Botryllus is a small, compound ascidian which is abundant around Woods Hole, Mass. It is found encrusting rocks, wharves, floats, and even the related genus Molgula. The daisy-like pattern, formed by the iridescent pigment bands extending between the siphons of the colony members, makes it easily recognized. Botryllus is viviparous.

June to September, although the reproductive period for any one colony seems to be relatively short (Grave and Woodbridge, 1924).

A. Care of Adults: These animals are easily maintained in large fingerbowls, supplied with a continuous gentle stream of sea water. Dead material should be removed, as it will rapidly foul the water.

B. Procuring Gametes: As is the case for other viviparous species of ascidians, artificial insemination in Botryllus has not proved successful.

C. Preparation of Cultures Early developmental stages must be dissected from the atrial cavity. This is done by slitting open the zooids and stripping the embryos from the atrial walls. They can be collected with a pipette and transferred to fingerbowls of sea water, where they will continue their development (Scott, (1934). In any one colony, all the embryos are at the same stage of development.

To obtain free-swimming larvae, a considerable number of adult colonies should be collected during the morning and placed in large fingerbowls of fresh sea water, near a window but out of direct sunlight. Sexually mature colonies contain zooids which are relatively thick and which tend to mat together. If they are fully ripe, some colonies will begin to release larvae within a few minutes. According to Grave (1937), the number of larvae released reaches a maximum at noon, with only an occasional tadpole being set free in the early morning or late evening.

For the study of metamorphosis and budding, tadpoles should be isolated in separate drops of sea water in watch glasses. When the larvae are firmly affixed, the dishes can be stored in an inverted position in wooden racks. These are in turn submerged in an aquarium supplied with a constant flow of sea water.

An easy way to collect tadpoles for the study of metamorphosis and budding is to stand slides around the inner wall of a fingerbowl containing Botryllus colonies. The tadpoles will attach to the slides, which may then be replaced in running sea water, in open slide-boxes. The advantage of collecting in this fashion lies in the fact that such slides of Botryllus may be killed, fixed, stained and mounted for further study.

D. Methods of Observation: Metamorphosing and budding individuals can conveniently be studied in the watch glasses to which they are attached; debris should be flushed out gently. The specimens may be slightly flattened if a coverslip is gently lowered to cover them.

NORMAL DEVELOPMENT

A. Egg Characteristics: The living egg measures 420 microns in diameter, according to Berrill (1937); when fixed, it is 215 microns in diameter (Scott, 1934). The yolk is in the form of small, evenly distributed granules. The mature egg is shed into the atrial cavity, at metaphase of the first maturation division. It is surrounded by a chorion and an inner and outer layer of follicle cells. The inner follicle cells are sparsely scattered within the narrow perivitelline space; the outer follicle cells become fused with the outer wall of the peribranchial cavity, thus holding the developing egg in a fixed position. Two to six eggs are found in a single individual.

B. Fertilization and Cleavage: Fertilization probably occurs when the egg is shed into the atrial cavity, and cleavage is virtually the same as in other ascidians. Gastrulation occurs between the sixth and seventh cleavages, and is similar to that of Styela (Scott, 1934).

C. Rate of Development: Development in this form is relatively rapid; a free swimming larva is produced in about 12 hours.

D. Later Stages of Development and Metamorphosis: The neural plate of the young embryo is wide in the future brain region and narrows posteriorly. The neural folds, which encircle it, are visible shortly before the round blastopore closes; as they fuse to form the neural tube, the tail becomes marked off from the trunk and turns sharply to the left. Into the tail bud grow the dorsal neural tube, lateral muscle bands, notochord, and a ventral strand of endoderm. As the tail develops, it encircles the body meridionally, and by the time it has grown halfway around the body, the neuropore (seen in the region of the brain vesicle in early stages) has closed. A clear region in the anterior, ventral portion of the trunk marks the position of the primitive enteric cavity.

Shortly after the closure of the neuropore, a rapid series of changes occurs in the brain vesicle, resulting in the formation of a sensory vesicle, with a single sense receptor, and an adjacent hypophysis and associated ganglia. A conspicuous dorsal groove is present in the epidermis. In later development this groove stretches between the siphons. The atrium is formed by a single dorsal invagination in the posterior region of the trunk. A row of vertical gill slits is formed on each side where the two lobes of this invagination come into contact with the posterior wall of the pharynx. The tunic, siphons, papillae, and ampullae develop relatively late in embryonic life. When fully formed, the larvae drop off into the atrium and are released through the atrial siphon. (See the paper by Scott, 1934, for further details. )

