MA Deyou, YANG Hongsheng, and SUN Lina

1) Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences (IOCAS), Qingdao 266071, P. R. China

2) University of Chinese Academy of Sciences, Beijing 10049, P. R. China

Profiling and Comparison of Color Body Wall Transcriptome of Normal Juvenile Sea Cucumber (Apostichopus japonicus) and Those Produced by Crossing Albino

MA Deyou1),2), YANG Hongsheng1),*, and SUN Lina1),2)

1) Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences (IOCAS), Qingdao 266071, P. R. China

2) University of Chinese Academy of Sciences, Beijing 10049, P. R. China

Sea cucumber (Apostichopus japonicus) is one of the most important aquaculture animals in China. Usually its normal body color is black that fits its living environment. The juvenile individuals obtained by crossing albino sea cucumber segregated in body color. To document the transcriptome difference between albino associating sea cucumber and the control, we sequenced their transcriptomes with RNA-seq. Approximately, 4.790 million (M) and 4.884 M reads, 200 nt in length, were generated from the body wall of albino associating sea cucumber and the control, respectively, from them, 9550 (46.81%) putative genes were identified. In total, 583 genes were found to express differentially between albino associating sea cucumber and the control. Of these differentially expressed genes (DEGs), 4.8% changed more than five-folds. The expression levels of eight DEGs were confirmed with real-time PCR. The changing trend of these DEGs detected with real-time PCR agreed well with that detected with RNA-seq, although the change degree of some DEGs was different. Four significantly enriched pathways were identified for DEGs, which included phagocytosis, Staphylococcus aureus infection, ECM-receptor interaction and focal adhesion. These pathways were helpful for understanding the physiological difference between albino associating sea cucumber and the control.

Apostichopus japonicus; sezparation; gene expression profiling; RNA-seq; real-time PCR

1 Introduction

Sea cucumber (Apostichopus japonicus Selenka) in Echinodermata, Holothuroidea inhabits the Asian coast from 35? to at least 44? north latitude, covering China, Japan, Korea and Russia. Due to its high nutritional and medicinal value, it has been exploited as an important fi shery resource in above Asian countries for decades (Fu et al., 2005; Du et al., 2012b). In recent years, the demand for beche-de-mer (dried sea cucumber) has been increasing rapidly. However, the supply of wild sea cucumber has been decreasing because of overexploitation and pollution (Hamel et al., 2001; Xia et al., 2012). Thus, the culture of holothurians, especially A. japonicus, was increased (Yuan et al., 2006). Nowadays, this species is one of the most economically important Echinoderm organisms farmed in many areas of Asia (Okorie et al., 2008; Zheng et al., 2012).

The color of wild and farmed sea cucumber is different as has been documented frequently in China, Korea and Japan (Kang et al., 2011). Color variation, one of the most important characteristics of this species, may affect its taste and market price in China. In general, the body color of A. japonicus farmed along Chinese coasts is dorsally tawny and ventrally fawny. Sea cucumber with an entire white body is rare and regarded as sacred by fishmen. It was expected that the offsprings of the albino of sea cucumber were all albino as albinism is generally regarded to be recessive. However, the juvenile offsprings from a cross between abino of sea cucumber in normal color appeared as were found in crossing practice, that is, the body color of the offsprings from albino segragate. Unfortunately, the reason under this observation is unknown.

Transcriptome profiling between normal individuals and albino of sea cucumbers may provide a new insight into the color segregation among thwe offsprings of the ablino of A. japonicus. Deep sequencing technology has become a revolutionary tool of understanding better the complicated eukaryote transcriptomes (Ansorge, 2009). Highly specific, sensitive and quantitative measurements insure the next generation sequencing technology to overcome the disadvantages of traditional hybridization-based approaches (Xu et al., 2011), including low coverage and insensitivity to low abundance transcripts(Canales et al., 2006). The deep sequencing technology provides ultrahigh-throughout data at a low cost (Haas and Zody, 2010). RNA-seq, a recently developed highthroughput sequencing method, can produce a genome wide transcription map that consists of both the transcriptional structure and expression level for each gene (Mortazavi et al., 2008; Wang et al., 2009). Furthermore, there is no strict requirement for a genome reference sequence (Lister et al., 2009). These advantages make RNA-seq the platform for transcriptome analysis without complete genomic sequences (Wang et al., 2010; Xiang et al., 2010; Dubey et al., 2011; Shi et al., 2011; Feng et al., 2012). As to sea cucumber A. japonicus, the genome sequence hasn’t been determined. Thus RNA-seq can be applied to profiling the transcriptome of the body wall of sea cucumber.

