Laboratory hybridization between two oysters: Crassostrea gigas and Crassostrea hongkongensis.
Article Type: Report
Subject: Crassostrea (Genetic aspects)
Oysters (Genetic aspects)
Hybridization (Methods)
Authors: Zhang, Yuehuan
Wang, Zhaoping
Yan, Xiwu
Yu, Ruihai
Kong, Jing
Liu, Jian
Li, Xiaoyu
Li, Yalin
Guo, Ximing
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Product: Product Code: 0913050 Oysters NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: China Geographic Code: 9CHIN China
Accession Number: 303011383
Full Text: ABSTRACT Interspecific hybridization is a useful tool in genetic improvement of agriculture and aquaculture species. The Pacific oyster (Crassostrea gigas) and the Hong Kong oyster (Crassostrea hongkongensis) are both important aquaculture species in China. To determine whether these 2 species can hybridize and produce viable offspring, we conducted 2 x 2 factorial crosses between them. Asymmetry in fertilization was observed when C. hongkongensis eggs were fertilized readily by C. gigas sperm, but the reciprocal cross resulted in no fertilization. Embryos from C. hongkongensis female x C. gigas male crosses developed normally without noticeable defects, although their survival rate to D-stage was less than embryos of the two intraspecific crosses. From D-stage to metamorphosis, larvae of hybrid crosses had slower growth and a lower survival rate than that of intraspecific crosses. Nevertheless, 0.57% of hybrid D-larvae survived to spat stage at day 90. Hybrid spat had good survival (78.9%) to 1 y of age, but were significantly (P < 0.001) smaller than oysters of intraspecific crosses. Gonadal development was absent or retarded in most hybrids at 1 y of age, although some hybrids (39.2%) produced mature gametes. Our results show that hybridization between C. gigas and C. hongkongensis is possible in one direction. Some hybrids are viable, partly fertile, and can be used potentially for gene introgression between these two species.

KEY WORDS: Crassostrea gigas, Crassostrea hongkongensis, hybridization, gene introgression, hybrid dysfunction, oyster, aquaculture

INTRODUCTION

Interspecific hybridization is a useful tool in genetic improvement of agriculture and aquaculture species (Briggs & Knowles 1967, Hulata 1995, Bartley et al. 2001). Hybrids may be used directly as stock for farming or they can be used for gene introgression between species. Studies on interspecific hybridization may also provide insight on mechanisms of prezygote and postzygote barriers to hybridization, which are important to our understanding of speciation and evolution (Pahimbi 1992, Palumbi 1994).

Oysters are important aquaculture species, and there is considerable need for genetic improvement of oysters because most cultured stocks are undomesticated and suffer from problems of slow growth, disease, and summer mortality (Guo 2009). Some genetic improvement of oyster stock has been achieved through selective breeding and chromosome set manipulation (Boudry 2009, Guo et al. 2009). Hybridization as a tool for oyster breeding has also received some attention. There has been strong interest in hybridizing Crassostrea virginica and Crassostrea gigas, because the latter is resistant to two lethal diseases of the former species (Calvo et al. 1999). Unfortunately, despite repeated attempts, C. virginica and C. gigas cannot be hybridized because of postzygotic barriers (Allen et al. 1993).

Many attempts at hybridization have been reported in Crassostrea species, although in most early studies the claims of hybridization were not supported by genetic confirmation of hybrids (Gaffney & Allen 1993). Sperm and larval contamination is a common occurrence in oyster spawning and larval culture. Claims of hybridization without genetic confirmation must be viewed with caution. The production of viable hybrids has been confirmed with genetic analysis in the following crosses: C. gigas x Crassostrea angulata (Soletchnik et al. 2002, Huvet et al. 2004), C. gigas x Crassostrea rivularis (Allen & Gaffney 1993, Que & Allen 2002), C. gigas x Crassostrea sikamea (Banks et al. 1994, Camara et al. 2008), and Crassostrea ariakensis x C. sikamea (Xu et al. 2009). Asymmetry in fertilization success has been reported in several crosses, providing opportunities for studying the evolution of the proteins involved in sperm-egg interaction.

