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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.

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.

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.

[ES] En este trabajo, enmarcado dentro de la seguridad alimentaria, se propone la determinación de la calidad microbiológica de muestras de alimentos presentes en el mercado. Concretamente, se analizaron muestras de moluscos bivalvos vivos. Se determinó la contaminación de microorganismos indicadores, así como la posible presencia de patógenos, de acuerdo con la legislación española para este tipo de productos. Se analizó también la presencia de patógenos emergentes del género Arcobacter. Las muestras se analizaron además por métodos moleculares, como la reacción en cadena de la polimerasa, PCR, y los resultados se compararon para obtener conclusiones sobre la calidad del producto que llega al consumidor y el método más adecuado para su determinación. Por último, se hizo un breve estudio sobre resistencia a antibióticos de las cepas aisladas., [EN] In this work, part of food security, the determination of the microbiological quality of the food samples on the market is proposed. Specifically, samples are analyzed bivalve molluscs. Pollution indicator organisms was determinate, and the possible presence of pathogens, according to the Spanish legislation for this type of products. The presence of emerging pathogens of the genus Arcobacter was also analyzed. Samples were also analyzed by molecular methods such as chain reaction polymerase, PCR, and results were compared to draw conclusions about the quality of the product that reaches the consumer and the most appropriate method for theis determination. Finally, a brief study on antibiotic resistance of isolated strains was made.

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.

Worldwide bivalve shellfish production reached 14.4 million tons in 2011, of which about 12.9 million tons came from aquaculture; therefore, this sector has an important role in ensuring food to humans and good occupational opportunities. Bivalves are obligate filter feeders, gaining nourishment from microalgae, bacteria and organic particles present in the surrounding water. In this way, they can also accumulate chemicals, marine biotoxins, bacteria and viruses that are pathogenic for humans. In order to protect consumers, two main actions are applied, although there are some differences depending on the country of production: monitoring of growing areas, and the treatment of bivalves after harvesting. Adequate heat treatments ensure the elimination of viruses and pathogenic bacteria, but they are obviously not applicable to bivalves when they are commercialized alive. In this case, to allow microbial contaminants to be purged, there are two alternatives: natural depuration in less-contaminated areas, the so-called “relaying”; and “controlled depuration” by immersion in tanks containing clean seawater, which is also known as purification. Depuration is the preferred option for bivalve shellfish sold alive, and is practiced extensively throughout the world, including Europe, North America, Asia and Australia; however, it is generally accepted that the results of this treatment are only adequate for shellfish with moderate levels of fecal contaminants, as it has very limited effectiveness on viruses and marine bacteria, such as Vibrio spp. This represents a criticism of the assurance of food safety, given that viruses, particularly those in the genus Norovirus (NoV) and Hepatitis A virus (HAV), as well as pathogenic vibrios, such as V. parahaemolyticus and V. vulnificus, are the most common illness-causing agents when shellfish are consumed raw, lightly cooked or have been cross-contaminated. This chapter presents an overview of the state-of-the-art analyses for the purification process, including our industrial research experiences, the sanitary relevance of the most threatening contaminants of bivalves, NoV, HAV, V. parahaemolyticus and V. vulnificus, and the advances in analytical methods for the control of such pathogens.

