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.

Roseovarius scapharcae sp. nov., isolated from ark shell Scapharca broughtonii.

A Gram-stain-negative, non-motile, aerobic and ovoid or rod-shaped bacterium, designated MA4-5T, was isolated from ark shell (Scapharca broughtonii) collected from the South Sea, South Korea. The novel strain grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining and maximum-likelihood phylogenetic trees based on 16S rRNA gene sequences showed that strain MA4-5T forms a coherent cluster with the type strains of Roseovarius albus, Roseovarius aestuarii and Roseovarius nubinhibens, sharing 97.0-99.2 % sequence similarity. It exhibited 16S rRNA gene sequence similarity of 93.1-96.1 % to the type strains of other Roseovarius species. Strain MA4-5T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids of strain MA4-5T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain MA4-5T was 53.8 mol% and its mean DNA-DNA relatedness values with the type strains of R. albus, R. aestuarii and R. nubinhibens were 11-26 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, indicated that strain MA4-5T is separate from recognized species of the genus Roseovarius. On the basis of the data presented, strain MA4-5T is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius scapharcae sp. nov. is proposed. The type strain is MA4-5T ( = KCTC 42703T = NBRC 111226T).

Aliisedimentitalea scapharcae gen. nov., sp. nov., isolated from ark shell Scapharca broughtonii.

A Gram-negative, aerobic, non-spore-forming, motile and ovoid or rod-shaped bacterial strain, designated MA2-16(T), was isolated from ark shell (Scapharca broughtonii) collected from the South Sea, South Korea. Strain MA2-16(T) was found to grow optimally at 30°C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain MA2-16(T) clustered with the type strain of Sedimentitalea nanhaiensis. The novel strain exhibited a 16S rRNA gene sequence similarity value of 97.1% to the type strain of S. nanhaiensis. In the neighbour-joining phylogenetic tree based on gyrB sequences, strain MA2-16(T) formed an evolutionary lineage independent of those of other taxa. Strain MA2-16(T) contained Q-10 as the predominant ubiquinone and C18:1 ω7c and 11-methyl C18:1 ω7c as the major fatty acids. The major polar lipids of strain MA2-16(T) were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA2-16(T) was 57.7 mol% and its DNA-DNA relatedness values with the type strains of S. nanhaiensis and some phylogenetically related species of the genera Leisingera and Phaeobacter were 13-24%. On the basis of the data presented, strain MA2-16(T) is considered to represent a novel genus and novel species within the family Rhodobacteraceae, for which the name Aliisedimentitalea scapharcae gen. nov., sp. nov. is proposed. The type strain is MA2-16(T) (=KCTC 42119(T) =CECT 8598(T)).

Rhodopirellula rosea sp. nov., a novel bacterium isolated from an ark clam Scapharca broughtonii.

A novel Gram-negative, motile, and ovoid-shaped strain, LHWP3(T), which belonged to the family Planctomycetaceae in the phylum Planctomycetes, was isolated from a dead ark clam Scapharca broughtonii collected during a mass mortality event on the south coast of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the isolate was most closely related to the type strain of Rhodopirellula baltica, with a shared 16S rRNA gene sequence similarity of 94.8%. The isolate grew optimally at 30°C in 4-6% (w/v) NaCl, and at pH 7. The major isoprenoid quinone was menaquinone-6 (MK-6). The dominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and unidentified polar lipids. The predominant cellular fatty acids were C16:0, C18:1 ω9c, and C18:0. The genomic DNA G+C content of strain LHWP3(T) was 53.0 mol%. Based on polyphasic taxonomic analyses, strain LHWP3(T) should be classified as a novel species in the genus Rhodopirellula in the family Planctomycetaceae, for which the name Rhodopirellula rosea sp. nov. is proposed. The type strain is LHWP3(T) (=KACC 15560(T) =JCM 17759(T)).

Blastopirellula cremea sp. nov., isolated from a dead ark clam.

