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Introduction Marine biotoxins are toxic substances that may cause illness and death in marine organisms and humans. This article disseminates the results of a 4-year study on the occurrence of marine biotoxins in raw bivalve molluscs purchased from Polish suppliers. Material and Methods A total of 256 samples of 8 different molluscs species were analysed for the presence of biotoxins using the ELISA method for paralytic shellfish poison, diarrhoetic shellfish poison, and amnaesic shellfish poison. Results The permitted limits of marine biotoxin content were not exceeded in any of the analysed samples and the majority of them were free from these compounds. Conclusion The results of the study indicate that the tested raw bivalve molluscs available in Poland were safe for consumers.

Ukraine has a unique natural resources potential and is one of the world's richest agricultural countries in terms of soil composition and land bioproductivity. More than 2/5 of its agricultural land is located within the steppe zone – a zone of unstable and insufficient humidification. The production of agricultural products under arid conditions largely depends on solving the problem of agricultural land artificial irrigation. Due to this, since the 1960s large-scale construction of amelioration facilities took place in Southern Ukraine, including construction of the Kakhovka Reservoir and the Kakhovka Irrigation System, the largest one in Europe. The quality of water intended for irrigation depends on a water source and internal physicochemical/biological processes observed in the amelioration system. The attached bivalve molluscs present an important biotic component of hydrotechnical amelioration systems. They form dense settlements on solid artificial substrates and are usually considered as a source of biological barriers that should be constantly fought against. However, their filtration and sedimentation role can exert influence on the processes of water self-purification. The study aims at evaluating the role of filtering molluscs fouling process when it comes to treatment of irrigation water from suspensions and removal of some heavy metals from the water column. The research is based on the results of field analysis of deposits sedimentation processes in the water column with the help of sedimentation traps at 5 stations along Kakhovka Main Channel. It was found that the total biomass of settlements of attached bivalve molluscs Dreissena polymorpha and Dreissena bugensis that are present in the channel constitutes about 370 – 463 tons and they contribute to precipitation of 11.6 – 27.5 tons of suspended matter to the bottom sediments during summer months. This process is manifested in a decrease of suspended matter concentration in the water along the channel. Together with suspended matter 1.8 – 3.9 kg of Cr, 0.3 – 0.6 kg of Ni, 0.09 – 0.24 kg of Co, 0.08 – 0.18 kg of Pb, 0.088 – 0,144 kg of Cd also join the bottom sediments every month. Thus, the settlements of molluscs attached to the hydraulic structures can be considered as biological amelioration agents contributing to the improvement of irrigation water quality.

Highly populated coastal environments receive large quantities of treated and untreated wastewater from human and industrial sources. Bivalve molluscs accumulate and retain contaminants, and their analysis provides evidence of past contamination. Rivers and precipitation are major routes of bacteriological pollution from surface or sub-surface runoff flowing into coastal areas. However, relationships between runoff, precipitation, and bacterial contamination are site-specific and dependent on the physiographical characteristics of each catchment. In this work, we evaluated the influence of precipitation and river discharge on molluscs' Escherichia coli concentrations at three sites in Central Italy, aiming at quantifying how hydrometeorological conditions affect bacteriological contamination of selected bivalve production areas. Rank-order correlation analysis indicated a stronger association between E. coli concentrations and the modelled Pescara River discharge maxima (r = 0.69) than between E. coli concentration and rainfall maxima (r = 0.35). Discharge peaks from the Pescara River caused an increase in E. coli concentration in bivalves in 87% of cases, provided that the runoff peak occurred 1–6 days prior to the sampling date. Precipitation in coastal area was linked to almost 60% of cases of E. coli high concentrations and may enhance bacterial transportation offshore, when associated with a larger-scale weather system, which causes overflow occurrence. HIGHLIGHTS Using a grid-distributed hydrological model to assess hydrometeorological conditions in the absence of direct measurements.; Analysis of a previous uninvestigated area.; New discharge threshold determining Escherichia coli concentration peaks found in Central Italy in order to allow the development of an early warning system for risk assessment.

International audience; Nowadays, pesticides and microplastics (MPs) are commonly found in coastal waters worldwide. Due to their widespread use, their persistence and toxicity, they may induce adverse effects on physiology and behaviour of marine organisms such as the Pacific oyster (Crassostrea gigas). This study explored the growth and valve activity of juvenile oysters exposed for 24 days to two frequently detected pollutants in the Pertuis Charentais (South West, France): a herbicide (chlortoluron, 85 µg.L−1) and high-density polyethylene microparticles (HDPE 20-25 µm, 112 MP.mL−1) alone or in combination (cocktail condition; 97 µg.L−1 of chlortoluron + 108 MP. mL−1). The valve activity of juvenile oysters recorded by using a High Frequency and Non-Invasive valvometer (HFNI) was characterized by three parameters: the number of valve micro-closures (VMC), the Valve Opening Amplitude (VOA), and the Valve Opening Duration (VOD). Additionally, daily shell growth and the oyster daily rhythm were assessed. The exposure to MPs of oysters led to a significant increase of VMC and a decrease of VOD and shell growth. The exposure to chlortoluron showed a significant increase of VOA and a decrease of VMC. In combination with MPs, chlortoluron still increased VOA and decreased VMC but also reduced the shell growth. Chronobiological analysis did not reveal any effects on the daily rhythm of both contaminants. This work highlighted significant effects of high environmental concentrations of MPs and Chlortoluron on the behaviour and growth of the Pacific oyster

