HUO Zhongming, WANG Zhaoping,, LIANG Jian, ZHANG Yuehuan, SHEN Jianping, YAO Tuo, SU Jiaqi, and YU Ruihai

1) Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Engineering Research Center for Shellfish Culture and Breeding of Liaoning Province, College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, P. R. China

3) Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, P. R. China

Effects of Salinity on Embryonic Development, Survival, and Growth of Crassostrea hongkongensis

HUO Zhongming1), WANG Zhaoping1),*, LIANG Jian2), ZHANG Yuehuan3), SHEN Jianping1), YAO Tuo1), SU Jiaqi1), and YU Ruihai1)

1) Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Engineering Research Center for Shellfish Culture and Breeding of Liaoning Province, College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, P. R. China

3) Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, P. R. China

This study examined the effects of salinity on embryonic development, survival, and growth of the Hong Kong oysterCrassostrea hongkongensis.The embryos, larvae, and juveniles ofC. hongkongensiswere held in artificial seawater at three different salinities (low, 15; medium, 23; and high, 30) to determine the optimum hatchery and nursery conditions for mass production of the seeds. Results showed that the percentage production of straight-hinged larvae from fertilized eggs was significantly lower at the high salinity than at the low- and medium-salinities (P <0.05). The survival rates of larvae and juveniles differed significantly among the three salinity trials, with the highest survival rate observed at the low salinity (P <0.05). The shell height of larvae was significantly larger at the low salinity than at the high and medium salinities from days 9 to 15 (P <0.05), whereas that of juveniles was significantly larger at the low salinity than at the high and medium salinities on day 70 (P <0.05). These results indicate that the larvae and juveniles ofC. hongkongensisare tolerant to a wide range of salinities (15 to 30), but show better growth and survival at relatively low salinities. Thus, it is recommended to use relatively low salinities in hatchery and nursery systems for improved yields ofC. hongkongensis.

salinity; embryonic development; survival; growth;Crassostrea hongkongensis

1 Introduction

The Hong Kong oysterCrassostrea hongkongensisis commercially cultured in southern China, yielding up to 100 metric tons per year (Lam and Morton, 2003; Liet al., 2011). Because of its unique flavor and fast growth, the demand for commercial aquaculture ofC. hongkongensishas been increasing in recent years. Traditionally,C. hongkongensisfarming is dependent on natural seeds collected from the wild. However, the natural seeds ofC. hongkongensisare often unreliable and their availability is limited to a short season. These issues have significantly constrained the cultivation ofC. hongkongensis. An alternative is to produce the seeds ofC. hongkongensisin hatcheries and nurseries and then transport them to the open sea for aquaculture use.

Large-scale production of an aquaculture species must be based on adequate knowledge of its ecological requirements for optimum development. Salinity is a major environmental element that affects biological activities, including those related to immune responses (Gagnaire, 2006; Tayloret al., 2007), physiological processes (Hildreth, 1980; Madrones-Ladja, 2002), development of embryos (Dos Santos and Nascimento, 1985; Tan and Wong, 1996), growth, and survival of larvae (Nell and Holliday, 1988; Liuet al., 1992; Soriaet al., 2011; Xuet al., 2012) and juveniles (Taylor, 2004). To the best of our knowledge, no study has addressed the salinity tolerance in relation to hatching, larval development, juvenile growth, and survival inC. hongkongensistill date.

The present study compares the hatchery potential ofC. hongkongensisin artificial seawater at three different salinities,i.e., low (15), medium (23), and high levels (30). The results will provide valuable data for developing proper hatchery and nursery techniques for mass production ofC. hongkongensisseeds in China.

2 Materials and Methods

2.1 Brood Stock

In mid-July, 2010, sexually matureC. hongkongensisindividuals were collected from the coastal sea of Guangxi Province, southern China. The animals werecultured at 25, 23–25℃ for one week in 60-m3indoor concrete tanks at the Dalian Aquaculture Company.

