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In Vitro Effects of Environmental Salinity and Cortisol on Chloride Cell Differentiation in Embryos of Mozambique Tilapia, OREOCHROMIS MOSSAMBICUS, Measured Using a Newly Developed ‘Yolk-Ball’ Incubation System

Kiyono Shiraishi¹, Junya Hiroi², Toyoji Kaneko^2,*, Manabu Matsuda¹, Tetsuya Hirano³ and Takao Mori¹

¹ Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo, Tokyo 113-0033, Japan,
² Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639, Japan and
³ Hawaii Institute of Marine Biology, University of Hawaii, Coconut Island, Kaneohe, HI 96744, USA

View larger version (109K): [in a new window]   Fig. 1. (A,B) An intact tilapia embryo 2 days before hatching (A), and an embryo whose yolk sac has been almost severed from the embryonic body (B). An arrow indicates the incision. (C–F) Incised wounds on the yolk-ball preparations at 0h (C,E) and 3h (D,F) after surgery stained with Trypan Blue (C,D) and observed by scanning electron microscopy (E,F). The wound (arrowheads) had almost healed after incubation for 3h in BSS. Note the symmetrical distribution of melanophores on the dorsal side of the yolk-sac membrane. Scale bars: A–D, 1mm; E,F, 100µm.

View larger version (66K): [in a new window]   Fig. 2. (A–D) Chloride cells in the yolk-sac membrane of intact tilapia embryos (A,B) and yolk-ball preparations (C,D) incubated in fresh water (A,C) and sea water (B,D) for 48h. Chloride cells were detected by whole-mount immunocytochemistry with FITC-labelled anti-Na+/K+-ATPase. (E,F) Chloride cells in the yolk-sac membrane of yolk-ball preparations incubated in fresh water (E) and sea water (F) for 48h, double-stained with FITC-labelled anti-Na+/K+-ATPase and propidium iodide. Chloride cells in sea water form multicellular complexes with accessory cells (arrowheads), as demonstrated by the presence of more than one propidium-iodide-labelled nucleus. Scale bars: A–D, 50µm; E,F, 50µm.

View larger version (13K): [in a new window]   Fig. 3. Changes in the surface area of chloride cells in the yolk-sac membrane of intact tilapia embryos (A) and yolk-ball preparations (B) incubated in fresh water (FW) and sea water (SW). Values are expressed means ± S.E.M. (N=4 samples). Asterisks indicate significant differences between freshwater and seawater groups (P<0.05).

View larger version (13K): [in a new window]   Fig. 4. Chloride cell surface area in yolk-ball preparations incubated for 48h in fresh water (FW) and sea water (SW) supplemented with cortisol. Values are expressed as means ± S.E.M. (N=8 samples). Two-way ANOVA showed a significant effect of salinity (between FW and SW, P<0.0001), but no significant effect of the dose of cortisol (P=0.2982).

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