* We are grateful to Sister Florence Marie Scott, for a review of this and the subsequent sections on tunicate development, and for much helpful information.

The adult colonies of this species form abundant dense clumps on rocks and piles, and may be collected from Lagoon Pond Bridge at Martha's Vineyard, Mass. They are conspicuous because of the orange color of the ascidiozooids, which shows through the tunics. The animals are hermaphroditic and viviparous.

Late June to early September. Maximum reproduction has been observed during July and August (Scott, 1945).

A. Care of Adults: When material is brought to the laboratory, the healthy, uninjured colonies should be placed, uncrowded, in large glass dishes with running sea water. At convenient intervals, the colonies should be inspected; healthy ones can be identified by their expanded oral and atrial siphons, and all others should be discarded. Such cultures may be kept in running sea water for days and, by daily removal of dead members, these "seasoned" cultures will produce abundant tadpoles for at least two weeks.

B. Procuring Gametes: Fertilization of ovarian eggs has not been successful in this species.

C. Preparation of Cultures: The eggs are retained in brood-spaces along the length of the ascidiozooid. Segmenting eggs are found in the posterior and lower portions of the abdomen, and tadpoles are packed into the thoracic region. With the aid of a microscope it is possible to dissect out eggs and larvae, but they may be obtained with greater ease by squeezing a mass of freshly collected adults over a fingerbowl containing a small amount of sea water. Many highly colored fragments will be ejected along with the embryos. Fill the dish with water and decant the coarse particles which whirl to the top. Tadpoles and eggs of all stages of development will be found on the bottom of the dish (Grave, 1921). Pregastrula stages will not develop outside the brood-spaces, but later embryos can be cultured in fingerbowls, provided the sea water is changed several times daily.

The larvae are normally released at dawn. Freshly collected, adult colonies should be placed in flowing sea water overnight, and transferred at daybreak to a container near a window. Shedding can be postponed to a more suitable hour if ripe colonies are kept in shrouded containers or in a dark room. Within 15-30 minutes after the time when these colonies are brought into the light, swarms of active tadpoles usually appear. Following this procedure at 9 A.M. yields about one-third of the available tadpoles; if it is delayed until mid-afternoon, the yield is approximately doubled. The tadpoles gather at the top of the water, near the side of the container which is exposed to the light. They may be collected with a pipette and isolated in separate drops of sea water in Syracuse dishes. When they have attached firmly to the dishes, they can be stored in an inverted position in wooden racks, which are submerged in an aquarium supplied continuously with fresh sea water.

D. Methods of Observation: Since the inner follicle cells divide and become closely packed within the perivitelline space, they obscure most of the developmental processes following the first few cleavages. The early embryos, therefore, are best studied in the form of sectioned material. -Temporary whole mounts may be made, however, and these are very useful. The material is fixed in Bouin's fluid and preserved and mounted in 70% alcohol in depression slides; coverslips should be sealed on with vaseline, to prevent evaporation. The eggs may be rotated, bringing all surfaces into view, by moving the coverslip. Since the yolk granules are now stained yellow, the relationship between the yolky cells and those containing only clear protoplasm is readily observed (Scott, 1945).

Metamorphosing and budding individuals may be examined in the watch glasses to which they are attached. Debris should be flushed out with care, and the specimens kept covered with sea water. They may be flattened, if necessary, by gently lowering a coverslip on them. Asexual reproduction begins in cultures about 17 days after the attachment of the tadpole.

Scott (1952) describes a method for making fixed and stained Feulgen preparations of metamorphosing individuals.

A. Egg Characteristics: The eggs, in metaphase of the first maturation division, are shed into brood-spaces. Because of the pressure there of surrounding eggs and larvae, they are often polyhedral in shape. When fixed, the egg measures 250 microns in diameter; it has a chorion, and an outer and inner layer of follicle cells. The outer follicle cells are tightly pressed against the chorion. The inner follicle cells multiply, as the egg cleaves, and completely fill the wide perivitelline space which formed after it was shed; the egg of Amaroucium contains more yolk than any of the ascidians whose embryology has been studied, and is therefore opaque ( Scott, 1945).

