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

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1 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 Abstract The tropical sea urchin, Diadema setosum belonging to the Family Diadematidae, is one of the regular echinoids, widely distributed throughout the warm Indo-West Pacific Ocean including the Malaysian intertidal reef. It has profound biological, ecological, aquacultural and pharmaceutical significance, but yet to be fully determined and explored. In order to examine the developmental basis of morphological changes in embryos, larvae, we have thoroughly studied the ontogeny of D. setosum in a controlled laboratory condition. Gametes were obtained from the sexually matured adult individuals and the eggs fertilized using limited concentration of dry sperm (10-5 dilution). Fertilization rate was estimated to be 96.8±1.3% and the resulting embryos were reared at o C. The first cleavage (2-cell), 4-cell, 8-cell, 16-cell, 32-cell and multi-cell (Morulla) stages were achieved at 01.20, 02.14, 02.44, 03.09, and h post-fertilization, respectively. Ciliated blastulae with a mean length of ± 1.88 µm hatched h following sperm entry. The Gastrulae attained at h postfertilization and the archenteron extended constantly, while the ectodermal red-pigmented cells migrated synchronously to the apical plate. The 4-arm pluteus larva formed with two well-developed postoral arms h following fertilization In this stage, pluteus larva experienced with complete digestive tract and was able to feed on unicellular algae (Chaetoceros calcitrans) in 2 d, grew continuously, and finally attained metamorphic competence at 35 d after fertilization. Settlement induction and metamorphosis took approximately 1 h 30 min from the attachment on the substratum followed by the complete resorption of larval tissues and the development of complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, the whole event usually took place within 1 d post-attachment. The newly formed juvenile ( ± 6.96 µm) with a complete adult structure (mouth, gut, anus, spine, tubefeet etc) then grew on coralline algae to 1-, 2- and 3-month old juvenile by increasing the overall juvenile body, spine and tube foot lengths. The 3-month old juvenile represents the sea urchin seed for stocking in grow-out culture. This study is the first successful investigation on embryonic, larval and juvenile development of D. setosum, the findings of which would immensely be helpful towards the development of induced breeding, seed M. Aminur Rahman*, Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia Fatimah Md. Yusoff and A. Arshad, Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia *Corresponding author's aminur1963@gmail.com. production and aquaculture of sea urchins in captive rearing condition. Keywords Sea urchin, Diadema setosum, Embryo, Larva, Juvenile, Development, Growth. I. INTRODUCTION HE black sea urchin, Diadema setosum (Leske, 1778) T(Echinodermata: Echinoidea: Diadematidae), is one of the regular echinoids widely distributed in the Indo-West Pacific Ocean, where it occurs from the Red Sea (Gulf of Suez, Gulf of Aqaba, Northern and Southern Red Sea), and the east coast of Africa to Japan and Australia [1] including Malaysia [2]. It has distinctively long black spines and five white spots on its aboral side. The orange ring around its anal cone completes the special visual features of this species. It has substantial biological, ecological, nutritional and medicinal significance. Gonads of sea urchins ( Roe or Uni ) have long been used as a priced delicacy in Asian, Mediterranean and Western Hemisphere countries [3]. At the same time, they are used as raw materials to produce foodstuff, in particular, the product of processing gonads [4 6]. Gonads of sea urchins have long been a luxury food in Japan [7]. Although, D. setosum has yet not been used as a commercially edible species in Malaysia, it has been reported that in Sabah, an indigenous tribe known as Bajau Laut consumes sea urchin roe with rice [2]. Sea Urchin gonads are also rich in valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs) and β-carotene [8]. PUFAs, especially eicosapentaenoic acid (EPA, C20:5) (n-3)) and docosahexaenoic acid (DHA C22:6 (n-3)), have significant preventative effects on arrhythmia, cardiovascular diseases and cancer [9]. On the other hand, the high levels of arachidonic acid (AA) and EPA recently detected in D. 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 been overfished [12]. However, the decrease in supply and the continued strong demand have led to a great increase in interest in aquaculture of sea urchins, particularly in those areas where their populations have been depleted [3, 13]. 80

