Dissertations / Theses on the topic 'Bivalves'

Tese (doutorado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia, Florianópolis, 2010
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Patógenos virais humanos têm sido associados a muitos episódios de gastrenterites, bem como a doenças relacionadas ao consumo de moluscos contaminados. O risco pode ser reduzido por um tratamento destes moluscos visando sua auto-limpeza previamente a sua comercialização. A depuração de moluscos reduz os níveis de microrganismos presentes na carne, diminuindo a chance de uma potencial infecção associada ao seu consumo in natura. Em um sistema de depuração, a água do mar pode ser recirculada pelo menos por 24 horas e, durante este ciclo, a água é tratada química ou fisicamente para eliminar a contaminação por microrganismos. Neste trabalho, um sistema fechado de depuração de moluscos foi testado para eliminar os patógenos virais em ostras. Além disso, a capacidade do cloro livre em inativar esses vírus, bem como a estabilidade viral em água do mar, com e sem radiação U.V. foram investigadas. Para os ensaios de depuração, ostras (Crassostrea gigas) foram artificialmente contaminadas em aquários, contendo água do mar semeada com Vírus da Hepatite A (HAV) e Adenovírus Humano (HAdV5). Em seguida, as ostras foram colocadas no tanque de depuração, e foram analisadas após 48h, 72h e 96h. Em cada amostragem, o trato gastrointestinal das ostras foi homogeneizado e processado visando a eluição das partículas virais. Para os estudos de desinfecção pelo uso do cloro, água do mar natural e artificial foram semeadas com Norovírus Murino 1 (MNV-1), HAdV2 e Poliomavírus JC (JCPyV) e tratadas com uma concentração inicial de cloro livre de 2,5 mg/l, por 60min. Para os ensaios de estabilidade viral em água do mar e desinfecção viral por luz U.V., 300L de água do mar foram semeadas com o HAV, MNV-1 e HAdV2 e tratados com U.V. (36W), em um mini-tanque de depuração, com recirculação por 120h. Um litro de água do mar foi coletado a cada 24h em até 120h e as amostras de água foram tratadas pelo método de floculação com leite acidificado para adsorção e concentração das partículas virais. O monitoramento viral nos tecidos das ostras e água do mar foi avaliado por métodos moleculares (PCR e q(RT)-PCR), e por métodos de cultura celular associados a métodos moleculares ou imunológicos (ICC-PCR, RT-PCR ICC, IFA, Citometria de Fluxo e Ensaio de Placas de Lise). A detecção por PCR, em amostras de ostras, mostrou que o genoma de HAdV5 foi detectado em todos os períodos de amostragem, e o genoma do HAV foi detectado até 72 h. Os testes envolvendo viabilidade viral por ICC-PCR, demonstraram uma inativação viral progressiva, nas ostras, ao longo das 96h de recirculação de água do mar tratada com luz U.V. Nos ensaios de desinfecção por cloro, após 30 minutos de tratamento de água do mar, foi observada uma redução de ~2log10 e ~3log10 para MNV-1 e HAdV2, respectivamente, com base nos resultados q(RT) PCR. Quando a infecciosidade viral foi analisada, uma redução de mais de 4log10 foi observada para MNV-1, enquanto HAdV2 apresentou uma redução de ~ 2.3log10, mantendo-se infeccioso após 60 minutos. JCPyV apresentou em média uma redução de 1,6 log10, quando avaliado por qPCR. Não houve diferenças na cinética de desinfecção viral observadas em água do mar natural e artificial. Para os ensaios de estabilidade viral em água do mar tratada ou não com luz U.V., com base nos resultados q(RT)-PCR, foram observadas cinéticas diferentes para cada vírus em 120 horas de contato. Reduções de ~ 5log10 e 3log10, em 120h, para HAdV2 e HAV, respectivamente; para MNV-1, foi observada uma redução de ~4,5 log10 em 72h sob tratamento com luz U.V. Apesar da detecção do genoma, HAdV2 foi capaz de permanecer infeccioso até 72h, de acordo com resultados de ICC-RT-PCR. Ensaios envolvendo estabilidade viral sem radiação U.V. demonstraram uma redução progressiva da carga viral ao longo das 120h de recirculação de água do mar para os três vírus (~ 2log10 para HAdV2; ~ 2,5 log10 para HAV e ~ 3log10 para MNV-1). Esta cinética diferente provavelmente está associada às espécies de cloro presentes, ao tempo de contato com a radiação U.V. e a características estruturais peculiares de cada um dos vírus. A diminuição natural da carga viral pode ser devido à existência de fatores ambientais, tais como força iônica e compostos encontrados naturalmente na água do mar. Este trabalho confirmou que HAdV é um patógeno viral particularmente resistente e exige mais tempo para inativação. Estes dados serão úteis para a otimização de desinfecção da água nos tanques de depuração de moluscos.
Viruses have been linked to nearly all episodes of gastroenteritis as well as outbreaks of illnesses related to consumption of contaminated shellfish. The risk may be reduced by appropriate treatment following harvesting as well as by depuration, which is a method that reduces the levels of microorganisms present in mollusk meat, decreasing the potential for infections associated with mollusk consumption in natura. In a depuration system, seawater may be recirculated for at least 24h, and during this cycle, the water must be chemically or physically treated to eliminate microbial contamination. In this work, a shellfish depuration closed system was tested to eliminate viral pathogens from oysters. Moreover, the chlorine capability to inactivate these viruses, as well as the viral stability and disinfection in seawater with and without U.V. irradiation were investigated. For depuration assays, oysters (Crassostrea gigas) were first artificially contaminated in aquariums containing seawater seeded with Hepatitis A virus (HAV) and Human Adenovirus (HAdV5). Then, the oysters were placed into the depuration tank, and were harvested after 48h, 72h and 96h. After each sampling, the gastrointestinal tracts were homogenized and the viral particles were eluted. To chorine disinfection assays, natural and artificial seawater were seeded with selected viruses (Murine Norovirus 1, MNV-1; HAdV2; JC Polyomavirus, JCPyV) and treated by adding initial free chlorine concentration of 2.5mg/l for up to 60min. For the stability assays, 300L of natural seawater were seeded with HAV, MNV-1 and HAdV2, and treated by 36W U.V. lamp, into the mini depuration tank, with recirculation, for up to 120h. One liter of viral seeded seawater was harvested every 24h and viral particles were concentrated by flocculation method using skimmed milk. The kinetics of viral decay in oysters and seawater was evaluated by molecular techniques (PCR and q(RT)-PCR), and by cell culture associated with molecular and immunological methods to access the viral infectivity (ICC-PCR, ICC RT-PCR, IFA, Flow Cytometry and Plaque Assay). The molecular detection by PCR for both viruses showed that the presence of HAdV5 genome was positive in all of the sampling periods, and the HAV genome was detected until 72 h. The tests involving viral viability by ICC-PCR, demonstrated a progressive viral inactivation along the 96h of seawater recirculation under U.V. light irradiation. After 30 minutes of treatment of natural seawater a ~2log10 and ~3log10 reduction where observed for MNV-1 and HAdV2, respectively, based on q(RT)PCR results. When viral infectivity was analyzed, a reduction of more than 4log10 was observed for infectious MNV-1, while HAdV2 presented ~2.3log10 reduction, remaining infective viruses present after 60 minutes. JCPyV presented a average reduction of 1,6 log10, analysed by qPCR. No differences in the disinfection kinetics have been observed between natural and artificial seawater. Based on qPCR results, the kinetics observed were different for each virus, reaching, at 120h of contact time, ~5log10 and ~3log10 reduction for HAdV2 and HAV, respectively; for MNV-1, was observed a ~4,5log10 reduction at 72h under U.V. treatment. Despite the genome detection, HAdV2 was able to remain infectious only up to 72h, according ICC-RT-PCR results. Assays involving viral stability without U.V. irradiation, demonstrated a progressive reduction of viral load along the 120h of seawater recirculation for three viruses (~2log10 for HAdV2; ~2,5log10 for HAV and ~3log10 for MNV). This different kinetics is probably associated to the chlorine species present, the contact time with the U.V. radiation and structural characteristic of each virus. The natural decreasing of viral load can be due to the existence of environmental factors, such as ionic strength and compounds naturally found in seawater. This work confirmed that HAdV is a particularly a resistant viral pathogen and requires longer periods for inactivation. These data will be subsequently useful to plan water disinfection in shellfish depuration tanks.

