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This study analyzed the function of different glutathione S-transferase (GST) isoforms and detoxification metabolism responses in Manila clam, Ruditapes philippinarum, exposed to 4 kinds of polycyclic aromatic hydrocarbons (PAHs) single, and their mixtures for 15 days under laboratory conditions. 13 kinds of GSTs in R. philippinarum were classified, and the results of tissue distribution indicated that 12 kinds of GSTs (except GST sigma 3) expressed most in digestive glands. We detected the mRNA expression levels of aryl hydrocarbon receptor signaling pathway, and detoxification system in digestive glands of clams exposed to benzo[a]pyrene (BaP), chrysene (CHR), benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), and BaP + CHR + BaA + BbF, respectively. Among these genes, we selected GST-sigma, GST-omega and GST-pi as potential indicators to BaP; GST-sigma, GST-A and GST-rho to CHR; GST-pi, GST-sigma, GST-A, GST-rho and GST-microsomal to BaA; GST-theta and GST-mu to BbF; while GST-pi and GST-mu to the mixture of BaP, CHR, BaA and BbF. Additionally, the bioaccumulation of PAHs in tissues increased remarkably over time, and showed an obvious dose-effect. Under the same concentration, the bioaccumulation in single exposure group was higher than that in mixture group, and the bioaccumulation of PAHs in tissues with different concentrations of stress was irregular. The results revealed the metabolic differences and bioaccumulation rules in clams exposed to four kinds of PAHs, and provided more valuable information for the PAHs risk assessment.

Polycyclic aromatic hydrocarbons (PAHs), as persistent toxic substances (PTS), have been widely monitored in coastal environment, including seawater and sediment. However, scientific monitoring methods, like ecological risk assessment and integrated biomarker response, still need massive researches to verify their availabilities. This study was performed in March, May, August and October of 2018 at eight sites, Yellow River estuary (S1), Guangli Port (S2), Xiaying (S3), Laizhou (S4), Inner Bay (S5), Outer Bay (S6), Hongdao (S7) and Hongshiya (S8) of Shandong Peninsula, China. The contents of 16 priority PAHs in local seawater and sediment were determined, by which ecological risk assessment risk quotient (RQ) for seawater and sediment quality guidelines (SQGs) were calculated to characterize the PAHs pollution. Meanwhile, multiple biomarkers in the digestive gland of clam Ruditapes philippinarum were measured to represent different biological endpoints, including ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), sulfotransferase (SULT), superoxide dismutase (SOD) and lipid peroxidation (LPO), by which integrated biomarker response (IBR) was calculated to provide a comprehensive assessment of environmental quality. Taken together, these results revealed the heaviest pollution at S2 as both PAHs concentrations and biomarkers responses reflected, and supported the integrated biomarker response as a useful tool for marine environmental monitoring, through its integration with SQGs.

The shell color of the Manila clam (Ruditapes philippinarum) is an economically important trait. We used high-throughput sequencing and transcriptome analysis to study the molecular mechanisms that underlie shell color formation and regulation in this species. We constructed small RNA libraries from mantle tissues from four shell color strains of Manila clam, subjected them to high-throughput sequencing. Notably, the results suggested that a number of pigment-associated genes including Mitf, HERC2, were negatively regulated by nvi-miR-2a, tgu-miR-133-3p, respectively. They might be involved in melanin formation via the activation of the melanogenesis pathway. And aae-miR-71-5p and dme-miR-7-5p linked to shell formation-related genes such as Calmodulin and IMSP3 were considered to participate in the calcium signaling pathway. We then used quantitative PCR to verify the candidate miRNAs and target genes in different shell color groups. Our results indicated that miR-7, miR-71, and miR-133 may regulate target mRNAs to participate in shell color pigmentation. These results provide the foundation to further characterize miRNA effects on the regulation of shell color and have significant implications for the breeding of new varieties of clams.

An investigative biomonitoring study was conducted along the coastal area of Laizhou Bay (China) to evaluate the impact of organic pollution on the clam Ruditapes philippinarum using bioaccumulation and multi-biomarker measurements. In addition, the polychlorinated biphenyls (PCBs), total petroleum hydrocarbons (TPHs) and nonylphenol (NP) content in surface sediment at the study sites were also analyzed. Concentrations of PCBs, TPHs and NP in the sediments of the study area were 1.90 ± 0.10 μg kg−1, 39.55 ± 2.42 mg kg−1, 9.23 ± 0.41 μg kg−1 dry weight, respectively, while the organic contaminants in the soft tissues of R. philippinarum were 14.81 ± 0.96 μg kg−1 for PCBs, 165.87 ± 5.03 mg kg−1 for TPHs and 86.16 ± 5.29 μg kg−1 for NP. Linear regression analysis on the levels of organic pollutants accumulated in R. philippinarum and in sediments showed no significant correlation. Multi-biomarkers including superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, total glutathione and lipid peroxidation were assayed in gills and digestive glands of R. philippinarum. Finally, the biomarkers in gills were selected to calculate the Integrated Biomarker Response (IBR) index and to evaluate the impact of the three organic contaminants on R. philippinarum collected from different sites. According to IBR results, the western coast and eastern coast exhibited higher environmental stress than the sampling sites along the southern coast of Laizhou Bay. Significant correlation was found between the level of organic contaminants in the sediments and IBR whereas no dependence was found between pollutants’ concentrations in sediments and separate biomarker responses. The results showed that PCBs and NP were the main organic pollutants among the three studied which have caused pollution pressure on R. philippinarum in Laizhou Bay coastal area.

