Both species are moderately abundant, and are obtained by dredging off Vineyard Haven, near East Chop, Martha's Vineyard, Mass. The most reliable feature for distinguishing between them is the shape of the arms or rays; in A. forbesi the rays are stout and cylindrical and tend to be blunt at the tip, and in A. vulgaris they are flattened and taper to a point. The sexes are separate but cannot be distinguished on the basis of external characteristics.

May, June and early July in the Woods Hole region. A few ripe females can be obtained in late July; however, it is not practicable to do experiments on a large scale after July 15. Formerly, starfish from the Hole produced viable gametes as late as the middle of August (R. S. Lillie, personal communication).

A. Care of Adults: Adults can be kept in good condition for a considerable period of time in aquaria supplied with running sea water, provided they are occasionally given mussels or small clams for food. If an animal sheds, it should be removed immediately from the aquarium, since it may induce shedding among the others. Dying or dead individuals should also be removed as promptly as possible.

B. Procuring Gametes: Shedding may sometimes be induced in mature animals by removing them from sea water and placing them on a board in the sun, or on the cement floor of the laboratory under the heat of a desk lamp. This method is reasonably effective in the case of the west coast starfish Patiria miniata, which normally sheds in the warm shallow water of tide pools at low tide. Usually, however, it is easier to obtain gametes as outlined below, than to depend on the occurrence of natural spawning.

Only animals with soft, bulging arms are fully ripe; it is a waste of time and material to open small, hard-skinned starfish in an attempt to obtain gametes. A small-bore pipette may be easily inserted into the arm of a ripe starfish, preferably near the central region, without causing injury; the few gametes thus obtained enable one to ascertain the sex of the animal rapidly.

After identifying a female, remove the punctured arm from the disc, and return the animal to an aquarium of running sea water to be used again. Injured animals will not, however, keep indefinitely; the gametes are rarely usable at the time the animal begins to undergo autotomy. Slit the detached arm along the mid-dorsal line to expose the bulging pair of ovaries, which are typically pale salmon in color. Then, with a pair of forceps carefully detach each plume-like ovary by grasping it near its point of attachment at the proximal end, thus closing the gonoduct; rinse it in a large fingerbowl of filtered sea water. Transfer the ovary to a second bowl and allow the eggs to exude from the blunt end. Do not cut up the ouary. At the end of five minutes, remove the gonads to another container or discard them; the best eggs are those which exude first. Gently stir the contents of the fingerbowl and allow the eggs to settle. When they have done so, pour off the supernatant fluid and carefully replace it with an equal volume of filtered sea water. Leave the eggs undisturbed for 20 to 30 minutes; during this time small samples may be removed with a pipette for examination. Eggs carefully obtained from a ripe female, kept under proper conditions of coolness and with an adequate oxygen supply from the time of collection, should show 85 to 90 per cent germinal vesicle breakdown at approximately the same time.

From the detached arm of a male remove a single testis, white or ivory in color, and rinse it in clean sea water. A small piece of the blunt end should be cut off and placed in 200 cc. of filtered sea water.

C. Preparation of Cultures: The optimum period for fertilization is after the breakdown of the germinal vesicle and before the extrusion of the first polar body. Eggs with intact germinal vesicles are non-fertilizable; they may elevate a fertilization membrane but will not develop further. It is convenient, therefore, to inseminate when the distal end of the first maturation spindle begins to protrude above the previously smooth surface of the oocyte in a moderate percentage of the eggs which have undergone germinal vesicle breakdown. To do so, add about five cc. of the sperm suspension, prepared as directed above, to a large fingerbowl of eggs. Immediately rotate the dish gently to ensure complete mixing. When the eggs have settled, the supernatant fluid should be decanted and an equal amount of sea water added. This procedure should be repeated at half-hour intervals, to eliminate the excess sperm which would otherwise foul the culture and prevent development of the late embryonic stages. There should be only one layer of well-spaced eggs on the bottom of a dish.

When the first swimming stages appear (after about 20 hours), pour off the upper half of the culture, containing the more normal swimming blastulae, into a series of tall battery jars. Add enough filtered sea water to fill the jars. Care must be taken to eliminate dead embryos or unfertilized eggs. In these tall jars evaporation is considerably reduced; however, the original level should be maintained by the addition of distilled water. It is essential that relatively few embryos be placed in a jar, if one wishes to raise older larvae. Aerate the cultures gently, using glass tubing rather than rubber hose for this purpose. Early bipinnaria may be obtained without special feeding, but diatoms must be added to the cultures in order to obtain brachiolaria or later stages. The culture jars should be kept at temperatures between 17 and 20û C., away from the direct sunlight (Larsen, 1937).

The egg of Asterias is very delicate as compared with most eggs used for routine laboratory work. Satisfactory results are not obtained unless adequate precautions are observed, including the following: (1) Avoid contamination of either type of gamete with perivisceral fluid; (2) avoid over-insemination; (3) avoid crowding of eggs; and (4) use only fresh, motile sperm.

