THE EFFECT OF SALINITY , TEMPERATURE AND GAMETE DENSITY ON THE EMBRYONIC DEVELOPMENT OF THE SLIPPER OYSTER

The study was designed to investigate effects of temperature, salinity and gamete density in the percentage of ernbryos that develop into normal D-larvae of Crassostrea iredalei 30 h after fertilization. Gametes were obtained by stripping mature oysters. It was found that the optimum salinity for embryonic development was relatively wide being between 25-35 ppt. Satisfaccory development of embryos into normal D-larvae was possible at salinities from 15-35 ppt. Normal development of embryos into the D-larval stage occured over a wide range of temperature (14"-34"C). The optimum temperature range for embryonic development was 19"29"C. Six levels of gamete density were tested to define the variation of embryonic development. The highest percentage of normal D-larvae was at density levels of 1x104, 2.5xld and 5x10 per litre; producing 81.7, 79.6 and 7L.4o/o respectively.


INTRODUCTION
The studies on culture methods for bivalves are extensive, not least because of commercial interests in certain species.The basic methods of rearing bivalve embryos and larvae are described in papers by Loosanoff and Davis (1963) and Walne (1974, f 981).Early embryonic stages of bivalves are more sensitive to environmental changes than adults (Calabrese & Davis,lgZ0).
Data on the influence of gamete density, salinity and temperature of C. rhizophorae for hatchery purposes has been reported by Dos Santos & Nascimento (1985).Kalyanasundaram & Ramam-oorthi (1986) observed the effect of temperature and salinity differences on embry- onic development of S. cuculloto.Early develop- ment studies in C. iredalei from the Philippines at ambient temperature and salinity have been described by Ver (1986).
The present study was designed to investigate effects of temperature, salinity and gamete density in the percentage of embryos that develop into normal D-larvae of C. iredalei.as well as the effects of temperature and salinity on the size of normal D-larvae, 30 hours after fertilization.

Water Treatment
Fine filtration of water was carried out using Sartorius capsule filter cartridge (1.2 pm and 0.20 pm) and sterilization was achieved by passing the water through an ultraviolet light unit.l,ow- salinity water was prepared by diluting filtered seawater with distilled water.High-salinity water was made by adding synthetic sea salts.All ') Researcher of Central Research Institute for Fisheries.Jakarta cultures were kept in constant temperature baths.
The pH level of seawater was 7.2-8.1,i'e.within the range regarded as optimal for oyster embryo's development (Calabrese & Davis, 1966).

Sources of Specimene
Adult oysters to be used for these experiment were collected from Lada Bay (West Java).These oysters were air-freighted to the U'K. and placed in a special quarantine room with a closed circuit seawater system.Space heaters were used to maintain a room ambient temperature of 24-27"C.
A mixture of cultured microalgae of two species Isochrysis galbana (Clone T'ISO) and Pauloua lutheri used as oyster diet.
Gametes were obtained by stripping mature oysters.Male and female gametes were placed separately in glass beakers.Seawater salinity was 25 ppt, at 24"+1fC.To eliminate excess pieces of gonadal tissue, eggs were sieved through 80 pm nylon mesh screens and collected on 35 pm mesh screens; sperm were sieved through 25 pm mesh screens.Egg densities were determined by counting three samples taken under agitation with a perfo-rated perspex disk on a rod.

Artificial Fertilization Experiments
The methods of fertilization were partly based on Walne (1974\ and f)os Santos and Nascimento (1985).The eggs were kept in test containers (250 ml glass beakers) under each experimental condi' tion for 24 h (30 h for measuring size of normal D-larvae).All the experiments were made in duplicate and repeated 3 times.No specific set of conditions can be considered as "control" conditions in this study.Fertilization took place in a 250 ml glass beaker after the appropriate volume of eggs and sperm suspension had been added' The suspension was gently stirred for a few minutes and then left without further stirring' Most gametes sank.After about 3 h two thirds of the seawater was decanted away and the volume made up with fresh seawater.Standard monitor- ing procedure was as follows.
After vigorous mixing a 10 ml sample was taken from each beaker and preserved with 5% formalin.Embryos are defined here as the stage between the fertilized egg and the ciliated trocho 2 pore.The normal larvae are defined as being perfectly D-shaped whilst abnormal larvae had irregular shaped shells (Dos Santos &   Nascimento, 1985).The numbers of embryos that developed normally and abnormally were counted.The eggs or larvae were counted and measured under a binocular microscope using a "Sedgewich Rafter" counting cell.About 5O eggs or larvae were measured for each treatment.
For the data analysis, the angular transformation (arcsin J"/o) was used for the percentage of normal D-larvae before analysis of variance.For comparison between means the Student-Newman-Keuls test (Sokal & Rohlf, 1981) was used.
Three kinds of experiments were conducted: (1) Determination of the appropriate salinity range for the maxirnum production of nor- mal D-laruae and effects of salinity differences on size of normal D-laruae.Eight different salinities between 5 and 40 ppt at 5 ppt interval were tested.Egg density and temperature were 104 per litre and ?4 C respectively.Numbers of sperm were ad- justed l0O per egg.
(2) Deterrnination of the appropriate tempera- ture range for the m.oximum production of normal D-laruae and effects of temperature on size of normal D-larvoe.Five different temperature, 14o, lg , 24 , n and ts4 C were tested.Salinity and egg density were 30 ppt and 104 per litre; and sperm were 100 per egg.
(3) Deterntination of the optimum density of eggs for the mo.ximum productinn of normal D-laruae.Six different egg densities per litre (1 x 104, 2.5 x 104, 1 x ld, 5 x ld and I x 106) were tested with 100 sperm per egg.Salinity and temperature were 30 ppt and 24"C.

