Interspecific Hybridization Between Crassostrea angulata and C. ariakensis

YAO Tuo, ZHANG Yuehuan, YAN Xiwu, WANG Zhaoping,, LI Dongchun, SU Jiaqi, and YU Ruihai

1)Fisheries College, Ocean University of China, Qingdao266003,P. R. China

2)Key Laboratory of Tropical Marine Bio-resources and Ecology,South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou510301,P. R. China

3)Engineering Research Center of Shellfish Culture and Breeding of Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian116023,P. R. China

Interspecific Hybridization Between Crassostrea angulata and C. ariakensis

YAO Tuo1), ZHANG Yuehuan2), YAN Xiwu3), WANG Zhaoping1),*, LI Dongchun3), SU Jiaqi1), and YU Ruihai1)

1)Fisheries College, Ocean University of China, Qingdao266003,P. R. China

2)Key Laboratory of Tropical Marine Bio-resources and Ecology,South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou510301,P. R. China

3)Engineering Research Center of Shellfish Culture and Breeding of Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian116023,P. R. China

Interspecific hybridization can generate heterosis, which is proven to be a useful tool in selective breeding programs for oyster culture.Crassostrea angulataandC. ariakensisare two important economic shellfish species in China. We conducted 2 × 2 reciprocal crosses to determine whether these two species can cross-fertilize and their hybrids can hatch, survive and perform heterosis. Fertilization was found symmetrical without delay. The rate of fertilization success ofC. angulata♀ ×C. ariakensis♂was lower than that ofC. ariakensis♀ ×C. angulata♂, and the success rate of both hybridizations was lower than that of two intraspecific crosses each. During the planktonic period, survival rate of the progeny was lower in the hybrid crosses than in the intraspecific crosses. On day 360, mean shell height of the progeny ofC. angulata♀ ×C. angulata♂was highest, which was followed by that ofC. angulata♀ ×C. ariakensis♂,C. ariakensis♀ ×C. ariakensis♂andC. ariakensis♀ ×C. angulata♂in a descending order. Morphology of adults produced by the hybrid crosses was similar to that ofC.angulata. Both hybrids underwent normal gonad development and produced mature gametes in the mating season. This study provided new insights into the quantitative traits in interspecific crosses ofCrassostreaspecies, thus being of guidance value for selective breeding of oyster.

Crassostrea angulata;Crassostrea ariakensis; interspecific hybridization; gonad development; heterosis

1 Introduction

Distant hybridization is an effective tool of obtaining hybrids between species or genera, which results in phenotypic and genotypic changes of the progenies (He, 1999). Hybridization involves the fusion of sex cells and the development of hybrids that combine the hereditary characteristics of both parents.

Heterosis, or hybrid vigor, is often performed by the first filial generation of different species, as was indicated by increased adaptive capacity and improved viability (Shull, 1908). Additionally, hybridization can affect various traits, penetrate species or category boundaries, expand genetic variation, and create new mutation types or even new species (He, 1999).

Intraspecific hybridization of bivalves has contributed to the genetic improvement of bivalve brood stocks or inbred lines, although the underlying genetic mechanisms are still poorly understood (Griffing, 1990; Hedgecocket al., 1996). Several attempts have been made to the interspecific hybridization between species of oysters with genetic confirmation, which includedCrassostrea gigas×C. rivularis(Allen and Gaffney, 1993),C. gigas×C. virginica(Allenet al., 1993),C. gigas×C. angulata(Huvetet al., 2002; Soletchniket al., 2002; Leit?oet al., 2007),C. gigas × C. hongkongensis(Zhanget al., 2012),C. rivularis×C. virginica(Allenet al., 1993),C. ariakensis × C. sikamea(Xuet al., 2009), andC. arikensis × C. hongkongensis(Huoet al., 2013). Literatures on their early development and genetic conformation (Tenget al., 2010; Biet al., 2005; Zhouet al., 2003) are rich; however little is known regarding the effects of hybridization on quantitative traits (e.g., growth, survival, and gonad development) of these interspecific hybrids of marine bivalves.

