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Effect of salinity on hatching, larval growth, survival and settling in the tropical oyster Crassostrea belcheri (Sowerby)
Aquaculture Aquaculture
145 ( 19%) 129- I39
Effect of salinity on hatching, larval growth, survival and settling in the tropical oyster Crassostrea belcheri ( Sowerby) Shau-Hwai Tan ay*, Tat-Meng Wong b a Centre forMarine and Coastal Studies, Universiti Sains Malaysia, 11800 Penang, Malaysia b Open Learning Institute of Hong Kong. 9-13 F, Trade Department Tower, 700 Nathan Road, Kowloon, Hong Kong Accepted 25 April
I!396
Abstract The effects of salinity (O-30 p.p.t.) on egg development, growth and survival of D-larvae to the plantigrade stage as well as settling in Crassosfrea befcheri were studied. Eggs were able to develop into D-larvae from 12 p.p.t. to 30 p.p.t. salinity. Optimum development of eggs to D-larvae occurred at salinities between 24 and 30 p.p.t. At these salinities, survival exceeded 75%. D-larvae were able to survive to the settling stage when cultured in salinities from 12 to 30 p.p.t., with the best survival occurring between 12 and 24 p.p.t. (20.3-22.6%). At 18 p.p.t. salinity, first settling occurred on the 17th day compared with the 19th day for larvae cultured at 12 and 24
p.p.t., and the 23rd day for those cultured at 30 p.p.t. Optimum settling occurred at salinities between 12 and 18 p.p.t. Keywords:
Crassostrea
belcheri; Hatching;
Larval growth;
Salinity;
Settling
1. Introduction The successful production of spat of the tropical oyster Crassostreu belcheri (Sowerby) in Malaysia via hatchery techniques was first reported by Wong et al. (1989). Since then efforts have been made to ‘scale up’ seed production to the commercial level. In the transition from laboratory investigations to large-scale hatchery production, research and development efforts must be directed towards defining optimal conditions
* Corresponding
author.
CW4-8486/%/$15.00 Copyright PII SOO44-8486(96)01338-S
6 1996 Elsevier Science B.V. All rights reserved.
130
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145 (1996) 129-139
for growth and survival as well as shortening the duration of the larval cycle. C. belcheri are naturally found in the intertidal zone attached to the aerial roots of mangroves. They are exposed to conditions of high temperature and variable salinity. The temperature in Malaysia is often uniformly high (25-32°C) with low diurnal changes. During the annual monsoon rains, salinity drops sharply. Under natural conditions C. b&her-i may be continuously exposed to almost fresh water (3-5 p.p.t.) for 7-8 days during the monsoonal floods. Lowered salinities have also been suggested to act as environmental triggers for spawning in C. belcheri in Sabah, Malaysia (Chin and Lim, 1975). Should spawning in fact occur under such conditions, then the fertilized eggs are likely to undergo embryonic and larval development under conditions of reduced salinity. Salinity tolerance in oysters has been reported for C. gigas (Helm and Millican, 1977), C. virginica (Loosanoff and Davis, 1963), C. iredulei and Saccosfreu cucullutu (Sudrajat, 1990); however, no study has addressed salinity tolerance changes with developmental stages in tropical oysters. This paper reports on the salinity tolerance of embryos and larvae of C. belcheri during the planktonic phase, as well as the effects on settling and metamorphosis.
2. Materials and methods 2.1. Spawning Mature C. belcheri oysters were collected from Telaga Nenas, Perak. The Telaga Nenas site is located approximately 12 km from the coast and experiences fairly stable salinity (25-30 p.p.t.) during most of the year. The oysters collected were acclimated in sand-filtered seawater (30 p.p.t.) in the laboratory for a few days. During this period, they were fed an algal mixture containing Chaetoceros cufcitruns and Isochrysis gulbunu (Tahitian strain: T-Iso). The oysters were induced to spawn with serotonin (Gibbons and Castagna, 1984). Egg handling and fertilization were as described by Loosanoff and Davis (1963). All experiments were conducted with pooled progeny from a number of parent broodstocks. 2.2. Algae The unicellular algae Isochrysis gulbunu (T-Iso) and Chuetoceros culcitrans were batch-cultured in 1 pm filtered seawater (salinity 30-32 p.p.t.) using F/2 medium (Guillard, 1972). Algal cultures were produced in 20-I glass carboys at 28 f 2°C. Only cultures in the log phase of growth were used. A haemocytometer was used to determine cell concentrations. 2.3. Test salinities Test salinities (0, 6, 12, 18, 24 and 30 p.p.t.) were prepared by mixing 1 pm filtered seawater with distilled water (0 p.p.t.). Salinities were determined by a refractometer (ATAGO Ltd.) precalibrated with distilled water. Salinity beyond 30 p.p.t. was not
S.-H. Tan, T.-M. Wong/Aquaculrure
145 (1996) 129-139
131
tested based on the range of salinity occurring in the natural environment of C. belcheri (25-30 p.p.t.1. Salinity of 0 p.p.t. was tested because the salinity drops sharply to 15 p.p.t. at Telaga Nenas and is sometimes even below 5 p.p.t., coinciding with the northeast monsoon which brings rain to the catchment at the source of the river. 2.4. Experiments 2.4.1. Development of embryos Fertilized eggs were gently aerated for 15-20 min until the first polar body was observed. The fertilized eggs were then transferred to different test salinities in l-l plastic aquaria at a stocking density of 10 embryos ml-’ (10000 fertilized eggs in 1000 ml). Slow aeration was supplied via air diffusers. The temperature was not controlled and varied from 27 to 29°C during the experimental period (24 h). All experiments were conducted in triplicate. At the end of 24 h, twenty 1- ’ ml samples were collected and the numbers of embryos which had developed into normal, D-hinge larvae were recorded. Normal larvae are defined here as being perfectly D-shaped at the prodissoconch I stage while abnormal larvae had irregular or misshapen shells either completely or incompletely formed. Shell heights of 30 normal larvae from each salinity were measured using a compound microscope (Olympus; phase-contrast BH) fitted with a micrometer eyepiece, and precalibrated with a 50 X 2 micron graticule. 