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2014-10-03T17:24:18Z No. of bitstreams: 1 dissertação completa corrigida_Isa _versão cd.pdf: 2410357 bytes, checksum: 77ffa404390e7378887c7388dd9bcce7 (MD5) Approved for entry into archive by Fatima Cleômenis Botelho Maria (botelho@ufba.br) on 2014-10-06T15:28:52Z (GMT) No. of bitstreams: 1 dissertação completa corrigida_Isa _versão cd.pdf: 2410357 bytes, checksum: 77ffa404390e7378887c7388dd9bcce7 (MD5) Made available in DSpace on 2014-10-06T15:28:52Z (GMT). No. of bitstreams: 1 dissertação completa corrigida_Isa _versão cd.pdf: 2410357 bytes, checksum: 77ffa404390e7378887c7388dd9bcce7 (MD5) CNPq, CAPES, FAPESB e PETROBRÁS O objetivo deste trabalho foi avaliar a distribuição de elementos essenciais e não essenciais em amostras de quatro espécies de moluscos bivalves e de sedimentos coletadas na Baía de todos os Santos (BTS), Bahia, Brasil, após digestão em forno de micro-ondas com cavidade. As amostras foram coletadas em sete localidades, entre setembro de 2010 e maio de 2011. As amostras foram analisadas para As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V e Zn por espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES) e espectrometria de massa com plasma indutivamente acoplado (ICP-MS ). Um procedimento de digestão assistida por micro-ondas otimizado, utilizando ácido nítrico diluído, foi utilizado para digerir as amostras de moluscos bivalves. As amostras de sedimentos foram digeridas em forno de micro-ondas seguindo o procedimento da Agência de Proteção Ambiental, EPA 3051A. A acumulação de metais em tecidos moles dos moluscos bivalves foi dependente da espécie. As amostras da espécie Lucina pectinata apresentaram as maiores concentrações de Cd, Cu e Pb quando comparadas com as espécies Anomalocardia brasiliana, Iphigenia brasiliana e Trachycardium muricatum. Todas as espécies apresentaram concentrações de As e Cr acima do limite máximo tolerável, especificados nas normas brasileiras. A análise exploratória dos dados por HCA e PCA mostrou uma tendência de formação de agrupamentos e caracterizações distintas entre as amostras das espécies Lucina pectinata e Trachycardium muricatum. Os resultados obtidos para as amostras de sedimentos indicaram que para a maioria dos elementos, as maiores concentrações foram encontradas na localidade de Tainheiros, sendo que todos os valores encontrados estão abaixo dos limites estabelecidos pela legislação americana e canadense. Este trabalho contribuiu para o fornecimento de informações inéditas sobre as concentrações de elementos essenciais e não essenciais nas espécies Lucina pectinata, Iphigenia brasiliana e Trachyrcardium muricatum amostradas em diferentes regiões da BTS. Além disso, foram obtidos dados complementares sobre a espécie Anomalocardia brasiliana e sedimentos, contribuindo assim para o monitoramento da região. The aim of this study was to evaluate the distribution of essential and nonessential elements in samples of four species of bivalve molluscs and sediments collected in the Todos os Santos Bay (TSB), Bahia, Brazil, after digestion in microwave oven cavity. The samples were collected from seven sites in the Todos os Santos Bay, Brazil, between September of 2010 and May of 2011. The samples were analyzed for As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn by inductively coupled plasma-optical emission spectrometry (ICP OES) and inductively coupled plasma mass spectrometry (ICP-MS). An optimized microwave-assisted digestion procedure, using nitric acid diluted, was used to digest the bivalve molluscs samples. The sediment samples were digested in a microwave oven following the procedure of the Environmental Protection Agency, EPA 3051A. Accumulation of metals in bivalve mollusks tissues was specie-dependent. The samples of the species Lucina pectinata showed the highest concentrations of Cd, Cu and Pb. All species showed As and Cr concentrations higher than the maximum tolerable limit, specified in the Brazilian regulation. The exploratory data analysis by HCA and PCA showed a tendency to form two groups between samples of the species Lucina pectinata and Trachycardium muricatum. For the most elements in the results obtained for sediment samples, the highest concentrations were found in Tainheiros, and the values obtained are below the limits set by the Canadian and U.S. law. This work contributed to provide new information about the concentrations of essential and nonessential elements in Lucina pectinata, Iphigenia brasiliana and Trachyrcardium muricatum species sampled in different regions of the TSB. Furthermore, additional data were obtained for Anomalocardia bras

Edible lamellibranch molluscs can be involved in foodborne disease and infections of varying severity. They are filter feeding animals able to retain and concentrate in their organism bacteria, parasites, viruses and biotoxins marine algae present in their external environment. Major shellfish harvesting and relaying areas from different areas in Sardinia region were defined and studied by analysing different physicochemical parameters in the water and the levels of Escherichia coli (E. coli), Norovirus (NoVs) genogroup I (NoVGI), NoVs genogroup II (NoVGII) and hepatitis A virus (HAV) in the shellfish harvested and farmed from 2009 to 2011. During that period the identification of the viral agents was carried out by one step real-time reverse transcriptase-polymerase chain reaction and Escherichia coli according to ISO TS 16649-3:2005 standard method. A total of 1266 shellfish samples were tested for NoVGI, NoVGII, HAV and faecal indicators. Norovirus contamination was found in 337 samples (26.6%); only one sample of mussels was positive for HAV (0.08%); while E. coli prevalence was 3.8% in shellfish. The probability of observing shellfish samples positive for NoVs, HAV and E. coli presence was associated with harvesting, growing and relaying areas, period of sampling, environmental parameters, animal species (P

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.

