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Larval development and juvenile growth of the sea cucumber Stichopus sp. (Curry fish)
Aquaculture 300 (2010) 73–79
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Larval development and juvenile growth of the sea cucumber Stichopus sp. (Curry fish) Chaoqun Hu a,b,⁎,1, Youhou Xu a,b,c,1, Jing Wen a,b,c,1, Lvping Zhang a,b, Sigang Fan a,b,c, Ting Su a,b,c a
Key Laboratory of Marine Bio-resources Sustainable Utilization (LMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China Key Laboratory of Applied Marine Biology of Guangdong Province and The Chinese Academy of Sciences (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China c Graduate University of Chinese Academy of Sciences, Beijing, 100049, China b
a r t i c l e
i n f o
Article history: Received 12 May 2009 Received in revised form 11 September 2009 Accepted 14 September 2009 Keywords: Curry fish Spawning Larvae Development Juvenile Growth Aquaculture
a b s t r a c t The global populations of Curry fish have been severely depleted over the past decade. This study describes spawning, fertilization, larval rearing, and juvenile growth in a commercially important Stichopus species. Data pooled from monthly trials conducted over 2 years indicate that, under optimal conditions, juveniles can be grown to a size of ca. 20 cm in length in 7 months. The survival rates are typically between 30 and 50%. Pilot research indicates that the growth of young sea cucumbers in abandoned abalone tanks has potential. Overall, this study demonstrates that Curry fish can be reared in captivity, thus providing an alternative to fisheries, or a way to maintain sustainable harvests and eventually contribute to the restoration of the natural populations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumbers (Echinodermata: Holothuroidea), a diverse group of marine invertebrates, known as ‘beche-de-mer’ or ‘trepang’, are generally consumed in Asia, where they are regarded as traditional medicine, delicacies and aphrodisiacs. Sea cucumbers present a high nutritional value due to their high protein, low fat content, aminoacid profile and are a rich source of trace elements (Chen, 2003). Curry fish is a yellow/brown sea cucumber, covered with small dark spots. It is a medium to large-sized species (300–700 mm), stout, thick and firm, with low papillae, and a moderately smooth tegument. Curry fish has a wide Indo–West Pacific distribution (Rowe and Gates, 1995), can be found on tropical reefs, and is considered edible with medicinal properties (Liao, 1997). The fast pace of development of sea cucumber fisheries to supply the growing international demand for beche-de-mer is placing most fisheries and many sea cucumber species at risk including Curry fish, which was highly abundant and is nowadays rare or locally extinct (FAO, 2008; Otero-Villanueva and Ut, 2007).
In China, the soaring price of Apostichopus japonicus has stimulated the development of aquaculture including sea ranching activities. Presently, China is successfully producing an estimated 10,000 t, dry weight, of A. japonicus from aquaculture, mainly to supply local demand. Chen (2004) noted that recovery plans for A. japonicus are in place together with the establishment of many conservation zones. Recently in Malaysia, great progress of research on aquaculture for Stichopus horrens has been achieved (Zaidnuddin, 2009). The reproductive biology of Stichopus variegates (presently revised to Stichopus herrmanni, Rowe and Gates, 1995) has been reported by Conand (1993). Tehranifard et al. (2006) reported the reproductive cycle of S. herrmanni from Iran. However, no published work is available on the larval development, juvenile growth and artificial propagation of Curry fish in China. This study presents data on the development of embryos, larvae and juveniles of Curry fish Stichopus sp. over a 16-month period at Weizhou Island, China. The results contribute to maintenance of sustainable use and will contribute to the restoration of natural populations.
2. Materials and methods ⁎ Corresponding author. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China. Tel.: +86 20 89023216; fax: +86 20 89023218. E-mail address: [email protected] (C. Hu). 1 These authors have equally contributed to this work. 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.09.033
2.1. Collection and maintenance of animals In total 648 adult sea cucumbers (mean wet weight > 500 g) were collected from nearby coastal areas of Weizhou Island (21°03′N,
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C. Hu et al. / Aquaculture 300 (2010) 73–79
Fig. 1. Dorsal ossicles of Stichopus sp.. a: Tables. b: C-shape rod. c: Rosette.
109°07′E) and maintained at the Weizhou Aquaculture Farm to serve as broodstock from April 2006 to October 2007. The adults were conditioned in the tanks for a few days prior to spawning. 2.2. Species identification Ossicles were prepared by scanning electronic microscopy (SEM). The dorsal body walls of ten sea cucumbers were obtained by dissection, and fixed in 95% ethanol at − 20 °C in the farm. In the laboratory, the body walls were treated with 10% sodium hypochlorite solution for 1–2 min to obtain ossicles, and carefully rinsed with distilled water at least three times following procedures by Massin et al. (2000). The specimens were critical-point dried and coated with gold–palladium in a sputter coater (Hitachi, E-1010) and ossicles were observed using 20 kv accelerating voltage and 1000–3000 magnifications under a Hitachi S-3400N SEM. Species identifications were based on the taxonomic descriptions of dermal ossicles by Liao (1997) and Massin (1999).
