The heteronereis form of Nereis limbata lives in the mud of Eel Pond at Woods Hole, Mass., and also, in smaller numbers, in the Fisheries Basin and in Great Harbor. The sexes are separate. During certain phases of the lunar cycle (from full to new moon), these worms swarm at the surface, beginning about an hour after sunset. The males can be recognized by their smaller size, more active movements, and more vivid coloration&emdash;they are bright red, with white posterior segments. The larger, more sluggish females are a pale yellow-green in color.

It is convenient to collect the animals from the Eel Pond floating dock of the Supply Department, at Woods Hole. The light of a 100-watt lamp is used to attract the worms (the dock being wired with electricity for the collecting lamps). A long-handled net (having a flat, oval-shaped head, about 10 inches in length in the long axis, and with gauze stretched tightly over the framework) is used to scoop the worms from the water. On nights when there is a "run," a few males will appear first, swimming in wide circles. The females appear later; fewer in number and swimming more slowly than the males, they are first seen at the outer boundary of the circle of light. As a female spirals slowly towards the illuminated surface of the water, males which approach within a certain orbit will deviate from their original spiral paths to swim actively around her in rapidly narrowing circles, shedding sperm as they do so. This action is stimulated by substances which originate in the eggs and which are given off by the body of the ripe female. In turn, the presence of sperm in the water is a stimulus which induces the female to circle and shed. The females sink slowly to the bottom when they are spent. If females are to be collected before any shedding occurs, it is necessary to obtain them before they begin to circle.

Exceptions to the "dark of the moon" swarming are as follows: (1) In the first run of June, the animals may swarm every night until the full moon of July. (2) On stormy or windy nights, or nights following very cloudy days, Nereis may fail to appear. (3) In late September on cold nights they do not swarm even in the dark of the moon. (4) The curve of swarming is bimodal, with a depression several days before new moon.

Other striking examples of lunar periodicity have been described by Clark and Hess (1940a, 1940b), Hempelmann (1911), Izuka (1903), Just (1914), Mayer (1908) and Woodworth (1907).

June through September, as discussed above (see, also, the paper by Lillie and Just, 1913).

A. Care of Adults: The animals should be collected one by one, and each female placed in a separate fingerbowl of sea water; several males may be kept together. It is best to prepare the fingerbowls in advance of the collection, placing a piece of Ulva (or a piece of paper towel, previously thoroughly soaked in clean sea water) in each dish, which is then half-filled with clean sea water. These dishes can be carried in a wooden tray to the collecting dock, and the animals placed directly in them. When they are brought back to the laboratory, the sea water should be changed and fingerbowls containing the animals should be covered and placed on a water table, surrounded by running sea water. Do not keep the worms in a refrigerator.

B. Procuring Gametes: An excess of sperm, which should be avoided, is usually obtained if the males and females are placed together in a dish, unless the male is removed as soon as it has shed its first cloud of sperm. Instead, gametes should be procured by pinching the animals with fine forceps, near the middle of the body; a single strong pinch should result in extrusion of the sex cells. The eggs from each female should be placed in a 250-ml. (or larger) fingerbowl, half-filled with clean sea water; no more than a single cell-layer of eggs should be present on the bottom of the dish, after the eggs have settled. "Dry" sperm are obtained by placing a male worm in a dry Syracuse dish and pinching the middle of the body. Adults should be removed from the dishes after shedding is completed.

Clipping the body with scissors is not a good practice, since it increases contamination of the gametes with coelomic fluid (which apparently adversely affects the normal fertilization reaction).

C. Preparation of Cultures: Add a few drops of dilute sperm suspension (one drop of "dry" sperm in 50 cc. of sea water) to the fingerbowl of eggs, and stir at once with a rapid circular movement of the dish. Care must be taken to avoid polyspermy, which results in interference with cleavage or in abnormal cleavage and development. In some forms, polyspermic eggs develop more rapidly than normally fertilized eggs, but those of Nereis usually fail to cleave.

To obtain later stages of development, allow the fingerbowl to remain undisturbed for about 30 minutes; then transfer the contents to a large dish and change the sea water. Keep the dish covered and on a sea water table, changing the water at least twice a day. After they leave the jelly, pour off the swimming trochophores to a clean dish of sea water; discard the jelly and dead eggs.

D. Methods of Observation: One or two minutes after insemination, place the eggs in a drop of thick Chinese ink suspension (made by rubbing a piece of the solid ink, wet with sea water, on a finely-ground glass surface). As the jelly is secreted by the eggs, it flows past the attached sperm, leaving a funnel-shaped cavity in which the ink particles remain, serving as an indicator of the point of sperm entrance.

Another method of studying sperm entrance is by the production of exaggerated entrance cones. Place a drop of eggs, inseminated 5 to 8 minutes earlier, in a slender dish containing 50 cc. of alkaline NaCI (pH 10.3-10.5) and mix rapidly and thoroughly. The vitelline membranes will elevate, due to a sudden inhibition of jelly release through the membrane, and a subsequent accumulation of the jelly in the perivitelline space (Costello and Young, 1939). The vitelline membrane remains permeable to water, which enters the perivitelline space as the jelly swells. The elevation of the membrane stretches out the sperm entrance cone between membrane and egg surface, forming a long filament which frequently causes a marked indentation of the membrane. It is sometimes necessary to use two or three changes of alkaline NaCl to obtain maximum exaggeration of the entrance cones. If the eggs (or the females from which they were obtained) have been kept in a refrigerator, they may become polyspermic when inseminated, and show numerous exaggerated entrance cones following this treatment. About ten minutes after treatment, the sperm head and middle piece may be seen moving across the perivitelline space to fuse with the egg surface. The membrane indentation is relaxed as soon as the sperm head has passed through. If the eggs are now carefully removed to sea water and washed several times, some will develop normally within the raised membranes. If they are left in the solution an optimum length of time before washing, and if the alkaline NaCl has been changed once or twice to remove most of the sea water, the eggs may be completely freed of their membranes. For further details of obtaining these denuded eggs, see the papers of Costello (1945a, 1949). For details of useful procedures for fixing, sectioning and staining Nereis eggs, consult the book by Just (1939b).

