Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Expression of ABCA3 transporter gene in Tegillarca granosa and its association with cadmium accumulation.

Exposure to cadmium (Cd), a heavy metal, can cause strong and toxic side effects. Cd can enter the body of organisms in several ways, leading to various pathological reactions in the body. Tegillarca granosa is a kind of bivalve shellfish favored by people in the coastal areas of China. Bivalve shellfish can easily absorb heavy metal pollutants from water bodies while filter feeding. T. granosa is considered a hyper-accumulator of Cd, and the TgABCA3 gene is highly expressed in individuals with a high content of Cd-exposed blood clam. However, it is unclear whether TgABCA3 is involved in Cd ion transport in blood clam and the molecular mechanism for the mechanism of the Cd-induced responses for maintaining cell homeostasis. In this study, the complete cDNA of the TgABCA3 gene was analyzed to provide insights into the roles of TgABCA3 in resistance against Cd in blood clam. The complete sequence of TgABCA3 showed high identity to that of TgABCA3 from other bivalves and contained some classical motifs of ATP-binding cassette transport proteins. TgABCA3 expression in different tissues was measured using real-time quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. The tissue-specific expression showed that TgABCA3 expression was highest in the gill tissue. The TgABCA3 expression in the gill tissue was silenced using the RNA interference technique. After TgABCA3 silencing, the TgABCA3 expression decreased, the Cd content increased, the oxygen consumption and ammonia excretion rates increased, and the ingestion rate decreased. These results showing that the extents of Cd accumulation and resulting toxic effects are related to expression levels and activity of TgABCA3 indicate that TgABCA3 has a protective function against Cd in the clam. This increase in Cd accumulation results in serious damage to the body, leading to the enhancement of its physiological metabolism. Therefore, the findings of the study demonstrated that TgABCA3 can participate in the transport of Cd ions in the blood clam through active transport and play a vital role in Cd detoxification.

RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams.

Dopamine beta-hydroxylase (DßH) plays an essential role in the synthesis of catecholamines (CA) in neuroendocrine networks. In the razor clam, Sinonovacula constricta a novel gene for DßH (ScDßH-α) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScDßH-α gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScDßH-α in adult clams using siRNA caused a reduction in the growth rate compared to control clams. Reduced growth was associated with strong down-regulation of gene transcripts for the growth-related factors, platelet derived growth factors A (PDGF-A) (P < 0.001) 24 h after ScDßH-α knock-down, vascular endothelial growth factor (VEGF1) (P < 0.001) and platelet derived growth factor B (PDGF-B-2) (P < 0.001) 24 h and 48 h after ScDßH-α knock-down and transforming growth factor beta (TGF-ß1) (P < 0.001) 48 h and 72 h after ScDßH-α knock-down. Taken together the results suggest that the novel ScDßH-α gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.

New insights into the Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges.

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum-P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.

Dopamine beta-hydroxylase and its role in regulating the growth and larval metamorphosis in Sinonovacula constricta.

Dopamine beta-hydroxylase (DßH) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The DßH gene was identified from the razor clam Sinonovacula constricta and referred to as ScDßH. The ScDßH gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScDßH transcript expression was abundant in liver and hemolymph. During early development, ScDßH expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of DßH were screened. After challenge with DßH inhibitor, the larval metamorphosis and survival rates, and juvenile growth were obviously decreased. Under the siRNA stress, the larval metamorphosis and survival rates were also significantly decreased. Therefore, ScDßH may play an important regulating role in larval metamorphosis and juvenile growth.

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta.

Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates and plays a critical role in protein metabolism and growth. In the present study, we investigated the full-length cDNA sequence of the LAP3 gene in Sinonovacula constricta (ScLAP3) using expressed sequence tags and rapid amplification of cDNA ends. The full-length ScLAP3 cDNA was 2885 bp, with a 1560 bp open reading frame encoding 519 amino acids. Sequence analysis revealed that ScLAP3 shared 70.9% identity with LAP3 from the blood clam Tegillarca granosa and 62.0-68.0% with other species. ScLAP3 was expressed in all six tested tissues, with significantly higher expression levels in the foot compared with mantle, adductor muscle, liver, gills, and siphon tissues in adults (P < 0.01). In the eight developmental stages, ScLAP3 expression gradually increased, with significantly higher levels in D-shaped larvae compared with other developmental stages (P < 0.01), suggesting that it may be involved in the formation of certain organs during early development. Association analysis identified three shared single nucleotide polymorphisms (SNPs), c.1073A > G, c.1139C > T and c.1154A > G in exons of ScLAP3 gene from 177 individuals of two groups, one selective strain and one wild population, which had significant effects on growth traits of S. constricta. The results provided candidate genetic markers to assist selective breeding of razor clams toward improved growth.

The role of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by PAHs in the calm Ruditapes philippinarum.

The NF-E2-related factor 2 (Nrf2) is a master regulator of cellular responses against environmental stresses. In this study we cloned the full-length cDNAs of the RpNrf2 encompassed 2823 bp from the clam Ruditapes philippinarum (R. philippinarum). Sequences alignment and phylogenetic analysis showed that Nrf2 was highly specific in the clams. RpNrf2 expression was detected in gill, digestive gland, mantle and adductor, which the highest transcription level was observed in gill and digestive gland. The gene expressions of RpNrf2, Kelch-like-ECH-associated Protein 1 (Keap1), Cul3-based E3 Ubiquitin Ligase (E3), Glutathione S-transferase (GST-pi), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive gland was evaluated by real-time PCR after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 µg/L) for 15 days, which showed that the expression of Nrf2 significantly increased at day 1 and day 6 after exposure (p < 0.05), and there was a negative relationship between the mRNA levels of Nrf2 and Keap1 that indicates the enhancement of Keap1 expression stimulating Nrf2 degradation. RNA interference experiments were conducted to examine the expression profiles of RpNrf2, antioxidant and detoxification genes (GST-pi, Cu/Zn-SOD, CAT and GPx) and Lipid Peroxidase (LPO) level in digestive gland exposed to BaP. The results showed that the mRNA level of Nrf2 was significantly decreased by 63.2%, and the changes of antioxidant and detoxification genes expression were consistent with the Nrf2 gene suggesting that Nrf2 is required for the induction of antioxidant and detoxification genes. Besides, the LPO levels expressed by malondialdehyde (MDA) contents were significant higher compared with the control group at 72 h post dsRNA-Nrf2 injection. In conclusion, our data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with E3 and Nrf2 signaling pathway plays an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.

Diamondoid naphthenic acids cause in vivo genetic damage in gills and haemocytes of marine mussels.

Diamondoids are polycyclic saturated hydrocarbons that possess a cage-like carbon skeleton approaching that of diamond. These 'nano-diamonds' are used in a range of industries including nanotechnologies and biomedicine. Diamondoids were thought to be highly resistant to degradation, but their presumed degradation acid products have now been found in oil sands process-affected waters (OSPW) and numerous crude oils. Recently, a diamondoid-related structure, 3-noradamantane carboxylic acid, was reported to cause genetic damage in trout hepatocytes under in vitro conditions. This particular compound has never been reported in the environment but led us to hypothesise that other more environmentally relevant diamondoid acids could also be genotoxic. We carried out in vivo exposures (3 days, semi-static) of marine mussels to two environmentally relevant diamondoid acids, 1-adamantane carboxylic acid and 3,5-dimethyladamantane carboxylic acid plus 3-noradamantane carboxylic acid with genotoxic damage assessed using the Comet assay. An initial screening test confirmed that these acids displayed varying degrees of genotoxicity to haemocytes (increased DNA damage above that of controls) when exposed in vivo to a concentration of 30 µmol L(-1). In a further test focused on 1-adamantane carboxylic acid with varying concentrations (0.6, 6 and 30 µmol L(-1)), significant (P < 0.05%) DNA damage was observed in different target cells (viz. gills and haemocytes) at 0.6 µmol L(-1). Such a level of induced genetic damage was similar to that observed following exposure to a known genotoxin, benzo(a)pyrene (exposure concentration, 0.8 µmol L(-1)). These findings may have implications for a range of worldwide industries including oil extraction, nanotechnology and biomedicine.

