JKAU: Mar. Sci., Vol. 21, No. 2, pp: 3-14 (2010 A.D. / 1431 A.H.) DOI : 10.4197/Mar. 21-2.1

The Early Development of the Sea Urchin Diadema setosum (Leske, 1778) in the region of North Jeddah, Red Sea

Adel N. Guirguis Faculty of Marine Science, King Abdulaziz University, Jeddah, Saudi Arabia [email protected].

Abstract. Changes in the gonads of the sea urchin, Diadema setosum in northern Jeddah on the Red Sea, were followed over a year. Maturation of gonads begins in April and spawning occurs mainly in early summer (from June to July). Spawning of some mature specimens was induced by KCl-injection. The optimal temperature for development was determined by incubation of batches of fertilized eggs at various temperatures (from 20 to 30oC). Early development proceeded rather rapidly at 25oC over a period of 3-5 days. The different stages of development from the onset of fertilization up to pluteus stage were described. Typical radial cleavage is recorded. Gastrulation is mainly by a combined process of embolic invagination and epiboly. Keywords: Early development, Sea Urchin, Diadema, Red Sea.

Introduction Diadema setosum (Leske, 1778), is the most abundant diadematid echinoderm in the Red Sea. It is well known by its extremely long needle-like (up to 20 cm) spines (Soliman, 2001). It is the most common benthic echinoid on the rocky beach as well as near reefs, characterized by its black colour. During the day, Diadema is inactive, either resting in crevices at the bases of the coral reefs or aggregated in groups. In the evening, animals leave their resting places and crawl about some meters away in order to forage on the rocky bottom. Diadema setosum possesses 3 4 Adel N. Guirguis

5 conspicuous white spots on the interambulacrals just above the gonads. The anal region is encircled with a bright orange ring (Clark, 1925; Price, 1983; Yokes & Galil, 2006). Diadema setosum is widely distributed in the West Indo-Pacific, from the Red Sea (Audouin, 1826; Fox, 1926; Clark & Owen, 1965; Clark, 1967; Magnus, 1967; Pearse, 1970; James & Pearse, 1971; Price, 1982), Arabian Gulf (Price 1982 & 1983), East coast of Africa (Muthiga, 2003), to India, Australia and Japan (James & Pearse, 1971; Lessios et al., 2001). Recently, Yokes and Galil (2006) collected this species from the Turkish Mediterranean coast at a depth of 15-18 m. D. setosum is of medical importance. Its brittle hollow spines, on penetrating the skin, remain embedded in the flesh of the victim, releasing venom. This causes redness, swelling and acute pain to swimmers, divers, fishermen, and collectors (Williamson et al. 1996; Yokes & Galil, 2006). Within the Echinoidea, gametogenesis have been described for several species: Diadema setosum (Yoshida, 1952), Lovenia elongata and Prionocidaris baculosa (Pearse, 1969a), Echinometra mathaei (Pearse, 1969b) and Heterocentrotus mammillatus (Dotan, 1990). Maturation and spawning of echinoids have been detected in Tripneustes gratila by Kidron et al. (1972). Spawning of D. setosum was first recorded by Mortensen (1901, cited in Hyman, 1955), then by Yoshida (1952), Pearse (1970) and more recently by Muthiga (2003). However, only a few studies were reported on the development of D. setosum. The aim of the present paper is thus to define the time of maturation and spawning of D. setosum in Jeddah coast, as well as to report on the events of the early development.

Material and Methods Specimens of Diadema setosum (Leske, 1778), of test diameter of 45-55 mm. and weight of 55-90 g were collected from Sharm Obhur (21°42’ 32.51’’N; 39°05’ 46.11’’E) near Obhur region of North Jeddah seashore, Red Sea. Samples of 10 large animals were monthly taken by snorkling from September 2008 to July 2009. The gonads were excised from the animals and fixed in Bouin's solution and examined histologically to follow up the maturation. The Early Development of the Sea Urchin Diadema setosum 5

Mature specimens collected from the field were kept in sea water- o filled aquaria, and maintained at constant temperature (about 25 C). Spawning was induced experimentally by injection of 0.1 ml of 0.5M potassium chloride through the peristome into the body cavity of tagged specimens, a method widely used to trigger spawning in sea urchin (Tyler & Tyler, 1966; Dotan, 1990). Ova were deposited at night on the periproct of two reared animals where they concentrate as dark masses hidden among the long needle-spines. Ova were then transferred to small glass vessels, containing sterilized sea water. Artificial fertilization was done by gently squeezing a small piece of testis tissue on the ripe ova. Fertilized eggs were pooled into five groups, each of approximately 50 . eggs. Each group was kept to develop at a fixed temperature For assessment of the optimal temperature for development, a range of o temperature incubation of 20, 23, 25, 28 and 30 C was used. The early stages were followed up by time and illustrated by both photographing and drawings.

