Adults of Hydroides hexagonus live in white, twisting, calcareous tubes which they secrete on old mollusc shells, stones, or timbers which are dredged in abundance from the harbor. For a description of features which distinguish this worm from Sabellaria, see p. 93 of this manual. The sexes are separate, but similar in appearance.

According to Grave (1933), this opens between June 10 and 15, and closes between October 1 and November 1. During the early part of the season, more than 50% of the shed eggs are immature and undersize; after the middle of July, nearly all the eggs are mature and fertilizable.

A. Care of Adults: If the worms are left in their tubes and placed in aquaria with a good supply of running sea water, they may be kept almost indefinitely in the laboratory.

B. Procuring Gametes: The gametes, which are carried in the coelomic cavity, will be released through the nephridiopores almost immediately after the worms are removed from their tubes. This is done by chipping away the tubes with forceps. Place each animal in a separate slender dish containing about 25 cc. of sea water, and observe shedding. Clouds of white sperm will flow from the male, while the female will release a large number of peach-colored eggs. This is a convenient way of distinguishing the sexes. Remove the male when the sea water is cloudy with sperm, and use this sperm suspension without dilution, as indicated below.

C. Preparation of Cultures: Eggs may be successfully fertilized as long as four hours after shedding. Sperm, once activated, remain viable for as long as eight hours. The sperm are inactive when first shed, but become activated slowly after dilution with sea water. For this reason it is best to delay insemination until at least half an hour after shedding. At this time examine a sperm sample under a compound microscope. If the sperm appear active, add five or six drops of the suspension to the eggs, which have been transferred to a slender dish of fresh sea water. Allow the inseminated eggs to stand undisturbed for about 30 minutes and then decant the upper layers of sea water, replacing it with fresh sea water. Cover the dish and place it on the water table. In ten hours or less, actively swimming gastrulae will be present; they should be decanted to a fingerbowl of fresh sea water. Discard the debris and the undeveloped eggs. The larvae are very hardy, and will develop for days without extra care, although if they are to be kept over long periods, they should be decanted daily to fresh sea water. They have been successfully reared through metamorphosis (about two weeks after hatching), but to do this, feeding with diatoms is necessary.

D. Methods of Observation: Since these eggs are small, they are best studied under high magnification, mounted on glass slides covered with unsupported coverslips. The older, moving larvae can be temporarily quieted by adding a drop of very dilute Janus green solution (1:1000 in sea water) to the mount. This will also serve to indicate clearly details of the digestive tract.

A. The Unfertilized Ovum: The eggs are small (67-72 microns in diameter), peach-colored, and have a thick, refractive, vitelline membrane. They are spherical and very opaque, due to the presence of a considerable amount of yolk, but the outline of the large germinal vesicle can be seen.

B. Fertilization and Cleavage: Insemination is not immediately followed by any noticeable changes, no entrance cone nor fertilization membrane being formed. Colwin, Colwin and Philpott (1956b) report that a narrow perivitelline space is present, however, after fertilization. The outline of the germinal vesicle becomes irregular and lobulated, and 15 minutes after insemination, it ruptures completely. Although the maturation spindle is not visible (because of the opacity of the egg), it is forming and moving to the periphery of the egg.

Prior to the appearance of the first polar body, two changes may be seen to occur. The egg flattens at the animal pole, and the vitelline membrane rises from the surface in this vicinity to form a cap-like space into which the first polar body is elevated. This polar body usually divides once, soon after its formation. Following this division, the second polar body is produced. A second cap-like space is formed at the vegetal pole, by the separation of the egg surface from the membrane. This precedes the first cleavage, which is equal; the AB cell cannot be distinguished from the CD cell. The second cleavage divides the egg further, into four approximately equal blastomeres, and very shortly after this, a dexiotropic, horizontal cleavage cuts off the first quartet of micromeres. Further cleavages follow the typical course of spiral cleavage, and produce a ciliated, moving blastula in about five hours. (See the paper by Shearer, 1911.)

C. Time Table of Development: The exact relationship between temperature and developmental rate has not been established in this form, but the following table will give an approximate chronology of stages observed at 24û to 25û C. The time is recorded from insemination.

Stage Germinal vesicle breakdown completed First polar body Second polar body First cleavage Second cleavage Third cleavage Swimming larva (blastula) Gastrula Well-formed trochophore Metamorphosis

Time 14 minutes 40 minutes 60-70 minutes 1 hour, 20 minutes 1 hour, 36 minutes 1 hour, 46-50 minutes 5-6 hours 9-12 hours 20 hours 12 days-2 weeks

D. Later Stages of Development and Metamorphosis: The process of gastrulation may be followed by observing embryos 6-10 hours after insemination. The vitelline membrane is not cast off when the cilia develop and the larvae start to swim; the cilia grow through it, and the membrane is not lost, eventually forming part of the cuticle of the worm body. In the young blastula, it is still possible to see the membrane raised from the surface in the polar area. The polar bodies, however, are no longer visible at this time. As gastrulation progresses, the elongated endodermal cells invaginate into the blastocoele to form the archenteron, which opens to the surface by way of the blastopore. In older gastrulae, the apical tuft and prototroch are well developed; they are therefore trochophores. (See the paper of Hatschek, 1886.)

