Manila Clam Venerupis Philippinarum Research Articles

C-type lectin (CTL) and sialic acid-binding lectin (SABL) from Venerupis philippinarum: Function on PAMP binding and opsonic activities in immune responses

Lectins are a superfamily of carbohydrate-recognition proteins that bind to specific carbohydrate structures and play significant roles in immune recognition and clearance of invaders. In the study, we investigated the potential mechanisms of PAMP binding and opsonic activities of a c-type lectin and a sialic acid-binding lectin from manila clam Venerupis philippinarum (designed as VpCTL and VpSABL). Both recombinant proteins (rVpCTL and rVpSABL) could bind LPS, PGN, glucan and zymosan in vitro. Coinciding with the PAMPs binding assay, a broad agglutination spectrum was displayed by rVpSABL including gram-positive bacteria Staphyloccocus aureus, gram-negative bacteria Escherichia coli, Vibrio parahaemolyticus, Vibrio harveyi, Pseudomonas putida, Proteus mirabilis and fungi Pichia pastoris, while no agglutinative activities on P. mirabilis and P. putida was observed in rVpCTL. Moreover, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rVpCTL and rVpSABL. More remarkable, VpCTL and VpSABL were highly detected in all the examined tissues, especially in gills and hepatopancreas. All the results showed that VpCTL and VpSABL could function as pattern recognition receptors (PRRs) with distinct recognition spectrum, perhaps involved in the innate immune responses of V. philippinarum.

Characterizing the habitat function of bivalve aquaculture using underwater video

Bivalve aquaculture is an expanding coastal industry with the potential to modify the habitat of fish and crab species, affecting their refuge, movement, and feeding. The habitat function of shellfish aquaculture is not yet well understood, in part due to difficulties in data collection using traditional methods. Underwater video was used to observe fish and crab species’ affiliations with cultured Pacific oyster Crassostrea gigas and Manila clam Venerupis philippinarum aquaculture sites in comparison to uncultured reference sediment and eelgrass habitats. Sites were monitored in 9 locations across 3 regions of Puget Sound, Washington, USA, in the summers of 2017 and 2018. Of the 3038 fish and crabs observed, 98% were represented by Embiotocidae (surfperch), crabs, three-spined stickleback Gasterosteus aculeatus, Cottidae (sculpins), and Pleuronectiformes (flatfish). Overall, the affiliations of fish and crabs with bivalve aquaculture varied by species groups, culture type, and regional environmental and habitat conditions. These interactions varied on a scale of approximately 150 km, highlighting variation of aquaculture-ecological interactions at a scale not previously recorded in Puget Sound. Species composition varied between aquaculture and non-aquaculture habitats in 2 of the 3 regions studied. Species diversity and richness in aquaculture habitats varied regionally, relative to reference habitats. Pelagic species were more abundant in aquaculture and reference sites that had vertical structure, but abundances of demersal and benthic species on aquaculture habitat relative to reference sites varied regionally. The availability of habitats within intertidal regions, including varying types of aquaculture, could determine community structure for marine organisms such as fish and crab.

Identification, antibacterial activities and action mode of two macins from manila clam Venerupis philippinarum

In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens.

The Effects of Temperature and Salinity Stressors on the Survival, Condition and Valve Closure of the Manila Clam, Venerupis philippinarum in a Holding Facility

We investigated the response of the Manila clam Venerupis philippinarum to possible temperature and salinity changes in a holding facility. First, clams were exposed to four temperatures for 15 days. Valve closure and survival of clams exposed to seawater at 18 °C were higher than that of those exposed to seawater at 24 °C. Second, clams were exposed to six salinities for 15 days. Survival of clams exposed to two salinity fluctuation conditions (24–30 and 27–24 psu) was lower than that of clams exposed to constant 30 psu conditions. Valve closures of clams exposed to constant low salinity conditions (24 psu) and two salinity fluctuation conditions (24–30 and 27–24 psu) were higher than those exposed to constant 30 psu conditions. Lastly, clams were exposed to two different temperatures and three different salinity conditions for 8 days. Valve closure and survival decreased significantly under the combination of 24 °C and 18 psu. These results suggest that an increase in temperature or a wider range of salinity fluctuations are detrimental to the survival of the Manila clam. The synergistic effect of temperature and salinity stressors may decrease the survival period of clams compared to the effect of a single stressor.

