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MicroRNA is an important regulatory factor at the post-transcriptional level. Previous miRNAomics analysis found that miRNA-375 was steadily upregulated in the clam Ruditapes philippinarum upon ammonia nitrogen exposure. However, we have no knowledge about its regulatory mechanism yet. In this study, the clams were challenged by the injection of miRNA-375 mimics/inhibitor in vivo. Then, a combined approach of qRT-PCR, enzyme assay, and ultrastructure observation was applied to investigate its regulatory effects on the related genes, cellular parameters, and histological structures, respectively. Results showed that increased expression of miRNA-375 interfered with the expression levels of both its target genes and ammonia toxicity-related genes, which would probably lead to oxidative stress, migration of damaged cells, apoptosis resistance, and increased possibility of tumor formation. In addition, miRNA-375 increased MDA content but decreased glutamate content and caused serious structure damage to the clam gills. Thus, increased miRNA-375 probably brings a disastrous fate to the clam R. philippinarum by inducing oxidative damage but promoting apoptosis resistance and cell migration. Overall, this study revealed for the first time the regulatory effects of miRNA-375 in the clams and gave valuable clues to understand the toxicological mechanisms of ammonia nitrogen on the marine bivalve. [ABSTRACT FROM AUTHOR]

The contamination of soil with arsenic (As) and heavy metal is an increasing global environmental concern. The objective of this study was to rehabilitate soil contaminated with As, Pb, and Zn using fishery by-products as stabilizers to achieve both soil restoration and waste resource recycling. Cockle shells (CS) and manila clam shells (MC), selected as fishery by-product stabilizers, were processed into −#10-mesh and −#20-mesh materials. Additionally, a −#10-mesh material was calcined at a high temperature to produce calcined cockle shells (CCS) and calcined manila clam shells (CMC). Contaminated soil was treated with 2–10 wt% of these stabilizers and subjected to wet incubation for 1–4 weeks. Subsequently, the concentrations of As, Pb, and Zn eluted by 0.1 M HCl were evaluated. Additionally, lettuce was grown in stabilized soil to evaluate the reduction in contaminant mobility. The stabilization treatment results indicated that the concentrations of eluted As, Pb, and Zn were significantly reduced when treated with the −#10-mesh and −#20-mesh CS and MC, and they were rarely detected when treated with the calcined materials (CCS and CMC). The Pb concentration in lettuce grown in the contaminated soil pot exceeded the criterion for leafy vegetables (0.3 mg/kg); however, Pb was not detected in lettuce from the stabilized soil pot. An X-ray diffraction (XRD) analysis revealed that CaCO3, the main component of CS and MC, was converted to CaO after calcination. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) and SEM elemental dot map analyses revealed that the immobilization of As was related to Ca–As precipitation and the immobilization of Pb and Zn to the pozzolanic reaction. Thus, recycling and processing CS and MC as stabilizers for contaminated soil can restore the agricultural value of the soil by immobilizing As, Pb, and Zn into safe forms, thus effectively preventing their uptake by crops. [ABSTRACT FROM AUTHOR]

The Manila clam is well known to be parasitized by the protist Perkinsus. However, other parasitic organisms affecting this clam remain poorly understood. In our previous study, 18S rRNA gene sequences (uncultured clones named WJR_C1 and WJR_C2) derived from two unknown ciliates were detected in the gills of Manila clams. Based on phylogenetic analyses, these sequences have been suggested to belong to the ciliate subclass Suctoria; however, their ecology remains unclear. In this study, along with re-evaluating the taxonomy of WJR_C1 and WJR_C2, we investigated the prevalence of both ciliates using a specific polymerase chain reaction (PCR)-based detection method. We also examined the localization of the ciliate represented by WJR_C1 in the host gill and its effect on the host using in situ hybridization and hematoxylin–eosin staining. Consequently, WJR_C1 and WJR_C2 were shown to belong not to Suctoria but to the subclass Rhynchodia, and to be particularly closely related to a parasite known as "mussel protozoan X" (MPX). The prevalence of these ciliates varied across regions and seasons, and the WJR_C1 ciliate was localized on the surface of host gills rather than inside the gills. The WJR_C1 ciliate did not appear to have any negative histological effects on host gills. [ABSTRACT FROM AUTHOR]

The activity levels of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) can reflect the immune status of an organism. The immune status may be affected by extreme changes in the weather, especially rapid declines in temperature. In this study, the SOD, CAT, LZM, ACP, and AKP activity levels of the Manila clam (Ruditapes philippinarum) were measured for 24 h while the seawater temperature rapidly decreased to the freezing point from 8 °C, 4 °C, and 2 °C to analyze its immunochemical response to temperature decline. The results showed that the enzyme activity levels fluctuated with time as the temperature declined. By fitting the data, a model was obtained to describe the variation in immune enzyme activity within a short time period as temperature declined. The mathematical description included the stress response and the direct temperature response. The enzyme activity was adjusted rapidly as a stress response in the short term as the temperature declined, before it tended to stabilize. The direct temperature response also caused the enzyme activity to change as the temperature declined to the freezing point. The correlation coefficient between the fitted model and the actual enzymatic activity levels exceeded 0.87, which demonstrated that the mathematical description was adequate. [ABSTRACT FROM AUTHOR]

The Manila clam (Ruditapes philippinarum) is one of the main products of Italian aquaculture and is primarily farmed in the north-Adriatic lagoons. To support this important farming sector, there is a need for increased production of clam seed pre-fattened to a size suitable for sowing in the lagoon, i.e., approximately 8–10 mm. The early pre-fattening up to 4–5 mm in shell length is critical and preferably requires controlled upwelling systems similar to those adopted by hatcheries. These could be implemented in the Italian "valli da pesca," which are semi-natural confined environments, historically exploited for extensive fish-farming and located in the innermost areas of the north-Adriatic lagoons. The implementation of clam pre-fattening activity in these environments of high naturalistic value, which are currently no longer profitable, presents an excellent opportunity to enhance their valorization and preservation. In this study, two pre-fattening trials were conducted, in autumn and in spring respectively, in an experimental "valle da pesca" equipped with an upwelling system. The aim was to define the optimal conditions of stocking density (approximatively 100–300 clams/cm2) and water flow rate (approximatively 10–20 mL/cm2) for clams with an initial shell length of 1.8–2.6 mm. The clam specific growth rate (SGR) varied between 2.3 and 5%/day, decreasing as stocking density increased and water flow rate decreased. The results suggest that a water flow rate per unit of biomass > 15 mL/min/g fresh weight (FW) (preferably > 20 mL/min/g FW) could support satisfactory clam growth rates. Chlorophyll-a maintained an average concentration of 1.3 mg/m3 in spring and 3.8 mg/m3 in autumn, respectively, but without a clear impact on the clam SGR. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) poses a major risk to shrimp aquaculture, and filter-feeding bivalves on shrimp farms may contribute to its persistence and transmission. This study investigated the bioaccumulation and vector potential of WSSV in Pacific oysters (Crassostrea gigas), blue mussels (Mytilus edulis), and manila clams (Venerupis philippinarum) cohabiting with WSSV-infected shrimp. Sixty individuals of each species (average shell lengths: 11.87 cm, 6.97 cm, and 5.7 cm, respectively) cohabitated with WSSV-infected shrimp (Penaeus vannamei, average body weight: 16.4 g) for 48 h. In the experiments, bivalves accumulated WSSV particles in both the gill and digestive gland tissues, with the digestive glands exhibiting higher viral load (average viral load, 3.91 × 104 copies/mg), showing that the viral concentrations in bivalve tissues are directly influenced by seawater WSSV concentrations, reaching levels sufficient to induce infection and 100% mortality in healthy shrimp using tissue homogenates. After a 168 h release period in clean water, the WSSV levels in bivalve tissues decreased below the detection thresholds, indicating reduced transmission risk. These results highlight the role of bivalves as temporary reservoirs of WSSV in aquaculture settings, with the transmission risk dependent on the viral concentration and retention period. Our findings suggest that the management of bivalve exposure in WSSV-endemic environments could improve the biosecurity of shrimp farms. [ABSTRACT FROM AUTHOR]