Hybrid sterility commonly occurs in distant hybridization. Progeny of such crosses generally have discordant physiological functions and disturbed reproductive systems, either losing the ability to reproduction or having low fertility (Lou, 1999). Thus, developing an efficient method for fertility analysis of hybrid progeny is of great significance to shellfish selective breeding. A reliable waywill be created for selective breeding, if gonad of hybrid progeny develops and even the backcross succeeds. For shellfish, many hybrids die at the nymphal stage; even though surviving progeny are obtained, the less number and purity to be researched make it lack statistical significance. Thus, the analysis of fertility of hybrids has been lacking.

In one of our studies, the two hybrid progeny stocks ofC. gigas×C. ariakensiswere completely fertile, and even hermaphroditic individuals were present among the hybrid offspring (Yao, 2014). Wang (2011) found that all hybrid progeny ofArgopecten irradians irradians×A. purpuratuswere female with only a single gonad. Asymmetry in fertilization was observed whenC. hongkongensiseggs were fertilized byC. gigassperm, but the reverse cross resulted in infertility (Zhanget al., 2012). To date, the most success of interspecific hybridization betweenCrassostreaspecies was met inC. hongkongensis♀ ×C. gigas♂ (Zhang, 2012) andC. hongkongensis♀ ×C. ariakensis♂ (Huo, 2013), where not only the fertility of hybrids was analyzed but also the backcross was realized.

SeveralCrassostreaspecies naturally occur along the coast of China, which includeC. ariankensis,C. angulata,C. gigas,C. hongkongensis, andC. sikamea(Wanget al., 2006). Of these,C. ariakensisandC. angulataare two important economic shellfish species and may present in the same estuary (Guoet al., 2008).C. ariakensisis one of the most important cultured oyster species because of its high market value in South China (Guoet al., 2006). This species is distributed widely from Liaoning to Guangxi Province, with populations centered in Guangdong Province (Wanget al., 2004). In 2004,C. ariakensiswas re-identified and assigned toC. rivularison the basis of morphologic and phylogenetic analyses (Wanget al., 2004).C. angulatais another economically important species with an annual production up to 1.3 million tons (Zhang, 2012). This species inhabits the southern part of Changing River and mainly concentrates on Fujian Province. In 2010,C. angulatawas defined as a subspecies ofC. gigas(Wang and Guo, 2010).C. angulatagrows fast whileC. ariakensisgrows substantially slow.If these two species can hybridize and produce viable offspring, then, the progeny may be capable of growing rapidly.

To examine the heterosis of hybrids betweenC. angulataandC. ariakensis, two reciprocal hybrid crosses and two pure crosses were made by pair mating. Heterosis of quantitative traits was examined in progeny cultured under the same conditions at the early growth stages. Maternal origin was analyzed throughout the cultivation period. Gonad development of hybrid progeny was examined, and the identity of parental species as well as their hybrids was confirmed genetically using a molecular marker.

2 Materials and Methods

2.1 Broodstock Maturation, Gamete Collection, and Fertilization

In August 2010, individuals ofC.angulataandC. ari-akensiswere purchased from Dalian and Shenzhen, China. The animals were identified as purebred individuals by molecular genetic testing using internal transcribed spacer 2 (ITS2) as a molecular marker (Wang and Guo, 2008b). Both species reproduced in August 2010 and July 2011 in Zhuanghe Ocean Shellfish Nursery, Dalian, China. The broodstocks ofC.angulataandC. ariakensisused in this study were the offspring produced in 2011, which were hung in collector bags in an ecological pool and reached synchronous sexual maturity in July 2012.

Gametes were obtained from mature broodstocks by dissection in July 2012. Dissection tools were thoroughly washed with freshwater between dissections to avoid cross-contamination. Before gamete collection, the animals were individually dissected. Gonadal tissues were sampled and then examined under a light microscope to determine gender. The gametes from each spawning were also examined microscopically. Based on gametic quality, three females and three males were selected each species for use in the following experiments. Eggs from each female were rinsed with sand-filtered seawater and pooled into one beaker per female. The egg suspension was passed through a 90-μm nylon screen to remove detritus, collected on a 25-μm nylon screen, and then resuspended in seawater. Sperm from each selected male were collected in separate beakers and then diluted to the same density before being pooled into a beaker in an equal volume (Xuet al., 2009). After gamete collection, tissues from spawned animals were fixed in ethanol for subsequent confirmation of species.