Molecular characterization, expression and immune functions of two C-type lectin from Venerupis philippinarum

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro, whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi, Vibrio splendidus and V. anguillarum, while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum.

Two c-type lectins from Venerupis philippinarum: Possible roles in immune recognition and opsonization

In the study, two c-type lectins were identified and characterized from the manila clam Venerupis philippinarum (designed as VpClec-1 and VpClec-2, respectively). Multiple alignments and phylogenetic analysis strongly suggested that they were new members of the c-type lectin superfamily. In normal tissue of clams, both VpClec-1 and VpClec-2 transcripts were highly expressed in the tissue of hepatopancreas. After Vibrio anguillarum challenge, the temporal expression of both VpClec-1 and VpClec-2 transcripts was up-regulated in the hemocytes of manila clams. The recombinant protein VpClec-1 (rVpClec-1) showed obvious binding activities to lipopolysaccharide (LPS), peptidoglycan (PGN), glucan and zymosan in vitro, while the recombinant protein VpClec-2 (rVpClec-2) could only bind LPS, glucan and zymosan. Coinciding with the PAMPs binding assay, both rVpClec-1 and rVpClec-2 displayed broad agglutination and antibacterial activities towards Vibrio harveyi, Vibrio splendidus, Vibrio anguillarum, Enterobacter cloacae and Aeromonas hydrophila. Moreover, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rVpClec-1 and rVpClec-2. Notably, the rVpClec-1 but not rVpClec-2 elicited a chemotactic response from hemocytes. All the results showed that VpClec-1 and VpClec-2 functioned as pattern recognition receptors (PRRs) with distinct recognition spectrum, and involved in the innate immune responses of manila clams.

Vibrio tapetis Displays an Original Type IV Secretion System in Strains Pathogenic for Bivalve Molluscs.

The Brown Ring Disease (BRD) caused high mortality rates since 1986 in the Manila clam Venerupis philippinarum introduced and cultured in Western Europe from the 1970s. The causative agent of BRD is a Gram-Negative bacterium, Vibrio tapetis, which is also pathogenic to fish. Here we report the first assembly of the complete genome of V. tapetis CECT4600T, together with the genome sequences of 16 additional strains isolated across a broad host and geographic range. Our extensive genome dataset allowed us to describe the pathogen pan- and core genomes and to identify putative virulence factors. The V. tapetis core genome consists of 3,352 genes, including multiple potential virulence factors represented by haemolysins, transcriptional regulators, Type I restriction modification system, GGDEF domain proteins, several conjugative plasmids, and a Type IV secretion system. Future research on the coevolutionary arms race between V. tapetis virulence factors and host resistance mechanisms will improve our understanding of how pathogenicity develops in this emerging pathogen.

Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum

Lysozymes play an important role in the innate immune responses with which mollusks respond to bacterial invasion through its lytic activity. In the present study, two c-type lysozymes (designed as VpCLYZ-1 and VpCLYZ-2, respectively) were identified and characterized from the manila clam Venerupis philippinarum. The full-length cDNA of VpCLYZ-1 and VpCLYZ-2 was of 629 and 736 bp, encoding a polypeptide of 156 and153 amino acid residues, respectively. The deduced amino acid sequences of VpCLYZs showed high similarity to other known invertebrate c-type lysozymes. Multiple alignments and phylogenetic relationship strongly suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family. Both VpCLYZ-1 and VpCLYZ-2 transcripts were constitutively expressed in a wide range of tissues with different levels. The VpCLYZ-1 transcript was dominantly expressed in hepatopancreas and hemocytes, while VpCLYZ-2 transcript was mainly expressed in the tissues of hepatopancreas and gills. Both the mRNA expression of VpCLYZ-1 and VpCLYZ-2 was significantly up-regulated at 12 h post Vibrio anguillarum challenge. The recombinant VpCLYZ-1 and VpCLYZ-2 (designed as rVpCLYZ-1 and rVpCLYZ-2) exhibited lytic activity against all tested bacteria, and rVpCLYZ-1 showed higher activities than rVpCLYZ-2 in killing Micrococcus luteus and V. anguillarum. Overall, our results suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of pathogens.

