Crassostrea ariakensis - Non-Native Oyster Research

OYSTER SEX WARS: EVIDENCE FOR A 'GAMETE SINK' IF CRASSOSTREA VIRGINICA AND CRASSOSTREA ARIAKENSIS SPAWN SYNCHRONOUSLY.
David Bushek, Andrea Kornbluh, Greg Debrosse, Haiyan Wang, Ximing Guo

OYSTERS AND OYSTER FARMING IN CHINA: A REVIEW.
Ximing Guo, Guofan Zhang, Lumin Qian, Haiyan Wang, Xiao Liu, Aimin Wang

A HISTOLOGICAL INVESTIGATION OF OYSTER PARASITES AND PATHOLOGY IN CHINA.
Emily Scarpa, Susan Ford, Ximing Guo, Lisa Ragone Calvo, David Bushek

DISTRIBUTION OF CRASSOSTREA ARIAKENSIS IN CHINA,
Haiyan Wang, Lumin Qian, Guofan Zhang, Xiao Liu, Aimin Wang, Yaohua Shi, Nianzhi Jiao, Ximing Guo

IDENTIFICATION OF CRASSOSTREA SPECIES FROM CHINA USING SNP-BASED MARKERS 104.
Haiyan Wang, Ximing Guo

IDENTIFICATION OF CRASSOSTREA ARIAKENSIS USING ITS LENGTH POLYMORPHISM,
Yongping Wang, Ximing Guo

DEVELOPMENT AND CHARACTERIZATION OF EST-SSR MARKERS IN THE EASTERN OYSTER CRASSOSTREA VIRGINICA,
Yongping Wang, Ximing Guo





OYSTER SEX WARS: EVIDENCE FOR A 'GAMETE SINK' IF CRASSOSTREA VIRGINICA AND CRASSOSTREA ARIAKENSIS SPAWN SYNCHRONOUSLY.
David Bushek (Haskin Shellfish Research Laboratory, Rutgers University), Andrea Kornbluh (Haskin Shellfish Research Laboratory, Rutgers University), Greg Debrosse (Haskin Shellfish Research Laboratory, Rutgers University), Haiyan Wang (Haskin Shellfish Research Laboratory, Rutgers University), Ximing Guo (Haskin Shellfish Research Laboratory, Rutgers University)

  Available data indicate spawning seasons for the Asian oyster Crassostrea ariakensis and the eastern oyster C. virginica overlap. Hybrids can form, but the larvae are not viable. If C. ariakensis are introduced into Chesapeake Bay and synchronous spawning occurs with C. virginica, hybridization could reduce the production of viable larvae (= gamete sink). We examined the effects of gamete age, sperm concentration, and ratios of heterospecific gametes on fertilization rate and hybridization of the two species. Hybrid fertilization rates were consistently lower than pure crosses. Fertilization rate decayed with gamete age, but occurred in gametes up to 8 hrs old. Fertilization rate also decayed with decreasing sperm density in both pure and hybrid crosses. Finally, fertilization rate declined by as much as 60% when sperm were (1) given a choice of eggs from each species to fertilize or (2) required to compete to fertilize eggs from a single species. Hence, a gamete sink will likely occur if these two species spawn synchronously. The magnitude of the gamete sink will depend in part on proximity of the two species, on gamete concentrations in the water column, and on the proportion of hybrids that form. Current efforts are enumerating the proportion of hybridization that occurred during these experiments. Molecular genetic methods to amplify ITS regions of the rRNA gene have been validated and yield two bands in pure crosses and four bands in hybrid crosses. Individual larvae are being typed to determine the proportion of hybrids formed under the various gamete mixtures.





OYSTERS AND OYSTER FARMING IN CHINA: A REVIEW
Ximing Guo (Haskin Shellfish Research Laboratory, Rutgers University), Guofan Zhang (Institute of Oceanology, Chinese Academy of Sciences, PRC), Lumin Qian (Third Institute of Oceanology, Oceanic Administration, PRC), Haiyan Wang (Haskin Shellfish Research Laboratory, Rutgers University), Xiao Liu (Institute of Oceanology, Chinese Academy of Sciences, PRC), Aimin Wang (Ocean College, Hainan University, PRC)