La croissance et la morphologie du test de gigantopecten ont ete suivies et analysees tant a l'echelon de l'individu qu'a celui de la population a l'aide de diverses approches biometriques. La valeur adaptive de certains caracteres morphologiques a ete discutee dans un essai d'analyse morpho-fonctionnelle. La plupart des gigantopecten semblent avoir ete infeodes aux plateformes carbonatees qui ont connu une grande extension pendant les periodes chaudes du miocene inferieur et superieur. Les relations entre les caracteres morphologiques de ces pecten geants et leur environnement ont ete etudiees. Un schema evolutif du phylum, s'appuyant sur le developpement ontogenique des coquilles ainsi que sur les relations morphologie-milieu, a ete propose: il conduit a exclure le sous-genre gigantopecten du genre chlamys et a le replacer dans le genre pecten

Le mollusque bivalve Argopecten purpuratus (Lamarck, 1819), de la famille des pectinidae se rencontre sur les cotes chiliennes depuis Arica jusqu'a Valparaiso. Il présente un intérêt commercial élevé du a une forte demande du marche international. Cependant, la surexploitation ayant entraîné une inquiétante diminution des bancs naturels, des centres d'élevages expérimentaux ont été construits en 1986. Malheureusement, jusqu'a ce jour, les résultats obtenus par ces ecloseries-nourriceries restent faibles en raison de la méconnaissance de divers aspects du cycle biologique de l'espèce. L’objectif de l'étude est de fournir des éléments fondamentaux permettant d'établir les bases biologiques de l'aquaculture de A. Purpuratus. Pour cela, des individus provenant de deux populations différentes: celle de la baie de mejillones et celle de la rinconada sont retenues et 5 aspects sont analyses: la reproduction, l'influence de facteurs environnementaux sur les cycles gamétiques, les caractéristiques biométriques, la morphogenèse des coquilles larvaires et la structure génétique des populations. Les données obtenues révèlent des différences concernant le nombre d'allèles, le polymorphisme enzymatique, le poids du muscle, le déroulement des cycles gamétiques et l'intensité des émissions. Outre une meilleure connaissance de la biologie de l'espèce, ces résultats devraient permettre de choisir les individus servant de géniteurs en écloserie industrielle et de pouvoir ainsi améliorer leurs performances.

Pour comprendre les fluctuations d'abondance de certaines especes marines il est important de ne pas negliger leurs stades larvaires planctoniques. Les representants de ces organismes sont difficiles a identifier car les outils specifiques adaptes sont peu nombreux. L'objet de ce travail est de proposer, dans le cadre du pndr (programme national sur le determinisme du recrutement), une approche methodologique qui facilite la reconnaissance des larves de bivalves. L'espece cible est la coquille saint-jacques, pecten maximus et le site d'etude, la rade de brest (france). La voie suivie est immunologique. Parmi les molecules potentiellement immunogenes, les proteines sont retenues pour induire la production de serum anti-larves de pecten maximus. Ce choix necessite le developpement d'une technique d'extraction proteique adaptee aux echantillons larvaires de bivalves marins. Les extraits proteiques obtenus a partir de differentes especes sont analyses en electrophorese sds-page et sont specifiques. Ils permettent de tester analytiquement, en immunotransfert, la specificite des serums anti-extrait proteique larvaire de ce pectinide. Ces serums reagissent fortement vis a vis de l'extrait proteique de larves de pecten maximus mais presentent egalement de nombreuses reponses croisees vis a vis des extraits proteiques appartenant aux autres especes etudiees (aequipecten opercularis, chlamys varia, ostrea edulis, crassostrea gigas, ruditapes decussatus, ruditapes philippinarum, mytilus edulis). Differentes solutions sont envisagees pour reduire le taux de reponses croisees. L'epuisement des serums contre des extraits proteiques de crassostrea gigas permet d'eliminer la quasi-totalite de ces reponses sans nuire a la reconnaissance des extraits proteiques de pecten maximus. Ces serums epuises sont utilises directement contre des lots de larves de bivalves pour valider leur pouvoir de reconnaissance des larves de pecten maximus. Trois marqueurs secondaires destines a la reconnaissance du couple larve de pecten maximus/anticorps sont testes : la phosphatase alcaline, les billes magnetiques (dynal), la fluorescence (fitc). La fluorescence donne les meilleurs resultats et c'est cette technique qui est retenue et mise en application pour permettre de visualiser les larves de pecten maximus contenues dans des echantillons de plancton en provenance de la rade de brest. Ce dernier aspect de l'etude permet sur une annee, d'identifier des larves de pecten maximus en rade de brest et de retrouver une concordance certaine entre la presence de ces larves et les phases de reproduction des adultes. L'outil immunologique detecte les larves de pecten maximus parmi les autres organismes planctoniques et pourrait etre developpe pour de nombreuses especes a condition de disposer de quantite d'extrait proteique monospecifique suffisante pour induire la production d'anticorps.

Neste estudo, 26 amostras de ostras (Crassostrea gigas) comercializadas na cidade de São Paulo e em alguns pontos do litoral de São Paulo, e 36 amostras de mexilhões (Perna perna) colhidas mensalmente em 3 pontos do litoral de Ubatuba - SP, foram submetidas à pesquisa de vibrios potencialmente patogênicos. As amostras desses moluscos eram submetidas a enriquecimento em água peptonada alcalina sem cloreto de sódio e com 1 por cento de cloreto de sódio, e GSTB. O isolamento foi realizado em ágar TCBS. Colônias sacarose positivas e negativas, sugestivas de espécies de Vibrio foram identificadas presuntivamente em meio de ágar ferro de Kligler, sendo confirmadas através de provas bioqufmicas complementares. Uma parte das amostras de vibrios potencialmente patogênicos isoladas foi submetida ao teste de Dean e teste de alça ligada em íleo de coelhos. Os vibrios potencialmente patogênicos encontrados em amostras de ostras foram V. alginolyticus (81 por cento ), V.parahaemolyticus (77 por cento ), V. cholerae não 0:1 (31 por cento ), V. fluvialis (27 por cento ), V. furnissii (19 por cento ), V. mimicus (12 por cento ) e V. vulnificus (12 por cento ) e em amostras de mexilhões foram V. alginolyticus(97 por cento ), V. parahaemolyticus(75 por cento ), V. fluvialis (47 por cento ), V. vulnificus (11 por cento ), V. cholerae não 0:1 (6 por cento ), V. furnissii (6 por cento ) e V. mimicus (6 por cento ). Observou-se acúmulo de fluido em alça ligada de íleo de coelho entre 0,25 e 0,49 ml/cm em 6,9 por cento das amostras, entre 0,5 e 0,99 ml/cm em 15,6 por cento e maior ou igual a 1 ml/cm em 15,1 por cento , e/ou intestino de camundongos lactentes (Teste de Dean) em 26,6 por cento das amostras testadas, confirmando o elevado potencial desses microrganismos em causar gastrenterite. Verificou-se ausência de variação sazonal e também, de correlação entre os vibrios potencialmente patogênicos isolados e os indicadores de contaminação fecal, confirmando que a presença desses microrganismos ocorre de forma autóctone e que, as condições climáticas foram favoráveis à sobrevivência dessas espécies em todas as épocas do ano. Considerando-se os resultados obtidos no presente estudo e o fato de que ostras e mexilhões são habitualmente ingeridos crus ou insuficientemente cozidos, pode-se concluir que sua ingestão constitui-se em um determinado grau de risco para a saúde do consumidor.
In this work, 26 oysters samples (Crassostrea gigas), found in the market of São Paulo city and some coastal areas of São Paulo State, and 36 mussels samples (Perna perna), that were collected monthly in 3 coastal areas of Ubatuba city - SP., were analyzed for the potential patogenic vibrios occurrence. Samples were enriched in alcalin peptone water with (1 per cent ) and without sodium cloride and GSTB. Isolation was performed on TCBS agar. suspect sacharosis positive and negative colonies, resembling vibrio species, were presumptively identified on Kligler iron agar, and confirmed by complementary biochemical tests. Some of this potential patogenic vibrios were submitted to suckling mouse assay and rabbit ileal loop assay. Potential patogenic vibrios isolated from oyster samples were: V. alginolyticus (81 per cent ), V. parahaemolyticus (77 per cent ), V. cholerae non 0:1 (31 per cent ), V. fluvialis (27 per cent ) I V. furnissii (19 per cent ), V. mimicus (12 per cent ) and V. vulnificus (12 per cent ) and from mussels samples were: V. a.1ginolyticus (97 per cent ), V. parabaemolyticus (75 per cent ), V. fluvialis (47 per cent ), V. vulnificus (11 per cent ), V. cholerae non 0:1 (6 per cent ), V. furnissii (6 per cent ) and V. mimicus (6 per cent ). It was found 6,9 per cent of samples between 0,25 and 0,49 ml/cm of fluid accumulation in ileal loop assay, 15,6 per cent between 0,5 and 0,99 ml/cm and 15,1 per cent was equal or higher than 1 ml/cm. Among the samples assayed for suckling mouse 26,6 per cent were positive. These results confirm the high potential of these microrganisms to induce gastroenteritis. Seasonal variation as well as correlation between the potential patogenic vibrios isolated and the fecal contamination indicators were not found, confirming that the presence of such microrganisms occurs autochthonously and that the climate conditions were favourable to these species survival during the whole year. with the results of this work and considering that oyster and mussels are usually ingested raw or insufficiently cooked, the conclusion is that the ingestion of such mollusks presents a certain degree of risk for the consumer\'s health.

Functional morphology analyzes the relationships between form and function in organisms. However, a comprehensive analysis of any organic structure requires an integrated approach to morphology. For this purpose constructional morphology was developed, where function, phylogeny and construction together explain form. This thesis investigates functional and constructional aspects of gastropods and bivalves; two groups of molluscs which are among the most common shell-bearing invertebrates. Their shell protects the soft parts and different morphologic specializations enhance this function. Morphology and mode of life are often closely coupled.

Comparison of the distantly related cardiid bivalves Cardium costatum and Budmania spp. reveals similar shell modifications. Both have prominent keels functional in anchoring the shell within the sediment. The straight keels in C. costatum indicate an additional strengthening function, whereas the keels in Budmania spp. often are deformed and do not. Other shell modifications include secondary resorption of shell material and hollow keel interiors, reducing shell weight. These similarities are explained by parallel evolution and a common cardiid Bauplan.

