These small green ascidians are found in abundance on sea-weed, wharf piles, etc., and may be collected from Lagoon Pond bridge at Martha's Vineyard, Mass. They are hermaphroditic and viviparous.

August and September, according to Berrill (1937). However, Grave and McCosh (1923) indicate a shorter breeding period for Woods Hole, namely, the first half of August. Bumpus (1898) reports that ripe specimens of this species were taken throughout July.

A. Care of Adults: The animals are relatively hardy and will continue to breed and produce larvae in the laboratory, provided they are kept in large dishes with a constant supply of fresh sea water.

B. Procuring Gametes: Artificial insemination, using gametes pipetted from the genital ducts, has not been successful for this species.

C. Preparation of Cultures: Fertilized eggs, in various stages of development, can be obtained from the atrial brood-chamber by slitting open the test of an adult. They are difficult to rear outside the parent, although some success has been attained using the thistle-tube apparatus described by Berrill (1935a). The mortality rate decreases with advanced stages.

Older larvae can be obtained by placing dishes of adults before a window. The number of larvae released starts to increase at 8 A.M., reaches a maximum about 10 A.M., and declines by 11 A.M.; only a few are released throughout the. rest of the day. This is a form well suited to the study of metamorphosis in ascidians; for such a study, the tadpoles should be isolated in separate drops of sea water in watch glasses. When the larvae have attached, the dishes may be stored in an inverted position in wooden racks which are submerged in aquaria constantly supplied with running sea water.

Bud formation is perhaps best observed in young cultures. If a small piece of the colony is affixed with vaseline to a watch glass which is stored in running sea water, stolons will be extended over the surface of the glass and new blastozooids formed at intervals along them. Within two weeks, a series of well-formed buds will be present.

D. Methods of Observation: To examine metamorphosing or budding individuals, remove a watch glass from the rack and gently flush out any debris; avoid exposing the surfaces of the animals to air during the examination. If specimens are growing upright, they may be flattened by gently lowering a coverslip on them.

NORMAL DEVELOPMENT

A. The Unfertilized Ovum: This egg measures 240 microns in diameter. It has a very thin membrane and practically no perivitelline space. The ovum is yellowish in color. The germinal vesicle ruptures and the first maturation spindle is formed when the egg enters the oviduct.

B. Fertilization and Cleavage: Perophora eggs leave the oviduct one at a time, already fertilized, and pass into the atrial brood-chamber, where they are retained throughout development. Cleavage and gastrulation are similar to those of Styela (see p. 222). Gastrulation is by a rather specialized form of invagination, and occurs between the sixth and seventh cleavages (Berrill, 1935a).

C. Rate of Development: Development is relatively slow, and at 16û C. the interval between successive cleavages is approximately four hours. Gastrulation begins about 45 hours after insemination, the blastopore closes after 60 hours, sensory pigment appears after 120 hours, and the rupture of the chorion occurs about 185 hours after insemination (Berrill, 1935a). Grave and McCosh (1923) reported that the average free-swimming period lasts five hours.

D. Later Stages of Development and Metamorphosis: The translucent green larvae are rather similar to those of Amaroucium, having three cup-like adhesive papillae and horizontal tail-fins. The attachment papillae have cones of secretion projecting from the center of the cups. The sensory vesicle is enormous and contains an eye with lens, as well as a statocyst. Hypophysis, definitive ganglion, and sub-neural gland are present. There are four rows of horizontal gill slits on the right side and six on the left. Both the siphons and endostyle are well formed. The heart is functional and shows a characteristic reversal of beat. Further details and diagrams are available in the paper by Grave and McCosh (1923); Berrill (1935a) gives some details of metamorphosis.

E. Asexual Reproduction: Perophora exhibits the type of budding designated as "septal." The stolons, which branch irregularly over the substrate, have central mesenchymal septa separating the outgoing and ingoing blood streams. In bud formation, there first appears a hypertrophy of the epidermis between the tip of the stolon and the last formed zooid. Beneath this evagination, the cells of the vascular septum proliferate and grow out to form a hollow vesicle within the epidermal bulge. The epidermal covering of the bud forms the epidermis of the new zooid, and the indifferent mesenchyme of the inner sac forms all the remainder of the blastozooid. In Perophora, the bud never loses its connection with the stolon and is, therefore, vascularized by the common blood stream of the colony. For further details see the papers by Huxley (1921) and Berrill (1935b).

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

BERRILL, N. J., 1935b. Studies in tunicate development. IV. Asexual reproduction. Phil. Trans. Roy. Soc., London, ser. B, 225: 327-379.

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

BUMPUS, H. C., 1898. The breeding of animals at Woods Holl during the months of June July and August. Science, 8: 850-858.

GRAVE, C., AND G. K. McCOSH, 1923. Perophora viridis (Verrill). The activities and structure of the free-swimming larva. Washington Univ. Stud., Sci. ser., 11: 89-116.

HUXLEY, J. S., 1921. Studies in dedifferentiation. II. redifferentiation and resorption in Perophora. Quart. J. Micr. Sci., 65: 643-697.

PITTER, W. E., 1897. Budding in compound ascidians, based on studies on Goodsiria and Perophora. J. Morph., 12: 149-238.