and concentration dictated by the daily observation of the digestive tract contents (the number of cells in the stomach). The water in each tank was changed every day by draining the tanks through a 53-μm sieve. After the first water change, the early auricularia larvae were stocked at a density of 0.2 larvae mL− 1. Seven days after fertilization, 100 sets of plastic settlement sieves were conditioned in an outdoor tank supplied with filtered running seawater to promote growth of a diatom film. Each set of substrates consisted of six sieves measuring 300 × 240 mm, stacked
Table 1 Number of spawning observations. Year
Month
Date
Lunar phase
na
Maleb
Femaleb
2006
May
26-05-2006 27-05-2006 28-05-2006 29-05-2006 10-06-2006 25-06-2006 26-06-2006 27-06-2006 28-06-2006 10-07-2006 24-07-2006 25-07-2006 26-07-2006 27-07-2006 08-08-2006 23-08-2006 24-08-2006 25-08-2006 26-08-2006 07-09-2006 21-09-2006 22-09-2006 23-09-2006 24-09-2006 14-06-2007 15-06-2007 16-06-2007 17-06-2007 28-06-2007 13-07-2007 14-07-2007 15-07-2007 16-07-2007 28-07-2007 12-08-2007 13-08-2007 14-08-2007 15-08-2007 27-08-2007 10-09-2007 11-09-2007 12-09-2007 13-09-2007
Last New New New Full Last New New New Full Last New New New Full Last New New New Full Last New New New Last New New New Full Last New New New Full Last New New New Full Last New New New
148 148 148 148 342 342 342 342 342 418 418 418 418 418 518 518 518 518 518 518 518 518 518 518 540 540 540 540 578 578 578 578 578 613 613 613 613 613 648 648 648 648 648
13 38 36 16 0 4 73 68 12 0 6 73 66 15 0 10 49 39 12 0 8 24 20 12 12 79 76 29 0 14 85 88 23 0 16 79 73 10 0 20 37 29 16
0 3 8 2 0 3 21 25 3 0 2 28 29 4 0 3 21 18 2 0 1 9 9 0 4 39 38 4 0 3 32 41 3 0 4 33 35 2 0 3 11 8 0
June
2.3. Spawning and fertilization On arrival (1800–2000 h) at the farm, the specimens were placed in aerated 2000-L tanks and monitored overnight for spawning activity. Spawning of sea cucumber was conducted by open-air drying followed flow-through filtered seawater stimulation. Males and females were isolated in plastic buckets. As soon as they showed signs of imminent spawning (spawning behavior, such as sway), they were isolated. Each female was then placed separately in a 300-L spawning tank and maintained until release of eggs. The female was removed, and the spermatozoa solution obtained from isolated spawning males, was added to the eggs. The best fertilization rates and lowest occurrence of polyspermy were obtained with a concentration of 5–10 spermatozoa per egg. After fertilization, the eggs were rinsed to remove excess sperm. 10 mL water was randomly collected after gentle stirring, and the number of eggs was counted, egg counts were performed at least three times and the amount was calculated. Individual female Stichopus sp. spawned a mean of 3.48 × 106 eggs (s.e. = 1.4 × 105, n = 5 females). Then the eggs were transferred to hatchery tanks at a density of 0.5 eggs mL− 1.
July
August
September
2007
July
2.4. Embryo and larval culture Several air stones positioned at the bottom of each culture tank provided sufficient aeration and ensured gentle water circulation. The larvae were reared at ambient seawater temperature (27–31 °C). In the wild, Curry fish lives under substrate with low light levels. Therefore, the photoperiod was designed as 0 h light:24 h dark in this experiment. When the auricularia had a functional gut, the larvae were fed every day using a wet mix of commercial marine yeast (Rhodotorula) and microalgae (dominated by Rhodomonas and Dunaliella) at a frequency
June
August
September
a b
Number of Stichopus sp. collected during each field trip. Number of spawning individuals.
C. Hu et al. / Aquaculture 300 (2010) 73–79
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Fig. 2. Spawning of Stichopus sp.. a: Pre-spawning posture; podia (pod), tentacle (ten). b: Ejaculation; papillae (pap), sperm (s). c: Pre-spawning posture; inflated gonopore (ig). d: Spawning; eggs (e).
with a 200-mm gap. When the late auricularia stage was reached, about 40 sets of substrates were placed in each rearing tanks. The larval feed was stopped after settlement. Continuous water exchange was maintained with filtered running seawater until the juveniles were detached. 2.5. Juvenile culture Juvenile sea cucumbers were fed ad libitum daily at 1900 h with sifted pieces of semi-dried Sargassum thunbergii (11.70% crude protein, 0.67% crude lipid, 12.89% ash, moisture b 1.00%; energy 8.55 kJ g− 1 dry mass) and the pieces (radius around 0.8 mm, weight around 2.5 mg) were insoluble in the sea water (An et al., 2007). The juvenile feed was increased as the individuals grew. Competitors such as copepods, ciliates, and protozoans were controlled using pesticide (Trichlorphon) and filtration. A concentration of 2 mL Trichlorphon m− 3 was applied every 2 h, and water was changed through a 53-μm sieve. 2.6. Specimen preparation for optical microscopy The spermatozoa, eggs, embryos, larvae, and juveniles were fixed in 2.5% glutaraldehyde in 2.0 μm filtered seawater. The specimens were viewed with a Leica DMR optical microscope. 3. Results and discussion 3.1. Species identification Three kinds of dermal ossicles were found in the dorsal body walls of Stichopus sp., tables, C-shaped rods and rosettes (Fig. 1). The size, shape and structure of these ossicles were in accordance with the descriptions of Curry fish by previous reports (Liao, 1997; Massin, 1999). 3.2. Spawning periodicity Close monitoring of the broodstock in the field over several months (Table 1) revealed that Stichopus sp. followed a predictable lunar spawning periodicity. They often spawned within the first
2 days following the new moons from May to August on Weizhou Island, even in captivity. This behavior has been observed for some aspidochirotid species including Holothuria scabra and Isostichopus fuscus (Hamel et al., 2002; Hamel et al., 2003). In contrast, some aspidochirotids including Isostichopus badionotus and Holothuria mexicana, are known to spawn within the first 5 days following the full moons (Guzman et al., 2003). Spawning during the warm season, the phenomenon was consistent with the observation by Conand (1993). However, the previous study did not investigate the lunar spawning periodicity. The spawning behavior of specimens in the field was consistent with previous observation on S. herrmanni (Conand, 1993). Pre-spawning behavior included rolling and twisting on the substrate (Fig. 2a). Males raised the anterior end prior to spawning and often swayed from side to side, or in a circular motion, as they released sperm. Most aspidochirotids lift the anterior end of the body from the substratum during spawning to facilitate dispersal of gametes and fertilization (McEuen, 1988). Male Stichopus sp. spawned 1–2 h before females, and spawned more bursts and longer. Male sea cucumbers are reported to spawn before females. This is suggested to be a proximal signal for synchronising females to spawn (McEuen, 1988). Male Stichopus sp. spawned soon after being placed in the spawning tank and released a steady stream of sperm from the single anterior gonopore (Fig. 2b). Males remained erect and spawned continuously for up to 3 h. They usually resumed spawning after artificial disturbance such as touch and carrying. Females typically started spawning by raising their bodies off the substrate, in a similar manner to males. It was possible to distinguish females from males shortly before spawning from the bulge around the gonopore (Fig. 2c). They remained erect for 15–30 min before releasing a short powerful burst of pale yellow to pink eggs, which lasted for 10– 15 s (Fig. 2d). The short powerful release of eggs by each species is typical of many holothurians, and forces eggs into the water column and aids dispersion and fertilization (Battaglene et al., 2002). Spermatozoa comprised a head with 2.6 μm (s.e. = 0.4 μm, n = 5 males) mean diameters and a 48 μm (s.e. = 3.8 μm, n = 5 males) long tail (Fig. 3a). The mean egg diameter of Stichopus sp. was 180 μm (s.e. = 2.8 μm, n = 5 females) (Fig. 3b), and the result agreed with the previous report (Conand, 1993).