2.4.2. Larval growth and survival D-hinge larvae were reared at each test salinity in 15-l glass carboys at a stocking density of 6-7 larvae ml-’ (90000-105 000 D-hinge larvae in 15 1). Aeration was supplied via air diffusers. All cultures were covered with PerspexTM sheets to prevent evaporation. The larvae were fed T-Is0 with an initial feed concentration of 5000 cells ml-‘. This was raised progressively as the larvae developed (Table 1). All test cultures received the same feed rations. The culture media were completely changed every 2 days, at which time samples were taken for microscopic examination, larval count and morphometric measurements. The shell heights of 30 larvae from each treatment were measured using a compound microscope. Three l.O-ml samples were collected and the mean value for larval density
Table 1 Concentration
of algal cells fed to C. belcheri in salinity experiments
Day
Concentrations
1-2 3-4 5-6 7-12 13-14 15-18 19-23 24-29
5000 1oGQo 2oooo 25000 3oocnl 35ocKl 4oocw 5oooo
of T-Is0 (cell ml-
’)
132
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145 (1996) 129-139
was calculated. When the larvae reached 250 p,rn (length) X 280 km (height) in size, a piece of clean polyvinylchloride (PVC) (145 mm X 9 mm X 2 mm) was suspended in each culture vessel to act as cultch. The experiments were terminated when settling of pediveliger was observed. 2.4.3. Settling Larvae of C. belcheri which had been cultured routinely at 25 p.p.t. salinity were used. Pediveligers which were retained on a 253 pm nitex sieve (height > 330 pm) were transferred into 1.0-I plastic aquaria containing the appropriate test salinity at a stocking density of 1 pediveliger ml-’ (1000 pediveligers in 1000 ml). Feed (T-Iso) was added at an initial density of 60000 cells ml-‘. A piece of clean PVC (145 mm X 9 mm X 2 mm) was used as cultch in each aquarium. Each aquarium was also lined by a sheet of transparent plastic. Aeration was provided via air diffusers. Triplicate tests were conducted at each salinity. At the end of 48 h, the cultch was removed and the remaining contents were siphoned through a 253 pm screen to retain any free swimming pediveligers. After washing, the larvae were transferred to fresh media of the same salinity and the cultch was replaced. Fresh feed was then added. Two days later the number of post-larva which had set on both the PVC plate and the plastic sheet were recorded using a binocular dissecting microscope. 2.5. Statistical analysis Data were analyzed with a one-way ANOVA to examine the effects of salinity on hatching, larval growth and survival or settling. If significant interactions were present, the data were then subjected to Duncan’s multiple range test to determine which treatments were significantly different.
3. Results 3.1. Development
of embryos
Fig. 1 shows the percentage of C. belcheri eggs that developed into normal D-larvae at different salinities. Eggs developed into D-larvae at 12-30 p.p.t. At 0 and 6 p.p.t. there was 100% mortality. Salinities between 24 and 30 p.p.t. yielded the highest percentages of normal D-larvae (78.0-83.2%) which did not differ significantly (P < 0.05). At 18 and 12 p.p.t.. the survival of embryos decreased significantly to 44.1 and 16.7%, respectively. At 12 p.p.t. numerous ciliated trochophores were still seen after 24 h whilst some 43.0% of D-larvae appeared deformed. The mean shell lengths and heights of normal D-larvae in different salinities are shown in Fig. 2. Growth (both shell length and shell height) was highest at 24 p.p.t_, although the difference in growth between larvae at 24 and 30 p.p.t. was not significant. The size of D-larvae decreased significantly at 18 and 12 p.p.t. salinity.
S.-H. Tan, T.-M. Wong/Aquaculture
133
145 (1996) 129-139
100 90
T
f
80 70 60 50 40 30 20 lo-
SALINITY Fig. 1. Percentage of Crassostrea belcheri eggs that developed Temperature, 28+ 1°C; pH, 7.9-8.5; egg density,10 ml- ‘.
(ppt) into normal
D-veliger
at different
salinities.
3.2. Larval growth and survival Figs. 3 and 4 show the percentage survival and shell growth (height), respectively, of C. belcheri larvae at different salinities. In all experiments the larvae suffered fairly high mortalities (45-60%) during the first 7 days. Thereafter, survival was reasonably good at suitable salinities whilst, those at 30 p.p.t. continued to show progressive die-offs right throughout the entire larval period. In 0 p.p.t. (freshwater), 100% mortality had occurred by the first observations made on Day 3. At 6 p.p.t., the percentage survival was reduced to 42% by Day 3 and 15% by Day 5. By Day 21 mortality reached 100%. Survival until settling occurred at 12, 18, 24 and 30 p.p.t., with the best survival (20.3-22.6%) occurring between 12 and 24 p.p.t. A one-way ANOVA showed that the
(A)
(B)
_. 6ALmrY
[PPU
ml.tWrY
Fig. 2. (A) Mean shell length and(B) mean shell height of 24-h Crassostrea b&hen’ Standard deviations are shown. The horizontal lines indicate mean egg diameters.
IPPG at different
salinities.
S.-H. Tan. T.-M. Wong/Aquculture
145 (1996) 129-139
.....
1
‘\
hi-y-
?----I---.-_
-
24 ppt 30 ppt
-!:p
*
I
I
I
I
I
I
I
I
I
I
I
3
5
7
9
11
13
15
17
19
21
23
AGE
w
6 PPt 12 ppt 18 ppt
-
I,
0
x---s -
(DAY)
Fig. 3. Percentage survival of Crussosfrea helcheri larvae reared at different value k s.d. of three sets of data. Temperature, 27-28°C; pH.7.S8.5.
salinities.
Points denote mean
differences between these three salinities were not significant (P < 0.05). The first settling occurred on Day 17 at 18 p.p.t. First settling was recorded from larvae grown in 12 and 24 p.p.t. on Day 19, whilst larvae grown in 30 p.p.t. only set on Day 23. Larvae
30 PPt setting
18 PP~ setting
I
3
5
7
9
I1
AGE
13
IS
17
12
PPt
24
ppt tting
“I’ 19
21
23
(DAY.)
Fig. 4. Mean shell height of Crussostreu belcheri larvae cultured value+ s.d. (n = 90). Temperature, 27-28°C; pH, 7.8-8.5.
in different
salinities.