Morphological shell characters in cerithiform gastropods have evolved independently in different taxonomic groups and multiple times within the same group. Shell characters are adaptive within five functional areas: defence from shell-peeling predators, burrowing and infaunal life, clamping, stabilization, and righting of the shell. Most characters are made feasible by determinate growth and a count-down programme.

In most environments predators that crush, peel and bore shells are present. As bivalves and gastropods grow by marginal accretion, and are able to replace lost shell material, traces of unsuccessful predation are preserved as scars. Experiments on the snail Nucella lamellosa show that repaired shells are just as strong as shells without damage. However, new scars follow old scar lines in 43% of tested specimens. This might be due to a higher organic content in this area.

The rudist bivalves were one of the many and varied groups of organisms to be extinguished at the end of the Cretaceous Period. They were a group of bivalves which evolved during Late Jurassic times to dominate the carbonate shelves on the margins of the Tethys Ocean during the Cretaceous Period. Through Late Cretaceous times their diversity climbed to a peak and then entered a period of rapid decline, resulting eventually in the complete extinction of the group. Theories as to the cause of that extinction should clearly be based upon a knowledge of the detailed pattern of the decline. Most important is the question of the timing of the extinction: How long did it take from the peak of diversity to the elimination of the entire group? Is there one main extinction event - or are there several - or is the pattern a gradual decline? In answering these questions this work adopts a new approach to dating end Cretaceous strata by using strontium isotope stratigraphy. The method works by measuring the 87 Sr/86Sr of palaeo-seawater preserved in marine carbonate, such as the thick low-Mg calcite layer of rudist shells. The 87Sr/86Sr of seawater was changing fairly rapidly through time in the latest Cretaceous. The pattern of change has been established in detail using samples from Boreal sequences of which the ages are known with respect to the belemnite stratigraphy. Using this as a standard graph, Tethyan rudist samples have been dated by a comparison of the Sr isotope ratio. The use of Sr isotope stratigraphy has enabled a time axis to be established, against which the ranges of rudist bivalves and of their facies have been ploued. From these data it can be clearly seen that the ranges of many of the established biostratigraphic markers are in error and that the stage boundaries, as defined by the belemnite and planktonic foraminiferal stratigraphies, are offseL When the pattern of rudist diversity is plotted against this time scale it can be seen that, at a specific level, the rudists were at their most diverse at the Campanian/Maastrichtian boundary. Their decline lasted until I almost the end of the Maastrichtian after which the only survivors are Tertiary forms. The decline is related to the disappearance of rudist facies with the end Cretaceous regression, though that graph is slightly displaced from that of rudist diversity. This shows that the rudist extinction is not merely due to non-exposure of terminal Maastrichtian strata.

Etude des principaux constituants biochimiques: lipides, cations. Role du glucose et de l'ion k+ dans la physiologie de la reproduction et des ions na+ et mg++ dans le maintien du volume cellulaire. Recommandation de deux indices statique et dynamique de la condition physiologique des adultes et des postlarves. Recommandations concernant l'evolution du cycle reproducteur

The focus of this study was to determine the effect of clam presence and behavior on the crossflow of the ambient horizontal flow and the effect of ambient horizontal flow characteristics influence the clam feeding behavior. Hence, there is a reciprocal relationship between organisms and the physical environment, and this study ultimately addressed the role of hydrodynamics in the predator-prey relationship between bivalve clams, Mercenaria mercenaria, and their predators, blue crabs and whelks. The study concludes that clams alter the chemical odorant source characteristics and control the transmission of the chemical signal through altering the crossflow. The first part of the study is a field experiment designed to quantify the effect of the presence and behavior of clams on the crossflow of the horizontal crossflow. The second part of this study is a two-part laboratory experiment designed to isolate the influence of environmental factors on clam behavior. One experiment quantifies the unsteadiness of the clam excurrent jet velocity time record according to environmental cues such as the horizontal crossflow velocity, the density of the clam patch, and the size of the clam. The second laboratory experiment quantifies the unsteadiness of the jet velocity values according to the presence of predator cues in the upstream flow. Clams are found, using an ADV system in the field, to alter the vertical distribution of velocity according to the sediment in which they are buried. Also, turbulence characteristics, such as Turbulent Kinetic Energy and Reynolds shear stress, are altered in the presence of clams according to the ambient horizontal crossflow velocity and treatment site. The laboratory flume PIV system captured vector plots for two-dimensional planes that bisect the clam excurrent siphons and clam jet velocity time records were extracted. A fractal analysis and a lacunarity analysis of the jet velocity time records found that clams alter their jet excurrent velocity unsteadiness according to the horizontal crossflow velocity. This behavioral change may contribute to the differences in the turbulence characteristics in the field experiment. Another result from the laboratory experiments is that the effect of clam patch density on the feeding activity was dependent on the size of the organism. This size/density dependent relationship suggests that predation by blue crabs dominates the system since larger clams are no longer susceptible to blue crab predation, whereas clams of all sizes are susceptible to whelk predation. Finally, clams increase the randomness of their excurrent jet velocity values when predator cues are located in the upstream flume flow. This suggests that the presence of predators elicits clam behavior that promotes the mixing and dilution of their chemical metabolites.

Mollusc shells are unique adaptations that serve to protect the organisms that make them, and are a defining feature of the phylum. However the molecular underpinnings of shell forming processes are still largely unexplored. To further understand mollusc shell formation, I studied three bivalve species in this project: the blue mussel Mytilus edulis, the Pacific oyster Crassostrea gigas, and the king scallop Pecten maximus. While previous analyses of the shell proteomes showed species specificity, transcriptomes of the mantle tissues revealed more commonalities. To reconcile these differences, I studied differential gene expression in shell damage-repair experiments and during the formation of the first larval shell, to produce a comprehensive overview of shell formation processes. Expression data showed large biological variability between individuals, requiring matched-pair experimental designs to detect differential gene expression during shell repair. Loci differentially expressed during shell repair and in the larvae encoded shell matrix proteins, transmembrane transporters, and novel transcripts. A large number of shell matrix proteins, encoded in differentially expressed loci, were common in all three species during shell formation, indicating that shell forming proteins between different species may be more common than previously thought. Differential expression of transmembrane transporters during shell repair indicated that the animals may be regulating bicarbonate ions during shell formation. Finally, the experiments revealed novel transcripts, with unknown annotations to public datasets, that may putatively be involved in shell formation.

Anomalodesmatans comprise a large, ancient and ecologically diverse group of marine bivalves, but are nonetheless inconspicuous in most extant shallow water communities. For various reasons, which include their present scarcity and a bewildering array of disparate morphologies, representatives of the group have always proved difficult to interpret, and their systematics lagged behind those of most other major bivalve taxa. Most of this dissertation reports the results of a comparative investigation on the shell morphology and anatomy of extant anomalodesmatans, which formed the basis for a reassessment of hypotheses of primary homology established by previous investigators and identification of novel characters for phylogenetic inference. Due to the chief role played by the hinge ligament in authoritative discussions of anomalodesmatan evolution, this organ was chosen as the focus of a more detailed treatment. Discontinuous ontogeny of fibrous ligament is shown to characterise several members of the group, with the implication that, in contrast to the prevailing model,not all anomalodesmatan adult ligaments may be considered homologous. Likewise, a system of multicellular glands concerned with sediment agglutination was studied with particular emphasis because it is both exclusive to and widespread within Anomalodesmata. Evidence of preserved glandular secretion is recorded for the first time in fossil material and the glands themselves found in extant laternulids and pholadomyids, thus considerably expanding their known taxonomic distribution. Finally, this volume also documents the largest cladistic analysis of extant anomalodesmatans performed to date, including morphological data compiled from both original observations and literature accounts. Among traditionally recognised superfamilies, Pholadomyoidea, Clavagelloidea and Septibranchia were found monophyletic. Taxa commonly referred to Pandoroidea and Thracioidea were recovered as part of two new clades, which are also supported by recent molecular studies. Interpreted in the light of the fossil record, reconstructed phylogenetic relationships favour the iterative evolution of shallow infaunal and epifaunal anomalodesmatans from deep-burrowing ancestors over previously advanced patterns for the history of the clade, namely ventral migration of the ligament and irreversible radiations into a deep infaunal life habit.

Human activities and climate change are causing irreversible changes in almost all marine environments. In these changing environments, it is the resilience of organisms that may decide whether they succeed or fail. Marine bivalves are habitat forming organisms, the species they support are intrinsically linked to their distribution. This thesis sought to determine whether habitat forming bivalves will be resilient to stressors at a magnitude predicted for the next 100 years. To answer this overarching question, experiments were done to test hypotheses on how stressors will interact to affect bivalves. Oysters currently exist in Sydney Harbour despite a multiple stressor environment including the influence of anthropogenic pollution and construction. These stressors were found to be causing biological impacts at a local scale and influencing patterns of distribution of both native and invasive oysters in Sydney Harbour. How current and future stressors will interact to impact bivalve populations remains unknown. Climate change is predicted too warm and acidify the oceans, which will potentially exacerbate existing environmental stressors. Separate manipulative experiments found that both pollution from anthropogenic sources, and emersion of oysters at low tide will interact with climate change to have synergistically negative effects on the physiology and reproduction of oysters. Intertidal organisms such as oysters are predicted to be resilient. Deep sea organisms are, however, predicted to have a low capacity for resilience. Manipulative experiments on the deep sea bivalve Acesta excavata found that pollution and climate change will interact to cause negative effects on the physiology and energy budget of A. excavata. These results represent important findings into how oyster and other bivalve populations may behave in a future environment that is heavily influenced by anthropogenic practices.