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

Klebsiella spp. are a major cause of both nosocomial and community acquired infections, with K. pneumoniae being responsible for most human infections. Although Klebsiella spp. are present in a variety of environments, their distribution in the sea and the associated antibiotic resistance is largely unknown. In order to examine prevalence of K. pneumoniae and related species in the marine environment, we sampled 476 batches of marine bivalve molluscs collected along the Norwegian coast. From these samples, K. pneumoniae (n = 78), K. oxytoca (n = 41), K. variicola (n = 33), K. aerogenes (n = 1), Raoultella ornithinolytica (n = 38) and R. planticola (n = 13) were isolated. The number of positive samples increased with higher levels of faecal contamination. We found low prevalence of acquired resistance in all isolates, with seven K. pneumoniae isolates showing resistance to more than one antibiotic class. The complete genome sequence of cefotaxime-resistant K. pneumoniae sensu stricto isolate 2016-1400 was obtained using Oxford Nanopore and Illumina MiSeq based sequencing. The 2016-1400 genome had two contigs, one chromosome of 5,088,943 bp and one plasmid of 191,744 bp and belonged to ST1035. The &beta, lactamase genes blaCTX-M-3 and blaTEM-1, as well as the heavy metal resistance genes pco, ars and sil were carried on a plasmid highly similar to one found in K. pneumoniae strain C17KP0055 from South-Korea recovered from a blood stream infection. The present study demonstrates that K. pneumoniae are prevalent in the coastal marine environment and that bivalve molluscs may act as a potential reservoir of extended spectrum &beta, lactamase (ESBL)-producing K. pneumoniae that may be transmitted through the food chain.

In 2015, tetrodotoxins (TTXs) were considered a potential threat in Europe since several studies had shown the presence of these toxins in European bivalve molluscs. In this study, we investigated the occurrence of TTXs in 127 bivalve samples (mussels and oysters) and in 66 gastropod samples (whelks) collected all along the French mainland coasts in 2017 and 2018. Analyses were carried out after optimization and in-house validation of a performing hydrophilic interaction liquid chromatography associated with tandem mass spectrometry (HILIC-MS/MS) method. The concentration set by European Food Safety Authority (EFSA) not expected to result in adverse effects (44 &micro, g TTX equivalent/kg) was never exceeded, but TTX was detected in three mussel samples and one whelk sample (1.7&ndash, 11.2 &micro, g/kg). The tissue distribution of TTX in this whelk sample showed higher concentrations in the digestive gland, stomach and gonads (7.4 &micro, g TTX/kg) than in the rest of the whelk tissues (below the limit of detection of 1.7 &micro, g TTX/kg). This is the first study to report the detection of TTX in French molluscs.

The objectives of this study were to present data on the presence of Salmonella enterica subsp. enterica and on the enumeration of Escherichia coli and faecal coliforms respectively in different species of bivalve molluscs and seawater and to conduct a retrospective evaluation to assess the capacity of E. coli in molluscs and faecal coliforms and S. enterica subsp. enterica in sea and brackish water to predict the presence of S. enterica subsp. enterica in bivalve molluscs, and therefore, the risk of exposure for consumers. Data were collected from 4972 seawater samples and 5785 live bivalve molluscs samples (2877 Ruditapes philippinarum , 2177 Mytilus galloprovincialis, 256 Chamelae gallina and 475 C. gigas and O. edulis ) collected in the molluscs production area of Ferrara, Northern Italy, from 1997 to 2015. An overall S. enterica subsp. enterica occurrence of 2.2% was reported in water and molluscs, with percentages varying depending on the type of sample and on the classification areas. All the 237 Salmonella strains were identified as genus Salmonella and a total of 53 different serovars were observed. Significant associations between the fecal indicators and presence of S. enterica subsp. enterica were observed both applying EU and USA criteria, but, it should be noted that the EU approach seems to be more stringent achieving the goal of identifying the most critical batches (94 out of the 100) whereas, following the USA approach, a not negligible and higher number of batches compliant for faecal coliforms but contaminated by S. enterica subsp. enterica has to be mentioned. In any case, the faecal indicators E. coli in molluscs and faecal coliforms in seawaters reflect only in part the presence of S. enterica subsp. enterica in molluscs and the consequent potential risk for consumers. Microbiological evaluation of seawaters seems to have a minor impact into the prediction of S. enterica subsp. enterica presence in molluscs.

The Toll/interleukin-1 receptor (TIR) domain has a fundamental role in the innate defence response of plants, vertebrate and invertebrate animals. Mostly found in the cytosolic side of membrane-bound receptor proteins, it mediates the intracellular signalling upon pathogen recognition via heterotypic interactions. Although a number of TIR-domain-containing (TIR-DC) proteins have been characterized in vertebrates, their evolutionary relationships and functional role in protostomes are still largely unknown. Due to the high abundance and diversity of TIR-DC proteins in bivalve molluscs, we investigated this class of marine invertebrates as a case study. The analysis of the available genomic and transcriptomic data allowed the identification of over 400 full-length sequences and their classification in protein families based on sequence homology and domain organization. In addition to TLRs and MyD88 adaptors, bivalves possess a surprisingly large repertoire of intracellular TIR-DC proteins, which are conserved across a broad range of metazoan taxa. Overall, we report the expansion and diversification of TIR-DC proteins in several invertebrate lineages and the identification of many novel protein families possibly involved in both immune-related signalling and embryonic development.