Stutzerimonas kunmingensis TFRC-KFRI-1, isolated from the gut of Manila Clam in the sediment of the West Sea of Korea, was investigated for its potential as a probiotic bacterium. This strain, belonging to the family Pseudomonadaceae, was previously classified as Pseudomonas kunmingensis but later reclassified to the genus Stutzerimonas, known for species with bioremediation and probiotic properties. To evaluate its genomic features and potential applications, we performed draft-genome sequencing and analysis. The genome of S. kunmingensis TFRC-KFRI-1 was assembled into a 4,756,396 bp sequence with a 62.8% GC content. Genomic analysis suggested potential genes for carbohydrate degradation and lactic acid production. The strain exhibited high average nucleotide identity (ANI) and 16S rRNA similarity with S. kunmingensis HL22-2T, further supporting its potential as a probiotic. This genome sequence provides valuable insights into the functional capabilities of S. kunmingensis TFRC-KFRI-1 and its potential applications in various industries, including aquaculture and food biotechnology. The genome sequence is available under GenBank accession number JBGJJB000000000.1, with related project information under BioProject PRJNA1147901 and Bio-Sample SAMN43173893. [ABSTRACT FROM AUTHOR]

Conservation efforts have raised awareness about the impact of small-scale fisheries on the distribution of seagrass plants. The patterns of recovery of the seagrass Zostera noltei and of the commercial bivalves Cerastoderma edule, Ruditapes decussatus and Ruditapes philippinarum after shellfish harvesting were studied in a field experiment in a shellfish bed in NW Spain. Sample plots were subjected to a single disturbance in two types of shellfish harvesting treatments in three zones characterized by different harvesting frequency and seagrass density. The photosynthetic efficiency (Fv/Fm), shoot density, leaf length and carbohydrate content of Z. noltei were monitored every three months during one year, and the C and N content of leaves and biomass of plants were measured one year after the disturbance. The abundance of adults, juveniles and recruits and the condition index of adult bivalves were quantified after the experimental harvesting. Shoot density and biomass of Z. noltei remained low during the ten months after the disturbance but recovered to control values after one year. Carbohydrate contents of apical rhizomes were lower in disturbed (treated) plots, whereas no effect was observed on Fv/Fm. Denser and more complex seagrass patches recovered faster. The abundance of adult bivalves below commercial size was lower in the disturbed plots, while the abundance of adults of commercial size, juveniles and recruits did not vary, indicating that abundance and condition were not hampered by harvesting pressure. The findings also suggest that Z. noltei meadows can recover within one year of the impact of shellfish harvesting if the harvesting areas are rotated and dense patches are preserved. [ABSTRACT FROM AUTHOR]

In order to produce gynogenetic diploid of Manila clam, Ruditapes philippinarum, we studied the optimal conditions of gynogenetic diploid induced by ultraviolet (UV). The irradiance of UV‐induced sperm was 230 μW (cm2·s), and the irradiation time was 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27 s. After the egg fertilized with genetically inactivated sperm, the fertilized eggs were treated with cytochalasin B (CB) at different concentrations of 0, 0.25, 0.50, 0.75, and 1.0 mg/L for 20 min. The ploidy of larvae under different induction concentrations were analyzed by flow cytometry, and the chromosome division process during sperm entry and cleavage was observed by fluorescence microscope. In addition, the ultrastructure of sperm, embryonic development, and chromosome were observed. The results showed that the fertilization rate and cleavage rate decreased gradually with the prolongation of UV irradiation time. We found UV irradiation for 9 s was the optimal time for haploid formation, with an induction rate of 100%. The CB treatment at 0.75 mg/L was the optimal concentration for the formation of D‐larvae, and the induction rate was 9.60% ± 5.10%. The results showed that the optimal conditions for inducing gynogenetic diploid of R. philippinarum were irradiation with UV intensity of 230 μW (cm2·s) for 9 s and inhibited second polar body release by CB at concentration of 0.75 mg/L. In this study, the conditions for inducing Manila clam gynogenetic diploid were obtained, which lays the basis for further genetic research on the gynogenesis of R. philippinarum. [ABSTRACT FROM AUTHOR]

In recent years, research on active polysaccharides has progressed significantly, particularly regarding their anticancer and immunomodulatory properties. Among these, clam polysaccharides, a type of marine-derived polysaccharide, exhibit notable biological activities, including both anticancer effects and immune modulation. The aims of this study are to investigate the anticancer and immunomodulatory effects of microwave-extracted clam polysaccharide (MCP) in vitro. Cell experiments demonstrated that MCP significantly inhibited both colony formation and migration of HT-29 cells. Furthermore, treatment with MCP led to the downregulation of Bcl-2 gene expression, a reduction in mitochondrial membrane potential, activation of cytochrome C gene and caspase-3 gene, and, finally, the induction of apoptosis in HT-29 cells, implying the involvement of the mitochondrial pathway. Additionally, MCP was found to prompt a phenotypic shift in macrophages from M2 to M1 subtype and from M0 to M1 subtype. MCP also decreased reactive oxygen species (ROS) levels within the cancer cells, thereby augmenting anticancer efficacy through a dual mechanism of immune activation and antioxidant enhancement. These findings suggest that MCPs present significant potential as natural antitumor agents and immunomodulators, especially in the development of functional foods or drugs. [ABSTRACT FROM AUTHOR]

