In the last few decades studies of Pectinaria gouldii have focused upon oocyte development and fertilization. These include identification of cytoplasmic organelles of the oocyte and their distribution during GVBD (Tweedell, 1962), the growth and development of the primary oocytes and the use of thymidine and uridine by the ovary and developing oocytes (Tweedell, 1966). The ovarian oogonia and developing embryos also show the uptake of 3H TTP (Tweedell, 1976). Studies on gamete migration, maturation and spawning have implicated a factor that reinstates meiosis, GVBD, and spawning (Tweedell, 1980).
The mitotic apparatus can be reversibly dispersed with dinitrophenol in shed oocytes (Sawada and Rebhun, 1969). Meiosis in unfertilized oocytes, normally arrested in metaphase I, can be reinitiated with the divalent cation ionophore, A23187; however, extracellular Ca2+ appears to be required (Anstrom and Summers, 1981). Sperm entry is not sufficient to reinitiate meiosis. When sperm are prematurely incorporated into immature oocytes, sperm transformation and pronuclear formation will not occur until after GVBD, implicating a released nuclear factor (Hylander et al., 1981).
Pectinaria specimens are found in mudflats beneath shallow water between the upper and lower tide marks. They are also dredged from 2 to17 fathoms in subtidal regions. Pectinaria gouldii range from Old Tampa Bay, Florida (Simon and Dauer, 1973), along Barnegat Bay, New Jersey (Busch and Loveland, 1975), and Ocean Pond, Fisher Island, New York (Whitlatch and Weinberg, 1982) to its northern limits on Cape Cod [although there are very early reports from Beaufort, North Carolina to Casco Bay, Maine; see Moore, 1900]. Early surveys from Woods Hole (Sumner et al., 1913) indicated that it occurred throughout the entire length of Buzzards Bay, Massachusetts, including the Wareham River. They were also reported in numerous sites along the northwest shores of Naushon and Nonamessett islands, the northeastern shore of Uncatena as well as Quicks Hole and Robinsons Hole in the Elizabeth Islands south of Woods Hole, Massachusetts. Two other sites along the east shore of Buzzards Bay were Scraggy Neck and Nyes Neck adjacent to Megansett Harbor (see Appendix 1).
More contemporary collection sites on Cape Cod include Barnstable Harbor (Gordon, 1966) on Cape Cod Bay for the largest specimens (John Valois, Woods Hole, Massachusetts, personal communication; see Appendix 2), Little Sippewissett Marsh on Buzzards Bay (Whitlatch, 1974) and from subtidal zones located in Wild Harbor off of West Falmouth on Buzzards Bay, Massachusetts (see Sanders et. al, 1980 for collection sites; Appendix 2). Small adults can also be taken from the Eel Pond in Woods Hole and Hadley Harbor in the Elizabeth Islands.
This worm lives in a beautiful cone shaped tube that is open at both ends and constructed of sand grains that are selected by size, then fitted and cemented together in a single layer (Fig. 1). The flat sloping head fills the wider end of the sand cone (see Appendix 3 for further external features). The animals live 2 to 3 inches beneath the sand surface with the wide oral end down and the apex of the tube generally uppermost. They ingest sand and detritus from a small subterranean chamber supplied with a small inlet tunnel. Further practical information on identification of burrows and collection of animals is provided in Appendix 2.
Adults obtained from the Marine Biological Laboratory in late March and early April were found to be devoid of mature oocytes, but coelomic fluid contained multiple packets of developing oocytes. Pre-spawning adults can be collected from June until mid August. During this period the coelomic cavity is filled with oocytes in sequential stages of development; multiple "ripe" oocytes dominate as spawning approaches (Fig. 2). Adult animals spawn naturally from mid-August to early September in the Cape Cod area.
It is quite likely that ovarian oogonial division and subsequent oocyte maturation is controlled by hormones. Extracts of the neural (subesophageal) ganglion will trigger the uptake of mature oocytes by the reproductive duct and ensuing GVBD in vivo (Tweedell, 1976). Normally, after maturation of the oocytes, spawning appears to be triggered by temperature, and spawning will be delayed if the seawater temperature stays low. For example, animals collected on August 12th one year already had mature gametes in their nephromixia (seawater temperature 23°C) but pregravid, non-spawning animals can be obtained from colder water until the beginning of September. Animals collected early in the season (June-July) can be held in refrigerated seawater at 12°C and used into September as a source of gametes.
