Halisarca dujardini Johnston, 1842

Ereskovsky, Alexander V. & Gonobobleva, Elisaveta L., 2000, New data on embryonic development of Halisarca dujardini Johnston, 1842 (Demospongiae, Halisarcida), Zoosystema 22 (2), pp. 355-368 : 357-362

Halisarca dujardini Johnston, 1842

( Ceractinomorpha, Halisarcida ) is a dioecious sponge. Males begin to produce spermatocysts with spermatocytes in approximately mid-December at water temperature of about – 0.1 °C. Early oocytes differentiate in the last decade of December when water temperature fluctuates around – 0.6 °C. Vitellogenesis starts in May at nearly + 2 °C. Eggs maturate by the end of May. Cleavage and larval development occur from the second half of June until the end of July when water temperature averages 10-12 °C. The volume of reproductive elements (embryos and prelarvae) reaches its maximum by the end of June – beginning of July and reaches to about 69.5% of sponge volume (Ereskovsky 2000). It is the time when mesohyl destruction and complete disorder of central and basal choanosome, which are largely filled with developing larvae, is taking place. Normal tissue organization remains only in the thin periphery of a sponge. In the studied population, larval emergence is swift, lasting for one and a half to two weeks in the second half of July at about 12 °C. Subsequently slow development of the new generation of sponges and postreproductive revival of maternal specimens is going on until December. The general scheme of the H. dujardini life cycle in the White Sea is illustrated on Fig. 1 .

Embryogenesis, larval development and metamorphosis of the studied population of H. dujardini is continuing generally for four weeks from late June till end of July.

The oocyte development of this viviparous species occurs inside temporary embryonic capsules formed before maturation divisions of oocytes at the expense of dedifferentiated choanocytes ( Korotkova & Apalkova 1975). Every capsule has a double layer structure: the cells of the embryonic capsule stretching along the embryo surface and the layer of collagen filaments extending parallel to the cell surface on the outside. The thickness of fiber layer is 1 ± 0.5 µm. Distal parts of embryonic capsule cells overlay each other forming a continuous cell layer. No special contacts between individual cells have been recorded ( Sizova & Ereskovsky 1997).

Before cleavage cell division, a mature egg is a slightly oval body of 129 × 105 µm size. A spherical nucleus about 28 µm in diameter with a distinctly marked nucleolus (8 to 12 µm) is located in the centre of an egg. Vitelline granules are evenly spread over the whole volume of egg ( Fig. 5A ).

Eggs cleavage in H. dujardini is complete, equal and asynchronous. If the egg pole where maturation divisions are taking place is conventionally considered to be the animal one, the first cleavage division in H. dujardini occurs in the meridional plane. The size of the first two blastomeres is about 75.3 × 105.6 µm. The nucleus diameter is 16.4 µm. During all cleavage stages, the interphase blastomeres nuclei contain one to three pronucleolar bodies of 3 to 8 µm diameter at their periphery. As a rule these structures are of rounded shape. During the second cleavage division, the relative position of spindles varies from nearly parallel to perpendicular to one to another, while the relative position of blastomeres resembles a tetrahedron.

Similarly during the third cleavage division the spindle position within the embryo is also varying. As a result of asynchronous cleavage, embryos at the stage of five, six and seven cells might be found in one sponge. The octacellular embryo is rounded or slightly oval-shaped in the cross section. All the blastomeres are oval ( Fig. 2A ).

Subsequent cleavage continues to be asynchronous but cleavage spindles in this case are positioned in parallel to the embryo surface; as a result division planes become radial. A single-layered coeloblastula consisting of 18 to 24 cells surrounding a small cavity is thus formed. Polarity of the embryo is indistinct. Large nuclei with nucleoli are located in the central part of the cells. Rounded vitelline granules of heterogeneous content and ranging between 0.2 to 2.4 µm in diameter occupy most of the volume of blastomere cytoplasm. These structures are pyroninephilous, their dimensions slightly increase with the distance from the nucleus. Beginning after the fourth to fifth cleavage cycle, polarization of the blastomeres occurs with the movement of nuclei in apical direction and vitelline granules in the basal direction ( Fig. 5C ). Cleavage planes of polarized blastomeres are radially directed resulting in the formation of blastula of 130 to 155 µm diameter with a small cavity (nearly 30 µm in diameter) limited by wedge-form cells. At the stage of about 100 cells, the apical-basal polarity of blastomeres is clearly indicated ( Fig. 5D ). Such cells are about 70 µm in length and their apical breadth is nearly 25 µm; the rounded nucleus (about 9 µm in diameter) contains a nucleolus (about 2.5 µm). Sparse cisterns of Golgi apparatus and minute mitochondria are arranged around the nucleus.