Points denote mean
S.-H. Tan, T.-M. Wong/Aquaculture
145 (1996) 129-139
135
50
40 35 30 r 25 I w 20 15 10 5 0
0
6
12
10
24
30
SALINITY (ppt) Fig. 5. Percentage of Crassostrea belcheri larvae which set at different salinities. Temperature, 27k 1°C; larvae density, 1 ml-‘; pH, 7.8-8.3.
grown in 6 p.p.t. were stunted and by Day 17 had only reached 160 ym compared with the 287-338 km achieved by larvae grown under more favourable salinities. Growth was highest at 18 p.p.t., although no significant differences (P < 0.05) were seen between larvae grown in 12, 18 and 24 p.p.t. throughout the entire larvae period. Larvae grown in 30 p.p.t_ showed no significant differences in shell height by Day 17 compared with larvae grown in 12-24 p.p.t. 3.3. Settling Fig. 5 summarises the mean percentage set at different salinities. The highest mean percentage set occurred in 18 p.p.t. salinity (38.6 f 2.5%), followed by 12 p.p.t. (35.8 f 4.8%). The difference between the mean percentage set between these two salinities was not significant (P < 0.05). Mean percentage set decreased to 25.6 + 2.7% at 24 p.p.t., 19.8 f 1.1% at 30 p.p.t. and 7.3 f 8.7% at 6 p.p.t. The differences in mean settling percentage at these salinities were significantly lower than those at 12 and 18 p.p.t. 100% mortality occurred at 0 p.p.t.
4. Discussion The salinity range where development of eggs to D-larvae is possible for C. belcheri is 12-30 p.p.t. The responses of C. belcheri embryos are comparable to those at 15-40 p.p.t. reported for Succostrea cucullata and those at lo-40 p.p.t. for C. iredalei (Sudrajat, 1990). In India, the salinity tolerance of S. cucullatu embryos was reported to be 15-35 p.p.t_ (Kalyanasundaram and Ramamoorthi, 1986). In Thailand, Jarayabhand (personal communication) reported that the salinity tolerance of 5. commercialis embryos was 20-40 p.p.t.
136 Table 2 Optimum
S.-H. Tan, T.-M. Wong/Aquaculture
salinities and tolerance
Species
Salinity (p.p.t.1 Tolerance
C. belcheri C. iredalei C. rhizophorae S. commercialis S. cucullara
range for development
range
12-30 IO-40 16-40 20-40 15-35 15-40
145 (1996) 129-139
of eggs of tropical oysters to D-larvae Reference
Optimum 24-30 25 27-37 25 25
This study Sudrajat, 1990 DOS Santos and Nascimento, 1985 P. Jarayabhand, personal communication, 1992 Kalyanasundaram and Ramamoorthi, 1986 Sudrajat, 1990
The optimum salinities for both growth and survival of C. belcheri embryos were between 24 and 30 p.p.t. Similar responses were reported for S. cucullufu (25 p.p.t.) and C. iredalei (25 p.p.t.) (Sudrajat, 1990), and for S. commercialis (2.5 p.p.t.1 (P. Jarayabhand, personal communication, 19921, while in the case of C. rhizophorue in Brazil, the optimum salinities for embryo development were 27-37 p.p.t. (DOS Santos and Nascimento, 1985) (Table 2). It is apparent that tropical oysters generally require almost full-strength seawater for optimal development of eggs to the D-larvae. Previous studies have shown that performance during embryo development is influenced by the salinity at which the broodstock is conditioned (Davis, 1958; Loosanoff and Davis, 1963). Broodstock used in this study were conditioned at 30 p.p.t. Further investigations are needed to determine whether this effect of broodstock conditioning salinity on embryo development also applies to C. belcheri. The results showed that C. belcheri larvae were capable of tolerating a wide range of salinities (6-30 p.p.t.) for extended periods. However, complete larval development to metamorphosis only occurred between 12 and 30 p.p.t. Growth was fastest at 18 p.p.t. This was accompanied by the earliest set (17 days), while at 12 and 18 p.p.t. first settling occurred on Day 19, and on Day 23 at 30 p.p.t.
Table 3 Optimum salinities and tolerance other soecies of ovsters Species
Salinity (p.p.t.) Tolerance
C. C. C. C. C.
belcheri virginica rivularis gigas iredalei
S. cucullata S. commercialis 0. edulis
range for larval growth and survival
12.0-30.0 10.0-27.5 17.0-20.0 15.0-34.0 10.0-35.0 18.0-30.0 15.0-35.0 20.0-27.0
range
of C. belcheri
larvae compared
Reference Optimum 12.0-24.0 17.5-27.0 20 25 20.0-30.0 24.0-30.0 25.0-30.0 20.0-30.0 22.5-27.0
This study Davis and Calabrese, I%4 Breese and Malouf, 1977 Helm and Millican, 1977 Sudrajat, 1990 Lau et al., 1992 Sudrajat, 1990 P. Jarayabhand, personal communication, Davis and Ansell, 1962
1992
with
S.-H. Tan, T.-M. Wong/Aqunculture Table 4 Optimum
salinities for settling and metamorphosis
145 (1996) 129-139
137
of various species of oyster
Species
Salinity (p.p.t.)
Reference
C. belcheri
12-18 15 18-24
This study Sahavachti et al., 1988 Tan, unpublished data Calabrese and Davis, 1970 Walne, 1956 Davis and Ansell, 1962 Sandison, 1966
C. iredalei C. uirginicu 0. edulis
9-10 24-33 23-25 30
c. gasar
The optimum salinity for larval growth and survival in C. belch-i is comparable to those reported for C. uirginicu (Davis and Calabrese, 1964) and C. riuuluris (Breese and Malouf, 1977), but lower than the 24-30 p.p.t. reported for C. iredalei (Lau et al., 500-a a--
500.
p 3
300-
E ,o 2 r:
200.
z
100.. El
I
I
I
12 18 SALINITY
6
-: ___
“\ ~-______t-J A: I__!?_O__p.:
A: B: c:
hbryo develomt Larval developlent Settingandmetamrpbsis
:
Optimusalinity Salinity tolerancerange
Fig. 6. Summary of salinity Crassostrea belcheri.
