Aquaculture 404–405 (2013) 47–54 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online Spawning, larval development and juvenile growth of the sea cucumber Stichopus horrens Chaoqun Hu a, b,⁎, 1, Haipeng Li a, b, c, 1, Jianjun Xia a, b, 1, Lvping Zhang a, b, Peng Luo a, b, Sigang Fan a, b, c, Pengfei Peng a, b, c, Haipeng Yang a, b, c, Jing Wen a, b, c a South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China Key Laboratory of Marine Bio-resources Sustainable Utilization (LMB), 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 University of Chinese Academy of Sciences , Beijing, 100049, China b a r t i c l e i n f o Article history: Received 15 April 2012 Received in revised form 14 March 2013 Accepted April 2013 Available online 17 April 2013 Keywords: Stichopus horrens Spawning Larval development Juvenile Growth Aquaculture a b s t r a c t Tropical commercial sea cucumber Stichopus horrens is extensively distributed throughout the tropical Indo-Pacific region, and wild stocks are severely depleted over the past decade This study firstly presented preliminary results on the spawning, the development of larvae and the growth of the juvenile of S horrens in Xisha Islands It was proved that S horrens followed a predictable lunar spawning periodicity and might reproduce throughout the year in Xisha Islands Dry stimulation was a simple and efficient way to induce S horrens to spawn The mean quantity of spawned eggs was 1.37 × 106 (s.e = 2.09 × 106, n = 8), and the mean egg diameter was 114 μm (s.e = 2.8 μm, n = 8) S horrens developed from fertilized eggs to pentactula larval after 19–27 days at 25–27 °C From oosperm to settled juvenile, the survival rate of S horrens was 1.0%–6.83% when enough live microalgae were supplied The mean growth rate of juveniles was 0.4 mm per day and the survival rate was about 23.3%–50.5% during 80 days culture after settlement Then some juveniles of S horrens were further cultivated in tanks and majority were released in the wild Ongoing observations showed good survival and growth of juvenile in the wild The study indicates S horrens are amenable to culture using standard techniques which may also be applied on the culture of other tropical species They exhibit a relatively quick growth both in indoor and wild culture Furthermore, the test results support possible aquaculture and stock restoration using hatchery produced stocks © 2013 Elsevier B.V All rights reserved Introduction Stichopus horrens (Echinodermata: Holothuroidea) is widespread in the tropical Indo-Pacific area (Hearn and Pinillos, 2006; Liao, 1997; Massin et al., 2002; Rasolofonirina et al., 2004) It usually hides in crevices, cracks and caves during the day, and emerges at night to feed (Hearn and Pinillos, 2006) The population status of this species is poorly understood The research of this species in the Galápagos Archipelago shows that the size of S horrens in the wild is in the range of 9–30 cm and no recruitment is observed, accounting for that the juvenile is highly cryptic or has spatial separation from adult (Hearn and Pinillos, 2006) As a commercial sea cucumber, S horrens is harvested for traditional medicine and delicacies, generally consumed in the world (Baine and Choo, 1999; Rasolofonirina et al., 2004), in China it is one of the edible tropical sea cucumbers (Liao, 1997) In the other countries, such as ⁎ Corresponding author at: South China Sea Institute of Oceanology, Chinese, Academy of Sciences, Guangzhou, 510301, China Tel.: +86 20 89023216; fax: +86 20 89023218 E-mail address: cqhu@scsio.ac.cn (C Hu) These authors have equally contributed to this work 0044-8486/$ – see front matter © 2013 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.aquaculture.2013.04.007 Madagascar (Rasolofonirina et al., 2004) and Malaysia (Baine and Choo, 1999), it is subject to commercial exploitation In the Galapagos Islands, it is illegal to fish S horrens, although its price is much lower than that of the local species Isostichopus fuscus (Hearn and Pinillos, 2006) Considering increasing demand in sea cucumbers, it is urgent for us to maintain the natural population and realize sustainable utilization in southern China Sea cucumber aquaculture would provide sufficient beche-de-mer product to satisfy the increasing market demand and enhance the declining wild sea cucumber population at the same time So far, the artificial propagation has been successful in some commercial holothurians (Archer, 1996; Chen and Chian, 1990; Dabbagh et al., 