Fig. 1. Bixafen exposure causes microcephaly. (A) Experimental setup used to analyze the body morphological parameters and brain size of zebrafish embryos exposed to 0.2 or 0.5 μM bixafen, from 6 hpf onward up to 36 or 66 hpf (brain analysis), or 96 hpf (morphological analysis). (B–D) Lateral and dorsal views of 96 hpf larvae treated with 0.1% DMSO (B) or 0.2 μM (C) or 0.5 μM bixafen (D). (E) Scheme depicting the different morphological parameters measured: body length (black double-arrow), head size (purple area), eye diameter (red double-arrow) and eye-otolith distance (green double-arrow). (F–I) Body length (F), head size (G), eye diameter (H) and eye-otolith distance (I), of 96 hpf larvae exposed to 0.1% DMSO (n = 14), or 0.2 μM (n = 15) or 0.5 μM (n = 12) bixafen. (J–O) Dorsal views of the brain of 36 (J–L) or 66 hpf transgenic Tg[huC:G/U:RFP] embryos (M-O), treated with 0.1% DMSO (J, M), or 0.2 (K, N) or 0.5 μM bixafen (L, O), and showing the microcephaly of embryos exposed to bixafen low/medium doses. (P-Q) Measurements of brain volume of 36 (P) or 66 hpf (Q) embryos exposed to 0.1% DMSO (n_36hpf = 8; n_66hpf = 5), or 0.2 μM (n_36hpf = 6; n _66hpf = 5) or 0.5 μM bixafen (n_36hpf = 8; n_66hpf = 4), further illustrate the microcephaly of embryos exposed to bixafen at 96 hpf LC30 and LOAEL concentrations. ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05; n.s., non-significant. Scale bar: (B–D) = 0.5 mm; (J–O) = 100 μm. Abbreviations: Tel, telencephalon; OT, optic tectum; Cer, cerebellum; SC, spinal cord.

Fig. 2. Bixafen exposure induces motor deficit and motor neuron axon defects. (A) Experimental setup used to analyze the motor activity and patterning of caudal primary motor neuron (CaPMN) axons and branches, in zebrafish embryos exposed to 0.2 or 0.5 μM bixafen, from 6 hpf onward up to 36 or 66 hpf (CaPMN axon morphology analysis), or 96 hpf (motor activity analysis). (B) Tracking plots illustrating the distance traveled by 96 hpf larvae treated with 0.1% DMSO (n = 85), or 0.2 (n = 72) or 0.5 μM bixafen (n = 77), during a 25 min time period. (C) Quantification of the distance traveled by 96 hpf larvae exposed to 0.1% DMSO, or 0.2 or 0.5 μM bixafen, during a 25 min time period. (D–I) Three-dimensional reconstruction of the organization of CaPMN axons and branches in 36 (D–F) or 66 hpf transgenic Tg[Olig2:eGFP] embryos (G-I′), exposed to 0.1% DMSO (D, G), 0.2 (E, H) or 0.5 μM bixafen (F, I), showing the disorganization of CaPMN axons and branches, following exposure to 0.2 and, even more, 0.5 μM bixafen (white arrow). (G′-I′) Higher magnifications of the morphology of CaPMN axons and branches, in embryos exposed to 0.1% DMSO (G′), or 0.2 (H′) or 0.5 μM bixafen (I′). (J, K) Quantification of the length of the primary CaPMN axons in 36 (J) and 66 hpf embryos (K), treated with DMSO (n_36hpf = 13; n_66hpf = 7), or 0.2 (n_36hpf = 8; n_66hpf = 6) or 0.5 μM bixafen (n_36hpf = 22; n_66hpf = 9), showing significantly shorter axons following exposure to bixafen at LOAEL (0.2 μM) and, even more, 96 hpf LC30 (0.5 μM). ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05. Scale bar: (D–I) = 100 μm; (G′-I′) = 50 μm.