tolerance
range
and optimum
I
2L
I O-veliger
s
30
(ppt)
salinity
at different
development
stages
of
138
S.-H. Tan, T.-M. Wong/Aquuculture
I45 (1996) 129-139
1992), as well as for S. cuculluta (Sudrajat, 1990), S. commercialis (P. Jarayabhand, personal communication, 1992), C. gigus (Helm and Millican, 1977) and Ostrea edulis (Davis and Ansell, 1962) (Table 3). Few studies have been reported on the effect of salinity on settling. The lowest salinity at which optimum settling occurred in C. belcheri (12-18 p.p.t.1 is different from the 24-33 p.p.t. recorded for 0. eduZis (Walne, 1956) and 30 p.p.t. for C. gasar (Sandison, 1966) (Table 4). C. belcheri larvae are able to set and metamorphose at lower salinities. The enhanced settling and metamorphosis at lower salinity displayed by C. belcheri larvae may be due to the high amount of secretion from the byssal glands when the salinity is between 15 and 20 p.p.t. (Prytherch, 1934). The ability of C. belcheri larvae to tolerate salinities as low as 6 p.p.t. for short periods appears to provide adaptive advantage for life in estuaries characterised by high daily salinity fluctuations. It is interesting to note that as the larvae develop from the embryo to the plantigrade stage, there is a progressive increase in tolerance to lower salinities (Fig. 6), which demonstrates how well adapted C. belcheri is towards exploiting the estuarine habitat where competition from the fully marine species is much reduced. The poor larval survival and settlin g at 30 p.p.t. may also explain why C. belcheri does not compete well against more marine species such as S. cucullata on exposed rocky shores facing open seas. Most hatcheries are located in a marine environment where full-strength seawater is used for larval culture, but these salinities appear unsuitable for C. belcheri culture. Whether this applies to other tropical oyster species is not clear. Similar studies should be conducted on other species in order to enhance seed production.
Acknowledgements This study was carried out at Muka Head Research Station, Universiti Sains Malaysia, with funding support from IDRC (Oysterseed Malaysia File No.: 3-P-88-0049) and the Government of Malaysia (No.: 123/3 101/2406). We record our sincere thanks to all project staff for technical support and to Puan Shahanum bt. Wan for typing this manuscript.
References Breese, W.P. and Malouf, R.E.. 1977. Hatchery rearing techniques for the oyster Crossosrrerr riouh-is Gould. Aquaculture, 12: 123-126. Calabrese, A. and Davis, H.C., 1970. Tolerances and requirements of embryos and larvae of bivalve mollusc. Helgol. Wiss. Meeresunters., 20: 553-564. Chin, P.K. and Lim, A.L., 1975. Some Aspects of Oyster Culture in Sabah, Kuala Lumpur, Malaysia. Ministry of Agriculture and Rural Development, Fisheries Bulletin 5, 14 pp. Davis, H.C., 1958. Survival and growth of clam and oyster larvae at different salinities. Biol. Bull., 14: 296-307. Davis, H.C. and Ansell, A.D., 1962. Survival and growth of larvae of the European oyster, Osrren edulis, at lowered salinities. Biol. Bull. Woods Holes, MA, 122(l): 33-39.
S.-H. Tan, T.-M. Wong/Aquaculture
145 (1996) 129-139
139
Davis, H.C. and Calabrese. A., 1964. Combined effects of temperature and salinity on development of eggs and growth of larvae of Mercenaria mercenaria and Crassosrrea uirginica. J. Fish. Biol., 63: 643-655. Dos Santos, A.E. and Nascimento, I.A., 1985. Influence of gamete density, salinity and temperature on the normal embryonic development of the mangrove oyster Crassostrea rhizophorae Guilding, 1828. Aquaculture, 41: 335-352. Gibbons, M.C. and Castagna, M., 1984. Serotonin as an inducer of spawning in six bivalve species. Aquaculture, 40: l89- 191. Guillard, R.R.L., 1972. The culture of phytoplankton for feeding marine invertebrates. In: W.L. Smith and M.H. Chanley (Editors), The Culture of Marine Invertebrate Animals. Plenum Press, New York and London, pp. 29-60. Helm, M.M. and Millican, P.F., 1977. Experiments in the hatchery rearing of Pacific oyster larvae (Crassosrrea gigas Thunberg). Aquacultnre, 47: 335-352. Kalyanasundamm, M. and Ramamoorthi, K., 1986. Temperature and salinity requirements for embryonic development of Saccosfrea cuculfara (Born). Mahasagar Bull. Nat]. Inst. Oceanogr., 19(l): 53-55. Lau, K.T., Wong, T.M. and Tan, S.H., 1992. Effect of salinity and feed concentration on larval growth and survival in the tropical oyster, Crussosfrea iredalei (Faustino). Paper presented at the 3rd Asian Fisheries Forum in Singapore, 26-30 November 1992. Asian Fisheries Society, Manila, Philippines. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve mollusks. In: Advances in Marine Biology, Vol. 1. Academic Press, London, pp. I - 136. Prytherch, H.F., 1934. The role of copper in the settling, metamorphosis, and distribution of the American oyster, Ostrea oirginicu. Ecol. Monogr., 4: 47- 107. K., Chawivanskom, V., Sahavacharin, S., Chindanond, A., Amomjaruchit, S., Nugrang, J., Silapajam, Limsurt, S., Angell, C.L., McCoy, E.W., Mutarasint, K. and Potaros, M., 1988. Hatchery techniques for tropical bivalve mollusc. In: E.W. McCoy and T. Chongpeepian (Editors), Bivalve Culture Research in Thailand. ICLARM Technical Report 19, pp 19-30. Sandison, E.E., 1966. The effect of salinity fluctuations on the life Gryphea gasar in Lagos harbor, Nigeria. J. Anim. Ecol., 35: 379-389. Sudrajat, A., 1990. Studies on the reproductive biology and culture of the rock oyster, Saccostrea cucullaru (Born) and slipper oyster, Crassoutreu iredalei (Faustino). Ph.D. Thesis, University College of Swansea. Walne, P.R., 1956. Experimental rearing of the larvae of Osrrea eduks L. in the laboratory. Fish. Invest. Ser. II, Xx(9): 23. Wong, T.M., Tan, S.H. and Cheong, T.K., 1989. Induced spawning larval development and early spat growth of the tropical oyster Crasvostrea belcheri in the laboratory. Paper presented at the 12th Marine Science Seminar at Institut Pengajian Tmggi, Universiti Malaya, Kuala Lumpur, 18 November 1989.
