Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis

In a recent study, we demonstrated the presence of defense factors, competent hemocytes and high enzymatic activities (peptidases, hydrolases, lytic, etc.), in the extrapallial fluid, located between the mantle and the shell, of the Manila clam, Ruditapes philippinarum. In Europe, this species is affected by brown ring disease, an epizootic disease caused by the bacterium Vibrio tapetis. The present work focused on the effect of the development of the disease on cellular and humoral defense parameters in the hemolymph and the extrapallial fluid of experimentally infected clams. Results indicate significant changes in total and dead hemocyte counts, as well as modifications in lysozyme activity and protein content, in the hemolymph and extrapallial fluid of challenged animals. Hemocyte counts and lysozyme activity increased significantly in the hemolymph, but particularly in the extrapallial fluid, where the highest values were observed. A healing (recalcification) process was observed 7 weeks following challenge, suggesting defense system efficiency at neutralizing the pathogen. These results are discussed with emphasis on the role of extrapallial fluids in the defense process against invading microorganisms.     

Virus-like particles associated with mortalities of the Manila clam Ruditapes philippinarum in England

Mortalities of the Manila clam Ruditapes philippinarum (Adams & Reeve, 1850) were reported in southern England (Kent and Poole Harbour) during late spring of 2008. In response to these reported mortalities, samples were collected from 5 sites across the south coast of England. Clams were sampled for both histology and electron microscopy. Transmission electron microscopy (TEM) revealed unenveloped virus-like particles within the connective tissue of the gills and surrounding the tubules in the digestive gland. The virus-like particles appeared to be free within the cytoplasm or associated with endoplasmic reticulum membranes and cytoplasmic vesicles. Particles were icosahedral in shape, with a diameter of 25 to 30 nm. The location, size and morphology of the virus-like particles suggest that they belong to the Picornaviridae family. This is the first report of this virus infection in wild and farmed R. philippinarum within the UK.     

Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacterium Vibrio tapetis

The Manila clam, Ruditapes philippinarum can become infected by the bacterium Vibrio tapetis which causing the Brown Ring Disease along North European Atlantic coasts. Variations in clam immune parameters have been reported in clam challenged with V. tapetis but no studies have been done on Nitric Oxide (NO) production. NO is a toxic agent to pathogens produced mostly by immune cells such as hemocytes in invertebrates. In this study, we demonstrated that NO production in hemolymph and extrapallial fluid of clams is dose dependent and increases with incubation time with V. tapetis. Moreover, the augmentation of NO production seems to be directly correlated to cell rounding and to the loss of pseudopods-forming capacity of hemocytes during the infection process.     

Molecular cloning and characterization of SOCS-2 from Manila clam Ruditapes philippinarum

Suppressor of cytokine signaling (SOCS) family members are key regulators of immunological homeostasis. In this study, we have discovered the SOCS-2 member from Manila clam Ruditapes philippinarum and further analyzed its immune responses against lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). Amino acid sequence of RpSOCS-2 consists of cytokine inducible SRC homology 2 (SH2) and SOCS box domains similar to vertebrate SOCS counterparts. It has the highest amino acid identity (41%) with Pacific oyster (Crassostrea gigas) SOCS-2 and showed close evolutional relationship with disk abalone (Haliotis discus discus) SOCS-2. Tissue specific expression results showed that RpSOCS-2 was constitutively expressed in all examined tissues with the highest level in gill tissue of un-challenged clams. RpSOCS-2 mRNA expression was up-regulated by LPS and poly I:C challenge in gills. Discovery of RpSOCS-2 homologue and expression analysis would support for understanding evolutional relationships and their role in innate immune responses in mollusks, respectively.     

Effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum

The effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum at the temperature of 19.6–21.6 °C, the salinity of 34‰ and pH of 8.0 were investigated from May 18 to July 18, 2006. In this study, the early, middle and late umbo-veliger larvae with the shell lengths of 100, 140, and 190 μm were subject to temporary food deprivation for up to 4.5, 20, and 25d at 0.5, 4, 5d intervals, followed by refeeding for the remaining of a 24, 20, 25d period, respectively. The results suggested that the larvae should have shown considerable tolerance to starvation due to their endogenous and exterior nutrition material, for larvae and time to the point-of-no-return (PNR: the threshold point during starvation after which larvae could no longer metamorphose even if food is provided) were calculated to be 4.25, 17.54, and 22.17d. As the starvation period prolonged, the mean shell length of larvae starved got close to constants at 1.5, 4, and 15d after starvation, which were different for larvae at different stages when starvation began, survival of larvae decreased, and was lower in treatments starved earlier in development than those starved later, for the early, middle and late umbo-veliger larvae, after 4.5, 20 and 25d of starvation period, few larvaes were alive. After starvation period, the alive larvaes were able to metamorphose and had a capability of compensatory growth when refeeding was given. Starvation not only affected metamorphosis rate, but also caused the delay in the time to metamorphosis and the decrease in the metamorphosed sizes. For example, for the continuously-fed larvae, duration to metamorphosis was 20.7d, for larvae with a size of 100-μm starved for up to 4d, larvae with a size of 140-μm starved for up to 16d, larvae with a size of 190-μm starved for up to 20d, duration to metamorphosis were 29.7, 31.7, and 37.7d, the delay in duration to metamorphosis were 9, 11, and 17d, respectively. Furthermore, importance of nutrition material for maintaining larval survival during starvation and the compensatory growth on larvae at the same feeding time were discussed.     

