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.     

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.     

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.     

Transcriptomic analysis provides insights into candidate genes and molecular pathways involved in growth of Manila clam Ruditapes philippinarum

Functional & Integrative Genomics - Growth is one of the most important traits of aquaculture breeding programs. Understanding the mechanisms underlying growth differences between individuals...     

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).     

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。     

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.     

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.     

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.     

Depuration processes affect the Vibrio community in the microbiota of the Manila clam,Ruditapes philippinarum

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.     

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.     

iTRAQ-based proteomic analysis reveals proteins associated with cold stress response in two different populations of Manila clam (Ruditapes philippinarum)

Water temperature is one of the key environmental factors for marine ectotherms and a change in temperature beyond and organism's capacity limits can cause a series of changes to physiological state and damage to the organism. Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. Ruditapes philippinarum is an ecologically and scientifically important marine bivalve species. To uncover the molecular mechanisms of acclimation of R. philippinarum to low-temperature stress, iTRAQ-based quantitative proteomics was conducted to compare the proteomes of the north and south populations of R. philippinarum under low-temperature stress. The results showed a total of 6355 and 6352 proteins were identified in two populations, respectively. Among these, 94 and 83 were differentially abundant proteins (DAPs), and most of DAPs were related to oxidation-process, protein binding, or an integral component of membrane. According to the results of KEGG pathway enrichment analysis, most of DAPs in both populations are involved in immune-related pathways, while other population-specific significant abundance proteins of south population and north population were enriched in biosynthesis of amino acids (Enolase, Glutamine synthetase) and unsaturated fatty acids pathways (3-ketoacyl-CoA thiolase, Stearoyl-CoA desaturase), respectively, indicating that two population of clams may have different cold-stress regulation mechanisms. Our study provides new insights into different cold stress tolerance mechanisms in northern and southern populations of R. philippinarum using iTRAQ-based proteomics. This work contributes to a better understanding of molecular basis on cold stress response and adaptations, which shed lights on evolutionary biology and general ecophysiology of R. philippinarum.     

Effects of starvation on growth, survival, and body biochemical composition among different sizes of Manila clam Ruditapes philippinarum

A study was conducted to investigate the impact of starvation on different sizes of Manila clam Ruditapes philippinarum (0.66 ± 0.11, 2.12 ± 0.38, and 11.65 ± 0.84 mm in shell length, respectively) in the summer of 2008. Different size clams were starved for 7, 15, 30, 45, and 60 d, respectively, and followed by refeeding for 30 d. During the study, the water temperature ranged 26.2–28.4 °C, salinity 22–24‰, and pH 7.80–8.12. Compensatory growth occurred in the smallest size-group after 7 and 15 d of starvation, respectively. The point-of-no-return (PNR₅₀) was determined to be 18.7 d. However, no compensatory growth was noted in the medium size-group, and the PNR₅₀ for this group was 25.2 d. The complete compensatory growth was observed for the largest size-group following food depravation for 7 and 30 d, respectively. In the same group, over-compensatory growth occurred 15 d post-starvation. The PNR₅₀ for the largest size-group was 46.3 d. The survival rate of different groups decreased as the starvation time prolonged. To discuss the change in body biochemical composition of individuals in the process of starvation and refeeding, the biochemical composition of the largest group individuals at different stages was determined. There were no significant differences in moisture and ash concentrations of the largest size-group during starvation and refeeding (P > 0.05). The relative body protein content increased as the starvation period prolonged and the level returned to normal after refeeding. The lipid content of the clam at the end of starvation was significantly lower than the initial level (P < 0.05), and remained below the initial level at end of the refeeding period.     

Genetic diversity and structure of Manila clam (Ruditapes philippinarum) populations from Liaodong peninsula revealed by SSR markers

Manila clam (Ruditapes philippinarum) is an important commercially marine bivalve, and its wild populations have been severely declining in the coast of China during the last decade. In this study, a set of 7 genomic simple sequence repeat (SSR), and 5 expressed sequences tag (EST)-derived SSR markers were analyzed on eight wild R. philippinarum populations to assess the genetic diversity and population differentiation. A total of 114 alleles were detected on 12 loci, and the number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 2.00 to 10.88 for each sample. The average of observed and expected heterozygosity ranged from 0.386 to 0.550, and from 0.533 to 0.707, respectively. Pairwise F_ST values indicated that all population pairs had significant genetic differentiation (overall F_ST = 0.242, P < 0.01). Cluster analysis using unweighted pair group method with the arithmetic mean (UPGMA) separated the eight populations into two groups. This study will shed light on the domestication and cultivation on population genetic diversity of R. philippinarum, and also provide the foundation for conservation of R. philippinarum germplasm resources in clam breeding practices.