Developing non-contact, non-destructive monitoring methods for marine life is crucial for sustainable resource management. Recent monitoring technologies and machine learning analysis advancements have enhanced underwater image and acoustic data acquisition. Systems to obtain 3D acoustic data from beneath the seafloor are being developed; however, manual analysis of large 3D datasets is challenging. Therefore, an automatic method for analyzing benthic resource distribution is needed. This study developed a system to estimate benthic resource distribution non-destructively by combining high-precision habitat data acquisition using high-frequency ultrasonic waves and prediction models based on a 3D convolutional neural network (3D-CNN). The system estimated the distribution of asari clams (Ruditapes philippinarum) in Lake Hamana, Japan. Clam presence and count were successfully estimated in a voxel with an ROC-AUC of 0.9 and a macro-average ROC-AUC of 0.8, respectively. This system visualized clam distribution and estimated numbers, demonstrating its effectiveness for quantifying marine resources beneath the seafloor. [ABSTRACT FROM AUTHOR]

Simple Summary: Clams have evolved the buried lifestyle, which depends on their unique soft tissue structure and their wedge-shaped muscular foot and long extendible siphons. However, the molecular mechanism of adaptative phenotype evolution remains largely unknown. The analysis of genomic data showed significant expansion or contraction in specific gene families and transposable elements. Comparative transcriptomics between different tissues reveals that extracellular matrix (ECM) receptors and neuroactive ligand receptors may play important roles in tissue structural support and neurotransmission in clams. The high-quality genome and transcriptome of Ruditapes philippinarum will provide valuable information on morphological novelties in mollusks. Bivalve mollusks, comprising animals enclosed in two shell valves, are well-adapted to benthic life in many intertidal zones. Clams have evolved the buried lifestyle, which depends on their unique soft tissue structure and their wedge-shaped muscular foot and long extendible siphons. However, molecular mechanisms of adaptative phenotype evolution remain largely unknown. In the present study, we obtain the high-quality chromosome-level genome of Manila clam R. philippinarum, an economically important marine bivalve in many coastal areas. The genome is constructed by the Hi-C assisted assembly, which yields 19 chromosomes with a total of 1.17 Gb and BUSCO integrity of 92.23%. The de novo assembled genome has a contig N50 length of 307.7 kb and scaffold N50 of 59.5 Mb. Gene family expansion analysis reveals that a total of 24 single-copy gene families have undergone the significant expansion or contraction, including E3 ubiquitin ligase and dynein heavy chain. The significant expansion of transposable elements has been also identified, including long terminal repeats (LTR) and non-LTR retrotransposons. The comparative transcriptomics among different clam tissues reveals that extracellular matrix (ECM) receptors and neuroactive ligand receptors may play the important roles in tissue structural support and neurotransmission during their infaunal life. These findings of gene family expansion and tissue-specific expression may reflect the unique soft tissue structure of clams, suggesting the evolution of lineage-specific morphological novelties. The high-quality genome and transcriptome data of R. philippinarum will not only facilitate the genetic studies on clams but will also provide valuable information on morphological novelties in mollusks. [ABSTRACT FROM AUTHOR]

Background: The deep-sea cold seep zone is characterized by high pressure, low temperature, darkness, and oligotrophy. Vesicomyidae clams are the dominant species within this environment, often forming symbiotic relationships with chemosynthetic microbes. Understanding the mechanisms by which Vesicomyidae clams adapt to the cold seep environment is significant. Acetylation modification of lysine is known to play a crucial role in various metabolic processes. Consequently, investigating the role of lysine acetylation in the adaptation of Vesicomyidae clams to deep-sea environments is worthwhile. So, a comparative study of lysine acetylation in cold seep clam Archivesica marissinica and shallow water shellfish Ruditapes philippinarum was conducted. Results: A total of 539 acetylated proteins were identified with 1634 acetylation sites. Conservative motif enrichment analysis revealed that the motifs -KacR-, -KacT-, and -KacF- were the most conserved. Subsequent gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted on significantly differentially expressed acetylated proteins. The GO enrichment analysis indicated that acetylated proteins are crucial in various biological processes, including cellular response to stimulation, and other cellular processes (p < 0.05 and false discovery rate (FDR) < 0.25). The results of KEGG enrichment analysis indicated that acetylated proteins are involved in various cellular processes, including tight junction, motor proteins, gap junction, phagosome, cGMP-PKG signaling pathways, endocytosis, glycolysis/gluconeogenesis, among others (p < 0.05 and FDR < 0.25). Notably, a high abundance of lysine acetylation was observed in the glycolysis/glycogenesis pathways, and the acetylation of glyceraldehyde 3-phosphate dehydrogenase might facilitate ATP production. Subsequent investigation into acetylation modifications associated with deep-sea adaptation revealed the specific identification of key acetylated proteins. Among these, the adaptation of cold seep clam hemoglobin and heat shock protein to high hydrostatic pressure and low temperature might involve an increase in acetylation levels. Acetylation of arginine kinase might be related to ATP production and interaction with symbiotic bacteria. Myosin heavy chain (Ama01085) has the most acetylation sites and might improve the actomyosin system stability through acetylation. Further validation is required for the acetylation modification from Vesicomyidae clams. Conclusion: A novel comparative analysis was undertaken to investigate the acetylation of lysine in Vesicomyidae clams, yielding novel insights into the regulatory role of lysine acetylation in deep-sea organisms. The findings present many potential proteins for further exploration of acetylation functions in cold seep clams and other deep-sea mollusks. [ABSTRACT FROM AUTHOR]

Human activities directly impact estuaries, where the biota is exposed to sediment contamination. A contamination assessment was carried out in several estuaries in the Southern Iberian Peninsula. Sediment samples were analyzed for the presence of metals/metalloids, and bioaccumulation tests were conducted with clams (Ruditapes philippinarum). Huelva Estuary had the highest contamination levels, while the inner bay of Cádiz and the outer stations of the estuaries from Guadiana, Guadalquivir, Palmones, and Guadarranque were the lesser contaminated. All sampling points (except Huelva) had low contamination levels of As and Cd, but they had high concentrations of Cu. The elements Pb, Cd, Zn, Cu, As, and Hg displayed correlations between the concentrations in sediments and the biota. High bioaccumulation of Zn, Cu, and As was observed in Huelva and Barbate. Important insights into the sediment contamination in Southern Iberian Peninsula estuaries suggest greater management and conservation efforts in these critical ecosystems. [ABSTRACT FROM AUTHOR]

Abstract Background Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems). Results Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score. Conclusions We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish.