For daily use, adults can be kept in shallow bowls supplied with vigorously running seawater. For longer storage adults should be kept in shallow trays containing 10 to 12 cm of sand bathed by continuously running unfiltered seawater. Animals can be stored in the pre-spawning condition if the trays are immersed in 12°C running unfiltered seawater tanks.
A dorsal dissection (Fig. 3) reveals two pairs of brownish nephromixia, combined excretory-reproductive ducts, located just inside the 2nd and 3rd body parapodia (segments) possessing setae; these parapodia also lack lateral "wings" found on more caudal segments. The nephridiopores are located in the 2nd and 3rd body segments just dorso-lateral and slightly caudal to the setae of the parapodia. These are only functional during spawning. Internally, the coiled nephromixia have large ciliated coelomostome funnels (Fig. 4) that receive the gravid oocytes at the time of spawning.
The four ovaries are narrow, transparent, and heavily vascularized ciliated structures surrounding prominent lateral blood vessels extending along the anterior bases of the 2nd and 3rd pairs of nephromixia. Oocytes are budded off from a free germinal area on the cephalic edge of each ovary directly into the coelomic fluid. The testes are found in a similar position in the male.
New coelomic oocytes are produced constantly during the breeding season, as indicated by the finding that ovarian preoocytes (oogonia) show nuclear uptake of 3H-thymidine and 3H-TTP (Tweedell 1966, 1976). Oocytes are budded off as packets from the free edge of the ovary into the coelom as small rosettes. The individual cells of each packet continue to increase in size. Typically, the coelomic fluid contains packets of 16 to 32 oocytes (cells 5.5 µm in diameter) which increase in size (cell diameter 10-15 µm) when the packets break apart. During the growth period of vitellogenesis, individual oocytes with a prominent germinal vesicle and a budding nucleolus grow vegetatively, increasing to about 10 times the diameter produced in the ovary. As spawning approaches, the mature coelomic oocytes have intact germinal vesicles. Prior to spawning, the mature disc shaped oocytes are guided into the nephromixia where they undergo germinal vesicle breakdown (GVBD). Subsequently these mature oocytes are released through lateral nephridiopores at spawning.
Females can be induced to shed oocytes prior to the spawning season, resulting in a series of varying developmental stages within the coelomic fluid. These oocytes exit from the coelom through posterior coelomoducts. Animals must first be removed from their protective sand test. Carefully insert a needle probe into the small, pointed posterior sand test opening to dislodge a pair of retaining hooks. If necessary, break off the tip end of the test. Immediately apply a rubber mouth tube at the narrow end and blow out the adult from the sand test into seawater. Animals can be sexed under a stereomicroscope by looking through the mid-ventral transparent body wall. Females can be identified by their translucent oval eggs free in the coelomic fluid; males have white sperm packets with obscure outlines. Pinching the body wall will generally cause the release of coelomic fluid containing oocytes in various stages of development (or sperm packets). A diverse selection of other cell types (amoebocytes, histiocytes) are also found in the coelomic fluid. One of the co-inhabitants with the developing oocytes in the coelomic fluid is a huge snow-white gregarine protozoan (adult vegetative stage) of Urospora sp. (Brasil, 1904). Its developmental stages are usually encysted and attached to the outer intestinal wall; Urospora can be confused with oocyte packets when free in the coelomic fluid.