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Fig. 4. Juvenile development of Stichopus sp.. a: Pentactula; tentacle (ten), ossicle (oss). b: Dorsal ossicles. c: 15-day juvenile; podia (pod), digestive tract (dt). d: 20-day juvenile; papillae (pap). e: 25-day juvenile. f: 30-day juvenile.
3.3. Embryonic and larval development The development of Stichopus sp. is initiated with the elevation of the fertilization envelope followed the expulsion of the first polar body occurs roughly 20 min after fertilization. The first cleavage was equal, radial and holoblastic, and divided the cell into two equal hemispheric blastomeres (Fig. 3c). The second cleavage again occurred along the animal–vegetal axis, yielding more spherical blastomeres (Fig. 3d–g). The blastula stage was reached by 3 h. By 6 h, the blastula had a conspicuous ciliary cover and continuously rotated within the envelope propelled by their cilia (Fig. 3h). Embryos hatched from the fertilization envelope as early gastrulae, ca. 14 h after fertilization (Fig. 3i). These early gastrulae swam with the help of cilia covering their entire surface; they elongated into full-size gastrulae after ca. 16 h (Fig. 3j–k). The auricularia larval stage was reached ca. 35 h after fertilization (Fig. 3l). Growing auriculariae could be observed during the next 4 days of culture. The buccal ciliated cavity, the mouth, the oesophagus, the intestine, as well as the cloaca were clearly visible (Fig. 4m–n). At this stage, they began to accumulate hyaline spheres (Fig. 4o). After 7–8 days, the auriculariae reached a maximum size of 1.4 mm, and they possessed an axohydrocoel (Fig. 3n–p). The auriculariae subsequently transformed to the doliolaria stage (Fig. 3p–r). In the course of this process, the larvae shrank down to nearly 50% of their initial size, the buccal ciliated cavity disappeared and the ten hyaline spheres became aligned (Fig. 3r). The doliolaria stage was reached ca. 9–10 days after fertilization (Table 2). The larvae stopped feeding. The cilia became aligned in five distinct bands along their cylindrical body. The digestive tract was obscure by the translucent body. Like other holothurians (Asha and Muthiah, 2005; Ramofafia et al., 2003; Hamel et al., 2003; McEuen and Chia, 1991), Stichopus sp.
transformed to the juvenile stage (Table 2). In most trials, the development, settlement and early growth of the larvae were somewhat asynchronous, and different stages and sizes could be found simultaneously in the culture. In extreme examples, auricularia and 6 mm long juveniles were in the same tanks. However, the developmental chronology in Table 2 was based on stages achieved by most of the culture. The larval stage of Stichopus sp. was relatively short, with newly metamorphosed juveniles appearing in the cultures as early as 15 days after fertilization. Other tropical holothurians such as H. scabra (Ramofafia et al., 2003), Holothuria spinifera (Asha and Muthiah, 2005), and I. fuscus (Hamel et al., 2003) also have a short development time. However, tropical species Actinopyga echinites, needs about 20 days to transform to the juvenile stage (Chen et al., 1991). The cold temperate species Psolus chitonoides and Psolidium bullatum, need more than 28 days to complete metamorphosis (McEuen and Chia, 1991).
3.4. Juvenile growth The doliolariae transformed into early pentactulae that possessed five buccal tentacles and dermal ossicles 9–10 days post fertilization (Fig. 4a–b). At this stage, the larvae began to swim close to the substrate and successively went through swimming and settling phases. Definitive settlement, with the complete loss of cilia, completion of metamorphosis and emergence of the two first ambulacral podia, occurred ca. 15 days after fertilization (Fig. 4c). Although the settled juveniles were observed as early as day 12 post fertilization, a majority of juveniles measured 1 to 1.5 mm in length recorded after 20 days of culture. At this stage, papillae developed along the dorsal body wall (Fig. 4d). The juveniles reached ca. 2–3 mm within 5 days (Fig. 4e), and 5 mm after ca. 30 days. They possessed
Fig. 3. Embryonic and larval development of Stichopus sp.. a: Spermatozoa. b: Fertilized egg. c–g: Cleavage stages. h: Blastula; envelope (env). i–k: Gastrula. l: Early auricularia. m: Mid auricularia; buccal ciliated cavity (bcc), oesophagus (oes), intestine (int), cloaca (clo). n–q: Late auricularia; mouth (mou), axohydrocoel (axo), hyaline sphere (hs). r: Doliolaria; cilia band (cb), digestive tract (dt).
