Classification of jinjiang oysters Crassostrea rivularis (Gould, 1861) from China, based on morphology and phylogenetic analysis

The jinjiang oyster Crassostrea rivularis [Gould, 1861. Descriptions of Shells collected in the North Pacific Exploring Expedition under Captains Ringgold and Rodgers. Proc. Boston Soc. Nat. Hist. 8 (April) 33-40] is one of the most important and best-known oysters in China. Based on the color of its flesh, two forms of C rivularis are recognized and referred to as the "white meat" and 11 red meat" oysters. The classification of white and red forms of this species has been a subject of confusion and debate in China. To clarify the taxonomic status of the two forms of C. rivularis, we collected and analyzed oysters from five locations along China's coast using both morphological characters and DNA sequences from mitochondrial 16S rRNA and cytochrome oxidase 1, and the nuclear 28S rRNA genes. Oysters were classified as white or red forms according to their morphological characteristics and then subjected to DNA sequencing. Both morphological and DNA sequence data suggest that the red and white oysters are two separate species. Phylogenetic analysis of DNA sequences obtained in this study and existing sequences of reference species show that the red oyster is the same species as C. ariakensis Wakiya [1929. Japanese food oysters. Jpn. J. Zool. 2, 359-367.], albeit the red oysters from north and south China are genetically distinctive. The white oyster is the same species as a newly described species from Hong Kong, C. hongkongensis Lam and Morton [2003. Mitochondrial DNA and identification of a new species of Crassostrea (Bivalvia: Ostreidae) cultured for centuries in the Pearl River Delta, Hong Kong, China. Aqua. 228, 1-13]. Although the name C. rivularis has seniority over C. ariakensis and C. hongkongensis, the original description of Ostrea rivularis by Gould [1861] does not fit shell characteristics of either the red or the white oysters. We propose that the name of C. rivularis Gould [1861] should be suspended, the red oyster should take the name C. ariakensis, and the white oyster should take the name C. hongkongensis. (C) 2004 Elsevier B.V. All rights reserved.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

Classification of common oysters from North China

Oysters are commonly found on rocky shores along China's northern coast, although there is considerable confusion as to what species they are. To determine the taxonomic status of these oysters, we collected specimens from nine locations north of the Yangtze River and conducted genetic identification using DNA sequences. Fragments from three genes, mitochondrial 165 rRNA, mitochondria! cytochrome oxidase I (COI), and nuclear 285 rRNA, were sequenced in six oysters from each of the nine sites. Phylogenetic analysis of all three gene fragments clearly demonstrated that the small oysters commonly found on intertidal rocks in north China are Crassostrea gigas (Thunberg, 1793), not C. plicatula (the zhe oyster) as widely assumed. Their small size and irregular shell characteristics are reflections of the stressful intertidal environment they live in and not reliable characters for classification. Our study confirms that the oysters from Weifang, referred to as Jinjiang oysters or C. rivularis (Gould, 1861), are C. ariakensis (Wakiya, 1929). We found no evidence for the existence of C. talienwhanensis (Crosse, 1862) and other Crassostrea species in north China. Our study highlights the need for reclassifying oysters of China with molecular data.

Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

Its length polymorphism in oysters and its use in species identification

In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between major 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 designed two pairs of primers and optimized PCR conditions for simultaneous amplification of ITS 1 and ITS2 in a single PCR. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS 1 in all Crassostrea and Saccostrea species, whereas they were about the same size in the three Ostrea species. No intraspecific variation in ITS length was detected. Among species, the length of ITS I and ITS2 was polymorphic and provided unique identification of 8 species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glonzerata, and O. angasi/O. edulis. 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.

A review of advances in research on marine molluscan antimicrobial peptides and their potential application in aquaculture

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. They are usually characterized by their small-size, heat-stability and broad range of antimicrobial activity. This review covers research advances on marine mollusc AMPs, specifically those isolated from mussels, scallops, oysters, venerid clams and abalone, which mainly include MGD, mytilin, myticin, mytimycin, big defensin, and RPD-1. Their structural characteristics, antibacterial activity, and expression pattern as well as peptide distribution and their release following microbial challenge are also discussed. In addition, the prospect of the application of AMPs as food additives or their use in immunostimulation to prevent diseases of aquatic animals, as well as their potential hazards, are also discussed.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids. with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned ANT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 mu g L-1 and 200 mu g L-1 CdCl2. The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment. (C) 2008 Elsevier Inc. All rights reserved.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