Eggs fromC. angulata(designated as A) were divided into two 2-L beakers in an equal volume. The eggs were examined microscopically to ensure no occurrence of uncontrolled fertilization (as indicated by the absence of polar bodies), and then fertilized with sperm fromC. angulataandC. ariakensis(designated as a), respectively. Sperm were added to the egg suspension until approximately 20-25 sperm surrounded an egg (as monitored under a light microscope). For interspecific crosses, approximately 20% more sperms were added (Xuet al., 2009). The same procedure was applied toC. ariakensisto achieve 2 × 2 factorial crosses, including two intraspecific hybrid groups [AA (C. angulata♀ ×C. angulata♂) and aa (C. ariakensis♀ ×C. ariakensis♂)] and two interspecific hybrid groups [Aa (C. angulata♀ ×C. ariakensis♂) and aA (C. ariakensis♀ ×C. angulata♂)]. Fertilization occurred within 60 min after gamete collection. Each treatment was replicated three times using different sets of parental oysters (i.e., three animals each sex each species). Fertilized eggs were sampled and held in beakers to evaluate fertilization success and survival to the D-stage. The remaining fertilized eggs were counted and placed in 60-L buckets at a density of 60 eggs mL-1for hatching with gentle aeration. Zygotes developed in filtered seawater at 27℃ and salinity 26.

2.2 Larval Rearing, Spat Nursery, and Grow-Out

Twenty-four hours after fertilization, each cohort of D-larvae were collected on a 40-μm sieve and rearedseparately in 60-L buckets. Three replicates were set up each cohort. The initial larval density was adjusted to 3 larvae mL-1in each culture vessel, and this density was maintained by adjusting the water volume. Seawater was completely changed every other day. The larvae were fedIsochrysis galbanaon days 0-6 andPlatymonas subcordiformisandI. galbana(volume ratio=1：1) afterward. The feeding rate was gradually increased from 2000 to 60000 cells mL-1d-1. The temperature and salinity of rearing water were maintained at 26-29℃ and 25-26, respectively.

After 15-18 days of larval rearing, the pediveligers showed eyespots, indicating the commencement of metamorphosis. Bunchy plastic corrugated plates were hung in the buckets as spat collectors. Three days later, spats were observed on the plastic plates. To accelerate growth, all newly settled spats were transferred to big concrete tanks (8.0 m × 8.0 m × 1.6 m). The settled spats were suspended in these tanks with aeration and fedChlorella vulgarisat a ratio of 80000-100000 cells mL-1d-1. Water was exchanged once a day at a rate of 30%, and water temperature was maintained at 26-28℃. On day 60, spats were detached and transferred into collector bags at a density of 60 animals per bag. The collector bags with the spats were hung in an ecological pool and periodically replaced with larger mesh bags from 1 to 3 mm in size.

During the outdoor nursery culture period (September to November), water temperature varied between 28 and 12℃ and salinity between 26 and 28. From December to March next year, all spats were transferred to indoor concrete tanks. During this period, the lowest water temperature was -1.4℃. When temperature increased to 9℃ in April, 2013, the oysters were again hung in the ecological pool. In June, oysters were diluted to 20 individuals each bag. At this time, some of the oysters had mature gonads.

2.3 Sampling and Measurements

Sixty minutes after fertilization, a 2-mL sample was collected each replicate to count the numbers of total eggs and fertilized eggs (i.e., those that had divided). The fertilization rate was calculated as the percentage of divided eggs among the total. The mean egg size was calculated by measuring 30 randomly selected eggs under an optical microscope fitted with an eyepiece calibrated micrometer. The same procedure was applied to D-stage larvae 24 h after fertilization. The survival rate of D-larvae was calculated based on the percentage of D-larvae among fertilized eggs.

During the larval stage, 30 larvae were randomly sampled each replicate to measure average shell height on days 3, 6, 9, 12, 15, and 18 using an electronic Vernier caliper; and the survival rate was calculated at the given time intervals. Metamorphic time was calculated as the time from day 0 to the day when 50% of the larvae had metamorphosed to pediveligers.

In the spat grow-out stage, 30 spats were randomly sampled each replicate to measure average shell height on days 90, 120, and 360; and the survival rate was assessed at the same time intervals. Spat survival rate was calculated as the percentage of spat present relative to the initial number of juveniles. Some surviving spats were sampled on day 90 and fixed in 95% ethanol for genetic confirmation of species identity.