Identification of Relevant Biomarkers in Mercury Exposed Clam Venerupis philippinarum.

This study assessed the impact of four different concentrations (2.5, 5, 7.5, 10 μg/L) of mercury exposure on Manila clam Venerupis philippinarum based on the dynamic characteristics of antioxidant related enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), acid phosphatase (ACP), alkaline phosphatase (ALP), total glutathione content, and malondialdehyde (MDA) content at 24 h and 48 h post exposure. The results showed that hemocyte SOD activity was inhibited while GR hemocyte activity significantly increased in the 2.5 μg/L group after 24 h. Hemocyte ALP activity and (GSH) content were also elevated at the 7.5 μg/L and 10 μg/L groups when exposed for 24 h, respectively. SOD activity, GR activity, ALP activity and GSH content in the hemocytes were relatively sensitive to different concentrations of mercuric mercury (Hg2+), suggesting that they may act as potential biomarkers in assessing environmental mercury exposure on Venerupis philippinarum. At different levels of exposure, antioxidant parameters showed differing responses in gill tissue, but no biomarkers were discovered in the samples examined. We found that hemocytes are more suitable biomarkers than proteins in gill tissue. Some hemocyte biomarkers are promising candidates for monitoring mercury pollution in marine clams.

Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams.

The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Qualitative network models in support of ecosystem approaches to bivalve aquaculture

Abstract Predicting the effects of aquaculture development for coastal ecosystems remains challenging, particularly for data-limited systems, and tools that account for complex ecological interactions are needed to support ecosystem approaches to aquaculture. Here, we used qualitative network models (QNMs) to examine the potential community effects of increasing bivalve aquaculture in South Puget Sound, a large estuarine system in Washington, United States. QNMs are formalized conceptual models that require only a qualitative understanding of how variables composing a system interact (that is, the sign of interactions: +, –, and 0) and are therefore well-suited to data-limited systems. Specifically, we examined community-wide responses to scenarios in which bivalve cultivation effort increased for three different bivalve species (Manila clam Venerupis philippinarum, Pacific oyster Crassostrea gigas, and geoduck Panopea generosa). Further, we evaluated community-wide responses to the removal of benthic bivalve predators, a future increase in nutrient loadings, and combinations of these scenarios acting simultaneously. The scenarios enabled identification of potential trade-offs between increased aquaculture and shifts in the abundance of community members and assessment of the possible effects of different management actions. We also analysed the QNM to identify key interactions that influence the sign outcome of community responses to press perturbations, highlighting potential points for management intervention and linkages deserving of more focused quantitative study. QNMs are mathematically robust and highly flexible, but remain underutilized. We suggest that they may serve as valuable tools for supporting ecosystem approaches to aquaculture.

Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis – Part II: Combined effect of temperature and two V. tapetis strains

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally infected with two different bacterial strains and challenged with two different temperatures. Bacterial strains used in this study were Vibrio tapetis strain CECT4600T, the causative agent of Brown Ring Disease (BRD) and V. tapetis strain LP2, supposed less virulent to V. philippinarum. V. tapetis is considered to proliferate at low temperatures, i.e. under 21 °C. In a global warming context we could hypothesize a decrease of mass mortalities caused by V. tapetis but these thermal changes could also directly impact the immune system of the host V. philippinarum. Thus, the aim of this study was to investigate the effects of the extrapallial injection with V. tapetis combined with temperature challenge on two enzymes activities in V. philippinarum. More precisely, after infection, phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immune response, were studied for 30 days in two compartments: the mantle and the hemolymph. Conchyolin Deposit Stages (CDS) and Shell Repair Stages (SRS) were also determined 30 days post-injection as a proxy of the virulence of the tested strains. In this study, we highlighted that host–pathogen interaction in a varying environment affects the enzymatic response of the host. The coupled effect of V. tapetis injection and temperature challenge was detected 30 days post injection and resulted in virulence differences. These findings were supported by CDS and SRS determination in clams and lead to the conclusion that clam's immunity could be enhanced at 22 °C while V. tapetis virulence is lowered at this temperature. Another result of our study was the increase of PO and SOD basal activities as clams are exposed to warmer temperature.

Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum

The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis – Part I: Spatio-temporal evolution of enzymes' activities post-infection

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally challenged with two Vibrio tapetis strains: CECT4600T, the causative agent of Brown Ring Disease (BRD); and LP2 supposedly non-pathogenic in V. philippinarum. Changes in phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immunity, were studied in two tissues, the mantle and hemolymph for 30 days after infection in the extrapallial cavity. Bacterial infection in V. philippinarum resulted in modulation of PO and SOD activities that was both tissue- and time-dependent. A response at early times was detected in the mantle and was associated with significant increases in PO and SOD activities in LP2- and CECT4600T-challenged clams 36 h post injection. This first response in the mantle could be explained by the proximity to the injection region (extrapallial cavity). In the hemolymph the response occurred at later times and was associated with an increase in PO activity and a decrease in SOD activity. As hemolymph is a circulating fluid, this response delay could be due to an “integration time” needed by the organism to counteract the infection. Injections also impacted PO and SOD activities in both tissues and confirmed a difference in pathogenicity between the two V. tapetis strains.

Induction of brown cells in Venerupis philippinarum exposed to benzo(a)pyrene

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) commonly present in the marine environment and responsible for carcinogenic, teratogenic and mutagenic effects in various animal species. In the present study, we investigated by both histochemical and immunohistochemical approaches the effect of an acute exposure to different concentrations of B(a)P in the Manila clam Venerupis philippinarum. The general morphology of the different clam tissues, which was investigated histologically, evidenced a significant increase in the number of intestinal brown cells after B(a)P exposure. An increasing trend response to B(a)P was detected. The histochemical analysis for lipofuscin revealed the presence of lipofuscin-like substances inside the cytoplasm of intestinal brown cells. The same cells exhibited a PAS positivity and a reactivity to Schmorl's solution for melanin pigment. Moreover, intestinal brown cells exhibited an immunopositivity to HSP70 antibody confirming the increasing trend response to B(a)P detected by the histochemical analysis. Our results suggest that histological tissue changes resulting from exposure to B(a)P can be an useful marker in biomonitoring studies.

Brown muscle disease: Impact on Manila clam Venerupis (=Ruditapes) philippinarum biology

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared.Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD.In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production.Most of the studied parameters underlined a cause–effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression.

Manila clam Venerupis philippinarum as a biomonitor to metal pollution

The Manila clam Venerupis philippinarum is a good biomonitor/bioindicator to marine metal pollution and is frequently used in aquatic toxicology. Two dominant pedigrees (white and zebra) of clam are distributed in the Bohai Sea; however, little attention has been paid to potential biological differences between these two pedigrees. In this study, we tested the sensitivity of both pedigrees to marine metal (cadmium and zinc) pollution biomonitoring and marine environmental toxicology. Results demonstrate significant biological differences in gills of white and zebra clams based on metabolic profiles and antioxidant enzyme activities. In addition, we found that hypotaurine, malonate and homarine were relatively high in white clam gills, while alanine, arginine, glutamate, succinate, 4-aminobutyrate, taurine and betaine were high in zebra clam gills. Zebra clam gills were also more sensitive to a mixture of Cd and Zn, as shown by antioxidant enzyme activities and metabolic profiles, but white clam gills could accumulate more Zn. Therefore, we suggest that the white pedigree can be used as a biomonitor to marine Zn pollution, whereas the zebra pedigree can be used for toxicology studies on Cd and Zn mixed pollution.