D. Methods of Observation: The presence of a jelly-hull around these eggs may be demonstrated by using dim illumination, or by adding a trace of Janus green. Vital staining with neutral red is helpful in studying the larval stages.

An aqueous (sea water) extract from squid egg-string jelly has proved useful in the Embryology Course al Woods Hole, for slowing down or immobilizing the larval forms of certain echinoderms. (It is apparently not effective for Callocardia veliger larvae, however. ) The extract is prepared as follows: Peel the outer covering from about four egg-strings, and cut up the strings in approximately 10 cc. of sea water. Allow the strings to remain in the sea water for one or two hours, then filter through a moderately coarse grade of filter paper. The extract will retain the ability to slow down or immobilize echinoderm larvae for at least two days, if it is kept under refrigeration. Two or three drops of the extract will quiet the larvae contained in one drop of sea water, in a depression slide. It is not necessary that the squid jelly used contain squid embryos; empty jelly-strings are equally effective for preparing extracts.

A. The Unfertilized Ovum: The egg of Asterias is very delicate and is surrounded by a jelly-hull. It is shed in the germinal vesicle stage, and on contact with sea water proceeds spontaneously to the first and second maturation divisions. The mature ovum contains a lightly pigmented yolk, pale yellow in color, through which (in later stages) the spindles of mitotic figures may sometimes be seen. The egg of A. forbesi measures about 110 microns in diameter (Fry, 1937).

B. Fertilization and Cleavage: Immediately after insemination, sperm may be seen on the jelly-surface of the eggs. Some will be attached by a tenuous filament to a fertilization cone which has arisen on the egg surface. The fertilization membrane elevates in a wave which begins at the cone and spreads rapidly around the egg. The sperm is drawn passively through the jelly to the cone, and after a pause the head piece is pulled through the membrane and enters the cone. Six minutes after insemination, the delicate sperm aster is formed and moves through the egg to fuse with the egg nucleus. It was in the egg of the starfish that Fol (1879) first observed the actual penetration of an egg by a spermatozoon. See the papers of Chambers (1930) and Colwin and Colwin (1956) for comments and additional observations.

The first two cleavages are meridional; they go through the animal and vegetal poles at right angles to each other. The third cleavage is horizontal; the eight cells of this stage are approximately equal in size. In the 16-cell stage, no definite arrangement of cells in rows occurs, and from this stage on, cleavage is irregular. Throughout the early cleavages the blastomeres exhibit a tendency to assume a spherical shape, resulting in a rather loose arrangement of cells. The perivitelline space is wider and the hyaline plasma membrane is thinner and weaker than in the Arbacia egg. These two conditions account, in part, for the loose connection between the blastomeres. Chambers has pointed out that in the absence of the fertilization membrane, the blastomeres tend to separate completely.

Eventually the cells become arranged in an epithelial wall enclosing the blastocoele. The surface cells acquire cilia and the blastula begins to rotate within the fertilization membrane. The two polar bodies are still visible, attached to the animal pole or Iying loose within the perivitelline space. The embryo hatches in the late blastula stage.

C. Developmental Rate: NO precise information is available. This is apparently due to the fact that few workers have obtained egg-batches showing uniform

germinal vesicle breakdown. According to Chambers and Chambers (1949), for example, the time of cleavage depends upon the exact time when the eggs are inseminated during the maturation stages. Their data indicate that if one waits until the egg is almost fully mature (i.e., shortly before the second polar body is to be extruded) before inseminating, the eggs cleave about 103.5 minutes later, at 16ûC.

Unfertilized Asterias eggs undergo maturation changes at 16-18û C., according to Chambers and Chambers (1949), as follows; the time is recorded from the moment of deposition of the eggs in sea water:

Stage Disappearance of nucleolus Formation of first polar body Formation of second polar body

Time 8-9 minutes 76-90 minutes 105-119 minutes

D. Later Stages of Development and Metamorphosis:

Gastrulation: The vegetal pole area thickens and flattens, and invagination begins. The blastopore is destined to become the anus. The larva elongates along the animal-vegetal axis and gradually becomes pear-shaped. The blind inner end of the archenteron becomes thin-walled and expands, and from it mesenchyme cells wander into the blastocoele. In a slightly later gastrula, two out-pocketings of the distal end of the archenteron can be seen; they are the primordia of the coelomic sacs. While they are forming, the first sign of the change from radial to bilateral symmetry may be seen, namely, the bending of the ciliated archenteron toward the future ventral side of the embryo.