B. Fertilization and Cleavage: The first polar body is extruded at fertilization. The basic pattern of mosaic development is essentially the same for all ascidians, but in Amaroucium, owing to the greater accumulation of yolk, the processes of cleavage and gastrulation are somewhat modified. The first cleavage is unequal. The four cells produced by the second cleavage are listed here in order of increasing size: right posterior, right anterior, left posterior, and left anterior. In the third cleavage, the dense yolky material becomes concentrated in the macromeres. Gastrulation occurs between the sixth and seventh cleavages. It is accomplished chiefly by epiboly, but this is accompanied by an involution of the mesoderm and a pseudo-invagination of the endoderm without the formation of an open archenteron. The closing blastopore is definitely skewed, due to a rapid growth of the right lip. (For further details, see the paper of Scott, 1945.)

C. Rate of Development Development is relatively slow, because of the large, inert yolk mass. However, no specific data pertaining to the rate of development are available, since pre-gastrula stages will not develop when removed from the parent.

D. Later Stages of Development and Metamorphosis: A free-swimming urodele-like tadpole is formed, with a relatively large trunk, measuring 600 microns in length, and a tail which is approximately twice as long. The lateral tail fins are well formed. Three cup-shaped adhesive papillae are visible. The tunic is transparent and glassy; embedded within it are a few scattered test cells. The sensory vesicle is conspicuous and the sense organs within it are well developed. The "eye" is a complex structure, consisting of sensory and pigment cells and a series of three lenses. There is a hypophysis, with its associated ganglia, and a nerve cord which extends into the tail and lies to the left of the notochord. The atria are fused posteriorly, connecting at this point with the atrial siphon. Four horizontal rows of gill slits (7 to 9 to a row) pierce the large pharynx on either side, in the posterior region where it is in contact with the two atria. The pharynx bears a conspicuous endostyle along its antero-dorsal border, and contains a large, central yolk mass. The U-shaped digestive tract is well developed. In the body cavity, antero-ventral to the yolk mass, is a small pericardial sac containing the developing heart. Both these structures originate from the floor of the pharynx. Complete descriptions and diagrams of all larval stages can be found in papers by Scott (1934, 1946, 1952); descriptions of the free-swimming tadpole are available in papers by Grave (1920, 1921). Some of the factors affecting metamorphosis have been described by Lynch (1956).

The swimming tadpole moves in irregular spurts, rotating on the longitudinal axis in a manner similar to that of a paramecium (Grave, 1920). When first released, the tadpoles show an immediate positive phototropism which is then reversed, at times so rapidly that before they reach the lighted side of the container, they become negative to the light stimulus. They are negatively geotropic and are always found near the surface of the water until the time of metamorphosis; then this tropism also reverses and they seek the lower levels of the container.

The length of the free-swimming period varies from 10 minutes to as long as 100 minutes (Grave, 1920). In a large vessel, most of the larvae will attach on the side of the dish near the bottom, but in small dishes they often fail to make an attachment although they will continue to metamorphose normally. Temporary attachment is made by the suckers which come in contact with a solid object; final attachment is effected by the secretion of an adhesive substance by an adhesive organ. A secretion within the cup adheres to the surface and the larva can detach itself and attach in another place numerous times before metamorphosis commences. At the time of attachment, or even before this occurs, the tail tissue buckles and is drawn into the trunk region; more extensive test is formed, and metamorphosis has begun. At the end of two days, metamorphosis is completed; sensory pigment is scattered through the body or is being eliminated through the digestive tract (which has reached its adult status) and the animal is feeding. Within the first hour of metamorphosis the heart assumes its characteristic reversal of beat. By four or five days, the zooid is well formed. All regions of the body are in full evidence: the spacious thorax with oral and atrial siphons and expanded pharynx; abdomen with the digestive loop, conspicuous in the bright orange tint of the stomach; and the post-abdomen, marked by a thin-walled, light orange epicardial tube throughout its length and a large heart at its distal tip. The zooid continues to grow through a period of about three weeks before asexual reproduction is initiated. During this time the post-abdomen increases in size and becomes filled with "blood" cells. The epicardium is the agent of asexual reproduction and colony formation. The process of metamorphosis is described by Scott (1952).