Background: Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems). Results: Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score. Conclusions: We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish. [ABSTRACT FROM AUTHOR]

Background: SmithRNAs (Small MITochondrial Highly-transcribed RNAs) are a novel class of small RNA molecules that are encoded in the mitochondrial genome and regulate the expression of nuclear transcripts. Initial evidence for their existence came from the Manila clam Ruditapes philippinarum, where they have been described and whose activity has been biologically validated through RNA injection experiments. Current evidence on the existence of these RNAs in other species is based only on small RNA sequencing. As a preliminary step to characterize smithRNAs across different metazoan lineages, a dedicated, unified, analytical workflow is needed. Results: We propose a novel workflow specifically designed for smithRNAs. Sequence data (from small RNA sequencing) uniquely mapping to the mitochondrial genome are clustered into putative smithRNAs and prefiltered based on their abundance, presence in replicate libraries and 5′ and 3′ transcription boundary conservation. The surviving sequences are subsequently compared to the untranslated regions of nuclear transcripts based on seed pairing, overall match and thermodynamic stability to identify possible targets. Ample collateral information and graphics are produced to help characterize these molecules in the species of choice and guide the operator through the analysis. The workflow was tested on the original Manila clam data. Under basic settings, the results of the original study are largely replicated. The effect of additional parameter customization (clustering threshold, stringency, minimum number of replicates, seed matching) was further evaluated. Conclusions: The study of smithRNAs is still in its infancy and no dedicated analytical workflow is currently available. At its core, the SmithHunter workflow builds over the bioinformatic procedure originally applied to identify candidate smithRNAs in the Manila clam. In fact, this is currently the only evidence for smithRNAs that has been biologically validated and, therefore, the elective starting point for characterizing smithRNAs in other species. The original analysis was readapted using current software implementations and some minor issues were solved. Moreover, the workflow was improved by allowing the customization of different analytical parameters, mostly focusing on stringency and the possibility of accounting for a minimal level of genetic differentiation among samples. [ABSTRACT FROM AUTHOR]

In our previous study, we demonstrated that the microalgae Phaeodactylum tricornutum can bioaccumulate bisphenol A analogues. Since this microalgae species is part of the diet of marine filter-feeding organisms, such as bivalves, in this study we tested the hypothesis that a diet based on exposed microalgae can exert negative effects on the clam Ruditapes philippinarum. Microalgae were exposed for 7 days to 300 ng/L of bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS), alone or as a mixture (MIX), to allow bioaccumulation. Microalgae were then supplied as food to bivalves. After 7 and 14 days of diet, the effects of exposed microalgae were evaluated on a battery of biomarkers measured in haemolymph/haemocytes, gills and digestive glands of clams. In addition, bioaccumulation of the three bisphenols was investigated in clams by UHPLC-HRMS. The results obtained demonstrated that total haemocyte count (THC) increased in clams following ingestion for 7 days of BPAF- and BPF-exposed microalgae, while BPS-exposed microalgae significantly reduced THC after 14 days of diet. MIX- and BPS-exposed microalgae increased haemocyte proliferation. The diet of exposed microalgae affected acid and alkaline phosphatase activity in clams, with an opposite response between haemolymph and haemocytes. Regarding antioxidants, an increase in catalase activity was observed in clams after ingestion of BPA analogue-exposed microalgae. The results also demonstrated marked oxidative stress in gills, the first tissue playing an important role in the feeding process. Oxidative damage was recorded in both the gills and digestive glands of clams fed BPA analogue-exposed microalgae. Alterations in epigenetic-involved enzyme activity were also found, demonstrating for the first time that BPA analogue-exposed food can alter epigenetic mechanisms in marine invertebrates. No bioaccumulation of BPA analogues was detected in clam soft tissues. Overall, this study demonstrated that a diet of BPA analogue-exposed microalgae can induce significant alterations of some important biological responses of R. philippinarum. To our knowledge, this is the first study demonstrating the effects of ingestion of BPA analogue-exposed microalgae in the clam R. philippinarum, suggesting a potential ecotoxicological risk for the marine food chain, at least at the first levels. [ABSTRACT FROM AUTHOR]

Previous studies have revealed that ammonia nitrogen has several adverse effects on clam Ruditapes philippinarum. However, knowledge is lacking regarding the related proteins involved in the toxicological responses, which is vital to elucidate the underlying mechanism of ammonia nitrogen. In this study, clams R. philippinarum were exposed to ammonia nitrogen for 21 d at two environmentally relevant concentrations. The tandem mass tags approach (TMT) was applied to assay the differentially expressed proteins (DEPs) in clam gill tissues on the 3rd and 21st day. Finally, a total of 7 263 proteins were identified. Bioinformatics analyses revealed that clam protein profiles changed in dose- and time-dependent manner after ammonia nitrogen exposure. We inferred that the clams may face heavy challenges after ammonia exposure, such as unbalanced gender ratio, lysosomal disease, energy lack, neurological disorders, altered glutamine metabolism, increased lipid synthesis, and impaired immunity. Variation profiles of enzyme activities of glutaminase and glutamine synthase provided direct evidence to verify the related inference from proteome data. Most of the inferred toxic effects merit further study. This study identified important proteins related to ammonia nitrogen toxicity in the clam and indicated the severe stress of marine ammonia pollution on the healthy development of mollusc aquaculture. [ABSTRACT FROM AUTHOR]

When bivalve aquaculture production relies on natural recruitment, yields may decline due to density‐independent constraints at multiple life stages. These life stage transitions include larval settlement, which is typically variable with rapid losses of newly settled individuals and the additional mortality from predation or abiotic factors (e.g., temperature, desiccation, and physical disturbance) as the bivalves grow. Recruitment monitoring and outplants were used to evaluate the potential limiting factors affecting different life stages in two nonnative clam species, Manila clams, Ruditapes philippinarum, and softshell clams, Mya arenaria in Willapa Bay, Washington, USA. Recruits (250–500 µm) did not differ in cumulative abundance in 2023 relative to prior years of monitoring (five recruitment seasons between 2011 and 2017). Recruits surviving to the end of the summer represented 12% or less of those that had arrived, but still showed spatial patterns consistent with rates of arrival. Manila clams outplanted across an elevation gradient suffered high mortality below mean lower low water regardless of size class (small: 8 mm and large: 20 mm), where native rock crabs (Cancer productus) were abundant. Clam growth declined at higher tidal elevations consistent with inundation time. European green crabs (Carcinus maenas) were relatively abundant at midtidal levels where commercial clam aquaculture typically occurs in the bay and where small clams were particularly reduced without predator protection from mesh. While these data support that green crabs may be reducing survival of 1‐year‐old clams at midintertidal elevations, they also identify earlier bottlenecks to repopulating commercial clam beds, which point out why seeding clams is used to maintain consistent production. Further exploration is needed before green crabs can be singled out as a new limit on clam yields relative to the roles of predation across the size classes of clams in Willapa Bay. [ABSTRACT FROM AUTHOR]

