Number and morphology measurements of hatching gland cells (HGCs) in zebrafish embryos exposed to triazoles. (A,B) Representative images of the pericardial area including the HGCs, labeled with GFP in cldnB:mGFP fish; (A) control, and (B) representative Triadimefon (FON) and Triadimenol (NOL)-treated 2 dpf embryo; (C) There was no difference in the number of HGCs between treated and control embryos at 1 or 2 dpf; (D) There was also no difference in cell size at 2 dpf; (E) Circularity index measurements were made on HGCs and were compared between treated embryos and controls. The shape of HGCs in treated animals with FON and NOL is more circular compared to untreated ones. Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with controls **** p < 0.0001). Scale bar: 50 μm.

Triazoles prevent secretion of the HGC granules. HGCs with granular content of representative 50–60 hpf (A) control, (B) FON, (C) NOL, and (D) 1,2,4-T-treated animals; (E,F) The number of HGCs containing granules in embryos exposed to a high concentration of triazoles for 48 h starting at blastula (E), or 1 dpf (F) stages, was counted and averaged. Scale bar: 20 µm. Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with controls * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Apoptosis in not activated by triazole exposure. Representative images (ventral views) of 2 dpf embryos. (A) control untreated; (B–D) embryos exposed to high concentrations of (B) FON, (C) NOL and (D) 1,2,4-T. White arrows indicate hatching gland area. Scale bar: 200 μm.