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2014-09-05T14:43:24Z No. of bitstreams: 1 Tese de Doutorado Gabriel Luiz dos Santos.pdf: 1820652 bytes, checksum: 813a41cbb9ae01d5c374c54cce45816c (MD5) Approved for entry into archive by Fatima Cleômenis Botelho Maria (botelho@ufba.br) on 2014-09-05T16:01:28Z (GMT) No. of bitstreams: 1 Tese de Doutorado Gabriel Luiz dos Santos.pdf: 1820652 bytes, checksum: 813a41cbb9ae01d5c374c54cce45816c (MD5) Made available in DSpace on 2014-09-05T16:01:28Z (GMT). No. of bitstreams: 1 Tese de Doutorado Gabriel Luiz dos Santos.pdf: 1820652 bytes, checksum: 813a41cbb9ae01d5c374c54cce45816c (MD5) CNPq O presente trabalho realizado em amostras coletadas na Baía de Todos os Santos, Bahia, Brasil, propõe estratégias para determinação multielementar e análise de especiação de vanádio em amostras de moluscos bivalves e sedimentos, empregando ICP OES e ICP-MS. Na primeira parte da tese, foram avaliadas as potencialidades de dois espectrômetros de massa com plasma indutivamente acoplado para determinação de As, Cr, Ni, Se e V em amostras de moluscos bivalves. Para efeito de comparação, três materiais de referência certificados (CRM) foram analisados: NIST 1566b SRM Oyster tissue, NRCC TORT-2 Lobster Hepatopancreas e NIST 2977 SRM Mussel tissue. A condição recomendada, que consistiu na determinação no modo padrão com adição de padrão interno, foi aplicada na determinação dos analitos em amostras de Macoma constricta coletadas em diferentes regiões da BTS. Os resultados apontaram que a maioria dos níveis de As, Cr, Ni, Se e V presentes na espécie Macoma constricta apresentaram valores acima dos níveis estabelecidos pela legislação vigente brasileira que são: 1,0 (As); 0,1 (Cr); 5,0 (Ni); 0,3 (Se) e 0,77 (V) g g-1 (peso úmido), o que representa risco à população e ao ecossistema. Na segunda parte da tese, foram investigadas as condições para a análise de especiação de vanádio em amostras de sedimentos, empregando extração alcalina e determinação por ICP OES. A concentração de V(IV) foi determinada por diferença, considerando a determinação de vanádio total, também realizada por ICP OES, após digestão das amostras com ácido nítrico em forno de micro- ondas com cavidade, usando o procedimento EPA 3051A. O procedimento foi validado através de testes de adição e recuperação e de análise do seguinte material de referência certificado: NIST 2702 (Inorganics in marine sediment). Pelos resultados obtidos pode-se concluir que os compostos contendo V(IV) são mais dominantes nas amostras dos sedimentos analisados. The present work conducted on samples collected in Todos os Santos Bay, Bahia, Brasil, proposes strategies for multielement determination and speciation analysis of vanadium in bivalve molluscs and sediments samples, using ICP OES and ICP-MS. In the first part of the thesis, the potential of two inductively coupled plasma mass spectrometers for determination of As, Cr, Ni, Se and V in bivalve molluscs samples were evaluated. For comparison, three certified reference materials (CRM) were analyzed: NIST 1566b SRM Oyster tissue, NRCC TORT-2 Lobster Hepatopancreas e NIST 2977 SRM Mussel tissue. The recommended condition, which consisted in determining the standard mode with addition of internal standard, was applied in the determination of analytes in samples of Macoma constricta collected in different regions of the BTS. The results showed that most levels of As, Cr, Ni, Se and V present in the species Macoma constricta have values above the levels established by the Brazilian legislation, that are: 1.0 (As); 0.1 (Cr); 5.0 (Ni); 0.3 (Se) and 0.77 (V) g g-1 (wet weight), which represents risk to the population and the ecosystem. In the second part of the thesis, the conditions for speciation analysis of vanadium in soil and sediment samples were investigated, using alkaline extraction and determining by ICP OES. The V (IV) concentration was determined by difference, considering the total vanadium determination, also performed by ICP OES, after the samples digestion with nitric acid in cavity microwave oven, using the procedure EPA 3051A. The procedure was validated through addition and recovery tests and analysis of the following certified reference material: NIST 2702 (Inorganics in marine sediment). From the obtained results it can be concluded that compounds containing V (IV) are majority in the sediment samples analyzed.