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Table 2 Development of Stichopus sp., from fertilization to juvenile. Stage
Size (μm)
Time
Fertilization Expulsion of the first polar body 2-cell 4-cell 8-cell 64-cell 128-cell Blastula Rotated blastula Early gastrula Gastrula Late gastrula Early auricularia Mid auricularia Auricularia Late auricularia Doliolaria Pentactula Juvenile
150–200 150–200 200–250 200–250 200–250 250–300 250–300 250–300 250–300 300–350 300–350 300–350 350–450 500–700 800–1000 1000–1300 400–500 400 400
0 20 (min) 45 (min) 51 (min) 88 (min) 137 (min) 149 (min) 3 (h) 12 (h) 14 (h) 16 (h) 20 (h) 35 (h) 3–4 (d) 5–6 (d) 7–8 (d) 9–10 (d) 10–11 (d) 12 (d)
several papillae, more podia and their intestine exhibited strong peristaltic movement (Fig. 4f). The juveniles continued to grow at a rate of ca. 1.0 mm per day for the next 3 to 4 weeks (Fig. 5). When they were ca. 5 mm in length, the juveniles started to accumulate orange-green pigment. In 8 mm long juveniles, the tip of the tentacles became ramified. After 40 days of culture, the juveniles were 1.8–2.0 cm long and 4 mm wide (Fig. 6a). The body wall became more opaque as the ossicle density and the tegument thickness increased. When the juveniles reached ca. 2 cm in length, the whitish colouration that characterised the early stages of life was gradually replaced by a brownish tinge similar to the one observed in adults. After approximately 60 days, the juveniles were ca. 4 cm long and 2 cm wide and were subsequently released in outdoor ponds, or into the field, to complete their growth (Fig. 6b). From settlement
(0.5–1.0 cm) onward, the juveniles were transferred to larger 12 m2 pre-conditioned flow-through tanks, with settlement substrates (stone stools). After about 210 days, some of the juveniles had reached sizes up to 20 cm (ca. 100 g; Fig. 6c), which is the typical harvest size. The survival rates were between 30 and 50%. Battaglene et al. (1999) reported that the juveniles of H. scabra stocked at 20 to 31 mm have a growth rate of an overall average of 0.5 mm day− 1 over 2 months. Dong et al. (2006) reported that the growth of A. japonicus juveniles varied at different constant temperatures and the maximum specific growth rate (SGR) was 1.48% day− 1 at 16–18 °C. The result indicated that the growth rate of Stichopus sp. juveniles was considerable. Ongoing research trials to transfer juveniles into growout ponds or to eventually release in the wild are being undertaken. Stichopus sp. juveniles survived in abandoned abalone tanks (18 m2 in area and 1.2 m in depth). Small Stichopus sp. juveniles collected from the Weizhou Island grew an average of 17 g/week in growout tanks with 88% survival rate. This suggests that abalone tanks along the coast can provide a good environment to cultivate Stichopus sp. juveniles to adult size for commercialization or stock enhancement. Further research on abalone pond growout techniques is currently being conducted. 4. Conclusions This study shows that aquaculture of Curry fish is feasible under the proper environmental conditions. With the improvement of the rearing techniques over the past year, a 50% survival rate has been achieved though the average success remains approximately 30% of juveniles developed from every larval culture. Aquaculture could thus be an alternative or complement to fisheries. It can facilitate restoration of natural populations. Further research to complement the present work is being conducted on the commercial-scale aquaculture of this highly prized sea cucumber, which is also a dominant feature of the tropical marine ecosystem. Currently, aquaculture and stock enhancement of Curry fish might provide part of the solution to the local sea cucumber crisis. Acknowledgments This work was supported by the National Key Technologies R&D Program (2009BAB44B02); the Science and Technology Program of Guangdong Province (A200901E01, A200899E02, 2009B091300155, 2007A020300007-15) and Guangxi Province (0815006-2). References
Fig. 5. Average growth of the juveniles of Stichopus sp.. Bars are s.e.
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Fig. 6. Juvenile development of Stichopus sp.. a: 40-day juvenile; ramified tentacle (rt), papillae (pap), podia (pod). b: 60-day juvenile. c: 210-day juveniles.
C. Hu et al. / Aquaculture 300 (2010) 73–79 Battaglene, S.C., Seymour, J.E., Ramofafia, C., Lane, I., 2002. Spawning induction of three tropical sea cucumbers, Holothuria scabra, H. fuscogilva and Actinopyga mauritiana. Aquaculture 207, 29–47. Chen, J., 2003. Overview of sea cucumber farming and sea ranching practices in China. SPC Beche-de-Mer Information Bulletin, vol. 18, pp. 18–23. Chen, J., 2004. Present status and prospects of sea cucumber industry in China. In: Advances in sea cucumber aquaculture and management. A. Lovatelli (comp./ed.); Conand, C; Purcell, S.; Uthicke, S.; Hamel, J-F; Mercier, A. (eds.). FAO Fisheries Technical Paper. Rome, 463: 25–38. Chen, C.P., Hsu, H.W., Deng, D.C., 1991. Comparison of larval development and growth of the sea cucumber Actinopyga echinites: ovary-induced ova and DTT-induced ova. Marine Biology 109, 453–457. Conand, C., 1993. Ecology and reproductive biology of Stichopus variegatus an IndoPacific coral reef sea cucumber (Echinodermata: Holothuroidea). Bulletin of Marine Science 52, 970–981. Dong, Y.W., Dong, S.L., Tian, X.L., Wang, F., Zhang, M.Z., 2006. Effects of diel temperature fluctuations on growth, oxygen consumption and proximate body composition in the sea cucumber Apostichopus japonicus Selenka. Aquaculture 255, 514–521. FAO, 2008. Sea cucumbers: a global review of fisheries and trade: FAO Fisheries and Aquaculture Technical Paper, Rome, vol. 516, pp. 1–317. Guzman, H.M., Guevara, C.A., Hernandez, I.C., 2003. Reproductive cycle of two commercial species of sea cucumber (Echinodermata: Holothuroidea) from Caribbean Panama. Marine Biology 142, 271–279. Hamel, J.F., Pawson, D.L., Conand, C., Mercier, A., 2002. The sea cucumber Holothuria scabra (Holothuroidea: Echinodermata): its biology and its exploitation as beche-de-mer. Advanced Marine Biology 41, 131–233. Hamel, J.F., Hidalgo, R.Y., Mercier, A., 2003. Larval development and juvenile growth of the Galapagos sea cucumber Isostichopus fuscus. SPC Beche-de-mer Information Bulletin 18, 3–8.