Identification and expression of antioxidant and immune defense genes in the surf clam Mesodesma donacium challenged with Vibrio anguillarum.

The immune system in marine invertebrates is mediated through cellular and humoral components, which act together to address the action of potential pathogenic microorganisms. In bivalve mollusks biomolecules implicated in oxidative stress and recognition of pathogens have been involved in the innate immune response. To better understand the molecular basis of the immune response of surf clam Mesodesma donacium, qPCR approaches were used to identify genes related to its immune response against Vibrio anguillarum infection. Genes related to oxidative stress response and recognition of pathogens like superoxide dismutase (MdSOD), catalase (MdCAT), ferritin (MdFER) and filamin (MdFLMN) were identified from 454-pyrosequencing cDNA library of M. donacium and were evaluated in mantle, adductor muscle and gills. The results for transcripts expression indicated that MdSOD, MdFLMN and MdFER were primarily expressed in the muscle, while MdCAT was more expressed in gills. Challenge experiments with the pathogen V. anguillarum had showed that levels of transcript expression for MdSOD, MdCAT, MdFER, and MdFLMN were positively regulated by pathogen, following a time-dependent expression pattern with significant statistical differences between control and challenge group responses (p<0.05). These results suggest that superoxide dismutase, catalase, ferritin and filamin, could be contributing to the innate immune response of M. donacium against the pathogen V. anguillarum.

The involvement of cysteine-rich intestinal protein in early development and innate immunity of Asiatic hard clam, Meretrix meretrix.

Cysteine-rich intestinal protein (CRIP), a Zn(2+)-binding protein, contains a single copy of the highly conserved double-zinc-finger structure known as the LIM (lin-11-isl-1-mec-3) motif. In this paper, a cDNA encoding MmCRIP was isolated from the Asiatic hard clam Meretrix meretrix. The full-length cDNA of MmCRIP consists of a 237-bp open reading frame that encodes a polypeptide of 78 amino acids with a predicted molecular weight (MW) of 8635.8 Da and theoretical isoelectric point (pI) of 9.01. Bioinformatics analysis showed that it belonged to a new member of the CRIP subfamily. Relationship analysis revealed that MmCRIP has high-levels of sequence similarity to many CRIPs reported in other animals, particularly in invertebrates. Real-time PCR analysis showed that the highest level of MmCRIP expression was in hemocyte tissue and at pediveligers stage. To investigate immune function, mature clams were challenged with Aeromonas hydrophila. During A. hydrophila infection, up-regulation of MmCRIP transcript in clam's hemocyte, gill and hepatopancreas was detected. DsRNAi (double-strand RNA interference) approach was employed to study the function of MmCRIP and the data showed that inactivation of the MmCRIP gene blocked larvae development and caused mass mortalities. The probable roles of MmCRIP in clam early development and innate immunity are presented for the first time.

Differential response of two ferritin subunit genes (VpFer1 and VpFer2) from Venerupis philippinarum following pathogen and heavy metals challenge.

As a principal extracellular iron storage molecule, ferritin plays an important role in the iron-withholding strategy of innate immunity and detoxification system. In this study, we cloned and characterized another ferritin from Venerupis philippinarum (designated as VpFer2), in addition to one previously reported (VpFer1). VpFer2 possessed all the conserved features critical for the fundamental structure and function of ferritin H subunit. VpFer1 and VpFer2 mRNA were both found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpFers, and VpFer2 showed more sensitive to Vibrio anguillarum infection. For heavy metals exposure, the expression level of VpFer1 was significantly induced by Cd at 48 h, but kept relatively constant after exposure to Cu. With regards to VpFer2, the expression level dropped significantly at 24 h, then began to increase to the peak value at 48 h under Cd exposure, while Cu exposure constantly depressed the expression level of VpFer2 throughout the time course. Similarly, VpFer2 seemed to be more sensitive to heavy metals exposure than VpFer1 as its mRNA level changed by higher magnitudes. All these results suggested that VpFers may be important proteins involved in host immune defense and heavy metals detoxification. The diverse expression patterns of VpFers demonstrated that VpFer2 was an early and sensitive responder to environmental stress in V. philippinarum.