2.4 Gonad Development

In the flourishing periods of the propagation, 60 interspecific hybrid individuals (Aa and aA separate) and 60 intraspecific hybrid individuals (AA and aa separate) randomly selected from each replicate were dissected to examine the development of the gonad and the sex ratio.

2.5 Genetic Confirmation of Species Identity

DNA was extracted from ethanol-fixed samples of the parents and the hybrid progeny using the TIANamp marine animals DNA extraction kit (Tiangen, China). Genetic confirmation of hybrids was conducted using an ITS2 marker (Wang and Guo, 2008a, b). Primer sequences for ITS2 were 5’-TCTCGCCTGATCTGAGG TCG-3’ (forward) and 5’-GCAGGACACATTGAACAT CG-3’ (reverse). PCR amplification was performed using a 25-μL volume containing 1.5 mmol L-1MgCl2, 0.2 mmol L-1dNTP (each), 0.2 μmol L-1primers (each), 20 ng template DNA, 1 U ofTaqDNA polymerase, 1 × buffer. The thermal cycler protocol consisted of an initial denaturation at 95℃ for 5 min, followed by 30 cycles of denaturing at 95℃ for 1 min, annealing at 62.5℃ for 1 min, and extending at 72℃ for 1 min, and a final extension at 72℃ for 5 min. Four controls were included in the experiment： one with DNA from an identifiedC. angulataparent, one with DNA from aC. ariakensisparent, and two with DNA from interspecific hybrids. All amplified DNA fragments were separated on 3% agarose gels containing 0.2 μg mL-1ethidium bromide. The separated DNA fragments were visualized under a Bio-RadUV transilluminator for species identification based on fragment length.

2.6 Statistical Analyses

All statistical analyses were performed using the Statistical Program for Social Sciences 16.0, and significance for all analyses was set toP＜ 0.05. The shell height was logtransformed to ensure normality and homoscedasticity (Neteret al., 1985). The differences in growth and survival among treatments were analyzed using one-way ANOVA followed by Duncan’s multiple comparison test.

3 Results

3.1 Fertilization and Hatching Rates

The mean diameter ofC. angulataeggs (49.57 μm) was significantly longer than that ofC. ariakensis(47.48 μm,P＜ 0.01). As expected, high fertilization rates were achieved for the two intraspecific crosses AA (76.27%) and aa (96.73%, Table 1). For the two interspecific crosses, eggs ofC. angulatawere fertilized by spermfromC. ariakensis, and eggs ofC. ariakensiswere fertilized by sperm fromC. angulata. However, fertilization rates of the two interspecific crosses were lower than those of the two intraspecific crosses. For the Aa cross, less than thousandth eggs can be seen fertilized by light microscopy in part repeating group. In contrast, the fertilization success of aA was markedly higher (19.1%) than that of Aa (2.13%,P＜ 0.01). After 20-24 h, larvae of the intraspecific and interspecific crosses reached the D-larval stage simultaneously. Mean D-larvae survival rates in the interspecific crosses were 56.17% for aA and 47.75% for Aa, both lower than those of the intraspecific crosses (58.86% for AA and 95.16% for aa). Shell height of initial D-larvae (24 h after fertilization) was significantly higher for AA larvae than for the other larvae groups (P＜ 0.01, Table 1).

Table 1 Egg diameter, fertilization success, percent survival of fertilized eggs reaching the D-stage, and initial D-larva shell height from 2 × 2 factorial crosses betweenCrassostrea angulata(A) andC. ariakensis(a)

3.2 Survival and Growth

Among the four groups, the growth rate of progeny was the highest in the inbred group AA, which had significantly greater shell height than the other groups on day 15 (P ＜0.05, Fig.1A). In addition, D-larvae of AA group reached metamorphosis in 15 days, earlier than progeny of the other groups. The shell height of Aa progeny was substantially higher than that of aa progeny for the first 14 days, but showed no statistically significant difference from day 15 to 18 (P ＞0.05). The growth rate of aA progeny was the lowest, with shell height consistently smaller than those of the other groups. The aA progeny began metamorphosis from day 23, later than the other three groups (day 15 for AA and day 18 for Aa and aa). The growth of AA offspring was faster than that of aa progeny, and the same characteristic emerged in the other two groups of Aa and aA. Taking the source of eggs into consideration, we consider that the hybrid groups had maternal effect.