2 Owing to the emerging importance of D. setosum, early life history information is an essential requirement for optimization of mass seed production, culture and management. A few studies on its abundance, distribution and population characteristics have recently been carried out [2, 14, 15], but no systematic studies have yet been conducted to optimize larval development, growth and survival. Therefore, an attempt was made to study the detailed embryonic, larval and juvenile development of D. setosum in a controlled rearing system. II. MATERIALS AND METHODS A total of 30 matured D. setosum, weighing from 80 to 150 g, were collected from the intertidal reef of Pulau Pangkor, Peninsular Malaysia during their natural breeding season in July September, Soon after collection, the live sea urchins were transported to the Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia (UPM) and maintained in well aerated sea water aquarium before use for the experiment. Gametes were obtained from adult individuals by 0.5 M KCl injection into the coelomic cavity. Eggs were collected by inverting female urchins over a glass beaker filled with filtered seawater (FSW), while dry sperm were pipetted off the genital pores of male urchins. Fertilization was done at limited sperm concentration (10-5 dilution of dry sperm) [16 19] and the resulting embryos were reared in glass bottles containing 500 ml FSW, which was stirred constantly by 10 rpm rotating motors. When the larvae attained feeding stage (four-armed pluteus), they were cultured in the same system (500 or 1000 ml glass bottles and supplemented with a laboratory cultured phytoplankton, Chaetoceros calcitrans at concentrations of 6,000-8,000 cells per ml [16]. When the matured larvae attained metamorphic competent were used for settlement tests. Induction of metamorphosis was performed on coralline algal extracts + Chaetoceros diatom (50:50) in the petri dishes (9.0 x 3.0 cm) containing FSW following the method of Rahman and Uehara [20] and Rahman et al. [18]. All the developmental stages of embryos and larvae were then observed at time intervals after insemination until they reached metamorphic competence. In each experiment, the times after insemination for 50% of the embryos to develop to 2-cell, 4- cell, 8-cell, blastula, gastrula, prism, 2-arm, early 4-arm, late 4-arm, POA-elongated pluteus and competent stages were estimated, following Fujisawa [21] and Rahman et al. [18, 22]. All morphometric measurements of embryo, larvae and juveniles were made on freshly prepared specimens, following McEdward [18, 23, 24] with slight modifications. Larvae were first killed in 5% formalin in FSW and were concentrated by settling to the bottom of a vial. After that, they were observed and finally measured and photographed under the compound microscope (Zeiss Axioskop 2) fitted with a software (Spot Advanced Verson 3.4). Each sample was observed four times to identify the developmental stages [18]. The newly formed juveniles were then reared on encrusting coralline red algal substratum in small aerated glass aquaria until 3 months by which time they attained stocking-sized seeds for culturing in grow-out system. III. RESULTS Detailed morphological changes occur during the embryonic development of D. setosum are summerized in Table I. The diameter of the unfertilized eggs of D. setosum was ranged between and µm (mean ± SD = ± 3.38 µm, n=30). The egg vitelline membrane was raised after sec of sperm entry and the fertilization membrane began to form. However, the complete formation of fertilization envelope took place within 5 min of insemination (Table I). First cell division was holoblastic and occurred h after fertilization. Second cleavage started h post-fertilization (Table I) and was meridional, dividing the embryo into 4 equal blastomeres. The third cleavage occurred at h and was equatorial, separating animal and vegetal blastomeres with 8 cells (Table I). During the 4 th division, micromeres originated equally from vegetal blastomeres, while 8 mesomeres were formed at h after fertilization (Table I). Equatorial division of mesomeres, meridional division of macromeres, and unequal micromere division formed embryos with 32 cells at h after fertilization (Table I). The seventh cleavage occurred without micromere