Embryonic and Larval Development of the Host Sea Anemones Entacmaea quadricolor and Heteractis crispa

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1 Reference: Biol. Bull. 213: (October 2007) 2007 Marine Biological Laboratory Embryonic and Larval Development of the Host Sea Anemones Entacmaea quadricolor and Heteractis crispa ANNA SCOTT* AND PETER L. HARRISON National Marine Science Centre, PO Box J321, Coffs Harbour, NSW 2450, Australia; and Coral Reef Research Centre, School of Environmental Science and Management, Southern Cross University, Lismore, NSW 2480, Australia Abstract. Little information is available on the sexual reproductive biology of anemones that provide essential habitat for anemonefish. Here we provide the first information on the surface ultrastructural and morphological changes during development of the embryos and planula larvae of Entacmaea quadricolor and Heteractis crispa, using light and scanning electron microscopy. Newly spawned eggs of E. quadricolor and H. crispa averaged 794 m and 589 m diameter, respectively, and were covered by many spires of microvilli that were evenly distributed over the egg surface, except for a single bare patch. Eggs of both species contained abundant zooxanthellae when spawned, indicating vertical transmission of symbionts. Fertilization was external, and the resulting embryos displayed superficial cleavage. As development continued, individual blastomeres became readily distinguishable and a round-toovoid blastula was formed, which flattened with further divisions. The edges of the blastula thickened, creating a concave-convex dish-shaped gastrula. The outer margins of the gastrula appeared to roll inward, leading to the formation of an oral pore and a ciliated planula larva. Larval motility and directional movement were first observed 36 h after spawning. E. quadricolor larval survival remained high during the first 4 d after spawning, then decreased rapidly. Introduction Received 21 October 2006; accepted 9 May * To whom correspondence should be addressed. In cnidarians, fertilization may take place within the parent (i.e., brooding) or externally in the water column after the gametes are shed (i.e., broadcast spawning) (Campbell, 1974; Harrison and Jamieson, 1999; Fautin, 2002). Species that broadcast-spawn their gametes provide a ready source of embryos for studies of early development, whereas for brooders the detection of early developmental stages is complicated by the retention of embryos within the parent (Dahan and Benayahu, 1998). Embryonic and larval development in the Actiniaria are diverse, often being influenced by the yolk content of the eggs, which varies among species (Uchida and Yamada, 1968; Mergner, 1971; Campbell, 1974; Spaulding, 1974; Conn, 1991). Species with small eggs and little or no yolk usually have complete and equal cleavage divisions. A coeloblastula is usually formed with the blastomeres comprising a single layered epithelium enclosing a large blastocoele (Nyholm, 1949; Mergner, 1971; Campbell, 1974; Spaulding, 1974; Fautin et al., 1989). Species with large eggs and substantial yolk typically have superficial cleavage that may be complete, equal, unequal, or meroblastic (Gemmill, 1920; Nyholm, 1949; Uchida and Yamada, 1968; Mergner, 1971; Chia and Spaulding, 1972; Campbell, 1974; Spaulding, 1974; Fautin et al., 1989). Superficial cleavage occurs in many actiniarian species including Actinia fragacea, Cribrinopsis fernaldi, Halcampa duodecimcirrata, and Actinostola spetsbergensis (Nyholm, 1949; Uchida and Yamada, 1968; Siebert and Spaulding, 1976; Riemann- Zürneck, 1976; Larkman and Carter, 1984). The large amount of yolk contained within the eggs often results in the formation of a stereoblastula that is solidly packed with cells (Uchida and Yamada, 1968). Gastrulation mechanisms vary widely between species and may involve unipolar or multipolar ingression, multipolar or secondary delamination, invagination, epiboly, or a combination of these processes (Cary, 1910; Uchida and Yamada, 1968; Fautin et al., 1989; Conn, 1991). Regardless 110

