Ark Shell Scapharca Broughtonii Research Articles

Identification and Expression of Heat Shock Protein 60 (HSP60) from the Ark Shell Scapharca broughtonii

Heat shock protein 60 (HSP60) is evolutionarily highly conserved, and it assists with protein folding in the mitochondria and serves as a molecular chaperone, increasing cell survival under various conditions. Studies of HSP60 in molluscs under stress conditions remain limited. Here, this study first characterize a full-length HSP60 cDNA from the ark shell Scapharca broughtonii (termed SbHSP60) containing a 1,725-bp open reading frame corresponding to a deduced protein of 574 amino acid residues with a predicted molecular mass of approximately 61.4 kDa. The deduced protein has 72.3%–80.3% amino acid identity with homologs from invertebrates and vertebrates. Phylogenetic analysis showed that SbHSP60 grouped with proteins from other molluscs, forming a sister cluster to the HSP60 proteins from vertebrates. The expression pattern of SbHSP60 as examined by quantitative real-time PCR suggested that these mRNA transcripts were distributed across all examined tissues. The highest levels of expression were found in the hepatopancreas and gill, with decreasing levels of expression in the mantle, foot, hemocytes, and adductor muscle. Expression of SbHSP60 after challenge with Vibrio anguillarum showed similar upregulation profiles in the hepatopancreas and gill, indicating that SbHSP60 is involved in the bacterial defense process as an acute-phase protein. Levels of SbHSP60 in the hepatopancreas and gill were higher in cadmium-exposed individuals than in controls. SbHSP60 was significantly upregulated by Cd stress in the gill, but expression was less affected in the hepatopancreas, implying different roles in cellular Cd stress protection. The effects of heat stress on SbHSP60 mRNA expression were duration- and tissue-specific. The induction of SbHSP60 in the heat-stressed group occurred predominantly in the hepatopancreas, suggesting that protein damage and the consequent requirement for the HSP60 protein are increased during the heat shock process.

Impact of nighttime hypoxia on ark shell Scapharca broughtonii mortality on a semi-enclosed embayment seabed

The ark shell Scapharca broughtonii population has decreased markedly in recent years in Nanao Bay, a semi-enclosed embayment in Japan. Continuous measurement of dissolved oxygen (DO) at the bottom of Nanao Bay was performed, and S. broughtonii survival rates were investigated experimentally and in situ. In summer the DO concentration showed diel variation, with generally sufficient oxygen during the day but concentrations < 2 mg/l at night. In rearing experiments to investigate their physiological state, ark shells were placed in a tank designed to produce hypoxic conditions at night. Blood succinate levels in the ark shells increased, and all individuals were dead after 35 days. The results of the study indicate that the main cause of mortality of S. broughtonii on the seabed in Nanao Bay is hypoxia at night.

Identification, Characterization and Expression of Heat Shock Protein 70 in Scapharca broughtonii

As one crucial member of heat shock proteins (HSPs) family, HSP70s play many important roles in a large amount of physiological processes including immune response. However, information regarding HSP70 in ark shell Scapharca broughtonii is still rather limited. Here the full-length cDNA of HSP70 gene (named SbHSP70) of S. broughtonii was identified by using reverse transcription PCR (RT-PCR) and rapid amplification ends (RACE) methods. The SbHSP70 cDNA was 2423 bp in length containing a 5&amp;prime; untranslated region (UTR) of 131 bp, 3&amp;prime;-UTR of 330 bp, and an open reading frame (ORF) of 1962 bp which encodes a peptide of 653 amino acids.The multiple alignment and phylogeny analysis showed that the SbHSP70 shared high homology sequence with other mollusk species, and clustered together with gastropods to form a sister group. The mRNA expression profiles of SbHSP70 in tissues of foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbHSP70 distributed in all the examined tissues, and the highest expression level was observed in foot, and a significant difference could be detected between gill and adductor muscle (p&amp;lt;0.05), no significant difference among the gill, mantle and hemocytes (p &amp;gt; 0.05). Its dynamic change during the early stage of larvae showed that it could be transferred from parent and may be involved in some key developmental process. What&amp;rsquo;s more, Vibrio anguillarum challenge resulted in regular change of expression of SbHSP70 mRNA, indicating SbHSP70 actively participated in the immune response process.

