This sea cucumber is abundant at Woods Hole, Mass. It lies almost completely buried in mud or sand in shallow water, and may be collected from many of the inlets in the vicinity. One recommended location is Bowen's Pond, near Falmouth Heights; another is in the Gutters at Hadley Harbor.

The sexes are separate and similar in appearance. The genital papilla, found between the two dorsal tentacles, is reportedly larger in males, but this has proved too unreliable a characteristic for sex determination. The sex ratio is approximately five females to six males (Colwin, 1948).

June and July, according to Bumpus (1898), but June and possibly earlier is suggested by Colwin (1948), who found that the capacity to spawn decreases markedly during the latter half of June. Mead (1898) observed that every female examined at the end of April was full of nearly ripe eggs, a fact which would certainly indicate a season beginning in May, but Just (1929) claimed that eggs obtained in April and May showed atypical responses to insemination. He believed they were unripe oocytes, capable of responding to insemination but unable to develop. Until this has been further investigated, Colwin (1948) recommends the first half of June for embryological experiments with this material.

A. Care of Adults: Any medium- or large-sized animal should be a suitable source of gametes. It is advisable to store the animals overnight in containers supplied continuously with fresh sea water, at a temperature between 15û and 17û C.; the latter precaution discourages shedding, and the former ensures that much of the debris, which Thyone pumps through its gut, will be removed, thus facilitating the recovery of gametes. The animals should then be isolated in containers with enough sea water to permit them to expand fully&emdash;6 X 71/2-inch battery jars are excellent. The sea water should be changed once or twice a day (Colwin, 1948).

B. Procuring Gametes: Although active sperm and apparently mature eggs can be teased from the gonadal lobes, artificial insemination of such eggs has not proved successful (Ohshima, 1925a; Just, 1939). This failure is perhaps explained by the fact that ovarian eggs contain a large germinal vesicle, while those shed normally are in metaphase of the first maturation division.

Spawning is preceded by full expansion and the so-called "feeding movements" of the tentacles. Ohshima (1925a, 1925b) noted that although shedding normally occurs late in the afternoon of the day of collection, it could be induced at any time by placing the animals in a dim light; it did not occur in darkness. However,

Colwin (1948) observed some normal shedding throughout the day and even in total darkness, although evening seemed to be the optimum time to obtain gametes. She discounts light intensity as a factor primarily involved in the shedding reaction. Any stimulus strong enough to cause a contraction of the animal will interrupt the process.

Colwin's method (1948) for obtaining naturally-shed gametes is to isolate the animals in sea water warmed to room temperature (20-22û C.) for about five hours. Shedding may begin immediately or after an interval; it may not occur until after the sea water has been re-cooled (16-18û C.). A period of warming is, however, apparently necessary for the occurrence of shedding in the laboratory. Other unknown factors may increase the frequency, but injections of KCl have no such effect. A concentrated sperm suspension is obtained by placing a shedding male into a fingerbowl containing only a very small amount of sea water. The adults are removed from their respective containers as soon as shedding ceases.

Colwin and Golwin (1956) have recently described another method for obtaining Thyone gametes, involving electrical stimulation of the adults. However, they state (p. 252) that they have not observed cleavage in inseminated cultures prepared from such gametes.

C. Preparation of Cultures: A small amount of sperm suspension should be added to the dish of eggs. After a short interval, the sea water should be changed, and the dish covered and placed on a water table. The sea water must be changed several times a day.

The larvae emerge in about three days. They may be successfully reared if placed in large battery jars of sea water, provided with some surface sand from a stock aquarium containing a diatom culture; then they may be left without further care. Details of the method for diatom culture may be found in a short paper by Grave (1902)

NORMAL DEVELOPMENT

A. The Unfertilized Ovum: The egg is hemispherical in shape, measuring 260 to 300 microns in the equatorial plane, and 200 to 300 along the egg axis, and is enclosed in a thick, striated, jelly hull, approximately 55 microns wide (Colwin and Colwin, 1956). It is opaque and yellow-brown in color. The ovum is shed at metaphase of the first maturation division, although Colwin and Colwin (1956) point out that in living eggs, the germinal vesicle often appears to be intact, perhaps due to persistence of its "residual substance."

B. Fertilization and Cleavage: The eggs are fertilized as soon as they are shed into the water, the sperm most frequently entering near the equator. The polar bodies are soon formed and extruded, the egg rounding up during their formation (Ohshima, 1925a). An equal and somewhat modified form of radial cleavage was briefly noted by Ohshima (1925a). Gastrulation is by invagination, and appears to be similar to that of Cucumaria (Ohshima, 1921). Illustrations of the fertilization cone and polar body formation may be found in papers by Ohshima (1925b), and by Colwin and Colwin (1956).

C. Time Table of Development: The following schedule is taken from the paper of Ohshima (1925a). The time is calculated from egg-laying, the temperature not being indicated.

Stage First polar body Second polar body First cleavage Second cleavage Third cleavage Gastrula Emergence

Time 20-30 minutes 50-60 minutes 120 minutes 150 minutes 180 minutes 18-20 hours 3-1/2 days

D. Later Stages of Development: The ciliated gastrula, with polar bodies still attached, enclosed, within its membrane, transforms into a dipleurula, and then, just before emergence, into a metadoliolaria larva. This type of development is similar to that of Holothuria floridana, described by Edwards ( 1909). The metadoliolaria has a large, overhanging pre-oral hood, five unbranched tentacles and a pair of ventral pedicels. This form creeps out of the egg membrane. During the course of later development, the pre-oral hood is resorbed (pentactula stage), the tentacles branch and increase in number to ten, the ventral pedicels increase in number, and calcareous plates appear in the skin. There is no freeswimming stage (Ohshima, 1925a).

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

COLWIN, L. H., 1948. Note on the spawning of the holothurian, Thyone briareus (Lesueur). Biol. Bull., 95: 296-306.

COLWIN, L. H., AND A. L. COI.WIN, 1956. The acrosome filament and sperm entry in Thyone briareus (Holothuria) and Asterias. Biol. Bull., 110: 243-257.

EDWARDS, C. L., 1909. The development of Holothuria floridana Pourtales with especial reference to the ambulacral appendages. J. Morph., 20: 211-230.

GRAVE, C., 1902. A method of rearing marine larvae. Science, 15: 579-580.

JUST, E. E., 1929. The production of filaments by echinoderm ova as a response to insemination, with special reference to the phenomenon as exhibited by ova of the genus Asterias. Biol. Bull., 57: 311-325.

JUST, E. E., 1939. Basic Methods for Experiments on Eggs of Marine Animals. P. Blakiston's Son & Co., Inc., Philadelphia.

MEAD, A. D., 1898. The breeding of animals at Woods Holl during the month of April, 1898. Science, 7: 702-704.

OHSHTMA, H., 1921. On the development of Cucumaria echinata v. Marenzeller. Quart. J. Micr. Sci., 65: 173-246.

OHSHIMA, H., 1925a. Notes on the development of the sea-cucumber, Thyone briareus. Science, 61: 420-422.

OHSHIMA, H., 1925b. Pri la maturigo kaj fekundigo ce la ovo de l'markukumoj. Bulteno Scienca de la Facultato Terkultura; Kyusu Imp. Univ., 1: no. 2, pp. 100-106 (in Japanese and Esperanto).