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Liao, Y., 1997. Fauna Sincia: Phylum Echinodermata Class Holothuroidea. Science Press, Beijing. 1–51. Massin, C., 1999. Reef-dwelling Holothuroidea (Echinodermata) of the Spermonde Archipelago (South-West Sulawesi, Indonesia). Zoologische Verhandelingen 329, 1–144. Massin, C., Mercier, A., Hamel, J.F., 2000. Ossicle change in Holothuria scabra with a discussion of ossicle evolution within the Holothuriidae (Echinodermata). Acta Zoologica 81, 77–91. McEuen, F.S., 1988. Spawning behaviour of northeast Pacific sea cucumbers (Holothuroidea: Echinodermata). Marine Biology 98, 565–585. McEuen, F.S., Chia, F.S., 1991. Development and metamorphosis of two psolid sea cucumbers, Psolus chitonoides and Psolidium bullatum, with a review of reproductive patterns in the family Psolidae (Holothuroidea: Echinodermata). Marine Biology 109, 267–279. Otero-Villanueva, M., Ut, V.T., 2007. Sea cucumber fisheries around Phu Quoc Archipelago: a cross-border issue between South Viet Nam and Cambodia. SPC Bech-de-mer Information Bulletin, 25, pp. 32–36. Ramofafia, C., Byrne, M., Battaglene, S.C., 2003. Development of three commercial sea cucumbers, Holothuria scabra, H. fuscogilva and Actinopyga mauritiana: larval structure and growth. Marine and Freshwater Research 54, 657–667. Rowe, F.W.E., Gates, J., 1995. Echinodermata. In: Wells, A. (Ed.), Zoological Catalogue of Australia, vol. 33: i–xiii. CSIRO Australia, Melbourne, pp. 1–510. Tehranifard, A., Uryan, S., Vosoghi, G., 2006. Reproductive cycle of Stichopus herrmanni from Kish Island, Iran. SPC Beche-de-Mer Information Bulletin, vol. 24, pp. 22–27. Zaidnuddin, I., 2009. Observation of the frst grow out activities with Stichopus horrens juveniles in Malaysia. SPC Beche-de-Mer Information Bulletin vol. 29, 48.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Larval development and juvenile growth of the sea cucumber Stichopus sp. (Curry fish) Chaoqun Hu a,b,⁎,1, Youhou Xu a,b,c,1, Jing Wen a,b,c,1, Lvping Zhang a,b, Sigang Fan a,b,c, Ting Su a,b,c a
Key Laboratory of Marine Bio-resources Sustainable Utilization (LMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China Key Laboratory of Applied Marine Biology of Guangdong Province and The Chinese Academy of Sciences (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China c Graduate University of Chinese Academy of Sciences, Beijing, 100049, China b
a r t i c l e
i n f o
Article history: Received 12 May 2009 Received in revised form 11 September 2009 Accepted 14 September 2009 Keywords: Curry fish Spawning Larvae Development Juvenile Growth Aquaculture
a b s t r a c t The global populations of Curry fish have been severely depleted over the past decade. This study describes spawning, fertilization, larval rearing, and juvenile growth in a commercially important Stichopus species. Data pooled from monthly trials conducted over 2 years indicate that, under optimal conditions, juveniles can be grown to a size of ca. 20 cm in length in 7 months. The survival rates are typically between 30 and 50%. Pilot research indicates that the growth of young sea cucumbers in abandoned abalone tanks has potential. Overall, this study demonstrates that Curry fish can be reared in captivity, thus providing an alternative to fisheries, or a way to maintain sustainable harvests and eventually contribute to the restoration of the natural populations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumbers (Echinodermata: Holothuroidea), a diverse group of marine invertebrates, known as ‘beche-de-mer’ or ‘trepang’, are generally consumed in Asia, where they are regarded as traditional medicine, delicacies and aphrodisiacs. Sea cucumbers present a high nutritional value due to their high protein, low fat content, aminoacid profile and are a rich source of trace elements (Chen, 2003). Curry fish is a yellow/brown sea cucumber, covered with small dark spots. It is a medium to large-sized species (300–700 mm), stout, thick and firm, with low papillae, and a moderately smooth tegument. Curry fish has a wide Indo–West Pacific distribution (Rowe and Gates, 1995), can be found on tropical reefs, and is considered edible with medicinal properties (Liao, 1997). The fast pace of development of sea cucumber fisheries to supply the growing international demand for beche-de-mer is placing most fisheries and many sea cucumber species at risk including Curry fish, which was highly abundant and is nowadays rare or locally extinct (FAO, 2008; Otero-Villanueva and Ut, 2007).
In China, the soaring price of Apostichopus japonicus has stimulated the development of aquaculture including sea ranching activities. Presently, China is successfully producing an estimated 10,000 t, dry weight, of A. japonicus from aquaculture, mainly to supply local demand. Chen (2004) noted that recovery plans for A. japonicus are in place together with the establishment of many conservation zones. Recently in Malaysia, great progress of research on aquaculture for Stichopus horrens has been achieved (Zaidnuddin, 2009). The reproductive biology of Stichopus variegates (presently revised to Stichopus herrmanni, Rowe and Gates, 1995) has been reported by Conand (1993). Tehranifard et al. (2006) reported the reproductive cycle of S. herrmanni from Iran. However, no published work is available on the larval development, juvenile growth and artificial propagation of Curry fish in China. This study presents data on the development of embryos, larvae and juveniles of Curry fish Stichopus sp. over a 16-month period at Weizhou Island, China. The results contribute to maintenance of sustainable use and will contribute to the restoration of natural populations.