2 EMBRYOGENESIS OF HOST SEA ANEMONES 111 of the mechanism, gastrulation results in the formation of two germ layers, the ectoderm and endoderm (Ball et al., 2002). When formation of the endoderm is nearly complete, the round gastrula elongates to form a ciliated free-swimming planula larva (Nyholm, 1949; Mergner, 1971; Campbell, 1974). Larvae are generally planktonic and serve as a means for dispersal prior to settlement (Chia, 1974; Harrison and Wallace, 1990; Martin and Koss, 2002; Harrison and Booth, 2007). Although embryonic development has been studied for a number of actiniarian species, some major gaps exist. This is particularly apparent for the 10 species of anemone that are known to provide essential habitat for obligate symbiotic anemonefish. Despite their ecological importance, no published scientific information on embryogenesis is currently available for these host sea anemone species. Accordingly, this paper describes the surface ultrastructural and morphological changes during the development of embryos of Entacmaea quadricolor (Rüppell and Leuckart, 1828) and Heteractis crispa (Ehrenberg, 1834), from early cleavage to planula larvae. The swimming speed and survival rates of E. quadricolor planulae are also quantified. Materials and Methods Thirty to forty individuals of Entacmaea quadricolor and Heteractis crispa were collected from the subtropical rocky reefs at North Solitary Island, Solitary Islands Marine Park, Australia (29 55 S, E) at 9 18 m depth. Species were maintained and monitored in separate flow-through outdoor seawater tanks at the National Marine Science Centre, Coffs Harbour. Eggs and developing embryos were cultured during the summer and autumn broadcast-spawning periods of E. quadricolor and H. crispa from 2003 to 2005 (Scott and Harrison, 2005; Scott and Harrison, in press). This allowed embryonic and larval development to be examined using a combination of light microscopy and scanning electron microscopy (SEM). Culture of embryos and larvae After the anemones had spawned, the gametes of both species were collected by using 250- m plankton mesh sieves. Eggs and sperm were placed into 60-l plastic tubs containing seawater; the number of eggs was adjusted to ensure development of healthy embryos and larvae, and excess sperm were flushed from the culture (after Harrison, 2006). For the first week, 10% 20% of the seawater was changed once or twice a day; subsequently this was done every 2 to 3 days. The tubs were continuously aerated with a slow stream of bubbles from a Pasteur pipette located in each corner. This maintained dissolved oxygen levels, provided water circulation, and ensured that the water quality remained suitable for development of the planulae (after Harrison, 2006). Tubs were placed indoors with a 12-h light/dark cycle. Seawater temperature ranged from 23.5 to 24.5 C. Analysis of embryonic stages and early planulae Samples of eggs, embryos, or planulae were collected at regular time intervals to study the development of E. quadricolor and H. crispa from early cleavage embryos to mature planulae. During the initial stages of development, samples were taken every hour for the first 8hand then at 10, 12, 15, 18, and 24 h after spawning. After this time, samples were taken once or twice a day until the larvae were 7 d old. E. quadricolor samples were collected after spawning on 21 March 2003 and 25 February 2005, and H. crispa samples after spawning on 9 March Live embryos and larvae were examined and photographed using a Leica S6D dissecting microscope equipped with a Nikon Coolpix 4500 digital camera prior to fixation with Bouin s fixative or 10% formalin in seawater. SEM was used to further elucidate the ultrastructural changes associated with embryo development. Scanning electron microscopy For each time-interval sample, about 40 embryos were selected and dehydrated through a graded ethanol series (25%, 50%, 75%, and 95% ethanol for 20 min each and then three changes of 100% ethanol for 30 min each). Specimens were placed in labeled 120- m polyporous pots and criticalpoint-dried with a Polaron 3100 critical point dryer using liquid CO 2. The chamber was chilled to 15 C and left for 30 min before the temperature was gradually raised to 36 C and 1200 psi. H. crispa embryos were mounted onto SEM stubs with double-sided sticky