Molecular and microscopic identification of Eomarteilia granula infection in Manila clam Ruditapes philippinarum off the south coast of Korea.

A report on the new species Eomarteilia (=Marteilia) granula infecting Manila clam Ruditapes philippinarum from Japan in 2014 suggests the possibility of E. granula infecting other Manila clam populations in the Northwest Pacific region, including Korea. In this study, we report the first infections by E. granula in Manila clams off the south coast of Korea. Histology revealed Marteilia-like plasmodia in the digestive tubule epithelia. Tissue imprints demonstrated that each parasite sporangium enclosed 4 spores and transmission electron microscopy (TEM) revealed ultrastructure of primary cells enclosing secondary cells, which contained spores. Mature spores consisted of 3 sporoplasms: outermost, intermediate, and innermost. The outermost sporoplasm showed a peripheral electron-dense monolayer characteristic of E. granula. The 18S rDNA amplified from the Marteilia-like parasite yielded 1784-bp PCR amplicon sequences which were 99.8% similar to that of E. granula previously reported (as M. granula) from Japan. In the molecular phylogenetic analysis, the novel Marteilia-like organism formed a well-supported clade with E. granula. Accordingly, we concluded that the novel Marteilia-like parasite that we found infecting some Korean Manila clams is Eomarteilia granula. Field surveys revealed that the infection was limited to clams of the south coast of Korea, with the prevalence ranging from 3.3 to 5.0%.

The linear mitochondrial genome of commensal hydroid Eutima japonica (Cnidaria, Hydrozoa, Eirenidae).

Here, we present the whole mitochondrial genome of commensal hydroid Eutima japonica McCrady 1859 (family Eirinidae); this is the first specimen of the family to have its mitogenome sequenced. The linear mitogenome is 15,315 bp in length and consists of 13 protein-coding genes (PCGs), large and small ribosomal subunits (rRNA), methionine and tryptophan transfer RNA (tRNA) genes (trnM and trnW), and a partial copy of cytochrome oxidase subunit I (cox1) pseudogene, as is typical for the class Hydrozoa. Nucleotide sequences of two cox1 genes at two ends of the linear mitogenome form a part of inverted terminal repeat. The overall genomic structure and gene arrangement of 13 PCGs were identical to the reported mitochondrial genomes of hydrozoans, except for the positions of two tRNA genes. Phylogenetic analysis of E. japonica 13 PCGs and other cnidarians recovers a closest relationship with the derived cluster of two hydrozoans, Laomedea flexuosa and Obelia longissimi within Leptothecata.

Molecular characterization of Urosporidium tapetis sp. nov., a haplosporidian hyperparasite infecting metacercariae of Parvatrema duboisi (Dollfus 1923), a trematode parasite of Manila clam Ruditapes philippinarum on the west coast of Korea.

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22-89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov.

A novel paramyxean parasite, Marteilia tapetis sp. nov. (Cercozoa) infecting the digestive gland of Manila clam Ruditapes philippinarum from the southeast coast of Korea.

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters.

First report of Perkinsus honshuensis in the variegated carpet shell clam Ruditapes variegatus in Korea.

The recent discovery of Perkinsus honshuensis, a new Perkinsus species infecting Manila clams Ruditapes philippinarum (Sowerby, 1852), in Japan, suggested that, based on proximity, P. honshuensis could also be in Korean waters, where to date, P. olseni was believed to be the only Perkinsus species present. Perkinsus sp. infections consistently occurred among Ruditapes variegatus clams on a pebble beach on Jeju Island, off the south coast of Korea. The typical 'signet ring' morphology of the parasite was observed in the connective tissue of the digestive gland, and infection intensity was comparatively low (3.3 × 103 ± 1.2 × 104 to 1.3 × 104 ± 6.1 × 104 cells g-1 gill weight). Further DNA analyses of internal transcribed spacer (ITS-1, 5.8S and ITS-2) and non-transcribed spacer (NTS) regions of the parasite showed 98.9-99.8 and 98.5-99.5% similarity to those of P. honshuensis from Japan, respectively. Phylogenetic analyses using ITS and NTS sequences indicated that Perkinsus sp. from Jeju formed a highly supported clade with P. honshuensis. This is the first report of P. honshuensis infections in clams in Korean waters and the first report of R. variegatus as a host for that parasite.

Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea.

Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern.

A galectin related protein from Oplegnathus fasciatus: Genomic, molecular, transcriptional features and biological responses against microbial pathogens.

