* We should like to express our appreciation to Dr. A. C. Clement for his helpful suggestions, and for much of the information on which this section is based.

This snail is commonly found on mud flats in shallow water, and is abundant at Woods Hole, Mass. The species is dioecious.

Egg-laying in the natural habitat at Woods Hole has been recorded from the last week in April (Mead, 1898) to mid-July. However, snails collected in June and kept in running sea water in the laboratory will continue to deposit egg-masses through August. Toward the end of the summer, unfertilized eggs are often deposited, and fertilized eggs may be fragile. Snails collected at Woods Hole from December 1 on through the spring months will, if kept in aquaria at room temperatures, lay abundantly. Difficulty has been encountered in obtaining eggs from snails collected between mid-summer and December (Clement, personal communication ) .

A. Care of Adults: These snails are very hardy and can be maintained in the laboratory in good breeding condition for a prolonged period. A new batch of adults usually takes several days to become acclimatized before they begin to spawn. As many as 250 animals can be kept in a large aquarium, provided there is a continuous flow of fresh sea water; however, 100 snails will provide an adequate supply of eggs for most purposes.

One or more minced clams should be provided for food every day.

A smaller number of adults (about 25 to 30) can be kept for at least a month in standing, aerated sea water, if they are removed to a separate container for feeding and then returned to the aquarium. This makes possible their use for certain types of embryological experiments at inland laboratories, where running sea water is not available. See "Special Comments" below.

If the wooden ends of the aquaria commonly used at Woods Hole are covered with removable glass plates, collection of egg capsules is greatly facilitated.

B. Procuring Fertilized Ova: Fertilization is internal, and 30 to 300 eggs are deposited in transparent capsules, which are produced singly or in groups. The snails usually climb up the sides of the aquarium to spawn, but congregate at the bottom of the tank to feed. If an irregular feeding schedule is followed, eggs will not be deposited on the day after the animals are fed.

Freshly-deposited capsules may be obtained if the snails are watched to determine the time of oviposition. The capsules are first visible at the pedal gland opening at the anterior, median part of the foot. After a capsule is fastened to the glass, the snail can be gently removed and the capsule transferred to a watch glass of pasteurized sea water (prepared by heating the water to 70 or 80û C. for 15 minutes, cooling it and shaking to aerate).

Clement (1952 and personal communication) describes the following methods for obtaining Ilyanassa eggs. A handy device for scraping the capsules from the aquarium walls is a razor blade mounted in a long wooden handle. The capsules are picked up and transferred with a long, wide-mouthed pipette. Excess jelly and the non-pasteurized sea water can be removed from the egg-capsule, before it is placed in the watch glass, by gently rolling it on a piece of paper towel or filter paper.

Using a binocular dissecting microscope, hold the capsule against the bottom of the watch glass, with a sharpened dissecting needle. With a pair of curved cuticle scissors, snip off the end of the capsule at a point where the eggs are not clustered. They are held in the capsule by a thick jelly which is soluble in water and which will ultimately dissolve. The eggs may be more rapidly freed, however, by exerting a very slow pressure on the capsule with a second needle.

C. Preparation of Cultures: Using a pipette, transfer the eggs to 35-mm. slender dishes (half-full of pasteurized sea water), placing about 10 to 20 eggs in a dish. Cover the dishes and place them in a large fingerbowl on a water table. Using this method, perfectly normal veliger larvae can be reared from decapsulated eggs (Clement, 1952).

D. Methods of Observation: Nuclear details of maturation, polar body formation and early cleavage stages are easily seen in fixed and stained, flattened, coverslip preparations. Accounts of two methods are given by Morgan (1933) and Tyler (1946).

The internal structure of the living veliger may be observed quite clearly in specimens mounted under a supported coverslip. The action of the velar cilia can be stopped by mounting the larvae in a 1% solution of urethane (Clement, 1952).

NORMAL DEVELOPMENT

A. The Ovum: The fertilized, uncleaved egg is spherical, and measures about 166 microns in diameter (Clement, 1952). NO membrane is visible. The ovum is very opaque, and contains a cap of granular cytoplasm around the animal pole, which is rich in mitochondria and lipid droplets (Clement and Lehmann, }956). The remainder of the ovum is filled with coarse yolk spherules, which are largest in the vegetal hemisphere. In sea water, the egg tends to orient with the animal pole floating upward.

B. Fertilization and Cleavage: The history of the egg from deposition to first cleavage has been described by Morgan (1933). In the majority of inseminated, freshly-deposited ova, the first polar spindle is forming deep within the cytoplasmic cap, in others, some variation may be observed. After its formation, the spindle moves toward the polar surface and takes up a radial position. The first polar body is extruded shortly after this. As it is forming, the first polar (yolk or anti-polar) lobe appears. This lobe is inconspicuous; the only indication of its presence is a slight elongation along the polar axis, which gives the egg a top-like appearance. It is soon withdrawn and the egg remains spherical for about ten minutes or less. A second polar lobe develops and continues to enlarge for some time after the second polar body is fully formed. The second lobe is very broad and is more constricted from the rest of the cytoplasm than the first lobe. After about an hour the second lobe is resorbed, the egg rounds up, and the pronuclei come together and enlarge.