There are at least 5 Crassostrea oyster species that occur naturally along the coast of China: C. gigas, C. ariakensis, C. sikamea, C. angulata, and Crassostrea hongkongensis (Wang et al. 2006, Guo et al. 2008). Often, some of these species overlap in distribution. In North China, C. ariakensis coexists with C. gigas. In central and southern China, C. ariakensis, C. hongkongensis, C. angulata, and C. sikamea may be found in the same estuary in various combinations. Studying hybridization among these sympatric species may contribute to our understanding of mechanisms of reproductive isolation and speciation.

Although C. gigas and C. hongkongensis do not overlap in distribution, both are major aquaculture species in China. The oyster C. gigas is traditionally cultured in North China, but it has been introduced to southern China for aquaculture production (Guo et al. 1999, Guo 2009). The oyster C. hongkongensis is the most important oyster species cultured in southern China (Wang et al. 2004, Guo et al. 2006). It is a large oyster species and commands high market prices. It is found in low-salinity estuaries from the Fujian to Guangxi provinces, with populations centered in the Guangdong province. Although C. hongkongensis thrives in the warm and low-salinity waters of southern China, C. gigas is adapted to the temperate and high-salinity waters of northern China. These local adaptations may have given them different abilities or unique physiological traits in environmental tolerance that may be useful for selective breeding. Tolerance of heat stress in C. hongkongensis, for example, would be a useful trait for C. gigas, which often suffers from summer mortality. The tolerance of high salinity of C. gigas, if transferred successfully may make C. hongkongensis grow better in high-salinity waters. Thus, hybridization between C. gigas and C. hongkongensis is potentially useful in the genetic improvement of these two important aquaculture species. However, no hybridization between these two species has been reported, because C. hongkongensis is a newly described species (Lain & Morton 2003, Wang et al. 2004).

To determine whether hybridization between C. gigas and C. hongkongensis is possible, we conducted 2 x 2 crosses between the two species. Here we report that hybridization between the two species is possible in one direction. Despite low survival, slow growth, and retarded gonadal development, some hybrids can reach sexual maturation and produce functional gametes, raising the possibility of gene introgression between these two species.

MATERIALS AND METHODS

Oysters, Gamete Collection, and Hybridization Crosses

The oyster C. hongkongensis was collected from Shenzhen (Guangdong, China), and C. gigas were collected from Yantai (Shandong, China). Oysters of both species were about 2 y old. They were transported to the hatchery of Qingdao Laodong Aquaculture Breeding Company for conditioning in February 2009. They reached sexual maturity in early April, and our experiments were conducted from mid April to mid May 2009.

Gametes from the two species were collected by dissecting gonads. Before gamete collection, the two species were identified by shell morphology and differences in gill tube structure as described in Wang et al. (2004). After gamete collection, a piece of adductor muscle from each parental oyster used for hybridization was fixed in 95% ethanol for subsequent confirmation with genetic markers (described later).

Egg suspension was passed through an 80-[micro]m nylon screen to remove large tissue debris, and eggs were caught and washed on a 20-[micro]m screen. For each species, eggs from 3 females were pooled and then divided equally into two 2-L beakers. Eggs were examined under a microscope to ensure no uncontrolled fertilization had occurred, as indicated by the absence of polar bodies. After confirming no uncontrolled fertilization, eggs in the 2 beakers were fertilized with pooled sperm from 3 C. gigas and 3 C. hongkongensis males, respectively. Thus, a 2 x 2 factorial cross was created producing 4 groups: C. gigas [female] x C. gigas [male] (GG), C. gigas [female] x C. hongkongensis [male] (GH), C. hongkongensis [female] x C. gigas [male] (HG), and C. hongkongensis [female] x C. hongkongensis [male] (HH). Sperm were added within 60 min after gamete collection to a density of about 20-25 sperm surrounding an egg in intraspecific crosses. For hybrid crosses, about 50% more sperm were added. Fertilization was conducted at 25[degrees]C in filtered seawater with a salinity of 30. The experiment was replicated 9 times using 9 sets of parents.

