全 文 ：Optimum dosage of KCl for spawning induction and the effects of water
temperature and salinity on embryonic and larval development in Asterias
amurensis
KANG Kyoung-ho1，2，ZHANG Zhi-feng1，SHAO Ming-yu1，LIM Sang-min2，KIM Jae-min2
(1. College of Marine Life Sciences，Ocean University of China，Qingdao 266003，China；2.Division of Marine Technology，
Chonnam National University，Yeosu 550-749，Korea )
Abstract：The basic information of reproductive biology is necessary for the effective control of starfish in
mariculture areas. In the present study，spawning induction of 0.5 mol/L KCl at various dosages (0 mL，1.0 mL，
3.0 mL，5.0 mL and 7.0 mL)，and the effects of water temperature and salinity on embryonic and larval rearing
were investigated in starfish Asterias amurensis. The optimum dosage group of 0.5 mol/L KCl on spawning
induction was assessed to be 3.0 mL injection group，with a maximum total percentage of spawning (80%)
and spawning quantity of per female 536.5×104 eggs，as well as a high fertilization rate (92.7±2.9)%. The
developmental rate of each stage increased with increasing of water temperature，and the suitable range of water
temperature for the embryonic and larval development was 15-20 ℃，and the optimum temperature was 20 ℃ .
The salinity range of 30-35，especially 35，was suitable for larval survival，development and growth.[Journal of
Fishery Sciences of China，2008，15(4)：677-682]
Key words：Asterias amurensis；spawning induction；embryonic and larval development；KCl；water
temperature；salinity
CLC number：Q178　　　Document code：A　　　Article ID：1005-8737-(2008)04-0677-06
Received date：2007-08-28；Revised date：2008-01-28.
Biography：KANG Kyoung-ho (1960-)，male；Ph D. Research field：marine biology. E-mail：mobidic@chonnam.ac.kr
中国水产科学
Journal of Fishery Sciences of China
第15卷第4期
2 0 0 8年 7月
Vol.15 No.4
J u l y 2 0 0 8
It is well known that starfish is the main enemy
of economic bivalve in aquaculture. For the effective
control of starfish in mariculture areas，the basic
information on its reproductive biology is necessary，
which also can be used to culture starfish larvae to
perform further experiments. In previous studies，
it has been reported on the artificial fertilization，
larval growth，ingestion of ultraplankton and large-
scale culture in several starfishes [1-4].
Asterias amurensis，subjected to Echinodermata，
Asteroidea，is a familiar species in the sea areas of
Korea and China. Kim [5] observed the histological
change of its gonad，and Yu et al. [6] investigated
temperature effects on its early development.
However，the spawning induction and effects of
environmental factors on embryonic and larval rearing
have not been studied in this species perfectly.
As a part of a broad project to completely
understand the propagation and development of
A. amurensis，and further develop a protocol for
controlling this harmful species in aquaculture，in this
study，spawning induction of KCl and the effects of
water temperature and salinity on its embryonic and
larval rearing were investigated.
1　Materials and methods
1.1　Materials
The optimum reproduc t ive season for A.
amurensis was at 5-6 months in a year. During the
reproductive season，adult starfishes (body length：
15.7-16.3 cm，arm length：8.5-8.9 cm，body weight：
96.1-99.9 g) were collected from Yeosu city，Korea
and maintained in the circulating aquaria at (15±
1)℃ in laboratory for a few days before experiment.
During the reproductive season，the average water
temperature is 17.3 ℃ in Yeosu coastal waters；The
678 第15卷中 国 水 产 科 学
average salinity is 35，and salinities range from 18 to
38 in Yeosu coastal waters due the effects of runoff
from land，rainfall and evaporation.
1.2　Spawning induction
Five dosages (0 mL，1.0 mL，3.0 mL，5.0 mL and
7.0 mL) of 0.5 mol/L KCl were respectively injected
into coelome at the base of arm to induce spawning.
Ten starfish were employed in each group，and
triplicates were conducted for each treatment. After
the starfish spawning，the percentage of spawning
individuals and spawning quantity per female were
counted，and meanwhile the eggs were fertilized
with sperm obtained from the males of the same
injected group. The fertilization rates were calculated
according to the appearance of 2-cell stage. The
fertilized eggs obtained from 3.0 mL injected group
were reared in tank at a density of 1 egg/mL to
perform the following experiment.