tape and sputter-coated with gold with a Balzers SCD 050 sputter-coater for 60 s (working distance 60 mm, current 30 ma). Samples were examined and photographed with a Leo 440 Stereoscan scanning electron microscope at Southern Cross University; a tungsten filament was used with the probe set at pa, EHT (accelerating voltage) at 10 kv, and a working distance of 16 to 25 mm. Due to problems encountered with the buildup of highvoltage charges and subsequent flaring of the early developmental stages in H. crispa, an alternative method of mounting and coating was employed for E. quadricolor. Specimens were mounted with double-sided carbon tape, sputter-coated with carbon thread at a working distance of 75 mm, and then sputter-coated with gold for 60 s (working distance 85 mm, current 30 ma), which reduced flaring. SEM settings were the same as those used for H. crispa. Development Larval motility. In 2005, experiments were done to determine the motility and swimming speed of E. quadricolor

3 112 A. SCOTT AND P. L. HARRISON planulae. Methods used were based on those developed by Reichelt-Brushett and Harrison (2004). In the laboratory, 20 larvae were placed in three replicate petri dishes (7.5-cm diameter) containing 10 ml of seawater (mean seawater temperature 23.8 C). Larvae were allowed to acclimatize for about 30 min. Plastic transparency film was placed over the dishes, and three moving larva from each replicate were haphazardly selected. The path traveled by each larva during a 30-s time period was traced onto the transparency film, using a fine-tipped marker pen, at 1, 1.5, 2, 7, and 14 d after spawning. Distance traveled was calculated using Image- Pro Discovery image analysis software, ver The distances recorded for each larva measured were averaged to determine the mean swimming speed of the larvae. Larval survival. The survival rates of E. quadricolor larvae were determined using methods based on those of Harrison (2006). Larvae used for the experiment were obtained from the spawning that occurred on 1 February Two days after spawning, five groups of 500 larvae were counted using a tally counter and a Bogorov plankton tray viewed under a dissecting microscope and illuminated with a fiberoptic light source. Each group of larvae was placed into one of five replicate 2-l glass jars, each containing 1.5 l of seawater. The jars were placed in a water bath with flowthrough seawater to maintain ambient water temperature, covered with clear plastic lids to prevent evaporation, aerated to maintain dissolved oxygen levels and provide circulation, and located outdoors to ensure natural lighting and photoperiod. The number of free-swimming larvae surviving in each jar was counted at set monitoring intervals to determine larval survival rates over time. At each monitoring interval, the water within each jar was changed to maintain suitable water quality. Results Embryogenesis Spawned eggs of Entacmaea quadricolor and Heteractis crispa were green to brown, with color varying slightly between females (Fig. 1). The spawned eggs of both species were predominately positively buoyant, but some were neutrally or negatively buoyant. E. quadricolor eggs were roughly spherical and had a mean diameter of 794 m (SE 6.0, n 20) when examined live with light microscopy; the diameter reduced to 609 m (SE 4.2, n 10) after the eggs were fixed and processed for SEM. H. crispa eggs were smaller than E. quadricolor eggs, averaging 589 m in diameter (SE 7.2, n 20) when live and 460 m in diameter (SE 4.1, n 10) after SEM processing. All other size measurements presented in this paper were determined using SEM. Eggs contained abundant zooxanthellae when spawned (Fig. 1). Algal cells also resided on the surface of some eggs Figure 1. Light micrograph of newly spawned Entacmaea quadricolor eggs showing zooxanthellae. Scale bar: 500 m. (Fig. 2a). Numerous spires of microvilli were evenly distributed over the egg surface, except for a single bare patch (Figs. 2b, 3a, 4a). Spacing between spires varied between eggs. Spires were closely spaced with the bases almost touching, or more widely spaced and interspersed with short microvilli. Microvilli spires on E. quadricolor eggs were wider and