2011; Hamel et al., 2003; Hu et al., 2010; James et al., 1988; Laxminarayana, 2005; Ramofafia et al., 1995, 2003), which offers an alternative to harvesting wild populations For example, the development of aquaculture including sea ranching activities has been stimulated by the high price of Apostichopus japonicus in northern China (Chen, 2003, 2004) For the tropical species, Holothuria scabra has been reared successfully in several locations throughout the Indo-Pacific areas (Battaglene et al., 1999; Eeckhaut et al., 2008; Giraspy and Ivy, 2005, 2010; Mercier et al., 2000) As to S horrens, little is known about its 48 C Hu et al / Aquaculture 404–405 (2013) 47–54 reproductive biology in southern China This species reproduces sexually throughout the year in Galapagos Islands (Toral-Granda, 2008) and possesses asexual reproduction in winter (Kohtsuka et al., 2005) It is reported that the juveniles of S horrens have been reared recently in Malaysia (Zaidnuddin, 2009) In this study, we present details of the spawning and the development of embryos, larvae and juveniles of S horrens in Xisha Islands, China for the first time The results will contribute to the stock enhancement, sustainable use and restoration of natural populations Materials and methods 2.1 Collection of animals Adult sea cucumbers (mean wet weight>500 g), S horrens were collected by snorkel (1–8 m depth) from nearby coastal areas of Xisha Islands (16°49′N, 112°19′E) monthly between May 2010 and Sep 2011 except months (Jan to Mar 2011, Jul 2011) The animals were maintained indoor in 4000-L fiberglass tanks to serve as brood stock at the Xisha Aquaculture Hatchery prior to inducing spawning These fiberglass tanks had been aerating all the day, and the water was changed twice everyday in the morning and evening respectively Animals were fed with Sargassum thunbergii powder (Dalian FengYuanDa Feed Co., LTD, Liaoning Province, China) at a rate of 10 g m −3 per day, and stopped feeding when they were stimulated to spawning 2.2 Spawning and fertilization Three methods had been carried out to induce spawning Thermal stimulation: The sea cucumbers were kept at 28 °C before stimulation, followed by raising water temperature by °C for about h, and then resumed to natural temperature Dry stimulation: let the water out of the tanks, left the broodstock in open-air for h, and added sea water Microalgae stimulation: Live microalgae (50,000–1,000,000 cells mL −1) for example, Dunaliella sp and Chaetoceros Mueller that could used for feeding larvae were added in the tanks for a day The specimens were monitored overnight The individual, which showed signs of imminent spawning behavior, was immediately moved to a 200 L plastic bucket, and they continued to spawn in the container When the female spawned over, it was taken away After the number of eggs was counted, spermatozoa (5–10 per eggs) were added to buckets After 30 min' fertilization time, the spawned eggs were rinsed to remove excess sperm Eggs were calculated and transferred to hatchery tanks (2 m or m 3) at the density of 1–1.5 eggs mL −1 2.3 Embryo and larval culture Several air stones were positioned at the bottom of each culture tank to provide sufficient aeration and ensure gentle water circulation The larvae were reared indoor at seawater temperature 25– 30 °C and the pH value between 7.9 and 8.2 The water in each tank was partially changed twice a day using a 60-μm mesh outlet screen insider the hatchery tanks The seawater was sand filtered, and then passed through 1-μm filter bag and finally sterilized by UV Deformed or dead larvae, faeces from larvae and excess food were removed at regular intervals by gently siphoning the tank base When the embryo had functional gut (43 h after fertilization), the feeding commenced two days post spawning A mixture of microalgae consisting of Chaetoceros mueller, Dunaliella sp., and Chlorella pynenoidosa (7:2:1) was provided as food of the larvae The food was fed regularly three times a day, and the concentration of food has been adjusted based on the daily observation of the digestive tract The amount of food was about 10,000–15,000 cells mL−1 in the early auricularia stage, then raised to 15,000–25,000 cells mL−1 in the mid auricularia stage, next got to 25,000–35,000 cells mL−1 in the auricularia stage, finally reached 35,000–45,000 cells mL−1 in the late auricularia stage Microalgae were cultured in 200 L plastic bucket using f/2 beta growth medium (Guillard and Ryther, 1962) at 25–30 °C with natural