1.3　Effects of water temperature and salinity on
embryonic and larval rearing
To estimate the effect of water temperature on
embryonic and larval development，the fertilized
eggs described above were reared at salinity 35 and
six temperature groups (5 ℃，10 ℃，15 ℃，20 ℃，
25 ℃ and 30 ℃ ) respectively，and temperature was
controlled by automatic heating controller. Otherwise，
in salinity groups，five salinity grads which were 20，
25，30，35 and 40 obtained by diluting filtered natural
seawater with distilled water or adding sea salt，were
designed and rearing were maintained at 15 ℃ . Four
thousand eggs per tank were used and triplicates were
conducted for every group. During the experiment，
100 random samples were collected in each group
termly to determine and count the developmental
stages (2-cell，8-cell，morula，blastula，gastrula and
bipinnaria) (Fig. 1)，the developmental rates (mean
time required to each developmental stage from
zygotes for all individuals) and the survival rates
under the microscope. Filtered seawater was renewed
by 2/3 every day and larvae were fed with Dunaliella
tertioklecta，D. primolecta and Phaeodactylum
tricornutum daily at a density of 2×104-5×105 cells/
mL during larval rearing.
Fig. 1　Developmental stages of A. amurensis
图 1　多棘海盘车的发育分期
2　Results
2.1　Effect of various dosages of 0.5 mol/L KCl on
spawning induction
The effect of various dosages of 0.5 mol/L KCl
on spawning induction was assessed in terms of
percentage of spawning starfish (Tab. 1)，spawning
quantity per female and fertilization rates (Tab. 2).
The maximum total percentage of spawning (80%)
and spawning quantity per female 536.5×104 eggs
was found in 3.0 mL injection group，accompanied
with a high fertilization rate (92.7±2.9)%. All
the other groups spawned less eggs，especially the
7.0 mL group (138.6×104 eggs/spawning female)，
which also had a low fertilization rate (76.3±6.5)%.
2.2　Effect of water temperature on embryonic
and larval rearing
The survival rates of embryos and larvae at
different water temperatures were showed in Fig. 2.
It indicated that the embryos and larvae could not
develop normally at 5 ℃，25 ℃ and 30 ℃ . In 10 ℃，
15 ℃ and 20 ℃ groups，developmental rates reaching
every stage became faster at 15 ℃ and 20 ℃ than at
10 ℃ (Tab. 3)，and the survival rate of 10 ℃ group
at each stage was also lower obviously than that of
15 ℃ and 20 ℃ groups. Figure 3 also showed that the
developmental rate of each stage was increased with
increasing water temperature，and the relationship
between them at each stage of embryonic development
第4期 679
KANG Kyoung-ho et al：Optimum dosage of KCl for spawning induction and the effects of water
temperature and salinity on embryonic and larval development in Asterias amurensis
Tab. 1　Percentage of spawning A. amurensis induced with various dosages of 0.5 mol/L KCl
表 1　不同剂量 0.5 mol/L KCl诱导多棘海盘车排放生殖细胞的个体百分比
KCl Dosage/mL
KCl 剂量
No. of specimens
个体总数
No. of spawning 排放个体数 Percentage of spawning /%
排放个体的百分比Female 雌 Male 雄
0 30 0 0 0
1.0 30 15 6 70
3.0 30 15 9 80
5.0 30 15 6 70
7.0 30 9 9 60
Tab. 2　Spawning quantity per female and fertilization rates of A. amurensis induced with various dosages of 0.5 mol/L KCl
表 2　不同剂量 0.5 mol/L KCl诱导多棘海盘车的排卵量和受精率 X±SD
KCl Dosage /mL
KCl 剂量 /mL
Spawning quantity /ten thousand
排卵量 /万粒
Average fertilization rate/%
平均受精率
0 0 0
1.0 192-260 (213.6 ± 51.7) 94.5 ± 2.3
3.0 429-621 (536.5 ± 78.9) 92.7 ± 2.9
5.0 233.9-385.4 (276.8 ± 63.3) 91.3 ± 2.7
7.0 125.8-154.7 (138.6 ± 14.8) 76.3 ± 6.5
Fig. 2　Survival rates of different developmental stages at various water temperatures in A. amurensis
图 2　多棘海盘车各发育时期在不同水温下的存活率
was notable. Therefore the suitable range of water
temperature for the embryonic development was
15-20 ℃，and the optimal temperature for survival，
development and growth was 20 ℃ .
