24 Spawning, larval development and juvenile growth of the sea cucumber Stichopus horrens

Aquaculture 404–405 (2013) 47–54

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Aquaculture
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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 9 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.

1. 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: (C. Hu).
1
These authors have equally contributed to this work.
0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
/>
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.
2. 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 4 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 3 °C for about
1 h, and then resumed to natural temperature.
Dry stimulation: let the water out of the tanks, left the broodstock
in open-air for 1 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 3 or 4 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 1 h and filtered by 60-μm
sieve, before feeding. The component rate was 1:1:2:5. Firstly, artificial feeding was added in at 1 g m −3. When the juvenile reached
10 mm, the feeding was increased at 5 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 3 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 1 km 2. 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.
3. 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 1. 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 3 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 7 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 3 h), and could repeat
for 3 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 3.
The zygote (Fig. 2a) cleaved into two equal hemispheric blastomeres
(Fig. 2b) ca 40 min after fertilization. Subsequently, the embryos

Table 2
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
4
5
18
6
19
5

20
21
22
3
4
17
5
6
3
2
12
3
4
5
4
1
2
2
3
2
1
2
2
2
3
2
2
2
3
4

5
3
4
5
2
3

a

Table 1
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 2 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 min. The blastula stage was reached by
200 min 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 4 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. 1. 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 3
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

0
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. 4 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. 5.


C. Hu et al. / Aquaculture 404–405 (2013) 47–54

51

Fig. 2. 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).

4. 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. 3. 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 4
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 3 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 7 day (27–30 °C), and the time of complete
settlement reduced 4 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. 4. 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. 5. 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 4 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, the hatchery-produced juveniles of S. horrens can be cultivated in captivity and be released to replenish stock in the wild,
which provide an alternative to fisheries and a way to the restoration
of the natural resource, and eventually contribute to maintain the natural population and realize sustainable utilization in southern China.
Acknowledgements
This work was supported by the National Key Technologies R&D Program (2012BAD18B03; 2011BAD13B02; 2009BAB44B02), Guangdong
Province and CAS Cooperation Program (2012B091100272), Natural
Science Foundation of Guangdong Province (S2011040000463),
Foundation for Distinguished Young Talents in Higher Education of
Guangdong, China (LYM11086), and Key Laboratory Program of Marine
Bio-resources Sustainable Utilization (LMB111004).
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