International audience; Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosis, while clearance was observed within 24 h p.i. A recruitment of hemocytes at the injection site was found in mussel PAM, together with marked morphological changes in the volume of muscular fibers, with a recovery of muscle tissue organization after 48 h p.i. A concomitant impairment in the osmoregulatory processes were observed in PAM by an initial inhibition of aquaporins and increased immunopositivity of Na+/K+ ATPase ionic pump, strictly related to the histological alterations and hemocyte infiltration detected in PAM. Accordingly, an intense cell turnover activity was also recorded following the infection event. Overall, results indicated the hemolymph as the system responsible for the physiological adaptations in mussels to stressful factors, such as pathogenicity, for the maintenance of homeostasis and immune defence. Also, the osmotic balance and cell turnover can be used as objective diagnostic criteria to evaluate the physiological state of mussels following bacterial infection, which may be relevant in aquaculture and biomonitoring studies.

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Human-induced climate change and ocean acidification are global environmental phenomena with a common driver: anthropogenic emissions of carbon dioxide. Both processes potentially threaten the Mediterranean bivalve mollusc aquaculture sector, which is economically relevant to several regions and countries. Detrimental effects on bivalve mollusc species might arise from the associated increase in sea surface temperature, pH reduction, higher frequency of extreme climatic events, and possible synergies with other nonclimatic stressors, such as harmful algal blooms and mollusc diseases. This paper presents the results of a questionnaire-based study of Mediterranean bivalve mollusc producers from 12 coastal regions and six countries, the latter including those with the highest production share in the Mediterranean region. This study aims to assess knowledge and perception of threat of climatic and nonclimatic environmental stressors within the Mediterranean aquaculture industry. Furthermore, it collects information about the (geographical) impacts of summer heat waves and ocean acidification. The results suggest that ocean acidification is still a relatively unknown phenomenon and generally poorly understood. Moreover, it is considered a secondary threat compared with other pressures. Summer heat waves are presently perceived as the highest threat, having been observed in a majority of the studied production sites in past years, with effects on seed (spat), adult mortality, and byssus attachment.

The aim of this study was to determine and compare the Most Probable Number (MPN) of Total Coliforms (TC), Escherichia coli and Enterococcus spp. and to characterize the antimicrobial resistance profiles of Enterococcus spp. isolated from oysters collected in the Barra de Guaratiba Mangrove, Rio de Janeiro, Brazil. The enumeration of E. coli has been used to indicate fecal contamination and hygienic-sanitary conditions of bivalve molluscs. Enterococci are capable to transfer several antimicrobial resistance genes to pathogenic bacteria, including those from Gram-negative group. The oysters were bought from local fishermen and a total of 123 individuals were analyzed. The TC, E. coli and Enterococcus spp. MPN mean were 26,300/100 g, 3,260/100 g and 2,820/100 g, respectively. The only correlation found was between TC and E. coli. Two strains of Enterococcus spp. were resistant to three different antimicrobial categories, including a high level resistance to streptomycin. One strain presented intermediate resistance to vancomycin. The E. coli levels exceeded the limits established by international legislation. This microbiological contamination in oysters reflects the water pollution and indicates a probable contamination of other seafood species from this mangrove, which can represent a risk for consumers and a threat to the environment and public health.

Algal biotoxins, chemical compounds produced by some microscopic algae, constitute the phytoplankton. The mussels, feeding on phytoplankton, can accumulate these compounds to become themselves toxic. There have been several cases of food poisoning by consumption of contaminated shellfish. Such food poisoning have pushed our health care system to provide monitoring of shellfish in the framework of the monitoring plans carried out by AASSLL. In this paper we report the results obtained monitoring the presence of ASP (Amnesic Shellfish Poisoning) biotoxins, like domoic acid (DA) and its isomers, produced by Pseudonitzschia algae. The analysis were carried out by using both the HPLC-UV official method and an experimental method performed with a Time of Flight mass spectrometer (ESI-TOF). The 100% of samples analysed by the official method have always been below the limits of sensitivity (except one sample), the 65% of samples analysed by ESI-TOF, showed the presence of domoic acid.

8 páginas, 6 figuras, 3 tablas., Metallothionein (MT) and other biomarker levels were measured in Scrobicularia plana clams to assess pollution of the Guadalquivir Estuary possibly affected by metals released from Aznalcóllar pyrite mine in 1998. After optimizing reagent concentrations for monobromobimane derivatization, MT levels were quantified by reversed-phase high-performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) in heated or unheated digestive gland extracts and compared to those obtained by differential pulse polarography (DPP). MT content assayed by RP-HPLC-FD in unheated samples was higher than that obtained by DPP and correlated better with metals and anti-oxidant activities. MT assay by RP-HPLC-FD in unheated extracts would be preferable for assessing metal pollution, due to its greater sensitivity and specificity. In addition to MT induction, glyoxalase II inhibition was well correlated with metal contents. Our results suggest that metals at the estuary do not originate from Aznalcóllar spill, but from those carried along by the river and deposited at its concave bank., This work was funded by grant CMT2006-08960-C02 from the Spanish Ministry of Education and Science, a grant from the Doñana 2005 Program, and grants CVI-151 and FQM-141 from the Andalusian Research Plan. A.R.-R. had a predoctoral fellowship from the Spanish Ministry of Education and Science.

