These tube-dwelling, polychaete worms are common at Woods Hole, Mass.; they live on old shells, stones, etc., which are dredged from the harbor bottom. The worms may be distinguished from Hydroides, which occurs in the same localities at Woods Hole, by their tubes and certain other adult features. The tubes of Sabellaria, often brown or pinkish in color, are formed by sand grains and are moderately soft and crumbly; those of Hydroides, greenish-gray or white in color, are calcareous and hard. The gill filaments of Sabellaria are filiform in general appearance, the "barbules" being very inconspicuous. Those of Hydroides, however, are much more brilliant, and may vary from purplish (sometimes striped) to brilliant scarlet in both males and females; each one has small "barbules" coming off the central shaft. Sabellaria has a tail-like abdomen which has no parapodia and which folds back on the thorax. The setae are more prominent than in Hydroides.

The sexes are separate. They are externally recognizable after the individuals have been removed from their tubes, but only if the worms are fully mature, containing large numbers of gametes. In these animals, the abdominal segments are swollen and appear opaque and white in the male, pink in the female. Individuals showing neither color distinctly may, however, shed abundantly. The sex of such animals can be ascertained by placing them in a few drops of sea water until shedding starts. Sperm will pour forth from the male in dense clouds, but egg masses will break up into small clumps on contact with water.

Sabellaria is said to spawn naturally in May and June, but ripe individuals may be obtained throughout the summer months (Waterman, 1934).

A. Care of Adults: If worms are left in their tubes and placed in aquaria with a good supply of running sea water, they will produce normally developing eggs for as long as nine weeks.

Uninjured animals are most easily obtained by removing the sand tubes from the substrate, chipping away enough of the tube to expose the head and tail of the worm, and then gently forcing out the animal by inserting a blunt probe into the anterior end of the tube&endash;the animal slowly withdraws, hind-end foremost, from the tube (Novikoff, 1939).

B. Procuring Gametes:

Female gametes: Place a female in a fingerbowl containing 200 cc. of sea water, in which it will shed if ripe. After it has shed for a few seconds, move it to a new spot and allow it to continue shedding. The eggs first shed should be discarded, as they may have been exposed to air when the worm was out of water. Since the eggs are expelled by active contractions of the body, it is easy to tell when shedding has ceased; the female should be removed and discarded at this time.

Male gametes: Sperm may be obtained by placing a male in a dish containing four drops of sea water. When shedding is completed, remove and discard the worm.

C. Preparation of Cultures: Allow the eggs to stand in sea water for about 15 minutes after shedding. Toward the end of this period, prepare a dilute sperm suspension as follows: Add one drop of concentrated sperm suspension to four drops of sea water, and add one drop of this diluted sperm suspension to a fingerbowl of sea water. To this fingerbowl add the eggs, using a narrow-mouth pipette to transfer them. This method of insemination prevents polyspermy and its accompanying abnormalities. If only small amounts of eggs and sperm are available, however, culturing can be done in slender dishes instead of fingerbowls. Allow the culture to stand undisturbed for an hour; then change the water, cover, and place on a water table. After 24 hours, decant the upper layers of water, which contain the more normal, top-swimming trochophores, to a clean fingerbowl. Repeat this procedure at least once a day, adding water each time. After a day or so, larvae should be fed on a pure culture of Nitzschia.

D. Methods of Observation: Because of the small size of these eggs, they are best examined using a high magnification (440 X ). The eggs are too opaque to reveal internal changes other than the breakdown of the germinal vesicle. The ciliation of the early larvae can best be seen with dark-field illumination. A very dilute solution of Janus green (1:1000 in distilled water) will partially inactivate older swimming forms.

E. Removal of Membranes: It is sometimes desirable for study or experimental work to obtain Sabellaria eggs without their tough vitelline membranes. The method devised by Hatt (1931), and modified by Novikoff (1939), consists of treating the eggs with alkaline NaCl. The details of this method, as described by Costello (1945a) for the egg of Nereis, are given on p. 84 of this manual. Sabellaria eggs from which the membranes have been removed may still retain some of the perivitelline jelly; this can be demonstrated readily by placing the eggs in a suspension of Chinese ink in sea water.

