Collection of coral colonies

Branches were collected from colonies of M. digitata and P. cylindrica a few days before the full moon in May, June, and/or July from 1995 to 2000, from the reefs at Sesoko Island and Bise, northern Okinawa Island, Japan. The branches were maintained in an outdoor tank supplied with unfiltered running seawater.

Observation of early development

While the gonochoric colonies of P. cylindrica released individual eggs or sperm, the hermaphroditic colonies of M. digitata released egg–sperm bundles at night a few days after the full moon (Heyward et al., 1987). To collect gametes, the branches were placed separately into plastic containers before the expected spawning time, at about 1930 h (M. digitata) and 2230 h (P. cylindrica). Released gametes were collected by sucking up seawater from the container with a large plastic pipette. The gametes from each species were placed in separate plastic beakers, with gametes from two or three colonies mixed in a single beaker (100–300 ml suspension each). Filtered (0.45 μm) seawater (FSW) was added to the beaker to a final volume of 1 or 2 L. For M. digitata, the concentration of gametes was about 1 bundle/ml (sperm: 10⁻⁴– 10⁻⁵ cells/ml). Fertilized eggs were kept in FSW at room temperature (28–30°C). Eggs and embryos were sampled and observed under a light microscope at intervals of 30 min to 1 h, and photomicrographs were taken (Nikon Microphot; Nikon, Tokyo, Japan).

Histology and transmission electron microscopy

Eggs and embryos were put into a microtube and allowed to sink to the bottom. The supernatant was discarded, and the specimens were fixed for at least 2 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 3% NaCl. The specimens were rinsed three times in the same buffer and post-fixed in 1% osmium tetroxide in the same buffer for 1 h on ice. The specimens were then dehydrated in a graded series of acetone, immersed in n-butyl glycidyl ether (QY1), and embedded in Spurr's resin. For light microscopic observation, sections (0.5–1 μm thick) were stained with 1% methylene blue and 1% azur II in 1% borax. For electron microscopy, silver or gold sections were stained with uranyl acetate and lead citrate and observed under a JEM-2000EX electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV.

Early development of Porites cylindrica

Fifteen of 16 branches of P. cylindrica spawned gametes repeatedly for 2 to 6 days after the full moon in June 2000. The branches began releasing gametes 3 h after sunset (2230 h), and spawning persisted for 1 to 1.5 h. Eleven of these 15 branches were female. The female colonies released buoyant eggs, which were about 220 μm in diameter and contained about 400 zooxanthellae (Table 1). Zooxanthellae occupied about 2.6% of the egg volume. Male colonies released sperm at almost the same time.

Table 1.

Various parameters of spawned eggs of Pocillopora verrucosa, P. eydouxi, Porites cylindrica and Montipora digitata in 1998–2000. Means±SD.

Zooxanthellae were located evenly in spawned eggs of P. cylindrica (Fig. 1A). Cleavage occurred at intervals of 30 to 40 min. The zooxanthellae in the embryo were delivered almost equally to the blastomeres (Fig. 1B). At the morula stage, each daughter cell contained only a few zooxanthellae (Fig. 1C). At 6 h after fertilization, a hollow blastula was formed (Fig. 1D). During gastrulation, blastomeres possessing zooxanthellae accumulated around the blastocoel, and the surface layer of the embryo was occupied by relatively large, columnar cells without zooxanthellae (Fig. 1E, F). At about 10 h after fertilization, a stereogastrula was formed (Fig. 1G). Blastomeres containing zooxanthellae and/or lipid droplets filled the inner part of the embryo and the endoderm, and the ectoderm without zooxanthellae appeared to differentiate, although the mesoglea was not clearly seen (Fig. 1H). A mouth opening was observed as an invagination of the ectoderm (Fig. 1H). Ciliated larvae started to swim 24 h after fertilization. At this stage, the ectoderm and endoderm were demarcated by the mesoglea, and zooxanthellae were located in the endoderm (Fig. 1I). A gastrovascular cavity was formed.

Fig. 1.

