Although it is possible in some species of echinoids to distinguish the sexes by external characteristics (see the papers by Marx, 1932, and Harvey, 1956b), no differentiating characteristics have, as yet, been described for the American sea urchin, Arbacia punctulata. The sexes are readily identified after the animals are opened, by the deep red or purple ovaries and the white testes, or, if unopened animals shed spontaneously, by the red eggs and the white sperm. Hermaphroditic animals are occasionally found (Boolootian and Moore, 1956).

At Woods Hole, Mass., the animals are obtained by dredging; they are, at the present time, rather scarce.

An exhaustive account of the life-history, embryology and metamorphosis of Arbacia is given by Harvey (1956a).

From the middle of June until the middle of August (Harvey, 1956a), although the season may vary somewhat from year to year.

A. Care of Adults: In the laboratory, animals can be kept in aquaria provided with running sea water; they should not be crowded.

B. Procuring Gametes: The following methods may be used, (4) or (5) being preferable because of the present scarcity of Arbacia at Woods Hole:

(1) Cut around the peristome (on the oral surface) and remove the Aristotle's lantern, taking care not to injure the gonads. The perivisceral fluid should then be drained from the body and the animal, aboral surface down, placed to shed on a Syracuse watch glass which has been moistened with sea water. After each animal is opened, the hands of the investigator and all instruments used should be washed with running fresh water, to avoid contamination of the gametes of one sex with those of the other, or with body fluids. If the eggs are shed through the gonopores of the female into the Syracuse dish, they should be transferred within ten minutes to a fingerbowl containing 200 cc. of sea water. The sperm are best kept "dry," just as they exude from the testes.

(2) Cut around the test, about halfway between the mouth and the equator, and proceed as in (1) above. Shedding is more frequently obtained by this method, but there is also more likelihood of cutting the gonads; and of contamination with the perivisceral fluid.

(3) Cut as in (2) above, pour out the body fluid and remove the gonads (at the gonoduct end) with blunt forceps, spatula or spoon. The ovaries should be placed in 200 cc. of sea water in a fingerbowl, and allowed to shed. If undisturbed, the eggs are extruded in compact clumps or strings, without ovarian tissue, and may be removed to a fresh dish by means of a wide-mouth pipette. If large quantities of eggs are desired, the ovaries should be allowed to shed for about 30 minutes, with occasional stirring; then pour them gently through cheesecloth (which has previously been washed in fresh water and soaked in filtered sea water) or bolting silk.

(4) Palmer (1937) induced spawning in Arbacia by injecting isotonic (0.53 M) KCl into the perivisceral cavity, and Tyler (1949) described a similar method, utilizing 0.5 cc. of 0.5 M KCl Ripe animals begin to shed in a few minutes; the eggs can be collected by inverting the female in a dish of sea water, or by washing them gently from the surface of the test with a pipette. The sperm should be collected "dry." This method has the advantage that one is enabled to sex animals without sacrificing them, but there is some evidence (Harvey, 1956b) that eggs obtained in this manner do not develop normally. The animals may be returned to an aquarium, and will produce another batch of gametes in about three weeks, if fed periodically.

(5) Harvey (1953) has reported an electrical method (similar to a technique devised by Iwata, 1950, for Japanese sea urchins) for sexing Arbacia and inducing shedding. An alternating current of 10 volts (reduced by a transformer from ordinary 60-cycle, 110-volt current) is applied, using lead electrodes, to any two points on the test of the animal, which is placed, aboral side up, in a dish and covered with sea water. Almost immediately after the current is passed, the gametes will be extruded from each of the gonopores; when the current is stopped, the shedding ceases, to be resumed when the current is again applied. The gametes should be removed and used at once. Harvey points out that this method is of great value in laboratories where sea urchins have become scarce, since only the quantity of gametes desired is obtained and the animals need not be sacrificed.

C. Preparing Cultures: Two drops (0.2 cc.) of "dry" sperm may be diluted with 10 cc. of sea water (Just, 1939) in a watch glass, just before insemination; do not use sperm which have been diluted more than 20 minutes, and avoid a high sperm concentration, which leads to polyspermy and abnormal cleavage. Two drops of diluted sperm are sufficient for a fingerbowl of eggs. Stir the dish immediately.

A. The Sperm: After dilution with sea water, the sperm become intensely active, although they are immobile in concentrated suspension due, presumably, to the effects of high concentrations of carbon dioxide. Concomitantly, their ability to fertilize eggs is lost more rapidly in dilute than in concentrated suspensions. (See the papers by Lillie, 1915; Cohn, 1918; Hayashi, 1945.) "Dry" sperm kept in the cold (2û C.) may remain usable for several days; at room temperatures, dilute sperm suspensions often lose their fertilizing power in an hour or less.