145 ( 19%) 129- I39
Effect of salinity on hatching, larval growth, survival and settling in the tropical oyster Crassostrea belcheri ( Sowerby) Shau-Hwai Tan ay*, Tat-Meng Wong b a Centre forMarine and Coastal Studies, Universiti Sains Malaysia, 11800 Penang, Malaysia b Open Learning Institute of Hong Kong. 9-13 F, Trade Department Tower, 700 Nathan Road, Kowloon, Hong Kong Accepted 25 April
I!396
Abstract The effects of salinity (O-30 p.p.t.) on egg development, growth and survival of D-larvae to the plantigrade stage as well as settling in Crassosfrea befcheri were studied. Eggs were able to develop into D-larvae from 12 p.p.t. to 30 p.p.t. salinity. Optimum development of eggs to D-larvae occurred at salinities between 24 and 30 p.p.t. At these salinities, survival exceeded 75%. D-larvae were able to survive to the settling stage when cultured in salinities from 12 to 30 p.p.t., with the best survival occurring between 12 and 24 p.p.t. (20.3-22.6%). At 18 p.p.t. salinity, first settling occurred on the 17th day compared with the 19th day for larvae cultured at 12 and 24
p.p.t., and the 23rd day for those cultured at 30 p.p.t. Optimum settling occurred at salinities between 12 and 18 p.p.t. Keywords:
Crassostrea
belcheri; Hatching;
Larval growth;
Salinity;
Settling
1. Introduction The successful production of spat of the tropical oyster Crassostreu belcheri (Sowerby) in Malaysia via hatchery techniques was first reported by Wong et al. (1989). Since then efforts have been made to ‘scale up’ seed production to the commercial level. In the transition from laboratory investigations to large-scale hatchery production, research and development efforts must be directed towards defining optimal conditions
* Corresponding
author.
CW4-8486/%/$15.00 Copyright PII SOO44-8486(96)01338-S
6 1996 Elsevier Science B.V. All rights reserved.
130
S.-H. Tan, T.-M. Wong/Ayuuculture
145 (1996) 129-139
for growth and survival as well as shortening the duration of the larval cycle. C. belcheri are naturally found in the intertidal zone attached to the aerial roots of mangroves. They are exposed to conditions of high temperature and variable salinity. The temperature in Malaysia is often uniformly high (25-32°C) with low diurnal changes. During the annual monsoon rains, salinity drops sharply. Under natural conditions C. b&her-i may be continuously exposed to almost fresh water (3-5 p.p.t.) for 7-8 days during the monsoonal floods. Lowered salinities have also been suggested to act as environmental triggers for spawning in C. belcheri in Sabah, Malaysia (Chin and Lim, 1975). Should spawning in fact occur under such conditions, then the fertilized eggs are likely to undergo embryonic and larval development under conditions of reduced salinity. Salinity tolerance in oysters has been reported for C. gigas (Helm and Millican, 1977), C. virginica (Loosanoff and Davis, 1963), C. iredulei and Saccosfreu cucullutu (Sudrajat, 1990); however, no study has addressed salinity tolerance changes with developmental stages in tropical oysters. This paper reports on the salinity tolerance of embryos and larvae of C. belcheri during the planktonic phase, as well as the effects on settling and metamorphosis.
2. Materials and methods 2.1. Spawning Mature C. belcheri oysters were collected from Telaga Nenas, Perak. The Telaga Nenas site is located approximately 12 km from the coast and experiences fairly stable salinity (25-30 p.p.t.) during most of the year. The oysters collected were acclimated in sand-filtered seawater (30 p.p.t.) in the laboratory for a few days. During this period, they were fed an algal mixture containing Chaetoceros cufcitruns and Isochrysis gulbunu (Tahitian strain: T-Iso). The oysters were induced to spawn with serotonin (Gibbons and Castagna, 1984). Egg handling and fertilization were as described by Loosanoff and Davis (1963). All experiments were conducted with pooled progeny from a number of parent broodstocks. 2.2. Algae The unicellular algae Isochrysis gulbunu (T-Iso) and Chuetoceros culcitrans were batch-cultured in 1 pm filtered seawater (salinity 30-32 p.p.t.) using F/2 medium (Guillard, 1972). Algal cultures were produced in 20-I glass carboys at 28 f 2°C. Only cultures in the log phase of growth were used. A haemocytometer was used to determine cell concentrations. 2.3. Test salinities Test salinities (0, 6, 12, 18, 24 and 30 p.p.t.) were prepared by mixing 1 pm filtered seawater with distilled water (0 p.p.t.). Salinities were determined by a refractometer (ATAGO Ltd.) precalibrated with distilled water. Salinity beyond 30 p.p.t. was not
S.-H. Tan, T.-M. Wong/Aquaculrure
145 (1996) 129-139
131
tested based on the range of salinity occurring in the natural environment of C. belcheri (25-30 p.p.t.1. Salinity of 0 p.p.t. was tested because the salinity drops sharply to 15 p.p.t. at Telaga Nenas and is sometimes even below 5 p.p.t., coinciding with the northeast monsoon which brings rain to the catchment at the source of the river. 2.4. Experiments 2.4.1. Development of embryos Fertilized eggs were gently aerated for 15-20 min until the first polar body was observed. The fertilized eggs were then transferred to different test salinities in l-l plastic aquaria at a stocking density of 10 embryos ml-’ (10000 fertilized eggs in 1000 ml). Slow aeration was supplied via air diffusers. The temperature was not controlled and varied from 27 to 29°C during the experimental period (24 h). All experiments were conducted in triplicate. At the end of 24 h, twenty 1- ’ ml samples were collected and the numbers of embryos which had developed into normal, D-hinge larvae were recorded. Normal larvae are defined here as being perfectly D-shaped at the prodissoconch I stage while abnormal larvae had irregular or misshapen shells either completely or incompletely formed. Shell heights of 30 normal larvae from each salinity were measured using a compound microscope (Olympus; phase-contrast BH) fitted with a micrometer eyepiece, and precalibrated with a 50 X 2 micron graticule. 2.4.2. Larval growth and survival D-hinge larvae were reared at each test salinity in 15-l glass carboys at a stocking density of 6-7 larvae ml-’ (90000-105 000 D-hinge larvae in 15 1). Aeration was supplied via air diffusers. All cultures were covered with PerspexTM sheets to prevent evaporation. The larvae were fed T-Is0 with an initial feed concentration of 5000 cells ml-‘. This was raised progressively as the larvae developed (Table 1). All test cultures received the same feed rations. The culture media were completely changed every 2 days, at which time samples were taken for microscopic examination, larval count and morphometric measurements. The shell heights of 30 larvae from each treatment were measured using a compound microscope. Three l.O-ml samples were collected and the mean value for larval density