In the present study, the transcriptome of the body wall of juvenile color sea cucumber individuals obtained by crossing albino (hereafter albino associating) and selecting from the normal (hereafter normal), respectively, was profiled and compared with RNA-seq technique, aiming to identify differentially expressed genes and differentially regulated pathways.

2 Materials and Methods

2.1 Animals

Ablino associating and normal juvenile color sea cucumber individuals were provided by The State Marine Seed Multiplication Farm affiliated to Shandong Oriental Ocean Sci-Tech Co., Ltd, Yantai, China. The sea cucumber was cultured in aerated water tanks with a flowthrough seawater supply. Sea cucumber was sampled after acclimation with mixed feed for 7 days. The tissue of body walls of albino associating and normal sea cucumber was peeled away carefully and quickly frozen in liquid nitrogen and stored at -80℃.

2.2 RNA Extraction, Library Construction and RNA Sequencing

Total RNA was isolated from mixed body wall tissue of 30 individuals using an RNeasy Mini Kit, which was treated with DNase using an RNase-free DNase Kit (Qiagen, Germany) according to manufacturer's instructions. The RNA concentration was determined using NanoDrop 1000 (Thermo) and the RNA integrity was confirmed using 2100 Bioanalyzer (Agilent Technologies). The mRNA enrichment, fragmentation, addition of adapters, size selection, PCR amplification and RNA sequencing were performed by Beijing Genome Institute (BGI) (Shenzhen, China). Magnetic beads with oligo(dT) were used to enrich poly(A) mRNA from the total. The mRNA was chopped into fragments approximately 200 bp in length. First strand cDNA was synthesized with random hexamer primers and reverse transcriptase (Invitrogen) while the second was synthesized using RNase H and DNA polymerase I. Sequencing adaptors were ligated to cDNA after being purified with a QiaQuick PCR Extraction Kit and washed with EB buffer. The cDNA ranging from 200 to 700 bp in length was purified by agrose gel electrophoresis and selected as the sequencing templates for PCR amplification. The sequence of two libraries was determined on Illumina HiSeq? 2000.

2.3 Sequence Annotation, Assessment and Gene Expression Levels

The original image data were transformed into sequence data by base calling, which were defined as the raw data or raw reads. The raw reads were cleaned by trimming the adaptor sequences and removing low quality reads and those with >10% unknown bases. The clean reads from two libraries were aligned to the references (Sun et al., 2011; Du et al., 2012a) using SOAPaligner/ SOAP2 (Li et al., 2009). A maximum of two mismatched bases between read and reference were allowed in alignment. In addition to the classification of raw reads, the statistical analysis of alignment, sequencing saturation analysis, distribution of reads on references and gene coverage were determine. The transcription level was calculated as the reads per kilobase per million reads (RPKM) (Mortazavi et al., 2008).

2.4 Identification of Differentially Expressed Genes (DEGs)

A rigid algorithm was applied to identify DEGs between albino associating and normal. The P-value was used to confirm differential gene expression. The false discovery rate (FDR) was set to determine the threshold of P-value used in multiple tests. We used FDR ≤ 0.001 and an absolute value of log2ratio ≥ 1 as the threshold for determining the significance of gene expression differences (Audic and Claverie, 1997).

2.5 Expression Pattern, Gene Ontology and Pathway Enrichment

Genes with similar expression patterns usually correct with each other functionally. All of the DEGs were mapped to gene ontology (GO) terms (the basic unit of GO) in the database (http://www.geneontology.org/) and KEGG (http://www.genome.jp/kegg/) (Kanehisa et al., 2008), by calculating the gene number and using a hypergeometric test to find significantly enriched GO terms and pathways among DEGs. GO terms that satisfied the condition of calculated P-value after Bonferroni correction as ≤ 0.05 were defined as significantly enriched terms among DEGs. The Q value was used to decide the threshold of P-value in multiple tests, and pathways with a Q value ≤ 0.05 were defined as significantly enriched.