B. Sequence of germinal vesicle breakdown (GVBD)
The fully developed oocyte measures 55 to 60 µm in diameter but has the shape of a flattened convex-concave disc measuring 25 µm in edge view. Under darkfield illumination long microvilli are found around two-thirds of the egg circumference but apparently they are absent in the animal-vegetal polar regions. The pale green transparent cell contains a large germinal vesicle and a prominent nucleolus with buds. Mature oocytes from the coelomic fluid change shape drastically and undergo GVBD upon contact with seawater (Fig. 5). Freshly shed disk-like oocytes (pre-GVBD) and spherical oocytes (post-GVBD) are shown in greater detail in scanning electron micrographs (Fig. 6 and Fig. 7) . At first the nuclear membrane becomes scalloped or crenulated within one to three minutes (Tweedell, l962). At three to five minutes the membrane becomes firm and round again. Shortly after, the nucleolus disappears at five to seven minutes and the egg changes from a disc to a sphere. Initiation of GVBD takes place from six to ten minutes after shedding and will be completed from 13 to 20 minutes depending upon the time of season. At this time the oocyte is in metaphase I and is receptive to normal fertilization.
After induced shedding of a female prior to the spawning season, a whole array of oocyte packets, individual oocytes in the vegetative growth phase and mature oocytes are released. Oocytes should be washed by light centrifugation two to three times in fresh seawater before further treatment while GVBD takes place.
Induced shedding of males by pinching the body wall produces sperm embedded head to head in diamond shaped packets (Fig. 8); these must be allowed to breakdown before being used for fertilization. Males can be shed in 50 ml of seawater; free swimming sperm are available in 15 to 20 minutes. The sperm packets and individual sperm are shown in greater detail in scanning electron micrographs (Fig. 9 and Fig. 10).
Fertilization of oocytes after GVBD can be done by adding six drops of sperm per 100 ml of filtered seawater in a culture dish containing a single layer of eggs. Eggs can stand in fresh seawater for several hours after GVBD before semination.
During sperm penetration (see Anstrom and Summers, 1981 for details) meiosis resumes and the second polar body is liberated. Meiosis can also be re-initiated without sperm, using the divalent cation ionophore A23187 in the presence of extracellular calcium (ibid, l981). The first polar division and advance to metaphase II starts after 30 to 35 min and the second polar body follows quickly. Following fertilization, pronuclear fusion takes place from 40 min (Costello et al., l957) to 50 or 60 min at 18°C after semination (Austin, l963).
The chronological sequence of development at 21 to 23°C is as follows (Tweedell, personal observations)
Polar bodies form - 30 min.
Fusion of pronuclei - 40 min.
2 Cell - 48-55 min.
4 Cell - 63-66 min.
8 Cell- 83-86 min.
Blastula- 5 l/2 hours
Gastrula- 10 hours
Trochophore larva - 22-24 hours
Normally, fully mature oocytes do not enter the nephromixia until spawning. An aqueous extract of the subesophageal ganglion-cement gland complex (Fig. 11) will allow the admission of only fully developed coelomic oocytes into the nephromixia (Tweedell, 1980). These coelomic oocytes first enter the ciliated funnel (coelomostome) of the nephromixia (Fig. 4). Once resident in these chambers, the oocytes undergo germinal vesicle breakdown, proceed to the first meiotic metaphase and are subsequently released through lateral nephridiopores (Fig. 3) at the time of spawning. A similar movement of male sperm packets results in the release of free swimming spermatozoa The cerebral ganglia (primitive brain) will also yield an active factor that works best in the males. After injection there is a selective movement of only the fully developed primary oocytes into the nephromixia where they undergo GVBD. At this point arrested (metaphase I) oocytes can be removed from the nephromixia (see Appendix 4). Animals will respond to this procedure from early June until the time of spawning.
A. The application of metabolic precursors in vivo and in vitro.
The effect of metabolic precursors, radioisotopes, spawning hormones, etc. can be assessed by injecting them into adults. Fertilized eggs and developing embryos will also take up 3H-thymidine or 3HTTP in vitro (Appendix 5). The nuclei of cells pulsed at the 2 cell stage can be traced easily into all of the gastrula's blastomeres and beyond. This tagging procedure can be used in blastomere recombination experiments (Tweedell, unpublished results).