Results Field observations during March have showing that of Diadema setosum had a tendency to move inshore and aggregate in numerous clusters (Fig. 1, B). At that time, the gonads were slightly lobulated, attaining full size. When ripe in April, the gonads changed from its faint yellow colour to pale cream or bright orange in females and white in males. All specimens examined were dioecious. The five gonads were suspended by mesenteric strands along the interambulacrals, opposite the five conspicuous white spots seen from aboral side. In July, mature ova and sperms were mixed in the laboratory and incubated at different temperatures. Various stages of development could be observed within 3-5 days, depending on the temperature, until attaining the pluteus stage (Fig. 2 & 3). The time required for the o development of each stage (at 25 C, the optimal temperature recorded) is shown in Fig. 2. The unfertilized egg (Fig. 2&3, A) is ellipsoidal in shape, small of about 0.1 mm in diameter and surrounded by a transparent jelly coat. At one end, it bears the micropyle (mi), through which the entry of sperm is made possible. Most of the eggs settled at the bottom. One hour after the addition of sperm, some of the eggs, being fertilized, became rounded 6 Adel N. Guirguis and with stiff coat (Fig. 2&3, B). The first cleavage was encountered within about one hour (Fig. 2&3, C&D), followed shortly by the second, both being perpendicular to one another (Fig. 2&3, E). The third cleavage was equatorial, bringing about the formation of eight nearly equal mesomeres and expressing radial cleavage (Fig. 2&3, F). All the succeeding divisions, till the blastula stage, follow the same pattern as in all deuterosomes (Fig. 2&3, G-H). The blastula is eventually set free from its surrounding envelope. Twenty four hours after fertilization, the vegetal pole of the blastula (0.2 mm in diameter), became slightly flattened and began proliferating mesenchymal cells into the blastocoel (Fig. 2&3, I, J). Meantime, a typical embolic invagination was in progress forming the archenteron of the gastrula (Fig. 2&3, K). Gastrulation was completed in one to two days. The transformation of the gastrula into the following four-armed stage (pluteus larva, Fig. 2&3, L) took another one to two days. It began by the elongation of the body into a conical shape. The apex of the cone, the aboral spike (as), represents the animal pole, and the base of the cone had the blastopore (future anus) in its centre. The pluteus larva was approximately 0.25 mm long, with four arms (a), each supported by an internal skeletal rod (sk). It acquired a pelagic existence near the water surface using its extensive ciliated bands on the arms. The archenteron was differentiated by constrictions into oesophagus, stomach (st) and rectum. It was not possible to follow the succeeding stages till attaining the benthic juvenile form.

A B

Fig. 1. Underwater photographs of live specimens of Diadema setosum, in crevice among corals (A), and in aggregation (B).

The Early Development of the Sea Urchin Diadema setosum 7

Fig. 2. Photographs showing the stages of development of Diadema setosum. (A) Unfertilized ovum; (B) Fertilized egg (1 h.); (C&D) Two blastomeres (2 h.); (E) Four blastomeres from top view (4 h.); (F) Eight blastomeres (8 h.); (G) Sixteen cells (12 h.); (H) Morula (18 h.); (I) Blastula (24 h.); (J) Beginning of gastrulation; (K) Gastrula (48 h.); (L) Pluteus stage (3 days old).

8 Adel N. Guirguis

Fig. 3. Diagrams illustrating the different stages of development in Diadema setosum. (A) Unfertilized ovum; (B) Fertilized egg; (C&D) 2-cell stage; (E) 4-cell stage, top view; (F) 8-cell stage; (G) 16-cell stage; (H) Morula;(I) Blastula; (J) Beginning of gastrulation; (K) Gastrula; (L) Pluteus stage. a, arm; ar, archenteron; as, aboral spike; b, blastocoel; bl, blastopore; e, envelope; m, mouth; mc, mesenchyme cells; me, mesomeres; mi, micropyle; sk, skeletal rod; st, stomach.