Early development of Porites cylindrica. (A) Spawned egg. Zooxanthellae are seen scattered throughout the cytoplasm. (B) Four-cell stage. The blastomeres contain an almost equal number of zooxanthellae (arrowheads). (C) Blastula. Each daughter cell contains a few zooxanthellae. (D) Section of an early blastula. A blastocoel has formed. (E) Section of a blastula showing multiple ingression of blastomeres containing zooxanthellae (arrowheads). (F) Section of part of a blastula during gastrulation. Blastomeres containing zooxanthellae (arrows) are in the process of ingression. (G) Gastrula. (H) Section of a gastrula. Blastomeres containing zooxanthellae (arrowheads) and those containing lipid droplets fill the inner space of the gastrula, forming a stereogastrula. (I) Section of a planula. Zooxanthellae (arrowheads) and lipid droplets are in the endodermal cells. Arrow indicates the mesoglea. bc, blastocoel; ec, ectoderm; en, endoderm; gv, gastrovascular cavity. Bars represent 100 μm in (A), (B), (C), and (G); 50 μm in (D), (E), (H), and (I); and 10 μm in (F).

Early development of Montipora digitata

Seven of 29 branches of M. digitata spawned egg–sperm bundles on the third to fifth night after the full moon during May–June 1999, and 11 of 40 branches spawned for 7 days after the full moon in June–July 1999. In 2000, all 12 branches collected spawned gametes for 10 days, beginning 1 day before the full moon in June. The spawning started about 1 h after sunset (between 2000 and 2100 h) and persisted for 1 to 1.5 h. Each egg–sperm bundle consisted of 10 to 15 eggs and sperm. The eggs were about 380 μm in diameter and contained about 1,400 zooxanthellae (Table 1), which occupied about 2.0% of the egg volume.

Zooxanthellae were distributed more or less homogeneously in the cytoplasm of M. digitata eggs (Fig. 2A). The first cleavage began about 2.5 h after fertilization. From the early cleavage stage to the morula stage, each blastomere contained an almost equal number of zooxanthellae (Fig. 2B, C). A hollow blastula was formed, although the blastocoel was filled with some acellular material (Fig. 2D). The blastula was composed of columnar blastomeres, and zooxanthellae were located in the basal region of the columnar cells. Gastrulation occurred by invagination, and one side of the embryo became concave (Fig. 2E, F). Two types of blastomeres (Fig. 2F) were observed, one containing a few large lipid droplets (arrows) and another containing many small lipid droplets (small arrows). The blastomeres of the latter type were aggregated on one side of the blastopore lip. At a later stage, the blastocoel disappeared, and the ectoderm and endoderm could be discerned (Fig. 2G). The ectoderm consisted of columnar cells, and the endoderm comprised cells containing large lipid droplets. Zooxanthellae were observed in both the ectoderm and endoderm. The ectoderm layer appeared to envelop the endodermal cells (Fig. 2G, H). The embryos then became spherical in shape again and formed a stereogastrula (Fig. 2I). Zooxanthellae were present in both the ectoderm and the endoderm, even in the late gastrula or early planula (Figs. 2J, 3A). Most of the zooxanthellae in the ectoderm were located in the basal region of the ectodermal cells (Fig. 3B). The mesoglea was very thin (about 0.3 μm) and was obscured or lost in some places (Fig. 3C, D). In these cases, cellular processes of ectodermal cells had entered the endoderm through a gap in the mesoglea (Fig. 3C), or zooxanthellae were located very close to the obscured mesoglea (Fig. 3D). As the planulae matured, zooxanthellae disappeared from the ectoderm, and were then seen only in the endoderm of mature planulae (Fig. 3K). Planulae settled and metamorphosed 3–4 days after fertilization. At about 6 days after settlement, the polyps began secreting a skeleton, and tentacles were discernible (Fig. 2L).

Fig. 2.

Early development of Montipora digitata. (A) Spawned egg. Zooxanthellae are distributed evenly in the egg. (B) Four-cell stage. The blastomeres contain an almost equal number of zooxanthellae. (C) The 64-cell stage. Zooxanthellae accompany each daughter cell. (D) Section of a blastula. The blastula is composed of large, columnar blastomeres, and zooxanthellae (arrowheads) are located in the basal region of the columnar cells. The central region of the blastula is filled with acellular material. Inset: Section of an early blastula at higher magnification. The cells contain a few zooxanthellae (z) and many lipid droplets. Bar=50 μm. (E) Early gastrula. One side of the embryo is concave. (F) Section of an early gastrula. Two types of blastomeres are seen, those containing small lipid droplets (smaller arrows) and those containing a few large droplets (larger arrows). Zooxanthellae (arrowheads) are present within some blastomeres. (G) Section of a gastrula. The ectoderm and endoderm are separated by mesoglea (arrow). The endoderm is discernible as an inner cell mass containing large lipid droplets. Zooxanthellae (arrowheads) are seen in both the ectoderm and endoderm. (H) Enlarged view of the ectodermal margin of the gastrula. The ectodermal layer appears to envelop the endoderm. (I) Late-stage gastrula. (J) Section of a late gastrula or early planula. Zooxanthellae (arrowheads) are distributed in both the ectoderm and endoderm. Arrow indicates the mesoglea. (K) Mature planula. The planula is completely ciliated at this stage. The inside of the planula appears dark because of the accumulation of zooxanthella-containing cells. (L) Primary polyp with tentacles. bp, blastopore; en, endoderm; ec, ectoderm; mo, mouth; te, tentacle; z, zooxanthella. Bars represent 100 μm except in (H), where bar represents 20 μm.