At this stage some cells of a blastula migrate into the cavity. This process is apolar. Before the beginning of migration, the nucleus is shifted to the basal part of the cell which in this case begins to expand ( Fig. 5C ). The examination of serial sections has indicated that two to four cells migrate simultaneously. In the central cavity of the blastula those cells become spherical (about 27 µm in diameter), the nucleus with nucleolus occupying the central position ( Fig. 2B ). The inner cells of embryo do not form a dense mass. Subsequently they start dividing with the ensuing differentiation into amoebocytes. At the same time flagella are formed at the apical end of external cells.

Flagellate cells continue to proliferate actively. Before its division, the cell becomes rounded and migrates to the periphery of the body where it starts to divide. The division plane is perpendicular to the larval surface ( Figs 3A ; 5E, F ). After the division, the daughter cells elongate and embed themselves into the columnar epithelium. At that time the epithelial cells are about 35 µm long and 5.6 µm wide near the nucleus, which itself is about 4.3 µm in diameter. Differentiation of cells of the posterior pole is occurring simultaneously with differentiation of anterior-lateral cells ( Fig. 5E ). No division of internal cells has been recorded at this stage.

During the period of active proliferation of the external cells, the larval surface becomes corrugated. The internal part of larva is a cavity surrounded by basal parts of flagellate cells. At the same developmental stage, granular eosinophilic (fuchsinophilic) amoebocytes migrate from the maternal sponge mesohyl into the larva via the embryonic capsule ( Figs 3B ; 5F ).

Gradually the larval surface becomes flattened, proliferation is discontinued and the cells acquire the shape and size characteristic of a mature larva. Before the surface becomes finally smoothed, a deep and narrow invagination of the flagellate layer occurs, closing near its mouth ( Fig. 2C ). Invagination of this layer proceeds perpendicular or at an angle to the anterior-posterior axis of the larva. A single-layered closed structure surrounding a small cavity is formed by the internalized flagellate cells as a result of this process. The blastocoel is reduced, persisting in the form of narrow gaps between the internal and external flagellate cell layers.

The form and shape of the closed structures newly formed by flagellate cells inside the larva may be widely variable. These formations have mostly the form of C- or ∑- shaped, curved cylinders; sometimes they are oval or spherical; their cross-sections are tubular-shaped. The inner cavity diameter ranges between 8.5 µm and 12.5 µm while inner cavity length varies from 28 µm to 61 µm. One or two cells may be often found inside the cavity.This stage terminates the larval development. The walls of embryonic capsules which contain newly formed larvae fuse with the walls of exhalant channels and larvae escape through the osculum. Milk white-colored larvae of H. dujardini emerged from the maternal organism are oval or have the shape of a spheroid slightly depressed in the anterior-posterior direction with flat or concave posterior pole ( Figs 2D ; 5G ). They are completely covered with flagella, however in the posterior pole region, the flagella are sparse. The diameter of flagella-sparse cell layer is about 45 µm. Anterio-lateral larval flagellate cells are nearly cylindrical, 38 to 45 µm long and 2.6 to 3.0 µm wide in the nucleus region. The prolate nucleus (2.6 × 6.6 µm) with small nucleolus and the flagellum emerging from a pocketshaped cytoplasm depression are located in their apical part ( Fig. 3D ). The basal two thirds of these cells is filled with abundant vitelline granules. This cell layer lacks a basal membrane.

In longitudinal section the flagellate cells of the posterior pole are wedge-shaped; they are shorter and wider than the anterio-lateral cells (26 µm long and 6-7 µm wide in the nucleus region). Numerous vitelline granules are spread not only in basal and central parts of the cell, but near the nucleus as well. Rounded or drop-shaped nuclei are about 5.3 × 4.2 µm in diameter and contain large nucleoli (near 1.6 µm) ( Fig. 3C ). A ring of tapering flagellate cells is arranged between cells of the posterior pole and covering flagellate cells. The length of transitional cells fluctuates around 30 µm, the width in the nucleus region is about 4 µm; nuclei with nucleoli are oval-shaped (3.5 × 4.9 µm) ( Fig. 3C ). The number of cells in the transitional zone does not exceed four to five. There are no specialized contacts between the larval cells.