No abstract

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.

Spatial variations in carbon and nitrogen stable isotopes (delta C-13 and delta N-15) 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). delta C-13 and delta N-15 values were measured in muscle tissues of four filter-feeding mollusc species (epifaunal: Pecten maxim us, 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 delta C-13 and delta N-15 values with increasing depth. At the Ushant front (similar to 130 m water depth), where there is a decrease in both bottom water chlorophyll a and suspended particulate matter, muscle delta C-13 and delta N-15 values decreased in all species. Although delta C-13 values of infaunal clams initially decreased at 30 m depth, delta C-13 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 delta N-15 values at the deepest stations (similar to 2 parts per thousand) were lower than expected considering the typical trophic enrichment value of 3-4 parts per thousand between prey and consumers. These low delta N-15 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.

Um levantamento sobre o nível de poluição dos Rios Cachoeira e Santana (Ilhéus, Bahia, Brasil) foi realizado durante um trimestre, por meio da avaliação da qualidade microbiológica da água e de frutos do mar (Crassostrea rhizophorae - ostra-do-mangue e Tagelus plebeius - moapem) extraídos desses rios. Tradicionais indicadores de poluição como coliformes totais (Ct) e coliformes termotolerantes (CT), além de contagem padrão de microrganismos, com isolamento e identificação das enterobactérias, foram determinados nas amostras dos rios, os quais exibiram diferentes níveis de poluição fecal. Foram isolados 68 microrganismos, distribuídos em dez espécies, dentre elas, Salmonella typhi, Escherichia coli e Shigella sp., alertando para o risco do consumo in natura desses moluscos, mesmo quando em conformidade com a legislação estabelecida para CT. A prospecção de moluscos para o acompanhamento dos índices de poluição não se mostrou efetiva considerando a pesquisa de CT em ostras e moapens. Verificou-se também o maior grau de contaminação no Rio Cachoeira, o qual serve de base para distribuição de água na região e como fonte de subsistência, por meio da atividade extrativista, para a população ribeirinha. A survey about the pollution level of the Cachoeira and Santana Rivers (Ilheus, Bahia, Brazil) was performed for a quarter, through the assessment of the microbiological quality of water and seafood (Crassostrea rhizophorae - oyster and Tagelus plebeius - razor clam) extracted from these rivers. Traditional indicators of pollution such as total coliforms (Ct) and thermtolerant coliforms (CT), and total count of micro-organisms, with isolation and identification of Enterobacteriaceae were determined in rivers samples, which exhibited different levels of faecal pollution. An amount of 68 micro-organisms was isolated, distributed in ten species, among them, Salmonella typhi, Escherichia coli and Shigella sp., warning about the risk of consuming these shellfish in nature, even when there is accordance with the laws established for CT. The bivalve prospection to monitor pollution levels was not effective considering CT detection in oysters and moapens. There was also the highest contamination degree in the Cachoeira River, which is used for water distribution in the region and as livelihood source through extractive activities for the riverside population.

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.