2.6 Real-Time PCR Analysis

Some DEGs were selected to validate RNA sequencing using RT-PCR. The RNA used to synthesizing cDNA was the same as that used to constructing RNA-seq libraries. The first strand cDNA was synthesized according to the operation manual (Promega). Specific primer pairs weredesigned for eight randomly selected genes (Table 4) using Primer Premier 5.0 based on the reference sequences.

The gene expression level was assayed using SYBR Green RT-PCR which was performed using an Eppendorf Mastercycler? ep realplex. The reaction mixture (25 μL) contained 12.5 μL of SYBR Green Master Mix (Takara), 0.5 μL (each) of forward and reverse primer (10 μmol L-1), 1 μL of 1:50 diluted cDNA, and 10.5 μL of RNase-free water. The thermal cycling program was as follows: one cycle at 95℃ for 5 s followed by 40 cycles of 95℃ for 10 s, 60℃ for 20 s and 72℃ for 30 s. The dissociation curve analysis of the amplification product was performed at the end of each PCR reaction. NDUFA13 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 13) was used as an internal control. The mRNA abundance was estimated using the threshold cycles with the ΔΔCT method (Livak and Schmittgen, 2001). All data were expressed as mean ± S.E. (n = 5). Difference was considered highly significant when P < 0.01 and significant at P < 0.05.

3 Results

3.1 Identification and Quantification of Reads

Totally 4789920 and 4884207 raw reads were obtained from libraries of albino associating and normal, respectively (Table 1), from which, a total of 4771690 and 4864162 clean reads were obtained (Table 1, Fig.1) after removing the low quality and those with either adaptors ＞10% unknown bases or ＞50% bases with a quality value≤ 5. The transcript sequences of nine sea cucumber transcriptome libraries generated with 454 cDNA sequencing technology (Sun et al., 2011; Du et al., 2012a) were used as the references. In total, 2012478 (42.18%) and 1989738 (40.91%) reads of the two libraries were mapped to 29668 unigenes as the references. From these reads, 1253804 and 1302204 unique reads were obtained (Table 1). There were 48400 unique reads in the albino associating less than those in the normal, which may reflect the differences in physiological status between albino associating and normal sea cucumbers.

Table 1 Statistics of RNA-seq reads of albino associating and normal libraries

Fig.1 Classification of raw reads of ‘Albino associating’ and ‘Normal’ libraries.

3.2 Sequencing Saturation and Randomness

The identification of unique tags determines sequencing saturation. Once the new unique reads are not detectable, the sequencing reaches its saturation. Our results indicated that ‘Albino associating’ and ‘Normal’ libraries have been sequenced to saturation and produced a global profile of transcripts (Fig.2), as no more new unique reads were identified when the number of reads reached a plateau after 5 millions.

Fig.2 Sequencing saturation of RNA-Seq in the two libraries.

Fig.3 Reads distribution of the two libraries in RNA-Seq.

The randomness was assessed according to the distribution of reads on reference genes. The reference genes are different in their lengths, thus the reads on these genes were transformed into the ratio of the reads on a gene to the length of the gene and the number of reads on each gene. It was expected that the reads evenly distribute on each gene if the sequencing is random. In this study, the reads of ‘Albino associating’ and ‘Normal’ libraries were found to distribute in this study was almost evenly (Fig.3).

3.3 Comparison of Transcript Abundance

The number of clean tags was calculated and normalized with RPKM method. The RPKM value represents the abundance of a gene transcript (expression level). FDR ≤ 0.001 and an absolute threshold value of log2ratio≥ 1 were used to determine the fold value of DEGs. In total, 583 DEGs were found differentially expressed between ‘Albino associating’ and ‘Normal’ libraries, of which 361 were up-regulated and 222 were down-regulated (Fig.4).

Fig.4 Comparison of transcript abundance between ‘Albino associating’ and ‘Normal’ libraries.

Table 2 List of DEGs 5 folds up-regulated in ‘Albino associating’ Library

Table 3 List of DEGs 5 folds down-regulated in ‘Albino associating’ Library compared with ‘Normal’ Library

Table 4 Genes selected for real-time RT-PCR analysis and their primers

There were 23 up-regulated genes and 29 down-regulated genes with ≥ 5-folds difference (Supplemental File 1). As shown in Table 2, the twelve annotated DEGs with > 5-folds up-regulation were related to immunological defense (four), metabolism (two), regulation (one), translation (one), signal transduction (one), transport (two) and transcription (one).