A. The Unfertilized Ovum: When first shed, the egg is very irregular in shape; usually it has a particularly deep indentation directly opposite the animal pole. This crater coincides with the point of former ovarian attachment. The small egg has a large, rather excentrically placed germinal vesicle, and a considerable amount of yolk distributed through the cytoplasm, making it appear opaque. A conspicuous vitelline membrane is very closely applied to the egg surface (Waterman, 1934).

A few minutes after shedding, a series of pre-maturation changes occurs. The germinal vesicle breaks down and its contents flow to one side of the egg, to form a clear, hyaline cap at the future animal pole. The first maturation spindle extends across not quite half the diameter of the egg. The egg rounds up, becoming spherical, and the vitelline membrane rises from the egg surface, leaving a perivitelline space about 12 microns wide. The surface changes accompanying this can

best be observed in the thin cytoplasm at the edge of the large, crater-like indentation. As the vitelline membrane elevates, a thin, transparent, hyaline plasma layer appears on the egg surface, presumably outside the egg's plasma membrane. The vitelline membrane and hyaline plasma membrane remain connected with one another by means of numerous fine, granule-free protoplasmic strands, which stretch across the perivitelline space. Elevation is due to the swelling of a transparent, dense jelly which is located between the membrane and the surface of the egg ( Hatt, 1931). At first the membrane is smooth, but as it elevates and stretches further, it becomes wrinkled. An interesting series of cortical changes accompanies membrane elevation. Upon contact with sea water, the small, refringent spherules Iying just beneath the vitelline membrane disappear, leaving a granule-free surface. It is this layer of hyaline material which is pulled out to form the radiating filaments. As elevation continues, many of the deeper cortical granules move toward the egg periphery and disappear, increasing the width of the cortical hyaline layer, the outer boundary of which becomes conspicuous (Waterman, 1936; Novikoff, 1939).

At the end of the pre-maturation stage, the egg is approximately 60 microns in diameter (Waterman, 1934).

The pre-maturation changes are very rapid, and are usually completed within ten minutes after shedding. To observe them, eggs should be transferred to a slide immediately after they are shed.

B. Fertilization and Cleavage: The phenomena of insemination uncomplicated by pre-maturation changes may be seen in eggs which have been allowed to stand in sea water for 15 minutes or longer. Except for the formation of an entrance cone and the withdrawal of the radiating filaments, the ovum is not visibly changed by insemination. No additional fertilization membrane is formed, and the cortical zone and existing membranes remain the same. When a spermatozoon attaches to the vitelline membrane, a large, rounded, hyaline entrance cone rises from the egg surface and pushes out toward the membrane. Novikoff (1939) and Waterman (1934, 1936) fail to agree on all details of sperm penetration, and those interested in the minor discrepancies are referred to the papers cited. According to Novikoff's account, when the entrance cone contacts the sperm, the head and middle piece separate from the tail and pass through the membrane. The discarded tail twitches a few times, and, after freeing itself from the membrane, may swim about with a rapid, whip-like motion for some time. The cone recedes, carrying the sperm head down into the egg cytoplasm. The filaments are withdrawn at this time, so that within 11 minutes of insemination, the egg surface is again smooth.

The formation of the first polar body occurs about twenty minutes after insemination, preceded by a distinct flattening of the egg in the polar region. The egg rounds up, but again flattens before the second polar body is produced. The egg then becomes spherical and remains in this condition until shortly before the first cleavage. At this time a large, first polar lobe is formed. A little more than an hour after insemination the first cleavage is completed, and the egg assumes a trefoil shape. Since the first cleavage plane passes just to one side of the polar bodies, the cleavage is slightly unequal. The polar lobe is soon resorbed into the larger CD blastomere. Soon a smaller second polar lobe is given off at the anti-polar region of this cell, and the second cleavage follows. At the completion of this division, the lobe flows into the D cell. The first quartet of micromeres (which

are almost as large as the macromeres) is given off by the usual dexiotropic division. During this division, a third polar lobe forms in the D cell, and afterwards it is incorporated into the larger basal 1D macromere. The later cleavages presumably follow the normal pattern of spiral cleavage, as exemplified by Nereis.