Made available in DSpace on 2014-06-11T19:26:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-05Bitstream added on 2014-06-13T19:54:17Z : No. of bitstreams: 1 neves_jp_me_rcla.pdf: 9688721 bytes, checksum: e459ad8bf7d442ee8695ccc1b4c3da9f (MD5) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Bivalves fósseis são comuns em rochas siliciclásticas e carbonáticas das formações Teresina e Rio do Rasto (Permiano Médio/Superior), Bacia do Paraná. Entretanto, a gênese das camadas de conchas carbonáticas ainda é pouco conhecida. Cinco camadas de calcários do centro-sul e norte do Estado do Paraná foram selecionados para estudo tafonômico: dois grainstones oolíticos a bivalves (Prudentópolis) e um grainstone a peloides e bivalves com intraclastos e oncoides (Rio Preto) da Formação Teresina; um packstone e um wackestone a bivalves e oncoides (Ribeirão Claro) da Formação Rio do Rasto. As camadas carbonáticas (≤ 45 cm de espessura), estão intercaladas entre rochas pelíticas, possuem contato basal erosivo, quantidade variável de intraclastos pelíticos, valvas caoticamente distribuídas com gradação vertical descontínua e empacotamento denso a disperso. As conchas encontram-se desarticuladas, comumente fragmentadas, às vezes incrustadas por cianobactérias, e correspondem a espécies alóctonas que viviam em substratos distintos. As assembléias de bivalves representam, portanto, tanatocenoses em tempestitos proximais amalgamados, gerados em água rasa com frequentes modificações por bioturbação e baixa taxa de sedimentação. A presente pesquisa corrobora outras evidências de que acumulações de conchas paleozóicas em mares epicontinentais resultaram de história tafonômica complexa com acentuada mistura temporal e espacial dos bioclastos sob influência de tempestades Bivalve fossils are common in siliciclastic and carbonate rocks of the Teresina and Rio do Rasto formations (Middle to Late Permian), Paraná Basin. However, little is known about shell concentrations in carbonates. Five limestone beds in south-central and northern Paraná State were selected for a taphonomic study: two oolite-bivalve grainstones at Prudentópolis and one peloid-bivalve grainstone with intraclasts and oncoids at Rio Preto, from Teresina Formation; one packstone and one wackestone with bivalves and oncoids at Ribeirão Claro, from Rio do Rasto Formation. The limestone beds (≤ 45 cm thick) are intercalated with pelitic rocks; the basal contact is sharp and erosive, with variable amount of pelitic intraclasts. Shells are randomly oriented (many nested/stacked), showing dense to disperse packing, and discontinuous grading. The shells are disarticulated, commonly fragmented, sometimes encrusted by stromatolites, and correspond to allochthonous specimens winnowed from distinct life-substrates. The bivalve concentrations were generated in shallow water settings punctuated by storms, under very low sedimentation rates, with frequent intrastratal bioturbation. Hence, the concentrations are amalgamated proximal tempestites. This work corroborates previous evidences that Paleozoic shell beds from the epeiric seas have complex taphonomic histories and result from strong temporal/spatial mixing of bioclasts under storm influence

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.