A. The Unfertilized Ovum: The egg of Nereis is approximately 140 microns in diameter and 100 microns high. Because of its shape, it tends to orient on a flat surface with the animal pole either above or below, only rarely to the side. It has a large germinal vesicle, with many small oil droplets and yolk spheres in the cytoplasm surrounding it. The egg has a cortex about seven microns thick, of jelly-precursor granules.

B. Fertilization and Cleavage: Very soon after insemination, a transparent jelly-layer is secreted by the egg, external to its vitelline membrane. This jelly arises from the cortical granules. In 20 minutes the zone of jelly will be as wide as the diameter of the egg it surrounds; its margin can often be observed with the aid of supernumerary spermatozoa or other particles (such as Chinese ink) at its edge or in the medium.

There is little visible change in the vitelline membrane at fertilization, although it is called the fertilization membrane after this event. However, a narrow perivitelline space is present shortly after insemination, resulting from the breakdown of the jelly-precursor granules and release of the jelly. The sperm entrance cone now becomes clearly visible, and is best seen in the profile view of an egg with a sperm at its periphery. In the course of the next 8 to 10 minutes, the vitelline membrane is indented slightly at its point of contact with the entrance cone, tending to obscure the sperm from view. About 20 minutes after insemination, the egg wrinkles, becoming distorted and almost amoeboid in appearance. The entrance cone has flattened considerably but is still present, and although the sperm is partially concealed from view, the entrance of its head into the egg is not completed until some time later (Just, 1912; Lillie, 1911, 1912). Its final penetration through the membrane (about 48 minutes after insemination) leaves the middle piece and tail outside.

The egg then rounds up, and elongates in a direction perpendicular to the polar axis. as the time approaches for the formation of the first polar body. The preparation should be shaken if no eggs lie so that the forming polar body is on the periphery. The polar body is given off into the space between the egg and the vitelline membrane, which is wider in the region of the animal pole than elsewhere. The second polar body forms under the first, thus lifting it away from the egg surface. The first polar body of Nereis rarely, if ever, divides.

The egg cleaves into two unequal blastomeres, and the second cleavage is also unequal. The third cleavage, from four to eight cells, produces four micromeres by spiral cleavage (Wilson, 1892; Costello, 1945a).

C. Time Table of Development: The following schedule is based on the development of 16 batches of eggs, at temperatures of 22-24û C.; times are calculated from insemination.

Stage Disappearance of membrane of germinal vesicle First polar body Sperm penetration Second polar body First cleavage Second cleavage Third cleavage Fourth cleavage Ciliated trochophore Pigmentation in trochophore

Time 10-15 minutes 42 minutes 48 minutes 58 minutes 81 minutes 108 minutes 132 minutes 162 minutes 8-10 hours 24-38 hours

There is a high temperature coefficient for the cleavage process; Lovelace (1949) gives data from which the following mean times have been calculated for first cleavage in 50% of the eggs in a given batch.

Mean temperature,û C. (within a 1û range) 20.0 21.3 22.1 23.1 24.1

Time after insemination 97 minutes 86.9 minutes 84.2 minutes 77.3 minutes 70.8 minutes

D. Later Stages of Development and Metamorphosis: Gastrulation is by epiboly. The products of the first three quartets of micromeres overgrow the four large oil-bearing cells, 3A, 3B, 3C and 4D. These four endodermal cells, after giving off a few small cells, persist unchanged for a relatively long period. The four large oil droplets (which result from coalescence of the smaller oil droplets of the egg) may be used as a criterion of normal development, since those in the C and D quadrants are larger than those in the A and B quadrants. For further details, consult the papers of Wilson (1892, 1898) and Costello (1945a).

The trochophore larva metamorphoses into a segmented worm in about seven days. The trochophore is somewhat atypical, and there is an abbreviated, "telescoped" larval development. The first signs of the segmented adult organization appear very early. To study, mount the larvae on a slide, and either entangle them in a few shreds of lens paper or quiet them by adding a drop of very dilute (1:1000) Janus green solution. Distinctive features of the trochophore larva at 40 hours (Wilson, 1892; Figure 84) include:

1. An equatorial prototroch consisting of 12 very large, ciliated cells, instead of the 16 typical of most annelidan and molluscan trochophores. There is the characteristic interruption in ciliation, in the mid-dorsal line. A narrow paratroch is present, near the vegetal pole.

2. Pigmentation, consisting of (a) a pair of red-pigmented eyespots in the pretrochal hemisphere; (b) orange-brown "prototrochal" pigment, in cells adjacent to the prototroch; (c) greenish-black anal pigment in the region of the proctodeum.

3. The four large macromeres (each still containing a single large oil drop) have not yet differentiated into the parts of the intestine. Short, blind ectodermal invaginations constitute stomodeum and proctodeum.

HOADLEY, L., 1934. Pulsations in the Nereis egg. Biol. Bull., 67: 484-493.

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

WOLTERECK, R.,- 1905. Zur Kopffrage der Anneliden. Verh. d. Deutsch Zool. Ges., 15: 154-186.