Larval stage: The larva is a typical annelid trochophore. For details of structure, the excellent figures of Hatschek (1886) and Shearer (1911) may be consulted. The larvae show positive phototaxis, and gather on the illuminated side of the dish. Trochophores, three to five days old, can be mounted on a slide with a few shreds of lens paper to entangle them, or they can be quieted with a drop of dilute Janus green (1:1000 in sea water). The larvae are transparent, and proper illumination (obtained by adjusting the microscope mirror and condenser) will help to bring out the details of structure. The apical tuft and the anal vesicle are landmarks for the animal and vegetal poles, respectively; the mouth is on the ventral side, the eye on the right.

The following may be observed:

1. The shape of the trochophore, with pre-trochal and post-trochal regions.

2. Apical tuft; several long cilia probably functioning as a sense organ.

3. Apical organ, a thickening of the ectoderm at the animal pole; a nerve center and the primordium of the cerebral ganglion.

4. The prototroch, an equatorial band of large cilia. In older trochophores, two rows will be found, with a row of short cilia anterior to the large cilia.

5. The metatroch (paratroch), a circular band of cilia in the middle of the posttrochal hemisphere.

6. A ciliated groove on the mid-ventral line connecting the mouth and the anus. It marks the line of closure of the blastopore, the mouth being the remnant of the blastopore, the anus a secondary opening at the lower end of the blastoporal slit.

7. One eyespot (with red pigment) on the right side in the pre-trochal hemisphere.

8. Two statocysts on the ventral side.

9. The digestive tract, consisting of mouth opening, stomodeum or oesophagus (ectodermal), enlarged stomach (endodermal), narrow intestine (endodermal, with the exception of terminal, ectodermal proctodeum ), and the anus, an opening dorsal to the vegetal pole. The whole tract is lined with cilia. The mechanism of food intake may be studied if the larvae are fed Chinese ink.

10. The anal vesicle, a large, vacuolated cell at the posterior end, not found in most other species of trochophores.

11. The cavity between the outer body wall and the intestine; not a true coelom but a primary body cavity, it is the persisting blastocoele.

12. The larval kidneys (paired), typical protonephridia with flame cells; they open near the anus, and appear as slender cords near the statocysts, extending between oesophagus and anus. (Consult the figures in the papers by Hatschek and Shearer. )

13. Muscles. Two fine strands may be seen bifurcating at the upper end of the larval kidney. One of them can be traced to its insertion at the apical plate, the other at the oesophagus. These are longitudinal muscles. Other longitudinal muscles extend from the stomach to points in the upper hemisphere. A strong circular muscle is located near the metatroch; the constriction of the larva caused by its contraction will be frequently observed. There are circular, sphincter muscles in the digestive tract.

14. Undifferentiated ectomesodermal cells, single or in small groups, can be seen attached to the stomach, to the inner body wall, near the apical organ, etc.

15. The important entomesodermal cells (derivatives of the 4d teloblasts), which give rise to the mesodermal structures of the worm body, are difficult to distinguish. They are small groups of cells near the lower end of the larval kidney.

COLWIN, A. L., L. H. COLWIN AND D. E. PHILPOTT 1956a. Sperm entry in Hydroides hexagonus (Annelida) and Saccoglossus kowalevski (Enteropneusta). Biol. Bull., 111: 289.

COLWIN, L. H., A. L. COLWIN AND D. E. PHILPOTT, 1956b. Electron microscope studies of the egg surfaces and membranes of Hydroides hexagonus (Annelida) and Saccoglossus kowalevskii (Enteropneusta). Biol. Bull., 111: 289-290.

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GRAVE, B. H., 1933. Rate of growth, age at sexual maturity, and duration of life of certain sessile organisms, at Woods Hole, Massachusetts. Biol. Bull., 65: 375-386.

GRAVE, B. H., 1937. Hydroides hexagonus. In: Culture Methods for Invertebrate Animals edit. by Galtsoff et al., Comstock, Ithaca, pp. 185-187.

HARGITT, C. W., 1910. Observations on the spawning habits of Hydroides dianthus. Amer. Nat., 44: 376-378.

HATSCHEK, B., 1886. Entwicklung der Trochophora von Eupomatus uncinatus, Philippi (Serpula uncinata). Arbeit. Zool. Inst. Wien, 6: 121-148.

SHEARER, C., 1911. On the development and structure of the trochophore of Hydroides uncinatus (Eupomatus). Quart. J. Micr. Sci., 56: 543-590.

WILSON, E. B., 1890. The origin of the mesoblast-bands in annelids. J. Morph., 4: 205-219.

ZELENY, C., 1905a. The rearing of serpulid larvae with notes on the behavior of the young animals. Biol. Bull., 8: 308-312.

ZELENY, C., 1905b. Compensatory regulation. J. Exp. Zool., 2: 1-102.

ZELENY, C., 1911. Experiments on the control of asymmetry in the development of the Serpulid, Hydroides dianthus. J. Morph., 22: 927-944.