Orientador: Regina Maura Bueno Franco Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Moluscos bivalves por serem eficazes organismos filtradores acumulam e concentram substâncias, partículas e até mesmo patógenos em seus tecidos, se os locais onde vivem ou são cultivados receberem cargas de efluentes contaminados. Estes animais atuam como bioindicadores de contaminação fecal e, ao serem ingeridos crus ou mal cozidos, podem transmitir os patógenos acumulados em seus tecidos. Diversos estudos em diferentes países reportaram a presença de protozoários patogênicos como Cryptosporidium e Giardia em moluscos bivalves destinados ao consumo humano, porém, no Brasil estes dados são inexistentes. Diante deste cenário, é interessante e relevante investigar a ocorrência destes protozoários neste tipo de alimento, consumidos em diversas regiões do país. Este estudo teve a finalidade de avaliar a contaminação natural por oocistos e cistos dos protozoários parasitos em moluscos bivalves localizados em um ambiente de estuário do litoral norte do Estado de São Paulo; detectar as formas parasitárias em água de rio e água do mar; verificar se existe correlação entre fatores climáticos, físico-químicos e microbiológicos e a ocorrência natural dos parasitos nestes ambientes aquáticos e verificar se existe correlação entre a qualidade das águas e a contaminação dos moluscos que vivem nesta região. Os exemplares de moluscos foram processados através da homogeneização de tecidos específicos e submetidos a centrífugo-concentração com éter-PBS e as amostras hídricas foram analisadas pela técnica de filtração em membranas. A visualização dos protozoários foi realizada através da reação de imunofluorescência direta e a confirmação da morfologia dos protozoários feita com a utilização de microscopia de contraste de fase e a incorporação de um corante vital (DAPI). Cistos de Giardia foram detectados em 8 das 12 amostras de água do rio e uma amostra continha oocistos de Cryptosporidium. Ambos os patógenos foram detectados na água do mar, onde Cryptosporidium esteve presente em 40,0 % e Giardia em 20,0 % do total amostrado. O pool de homogenados teciduais de brânquias foi positivo para oocistos e cistos em dois períodos, com concentrações de 12/oocistos/animal e 50 cistos/animal. Não houve associação significativa entre os fatores climáticos, físico-químicos e microbiológicos e o número de oocistos detectados na água do rio e na água do mar; para os cistos, foi encontrada uma correlação significativa com coliformes termotolerantes (p = 0,007) na água do mar. Ademais, não foi possível estabelecer correlação entre a qualidade da água e o número de cistos e oocistos detectados nos tecidos das ostras. Esta é a primeira vez que oocistos de Cryptosporidium e cistos de Giardia foram encontrados em ostras de uma região costeira do país e também na América Latina. Estes achados têm importância para a saúde pública, pois refletem a extensão da contaminação dos ambientes e dos alimentos de origem marinha, requerendo a padronização de um controle de qualidade. Palavras-chave: Cryptosporidium spp.; Giardia spp.; moluscos bivalves; Crassostrea rhizophorae; água do mar; água de rio; estuário; esgoto Abstract: Bivalve molluscs are great filter feeders that may accumulate and concentrate substances, particles and even pathogens in their tissues, if the places that they inhabit or are cultivated receive contaminated effluents dispose. These animals, which serve as biological indicators of faecal contamination, may transmit the protozoa accumulated in their tissues, whenever they are ingested in a raw or lightly cooked form. Several studies in different countries reported the presence of parasite protozoa like Cryptosporidium and Giardia in bivalve molluscs destined for human consumption, however, in Brazil, these data are non-existent. For this reason, it is interesting and relevant the investigation of the occurrence of these protozoan in this type of food, consumed in different regions of the country. The aim of this study was to evaluate the natural contamination of oocysts and cysts of parasite protozoan in bivalve molluscs located in an estuarine environment in the Northern coast of São Paulo State; detect this protozoan in river and seawater; verify if there is a correlation between climatic, microbiological, physical and chemical factors with the natural occurrence of parasites in aquatic environments and verify if there is a correlation between water quality and the contamination of bivalve molluscs present at the region. Bivalve molluscs were processed through the homogenization of specific tissues and submitted to centrifugal concentration with ether-PBS, and water samples were analyzed by membrane filtration technique. Protozoa were visualized with direct immunofluorescence antibody test and to confirm the morphology of the parasites, contrast-phase microscopy and the incorporation of a vital dye (DAPI) were used. Giardia cysts were detected in 8 of 12 samples of river water and one sample contained Cryptosporidium oocysts. Both pathogens were detected in marine water where Cryptosporidium were present in 40,0% and Giardia in 20,0% of all samples analyzed. Gill pools homogenates of oysters were positive for oocysts and cysts in two periods with a mean concentration of 12 oocysts/oyster and 50 cysts/oyster. There was no significant association between the climatic, physical, chemical and microbiological factors with the number of oocysts detected in river water and in seawater; for cysts, it was found a high association only with thermotolerant coliforms (p = 0,007) in seawater. Moreover, it was not possible to establish correlation between water quality and the number of oocysts and cysts detected in oysters tissues. This is the first time that Cryptosporidium oocysts and Giardia cysts were found in oysters from the coastal region of Brazil, and it is also the first report in Latin America to date. These findings might be of public health importance, reflecting the extension of the contamination in marine environment and on seafood, requiring a need for quality control standards. Key-words: Cryptosporidium spp.; Giardia spp.; bivalve molluscs; Crassostrea rhizophorae; seawater; river water; estuary; sewage Mestrado Parasitologia Mestre em Parasitologia