Fig. 3.

Transmission electron micrographs of early planulae in Montipora digitata. (A) Zooxanthellae residing in the ectoderm and endoderm of an immature planula. Arrows indicate the mesoglea. (B) Zooxanthella in the cellular process of an ectoderm cell. The zooxanthellae are located at the basal end of the cell. (C) Zooxanthella in an ectoderm cell near the mesogleal layer. A cellular process (arrowheads) of an ectoderm cell has entered the endoderm through a gap in the mesogleal layer (arrows). (D) Zooxanthella in the endoderm near the mesogleal layer. The mesogleal layer (arrows) near the zooxanthella is obscured. en, endoderm; ec, ectoderm; li, lipid droplet; zo, zooxanthella. Bars represent 5 μm in (A), (B), and (D) and 2 μm in (C).

Early cleavage and distribution of zooxanthellae

In P. verrucosa and P. eydouxi, zooxanthellae moved toward the animal pole 1–2 days before spawning and became concentrated in one hemisphere of the oocytes (Hirose et al., 2001). The first cleavage apportioned zooxanthellae more or less equally between the first two blastomeres. At the second cleavage, however, two of the four blastomeres received almost all of the zooxanthellae, and the other two had few or none. This uneven distribution of zooxanthellae persisted until the zygotes developed into gastrulae (Hirose et al., 2000). In contrast to the two species of Pocillopora, P. cylindrica and M. digitata exhibited an even distribution of zooxanthellae in their eggs, and the zooxanthellae were delivered almost equally to the blastomeres until blastula formation.

Blastoderm differentiation in the two species

In both P. cylindrica and M. digitata, fertilized eggs developed into a hollow blastula, as occurs in the two species of Pocillopora (Hirose et al., 2000). In P. cylindrica, when the blastocoel disappeared and an acoel embryo was formed (Fig. 1G, H), the ectoderm and endoderm appeared to be at least partially differentiated, although they were not clearly demarcated by a mesogleal layer. The embryo at this stage might be considered a gastrula rather than a blastula. A mouth later formed by invagination of the ectoderm, and the ectoderm and endoderm became separated by a mesogleal layer. This process is considered to be planula development rather than gastrulation (Harrison and Wallace, 1990).

In M. digitata, the blastocoel was filled with some acellular material, which probably consisted of lipid droplets that had been expelled from blastomeres and accumulated in the blastocoel. This material was likely incorporated later by endodermal cells in the course of invaginating gastrulation. When the blastocoel disappeared upon invagination, the ectoderm and endoderm differentiated, and the endodermal cells became filled with large lipid droplets.

Gastrulation and zooxanthella relocation

In P. cylindrica as well as the two species of Pocillopora, blastomeres containing zooxanthellae and/or lipid droplets detached from the outer layer and dropped into the blastocoel until it was completely filled, resulting in a stereogastrula. In these corals, zooxanthella localization to the endoderm occurred during gastrulation (Fig. 4). Gastrulation was by multipolar ingression or delamination rather than invagination. Gastrulation through delamination has been suggested in Astrangia danae (Szmant-Froelich et al., 1980), Favia fragum (Szmant-Froelich et al., 1985), and M. verrucosa (Maté et al., 1998).

Fig. 4.

A schematic illustration showing the timing and mechanism of zooxanthella localization. Developmental stages are indicated on the left. Boxes indicate the first stage during which zooxanthellae are localized in the oocyte or embryo. The timing of zooxanthella localization and type of gastrulation differ among the three genera.

In M. digitata, gastrulation appeared to occur via the combined processes of invagination and epiboly. Zooxanthellae were present in both the endoderm and ectoderm of the late gastrula or early planula. Some presumptive endoderm cells may remain in the ectoderm of M. digitata planulae, or the specificity of zooxanthellae toward presumptive endoderm cells may be low or develops at a later stage in M. digitata compared to P. cylindrica and the two Pocillopora species. The zooxanthellae gradually disappeared from the ectoderm as the planulae matured.

