Melatonin concentration in zebrafish larvae. asmt1^+/−, asmt1^−/−, and asmt2^+/− showed a significant reduction of 50% compared to WT. asmt2 ^−/− revealed an even greater decrease compared to the WT and the other mutant lines. Data are presented as mean ± SEM (n = 5 larvae/group). γγγ p < 0.001 vs. WT; * p < 0.05 vs. asmt2^−/−; ** p < 0.01 vs. asmt2^−/−. One-way ANOVA with Tukey’s post hoc test.

Total distance traveled during 24 h, 14 h light and 10 h darkness, by zebrafish larvae. (A) The WT group exhibited a normal activity pattern, with more activity during the day than during the night. (B) asmt1^+/− experienced an advance in the activity phase, even at night. (C) asmt1^−/−, (D) asmt2^+/−, and (E) asmt2^−/− did not show an activity rhythm. (F) The total distance traveled decreased significantly in asmt2^−/− compared to the other groups. (G) asmt1^−/− and asmt2^+/− significantly decreased the distance traveled during the day compared to asmt1^+/− and asmt2^−/− compared to the rest of the groups. (H) Nighttime activity was significantly lower in asmt2^−/− than in the WT and the other mutant lines. Data are presented as mean ± SEM (n = 12 larvae/group). # p < 0.01 vs. asmt1^+/−; * p < 0.05 vs. asmt2^−/−; ** p < 0.01 vs. asmt2^−/−; *** p < 0.001 vs. asmt2^−/−. Unpaired t test.

Locomotor activity recorded in adult zebrafish. (A) asmt1^−/− showed a significant reduction in the distance traveled compared to WT, asmt1^+/−, asmt2^+/−, and asmt2^−/−. (B) Both asmt1^−/− and asmt2^−/− showed a slight decrease in maximum speed compared to WT and heterozygotes, but only asmt2^−/− was significant. (C) The mean speed only decreased significantly in asmt1^−/− compared to the other groups. (D) asmt1^−/− increased the resting time more than WT, asmt1^+/−, asmt2^+/−, and asmt2^−/−. Data are presented as mean ± SEM (n = 5 larvae/group). # p < 0.05 vs. asmt1^+/−; $ p < 0.05 vs. asmt1^−/−; $$ p < 0.01 vs. asmt1^−/−. Unpaired t test.

Morphometric aspects of zebrafish larvae. (A) The total length was significantly lower in asmt1^+/−, asmt1^+/−, asmt2^+/−, and asmt2^−/− than in WT. The asmt1^+/− mutant line showed a significant reduction in (B) width, (C) eye length, (D) ear length, and (E) swim bladder length compared to WT and the other mutant groups. Data are presented as mean ± SEM (n = 5 larvae/group). γγ p < 0.01 vs. WT; γγγ p < 0.001 vs. WT; ### p < 0.001 vs. asmt1^+/−. One-way ANOVA with Tukey’s post hoc test.

Malformations in 5 dpf zebrafish larvae and 2-year-old adult zebrafish. (A) Some asmt1^+/− zebrafish showed edema, while in asmt1^−/−, all three types of malformation were found, and tail malformation was observed in asmt2^−/−, contrasting with WT and asmt2^+/−, which did not show any malformations. (B) Malformations were found to increase in the asmt1^+/− group, which were even greater in the homozygous lines. Scoliosis predominated in asmt1^−/− and caudal fin malformation in asmt2^−/−. However, asmt2^+/− did not exhibit any malformations. Data are presented as mean ± SEM (n = 5 larvae/group).