photoperiod Only microalgae in the logarithmic growth phase were used As the larvae reached late auricularia phase, the sets of plastic sheets were immersed in outdoor tanks with f/2 beta growth medium added and benthic diatoms inoculated to promote growth of biological film and diatoms Each set of settlement plate is consisted of 10 wavy polycarbonate plates measuring 400*300 mm, stacked with a 25 mm gap 20 sets of cultured substrates were placed in each rearing tank when 5% of the late auricularia had metamorphosed into doliolaria The larval feed was stopped after settlement 2.4 Juvenile culture Early juveniles consumed the benthic diatoms on the settlement plates From the 5th day after settlement, they were fed twice daily (08:00 and 19:00) The food was included Oceanic Red Yeast (Beihai Qunlin bioengineering Co., LTD, Guangxi Province, China), microalgae powder of Dunaliella sp (Neimenggu Lantai bioengineering Company, Neimenggu province, China), powder of spirulina sp (South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou province, China), algae powder of Sargassum thunbergii (Dalian FengYuanDa Feed Co., LTD, Liaoning Province, China) The mixture was immersed in sea water for h and filtered by 60-μm sieve, before feeding The component rate was 1:1:2:5 Firstly, artificial feeding was added in at g m −3 When the juvenile reached 10 mm, the feeding was increased at g m −3 The frequency of feeding was increased as the individuals grew The main predator copepods were controlled and eliminated by using Trichlorphon at a dosage of 1.5 g m −3, and changing water h later Aeration was provided continuously and half of the water was exchanged daily During the course of the rearing, the water pH varied from 7.9 to 8.2, ammonia was less than 0.2 mg L −1, and nitrite was less than 0.1 mg L −1 The temperature of seawater was varied from 24 °C to 30 °C When they reached 30 mm, the majority of juvenile was released in wild to enrich the wild population The rest was cultured in the tanks The growth of juvenile in the wild was studied There were sixty cages that were released to the wild (16°49′56′′N, 112°19′42′′E) in Jan 2011 The other juveniles were sowed in the same place It was a rectangle region, an area of km Each cage was numbered by a plastic card Corallites and polyvinyl chloride pipes were added in the cages to provide settlement for juveniles Forty juveniles were stocked in each cage The growth of sea cucumber in the wild was studied by monitoring individuals every 2–3 months The cages were recaptured by diver, and put them back where we found them The sea cucumbers were measured by a ruler graduated in millimeter when they were completely relaxed in the water The weight of sea cucumber were measured using an electronic balance (up to 0.01 g), after the excess water was removed from anus by putting they straight and the external water was blot dried 2.5 Specimen preparation for optical microscopy The fertilized eggs, embryos, larvae and early juveniles were viewed and recorded with a Nikon (Eclipse 50i) optical microscope Results 3.1 Spawning periodicity From May to Sep 2010, the stimulation spawning trials were taken Batches of twenty to fifty animals were kept in a 2000 L tanks to induce spawning The successful experiment was present in Table In C Hu et al / Aquaculture 404–405 (2013) 47–54 reproduction time (except full lunar phase), 28 individuals spawned successfully by thermal stimulation, 45 by dry stimulation, and by microalgae stimulation The average spawning rate of dry stimulation in trial was 28.6%, while thermal stimulation was 17.9%, and microalgae stimulation was 3.75% Compared with the three methods, dry stimulation was the simplest and efficient way hence used in the later experiments The spawning trials (Table 2) showed that S horrens followed a lunar spawning periodicity and usually reproduced during crescent days on Xisha Islands Spawning of S horrens was successfully induced in every month except for May and July 2010, during which the individuals used were maintained indoor longer The individuals spawned were those cultured for less than days in captivity Moreover, no spawning was observed for those kept for more than 12 days at the hatchery There were 1222 of S horrens used in the spawning trials, among which 188 females ovulated and 320 males ejaculated S horrens usually climbed to the water surface, and kept moving, rolling and twisting on the substrate prior to spawning Males swayed their anterior end body from side to side