  In an effort to survey Crassostrea ariakensis populations in China, we conducted literature reviews and visited over 37 sites along China's coast. Here we present our findings about oysters and oyster farming in China in light of recent taxonomic revisions. Seventeen species of oysters have been reported along China's coast. Many of the species occur in southern China and are relatively rare. There is considerable confusion about the classification of C. ariakensis and three other species. Numerous oyster reefs, both ancient and living, exist along the coast, and C. ariakensis was present in all four live reefs that we saw. In three of the reefs, C. ariakensis is the dominant or founding species, where large oysters on the bottom are C. ariakensis and small oysters on top are other species. Oyster farming is primarily for C. hongkongensis in the south (Guangxi and Guangdong), C. gigas in the north (Shandong and Liaoning), and C. angulata in the middle (Fujian and Zhejiang). C. hongkongensis, also known as the white oyster or C. rivularis, is one of the most important species cultured in China. C. hongkongensis culture is based on natural seeds with one estuary in Guangxi supplying about 2.5 billion. Most of the published literature on C. rivularis from southern China is for C. hongkongensis, not for C. ariakensis, which is not intentionally cultured. It is present at low frequencies in natural C. hongkongensis seeds and selected against by farmers. Pollution has devastated oyster populations in at least two estuaries.





A HISTOLOGICAL INVESTIGATION OF OYSTER PARASITES AND PATHOLOGY IN CHINA
Emily Scarpa(Haskin Shellfish Research Laboratory, Rutgers University), Susan Ford(Haskin Shellfish Research Laboratory, Rutgers University), Ximing Guo(Haskin Shellfish Research Laboratory, Rutgers University), Lisa Ragone Calvo(Haskin Shellfish Research Laboratory, Rutgers University), David Bushek(Haskin Shellfish Research Laboratory, Rutgers University)


  The proposed introduction of Crassostrea ariakensis into Chesapeake Bay as a means to restore oyster populations presents a number of potential risks, such as pathogens and pathological conditions that require careful examination and research before approval. Parasites occurring, even at low prevalence, in C. ariakensis within its native distribution may seriously impact this or other species in a different environment. Pathogens present in oyster species coexisting with C. ariakensis in its native habitat may also present problems if they are able to use C. ariakensis as a host or reservoir.
  In this survey, 27 samples were collected from 8 provinces along China's coastline. Cross-sections of individual oysters were preserved in Davidson's fixative. Genetic analysis of ethanol preserved gill samples was conducted to identify species of individual oysters. Nine samples were selected based on presence of C. ariakensis. These samples, representing 6 sites and consisting of C. ariakensis and a number of coexisting oysters, were processed by normal histological procedures and examined. Individual C. ariakensis were also examined using a fluorescence immunostaining technique to identify presence of Perkinsus sp. infections.
  Observed parasites included ciliates, such as Sphenophyra-like ciliates and trichodinids; crustaceans, including intestinal copepods; coccidians, including Nematopsis and a coccidian-like organism; rickettsia/chlamydia-like organisms; and trematodes. Overall prevalence of any particular parasite did not reach above 3% and averaged less than 1%. However, prevalences of coccidian-like and Nematopsis species were as high as 45% and 64%, respectively, at specific sites. To date, no evidence of a significant pathological impact has been observed.
  Continuing research will utilize molecular techniques to identify Haplosporidium nelsoni and Bonamia infections.





DISTRIBUTION OF CRASSOSTREA ARIAKENSIS IN CHINA
Haiyan Wang (Haskin Shellfish Research Laboratory, Rutgers University), Lumin Qian (Third Institute of Oceanology, Oceanic Administration, PRC), Guofan Zhang (Institute of Oceanology, Chinese Academy of Sciences, PRC), Xiao Liu (Institute of Oceanology, Chinese Academy of Sciences, PRC), Aimin Wang (Ocean College, Hainan University), Yaohua Sh i (Ocean College, Hainan University), Nianzhi Jiao (Center for Marine Environmental Sciences, Xiamen University), Ximing Guo (Haskin Shellfish Research Laboratory, Rutgers University)


  It is commonly assumed that Crassostrea ariakensis is synonymous with C. rivularis that, according to the literature, is abundant and widely distributed in China. However, at least three species, C. gigas, C. hongkongensis and C. ariakensis, have been reported as C. rivularis in China, creating uncertainties about the distribution of C. ariakensis. To determine the distribution of true C. ariakensis in China, we collected and classified 2,624 oysters from 50 locations along China's coast using species-specific DNA markers. C. ariakensis was found at 10 sites ranging from northern Shandong to Guangxi. While C. ariakensis had a wide geographical distribution, its occurrence within its range is patchy or scarce. Overall, C. ariakensis accounted for only 9.5% of all oysters collected. Large C. ariakensis populations were found in only three areas: Jiulong River in Fujian, Dongzao Harbor near Yangtze River and Yellow River basin in Bohai Sea, with none observed in-between. In Guangxi and Guangdong, C. ariakensis was present in all samples collected at low frequencies (0.5-17.5%). All three major populations are found in or near large rivers, and the absence of rivers may be a factor contributing to the fragmented distribution. At all sites, C. ariakensis co-existed with other species: C. gigas in Bohai Sea; C. sikamea at Dongzao Harbor; and C. hongkongensis, C. angulata, C. sikamea and Saccostrea species in southern China. C. ariakensis tended to occur subtidally, while other species were often found intertidally.