Abundant clam populations provide ecosystem services and commercial and recreational shellfish harvests worldwide. Two non‐native clams (Manila clams, Ruditapes philippinarum; eastern soft‐shell clams, Mya arenaria) commonly populate mid‐intertidal zones of coarse‐sediment beaches in Washington State, USA. To better understand factors influencing their abundance, we used passive mid‐intertidal spat collectors to determine the density and distribution of clam recruits (150–500 μm shell length) at eight beaches spanning over 500 km and over two reproductive seasons (2016 and 2017). Cumulative seasonal recruitment (clams <500 μm) in 2016 and 2017 at +1.0 m MLLW ranged from 800 to 125,000 m−2 for Ruditapes philippinarum and 50–85,000 m−2 for M. arenaria, and did not differ by substrate type at small scales (gravel vs. crushed shell). At larger scales, different interannual recruitment patterns across sites did not dramatically shift the ranked order of highest and lowest recruitment sites. Cumulative recruitment tended to be lower at warmer sites, while timing of peak recruitment was not consistently related to temperature. Sites also differed in the relative recruitment of the two clam species, but most beaches had greater densities of R. philippinarum than M. arenaria at both recruitment and juvenile stages. Although recruitment densities differed by two orders of magnitude across sites, post‐settlement factors eliminated initial spatial variation within a year, dampening recruitment variability among these beaches that all contribute to commercial and recreational clam production. [ABSTRACT FROM AUTHOR]

Coastal lagoons are environments with an important ecological richness, which makes them socio-economically highly important environments. However, they are often subject to anthropogenic pressures that may seriously threaten them. The phenomenon of anoxia is regularly the cause of massive mortality and loss of biodiversity. For example, in the Berre lagoon (France), in 2018, an anoxic crisis decimated the Zostera meadow and the stock of Manila clams, Ruditapes philippinarum (A. Adams & Reeve, 1850). As clams are a fished species, this massive mortality has resulted in a fishing cessation and thus is having a significant economic impact. This study is a contribution to our understanding of the resilience of species after a strong perturbation. In our case, we aim to understand the time needed for the stock of R. philippinarum to recover after the ecological crisis of 2018 by providing an analysis of the population dynamics between October 2018 and October 2020. The gonad development stage and condition index highlighted that clam size at sexual maturity was between 20 and 23 mm. The clam's reproduction season is continuous between March and October, with differences observed between sites, and between years at the same site in the Berre lagoon. Long-term field monitoring (in situ cages coupled with sclerochronology) was performed to estimate the growth rate and the mortality. von Bertalanffy growth function parameters show a high growth rate (K  = 1.42 yr−1) with a small |${{L}_\infty }$| (35.70 mm). This means that clams need 1.5 yr to reach 30 mm in length: the minimum catch size for the fishery in the Berre lagoon. The mortality rate was 2.52 yr−1, which is possibly due to significant variations in water temperature and episodes of hypoxia and anoxia. These data on the population dynamics of R. philippinarum in the Berre lagoon show that the clam's recovery time can be relatively fast (some years) only if the environment becomes favourable and the anoxic conditions are absent. [ABSTRACT FROM AUTHOR]

In the Ebro Delta (Catalonia, Spain), the abundance of burrowing bivalves has dramatically decreased, with the blue crab, Callinectes sapidus, being blamed by shellfish collectors. Trends from 2010 evidence a decrease in the capture of clams (Ruditapes spp.) before 2016 (start of blue crab fisheries), although a further decline in both clams and cockles (Cerastoderma glaucum) occurred in 2018. In contrast, captures of razor clams (Ensis siliqua) have increased by 3.6-fold since 2016. Predation risk for these taxa, with contrasting burrowing capacities (1.7 ± 0.3 cm, 0.4 ± 0.2 cm, and 26.3 ± 0.1 cm, respectively), was assessed using predation preference (N = 5 tanks; 5 individuals of each species) and no-choice experiments (N = 5 tanks; 15 individuals of the same taxa) in the absence and presence of sand. The results showed that, in the absence of sand, razor clams were fully preyed upon in 24 h, clams in 96 h, and cockles reached 60% after 144 h. Conversely, when sand was present, only 4% of razor clams were predated, while clams and cockles reached 60–100% in 120–144 h. The no-choice results featured similar patterns, depending on substrate availability. Overall, clams and cockles appear to be greatly vulnerable to blue crab predation, whereas razor clams may escape thanks to their deeper burrowing capacity. [ABSTRACT FROM AUTHOR]

Morphology of bivalves, a kind of quantitative character, is closely connected with the ecological and physiological behaviors of bivalves, including growth, survival, metabolism and reproduction. In this study, a multivariate statistical method was used to analyze seven indices for shell shape evaluation of Ruditaps philippinarum (R. philippinarum). Polymorphism of R. philippinarum shell shape across seven geographical populations from the southern and northern regions of China was revealed, and decisive effect of shell shape in determining the trait of weight was discussed. In total, three types of R. philippinarum shell shape were observed: globular, flat, and lateral-view round. The Laizhou (LZ) population from the north of China displayed an approximately globular shell shape, with a shell-width-toshell-length ratio of 0.5496 and an ellipse index ventral view (EIV) value of 0.9543. The Lianjiang (LJ) population from the south of China displayed a flat shell shape, with a shell-width-to-shell-length ratio of 0.4044, an ellipse index lateral view (EIL) value of 1.2518, and an EIV value of 0.7063. The Zhangzidao (ZZD) population from the north of China displayed a lateral view round shell shape, with a shell-width-toshell-length ratio of 0.7476 and a circle index lateral view (CIL) value of 0.7195. According to the results of principal component analysis (PCA), discriminant analysis and cluster analysis on shell shape parameters, LJ population and LZ population could be grouped separately. However, despite the long geographical distance, Rongcheng (RC) population was highly similar with ZZD population in terms of shell shape traits, implying that these two populations might be more closely related. Lowest degree of divergence in shell shapewas observed in Beihai (BH) population and Yingkou (YK) population, both of which belonged to Zuidong (ZD) population. Weight of globular Manila clams was significantly higher than that of flat clams (P < 0.05). This study not only lays a foundation for conservation of R. philippinarum germplasm resources, but also highlights the importance of considering shell morphology in the breeding and culture of R. philippinarum. [ABSTRACT FROM AUTHOR]