C. Time Table of Development: The exact relationship of temperature and developmental rate has not been worked out in detail. The following schedule is approximate for laboratory temperatures varying between 19û and 25û C. (Novikoff. 1937). Times are recorded from insemination.

Stage First polar body Second polar body First polar lobe First cleavage Second polar lobe and cleavage Swimming larva Apical tuft and prototroch Metamorphosis

Time 19-23 minutes 28-34 minutes 50-55 minutes 65-70 minutes 80-85 minutes 5-1/2 hours 8 hours 7 weeks

D. Later Stages of Development and Metamorphosis: Consult the papers by Novikoff (1938a) and Wilson (1929) for details of development, and for illustrations of larvae 5 to 29 hours old. The larvae of Sabellaria are interesting because they show very long bristles which have probably both a suspensory and a protective function. In Sabellaria trochophores two days old and older, some of the distinctive features include:

1. Stiff cilia in the apical region, which develop before the disappearance of the apical tuft (Novikoff, 1938a).

2. The prototroch, consisting of three rows of cilia with a gap on the dorsal side. 3. The neurotroch in the mid-ventral line.

4. One eye on the left side; more eyespots develop later.

5. Very long bristles with a fine structure, which develop in seta sacs. Ten pairs are formed, one after another. At metamorphosis they are replaced by ordinary setae.

6. The digestive tract, which is internally ciliated.

FAURÉ-FREMIET, E., 1924. L'oeuf de Sabellaria alveolata L. Arch. d'Anat. Micr., 20: 211-342.

HARRIS, J. E., 1935. Studies on living protoplasm. I. Streaming movements in the protoplasm of the egg of Sabellaria alveolata (L.). J. Exp. Biol., 12: 65-79.

HATT, P., 1931. La fusion expérimentale d'oeufs de "Sabellaria alveolata L." et leur developpement. Arch. Biol., 42: 303-323.

HATT, P., 1932. Essais experimentaux sur les localisations germinales dans l'oeuf d'un Annelide (Sabellaria alveolata L.). Arch. d'Anat. Micr., 28: 81-98.

NOVIKOFF, A. B., 1937. Sabellaria vulgaris. In: Culture Methods for Invertebrate Animals, edit. by Galtsoff et al., Comstock, Ithaca, pp. 187-191.

NOVIKOFF, A. B., 1938a. Embryonic determination in the annelid, Sabellaria vulgaris. I. The differentiation of ectoderm and endoderm when separated through induced exogastrulation. Biol. Bull., 74: 198-210.

NOVIKOFF, A. B., 1938b. Embryonic determination in the annelid, Sabellaria vulgaris. II. Transplantation of polar lobes and blastomeres as a test of their inducing capacities. Biol. Bull., 74: 211-234.

NOVIKOFF, A. B., 1939. Surface changes in unfertilized and fertilized eggs of Sabellaria vulgaris. J. Exp. Zool., 82: 217-237.

NOVIKOFF, A. B., 1940. Morphogenetic substances or organizers in annelid development. J. Exp. Zool., 85: 127-155.

WATERMAN, A. J., 1934. Observations on reproduction, pre-maturation, and fertilization in Sabellaria vulgaris. Biol. Bull., 67: 97-114.

WATERMAN, A. J., 1936. The membranes and germinal vesicle of the egg of Sabellaria vulgaris. Biol. Bull., 71: 46-58.

WILSON, D. P., 1929. The larvae of the British Sabellarians. J. Mar. Biol. Assoc., 16: 221268.