LIVING MATERIAL :

These clams are found on sandy bottoms in shallow water, and may be collected by digging at low tide. Large individuals are obtainable at Barnstable, Mass. The sexes are separate, but are similar in appearance.

Ripe animals can be secured at least throughout the summer months (Schechter, 1941). They have been collected off the New Jersey coast as late as December (Heilbrunn, personal communication), and Allen (1953) reports that eggs are obtainable from animals collected in New Jersey waters at any time from early spring until late autumn. In the Woods Hole region, Allen (1953) describes the season as beginning late in May and extending into September.

A. Care of Adults: Clams kept in large aquaria, with a good supply of running sea water, will retain their gametes in a viable condition for several weeks. It is advisable to segregate the sexes. Dead or dying individuals should be removed immediately.

The sex of an individual may be ascertained, without injury, as follows: Place a wooden peg between the open valves to keep them slightly apart. Now insert, as near the hinge as possible, a hypodermic needle (2-l/2-inch, 20-gauge), attached to a syringe containing a small amount of sea water. Inject a few drops into the region of the gonad (anterior to, and below, the pedal retractor muscle), and then draw the fluid back into the syringe. If the animal is mature, this withdrawn material will contain either eggs or sperm, which are readily identifiable.

B. Procuring Gametes: Both male and female gametes may be procured by either of the following methods: 1) Select an animal, rinse it in fresh water and dry. In a few seconds, the valves will gape slightly and a strong, sharp scalpel can be introduced to cut the adductor muscles. Remove the animal from the shell and excise the gonad (which lies beneath the gills, mantle and heart). Eggs should be strained through several thicknesses of cheesecloth (previously washed in fresh water and then saturated with sea water) into a large volume of filtered sea water. When the eggs have settled, decant the supernatant fluid and replace it with fresh filtered sea water (Allen, 1953); this process of washing should be repeated at least three times, in order to remove all blood and body fluids, which apparently have a deleterious effect on fertilization. 2) Sometimes the animals shed spontaneously when they are removed from the shell.

C. Preparation of Cultures: Inseminate a fingerbowl of eggs with four or five drops of sperm suspension and gently rotate the dish once. Allow it to stand undisturbed for about 30 minutes; then change the water and cover the dish with a glass plate. Place the culture on a water table, and after about 10 hours, decant the upper layers of water containing the vigorous top-swimming larvae to a clean fingerbowl. Add fresh sea water to the decanted larvae, and repeat this procedure at least once a day. After two or three days, diatoms should be added to the culture. Details for the maintenance of larval cultures are given by Prytherch (1937) and by Loosanoff (1954).

D. Methods of Observation: Nuclei will stand out very sharply if the eggs are placed on an ordinary slide, covered with a coverslip, and flattened slightly by removing some of the water. Both eggs and swimming larvae can be observed on depression slides, with or without coverslips. The larvae may be quieted by adding a few drops of dilute Janus green to the preparations. This paralyzes the cilia, and the animals, although living, are held motionless.

Normal DEVELOPMENT

A. The Unfertilized Ovum: When freshly shed, the egg is irregular in shape due to pressure within the ovary, but on standing it soon becomes spherical. It is small, measuring 53 to 56 microns in diameter. The center of the egg is almost completely filled by an enormous germinal vesicle (30 microns in diameter), containing a prominent nucleolus. There is a thin layer of clear cortical protoplasm, surrounding some densely packed yolk. The eggs will retain this appearance for many hours, unless inseminated.

B. Fertilization and Cleavage: Within a few minutes after insemination, the germinal vesicle begins to break down, and soon only a light area in the center of the egg marks its former position. A thin fertilization membrane is elevated a very short distance from the egg surface. The first polar body forms shortly after germinal vesicle breakdown, and the second polar body is extruded directly beneath it. Their position marks the plane of the first cleavage.

The male and female pronuclei become visible, approach each other and fuse. Cleavage occurs soon after; it is unequal, and the first two blastomeres differ greatly in size. The second cleavage follows and, in the case of the larger cell, is again unequal, producing one large cell and three smaller ones. The subsequent cleavages are rapid. They are of a spiral type, but because of the size differences of the blastomeres, this is more difficult to follow than in Crepidula. The small, rapidly dividing ectodermal cells spread over the larger, yolk-filled endodermal cells, leaving an uncovered region, the blastopore. Thus, gastrulation is by epiboly; a swimming gastrula is formed.