RESULTS
Effect of Salinity on the Embryonic Develop- ment Table I shows that at salinity 5 ppt no em- bryos developed to normal D-larvae.ANOVA results indicated significant differences in the percentage of normal D-larvae between treat- ments (P<0.05).The optimum salinity for embry- onic development was relatively wide betwen 25-35 ppt (Figure 1).Although the data indicate that 30 ppt may be near the optimum, differences from neighbouring salinities were not significant.IFR Journal Vol.II No.1, 1996 The percentages of normal D-larvae produced at salinities of 25 and 35 ppt were not signi{icantly different (b0.05).Satisfactory development of embryos into normal D-larvae was possible at salinities from 15-35 ppt (59.2 tn 81.7o/o normal D- larvae).
Table l.Artifrcial fertilization of C. iredalei.Percentages of embryos developing to normal D-larvae at different salinities, 24 hours after fertilization.In all treatments, egg density and temperature were lxlo{ per litre and 24'C respectively.The highest mean length was recorded at salinity of 25 ppt (Figure 2).There was a signifi- cant difference between mean length of normal D-larvae at salinity of 25 and those at salinities of 20, 30 and 35 ppt (P<0.05).

Effect of Temperature on Embryonic Develop- ment
Normal development of embryos into the D-larvae stage occurred over a wide range of temperature ( 1 4'-34"C).ANOVA results indicated significant differences in the percentage of normal D-larvae between treatments (Table 2).The optimum temperatures for embryonic develop- ment to normal D-larvae were 19n to 29 C, and resulted in high percentages of normal D-larvae (74.0 to 81.0%) (Figure 3).The percentages of normal D-larvae at temperatures of 14'C and 34oC were low (5.6 and 41.8%).
The mean length of normal D-larvae at differ-ent temperature (30 h after fertilization) are shown in Figure 4.There was a significant differ- ence (P<0.05) in size of normal D-larvae between temperature 19o,24" and 29"C.The highest mean length recorded was at temperature of 2g'C .mean length = 70.3pm).

Deaelopment
There were 6 levels of gamete density in this experiment to test variations of embryonic devel- opment into normal D-larvae.The highest percentages of normal D-larvae of C. iredalei were at egg densities of I x 104, 2.5 x ld and 5 x 1d per litre (Table 3 and Figure 5); it recorded 8f .7,79.6 and.71.4o/o respectively.These values were not significantly different (F>0.05).The percentage of normal D-larvae decreased to 60.9, 10.5 and 6.5% when the egg densities were increased to I x lO5, 5 x 105 and 1 x 106 per litre.Temperature and egg density were 24oC and 104 per litre.Standard deviations are also shown.
Table 2. Artificial fertilization of C. iredalei.Percentages of embryos developing to normal D-larvae at different temperatur es, 24 h after fertilization.In all treatments, egg density and salinity were lx10a per litre and 30 ppt respectively.Temperature ('C) Experiments ------mean egg diameter

DISCUSSION
Effecte of Salinity and.Temperature The early embryonic stages are easily influenced by the environment.Salinity and tempera- ture are two environmental factors of primary importance for successful development of oyster Table 3. Artificial fertilization of C. iredalei.Percentages of embryos developing to normal D-larvae at different egg densities,24 h after fertilization.In all treatments, temperature and salinity  (Galtsoff, 1964).Studies on the effects of salinity and temperature on embryonic develop- ment on temperate oysters have been reported by Amemiya (1928), Davis and Calabrese (1964), Helm & Millican (1977).For tropical oysters, Dos Santos and Nascimento (1985)   Davis (1958), Loosanoff & Davis (1963) and Lough & Gonor (1971) stated that temperature and salinity conditions, at which the parent stock develop gonads and spawn, influences the tolerances of the embryos.If this is so for the present species the conditioning of adult oysters in the laboratory could affect the tolerance limit of salinity and temperature for embryonic develop- ment.
It is therefore apparent that the optimal temperature for embryonic development for practical purposes in the tropics is best around ambient temperature,24o to 29'C, and at salinity about 28 to 32 ppt.Ver (1986) and Kalvana- sudaram & Ramamoorthi (1986) report similar findings.
Observations on D-larvae 30 h after fertilization (i.e.before onset of feeding) showed that there were significant differences of mean length of larvae between different salinities and tempe- rature (20 tn 35 ppt) and 19o to 29"C).The highest mean length was recorded at a salinity of only 25 ppt and at temperature of 29"C.Davis (1958) reported that for C. uirginica larvae a salinity of 17.5 ppt.was optimum for growth even though the adults occur in higher salinities.The tempera- ture (29'C) for maximum growth of the present species was within the range of ambient tempera- ture.

Salinity
Figure l.C. iredalei.Percentage of eggs that developed into normal D-larvae at different salinities.
Journal Vol.II No.l.199634"C.Optimal salinity and temperature for em- bryonic development of this species was 2b-Bb ppt and at temperatures lg-Zg"C.Thus optimal salinity and temperature for embryonic develop- ment in C. iredalei was wide.The habitat of C. iredalei is in estuarine areas, the salinity is a Percentage of eggs that developed into normal D-larvae at different egg densities, 24 h after fertilization.Salintiy and temperture were 30 o/oo and 24'C.Standard deviations are also shown. IFR