shorter when compared to those covering the surface of H. crispa eggs. E. quadricolor spires averaged 8.7 m (SE 0.2) in length and 14.8 m (SE 0.4) in width, whereas H. crispa spires averaged 12.9 m (SE 0.5) in length and 9.3 m (SE 0.3) in width (n 5, with 4 spires per egg measured for both species). The bare patch was elliptical, measuring 52.5 by 81.9 m (SE 7.6, 10.4) for E. quadricolor, and 36.9 by 55.6 m (SE 8.3, 10.0) for H. crispa (n 5). E. quadricolor sperm were found singly or in clusters on some of the eggs and embryos (Fig. 5). These sperm possessed an ovoid head about 2 m in length, and a flagellum of about 30 m. H. crispa sperm were not observed under SEM, but observations via light microscopy revealed that most sperm were inactive when spawned, with the exception of some movement of the sperm head. Most sperm started to swim about 45 min after spawning. Fertilization was external for E. quadricolor and H. crispa. The resulting embryos displayed superficial cleavage. During early cleavage stages, the embryo changed shape from spherical to having slight lumps or furrows; however, no cell separation was evident on the surface (Figs. 3b, 4b). Individual blastomeres started to become visible about 4 5 h after spawning, tending to become visible on one side of the embryo before the other (Figs. 3c, 4c). Cytoplasmic separation among the blastomeres was still incomplete, and the blastomeres first became readily distinguishable at the 64-cell stage for E. quadricolor (Fig. 3d) and the 32-cell stage for H. crispa (Fig. 4d). Blastomeres were usually of equal size (Figs. 3d, 4e); how-

4 EMBRYOGENESIS OF HOST SEA ANEMONES 113 Figure 2. Surface detail of a newly spawned Heteractis crispa egg: (a) numerous algal cells residing on the surface; (b) bare patch. Scale bars: a, 15 m; b, 25 m. ever, divisions were not always synchronous, resulting in blastulae with irregular numbers of cells (Figs. 3e, 4f). The spires of microvilli began to unravel, and became less regularly arranged on the embryo surface at this stage (Figs. 6, 7). Initially, the blastula was round to ovoid (6 7 h after spawning), remaining at about the same size as the undivided egg. As divisions proceeded, the blastula started to flatten, becoming disc-shaped about 7 8 h after spawning and much wider when compared to the original diameter of the egg (E. quadricolor 34% wider; H. crispa 29% wider) (Figs. 3f, 4g, h). The edges of the embryo then started to thicken, moving inward and upward, creating a concaveconvex dish-shaped gastrula (10 12 h after spawning) (Figs. 3g, h, 4i k). With this development the circumference decreased, the embryo became more spherical, and cell division led to a smooth surface. Short microvilli covered the surface of the late gastrula prior to the development of a ciliated planula larva (Figs. 3i, 4l, 6d, 7d). E. quadricolor and H. crispa embryos first developed into early planulae about 14 h and 22 h after spawning, respectively. The surface of E. quadricolor planulae had small round porelike structures that ranged from 1 to 3 m in diameter and were randomly distributed amongst the cilia (Figs. 6d, 8). Pore-like structures were not noted on the surface of H. crispa planulae. Larval development. The early larval stages of E. quadricolor and H. crispa were round to pear-shaped. These larvae were similar in color to the newly spawned eggs, being green to brown due to the presence of zooxanthellae inherited from the female parent. As development proceeded the larvae elongated along the oral-aboral axis. Although most commonly elongate, larvae were able to change shape quite quickly and showed considerable variation in form, with barrel- and pear-shaped forms also being observed. Elongate E. quadricolor larvae measured 1120 m by675 m (SE 42.9, 27.4 respectively), whereas H. crispa larvae were slightly smaller, measuring 740 mby590 m (SE 32.2, 47.8 respectively). Color changed with larval age, from the original green-brown to orange-brown in E. quadricolor, and to lighter green in H. crispa. Initially, the larvae were concentrated near the water surface due to their positive buoyancy and lack of motility. However, with increasing age they became more evenly distributed throughout