The timing of zooxanthella localization differed among P. cylindrica, M. digitata, and the two species of Pocillopora (Fig. 4). This difference in timing may be related to the mode of gastrulation. However, in the temperate sea anemone Anthopleura ballii, which produces zooxanthellate eggs, zooxanthellae are localized at one end of the blastula, and the blastomeres containing zooxanthellae invaginate during gastrulation, restricting the zooxanthellae to the endoderm of planulae (Davy and Turner, 2003). The uneven distribution of zooxanthellae in embryos could indicate an earlier determination of presumptive endodermal cells. Zooxanthellae might be delivered more or less exclusively to presumptive endodermal cells during early developmental stages in the two Pocillopora species and the sea anemone A. ballii. It is likely that zooxanthella localization in embryos is related to the timing of the determination of presumptive endodermal cells, with the localization of zooxanthellae to the endoderm occurring no earlier than gastrulation.

Possible mechanisms of zooxanthella translocation from ectoderm to endoderm

Zooxanthellae have been observed in the ectoderm of the early planulae of some corals (F. fragum: Szmant-Froelich et al., 1985; Fungia scutaria: Schwarz et al., 1999), soft corals (Xenia umbellata: Benayahu et al., 1988; Litophyton arboreum: Benayahu et al., 1992, Benayahu, 1997; A. glauca: Benayahu and Schleyer, 1998), and the scyphozoan L. unguiculata (Montgomery and Kremer, 1995). In these cnidarians, zooxanthellae generally reside only in the endoderm in later stages of the life cycle; thus, zooxanthellae in the ectoderm might be translocated to the endoderm or removed from the ectoderm. Several mechanisms have been suggested for the translocation of zooxanthellae from the ectoderm to the endoderm (Montgomery and Kremer, 1995; Benayahu, 1997; Benayahu and Schleyer, 1998). In the jellyfish L. unguiculata, ectodermal cells infected by zooxanthellae can migrate to the endoderm of planulae (Montgomery and Kremer, 1995). In the soft coral A. glauca, zooxanthellae within a vacuole in the detached ectodermal cytoplasm pass through temporarily opened gaps in the mesoglea toward the endoderm (Benayahu, 1997; Benayahu and Schleyer, 1998). In M. digitata, we could not observe zooxanthellae in the process of entering the endoderm through a gap in the mesoglea. Some zooxanthellae in the endoderm, however, were located just below the mesogleal layer, which was more or less obscured, and appeared to have been translocated from the ectoderm (Fig. 3C). Most zooxanthellae in the ectoderm were contained in cellular processes and were located close to the mesogleal layer. It is not clear whether a whole zooxanthella-containing cell migrates to the endoderm, or whether only the zooxanthellae, together with small amounts of cytoplasm, move to the endoderm, as suggested in some soft corals (Benayahu, 1997; Benayahu and Schleyer, 1998).

We observed no degraded zooxanthella particles in the surface layer of embryos or early planulae of M. digitata. In contrast, Davy and Turner (2003) only occasionally found zooxanthellae in the ectoderm of the late gastrula or early planula and suggested that these stray zooxanthellae may degrade in the host or be expelled. It has been proposed that degraded zooxanthella particles are produced by partial digestion of zooxanthellae by the host (Titlyanov et al., 1996, 1998; Mise and Hidaka, 2005). Few zooxanthellae were observed at the bottom of beakers incubating embryos of M. digitata. The absence or scarcity of degraded and expelled zooxanthellae may indicate that those in the ectodermal layer of planulae are transferred to the endoderm during the maturation of planulae.

Vertical transmission via zooxanthellate eggs

The three genera Porites, Montipora, and Pocillopora, which contain species that spawn zooxanthellate eggs, belong to different families and occur in different lineages in phylogenetic trees of scleractinian corals (Veron, 1995; Romano and Palumbi, 1996, 1997; Romano and Cairns, 2000; Fukami et al., 2000; Kerr, 2005). Furthermore, the same family can contain some species that release zooxanthella-containing eggs and others that produce zooxanthella-free eggs. For example, most Montipora species produce eggs containing zooxanthellae, whereas Acropora species, which belong to the same family, release eggs without zooxanthellae (Fadlallah, 1983). This together with the present finding that the timing of zooxanthella localization and types of gastrulation were different among the three genera (Pocillopora, Porites, and Montipora) suggests that vertical transmission of symbionts via zooxanthellate eggs evolved independently in the three genera studied.