The spermatozoon consists of three parts: head, middle piece and tail. These are 3.25 microns, 0.75 micron and 45 microns in length, respectively (Harvey and Anderson, 1943). The fibrillar axial filament of the tail protrudes a short distance beyond the end of the sheath.

B. The Unfertilized Ovum: A good batch of eggs from a ripe female should show uniformity of size, perfectly spherical form and complete absence of immature

eggs (which are in the germinal vesicle stage). Both meiotic divisions are completed while the eggs are still in the ovary, and the polar bodies very seldom remain attached when the eggs are shed (Hoadley, 1934). Occasionally (especially from relatively unripe animals, or after maceration of the ovaries), eggs may be found that are in the germinal vesicle stage; this is recognizable in the living state by the large clear nucleus (as opposed to the small nucleus of the ripe egg) and nucleolus. Such eggs may exhibit some surface response to sperm (including the formation of "papillae"), but they do not develop after insemination. The ripe egg, 72 to 75 microns in diameter, has a small, clear nucleus; it contains uniformly dispersed, pale yolk granules, and slightly larger red granules containing echinochrome pigment, which is a substituted naphthoquinone related to the K vitamins (Ball, 1936; Hartmann et al., 1939; Tyler, 1939). The nucleus is usually excentric in location. Since the polar bodies are not ordinarily present, the position of the nucleus with respect to the polar axis is not readily determined; occasionally, however, batches of eggs are obtained in which the polar bodies are still attached, and in these, observations by Hoadley (1934) have shown that the nucleus may lie in any part of the cytoplasm between the cortex and the center. In the transparent, gelatinous coat of the egg (about 30 microns wide), there is a funnel shaped space which usually lies in the polar axis. The funnel is rendered visible by staining the jelly with Janus green, or by placing the eggs in a suspension of Chinese ink. For this purpose, the eggs should be taken immediately after shedding, because the "micropyle" (funnel) may disappear as the jelly swells after the eggs are shed into sea water. See the diagrams by Harvey and Dan (Harvey, 1956a, p. 84) of the membranes and layers of the fertilized, as compared with the unfertilized, egg.

C. Fertilization: Sperm penetration occurs very rapidly (apparently at any point on the egg), and it is usually difficult to study. Within a few seconds after insemination, the cortical responses of the egg begin; the vitelline membrane starts to elevate rapidly from the egg surface (in about five seconds), leaving a perivitelline space. This membrane hardens and thickens during the next five minutes, and, after alteration, is called the fertilization membrane. At the protoplasmic egg surface (which is at first slightly disrupted by the elevation of the vitelline membrane), a new, clear layer is formed about ten minutes after fertilization: the hyaline plasma membrane, which is apparently a calcium-proteinate, acting as a cement to hold the blastomeres together after cleavage. It disappears when the eggs are placed in calcium-free sea water. After insemination, the jelly-layer is often clearly delimited by the supernumerary sperm trapped near its surface. Moser (1939, 1940) has correlated the elevation of the vitelline membrane with the breakdown of certain cortical granules. These granules are embedded in the cortex, and are not easily displaced by centrifugation (as are the granules of the underlying fluid endoplasm) . Runnstrom et al. ( 1944) state that the cortical granules contribute to the formation of the fertilization membrane.

D. Cleavage: About 15 minutes after insemination (at 20û C.) a sperm aster is visible as a spherical region containing clear rays which extend from a clear center; this configuration attains its maximum development 20 to 30 minutes after insemination. Then a clear streak appears in the egg, slightly above the equator, and 45 to 50 minutes after insemination, it is replaced by two clear areas, the asters of the first cleavage spindle. The first three cleavages divide the egg into eight blastomeres of equal size. The planes of the first two cleavages are meridional (in the polar axis), while that of the third is equatorial or horizontal (at right angles to the polar axis). The progress of the cleavage furrows in dividing eggs can be followed; the hyaline layer forms the surface of the furrow and later, when the cells flatten against one another, it forms the boundary between them. At the fourth cleavage, the four upper cells divide meridionally, forming eight equal mesomeres, while the lower four cells divide unequally and horizontally, to form four large macromeres; below them, at the vegetal pole, are four small, clear micromeres.

At the fifth division, the eight mesomeres divide equally and horizontally, forming two tiers of cells termed an1 (at the animal pole) and an2 (see the paper by Hörstadius, 1939), while the macromeres and micromeres divide meridionally. At the sixth cleavage, the an1 and an2 cells divide in a more or less radial direction, while the macromeres divide horizontally to form the veg1, and veg2 tiers of cells. Veg2 cells are next to the micromeres, which have also divided at this time but which do not form distinct layers. Tiers of cells are not readily distinguished in later cleavage stages, and no special cell-layer designation is used after the 64-cell stage. It has been shown by Hörstadius (1939) that the an,, an2 and veg1 cells form the ectoderm, the veg2 cells form endoderm (gut) and part of the mesoderm (coelom), while the micromeres form the mesodermal skeleton components.

