568 Phylum Chordata 



active stigmata, no food at all from external sources is needed. 

 Oviparous forms (Ciona, Ascidia, etc.) . Eggs and sperm are extracted 

 from ducts after removal of test, followed by artificial fertilization. Some 

 species are self-fertile, others self-sterile, others, e.g. Ciona, vary with 

 locality. In any case better cultures are obtained by cross fertilizing. A 

 small quantity of sperm is mixed with the eggs in a finger bowl of sea- 

 water and proper mixing ensured by pouring from one bowl to another 

 several times. After 15 or 20 minutes the eggs should have been 

 fertilized and will lie at the bottom of the vessel. As much of the 

 supernatant water as possible should then be siphoned off and replaced 

 with clean water; the process of allowing the eggs to settle and replacing 

 the water is repeated several times to ensure complete elimination of all 

 surplus spermatozoa. Failure to do so leads to abnormal development. 

 Development up to the tadpole and beyond may take place in finger- 

 bowls, but vessels holding a larger volume of water are much more 

 satisfactory. If large cultures are desired in a limited volume of water, 

 bubbling air continuously through the water should ensure normal 

 development. In the case of Ciona such procedure is advisable in any 

 case, as development in this form is very prone to become abnormal 

 during the period of tail elongation. 



If the early development of Ciona or Ascidia is to be studied, the 

 egg membranes may be removed in the following manner (Berrill, 1933). 

 Unfertilized eggs are placed in a vessel of seawater containing crustacean 

 stomach juice (not liver extract) in a proportion of about one part 

 stomach juice to 50 or 100 parts of seawater. This medium digests off 

 the membranes in the course of a very few hours (usually 2 or 3) and 

 on complete replacement of the water by clean seawater the eggs may 

 be fertilized. 



To obtain suitable crustacean stomach juice medium -sized decapods should be 

 used, preferably after a day or so in an aquarium when the stomach should be empty 

 of food and contain only a clear reddish brown fluid. The animal may be killed 

 or immobilized by shallow stabbing with a scalpel along its mid-ventral surface. It 

 should then be placed with its ventral side downwards and the carapace cut away 

 with strong scissors so that the upper surface of the stomach is exposed. A small 

 incision is made in the upper wall of the stomach and the contents withdrawn 

 with a fine pipette. If digesting food is present in the fluid thus obtained it should 

 be allowed to settle and the clear supernatant fluid pipetted off and used. 



Eggs remain viable for about 18 hours after extraction. The method 

 will not work with eggs that have been fertilized as it affects the surface 

 tension of the dividing eggs. Slight shaking, or decanting from one ves- 

 sel into another, during early cleavage stages will suffice to separate 

 blastomeres without resort to the use of calcium-free seawater. 



Viviparous forms. The embryos of viviparous ascidians extracted 

 before the attainment of the tadpole stage usually stop developing. A 