小庙洪牡蛎礁巨蛎属牡蛎间生殖隔离研究

中国牡蛎种类丰富，但是由于贝壳可塑性强，表型变化大，分类学及系统发生学研究进展缓慢，存在诸多争议。本文在全国部分海区采集牡蛎样品，并通过表型和分子生物学方法进行鉴定，重点调查潮下带牡蛎的分布情况。选取江苏南通小庙洪牡蛎礁作为典型海域，研究亲缘关系接近的巨蛎属牡蛎之间在繁殖上如何维持相互之间的关系。本研究的目的在于摸索牡蛎物种鉴定的实用方法，并阐明熊本牡蛎和近江牡蛎生殖隔离是如何维持的。研究结果对牡蛎等海洋贝类的分类及系统发生学研究具有一定的借鉴作用，为牡蛎礁的生态保护提供生物学参考依据。 在辽宁营口、海洋岛，河北秦皇岛，山东西霞口、潍坊、青岛，江苏南通，浙江舟山、奉化，福建莆田，广东汕头等地区采集牡蛎样品，重点采集潮下带的牡蛎，通过地理分布、表型、DNA含量测定和COI种特异性探针鉴定对各地区的牡蛎物种组成进行分析。选取了南通小庙洪牡蛎礁作为研究牡蛎物种间相互关系的最佳地点。该地区牡蛎由主要分布在潮间带的熊本牡蛎和主要分布在潮下带的近江牡蛎组成。 对南通小庙洪牡蛎礁上两种巨蛎属牡蛎在空间上的分布进行调查；定期取样，制作性腺切片，观察两种牡蛎的性腺发育情况；同时，定期在海区挂板，采用半人工采苗的方式采集海区牡蛎幼体，通过ITS1基因鉴定采集的稚贝所属物种。通过调查发现二者的分布存在重叠区域；两种牡蛎在2007年6月中旬至9月中旬存在两个明显的繁殖高峰期，在每个繁殖期内两种牡蛎同时发生大量繁殖行为的可能性极高。结果表明，两种牡蛎不存在明显的生态隔离和季节隔离等交配前生殖隔离机制。 在室内对两种牡蛎进行2×2完全双列杂交，并通过ITS1基因的克隆测序验证杂种后代。双列杂交受精实验采用多亲本混合交配的方式（即每个物种的卵子或精子均来自多个个体），共进行7次重复。结果表明，熊本牡蛎的卵子能够被近江牡蛎的精子以较低的受精率（24.6%）受精，且形成的后代确实为两个物种杂交产生，而反方向不能受精。 幼虫和稚贝生长存活的双列杂交实验共进行3次重复。估测受精率、受精卵的孵化率，第1至29日每两天测量一次D形幼虫的壳高和密度。结果表明杂交幼虫（熊本牡蛎♀与近江牡蛎♂的杂交，SA）的受精率（12.5%）显著低于（P < 0.05）熊本牡蛎（75.4%）和近江牡蛎（84.5%）纯种对照组；而孵化率与对照组无显著差异（P > 0.05）；自受精至29日龄，杂交幼虫壳高的生长速度明显低于纯种对照组（P < 0.05）；但是存活率与熊本牡蛎无显著差异（P > 0.05）。杂交幼虫附着变态期间死亡率高；93日龄，熊本牡蛎有10.08%的匍匐期面盘幼虫存活至稚贝，显著大于（P < 0.05）近江牡蛎（2.18%）和杂交幼虫（0.76%）；杂交稚贝壳高显著小于（P < 0.05）纯种对照组。稚贝在海区挂养3个月。与挂养前相比，杂交稚贝在潮下带的存活率为19.2%，125个稚贝仅存24个，壳高平均增加2.15 mm。熊本牡蛎在潮间带的存活率达到65%，壳高平均增加3.99 mm。近江牡蛎在潮下带的存活率达到85.7%，壳高平均增加5.22 mm，在潮间带的存活率仅10.9%，壳高平均增加3.28 mm。受精6个月后，所有杂交稚贝仅剩3枚，运回实验室暂养一段时间后全部死亡。 设置20、25、30、35共4个盐度梯度，估算双列杂交组合在不同盐度下的受精率、孵化率、7日内的存活率和壳高生长，实验共进行3个重复。结果表明，杂交组合在4个盐度下的受精率（平均为11.2%）均显著低于（P < 0.05）熊本牡蛎（75.0%）和近江牡蛎（77.5%）纯种对照组；而孵化率与对照组无显著差异（P > 0.05）；杂交组合在盐度35时的孵化率（85.0%）显著大于20（49.6%）、25（51.3%）和30（55.4%）三个盐度梯度。7日龄杂交幼虫（10.5%）和近江牡蛎（11.4%）的存活率显著小于（P < 0.05）熊本牡蛎（30.3%）；杂交幼虫的壳高（75.7 μm）显著小于（P < 0.05）熊本牡蛎（81.5 μm）和近江牡蛎（85.6 μm）对照组。两因素方差分析表明，杂交组合方式对受精率、孵化率、7日龄存活率和7日龄壳高均有显著影响，盐度对7日龄壳高的影响显著。 实验结果表明，江苏小庙洪牡蛎礁上的熊本牡蛎和近江牡蛎之间在分布空间上存在重叠区域，繁殖时间也存在重叠，并未产生明显的生态隔离和季节隔离。二者之间存在不对称性杂交，熊本牡蛎的卵子能够与近江牡蛎的精子以较低的受精率受精，而反方向完全不亲和。杂交幼虫在受精率上存在显著劣势；但是受精卵的孵化率与纯种受精卵无显著差异；杂交幼虫在生长上也存在显著劣势；而存活率与熊本牡蛎无显著差异；杂交幼虫附着变态期间大量死亡，存活下来的稚贝生长缓慢，死亡率高。表明熊本牡蛎和近江牡蛎之间即存在交配前的配子不亲和性隔离，又存在交配后隔离，主要表现为杂种不活。