Sixteen of the 29 down-regulated genes with ≥ 5-folds change were annotated (Table 3). They were relevant to acute phase (two), signal transduction (five), cell adhension (one), regulation (three), transport (one), growth and development (three) and translation (two).

3.4 Real-Time PCR Analysis

The steady-state transcription level of eight DEGs was analyzed to validate the performance of RNA-seq (Table 4). NDUFA13 was confirmed as stable in our previous studies, so it was used as the internal control. The expression trends of the selected genes were similar to that detected with RNA-seq (Fig.5).

3.5 Gene Ontology Analysis

In total, 808 genes were categorized into 35 functional groups belonging to three main GO ontologies, biological process, cellular component and molecular function (Supplemental File 2). In comparison with CON, the terms ‘binding’ (in molecular function), ‘cell’ and ‘cell part’ (in cellular component) and ‘cellular process’ and‘metabolic process’ (in biological process) were more abundant in ‘Albino associating’ Library (Fig.6).

Fig.5 RT-PCR validation of eight DEGs in normal (gray) and albino associating (black) sea cucumbers. a, four up-regulated; and b, four down-regulated genes in ‘Albino associating’ Library as were identified with RNA-seq;*and**, significantly different at P < 0.05, and P < 0.01, respectively.

Fig.6 GO classification of unigenes changed in their transcript abundance in ‘Albino associating’ Library in comparison with CON.

3.6 Pathway Enrichment of DEGs

The DEGs were significantly enriched in metabolic and signal transduction pathways. It was found that 100 pathways were affected by up-regulated DEGs and 32 by down-regulated DEGs (Supplemental Files 3). The top ten enriched pathways are listed in Table 5. Pathways with a Q value < 0.05 were determined as significantly enriched. There were only two pathways significantly enriched, which involved in phagocytosis and pathogen infection. ECM-receptor interaction and focal adhesion were significantly affected by down-regulated DEGs (Q ＜ 0.05).

Table 5 Pathways of enriching DEGs significantly in ‘Albino associating’ Library compared with ‘Normal’ Library

4 Discussion

Transcriptome is frequently profiled in model organisms by referring to their genome sequences (Xia et al., 2011). Less transcriptomes of non-model organisms were profiled than the model ones because of the unavailability of genome references. RNA-seq is a recently developed technology which can be employed in studying the species without genome sequence (Br?utigam et al., 2011). It has evolved as the main platform of gene expression analysis, differential splicing documentation and genic SNP identification (Parchman et al., 2010; Xu et al., 2011; Feng et al., 2012). In the present work, the transcriptomeof the body wall of albino associating sea cucumber was profiled and compared with that of the control for the first time. Owing to the deficiency of genomic sequence, about 2.78 (57.82%) and 2.51 million (59.09%) reads of ‘Albino associating’ Library and CON were not mapped on reference genes, respectively, and some of them significantly changed at expression level (P ＜ 0.05). These reads should be kept an eye on in future studies (Supplementary data 1).

The expression level of a set of selected genes was evaluated by RT-PCR. The difference in gene expression did not match the magnitude of those detected with RNA-seq, but the trend of up- and down-regulation was similar each other. The low expression level detected with RTPCR may be attributed to the difference in the sensitivity of two techniques. RNA-seq is likely to be more sensitive, especially for low-abundant transcripts, than either microarray or RT-PCR (’t Hoen et al., 2008; Tang et al., 2009).