C. Time Table of Development There is considerable variation in developmental rate, depending on temperature and other environmental factors; however, the following schedules (which are obviously discrepant) give some idea of the chronology at 21û C. (Allen, 1953) and 25û C. (Schechter, 1941). Times are recorded from insemination.

Stage Germinal vesicle breakdown Formation of polar bodies Pronuclei visible First cleavage Second cleavage Swimming forms

Time (21û C.) 6-7 minutes 29 minutes 50 minutes 74 minutes 99 minutes Within 24 hours

Time (25û C.) 10 minutes 30 minutes 50 minutes 65 minutes 95 minutes 5 hours

D. Later Stages of Development and Metamorphosis: The young swimming larva (5-6 hours after insemination) has on the future dorsal side a plate of large cells, the primordium of the shell gland. Internally, two large dark cells, the mesodermal teloblasts, are often visible. By 9 hours, the embryos have lost their somewhat barrel-shaped appearance and are pyramidal trochophores. The expanded base of the pyramid is the region in which the velum will form. The cilia are not markedly visible at this time. On the ventral side a small indentation marks the blastopore, and on the dorsal surface the invagination of the shell gland appears as a conspicuous concavity. By 14 hours the shell gland has evaginated, and this hollow cannot be seen. The cilia of the velum and the apical flagellum are well developed.

The young veliger (18-19 hours) has a well-deveIoped bivalved shell, with a straight hinge line. On the side opposite the apical flagellum a telotroch has formed. The stomodeal invagination lies just below the velum, but as yet there is no proctodeal invagination. The internal structures are obscured by a mass of undifferentiated endodermal and mesodermal cells which fills most of the postvelar area.

The two-day larva has both mouth and anal openings, and clearly defined oesophagus, stomach, intestine and liver. The movements of the digestive tract and the cilia which develop along its entire length are easily observed. Three groups of retractor muscles converge dorsally, and the anlage of the anterior adductor muscle can be seen as a small clump of cells anterior and dorsal to the stomach.

In about five days the velum gradually disappears, giving place to a slender, active foot (Belding, 1910). This functions first as a swimming and later as a crawling organ. The young clam is now ready to settle to the bottom.

The development of Dreissensia is very similar to that of Mactra; it has been described and figured by Meisenheimer (1901).

ALLEN, R. D., 1953. Fertilization and artificial activation in the egg of the surf-clam, Spisula solidissima. Biol. Bull., 105: 213-239.

BELDING, D. L., 1910. The growth and habits of the sea clam, Mactra solidissima. Ann. Rep. Comm. Fish and Game, Massachusetts, 1909, pp. 26-41.

KOSTANECKI, K., 1902. Ueber künstliche Befruchtung und künstliche parthenogenetische\ Furchung bei Mactra. Bull. de l'Acad. des Sci., Cracovie, Cl. des Sci. Math. et Natur., Juillet, 1902, pp. 363-387.

KOSTANECKI, K., 1904. Cytologische Studien an künstlich parthenogenetisch sich entwickelnden Eiern von Mactra. Arch. f. mikr. Anat., 64: 1-98.

KOSTANECKI, K., 1908. Zur Morphologie der künstlichen parthenogenetischen Entwicklung bei Mactra. Arch. f. miter. Anat., 72: 327-352.

KOSTANECKI, K., 1911. Experimentelle Studien an den Eiern von Mactra. Bull. de l'Acad. des Sci., Cracovie, Cl. des Sci. Math. et Natur., ser. B, Mars, 1911, pp. 146-161.

LOOSANOFF, V. L., 1954. New advances in the study of bivalve larvae. Amer. Sci., 42: 607-624.

MEISENHEIMER, J., 1901. Entwicklungsgeschichte von Dreissensia polymorpha Pall. Zeitschr. f. wiss. Zool., 69: 1-137.

PRYTHERCH H. F., 1937. The cultivation of lamellibranch larvae. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 539-542.

SCHECHTER, V., 1936. Comparative hypotonic cytolysis of several types of invertebrate egg cells and the influence of age. Biol. Bull., 71: 410.

SCHECHTER, V., 1937. Calcium and magnesium in relation to longevity of Mactra, Nereis and Hydroides egg cells. Biol. Bull., 73: 392.

SCHECHTER, V., 1941. Experimental studies upon the egg cells of the clam, Mactra solidissima, with special reference to longevity. J. Exp. Zool., 86: 461-477.

SCLUFER, E., 1955. The respiration of Spisula eggs. Biol. Bull., 109: 113-122.