Ark shell Scapharca broughtonii hemocyte response against Vibrio anguillarum challenge

Scapharca broughtonii is one of the most important Arcidae aquaculture species in the Asia-Pacific region. We aimed to investigate the immune responses of hemocytes from ark shell S. broughtonii hemolymph against pathogens. Hemocyte ultrastructure and immunological activity in response to Vibrio anguillarum challenge were observed by scanning and transmission electron microscopy. Before ultrastructure observation, we used the API ZYM semi-quantitative kit to evaluate the levels of hydrolytic enzymes in the plasma and hemocytes following V. anguillarum infection. An enzyme-linked immunosorbent assay kit was used to investigate the variation in the lysozyme activity and hemocytes following bacterial infection. The results showed that hemocytes were the main defense cells against bacterial infection, whereas plasma played a role in the transport and support of hemocytes. It was presumed that an important function of lysozymes and hydrolytic enzymes in lysosomes was for bacterial digestion. Three major types of hemocytes were observed, namely, red blood cells (RBCs), white blood cells (WBCs), and thrombocytes (TCs). Scanning electron microscopy showed that the normal RBCs appeared pie-shaped with 10 μm diameter and 4 μm central thickness, whereas WBCs were spherical in shape with varying sizes, 4–8 μm diameter, and included small lymphocytes. TCs were long, spindle-shaped, and 12–20 μm in length. The cell membrane surface was smooth and even for all cells before pathogen challenge. Under transmission electron microscopy, RBCs displayed a limited ability to devour and digest bacteria adherent to the cell surface following infection. Many hemoglobin particles were observed in the RBC cytoplasm. WBCs were very active against bacterial invasion and showed a strong ability to digest and decompose infected and wrapped V. anguillarum through phagocytosis and lysosome fusion. Digestive vacuoles rapidly became transparent and were thought to contain increasing quantities of pathogen-induced lysozymes. WBCs that devoured pathogenic bacteria were prone to deformation as well as adhesion to each other. TCs were rich in endoplasmic reticulum (ER) content in their cytoplasm and were widely connected in a net-shaped structure. Mitochondria in TCs formed clusters upon invasion of V. anguillarum in the hemolymph. TCs disintegrated to release the ER into the plasma to form a mesh that facilitated clotting. The ability of circulating hemocytes to quickly modify their morphologies and stainability suggests that S. broughtonii is endowed with highly dynamic hemocyte populations capable of coping with environmental changes and rapidly growing pathogens.

먹이생물에 따른 피조개 Scapharca broughtonii 유생의 발달단계별 성장과 생존

먹이생물에 따른 피조개 Scapharca broughtonii 유생의 발달단계별 성장과 생존

Molecular cloning, expression and biochemical characterization of hemoglobin gene from ark shell Scapharca broughtonii

Hemoglobin, the main component of haemolymph, is widely distributed in animals. Although its important oxygen transport functions has been extensively reported, studies on the immune function of hemoglobin in mollusc are few. Research on immune of hemoglobin of ark shell Scapharca broughtonii attracted more and more attention due to its ownership of erythrocyte comparing with many other shellfish. In this study, the hemoglobin cDNA of S. broughtonii was cloned by EST and RACE methods (named as SbHb). Sequence analysis revealed that the cDNA was 946 bp in length, including an open reading frame (ORF) of 459 bp which encoded a polypeptide of 152 amino acid residues, and a 5′-untranslated region (UTR) of 313 bp, a 3′-UTR of 174 bp. Sequence and homology analysis showed that the SbHb shared similarity with that of other related species. The mRNA expression profiles of SbHb in tested tissues analyzed by quantitative real-time PCR (qRT-PCR) revealed that the mRNA of SbHb could be all detected in foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas, and the highest level was found in the haemocytes, which is 163.2 times higher than that in adductor muscle. Vibrio anguillarum stimulation and hypoxia treatment both had significant impact on the expression of SbHb, which showed the same trends as increasing first to the highest at 16 h after treatment and then followed by declining. Recombinant protein of SbHb (rSbHb) was successfully obtained by prokaryotic expression, and further function analysis indicated obviously that the rSbHb had very strong phenoloxidase-like activity (PO-like activity) and it could remarkably inhibit growth of gram-negative bacteria V. anguillarum. All the data suggested that the SbHb plays a significant role in the process of antibacterial and anoxia tolerance reaction in S. broughtonii, providing the evidence that SbHb is a key immune factor.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response.