Table 1 Concentration
of algal cells fed to C. belcheri in salinity experiments
Day
Concentrations
1-2 3-4 5-6 7-12 13-14 15-18 19-23 24-29
5000 1oGQo 2oooo 25000 3oocnl 35ocKl 4oocw 5oooo
of T-Is0 (cell ml-
’)
132
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145 (1996) 129-139
was calculated. When the larvae reached 250 p,rn (length) X 280 km (height) in size, a piece of clean polyvinylchloride (PVC) (145 mm X 9 mm X 2 mm) was suspended in each culture vessel to act as cultch. The experiments were terminated when settling of pediveliger was observed. 2.4.3. Settling Larvae of C. belcheri which had been cultured routinely at 25 p.p.t. salinity were used. Pediveligers which were retained on a 253 pm nitex sieve (height > 330 pm) were transferred into 1.0-I plastic aquaria containing the appropriate test salinity at a stocking density of 1 pediveliger ml-’ (1000 pediveligers in 1000 ml). Feed (T-Iso) was added at an initial density of 60000 cells ml-‘. A piece of clean PVC (145 mm X 9 mm X 2 mm) was used as cultch in each aquarium. Each aquarium was also lined by a sheet of transparent plastic. Aeration was provided via air diffusers. Triplicate tests were conducted at each salinity. At the end of 48 h, the cultch was removed and the remaining contents were siphoned through a 253 pm screen to retain any free swimming pediveligers. After washing, the larvae were transferred to fresh media of the same salinity and the cultch was replaced. Fresh feed was then added. Two days later the number of post-larva which had set on both the PVC plate and the plastic sheet were recorded using a binocular dissecting microscope. 2.5. Statistical analysis Data were analyzed with a one-way ANOVA to examine the effects of salinity on hatching, larval growth and survival or settling. If significant interactions were present, the data were then subjected to Duncan’s multiple range test to determine which treatments were significantly different.
3. Results 3.1. Development
of embryos
Fig. 1 shows the percentage of C. belcheri eggs that developed into normal D-larvae at different salinities. Eggs developed into D-larvae at 12-30 p.p.t. At 0 and 6 p.p.t. there was 100% mortality. Salinities between 24 and 30 p.p.t. yielded the highest percentages of normal D-larvae (78.0-83.2%) which did not differ significantly (P < 0.05). At 18 and 12 p.p.t.. the survival of embryos decreased significantly to 44.1 and 16.7%, respectively. At 12 p.p.t. numerous ciliated trochophores were still seen after 24 h whilst some 43.0% of D-larvae appeared deformed. The mean shell lengths and heights of normal D-larvae in different salinities are shown in Fig. 2. Growth (both shell length and shell height) was highest at 24 p.p.t_, although the difference in growth between larvae at 24 and 30 p.p.t. was not significant. The size of D-larvae decreased significantly at 18 and 12 p.p.t. salinity.
S.-H. Tan, T.-M. Wong/Aquaculture
133
145 (1996) 129-139
100 90
T
f
80 70 60 50 40 30 20 lo-
SALINITY Fig. 1. Percentage of Crassostrea belcheri eggs that developed Temperature, 28+ 1°C; pH, 7.9-8.5; egg density,10 ml- ‘.
(ppt) into normal
D-veliger
at different
salinities.
3.2. Larval growth and survival Figs. 3 and 4 show the percentage survival and shell growth (height), respectively, of C. belcheri larvae at different salinities. In all experiments the larvae suffered fairly high mortalities (45-60%) during the first 7 days. Thereafter, survival was reasonably good at suitable salinities whilst, those at 30 p.p.t. continued to show progressive die-offs right throughout the entire larval period. In 0 p.p.t. (freshwater), 100% mortality had occurred by the first observations made on Day 3. At 6 p.p.t., the percentage survival was reduced to 42% by Day 3 and 15% by Day 5. By Day 21 mortality reached 100%. Survival until settling occurred at 12, 18, 24 and 30 p.p.t., with the best survival (20.3-22.6%) occurring between 12 and 24 p.p.t. A one-way ANOVA showed that the
(A)
(B)
_. 6ALmrY
[PPU
ml.tWrY
Fig. 2. (A) Mean shell length and(B) mean shell height of 24-h Crassostrea b&hen’ Standard deviations are shown. The horizontal lines indicate mean egg diameters.
IPPG at different
salinities.
S.-H. Tan. T.-M. Wong/Aquculture
145 (1996) 129-139
.....
1
‘\
hi-y-
?----I---.-_
-
24 ppt 30 ppt
-!:p
*
I
I
I
I
I
I
I
I
I
I
I
3
5
7
9
11
13
15
17
19
21
23
AGE
w
6 PPt 12 ppt 18 ppt
-
I,
0
x---s -
(DAY)
Fig. 3. Percentage survival of Crussosfrea helcheri larvae reared at different value k s.d. of three sets of data. Temperature, 27-28°C; pH.7.S8.5.
salinities.
Points denote mean
differences between these three salinities were not significant (P < 0.05). The first settling occurred on Day 17 at 18 p.p.t. First settling was recorded from larvae grown in 12 and 24 p.p.t. on Day 19, whilst larvae grown in 30 p.p.t. only set on Day 23. Larvae
30 PPt setting
18 PP~ setting
I
3
5
7
9
I1
AGE
13
IS
17
12
PPt
24
ppt tting
“I’ 19
21
23
(DAY.)
Fig. 4. Mean shell height of Crussostreu belcheri larvae cultured value+ s.d. (n = 90). Temperature, 27-28°C; pH, 7.8-8.5.
in different
salinities.
