In Vitro Fertilization of Ciona intestinalis Ascidia

Many species of ascidian have been studied by developmental biologists; here we cover only C. intestinalis. This species is cosmopolitan and common; in the United Kingdom it is abundant in the vicinity of several major marine biology laboratories, including Plymouth, Southampton, and Millport. As with many other ascidian species, if adults are kept in constant illumination, they can be induced to spawn by moving them to the dark (2); animals can also be kept in the dark, and spawned by moving to the light. An alternative procedure to obtain embryos involves in vitro fertilization using gametes obtained by dissecting gonoducts. This method is convenient, since embryos can be obtained immediately after collection of animals. They can be spawned throughout the year or throughout summer in northern Europe.

1. Adults should be kept in seawater aquaria (12-18°C or temperature at collection site), in constant dark or constant illumination to prevent spontaneous spawning.

2. Select several mature adults; these can be recognized by the white sperm duct visible through the body wall. The yellow or brown oviduct is also sometimes visible.

3. Using sharp scissors, cut the test and body wall open longitudinally. The white sperm duct is usually obvious in mature animals; the oviduct, which lies parallel, is sometimes less clear. A dissecting microscope is sometimes required to see eggs within the oviduct.

4. Carefully cut through the oviduct, and squeeze eggs out using fine forceps. Transfer eggs to a Petri dish using a Pasteur pipet. Only use eggs from the oviduct; do not dissect from the ovary.

5. Now cut through the sperm duct and squeeze out sperm in a similar manner. Transfer sperm to a separate Petri dish, before it becomes dilute.

6. Repeat steps 3-5 for at least one further animal.

7. Add a drop of sperm from one animal to several hundred eggs collected from another. After 10 min, wash away excess sperm with several changes of filtered seawater.

8. At 16°C, first cleavage occurs after 1 h, gastrulation at 5 h, and hatching after about 24 h. Ciona has a nonfeeding tadpole larva that swims for 12-24 h before settling and metamorphosis.

9. If prehatching stages are required, e.g., for in situ hybridization to embryos, the chorion must be removed before fixation. This membrane surrounding the egg is associated with large follicle cells projecting from its outer surface and test cells on its inner surface. The chorion and associated cells can be removed manually using sharp tungsten needles; however, this is difficult and time-consuming owing to the small perivitelline space.

10. An alternative method is to place fertilized eggs, prior to first cleavage, into dechorionating solution. Observe under a dissecting microscope until most eggs are dechorionated (10-20 min), and then gently wash with several changes of filtered seawater. This procedure can also be performed prior to fertilization.

11. After dechorionation, embryos must be cultured in agar-coated Petri dishes.

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