These large, solitary ascidians can sometimes be collected from the wharf piles at Oak Bluffs and Vineyard Haven, Mass., and from the salt water tanks on the roof of the Marine Biological Laboratory, at Woods Hole, when the tanks are emptied in September; they are not always obtainable. The animals are hermaphroditic and, under some conditions at least, self-fertile (Just, 1934a, 1934b). They are oviparous.

Almost any time of the year, since Ciona is sexually mature above a certain size limit. Reproduction is seasonal only to the extent of the rhythm of the growth cycle (Berrill, 1937).

A. Care of Adults: Mature adults will continue to produce normal eggs for several days in the laboratory, provided they are not crowded. Place only a few together in a large fingerbowl and insure a constant supply of fresh sea water.

B. Procuring Gametes: The eggs are normally shed at daybreak, but Rose (1939), using Styela, has developed a method for postponing this until a more convenient time. The adults are stored in a dark place, such as a desk drawer, until eggs are needed; shedding occurs almost immediately upon return of the animals to light.

Artificial insemination in this species is highly successful, since the genital ducts contain only ripe gametes. These may be obtained by slitting open the test and pipetting the eggs and sperm from the oviduct and sperm duct, respectively. If necessary, fine scissors can be used to puncture the ducts. The eggs remain viable for 18 hours after removal. They should be passed through several changes of sea water before insemination, to free them of perivisceral fluid.

C. Preparation of Cultures: Naturally-spawned eggs should be collected with a small-mouthed pipette and placed in fingerbowls of fresh sea water. The artificially-obtained eggs should be inseminated with a sperm suspension sufficiently concentrated to impart a faint milkiness to the sea water in which the eggs are contained. Berrill (1937) routinely uses eggs and sperm from different individuals, which is a wise precaution even in this self-fertile species. After fertilization, the essential requirements for normal development are the complete removal of excess sperm and oviducal fluid, and, above all, the use of glassware chemically and organically clean, as Morgan (1945) has demonstrated.

The development of Ciona tends to become abnormal during the period of tail elongation; placing the cultures in a larger volume of water prevents this tendency

and makes it possible to rear individuals through metamorphosis to maturity. The sea water should be replaced three or four times during the course of development. Feeding must be initiated once the small ascidiozooid has attached. Ciona, according to Berrill (1947), grows readily in an inverted bell jar or in a battery jar, equipped with an aerator. The diatom Nitzschia is used as a basic food, and its culture, within the bell jar, is regulated by controlling the amount of light with a dark paper shield. Nutrient salts are added from time to time.

D. Removal of the Chorion: Berrill (1932, 1937) gives several methods for the removal of the chorion for experimental purposes; this technique involves the use of crab stomach juice or proteolytic enzymes. Berg (1956) digested the chorion off unfertilized eggs with a 3% solution of protease in sea water. For observation, the chorion may be removed by simply rolling the eggs under a coverslip (Conklin, 1905).

A. The Unfertilized Ovum: The diameter of this egg is between 150 and 170 microns, according to Conklin (1905) and Berrill (1935). The egg is surrounded by a chorion, and inner and outer layers of follicle cells, the latter being elongated and pyramidal in shape (Berrill, 1929). There is a perivitelline space (Conklin, 1905). The egg has a clear, transparent cortical layer, and either green or red pigment in the yolk granules; the former color indicates a physiologically young egg, the latter a physiologically old one (Berrill, 1929). The oöcyte proceeds to the metaphase of the first maturation division when it enters the oviduct, and is shed into the water at this stage.

B. Fertilization and Cleavage: Although possible, self-fertilization is not at all common in Ciona. Following insemination, two polar bodies are extruded. They are larger than the inner follicle cells and remain attached to, or embedded in, the egg, thus constituting an important landmark. The first two cleavages are equal and divide the egg into future right and left halves.

Berg (1956) isolated Ciona blastomeres at the four-cell stage, and by spectrophotometric methods demonstrated that the cytochrome oxidase activity of posterior blastomeres is about 2.7 times that of anterior blastomeres. He interprets his results to indicate a localization of mitochondria in the posterior blastomeres.

Gastrulation is by invagination and epiboly (Castle, 1896). (For further details of development, consult the papers by Conklin, 1905, and Duesberg, 1915.)