2.2 Experimental Design

Gametes were obtained following the method of Allen and Gaffney (1993). All instruments, as well as the surface of the oysters, were cleaned with fresh water. Each oyster was opened using disposable tools, and the gonad was examined microscopically to identify male, female, and hermaphroditic individuals. Individuals with mature gametes were chosen for fertilization. Oocytes were dissected from gonad tissue of each individual and placed directly into 2-L individual containers. The oocytes from three femaleC. hongkongensiswere mixed together and held in a container, and the sperms from three maleC. hongkongensiswere added to the container with unfertilized eggs. Artificial sea water with different salinities was prepared by adding sea salts to freshwater. The salinity was measured using a refractometer (HANNA/HI9820 3D). The experimental groups were hatched at salinity 15 (S 15), 23 (S 23), or 30 (S 30). After 40 min, all containers were rinsed with seawater pre-filtered through a 30-μm nylon sieve and then transferred to 5-L containers with fresh seawater at the same temperature (25–27℃) and intended salinity. The experiment was replicated three times using different sets of parental oysters and beaker matrices.

2.3 Rearing and Measurement

After about 24 h, the fertilized eggs developed into straight-hinged larvae (D-larvae). The D-larvae were collected using a 40-μm nylon sieve and then transferred into 60-L tanks for larval rearing in seawater at the corresponding salinity. Each D-larva group was placed in 60-L containers with an initial density of 5 larvae per milliliter. During larval rearing, water was completely exchanged with sand-filtered seawater of the corresponding salinity every 2 d, and the temperature was maintained at 26–28℃. The larvae were fed withIsochrysis galbanaduring days 1–3 and a mixture ofI. galbanaandChlorellaspp. (2:1) from day 4 to the juvenile stage. The feeding ration was increased as the larval development progressed; the larvae were fed a ration of 2000–10000 cells mL-1d-1on days 1–3 and 30000–60000 cells mL-1d-1from day 4 to the juvenile stage. The same density of each group of larvae was ensured by adjusting water volume every 4 d. One milliliter of water samples was taken and the numbers of eggs and live larvae counted under a microscope for estimating the density. The hatching rate was calculated based on the production of D-larvae from fertilized eggs. The larval survival rate was calculated based on the total number of live larvae on day 15 after the D-larval stage. Shell heights of 30 randomly selected larvae were preserved with 4% formaldehyde and then measured using a microscope on days 3, 6, 9, 12, and 15. The larval density was reduced with larval growth from the initial 5 larvae per milliliter to 1–2 larvae after metamorphosis. After 15–18 d, when the juveniles began to attach to the settlement board, they were placed in bags (25 cm×20 cm) of 700-μm mesh size with 200 juveniles per bag and reared at the same salinity as the larvae in 60-L containers for 50 d post-fertilization. The juvenile shell heights were measured with an electronic Vernier caliper for 30 randomly chosen individuals from each bag on days 50, 70, and 90. The juvenile survival rate was calculated based on the total number of live juveniles on days 50 and day 90.

2.4 Statistical Analysis

Data were transformed (log or arcsine) if necessary to meet the assumptions of the statistical methods employed. Effects among the low, medium, and high salinities (15, 23, 30) were analyzed using one-way ANOVA as implemented in R statistical software. Effects of salinity on embryonic development, survival, and growth ofC. hongkongensiswere analyzed by Tukey multiple comparisons as implemented in R statistical software. Significance level for all analysis was set atP <0.05.

3 Results

3.1 Embryonic Development

Fig.1 The survival rates of Crassostrea hongkongensis eggs at the D-larvae stage, D-larvae on day 15, and juveniles from days 50 to 90 at three different salinities. Each point represents one replicate.

Table 1 Statistical analysis of the salinity effects on survival of Crassostrea hongkongensis

Among the three salinity trials, the percentage of eggs surviving to the D-larvae stage was significantly lower atthe high salinity (30, 53.56% ± 5.08%) than at the low (15, 78.66% ± 7.31%) and medium salinities (23, 77.56% ± 4.48%). No significant difference was observed in the percentage of eggs surviving to the D-larvae stage between the low and medium salinity trials (Fig.1, Table 1).

3.2 Survival

The larval survival rate of D-larvae on day 15 differed significantly among the three salinity trials,i.e., 80.00% ± 5.10% at the low salinity, 55.44% ± 8.25% at the medium salinity, and 32.78% ± 5.24% at the high salinity (Fig.1, Table 1). Relatively higher survival was observed inC. hongkongensisjuveniles at the low salinity from days 50 to 90. Tukey multiple comparisons showed that the survival rate ofC. hongkongensisjuveniles differed significantly among the three salinity trials,i.e., 79.33% ± 2.65% at the low salinity, 55.78% ± 4.49% at the medium salinity, and 42.11% ± 6.31% at the high salinity (Fig.1, Table 1).