the water column. Larvae were observed to occasionally release mucus-like material from the oral pore. An apical tuft of cilia was not visible at any stage of larval development. Larval motility. The first sign of larval motility in E. quadricolor was slow spinning due to uncoordinated ciliary beats. As the ciliary beat became coordinated, the planulae became mobile, with directional movement being recorded 36 h after spawning (Fig. 9). Locomotion occurred with the aboral pole pointing forward, and with clockwise rotation around the longitudinal axis when viewed from the oral pole. The direction of rotation occasionally varied. As the larvae grew older, rotation around the longitudinal axis was replaced by directional movement without spinning, and swimming speed greatly increased. The mean distance traveled in 30 s by 1-week-old E. quadricolor planulae was 4.65 cm (SE 0.9, n 9), increasing to 6.38 cm in 30 s (SE 0.8, n 9) 2 weeks after spawning (Fig. 9). Larvae generally swam forward continuously, although short bursts of acceleration or stationary periods that included temporary attachment to the substratum by the aboral end were also common. Development of motility and the method of locomotion in H. crispa larvae were the same as those described for E. quadricolor. Larval survival. Survival of E. quadricolor planulae was initially high, with 91.3% of the larvae remaining at 4 d of age (Fig. 10). This was followed by a rapid decrease in

5 114 A. SCOTT AND P. L. HARRISON Figure 3. Scanning electron micrograph sequence of the embryonic development of Entacmaea quadricolor: (a) newly spawned egg showing spires of microvilli; (b) embryo undergoing superficial cleavage; (c) individual blastomeres becoming visible on the embryo surface; (d) blastula with 64 cells; (e) late blastula; (f) early gastrula; (g h) basin-shaped gastrula; (i) ciliated planula larva with oral pore clearly visible, 14 h after spawning. Scale bars: 150 m. percentage survival. Mortality was about 50% by the time the larvae were 7 d old, and 21.9% of those that remained had started to metamorphose in the water column. By the time the planulae were 14 d old, only a few swimming larvae remained. Numerous larvae (estimated at more than 50 in some replicates) had metamorphosed into juvenile anemones that were found strongly attached to the bottom and sides of the jars. Formal monitoring of the experiment ceased at this point; however, when the larvae were 59 d old, a few elongate free-swimming larvae still remained in some of the replicate jars. Discussion Embryonic and larval development in the order Actiniaria are very diverse (Spaulding, 1974). Many workers have studied embryogenesis in various species of sea anemone (e.g., Cary, 1910; Gemmill, 1920, 1921; Nyholm, 1943; Chia and Spaulding, 1972; Siebert, 1973, Siebert, 1974; Spaulding, 1974; Schäfer, 1985; Fukui, 1991; Davy and Turner, 2003; Kraus and Technau, 2006). Despite this research, our understanding of embryogenesis for many species of sea anemone is still incomplete. A major reason for this lack of information is the difficulty in predicting and controlling spawning (Spaulding, 1974). The discovery of annual spawning periods of Entacmaea quadricolor and Heteractis crispa by Scott and Harrison (2005) at the Solitary Islands Marine Park provided access to large numbers of spawned gametes. Here we present the first account of the embryonic and larval development of these ecologically important species of host sea anemone. E. quadricolor and H. crispa broadcast-spawn eggs and sperm into the water column for external fertilization (Scott and Harrison, 2005, Scott and Harrison, in press), which is considered the ancestral pattern for the phylum Cnidaria (Fau-