Galectins, a family of ß-galactoside-binding lectins, are pattern recognition receptors that recognize pathogen-associated molecular patterns and are subsequently involved in the opsonization, phagocytosis, complement activation, and killing of microbes. Here, we report a novel galectin related protein (GRP) identified from rock bream (Oplegnathus fasciatus), designated OfGal like B. The cDNA of OfGal like B is 517 bp with an open reading frame (ORF) of 438 bp, encoding 145 amino acids, with a single carbohydrate recognition domain (CRD). However, only two of the seven critical residues responsible for carbohydrate recognition were identified in the CRD. There was no signal peptide identified in the OfGal like B protein. The genomic structure of OfGal like B, determined using a bacterial artificial chromosome (BAC) genomic library, consists of four exons and three introns. Homology assessment, multiple sequence alignment, and phylogenetic analysis indicated that OfGal like B is an evolutionarily conserved lectin that is closely related to the proto-type galectins. OfGal like B mRNA was constitutively expressed in a wide range of tissues in healthy rock breams. When challenged with bacterial or viral stimulants, OfGal like B was up-regulated in the gills and spleen of rock breams, indicating that it likely plays an important role during bacterial and viral infections. Furthermore, recombinant OfGal like B (rOfGal like B) lacked carbohydrate-binding activity but was able to recognize and agglutinate bacteria, including Streptococcus iniae, Listeria monocytogenes, Vibrio tapetis, Escherichia coli, and Edwardsiella tarda, and a ciliate parasite, Miamiensis avidus. These results collectively suggest that OfGal like B is involved in pathogen recognition and plays a significant role(s) in the innate defense mechanism of rock bream.

First report of Urosporidium sp., a haplosporidian hyperparasite infecting digenean trematode Parvatrema duboisi in Manila clam, Ruditapes philippinarum on the west coast of Korea.

In this study, we first report on the occurrence of Urosporidium sp., a haplosporidian hyperparasite infecting the trematode, Parvatrema duboisi, which parasitizes Manila clams, Ruditapes philippinarum on the west and south coasts of Korea. The larval P. duboisi infected by the sporocyst stage of Urosporidium sp. demonstrated numerous small yellowish spores in their tissues. The heavily infected metacercariae exhibited degenerate bodies and the larvae were often motionless. Clams heavily infected by the metacercariae of P. duboisi also displayed abnormal golden spots on the mantle tissue. In histology, different life stages of Urosporidium sp. could be identified, including the uni-nucleate, plasmodial, sporogonic stages and the acid fast mature spores released from the cyst. In scanning electron microscopy (SEM), the mature spore exhibited a semi-circular rim around the apical end and the orifice was covered internally with a flap. Loop-like filaments ornamentation was also identified from Urosporidium sp. in SEM, suggesting that Urosporidium sp. found in this study is a new member in the genus. Prevalence of Urosporidium sp.-infected trematodes in this study ranged from 2.5% to 24.0% in April 2010 and the infection was observed from 8 sampling sites out of the 26 sites surveyed on the west and south coasts.

Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: morphology and functional aspects.

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses.

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea.

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.

Identification of scuticociliates (Pseudocohnilembus persalinus, P. longisetus, Uronema marinum and Miamiensis avidus) based on the cox1 sequence.

Scuticociliatosis is characterized as highly histophagous, causing systemic tissue destruction and high mortality in cultured marine fish. Some of the scuticociliates have been implicated as the causative agents of scuticociliatosis. Here, we describe our study to differentially identify various species in complex animal-sourced samples, namely olive flounder Paralichthys olivaceus and black rockfish Sebastes schlegelii suffering from scuticociliatosis. The mitochondrial cytochrome c oxidase 1 (cox1) gene from the scuticociliates was amplified and sequenced. The divergence percentage of small subunit ribosomal DNA sequence between average scuticociliate species was found to be low (8.3%) but the genetic divergence of cox1 sequence reached 23.5%, suggesting that a hyper-variable region of the cox1 gene could be used as a diagnostic DNA barcoding region. Thus, we developed species-specific primers for use in multiplex PCR of complex (pooled) samples. The primers yielded species-specific fragments (of distinct size) that allowed for simple, rapid, and effective identification and differentiation of multiple species present in a single sample.

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum).

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.

Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea.