Dipleurula larva: By the time this larva is fully formed, the blind end of the archenteron has made contact with an ectodermal depression, the stomodeum, on the ventral body wall and has broken through to form the mouth. Overhanging this is the oral lobe. The ventral side of the dipleurula is convex in shape. The entire surface is finely ciliated, and in addition there is a continuous ciliary band which is longitudinal with two cross-bars. The longitudinal bands above the upper cross-bar loop toward the mid-line where they eventually meet. Thus a frontal field, the pre-oral ciliary band, is separated on the upper ventral part of the larva, overhanging the oral field. This separate frontal field is characteristic of asteroid larvae. The alimentary tract consists of three parts, characteristic of echinoderm larvae: oesophagus (with a constriction near the entrance to the stomach), stomach, and intestine. In lateral views, the bend of the intestine can be seen. Ciliation occurs in the oral field and certain other parts of the tract. The two coelomic vesicles are clearly visible at the lower end of the oesophagus, near its entrance into the stomach. A subdivision of the vesicles is not yet clearly demarcated, but the narrow tube connecting the larger left coelomic vesicle with the dorsal body wall, the pore canal, and its opening, the madreporic pore, can be readily seen. Loose mesenchyme cells are scattered in the body cavity, which is the persisting blastocoele.

The dipleurula (early bipinnaria) larva represents an early larval type common to Asteroidea, Echinoidea, Ophiuroidea, and Holothuroidea (see the book of Korschelt and Heider, 1936, vol.l, p. 499).

Bipinnaria larva: This larva is characterized by a number of pairs of lobes or arms which grow out from the margin Or the ectoderm and which carry along the ciliary band. They are not supported by a skeleton, and young stages may not have all the arms developed. Pairs of arms follow each other in quick succession; unpaired median dorsal, and paired antero-dorsal, postero-dorsal, postero-lateral, post-oral and pre-oral arms can be identified. The coelomic vesicles grow out into long tubes and fuse in the anterior part of the larva. No further subdivisions have yet occurred. Brachiolaria larva: Unlike the bipinnaria arms, which are long hollow tubes, those of the brachiolaria, the brachia, are short, and contain diverticula of the coelom. They are not ciliated but bear small papillae, differentiated from their end discs; they can adhere to the substrate. A sucker, the gland cells of which secrete a sticky substance, is formed between the brachia. The developing disc, the future young starfish, can be seen. Metamorphosis:: In late stages of metamorphosis, the anterior part of the brachiolaria, in front of the disc, shrinks to form a stalk. This attaches firmly to the substrate by means of the sucker and brachia, and bears the Asterias anlage at its distal end. About one week is required for the young starfish to complete development, rupture the stalk, and crawl away.

For further details of these stages consult papers by Agassiz (1877), Gemmill (1914) and the text-book of MacBride (1914), all of which contain figures.

AGASSIZ, A., 1877. North American starfishes. I. Embryology of the starfish. Mem. Mus. Comp. Zool., Harvard, 5: no. 1, pp. 1-83. First published in 1864.

CHADWICK, H. C., 1923. Asterias. Liverpool Mar. Biol. Comm. Mem., no. 25, pp. 1-63.

CHAMBERS, R., 1930. The manner of sperm entry in the starfish egg. Biol. Bull., 58: 344-369.

CHAMBERS, R., AND E. L. CHAMBERS, 1949. Nuclear and cytoplasmic interrelations in the fertilization of the Asterias egg. Biol. Bull., 96: 270-282.

COLWIN, L. H., AND A. L. COLWIN, 1955. Some factors related to sperm entry in two species of Asterias. Biol. Bull., 109: 357.

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

COSTELLO, D. P., 1935. Fertilization membranes of centrifuged Asterias eggs. I. The effects of centrifuging before fertilization. Physiol. Zool., 8: 65-72.

DELAGE, Y., 1904. Élevage des larves parthenogen~etiques d'Asterias glacialis. Arch. de Zool. expert, 4e ser., 2: 27-42.

FOL, H., 1879. Recherches sur la fecondation et le commencement de l'henogenie chez divers animaux. Mem. Soc. Phys. et Hist. Nat., Geneve, 26: 12-397.

FRY, H. J., 1937. Asterias forbesi. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 547-550.

GEMMILL, J. F., 1914. VII. The development and certain points in the adult structure of the starfish Asterias rubens, L. Phil. Trans. Roy. Soc., London, ser. B, 205: 213-294.

GOTO, S., 1898. The metamorphosis of Asterias pallida, with special reference to the fate of the body cavities. J. Coll. Sci., Imp. Univ., Japan, 10: 239-278.

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

KORSCHELT, E., AND K. HEIDER, 1936. Vergleichende Entwicklungsgeschichte der Tiere. Vol. 1. G. Fischer, Jena.

LARSEN, E. J., 1937. The laboratory culture of the larvae of Asterias forbesi. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 550-553.

LILLIE, R. S., 1941. Further experiments on artificial parthenogenesis in starfish eggs, with a review. Physiol. Zool., 14: 239-267.

MacBride, E. W., 1914. Text-Book of Embryology. Vol. I. Invertebrata. Macmillan and Co., Ltd., London.

NEWMAN, H. H., 1925. An experimental analysis of asymmetry in the starfish, Patiria miniata. Biol. Bull., 49: 111-138.