Larval Rearing

Fertilized eggs were sampled and held in beakers to evaluate fertilization success. The remaining eggs from each group were counted and cultured in a 60-L bucket for incubation at a density of 30-40 eggs/mL. At 27 h after fertilization, D-larvae from each group were collected on a 40-[micro]m screen and reared in a 60-L bucket at 5 larvae/mE Larvae were fed with Isochrysis galbana on days 0-6, and a mixture of Platymonas subcordiformis and I. galbana (1:1) after day 6. Feeding was increased gradually from 6,000-80,000 cells/mL/day. Seawater was changed completely every 3 days. The culture seawater was maintained at 24.8 26.0[degrees]C.

Settlement, Nursery, and Grow-out

When most larvae developed an eye spot and foot, strings of corrugated plastic plates were placed in buckets as cultch. Larvae set within 8 days, and newly settled spat were nursed in buckets for 2 wk to prevent contamination from wild spat. Subsequently, all spat were transported to concrete tanks (8.0 x 5.0 x 1.2 m) and fed with Chlorella vulgaris at 80,000-10,000 cells/ mL/day. Water was changed 30% once daily. At day 60, spat were detached, transferred into spat bags, and hung on suspended longlines in large shrimp ponds. Spat bags were changed periodically from small to large mesh sizes. During the grow-out period from July 2009 to June 2010, water temperature ranged from 6.0-30.2[degrees]C and salinity ranged from 24-30.

Sampling, Measurements, and Data Analysis

Sixty minutes after fertilization, a 2-mL sample was collected from each group, and the number of fertilized (as indicated by polar bodies or cell division) and unfertilized eggs were counted. Fertilization level was calculated as the percentage of fertilized eggs to the total number of eggs. Egg diameter was determined by measuring 90 eggs under a microscope fitted with a calibrated eyepiece micrometer. Larval size was measured the same way for 30 larvae per group at D-stage and every 3 days thereafter. At 27 h postfertilization, the number of D-stage larvae was determined for each group, and percent survival of fertilized eggs to D-stage was determined. Subsequently, larval survival was determined as a percentage of D-stage larvae surviving to different days postfertilization.

During nursery and grow-out, 30 spat or oysters were sampled randomly and their shell height was measured with an electronic Vernier caliper at days 90, 180, and 360. Cumulative survival was assessed on the same days. Whole wet weight was measured with an electronic scale to 0.001 g. Also at day 360, oysters from all 3 groups were sampled for genetic confirmation and assessment of gonadal development.

All statistical analyses were performed with Statistical Program for Social Sciences (SPSS) 16.0, and significance for all analyses was set at P < 0.05 unless noted otherwise. Shell height and wet weight were transformed to logarithms to ensure normality and homoscedasticity. Fertilization level and survival data were arcsine-transformed before analysis. Differences in growth and survival among 3 groups (GG, HG, and HH) were analyzed with l-way ANOVA, followed by multiple comparison tests (LSD).