The epidermis layer of aquatic animals is in direct contact with the external environment and transmits the internal response of organisms to it. Consequently, changes in gene expression in body surface tissues reflect the effect of external conditions on organisms, that is, differentially expressed genes represent the difference in their physiological condition. In this study, genes that were closely related to defense were significantly up- regulated. For instance, macrophage mannose receptor- like 1 (MRC-like 1), which recognizes infectious agents and mediates endocytosis and phagocytosis in host defense (Ezekowitz et al., 1990; Stahl and Ezekowitz, 1998), was highly activated at the transcriptional level in ‘Albino associating’ Library compared to that in CON. As one member of C-type lectin superfamily, MRC1 is believed to play an important role in innate immune response (Weis et al., 2006). It is reasonable to conclude that the albino associating sea cucumber was easier to be intruded by pathogenic microorganisms than the control under the same cultivation condition. The hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development (McClay and Fink, 1982). Its major constituent is the protein hyalin (Wessel et al., 1998). The gene encoding this component was significantly up-regualted in ‘Albino associating’ Library. Harmful organisms may cause thickening of the body wall of albino associating sea cucumber. Cathepsin D, a ubiquitous lysosomal endoprotease, participates in phagocytic endocytosis responding to external stimuli (Faust et al., 1985; Diment et al., 1988). The significantly up-regulated expression of cathepsin D gene seemed to indicate that the albino associating sea cucumber lays an uncomfortable state thus makes an immune response to exogenous condition.

The down-regulated genes generally involved in acute phase, signal transduction and growth and development (Table 3). Fibrinogen is the principal protein of vertebrate blood clotting. In this study, fibrinogen-like protein A was detected. This finding indicated that some invertebrates really have clotting systems (Xu and Doolittle, 1990). The potentiality wound recovery in the body wall of albino associating sea cucumber may be decreased due to significant down-regulation of fibrinogen-like protein A encoding gene. Angiopoietin-like protein participates in angiogenesis signal transduction, whose expression was 5.03-folds down-regulated. Additionally, the angiopoietin-1 receptor regulates a series of important biological processes including angiogenesis, endothelial cell survival, proliferation and reorganization of actin cytoskeleton (Gale and Yancopoulos, 1999; Kim et al., 2000; Papapetropoulos et al., 2000; Fujiyama et al., 2001), and its expression was 12.73-folds down-regulated in ‘Albino associating’ Library. No optimum external condition may result in the low growth of the juvenile albino associating sea cucumber.

The ‘phagosome related’ pathway was most affected by the up-regulated DEGs (Q ＜ 0.01). This implied that harmful organisms are more inclined to affect the albinoassociating sea cucumber, than the normal because phagocytosis was significantly more vigorous in ‘Albino associating’ Library library than in CON. The second affected was the ‘Staphylococcus aureus infection’ pathway in which genes encoding complement 4 (C4) were more prevalent in ‘Albino associating’ Library than in CON. Moreover, C4 is necessary for innate immunity and inflammatory response. The significantly enriched pathways for down-regulated genes prevalent in ‘Albino associating’ Library contained ECM-receptor interaction and focal adhesion. In the Holothuroidea, the regeneration of skin, intestines, and the respiratory tree are fascinating characteristics (Quinones et al., 2002; Sun et al., 2011). The remodeling of extracellular matrix (ECM) is closely related to organ morphogenesis and regeneration as were observed in many species (Yannas et al., 1989; Onda et al., 1990; Kim et al., 1997; Goetsch et al., 2003). The significant down-regulation of ECM-receptor interaction in albino associating sea cucumber seemed to show that their regeneration potentiality may be weaker than the normal. Focal adhesions have been proved to be the sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provide a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control (Burridge and Chrzanowska-Wodnicka, 1996). The analysis of significantly enriched pathways indicated that the immune status is likely to be activated, while the growth rate may be restricted in albino associating sea cucumber.

4.1 Immune Defense Genes

Macrophages mediate the interactions with natural and altered-self components of the host as well as a range of microorganisms through a broad range of plasma membrane receptors (Taylor et al., 2005). The mannose receptors are integral membrane proteins expressed on the surface of tissue macrophages, mediating endocytosis and phagocytosis of the bound ligands by macrophages (Ezekowitz et al., 1990). As a result, mannose receptors (MR) play a key role in antigen adherence and presenta-tion (Schlesinger, 1993; Stahl and Ezekowitz, 1998). In this study, we detected two dramatically up-regulated reads that were matched to MRC1 in the ‘Albino associating’Library, which seemed to show that these albino associating sea cucumber might be affected badly by pathogens.