680 第15卷中 国 水 产 科 学
Tab. 3　Time required from fertilized egg to each developmental stage at various water temperature in A. amurensis
表 3　不同水温下多棘海盘车受精卵发育至各时期所需时间　　　　　　　　　　　X±SD；h　
Developmental stage
发育时期
Water temperature /℃ 水温
5 10 15 20 25
2-Cell 2- 细胞 9.8 ± 0.9 3.8 ± 0.5 0.9 ± 0.2 0.6 ± 0.1 0.3 ± 0.1
8-Cell 8- 细胞 17 ± 1.2 7.7 ± 0.6 2.3 ± 0.3 1.4 ± 0.3 -
Morula 桑椹胚 42 ± 3.6 13.1 ± 1.6 3.8 ± 0.8 3.85 ± 0.7 -
Blastula 囊胚 58 ± 7.7 24.5 ± 2.1 13.1 ± 1.1 13.3 ± 1.2 -
Gastrula 原肠胚 114.9 ± 11.5 42 ± 5.6 24.8 ± 2.6 22.7 ± 2.3 -
Bipinnaria 羽腕幼虫 - 72.1 ± 6.6 53.8 ± 3.8 51 ± 4.1 -
Fig. 3　Relationship between water temperature （Tw） and time （t，h） required to each developmental stage after fertilization
图 3　水温（Tw）和受精卵发育至各时期所需时间（t，h）的关系
2.3　Effect of salinity on embryonic and larval
rearing
The survival rates of embryos and larvae
at different salinity were showed in Fig. 4. The
bipinnaria only survived at salinities 30 and
35 groups，and the difference of developmental rates
at each developmental stage (Tab. 4) between 30 and
35 was not distinct. Therefore the salinity range of 30
~35，especially 35，was suitable for larval survival，
development and growth.
Fig. 4　 Survival rates of embryos and larvae at different
salinity in A. amurensis
图 4　多棘海盘车胚胎和幼虫在不同盐度下的存活率
第4期 681
KANG Kyoung-ho et al：Optimum dosage of KCl for spawning induction and the effects of water
temperature and salinity on embryonic and larval development in Asterias amurensis
3　Discussion
Although eggs obtained by dissection of gonad
can also be fertilized artificially，spawned eggs by
nature or induction are better for fertilization because
fewer immature eggs are shed [3]. The injection of
10-3 mol/L 1-methyl-adenine is often used to induce
the spawning of starfish and oocyte maturation [3，7-8].
However，KCl，which is used in spawning induction
of sea urchin [9]，is more common and cheaper and is
testified that 0.5 mol/L KCl could induce spawning
of A. amurensis in this study. The maximum total
percentage of spawning (80%) and number of eggs
per spawning female (536.5×104 eggs/spawning
female) were found in 3.0 mL injection group with a
high fertilization rate (92.7±2.9)%，and numbers of
eggs per spawning in 1.0 and 5.0 ml injection groups
were approximate half of the former，although the
difference in percentage of spawning female and
fertilization rate between them was not significant.
In contrast，the spawning performance of 7.0 ml
injection group was worse obviously. So the injection
of 3.0 ml is the optimum dosage of 0.5 mol/L KCl to
induce the spawning of A. amurensis.
Water temperature is thought as one of critical
environmental factors in the embryonic and larval
development of marine invertebrates. The results in
this paper showed the influence of water temperature
on development of A. amurensis was obvious. At 30
and 25 ℃，the fertilized eggs could not perform the
first and third cleavage，and the bipinnaria could not
survive at 5 ℃ . The suitable range of water tempera-
ture for the embryonic and larval development was
15-20 ℃，and the optimal temperature for survival，
development and growth was 20 ℃，which is similar
with the results of Yu et al. [6].
The effect of salinity on development and growth
of starfish is seldom studied. Sarantchova [10] tested
the adaptive responses to salinity changes in sea
starfish Asterias rubens L. at different ontogenetic
stages and showed the narrow limits of tolerance for
salinity at initial ontogenetic stages of the starfish，
such as the stage of fertilized egg and the gastrula.
However，this characteristic of tolerance for salinity
was not observed in this study. The survival range of
salinity for bipinnaria was 30-35，but that of morula
was 25-40，although the developmental rates in 25
and 40 groups were obviously lower. Therefore the
suitable range of salinity was 30-35，and 35 was the
optimum salinity for larval survival，development
and growth of A. amurensis.
