MnCl₂ exposure causes morphological changes in zebrafish larvae at 5 dpf (a(I–IV)), affecting body length (b), eye diameter (c) and olfactory organ size (d). (a(I–IV)) Representative images of zebrafish larvae at 5 dpf unexposed (I) and exposed to 500 µM MnCl₂ at the given durations (II–IV). One-way ANOVA (b,d) *** p < 0.001; (c) *** p = 0.003; ns. not significant, °correspond to outliers. n = 8 larvae per condition. Scale bar: 100 µm. Data presented as mean ± standard deviation (SD).

MnCl₂ alters the morphology of the olfactory organ of larval zebrafish at 5 dpf. (a,b), Z-projection of confocal images from the rostral top of the head showing the olfactory organ (thick arrows) immunochemically stained against alpha-tubulin in untreated (a) and Mn exposed (500 µM, from 2.5 hpf to 5 dpf) 5 dpf larvae (b). (c,d) Scanning electron microscope images of the olfactory epithelium of untreated (c) and Mn exposed (500 µM, from 2.5 hpf to 5 dpf) larvae (d), showing the morphological changes in the olfactory pit opening and a detailed view of the apical processes (arrows) of the olfactory cells. Scale bars: 50 µm (a,b); 10µm (c,d).

MnCl₂ exposure does not lead to changes in the area of the lateral profile of the whole brain or the telencephalic regions at 5 dpf. (a,b) Lateral views of the telencephalon after immunostaining against tubulin in (a) negative control larva and (b) larva exposed to MnCl₂ (500 µM). Hb, habenula; OB, olfactory bulb; OT, optic tectum; Tel, telencephalic lobe. Scale bars: 50 µm. Area of (c) whole brain, (d) telencephalon, (e) olfactory bulbs and (f) telencephalic lobes in lateral view. The duration of MnCl₂ exposure (500 µM) was 2.5 hpf to 5 dpf. One-way ANOVA. (a) p = 0.821; (b) p = 0.895; (c) p = 0.410; (d) p = 0.323. n = 8 larvae per condition. Data presented as mean ± SD. (d-f) Area normalized to body length. °, * correspond to outliers.

MnCl₂ exposure reduces Nrgn expression in the brain. (a–c) Representative images of Nrgn immunostaining of brains at 5 dpf (lateral views). (a) Negative control (NC)—unexposed larva. (b) Larva exposed to MnCl₂ (500 µM) from 2.5 hpf to 5 dpf. (c) Larva exposed to MnCl₂ (500 µM) from 2.5 hpf to 2 dpf. ca. anterior commissure. Ce, cerebellum; H, hypothalamus; NC, negative control; MO, medulla oblongata; OB, olfactory bulbs; OT, optic tectum; Po, preoptic area; Tel, telencephalon. Scale bar: 100 µm. (d) Neurogranin mean fluorescence intensity levels (Relative Unit Intensity (RUI)) at 5 dpf following MnCl₂ exposure (500 µM). One-way ANOVA. * p = 0.048; ns, not significant. n = 8 larvae per condition. Data presented as mean ± SD. (e) mRNA expression of nrgna and nrgnb at 4 dpf upon MnCl₂ exposure (500 µM) from 2.5 hpf. Student’s two-tailed t-test. nrgna: ** p = 0.026; nrgnb: *** p = 5.51 × 10⁻⁵. n = 6 per condition. Data presented as mean ± SD.

MnCl₂ exposure causes dose-dependent Mn accumulation in whole larval zebrafish, accompanied by pigmentation changes. (a) Mn concentrations at 6 dpf determined by ICP-MS in untreated and MnCl₂-treated (100 µM) larvae at the given exposure durations. Data are presented as mean ± s.d. (One-way ANOVA with Tukey’s posthoc test; * p < 0.05; **** p < 0.0001; ns, not significant). (b,c) Pigmentation pattern of MnCl₂ exposed zebrafish larvae at the given exposure durations at (b) 4 and (c) 6 dpf. All images are dorsal views of zebrafish larvae. White arrows indicate larvae with abnormal pigmentation compared to control, and hence abnormal visual background adaptation. Scale bar: 250 µm.

MnCl₂ exposure effects on zebrafish larval behavior during light and dark conditions, as demonstrated by (a) bout number. One-way ANOVA. *** p < 0.001; ** p = 0.020; (b) mean bout velocity (** p = 0.020) and (c) displacement; ns, not significant. Data presented as mean ± standard error of the mean (SEM); (d) fraction of time spent in the illuminated half field, separated in time bins of 1 min each for untreated, Mn 2–6 dpf, Mn 3–6 dpf and Mn 2–4 dpf treated larvae; (e left) fraction of correct turns for untreated and MnCl₂ exposed larvae (when subjected to left- and right-oriented whole field motion stimuli). Bar heights represent the means across all fish in each group; error bars represent the SEM. Only directional bouts (i.e., left- and rightward swims, without forward swims) were considered for quantification. One-way ANOVA is not significant. (e right) The number of swim bouts during pause intervals and whole field motion stimuli for unexposed and Mn-exposed larvae. Presented as means ± SEM. Untreated larvae: (a–c) n = 36; (d) n = 68; (e) n = 71. Mn 2–6 dpf: (a–d) n = 32; (e) n = 52. Mn 3–6 dpf: (a–d) n = 36; (e) n = 55. Mn 2–4 dpf: (a–d) n = 64.

MnCl₂ exposure effects on zebrafish larval behavior during light and dark conditions, as demonstrated by (a) bout number. One-way ANOVA. *** p < 0.001; ** p = 0.020; (b) mean bout velocity (** p = 0.020) and (c) displacement; ns, not significant. Data presented as mean ± standard error of the mean (SEM); (d) fraction of time spent in the illuminated half field, separated in time bins of 1 min each for untreated, Mn 2–6 dpf, Mn 3–6 dpf and Mn 2–4 dpf treated larvae; (e left) fraction of correct turns for untreated and MnCl₂ exposed larvae (when subjected to left- and right-oriented whole field motion stimuli). Bar heights represent the means across all fish in each group; error bars represent the SEM. Only directional bouts (i.e., left- and rightward swims, without forward swims) were considered for quantification. One-way ANOVA is not significant. (e right) The number of swim bouts during pause intervals and whole field motion stimuli for unexposed and Mn-exposed larvae. Presented as means ± SEM. Untreated larvae: (a–c) n = 36; (d) n = 68; (e) n = 71. Mn 2–6 dpf: (a–d) n = 32; (e) n = 52. Mn 3–6 dpf: (a–d) n = 36; (e) n = 55. Mn 2–4 dpf: (a–d) n = 64.