetic model and for developmental research, little is known about its natural behavior (e.g., spatial distribution and social interactions). In the laboratory, zebrafish are usually kept in small aquaria under high densities. The mating behavior of the zebrafish seems to be influenced by the exposure of mating partners to one another several to 24 h before spawning begins at sunrise; female gonadal hormones apparently stimulate males to perform courtship behavior (1, 2). Our preliminary experiments (unpubl. data) showed reproductive suppression of female zebrafish kept together for 4 days before they were separated and mated with a single male overnight. On average, each of these females (n 52) laid only 45 12 (SE) eggs, in comparison to 200 12 (SE) eggs of a female kept isolated for 4 days and then mated with one male (n 15; 150 12 (SE) eggs) or two males (n 30; 180 12 (SE) eggs) overnight. Females kept together in a aquarium behaved aggressively toward each other and developed a dominance hierarchy. These results led us to assume that females would avoid other females if they could. The aim of this study was to investigate spatial and social distribution of female zebrafish in a large aquarium where they could choose between different localities and partners. We observed wild-type zebrafish, aged 10 months, which were kept in 30-l aquaria under a day/night cycle of 14/10 h with light on at 9:00 am before the experiments were started. Four males and four females were selected for each experiment. The eight fish were tagged individually behind the dorsal fin with Floy fish tags, all bearing a different color. These tags did not seem to affect the behavior of the fish. The fish were placed in a large 195-cm 195-cm aquarium filled with 600 l (water height: 16 cm) of formulated freshwater. A grid system of 16 equal squares was drawn on the bottom of the aquarium. In 7 of the 16 squares, artificial plants were placed in order to imitate zebrafish habitat. The fish were left to adjust to their new environment for about 3 h. Each of the five experiments lasted for 5 days. Half-hour observations were made twice daily, with at least 1 h between observation sessions. The observation sessions were split into 1-min intervals (31 intervals per observation), and the location of each fish was noted at every time interval. Newly released fish showed a strong tendency for shoaling, but after about 2–3 h they dispersed to different parts of the aquarium. Thereafter, they spent 99% of their time in one of the seven squares containing artificial plants. We evaluated how often each female was seen either alone or in the presence of only males (1 or 2 males) or with other females without any males (Fig. 1). In addition, we calculated how often females were expected to be found either alone or in these groups, assuming a random distribution (expected values). The comparison of observed and expected values showed that females avoided staying alone ( 2 4.8, df 1, P 0.05), but they preferred to stay with a single male ( 2 3.9, df 1, P 0.05) or with two males ( 2 142, df 1, P 0.001) when no other females were around. They spent only 5% of the observation time with femaleonly groups ( 2 5.7, df 1, P 0.05). Females showed no pairing with specific males. On average, each female was observed with 1.6 males per observation period. Our study indicates that under more normal conditions female zebrafish might live with one or two males but separated from other females. Males seemed to change female partners on a daily basis. We assume that they were probably attracted by female pheromones indicating female reproductive stage. Bloom and Perlmutter (3) showed that zebrafish, when placed in a T-maze, demonstrated either a preference for or an avoidance of donor water produced when specified numbers of conspecifics of either sex were kept in holding systems for set lengths of time. Their results and ours are consistent with the higher egg production we observed in females kept with one or two males. The conditions under which zebrafish are often kept in laboratories seem to conflict with their natural preferences. Egg production could be increased by considering these observed behavioral patterns.