3.3 Growth Rate

The larval shell height was significantly larger at the low salinity than at the high and medium salinities from day 9. The difference in larval shell height was not significant at the high and medium salinities except on day 9 (Fig.2, Table 2).

Fig.2 Larval shell height of Crassostrea hongkongensis at three different salinities. Each point represents one replicate. sh, shell height.

Table 2 Statistical analysis of the salinity effects on larval growth of Crassostrea hongkongensis

The mean shell height ofC. hongkongensisjuveniles was larger at the low salinity compared to those at the high and medium salinities (Fig.3, Table 3). Tukey multiple comparisons showed that the difference in shell height ofC. hongkongensisjuveniles was significant among the three salinity trials on days 70 and 90 but not on day 50.

Fig.3 Juvenile shell height of Crassostrea hongkongensis at three different salinities. Each point represents one replicate. sh, shell height.

Table 3 Statistical analysis of the salinity effects on juvenile growth of Crassostrea hongkongensis

4 Discussion

Embryonic development is an important step that needs to be controlled carefully in oyster hatchery operations for high yields of D-larvae. Previously, embryos ofC. rhizophraeincubated at salinities lower than 19 were reported to exhibit abnormal development at the D-larval stage (Dos Santos and Nascimento, 1985). Madrones-Ladjia (2002) found thatPlacuna placentadid not completely develop at salinities lower than 22. In the present study, embryos ofC. hongkongensissuccessfully developed to normal D-larvae at salinities of 15, 23, and 30 but performed better at the low and medium salinities than at the high salinity. Together these findings provide a guide for improving D-larvae production ofC. hongkongensis.

In the present study, rearingC. hongkongensisin low (15), medium (23), and high salinities (30) had significant effects on larval and juvenile survival and growth. Larvae and juveniles survived and grew best at the low salinity while showing lower growth and survival rates at the medium and high salinities. A number of previous studies of salinity tolerance in oysters indicate thatCrassostreaspecies are generally euryhaline (Li and Qi, 1994; Guoet al., 2008) but may exhibit better growth and survival at an optimal salinity. For example, high growth and survival rates of larvae have been observed inOstrea talienwhanensisat an optimum salinity of 25–34 (Liuet al.,1992),C. virginicaat 9–10 (Davis and Calabrese, 1964),C. gasarat 30 (Sandison, 1966), andO. edulisat 24–33 (Walne, 1956). The growth rate ofC. gigaswas the highest at salinity of 19 to 27, with relatively high survival rate at all salinities tested (15–39) (Nell and Holliday, 1988).C. belcherisettled well in salinities between 12 and 24 but grew and survived better at salinities of 12–18 (Tan and Wong, 1996).

The mechanisms through which salinity affects marine mollusks remain unclear. Previous studies indicate that isosmotic intracellular regulation plays an important role in marine bivalves (Lange, 1970; Strand, 1993; Singoretbrailovsky, 1996; Damrongpholet al., 2001; Laing, 2002; Wanget al., 2011a, b). In addition, Hosoiet al.(2003) reported that Ala, Gly, Pro, Tau, and Glu are typically the dominant free acid amino acids (FAAs) in mollusks, which contribute to intracellular osmolalitic processes and play an important role in salinity tolerance. Further studies of FAA genes inC. hongkongensiscultured at different salinities may help identify the salinity effects onC. hongkongensis.

The results of this study clearly demonstrate that the larvae and juveniles ofC. hongkongensiscan tolerate a wide range of salinities (15–30) but exhibit larger shell height and better survival at the lower salinities. Hence, hatchery and nursery culturing of this species at lower salinities will likely produce a higher yield than at higher salinities. Use of an optimally controlled suitable salinity for large-scale artificial breeding shall increase the seed production ofC. hongkongensisand decrease the breeding time in aquaculture systems in China.

Acknowledgements

The authors thank the Dalian Hairi Fisheries Food Limited Corporation for technical support and Chuanwen Song, ShitianSang, and Wenxue Guo for assistance with the hatchery operation. This research was supported by grants from the National Natural Science Foundation of China (31172403) and the National Basic Research Program of China (2010CB126406).

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(Edited by Qiu Yantao)

(Received November 2, 2012; revised December 25, 2012; accepted November 14, 2013)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. E-mail: zpwang@ouc.edu.cn