The free-swimming tadpole is smaller than that of Amaroucium, having a body length of only 320-400 microns. The translucent tunic contains scattered cells, and extends out over the tail in the form of vertical fins. At the anterior end of the trunk can be seen eight conspicuous, sac-like outgrowths of the mantle, which are destined to be parts of the still non-functional and incomplete circulatory system (Grave and Woodbridge, 1924). Also extending from the anterior region of the mantle are three projections arranged in the form of a triangle. Each of these contains a basal ganglion connected to the central nervous system, and they are believed to be sensory (rather than adhesive) in function (Scott, 1934). The siphons are inconspicuous and non-functional during the free-swimming period. The deep dorsal groove is visible between the siphons. The sensory vesicle appears as a large, clear sac located just behind the ampullae; suspended within it by a slender stalk is the statolith, a dense black cup associated with light-sensitive elements (Grave and Riley, 1935). The pharynx is large and contains a prominent endostyle along its anterior border. It extends posteriorly around the sensory vesicle in the form of two lateral lobes, each of which is perforated by a vertical row of four to six gill slits. The mass of yolk, which is so conspicuous in the pharyngeal floor of the Amaroucium tadpole, is completely lacking in the tadpole of Botryllus. A small, undeveloped heart lies below the pharynx. The short oesophagus leads to a sac-like stomach which narrows to a small intestine, coursing upward to the atrium. In the tail, the central notochord, dorsal neural tube, lateral muscle bands, and ventral cord of endoderm are clearly visible.

When first released, the tadpoles are strongly attracted to light; this attraction lasts throughout the greater portion of the free-swimming life. There is a period of indifference to light stimulus before metamorphosis, and some indication of a negative phototropism immediately before fixation (Grave and Woodbridge, 1924). The initial response to gravity is negative, but this decreases as metamorphosis approaches.

The length of the free-swimming period varies from 13 minutes to 27 hours, although on the average metamorphosis occurs in about two hours. Grave (1935) and Grave and Nichol (1939) have done some interesting work in an attempt to analyze the conditions which influence the onset of fixation. The anterior end of the tadpole attaches and metamorphosis is extremely rapid. One of the most striking features of the process is the unfolding of the ampullae, which spread out around the base of the developing tunicate like the petals of a flower.

Tadpoles which have attached and have been growing for two days are usually oriented so that the oral and atrial siphons are directed away from the substrate. The large pharynx, shaped like a truncate cone, bears three rows of stigmata (visceral clefts) which allow water to pass out into the atrial cavity on either side. A rod-like endostyle lies on the underside of the pharynx. The stomach ordinarily appears as a yellow body under the atrial opening. The intestine, near its junction with the stomach, turns to one side and loops to empty near the atrium.

E. Asexual Reproduction: Colony formation in Botryllus is often accomplished by the so-called "atria!" type of budding. The first bud, or blastozooid, is formed by an invagination of one side of the atrium, and its subsequently differentiated parts are thus derived solely from ectoderm. It is furnished with a blood supply. This first blastozooid is single, but all the later buds are formed in symmetrical pairs. By one week after attachment, four rows of stigmata have developed in the pharynx of the oözooid, and probably three or four rows in the blastozooid. Buds of the second and third order may have formed. The same organ structures are visible in all these individuals, notwithstanding their diverse embryology, with the minor exception that the oözooid does not develop gonads. By re-orientation

of the individuals, the completed colony develops a common atrial pit at its center, and separate pharyngeal openings at the periphery. (For further details consult the papers of Pizon, 1893; Berrill, 1941a, 1941b; Watterson, 1945.)

Recently, Oka and Watanabe (1957) have described a process of "vascular budding" in this form.

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., 1941a. The development of the bud in Botryllus. Biol. Bull., 80: 169-184.

BERRILL, N. J., 1941b. Size and morphogenesis in the bud of Botryllus. Biol. Bull., 80: 185193.

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

GRAVE, C., 1937. Notes on the culture of eight species of ascidians. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 560-564.

GRAVE, C., AND P. A. NICHOL, 1939. Studies of larval life and metamorphosis in Ascidia nigra and species of Polyandrocarpa. Pap. Tortugas Lab., 32: 1-46. (Carnegie Inst., Wash., Publ. no. 517.)

GRAVE, C., AND G. RILEY, 1935. Development of the sense organs of the larva of Botryllus schlosseri. J. Morph., 57: 185-211.

GRAVE, C., AND H. WOODBRIDGE, 1924. Botryllus schlosseri (Pallas): The behavior and morphology of the free-swimming larva. J. Morph., 39: 207-247.

HERDMAN E. C., 1924. Botryllus. Mem. Liverpool Mar. Biol. Comm., 26: 1-40.

OKA, H., AND H. WATANABE, 1957. Vascular budding, a new type of budding in Botryllus. Biol. Bull., 112: 225-240.

PIZON, A., 1893. Histoire de la blastogenese chez les Botryllides. Ann. Sci. Nat., Zool., ser. 7, 14: 1-386.

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.

WATTERSON R. L., 1945. Asexual reproduction in the colonial tunicate, Botryllus schlosseri ( Pallas ) Savigny, with special reference to the developmental history of intersiphonal bands of pigment cells. Biol. Bull., 88: 71-103.