We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA 100 ) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5 μg of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA 100 , led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations.

Herpes-like viral infections have been reported in different bivalve mollusc species throughout the world. High mortalities among hatchery-reared larvae and juveniles of different bivalve species have been associated often with such infections. The diagnosis of herpes-like viruses in bivalve molluscs has been performed traditionally by light and transmission electron microscopy. The genome sequencing of one of these viruses, oyster herpesvirus 1 (OsHV-1), allowed the development of DNA-based diagnostic techniques. The polymerase chain reaction (PCR) has been used for the detection of OsHV-1 DNA in bivalve molluscs at different development stages. In addition, the PCR used for detection of OsHV-1 has also allowed the amplification of DNA from an OsHV-1 variant. The literature on DNA extraction methods, primers, PCR strategies, and confirmatory procedures used for the detection and identification of herpesviruses that infect bivalve molluscs are reviewed. (c) 2006 Elsevier B.V. All rights reserved.

The aim of the present survey was to investigate the correct enforcement of the Community rules on the labelling and marketing of bivalve and gastropod molluscs retailed in Sardinia, Italy between 2009 and 2013. A total of 1500 packages and labels for live bivalve and gastropod molluscs were considered. A total of 375 labels (25%) presented non-compliance concerning the wrong trade name and additional wrong or missing information. The highest percentage of anomalous labels has been detected in small-scale retail shops (35%) and open-air markets (25%) compared with the big retailing chains (20%). The 5% of packages were not in compliance with the European Community rules on packaging of bivalve and gastropod molluscs. The high percentage of non-compliance with the European regulations on labelling results is a strong limitation for the consumers and highlights the need to improve the control system about labelling of seafood products.

22 pages, 1 figure, 1 plate, 2 tables., The genus Perkinsus includes protistan parasites infecting marine molluscs throughout the world, some of which are associated with mass mortalities. Life cycle involves vegetative proliferation within the host, by which a cell named trophozoite undergoes successive bipartitioning. Other stages have been observed in vitro or in vivo, depending on the species: hypnospore, zoosporangium and zoospore. Molecular taxonomy supports a close affinity between dinoflagellates and Perkinsus spp. Six species of Perkinsus are currently considered valid: P. marinus, P. olseni, P. qugwadi, P. chesapeaki, P. andrewsi and P. mediterraneus. Histology and, above all, incubation of host tissues in Ray's fluid thioglycollate medium (RFTM) are classic diagnostic methods. In addition, more sensitive and quicker molecular diagnostic techniques based on either immunoassays or PCR have been developed for Perkinsus spp. Epizootiological studies have shown a marked influence of water temperature and salinity on P. marinus infection in oysters Crassostrea virginica, thus determining parasite geographical range and temporal disease dynamics (seasonality). In vitro cultures have been established for four Perkinsus spp. Immune response to infection varies depending on host and involves phagocytosis or encapsulation of the parasite cells by host haemocytes. A polypeptide is secreted by clam Tapes philippinarum haemocytes that could kill the parasite. In vitro cultured P. marinus cells secrete proteases that are likely involved in degradation of host tissues. P. marinus can suppress the toxic oxygen radicals produced by host haemocytes. In addition to host death, sublethal effects caused by Perkinsus spp. (reduction of fecundity, growth, and condition) may have significant ecological and economic implications. Various strategies have been assayed to mitigate the consequences of P. marinus epizootics on the oyster industry: modifications of management/culture procedures, selective breeding to obtain resistant oyster strains, and the use of triploid oysters and allochthonous oyster species. Some chemotherapeutants have been proved to inhibit or kill parasite cells in vitro.