The paper assesses methyl and total mercury concentrations in three mollusc species (Perna perna - common mussel, Crassostrea rhizophorae - mangrove oyster, Anomalocardia brasiliana - clam) from two estuaries in Rio de Janeiro State, both impacted by organic matter and heavy metals. Mussels showed higher capacity to accumulate mercury compared to the other mollusc species (oyster, clam). A significant difference was observed between mercury concentration in the female mussel organisms and in males (81 ± 1 µg kg-1 dry wt. and 70 ± 5 µg kg-1 dry wt. respectively) with similar total shell length. No significant difference was observed among the % MeHg in the female (64%) and male (63%) mussel organisms. Even though the feeding habits of the molluscs are similar; mussels presented higher mercury and methylmercury concentrations in their soft tissues (76 ± 7 µg kg-1 dry wt. and 48 ± 5 µg kg-1 dry wt.) than oysters (19 ± 4 µg kg-1 dry wt. and 6 ± 1 µg kg-1 dry wt.). This is possible related to their capacity to select particle size and the composition of the ingested food they assimilate, and also reflects the greater ability of mussels to concentrate and excrete methylmercury and also to reflect their environmental conditions. Mercúrio total e metilmercúrio foram avaliados em três espécies de moluscos (Perna perna - mexilhão, Crassostrea rhizophorae - ostra, Anomalocardia brasiliana - vôngole) provenientes de dois estuários no estado do Rio de Janeiro, impactados por matéria orgânica e metais pesados. O mexilhão foi a espécie que apresentou melhor capacidade para acumular o mercúrio dentre as espécies de molusco estudadas. Foi observada uma diferença significante nas concentrações de mercúrio entre os organismos de mexilhão, fêmeas e machos (81 ± 1 µg kg-1 e 70 ± 5 µg kg-1 em peso seco respectivamente), com comprimento de concha semelhante. Entretanto, não foi observada diferença significante na percentagem de metilmercúrio entre fêmeas (64%) e machos (63%) de mexilhão. Apesar de possuírem o hábito alimentar semelhante, o mexilhão apresentou concentrações de mercúrio total e metilmercúrio superiores (76 ± 7 µg kg-1 e 48 ± 5 µg kg-1 peso seco) às da ostra (19 ± 4 µg kg-1 e 6 ± 1 µg kg-1 peso seco). Este fato pode estar relacionado com diferenças na capacidade em selecionar o tamanho de partículas e os alimentos ingeridos; também, estar refletindo a maior habilidade do mexilhão em concentrar e excretar o metilmercúrio dos seus tecidos, bem como refletindo as condições ambientais.

The macro and micro element contents of soft tissues from Tapes philippinarum, Callista chione, and Mytilus galloprovincialis, either with (CL) or without (SL) shell liquor, were investigated both at the raw and cooked state, to gain information on true (TR) and apparent (AR) retention factors of the mineral component of these bivalve molluscs. On a per-serving basis, SL molluscs were more significant sources of several minerals than their CL counterparts. TR proved to be a more reliable coefficient than AR in the estimation of nutrient composition of cooked molluscs based on data from the raw ones, probably due to the importance of the cooking yield for this kind of food