6 EMBRYOGENESIS OF HOST SEA ANEMONES 115 Figure 4. Scanning electron micrograph sequence of the embryonic development of Heteractis crispa: (a) newly spawned egg showing spires of microvilli; (b) embryo undergoing superficial cleavage; (c) individual blastomeres becoming visible on the embryo surface; (d) 32-cell stage, blastomeres becoming more defined; (e) early blastula with 32 cells, individual blastomeres; (f) blastula with increased number of cells, separated and clearly defined; (g) 64-cell stage; (h) disc-shaped early gastrula with 256 cells; (i k) basin-shaped gastrula; (l) ciliated planula larva with oral pore clearly visible, 48 h after spawning. Scale bars: 150 m. tin et al., 1989). The size of the newly spawned E. quadricolor and H. crispa eggs is similar to that of Tealia crassicornis (see Chia and Spaulding, 1972) and falls within the 110- to m size range previously recorded for other actiniarian eggs (Gemmill, 1921; Spaulding, 1972; Clark and Dewel, 1974). The green-to-brown color of E. quadricolor and H. crispa eggs varies slightly between individuals. Gemmill (1920) also noted variation in the color of eggs released by different Adamsia palliata individuals. Egg color is not only variable within species, but can also show marked variation between species (Gemmill, 1920, 1921; Nyholm, 1949; Chia and Spaulding, 1972; Siebert, 1973, 1974; Dunn, 1975; Siebert and Spaulding, 1976). The morphology of the anemone sperm is similar to the primitive sperm type seen in some other

7 116 A. SCOTT AND P. L. HARRISON Figure 5. Entacmaea quadricolor sperm, showing the shape of the sperm head and long flagellum. Scale bar: 3 m. actiniarian and anthozoan groups (reviewed by Harrison and Jamieson, 1999). Gamete buoyancy can be critical to reproductive success. For example, negative buoyancy of Epiactis prolifera eggs is a necessity because the young are attached to, or enfolded by, the exterior surface of the parent after spawning (Dunn, 1975). The buoyant nature of E. quadricolor and H. crispa eggs in conjunction with spawning synchrony between male and female anemones may increase fertilization success. Many scleractinian reef corals and gorgonians have buoyant gametes that accumulate at the sea surface (Harrison et al., 1984; Harrison and Wallace, 1990; Oliver and Babcock, 1992; Coma and Lasker, 1997). Such accumulation lowers dilution rates and increases the chances of egg and sperm encounter (Oliver and Babcock, 1992; Coma and Lasker, 1997). Spawned E. quadricolor and H. crispa eggs were covered by numerous spires of microvilli, a morphological characteristic that is unique to many actiniarian eggs (Chia and Spaulding, 1972; Dewel and Clark, 1974; Spaulding, 1974; Figure 6. Scanning electron microscopy images showing changes in surface structure of Entacmaea quadricolor during embryogenesis: (a) clumped spires of microvilli, 10 min after spawning; (b) spires begin to slowly unravel, 6 h after spawning; (c) spires unravelled, 12 h after spawning; (d) cilia begin to appear 14 h after spawning, and pore-like structures become visible. Scale bars: 10 m.

8 EMBRYOGENESIS OF HOST SEA ANEMONES 117 Figure 7. Scanning electron microscopy images showing changes in surface structure of Heteractis crispa during embryogenesis: (a) clumped spires of microvilli, 10 min after spawning; (b) spires begin to slowly unravel, 10 h after spawning, individual blastomeres visible below microvilli; (c) spires unravelled, 12 h after spawning; (d) cilia appear, 22 h after spawning. Scale bars: 10 m. Figure 8. Detail of the pore-like structure found on the surface of Entacmaea quadricolor larvae. Scale bar: 5 m. Schmidt and Schafer, 1980; Schroeder, 1982; Schäfer, 1985). These spires were evenly distributed over the entire egg surface except for a single bare patch that indicates the region of the egg nucleus (Spaulding, 1972; Fautin et al., 1989) and thus the likely site of fertilization. H. crispa spires were similar in size to the spires found on other anemone eggs that range from 10 to 25 m in length and 8 to 10 m in width (Chia and Spaulding, 1972; Clark and Dewel, 1974; Jennison, 1979; Schroeder, 1982). In contrast, E. quadricolor spires were both shorter and wider than those of other actiniarians studied to date. Although various functions have been proposed, the purpose of these spires remains unknown (Chia and Spaulding, 1972; Siebert, 1973; Spaulding, 1974; Fautin and Mariscal, 1991). Spires could provide a barrier to sperm penetration, thus preventing polyspermy (Siebert, 1973); they could protect the egg from abrasion and mechanical damage (Schroeder, 1982; Larkman and Carter, 1984; Fautin et al., 1989); or they could combine both functions.