The Early Development of the Sea Urchin Diadema setosum 9

Discussion In the study area, Diadema setosum has one reproductive cycle per year, similar to other echinoids inhabiting the Red Sea, such as Prionocidaris baculosa (Pearse, 1969a), Echinometra mathaei (Pearse, 1969b; 1970) and Heterocentrotus mammillatus (Dotan, 1990). Its reproduction tended to be fairly synchronous, and all specimens examined were apparently at the same reproductive stage. Ripening of gonads did not actually occur among some individuals. The beginning of gonadal ripening was restricted during March and April and maximum size of the gonads was attained a few months before maturity which reached in June and July. This relatively long period may be due to the mechanism of the accumulation of nutritive materials in the gonads (Dotan, 1990), thus increasing the amount of gametes subsequently produced (Lawrence & Lane, 1982). The maturation of gonads in echinoids at different geographical habitats was generally reviewed by Hyman (1955). She recorded that some echinoids ripen in the Red Sea from April to June while others from July to September, in Japan from April to July but others in July and August, in Hawaii in April, in Panama in December and January, in Java Sea in May, and in Port Jackson (Australia) in February. Hyman (1955) attributed the onset of spawning to the rise in temperature. Pearse & Lockard (2004) agreed with Hyman (1955) and showed that most metabolic activities are adjusted to changes in temperature. Specimens of Diadema antillarum spawn during spring in West India (Millott, 1953) and Barbados (Lewis, 1966), but in July in Bermuda (Mortensen, 1937). The breeding season of D. setosum was first observed by Mortensen in 1901 (as cited by Hyman, 1955) during April and May in Java Sea, then by Fox (1922, 1924a & b) in July and August at Suez, by Yoshida (1952) from May to September in Japan, by Kobayashi & Nakamura (1967) in summer at Seto, by Pearse (1970) from April to September in the Gulf of Suez, and by James & Pearse (1971) from early June to mid-September in both the Gulf of Suez and northern Red Sea. It was concluded that not only temperature is the critical factor for breeding and spawning of the species but also many biotic and abiotic factors may bring about the same effect. 10 Adel N. Guirguis

In Kenya, Muthiga (2003) determined the annual reproductive cycle of D. setosum and D. savignyi by measuring the gonad indices (G.I.) and stated that both species exhibited a long breeding period, where gametogenesis started in February (G.I.= 8%), peaked in May (9.7%) and rapidly decreased in June. He also showed gonad indices of a few specimens remained above 7% through much of the year. This is in fact the first record of continuous reproduction of the species in question in the Red Sea. He added that samples of larger sizes are, however, needed before any definite conclusions concerning reproductive periodicity can be established, since not all mature specimens spawn (probably due to reabsorption of the gonads). It was also mentioned by Moore (1966) that during most of the year, the gonadal size is determined by the physiological condition of each individual and do not show direct association with the total body weight. Casual observation of clumped grouping in Diadema setosum was the case during spring near Jeddah shore at depths of about 0.5 to 1.0 meter. Such aggregations of some diadematid sea urchins during maturation have been reported also by Fox (1924a), Thornton (1956), Pearse & Arch (1969) and Pearse (1972). It has been suggested that this is a widely occurring phenomenon among many other species (Elmhirst, 1922; Schuel, 1984; Pennington, 1985). Kidron et al. (1972) reported that spawning was induced immediately by injection of KCl into the body cavity of the sea urchin Tripneustes gratilla, collected in January from Eilat, Red Sea. Lessios (1985) also stated that injection of isotonic KCl is a simple, non- destructive and quick method for studying the reproductive state of echinoderms. The results of the present study have shown that most individuals did not react to KCl injection except during the time of maturation. It may be used as indicator for the gonad ripeness of the specimen. Similar results were observed for some echinoids by Dotan (1990). However the effect of KCl on maturation and spawning has not been given a satisfactory interpretation from the physiological point of view. The development of the ova of Diadema setosum, as illustrated in the present work, agrees with the previous reports on other echinoids, such as those of Barker (1985) on Goniocidaris umbraculum and Bosch et al. (1987) on Sterechinus neumayeri. As regards cleavage, blastula The Early Development of the Sea Urchin Diadema setosum 11 formation and gastrulation, encountered in this study, by all means follow the same pattern prevailing among sea urchins. Many workers on sea urchins noticed the presence of mesenchyme cells during blastulation and gastrulation in the sea urchins (Morrill & Santos, 1985; Burke et al., 1991; Cherr et al., 1992; Malinda et al., 1995; Berg et al., 1996). Cherr et al. (1992) and Berg et al. (1996) detected also an extracellular blastocoelic matrix of the sea urchin late blastula. They expected that this matrix has an important role in gastrulation. In the present study the larvae obtained in laboratory did not develop beyond the four-armed-stage and died shortly. Hyman (1955) stated that the plutei of Diadema remain in this stage until reaching about 1 mm, and then the two post-oral arms gradually shifted to horizontal position. Ebert (2001) studied the survival of the post-settlement stages of sea urchins, and concluded that it needs special precautions in addition to the presence of diatoms and planktonic organisms. Larvae in nature may be protected inside the forest of spines of sea urchins. Brooding by parents is largely expected for the sake of protecting such tiny larvae.

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