C. Rate of Development: Duesberg (1915) states that cleavage begins one hour after insemination, and that hatching occurs at 19 hours, whereas Conklin (1905) reported that the latter event took place 12 hours after insemination; the temperature was not recorded in these papers. At 16û C., hatching occurs at about 25 hours, according to Berrill (1935). He also noted gastrulation at 7 hours, closure of the blastopore at 11 hours, and the appearance of sensory pigment 19 hours after insemination at 16ûC.

D. Later Stages of Development and Metamorphosis: The tadpole, which hatches by means of a proteolytic enzyme (Berrill, 1932), is urodele-like in appearance. It has vertical tail fins, three adhesive papillae for attachment, a sensory vesicle with both a statocyst and a light-sensitive organ, and a short intestine. The siphons are not prominent. The endostyle is easily seen. (See the diagrams by Willey, 1893; MacBride, 1914; Berrill, 1929.)

The free-swimming period may last from 6 to 36 hours, usually more than 12 hours (Berrill, 1935). At metamorphosis the tail is resorbed and the mouth and atrial siphons rotate to a dorsal position. A heart and two primary gill slits appear soon after attachment. The affixed anterior region of the tadpole grows out to form a stalk which lifts the trunk away from the substrate. Details and figures of metamorphosis are given by Willey (1893) and Berrill (1929).

BERG, W. E., 1956. Cytochrome oxidase in anterior and posterior blastomeres of Ciona intestinalis. Biol. Bull., 110: 1-7.

BERRILL, N. J., 1929. Studies in tunicate development. I. General physiology of development of simple ascidians. Phil. Trans. Roy. Soc., London, ser. B, 218: 37-78.

BERRILL, N. J., 1932. The mosaic development of the ascidian egg. Biol. Bull., 63: 381-386.

BERRILL, N. J., 1935. Studies in tunicate development. III. Differential retardation and acceleration. Phil. Trans. Roy. Soc.. London, ser. B, 225: 255-326.

BERRILL N. J., 1937. Culture methods for ascidians. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 564-571.

BERRILL, N. J., 1947. The development and growth of Ciona. J. Mar. Biol. Assoc., 26: 616625.

CARLISLE, D. B., 1951. On the hormonal and neural control of the release of gametes in ascidians. J. Exp. Biol., 28: 463-472.

CASTLE, W. E., 1896. The early development of Ciona intestinalis, Flemming (L.). Bull. Mus. Comp. Zool., Harvard, 27: 201-280.

CONKLIN, E. G., 1905. The organization and cell-lineage of the ascidian egg. J. Acad. Nat. Sci., Philadelphia, ser. 2, 13: 1-119.

DUESBERG, J., 1915. Recherches cytologiques sur la fecondation des Ascidiens et sur leur developpement. Contrib. to Embryol., 3: 33-70. (Carnegie Inst., Wash., Publ. no. 223.)

HARVEY, L. A., 1927. The history of the cytoplasmic inclusions of the egg of Ciona intestinalis (L.) during oogenesis and fertilisation. Proc. Roy. Soc., London, ser. B, 101: 136-162.

JUST, E. E., 1934a. On the rearing of Ciona intestinalis under laboratory conditions to sexual maturity. Carnegie Inst., Wash., Year Book, 33: 270.

JUST, E. E., 1934b. Zoological researches. Carnegie Inst., Wash., Year Book, 34: 280-284.

MAcBRIDE, E. W., 1914. Text-Book of Embryology. Vol. I. Invertebrata. Macmillan and Co., Ltd., London.

MORGAN, T. H., 1945. The conditions that lead to normal or abnormal development of Ciona. Biol. Bull., 88: 50-62.

ROSE, S. M., 1939. Embryonic induction in the Ascidia. Biol. Bull., 77: 216-232.

WILLEY, A., 1893. Studies on the Protochordata. I. On the origin of the branchial stigmata, praeoral lobe, endostyle, atrial cavities etc., in Ciona intestinalis, Linn., with remarks on Clavelina lepadiformis. Quart. J. Micr. Sci., 34: 317-360.

ZEIJTHEN, E., 1955. Mitotic respiratory rhythms in single eggs of Psammechinus miliaris and of Ciona intestinalis. Biol. Bull., 108: 366-385