division and the embryos attained Merulla stage with 108 cells after h following fertilization, (Table I). In blastula stage, cells acquired a polygonal shape. Soon before hatching, the vegetal plate thickened and cilia were formed on the perimeter at h after fertilization (Table I). TABLE I EMBRYONIC DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION EXPERIMENTS WERE CONDUCTED AND FOR EACH DEVELOPMENTAL STAGE, 10 EMBRYOS FROM EACH REPLICATE WERE USED FOR THE OBSERVATION AND MEASUREMENT OF EMBRYOS Time after Developmental stages Diameter (µm) insemination h Fertilized eggs with the ± 2.93 formation of fertilization membrane h Fertilized eggs with complete ± 3.88 fertilization membrane h 2-cell stage ± h 4-cell stage ± h 8-cell stage ± h 16-cell stage ± h 32-cell stage ± h Multi-cell (Morulla) stage ± h Hatching Blastula ± 1.88 The morphological events occur during the larval development of D. setosum are presented in Table II. The gastrula formed h post-fertilization (Table II). At the beginning of this stage, larva experienced with primary mesenchyme cells (PMC) which were detached from the vegetal pole. In the course of this stage, red-pigmented cells were first observed on the vegetal pole and then migrated through the epithelium, simultaneously with PMC, towards the apical plate. Secondary mesenchyme cells (SMC) originated on the vegetal pole, extending cytoplasm projections towards the blastocoel during archenteron invagination. SMC on the archenteron then reached the anterior pole while redpigmented epithelial cells reached the anterior pole. The Prism stage started in h after fertilization (Table II). During the complete development of prism larva, the surface of the 81

3 embryo was covered by cilia with an apical tuft on the anterior pole and a ciliated ring around the anus. The 2-arm pluteus stage was developed h after fertilization (Table II). In this stage, the mouth opened, but the larvae were unable to feed as the digestive system was not functionally active. The 4- arm pluteus larva having a mean length of ± µm was formed with two well-developed postoral arms h post-fertilization. Larva experienced with a complete digestive tract and was able to feed on unicellular algae. The 4-arm pluteus larva was further grew and developed to Late-4 arm pluteus ( ± 13.52), POA (postoral arms)-elongated stage-1( ± 15.4) and POA-elongated stage-2 ( ± µm) by increasing the overall larval lengths within 10, 16 and 22 days post-fertilization, respectively (Table II). The precompetent larval stage started to form at d after fertilization (Table II). During this stage, the basal portion of the larva was enlarged and the pigmented arches appeared to form, and the pedicellaria was encircled with a ciliated ring. In mature (competent) larval stage, the rudiment developed tubefeet and spines, which became active still inside the larval body. Larval structures were discarded or absorbed at this point. Under the temperature of o C, competent stage was reached at approximately 35 d post-fertilization (Table II). TABLE II LARVAL DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION EXPERIMENTS WERE CONDUCTED AND FOR EACH DEVELOPMENTAL STAGE, 10 LARVAE FROM EACH REPLICATE WERE USED FOR THE OBSERVATION AND MEASUREMENT OF LARVAE Time after Developmental stages Length (µm) insemination h Gastrula ± h Prism ± h 2-arm pluteus ± h 4-arm pluteus ± d Late 4-arm pluteus ± d POA-elongated stage ± d POA-elongated stage ± d Pre-competent larva ± d Competent larva ± Induction of metamorphosis occurred when larvae attached firmly to the bottom with the protruding tubefeet and the larval tissues began to regress and accumulate on the aboral surface of the rudiment. Metamorphosis was followed by the resorption of larval tissues and the development of a complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, and the whole event usually took place within 1 d post-settlement (Table III). Early postlarval juveniles had no skeleton on the aboral surface, except for the remnants of larval rods. The gut was not yet formed and neither mouth nor anus was present. During the resorption of larval tissues, the rudiments of Aristotle s lantern and teeth were visible in the oral region. The newly formed juvenile with a complete adult structure (mouth, gut, anus, spine, tubefeet etc) then grew on coralline algae to 1-, 2- and 3-month old juvenile by increasing the overall juvenile body, spine and tube foot lengths (Table III). The 3-month old juvenile produced through the above developmental and growth