Doubly Uniparental Inheritance and beyond: The contribution of the Manila clam Ruditapes philippinarum

The Manila clam, Ruditapes philippinarum (Adams & Reeve, 1850), is a widespread and commercially important bivalve species showing a peculiar way of mitochondrial inheritance known as Doubly Uniparental Inheritance (DUI), which is different from the strict maternal inheritance found in the broad majority of metazoans. DUI in R. philippinarum was discovered later than in mytilids and unionids. Nevertheless, this case keeps providing interesting data pertinent to the mechanism of this inheritance system. In this review, we discuss the contribution of this species, both in the context of the available knowledge on DUI and in the broader context of metazoan mitochondrial biology. Indeed, thanks to its unusual features, DUI can shed light on mitochondrial inheritance and biogenesis and, above all, on the relationship between mitochondria and germ line. Moreover, DUI is a unique experimental system for studying mitochondrial heteroplasmy, and two processes that shape genome evolution: genomic conflicts and mito-nuclear coevolution, which are at the very root of eukaryotic life.     

Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea

The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study. A method was developed for the isolation of the Manila clam lectin (MCL). Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation. SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits. The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography. The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM. MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation. The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc. In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins. MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria. It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans. Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.     

Genetic diversity of introduced Manila clam Ruditapes philippinarum populations inferred by 16S rDNA

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.     

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.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl+alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl+alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.     

大蒜对菲律宾蛤仔早期生长发育的影响

实验了不同大蒜汁浓度(0、2、4、8、16、32 mg/L)对菲律宾蛤仔受精卵孵化率,幼虫生长、存活、变态及稚贝生长与存活的影响,总结了室内大规模人工育苗过程中大蒜防病效果。结果表明:随着大蒜汁浓度的增加,孵化率降低;大蒜汁浓度达到16mg/L,胚胎发育延迟;达到32mg/L,受精卵不能孵化为正常幼虫。浮游期间,幼虫的生长受大蒜汁抑制,幼虫的存活率则随着大蒜汁浓度增加先升高后降低;幼虫的变态率随着大蒜汁浓度的增加先升高后降低,以16mg/L为最适浓度;变态规格随着大蒜汁浓度增加而减小。室内培育期间,稚贝生长与存活随着大蒜汁浓度的增加先升高后降低,以8mg/L为最适浓度;室内大规模人工育苗过程中,使用浓度为8-10mg/L大蒜汁可以起到较好的防病效果。     

Spatio-temporal patterns of perkinsosis in the Manila clam Ruditapes philippinarum from Arcachon Bay (SW France)

Pathogens belonging to the genus Perkinsus infect many bivalve molluscan species around the world, including the Manila clam Ruditapes philippinarum. We investigated the spatial distribution of this parasite at 34 stations throughout Arcachon Bay (SW France). Prevalence of perkinsosis was 93% and mean infection abundance was 96 x 10(3) cells g(-1) wet gill. Lowest mean abundances were found close to the Leyre River mouth and a significant negative correlation was observed between mean abundance and salinity. Perkinsosis was rare at the oceanic site where salinities and other environmental parameters were stable. A second aim of this study was to survey perkinsosis during annual cycles at 4 sites within Arcachon Bay. Prevalence and intensities (+/- SE) of the disease were high, on average between 70 and 100%, and 130 x 10(3) +/- 6.7 x 10(3) cells g(-1) wet gill. No seasonal cycle was evident. Clams were infected at 9 mm shell length and infection increased with clam size. The third objective was to determine the disinfection and infection kinetics through a 21 mo reciprocal transplantation between a nearly Perkinsus sp.-free area and a highly affected site. Disinfection appeared to be a very slow process and was similar at the site with favorable conditions for Perkinsus sp. as at the site with unfavorable conditions. Conversely, infection acquisition appeared to be episodic with spatially defined areas. Consequently, the overall lack of a clear seasonal infection pattern is interpreted as the combination of episodic infection events and slow disinfection kinetics.     

Lectin from the Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni

Glycan-binding proteins (lectins) are widely expressed in many invertebrates, although the biosynthesis and functions of the lectins are not well understood. Here we report that Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) upon infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a approximately 74-kDa precursor and secreted into hemolymph where it is converted to 30- and 34-kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by one or more factors in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite, and this binding is inhibitable by either EDTA or GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL is concentrated within cyst-like structures. To define the glycan binding specificity of MCL we examined its binding to an array of biotinylated glycans. MCL recognizes terminal non-reducing beta-linked GalNAc as expressed within the LacdiNAc motif GalNAcbeta1-4GlcNAcbeta1-R and glycans with terminal, non-reducing beta-linked Gal residues. Our results show that the synthesis of MCL is specifically up-regulated upon parasite infection of the clams and may serve as an opsonin through recognition of terminal GalNAc/Gal residues on the parasites.     

Effect of temperature on defense parameters in manila clam Ruditapes philippinarum challenged with Vibrio tapetis

Brown Ring Disease (BRD), a vibriosis affecting the clam Ruditapes philippinarum, is present on the Atlantic coasts of Western Europe and is considered to be a cold water disease. The present work investigated the effect of temperature on immune response and its relationships with BRD development. Clams maintained at different temperatures (8, 14 and 21 degrees C) were experimentally challenged with the pathogen Vibrio tapetis, the etiologic agent of BRD. Results demonstrated significant effects of temperature on disease development and on hemolymph immune parameters including total and viable hemocyte counts, lysozyme and leucine aminopeptidase activities. Thirty days after challenge, clams maintained at 21 degrees C displayed significantly higher values for all the measured immune parameters in comparison to specimens incubated at 14 degrees C. Improved performance of the immune system was associated with a low BRD prevalence. The recovery process, which occured mainly at 21 degrees C, was associated with high percentages of viable hemocytes and high activities of leucine amino-peptidase and lysozyme. This laboratory study clearly demonstrates that temperature strongly affects BRD development and clam immune response during infection. Favourable immune status at higher temperature may confer upon the clam a better capacity to fight the disease agent, and therefore to recover more easily.