As the first cleavage spindle forms, the third polar lobe appears. In development and form, it is much like the second, although it ultimately becomes more constricted from the rest of the cell. After it has reached maximum size, the upper hemisphere of the egg broadens and flattens at the pole. A constriction appears in the region of the polar bodies, indicating the approach of the first cleavage. As the cleavage furrow cuts deeper between the blastomeres, the constriction delimiting the polar lobe also grows deeper, until a stage is reached where it is impossible to tell into which of the two blastomeres the lobe will flow. This is the trefoil stage. It lasts for several minutes; then a short, clear, yolk-free stalk can be seen connecting the polar lobe to one of the blastomeres. The stalk soon broadens as the contents of the lobe flow into the blastomere. The resulting blastomeres are unequal in size, for the larger CD cell contains the contents of the polar lobe. The fourth and final polar lobe appears during second cleavage in the vegetal region of the CD cell. The fourth lobe is of the same size as the third. After the cleavage is completed, there are three small cells: A, B and C, and a large cell, D, which contains the lobe substance. The first quartet of small, clear micromeres is cut off by a typical dexiotropic division; further divisions are very similar to those of Crepidula. The papers of Crampton (1896) and Clement (1952) may be consulted for further details.

C. Time Table of Development: Due to differences in maturity of the freshly deposited ova, it is difficult to establish an exact chronology of development. After extrusion of the first polar lobe (between 15 and 50 minutes after egg deposition, according to Morgan, 1933), development is quite constant. The following table gives approximate rates of development at a room temperature of 23 to 24û C. The time is recorded from the initial appearance of the first anti-polar lobe.

Stage Egg spherical, first lobe withdrawn Beginning of second polar lobe Egg spherical, second lobe withdrawn Appearance of third polar lobe First cleavage Second cleavage Third cleavage Ciliated, rotating embryo Veliger

Time 11 minutes 21 minutes 1 hour, 21 minutes 1 hour, 59 minutes 2 hours, 59 minutes 4 hours, 9 minutes 5 hours, 9 minutes 2 days 4-5 days

D. Later Stages of Development: The very young embryo is ciliated and rotates within the capsule; it contains a mass of opaque endoderm. There is no trochophore stage. The young veliger (five or six days) has a pigmented oesophagus, stomach and intestine. The velum bears large cilia and has a pigmented rim. Shell, operculum, foot, eyes, otocysts and heart are easily seen. At hatching (about seven to eight days), the larva swims actively by means of the velar cilia. The yolk is soon absorbed and details of the digestive tract may be seen (Clement, 1952).

SPECIAL COMMENTS:

This material can readily be used for class experiments in non-marine locations, since the animals breed readily after shipment in damp-pack. Slices of commercial frozen shrimp are a convenient and satisfactory food.

ANKEL, W. E., 1929. Über die Bildung der Eikapsel bei Nassa-Arten. Verh. d. Deutsch. Zool. Ges., 33: 219-230.

BUTROS, J. M., 1956. Simultaneous effects of metabolic inhibitors on the viscosity, surface rigidity and cleavage in Ilyanassa eggs. J. Cell. Comp. Physiol.. 47: 341-356.

CLEMENT, A. C., 1935. The formation of giant polar bodies in centrifuged eggs of Ilyanassa. Biol. Bull., 69: 403-414.

CLEMENT, A. C., 1952. Experimental studies on germinal localization in Ilyanassa. I. The role of the polar lobe in determination of the cleavage pattern and its influence in later development. J. Exp. Zool., 121: 593-625.

CLEMENT, A. C., 1956. Experimental studies on germinal localization in Ilyanassa. II. The development of isolated blastomeres. J. Exp. Zool., 132: 427 116. -

CLEMENT, A. C., AND F. E. LEHMANN, 1956. Über des Verteilungsmuster von Mitochondrien und Lipoidtropfen während der Furchung des Eies von Ilyanassa obsoleta ( Mollusca, Prosobranchia). Naturwiss., 43: 478-479.

CRAMPTON H. E., JR., 1896. Experimental studies on gasteropod development. Arch. f. Entw., 3: 1-19.

DAN, K., AND J. C. DAN, 1942. Behavior of the cell surface during cleavage. IV. Polar lobe formation and cleavage of the eggs of Ilyanassa obsoleta Say. Cytologia, 12: 246-261.

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

MORGAN, T. H., 1933. The formation of the antipolar lobe in Ilyanassa. J. Exp. Zool., 64: 433-467

MORGAN, T. H., 1935a. Centrifuging the eggs of Ilyanassa in reverse. Biol. Bull., 68: 268279.

MORGAN, T. H., 1935b. The separation of the egg of Ilyanassa into two parts by centrifuging. Biol. Bull., 68: 280-295.

MORGAN, T. H., 1935c. The rhythmic changes in form of the isolated antipolar lobe of Ilyanassa. Biol. Bull., 68: 296-299.

MORGAN, T. H., 1936. Further experiments on the formation of the antipolar lobe of Ilyanassa. J. Exp. Zool., 74: 381-425.

MORGAN, T. H., 1937. The behavior of the maturation spindles in polar fragments of eggs of Ilyanassa obtained by centrifuging. Biol. Bull., 72: 88-98.

PELSENEER, P., 1911. Recherches sur l'embryologie des Gastropodes. Mem. Acad. Roy., Belgique, ser. 2, Coll. IN-4•, 3: 1-167.

TYLER, A., 1946. Rapid slide-making method for preparation of eggs, protozoa, etc. Coll. Net, 19:50.