E. Time Table of Development: The following schedules of development have been observed at temperatures of 23û C. (data from Harvey, 1956a) and 20û C.; the time is recorded from insemination.

Stage Formation of fertilization membrane Formation of hyaline layer Sperm aster appears "Streak" stage Cleavage asters First cleavage Second cleavage Third cleavage Blastula Hatching of blastula Gastrula Pluteus Metamorphosis

20û C. 5 minutes 10 minutes 15 minutes 35 minutes 45 - 50 minutes 56-67 minutes 107 minutes 145 minutes 6 hours 10 hours 20 hours 48 hours 2 weeks

23û C. 2 minutes 2 minutes 8 minutes 20-35 minutes 35 minutes 50 minutes 78 minutes 103 minutes 7-8 hours 12-15 hours 24 hours

Different batches of eggs vary slightly (1-2%) in average cleavage times; within a batch, most eggs will develop at the average rate, but some may vary by about 10% Temperatures above 30-32û C. are lethal for Arbacia eggs.

F. Later Stages of Development: At the eight-cell stage, there is a very small central cavity which enlarges as cleavage continues, to form the blastocoele. About six hours after fertilization, a smooth-surfaced, spherical young blastula is formed, the wall of which is one cell in thickness. Cilia soon develop on the surface, and the blastula is rotated by their action within the fertilization membrane. The blastula hatches out of the fertilization membrane in about ten hours. It has been shown that the blastula releases a "hatching enzyme" at this time, which weakens and dissolves the membrane so that the blastula can break through. A tuft of long cilia develops at the animal pole of the blastula, which is the forward end when the embryo is swimming. At the base of this apical tuft the blastula wall is thickened, forming the apical plate. At the vegetal pole, the blastula wall becomes flattened, and the micromeres migrate into the blastocoele, forming the rudimentary mesenchyme which gives rise to the skeleton.

About 20 hours after fertilization, the cells at the vegetal pole invaginate to form a blind tube, the archenteron. This reaches the opposite end of the blastocoele in about five hours. The gastrula contains approximately one thousand cells, and its outer wall, as well as the wall of the archenteron, has a single layer of cells. The primary mesenchyme cells form a ring around the blastoporal end of the archenteron. Secondary mesenchyme and, later, the coelom are budded off from the tip of the archenteron.

At the completion of gastrulation, the tip of the archenteron turns to one side of the gastrula, which becomes flattened over an area extending from the animal pole nearly to the blastopore. This is the first sign of bilateral symmetry, the flattened area representing the ventral side of the embryo. The primary mesenchyme cells aggregate in two groups, one on each postero-ventral side, and each group secretes a triradiate spicule, the beginning of the skeleton. Where the tip of the archenteron touches the ectoderm, there is formed a depression which later acquires an opening into the archenteron to become the stomodeum. The archenteron becomes divided by two constrictions into oesophagus, stomach and intestine. The apical tuft disappears, a ciliated band surrounds the oral field, the embryo begins to elongate in the dorso-ventral axis, and the direction of swimming changes, so that the ventral side is forward.

After about 48 hours, the embryo enters the pluteus stage, which is fully developed by the end of the third day. The original apical plate grows out in a ventral direction, to form the oral lobe which includes the stomodeum and the anterior part of the oesophagus. Two short outgrowths, the oral (antero-lateral) arms, are formed on the oral lobe, and, at the anal side, two longer anal (aboral or post-oral) arms grow out in the same general direction. The original triradiate spicules form skeletal rods which extend into the oral arms (oral rods), the anal arms (anal rods), dorsally through the body (body rods) and laterally (ventral' transverse rods). Each of the rods is made up of three or four parallel parts joined by cross-bars. Different species of sea urchins differ in this regard, so that the structure of the skeletal rods is a useful characteristic in hybridization studies.

The embryo continues to elongate in the dorso-ventral direction, and becomes pointed at the postero-dorsal end where the body rods meet. The axis running through oesophagus, stomach and intestine becomes J-shaped. The stomach expands, to become a spherical structure which fills a large part of the body of the pluteus; sphincter muscles connect it with the oesophagus and intestine. The two coelomic sacs extend postero-laterally from the oesophagus; the one on the left side becomes larger and later acquires a dorsal opening called the pore canal. The right coelomic sac buds off cells to form the madreporic vesicle; but otherwise remains rudimentary. The left coelom undergoes extensive later development in the formation of structures of the adult sea urchin. These changes do not occur until the second week, when metamorphosis begins in properly fed larvae. The adult organs are built up in and around a structure called the "echinus rudiment," which is formed by the fusion of an invagination (the amniotic invagination) of the ectoderm on the left side with the mid-portion (hydrocoele) of the left coelom. The left side of the pluteus becomes, then, the future oral surface of the adult.