Haemoglobins (Hbs) are among the most well investigated proteins due to their oxygen carrying capacity. These proteins are found in many different animals where they exhibit extraordinary diversity of form, yet little is known about their distribution, function, and evolution in invertebrate lineages. This study demonstrates that multiple Hb genes are expressed in bivalve molluscs, that some of these duplicated genes show tissue specific expression, and that they may have undergone neofunctionalisation through gene duplication. This study also describes the generation of a comprehensive resource for the bivalve molluscs and the presence of Hb-like encoding genes in these organisms.

La genetique des populations de bivalves marins est caracterisee par deux observations recurrentes: 1) une correlation positive entre l'heterozygotie individuelle et la valeur selective, 2) des deficits en heterozygotes inexpliques au regard du mode de reproduction (fecondation externe, dispersion larvaire). Le polymorphisme genetique des populations naturelles du bivalve spisula ovalis a ete etudie par electrophorese enzymatique pour rechercher l'origine de ces phenomenes. Des correlations positives entre l'heterozygotie et la survie d'une part, entre l'heterozygotie et la croissance d'autre part, ont ete mises en evidence chez cette espece. La comparaison des observations avec les attendus theoriques suggere que les locus enzymatiques utilises sont neutres, et que leur heterozygotie est correlee a celle d'autres locus responsables des variations de croissance et de survie. La structure genetique des populations de s. Ovalis montre des deficits en heterozygotes, ainsi qu'une heterogeneite spatio-temporelle fine. Les deficits en heterozygotes sont variables entre locus et pourraient etre dus a une sous-estimation de l'heterozygotie par la technique d'electrophorese (alleles nuls). La structure genetique fine a ete mise en relation avec la demographie de l'espece, caracterisee par une segregation spatiale entre classes d'age, pouvant restreindre les echanges genetiques

L'ère Cénozoïque est caractérisée par le dernier passage d'un mode climatique "greenhouse" à un mode climatique "icehouse" avec la mise en place de la calotte Antarctique. Les premières traces de glaciations ont pu être mises en évidence à la Transition Éocène-Oligocène (EOT ; ~33.9 Ma) qui est ensuite suivie d'une période d'instabilité de la calotte correspondant à des phases de glaciations et de déglaciations jusqu'à l'établissement de la calotte polaire Antarctique moderne au cours de la Transition Climatique du Miocène Moyen (MMCT ; ~14 Ma). Cependant, les processus à l'origine de ces phases de glaciation-déglaciation restent aujourd'hui mal contraint et la pCO2 comme facteur principal n'explique pas la fonte de la calotte au cours de l'Optimum Climatique du Miocène Moyen (MMCO ; ~16 Ma) avec des pCO2 reconstruites comprises entre 380 et 500 ppm. Cette thèse a pour objectif d'apporter de nouvelles informations paléoclimatiques et paléoenvironnementales au cours de cette période charnière par l'acquisition de nouvelles données de température et de salinité en domaine côtier à l'aide d'une combinaison de traceurs géochimiques, delta(18)O, DELTA(47) et (87)Sr/(86)Sr, mesurés sur des coquilles de bivalves. Le couplage delta(18)O-DELTA(47) a tout d'abord été testé dans des différents types d'environnement allant d'environnements restreints tels que des lagunes et baies à des environnements côtiers ouverts. Les salinités calculées sont cohérentes avec les gammes de tolérance des organismes présents dans les différents faciès rencontrés. Les résultats obtenus montrent que ce couplage delta(18)O-DELTA(47) peut être utilisé avec succès pour déconvoluer l'évolution de la température et du delta(18)O de l'eau de mer locale dans des environnements allant de marin franc à saumâtre. Les résultats en (87)Sr/(86)Sr ont également pu être reliés à l'activité du lessivage continentale et à la salinité lorsque celle-ci est assez basse et correspondant à un environnement saumâtre. Les données acquises montrent une chute de 10°C des températures côtières dans l'est de l'Atlantique Nord (Angleterre) pour l'accident EOT-1 ainsi qu'une baisse de salinité et une modification du réseau de drainage des rivières, davantage affectés par la chute du niveau marin associé à la mise en place de la calotte Antarctique. Une chute de ~8°C a également été mise en évidence dans des environnements marins ouverts dans l'ouest de l'Atlantique Nord (Floride) au cours de l'EOT associée à un contraste saisonnier de température et de salinité plus prononcé. Les données acquises pour le Miocène Moyen a mis en évidence une large gamme d'expression du MMCO, avec un refroidissement de ~10°C en Mer Méditerranée et un réchauffement concomitant de ~6°C dans l'Océan Atlantique Nord pour des sites situés à latitudes similaires mais dans des environnements côtiers et des bassins différents. Ces nouvelles données mettent en évidence la diversité de la réponse locale des environnements côtiers en réponse à un changement climatique global, qui complexifie l'utilisation d'archives provenant de ce type d'environnement pour étudier l'évolution du climat
The Cenozoic era is characterized by the last transition from a "greenhouse" climate mode to an "icehouse" climate mode with the Antarctic ice-sheet build up. The first traces of glaciation are highlighted at the Eocene-Oligocene Transition (EOT; ~ 33.9 Ma) which is followed by a period of instability of the ice-sheet corresponding to glaciation and deglaciations phases until the terminal glaciation of the Antarctic ice cap during the Middle Miocene Climate Transition (MMCT). However, the processes governing these phases of glaciation-deglaciation remain poorly constrained today and pCO2 fluctuations are not sufficient to explain ice-sheet melting during the Middle Miocene Climate Optimum (MMCO) with pCO2 ranging from 380 to 500 ppm. The main aim of this PhD is to provide new paleoclimatic and paleoenvironmental information during this key period, from coastal environments using a combination of different geochemical proxies, delta(18)O, DELTA(47) and (87)Sr/(86)Sr, measured on bivalve shells. The delta(18)O-DELTA(47) coupling was tested in a variety of environments ranging from restricted environments such as lagoon and bay to open coastal environments. The calculated salinities are in good agreement with the tolerance of the organisms present in the different facies encountered. The results obtained with delta(18)O-DELTA(47) coupling can be used successfully to deconvolve the evolution of temperature and local seawater delta(18)O in brackish to marine environments. The (87)Sr/(86)Sr results could also be linked to runoff activity and for low salinities corresponding to a brackish environment. The new data acquiring for the EOT revealed a 10°C drop in temperatures for the EOT-1 accident in the eastern North Atlantic (England) as well as a salinity drop and a river drainage network modification more affected by the sea level fall occurring during the EOT. A drop of ~8°C was also recorded in open marine environments from western North Atlantic (Florida) during the EOT, associated to an increase in seasonal contrast in temperature and salinity. The new data acquired for the Middle Miocene highlight marks differences in the MMCO expression with a ~10°C cooling appearing during this climate optimum in the Mediterranean Sea and a contemporaneous warming of ~6°C in the North Atlantic Ocean for sites located at the same latitude but in different coastal environments and basins. These new data highlight the diversity in the local response of coastal environment in response to global climate change, that impedes the use of archives from this type of environment to study climate evolution

Le but de ce travail de doctorat est d’évaluer la possibilité d’utiliser Haslea ostrearia et la marennine en ostréiculture. Les objectives de ce projet sont : (1) l'évaluation du comportement alimentaire de l'huître creuse Crassostrea gigas sur des cellules d’H. ostrearia de différentes tailles et les conséquences potentielles sur les populations algales; (2) la caractérisation du verdissement par la marennine et ses conséquences sur la physiologie de C. gigas; (3) les conséquences du verdissement sur les traits comportementaux, physiologiques et biochimiques de plusieurs espèces de bivalves; (4) l'utilisation conjuguée d’H. ostrearia avec d'autres microalgues d’importance en aquaculture. Nos résultats suggèrent que la taille des cellules affecte considérablement le processus de selection d’H. ostrearia par l’huître. Cette étude démontre également que la forme extracellulaire de la marenninne contribue significativement au verdissement dans les mucocytes des branchies. Mis à part le verdissement des organes palléaux des bivalves, une concentration modérée de marennine (2 mg L-1) affecte les performances comportementales, physiologiques et biochimiques des bivalves. Néanmoins, ces effets pourraient être compensés par ses activités biologiques comme agent antibactérien naturel et source d’alimentation mixte d’algues en conchyliculture
This Ph.D. thesis focuses on several assessments to achieve the optimum benefit of utilization of marennine in the field of aquaculture. The study covers: (1) the assessment in feeding behavior of the Pacific oyster Crassostrea gigas on different sizes of Haslea ostrearia and its ecological consequence; (2) the characterization of the greening by marennine and its consequences on some physiological traits of on C. gigas. (3) the consequence of greening by marennine on behavioral, physiological and biochemical traits of bivalves; (4) the utilization of H. ostrearia and marennine in a combination diet with other microalgae relevant to aquaculture.Our results suggest that cell size impacts considerably the selection process of H. ostrearia by oyster. This study also demonstrates that the extracellular form of marennine contributes significantly to the greening in the mucocytes of the gills. Apart from greening the pallial organs of bivalves, marennine (2 mg L-1) affects the behavioural, physiological and biochemical performance. Nevertheless, these effects can be compensated for its biological activities such as natural antibacterial agent and use as a mixed algal diet for bivalve aquaculture