To the Editor: Bivalve mollusks (shellfish), such as mussels and oysters, are filter feeders; they concentrate microorganisms of human and animal origin (up to 100×) from the surrounding environment. Several recent reports have linked the incidence of human infection with hepatitis E virus (HEV) to consumption of undercooked pork, game products, and shellfish (1,2). Infectious HEV has been found in swine manure and wastewater (3); therefore, application of manure to land and subsequent runoff could contaminate coastal water, leading to contamination of shellfish and, subsequently, possible human infection. Because they are filter feeders, bivalve mollusks are biologically relevant sentinels and can indicate potential pathogens that are contaminating the environment. It is essential to ensure that this sustainable resource of coastal areas, where mussels and oysters are farmed or collected wild, is not subjected to environmental contamination that could lead to public health risks. Risk management for bivalve mollusks, aimed at control of fecal pollution, relies heavily on the use of Escherichia coli as an indicator of fecal (sewage) contamination and is enacted under European food regulations (Regulation 854/2004, www.cefas.co.uk/media/455777/extract_reg_no_854_2004.pdf). However, although these regulations probably reduce the number of infections, especially bacterial infections, they are not viewed as adequately controlling the risk for viral infections. Specific risks are posed by the robustness of viruses in the environment and the different behavior of viruses within bivalve mollusks compared with behavior within bacterial fecal indicators. HEV is deemed to be inactivated during processing procedures used to prepare mussels for consumption; however, HEV is only 50% inactivated at 56°C and 96% at 60°C for 1 hour, it is stable when exposed to trifluorotrichloroethane, and it is resistant to inactivation by acidic and alkaline conditions (4). Most shellfish are usually eaten raw, but viable virus can also pose a risk to public health in shellfish that are lightly steamed or preserved by smoking and/or in acetic acid. Indeed, a recent study by the Food Standards Agency, in which >800 oyster samples from 39 growing beds in the United Kingdom were collected and screened during 2009–2011, found norovirus at low levels in at least 76% of oysters (5). Other studies identified hepatitis A virus and norovirus in shellfish production areas and in ready-to-eat products in the United Kingdom (1,6). In fact, depuration experiments demonstrated no decrease in titers against hepatitis A virus over a 23-hour cleansing period (7). In addition, acute HEV infection attributed to consumption of shellfish was diagnosed for 33 passengers who recently returned from a cruise (2). However, data have been restricted to questionnaires implicating consumption of shellfish as a source of transmission; no follow-up analyses of the contaminated foodstuff have been conducted. Thus, possible transmission routes for HEV remain poorly studied in the United Kingdom (2). To determine whether HEV is present in mussels collected locally for human consumption, we examined 48 mussels from 5 tidal locations in Scotland. We collected closed mussels from the west coast of Scotland (11 at Lunderston Bay and 28 at Ardrossan) and the east coast of Scotland (9 at Stannergate, Dundee; Ferryden, Montrose; and the Ythan Estuary at Newburgh). The site at Ardrossan was near a slaughterhouse and a meat preparation purification plant that processes pigs. The plant was considered a potential source of contamination, and mussels were collected in a 10-m2 area around an outfall (drain/sewage pipe) directly in line with the processing plant. A total of 36 (92%) of the 39 mussels from the west coast were positive by PCR for HEV, and 5 (55%) of the 9 from the east coast were positive. The mean value of HEV RNA detected in the samples was 4.25 log10 IU/mL (range 3.73–5.2 log10 IU/mL), and the assay was validated by using the current candidate HEV World Health Organization standard (http://whqlibdoc.who.int/hq/2011/WHO_BS_2011.2175_eng.pdf). Phylogenetic analysis showed that most bivalve mollusk sequences clustered with HEV genotype 3 from humans and swine (Figure; Technical Appendix). Also, HEV sequences isolated specifically from a UK human source corresponded with sequences isolated from the bivalve mollusks. The presence of a swine-like HEV genotype 3 in freshwater bivalve mollusks has also been reported in Japan and South Korea (1,9). Figure Phylogenetic analysis of HEV open reading frame 2 sequences isolated from Mytilus spp. RNA was isolated from 50–100 mg of digestive gland or gill. Tissue was homogenized in 300 μL phosphate-buffered saline, and viral RNA was isolated by ... Worldwide, an estimated 40,000 persons die and another 40,000 experience long-term disability as a result of consuming raw or undercooked shellfish (10). This study, demonstrating the presence of HEV in mussels collected locally in Scotland for human consumption, raises concern as to whether these shellfish are a potential source of infection, as reported (2). The association between environmental contamination with HEV and possible transmission by eating shellfish warrants investigation. Technical Appendix: ClustalW alignment of sequences used to generate the phylogenetic tree in the Figure. Click here to view.(218K, pdf)

Biology and population dynamics of the suspension-feeding wedge clam Donax trunculus (Linnaeus, 1758) were studied for 13 months (November 1994–April 1996) along the Italian Southern Adriatic coast near the Lagoon of Lesina. Specimens were found at depths between 0 and 2 m, mainly in fine grain bottoms. The spatial coastal distribution showed an intraspecific segregation between young and adult wedge clams. A unimodal recruitment (length >4 mm) occurred in winter (December–February). Length frequency distributions were used to determine age and growth rate. Three year classes were regularly observed and their growth pattern defined. The population showed a maximum length of 37 mm and a longevity of 4 years. Analysis of seasonal variations in the reproductive cycle showed that gametogenesis occurred in spring in females. After the spawning season (March–July) females of D. trunculus remained in a resting stage from August to January.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Despite the large number of animal complete mitochondrial genomes currently available in public databases, knowledge about mitochondrial genomics in invertebrates is uneven. This paper reports, for the first time, the complete mitochondrial genome of the grooved carpet shell,Ruditapes decussatus, also known as the European clam.Ruditapes decussatusis morphologically and ecologically similar to the Manila clamRuditapes philippinarum, which has been recently introduced for aquaculture in the very same habitats ofRuditapes decussatus, and that is replacing the native species. Currently the production of the European clam is almost insignificant, nonetheless it is considered a high value product, and therefore it is an economically important species, especially in Portugal, Spain and Italy. In this work we: (i) assembledRuditapes decussatusmitochondrial genome from RNA-Seq data, and validated it by Sanger sequencing; (ii) analyzed and characterized theRuditapes decussatusmitochondrial genome, comparing its features with those of other venerid bivalves; (iii) assessed mitochondrial sequence polymorphism (SP) and copy number variation (CNV) of tandem repeats across 26 samples. Despite using high-throughput approaches we did not find evidence for the presence of two sex-linked mitochondrial genomes, typical of the doubly uniparental inheritance of mitochondria, a phenomenon known in ∼100 bivalve species. According to our analyses,Ruditapes decussatusis more genetically similar to species of the Genus Paphia than to the congenericRuditapes philippinarum, a finding that bolsters the already-proposed need of a taxonomic revision. We also found a quite low genetic variability across the examined samples, with few SPs and little variability of the sequences flanking the control region (Largest Unassigned Regions (LURs). Strikingly, although we found low nucleotide variability along the entire mitochondrial genome, we observed high levels of length polymorphism in the LUR due to CNV of tandem repeats, and even a LUR length heteroplasmy in two samples. It is not clear if the lack of genetic variability in the mitochondrial genome ofRuditapes decussatusis a cause or an effect of the ongoing replacement ofRuditapes decussatuswith the invasiveRuditapes philippinarum, and more analyses, especially on nuclear sequences, are required to assess this point.