The Manila Clam is an important economic shellfish in China’s seafood industry. In order to improve the design of juvenile Manila Clam seeding equipment, a juvenile clam discrete element method (DEM) particle shape was established, which is based on 3D scanning and EDEM software. The DEM contact parameters of clam-stainless steel, and clam-acrylic were calibrated by combining direct measurements and test simulations (slope sliding and dropping). Then, clam DEM simulation and realistic seeding tests were carried out on a seeding wheel at different rotational speeds. The accuracy of the calibrated clam DEM model was evaluated in a clam seeding verification test by comparing the average error of the variation coefficient between the realistic and simulated seeding tests. The results showed that: (a) the static friction coefficients of clam-acrylic and clam-stainless steel were 0.31 and 0.23, respectively, (b) the restitution coefficients of clam-clam, clam-acrylic, and clam-stainless steel were 0.32, 0.48, and 0.32, respectively. Furthermore, the results of the static repose angle from response surface tests showed that when the contact wall was acrylic, the coefficient rolling friction and static friction of clam-clam were 0.17 and 1.12, respectively, and the coefficient rolling friction of clam-acrylic was 0.20. When the contact wall was formed of stainless steel, the coefficient rolling friction and static friction of clam-clam were 0.33 and 1.25, respectively, and the coefficient rolling friction of clam-stainless steel was 0.20. The results of the verification test showed that the average error between the realistic and simulated value was &lt, 5.00%. Following up from these results, the clam DEM model was applied in a clam seeding simulation.

The asari (Manila) clam Ruditapes philippinarum is a commercially important bivalve that lives on tidal flats, but the clam landings have decreased in many estuaries in Japan. To increase the production of the clam, we conducted pilot translocation experiments at small spatial scales within the estuarine system of Matsukawaura Lagoon, Japan. Two scales were used: one was a horizontal translocation from an area with low clam productivity to an area with high productivity, and the other was a vertical translocation from the upper area to the lower area of the tidal flat. Our caging experiment with the horizontal translocation revealed that the growth rate and somatic condition of the clams translocated to favorable conditions were significantly better than those remaining in the original habitat. On a sandy fishing ground, vertical translocation of clams from the upper intertidal zone to the lower intertidal zone resulted in higher growth rates and better somatic conditions for the clams. Moreover, the clams from sandy ground showed larger growth increases than those from muddy-sand ground, indicating that the origin of clams affected translocation effectiveness. We found that clam biomass increased by 42%– 53% with the vertical translocation of small (

A total of 1338 samples were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of lipophilic marine toxins in bivalve mollusks collected from the southeast coast of China from 2017 to 2020. The most abundant toxin was HomoYTX, followed progressively by YTX and PTX2. Low proportions of OA, DTX-1, and DTX-2 were found. No AZA1, AZA2, and AZA3 were quantified above limit of quantitation (LOQ). The highest concentrations of HomoYTX, YTX, PTX2, OA, DTX-1, and DTX-2 were 429, 98.0, 40.3, 33.0, 22.6, and 26.5 μg/kg, respectively. Mussels (Mytilus galloprovincialis, Perna viridis), scallop (Chlamys farreri) and clam (Atrina pectinate) accumulated higher toxin levels than clams (Sinonovaculla Constricta, Ruditapes philippinarum), oyster (Crassostrea gigas) and scallop (Arca granosa). Homo YTX and PTX2 levels reached the maximum in July and June, respectively, and the OA-group peaked in August. The results provide a reliable basis for monitoring marine toxins and protecting the health of aquatic consumers.

Culture-dependent techniques only permit the study of a low percentage of the microbiota diversity in the environment. The introduction of next generation sequencing (NGS) technologies shed light into this hidden microbial world, providing a better knowledge on the general microbiota and, specifically, on the microbial populations of clams. Tissue-associated microbiota of Ruditapes decussatus and Ruditapes philippinarum (mantle, gills, gonad and hepatopancreas) was analysed in two different locations of Galicia (northwest of Spain) during Spring (April) and Autumn (October), employing a metataxonomic approach. High bacterial diversity and richness were found in all samples where a total of 22,044 OTUs were obtained. In most samples, phylum Proteobacteria was most frequently retrieved, although other phyla as Actinobacteria, Bacteroidetes, Tenericutes, Firmicutes or Chlamydiae also appeared at high relative abundances in the samples. At genus level, great variation was found across tissues and sampling periods. A Nonmetric Multidimensional Scaling (NMDS) and a hierarchical clustering analysis allowed to further analyse the factors responsible for the differences among groups of samples in the different sites. Results showed sample ordination based on tissue origin and sampling periods, pointing out that the microbiota was influenced by these factors. Indeed, predominance of certain genera was observed, such as Endozoicomonas or Methylobacterium in gills and gonads, respectively, suggesting that selection of specific bacterial taxa is likely to occur. So far, this study provided a general picture of the tissue associated microbial population structure in R. decussatus and R. philippinarum clams, which, ultimately, allowed the identification of specific tissue-related taxa.