Molecular cloning and expression analysis of a selenium-independent glutathione peroxidase identified from Manila clam Venerupis philippinarum

Glutathione peroxidase (GPx) is a key enzyme of cellular detoxification systems that defend cells against reactive oxygen species. It can be divided into selenium-dependent GPx (Se-GPx) and selenium-independent GPx (non-Se-GPx) subfamilies. In this study, the full-length cDNA of a non-Se-GPx was identified from Venerupis philippinarum (denoted as VpGPx) using EST analysis and RACE approaches. The full-length cDNA of VpGPx encoded a polypeptide of 226 amino acids with the predicted molecular mass of 25.41 similar to kDa. Phylogenetic analysis indicated that VpGPx had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the GPx protein family. Spatial expression analysis found that VpGPx transcript was most abundantly expressed in hepatopancreas, gills and haemocytes, and weakly expressed in the tissues of mantle. After Vibrio anguillarum challenge, the expression of VpGPx transcript in overall haemocyte population was down-regulated in the first 6 similar to h, and increased to the peak at 48 similar to h. As time progressed, the expression level dropped to 0.5-fold of the control level at 96 similar to h. All these results indicated that VpGPx was perhaps involved in the immune response against microbe infection and might be contributed to the clearance of bacterial pathogens.

Cloning and characterization of a sialic acid binding lectins (SABL) from Manila clam Venerupis philippinarum

Sialic acid binding lectin (SABL) is a member of immunoglobulin-like lectins family that are thought to promote cell–cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. In the present study, the full-length cDNA of SABL was identified from Manila clam Venerupis philippinarum (denoted as VpSABL) by cDNA library and RACE approaches. The cDNA of VpSABL consisted of a 5′terminal untranslated region (UTR) of 62 bp, a 3′ UTR of 354 bp with a poly (A) tail, and an open reading frame (ORF) of 588 bp encoding a polypeptide of 195 amino acids with a typical C1q domain in the C-terminus. Multiple alignment analysis indicated that the deduced amino acid of VpSABL shared higher positive to other SABLs and C1q-contained proteins and should be adopted typical 10 β-strand jelly-roll folding topology common to all C1q-TNF family. Spatial expression analysis indicated that mRNA transcript of VpSABL was predominantly detectable in tissues of mantle, hepatopancreas and gill, and to a lesser degree in the tissues of muscle and haemocytes. After challenged by Vibrio anguillarum, the mRNA level of VpSABL in overall haemocytes population was recorded by quantitative real-time RT-PCR. VpSABL mRNA was down-regulated in the first 24 h post-infection. Then, the expression level increased to the peak at 72 h and recovered to the original level at 96 h. All these results indicated that VpSABL was involved in the immune response against microbe infection and might be contributed to the recognition of bacterial pathogens.

Mineral phase in shell repair of Manila clam Venerupis philippinarum affected by brown ring disease

The mineral phase of shell repair in the Manila clam Venerupis philippinarum affected by brown ring disease (BRD) was characterised at various scales and at various stages of shell repair by confocal Raman microspectrometry and scanning electron microscopy. Spherulitic and quadrangular aragonite microstructures associated with polyene pigments were clearly observed. Von Kossa staining showed that at the beginning of shell repair, hemocytes are filled with insoluble calcium carbonate salts in all fluids and then are transported toward the extrapallial fluids and the repair sites. Our analyses suggest that after a Vibrio tapetis attack and BRD deposit some clams rapidly cover the deposit, resulting in a modification in the microstructure, which could be produced by the participation of both the mantle and hemocytes.