Fig.1 Growth (A) and survival rate (B) of oyster hybrid and control larvae at different time intervals post fertilization. Survival data were standardized by setting initial day at 100%. Error bars indicate standard deviation of the mean (n= 3).

Overall, the two hybrid groups had notably lower survival than the two inbred groups (Fig.1B). The former groups had a higher survival rate within the first 3 days, which declined significantly afterward possibly due togamete compatibility problems. The survival rates of Aa and aA were 18.1% and 0.76% on day 18, respectively. The fertilization rate and hatchability of aA were higher than those of Aa, but the reverse was true for the survival rate of D-stage larvae.

After metamorphosis, the growth of spat accelerated and few dead individuals were found in all groups. On day 90, AA progeny were markedly larger than progeny of the other groups. Size of aa progeny was not significantly different from that of aA and Aa progeny, but Aa progeny were notably larger than aA offspring (P ＜0.05) (Fig.2). From day 90 to 120, all juveniles grew slowly in water at low temperature. On day 360, shell height followed the order of AA ＞ Aa ＞ aa ＞ aA, showing significant differences among all groups (P ＜0.05).

Fig.2 Juvenile and adult oyster body size measured as shell height. Error bars indicate standard deviation of the mean (n= 3). Different letters above the column denote significant differences (P＜ 0.05) as determined by Duncan’s multiple comparison test.

3.3 Gonad Development

To analyze the fertility of the hybrids, many oysters of four groups were dissected in July. The gonads of specimens from each of the four groups were developed in full (Fig.3). Part of the gonad tissue was sampled and evaluated under an optical microscope. Shaking fiercely sperm, round and pear-shaped eggs could be observed. Males were a little more common than females, and the sex ratios of two hybrid groups were 1.22：1 (aA) and 1.31：1 (Aa) (Table 2). The shell was round and fat in the aa group but round or long and thick for the AA group. The shell shape of progeny from the Aa and aA crosses resembled that of AA progeny.

Fig.3 Photos of mature gonads for aa (Crassostrea ariakensis), AA (C. angulata), aA (C. ariakensis♀ ×C. angulata ♂), and Aa (C. angulata♀ ×C. ariakensis ♂).

Table 2 Sex ratio of offspring of different oyster crosses

3.4 Genetic Confirmation of Species Identity

All parents used in this study were unambiguously identified asC. angulataorC. ariakensisby PCR assay using the ITS2 marker (Fig.4). All DNA bands were excised and sequenced (‘a(chǎn)’, ‘A’, ‘a(chǎn)u’, ‘a(chǎn)d’, ‘Au’, and ‘Ad’; Fig.4). Spat ofC. angulata(AA) produced a single DNA band at approximately 650-660 base pairs (bp), whereas spat ofC. ariakensis(aa) produced a single band at 570-580 bp. The sequence from band ‘A’ produced a 660-bp fragment, which matched the ITS2 sequence ofC. angulata(‘A’) (KF662358.1; e-value = 0.0, identity = 100%). The sequence from band ‘a(chǎn)’ produced a 579-bp fragment containing a 504-bp ITS2, which matched the ITS2 sequence ofC. ariakensis(‘a(chǎn)’) (EU252081.1; e-value = 0.0, identity = 99%). All interspecific crosses produced two similar bands, one between 600 and 640 bp and the other at approximately 680 bp (Fig.4), different from the DNA bands produced by their parents. So it can be seen that, intraspecific cross produce one band while interspecific cross produce two, when ITS2 marker was used.

Fig.4 ITS2 length polymorphisms. Lanes M, marker DL100; 1-3, aa; 4-6, hybrid aA; 7-9, hybrid Aa; and 10-12, AA.

4 Discussion

Results of this study clearly demonstrated that the hy-bridization betweenC. angulataandC. ariakensisis possible and these twoCrassostreaspices can fertilize reciprocally. Similar results have previously been reported for hybridization betweenC. gigasandC. rivularis(Allen and Gaffney, 1993). In the current study, the aA cross had higher fertilization success than the Aa cross. Although the fertilization rate was low for the Aa cross, it had a relatively higher survival rate than the aA cross (Table 1). On day 6, the two hybrid groups suffered catastrophic mortality, likely due to genome incompatibility between the two species. However, lower fertilization and hatch success were only found in hybrid groups but not in inbred groups (Table 1). Survival was influenced by gametic and genetic incompatibility, as abnormality rate was higher while survival to the veliger stage was lower in the interspecific crosses (Downing, 1988).