As an emerging pollutant, the presence of microplastics in marine organisms has been concerned increasingly. Shellfish, which are both economically and ecologically important, are of particular concern. In this study, we investigated the microplastic pollution in wild and farmed oysters (Crassostrea gigas) and clams (Ruditapes philippinarum) in the Jiaozhou Bay, China, for the first time. We found the microplastic pollution in shellfish in Jiaozhou Bay was at a moderate level. The abundance of microplastics in shellfish ranged from 0.16 to 12.09 items/g (wet weight, ww) or 1 to 9 items/ind. The average abundance of the ingested microplastics was 1.21 items/g (or 2.17 items/ind.) in all shellfish, 1.51 items/g (or 2.00 items/ind.) in clams and 0.92 items/g (or 2.34 items/ind.) in oysters. The abundance of microplastics in clams was significantly higher than that in oysters. Most microplastics (92.97%) were fibers, followed by fragments. The predominant color of the microplastics was black (42.97%), followed by blue, transparent, and red. Cellophane and polyethylene terephthalate (PET) dominated the microplastic composition. According to shellfish consumption, it can be inferred that the average microplastic consumption through Chinese diet is 1.27×103 items per capita per year.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.

Inflammation is a form of innate immune response of living organisms to harmful stimuli. In marine bivalves, inflammation is a common defense mechanism. Several studies have investigated the morphological features of inflammation in bivalves, such as hemocyte infiltration. However, the molecular and biochemical responses associated with inflammation in marine bivalves remain unexplored. Here, we investigated changes in nitric oxide (NO) levels, cyclooxygenase 2 (COX-2) activity, and allograft inflammatory factor-1 (AIF-1) gene expression levels in hemolymph samples collected from Manila clam (Ruditapes philippinarum) exposed to pro- and anti-inflammatory substances. These included the pro-inflammatory agent lipopolysaccharide (LPS), and the nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac, all widely used in vertebrates. Our study showed that NO levels, COX-2 activity, and AIF-1 expression increased in response to the treatments with LPS and decreased in response to the treatments with NSAIDs in a concentration-dependent manner. These results suggest that the mechanism of inflammatory responses in bivalves is very similar to that of vertebrates, and we propose that inflammatory responses can be quantified using these techniques and used to determine the physiological status of marine bivalves exposed to biotic or abiotic stresses.

The occurrence of some carcinogenic metals; Cu, Ni, Pb and Cd was investigated in sediment samples collected from 12 stations at Great Bitter and Timsah lakes. In the same time, two of the most famous edible bivalve species (Ruditapes decussatus and Paratapes undulatus) live buried in these sediments were chosen for measuring the same metals within their edible tissues in order to investigate the permissibility limits for human consumption. The bio-available forms of these metals were measured in the bulk sediments and the finest fractions (O3, O4 and O5) and in the edible tissues (flesh) of the selected bivalve individuals using a flame atomic absorption spectrophotometer (AAS). The finest fraction (O5) recorded the highest averages of most carcinogenic metals at Great Bitter and Timsah lakes with significant occurrences of Cd at both lakes. Carcinogenic Cd in the edible tissues of P. undulates and R. decussatus showed neglected low contents at the Great Bitter and Timsah lakes in spite of its very high contents in the surrounding sediment layer. Ni, Cu, and Pb averages in the edible tissues of P. undulates at the Great Bitter lake were 1.32, 0.23 and 0.37 µg/g wet wt. respectively. The averages of Ni, Cu and Pb in R. decussatus at Timsah Lake, were; 1.30, 0.24 and 0.95 µg/g wet wt. and in P. undulates were; 0.79, 0.14, and 1.15 µg/g wet wt. respectively. Bio-sediment accumulation factor (BSAF) in the different bivalve species at the Great Bitter and Timsah lakes were much lower than unity (<<

The high load of protozoan parasites in marine bivalves often leads to mass mortalities of the hosts. On the west coast of Korea in the Yellow Sea, the protozoan parasite Perkinsus olseni has been identified as the agent causing mass mortality of Manila clam Ruditapes philippinarum. During August and September 2004, mass mortality of clam occurred at Hwangdo (HD) tidal flat in Anmyeondo Island on the west coast, resulting in a 50% reduction in the clam landings. Shortly after the mortality event, we examined pathology, and the fitness of the survived clams from HD to elucidate the impacts of P. olseni infection. Histology revealed that clams collected from HD in October 2004 were infected by P. olseni. In histology, P. olseni could be observed from all types of tissues of clams from HD, and severe inflammation was observed in the gills. Ray's fluid thioglycollate medium assay (RFTM) indicated that the infection intensity in clams from HD (1.738 × 106 cells/g gills in October and 1.476 × 106 cells/g gills in December) was significantly higher than the levels in clams from the neighboring tidal flats (0.001 to 0.622 × 106 cells/g gills, P