Mestrado em Biologia Marinha
A contaminação microbiológica de moluscos bivalves constitui uma das principais preocupações inerentes à segurança alimentar desta importante fonte de receitas para a economia portuguesa. Os bivalves capturados ou produzidos em zonas estuarinas ou lagunares devem passar por um processo de depuração, para redução dos níveis de contaminação microbiológica, antes da sua comercialização. Este trabalho teve como principal objetivo o desenvolvimento de sistemas modulares de depuração, que apresentem vantagens em relação aos sistemas convencionais, tais como: baixos custos de operação, maior facilidade de controlo do processo de depuração, maior eficácia na desinfeção da água de depuração e maior facilidade de manutenção dos sistemas. Numa primeira fase, foram realizadas três experiências para a otimização de parâmetros de depuração: 1) Otimização da desinfeção da água de depuração dos moluscos bivalves pela manipulação da intensidade (μW.s.cm-2) da radiação UV-C aplicada; 2) Otimização da carga biológica a depurar (kg.L-1) e do tempo de depuração; e 3) Avaliação da temperatura e salinidade ótimas para depuração dos moluscos bivalves estudados: berbigão (Cerastoderma edule), ostra do pacífico (Crassostrea gigas) e amêijoa macha (Venerupis corrugata). Posteriormente, testou-se a capacidade máxima de funcionamento da depuradora modular desenvolvida e avaliaram-se os resultados obtidos na depuração, através da comparação da contaminação microbiológica destes bivalves com os bivalves depurados pelos processos convencionais. De forma a avaliar a otimização de cada parâmetro foi estudado o espectro total de contaminação microbiológica e a presença específica de bactérias patogénicas Escherichia coli, Salmonella spp. e Vibrio spp. Através da contagem de UFC do espectro total de contaminação, expressas em UFC/100g, foi possível concluir que de um modo geral as 12h de depuração foram suficientes para redução eficaz de bactérias presentes. Foi unânime às três espécies o valor otimizado para intensidade de radiação UV, 6.000 μW.s/cm2, densidade de estabulação, 98 g/L para berbigão e amêijoa e 120 unidades para ostra e a combinação de temperatura 21 °C com salinidade 30 mostrou-se ótima para o berbigão e ostra, sendo que 21 °C com salinidade 35 provou ser a melhor combinação para a amêijoa. Foi possível comprovar o bom funcionamento da depuradora modular com capacidade máxima, 360 L, com equipamento de desinfeção UV de 25 W para ambas as espécies. Estudos desta natureza são essenciais para tornar o processo de depuração o mais eficiente possível, de acordo com as especificidades de cada espécie.
Microbiological contamination of bivalve mollusks is one of the main concerns of alimentary safety of this important source of revenue for Portuguese economy. Bivalves that have been captured or grown in estuaries and lagoons must go through a process of depuration in order to reduce their levels of microbiological contamination, before they can be sold. The main goal of this work is the development of a modular depuration system with advantages over conventional systems, such as: lower implementation costs, higher control of depuration process, greater efficiency in the disinfection of water used for depuration, and easier maintenance. In the first step, three experiments were undertaken so that depuration parameters could be optimized: 1) Optimization of disinfection of the water used for depuration of bivalve mollusks through intensity manipulation (μW.s.cm-2) of the applied UVC radiation; 2) Optimization of bivalve stocking density (kg.L-1) and of the depuration time; 3) Evaluation of the optimal temperature and salinity levels for the depuration of the studied species: edible cockle (Cerastoderma edule), pacific oyster (Crassostrea gigas) and pullet carpet shell (Venerupis corrugata). After, the maximum operation level of the developed modular depurator was tested, and the results of depuration were evaluated by comparing the microbiological contamination of these bivalves with the bivalves depurated through conventional procedures. In order to evaluate the optimization of each parameter, the total spectrum of microbiological contamination was studied, along with the specific presence of pathogenic bacteria Escherichia coli, Salmonella spp. and Vibrio spp. Through UFC assemblage of total contamination spectrum, in UFC/100g, it was possible to conclude that overall 12h of depuration are enough to an efficient reduction of bacteria. It was possible to conclude that the optimal value of UV radiation was 6.000 μW.s/cm2 for the studied species, and the stocking density, 98 g/L for edible cockle and clams, and 120 units for pacific oyster; the optimal combination of temperature and salinity for depuration of cockles and oysters was 21 °C and 30, respectivelly, whereas the depuration of clams was more effective under 21 °C, with salinity of 35. It was possible to prove the effectiveness of depuration in the developed systems optimized under maximum capacity of 360 L, with 25 W UV equipment, for both species. Studies of this matter are essencial to transform depuration process into more efficient as possible, in accordance with specifications of each specie.

Doutoramento em Biologia
Os moluscos bivalves constituem um recurso haliêutico de elevada importância na economia (inter)nacional pelas suas características organolépticas, valor nutritivo e relevância na gastronomia tradicional. Não obstante, representam um produto alimentar de elevado risco para a saúde pública. A contaminação microbiológica (autóctone e antropogénica), sendo crónica nos bancos de bivalves das zonas estuarino-lagunares, constitui uma das principais preocupações associadas à segurança alimentar. Aquando da filtração inerente aos processos de respiração e alimentação, os bivalves bioacumulam passivamente microrganismos incluindo os patogénicos. A sua colocação no mercado impõe pois, prévia salubrização para níveis microbiológicos compatíveis com a legislação em vigor, salvaguardando a saúde pública. Apesar da monitorização das áreas de apanha e produção, das medidas de prevenção e da depuração, a ocorrência de surtos associados ao consumo de bivalves tem aumentado. Tal deve-se à insuficiente monitorização da contaminação microbiológica dos bivalves, contribuindo para uma gestão ineficaz do produto e consequente sub-valorização. O presente trabalho pretendeu caracterizar o estado de desenvolvimento do sector de exploração de bivalves em Portugal do ponto de vista da segurança alimentar, e analisar os aspectos cruciais da monitorização e da depuração do produto apresentando alternativas abrangentes e aplicáveis ao sector. Assim, desenvolveu-se uma metodologia de base molecular passível de adaptação à monitorização dos bivalves das zonas conquícolas, como alternativa ao método de referência vigente do Número Mais Provável que é baseado apenas na quantificação de Escherichia coli. O mexilhão (Mytilus edulis) da Ria de Aveiro, bivalve de interesse comercial a nível (inter)nacional serviu de modelo para a comparação de protocolos de extração de DNA. Esta metodologia foi desenvolvida de modo a que os métodos de extração de DNA sejam passíveis de aplicação a outras matrizes biológicas ou ambientais. Para além da detecção e quantificação directa de bactérias patogénicas, esta metodologia poderá ser aplicada à monitorização da transferência vertical microbiana nos bancos de bivalves bem como à caracterização da dinâmica espacio-temporal das populações microbianas no ambiente e à monitorização dos processos de depuração. Foi ainda abordado o potencial da aplicação de bacteriófagos ou de enzimas líticas para a optimização dos processos de purificação. O trabalho realizado e as perspectivas futuras propostas pretendem contribuir para a dinamização e requalificação do sector de exploração de bivalves através da melhoria do nível de segurança alimentar dos moluscos bivalves comercializados para alimentação humana, valorizando este recurso.
Bivalve molluscs stand out among fishery products, because of their (inter)national economic importance. For organoleptic and nutritional characteristics, because of culture, tradition and food availability reasons, bivalves are highly appreciated by consumers. Notwithstanding they represent a high risk food product. Microbial contamination (autochthonous and anthropogenic) is a major concern associated with the food safety of shellfish since it is known to be chronic in the shellfish beds of estuarine areas. By filter-feeding, bivalves passively accumulate microorganisms including pathogens. Products safe for sale and consumption must accomplish legislation standards preventing infections in the consumers and safeguarding public health. Despite monitoring of shellfish harvesting and production areas, the imposed preventative measures and the depuration processes, the occurrence of outbreaks associated with consumption of shellfish has increased. This may be due to underestimated assessment of microbial contamination of shellfish leading to low quality products. This work aimed to characterize the state of development of the sector of exploitation of bivalves in Portugal and to analyze the some aspects of bivalve monitoring and purification that lead to underestimation of microbiological contamination, in the perspective of providing comprehensive alternatives applicable to the sector. A molecular based approach that can be used in the monitoring of shellfish was developed as an alternative to the current reference method, the Most Probable Number based on the quantification of Escherichia coli only. Mussels (Mytilus edulis) from the Ria de Aveiro were used as model bivalves because they are widely represented in the national and international markets. This methodology used an innovative processing of bivalves along with extraction and detection methods that can be used in other biological and environmental matrices. The proposed methodology can be applied not only to direct detection and quantification of pathogenic bacteria, but also to the analysis of vertical tranference of microorganisms in growing beds and to the assessment of spatial and temporal dynamics of microbial populations. Also, it can be used to the classification of harvesting and production areas and to the evaluation of current purification processes. Also, it was suggested the use of bacteriophages and litic enzymes as an adjuvant for the improvement of the actual depuration processes. The revision of data on this field of activity and the experimental results were integrated in the perspective of providing scientific basis for the sustainable development of bivalve exploitation and valorization of the products in terms of food safety and economic profitability.