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii.

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a "QPS" and an invariant "WND" motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.

The link between selenium binding protein from Sinonovacula constricta and environmental pollutions exposure.

Selenium binding proteins (SeBPs) play a crucial role in controlling the oxidation/reduction in many physiological processes. Here we reported the isolation and characterization of a cDNA of SeBP gene from Sinonovacula constricta (denoted as ScSeBP). The full-length cDNA of ScSeBP was of 2345 bp, consisting of a 5'UTR of 246 bp, a 3' UTR of 626 bp, and a complete ORF of 1473 bp encoding a polypeptide with 491 amino acid residues. The predicted molecular mass of deduced amino acid of ScSeBP was 54.85 kDa and the theoretical pI was 6.44. Tissue distribution analysis of the ScSeBP revealed that the mRNA transcripts of ScSeBP were constitutively expressed in all examined tissues with the higher expressions in gill, gonad and the haemocytes. The temporal expression of ScSeBP in gill and haemocytes after B[α]P and heavy metals exposure were recorded by qPCR. B[α]P exposure at 0.5 and 5 mg L(-1) caused significant increase in mRNA expression of ScSeBP in haemocytes, but down-regulated ScSeBP mRNA expression in gill. Concerning heavy metals stresses, the suppressed expression patterns were detected in gill and haemocyte except lower concentration of PbCl2 exposure in haemocytes at 12 h. All our results indicated that ScSeBP was one of key effectors in mediating B[α]P and heavy metals exposure.

Cloning, promoter analysis and expression of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in Japanese scallop (Mizuhopecten yessoensis).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key adaptor molecule for the tumor necrosis factor superfamily and Toll-like/interleukin-1 receptor superfamily. It plays an important role in innate and adaptive immunity. The TRAF6 of Japanese scallop Mizuhopecten yessoensis (designated as MyTRAF6) was identified and characterized in this study. The full-length cDNA of MyTRAF6 was 2,407 bp, which consisted of 239-bp 5'-terminal untranslated region, 1,974-bp open reading frame encoding a polypeptide of 657 amino acids, 194-bp of 3'-terminal untranslated region followed by a canonical polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of MyTRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. It had an overall identity of 43-96% with those of other TRAF6s, the highest identity (96%) with Chlamys farreri TRAF6, and the least identity (43%) with Meleagris gallopavo TRAF6. Phylogenetic analysis classified MyTRAF6 as a true TRAF6 ortholog. In addition, the promoter of MyTRAF6 was also identified by genome walking. It contained several potential transcription factor-binding sites and three single nucleotide polymorphisms. qRT-PCR analysis revealed that MyTRAF6 was highly expressed in hemocytes of adult M. yessoensis. MyTRAF6 transcript level in the hemocytes reached a maximum 6 h after Vibrio anguilarum challenge. The results indicated that MyTRAF6 may fulfill an important function during M. yessoensis bacterial infection. It could be a key effector molecule involved in the innate defense of molluscs.

Identification and in silico analysis of a novel troponin C like gene from Ruditapes philippinarum (Bivalvia: Veneridae) and its transcriptional response for calcium challenge.

Troponin C (TnC) is one of the subunits composing the troponin complex, which is primarily expressed in muscle tissue and plays a major role in regulating contractility. We have identified a novel TnC-like gene (RpTnC) from the Ruditapes philippinarum Manila clam. Sequence analysis indicated that RpTnC has a 450bp coding sequence, encoding a 150 amino acid protein with a molecular mass of 17.4 kDa. The RpTnC protein consisted of four EF-hand motifs (I-IV), each with a Ca2+-binding site. In silico comparative analysis of protein sequence showed that only site IV, demonstrating a conserved stretch (DxDxSx6E), is functionally active for Ca2+-coordination. Moreover, RpTnC was homologically (61.3% identity) and phylogenetically closest to Japanese flying squid TnC. The mRNA expression analysis using quantitative real-time PCR revealed a differential basal-expression of RpTnC transcripts in six different clam tissues, with higher levels in adductor muscle and mantle. Intramuscular administration of CaCl2 caused a prominent upregulation of RpTnC transcripts in adductor muscle (~5 fold). Collectively, our findings suggest that the TnC homolog of Manila clam identified in this study may be involved in important role(s) in clam physiology, mainly in its muscle tissues, and its transcription could be significantly influenced by increased Ca2+ levels.