Ruditapes philippinarum is an economically important marine shellfish aquaculture species, and it has the ability to regenerate its siphons. To gain a greater understanding of the molecular mechanisms at work during siphon regeneration and to provide evidence for morphological regeneration, we examined transcriptome responses of siphon tissue of R. philippinarum during regeneration and observed regenerative siphons under the stereomicroscope. The overall process of siphon regeneration was dissected based on the morphological changes of siphon and the identification of up-regulated key differentially expressed genes (DEGs). The protein biosynthesis and metabolism played important roles in wound healing and siphon regeneration of R. philippinarum. Transcriptomic analysis identified the Wnt and TGF-β signaling pathways by focusing on the function and expression pattern of genes in these pathways during siphon regeneration. In addition, we carried out a genome-wide identification and phylogenetic analysis of TGF-β superfamily in R. philippinarum. The expression profiles of the TGF-β superfamily genes were analyzed in eight adult tissues (adductor muscle, mantle, foot, gill, siphon, digestive gland, gonad, and labial palp) and regenerative siphon. This study shed new light on the process of morphological regeneration and regenerative mechanism of siphon of R. philippinarum.

Asari clam (or Manila clam)Ruditapes philippinarumis an important bivalve for local fisheries. This species exhibits a large variation in shell morphology, and the shell roundness tends to be greater in more unsuitable habitats. To test whether the increments in shell size parameters (length, height and width) were affected solely by environmental conditions or by internal factors such as initial shell shapes or growth rate, a field caging experiment was conducted at two different sites of unsuitable and suitable habitats in Matsukawaura Lagoon, Japan, where shell shapes of wild clams were significantly different between the habitats. In the experiment, clams were released from the two sites to the same site or to the other site and were re-collected after 3, 6 and 12 months of caging. Caged clams originating from unsuitable habitats and released to suitable habitats showed a reduction in shell height relative to shell length, while clams from suitable habitats introduced to unsuitable habitats showed marked increases in both shell height and width. Generalized linear mixed models suggested that the increase in shell height was affected largely by the release habitat (environment) whereas the increase in shell width was affected largely by the individual growth rate. These results suggest that marginal growths in shell height and width respond differently to external and internal factors of clams, resulting in plasticity in their shell shapes according to the environments to which they are translocated.

Shell-color polymorphism is a common phenomenon in several mollusk species and has been associated with thermal capacity, developmental stability, shell strength, and immunity. Shell-color polymorphism has been related to the differential expression of genes in several signal transduction pathways; however, the functions of micro-RNAs (miRNAs) in shell-color formation remain unclear. In the present study, we compared high-quality, small-RNA transcriptomes in three strains of the Manila clam Ruditapes philippinarum with specific shell-color patterns, artificially selected for six generations. Totals of 114 known and 208 novel miRNAs were identified by high-throughput sequencing, of which nine known and one novel miRNA were verified by stem-loop quantitative real time-polymerase chain reaction. Predicted miRNA targets were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. miR-137 and miR-216b and the Hedgehog signaling pathway and Wnt signaling pathway were identified as being potentially involved in pigment formation and regulation in R. philippinarum. These results may help to clarify the role of miRNAs in shell coloration and shed light on the mechanisms regulating color formation in bivalve shells.

Filter-feeder bivalves such as non-indigenous Ruditapes philippinarum absorb and accumulate metals, resulting in multi-element profiles. The goal of this study was to analyse spatial and temporal distributions of the multi-element signatures in R. philippinarum populations of the Tagus and Sado estuaries (SW coast, Portugal). The clam and sediment samples were collected at three sampling sites in each estuary, on three sampling occasions, and the analysis were done by ICP-MS (inductively coupled plasma mass spectrometry). The chemical elements were categorized according to estuarine geomorphology sources (Se, Co, Ni and Cu), elements with function in metabolic processes of the clams (Mn, Fe, Zn and Cr) and elements derived from the anthropogenic inputs (As, Pb and Cd). Zinc, Co, Ni and Pb were the main contributors for the chemical signatures of Tagus estuary populations, whilst for the Sado estuary populations were Cu, Fe, Cr, As and Cd. They were representative of all elemental categories and proved to be spatial and temporal habitat discriminators of bivalves' estuarine populations. The multi-element signatures of R. philippinarum as a natural tag derived from the physical and chemical conditions of its habitat is a potential rapid tool to use in ecological monitoring and habitat assessment.

With a decrease in the domestic catches of Ruditapes philippinarum, suspended culture is a potential approach to increase its production. We investigated the establishment of suspended clam culture in Suttsu fishing port, Hokkaido. Cages accommodating 100 clams (mean shell length 28 mm) were suspended in the “stocking area” at the back of the port and the “fairway area” at the center of the port. Rearing periods were November 2017–October 2018 in the former and November 2018–October 2019 in the latter. Shell length was measured every month; water temperature, chlorophyll-a concentration, and flow velocity around cages were measured regularly. The mean shell length at the end of the experiments was 34 mm in the stocking area and 41 mm in the fairway. Although the water temperature, chlorophyll-a concentration, and advective components of flow velocity did not differ between the sites, the fluctuating component of flow velocity was higher in the fairway than in the stocking area. This suggests the former is more suitable for clam growth than the latter in terms of food supply. Therefore, the introduction of suspended culture in the fairway area of Suttsu fishing port is expected to improve the production efficiency of clam fishery.