4.2 Metabolism

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex assembly factor 3 (NDUFAF3), an essential factor for the assembly of mitochondrial respiratory chain complex I, involves in electron transport to generate energy (Saada et al., 2009). The expression of the reads similar to NDUFAF3 was checked to be remarkably up-regulated in‘Albino associating’ Library. It could be deduced that the need for energy in this type of sea cucumber was urgent. Sortilin-related receptor (SORL) is a member of low density lipoprotein receptor family, mediating lipid metabolism. It could be concluded that the albino associating sea cucumber had consumed lipid for meeting the need of energy.

4.3 Transportation

In the present work, three DEGs with ＞ 5-folds upregulation were tested to be associated with transportation in ‘Albino associating’ Library. The most up-regulated gene was an isoform of one dynactin subunit. Dynactin is required for most types of cytoplasmic dynein activity in eukaryotes, because it is a dynein activator together with dynein involving in several cellular functions including cell division and intracellular transport (Karki and Holzbaur, 1999; Schroer, 2004). The coatomer joins in the protein transportation as one component of COP I coat (Futatsumori et al., 2000). The gene mapped to coatomer subunit gamma-2 (COPG2) was notably up-regulated in ‘Albino associating’ Library, which indicated that protein transportation may be vigorous in ‘Albino associating’ Library.

4.4 Acute-Phase Response Related Genes

Fibrinogen is known to be one of the acute-phase proteins in inflammatory response (Gabay and Kushner, 1999). In this study, we found the transcriptional level of fibrinogen-like protein A decreased obviously in ‘Albino associating’ Library. Hypothetical protein BRAFLDRAFT_234302 is considered to have scavenger receptor activity and to recognize and clear some pathogenic bacteria. The expression of this putative protein was notably downregulated as well. It is inferred that the rapid response capability for pathogen may be lower in albino associating sea cucumber than that of the control.

4.5 Growth and Development Related Genes

The angiopoietin-1 receptor TEK-2, one of endothelial cell-specific tyrosine kinase receptors, involves in regulating blood vessels angiogenesis under physiological and pathologic conditions with ligands of angiopoietins (Suri et al., 1996; Luo and Wei, 2006). Angiopoietin plays an important role in a broad range of growth events, including regulation of angiogenesis, endothelial cell survival, proliferation, migration, and reorganization of the actin cytoskeleton (Hayes et al., 1999; Dunk et al., 2000). In‘Albino associating’ Library, the encoding genes of the two proteins were significantly down-regualted at transcriptional level. The expression of AAC4 protein gene that is highly expressed during multicellular development markedly declined as was observed in this study. Thus it is reasonable to suggest that albino associating sea cucumber should grow slower than the control.

4.6 Signal Transduction

In this work, five DEGs (＞ 5-folds down-regulated) may involve in signal transduction according to the functional annotation in Branchiostoma floridae. Their special effects in regulating some pathways should be studied further.

Neither significant changes (≥ 5-fold) of DEGs or enriched pathways were found in correlation with pigment synthesis, especially melanin. We could infer that there had been no difference in the body surface color between albino associating sea cucumber and the control, because they were indeed almost the same in appearance.

Due to the scarceness of albino sea cucumber in wild, albinism of this species is assumed to be a recessive character. However, it is still undecided whether albinism is caused by a single gene or multiple genes because of insufficient genome information. Further statistical and genetic study on albino offspring should be carried out. As to the experimental design, there were only two libraries were sequenced without biological or technical replicates in this study. In order to make the results more convincing, replicates will be studied.

5 Conclusions

RNA-seq deep sequencing technology can be utilized to analyze DEGs. The gene expression level in the body wall of albino associating juvenile sea cucumber changed significantly compared with the control. This analysis of DEGs and enriched pathways between albino associating and control sea cucumber is valuable for understanding their physiological difference.

Acknowledgements

We thank Beijing Genomics Institute at Shenzhen for technical assistance on data analysis. This work was funded by the National Natural Science Foundation of China (No. 40976089), the National Key Technology Support Program of China (No. 2011BAD13B02), the National Oceanic Public Welfare Industry Special Scientific Research of China (No. 201205023) and the Chinese National 863 Project (2012AA10A412).

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(Edited by Qiu Yantao)

(Received February 21, 2013; revised April 19, 2013; accepted April 30, 2014)

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

* Corresponding authors. Tel: 0086-532-82898610

E-mail: hshyang@qdio.ac.cn