We conducted the interspecies cross experiment for three years, and found that diluting the density of D-larvae is the key to improve the survival of hybrid offspring. High density leads to higher mortality and a lower growth rate of D-larvae, as they tend to assemble at the bottom of the rearing bucket. D-larvae often die at high density, even when those settled to the bottom are removed. Additionally, it is difficult to feed the floating offspring.

In this study, the growth rate of the progeny was highest in the AA cross and lowest in the aA cross, with values of the Aa and aa crosses falling in the middle. The same pattern was found for the timing of metamorphosis (Fig.1). These results suggest a strong maternal effect, which is often an important source of variation in early growth and survival (Zhanget al., 2009). The juveniles are subject to a maternal environment during the first stages of life, leading to changes in the phenotypic values of many metric characteristics, even when measured in adults (Xuet al., 2009). These maternal effects include cytoplasmic inheritance, maternal nutritionviathe egg, nutrition by pre- and post-natal feeding, and imitative behavior (Bradfor, 1972). In oysters, a clear maternal effect on hybrid growth and survival has been reported forC. gigas × C. angulata(Soletchniket al., 2002; Leit?oet al., 2007; Zhenget al., 2012),C. ariakensis × C. sikamea(Xuet al., 2009), andC. gigas × C. hongkongensis(Zhanget al., 2012).

In the present study, aA progeny were smaller than those of the other crosses on day 360 (Fig.2), indicating a lack of heterosis in this cross. Aa offspring were smaller than AA progeny but notably larger than aa offspring (Fig.2), so the Aa cross showed heterosis in maternal inheritance. Positive heterosis of growth rate and yield has been reported in interspecific crosses of several bivalve mollusks, includingSpisula solidissima×Mulinia lateralis(clams, Lindellet al., 2006) andA. irradiansconcentricusSay× A. irradians irradiansLamarck (Zhenget al., 2007), andA. irradians irradians × A. purpuratus(scallops, Wanget al., 2011). However, most of these reports have focused on the early stages of hybrid development (fertilization and hatching success), whereas few studies have examined the impact of gametic incompatibility on later stages of development in marine bivalve species (Rawsonet al., 1999). Similar results have been reported in hybridization betweenC. gigasandC. rivularis, as few viable hybrids were obtained fromC. rivularis ♀ ×C. gigas ♂(Allen and Gaffney, 1993). Thus, it is not surprising that gametic compatibility divergence led to the asymmetric heterosis observed in our study.

There is no doubt that both Aa and aA were hybrids, as ITS2 PCR generated two fragments (Fig.4). Xuet al. (2009) reported similar results in the hybridization ofC. ariakensis × C. sikamea. Once the D-larvae settled, their survival rate did not vary among groups, and few individuals were found dead in either the hybrid or inbred groups (Fig.1). Thus, gametic incompatibility mainly affected fertilization of eggs and survival of larvae in the early developmental stages. Once settled, juveniles were able to adapt to gametic incompatibility, and gonads developed normally. Both Aa and aA crosses were able to generate individuals with mature gametes, and their sex ratios were similar to those of the pure species (Table 2). Similarly, Huvetet al. (2002) found that progeny ofC. gigasandC. angulatacrosses were able to produce mature gametes.

In this study, the growth and survival rates were lower in interspecific crosses than in intraspecific crosses ofCrassostrea. However,C. ariakensisinhabiting estuarine areas is adapted to low-salt environment (Wanget al., 2006) whileC. angulatais accustomed to high-salt environment (Zhang, 2012). Whether their hybrids could survive over a larger range of salinity should be studied further. Meanwhile, perfect effect of genetic improvement can be achieved by interspecific backcross (Lou, 1999). Thus, backcrossing the hybrids to their parents is another urgent issue.

Acknowledgements

The authors thank Drs. Liqiang Zhao (Dalian Ocean University) and Zhongming Huo (Ocean University of China) for critical comments. We also thank the anonymous referees for offer oysters in the research. This work was supported by the National Natural Science Foundation of China (Grant No. 31172403) and the National Key Basic Research Development Planning Project (Grant No. 2010CB126406).

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(Edited by Qiu Yantao)

(Received November 26, 2013; revised February 6, 2014; accepted April 13, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-82031623 E-mail： zpwang@ouc.edu.cn