Green tides caused by Ulvales group macroalgae have been reported from many eutrophic waters around the world. The asari clam Ruditapes philippinarum is an important fisheries resource in Japan and co-exists in coastal waters with Ulvales macroalgae. This study examined a possible impact of fresh Ulva fronds on the growth of juvenile, adult, and spat asari clams by conducting rearing experiments. Significant inhibition of juvenile and spat growth, but not adult growth, was observed when fresh Ulva fronds were floated on rearing water at 42.7 g/l (or 0.68 g/cm2), a density per area corresponding to the vegetation at Ajina flat on June 2006. This growth inhibition was experimentally observed at a level of 1.8 mg C/l (or a 0.47 mg C/l increase against blank rearing water) of dissolved organic matter (DOM) released from Ulva fronds. On the other hand, coastal waters with Ulva blooms contained higher levels of DOM (mean ± SD) at 2.3 ± 0.8 mg C/l (or 1.6 mg C/l higher than neighboring waters with sparse Ulva vegetation), suggesting that similar inhibition of asari clam growth may be occurring in the natural water environment with prevalent Ulva blooms.

Human beings are confronted with a serious health hazard when ingesting Ruditapes philippinarum contaminated with heavy metals, and thus it is significantly necessary to identify heavy metal contaminated Ruditapes philippinarum. This study investigates the feasibility of hyperspectral imaging to identify heavy metal contamination in Ruditapes philippinarum rapidly. To reduce the effects of noise, four different spectral pretreatments were performed on the original spectra. To select characteristic wavebands for identification, four waveband selection algorithms based on neighbourhood rough set theory were proposed, namely, mutual information, consistency measure, dependency measure, and variable precision. The selected wavebands were input to an extreme learning machine to construct classification models. The results demonstrated that multiplicative scatter correction pretreatment was suitable for Ruditapes philippinarum hyperspectral imaging datasets. The identification models exhibited satisfactory performance to distinguish healthy Ruditapes philippinarum from those contaminated by both individual and multiple heavy metals. The identification results of Cd and Pb contaminated samples were more accurate than those of Cu and Zn contaminated samples. When the number of training samples decreased the identification performance decreased, but not significantly. The results showed that combined with pattern recognition analysis hyperspectral imaging technology can be used to distinguish healthy Ruditapes philippinarum samples from those contaminated by heavy metals, even with only a small number of training samples. This model is suitable for applications in analysing many shellfish rapidly and non-destructively.

Arcachon Bay, in the southern Bay of Biscay, is the first French production area of Manila clam (Ruditapes philippinarum) with an annual commercial fisheries production around 400 tons in recent years. This bivalve mollusc was introduced into the bay in the 1980s for aquaculture purpose and quickly succeeded in natural settlement so that its high market value led to the progressive establishment of a perennial fishery from the mid-1990s. Local management plans applied to this species are based on a licensing system for the commercial, protected fishery areas and days of prohibited fishing for both commercial and recreational fishermen. A minimum catch size is also prescribed by European regulation, and so growth rates are an important matter in this context. This work focuses on studying shell growth from the recapture of shells, previously marked using a chemical marker (calcein) and then grown in natural conditions, and was performed in order to determine shell growth patterns. Manila clams were marked by immersion in situ in seawater containing calcein. In order to study the limitations of calcein on the mark readability in situ and manila clam mortality, various exposure times, from 30 minutes to 1 hour, and concentrations, from 50 mg.L-1 to 200 mg.L-1 were tested. After a period of 35 days, 69 growth micro-increments were observed, and, it was found that increment deposition in manila clams occurs with a tidal periodicity. Moreover, this study showed that a method for fast-marking of manila clams, and potentially other species, is feasible in situ.

The scuticociliatid ciliates Ancistrum haliotis and A. crassum are parasites that may cause high mortality in the cultured abalone Haliotis spp. and the bivalve Ruditapes philippinarum. Traditional identification with silver staining methods is hampered by their morphological similarities to closely related species and the complicated procedures of morphological analysis. We designed two SSU rRNA-targeted oligonucleotide probes labeled with a fluorochrome, and optimized the fluorescence in situ hybridization (FISH) protocols for identification of A. halioti and A. crassum, respectively. The assays resulted in a clear identification by strong fluorescence signals from the oligonucleotide probes. The method can be used for quick and accurate quantitative analysis of A. haliotis and A. crassum infections on host molluscs.

Intensive commercial harvesting of the wedge clam Donax trunculus and the striped venus Chamelea gallina was conducted along the southern coast of the Marmara Sea using a mechanical dredge (MD) and a hand dredge (HD). The purpose of this study was to characterize and quantify the bycatch and discards of both types of dredge. The dredge tows were performed along sandy bottoms at depths ranging from 0 to 2 m between August 2011 and July 2012. A total of 29 species belonging to 20 families were collected from 137 tows during 67 fishing trips. The total catch in terms of biomass was 107,752.69 g for the MD and 94,257.40 g for the HD, of which 10.78% and 5.08%, respectively, comprised commercial bycatch species, discards, individuals of the target species smaller than the minimum legal size (