Orientadores: Solange Cadore, Sergio Luis Costa Ferreira Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica Resumo: A poluição dos mares, baías e estuários provocada pela emissão de efluentes industriais e residenciais contendo íons metálicos é um dos principais fatores que favorece a contaminação dos moluscos bivalves. Esses organismos sofrem de maneira intensa a bioacumulação de íons metálicos, mesmo em baixas concentrações, tornando-se muitas vezes impróprios para o consumo humano e, além disso, podem refletir o grau de contaminação ambiental. Métodos de digestão com aquecimento assistido por microondas (MW) e com bloco digestor (BD) foram desenvolvidos para a determinação de íons metálicos em tecido de moluscos bivalves, por Espectrometria de Emissão Óptica em Plasma com Acoplamento Indutivo (ICP OES). Um planejamento de misturas do tipo simplex-centróide foi utilizado para estudar o efeito dos reagentes HNO3, HCl e H2O2 nos dois métodos. A análise de material de referência certificado de tecido de ostra (SRM 1566b - NIST) em condições instrumentais otimizadas e nos comprimentos de onda selecionados, apresentaram recuperação dos íons estudados entre 90 - 105%, para o método de digestão com aquecimento assistido por MW, e entre 90 - 100% para o método de digestão com aquecimento no BD. A eficiência das digestões foi avaliada pela determinação do carbono orgânico total (TOC) presente na solução, obtendo-se uma variação de 0,05 a 0,64%. A acidez das soluções variaram de 1,3 a 5,6% sendo que as soluções provenientes da digestão em forno de MW apresentaram menores valores comparados com a decomposição no bloco digestor. Usando o modelamento de misturas foi possível ajustar modelos matemáticos e determinar a composição adequada da mistura HCl - HNO3 - H2O2 para a decomposição de tecidos de moluscos bivalves. As proporções dos componentes foram 0,333: 0,333: 0,334 e 0,200: 0,200: 0,600 para digestão assistida por microondas e em bloco digestor, respectivamente. Os métodos empregados permitiram determinar macro e microconstituintes em tecidos de moluscos, constituindo-se em importantes ferramentas a serem empregadas para a solução de problemas ambientais Abstract: The pollution of oceans, bays and estuaries as a result of industrial and domestic effluents that contain metallic ions is one of the most important factors that lead to the contamination of bivalve molluscs. These organisms are greatly affected by the bioaccumulation of inorganic species, even at low concentrations, reflecting the environmental contamination level. Under these conditions the ingestion of this kind of food is not recommended. Digestion methods based on heating in a microwave oven (MW) or with a digester block BD) for the subsequent determination of metallic ions in bivalve tissues by Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) were developed. Mixtures planned using a simplex-centroide program were to study the effect of the reagents HNO3, HCl e H2O2 in both methods. The analysis of a certified reference material of oyster tissue (SEM 1566b - NIST) under the optimized instrumental conditions at the selected wavelengths showed recoveries of 90 -105%, for digestion by MW and of 90 -100%, for digestion by BD. The efficiency of digestion was evaluated by the determination of total organic carbon (TOC) remaining in the solution and values between 0.05 and 0.64% were obtained. The digestion using MW heating showed lower values compared with BD decomposition. Using the modelling of the mixtures it was possible to determine the most adequate composition of HCl-HNO3-H2O2 mixture for the decomposition of bivalve mollusc tissues. The ratios of mixtures were: 0.333:0.333:0.334, for MW digestion and 0.200:0.200:0.600 for BD digestion. The developed methods allowed the determination of macro and microconstituents in mollusc tissues and are important tools for environmental studies Doutorado Química Analítica Doutor em Ciências

Characterization of a novel satellite DNA family from the bivalve mollusc Donax trunculus revealed extensive grouping of monomer sequence variants and formation of subfamilies identified by distinctive combinations of diagnostic nucleotides. In addition to the tandemly organized 169 bp long monomers, at least one subfamily was created through amplification of two adjacent repeats in a higher order register, while sequence rearrangements led to the creation of a complex satellite unit. Irrespective of the differences that exist between the constituent subunits, higher order repeats can also be further divided into variant subgroups. The localization pattern of diagnostic changes indicates that sequence homogenization mechanisms are not only effective in the spread of the higher order repeat as a whole, but also act on a lower scale. Nucleotide diversity analysis of all variants of the entire satellite family revealed the persistence of conserved sequence segments which stands in contrast to the sequence and organizational divergence of various groups, and may indicate functional constraints on the evolution of this satellite.

8 páginas, 1 figura, 3 tablas. VIII Congreso Nacional de Acuicultura: Acuicultura y desarrollo Sostenible (2001, 22-25 mayo, Santander), [EN] We present a histomorphological description of the digestive tract of the oyster Crassostrea angulata (Lamarck, 1819), as well as a histochemical study of its carbohydrate distribution. The study shows that, in general, glycogen is found in the connective tissue, and neutral mucopolysaccharides/glycoproteins and/or acid mucosubstances in the epithelium and basal cell layers. Glycogen was not detected in the digestive gland, possibly because of its mobilization to the connective tissue for reproduction., [ES] Se realiza una descripción histomorfológica del aparato digestivo del ostión Crassostrea angulata (Lamarck, 1819) y se pone de manifiesto la distribución de carbohidratos mediante técnicas histoquímicas. La pauta general observada es la presencia de glucógeno en el tejido conjuntivo y de mucopolisacáridos/glicoproteínas neutras y/o ácidas en las membranas basales y el epitelio. En la glándula digestiva no se observa glucógeno debido, posiblemente, a su movilización hacia el tejido conjuntivo para su posterior uso con fines reproductivos.