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.     

Quantitative assessment of reproductive effort of the Manila clam Ruditapes philippinarum in a lagoon on Jeju Island (Korea) using enzyme-linked immunosorbent assay

We investigated gonad development and reproductive effort (RE) of the Manila clam Ruditapes philippinarum at Jeju Island, Korea. Gonad maturation and RE were determined using histology and an indirect enzyme-linked immunosorbent assay (ELISA). In June 2006, most of the clams (80%) in the lagoon were in the resting stage. Spawning clams first appeared in late July, and most clams spawned from early August to mid-September. The condition index increased gradually from early July to late August, then declined from early to mid-September, suggesting that spawning occurred during this period. The gonadosomatic index assessed by ELISA also increased dramatically from June (0.9), peaked in early August (19.7) then declined from late August to mid-September, indicating that clams at the study site had only one spawning pulse during the spawning period. Spawning at Jeju Island was one month later than Manila clams on the west coast of Korea. The delayed spawning and low RE of the clams could be in part, be explained by lower food availability, as the level of chlorophyll-a recorded in this study was much lower than that found in water from the west and south coast.     

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.     

Isolation and characterization of microsatellite markers for the clam Ruditapes philippinarum and cross-species amplification with the clam Ruditapes variegate

Ruditapes philippinarum and R. variegate are commercially important shellfish in Korea. In order to understand the processes and organization of genetic diversity and genetic resources for the sustainable management of fisheries resources we developed 13 primer pairs for microsatellite loci in R. philippinarum and tested their cross-amplification in R. variegate. Twelve primers amplified in both species. Nine loci were polymorphic in R. philippinarum and eight in R. variegate, each with nine to 26 alleles per locus. Unbiased expected heterozygosity levels varied from 0.73 to 0.94 in R. philippinarum. All polymorphic loci possessed species-specific alleles. No linkage disequilibrium was found. Results indicated that these microsatellite were highly polymorphic and will be useful for conservation genetics of both species.     

In vitro propagation of two Perkinsus spp. parasites from Japanese Manila clams Venerupis philippinarum and description of Perkinsus honshuensis n. sp

Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, and Spain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April 2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate- and high-intensity infections. Perkinsus sp. cells in tissue samples were enlarged in alternative Ray's fluid thioglycollate medium, before propagation in DME:Ham's F-12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, which gave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolates comprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morphotype, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes, were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of all described Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents a new species, Perkinsus honshuensis n. sp.     

菲律宾蛤仔基因组DNA的提取及其RAPD分析

对菲律宾蛤仔(Ruditapes philippinarum)的三种组织,即性腺、斧足和内脏团的基因组DNA提取方法进行了研究,并对所提取的DNA作RAPD分析。结果表明性腺的DNA的得率明显比斧足和内脏团高。并且探讨了用于对基因组DNA进行RAPD研究的4种引物及最佳PCR条件,得到了较好的RAPD结果。     

庄河海域菲律宾蛤仔底播增殖区自身污染

采用生物沉积物捕集器和封闭式代谢瓶,周年现场研究了庄河海域菲律宾蛤仔的生物沉积速率、排氨率和排磷率.结果表明:菲律宾蛤仔的生物沉积速率、排氨率和排磷率均具有明显的季节变化.生物沉积速率为0.15～1.47 g·ind-1·d-1(年均0.61 g·ind-1·d-1);其排氨率及排磷率分别为0.02～0.40 mg·ind-1·d-1(年均0.17 mg·ind-1·d-1)和0.01～0.39 mg·ind-1·d-1(年均0.13 mg·ind-1·d-1).根据以上结果,估算庄河海域底播增殖菲律宾蛤仔每年产生的生物沉积物达到5.46×107t(干质量),折合有机物9.07×106t、有机碳1.00x106t和有机氮1.18xlO5t;而氨氮和磷酸盐分别为1.49x104t和1.15x104t.表明浅海高密度、规模化菲律宾蛤仔增养殖区自身污染严重,其对环境的影响不可忽视.     

