. Immunomodulation response assessment of Tg(mpo:GFP) zebrafish larvae after PcShK3 treatment. (A) Neutrophils (mpo+) accumulated within the fin-cut region; (B) No significant changes of neutrophils accumulation could be observed after PcShK3 treatment.

Mortality rate and distribution of P. caribaeorum ShK toxin-like peptide. (A) Fluorescent images of in vivo distribution of rhodamine B-conjugated peptide in Tg(fli1:EGFP)y1 zebrafish. Bright field pattern of zebrafish (6 dpf) treated with 20 μM of RhoB-PcShK3 (A-1) and free rhodamine B as negative control (A-2) for 24 h. Green fluorescence pattern of EGFP expression in transgenic Tg(fli1:EGFP)y1 zebrafish (B-3,B-4). Fluorescent images (A-5,A-6) from zebrafish (6 dpf) after incubation with rhodamine B-conjugated PcShK3 (20 μM) and free rhodamine B (20 μM) for 24 h. Merged images (A-7) of blood vessels (green) and rhodamine B conjugated PcShK3 (red). Merged fluorescent images (A-8) of blood vessels (green) and free rhodamine B (red); (B) Mortality rate of zebrafish larvae after exposure to peptide. The mortality rate reached 50% when zebrafish larvae was exposed to peptide (40 µM) for 8 h. The mortality rate reached 100% of zebrafish larvae exposed to peptide (75 µM) for 48 h. The LD₅₀ is estimated to 43.53 ± 6.45 µM.

Evaluation of cardiac malformations of zebrafish larvae upon PcShK3 peptide exposure. (A) Time and dose-dependent change in developmental cardiac malformations in larvae after exposure to peptide (30 μM) from 4 to 48 hpt. The blood accumulation (ba) appeared after 4 h of drug treatment and decreased after 24 h. The rate of pericardial edema (pe) increased from 24 hpt in a dose-dependent fashion. Values are shown as the mean ± S.D. of three replicates each with 15 larvae; (B) Phenotype plot of cardiac abnormalities induced by the peptide.

PcShK3 protects against 6-OHDA-induced dopaminergic neuron loss in zebrafish. (A) Representative morphology of DA neurons in zebrafish brain indicated by TH staining. TH⁺ neurons in diencephalic region are indicated by yellow brackets; (B) quantitative analysis of the area of TH⁺ neurons of each group, ### p < 0.001 versus control group, ** p < 0.01 versus 6-OHDA group.