These worm-like animals are 15 cm. or more in length; the proboscis is pinkish yellow in color and the body orange-yellow. They are collected from sand flats in the Woods Hole, Mass., area. The sexes are separate.

No specific information on this subject has been recorded, for animals in the Woods Hole region. Presumably they are ripe during the latter half of July, and in August.

A. Care of Adults: The animals may be kept in fingerbowls supplied with a layer of clean sand. A gentle stream of running sea water should be provided

B. Procuring Gametes: Colwin and Colwin (1953) report that females will shed spontaneously in the laboratory, the number of eggs spawned ranging from a few dozen to more than a thousand; the usual number (depending somewhat on the size of the female) is 100-300. Sperm may be obtained by cutting into the testis of a ripe male and collecting the sperm, which ooze out, in a pipette.

C. Preparation of Cultures: Colwin and Colwin (1953) placed naturally-shed eggs in small glass dishes containing sand-filtered sea water; spermatozoa (obtained as described above) were added directly from the pipette in which they were collected (Colwin and Colwin, 1950). Several changes of fresh, sand filtered sea water should be made following insemination, and at least two or three times daily thereafter. The egg of Saccoglossus is sensitive to a number of environmental factors, including temperature, and the Colwins routinely took precautions to assure that the temperature of the sea water did not rise above 25û C. The eggs should not be crowded. It is advisable to keep the culture dishes in moist-chambers which are surrounded by running sea water.

Pre-hatching larvae should be transferred to dishes containing small amounts of fine, clean sand. If this is not done, free-swimming larvae tend to become caught in the surface film of the water, and the older, crawling larvae may become trapped by the adhesion to glass surfaces of the mucus which they secrete. Feeding is apparently not necessary; Colwin and Colwin (1953) supplied older cultures with unfiltered sea water, which contains an adequate amount of food to supply the larvae for at least as long as 36 days.

A. The Unfertilized Ovum: The eggs are opaque, and vary in color from whitish-gray to dark grey-blue (Colwin and Colwin, 1953); they are rather irregular in shape before fertilization, and measure, on the average, about 330 microns by 420 microns. The germinal vesicle usually breaks down before the eggs are shed, and is at the metaphase of the first maturation division when it passes from the body of the female. The egg may remain fertilizable for a considerable period of time after shedding.

B. Fertilization and Cleavage: Fertilization takes place at the metaphase of the first maturation division, the sperm apparently entering at any point on the egg surface (Colwin and Colwin, 1953). The first polar body is given off about ten minutes after insemination, and the second 30 to 40 minutes later. The position of the polar bodies bears no very constant relation to the exact position of the animal pole. The details of fertilization (including the formation of the fertilization cone and membrane elevation) are described by Colwin and Colwin (1954a, 1954b).

After fertilization, the eggs become spherical in shape and are about 350 microns in diameter; soon after the first polar body appears, a broad, shallow "girdle" constricts the equatorial zone, and 15-30 minutes after insemination, a pear-shaped stage is attained, the vegetal hemisphere constituting the large, blunt end and the animal hemisphere the narrower portion. Shortly before the second polar body appears, the egg again becomes spherical, and after the second polar body is given off, a "reversed pear-shape" is evident, in which the animal hemisphere is the large, blunt end and the vegetal hemisphere is the more pointed one. Once again, the egg rounds up, and shortly before the first cleavage, there is a shortening of the animal-vegetal axis.

The first cleavage is usually approximately equal, the furrow passing from the animal to the vegetal pole; the same holds true for the second division. The third cleavage is latitudinal, resulting in an animal and a vegetal tier of cells; the size ratio of the two tiers, with respect to one another, is variable. At the fourth cleavage, the animal tier of cells divides somewhat sooner than the vegetal tier, so that a transitory 12-cell stage is present, followed shortly by a 16-cell stage after the vegetal cells divide. The eight animal cells are divided into a single tier composed of two more or less parallel rows, each consisting of two large central cells with a smaller cell at each end. These two long rows of cells represent the dorsal and ventral sides, respectively, of the embryo, and their two ends are the future left and right sides of the embryo (Colwin and Colwin, 1953). The vegetal cells at the 16-cell stage are arranged in an upper tier of four large cells and a lower tier of four small cells.

The early blastula is somewhat flattened at the vegetal end, but later it becomes more nearly spherical. Gastrulation is by invagination, and the embryo rotates actively before closure of the blastopore begins; cilia are present (as a transverse ciliated band) in a ring around the blastopore.

C. Time Table of Development: The following schedule is based on data from the paper by Colwin and Colwin (1953); temperatures were from 20û to 25û C., and the times were recorded from insemination.

Stage First polar body "Equatorial girdle" Pear-shape Second polar body "Reversed pear-shape" Sphere-shape First cleavage Second cleavage Third cleavage Fourth cleavage Blastula Gastrula Elongation of gastrula Appearance of first pair of gill slits Hatching

Time 10 minutes 12-17 minutes 15-20 minutes 40-50 minutes 40-55 minutes 50-70 minutes 1-3/4 to 2-1/2 hours 2-1/2 to 3-1/4 hours 3-1/2 to 3-3/4 hours 3-3/4 to 4-1/2 hours 6-15 hours 13-24 hours 18 hours 3 days 7 days

D. Later Stages of Development and Metamorphosis: About 18 hours after insemination, the late gastrula begins to elongate in an antero-posterior direction. The division of the body into proboscis, trunk and collar regions is next accomplished; this process usually is complete 48 hours after insemination. The larva acquires pigmentation except in the area of the posterior collar groove. The first pair of gill slits appears when the pharyngeal pouches become perforated at three to four days, and two or three additional pairs subsequently develop. Emergence of the larva from its membranes occurs by the seventh day, and an adhesive sucker is developed. Further details, and photographs of many stages in the development of this form, are to be found in the paper by Colwin and Colwin (1953).

BATESON W., 1884. The early stages in the development of Balanoglossus (sp. incert.). Quart. J. Micr. Sci., 24: 208-236.

BATESON, W., 1885. The later stages in the development of Balanoglossus kowalevskii, with a suggestion as to the affinities of the Enteropneusta. Quart. J. Micr.. Sci., 25: Suppl.. 81-122.

COLWIN, A. L., AND L. H. COLWIN, 1950. The developmental capacities of separated early blastomeres of an enteropneust, Saccoglossus kowalevskii. J. Exp. Zool., 115: 263-295.

COLWIN A. L., AND L. H. COLWIN, 1951. Relationships between the egg and larva of Saccoglossus kowalevskii (Enteropneusta): Axes and planes; general prospective significance of the early blastomeres. J. Exp. Zool., 117: 111-137.

COLWIN, A. L., AND L. H. COLWIN, 1953. The normal embryology of Saccoglossus kowalevskii (Enteropneusta). J. Morph., 92: 401-453

COLWIN, L. H., AND A. L. COLWIN, 1954a. Fertilization changes in the membranes and cortical granular layer of the egg of Saccoglossus kowalevskii (Enteropneusta). J. Morph., 95: 1-45

COLWIN, L. H., AND A. L. COLWIN, 1954b. Sperm penetration and the fertilization cone in the egg of Saccoglossus kowalevskii (Enteropneusta). J. Morph., 95: 351-371.