References：
[ 1 ]　 Ayukal T. Ingestion of ultraplankton by the planktonic larvae of
the crown of thorns starfish，Acanthaster planci [J]. Biol Bull，
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[ 2 ]　 Benzie J A H，Dixon P. The effects of sperm concentration，
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plasticity in a forcipulate seastar [J]. J Exp Mar Biol Ecol，
Tab. 4　Time required from fertilized egg to each developmental stage at different salinity in A. amurensis
　　表 4　不同盐度下多棘海盘车受精卵发育至各时期所需时间 X±SD；h　
Developmental stage
发育时期
Salinity 盐度
20 25 30 35 40
2-Cell 2- 细胞 3.9 ± 0.8 3.5 ± 0.5 1.0 ± 0.3 0.9 ± 0.2 2.81 ± 1.2
8-Cell 8- 细胞 7.6 ± 1.5 7.1 ± 1.0 2.5 ± 0.7 2.4 ± 0.5 5.5 ± 2.4
Morula 桑椹胚 - 13.2 ± 2.1 4.0 ± 0.6 3.9 ± 0.6 9.5 ± 3.8
Blastula 囊胚 - - 13.4 ± 1.1 13.2 ± 1.2 -
Gastrula 原肠胚 - - 25.9 ± 3.2 24.9 ± 2.7 -
Bipinnaria 羽腕幼虫 - - 55.4 ± 4.9 52.7 ± 4.4 -
682 第15卷中 国 水 产 科 学
多棘海盘车诱导排卵最佳 KCl剂量以及水温和盐度对胚胎和幼虫发育的影响
康庆浩 1，2，张志峰 1，邵明瑜 1，林相民 2，金在敏 2
(1. 中国海洋大学 海洋生命学院，山东 青岛 266003 ；2. 全南国立大学校 海洋技术学部，韩国 丽水 550-749)
摘要：海星的大量繁殖与入侵可对滩涂养殖贝类造成极大威胁。对海星繁殖生物学的研究可为有效控制海星过度增
殖提供信息，同时也可为其胚胎和幼虫培育以及发育生物学的深入研究奠定基础。本研究以韩国和中国沿海常见的多
棘海盘车（Asterias amurensis）为材料，研究 KCl 诱导海盘车排放生殖细胞的最佳剂量，以及水温和盐度对胚胎和幼虫
发育的影响。采用不同剂量 (1.0 mL、3.0 mL、5.0 mL 和 7.0 mL)0.5 mol/L KCl 注入性成熟海盘车体腔，皆可诱导生殖
细胞的排放，3 mL 注射组的排放个体比例最高 (80%)，雌性个体排卵量最大 (536.5×104 个 /ind)，同时用诱导获得的
精 / 卵进行人工授精后也获得了较高的受精率 (92.7±2.9)%。水温和盐度对多棘海盘车胚胎和幼虫的存活率具有显著
的影响。在盐度为 35 时，20 ℃时多棘海盘车羽腕幼虫的存活率最高 (90.1±2.1)%，15 ℃时次之 (84.4±5.2)%。在温
度为 15 ℃时，盐度为 35 时幼虫培养 60 h 后存活率最高 (87.6±4.1)%，盐度为 30 时次之 (85.4±4.0)%。多棘海盘车各
期胚胎和幼虫的发育速度 (1/t，h-1) 随水温升高而加快，在一定温度范围内有明显的正相关性，其关系式为：至 2- 细胞：
1/t=0.110 8Tw-0.599 7 (r
2=0.946 5)；至 8- 细胞：1/t=0.045 4Tw-0.233 4 (r
2=0.947 7)；至桑椹胚：1/t=0.017 9Tw-0.067 9
(r2=0.868 7)；至囊胚：1/t=0.004 2Tw (r
2=0.89)；至羽腕幼虫：1/t=0.000 6Tw-0.008 7 (r
2=0.878 4)。盐度为 35 时，胚胎和幼虫
的发育速度最快，发育至羽腕幼虫需 55.4 h。根据不同水温和盐度条件下，胚胎和幼虫的存活率和发育速率，确定 15~20 ℃
是多棘海盘车发育的适宜温度，最适温度为 20 ℃；适宜盐度为 30~35，最适盐度为 35。[中国水产科学，2008，15(4)：
677-682]
关键词：多棘海盘车；排卵诱导；胚胎和幼虫发育；KCl；水温；盐度
1999，237：203-224.
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