2. Materials and methods ⁎ Corresponding author. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China. Tel.: +86 20 89023216; fax: +86 20 89023218. E-mail address: [email protected] (C. Hu). 1 These authors have equally contributed to this work. 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.09.033
2.1. Collection and maintenance of animals In total 648 adult sea cucumbers (mean wet weight > 500 g) were collected from nearby coastal areas of Weizhou Island (21°03′N,
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Fig. 1. Dorsal ossicles of Stichopus sp.. a: Tables. b: C-shape rod. c: Rosette.
109°07′E) and maintained at the Weizhou Aquaculture Farm to serve as broodstock from April 2006 to October 2007. The adults were conditioned in the tanks for a few days prior to spawning. 2.2. Species identification Ossicles were prepared by scanning electronic microscopy (SEM). The dorsal body walls of ten sea cucumbers were obtained by dissection, and fixed in 95% ethanol at − 20 °C in the farm. In the laboratory, the body walls were treated with 10% sodium hypochlorite solution for 1–2 min to obtain ossicles, and carefully rinsed with distilled water at least three times following procedures by Massin et al. (2000). The specimens were critical-point dried and coated with gold–palladium in a sputter coater (Hitachi, E-1010) and ossicles were observed using 20 kv accelerating voltage and 1000–3000 magnifications under a Hitachi S-3400N SEM. Species identifications were based on the taxonomic descriptions of dermal ossicles by Liao (1997) and Massin (1999).
and concentration dictated by the daily observation of the digestive tract contents (the number of cells in the stomach). The water in each tank was changed every day by draining the tanks through a 53-μm sieve. After the first water change, the early auricularia larvae were stocked at a density of 0.2 larvae mL− 1. Seven days after fertilization, 100 sets of plastic settlement sieves were conditioned in an outdoor tank supplied with filtered running seawater to promote growth of a diatom film. Each set of substrates consisted of six sieves measuring 300 × 240 mm, stacked
Table 1 Number of spawning observations. Year
Month
Date
Lunar phase
na
Maleb
Femaleb
2006
May
26-05-2006 27-05-2006 28-05-2006 29-05-2006 10-06-2006 25-06-2006 26-06-2006 27-06-2006 28-06-2006 10-07-2006 24-07-2006 25-07-2006 26-07-2006 27-07-2006 08-08-2006 23-08-2006 24-08-2006 25-08-2006 26-08-2006 07-09-2006 21-09-2006 22-09-2006 23-09-2006 24-09-2006 14-06-2007 15-06-2007 16-06-2007 17-06-2007 28-06-2007 13-07-2007 14-07-2007 15-07-2007 16-07-2007 28-07-2007 12-08-2007 13-08-2007 14-08-2007 15-08-2007 27-08-2007 10-09-2007 11-09-2007 12-09-2007 13-09-2007
Last New New New Full Last New New New Full Last New New New Full Last New New New Full Last New New New Last New New New Full Last New New New Full Last New New New Full Last New New New
148 148 148 148 342 342 342 342 342 418 418 418 418 418 518 518 518 518 518 518 518 518 518 518 540 540 540 540 578 578 578 578 578 613 613 613 613 613 648 648 648 648 648
13 38 36 16 0 4 73 68 12 0 6 73 66 15 0 10 49 39 12 0 8 24 20 12 12 79 76 29 0 14 85 88 23 0 16 79 73 10 0 20 37 29 16
0 3 8 2 0 3 21 25 3 0 2 28 29 4 0 3 21 18 2 0 1 9 9 0 4 39 38 4 0 3 32 41 3 0 4 33 35 2 0 3 11 8 0
June
2.3. Spawning and fertilization On arrival (1800–2000 h) at the farm, the specimens were placed in aerated 2000-L tanks and monitored overnight for spawning activity. Spawning of sea cucumber was conducted by open-air drying followed flow-through filtered seawater stimulation. Males and females were isolated in plastic buckets. As soon as they showed signs of imminent spawning (spawning behavior, such as sway), they were isolated. Each female was then placed separately in a 300-L spawning tank and maintained until release of eggs. The female was removed, and the spermatozoa solution obtained from isolated spawning males, was added to the eggs. The best fertilization rates and lowest occurrence of polyspermy were obtained with a concentration of 5–10 spermatozoa per egg. After fertilization, the eggs were rinsed to remove excess sperm. 10 mL water was randomly collected after gentle stirring, and the number of eggs was counted, egg counts were performed at least three times and the amount was calculated. Individual female Stichopus sp. spawned a mean of 3.48 × 106 eggs (s.e. = 1.4 × 105, n = 5 females). Then the eggs were transferred to hatchery tanks at a density of 0.5 eggs mL− 1.
July
August
September
2007
July
2.4. Embryo and larval culture Several air stones positioned at the bottom of each culture tank provided sufficient aeration and ensured gentle water circulation. The larvae were reared at ambient seawater temperature (27–31 °C). In the wild, Curry fish lives under substrate with low light levels. Therefore, the photoperiod was designed as 0 h light:24 h dark in this experiment. When the auricularia had a functional gut, the larvae were fed every day using a wet mix of commercial marine yeast (Rhodotorula) and microalgae (dominated by Rhodomonas and Dunaliella) at a frequency
June
August
September
a b
Number of Stichopus sp. collected during each field trip. Number of spawning individuals.