As a suspension feeder, Manila clam Ruditapes philippinarum (A. Adams and Reeve 1850) plays a crucial role in the coastal soft bottom ecosystem in the temperate region, linking the benthic primary production to the upper trophic level. Manila clam density on tidal flats on the west coast of Korea has been declining for the past decades, and infection by the protozoan parasite Perkinsus olseni (Lester and Davis 1981) is one of the major causes for the decline. Recent studies carried out in Japan revealed that P. olseni induces mortalities of the juveniles in their natural habitats, which may lead to the recruitment failure and subsequent decline in the clam population. In this study, we surveyed P. olseni infection in juvenile Manila clam occurring on two tidal flats on the Taean coast. Ray’s fluid thioglycollate medium assay (RFTM) revealed that P. olseni infection was not limited to the adult clams, and the juvenile and small-sized clams are also infected by P. olseni. As young as four-month-old juveniles from Jugyo tidal flat were infected by P. olseni, with the prevalence (i.e., percentage of the infected individuals) of 75.0% and the intensity of 7.77 × 105 cells g−1 wet tissue weight (WT). The adult Manila clams (SL > 30 mm) from Jugyo tidal flat showed a prevalence of 96.0%, and the intensity as 5.80 × 10 cells g−1 WT. The observed infection prevalence and intensity of the juvenile are somewhat comparable to those of the adult clams, suggesting that a high level of P. olseni infection in the juveniles may lead to mortality and a long term decline in the clam population density.

Feeding periodicities and rhythms of suspension-feeding bivalves in coastal waters are closely related to diel and tidal cyclic environmental changes, such as food availability and predation risk. Although such feeding periodicities of bivalves are important for understanding how their feeding strategies adapt to localized habitats, the feeding periodicity in bivalves in the field remains to be clarified, especially in infaunal species. The present study focused on the morphological condition of the crystalline style as an indicator of the feeding activity of the infaunal bivalve Manila clam, Ruditapes philippinarum, and estimated their feeding periodicity in the field. First, the effects of feeding (siphon-extending) behaviour, food and the light/dark cycle on the condition of the crystalline style (defined based on its morphology and hardness) were investigated in the laboratory. Results of the ordered logistic models and model selection showed that clams extending their siphons kept their crystalline styles hard. Furthermore, the effects of the light/dark cycle and tidal phase on the condition of the crystalline style were investigated in natural intertidal and subtidal zones and assessed using the results of the ordered logistic models. In the intertidal zone, the effect of the tidal phase was significant, whereas the effect of the light/dark cycle was not, and feeding was estimated to be active at ebb and flood tides, when the density of food particles was likely to be high. On the other hand, in the subtidal zone, the results of the model selection were not clear because of significant effects of interactions between the light/dark cycle and tidal phase. However, when the interactions were removed from the models, the effect of the light/dark cycle was significant, and feeding was estimated to be active at night when the predation risk from visual predators would be relatively low. These results confirm that R. philippinarum possesses behavioural plasticity in feeding periodicity, which is adaptable to both intertidal and subtidal zones.

To access the nutritional quality of the Ruditapes philippinarum, a comprehensive quality evaluation procedure is always important to be established. In this study, fifteen nutritional quality evaluation indicators of R. philippinarum from 7 months were analyzed, and the most important indicators were determined using a combination of multiple chemometric methods such as correlation analysis (CA), principal component analysis (PCA), and system cluster analysis (SCA). Significant differences in nutritional quality were observed across the 7 months, as per the ANOVA results (P 1 were obtained with PCA, and a cumulative contribution of 92.11% was achieved. In addition, four essential quality indicators were extracted using SCA. Using these four indicators, a simple and efficient procedure can be applied for quality control in aquaculture. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-020-04796-6) contains supplementary material, which is available to authorized users.

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.

Paratectonatica tigrina employs three predatory tactics against bivalve prey: wall-drilling, edge-drilling and non-drilling (suffocation) tactics. In this study, we conducted laboratory experiments to examine the predator–prey interactions between the naticid gastropod P. tigrina and two species of bivalve prey, Ruditapes philippinarum and Meretrix lusoria, and to investigate how predatory tactics differ for the bivalve species. Predation success increased with the shell weight ratio between predator and prey, however, there was no significant difference among prey species. The shell weight ratio significantly differed (P < 0.05) between predatory tactics against R. philippinarum, but not against M. lusoria, implying that P. tigrina determines its predatory tactics based on the shell weight ratio for R. philippinarum. However, we observed that feeding duration was not influenced by shell weight ratio or prey species, even though data were separated for each predatory tactic. These results suggest that there is no difference in feeding efficiency based on shell weight ratio between prey species and between predatory tactics. Paratectonatica tigrina may alter its predatory tactics suitably depending on the prey species and the relative size difference, despite the same feeding efficiency.