Water pollution represents a threat of increasing importance to human health. Bivalve mollusks are filter-feeding organisms that can accumulate chemical and microbiological contaminants in their tissues from very low concentrations in the water or sediments. Consumption of contaminated shellfish is one of the main causes of seafood poisoning. Thus, marine bivalves are normally depurated in sterilized seawater for 48. h to allow the removal of bacteria. However, this depuration time might be insufficient to eliminate chemical contaminants from their tissues. We have developed a novel technology that accelerates up to fourfold the excretion rate of xenobiotics in bivalves by treatment with the antioxidant and glutathione (GSH) pro-drug N-acetylcysteine (NAC) during the depuration period. NAC improved dose-dependently the detoxification of the organophosphate (OP) pesticide fenitrothion in the mussel Mytilus galloprovincialis, diminishing its levels up to nearly a hundred fold compared to conventional depuration, by enhancing the glutathione S-transferase (GST) activity and inducing the GSH anabolism (GSH synthesis and reduction by glutathione reductase). Notably, this induction in GSH anabolism and GST activity was also observed in uncontaminated bivalves treated with NAC. As the GSH pathway is involved in the detoxification of many pollutants and biotoxins from harmful algal blooms, we validated this proof of principle in king scallops (. Pecten maximus) that naturally accumulated the amnesic shellfish poisoning (ASP) toxin domoic acid. We illustrate here a method that enhances the elimination of organic contaminants in shellfish, opening new avenues of depuration of marine organisms. © 2014., S.P-L. was partially supported by a Postdoctoral Fellowship from Generalitat Valenciana (Conselleria de Empresa, Universidad y Ciencia, Spain, CTBPDC/2003/035). E.B. was supported by a pre-doctoral fellowship from University Jaume I. This work has been developed in the framework of the Research Unit of Marine Ecotoxicology IATS (CSIC)-IUPA (UJI) and was supported by a grant from the Spanish Government (Ministerio de Educación y Ciencia, PTR95-0991.OP) to J.B.P. and R.S. and from Generalitat Valenciana as a research group of excellence (PROMETEO/2009/054).

Sapovirus (SaV), etiologic agent of human gastroenteritis, is a RNA virus from the Caliciviridae family, mostly detected in Asia. It has been classified by the U.S. Environmental Protection Agency as an emergent human pathogen with risk for public health in aquatic environments. Bivalves, due to their filtrating feeding, concentrate viral particles presented in seawater, and it has been demonstrated that the bioaccumulated viruses can resist depuration treatments to which bivalves are submitted. In addition, the traditional way of bivalve consumption increases the probability of infection, making them a high risk food [1]. The main objective of this work was to detect and quantify SaV in bivalves from Galicia, Northwestern Spain. A total of 168 samples, including mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and V. decussata) and cockles (Cerastoderma edule) were obtained during a period of 18 months from two Galician Rías (Ría of Ares-Betanzos and Ría of Vigo, respectively at North and South of the region). Samples consisted into 10 or 20 individuals, which were dissected under aseptic conditions to obtain their digestive tissue. The viral RNA was extracted using Nucleospin RNA Virus Kit (Macherey-Nagel; Dürem, Germany). Subsequently, viral detection and quantification by real time RT-PCR was performed using Platinum Quantitative RT-PCR Thermoscript One-Step System Kit (Invitrogen, Saint Aubin, France), according to the recently developed standard method ISO/TS 15216:2013. Primers SaV124F and SaV124R, and probe SaV124TP designed to amplify human genogroups I, II and IV were used [2]. Both through viral RNA extraction and viral detection and quantification, internal controls were used to avoid false positive or negative results, and the possible presence of inhibitors. SaV was detected in 30 of the 168 samples (17,85%). There was no significant difference between the two Rías in terms of detections, although it was observed a seasonality increment of positive samples from November 2011 until April 2012 in both Rías due to the rainy season and the decrease of seawater temperature. Total quantification ranged between 103 and 105 copies of viral RNA/g of digestive tissue (c/g), being mussels the specie with lower main quantification (3,1 x 104 c/g) and clams the species with higher medium rates (2,0 x 105 c/g). This represents the first study out of Japan in which human Sapovirus was detected and quantified into human food intended for consumption.

