Ascidian Embryology: Phallusia and Ciona Protocols

Specimens of the ascidian species Phallusia philippinensis (misidentified as P. nigra in previous papers [39 (link)]) were collected from the Ginowan Fishery Bay on the west coast of Okinawa Island, Japan. Ciona intestinalis (type A: also called as C. robusta) were supplied by National Bio-Resource Project at Misaki Marine Biological Station, University of Tokyo. P. philippinensis specimens were kept in an aquarium at 20–25 °C in the dark, whereas C. intestinalis specimens were kept at 16 °C under constant light until experimental use. Eggs and semen were collected from the oviduct and spermiduct, respectively, via dissection. Semen was stored on ice or at 4 °C until further use. Eggs were placed in either artificial seawater (ASW) (pH 8.2) or measuring medium after collection and immediately used for experiments. Where dechorionated eggs were required, the vitelline coat and accessory cells were manually removed from the eggs using a sharpened insect pin and fine blade (Shiga Konchu, Tokyo, Japan). Dechorionated eggs were kept in a 1.5% agar-coated dish to avoid disruption.
The ASW consisted of NaCl (462 mM), KCl (9.2 mM), CaCl₂ (9 mM), MgSO₄ (28 mM), MgCl₂ (22 mM), and 10 mM HEPES (Dojindo, Kumamoto, Japan) (pH 8.2). The measuring media were: ASW, whose pH was adjusted with 10 mM good buffers (Dojindo): MES (pH 5.0–6.5); pH PIPES (pH 6.5–7.2); HEPES (pH 7.2–8.2); TAPS (pH 8.2); CHES (pH 9.5) instead of HEPES (pH 8.2).
This study using invertebrates is not regulated by animal welfare, but experiments were performed in accordance with the principle of animal welfare.