Ascidiacea 563 



upon the extent to which they were crowded. Without attempting to 

 ascertain just what constitutes crowding and what quantity of eggs in a 

 50 cc. beaker is the optimum for normal development, it was found 

 empirically that abnormalities are likely to occur when the bottom of the 

 beaker is covered by a layer more than one egg in thickness. To free 

 the eggs from excess sperm and prevent the accumulation of injurious 

 by-products of development, the surface stratum of water in each beaker 

 was decanted and replaced with fresh seawater as soon and as frequently 

 during the first three or four hours after fertilization as settling of the 

 eggs would permit. After this, less frequent changes in the water were 

 similarly made. (Grave, 1935.) 



BOTRYLLUS SCHLOSSERI 



To obtain free-swimming larvae of this compound ascidian, collect a 

 considerable number of adult colonies during the early morning and 

 place in the quiet water of a large aquarium jar before a window but not 

 in direct sunlight. In sexually mature colonies the zooids are relatively 

 thick and tend to mat together. Colonies in which the zooids are thin 

 and clean do not usually liberate larvae. If the colonies contain an 

 abundance of sexually mature zooids, swimming larvae may be expected 

 to appear within a few minutes in the part of the jar most illuminated. 

 When first liberated the larvae are positive in orientation to light and 

 negative to gravity and may be collected easily with a pipette as they 

 swarm at the most illuminated part of the water surface. There is no 

 well marked periodicity in the liberation of larvae but light seems to 

 be an important factor. Observations made during the summer of 192 1 

 at Woods Hole showed that no larvae are given off in the early morning 

 hours; that they appear in greater number as the day advances, finally 

 reaching a maximum about or shortly after noon ; that during the after- 

 noon the number of larvae liberated decreases gradually until the eve- 

 ning when an occasional larvae only is set free. Botryllus larvae vary 

 greatly in the duration of the free-swimming period. Some attach and 

 metamorphose within ten minutes after liberation. A small percentage 

 continue to swim intermittently and retain the tadpole form for twelve 

 hours or more, but the greater number may be expected to metamorphose 

 after a free-swimming period of about two hours. Under more normal 

 conditions the larvae tend to become attached to the side or bottom 

 of the container immediately before metamorphosis begins but under 

 laboratory conditions in small quantities of water in small vessels, many 

 larvae fail to attach and a small percentage do not metamorphose. Seg- 

 menting eggs and stages in development of the larvae can only be secured 

 by dissection of sexually mature zooids and removal from the brood 

 chamber in the atrium. (Grave and Woodbridge, 1924; Grave, 1932.) 



