Chitons are usually found clinging to shells or rocks dredged from Vineyard Sound at Woods Hole, Mass.; they are quite abundant. The sexes are separate, but there is no way of distinguishing them externally.

The breeding season begins about June 20 and continues to the end of September, reaching its height from about July 10 to August 20. There is evidence of a lunar periodicity; and although the eggs are obtainable at any time during the breeding period, they are shed most abundantly between full moon (or a few days before) and third quarter (Grave, 1932).

A. Care of Adults: When brought into the laboratory, the chitons should be removed from the shells and rocks to which they cling, and placed in large fingerbowls (25 to 30 animals per dish) supplied with running sea water. Ripe animals seem to shed most abundantly during their second evening in the laboratory, with a pronounced decrease during subsequent nights. For this reason, it is advisable to obtain fresh animals after about three days.

B. Procuring Gametes: If naturally-inseminated eggs are desired, toward evening wash the adults free of sediment and replace them in a large fingerbowl. Half-fill it with fresh sea water, and allow to stand undisturbed. The males extrude sperm at about 8 P.M., and about half an hour later a few females may spawn. Shedding by the males seems to have a stimulating effect on the females, although isolated females do shed. Unfertilized eggs and sperm may be collected separately by isolating the animals at dusk, in small fingerbowls half-filled with sea water (Grave, 1937).

C. Preparation of Cultures: If the animals were isolated before spawning, several pipettes-fur of sperm suspension may be added to a fingerbowl of eggs. Many of the eggs remain fertilizable for as long as 24 to 48 hours after shedding (Grave, 1932). The inseminated eggs should be left undisturbed for 30 minutes, and the sea water then changed. When spawning is completed, naturally-inseminated eggs can be collected in a pipette and transferred to fingerbowls of fresh sea water.

All cultures should be kept covered on the water table. Decant the upper layers of sea water and refill the dishes several times during the first 24 hours. The larvae, which hatch in about 25 to 30 hours, should be transferred twice daily to fresh sea water, using a pipette to collect them. Since they are well supplied with yolk, there is no need to feed the larvae, although diatom feeding, initiated on the sixth day of larval life, often hastens the onset of metamorphosis

Young metamorphosed chitons can be kept indefinitely if sea water is allowed to flow through their dishes for a part of each day. Special feeding is not necessary, and if they are occasionally washed free of sediment, the young animals will thrive.

D. Methods of Observation: Because of the size and opacity of the egg, the early development is best studied by mounting egg samples in depression slides. A dilute solution of Janus green will slow down the moving larvae.

A. The Unfertilized Ovum: The egg is spherical and measures 180 to 190 microns in diameter. It is opaque, due to its high yolk content and to the chorion which surrounds it (Grave, 1932); the chorion is tough, ornate and bristly. When the eggs emerge from the oviduct, they are embedded in a viscid, jelly-like secretion which spreads over the bottom of the dish in a thin film. Although it cannot be seen in the living egg, sections show that the ovum is usually developing the first maturation spindle when it is shed.

B. Fertilization and Cleavage: There are no visible changes at the time of fertilization; no fertilization membrane is elevated and the egg does not change shape. Two transparent polar bodies are formed, but no polar lobes. The first noticeable change occurs shortly before first cleavage when there is a slight flattening of the egg at the animal pole.

The first cleavage furrow usually divides the egg into two equal blastomeres, but in a small percentage of eggs one blastomere is perceptibly larger. The second cleavage is at right angles to the first, and again in some cases the D cell is slightly larger than the others. The cells of the first quartet of micromeres, given off by the dexiotropic third cleavage, are distinguishable from the larger macromeres. Further divisions follow the regular pattern of spiral cleavage. Four quartets of micromeres are produced. The first three give rise to ectoderm, nervous system and stomodeum, while the fourth quartet, except for 4d, becomes part of the endoderm, along with the macromeres; the 4d cell gives rise to mesoderm and to some endoderm.

C. Time Table of Development: The following schedule, based on a batch of eggs developing at 23û to 24û C., is offered as an approximate outline of developmental rate. Metamorphosis seemed to occur early in this particular batch, the usual time being 6 to 10 days (Grave, 1932). The times are recorded from insemination.

Stage First polar body Second polar body First cleavage Second cleavage Third cleavage Gastrulation Beating cilia Rotation within capsule Hatching Free-swimming trochophores Metamorphosis

Time 30 minutes 55 minutes 1 hour, 30 minutes 2 hours 2 hours, 40 minutes About 13 hours 14 hours 20 hours 36 hours 2-1/2 to 3 days 4 days

D. Later Stages of Development and Metamorphosis: The young trochophore (40 to 60 hours old) is propelled rapidly through the water by a band of powerful cilia, the prototroch. The larva rotates on its longitudinal axis, following a spiral path. Crowning the pre-trochal hemisphere (the head vesicle) is a clump of very long cilia, the apical tuft, which is apparently sensory in function. There are two lateral, reddish-brown eyes; the mouth lies just below the prototroch. Other regions of the digestive tract are obscured by the yolk mass.

As the larva develops, there is an elongation of the body, especially of the post-trochal hemisphere. By three to four days, the mouth and archenteron are visible, due to reduction in the quantity of yolk. An anus is formed, and the shell segments begin to appear on the dorsal surface, gradually extending anteriorly to the region of the head vesicle. A contractile foot develops on the ventral surface, just posterior to the mouth. The larvae are still propelled by the prototrochal cilia, although slightly older stages may creep along by means of the foot.

Metamorphosing larvae are found on the bottoms of the culture dishes. The prototroch and the apical cilia are lost during metamorphosis. There is a sudden dorso-ventral flattening of the body, and the larva now creeps about by the well-developed foot. The shell plates increase in number, although at this time, the full adult set is not complete. The mantle, a fold of the body wall, develops just dorsal and lateral to the foot.

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GRAVE, B. H., 1932. Embryology and life history of Chaetopleura apiculata. J. Morph., 54: 153-160.

GRAVE, B. H., 1937. Chaetopleura apiculata. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 519-520.

HEATH, H., 1899. The development of Ischnochiton. Zool. Jahrb., abt. Anat. Ontog. Thiere, 12: 567-656.

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KOWALEVSKY, A. O., 1882. Weitere Sudien [sic] uber die Entwickelung der Chitonen. Zool. Anz., 5: 307-310

KOWALEVSKY, M. A.; 1883. Embryogenie du Chiton polii (Philippi) avec quelques remarques sur le developpement des autres Chitons. Ann. Mus. Hist. Nat., Marseilles, 1: (Mem. 5) pp. 1-46.

LOVEN, S. 1856. Ueber die Entwickelung von Chiton. Arch. f. Naturgesch., 22 Jahrg., 1: 206-210.

METCALF, M. M., 1893. Contributions to the embryology of Chiton. Stud. Biol. Lab., Johns

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