or stay on tanks, and then they released sperm (Fig 1b) Males spawned before females, and spawned more often and for longer (up to h), and could repeat for days Females commonly started spawning about 0.5–1 h after the first male spawning Females often moved around, raised their anterior body off the substrate, in a stronger manner compared to males Gonepore became cone-shaped apical papillae which was the most important character before spawning, an easy way to distinguish females from males (Fig 1a, c) Females spawned once in one month and expulsed eggs in a short powerful spurt, which only lasted for several seconds (Fig 1d) The quantity of spawned eggs varied among the individuals, with a mean of 1.37 × 106 eggs (s.e =2.09 × 106, n = 8) The mean egg diameter was 114 μm (s.e =2.8 μm, n = 8) 3.2 Embryonic and larval development The chronology of development for S horrens was shown in Table The zygote (Fig 2a) cleaved into two equal hemispheric blastomeres (Fig 2b) ca 40 after fertilization Subsequently, the embryos Table Numbers of observed spawning individuals Time Lunar phase Numbera Male/femaleb Daysc 13-05-2010 14-05-2010 15-05-2010 28-05-2010 11-06-2010 11-06-2010 12-06-2010 12-06-2010 26-06-2010 11-07-2010 12-07-2010 13-07-2010 26-07-2010 9-08-2010 9-08-2010 10-08-2010 11-08-2010 24-08-2010 7-09-2010 7-09-2010 8-09-2010 9-09-2010 10-09-2010 22-09-2010 8-10-2010 9-10-2010 22-10-2010 7-11-2010 20-11-2010 5-12-2010 6-12-2010 20-12-2010 2-04-2011 3-04-2011 17-04-2011 2-05-2011 17-05-2011 30-7-2011 31-07-2011 1-08-2011 28-08-2011 29-08-2011 30-08-2011 27-09-2011 28-09-2011 Last New New Full Last Last New New Full Last New New Full Last Last New New Full Last Last New New New Full New New Full New Full Last New Full Last New Full Last Full Last New New Last New New New New 36 36 36 32 15 32 15 32 40 40 40 40 32 46 30 46 46 26 64 50 64 64 64 45 46 46 32 28 33 51 51 29 28 28 25 40 43 50 50 50 258 258 258 158 158 0/0 0/0 0/0 0/0 1/0 0/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 6/1 0/0 8/0 7/8 0/0 4/1 0/0 4/1 19/2 14/1 0/0 16/6 7/1 0/0 13/1 0/0 5/1 11/4 0/0 6/1 4/1 0/0 11/5 0/0 3/0 18/6 6/2 9/50 103/56 36/17 1/15 6/8 12 13 14 18 19 20 21 22 17 12 2 2 2 2 5 a Table Three stimulation ways were used in the trial (dry stimulation, thermal stimulation and microalgae stimulation), and the individuals for each experiment group was kept in a m3 tank Time Lunar phase Way Numbera Male/femaleb %c 9-08-2010 Last 10-08-2010 New 11-08-2010 New 7-09-2010 Last 8-09-2010 New 9-09-2010 New 10-09-2010 New 22-09-2010 Full Dry Thermal Dry Thermal Dry Thermal Dry Thermal Microalgae Dry Thermal Microalgae Dry Thermal Microalgae Dry Thermal Microalgae Dry Thermal 23 23 23 23 23 23 22 22 20 22 22 20 22 22 20 22 22 20 23 22 4/1 2/0 6/0 2/0 4/5 3/3 3/1 1/0 0/0 3/0 1/1 0/0 9/1 8/1 2/0 7/1 6/0 1/0 0/0 0/0 21.7% 8.7% 26.1% 8.7% 39.1% 26.1% 18.2% 4.5% 0% 13.6% 9.1% 0% 45.5% 40.9% 10.0% 36.4% 27.3% 5.0% 0% 0% a b c : Numbers of sample : Numbers of spawning individual : The percentage of spawning individual 49 b c : Numbers of sample : Numbers of spawning individual : The days of sea cucumber maintained indoor divided roughly once per 20 The blastula stage was reached by 200 post fertilization (Fig 2c) The embryos hatched from fertilization envelope and developed into early gastrulae, ca 12–14 h after fertilization (Fig 2d) About 43 h later, the early auricularia stage was reached (Fig 2e) With further development, they grew gradually in size and began to accumulate hyaline spheres (Fig 2f–g) After 13 days, the auricularia reached a maximum size of 1.2–1.4 mm, and they possessed an anxohydrocoel and six pairs of obvious hyaline spheres (Fig 2h) In the following hours, auricularia initiated the metamorphosis to the doliolaria (Fig 2i) In this process, the larvae shrank down to nearly half of their initial size, the buccal cavity disappeared and the hyaline spheres closed together The sea cucumber started metamorphosis, transformed to pentactulae larvae after 19 days (Fig 3a) There were about 40,000 larvae in each tank which reared to the juvenile stage in the trial of October 2010 In the later trial (Dec 2010–Jul 2011), only a few larvae developed into juvenile due to lacking of microalgae as food source In Sep 2011, juveniles, about 110,000, were survived in each tank Table showed the survival of S horrens at different development phases from early auricularia to settled juvenile 50 C Hu et al / Aquaculture 404–405 (2013) 47–54 Fig Spawning of Stichopus horrens a: Pre-spawning