Mestrado em Química - Química Analítica e Qualidade
The main objective of this work was the application of an isocratic high performance liquid chromatography (HPLC) method coupled with a fluorescence detector to find the best conditions for the separation and quantification of 17β-estradiol (E2) and 17α-ethinyl estradiol (EE2) hormones in bivalve samples. A reversed phase column (Kromasil 100 C18, 5μm, 15 x 0.21cm), was employed with a fluorescence detector operating at excitation and emission wavelengths of 280nm and 305nm, respectively. A calibration curve was obtained with a good correlation coefficient for each compound. During the chromatographic analysis of the bivalve extracts, there were contamination problems, originated from glassware containers, E2 and EE2 standards preparation, and during hormones extraction from the samples. Thus, in a first step, a special attention was given to the elimination of these contamination problems in order to obtain the lowest limits of quantification for both E2 and EE2, and achieve their quantification in oyster samples. Therefore, after the washing process routinely applied in the laboratory, all glassware employed in samples preparation were washed with acetonitrile followed by the mobile phase used in the chromatographic analysis. After this washing procedure, and the use of a pre-column in the chromatographic separation process, the identification and quantification of hormone E2 was successfully accomplished for the oyster samples. An average value of 24.59 ng/g was obtained for oyster samples collected at an aquaculture location in Aveiro.
O principal objetivo deste trabalho foi a aplicação de um método de cromatografia líquida de alta eficiência (HPLC, sigla inglesa para High-Performance Liquid Chromatography) acoplada a um detetor de fluorescência para encontrar as condições mais adequadas para a separação isocrática das hormonas 17β-estradiol (E2) e 17-α-etinilestradiol (EE2), em amostras de bivalves. Foi utilizada uma coluna com uma fase reversa (Kromasil 100 C18, 5μm, 15 x 0,21 cm) e um detetor de fluorescência operando a comprimentos de onda de excitação e de emissão de 280nm e 305nm, respectivamente. Obtiveram-se curvas de calibração com bom coeficiente de correlação tendo-se contudo verificado problemas de contaminação durante as análises de amostras de bivalves. Estes problemas de contaminação tiveram origem, principalmente, no vasilhame de vidro usado, na preparação dos padrões e durante o procedimento de extração das hormonas E2 e EE2 das amostras. Assim, e numa primeira fase, procedeu-se à eliminação dos vários fatores potencialmente na origem dos problemas de contaminação de modo a ser possível obter os menores limites de quantificação possíveis para E2 e EE2, bem como proceder à respetiva quantificação nas amostras de bivalves. Assim, todo o material utilizado teve de ser cuidadosamente lavado com acetonitrilo e fase móvel utilizada na separação cromatográfica, posteriormente ao processo de lavagem habitualmente aplicado no laboratório. Depois deste processo de lavagem, e a utilização de uma pré-coluna no procedimento de separação cromatográfica, conseguiu-se identificar e quantificar a hormona E2 nos cromatogramas obtidos para as amostras de bivalves. Obteve-se um valor médio de 24.59 ng/g para as amostra de bivalves recolhidas num local de aquacultura em Aveiro.

No presente estudo é realizada uma análise morfofuncional (paleoautoecológica) dos bivalves das formações Serra Alta, Terezina e Corumbataí, Grupo Passa Dois, Neopermiano, que ocorrem nas assembléias de Anhembia froesi, Pinzonella illusa e Pinzonella neotropica, no Estado de São Paulo. O estudo teve por objetivo a reconstrução do habito de vida destes invertebrados como ponto de partida para a discussão de aspectos paleoecológicos mais amplos. Na assembléia de Anhembia froesi (Formação Serra Alta e base da Formação Corumbataí), ocorrem bivalves escavadores rasos, lentos (Anhembia froesi, Tambaquyra camargoi, Mendesia piracicabensis, Maackia contorta), de semi-infauna (Barbosaia angulata) e epifauna (?Anthraconaia mezzalirai) bissadas. Na assembléia de Pinzonella illusa (formações Serra Alta e Corumbataí), com maior diversidade de guildas, predominam os bivalves escavadores rasos, lentos (Pinzonella illusa, Plesiocyprinella carinata, Ferrazia cardinalis, Terraiopsis aequilateralis e Othonella araguaiana), sendo encontrados, também, escavadores rasos, rápidos (Favalia arcuata, Holdhausiella elongata e Runnegariella fragilis) ou escavadores intermediários (Casterella gratiosa, Itatamba paraima). Evidências morfofuncionais (e.g., obesidade da concha) sugerem que estas espécies tiveram preferência por substrato arenoso, estável. Formas escavadoras intermediárias, rápidas (Cowperesia anceps, Angatubia cowperesioides), em substratos finos também ocorrem, sendo incomuns os escavadores profundos (Roxoa corumbataiensis) e de epifauna bissada (Coxesia mezzalirai). A assembléia de Pinzonella neotropica (formações Corumbataí e Terezina) inclui bivalves escavadores rasos, lentos, em substrato arenoso, estável (Pinzonella neotropica, Jacquesia brasiliensis), escavadores intermediários, rápidos, em substrato fino (Cowperesia anceps) e escavadores profundos, em substrato estável (Roxoa intricans). Além destes, bivalves da semi-infauna bissada (Naiadopsis lamellosus) são também freqüentes. A baixa proporção de bivalves da epifauna nas malacofaunas estudadas pode ser explicada pela interação de fatores, como a baixa disponibilidade de substratos grossos, duros; as condições de águas rasas, freqüentemente afetadas por tempestades e o alto grau de estresse ambiental (variação na salinidade) que marcam o intervalo estratigráfico de ocorrência dos bivalves. Nenhuma das 25 espécies estudadas apresenta características anatômicas (e.g., claustrum, tubérculos, torção das valvas) ou feições tafonômicas (e.g., pontos de dissolução do umbo, fraturas regeneradas) tipicamente encontradas nas conchas dos bivalves dulcícolas. Por outro lado, feições morfológicas exclusivas de bivalves marinhos foram observadas em Ferrazia cardinalis (e.g, costelas radiais), Runnegariella fragilia (e.g., concha anteriormente expandida) e Cowperesia anceps (e.g., ornamentação concêntrica). Além disso, características bioestratinômicas típicas de conchas de bivalves marinhos ou de águas salobras, como predação por organismos durófagos foram verificadas em valvas de Plesiocyprinella carinata e, principalmente, de Pinzonella illusa. Os bivalves do Grupo Passa Dois (exclusive Formação Irati) não foram, portanto, organismos dulcícolas. Essa hipótese encontra respaldo também em análises cladísticas que mostram que a maior parte, se não todos os bivalves do intervalo estudado, são afins à famílias marinhas (e.g., Família Megadesmidae). Entretanto, precisa ficar claro que o teor preciso de salinidade do ambiente não pode ser determinado com base exclusivamente na análise paleoautoecológica. Neste estudo, sugere-se, pela primeira vez, que Anhembia froesi e Tambaquyra camargoi (formações Serra Alta e Corumbataí) são excelentes candidatos permianos para figurarem no rol de bivalves quimiossimbiontes, como indicado pela presença de rostrum e gigantismo nas suas conchas e pelo fato de serem encontradas em sedimentos finos depositados abaixo ou junto ao nível de base das ondas de tempestades, em ambiente com teor variável de oxigênio, o que pode ser corroborado também por outras evidências estratigráficas e tafonômicas (e.g., presença de horizontes com nódulos fosfáticos nos sedimentos associados). No geral, os bivalves estudados ocorrem em concentrações fossilíferas internamente complexas, mormente representadas por tempestitos proximais (predominantes) e distais. Por exemplo, bivalves preservados em posição de vida são muito raramente encontrados nas concentrações fossilíferas examinadas. Geralmente, os bivalves escavadores rasos, de semi-infauna e epifauna, representam elementos parautóctones a alóctones nas acumulações esqueléticas, exibindo maior grau de mistura temporal e baixa resolução espacial (time-averaging), do que os bivalves escavadores profundos e intermediários. Isso se deve, possivelmente, a estratégia de vida mais exposta à ação de distúrbios físicos (e.g., correntes tracionais de fundo) exibida pelas formas de epifauna, semi-infauna e infauna rasa. As características morfofuncionais e tafonômicas das conchas dos bivalves podem constituir, em conjunto, importantes ferramentas para a determinação da dinâmica sedimentar (e.g., taxa de sedimentação). Por exemplo, em uma concentração coquinóide (tempestito), encontrada no topo da Formação Corumbataí (assembléia de Pinzonella neotropica), há a presença de conchas articuladas fechadas de bivalves escavadores rasos (Pinzonella neotropica), intermediários (Cowperesia anceps) e de semi-infauna bissada (Naiadopsis lamellosus). Valvas desta última espécie ocorrem desarticuladas, fragmentadas e caoticamente distribuídas na matriz, contribuindo significativamente com a composição da acumulação esquelética. Entretanto, valvas articuladas e em posição de vida ocorrem também no topo da concentração, permitindo não apenas o reconhecimento do processo de retroalimentação tafonômica, como também a identificação de diferentes eventos de não deposição de sedimentos, seguidos de episódios de rápida deposição de finos, possivelmente associados às tempestades. Todos os dados paleoautoecológicos e tafonômicos obtidos mostram, consistentemente, que reconstruções paleossinecológicas não podem ser estabelecidas sem o prévio conhecimento desses dados. Neste contexto, um protocolo contendo 7 etapas distintas (e.g., atividades preliminares como a delimitação do escopo de estudo e hipóteses, atividades de coleta, atividades de laboratório, análise qualitativa, análise quantitativa, interpretação dos dados e apresentação dos resultados) é sugerido, como ponto de partida para a análise e verificação mais rigorosa de hipóteses paleossinecológicas.
In this study a detailed paleoautoecological analysis of the Permian bivalves of Passa Dois Group (Serra Alta, Terezina and Corumbataí Formations), Paraná Basin, was carried out. Its main goal is the reconstruction of the mode of life of these bivalves as a starting point for broad paleoecological discussions. The Anhembia froesi assemblage (Serra Alta and Corumbataí Formations) is dominated by shallow and slow burrowing bivalves (Anhembia froesi, Tambaquyra camargoi, Mendesia piracicabensis, Maackia contorta). Semi-infaunal (Barbosaia angulata) and epifaunal, byssate (?Anthraconaia mezzalirai) bivalves are also common. On the other hand, a large number of shallow and slow burrowers (Pinzonella illusa, Plesiocyprinella carinata, Ferrazia cardinalis, Terraiopsis aequilateralis and Othonella araguaiana) dominate the Pinzonella illusa assemblage (Serra Alta and Corumbataí Formations). Rapid burrowers (shallow or intermediate) are, however, represented by the shallow burrowers Favalia arcuata, Holdhausiella elongata and Runnegariella fragilis, and the intermediate burrowers Cowperesia anceps, Angatubia cowperesioides, respectively. Slow, intermediate burrowers (Casterella gratiosa, Itatamba paraima) are less common, while deep burrowers (Roxoa corumbataiensis) and epifaunal byssate (Coxesia mezzalirai) bivalves are rare. Several lines of morphological evidence (e.g., shell morphology, obesity) strongly suggest that the shallow and deep burrowers as well as epifaunal byssate bivalves colonized stable (sandy) substrates. The same ecological guilds that are represented in this assemblage can be recognized in the Pinzonella neotropica assemblage (upper part of Corumbataí and Terezina Formations) such as: shallow and slow burrowers in soft and stable substrates (Pinzonella neotropica, Jacquesia brasiliensis); rapid and intermediate burrowers in soft bottoms (Cowperesia anceps); deep burrowers (Roxoa intricans) and endobyssate elements (Naiadopsis lamellosus) in soft, but bioclastic-rich sediments. All of them were suspension-feeding bivalves. The low percentage of epifaunal, byssate bivalves in these assemblages is noteworthy. An interplay of abiotic and biotic factors could explain this feature. For example, during the deposition of the Corumbataí and Terezina Formations, condition of shallow waters, with soft and unstable substrate, frequently affected by storm events prevailed. Morphologic (e.g., claustrum, tubercles, valve torsion) and taphonomic features (e.g., dissolution pits, regenerated breakages) commonly observed in freshwater bivalves were not found in the studied species (n= 25). On the other hand, morphologic features typically observed in marine bivalves were noted in Ferrazia cardinalis (e.g., radial ribs), Runnegariella fragilis (e.g., anteriorly expanded shells) and Cowperesia anceps (e.g., concentric ornamentation). Additionally, some specimens of Plesiocyprinella carinata, and particularly Pinzonella illusa, show signs (drill holes) of drilling predation. Drilled bivalves are unknown among freshwater forms. The Passa Dois Group bivalves (not including the Irati Formation) were not freshwater bivalves, which is in accordance with recent cladistic analysis that indicates affinities with marine bivalve families. In addition, Anhembia froesi and Tambaquyra camargoi (Serra Alta and Corumbataí) are interpreted, for the first time, as chemosymbiotic bivalves. This is supported by the occurrence of a rostrum (Anhembia froesi) and the large body size of their shells. These bivalves are also found in fine, soft, oxygen deficient substrates, deposited below or near storm wave base. The examined bivalves are often preserved in internally complex fossil concentrations (proximal and distal tempestites). In all of these bivalves in life position are rare. The occurrence of disharmonious time-averaging is remarkable, particularly among the epifaunal, semi-infaunal and shallow burrowers. This is because the shells of bivalves with exposed strategy are more prone to spatial and temporal mixing. The paleoautoecologic and taphonomic analysis could be an important tool for the study of sedimentary dynamics (e,g., sedimentation rate). For example, life positioned specimens of Naiadopsis lamellosus with the comissure plane inclined about 45 degrees to the bedding plane occur on the top of a thin (~10 cm) bioclastic-rich concentration, intercalated in siltstones. The underlying shell layer was buried to a depth of ~1.6 cm. Normally, the shells are buried up to their umbonal carena, the anterior margin (buried portion) of closed articulated shells being slightly compressed dorso-ventrally. The original substrate provided by the underlying accumulation, including a great amount of bioclasts and also some muddy parts, seems suitable for the setting of Naiadopsis lamellosus larvae. Still, the presence of disarticulated, complete or fragmented, and abraded or pristine shells of Naiadopsis lamellosus with different size classes suggests different episodes of substrate colonization and reworking, indicating the occurrence of different periods of extensive taphonomic feedback. Although preserved in life position taphonomic evidence indicates that the temporal resolution of these concentrations is variable, often preserving a time-averaged record of the benthic marine populations. In the case of Naiadopsis lamellosus, different periods of colonization and disruptment have contributed to the bioclast enrichment of the substrate, at different times, corroborating the idea that the genetic processes that are responsible for the origin of a particular concentration are less important for the time-averaging phenomenon than the presence of old shells or bioclasts in a given depositional system. The obtained data have shown consistently that paleossynecological reconstruction that are not based on detailed paleoautoecologic and taphonomic studies are not justifiable. In this context, a seven step protocol is proposed as an alternative approach for a more accurate test of paleossynecological hypothese.