Genetic Confirmation

The identity of parents and selected progeny were confirmed using the ITS2 (internal transcribed spacer 2) marker as described by Wang and Guo (2008). DNA was extracted from ethanol-fixed samples using the TIANamp Marine Animals DNA kit (Tiangen). Primer sequences for ITS2 were 5'-GGG TCGATGAAGAACGCAG (5.8S forward) and 5'-GCTC TTCCCGCTTCACTCG (18S reverse). PCR was performed in a 25-[micro]L volume containing 1.5 mM MgC12, 0.2 mM dNTP, 0.2 [micro]M of each primer, 20 ng template DNA, 1 U Taq polymerase, 2.5 [micro]L 10 x PCR buffer, and 0.4 mg/mL BSA. The thermal cycler protocol consisted of an initial denature at 95[degrees]C for 5 min, 30 cycles of 95[degrees]C for 1 min, 62.5[degrees]C for 1 rain, and 72[degrees]C for 1 min, with a final extension at 72[degrees]C for 5 min. Three controls were included in PCR- one with DNA from a known C. hongkongensis, one with DNA from a known C. gigas, and the other with mixed DNA of the 2 species. Amplified fragments were separated on 2% agarose gels containing 0.2 [micro]g/mL ethidium bromide, and were visualized under a UV transilluminator (BIORAD). Species are identified by fragment size difference (Wang & Guo 2008).

PCR fragments were cloned and sequenced to confirm species identity further. PCR products were purified using U-NIQ-5 Column PCR Product's Purification Kit (San-gon, Shanghai), ligated into pMD 18-T vector following the instructions of the Takara DNA Ligation Kit ver. 2 and were used to transform competent JMI09 Escherichia coli cells using standard protocols. Recombinant colonies were identified by blue white screen. Inserts of the expected size were detected via restriction enzyme digestion (Eco RI and Hind III). Vector DNA containing the desired inserts was purified further using the Pharmacia EasyPrep Kit, and sequencing was performed in both directions on an ABI PRISM 377XL DNA Sequencer using the ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS (Perkin Elmer).

RESULTS

Fertilization and Embryonic Development

A notable difference between C. gigas and C. hongkongensis is the size of their eggs. The eggs of C. gigas were larger than those of C. hongkongensis. The eggs of C. gigas had an average diameter of 50.6 [micro]m, those of C. hongkongensis had a diameter of 40.6 [micro]m (Table 1), and the difference is highly significant (P < 0.001).

Of 9 replicates of 2 x 2 crosses, 4 replicates were incomplete because of poor gamete quality and problems during larval culture that seriously affected intraspecific controls, and abandoned. Data from 5 complete replicates are presented here. Fertilization level in the 2 intraspecific crosses was high, ranging from 96.0-99.1% in C. gigas and 67.2-92.6% in C. hongkongensis. Eggs of C. hongkongensis could be fertilized readily by C. gigas sperm in the HG cross, although the fertilization level (61.6%) was lower than that in intraspecific crosses (Table 1). On the other hand, no fertilization was observed in the reciprocal cross, C. gigas eggs x C. hongkongensis sperm (GH cross), despite adding 50% more sperm.

Embryonic development in the HG hybrid cross appeared normal, and no apparent abnormalities were noticed. Embryos in all 3 crosses except GH, which that had no fertilization, reached D-stage at 24-27 h postfertilization. Survival of fertilized eggs to D-stage larvae in the HG hybrid cross was 56.3%, significantly (P < 0.001) lower than in HH (75.6%) or GG (97.1%: Table 1). Survival to D-stage of the 2 intraspecific crosses was also significantly (P < 0.01) different.

Survival and Growth

At day 1, or 24-27 h postfertilization, the height of D-larvae differed significantly (P = 0.005) among the 3 groups: 62.8 [micro]m in HH, 61.5 [micro]m in HG, and 70.9 [micro]m in GG, closely following differences in egg size. From day 1, larvae in the HG hybrid cross were always smaller than those of the 2 intraspecific crosses (Fig. 1 A). Growth of HH larvae was slower than GG larvae during the first 10 days, but no significant difference was observed from day 13-19.