Lee, H.-J.; Jeung, H.-D.; Song, J.-H.; Hur, Y.B., and Hong, H.-K., 2024. Enhancing seed harvest of wild Manila clam (Ruditapes philippinarum) through fiberglass mesh installation in the tidal flat of Jugyo, west coast of Korea. Journal of Coastal Research, 40(3), 613–616. Charlotte (North Carolina), ISSN 0749-0208. The Manila clam, Ruditapes philippinarum (Bivalvia: Veneridae), is a commercially important species cultivated in tidal flats along the coasts of Korea. However, the local clam industries on the west coast face challenges due to a shortage of seed clams, primarily relying on catching wild juveniles or importing seeds from China. This study aimed to enhance the natural seed harvest of Manila clams by installing fiberglass mesh on the tidal flats of Jugyo, Korea. Fiberglass mesh was installed at three different stations on the tidal flats in October 2017 and clam density was monitored monthly from November 2017 to January 2018. Sediment samples were collected at each station three times using stainless steel quad-rats, and clams were categorized by size (<5 mm, 5–10 mm, 10–20 mm, 20–30 mm, and >30 mm). The results revealed spatial and temporal variations in clam density, with higher concentrations observed closer to the tidal waterline. Clam size distribution also differed across stations, with smaller clams dominating certain areas. The mesh installation in November 2017 did not significantly affect clam density compared with the control group. However, clam density substantially increased in December 2017 and January 2018 because of mesh installation, especially at the lowest station. These findings suggest that installing fiberglass mesh can enhance the survival of newly settled Manila clams, providing a potential solution to the seed shortage the local clam industries face. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are implicated in various pathological conditions due to their ability to induce oxidative damage to cellular components. In this study, we investigated the antioxidant properties of a peptide isolated from the hydrolysate of Manila clam (Ruditapes philippinarum) muscle. Purification steps yielded RPTE2-2-4, exhibiting potent scavenging activities against DPPH•, HO•, and O2•−, akin to Vitamin C. Structural analysis showed that the isolated peptide, LFKKNLLTL, exhibited characteristics associated with antioxidant activity, including a short peptide length and the presence of aromatic and hydrophobic amino acid residues. Moreover, our study demonstrated the cytoprotective effects of the peptide against H2O2-induced oxidative stress in HepG2 cells. Pretreatment with the peptide resulted in a dose-dependent reduction in intracellular ROS levels and elevation of glutathione (GSH) levels, indicating its ability to modulate cellular defense mechanisms against oxidative damage. Furthermore, the peptide stimulated the expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), further reinforcing its antioxidant properties. Overall, our findings highlight the potential of the Manila clam-derived peptide as a natural antioxidant agent with therapeutic implications for oxidative stress-related diseases. Further investigation into its mechanisms of action and in vivo efficacy is warranted to validate its therapeutic potential. [ABSTRACT FROM AUTHOR]

Stutzerimonas kunmingensis TFRC-KFRI-1, isolated from the gut of Manila Clam in the sediment of the West Sea of Korea, was investigated for its potential as a probiotic bacterium. This strain, belonging to the family Pseudomonadaceae, was previously classified as Pseudomonas kunmingensis but later reclassified to the genus Stutzerimonas, known for species with bioremediation and probiotic properties. To evaluate its genomic features and potential applications, we performed draft-genome sequencing and analysis. The genome of S. kunmingensis TFRC-KFRI-1 was assembled into a 4,756,396 bp sequence with a 62.8% GC content. Genomic analysis suggested potential genes for carbohydrate degradation and lactic acid production. The strain exhibited high average nucleotide identity (ANI) and 16S rRNA similarity with S. kunmingensis HL22-2T, further supporting its potential as a probiotic. This genome sequence provides valuable insights into the functional capabilities of S. kunmingensis TFRC-KFRI-1 and its potential applications in various industries, including aquaculture and food biotechnology. The genome sequence is available under GenBank accession number JBGJJB000000000.1, with related project information under BioProject PRJNA1147901 and Bio-Sample SAMN43173893.

Abstract Background The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam’s microbial communities. Results Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments’ chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities. Conclusions Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon.

Background: Semi-enclosed bays are important links in the material cycle and energy flow between terrestrial and marine ecosystems. They are also areas of intense human activities and heavily influenced by such activities as aquaculture, industrial and agricultural wastewater discharge. The rate of biodiversity loss and changes in ecosystems have prompted research into the relationship between species diversity and ecosystem functioning. Studies have shown that application of functional diversity indices is useful for assessing the status of ecosystem functioning. We quantitatively sampled macrofauna in a semi-enclosed bay in four seasons and analyzed the relationship between species, functional diversity and secondary production, biomass and feeding functional groups of macrofauna. Results: The annual secondary production was 325.01 kJ m−2 year−1. Detritivorous, carnivorous and planktophagous feeders were the main functional groups of macrofauna. Differences in the spatial–temporal distribution of functional groups were influenced by Ruditapes philippinarum and Hemileucon bidentatus. Functional richness had significant negative correlations with macrofaunal biomass and secondary production. Functional divergence, functional dispersion and Rao's quadratic entropy had highly significant negative correlations with macrofaunal biomass and secondary production. Conclusion: The results showed that high overlap of ecological niches can increase competition for habitat resources, leading to a decline in biomass and secondary production. In addition, aquaculture could promote the use of habitat resources to some extent, while it could increase competition for ecosystem resources (including habitat resources, atmospheric resources, water resources, etc.). Functional diversity is a good indicator for the ecosystem functioning and the competition status for habitat resources, which can provide insights into the current state of ecosystem function. [ABSTRACT FROM AUTHOR]

In the last few decades, there has been a growing interest in understanding the behavior of personal care products (PCPs) in the aquatic environment. In this regard, the aim of this study is to estimate the accumulation and effects of four PCPs within the clam Ruditapes philippinarum. The PCPs selected were triclosan, OTNE, benzophenone-3, and octocrylene. A progressive uptake was observed and maximum concentrations in tissues were reached at the end of the exposure phase, up to levels of 0.68 µg g−1, 24 µg g−1, 0.81 µg g−1, and 1.52 µg g−1 for OTNE, BP-3, OC, and TCS, respectively. After the PCP post-exposure period, the removal percentages were higher than 65%. The estimated logarithm bioconcentration factor ranged from 3.34 to 2.93, in concordance with the lipophobicity of each substance. No lethal effects were found although significant changes were observed for ethoxyresorufin O-demethylase activity, glutathione S-transferase activity, lipid peroxidation, and DNA damage. [ABSTRACT FROM AUTHOR]

Previously, we have prepared clam peptides (RBPs) that exhibited potential antihypertension and kidney protective effects in SHR rats by fermentation of Ruditapes philippinarum with Bacillus natto. In this study, we established an adenine-induced chronic kidney disease (CKD) in SD rats and investigated the improvement effects of RBPs on CKD. The results showed that compared with the model group, 8 weeks gavage of RBPs restored the structure of the kidney and reduced serum levels of CKD-related biochemical indicators, including NGAL, CR, Ca, UA, TG, IP, L-FABP, BUN, CHO, and cystatin C (p < 0.05). RBPs treatment also reduced serum levels of MDA, IL-1β, TGF-β1, and TNF-α and increased those of CAT and T-AOC (p < 0.05). In addition, the results of intestinal microbiota analysis showed that RBPs improved the intestinal flora composition by downregulating the proportion of Firmicutes and Bacteroidetes, decreasing the relative abundance of Romboutsia, Ruminococcaceae_UCG-005, and Christensenellaceae_R-7_group and increasing that of Lactobacillus and Muribaculaceae (p < 0.05). The results of this study indicated that RBPs can also be used as bioactive substances in marine drugs or functional foods to alleviate CKD. [ABSTRACT FROM AUTHOR]