of male; gonopore (g) b: Ejaculation; sperm (s) c: Pre-spawning of female; inflated gonopore (ig) d: Ovulation; egg (e) stage in the successful trials The embryo hatched and reached the early auricularia stage was above 90.9% of the fertilized eggs The larval survival rate decreased in each development progression However, the sharp decline occurred in metamorphosis stage which was from auricularia to settlement Overall, there were 1%–6.85% of the initially embryo reached to juvenile 3.3 Juvenile growth After 30 days' culture, majority of juveniles reached 1.0–2.0 mm in length The primary podium occurred at the rear end, and initial papillae developed along the dorsal body wall Subsequently, juvenile developed more podia, papillae and tentacles The juvenile endoskeleton Table Development of Stichopus horrens from fertilization to juvenile at 25–27 °C Five samples were measured to obtain the mean size of larvae in each stage Stage Size(μm) Fertilization 2-cell 4-cell 8-cell 16-cell 128-cell Blastula Early gastrula Late gastrula Early auricularia Mid auricularia auricularia Late auricularia Doliolaria Pentactula Juvenile 120.60 139.86 145.54 172.41 185.43 197.59 201.76 232.36 326.71 402.55 502.65 1005.44 1437.35 746.0 398.52 1682.32 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Time 11.53 11.54 7.48 9.88 6.38 5.17 6.51 13.47 6.04 67.92 71.42 129.45 43.33 82.10 7.38 320.58 40–50(min) 70–80(min) 100(min) 120(min) 180(min) 220(min) 8–9(h) 25(h) 45(h) 6(d) 8(d) 13(d) 18–26(d) 19–27(d) 30(d) developed gradually and mainly was the table ossicle that distributed all around of the body (Fig 3b) When they were ca 10 mm in length, the juveniles started to accumulate pigments The growth rate and survival rate of juvenile (Oct 2010) were given in Fig during the 80 days after complete settlement In the initial two weeks, the juveniles grew faster at mean rate of 0.6 mm per day, and could reach about 10.0 mm in length However, the growth of juvenile fell notably and was ca 0.2 mm per day during the following ca one month Subsequently, the rate increased again, and the average growth rate was 0.4 mm per day during 80 days They reached a mean of 32.0 mm in length and 1.2 g in weight The survival rate was about 23.3% by day 80 post settlement A high mortality of juvenile occurred and lasted about two weeks from day 34 to 46 after settlement In this time, a lot of the predator copepods were found in the tank, especially on the juvenile We found the tegument of juvenile was ruptured After the insecticide killed out most of copepods, mortality rate was decreased In the later experiment (Sep 2011), without the outbreaks of copepod, the survival rate of juvenile was reached 50.5% in day 80 post settlement After 80 days' culture after complete settlement, majority of the juveniles were released to the wild In the first observation for the juveniles in the wild, there were six cages which were found by snorkel in the released area after 50 days The mean individuals in the cage were 12.66 (s.e = 6.62, n = 6) The juveniles in the cages reached average 4.7 cm (s.e = 0.72, n = 13) and 2.89 g (s.e = 1.39, n = 13) All of the cages had some flaws or holes, because of irrevocably abrasion in seabed After 110 days and 180 days in the wild, no cage was found in this region, and some debris of cages was discovered We found some S horrens in released area in night The remaining individuals (about 3000) were still reared in a tank for the further studying The survival rate of juvenile indoor was 95.5% And the growth of juvenile maintained in the indoor was given in the Fig C Hu et al / Aquaculture 404–405 (2013) 47–54 51 Fig Embryonic and larval development of Stichopus horrens a: Fertilized egg b: Cleavage stages c: Blastula d: Gastrula e: Early auricularia; buccal ciliated cavity (bcc), cloaca (clo), intestine (int), oesophagus (oes) f: Mid auricularia g: Auricularia h: Late auricularia; anxohydrocoel (axo), hyaline sphere (hs) i: doliolaria; cilia band (cb), digestive tract (dt), hyaline sphere (hs) Discussion 4.1 Spawning periodicity Much research considered that temperature stimulation is an effective way to induce the spawning of sea cucumber (Battaglene et al., 2002; Costelloe, 1985; Ramofafia et al., 2003) Temperature increase is an indispensable condition for gonad maturation (Guzman et al., 2003; Muthiga and Kawaka, 2009; Tehranifard and Uryan, 2006) In this paper, dry stimulation seemed to be the best method to induce S horrens to spawn in three ways The operation was