The susceptibility of Irish-grown and Galician-grown Manila clams, Ruditapes philippinarum, to Vibrio tapetis and Brown Ring Disease

Brown Ring Disease (BRD), which affects the Manila clam in Europe, is caused by the bacterium, Vibrio tapetis. BRD has been diagnosed in Ireland on only one occasion (1997) although the aetiological agent has recently been detected in apparently healthy Manila clams from a number of sites around the Irish coast. The present work investigated the susceptibilities to BRD of two stocks of Manila clams, one from Ireland and the second from Galicia, north-western Spain, where BRD has been reported on a number of occasions. Exposure of the clams was by addition of V. tapetis to the holding waters. Development of BRD was assessed by the appearance of brown ring signs on the host shells, by bacterial isolation and characterization, and by detection of the bacterium by PCR. The pathogen was recovered from infected individuals and confirmed as V. tapetis by biochemical tests and a slide agglutination test. Galician clams experienced significantly higher mortalities, BRD prevalences and V. tapetis levels than Irish clams. Background infection with V. tapetis in the control stocks prevented conclusions being drawn on comparative susceptibility of the two stocks. Irish clams were significantly affected by the experimental challenge, as demonstrated by the development of BRD and an increase in V. tapetis levels. Results illustrate the vulnerability of Irish clams to BRD and have implications for the movement and transfer of clam seed in Ireland.     

Effects of shell morphological traits on the weight trait of the orange strain of the Manila clam

A new strain of Manila clam with orange shell color was produced after selection within a full-sib family for two generations. In the present study, the shell length, height, and width, and the live body weight of the orange strain were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and the live body weight was used as the 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 were all highly significant (P < 0.01). The correlation indices (R²) of morphological traits against the live body weight of clams were larger than 0.85, indicating that the morphology traits were the main factors affecting the body weight. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), and shell width X₃ (cm) against live body weight Y (g): Y = ? 2.62 + 0.34 X₁ + 0.145 X₂, (X₁ < 0.05, X₂ < 0.05). The results suggest that the shell length could be used as the main trait for selective breeding and could indirectly make a large improvement in the weight trait.     

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.     

Experimental uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in two species of clams: Ruditapes decussatus and Ruditapes philippinarum

Aims: To describe uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in Ruditapes decussatus and Ruditapes philippinarum. Methods and Results: Ruditapes decussatus accumulated greater concentrations of pathogenic and nonpathogenic V. parahaemolyticus than R. philippinarum. These concentrations decreased earlier in R. decussatus. Nonpathogenic V. parahaemolyticus reached higher concentrations (approx. 1 log CFU g^?1 in tissues of R. decussatus and more than 1 log CFU g^?1 in R. philippinarum) than pathogenic V. parahaemolyticus at similar times. It also persisted longer in both species of clams (72 h in R. decussatus and 96 h in R. philippinarum), while pathogenic V. parahaemolyticus persisted 48 h in R. decussatus and 72 h in R. philippinarum. Conclusions: Ruditapes decussatus incorporated both isolates of V. parahaemolyticus faster than R. philippinarum and it eliminated both isolates earlier than R. philippinarum. Under same conditions, nonpathogenic V. parahaemolyticus might survive better than pathogenic V. parahaemolyticus within both species of clam. Significance and Impact of the Study: Pathogenic V. parahaemolyticus is an important cause of foodborne illnesses. This study shows it may be possible to use nonpathogenic V. parahaemolyticus to perform experimentation to evaluate bacterial evolution in clams, developing an in vivo model useful for risk analysis.     

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.     

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is infected with 2 Perkinsus species, Perkinsus olseni and P. honshuensis, in Japan. The latter was described as a new species in Mie Prefecture, Japan, in 2006. Ray's Fluid Thioglycollate Medium (RFTM) assay has been most commonly used to quantify Perkinsus infection. However, this assay cannot discriminate between species that resemble one another morphologically. We developed real-time PCR assays for the specific quantification of P. olseni and P. honshuensis. DNA was extracted using Chelex resin. Cultured P. olseni and P. honshuensis cells were counted and spiked into uninfected clam gill tissue prior to DNA extraction to generate standard curves, which allowed quantification based on the PCR cycle threshold values. We compared the RFTM assay with both real-time PCR assays by quantifying Perkinsus spp. in gill tissue samples from the same individual clams obtained from various localities in Japan. Infection intensities estimated by both assays were significantly correlated (r2 = 0.70). Our results suggest that the prevalence and infection intensity of P. honshuensis are much lower than for P. olseni in Manila clams.