Shoaling develops with age in Zebrafish (Danio rerio)

Abstract

The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. from a tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities.

Fig. 1. The average distance between all pairs of fish within the shoal significantly decreases with the age of the fish, a longitudinal analysis

Mean ± SEM is shown. n = 19 shoals (each shoal consisting of 10 fish) were tested from 7dpf to 76dpf of age, i.e. on nine occasions. The arena size was kept proportional to the average body length of the fish (28X the body length) and the average distance between all different pairs of fish is also expressed in body lengths. The inset shows the results of a Monte Carlo simulation and shows the distribution of average distance between all pairs of fish in 10-fish shoals in case of random positioning of shoal members within the shoal. The mean of this distribution, i.e. random chance, is indicated on the main graph by the dashed line. Comparison between particular age points is indicated above the X-axis (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). For further details see Methods and Results.

Fig. 2. The average distance between all pairs of fish within the shoal significantly decreases with the age of the fish, a cross sectional analysis

Mean ± SEM is shown. n = 8 shoals with each shoal consisting of 10 fish. Each shoal was tested once. The arena size was kept proportional to the average body length of the fish (28X the body length) and the average distance between all different pairs of fish is also expressed in body lengths. For further details, including the results of post hoc Tukey HSD test, see Methods and Results.

Fig. 3. Increasing tank size does not decrease the average distance between all pairs of fish within the shoal

Mean ± SEM is shown. Two age groups, 30 dpf juveniles (J) and 60 dpf adults (A) were exposed to different tank sizes in a randomized manner. n = 10 shoals (with 10 fish in each shoal) were tested for each age group. Each shoal was tested in five differently sized tanks (XS, S, M, L, and XL). The medium (M) sized tank was identical to what was used in previous experiments, i.e. it was 28X the average body length of the fish tested. The arena sizes for juveniles were as follows: XS = 10 × 10cm, S= 12 × 12 cm, M = 15 × 15 cm, L=20 × 20cm, XL = 25 × 25 cm. The arena sizes for adults were as follows: XS=30 × 30 cm, S= 40 × 40 cm, M= 45 × 45 cm, L=50 × 50 cm, and XL=60 × 60 cm. Note that increasing tank sizes led to significantly increased (but not decreased) average distance between all pairs of fish in the juveniles and increasing tank sizes had no significant effect on adults. For additional details see Methods and Results.