International Conference on Agriculture, Food and Environmental Engineering (ICAFEE'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

First Successful Documentation on the Embryonic, Larval and Juvenile Development of the Tropical Sea Urchin, Diadema Setosum (Leske, 1778)

M. Aminur Rahman, Fatimah Md. Yusoff, and A. Arshad

production and aquaculture of sea urchins in captive rearing Abstract—The tropical sea urchin, Diadema setosum belonging condition. to the Family Diadematidae, is one of the regular echinoids, widely distributed throughout the warm Indo-West Pacific Ocean including Keywords— Sea urchin, Diadema setosum, Embryo, Larva, the Malaysian intertidal reef. It has profound biological, ecological, Juvenile, Development, Growth. aquacultural and pharmaceutical significance, but yet to be fully determined and explored. In order to examine the developmental I. INTRODUCTION basis of morphological changes in embryos, larvae, we have thoroughly studied the ontogeny of D. setosum in a controlled HE black sea urchin, Diadema setosum (Leske, 1778) laboratory condition. Gametes were obtained from the sexually T(Echinodermata: Echinoidea: Diadematidae), is one of the matured adult individuals and the eggs fertilized using limited regular echinoids widely distributed in the Indo-West concentration of “dry” sperm (10-5 dilution). Fertilization rate was Pacific Ocean, where it occurs from the Red Sea (Gulf of estimated to be 96.8±1.3% and the resulting embryos were reared at Suez, Gulf of Aqaba, Northern and Southern Red Sea), and the o 24-25 C. The first cleavage (2-cell), 4-cell, 8-cell, 16-cell, 32-cell east coast of Africa to Japan and Australia [1] including and multi-cell (Morulla) stages were achieved at 01.20, 02.14, 02.44, Malaysia [2]. It has distinctively long black spines and five 03.09, 03.32 and 03.54 h post-fertilization, respectively. Ciliated white spots on its aboral side. The orange ring around its anal blastulae with a mean length of 111.47 ± 1.88 µm hatched 08.45 h following sperm entry. The Gastrulae attained at 16.36 h post- cone completes the special visual features of this species. It fertilization and the archenteron extended constantly, while the has substantial biological, ecological, nutritional and medicinal ectodermal red-pigmented cells migrated synchronously to the apical significance. plate. The 4-arm pluteus larva formed with two well-developed Gonads of sea urchins (“Roe” or “Uni”) have long been postoral arms 48.30 h following fertilization In this stage, pluteus used as a priced delicacy in Asian, Mediterranean and Western larva experienced with complete digestive tract and was able to feed Hemisphere countries [3]. At the same time, they are used as on unicellular algae (Chaetoceros calcitrans) in 2 d, grew raw materials to produce foodstuff, in particular, the product continuously, and finally attained metamorphic competence at 35 d of processing gonads [4−6]. Gonads of sea urchins have long after fertilization. Settlement induction and metamorphosis took been a luxury food in Japan [7]. Although, D. setosum has yet approximately 1 h 30 min from the attachment on the substratum followed by the complete resorption of larval tissues and the not been used as a commercially edible species in Malaysia, it development of complete juvenile structure with adult spines, has been reported that in Sabah, an indigenous tribe known as extended tubefeet and well-developed pedicellaria, the whole event “Bajau Laut” consumes sea urchin roe with rice [2]. Sea usually took place within 1 d post-attachment. The newly formed Urchin gonads are also rich in valuable bioactive compounds, juvenile (473.16 ± 6.96 µm) with a complete adult structure (mouth, such as polyunsaturated fatty acids (PUFAs) and β-carotene gut, anus, spine, tubefeet etc) then grew on coralline algae to 1-, 2- [8]. PUFAs, especially eicosapentaenoic acid (EPA, C20:5) and 3-month old juvenile by increasing the overall juvenile body, (n-3)) and docosahexaenoic acid (DHA C22:6 (n-3)), have spine and tube foot lengths. The 3-month old juvenile represents the significant preventative effects on arrhythmia, cardiovascular “sea urchin seed” for stocking in grow-out culture. This study is the diseases and cancer [9]. On the other hand, the high levels of first successful investigation on embryonic, larval and juvenile development of D. setosum, the findings of which would immensely arachidonic acid (AA) and EPA recently detected in D. be helpful towards the development of induced breeding, seed setosum supported the development of aquaculture of this urchin [10], since PUFAs are important for human nutrition [11]. Sea urchin fisheries have expanded so greatly in recent years that the populations of sea urchins around the world have M. Aminur Rahman*, Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, been overfished [12]. However, the decrease in supply and the Malaysia continued strong demand have led to a great increase in Fatimah Md. Yusoff and A. Arshad, Department of Aquaculture, Faculty interest in aquaculture of sea urchins, particularly in those of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, areas where their populations have been depleted [3, 13]. Malaysia *Corresponding author's E-mail: [email protected].