Les mecanismes vitellogenetiques et les phenomenes de lyse ovocytaire sont decrits, une etude histochimique des reserves ovocytaires et realisee. Differentes activites enzymatiques en relation avec la resorption du vitellus et les processus d'autolyse cellulaire ont ete localisees dans l'ovocyte. Des facteurs neuroendocriniens controlent l'ovogenese et agissent sur le metabolisme des acides nucleiques et des lipides

Dans les ecosystemes aquatiques, les variations de facteurs d'origine naturelle ou anthropique sont susceptibles d'affecter les interactions souvent complexes des organismes avec leur milieu. En reponse aux perturbations environnementales, les fonctions biologiques et physiologiques fondamentales des organismes marins peuvent etre modifiees, generant des situations regroupees dans la notion de stress. Ainsi, toute atteinte des fonctions de defense d'un hote, toute diminution de l'immunocompetence, peut augmenter la sensibilite aux agressions d'origine parasitaire ou microbienne induisant dans le cas le plus defavorable des mortalites massives. L'etude realisee au laboratoire chez la palourde ruditapes philippinarum a permis de mieux connaitre l'origine de variations de parametres hemolymphatiques et, dans les conditions pathologiques, de suivre la cinetique de la reponse cellulaire. Ainsi, des modifications de parametres cellulaires et biochimiques de l'hemolymphe ont ete observees chez cette espece de bivalve en cas de stress physiologique (jeune, stress osmotique) ou en condition pathologique (exposition a une bacterie pathogene). Les variations de la concentration en hemocytes circulants (chc) et de composition biochimique de l'hemolymphe (activite enzymatique, taux d'acides amines libres) enregistrees chez ce bivalve marin revelent l'influence de facteurs environnementaux et d'un pathogene sur des parametres lies a la capacite de defense interne. D'autre part, les reponses observees a court terme chez r. Philippinarum apres injection dans la cavite palleale de vibrio p1, bacterie responsable de la maladie de l'anneau brun, confirment que l'hemocytose (augmentation de la chc) et la modification de la formule hemocytaire (fhc) sont des manifestations precoces de la reponse immunitaire a mediation cellulaire. Au laboratoire, l'exposition d'huitres (crassostrea gigas) a des doses sub-letales de micropolluants (metaux lourds, pesticides, tributyletain) a induit d'une part une augmentation de la chc et d'autre part, des modifications remarquables de la fhc. Un bio-test d'agregation hemocytaire spontanee (ahs) in vitro a ete developpe au laboratoire et pris en compte dans le diagnostic d'immunomodulation chez des bivalves exposes in vivo ou in situ a des contaminants. Cette capacite hemocytaire qui constitue l'une des etapes de la defense interne, a ete alteree chez des huitres et des moules (mytilus edulis) par la mise en contact in vitro des hemocytes avec des ions metalliques ou le biocide tributyletain. Sur le terrain, la mesure de parametres hemolymphatiques dans des lots d'huitres et de moules transplantees a revele une atteinte du fonctionnement du systeme d'immunodefense (sid) dans les sites les plus exposes a des contaminants avec notamment, des modifications de la chc, la fhc et l'ahs. Ces reponses d'ordre physiologiques permettent de craindre une plus grande sensibilite aux agressions d'origine microbienne et parasitaire et, parallelement, une alteration a court terme de l'etat sanitaire des populations de mollusques naturelles ou en elevage. Il apparait desormais que les perturbations environnementales et les stimuli pathogeniques peuvent induire des modifications de parametres lies au fonctionnement du sid. L'exposition aux contaminants entraine egalement des variations de parametres cellulaires de l'hemolymphe (chc, fhc et ahs) chez les mollusques bivalves. Ces reponses mettent en evidence le haut degre de sensibilite des cellules hemolymphatiques aux micropolluants. Ainsi, dans les etudes de surveillance d'ecosysteme marin, l'utilisation de parametres lies a l'activite hemocytaire tel l'ahs apparait comme un outil tres sensible pour l'evaluation des effets biologiques de contaminants d'origine anthropique, compte tenu de l'influence d'autres facteurs physico-chimiques et des cycles saisonniers sur les parametres cytometriques de l'hemolymphe (par exemple la chc)