Survival of D-larvae to different days postfertilization differed significantly among groups and always followed the order of GG > HH > HG (Fig. 1 B). Only 2.8% of D-larvae in the HG cross survived to day 19, compared with 43.9% in the HH cross and 63.6% in the GG cross (Table 1). HG hybrids also had lower metamorphosis success, with only 0.6% of D-larvae reaching spat stage at day 90 compared with 36.2% in HH and 49.4% in GG. From day 90-360, survival of hybrid spat (78.9%) was actually higher than that of HH (56.5%) and GG (64.1%) spat (Table 1). During the same period, spat from the HG hybrid cross were consistently smaller than those from the HH and GG crosses (Fig. 2). At day 360, HG oysters were 62.6 mm in shell height and 64.1 g in whole weight compared with 81.3 mm and 92.4 g for HH oysters, and 114.6 mm and 125.0 g for GG oysters. The differences were highly significant (P < 0.001 ; Fig. 2).

Genetics Confirmation

At day 360, we sampled oysters from the 3 crosses for genetic confirmation and to assess gonadal development. All parents used in the 5 replicates were unambiguously identified as C. gigas or C. hongkongensis with the ITS2 marker (Fig. 3). Amplification in C. gigas and C. hongkongensis produced single bands at about 800 bp and 720 bp, respectively. All hybrid spat produced 2 bands, corresponding to the 2 parental species.

[FIGURE 1 OMITTED]

To confirm that the 2 bands are indeed from C. gigas and C. hongkongensis, we sequenced fragments amplified from C. gigas, C. hongkongensis, and the 2 fragments from hybrids. Sequences obtained from the 800-bp fragment from C. gigas and hybrids both matched ITS2 of C. gigas in GenBank AF280610.1 (e-value = 0.0, identities = 99%). There was no ITS2 sequence for C. hongkongensis in public databases. From the 720-bp fragment amplified from C. hongkongensis, we obtained a 720-bp sequence and deposited it in GenBank under accession no. GU338879. From the 720-bp fragment amplified from hybrids, we obtained a 719-bp ITS2 sequence that matched the ITS2 of C. hongkongensis we previously obtained: GU338879 (e-value 0.0, identities = 100%). Although there are some minor variation in sequences between different oysters and clones (because ITS2 has many copies per genome and can be variable), genetic analysis indicates clearly that oysters from the HG hybrid crosses contain ITS2 of both C. gigas and C. hongkongensis, and therefore are true hybrids.

To assess the reproductive potential of HG hybrids, we examined gonadal samples microscopically for the presence of gametes (mature eggs and mobile sperm). All oysters from the GG and HH crosses were fully mature and had either eggs or sperm (Fig. 4). Of the 153 hybrid oysters, 90% or 60.8% had no eggs or mobile sperm (respectively), 45 had mature eggs, 12 had mobile sperm, and 3 were hermaphrodites (Table 2). Even when hybrids contained mature gametes, most of them showed retarded gonadal development in appearance (Fig. 4) and relatively few gametes compared with oysters from intraspecific crosses.

DISCUSSIONS

This study demonstrates clearly that hybridization between C. hongkongensis and C. gigas is possible, but only in one direction: C. hongkongensis female x C. gigas male. No fertilization occurred in the reciprocal cross, showing clear asymmetry in fertilization and gamete compatibility. Asymmetry in fertilization has been observed in several hybrid crosses in oysters, including C. sikamea x C. gigas (Banks et al. 1994, Camara et al. 2008), C. rivularis x C. virginica (Allen et al. 1993), and C. sikamea x C. ariakensis (Xu et al. 2009). It is worth noting that C. gigas sperm can fertilize C. sikamea eggs, but C. gigas eggs cannot be fertilized by C. sikamea sperm (Banks et al. 1994). Similarly in this study, C. gigas sperm can fertilize C. hongkongensis eggs, but C. gigas eggs cannot be fertilized by C. hongkongensis sperm.