Studying the morphological features of the invasive spiny file clam Lima lima was conducted for the first time in the expansion channel of the Suez Canal. Morphological descriptions were based on their exterior morphology and number of radial ribs (mean = 18). Various measurements were recorded, and biometric relationships were examined, particularly the correlations between the independent parameter shell length (SL) and various dependent parameters. Notably, the relationships with shell height and shell inflation demonstrated negative allometric growth. Moreover, regression that describes the relationship between shell length (SL) and soft body weight had a coefficient of determination R2= 0.709, showing a high significant correlation and a negative growth. Molecular analysis showed that the mantle had the highest quantity and optimum quality of gDNA, while the muscles had low value of DNA concentration. Integration of the morphological characteristics by molecular examination using DNA barcoding was designed to confirm the identification of the anti-lessepsian migrant clam L. lima collected from the expansion channel of the Suez Canal. [ABSTRACT FROM AUTHOR]

The maintenance of stocks of estuarine species strongly depends on the ability of the species to cope with environmental stress. In NW Spain, commercial clam beds, which are usually co-occupied by the seagrass Zostera noltei , are often exposed to reduced salinity caused by intense rainfalls. Our goals were to evaluate the effects of low salinity events on both juvenile clams and Z. noltei , including their interactions. A mesocosm experiment was performed to simulate three salinity decreases (35–35, 25–10, and 20–5), and several indicators of clam and seagrass performance were measured after 3 and 6 days of exposure and again after a recovery period of 4 days. No differences were observed in the non-native clam Ruditapes philippinarum , while oxygen consumption, clearance rate and growth decreased significantly in the native clams R. decussatus and Venerupis corrugata in response to low salinity stress. Zostera noltei indicators did not vary in response to low salinity exposure, except the sucrose content, which decreased. Moreover, the seagrass buffered juvenile clams from salinity fluctuations in the short term, although the interactions were weak. The species-specific sensitivity to low salinity should be a major concern in future management plans for the shellfish beds in the context of climate change. [ABSTRACT FROM AUTHOR]

The alveolate protist Perkinsus olseni infections impose considerable stresses on the host Manila clam Ruditapes philippinarum, often reducing their tissue weight due to the drain on energy resources. However, the relationship between condition factors and P. olseni infection exhibits inconsistency in different scenarios, as it is influenced by various factors such as environmental conditions, food availability, and reproductive conditions. To gain deeper insights, we surveyed Manila clams occurring on tidal flats in Anmyeondo Island, Cheonsu Bay to assess P. olseni intensity and its effects on the fitness of Manila clams. The Ray's fluid thioglycollate medium assay revealed that P. olseni infection in clams from inner Cheonsu Bay had considerably greater P. olseni intensities in May and September 2021 than those from outer Cheonsu Bay and reference sites (P < 0.05). Despite the high level of P. olseni infection, condition index (CI), a ratio of the flesh weight to the shell weight, in clams from inner Cheonsu Bay was also significantly higher than in clams from outer Cheonsu Bay and reference sites (P < 0.05). A noteworthy finding of this study was the positive association between P. olseni infection intensity and CI in clams, which suggests that despite the high parasite load, clams were able to maintain a higher CI, possibly due to the abundance of nutrients and organic matter present in the inner bay. The P. olseni infection intensities measured in September were significantly higher at most of the sampling sites in the inner and outer bay, which could be associated with the poor health condition of clams during a post-spawning period. These results highlight the complexity of bivalves' ecological relationships in Cheonsu Bay and offer new insights into their adaptability accompanied by other factors to parasite stressors. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we performed a simplified genome sequencing of 301 individuals from 10 populations of Ruditapes philippinarum in coastal China using restriction site-associated DNA sequencing (RAD-seq) technology, and obtained a large number of single-nucleotide polymorphism (SNP) markers of Manila clam. Eighty-eight SNP markers were successfully developed using high-resolution melting (HRM) analysis, and the genetic structure and genetic diversity of two populations were analyzed. SNP markers provide a valuable resource for population and conservation genetics studies of this commercially important species. Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam Ruditapes philippinarum using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of FIS was estimated to be from −0.9643 to 1.0000. The global Fst value was 0.1248 (p < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy–Weinberg equilibrium (p < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species. [ABSTRACT FROM AUTHOR]

Aquatic animals are popular for their unique umami and high-quality protein. However, under the realistic background of increasing marine pollution, whether it affects the aquatic animal tastes, and what the interference mechanism is still remains unknown. Benzo[a]pyrene (B[a]P) is a typical Polycyclic aromatic hydrocarbons (PAHs) with high toxicity. In this study, we investigated the effects of B[a]P (0, 0.8, 4 and 20 μg/L) on the content and taste evaluation of Ruditapes philippinarum taste substances, and clarified the interference mechanism of B[a]P on taste substance metabolisms with transcriptome analysis. The results demonstrated that B[a]P significantly altered the contents and taste activity values (TAVs) of free amino acids (FAAs), 5′-nucleotides, organic acids, flavor peptides, organic bases, sugars and inorganic ions, as well as the gene expressions within their synthesis and decomposition, indicating that B[a]P affected these taste substance contents by interfering with their metabolisms, thereby changing the clam tastes (decreases of umami and sweetness, and increase of bitter taste). This study provided scientific basis for quality assurance of bivalve cultivation and control of marine pollution. [ABSTRACT FROM AUTHOR]

Understanding the density-dependent impacts of an invasive predator is integral for predicting potential consequences for prey populations. Functional response experiments are used to assess the rate of prey consumption and a predator's ability to search for and consume prey at different resource densities. However, results can be highly context-dependent, limiting their extrapolation to natural ecosystems. Here, we examined how simulated habitat complexity, through the addition of substrate in which prey can escape predation, affects the functional response of invasive European green crabs (Carcinus maenas) foraging on two different bivalve species. Green crabs feeding on varnish clams (Nuttallia obscurata) shifted from a Type II hyperbolic functional response in the absence of substrate to density-independent consumption when prey could bury. Green crabs ate few Japanese littleneck clams (Venerupis philippinarum) under all densities, such that no functional response curve of any type could be produced and their total consumption was always density independent. However, the probability of at least one Japanese littleneck clam being consumed increased significantly with initial clam density and crab claw size across all treatments. At mean crab claw size and compared to trials without substrate, the proportion of varnish clams consumed were 4.2 times smaller when substrate was present, but substrate had a negligible effect (1.2 times) on Japanese littlenecks. The proportion of varnish clams consumed increased with crab claw size and were higher across both substrate conditions than the proportion of Japanese littlenecks consumed; however, the proportion of Japanese littleneck clams consumed increased faster with claw size than that of varnish clams. Our results suggest that including environmental features and variation in prey species can influence the density-dependent foraging described by functional response experiments. Incorporating replicable features of the natural environment into functional response experiments is imperative to make more accurate predictions about the impact of invasive predators on prey populations. [ABSTRACT FROM AUTHOR]