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n = 20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n = 30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n + 1, 2n + 2, 2n + 3, 3n - 2 and 3n - 1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n + 3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.

Seasonal variation in metabolism of cultured Pacific oyster, Crassostrea gigas, in Sanggou Bay, China

Rates of respiration and excretion of the Pacific oyster, Crassostrea gigas, were measured seasonally from June 2002 to July 2003 under ambient conditions of food, water temperature, pH, and salinity in Sanggou Bay, an important mariculture coast in north China. The aim of this study is to obtain fundamental data for further establishing an energy budget model and assessing the carrying capacity for cultivation of C. gigas in north China. Oysters were collected monthly or bimonthly from the integrated culture areas of bivalve and kelp in the bay. Oxygen consumption and ammonium and phosphorus excretion rates were measured, and ratios of O/N and NIP were calculated. One-way ANOVA was applied to determine differences among these parameters that act as a function of seasonal variation. All the physiological parameters yielded highly significant variations with season (P<0.01) The rate of respiration varied seasonally, with the highest oxygen consumption rate in July and the lowest rate in January, ranging from 0.07 to 2.13 mg O-2 h(-1) g(-1) dry tissue weight (DW). Maximum and minimum ammonium excretion rates were recorded in August and January, respectively, ranging from 0.51 to 5.40 mu mol NH4-N h(-1) g(-1) DW. Rates of phosphorus excretion varied from 0.11 (in January) to 0.64 (in July) mu mol PO4-P h(-1) g(-1) DW. The O/N and N/P ratios changed from 9.2 (in January) to 59.8 (in July) and from 4.6 (in January) to 10.9 (in August), respectively. For each season, the allometric relationship between the physiological response (e.g., rate of oxygen consumption, ammonium and phosphorus excretion) and DW of the animal was estimated using the formula: Y=a x DWb. (C) 2005 Elsevier B.V. All rights reserved.

Tandem duplication-random loss is not a real feature of oyster mitochondrial genomes

Duplications and rearrangements of coding genes are major themes in the evolution of mitochondrial genomes, bearing important consequences in the function of mitochondria and the fitness of organisms. Yu et al. (BMC Genomics 2008, 9: 477) reported the complete mt genome sequence of the oyster Crassostrea hongkongensis (16,475 bp) and found that a DNA segment containing four tRNA genes (trnK(1), trnC, trnQ(1) and trnN), a duplicated (rrnS) and a split rRNA gene (rrnL5') was absent compared with that of two other Crassostrea species. It was suggested that the absence was a novel case of "tandem duplication-random loss" with evolutionary significance. We independently sequenced the complete mt genome of three C. hongkongensis individuals, all of which were 18,622 bp and contained the segment that was missing in Yu et al.'s sequence. Further, we designed primers, verified sequences and demonstrated that the sequence loss in Yu et al.'s study was an artifact caused by placing primers in a duplicated region. The duplication and split of ribosomal RNA genes are unique for Crassostrea oysters and not lost in C. hongkongensis. Our study highlights the need for caution when amplifying and sequencing through duplicated regions of the genome.

Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.