남해 연안 피조개 (Scapharca broughtonii) 양식장의 환경특성

남해 연안 피조개 (Scapharca broughtonii) 양식장의 환경특성

남해안 패류양식장에서 분리한 Vibrio parahaemolyticus의 병원인자 분포 및 항균제 내성

From 2013 through 2015, we investigated the contamination status and antimicrobial resistance patterns of pathogenic Vibrio parahaemolyticus in commercially valuable seawater and shellfish (Oyster Crassostrea gigas, short-neck clam Venerupis philippinarum, ark shell Scapharca broughtonii and mussel Mytilus galloprovinciallis) from the southern coast of Korea. The detection rate of V. parahaemolyticus was highest in short-neck clams (23.7%), followed by ark shells (19.2%), oysters (15.9%), mussels (13.6%), and seawater (8.6%). The following percentages of PCR assays of shellfish were positive for the thermostable direct hemolysin-related hemolysin gene (trh) : oysters (12.8%), short-neck clams(11.8%), and ark shells (3.4%). Similar assays for the thermostable direct hemolysin gene (tdh) resulted in positive results for short-neck clams (5.9%) and ark shells (3.4%). Antimicrobial resistance was present in 100% of 8 tdh (+) and 2 trh (+) V. parahaemolyticus isolates challenged with ampicillin. However, all pathogenic V. parahaemolyticus were sensitive to 14 other antibiotics. To ensure the safety of shellfish consumption, the continuous monitoring of the prevalence and distribution of virulence factors of V. parahaemolyticus in shellfish farms is needed.

Comparative Genetic Characterization of Ark ShellScapharca broughtoniiin Northeast Asia

ABSTRACT Ark shell Scapharca broughtonii is one of the most commercially important bivalve resources and is widely distributed in northeastern Asia. The aim of this study was to determine its genetic structure in Japan, China, and Korea by means of nucleotide sequence analysis of a 507-bp portion of themitochondrial DNA cytochrome c oxidase subunit I gene. Of 180 individuals collected from three localities in Japan and one locality each from China and Korea, a total of 66 haplotypes were found on the basis of 56 variable nucleotides. Although Japan and Korea populations shared six common haplotypes, there was no common haplotype between Japan/Korea and China. The pairwise FST and genetic distance values were comprehensively high, with significant differences between Japan/Korea and China, indicating that the divergence had reached subspecies level. In conclusion, haphazard transplantation and import of seedlings and/or adults of S. broughtonii and its related species should be absolutely avoided as S. bro...

Development of three multiplex PCR primer sets for ark shell (Scapharca broughtonii) and their validation in parentage assignment

Scapharca broughtonii is a commercially important and over-exploited species. In order to investigate its genetic diversity and population structure, 43 novel polymorphic microsatellites were isolated and characterized. The number of alleles per locus ranged from 3 to 22 with an average of 6.93, and the observed and expected heterozygosities varied between 0.233 and 1.000, and 0.250 and 0.953, with an average of 0.614 and 0.707, respectively. Three highly informative multiplex PCRs were developed from nine of those microsatellites for S. broughtonii. We evaluated and validated these multiplex PCRs in 8 full-sib families. The average polymorphism information content (PIC) was 0.539. The frequency of null alleles was estimated as 3.13% of all the alleles segregation based on a within-family analysis of Mendelian segregation patterns. Parentage analysis of real offspring demonstrated that 100% of all offspring were unambiguously allocated to a pair of parents based on 3 multiplex sets. Those 43 microsatellite loci with high variability will be helpful for the analysis of population genetics and conservation of wild stock of S. broughtonii. The 3 sets of multiplex PCRs could be an important tool of pedigree reconstruction, population genetic analysis and brood stock management.