[FIGURE 2 OMITTED]

Sperm-egg interaction involves gamete recognition proteins (GRPs), and one of the GRPs is bindin, which is found in the acrosome of sperm (Vacquier & Moy 1977, Vacquier 1998). Bindins are lectins that bind specifically to receptors on the surface of eggs. Assuming all Crassostrea species are evolved from a common ancestor, the fact that C. hongkongensis and C. sikamea sperm cannot fertilize eggs of C. gigas suggests that bindins of C. hongkongensis and C. sikamea or receptors on eggs of C. gigas may have gone through significant changes. The former scenario may be more plausible because C. sikamea sperm have also lost their ability to fertilize eggs of C. ariakensis (Xu et al. 2009). It has been shown that bindins of C. gigas are extremely diverse (Moy et al. 2008). The high diversity of bindin may have given C. gigas sperm the ability to fertilize eggs of several Crassostrea species, including C. hongkongensis (this study), C. sikamea (Banks et al. 1994), and C. virginica (Allen et al. 1993). Such interpretation is largely speculative, because hybridization and bindin diversity have not been studied extensively in oysters. Further studies are needed to elucidate the role of GRPs in oyster speciation.

[FIGURE 3 OMITTED]

It is not surprising that hybrid larvae show slow growth and reduced survival compared with larvae of parental species. The oysters C. gigas and C. hongkongensis are believed to have diverged 28.8 million y ago (Ren et al. 2010). Some genome incompatibility may have developed during this long divergence, causing some hybrid dysfunction and affecting the growth and viability of hybrid larvae. Hybrid crosses between other Crassostrea species have also exhibited lower survival and poor growth compared with intraspecific crosses (Allen et al. 1993, Xu et al. 2009). Hybrid juveniles and adults were also smaller than parental species in this study, which is also true for C. sikamea x C. ariakensis hybrids (Xu et al. 2009). This is clearly different from the usually positive heterosis observed in intraspecific hybrids (Cruz & Ibarra 1997, Zheng et al. 2006, Hedgecock & Davis 2007).

The observation that most hybrid oysters did not produce mature gametes at 1 y of age when all C. gigas and C. hongkongensis were fully mature suggests that genome incompatibility between C. gigas and C. hongkongensis also affected the reproductive potential of some hybrids. On the other hand, some hybrids produced some functional gametes and may be partly fertile. Fertile hybrids have been observed between C. gigas and C. sikamea (Camara et al. 2008), and between C. gigas and C. angulata (Huvet et al. 2002), although whether the latter 2 species are different species has been a subject of debate (Menzel 1974, Huvet et al. 2002). A recent study has classified them as 2 subspecies, C. gigas gigas and C. gigas angulata, based on their level of divergence (Wang et al. 2010). The finding of fertile hybrids opens up the possibility of gene introgression between different species. Whether HG hybrids can be backcrossed to their parental species and produce viable offspring requires further investigation.

[FIGURE 4 OMITTED]

In conclusion, our study demonstrates that both prezygotic and postzygotic barriers to hybridization exist between C. hongkongensis and C. gigas, although none of them are complete. Fertilization is possible in one direction but not in the other direction. Hybrids exhibit slow growth, reduced survival, and retarded gonadal development, although some can survive to sexual maturation and produce some functional gametes. These fertile hybrids may be valuable in gene introgression between C. gigas and C. hongkongensis, both of which are major aquaculture species. On a theoretical note, these findings raise the issue of whether the level of hybridization barrier observed is sufficient to maintain species integrity should the 2 species become sympatric again.

ACKNOWLEDGMENTS

We thank Xirui Guo, Qiang Ma, and Junwei Sun of Laodong Aquaculture Breeding Company for operational support; Fei Xu for providing oyster broodstock; Xue Yi and Chenchen Zhang for their help with larval rearing; and Jiaqi Su, Hui Zhang, Huanqiang Sun, Liqiang Zhao, Xin Sun, Shaowen Li, and Yan Wang for assistance in the hatchery and grow-out. We thank Haiyan Wang, Jinhai Wang, Zhifei Yu, Changwei Shao, and Feng Gao for their kind assistance with molecular identification. This research was supported by grants from the National Natural Science Foundation of China (31172403) and the National Basic Research Program of China (2010CB 126406).

