Styela is a simple ascidian with a hard tunic and small granular tubercles on and about the papillae bearing the apertures. It is brownish or yellow in color, and up to 25 mm. in length. Occasionally, the animals are found in groups although, as noted above, the form is not a colonial one. They are quite common around Woods Hole, Mass.

June to September, according to Berrill (1937).

A. Care of Adults: The animals live well in the laboratory, if they are adequately supplied with sea water. High temperatures should be avoided.

B. Procuring Gametes: Although it is hermaphroditic, Styela is ordinarily self-sterile. Eggs and sperm are shed between 4 and 7 P.M., and fertilization takes place when the ripe gametes from two different individuals are mixed. The usual method of obtaining Styela eggs and embryos has been to mince the gonads from a large number of individuals, in a dish of sea water. This liberates all stages in the maturation of eggs and sperm, and usually at least a few eggs will be fertilized (whatever the time of day or night) and will begin normal development.

Rose (1939) has described a method of controlling natural spawning in the laboratory; it works well except for a few weeks in mid-summer, when the animals are spent. The adults are kept in the dark until eleven or twelve hours before fertilization is desired; then an artificial day is started, by turning on a 40-watt electric light, placed about 18 inches from the animals. Eggs and sperm are discharged in clouds at the desired time. The same batch of animals can be induced to shed a number of times on successive days.

A. The Unfertilized Ovum: The mature unfertilized egg is approximately 150 microns in diameter, and has a tough membrane, the chorion, to which a few follicle cells adhere at the outer surface. Between the chorion and the egg surface, there are small, spherical inner follicle cells ("nurse cells"), which contain yellow granules. The peripheral layer of the egg is clear and contains minute yellow granules, and the central part of the egg consists of grey yolk platelets. The germinal vesicle is large and clear, and is excentrically placed, near the animal pole; it ruptures and maturation begins at about the time when the eggs are discharged. The maturation spindle remains at the metaphase of the first division until the sperm enters.

B. Post-Fertilization Changes: The sperm enters at or near the vegetal pole (Conklin, 1905a); maturation continues, and two polar bodies are given off. An extensive rearrangement of the cytoplasm now occurs: within two to eight minutes after fertilization, the clear, yellowish peripheral material streams to the lower pole, over the yolk, followed by the clear protoplasm from the animal pole. This process is best studied using daylight for illumination; the microscope diaphragm should be open as far as possible.

The grey yolk rises to occupy the upper pole, except for the space which surrounds the maturation spindle. Soon the yellow substance accumulates on one part of the lower hemisphere, where it assumes a crescentic form. Immediately above the broad part of the yellow crescent, there is a layer formed by the clear cytoplasm.

The different pigmented regions of the egg correspond closely to the various embryonic areas with specific presumptive developmental fates. The yellow pigment area, at the posterior vegetal region, forms the "yellow crescent," which is presumptive mesoderm. The ventral and anterior portion of the vegetal hemisphere, which has the slate grey color of the yolk, forms endoderm and small amounts of mesoderm; it also contributes to a portion of the neural plate. The animal hemisphere material, which is light grey in color because of the presence of clear protoplasm beneath the peripheral yolk, forms the body epidermis and a portion of the neural plate. The animal pole becomes the ventral-anterior side of the larva, while the vegetal pole is the future dorsal side.

C. Cleavage and Gastrulation: The first cleavage is equal, separating the two "horns" of the yellow crescent from one another and bisecting the clear protoplasm anterior to the yellow region. The second cleavage is nearly equal, vertical, and at right angles to the first. The two posterior cells contain only a small amount of yolk and practically all the yellow crescent substance. The two anterior cells, on the other hand, contain much yolk and almost no yellow crescent material. There is an equal division of the clear protoplasm to the four cells. At the third cleavage, which is horizontal, the yellow crescent substance is almost entirely confined to the two posterior dorsal cells. The planes of cleavage at the fourth division vary in different quadrants, but the cells do not overlap the sagittal plane of the embryo. Two of the antero-dorsal cells and two of the postero-ventral cells of the 16-cell embryo are crowded away from this sagittal plane, but all the other cells touch it. The dorsal and ventral hemispheres at this stage are mirror images of one another. The yellow pigment lies in four posterior cells. Division in the dorsal (vegetal) hemisphere precedes that in the ventral (animal) hemisphere at the fifth cleavage, and cleavage in the anterior part of each hemisphere precedes that in the posterior part. When the 32-cell stage is reached, the yellow substance is almost entirely confined to six dorso-posterior cells, three on each side of the midline. They give rise to mesoderm and mesenchyme. Six yolk-filled cells at the vegetal pole, anterior to the yellow mesoderm cells, give rise to endoderm. Four cells at the anterior border of the embryo (just below the equator) and two just above the equator produce the notochord and neural plate. All the other cells are ectodermal.

Gastrulation is by epiboly. The gastrula passes through disc-shaped, saucer shaped and cup-shaped stages, starting at the seventh cleavage. As it finally becomes egg-shaped, the blastopore assumes the form of a "T," the stem of the "T" being bordered by the yellow mesoderm-mesenchyme cells. The cells overhanging the cross-bar of the T-shaped blastopore constitute its dorsal lip. They overgrow it, finally engulfing the yellow cells which are then seen only dimly through the translucent ectoderm.

D. Time Table of Development: The following approximate schedule for the development of normally-shed Styela eggs is from the classic monograph of Conklin (1905a). If eggs are obtained from "minced" cultures, cleavage is delayed, the eggs apparently maturing at variable intervals after coming into sea water. Time is recorded from insemination; the temperature is not specified, although Conklin states that these observations were made during the evening hours.

Stage First cleavage Second cleavage Third cleavage Fourth cleavage Fifth cleavage Sixth cleavage Seventh cleavage (beginning of gastrulation) Eighth cleavage Neural plate Fully-formed tadpole

Time 40 minutes 70 minutes 100 minutes 120 minutes 140 minutes 160 minutes 180 minutes 200 minutes 5 hours 12 hours

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

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COHEN, A., AND N. J. BERRILL, 1936. The early development of ascidian eggs. Biol. Bull., 70: 78 88.

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

CONKLIN, E. G., 1905b. Mosaic development in ascidian eggs. J. Exp. Zool., 2: 145-223.

CONKLIN, E. G., 1905c. Organ-forming substances in the eggs of ascidians. Biol. Bull., 8: 205-230.

CONKLIN, E. G., 1931. The development of centrifuged eggs of ascidians. J. Exp. Zool., 60: 1-120.

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