IDENTIFICATION OF CRASSOSTREA SPECIES FROM CHINA USING SNP-BASED MARKERS 104
Haiyan Wang (Haskin Shellfish Research Laboratory, Rutgers University), Ximing Guo (Haskin Shellfish Research Laboratory, Rutgers University)


  China is home to 17 species of oysters and among them, five Crassostrea species, C. hongkongensis, C. angulata, C. gigas, C. sikamea and C. ariakensis are most common and commercially important. These five species often co-exist in the same estuary, and their identification using morphological characteristics is problematic. Genetic markers are needed for rapid and reliable identification of these oysters. Single nucleotide polymorphisms (SNPs) are simple and powerful markers for various genetic analyses. In this study, we developed species-specific SNP markers for the identification of common oysters from China. The mitochondrial cytochrome oxidase I (COI) gene and the nuclear 28S ribosomal RNA gene were used for marker development. DNA sequences from different species were either obtained by direct sequencing or downloaded from GenBank. Sequences were aligned, and species- and genus-specific SNPs were identified. Primers were designed for species/allele-specific amplification to generate fragments of different sizes in each species. A multiplex set of species-specific markers from COI was able to distinguish all five Crassostrea species in a single-tube PCR. It also separated Ostrea and Saccostrea species from Crassostrea species with the exception of C. virginica and C. rhizophorae. The 28S primer set was able to separate C. hongkongensis, C. ariankensis from other species, as well as Saccostrea and Ostrea species from Crassostrea species (except C. virginica and C. rhizophorae). The SNP-based markers do not require fluorescencelabeling or post-PCR digestion, providing a simple, fast and reliable method for oyster identification.





IDENTIFICATION OF CRASSOSTREA ARIAKENSIS USING ITS LENGTH POLYMORPHISM
Yongping Wang (Haskin Shellfish Research Laboratory, Rutgers University), Ximing Guo (Haskin Shellfish Research Laboratory, Rutgers University)


  Oysters cannot be reliably identified using morphological characteristics alone. In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between ribosomal RNA genes in 12 common species of Ostreidae: Crassostrea virginica, C. rhizophorae, C. gigas, C. angulata, C. sikamea, C. ariakensis, C. hongkongensis, Saccostrea echinata, S. glomerata, Ostrea angasi, O. edulis, and O. conchaphila. We downloaded and aligned ribosomal RNA sequences from all oyster and some other bivalve species to identify conserved sequences flanking ITS1 and ITS2. We designed two pairs of primers and optimized PCR conditions for simultaneously amplification of ITS1 and ITS2 in a single tube. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS1 in all Crassostrea and Saccostrea species, while they were about the same size in three Ostrea species. No intraspecific 105 variation in ITS length was detected. Among species, the length of ITS1 and ITS2 was polymorphic and provided unique identification of eight species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glomerata, and O. angasi/O. edulis. Two species within a pair were not distinguishable by ITS length. The ITS assay provides simple, rapid and effective identification of C. ariakensis and several other oyster species. Because the primer sequences are conserved, the ITS assay may be useful in the identification of other bivalve species.





DEVELOPMENT AND CHARACTERIZATION OF EST-SSR MARKERS IN THE EASTERN OYSTER CRASSOSTREA VIRGINICA
Yongping Wang (Rutgers University), Ximing Guo (Rutgers University)


  Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs that contained at least eight units of di-nucleotide repeats or five units of tri-, tetra-, penta- and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSRcontaining ESTs with good and sufficient flanking sequences. PCR amplification was successful for 71 (81%) of the primer pairs including 19 pairs amplified fragments that were significantly longer than the expected size, probably due to introns. Sixty-six pairs that produced fragments size shorter than 800 bp were screened for polymorphism in five oysters from three populations using polyacrylamide gels, and 53 of them (80%) were polymorphic. Forty-five SSRs were labeled and genotyped in 30 oysters from three populations using an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting that they belonged to duplicated loci, but they can still be used for segregation analysis. The remaining 40 SSRs had two alleles per individual including seven with null-alleles. In the 30 oysters analyzed, the SSRs had an average of 9.6 alleles per locus, ranging from 2 to 24. All 45 markers were used for segregation analysis in a family with 34 progeny. None of the 45 loci showed significant deviation from Mendelian ratios. This study demonstrates that ESTs are valuable resources for the development of genetic markers in the eastern oyster.