Top view of the experimental apparatuses used in this study. (A) Experiment 1a (spontaneous colour preference). Larvae were observed in a petri dish subdivided into four equal sectors. (B) Experiment 1b (NOR test). Larvae were familiarized to two objects of the same colour and tested with one familiar object and one of a different colour. (C) Experiment 2 (NOR test). Larvae were familiarized to two objects of the same shape and tested with one familiar object and one of a different shape. (D) Experiment 3 (NOR test). Larvae were collectively familiarized to one printed geometric figure and then individually tested in a rectangular arena with one familiar geometric figure and a novel one. (E) Experiment 4 (neophobic response test). Larvae were placed in a rectangular arena containing an unfamiliar object (a black cone), then we measured the time spent in the vicinity of the object.

(A) Percentage of time (mean ± standard error) spent in each coloured sector in 7, 14 and 21-dpf larvae. Larvae showed a preference for the blue sectors compared to the other colours (all P < 0.001). There was no difference amongst the three ages in the time spent in the four sectors (P = 0.705). (B–D) Temporal pattern of time spent in the four sectors for each age. The preference for blue decreased during the trial (P < 0.001). Dotted lines represented the expected proportion of time in each sector by chance (25%).

(A) Memory for object’s colour. Larvae did not show a preference for the familiar or the novel stimulus (P = 0.559), and there was no difference amongst the ages (P = 0.884). (B) Memory for object’s shape. Larvae showed an overall preference for the novel stimulus (P = 0.043), and there was no difference amongst the ages (P = 0.389). (C) Memory for the shape of a bi-dimensional geometrical figure. Larvae did not show an overall preference for the familiar or the novel stimulus (P = 0.233), but the three ages showed a significant difference in preference (P = 0.032). Dotted lines represented the expected proportion of time by chance (50%) and asterisks indicated significant differences from chance (P < 0.05).

Overall, time close to the stimuli decreased with age (P < 0.001; linear trend: P = 0.036) with a significant difference amongst experiments (P < 0.001) and interaction.

(A) Subjects showed a spontaneous preference for the red colour, and they tended to prefer the red colour for both familiar and novel objects. (B), (C) No difference was found in the two other NORt experiments. Dotted lines represented the expected proportion of time by chance (50%) and asterisks indicated significant differences between the two conditions (P < 0.05).

(A) Time near a new stimulus decreases with increasing age (P = 0.048; linear trend: P < 0.001). (B–D) Tendency to approach the stimulus for each age increased throughout the test (P < 0.001). Dotted lines represented the expected proportion of time by chance (25%).