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YUEHUAN ZHANG, (1) ZHAOPING WANG, (1) * XIWU YAN, (2) RUIHAI YU, (1) JING KONG, (1) JIAN LIU, (1) XIAOYU LI, (3) YALIN LI (3) AND XIMING GUO (4) *

(1) Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao 266003, China, (2) Engineering Research Center of Shellfish Culture and Breeding of Liaoning Province, College of Fisheries and Life Science, Dalian Ocean University, 22 Heishjiao Street, Dalian 116023, China," (3) Qingdao Laodong Aquaculture Breeding Company, Wanggezhuang Fanling, Qingdao 266105, China," (4) Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller A venue, Port Norris, NJ08349

* Corresponding author. E-mail: zpwang@ouc.edu.cn, xguo@hsrl. rutgers.edu

DOI: 10.2983/035.031.0304
TABLE 1.
Egg diameter, fertilization level, percent development to
D-stage, and cumulative survival of D-larvae to different days
postfertilization of Crassostrea gigas (GG), C. hongkongensis
(HH), and their reciprocal hybrid crosses (GH, HG, female species
first).

                    Egg Diameter
Group   Replicate    ([micro]m)    Fertilization (%)   D-stage (%)

GG          1           50.4             98.0             99.1
            2           50.6             99.2             97.6
            3           50.8             98.5             96.0
            4           50.7             99.0             96.5
            5           50.7             98.9             96.5
          Mean        50.6 (a)         98.7 (a)         97.1 (a)
GH          1            --                0               --
            2            --                0               --
            3            --                0               --
            4            --                0               --
            5            --                0               --
          Mean           --                0               --
HG          1            --              79.2             58.9
            2            --              51.9             52.3
            3            --              53.5             64.4
            4            --              75.7             45.1
            5            --              48.0             60.6
          Mean           --            61.6 (c)         56.3 (c)
HH          1           40.5             87.7             73.7
            2           40.6             80.7             67.7
            3           40.7             88.1             79.3
            4           40.4             92.6             70.2
            5           40.8             67.2             87.1
          Mean         40.66           83.3 (b)         75.6 (b)

Group   Replicate   Day 19 (%)   Day 90 (%)   Day 360 (%)

GG          1         63.00        36.95         25.43
            2         47.95        45.46         32.56
            3         77.12        52.11         37.35
            4         62.53        50.84         22.70
            5         67.30        61.39         40.18
          Mean      63.58 (a)    49.35 (a)     31.64 (a)
GH          1           --           --           --
            2           --           --
            3           --
            4           --                        --
            5                                     --
          Mean                       --           --
HG          1          4.54         0.89         0.82
            2          2.71         0.63         0.51
            3          2.03         0.31         0.27
            4          3.49         0.52         0.46
            5          1.18         0.50         0.19
          Mean       2.79 (c)     0.57 (c)     0.45 (c)
HH          1         50.81        36.41         24.93
            2         34.04        23.64         12.37
            3         41.87        31.39         20.59
            4         42.76        40.76         17.76
            5         50.12        48.80         26.70
          Mean      43.92 (b)    36.20 (b)     20.47 (b)

Means with different letter denote significant differences at P < 0.05.

TABLE 2.
Number and percentage of male, female, hermaphrodite, and
undifferentiated (no gamete) oysters in 1-year old C. gigas
(GG), C. hongkongensis (HH), and their hybrids (HG).

Item                 GG            HG            HH

Female           33 (55.0%)    45 (29.4%)    32 (53.3%)
Male             27 (45.0%)    12 (7.8%)     28 (46.7%)
Hermaphrodite     0             3 (2.0%)      0
No gamete         0            93 (60.8%)     0
Total            60 (100%)    153 (100%)     60 (100%)
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