NOTICE: this is the author’s version of a work that was accepted for publication in Toxicon. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicon, Volume 65, April 2013, Pages 81–89, doi:10.1016/j.toxicon.2013.01.010, The relationship between azaspiracid shellfish poisoning and a small dinoflagellate, Azadinium spinosum, has been shown recently. The organism produces AZA1 and -2, while AZA3 and other analogues are metabolic products formed in shellfish. We evaluated whether mussels were capable of accumulating dissolved AZA1 and -2, and compared the toxin profiles of these mussels at 24 h with profiles of those exposed to live or lysed A. spinosum. We also assessed the possibility of preparative production of AZA metabolites by exposing mussels to semi-purified AZA1. We exposed mussels to similar concentration of AZAs: dissolved AZA1 + 2 (crude extract) at 7.5 and 0.75 μg L−1, dissolved AZA1+2 (7.5 μg L−1) in combination with Isochrysis affinis galbana, and lysed and live A. spinosum cells at 1 × 105 and 1 × 104 cell mL−1 (containing equivalent amounts of AZA1 + 2). Subsequently, we dissected and analysed digestive glands, gills and remaining flesh. Mussels (whole flesh) accumulated AZAs to levels above the regulatory limit, except at the lower levels of dissolved AZAs. The toxin profile of the mussels varied significantly with treatment. The gills contained 42–46% and the digestive glands 23–24% of the total toxin load using dissolved AZAs, compared to 3–12% and 75–90%, respectively, in mussels exposed to live A. spinosum. Exposure of mussels to semi-purified AZA1 produced the metabolites AZA17 (16.5%) and AZA3 (1.7%) after 4 days of exposure, but the conversion efficiency was too low to justify using this procedure for preparative isolation.

The bivalve molluscs represent an important matrix to be studied for several reasons. Their nutritional properties make them valuable to the consumers, so that their consumption and commercial value has risen worldwide. Simultaneously, their significant water-filtering capability and their persistence in the same place make them good bio-indicators of marine ecosystems. The presence of the heavy metal contaminants, as Cd, Pb and Hg, was investigated in bivalve molluscs such as mussels (Mytilus galloprovincialis), clams (Venus gallina) and oysters (Ostrea edulis). In the present study, a survey was carried out on 334 samples addressed to the Istituto Zooprofilattico Sperimentale - Puglia e Basilicata, Foggia (ITALY) between 2009-2011, and collected by official authorities along the coasts of Apulia Region. The conformity of heavy metal content in bivalve molluscs was verified, in according to EC Reg. 1881/2006. The compliance was found for the total amount of samples. The obtained data on heavy metals concentration in bivalve molluscs were compared with data found in monitoring studies on the incidence of heavy metals in 1981 in North-Western Mediterranean Sea, in 2003 in Tyrrhenian Sea and in 2010 in Pacific Ocean (Chile), reported in literature. The information obtained from this work offer an essential database, not only for the authorities involved in food control, but also for the official institutions responsible of a constant control of the marine ecosystem pollution.

Cette etude consiste a evaluer le biotransfert du tributyletain (TBT) par le filtreur bivalve Mya arenaria, a partir d'une culture semi-continue de la diatomee Phaeodactylum tricornutum exposee a un niveau de contamination n'arretant pas sa croissance a long terme. La dose du contaminant employee (100 ng l -1 est de l'ordre de grandeur de celles decelees dans certaines regions contaminees, telles les marinas et les regions portuaires. Dans ces conditions, la culture de P. tricornutum a maintenu son equilibre de croissance durant plus de 50 jours. Sa productivite etait legerement inferieure (de 4 %) a celle de la culture temoin (sans contaminant). L'ingestion des diatomees contaminees, sur une periode de 90 jours, a entraine une bioaccumulation de TBT et DBT (dibutyletain) dans la chair du mollusque, atteignant des concentrations respectives de 133 et 128 ng g -1 (poids humide). L'etude suggere que le biotransfert du TBT et du DBT associes a des micro-algues en croissance continue ou semi-continue pourrait constituer un facteur de bioamplification important du biocide en milieu naturel.

Okadaic acid (OA) and dinophysistoxins (DTXs) are the main toxins responsible for diarrhetic shellfish poisoning (DSP) intoxications during harmful algal blooms (HABs). Although the genotoxic and cytotoxic responses to OA have been evaluated in vitro, the in vivo effects of these toxins have not yet been fully explored. The present work fills this gap by evaluating the in vivo effects of the exposure to the DSP-toxin-producing dinoflagellate Prorocentrum lima during the simulation of an early HAB episode in the mussel Mytilus galloprovincialis. The obtained results revealed that in vivo exposure to this toxic microalgae induced early genotoxicity in hemocytes, as a consequence of oxidative DNA damage. In addition, the DNA damage observed in gill cells seems to be mainly influenced by exposure time and P. lima concentration, similarly to the case of the oxidative damage found in hemocytes exposed in vitro to OA. In both cell types, the absence of DNA damage at low toxin concentrations is consistent with the notion suggesting that this level of toxicity does not disturb the antioxidant balance. Lastly, in vivo exposure to growing P. lima cell densities increased apoptosis but not necrosis, probably due to the presence of a high number of protein apoptosis inhibitors in molluscs. Overall, this work sheds light into the in vivo genotoxic and cytotoxic effects of P. lima. In doing so, it also demonstrates for the first time the potential of the modified (OGG1) comet assay for assessing oxidative DNA damage caused by marine toxins in marine invertebrates. Ministerio de Economía y Competitividad; AGL2012-30897