9 118 A. SCOTT AND P. L. HARRISON Figure 9. Mean distance traveled (cm) in 30-s periods by Entacmaea quadricolor larvae at different stages of development (n 9, standard error). When spawned, E. quadricolor and H. crispa eggs contain zooxanthellae, which are therefore acquired via vertical transmission (Trench, 1997). This is in contrast to Anthopleura elegantissima, Anthopleura xanthogrammica, and Aiptasia tagetes, in which direct transmission of zooxanthellae from the host to the eggs does not occur (Siebert, 1974; Riggs, 1988). In these species, symbionts must be acquired from the environment via horizontal transmission during larval or adult stages (Trench, 1997). Anthopleura ballii has vertical transmission of zooxanthellae, which Davy and Turner (2003) suggested were acquired at, or just prior to, release of the eggs. Algal cells were observed on the surface of newly spawned E. quadricolor and H. crispa eggs and may have been in the process of being incorporated into the eggs, indicating that these eggs may also acquire zooxanthellae near the time of spawning. Benayahu et al. (1988) found that algal symbionts of the internally brooding soft coral Xenia umbellata are most probably transmitted by surface adherence, and they suggested that early symbiont acquisition may help support energy requirements during development. The zooxanthellae in E. quadricolor and H. crispa eggs are also likely to provide energy for larvae during their planktonic developmental phase. Cleavage is the first morphogenetic process in the embryonic development of E. quadricolor and H. crispa, and it occurs in a manner similar to that of other species of sea anemone that have large yolky eggs, in that the early divisions are superficial (Uchida and Yamada, 1968; Mergner, 1971; Campbell, 1974; Fautin et al., 1989). Blastomeres tended to become visible on one side of the embryo before the other. Gemmill (1921) suggested this may indicate the side of the fertilized nucleus. Cytoplasm of the developing embryo became segmented at the 32-cell stage in H. crispa, and at the 64-cell stage in E. quadricolor. The number of nuclear divisions that occur prior to cytokenesis may be related to the size of the anemone egg, with larger eggs having a greater number of divisions prior to division of the cytoplasm (Spaulding, 1974). H. crispa eggs are smaller than E. quadricolor eggs, which further supports Spaulding s suggestion. However, it is possible that cell divisions proceed rapidly after cytoplasmic separation for E. quadricolor, and earlier stages were not found due to the interval between sampling times. Changes in surface ultrastructure occur in E. quadricolor and H. crispa embryos during different stages of development. At the late blastula stage, the spires of microvilli covering the surface of the eggs and early embryos appear to unravel and become less regularly arranged. During gastrulation the surface spires disappear and the embryo surface becomes smooth and covered by short microvilli before development of the ciliated planula larvae. Sea anemones such as T. crassicornis, Peachia quinquecapitata, and A. ballii undergo similar changes during embryogenesis (Chia and Spaulding, 1972; Spaulding, 1972; Davy and Turner, 2003). Corresponding to the larger egg sizes, the larvae of E. quadricolor were larger than those of H. crispa. Larvae of both species were ciliated, and they rapidly contracted into different shapes and sizes. Change in planula shape and size has previously been attributed to muscle contraction (Widersten, 1968). E. quadricolor and H. crispa larvae moved in slow circles before developing the ability to swim actively at about 36 h after spawning. The aboral pole pointed forward during locomotion, with rotation generally being clockwise around the longitudinal axis, which is typical of most sea anemone planulae (Gemmill, 1920; Widersten, 1968; Chia et al., 1989; Hand and Uhlinger, 1992). Widersten (1968) found that rotation around the longitudinal axis Figure 10. Mean percentage survival of Entacmaea quadricolor larvae over time. Number in brackets indicates the mean percentage of remaining individuals that had started to metamorphose in the water column (n 5, standard error).