E. Asexual Reproduction: Asexual reproduction is accomplished in this species by strobilization, i.e., segmentation of the post-abdomen which contains the epicardial strand. It is known as "pharyngeal" or "epicardial" budding. At the time of constriction, the buds consist of an inner vesicle of epicardial origin and an outer covering of parental epidermis. The cavity between these layers is congested with body ("blood") cells of various kinds, predominant among which are the large nutritive cells. Present, also, in each segment is a portion of the tube of neural tissue which develops in the post-abdomen as an extension of the abdominal nerve and which increases in size with growth of the post-abdomen. All-internal organs of the new individual form from epicardial tissue, a pharyngeal derivative, and are, therefore, endodermal in origin. All other organs originate from neural and epidermal tissues.

During strobilization of the post-abdomen of the parent, the heart is isolated in the terminal bud where it persists as the heart of that member; all other members regenerate a new heart.

The buds, while developing into new zooids, move up and take their place around the parent, thus either forming a new colony around a metamorphosed individual or increasing the area of an old one. Swarms of buds in all stages of growth and migration can usually be found at the bases of the tiniest finger-like projections of a large healthy colony. (Details of this process are described by Kowalevsly, 1874; Berrill, 1935; and Korschelt, 1936.)

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

BRIEN P. 1930. Contribution á l'etude de la regeneration naturelle et experimentale chez les Ciaveiinidae. Ann. Soc. Roy. Zool. Belg., 61: 19-112.

GRAVE, C., 1920. Amaroucium pellucidum (Leidy) form constellatum (Verrill). I. The activities and reactions of the tadpole larva. J. Exp. Zool., 30: 239-257.

GRAVE, C., 1921. Amaroucium constellatum (Verrill). II. The structure and organization of the tadpole larva. J. Morph., 36: 71-91.

GRAVE, C., 1935. Metamorphosis of ascidian larvae. Pap. Tortugas Lab., 29: 209-291. (Carnegie Inst., Wash., Publ. no. 452.)

KOWALEVSKY, A., 1874. Über die Knospung der Ascidien. Arch. f. miter. Anat., 10: 441-470.

KORSCHELT, E., 1936. Vergleichende Entwicklungsgeschichte der Tiere, vol. 2. Jena.

LYNCH, W. F., 1956. Factors inhibiting metamorphosis in tadpoles of the tunicate Amaroecium constellatum. Biol. Bull., 111: 308.

MAST, S. O., 1921. Reactions to light in the larvae of the ascidians, Amaroucium constellatum and Amaroucium pellucidum with special reference to photic orientation. J. Exp. Zool., 34: 149-187.

SCOTT, SISTER FLORENCE MARIE, 1934. Studies on the later embryonic development of Tunicata: Botryllus schlosseri and Amaroecium constellatum. Ph.D. Dissertation, Columbia Univ., pp. 1-53.

SCOTT, SISTER FLORENCE MARIE, 1945. The developmental history of Amaroecium constellatum. I. Early embryonic development. Biol. Bull. 88: 126-138.

SCOTT, SISTER FLORENCE MARIE, 1946. The developmental history of Amaroecium constellatum. II. Organogenesis of the larval action system. Biol. Bull., 91: 66-80.

SCOTT, SISTER FLORENCE MARIE, 1952. The developmental history of Amaroecium constellatum. III. Metamorphosis. Biol. Bull., 103: 226-241.