simple and convenient Drying was like the tides at their highest level in each month, which is the same as to new moons at night Fig Juvenile development of Stichopus horrens a: Pentactula b: 30-day juvenile; tentacle (ten), papillae (pap), ossicle (oss), podia (pod), digestive tract (dt) 52 C Hu et al / Aquaculture 404–405 (2013) 47–54 Table Survival rate of Stichopus horrens from early auricularia to juvenile Data is expressed as the mean % survival rate s.e = Standard error, n = numbers of tank in trial Time Early auricularia Mid auricularia Auricularia Late auricularia Doliolaria Juvenile 2010-Oct 94.4 (s.e = 0.91, n = 3) 90.6 (s.e = 6.32, n = 3) 90.9 (s.e = 18.2, n = 3) 84.8 (s.e = 9.09, n = 3) 71.7 (s.e = 3.12, n = 3) 78.7 (s.e = 8.22, n = 3) 29.4 (s.e = 5.24, n = 3) 63.6 (s.e = 10.9, n = 3) 20.0 (s.e = 5.21, n = 3) 36.0 (s.e = 0.52, n = 3) 1.0 (s.e = 5.24, n = 3) 6.83 (s.e = 1.05, n = 3) 2011-Sep S horrens spawned in new moon days and followed a predictable lunar spawning periodicity deduced from all of the spawning trials in Xisha Islands This behavior has been observed in some tropical sea cucumbers including Stichopus sp and Holothuria scabra (Hamel et al., 2002; Hu et al., 2010) On the contrary, some species including Stichopus chloronotus, Isostichopus badionotus and Bohadschia argus were observed to spawn after full moon days during the breeding season (Babcock et al., 1992; Guzman et al., 2003) It is believed that lunar periodicity probably influences spawning by stimulating endogenous cues in sea cucumbers (Kubota and Tomari, 1998) In our study, the maintaining time of S horrens before inducing spawning influenced the release of gametes, which was also found in H fuscogilva (Battaglene et al., 2002) The longer maintaining days caused the failure of spawning of S horrens during May and July 2010 The spawned periods of S horrens covering the summer and winter seasons indicated that S horrens might reproduce throughout the year in Xisha Islands, although this point needs to be confirmed by further studies It was reported that this species can reproduce throughout the year in Galapagos Islands (Toral-Granda, 2008) The spawning time of S horrens is always at deep night (concentrated on 00:00–02:00), similar to that of Stichopus sp (Hu et al., 2010) Moreover, the females always spawned after the males The sperm released might have induced the females to spawn The pre-spawning behavior in S horrens was not intensive especially for males, comparing with other sea cucumbers, such as Stichopus sp., H spinifera, H scabra and Actinopyga mauritiana (Asha and Muthiah, 2002; Battaglene et al., 2002; Hu et al., 2010) 4.2 Embryonic and larval development The fertilization and the development from oosperm to early auricularia were almost the same to those of the other tropical holothurians (Asha and Muthiah, 2002; Hamel et al., 2003; Hu et al., 2010; Ramofafia et al., 2003) From then on, the development differentiated among diverse species (Asha and Muthiah, 2002; Hamel et al., 2003; Hu et al., 2010; Ramofafia et al., 2003) This revealed that the embryo development in sea cucumbers depended on endogenous factors and environmental conditions The larvae of S horrens developed slower from mid auricularia to doliolaria stage compared with Stichopus sp., (Hu et al., 2010) The time taken to reach the doliolaria stage was 18 days in S horrens, similar to that in I fuscus and H atra (Hamel et al., 2003; Ramofafia et al., 1995), but 13 days less than that of other sea cucumbers including H spinifera and H scabra (Asha and Muthiah, 2002; James et al., 1994; Ramofafia et al., 2003) Environmental conditions especially the temperature had been demonstrated that they played an important role in sea cucumber larval development (Asha and Muthiah, 2005; Li and Li, 2010) The optimum temperature for larvae development was at 27–30 °C in tropical sea cucumber, such as H scabra, A echinites and H atra (Chen and Chian, 1990; James et al., 1994; Ramofafia et al., 1995) The larvae of S horrens in this experiment were cultured at 25–27 °C The lower culture temperature prolonged the developmental time of the larvae Table gives the development of S horrens at 25–27 °C (Dec 2010 and Sep 2011) In the high temperature season (Jun–Aug 2010 and 2011), the larvae of S horrens developed to auricularia in day (27–30 °C), and the time of complete settlement reduced day In our study, the failed trials (Sep 2010 and Nov 2010–Aug 2011) with poor survival were caused due to the non-availability