The morphology, infraciliature, silverline system, and the small subunit ribosomal RNA (SSU rRNA) of the little-known marine scuticociliate Pseudocohnilembus longisetusThompson, 1965 from the diseased black rockfish Sebastes schlegelii in Korea were studied. This scuticociliate possessed the typical characteristics of the genus Pseudocohnilembus, but could be discriminated from Pseudocohnilembus hargisi, and Pseudocohnilembus persalinus in terms of the body size, shape, the number of somatic kineties and kinetids in somatic kinety 1, and the number/position of contractile vacuole pores. The SSU rRNA gene of P. longisetus was sequenced in order to gain a better understanding of appropriate phylogenetic classification. The SSU rRNA was 1754 bp and the sequence was deposited in GenBank under accession number FJ899594. The SSU rRNA gene sequences of P. longisetus had an identity of 98.1%, 96.8% and 95.3% with P. hargisi, P. persalinus, and Pseudocohnilembus marinus SSU rRNA sequences, respectively. Our population of P. longisetus belonged to the genus Pseudocohnilembus and was in an isolated position based on the SSU rRNA gene tree, which was consistent with the conclusions based on the morphological studies. However, further investigation is required to determine the pathogenicity of this species.

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques.

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.

Pathogenicity of Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum (Ciliophora, Scuticociliatida).

The scuticociliates Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum were cloned and identified using morphological characteristics and the small subunit ribosomal RNA gene (SSU rRNA). M. avidus strains YS1, WS1, YK1 and JJ3 from southern coastal areas and Jeju Island in Korea were pathogenic to olive flounder Paralichthys olivaceus (80 to 100% mortality in 8 to 10 g fish) when inoculated intraperitoneally (i.p.) with 1.0 to 1.4 x 10(6) ciliates fish(-1). Mortality was lower (10 to 45%) when the inoculum was 1.0 to 1.4 x 10(4) ciliates fish(-1) in the i.p.-injected group. The M. avidus strains of YS1, WS1, YK1 and JJ3 caused 60 to 100% mortality by immersion infection with 3.2 to 4.2 x 10(3) ml(-1) in 8 to 10 g fish and 3.0 to 4.0 x 10(3) ml(-1) in 30 to 40 g fish. M. avidus strain Mie0301 from the Mie prefecture in Japan caused 70% mortality by immersion infection with 4.4 x 10(3) ml(-1) in 30 to 40 g fish. The predominant sign was severe abdominal distension in i.p.-injected fish, and extensive ulcer lesions in the skeletal muscle in immersion-infected fish. Numerous ciliates were observed in the ascetic fluid, ulcers, haemorrhagic lesions, gills and brain of infected fish. However, P. persalinus (strain SCL-A), P. hargisi (strain SCL-B) and U. marinum (strain JK3) showed less than 30% mortality from both i.p. and immersion challenges, with no ciliate invasion in the skin, gills or brain. M. avidus-infected fish showed many ciliates in gills, fins, skin muscle, brain and intestine accompanied by necrosis and haemorrhages. However, no histological changes were observed in P. persalinus-, P. hargisi- or U. marinum-infected fish.

Molecular characterization and expression analysis of regucalcin in disk abalone (Haliotis discus discus): intramuscular calcium administration stimulates the regucalcin mRNA expression.

Regucalcin is a novel calcium (Ca(2+)) binding protein and it has been demonstrated to play a multifunctional role in many organisms. Here, we report the molecular cloning of invertebrate regucalcin cDNA from disk abalone Haliotis discus discus. The full length cDNA showed 1321 bp of nucleotides with a polyadenylated sequence (AATAAA). Abalone regucalcin (HdReg) open reading frame (ORF) consists of 918 nucleotides encoding 305 amino acids (aa). Estimated molecular mass was 33 kDa and predicted isoelectric point (pI) was 4.9. The HdReg aa sequence did not contain the EF-hand motif as a Ca(2+) binding domain, suggesting a novel class of Ca(2+) binding protein. Moreover, it showed 45% identity to chicken and zebrafish, and 44% to rat and mouse regucalcin in deduced aa level. The tissue expression analysis of HdReg mRNA was investigated by RT-PCR and it was expressed in all the tissues tested such as gill, mantle, digestive tract, and abductor muscle. Semi-quantitative RT-PCR results showed that an intramuscular administration of calcium chloride (CaCl(2)) (0.5 mg CaCl(2)/g of abalone) could significantly induce regucalcin mRNA in abductor muscle after 30 min of administration and reached maximum after 1 h. Subsequently, the expression level was decreased after 2 h. This indicates that the expression of regucalcin mRNA is constitutive, and specifically up regulated in abalone abductor muscle by Ca(2+) administration.

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase.

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.

Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis.

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus).

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.

Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus).

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O(2)(-) into the stabler H(2)O(2). In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 degrees C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 degrees C and completely heat inactivated at 80 degrees C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.