80 International Conference on Agriculture, Food and Environmental Engineering (ICAFEE'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

Owing to the emerging importance of D. setosum, early life III. RESULTS history information is an essential requirement for optimization Detailed morphological changes occur during the embryonic of mass seed production, culture and management. A few development of D. setosum are summerized in Table I. The studies on its abundance, distribution and population diameter of the unfertilized eggs of D. setosum was ranged characteristics have recently been carried out [2, 14, 15], but between 82.20 and 88.63 µm (mean ± SD = 85.75 ± 3.38 µm, no systematic studies have yet been conducted to optimize n=30). The egg vitelline membrane was raised after 40-50 sec larval development, growth and survival. Therefore, an attempt of sperm entry and the fertilization membrane began to form. was made to study the detailed embryonic, larval and juvenile However, the complete formation of fertilization envelope development of D. setosum in a controlled rearing system. took place within 5 min of insemination (Table I). First cell division was holoblastic and occurred 01.20 h after II. MATERIALS AND METHODS fertilization. Second cleavage started 02.14 h post-fertilization A total of 30 matured D. setosum, weighing from 80 to 150 (Table I) and was meridional, dividing the embryo into 4 equal g, were collected from the intertidal reef of Pulau Pangkor, blastomeres. The third cleavage occurred at 02.44 h and was Peninsular Malaysia during their natural breeding season in equatorial, separating animal and vegetal blastomeres with 8 July–September, 2012. Soon after collection, the live sea cells (Table I). During the 4th division, micromeres originated urchins were transported to the Laboratory of Marine equally from vegetal blastomeres, while 8 mesomeres were Biotechnology, Institute of Bioscience, Universiti Putra formed at 03.09 h after fertilization (Table I). Equatorial Malaysia (UPM) and maintained in well aerated sea water division of mesomeres, meridional division of macromeres, aquarium before use for the experiment. Gametes were and unequal micromere division formed embryos with 32 cells obtained from adult individuals by 0.5 M KCl injection into at 03.32 h after fertilization (Table I). The seventh cleavage the coelomic cavity. Eggs were collected by inverting female occurred without micromere division and the embryos attained urchins over a glass beaker filled with filtered seawater Merulla stage with 108 cells after 03.54 h following (FSW), while “dry” sperm were pipetted off the genital pores fertilization, (Table I). In blastula stage, cells acquired a of male urchins. Fertilization was done at limited sperm polygonal shape. Soon before hatching, the vegetal plate concentration (10-5 dilution of “dry” sperm) [16−19] and the thickened and cilia were formed on the perimeter at 09.15 h resulting embryos were reared in glass bottles containing 500 after fertilization (Table I). ml FSW, which was stirred constantly by 10 rpm rotating motors. When the larvae attained feeding stage (four-armed TABLE I pluteus), they were cultured in the same system (500 or 1000 EMBRYONIC DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION EXPERIMENTS WERE CONDUCTED AND FOR EACH ml glass bottles and supplemented with a laboratory cultured DEVELOPMENTAL STAGE, 10 EMBRYOS FROM EACH REPLICATE WERE USED phytoplankton, Chaetoceros calcitrans at concentrations of FOR THE OBSERVATION AND MEASUREMENT OF EMBRYOS 6,000-8,000 cells per ml [16]. When the matured larvae Time after Developmental stages Diameter (µm) attained metamorphic competent were used for settlement insemination 00.01 h Fertilized eggs with the 88.82 ± 2.93 tests. Induction of metamorphosis was performed on coralline