Ferritin has an important immune function in the ark shell Scapharca broughtonii

Ferritin, the principle cytosolic iron storage protein in the majority of living organisms, has important roles during immune process in invertebrates. Detailed information about ferritin in the ark shell Scapharca broughtonii, however, has been very limited. In this study, full-length ferritin (termed SbFer) was cloned by the rapid amplication of cDNA ends (RACE) method based upon the sequence from the transcriptome library. The cDNA contained a 182 bp 5′-untranslated region, a 519 bp open reading frame encoding a polypeptide of 172 amino acids, a 229 bp 3′-untranslated region, and three introns (902, 373 and 402 bp) embedded in four exons. There was an iron response element (IRE) in the 5′-untranslated region. The deduced amino acid sequence of SbFer possessed many characteristics of vertebrate H type ferritin, shared 63%–91% identity with mollusks and greater identity with vertebrate H type ferritin compared to the L type. The SbFer gene expression pattern examined by quantitative real-time PCR showed ferritin mRNA was expressed in all ark shell tissues examined. The highest levels of expression were found in hemocytes with decreasing levels of expression in foot, mantle, gill, adductor muscle and hepatopancreas. A challenge with Vibrio anguillarum resulted in time-dependent significant upregulation of SbFer mRNA, indicating SbFer participated actively in the bacterial defense process. Further analysis of the antibacterial activity indicated recombinant SbFer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria. Taken together, these results suggested strongly that ferritin of the ark shell is involved in immune defense against microbial infection and it is a constitutive and inducible acute-phase protein.

A manganese superoxide dismutase (MnSOD) from ark shell, Scapharca broughtonii: Molecular characterization, expression and immune activity analysis

Manganese superoxide dismutase (MnSOD) is one of the key members of the antioxidant defense enzyme family, however, data regarding to the immune function of MnSOD in mollusks still remain limited now. In this study, a full-length MnSOD cDNA was identified by rapid amplification of cDNA ends (RACE) method from cDNA library of ark shell Scapharca broughtonii (termed SbMnSOD). The cDNA contained an open reading frame (ORF) of 696 bp which encoded a polypeptide of 232 amino acids, a 5′-UTR with length of 32 bp and a 3′-UTR of 275 bp. Four putative amino acid residues (His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of SbMnSOD and the signature sequence (DVWEHAYY) also existed in SbMnSOD. The deduced amino acid sequence of SbMnSOD shared high homology to MnSOD from other species. All those data revealed that the SbMnSOD was a novel member of the MnSOD family. The mRNA expression profiles of SbMnSOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbMnSOD distributed in all the examined tissues. Importantly, Vibrio anguillarum challenge resulted in the increased expression of SbMnSOD mRNA with a regular change trend in all examined tissues, indicating SbMnSOD actively participated in the immune response process. What's more, further analysis on the antibacterial activity of the recombinant SbMnSOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria. The present results clearly suggested that SbMnSOD was an acute phase protein involved in the immune reaction in S. broughtonii.

Genetic diversity and population structure of the ark shell Scapharca broughtonii along the coast of China based on microsatellites

As a commercially important species in East Asia, the natural resources of Scapharca broughtonii have been suffering from severe population decline across its main habitats. In China, recovery efforts for S. broughtonii are in progress. To provide scientific bases for fisheries management and conservation program, genetic diversity and population structure of seven wild populations of S. broughtonii from the northern China coast was assessed using seven microsatellite loci in this study. High genetic diversity was present in all the seven populations, as observed in mean allelic richness per locus (11.3–12.5), and average expected heterozygosity (0.835–0.867). No significant difference in allelic richness or expected heterozygosity was observed among the seven populations. Pairwise FST estimates and NJ tree topologies based on DC distances indicated that the seven populations fell into two groups, showing a clear division between the populations from the south and north of the Shandong Peninsula. Genetic differentiation was further analyzed using AMOVA and assignation tests. Genetic barrier analysis using Monmonier algorithm also confirmed that the Shandong Peninsula was the putative barrier separating the northern and southern populations. In addition, marine currents probably play an important role in high gene flow among three populations from the same marine gyre.

Age and Growth of the Ark ShellScapharca broughtonii(Bivalvia, Arcidae) in Japanese Waters

ABSTRACT Age and growth of the ark shell Scapharca broughtonii in 3 bays in Japan (Mutsu, where ark shells are cultured, and Nanao and Sendai, where they occur naturally) were examined using a shell sectioning and staining method. When a water-based marking pen was used to stain the opaque zones only in shell cross-sections, unstained translucent zones (i.e., growth rings) were clearly observed. Based on seasonal changes in marginal structures of shell cross-sections and marginal growth indices (MGIs), growth rings were estimated to be formed annually in summer when shell growth ceased. The estimated maximum ages for ark shells from Nanao and Sendai bays were 6 y and 20 y, respectively, whereas for Mutsu Bay the maximum age was not calculated because we had only young, cultured specimens, von Bertalanffy growth models were estimated as Lt = 80.1(1 -e -0.365(t+ 0-160) for Mutsu Bay, Lt = 115.3(1-e -0.478t-0162) for Nanao Bay, and Lt = 84.8(1 -e -0.473(t + 0.222) for Sendai Bay. There was no significant dif...

