LIVING MATERIAL

The common sand dollar is found in isolated areas on sandy bottoms below low tide level, and is obtained in abundance by dredging at West Barnstable on Cape Cod Bay, Mass.

The sexes are separate, but it is impossible to distinguish the male from the female by superficial examination.

The animals breed abundantly at Woods Hole, Mass., from at least the last week of March to the end of July; even in August, a limited amount of ripe gametes may be obtained (Bumpus, 1898a, 1898b; Mead, 1898).

A. Care of Adults: The adults may be kept indefinitely in an aquarium provided with a layer of clean sand and a continuous supply of fresh sea water. They can be fed on kelp, mussels, and other marine plants and animals.

B. Procuring Gametes: Tyler's method (1949) for procuring gametes is to inject isosmotic KCI (0.5 M) with a hypodermic syringe into the body cavity of the sand dollar, inserting the needle (25-27 gauge, 1/2- to 3/4-inch) through the mouth in a direction as nearly parallel as possible to the oral surface. A single injection of 0.5 cc. of 0.5 M KCI into an animal about 30 cc. in volume will induce shedding of virtually all the ripe eggs or sperm. For smaller animals, the dose should be proportionately smaller (0.5-0.2 cc.). Shedding starts within a few seconds and is completed in 5 to 15 minutes. Unripe animals usually fail to respond. The eggs can be collected readily by immersing the animal in a large fingerbowl containing about 250 cc. of sea water. The sperm can be removed "dry" or in concentrated suspension.

This method does not injure the animals. More gametes are matured, provided the animals are well fed, and successive sheddings may be obtained at twoweek intervals during the season. If only a small number of gametes is desired, a correspondingly small amount of KCI should be injected, and the animals may then be kept for further use.

The eggs of Dendraster excentricus, the common sand dollar of the Pacific Coast, may be obtained by this same method (Tyler, 1949). Their development is similar to that of Echinarachnius parma.

C. Preparation of Cultures: The eggs should be washed several times in sea water. To them add one drop of sperm suspension (one drop "dry" sperm in 10 cc. of sea water) and mix thoroughly. After about 10 minutes, when the eggs have settled, decant the sea water to remove the excess sperm, and add fresh sea water. Store the fingerbowls on the water table. The sea water should be changed once a day. Normal development will not take place at temperatures above 24û C.

The larvae can be reared to metamorphosis if they are transferred to large battery jars of sea water and provided with some surface sand from a stock aquarium containing a diatom culture. No further care is necessary. Details of the method for diatom culture may be found in a short paper by Grave (1902).

D. Removal of Membrane: The fertilization membrane may be easily removed by shaking the eggs in a test-tube, half-full of sea water, a few seconds after the membrane has elevated. Echinarachnius eggs are fragile and will not stand as much shaking as those of Arbacia.

NORMAL DEVELOPMENT

A. The Unfertilized Ovum: The mature egg of this species measures approximately 135 microns in diameter, and is surrounded by a thick jelly-layer in which are suspended fine red pigment granules. The diameter across the jelly varies from 220 to 250 microns (Fewkes, 1886). The egg itself, free of the jelly, is yellow due to its yolk content.

B. Fertilization and Cleavage: In a matter of seconds (7-22) after sperm penetration, the vitelline membrane begins to elevate in a wave from the entrance point of the sperm around the egg cortex (Just, 1919a). The resulting perivitelline space is quite large. Cleavage is usually equal and regular until the 8-cell stage (Fewkes, 1886). There is a thinner hyaline plasma layer than in the Arbacia egg, and less regularity of cleavage. However, the cleavage pattern is not markedly different from that of Arbacia. A hollow, ciliated blastula, which rotates slowly within the fertilization membrane, is produced. Gastrulation is by invagination.

C. Time Table of Development: No temperature was recorded for the following schedule of development observed by Fewkes (1886). The times are calculated from fertilization.

Stage First cleavage Second cleavage Third cleavage Blastula

Time 1-1/2 hours 2 hours 3 hours 10 hours

D. Later Stages of Development and Metamorphosis: The early larva is nearly spherical in shape and rests on a tripod formed by the two posterior arms and the single anterior lobe. Pigment can be seen at the anal pole. A rudimentary skeleton, primitive alimentary tract, and mouth and anal openings are present. As the larva matures, it becomes more and more helmet-shaped. At four days the oral, or anterior, arms begin to form, and by the end of a week the anterolateral and antero-internal arms have been added.

The body of the older pluteus is elongated, and rounded at the anal region. The four pairs of ciliated arms are of equal length; all except the antero-internal pair contain clusters of dark red pigment granules at the distal ends of the calcareous supporting rods. The pigmentation extends along the arms and over the body wall. The pluteus is propelled freely, but not rapidly, through the water.

The young sand dollar, which is very different in shape from the adult, is formed about the left hydrocoele of the pluteus, which is gradually absorbed. It is almost completely opaque, due to the formation of pigment, spines, calcareous rods and plates. The body is rounded and plump, and the spines are proportionately large when compared with those of the adult. Further details and illustrations of the larval development can be found in a paper by Fewkes (1886)

BUMPUS, H. C., 1898a. The breeding of animals at Woods Holl during the month of May, 1898. Science, 8: 58-61.

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

CHILD, C. M., 1950. Differential modification of sand-dollar development in relation to temperature. Physiol. Zool., 23: 140-168.

FEWKES, J. W., 1886. Preliminary observations on the development of Ophiopholis and Echinarachnius. Bull. Mus. Comp. Zool., Harvard, 12: 105-152.

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

JUST, E. E., 1919a. The fertilization reaction in Echinarachnius parma I. Cortical response of the egg to insemination. Biol. Bull., 36: 1-10.

JUST, E. E., 1919b. II. The role of fertilizin in straight and cross fertilization. Biol. Bull., 36: 11-38.

JUST, E. E., 1919c. III. The nature of the activation of the egg by butyric acid. Biol. Bull., 36: 39-53.

Just, E. E., 1920. IV. A further analysis of the nature of butyric acid activation. Biol. Bull., 39: 280-305.

JUST, E. E., 1922. V. The existence in the inseminated egg of a period of special susceptibility to hypotonic sea-water. Amer. J. Physiol., 61: 516-527.

JUST, E. E., 1923a. VI. The necessity of the egg cortex for fertilization. Biol. Bull., 44: 1-9.

JUST, E. E., 1923b. VII. The inhibitory action of blood. Biol. Bull., 44: 10-16

JUST, E. E., 1923c. VIII. Fertilization in dilute sea-water. Biol. Bull., 44: 17-21.

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

PEASE, D. C., 1941. Echinoderm bilateral determination in chemical concentration gradients. I. The effects of cyanide, ferricyanide, iodoacetate, picrate, dinitrophenol, urethane, iodine, malonate, etc. J. Exp. Zool., 86: 381-404.

PEASE, D. C., 1942a. Echinoderm bilateral determination in chemical concentration gradients. II. The effects of azide, pilocarpine, pyocyanine, diamine, cysteine, glutathione, and lithium. J. Exp. Zool. 89: 329-345.

PEASE, D. C., 1942b. Echinoderm bilateral determination in chemical concentration gradients. III. The effects of carbon monoxide and other gases. J. Exp. Zool., 89: 347-356.

TYLER, A., 1949. A simple, non-injurious method for inducing repeated spawning of sea urchins and sand-dollars. Coll. Net, 19: 19-20.