Appendix 1 : Induced spawning method

Light-induced spawning is a procedure utilized when adults are needed to be kept alive for repeated embryo collection. It is based on the observation that a number of ascidians spawn in response to light following darkness (see Lambert and Brandt, 1967). This method is similar to that successfully utilized for genetic screens (Moody et al., 1999; Nakatami et al., 1999).

1. Select gravid individuals by their full oviducts (observed by organism transparency).
2. Place these animals under continuous illumination (until the dark adaptation period, next step) to prevent spontaneous spawning.
3. Place them in the dark for one hour.
4. Re-expose animals to light; spawning generally occurs within about 30 min after the onset of illumination.
5. Remove the animals and collect gametes. Use a single animal per vessel for self-fertilization. Use more animals (2 or more) per vessel for cross-fertilization.