C. Hu et al. / Aquaculture 300 (2010) 73–79
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Fig. 2. Spawning of Stichopus sp.. a: Pre-spawning posture; podia (pod), tentacle (ten). b: Ejaculation; papillae (pap), sperm (s). c: Pre-spawning posture; inflated gonopore (ig). d: Spawning; eggs (e).
with a 200-mm gap. When the late auricularia stage was reached, about 40 sets of substrates were placed in each rearing tanks. The larval feed was stopped after settlement. Continuous water exchange was maintained with filtered running seawater until the juveniles were detached. 2.5. Juvenile culture Juvenile sea cucumbers were fed ad libitum daily at 1900 h with sifted pieces of semi-dried Sargassum thunbergii (11.70% crude protein, 0.67% crude lipid, 12.89% ash, moisture b 1.00%; energy 8.55 kJ g− 1 dry mass) and the pieces (radius around 0.8 mm, weight around 2.5 mg) were insoluble in the sea water (An et al., 2007). The juvenile feed was increased as the individuals grew. Competitors such as copepods, ciliates, and protozoans were controlled using pesticide (Trichlorphon) and filtration. A concentration of 2 mL Trichlorphon m− 3 was applied every 2 h, and water was changed through a 53-μm sieve. 2.6. Specimen preparation for optical microscopy The spermatozoa, eggs, embryos, larvae, and juveniles were fixed in 2.5% glutaraldehyde in 2.0 μm filtered seawater. The specimens were viewed with a Leica DMR optical microscope. 3. Results and discussion 3.1. Species identification Three kinds of dermal ossicles were found in the dorsal body walls of Stichopus sp., tables, C-shaped rods and rosettes (Fig. 1). The size, shape and structure of these ossicles were in accordance with the descriptions of Curry fish by previous reports (Liao, 1997; Massin, 1999). 3.2. Spawning periodicity Close monitoring of the broodstock in the field over several months (Table 1) revealed that Stichopus sp. followed a predictable lunar spawning periodicity. They often spawned within the first
2 days following the new moons from May to August on Weizhou Island, even in captivity. This behavior has been observed for some aspidochirotid species including Holothuria scabra and Isostichopus fuscus (Hamel et al., 2002; Hamel et al., 2003). In contrast, some aspidochirotids including Isostichopus badionotus and Holothuria mexicana, are known to spawn within the first 5 days following the full moons (Guzman et al., 2003). Spawning during the warm season, the phenomenon was consistent with the observation by Conand (1993). However, the previous study did not investigate the lunar spawning periodicity. The spawning behavior of specimens in the field was consistent with previous observation on S. herrmanni (Conand, 1993). Pre-spawning behavior included rolling and twisting on the substrate (Fig. 2a). Males raised the anterior end prior to spawning and often swayed from side to side, or in a circular motion, as they released sperm. Most aspidochirotids lift the anterior end of the body from the substratum during spawning to facilitate dispersal of gametes and fertilization (McEuen, 1988). Male Stichopus sp. spawned 1–2 h before females, and spawned more bursts and longer. Male sea cucumbers are reported to spawn before females. This is suggested to be a proximal signal for synchronising females to spawn (McEuen, 1988). Male Stichopus sp. spawned soon after being placed in the spawning tank and released a steady stream of sperm from the single anterior gonopore (Fig. 2b). Males remained erect and spawned continuously for up to 3 h. They usually resumed spawning after artificial disturbance such as touch and carrying. Females typically started spawning by raising their bodies off the substrate, in a similar manner to males. It was possible to distinguish females from males shortly before spawning from the bulge around the gonopore (Fig. 2c). They remained erect for 15–30 min before releasing a short powerful burst of pale yellow to pink eggs, which lasted for 10– 15 s (Fig. 2d). The short powerful release of eggs by each species is typical of many holothurians, and forces eggs into the water column and aids dispersion and fertilization (Battaglene et al., 2002). Spermatozoa comprised a head with 2.6 μm (s.e. = 0.4 μm, n = 5 males) mean diameters and a 48 μm (s.e. = 3.8 μm, n = 5 males) long tail (Fig. 3a). The mean egg diameter of Stichopus sp. was 180 μm (s.e. = 2.8 μm, n = 5 females) (Fig. 3b), and the result agreed with the previous report (Conand, 1993).
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Fig. 4. Juvenile development of Stichopus sp.. a: Pentactula; tentacle (ten), ossicle (oss). b: Dorsal ossicles. c: 15-day juvenile; podia (pod), digestive tract (dt). d: 20-day juvenile; papillae (pap). e: 25-day juvenile. f: 30-day juvenile.
3.3. Embryonic and larval development The development of Stichopus sp. is initiated with the elevation of the fertilization envelope followed the expulsion of the first polar body occurs roughly 20 min after fertilization. The first cleavage was equal, radial and holoblastic, and divided the cell into two equal hemispheric blastomeres (Fig. 3c). The second cleavage again occurred along the animal–vegetal axis, yielding more spherical blastomeres (Fig. 3d–g). The blastula stage was reached by 3 h. By 6 h, the blastula had a conspicuous ciliary cover and continuously rotated within the envelope propelled by their cilia (Fig. 3h). Embryos hatched from the fertilization envelope as early gastrulae, ca. 14 h after fertilization (Fig. 3i). These early gastrulae swam with the help of cilia covering their entire surface; they elongated into full-size gastrulae after ca. 16 h (Fig. 3j–k). The auricularia larval stage was reached ca. 35 h after fertilization (Fig. 3l). Growing auriculariae could be observed during the next 4 days of culture. The buccal ciliated cavity, the mouth, the oesophagus, the intestine, as well as the cloaca were clearly visible (Fig. 4m–n). At this stage, they began to accumulate hyaline spheres (Fig. 4o). After 7–8 days, the auriculariae reached a maximum size of 1.4 mm, and they possessed an axohydrocoel (Fig. 3n–p). The auriculariae subsequently transformed to the doliolaria stage (Fig. 3p–r). In the course of this process, the larvae shrank down to nearly 50% of their initial size, the buccal ciliated cavity disappeared and the ten hyaline spheres became aligned (Fig. 3r). The doliolaria stage was reached ca. 9–10 days after fertilization (Table 2). The larvae stopped feeding. The cilia became aligned in five distinct bands along their cylindrical body. The digestive tract was obscure by the translucent body. Like other holothurians (Asha and Muthiah, 2005; Ramofafia et al., 2003; Hamel et al., 2003; McEuen and Chia, 1991), Stichopus sp.
transformed to the juvenile stage (Table 2). In most trials, the development, settlement and early growth of the larvae were somewhat asynchronous, and different stages and sizes could be found simultaneously in the culture. In extreme examples, auricularia and 6 mm long juveniles were in the same tanks. However, the developmental chronology in Table 2 was based on stages achieved by most of the culture. The larval stage of Stichopus sp. was relatively short, with newly metamorphosed juveniles appearing in the cultures as early as 15 days after fertilization. Other tropical holothurians such as H. scabra (Ramofafia et al., 2003), Holothuria spinifera (Asha and Muthiah, 2005), and I. fuscus (Hamel et al., 2003) also have a short development time. However, tropical species Actinopyga echinites, needs about 20 days to transform to the juvenile stage (Chen et al., 1991). The cold temperate species Psolus chitonoides and Psolidium bullatum, need more than 28 days to complete metamorphosis (McEuen and Chia, 1991).