A Study on Survival and Growth of Ark shell, Scapharca broughtonii with differential density during hanging culture

여수 횡간도 지역에서 밀도별 피조개 각장 과 전중량의 성장은 밀도와 역 상관관계를 보였다. 밀도별 각장 평균성장은 10마리구가 5.21 mm로 가장 양호하였으며 밀도가 높을수록 각장 성장은 낮아졌다. 밀도별 전중량의 증가도 같은 결과를 보여 전반적으로 10-20마리구의 평균각장 및 평균 전중량의 증가가 30-40마리구에 비하여 양호하게 나타났다. 남해해역에서 밀도별 피조개 각장과 전중량의 성장도 밀도와 역 상관관계를 보였으나 여수해역보다 약한 역 상관관계를 보였다. 밀도별 각장 평균성장은 10마리구가 4.55 mm로 가장 양호한 결과를 보였으나, 20-40마리구의 밀도별 평균각장의 성장은 3.11, 3.36, 3.27 mm로 비슷하게 나타났다. 반면에 밀도별 전중량의 증가는 10-20마리구가 30-40마리구보다 많았다. 결론적으로 피조개의 수하양성시 양성밀도는 20마리/<TEX>$0.12m^2$</TEX> (166마리/<TEX>$1m^2$</TEX>) 가 적당하다고 사료되었다. 여수해역에서 피조개의 생존율은 밀도별로 각각 68%-87%, 로 밀도의존적 결과를 보였으나, 먹이생물인 식물플랑크톤이 풍부한 남해해역의 생존율은 91%-100%로 월등히 양호한 결과를 보였는데, 이는 여수해역의 식물플랑크톤의 양이 남해해역의 55.89%에 불과하였기 때문이라 사료된다. 수하 양성 해역별의 수온범위는 <TEX>$16.22-25.66^{\circ}C$</TEX>였고, 염분은 30.07-33.48였으며, pH는 8.10-8.45범위였다. 그리고 용존산소 (DO) 는 4.49-9.65 mg/L 였다. Ark shell Scapharca broughtonii length and total weight growth by density, which was hanging-cultured near the sea area of Yeosu-city, Hoenggan-island (below is Yeosu), showed inverse correlation with density. As for the average growth of shell length by density, the test area of 10 shells showed the best result as 5.21 mm, and it became lower as density became higher. Also, the increase of total weight by density showed the same result, thus on the whole, the increase of average shell length and total weight of 10-20 shell test area showed the better result than the test area of 30-40 shells. In addition, even if the parashell's shell length by density and total weight growth in the sea area of Namhae-gun, Mijo-fishing port (below is Namhae) showed inverse correlation with density, it showed the slighter inverse correlation than the sea area of Yeosu. As for the average growth of shell length by density, the test area of 10 shells showed the best result as 4.55mm, but the average growth of shell length by density in the test area of 20-40 shells appeared to be similar as 3.11, 3.36 and 3.27 mm. On the other hand, as for the increase of total weight by density, the test area of 10-20 shells showed the more increase than the test area of 30-40 shells. In conclusion, it is guessed that the culture density of 20 shells/<TEX>$0.12m^2$</TEX> (166 shells / <TEX>$1m^2$</TEX>) would be appropriate when hanging-culturing the shells. At the sea area of A, the survival rate of Scapharca broughtonii parashell was 68-87% by density respectively, and it showed the density-dependent result, while the survival rate of sea area of Namhae, where phytoplankton as prey is abundant, showed the much better result as 91-100%. It is guessed that this may be due to the fact that the quantity of phytoplankton in sea area of Yeosu is just 55.89% of sea area of Namhae. When hanging-culturing the shells, the water temperature by sea area ranged from 16.22 to <TEX>$25.66^{\circ}C$</TEX>, salinity 30.07-33.48, pH 8.10-8.45, and DO 4.49-9.65 mg/L.