B. Vital dyes and fluorescent tagging of oocytes and early embryos.
Living eggs can be stained with vital dyes and vital fluorochromes before and after germinal vesicle breakdown or fertilization (Tweedell, 1962). One fluorochrome, acridine orange, has been used to follow development into the larval stage (Austin, 1963, Tweedell, l962). Either pre- or post-fertilized eggs are exposed to the fluorescent dye and monitored by a fluorescence microscope. [To avoid a phototoxic reaction, embryos should be kept in the dark.] Free swimming, rotating blastulae and gastrulae can occur in 100% of the exposed eggs, exhibiting inclusions with yellow, green and red-orange fluorescence. Rhodamine B is also useful for continuous observations. A variety of cytoplasmic and nuclear granules, inclusions and organelles can be stained in the living eggs and embryos (Appendix 6).
Certain vital dyes, including toluidine blue, Nile blue sulfate and crystal violet, are useful for tagging intranuclear and perinuclear granules, nucleoli, mitochondria, protein yolk, lipids, lipid granules and astral granules (Appendix 6).
C. Stratification of organelles by centrifugation
Cytoplasmic granules and inclusions, and cellular organelles (mitochondria, etc.) can be identified by centrifugation of vitally stained organisms. Stratification of the cellular components permits tentative identification before GVBD (Fig. 12) or after GVBD (Fig. 13). Additional specific cytochemical tests can be applied to eggs after fixation for further identification (see Appendix 6). A direct comparison of these inclusions can be made before and after GVBD. Once selected, the tagged components can be followed through embryonic development.
Intact oocytes, stages in GVBD, fertilization and cleavage can be recovered after shedding and prepared for light microscopy, fluorescence, autoradiography, or S.E.M. and autoradiography. Specimens are fixed directly on coverslips (Appendix 7).
B. Preparation of adults for in vivo oocyte studies.
Prior to cytological studies of cells developing in the coelomic fluid, it is necessary to remove sand grains from the digestive tract in preparation for embedding and sectioning. Once this is accomplished, the entire sequence of ooctyte formation, vitellogenesis and GVBD can be analyzed by light microscopy or autoradiography (Appendix 7).
I wish to thank John Valois, former Superintendent of Marine Resources at the Marine Biological Laboratory, for aid and help about collecting specimens and David Remsen of Information Systems at the MBL for assistance in programming. Original research support was from Biomedical Sciences Support from General Research Resources, N.I.H. and the Faculty Research Fund, University of Notre Dame.
Appendix 1
Distribution of Pectinaria near Woods Hole
Appendix 2
Local Pectinaria Collection Sites
Appendix 3
External Features of Adult Pectinaria
Appendix 4
Preparation of Meiosis Inducing Factor
Appendix 5
Injection of Isotopes or Maturation Factor Into Adults
Appendix 6
Identification of Egg Inclusions with Fluorochromes, Vital Dyes and Stratification
Appendix 7
Cytological Preparations
Appendix 8
Fixatives and Solutions
Fig. 1: Lateral view of adult Pectinaria alongside its protective test constructed from sand grains.
Fig. 2: Ventral view of living adult Pectinaria gouldii with gametes filling the coelom.
Fig. 3: Diagram of dorsal dissection of an adult female Pectinaria.
Fig. 4: An anterior diagrammatic view of a single nephromixium and attached ovary.
Fig. 5: Living coelomic oocytes freshly shed into seawater.
Fig. 6: Scanning electron micrograph of freshly shed coelomic oocytes prior to GVBD.
Fig. 7: Scanning electron micrograph of a mature spherical post-GVBD coelomic oocyte.
Fig. 8: Living sperm packets of Pectinaria.
Fig. 9: A rosette of sperm cells, as seen in scanning EM.
Fig. 10: Detail of spermatozoa from a rosette.
Fig.11: Diagram of dissection of the nervous-cement gland complex of Pectinaria gouldii.
Fig. 12: Diagram of a centrifuged immature oocyte, showing particle distribution in relation to the intact germinal vesicle.
Fig. 13: A composite diagram of the centrifuged oocyte of Pectinaria after GV breakdown.
Fig. 14: Location of a Pectinaria collection site at Barnstable Harbor (map).
Fig. 15: General photographic view of a Pectinaria collection site.
Fig. 16:. Close-up photographic view of a Pectinaria collection site.