Background The manila clam Ruditapes philippinarum and the wedge clam Donax trunculus are economic bivalve species which constitute an important part of the natural bivalve beds in the Marmara Sea, Turkey. Toxic chemicals such as, dioxins, dioxin-like polychlorinated biphenyls (PCBs), polycyclic hydrocarbons (PAHs), and toxic metals are recognized as important health risk factors that threaten public health via food or directly in the environment. In the present study, we aimed to determine and monitor levels of these toxic chemicals in both clam species, R. philippinarum and D. trunculus, between 2013 and 2017. Results According to the results, maximum levels of dioxins, PAHs, and toxic metals (lead, cadmium, mercury) during the period were determined as 0.18 pg g-1 , 2.43 mg kg-1 , and 0.44-0.53-0.1 mg kg-1 , respectively. The level of contaminants in both clam species were determined to be below the threshold or tolerable daily intake values established by the European Commission, European Food Safety Authority (EFSA), and Food and Agriculture Organization/World Health Organization (FAO/WHO). Seasonal fluctuations were observed to be similar in both species and also in seasons, but elevated levels of PCBs and PAHs were detected in warmer months during the period. Conclusion As a consequence, toxic chemicals in both clam species were found at low concentrations in this study from the point of view of public health concerns; however, the contaminants should be closely monitored in the future due to their elevated levels in samples. © 2020 Society of Chemical Industry.

Tidal flats are ecologically important as they support a large community of animals (e.g., crabs, mollusks, and polychaetes) and restore water quality. However, information regarding microbial eukaryotes, most of which are protists, associated with animals inhabiting these environments is limited. In the present study, utilizing polymerase chain reaction techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene of protists from the gill tissues of the Manila clam Ruditapes philippinarum in the Wajiro tidal flat, Hakata Bay, Japan. Based on the phylogeny of the SSU rRNA gene, the detected organisms were shown to belong to not only Perkinsozoa but also to Ciliophora, Kinetoplastida, and Haplosporidia. For the protist affiliated with Perkinsozoa, the internal transcribed spacer (ITS) of the rRNA genes was retrieved from the same gill tissue samples for precise identification at the species level. The ITS sequence obtained was identical to that of Perkinsus olseni, which is highly prevalent in Manila clam populations in Japan. Our findings suggest that protists potentially parasitic on this representative mollusk in tidal flats are phylogenetically diverse, and that the organisms detected may affect the population dynamics of the host clams.

Aquaculture and capture production of clam species have great economic value in Europe. The production is dominated by the introduced species, Manila clam, Ruditapes philippinarum, which has displaced some of the native species of clams. In Europe, landings of autochthonous clams have decreased in the recent years due to overfishing, failure in recruitment, abiotic stress and diseases. Due to aforementioned factors, seed production in hatcheries has become essential to ensure the sustainability of the fisheries and aquaculture production of European native clams. Many studies have focused on the different steps involved in hatchery rearing, but the volume of seed of native species produced in commercial hatcheries is still relatively low in comparison to the exotic Manila clam. Presently, in European hatcheries, seeds from only two native species, Ruditapes decussatus and Venerupis corrugata, are produced at industrial scale. The present review analyses the state of hatchery culture of European native clam species, and the aims are to transfer information to the industry and identify critical gaps that might be impeding the development of hatchery production.

The suitability of fermented products of Ulva fronds as diets for juvenile asari clams was tested. Ulva fronds were transformed by fermentation into a single-cell product (Ulva marine silage; UMS) of suitable size for ingestion by the clams. No dietary effect was seen in clams fed UMS alone. Furthermore, combined feeding with UMS and Chaetoceros calcitrans, which is considered a suitable diet for clams, resulted in a reduction in growth. An aqueous suspension of Ulva fronds prepared without fermentation showed inhibitory effects on clam growth, which indicated that the presumed inhibitory component(s) originated from the Ulva fronds. Further feeding tests indicated that the component(s) that inhibited clam growth were water soluble and had a molecular weight of < 1000 Da. An authentic product of glucuronic acid, one of the major components of the water-soluble fraction of UMS, induced inhibitory effects on shell growth. However, further study is necessary to identify the components of Ulva fronds that inhibit growth in the asari clam. The results indicate that the utilization of Ulva fronds as marine silage for the asari clam is problematic because they appear to contain component(s) that inhibit clam growth.

Seafood is increasingly used worldwide due to its nutritional value. It is rich in essential minerals, liposoluble vitamins, and high-quality proteins. However, many seafood species tend to accumulate metals, making them a threat to human health. We chose Xincun Lagoon and Li-an Lagoon, which have been seriously polluted by local activities, and the relatively clean Tufu Bay as the research areas to investigate whether trace metals in economic seafood species in these areas threaten the food safety of consumers. The results show that the mean concentrations (mg kg-1 ww) of trace metals were Cu 0.22 ± 0.18, Pb 0.040 ± 0.028, Zn 5.25 ± 1.60, Cd 0.009 ± 0.004, Hg 0.0072 ± 0.0031, As 0.21 ± 0.12, and Cr 0.084 ± 0.048 in fish samples (Trachinotus ovatus); Cu 4.00 ± 0.96, Pb 0.075 ± 0.030, Zn 19.10 ± 2.64, Cd 0.050 ± 0.024, Hg 0.0131 ± 0.0035, As 0.91 ± 0.27, and Cr 0.216 ± 0.095 in crab samples (Charybdis natator); and Cu 5.37 ± 1.39, Pb 0.095 ± 0.023, Zn 21.38 ± 3.59, Cd 0.096 ± 0.026, Hg 0.0208 ± 0.0052, As 1.65 ± 0.59, and Cr 0.406 ± 0.117 in bivalve samples (Ruditapes philippinarum), respectively, with species specific (fish < crab < bivalve) and significant seasonal and spatial variation (p < 0.05). However, health risk assessment (EDI, THQ, TTHQ, CR) indicated that the consumption of the analyzed seafood from the study areas is safe for local consumers. But there is a potential risk for fishermen and those who prefer seafood.