Tandem repeat arrays in the mitochondrial genome as a tool for detecting genetic differences among the ark shell Scapharca broughtonii

AbstractWe found that tandem repeat (TR) arrays were present in the ark shell Scapharca broughtonii mitochondrial genome, and we demonstrated the efficiency of using this TR region as a tool for the detection of genetic differentiation. In samples collected from seven populations off the Japanese, Chinese, Russian and Korean coasts, five TR types with length ranging from 481 to 601 bp (base pairs) were found, as were 18 haplotypes (n = 155). All the Japanese samples from SD, IK, AM and KG, which represented four geographically distinct populations, had the same type of tandem repeat array, 481‐TR. All the samples from China and all but one sample from Russia had the same TR type, 511‐TR; these finding may be strong evidence that the ark shell populations in China and Russia are very similar. Based on a neighbor‐joining dendrogram of haplotypes, the Korean population was divided into two groups as follows: samples with haplotype H481‐3 and samples with haplotypes H601‐1, H601‐2, H601‐3 or H541‐1. Based on the haplotype diversity and nucleotide diversity, the Chinese, Russian and Korean populations showed a higher diversity than Japanese populations (P &lt; 0.05).

Identification and expression analysis on bactericidal permeability-increasing protein (BPI)/lipopolysaccharide-binding protein (LBP) of ark shell, Scapharca broughtonii

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are the numbers of the lipid transfer protein/lipopolysaccharide-binding protein family and play crucial roles in the innate immune response to Gram-negative bacteria. A novel Sb-BPI/LBP1 from ark shell Scapharca broughtonii was isolated by expressed sequence tag (EST) and RACE techniques. The Sb-BPI/LBP1 cDNA encoded a polypeptide of 484 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 463 amino acids. The deduced amino acid sequence of Sb-BPI/LBP1 contained an N-terminal BPI/LBP/CETP domain BPI1 with three functional regions that display LPS-binding activity, and a C-terminal BPI/LBP/CETP domain BPI2. In structure and sequence, Sb-BPI/LBP1 showed highly similar to those of the BPI/LBPs from invertebrate and non-mammalian vertebrate, the LBPs and BPIs from mammal. By quantitative real-time RT-PCR, Sb-BPI/LBP1 transcripts could be detected in all normal tested tissues, including hepatopancreas, adductor muscle, mantle margin, heart, gonad, gill and hemocytes, and was universally up-regulatable at 24 h post LPS challenge. The mRNA expression of Sb-BPI/LBP1 in hemocytes was the most sensitive to LPS challenge, significantly up-regulated at 12 h post LPS challenge and peaked at 24 h (16.76-fold, P < 0.05). These results suggested that Sb-BPI/LBP1 was a constitutive and inducible acute-phase protein contributing to the host immune defense against Gram-negative bacterial infection in ark shell S. broughtonii.

Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: An ultra-large metazoan mitochondrial genome

The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47kb to about 50kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8–10 length variants. The motifs of the HTR arrays are about 353–362bp and the number of repeats ranges from 1 to 11.

Molecular characterization and expression of a novel big defensin (Sb-BDef1) from ark shell, Scapharca broughtonii

Big defensins, endogenous cysteine-rich antimicrobial peptides (AMPs) with antimicrobial activity and immunomodulatory property, play crucial roles in host defense against various microbial pathogens. A novel big defensin (Sb-BDef1) of ark shell Scapharca broughtonii was identified by expressed sequence tag (EST) and RACE techniques. The Sb-BDef1 cDNA contained an open reading frame (ORF) of 336-bp encoding a polypeptide of 111 amino acids with a putative signal peptide of 21 amino acid residues, followed by a putative propeptide of 11 residues and a putative mature peptide of 79 residues. The mature peptide shared the common features of big defensins, including a high hydrophobic residues region (59%) in the N-terminus, a defensin domain in the C-terminus, which perfectly corresponds to the six conserved disulfide-bonded cysteine residues involved in the formation of the internal disulfide bridges (C1–C5, C2–C4 and C3–C6) in all big defensins from mollusk, horseshoe crab and amphioxus. Quantitative real-time PCR analysis revealed that the expression of Sb-BDef1 transcript was detected in all the tissues examined from normal ark shells, and the temporal expression of Sb-BDef1 mRNA was remarkably up-regulated at 8, 16 h in hemocytes, and at 16, 24 h in hepatopancreas after Vibrio anguillarum-challenge, respectively. These results suggested that Sb-BDef1 was a constitutive and inducible acute-phase protein and should be involved in immune response of Gram-negative microbial infection in ark shell S. broughtonii.