G. Special Methods of Observation: Dark-field illumination shows a bright reddish "luminous" layer on the surface of the unfertilized egg. The luminosity diminishes and becomes paler after fertilization (Runnstrom, 1928; Ohman, 1945). The skeletal spicules and rods are best demonstrated by the use of a polarization microscope.

BALL, E. G. 1936. Echinochrome, its isolation and composition. J. Biol. Chem., 114: vi.

BOOLOOTIAN R. A., AND A. R. MOORE, 1956. Hermaphroditism in echinoids. Biol. Bull., 111: 328-335

COHN, E. J., 1918. Studies in the physiology of spermatozoa. Biol. Bull., 34: 167-218.

DAN, K., 1954. The cortical movement in Arbacia punctulata eggs through cleavage cycles. Embryologia, 2: 115-122.

FRY, H. J., 1936. Studies of the mitotic figure. V. The time schedule of mitotic changes in developing Arbacia eggs. Biol. Bull., 70: 89-99.

HARTMANN M., O. SCHARTAU, R. KUHN AND K. WALLENFELS 1939. Über die Sexualstoffe der Seeigel. Naturwiss., 27: 433.

HARVEY, E. B., 1939. A method of determining the sex of Arbacia, and a new method of producing twins, triplets and quadruplets. Coll. Net, 14: 211.

HARVEY, E. B., 1940. A note on determining the sex of Arbacia punctulata. Biol. Bull., 79: 363

HARVEY, E. B., 1953. A simplified electrical method of determining the sex of sea urchins and other marine animals. Biol. Bull., 105: 365.

HARVEY, E. B., 1956a. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, N. J.

HARVEY, E. B., 1956b. Sex in sea urchins. Pubbl. Stas. Zool., Napoli, 28: 127-135.

HARVEY, E. B., AND T. F. ANDERSON,, 1943. The spermatozoon and fertilization membrane of Arbacia punctulata as shown by the electron microscope. Biol. Bull., 85: 151-156.

HAYASHI, T., 1945. Dilution medium and survival of the spermatozoa of Arbacia punctulata. I. Effect of the medium on fertilizing power. Biol. Bull., 89: 162-179.

HOADLEY, L., 1934. The relation between the position of the female pronucleus and the polar bodies in the unfertilized egg of Arbacia punctulata. Biol. Bull., 67: 220-222.

HÖRSTADIUS, S., 1939. The mechanics of sea urchin development, studied by operative methods. Biol. Rev., 14: 132-179.

IWATA, K. S., 1950. A method of determining the sex of sea urchins and of obtaining eggs by electric stimulation. Annot. Zool. Jap., 23: 39-42.

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

LILLIE, F. R., 1915. Studies of fertilization. VII. Analysis of variations in the fertilizing power of sperm suspensions of Arbacia. Biol. Bull., 28: 229-251.

MARX, W., 1932. Zum Problem der Determination der Bilateralität im Seeigelkeim. (Nebst einem Beitrag zur Kenntnis des Geschlechtsdimorphismus einiger Seeigel.) Arch. f. Entw., 125: 96-147.

MOSER, F., 1939. Studies on a cortical layer response to stimulating agents m the Arbacia egg. Parts I and II. J. Exp. Zool., 80: 423-471.

MOSER, F., 1940. Studies on a cortical layer response to stimulating agents in the Arbacia egg. Parts III and IV. Biol. Bull., 78: 68-91

OHMAN, L. -O., 1945. On the lipids of the sea-urchin egg. Arch. f. Zoologi, 36A: no. 7, 1-95. PALMER, L., 1937. The shedding reaction in Arbacia punctulata. Physiol. Zool., 10: 352-367.

RUNNSTRÖM J., 1928. Die Veranderung der Plasmakolloide bei der Entwicklungserregung des Seeigeleis. Protoplasma, 4: 388-514.

RUNNSTRÖM, J., L. MONNÉ AND E. WICKLUND, 1944. Mechanisms of formation of the fertilization membrane in the sea urchin egg. Nature, 153: 313-314.

TYLER, A., 1939. Crystalline echinochrome and spinochrome: Their failure to stimulate the respiration of eggs and of sperm of Strongylocentrotus. Proc. Nat. Acad. Sci., 25: 523528.

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

ZEUTHEN, E., 1955. Mitotic respiratory rhythms in single eggs of Psammechinus miliaris and of Ciona intestinalis., Biol. Bull., 105: 366-385.