Points denote mean
S.-H. Tan, T.-M. Wong/Aquaculture
145 (1996) 129-139
135
50
40 35 30 r 25 I w 20 15 10 5 0
0
6
12
10
24
30
SALINITY (ppt) Fig. 5. Percentage of Crassostrea belcheri larvae which set at different salinities. Temperature, 27k 1°C; larvae density, 1 ml-‘; pH, 7.8-8.3.
grown in 6 p.p.t. were stunted and by Day 17 had only reached 160 ym compared with the 287-338 km achieved by larvae grown under more favourable salinities. Growth was highest at 18 p.p.t., although no significant differences (P < 0.05) were seen between larvae grown in 12, 18 and 24 p.p.t. throughout the entire larvae period. Larvae grown in 30 p.p.t_ showed no significant differences in shell height by Day 17 compared with larvae grown in 12-24 p.p.t. 3.3. Settling Fig. 5 summarises the mean percentage set at different salinities. The highest mean percentage set occurred in 18 p.p.t. salinity (38.6 f 2.5%), followed by 12 p.p.t. (35.8 f 4.8%). The difference between the mean percentage set between these two salinities was not significant (P < 0.05). Mean percentage set decreased to 25.6 + 2.7% at 24 p.p.t., 19.8 f 1.1% at 30 p.p.t. and 7.3 f 8.7% at 6 p.p.t. The differences in mean settling percentage at these salinities were significantly lower than those at 12 and 18 p.p.t. 100% mortality occurred at 0 p.p.t.
4. Discussion The salinity range where development of eggs to D-larvae is possible for C. belcheri is 12-30 p.p.t. The responses of C. belcheri embryos are comparable to those at 15-40 p.p.t. reported for Succostrea cucullata and those at lo-40 p.p.t. for C. iredalei (Sudrajat, 1990). In India, the salinity tolerance of S. cucullatu embryos was reported to be 15-35 p.p.t_ (Kalyanasundaram and Ramamoorthi, 1986). In Thailand, Jarayabhand (personal communication) reported that the salinity tolerance of 5. commercialis embryos was 20-40 p.p.t.
136 Table 2 Optimum
S.-H. Tan, T.-M. Wong/Aquaculture
salinities and tolerance
Species
Salinity (p.p.t.1 Tolerance
C. belcheri C. iredalei C. rhizophorae S. commercialis S. cucullara
range for development
range
12-30 IO-40 16-40 20-40 15-35 15-40
145 (1996) 129-139
of eggs of tropical oysters to D-larvae Reference
Optimum 24-30 25 27-37 25 25
This study Sudrajat, 1990 DOS Santos and Nascimento, 1985 P. Jarayabhand, personal communication, 1992 Kalyanasundaram and Ramamoorthi, 1986 Sudrajat, 1990
The optimum salinities for both growth and survival of C. belcheri embryos were between 24 and 30 p.p.t. Similar responses were reported for S. cucullufu (25 p.p.t.) and C. iredalei (25 p.p.t.) (Sudrajat, 1990), and for S. commercialis (2.5 p.p.t.1 (P. Jarayabhand, personal communication, 19921, while in the case of C. rhizophorue in Brazil, the optimum salinities for embryo development were 27-37 p.p.t. (DOS Santos and Nascimento, 1985) (Table 2). It is apparent that tropical oysters generally require almost full-strength seawater for optimal development of eggs to the D-larvae. Previous studies have shown that performance during embryo development is influenced by the salinity at which the broodstock is conditioned (Davis, 1958; Loosanoff and Davis, 1963). Broodstock used in this study were conditioned at 30 p.p.t. Further investigations are needed to determine whether this effect of broodstock conditioning salinity on embryo development also applies to C. belcheri. The results showed that C. belcheri larvae were capable of tolerating a wide range of salinities (6-30 p.p.t.) for extended periods. However, complete larval development to metamorphosis only occurred between 12 and 30 p.p.t. Growth was fastest at 18 p.p.t. This was accompanied by the earliest set (17 days), while at 12 and 18 p.p.t. first settling occurred on Day 19, and on Day 23 at 30 p.p.t.
Table 3 Optimum salinities and tolerance other soecies of ovsters Species
Salinity (p.p.t.) Tolerance
C. C. C. C. C.
belcheri virginica rivularis gigas iredalei
S. cucullata S. commercialis 0. edulis
range for larval growth and survival
12.0-30.0 10.0-27.5 17.0-20.0 15.0-34.0 10.0-35.0 18.0-30.0 15.0-35.0 20.0-27.0
range
of C. belcheri
larvae compared
Reference Optimum 12.0-24.0 17.5-27.0 20 25 20.0-30.0 24.0-30.0 25.0-30.0 20.0-30.0 22.5-27.0
This study Davis and Calabrese, I%4 Breese and Malouf, 1977 Helm and Millican, 1977 Sudrajat, 1990 Lau et al., 1992 Sudrajat, 1990 P. Jarayabhand, personal communication, Davis and Ansell, 1962
1992
with
S.-H. Tan, T.-M. Wong/Aqunculture Table 4 Optimum
salinities for settling and metamorphosis
145 (1996) 129-139
137
of various species of oyster
Species
Salinity (p.p.t.)
Reference
C. belcheri
12-18 15 18-24
This study Sahavachti et al., 1988 Tan, unpublished data Calabrese and Davis, 1970 Walne, 1956 Davis and Ansell, 1962 Sandison, 1966
C. iredalei C. uirginicu 0. edulis
9-10 24-33 23-25 30
c. gasar
The optimum salinity for larval growth and survival in C. belch-i is comparable to those reported for C. uirginicu (Davis and Calabrese, 1964) and C. riuuluris (Breese and Malouf, 1977), but lower than the 24-30 p.p.t. reported for C. iredalei (Lau et al., 500-a a--
500.
p 3
300-
E ,o 2 r:
200.
z
100.. El
I
I
I
12 18 SALINITY
6
-: ___
“\ ~-______t-J A: I__!?_O__p.:
A: B: c:
hbryo develomt Larval developlent Settingandmetamrpbsis
:
Optimusalinity Salinity tolerancerange
Fig. 6. Summary of salinity Crassostrea belcheri.