A most striking feature of the larval structure of H. dujardini is the presence of a large closed formation composed of flagellate cells (derivative of the prelarval flagellate layer) inside the larva. This structure is spherical and has a cavity delimited by the apical tips of the internalized flagellate cells. The external diameter (over basal cell apexes) of spherical layer ranges from 49 µm to 54 and the diameter of its cavity from 13.8 µm to 17.2 µm ( Fig. 2D ). The cells forming this structure are wedge-shaped with the extended basal part concentrating numerous vitelline granules. the flagellum, submerged into a pocket-shaped depression, and the nucleus are located near the narrow apical pole. Cell length varies from 13.8 µm to 17.2 µm, cells width from 3.4 µm to 5.7 µm. Drop-shaped or egg-form nuclei (2.3 µm × 5,8 µm) in these cell contain clearly distinguished nucleoli of about 0.9 µm diameter ( Fig. 4A ).

Before larval emergence out of maternal tissues, the interior chambers become spherical and their dimensions decrease. The process of cell ejection seems to be the cause of these changes. Numerous intermediate cells are located among internal cells in the space between the internal and external epithelia. They are oval-shaped, one pole has a flagellum, near which a rounded nucleus is located. The cytoplasm contains numerous vitelline granules ( Fig. 4B ).

Interspaces between outer and inner flagellate cells are occupied by the internal cells of the larva: nucleolate amoebocytes. In addition, granular eosinophilic cells of maternal origin are included into the larva. They may be located in different zones: apical, central, basal regions of flagellate layer, in the central part of larva, between the cells of the posterior pole.

Larvae have a spiral swimming movement counter-clockwise at the same time rotating themselves clockwise around the anterior-posterior axis. Sometimes they perform twirling movements. The free-living period of the larva lasts for 4 to 36 hours.

Larvae settle on a substrate with their anterior pole. However, even on a substrate, larvae continue to rotate around their anterior-posterior axis for 30 to 45 minutes before attachment. This rotation is finished after complete attachment.

Preparation for metamorphosis begins already during the swimming period as evidenced in larval behaviour and structure. Premetamorphic larvae are slower in their swimming, may attach themselves to surface film or sink and slowly move over the substrate. Whether attached to surface film or moving over the substrate, the larvae proceed slowly rotating.

Shape variation of the external flagellate cells and violation of their mutual contacts may primarily be observed on ultrastructural level. The lateral surfaces of cells become corrugated and the cells are contracted in the apical-basal direction. However, flagellate cells remain interconnected in their apical parts. In this state larvae settle down on substrate and attach to it.

During the first stages of settling and metamorphosis, the larva slightly flattens in the anterior-posterior direction; its anterior pole flattens on the substrate, while the posterior one remains rounded. The total disintegration of the internal spherical chamber and external flagellate layer takes place during the first 12 hours after the attachment.

Subsequent processes associated with metamorphosis will be described in a special study.

Figures

FIG. 1. — Life-history scheme of Halisarca dujardini in the White Sea.

FIG. 5. — Scheme of H. dujardini embryonic development; A, mature egg; B, early cleavage; C, beginning of blastomere polarization; D, multipolar immigration; E, stage larval polarization; F, formation of internal sphere and migration of maternal eosinophilic granular amoebocytes into larva; G, disphaerula, larva of H. dujardini. Abbreviations: bi, blastocoel; ec, embryonic capsule; efa, eosinophilic amoebocytes; ic, internal cells; isp, internal sphere; mc, migrated cells; ppc, posterior pole cells; tc, transition cells; yg, yolk granules.

FIG. 2. — A, cleaving embryos in 8-cell stage; B, blastula after immigration of external cells; C, stage of invagination of flagellate cells forming internal sphere; D, disphaerula of H. dujardini. Abbreviations: ec, embryonic capsule; ic, internal cells; n, nucleus; efc, external flagellate cells; b, blastomere; bi, blastocoel; ifc, internal flagellate cells; is, internal sphere; ppc, posterior pole cells; tc, transition cells. Scale bars: A, 20 µm; B, 15 µm; C, 20 µm; D, 10 µm.

FIG. 3. — A, division of external flagellate cells in prelarva; B, maternal eosinophilic granular amoebocyte between internal cells of disphaerula; C, posterior pole of larva; D, external flagellate epithelium of larva. Abbreviations: ba, bacteria; ic, internal cells; ec, embryonic capsule; ppc, posterior pole cells; tc, transition cells; dc, divided cell; ecc, embryonic capsule cell; f, flagellum; n, nucleus; yg, yolk granules. Scale bars: A, 5 µm; B, 5 µm; C, 10 µm; D, 5 µm.

FIG. 4. — A, internal flagellate cells of disphaerula; B, flagellated cell that migrated from internal sphere. Abbreviations: f, flagellum; isc, internal sphere cavity; n, nucleus; yg, yolk granules; ba, bacteria. Scale bars: A, 4 µm; B, 1 µm.