The Yellow-Bohai Sea is an important semi-enclosed continental shelf marginal seas with an intensive aquaculture industry in China. The current study analyzed the contamination status and the time variations of paralytic shellfish toxins (PSTs) in shellfish between 2019 and 2020 from the Yellow-Bohai Sea in the Dalian area and estimated the acute health risks to consumers in China. A total of 199 shellfish samples (including 34 Pacific oysters, 25 Mediterranean blue mussels, 34 Manila clams, 36 bay scallops, 34 veined rapa whelks and 36 bloody clams) were analyzed from four representative aquaculture zones around the Yellow-Bohai Sea in Dalian. Among the samples, scallops and blood clams were the shellfish species with the highest detection rate of PSTs (94.4%), and the highest level of PSTs was detected in scallops with 3953.5 μg STX.2HCl eq./kg (μg STX.2HCL equivalents per kg shellfish tissue), followed by blood clams with 993.4 μg STX.2HCl eq./kg. The contents of PSTs in shellfish showed a time variation trend, and autumn was the season of concern for PST contamination in Dalian. For general Chinese consumers, the probability of acute health risks to shellfish consumers from dietary exposure to PSTs was around 13%. For typical consumers in coastal areas of China, especially those with higher shellfish intake, there was an acute health risk associated with exposure to PSTs through shellfish consumption during the occurrence of harmful algal blooms. It is suggested that the government continue to strengthen the monitoring of the source of PSTs and the monitoring of harmful algal blooms and give reasonable advice on shellfish consumption for consumers in coastal areas, such as not eating scallop viscera. [ABSTRACT FROM AUTHOR]

Introduction: The deep-sea environment is always characterized by high hydrostatic pressure, fluctuating temperatures, heavy metals, darkness, and others. The Vesicomyidae inhabit cold seep zones, hydrothermal vents, and other chemically reduced environments. Methods: To enhance the understanding of the adaptation mechanisms of clams in extreme environments, a comprehensive proteomic study was conducted on the cold seep clam Archivesica marissinica and shallow water clam Ruditapes philippinarum. Results: A total of 4,557 proteins were identified from the comparative groups. The Gene Ontology results indicated that the differentially expressed proteins (DEP) for the comparative group Rpgill vs. Amgill were enriched in the nitrogen compound metabolic process, and others. The comparative analysis for Amfoot vs. Amgill and Ammantle vs. Amgill revealed significant enrichment of the differential proteins that were involved in metal ion transport, divalent inorganic cation transport, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the comparative group Rpgill vs. Amgill was significantly enriched in the regulation of actin cytoskeleton, lysosome, and others. The proteins that exhibited differential expression in the cold seep clam’ different tissues were also enriched in important pathways, such as lysosome, fatty acid degradation, nitrogen metabolism. Discussion: The further analysis identified crucial response proteins involved in various biological pathways. For example, the pattern recognition receptors, such as galectin and peptidoglycan recognition protein, participated in recognition of symbiotic microorganisms. The lysosome pathway members, such as cathepsin and saposins, were engaged in the degradation process of symbiont proteins during symbiont digestion. Profilin and gelsolin from actin cytoskeleton pathway might be pressure-related proteins. Furthermore, carbonic anhydrases from nitrogen metabolism KEGG pathway provide inorganic carbon for symbiotic bacteria. Additionally, Mn superoxide dismutase plays a role in the scavenging of superoxide anion radicals and antioxidant activity. Then, arginine kinases facilitate the low temperature adaptation of deep-sea shellfish with its cold adaptation characters. These findings offer novel perspectives on the proteins that are implicated in A. marisica’s response to cold seep environments, thereby contributing to the understanding of deep-sea biological adaptation and the preservation of deep-sea ecosystems. [ABSTRACT FROM AUTHOR]

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a "one parasite-one disease" perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46-84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g-1 of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations. [ABSTRACT FROM AUTHOR]

Aquaculture located in urban river estuaries, where other anthropogenic activities may occur, has an impact on and may be affected by the environment where they are inserted, namely by the exchange of antimicrobial resistance genes. The latter may ultimately, through the food chain, represent a source of resistance genes to the human resistome. In an exploratory study of the presence of resistance genes in aquaculture sediments located in urban river estuaries, two machine learning models were applied to predict the source of 34 resistome observations in the aquaculture sediments of oysters and gilt-head sea bream, located in the estuaries of the Sado and Lima Rivers and in the Aveiro Lagoon, as well as in the sediments of the Tejo River estuary, where Japanese clams and mussels are collected. The first model included all 34 resistomes, amounting to 53 different antimicrobial resistance genes used as source predictors. The most important antimicrobial genes for source attribution were tetracycline resistance genes tet(51) and tet(L); aminoglycoside resistance gene aadA6; beta-lactam resistance gene blaBRO-2; and amphenicol resistance gene cmx_1. The second model included only oyster sediment resistomes, amounting to 30 antimicrobial resistance genes as predictors. The most important antimicrobial genes for source attribution were the aminoglycoside resistance gene aadA6, followed by the tetracycline genes tet(L) and tet(33). This exploratory study provides the first information about antimicrobial resistance genes in intensive and semi-intensive aquaculture in Portugal, helping to recognize the importance of environmental control to maintain the integrity and the sustainability of aquaculture farms. [ABSTRACT FROM AUTHOR]

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a “one parasite-one disease” perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46–84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g−1of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC–MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6 h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72 h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum. [ABSTRACT FROM AUTHOR]

Studies have shown that the shellfish have innate immune system, which is a very important immune form of shellfish, and they rely on the innate immune system to resist diseases. As a transcription factor, Microphthalmia-associated transcription factor (MITF) plays a regulatory role in immune response and the shell color is also an important index for the breeding of excellent varieties of R. philippinarum. The research on immune response mechanism of RPMITFs can provide important reference data for the breeding of excellent clam varieties. In the genome of R. philippinarum, the RPMITF genes family of shell color-related gene family was selected as the target gene of this experiment. There are 12 RpMITF genes named RpMITF1, RpMITF2, RpMITF3, RpMITF4, RpMITF5, RpMITF6, RpMITF7, RpMITF8, RpMITF9, RpMITF10, RpMITF11, and RpMITF12. The open reading frame length is 639, 1233, 996, 1239, 675, 624, 816, 1365, 612, 1614, 1122, and 486 bp, encoding 212, 410, 331, 412, 224, 207, 271, 454, 203, 537, 373, and 161 aa, respectively. The predicted molecular weight range of amino acids is 18.85–62.61 kda, and the isoelectric point range is 5.26–9.44. Real-time quantitative PCR was used to detect the gene expression of RpMITF gene family in hepatopancreas tissues of two populations of Manila clam at 6 time points (0, 3, 6, 12, 24, and 48 h) after Vibrio anguillarum stress. The results show that RpMITF gene family was significantly expressed in hepatopancreas of two clam populations after V. anguillarum stress (P < 0.05). [ABSTRACT FROM AUTHOR]