tolerance
range
and optimum
I
2L
I O-veliger
s
30
(ppt)
salinity
at different
development
stages
of
138
S.-H. Tan, T.-M. Wong/Aquuculture
I45 (1996) 129-139
1992), as well as for S. cuculluta (Sudrajat, 1990), S. commercialis (P. Jarayabhand, personal communication, 1992), C. gigus (Helm and Millican, 1977) and Ostrea edulis (Davis and Ansell, 1962) (Table 3). Few studies have been reported on the effect of salinity on settling. The lowest salinity at which optimum settling occurred in C. belcheri (12-18 p.p.t.1 is different from the 24-33 p.p.t. recorded for 0. eduZis (Walne, 1956) and 30 p.p.t. for C. gasar (Sandison, 1966) (Table 4). C. belcheri larvae are able to set and metamorphose at lower salinities. The enhanced settling and metamorphosis at lower salinity displayed by C. belcheri larvae may be due to the high amount of secretion from the byssal glands when the salinity is between 15 and 20 p.p.t. (Prytherch, 1934). The ability of C. belcheri larvae to tolerate salinities as low as 6 p.p.t. for short periods appears to provide adaptive advantage for life in estuaries characterised by high daily salinity fluctuations. It is interesting to note that as the larvae develop from the embryo to the plantigrade stage, there is a progressive increase in tolerance to lower salinities (Fig. 6), which demonstrates how well adapted C. belcheri is towards exploiting the estuarine habitat where competition from the fully marine species is much reduced. The poor larval survival and settlin g at 30 p.p.t. may also explain why C. belcheri does not compete well against more marine species such as S. cucullata on exposed rocky shores facing open seas. Most hatcheries are located in a marine environment where full-strength seawater is used for larval culture, but these salinities appear unsuitable for C. belcheri culture. Whether this applies to other tropical oyster species is not clear. Similar studies should be conducted on other species in order to enhance seed production.
Acknowledgements This study was carried out at Muka Head Research Station, Universiti Sains Malaysia, with funding support from IDRC (Oysterseed Malaysia File No.: 3-P-88-0049) and the Government of Malaysia (No.: 123/3 101/2406). We record our sincere thanks to all project staff for technical support and to Puan Shahanum bt. Wan for typing this manuscript.
References Breese, W.P. and Malouf, R.E.. 1977. Hatchery rearing techniques for the oyster Crossosrrerr riouh-is Gould. Aquaculture, 12: 123-126. Calabrese, A. and Davis, H.C., 1970. Tolerances and requirements of embryos and larvae of bivalve mollusc. Helgol. Wiss. Meeresunters., 20: 553-564. Chin, P.K. and Lim, A.L., 1975. Some Aspects of Oyster Culture in Sabah, Kuala Lumpur, Malaysia. Ministry of Agriculture and Rural Development, Fisheries Bulletin 5, 14 pp. Davis, H.C., 1958. Survival and growth of clam and oyster larvae at different salinities. Biol. Bull., 14: 296-307. Davis, H.C. and Ansell, A.D., 1962. Survival and growth of larvae of the European oyster, Osrren edulis, at lowered salinities. Biol. Bull. Woods Holes, MA, 122(l): 33-39.
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Davis, H.C. and Calabrese. A., 1964. Combined effects of temperature and salinity on development of eggs and growth of larvae of Mercenaria mercenaria and Crassosrrea uirginica. J. Fish. Biol., 63: 643-655. Dos Santos, A.E. and Nascimento, I.A., 1985. Influence of gamete density, salinity and temperature on the normal embryonic development of the mangrove oyster Crassostrea rhizophorae Guilding, 1828. Aquaculture, 41: 335-352. Gibbons, M.C. and Castagna, M., 1984. Serotonin as an inducer of spawning in six bivalve species. Aquaculture, 40: l89- 191. Guillard, R.R.L., 1972. The culture of phytoplankton for feeding marine invertebrates. In: W.L. Smith and M.H. Chanley (Editors), The Culture of Marine Invertebrate Animals. Plenum Press, New York and London, pp. 29-60. Helm, M.M. and Millican, P.F., 1977. Experiments in the hatchery rearing of Pacific oyster larvae (Crassosrrea gigas Thunberg). Aquacultnre, 47: 335-352. Kalyanasundamm, M. and Ramamoorthi, K., 1986. Temperature and salinity requirements for embryonic development of Saccosfrea cuculfara (Born). Mahasagar Bull. Nat]. Inst. Oceanogr., 19(l): 53-55. Lau, K.T., Wong, T.M. and Tan, S.H., 1992. Effect of salinity and feed concentration on larval growth and survival in the tropical oyster, Crussosfrea iredalei (Faustino). Paper presented at the 3rd Asian Fisheries Forum in Singapore, 26-30 November 1992. Asian Fisheries Society, Manila, Philippines. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve mollusks. In: Advances in Marine Biology, Vol. 1. Academic Press, London, pp. I - 136. Prytherch, H.F., 1934. The role of copper in the settling, metamorphosis, and distribution of the American oyster, Ostrea oirginicu. Ecol. Monogr., 4: 47- 107. K., Chawivanskom, V., Sahavacharin, S., Chindanond, A., Amomjaruchit, S., Nugrang, J., Silapajam, Limsurt, S., Angell, C.L., McCoy, E.W., Mutarasint, K. and Potaros, M., 1988. Hatchery techniques for tropical bivalve mollusc. In: E.W. McCoy and T. Chongpeepian (Editors), Bivalve Culture Research in Thailand. ICLARM Technical Report 19, pp 19-30. Sandison, E.E., 1966. The effect of salinity fluctuations on the life Gryphea gasar in Lagos harbor, Nigeria. J. Anim. Ecol., 35: 379-389. Sudrajat, A., 1990. Studies on the reproductive biology and culture of the rock oyster, Saccostrea cucullaru (Born) and slipper oyster, Crassoutreu iredalei (Faustino). Ph.D. Thesis, University College of Swansea. Walne, P.R., 1956. Experimental rearing of the larvae of Osrrea eduks L. in the laboratory. Fish. Invest. Ser. II, Xx(9): 23. Wong, T.M., Tan, S.H. and Cheong, T.K., 1989. Induced spawning larval development and early spat growth of the tropical oyster Crasvostrea belcheri in the laboratory. Paper presented at the 12th Marine Science Seminar at Institut Pengajian Tmggi, Universiti Malaya, Kuala Lumpur, 18 November 1989.