formation of fertilization algal extracts + Chaetoceros diatom (50:50) in the petri dishes membrane (9.0 x 3.0 cm) containing FSW following the method of 00.05 h Fertilized eggs with complete 93.30 ± 3.88 Rahman and Uehara [20] and Rahman et al. [18]. All the fertilization membrane 01.20 h 2-cell stage 99.13 ± 7.17 developmental stages of embryos and larvae were then 02.14 h 4-cell stage 103.47 ± 1.76 observed at time intervals after insemination until they reached 02.44 h 8-cell stage 105.96 ± 1.71 metamorphic competence. In each experiment, the times after 03.09 h 16-cell stage 107.60 ± 1.52 insemination for 50% of the embryos to develop to 2-cell, 4- 03.32 h 32-cell stage 109.05 ± 1.63 cell, 8-cell, blastula, gastrula, prism, 2-arm, early 4-arm, late 03.54 h Multi-cell (Morulla) stage 110.50 ± 2.24 09.15 h Hatching Blastula 111.47 ± 1.88 4-arm, POA-elongated pluteus and competent stages were estimated, following Fujisawa [21] and Rahman et al. [18, 22]. The morphological events occur during the larval All morphometric measurements of embryo, larvae and development of D. setosum are presented in Table II. The juveniles were made on freshly prepared specimens, following gastrula formed 16.36 h post-fertilization (Table II). At the McEdward [18, 23, 24] with slight modifications. Larvae were beginning of this stage, larva experienced with primary first killed in 5% formalin in FSW and were concentrated by mesenchyme cells (PMC) which were detached from the settling to the bottom of a vial. After that, they were observed vegetal pole. In the course of this stage, red-pigmented cells and finally measured and photographed under the compound were first observed on the vegetal pole and then migrated microscope (Zeiss Axioskop 2) fitted with a software (Spot through the epithelium, simultaneously with PMC, towards the Advanced Verson 3.4). Each sample was observed four times apical plate. Secondary mesenchyme cells (SMC) originated to identify the developmental stages [18]. The newly formed on the vegetal pole, extending cytoplasm projections towards juveniles were then reared on encrusting coralline red algal the blastocoel during archenteron invagination. SMC on the substratum in small aerated glass aquaria until 3 months by archenteron then reached the anterior pole while red- which time they attained stocking-sized seeds for culturing in pigmented epithelial cells reached the anterior pole. The Prism grow-out system. stage started in 22.53 h after fertilization (Table II). During the complete development of prism larva, the surface of the

81 International Conference on Agriculture, Food and Environmental Engineering (ICAFEE'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia) embryo was covered by cilia with an apical tuft on the anterior represents the “sea urchin seed” for stocking in grow-out pole and a ciliated ring around the anus. The 2-arm pluteus aquaculture. stage was developed 34.35 h after fertilization (Table II). In this stage, the mouth opened, but the larvae were unable to TABLE III feed as the digestive system was not functionally active. The 4- JUVENILE DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION EXPERIMENTS WERE CONDUCTED AND FOR EACH DEVELOPMENTAL STAGE, arm pluteus larva having a mean length of 242.88 ± 12.25 µm 10 JUVENILES FROM EACH REPLICATE WERE USED FOR THE OBSERVATION was formed with two well-developed postoral arms 48.30 h AND MEASUREMENT OF JUVENILES post-fertilization. Larva experienced with a complete Developmental stages Body length Spine length Tube foot digestive tract and was able to feed on unicellular algae. The (mm) (mm) length (mm) 4-arm pluteus larva was further grew and developed to Late-4 Juvenile (1 day after 0.47 ± 0.01 0.48 ± 0.02 0.51 ± 0.03 arm pluteus (580.11 ± 13.52), POA (postoral arms)-elongated metamorphosis) stage-1(1011.76 ± 15.4) and POA-elongated stage-2 (1186.67 Juvenile (1 month after 3.91 ± 0.24 14.29 ± 4.98 ± 0.32 ± 18.39 µm) by increasing the overall larval lengths within 10, metamorphosis) 0.40 16 and 22 days post-fertilization, respectively (Table II). Juvenile (2 month after 6.22 ± 0.31 28.81 ± 6.15 ± 0.51 metamorphosis) 2.54 The precompetent larval stage started to form at 28.00 d Juvenile (3 month after 9.52 ± 0.51 42.88 ± 7.27 ± 0.62 after fertilization (Table II). During this stage, the basal metamorphosis) 3.81 portion of the larva was enlarged and the pigmented arches appeared to form, and the pedicellaria was encircled with a IV. DISCUSSION ciliated ring. In mature (competent) larval stage, the rudiment The development of embryo and larva of D. setosum were developed tubefeet and spines, which became active still inside more or less similar to those reported in other echinoids [18, the larval body. Larval structures were discarded or absorbed o 25−28] except for those in which larva grows with only two at this point. Under the temperature of 24–25 C, competent very long and well-developed postoral arms until attaining stage was reached at approximately 35 d post-fertilization competent stage [29]. The developmental timing of hatching (Table II). blastulae took longer period (09.15 h at 24oC) than those in Lytechinus variegatus (6 h at 23oC) [30] and in Salmacis TABLE II o LARVAL DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION sphaeroides (08.45 h at 24 C) [18]. Developmental timing of EXPERIMENTS WERE CONDUCTED AND FOR EACH DEVELOPMENTAL STAGE, later stages followed the same trends but slightly differed from 10 LARVAE FROM EACH REPLICATE WERE USED FOR THE OBSERVATION AND those of Caribbean species of L. variegatus [30]. Gastrulation MEASUREMENT OF LARVAE occurs with the correlation between the types of gastrulation Time after Developmental stages Length (µm) insemination and the pattern of migration of red-pigmented cells in D. 16.36 h Gastrula 117.58 ± 1.79 setosum, as that also reported in S. sphaeroides [18]. 22.53 h Prism 122.64 ± 2.62 Moreover, the triradiate spicules (the first sign of larval 34.35 h 2-arm pluteus 207.07 ± 10.48 skeleton), were formed during gastrulation in D. setosum, 48.30 h 4-arm pluteus 242.88 ± 12.25 which were more or less similar to those observed in other 10.00 d Late 4-arm pluteus 580.11 ± 13.52 16.00 d POA-elongated stage-1 1011.76 ± 15.44 echinoids [18, 27, 28]. 22.00 d POA-elongated stage-2 1186.67 ± 18.39 The competent larvae of D. setosum demonstrated substrate- 28.00 d Pre-competent larva 894.28 ± 14.82 test behavior, which was similar to those documented in other 35.00 d Competent larva 752.26 ± 13.95 echinoid species [18, 28, 31, 32]. Larval arms in newly metamorphosed juvenile were completely absorbed along with Induction of metamorphosis occurred when larvae attached the skeletons and epidermis, as similar to those observed in S. firmly to the bottom with the protruding tubefeet and the larval sphaeroides [18]. Subsequent to the induction of settlement tissues began to regress and accumulate on the aboral surface and complete metamorphosis, D. setosum juveniles had 4 of the rudiment. Metamorphosis was followed by the primary spines per interambulacrum (20 totals), similar to resorption of larval tissues and the development of a complete those documented in P. lividus [31], Strongylocentrotus juvenile structure with adult spines, extended tubefeet and purpuratus [33] and S. sphaeroides [18]. The newly well-developed pedicellaria, and the whole event usually took metamorphosed juveniles of D. setosum had one tubefoot per place within 1 d post-settlement (Table III). Early postlarval ambulacrum, as similar to that reported in S. fanciscanus and juveniles had no skeleton on the aboral surface, except for the S. purpuratus [33], P. lividus [31], E. cordatum [32] and S. remnants of larval rods. The gut was not yet formed and sphaeroides [18]. The competent larvae of D. setosum had neither mouth nor anus was present. During the resorption of pedicellariae during the late larval period and after larval tissues, the rudiments of Aristotle’s lantern and teeth metamorphosis as those documented in other regular urchins, were visible in the oral region. The newly formed juvenile with P. lividus [31], S. fanciscanus [33] and S. sphaeroides [18]. a complete adult structure (mouth, gut, anus, spine, tubefeet On the contrary, competent larvae of E. cordatum do not etc) then grew on coralline algae to 1-, 2- and 3-month old exhibit spines or pedicellariae [32], while C. subdepressus do juvenile by increasing the overall juvenile body, spine and have spines but devoid of any pedicellariae at all [28]. tube foot lengths (Table III). The 3-month old juvenile The newly metamorphosed juvenile of D. setosum has produced through the above developmental and growth stages neither a mouth nor an anus and no guts either. Similar phenomenon was also observed in other sea urchins [18, 27]

82 International Conference on Agriculture, Food and Environmental Engineering (ICAFEE'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia) and sea biscuits [28, 34]. The digestive system and probably [13] Lawrence, J.M., Lawrence, A.L., McBride, S.C., George, S.B., Watts, other internal organs appear at about 4-5 days after settlement S.A. and Plank, L.R. 2001. Developments in the use of prepared feeds in sea-urchin aquaculture. World Aquaculture, 32(3): 34–39. and then the urchin begins to feed and passes through [14] Kee, A.A.A. 2003. Biology and ecology aspects of sea urchin (Diadema subsequent juvenile stages, as those documented in P. lividus setosum) in Tioman Islands. Master’s thesis, National University of [31], Colobocentrotus mertensii [27] and S. sphaeroides [18]. Malaysia, Selangor, Malaysia, 220 p. [15] Rahman, M. A. and Yusoff, F. M. 2010. Sea urchins in Malaysian coastal waters. The Oceanographer, 4(1): 20–21. V. CONCLUSION [16] Rahman, M.A., Uehara, T. and Aslan, L.M. 2000. Comparative viability This study demonstrates the first successful investigation on and growth of hybrids between two sympatric species of sea urchins (genus Echinometra) in Okinawa. Aquaculture, 183: 45–56. the embryonic, larval and post-metamorphic juvenile (until 3- [17] Rahman, M.A., Uehara, T. and Lawrence, J.M. 2005. Growth and month-old) development of D. setosum under a captive heterosis of hybrids of two closely related species of Pacific sea urchins laboratory condition. The findings emerged from the present (genus Echinometra) in Okinawa. Aquaculture, 245: 121–133. study would greatly be helpful towards the understanding of [18] Rahman, M.A., Yusoff, F.M., Arshad, A. Shamsudin, M.N. and Amin, S.M.N. 2012b. Embryonic, larval, and early juvenile development of the ontogeny and life-history strategies, which will eventually tropical sea urchin, Salmacis sphaeroides (Echinodermata: Echinoidea). assist us in the development of breeding, larval rearing, seed The Scientific World Journal, 2012: 1–9. production and aquaculture of sea urchins in captive [19] Rahman, M.A., Arshad, A., Yusoff, F.M. and Amin, S.M.N. 2013. conditions. Hybridization and growth of tropical sea urchins. Asian Journal of Animal and Veterinary Advances, 8(2): 177–193. [20] Rahman, M.A. and Uehara, T. 2001. Induction of metamorphosis and ACKNOWLEDGEMENTS substratum preference in four sympatric and closely related species of We would like to extend our sincere thanks and sea urchins (Genus Echinometra) in Okinawa. Zoological Studies, 40(1): 29–43. appreciations to Universiti Putra Malaysia for financial [21] Fujisawa, H. 1993. 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