10 EMBRYOGENESIS OF HOST SEA ANEMONES 119 was replaced by slow gliding over the substratum in Metridium senile larvae. Rotational movement in E. quadricolor and H. crispa was also replaced with directional movement without spinning; however, the larvae became more motile and swimming speed subsequently increased over time. When directional movement in E. quadricolor larvae was first recorded, the larvae had a mean swimming speed of 2.73 cm in 30 s, which increased to 6.38 cm in 30 s when they were 2 weeks old. Swimming speed for other sea anemone planulae has been described as slow for some species and as remarkably rapid in others (Widersten, 1968). Larval swimming speeds have not been previously quantified for other actiniarians and therefore comparison between species is not possible. Larval swimming speed has been quantified for some other cnidarians. The swimming speed of E. quadricolor larvae is similar to that of the larvae of the octocoral Dendronephthya hemprichi (see Dahan and Benayahu, 1998) and the larvae of the reef corals Acropora millepora and Oxypora lacera (R. Babcock, unpubl. data cited in Mundy and Babcock, 1998). In contrast, the larvae of the other reef corals, including Goniastrea aspera and Platygyra daedalea, and the ahermatypic coral Caryophyllia smithii, swim much faster, at about 25, 21, and 90 cm per 30 s, respectively (Tranter et al., 1982; Reichelt-Brushett and Harrison, 2004). Although the swimming capability of E. quadricolor larvae would not permit continuous movement against water currents, it could allow larvae to position themselves within the water column or to search for suitable habitat prior to settlement on the substratum. Trails of mucus are used by some anemone and coral larvae as a method of feeding. Particles come into contact with, and subsequently become attached to, the mucous trail, which is then drawn into the mouth and ingested (Siebert, 1974; Tranter et al., 1982; Schwarz et al., 2002). Occasionally mucus-like material was found trailing from the oral pore of the swimming E. quadricolor and H. crispa larvae, indicating that these species may also use this mechanism for feeding. The rapid decrease in survival of E. quadricolor planulae contrasts with the gradual decrease in survival found for larvae from the broadcast-spawning reef corals Favites chinensis and G. aspera (see Nozawa and Harrison, 2005) and the octocoral D. hemprichi (see Dahan and Benayahu, 1998). However, D. hemprichi larvae were maintained in filtered seawater with ampicillin, which would have increased survival rates and may have prevented bacterially induced metamorphosis (Dahan and Benayahu, 1998). Small numbers of free-swimming larvae were noted in some of the survival jars 59 d after spawning, and were also found during settlement experiments 45 d after spawning (Scott, unpubl. data). This is considerably longer than the maximum survival times of 26 d and 35 d recorded for the larvae of the sea anemones Haliplanella lineata and Aiptasia tagetes, respectively (Riggs, 1988; Fukui, 1991). Planktonic larvae such as those produced by E. quadricolor and H. crispa serve as a means of dispersal for many cnidarian species (Chia, 1974; Harrison and Wallace, 1990; Leitz, 1997; Miller and Ball, 2000; Martin and Koss, 2002; Nozawa and Harrison, 2002; Harrison, 2006). Given the development rates of planulae from externally fertilized gametes and the longevity of some planulae, it is possible that the majority of larvae produced might be dispersed away from the Solitary Islands. This pattern has also been suggested for subtropical corals found in the region (Wilson and Harrison, 1998). Oceanographic currents are highly variable in this region, with water movements being influenced by the southward-flowing East Australian Current, by northward-flowing inshore currents, and occasionally by upwellings that produce a nearshore, northward-flowing counter current (Commonwealth of Australia, 2001). E. quadricolor and H. crispa larvae are likely be transported rapidly southward and offshore when the East Australian Current is close to shore (Harriott and Banks, 1995; Wilson, 1998). At other times, the larvae may be transported slowly northward, or possibly even retained at the Solitary Islands or nearby reefs when the currents are weak and oscillate in a north-south direction along the coast (Wilson, 1998; Wilson and Harrison, 2003). Further research is needed to accurately determine E. quadricolor and H. crispa larval longevity, which in combination with settlement competency experiments would allow a greater understanding of the potential dispersal capabilities of these species. Knowledge of the reproductive biology of sea anemones that host anemonefish not only provides important information on the processes that serve to maintain and renew populations of these species, but also has important implications for anemonefish populations that depend on their hosts for survival. Acknowledgments Thanks to Maxine Dawes (SEM Laboratory, Environmental Science and Management, Southern Cross University) for her invaluable help with SEM images and training. We are grateful to all those who volunteered their time during field and laboratory work, especially C. Damiano and A. Carroll, and the staff at the National Marine Science Centre, Coffs Harbour, for their assistance. 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