These worms live in parchment-like, U-shaped tubes in sand just below tide level; they can be dug only at low tide. The sexes are separate, and are distinguished by the parapodia on the posterior (sexual) segments. These parapodia are uniformly ivory white in the male, but in the female they contain yellow coils, which are the ovaries with their enclosed eggs.

June, July, and sometimes the first two weeks in August.

A. Care of Adults: When brought into the laboratory, the animals are often still in their leathery tubes, which can be slit with scissors so that the worms can be removed gently. The sexes should be segregated, with no more than two or three animals per large fingerbowl. The dishes should be placed on a water table, and supplied with a constant, gentle stream of sea water. One or two females and one ripe male will give an adequate supply of eggs and sperm for ordinary embryological experiments.

B. Procuring Gametes: Animals may be kept in the laboratory for several days and parapodia removed as needed.

Female gametes: Unless the sexes have been kept separate for at least two days, rinse the female for a few seconds under a gentle stream of fresh water, to kill any sperm which may have adhered to the mucous film on the body. Cut off one or two parapodia and transfer them to a double layer of cheesecloth (which has been rinsed well in fresh water and then in sea water), allowing the eggs to filter into a fingerbowl of freshly filtered sea water. The straining will remove debris and most of the mucous matrix around the eggs. The parapodia may be teased apart, if necessary, to release the eggs.

Male gametes: Scissors are used to remove a posterior parapodium from a male, the tip of the segment being held with forceps. Allow the sperm to flow into a slender dish containing 10 cc. of filtered sea water. A drop of this suspension examined microscopically should contain highly motile sperm. If large numbers of motionless sperm are present, the suspension should be discarded and the procedure repeated with another male.

C. Preparation of Cultures: Procure eggs as directed above, and about 10 minutes later prepare the sperm suspension. Fifteen minutes after they are obtained, the eggs should be inseminated with one drop of the sperm suspension. This allows time for germinal vesicle breakdown. Thirty minutes after insemination, the eggs should be transferred to a fingerbowl of fresh sea water and placed on a water table. The bowl should be covered and the water changed at least twice a day after trochophores develop.

D. Methods of Observation: No special technique is required for observing living Chaetopterus eggs, but it is often desirable to prepare permanent slides of various stages. For whole mounts, which are useful for determining stages of mitosis, fertilization, etc., see the paper by Henley and Costello ( 1957). For sectioning these eggs, consult the very complete directions given by Just (1939, p. 88).

A. The Unfertilized Ovum: This egg is rather dark and granular, from the contained yolk-spheres. It is slightly more than 100 microns in diameter, and is often not quite spherical. When taken from the female the oocyte, like that of Nereis, contains a large, central, immature nucleus, the germinal vesicle. However, in the egg of Chaetopterus maturation proceeds spontaneously to the metaphase of the first polar division after exposure to sea water. At this stage development is arrested until activation or death (Lillie, 1906; Pasteels, 1935). The spindle cannot be distinguished as such in the living egg without considerable flattening; the relatively clear region containing it is located quite excentrically. The spindle is attached to the egg surface in the region where the first polar body subsequently will be given off.

B. Fertilization and Cleavage: A few sperm may be seen adhering to the eggs almost immediately after insemination. Within five to six minutes the vitelline membrane separates slightly from the egg surface, and may now be called the fertilization membrane. Membrane elevation is inconspicuous in the egg of Chaetopterus, and there is little or no change in the membrane itself at this time; thickening and hardening do not occur. Later, however, the membrane undergoes a series of wrinklings which are quite pronounced (Pasteels, 1950). Ten to twelve minutes after insemination, the eggs, which become almost spherical after fertilization, elongate along an axis perpendicular to the polar axis. This is preparatory to the formation of the first polar body. In this division the egg thus assumes approximately the shape of a blastomere, although the polar body which results is a vestigial cell. The egg now rounds up, but elongates again in the same manner to produce a second polar body, which is usually formed under the first, pushing it away from the egg surface. The egg rounds up again, and the egg pronucleus may sometimes be seen migrating toward the center of the egg; occasionally, the sperm nucleus may be detected. The clear zone extends from the polar region toward the equator of the egg. A typical "pear-shaped" stage is reached, with the polar bodies in a position corresponding to that of the stem attachment in a pear. The bulge which forms the polar lobe appears quite suddenly at the antipolar end of the egg, reversing its shape.

The first cleavage furrow begins at the animal pole and passes to one side of the polar lobe, which thus becomes incorporated into one of the two smooth, unequal blastomeres. Abnormal three-celled eggs, resulting from polyspermy, may be seen. The two blastomeres become closely apposed, and about 10 minutes later the second cleavage occurs. The large blastomere again forms a polar lobe, and a four-cell stage results, in which one blastomere is larger than the other three.