The role of catalase in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix.

Catalase (CAT) can effectively eliminate H(2)O(2) and maintain the redox balance of immune system, which is essential for innate immunity. A catalase gene was cloned and its potential role in immune system was investigated in the clam, Meretrix meretrix. The catalase (MmeCAT) gene had an open reading frame of 1533 bp encoding 511 amino acids which showed high identity with that of molluscs. The distribution of MmeCAT in clam tissues was examined and the mRNA, protein expression and CAT activity paralleled with each other, with the highest expression in hepatopancreas. In response to H(2)O(2) challenge, MmeCAT mRNA showed significantly higher expression at 12 h and 24 h post-challenge in experimental clams than in control clams (P < 0.05). Meanwhile, the protein expression in experimental clams was increased to about 3 times as much as that in control clams at 6 h post-challenge. After injection with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeCAT mRNA was significantly up-regulated at 12 h and 24 h post-injection (P < 0.05). It suggested that MmeCAT might be involved in the immune response to Vibrio infection. To better understand the role of MmeCAT in immune system, its mRNA expression was compared between a Vibrio-resistant population and a control population after immersion challenge with MM21. The continuously increased transcription in resistant population suggested MmeCAT could benefit the immune system of clams to defend against pathogen infection. Our study indicated that the redox balance was essential for M. meretrix to resist pathogen infection.

Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis.

The response of the defense components lysozyme (LYZ), metallothionein (MT), and superoxide dismutase (SOD) to combined exposure to heavy metals and bacteria was assessed at transcriptional level in the surf clam Mactra veneriformis. First, the full-length LYZ cDNA containing 808 nucleotides and encoding 194 deduced amino acids was identified from the clam. Multiple alignments revealed that MvLYZ had a high identity with invertebrate-type LYZs from other mollusks. Next, clams were exposed to Vibrio parahaemolyticus and a mixture of cadmium and mercury, alone or in combination, for 7 days. Cumulative mortality of clams and mRNA expressions of the three defense components were analyzed. The highest cumulative mortality took place in the combined treatment on day 7. The expression of the three genes was up-regulated in response to treatments compared to the control with different response times and transcriptional levels; the response to combined exposure occurred earlier than to single exposure. Among the experimental groups, MvLYZ expression and MvSOD expression peaked in the combined treatment on day 3, whereas MvMT expression peaked in heavy metals treatment on day 5. Furthermore, interactive effects of heavy metals and Vibrio on transcriptional response changed over the exposure time. Therefore, transcriptional regulation of the three genes under combined exposure was more complex than under single exposure.

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity.

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities.

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(•-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20 °C, under alkaline condition (pH 9) with a specific activity of 3299 U mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.

Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii.

Calmodulin (CaM) is a multifunctional intracellular calcium ion receptor protein that participates in a range of cellular processes, including calcium metabolism in mussels. To investigate the role of CaM in freshwater mollusk shell calcium metabolism, the full-length CaM cDNA was isolated from the freshwater pearl mussel, Hyriopsis schlegelii (referred to as hsCaM) using SMART RACE technology. The full-length hsCaM was 855 bp in size, containing a 70-bp 5'-untranslated sequence, a 447-bp open reading frame, a 309-bp 3'-untranslated sequence, and a 26-nucleotide long poly(A) tail. The hsCaM mRNA expression in different mussel tissues was examined using real-time PCR. The hsCaM mRNA was found to be ubiquitously expressed, but far more abundant in the gill, foot, and mantle than in the posterior adductor muscle. Real-time PCR was also used to determine hsCaM mRNA expression levels in mantle tissues of H. schlegelii at different ages. No significant differences between one-, two-, and three-year-old mussels were detected, but expression increased in four-year-old mussels and then decreased in five-year-old mussels. CaM appears to be involved in calcium regulation of the mantle in four-year-old mussels, which may secrete more mother of pearl during pearl culture.