stages represents the sea urchin seed for stocking in grow-out aquaculture. TABLE III JUVENILE DEVELOPMENT OF D. SETOSUM. THREE REPLICATE FERTILIZATION EXPERIMENTS WERE CONDUCTED AND FOR EACH DEVELOPMENTAL STAGE, 10 JUVENILES FROM EACH REPLICATE WERE USED FOR THE OBSERVATION AND MEASUREMENT OF JUVENILES Developmental stages Juvenile (1 day after Juvenile (1 month after Juvenile (2 month after Juvenile (3 month after Body length Spine length Tube foot length 0.47 ± ± ± ± ± ± ± ± ± ± ± ± 0.62 IV. DISCUSSION The development of embryo and larva of D. setosum were more or less similar to those reported in other echinoids [18, 25 28] except for those in which larva grows with only two very long and well-developed postoral arms until attaining competent stage [29]. The developmental timing of hatching blastulae took longer period (09.15 h at 24 o C) than those in Lytechinus variegatus (6 h at 23 o C) [30] and in Salmacis sphaeroides (08.45 h at 24 o C) [18]. Developmental timing of later stages followed the same trends but slightly differed from those of Caribbean species of L. variegatus [30]. Gastrulation occurs with the correlation between the types of gastrulation and the pattern of migration of red-pigmented cells in D. setosum, as that also reported in S. sphaeroides [18]. Moreover, the triradiate spicules (the first sign of larval skeleton), were formed during gastrulation in D. setosum, which were more or less similar to those observed in other echinoids [18, 27, 28]. The competent larvae of D. setosum demonstrated substratetest behavior, which was similar to those documented in other echinoid species [18, 28, 31, 32]. Larval arms in newly metamorphosed juvenile were completely absorbed along with the skeletons and epidermis, as similar to those observed in S. sphaeroides [18]. Subsequent to the induction of settlement and complete metamorphosis, D. setosum juveniles had 4 primary spines per interambulacrum (20 totals), similar to those documented in P. lividus [31], Strongylocentrotus purpuratus [33] and S. sphaeroides [18]. The newly metamorphosed juveniles of D. setosum had one tubefoot per ambulacrum, as similar to that reported in S. fanciscanus and S. purpuratus [33], P. lividus [31], E. cordatum [32] and S. sphaeroides [18]. The competent larvae of D. setosum had pedicellariae during the late larval period and after metamorphosis as those documented in other regular urchins, P. lividus [31], S. fanciscanus [33] and S. sphaeroides [18]. On the contrary, competent larvae of E. cordatum do not exhibit spines or pedicellariae [32], while C. subdepressus do have spines but devoid of any pedicellariae at all [28]. The newly metamorphosed juvenile of D. setosum has neither a mouth nor an anus and no guts either. Similar phenomenon was also observed in other sea urchins [18, 27] 82

4 and sea biscuits [28, 34]. The digestive system and probably other internal organs appear at about 4-5 days after settlement and then the urchin begins to feed and passes through subsequent juvenile stages, as those documented in P. lividus [31], Colobocentrotus mertensii [27] and S. sphaeroides [18]. V. CONCLUSION This study demonstrates the first successful investigation on the embryonic, larval and post-metamorphic juvenile (until 3- month-old) development of D. setosum under a captive laboratory condition. The findings emerged from the present study would greatly be helpful towards the understanding of ontogeny and life-history strategies, which will eventually assist us in the development of breeding, larval rearing, seed production and aquaculture of sea urchins in captive conditions. ACKNOWLEDGEMENTS We would like to extend our sincere thanks and appreciations to Universiti Putra Malaysia for financial support through Research Management Centre (RMC) under Research Universiti Grant Scheme (RUGS) vide Project No RU. REFERENCES [1] Lessios, H.A., Kessing, B.D. and Pearse, J.S Population structure and speciation in tropical seas: global phylogeography of the sea urchin diadema. Evolution, 55(5): [2] Rahman, M.A., Amin, S.M.N., Yusoff, F.M., Arshad, A., Kuppan, P. and Shamsudin, M.N. 2012a. Length weight relationships and fecundity estimates of long-spined sea urchin, Diadema setosum, from the Pulau Pangkor, Peninsular Malaysia. Aquatic Ecosystem Health and Management, 15: [3] Lawrence, J.M., Olave, S., Otaiza, R., Lawrence, A.L. and Bustos, E Enhancement of gonad production in the Sea Urchin Loxechinus albus in Chile fed extruded feeds. 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