The four clear nuclei become visible, and shortly after this the third division takes place, forming four relatively large micromeres. A profile view shows the rotated displacement of the micromeres resulting from spiral cleavage, although this displacement is neither great nor conspicuous in the egg of Chaetopterus.

The polar bodies are larger than those of Nereis. The inequality of the first two cleavage blastomeres is due to two factors: 1) an inequality of the poles and asters of the first cleavage spindle, and 2) the addition of the polar lobe material to the CD blastomere (Mead, 1897; Lillie, 1906).

C. Time Table of Development: Chaetopterus eggs develop rapidly. If eggs are fertilized after the partial maturation in sea water has been completed, they develop as rapidly as eggs inseminated when first placed in sea water 12 to 15 minutes earlier. A rise in temperature increases the rate of development, but temperatures above 26û C. are not desirable.

The following table includes a summary of the development of many batches of Chaetopterus eggs, at temperatures of 22-23û C. and 24-26û C. The times are calculated from insemination, and represent the averages of data obtained over a period of several years.

Stage First polar body Second polar body "Pear" stage Polar lobe First cleavage Second cleavage Swimming trochophore

Time at 22-23ûC 14 minutes 28 minutes 42 minutes 47 minutes 51 minutes 71 minutes 22-24 hours

Time at 24-26ûC 11 minutes 18 minutes 36 minutes 41 minutes 42 minutes 59 minutes 8-20 hours

D. Later Stages of Development Chaetopterus larvae differ from typical trochophores in having no pre-oral prototroch. A prominent apical flagellum (single except in rare cases) is present. In slightly older larvae, a second band of cilia, the mesotroch, is found below the prototroch (Wilson, 1882, 1929).

In the late trochophore, two to six days old, there is a gradual disappearance of yolk. The various regions of the digestive tract can be identified: the wide, slit-like mouth on the ventral surface, which leads to a short, ciliated oesophagus; the large, clear, sac-like stomach, which is separated from the short intestine by a double fold of endoderm; the anus which opens on the dorsal side, just anterior to the terminal papilla or holdfast. The mesotroch of the early larva is replaced in the older animal by a pair of lateral flagella, and a second ciliated band, the paratroch, appears in the region of the posterior boundary of the intestine. In the anterior region (the head vesicle) the apical flagellum is retained and a pair of lateral eyespots is now visible. (See the paper of Wilson, 1882, and Figures 49 and 55 in the paper of Wilson, 1929).

GOLDSTEIN, L., 1953. A study of the mechanism of activation and nuclear breakdown in the Chaetopterus egg. Biol. Bull., 105: 87-102.

HENLEY, C., AND D. P. COSTELL, 1957. The effects of x-irradiation on the fertilized eggs of the annelid, Chaetopterus. Biol. Bull., 112: 184-195

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

LILLIE, F. R., 1902. Differentiation without cleavage in the egg of the annelid Chaetopterus pergamentaceus. Arch. f. Entw., 14: 477-499.

LILLIE, F. R., 1906. Observations and experiments concerning the elementary phenomena of embryonic development in Chaetopterus. J. Exp. Zool., 3: 153-268.

MEAD, A. D., 1897. The early development of marine annelids. J. Morph., 13: 227-326.

PASTEELS, J., 1935. Recherches sur le determinisme de ['entree en maturation de l'oeuf chez divers Invertebres marine. Arch. Biol., 46: 229-262.

PASTEELS, J., 1950. Mouvements localises et rythmiques de la membrane de fecondation chez des oeufs fecondes ou actives (Chaetopterus, Mactra, Nereis). Arch. Biol., 61: 197-220.

TITLEBAUM, A., 1928. Artificial production of Janus embryos of Chaetopterus. Proc. Nat. Acad. Sci., 14: 245-247.

TYLER, A., 1930. Experimental production of double embryos in annelids and mollusks. J. Exp. Zool., 57: 347-407.

WHITAKER D. M., 1933. On the rate of oxygen consumption by fertilized and unfertilized eggs. IV. Chaetopterus and Arbacia punctulata. J. Gen. Physiol., 16: 4/5-495.

WILSON, E. B., 1882. Observations on the early developmental stages of some polychaetous Annelides. Stud. Biol. Lab., Johns Hopkins Univ., 2: 271-299.

WILSON, E. B., 1929. The development of egg-fragments in annelids. Arch. f. Entw., 117: 179-210.