Azadinium spinosum , a small toxic dinoflagellate, was recently isolated and identified as a primary producer of azaspiracid toxins (AZAs). Previous experiments related to AZA accumulation in blue mussels upon direct feeding with A. spinosum revealed increased mussel mortality and had negative effects on the thickness of the digestive gland tubules. Therefore we conducted follow up experiments in order to study effects of A. spinosum on mussel feeding behaviour. Individual assessment of mussel feeding time activity (FTA), clearance rate (CR), filtration rate (TFR), absorption rate (AR), faeces and pseudofaeces production were carried out on mussel fed either toxic ( A. spinosum ) or non-toxic ( Isochrisis aff. galbana (T- Iso )) diets. Furthermore, AZA accumulation and biotransformation in mussels were followed using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). A. spinosum had a significant effect on mussel feeding behaviour compared to T- Iso : CR was lower by a factor of 6, FTA by a factor of 5, TFR by a factor of 3 and AR even decreased to negative values for the last day of exposure. Even so, a rapid AZA accumulation was observed during the first hours of the trial; less than 6 h of feeding were required to reach AZA concentration in mussel above regulatory level. In consistence with physiological observations, AZA concentration of about 200 μg kg −1 did not increase further until the end of the study. AZA bioconversion was also found to be a fast process: after 3 h of exposure AZA17, -19 and AZA7-10 were already found, with a proportion of AZA17 equal to AZA2. These results show a negative effect of A. spinosum on blue mussel feeding activity and indicate a possible regulation of AZA uptake by decreasing filtration and increasing pseudofaeces production.

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum. Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL−1 of A. spinosum and a mixture of 5000 cells mL−1 of A. spinosum with 5000 cells mL−1 of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL−1 of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules. M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1. Highlights ► Azadinium spinosum was cultured and fed to blue mussels (Mytilus edulis). ► Mussels accumulated azaspiracids in less than 6 h to greater than legal limit. ► Biotransformation of algal toxins into shellfish metabolites was also rapid with >25% metabolites observed after 6 h. ► Detoxification speed was comparable with other lipophilic toxins (half-life ca. 11 days). ► Azaspiracids-6, -17 and -19 should also be considered in legislation.

International audience; Spatial variations in carbon and nitrogen stable isotopes (δ13C and δ15N) of benthic filter feeders were investigated on an inshore-offshore gradient (0-250 km) along the continental shelf of the northern Bay of Biscay (NE Atlantic Ocean). δ13C and δ15N values were measured in muscle tissues of four filter-feeding mollusc species (epifaunal: Pecten maximus, Aequipecten opercularis; infaunal: Glycymeris glycymeris, Venus casina) and in benthic particulate organic matter along a corresponding water-depth gradient from 6 to 220 m. All four species showed a decreasing pattern of muscle δ13C and δ15N values with increasing depth. At the Ushant front (∼130 m water depth), where there is a decrease in both bottom water chlorophyll a and suspended particulate matter, muscle δ13C and δ15N values decreased in all species. Although δ13C values of infaunal clams initially decreased at 30 m depth, δ13C values of epifaunal scallops decreased around 120 m depth, far below the expected depth reduction in microphytobenthic production suggesting that carbon isotopes might not simply track microphytobenthic utilization. The difference between infaunal and epifaunal bivalve stable isotope values may reflect differences in feeding strategies. Muscle δ15N values at the deepest stations (∼2‰) were lower than expected considering the typical trophic enrichment value of 3-4‰ between prey and consumers. These low δ15N values may result from low metabolic rates and suggest the classic trophic enrichment may not hold true in species inhabiting deep waters. Stable isotopes in benthic filter feeders can reveal much information regarding their ecology and environment, but are not straightforward recorders of stable isotope baseline variations as is often assumed.

Different sources were consulted to obtain information on the occurrence of viruses in bivalve molluscs on the European market. Twenty-six peer-reviewed articles were identified reporting on the molecular detection of viral RNA in 4,260 samples in total. The data obtained will be presented geographically on virus types detected, the origin and treatment of the shellfish, and the detection technique applied. The data demonstrate that viral RNA can be detected in shellfish from polluted areas, in depurated shellfish as well as those for human consumption. The European Rapid Alert System for Food and Feed (RASFF) database was consulted as another source. Twenty-eight notifications were identified on the presence of hepatitis A virus or norovirus in shellfish on the European market. The most recent report of the European laboratory network was referred to, to gain insight into the laboratory capability at present for the analyses of shellfish on the presence of viruses. Approximately 67% of 27 participating laboratories obtained intended results for all samples, consisting of lenticules loaded with 103 copies norovirus (genogroup I (GGI) and/or genogroup II (GGII)) and/or 1 × 105–8 × 104 copies of hepatitis A virus. From 1993, there has been a continuous development of molecular detection techniques and tools have been described to ensure quality assurance. End product testing will, however, not be achievable. As depuration has been shown not to be effective for the complete elimination of viruses, shellfish should not be in contact with faecal contaminated water in order to minimise the risk of shellfish-transmittable viral diseases.

1.Freshwater mussels or naiads are generally considered to thrive in river habitats, provided the ecological conditions are good. The presence of populations of these bivalves in artificial channels and ditches with natural bottoms has only scarcely been reported. The aim of this paper was to present the idea that these ‘channel’ and ‘ditch’ habitats could in fact be a sanctuary for naiads.2.Approximately 80 km of several of these waterways fed by the mid Ebro River were sampled in Spain to monitor their naiad populations. Observations indicate that these habitats harbour substantial colonies of freshwater mussels (including two populations of adult specimens of the endangered species Margaritifera auricularia), much more so than the corresponding river.3.The authors wish to alert conservation authorities and freshwater mussel experts to the extreme fragility and importance of this kind of habitat for the long-term conservation of these imperilled molluscs.