3.4. Juvenile growth The doliolariae transformed into early pentactulae that possessed five buccal tentacles and dermal ossicles 9–10 days post fertilization (Fig. 4a–b). At this stage, the larvae began to swim close to the substrate and successively went through swimming and settling phases. Definitive settlement, with the complete loss of cilia, completion of metamorphosis and emergence of the two first ambulacral podia, occurred ca. 15 days after fertilization (Fig. 4c). Although the settled juveniles were observed as early as day 12 post fertilization, a majority of juveniles measured 1 to 1.5 mm in length recorded after 20 days of culture. At this stage, papillae developed along the dorsal body wall (Fig. 4d). The juveniles reached ca. 2–3 mm within 5 days (Fig. 4e), and 5 mm after ca. 30 days. They possessed
Fig. 3. Embryonic and larval development of Stichopus sp.. a: Spermatozoa. b: Fertilized egg. c–g: Cleavage stages. h: Blastula; envelope (env). i–k: Gastrula. l: Early auricularia. m: Mid auricularia; buccal ciliated cavity (bcc), oesophagus (oes), intestine (int), cloaca (clo). n–q: Late auricularia; mouth (mou), axohydrocoel (axo), hyaline sphere (hs). r: Doliolaria; cilia band (cb), digestive tract (dt).
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Table 2 Development of Stichopus sp., from fertilization to juvenile. Stage
Size (μm)
Time
Fertilization Expulsion of the first polar body 2-cell 4-cell 8-cell 64-cell 128-cell Blastula Rotated blastula Early gastrula Gastrula Late gastrula Early auricularia Mid auricularia Auricularia Late auricularia Doliolaria Pentactula Juvenile
150–200 150–200 200–250 200–250 200–250 250–300 250–300 250–300 250–300 300–350 300–350 300–350 350–450 500–700 800–1000 1000–1300 400–500 400 400
0 20 (min) 45 (min) 51 (min) 88 (min) 137 (min) 149 (min) 3 (h) 12 (h) 14 (h) 16 (h) 20 (h) 35 (h) 3–4 (d) 5–6 (d) 7–8 (d) 9–10 (d) 10–11 (d) 12 (d)
several papillae, more podia and their intestine exhibited strong peristaltic movement (Fig. 4f). The juveniles continued to grow at a rate of ca. 1.0 mm per day for the next 3 to 4 weeks (Fig. 5). When they were ca. 5 mm in length, the juveniles started to accumulate orange-green pigment. In 8 mm long juveniles, the tip of the tentacles became ramified. After 40 days of culture, the juveniles were 1.8–2.0 cm long and 4 mm wide (Fig. 6a). The body wall became more opaque as the ossicle density and the tegument thickness increased. When the juveniles reached ca. 2 cm in length, the whitish colouration that characterised the early stages of life was gradually replaced by a brownish tinge similar to the one observed in adults. After approximately 60 days, the juveniles were ca. 4 cm long and 2 cm wide and were subsequently released in outdoor ponds, or into the field, to complete their growth (Fig. 6b). From settlement
(0.5–1.0 cm) onward, the juveniles were transferred to larger 12 m2 pre-conditioned flow-through tanks, with settlement substrates (stone stools). After about 210 days, some of the juveniles had reached sizes up to 20 cm (ca. 100 g; Fig. 6c), which is the typical harvest size. The survival rates were between 30 and 50%. Battaglene et al. (1999) reported that the juveniles of H. scabra stocked at 20 to 31 mm have a growth rate of an overall average of 0.5 mm day− 1 over 2 months. Dong et al. (2006) reported that the growth of A. japonicus juveniles varied at different constant temperatures and the maximum specific growth rate (SGR) was 1.48% day− 1 at 16–18 °C. The result indicated that the growth rate of Stichopus sp. juveniles was considerable. Ongoing research trials to transfer juveniles into growout ponds or to eventually release in the wild are being undertaken. Stichopus sp. juveniles survived in abandoned abalone tanks (18 m2 in area and 1.2 m in depth). Small Stichopus sp. juveniles collected from the Weizhou Island grew an average of 17 g/week in growout tanks with 88% survival rate. This suggests that abalone tanks along the coast can provide a good environment to cultivate Stichopus sp. juveniles to adult size for commercialization or stock enhancement. Further research on abalone pond growout techniques is currently being conducted. 4. Conclusions This study shows that aquaculture of Curry fish is feasible under the proper environmental conditions. With the improvement of the rearing techniques over the past year, a 50% survival rate has been achieved though the average success remains approximately 30% of juveniles developed from every larval culture. Aquaculture could thus be an alternative or complement to fisheries. It can facilitate restoration of natural populations. Further research to complement the present work is being conducted on the commercial-scale aquaculture of this highly prized sea cucumber, which is also a dominant feature of the tropical marine ecosystem. Currently, aquaculture and stock enhancement of Curry fish might provide part of the solution to the local sea cucumber crisis. Acknowledgments This work was supported by the National Key Technologies R&D Program (2009BAB44B02); the Science and Technology Program of Guangdong Province (A200901E01, A200899E02, 2009B091300155, 2007A020300007-15) and Guangxi Province (0815006-2). References
Fig. 5. Average growth of the juveniles of Stichopus sp.. Bars are s.e.
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Fig. 6. Juvenile development of Stichopus sp.. a: 40-day juvenile; ramified tentacle (rt), papillae (pap), podia (pod). b: 60-day juvenile. c: 210-day juveniles.
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