Strain LHWP2(T), a novel, aerobic, budding, motile and ovoid bacterium belonging to the phylum Planctomycetes, was isolated from a dead ark clam (Scapharca broughtonii) from the south coast of Korea. Strain LHWP2(T) grew optimally at 30 °C, in the presence of 4% (w/v) NaCl, and at pH 7. The predominant cellular fatty acids were C16:0, C18:1ω7c and/or C18:1ω6c (summed feature 8) and C18:1ω9c. The major isoprenoid quinone was menaquinone-6 (MK-6). The dominant polar lipid was identified as phosphatidylglycerol. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain was most closely related to Blastopirellula marina DSM 3645(T), with a 16S rRNA gene sequence similarity of 94.1%. The genomic DNA G+C content of strain LHWP2(T) was 49.5 mol%. Strain LHWP2(T) was distinguished from B. marina DSM 3645(T) based on its optimum salinity, acid production from substrates, assimilation of substrates and DNA G+C content. Overall, these phenotypic, genotypic and phylogenetic data suggest that strain LHWP2(T) should be classified as a novel species belonging to the genus Blastopirellula, for which the name Blastopirellula cremea sp. nov. is proposed. The type strain is LHWP2(T) (=KACC 15559(T)=JCM 17758(T)).

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.

Ruegeria conchae sp. nov., isolated from the ark clam Scapharca broughtonii.

A slightly halophilic, Gram-negative, strictly aerobic, non-motile rod, designated TW15(T), was isolated from an ark clam in South Korea. Growth occurred at 10-37 °C, with 1-5% (w/v) NaCl and at pH 7.0-10.0. Optimal growth occurred at 25-30 °C, with 2% (w/v) NaCl and at pH 8.0. Strain TW15(T) exhibited both oxidase and catalase activities. The major fatty acids of strain TW15(T) were summed feature 8 (consisting of C(18:1)ω7c and/or C(18:1)ω6c) and 11-methyl C(18:1)ω7c. The predominant isoprenoid quinone was ubiquinone-10 (Q-10). The polar lipids of strain TW15(T) comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and five unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TW15(T) was most closely related to Ruegeria lacuscaerulensis DSM 11314(T) (98.0% 16S rRNA gene sequence similarity). DNA-DNA relatedness with closely related strains was <52 ± 3%. The DNA G+C content was 55.7 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain TW15(T) represents a novel species of the genus Ruegeria, for which the name Ruegeria conchae sp. nov. is proposed. The type strain is TW15(T) ( = KACC 15115(T)  = JCM 17315(T)).

Kistimonas scapharcae sp. nov., isolated from a dead ark clam (Scapharca broughtonii), and emended description of the genus Kistimonas.

A Gram-negative, motile, facultatively anaerobic rod, designated A36(T), was isolated from a dead ark clam found on the south coast of Korea. The isolate was catalase- and oxidase-negative. 16S rRNA gene sequence analysis indicated that strain A36(T) was most closely related to Kistimonas asteriae KMD 001(T), with which it shared 98.2% 16S rRNA gene sequence similarity. Strain A36(T) grew optimally at 30-37 °C, with 1% (w/v) NaCl and at pH 8.0. The major respiratory quinone was ubiquinone-9 (Q-9). The major polar lipids were phosphatidylserine, phosphoethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15) 2-OH) and C(16:0). The genomic DNA G+C content was 47.3 mol%. DNA-DNA relatedness between the isolate and K. asteriae JCM 15607(T) was <25 ± 3%. Strain A36(T) represents a novel species of the genus Kistimonas, for which the name Kistimonas scapharcae sp. nov. is proposed. The type strain is A36(T) ( = KACC 16204(T)  = JCM 17805(T)). An emended description of the genus Kistimonas is also provided.

Hemoglobin of the bloody clam Tegillarca granosa (Tg-HbI) is involved in the immune response against bacterial infection.

Hemoglobins (Hb) are the major protein components of erythrocytes circulating in the red blood, but can serve additional functions besides the transport of oxygen. Here, the cDNA of the bloody clam (Tegillarca granosa) Hb dimer (designated Tg-HbI) was cloned and was found to be 748 bp in length, consisting of an open reading frame of 441 bp encoding a polypeptide of 147 amino acids. The deduced amino acid sequence of Tg-HbI shared 81.6% similarity with HbI from two species of the genus Scapharca and 46-51% similarity with the Hb proteins from other mollusks. The 3D structure of bloody clam Tg-HbI was predicted by the SWISS-MODEL Protein Modelling Server and compared with that of Scapharca kagoshimensis. The mRNA transcript of Tg-HbI was detected in all of the clam cells/tissues examined, including haemocytes, the adductor muscle, foot, hepatopancreas, gill and mantle. The mRNA expression of Tg-HbI was significantly up-regulated after Vibrio parahaemolyticus, lipopolysaccharide and peptidoglycan challenge, indicating that Tg-HbI was involved in the immune defence responses against bacterial infection and exposure to bacterial pathogenic factors. As the first functional research on the Hb protein in bloody clam, our findings provide new insight into the innate immune defence mechanisms of T. granosa and other mollusks.