Ruditapes philippinarum is an important marine bivalve species. In this study, we conducted the RNA-seq of four different shell color strains of the R. philippinarum and investigated the analysis of the differential expression patterns of specific genes associated with pigmentation. The maximum different genes was 13 between WZ vs O, WZ vs W and WZ vs O have same numbers of different genes, was 5, Z vs W has 4 genes of 18 DEGs, W vs O just have two DEGs, while there is no DEGs between WZ vs Z. The synthesis of melanin plays important roles in the pigmentation of the shell and is closely related to the formation of the surface pattern. We speculate the possible involvement of porphyrin and chlorophyll metabolism combined with calcium signaling pathway in shell color determination. This study sheds light on the pigmentation and coloration mechanism of the Manila clam.

In coastal systems, pollutants as pharmaceutical drugs exert changes from the molecular to the organism level in marine bivalves. Besides pollutants, coastal systems are prone to changes in environmental parameters, as the alteration of salinity values because of Climate Change. Together, these stressors (pharmaceutical drugs and salinity changes) can exert different threats than each stressor acting individually; for example, salinity can change the physical-chemical properties of the drugs and/or the sensitivity of the organisms to them. However, limited information is available on this subject, with variable results, and for this reason, this study aimed to evaluate the impacts of salinity changes (15, 25 and 35) on the effects of the antiepileptic carbamazepine (CBZ, 1 μg/L) and the antihistamine cetirizine (CTZ, 0.6 μg/L), when acting individually and combined (CBZ + CTZ), in the edible clam Ruditapes philippinarum. After 28 days of exposure, drugs concentrations, bioconcentration factors and biochemical parameters, related to clam's metabolic capacity and oxidative stress were evaluated. The results showed that clams under low salinity suffered more changes in metabolic, antioxidant and biotransformation activities, in comparison with the remaining salinities under study. However, limited impacts were observed when comparing drug effects at low salinity. Indeed, it seemed that CTZ and CBZ + CTZ, under high salinity (salinity 35) were the worst exposure conditions for the clams, since they caused higher levels of cellular damage. It stands out that salinity changes altered the impact of pharmaceutical drugs on marine bivalves.

This study reports on the evaluation of the antioxidant of ethanolic extracts of Mytilus galloprovincialis and Ruditapes philippinarum collected from the Marmara Sea. The antioxidant activity of the two bivalve species was evaluated using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. The total carotenoid contents were also determined. In the DPPH method, all extracts exhibited low radical scavenging activity when compared to the standards used, i.e. -tocopherol and butylated hydroxytoluene (BHT) with the inhibition percentage in the range of 35.87±0.05 – 36.27±0.02%. In particular, M. galloprovincialis exhibited the highest radical scavenging activity with an IC50 value of 29.55±0.02 mg/g Ext. However, there are no statistically significant differences in the IC50 values of two species. The highest total carotenoid content was found in M. galloprovincialis as 1.13±0.02 µg/g sample. The statistically significant differences were found in total carotenoid contents of two bivalves. In this study in M. galloprovincialis was also found to be high in carotenoid content, while R. philippinarum was found to be almost nonexistent. More detailed studies are needed to determine relationship between the bioactive compounds of crustaceans and their antioxidant activities, distributed in our country.

Differences in taste and odour between three kinds of clam with the highest aquacultural production in China were investigated. Meretrix petechialis, Mactra veneriformis and Ruditapes philippinarum (fresh and dried product) were analysed firstly by electronic tongue and electronic nose. Fresh and dried clams could be easily distinguished, and there was little difference between uncooked fresh clams, while greater difference occurred between R. philippinarum and other fresh clams after cooking. The total free amino acid (FAA) content of uncooked clams increased after cooking but decreased in dried clam; the highest proportion was of sweet taste FAAs. Ala, Glu, Arg, 5′‐adenosine monophosphate and 5′‐inosine monophosphate were the most important active taste compounds. The umami intensity order was found to be fresh R. philippinarum, followed by M. petechialis and M. veneriformis, and finally dried R. philippinarum. The greatest number of volatile compounds was found in dried clam, while M. petechialis and M. veneriformis had the most compounds in common.

International audience; Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.

To evaluate the feasibility of particulated microalgae composite (PMC) as live microalgae substitute in artificial breeding of Manila clam Ruditapes philippinarum, effects of replacing Dicrateria inornata with partial or total PMC on the growth, digestive enzyme activities, nutritional ingredient composition and microbial composition in faeces of juvenile clams were investigated. The results showed that 50 and 100% PMC replacement did not affect the mean shell length, mean body weight and survival rate of juvenile clams (p>0.05). Juvenile clams fed with 100% PMC replacement had higher lipase and pepsin activities than those fed with 100% D. inornata (p 0.05). Significant increase in delicious amino acid contents (p