live microalgae to feed However, with proper supply of live feed, the larvae maintained a high survival rate until they reached late auricularia In October 2010, two sharp declines of survival occurred during larval development: 41.0% auricularia reached late auricularia and only about 5.0% doliolaria successfully achieved settlement And in Sep 2011 only 56.6% late auricularia reached doliolaria and about 19.1% doliolaria developed to juvenile The settlement rate of S horrens was lower than other holothurians (Dabbagh et al., 2011; Laxminarayana, 2005; Ramofafia et al., 2003) The sharp decrease of larvae number appeared in the metamorphosis stage which is the key process that determines the success to multiplication of sea cucumber Nutritive condition was considered as the important factor in this stage (Asha and Muthiah, Fig Mean survival and growth of the juveniles of Stichopus horrens Bars are standard errors C Hu et al / Aquaculture 404–405 (2013) 47–54 53 Fig Further growth of the juveniles of Stichopus horrens in the tanks Bars are standard errors 2005; Ramofafia et al., 2003) The mixed microalgae used in this study could meet the requirement of the nutrition for the development of S horrens The formation of the hyaline spheres which would store and provide essential energy for the non-feeding metamorphic period of the larvae, was a characteristic feature of the larvae developing into its late auricularia of sea cucumber (Ramofafia et al., 2003; Sewell and McEuen, 2002) Those larvae that could not form the hyaline spheres would die, resulting in decreased survival rate It is usually described that there were only five pairs of hyaline spheres in other holothurians (Hamel et al., 2003; Hu et al., 2010; Liao, 1997; Ramofafia et al., 2003) However in this study, six pairs of hyaline spheres were clearly observed at the late auricularia and doliolaria stages of S horrens The hyaline spheres at the preanal arms locating venter were difficult to be viewed in contrast with the others Hence, this pair of hyaline spheres may be neglected in other species Whether they were overlooked in other species or this was the unique characteristic for S horrens needs further research in other species 4.3 Juvenile growth The growth rate of S horrens juveniles varied at different culture periods which may relate to the food In the first two weeks after settlement, juveniles of S horrens showed a rapid growth and reached 0.6 mm per day, which was close to Stichopus sp.(Hu et al., 2010) and I fuscus (Hamel et al., 2003) during the same period Plenty of edible diatoms on the plates provided enough nutrition food for them shown by translucent rings that appeared around the sea cucumber at the settlement When the juveniles grew to a mean size of 10.0 mm in length after two weeks, little diatoms existed on the settlement and they began to feed mainly on the artificial food The transition of feeding might delay the growth of the juveniles Consequently, after acclimatization to food, the growth rate gradually increased The same with other sea cucumbers, the different size juveniles in one culture tank had larger disparity in size as the culture progressed The competition on food resulted in different growth rate among individuals The survival of Holothuria scabra juveniles reared on hard substrates was 34.4% after weeks (Battaglene et al., 1999) In contrast, juvenile of S horrens survival rate during that period was higher and achieved 73.6% Unfortunately, the outbreak of the predator copepods was not eliminated in time, which directly resulted in the high juvenile mortality in two weeks From then on, survival of the juveniles improved as they grew Mass reproduction of the predator copepods in the tanks was the critical factor resulting in the low survival of juvenile S horrens, despite that they could be controlled and eliminated by pesticide Considering that some juveniles might escape from the cage through the flaws or holes, the real survival rate of juveniles was supposed to be higher than that we got (31.65%) in the first tracing observation The growth rate of juvenile in the cages was slower compared with that in the tank Lack of food in the cages would be the primary reason The ongoing observation of cages was suspended because the cages were abraded in seabed We found some juvenile in this region in night, and none in day The investigation before released showed that no S horrens existed in this region Overall, 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