Le couplage des méthodes CLHP dites " sans acide " et " avec acide " a permis la séparation des sous-classes PEplasmalogène, PSplasmalogène et PCplasmalogène. Chez les 38 espèces de bivalve analysées, les acides gras NMI, le 20:1(n-11) ou le 22:4(n-9)trans13 sont préférentiellement incorporées dans les plasmalogènes et plus spécifiquement dans la PSplsm et la PEplsm. Cette association semble très caractéristique car ces acides gras sont les rares acides gras biosynthétisés par les bivalves marins. L'importance des aminophospholipides plasmalogènes (PS et PE) vis à vis de la PC indique une localisation préférentielle des plasmalogènes et donc des acides gras biosynthétisés sur la face interne des membranes cellulaires. La présence de l'un ou l'autre de ces acides gras pourrait être liée à l'évolution des bivalves. Concernant les cardiolipides des bivalves marins, la structure moléculaire de cette classe a été confirmée à partir d'analyses chromatographiques et structurales. (RMN et ESI+/MS). Les résultats de l'investigation effectuée sur 38 espèces ont démontré qu'il était possible de regrouper les cardiolipides en trois grands types caractéristiques. Un " type1 " correspondant à une composition contenant presque exclusivement du 22:6(n-3), un " type 2 " caractérisé par la présence conjointe du 22:6(n-3) et du 20 :5(n-3), le " type3 " correspondant à celle conjointe du 22 :6(n-3), du 18 :2(n-6) et du 18 :3(n-3). Ces différences apparaissent comme étant corrélés avec les grands taxons phylogénétiques établis chez les bivalves : les Filibranches Euptèriomorphes (CL de type 1), les Filibranches Ptériomorphes (CL de type 3) et les Eulamellibranches Hérédontes (CL de type 2)

Chez les métazoaires, la coquille des mollusques constitue un objet d'étude de référence pour comprendre les phénomènes de formation des biominéralisations carbonatées. La coquille, sécrétée par l'épithélium minéralisant du manteau, est constituée à plus de 95% de carbonate de calcium - calcite et/ou aragonite, et de moins de 5% d’une matrice organique composée de protéines, de glycoprotéines et de polysaccharides. Cette matrice calcifiante est directement impliquée dans les processus de formation du biominéral. Ce travail de thèse consiste en l’étude de la matrice organique associée à la couche nacrée. Chez les mollusques, la nacre est présente dans les coquilles de certains représentants actuels des bivalves, des gastéropodes, des céphalopodes, mais aussi des monoplacophores. La très grande majorité des données publiées sur les constituants macromoléculaires des matrices associées à la nacre concerne exclusivement les genres Pinctada et Pinna, pour les bivalves, et le genre Haliotis, pour les gastéropodes. Ce travail met en œuvre une approche comparative de ces composés à travers la caractérisation biochimique des matrices de deux nouveaux modèles. Nous considérons que cette approche comparative nous permettra de proposer de nouvelles hypothèses quant aux mécanismes de formation de la nacre, mais également quant à l’évolution de ces constituants organiques au sein des mollusques nacriers. Le premier modèle étudié est le mollusque d'eau douce Unio pictorum, un bivalve à coquille nacro-prismatique très commun des cours d'eau bourguignons. La matrice organique acido-soluble extraite de la couche nacrée présente une activité enzymatique de type anhydrase carbonique, une enzyme essentielle aux processus de calcification, déjà observée par ailleurs chez Pinctada sp. . Des électrophorèses réalisées en conditions dénaturantes sur cette matrice acido-soluble montrent la présence de cinq protéines majoritaires de masses moléculaires apparentes 95, 50, 29, 16 et 12 kDa. L'étude de la glycosylation de ces protéines nous a montré que les protéines de masses moléculaires 95, 50 et 29 kDa, étaient des glycoprotéines fortement glycosylées et que leurs ramifications saccharidiques étaient directement impliquées dans les processus de minéralisation. Notamment, la glycoprotéine de 95 kDa, spécifique de la couche nacrée, porte une quantité remarquable de sucres sulfatés qui sont impliqués dans sa capacité à lier les ions Ca2+ ou à interagir avec la précipitation du CaCO3 in vitro. Des séquences partielles internes ont pu être obtenues pour les différentes protéines de la matrice acido-soluble de la nacre grâce à des analyses par spectrométrie de masse. Le second modèle est le céphalopode Nautilus macromphalus dont la coquille nacro-prismatique est entièrement composée d'aragonite. Des électrophorèses de la matrice acido-soluble ont montré qu’elle est composée de polysaccharides de haut poids moléculaire, de glycoproteines migrant aux alentours de 60-50 kDa et de 3-4 protéines de masses moléculaires apparentes comprises entre 20 et 10 kDa, capables de lier le calcium in vitro. Sur gel d’électrophorèse à deux dimensions, les différents constituants organiques de la matrice acido-soluble migrent soit à des valeurs de pI très acides (inférieur ou égal à 3 unités), soit à des pI très basiques (supérieur à 9), alors que chez les autres mollusques non céphalopodes, les protéines de nacre sont faiblement acides ou neutres. Des séquences partielles de ces protéines ont été obtenues par séquençage de novo à partir de protéines purifiées par électrophorèses 2-D et de matrice complète analysées par spectrométrie de masse après digestion trypsique. Les nouvelles séquences observées présentent des similitudes avec des protéines de nacre décrites chez les bivalves Pteriomorphia, mais aucune homologie n’a pu être détectée avec les protéines décrites chez les gastéropodes. Nous avons également décrit, dans une étude préliminaire, les matrices acido-solubles extraites des coquilles de brachiopodes Rhynchonelliformea. Ce travail montre que, chez ce groupe externe aux mollusques, les mécanismes moléculaires de calcification impliquent également la production d’une matrice calcifiante composée de macromolécules aux propriétés biochimiques diverses.

Le recrutement est un processus clé du cycle de vie des invertébrés marins benthiques, dont les bivalves. Si la phase larvaire et l’étape de fixation/métamorphose sont largement étudiées, les processus post-fixation, dont font partie les migrations secondaires, restent relativement ignorés ou du moins sous-estimés.Ces migrations secondaires ou post-fixations résultent de processus hydrodynamiques modulés par des réponses éco-éthologiques.Le présent projet proposait d’étudier les mécanismes de migrations secondaires des recrues de bivalves en développant une approche éco-étho-physiologique afin de répondre à trois objectifs : i) Estimer le poids potentiel des migrations secondaires dans les habitats de sédiments grossiers ii) Identifier des facteurs environnementaux, et plus spécifiquement ceux de l’environnement trophique en relation avec l’hydrodynamisme, qui contrôlent ces processus iii) Évaluer les potentielles interactions entre migrations secondaires et physiologie des recrues. Dans un premier temps, nous avons développé une étude in-situ des migrations secondaires des recrues de bivalves dans un habitat de sédiments grossiers dans l’archipel de Chausey (Normandie, France) via l’utilisation de pièges à recrues. En parallèle, un suivi de nombreux paramètres environnementaux (conditions hydrologiques, hydrodynamiques et trophiques) a été mis en place. Puis, nous avons étudié en conditions expérimentales, le potentiel de dispersion post-fixation de différentes espèces de bivalves exploitées et l’influence de la physiologie (en termes de réserves énergétiques) sur les migrations secondaires. Pour ce faire, nous avons utilisé des outils innovants, tels un tube de vitesse de chute et un canal hydrodynamique tout en développant la zootechnie de l’élevage larvaire et poste-larvaire de la praire, Venus verrusosa. Nos résultats mettent en évidence l’importance des migrations secondaires des recrues de bivalves dans les habitats de sédiments grossiers avec près de 24 taxons différents de bivalves identifiés en migration. Certaines de ces migrations post-fixations correspondraient à des changements ontogéniques d’habitats, et les habitats de sédiments grossiers constitueraient une zone de nurserie pour plusieurs espèces. De plus, ce projet démontre, pour la première fois, le rôle de la physiologie et de l’environnement trophique dans le contrôle des migrations secondaires et cela à différents niveaux. Nous avons observé une synchronisation entre un événement massif de migrations secondaires actives et un changement de la composition de la communauté phytoplanctonique, plus particulièrement un bloom de nanoeucaryotes. Cette réponse des recrues de bivalves à un signal trophique pélagique («trophic migration trigger», TMT), pourrait être due au coût énergétique supplémentaire associé aux comportements actifs de migration. En effet, nous avons ensuite constaté en conditions expérimentales que la capacité des recrues à contrôler leur dispersion par leur comportement est directement corrélée avec leur profil physiologique. Les recrues avec le plus de réserves énergétiques ont une meilleure capacité d’accroître leur potentiel de dispersion par leur comportement. Aussi les recrues migreraient quand leur principale source de nourriture est disponible dans le milieu afin de mieux supporter le coût des comportements de migrations secondaires actives comme la dérive byssopélagique. De plus, le potentiel de migration post-fixation apparait comme fortement lié à la synchronicité entre la période d’arrivée de la larve sur le sédiment et le cycle de marée (morte-eau ou vive-eau), mais également aux taux de croissance post-larvaires qui dépendent eux-mêmes de l’environnement trophique. Les patrons de migrations secondaires des recrues bivalves résultent donc d'un étroit couplage physico-biologique impliquant l'hydrodynamique, mais aussi des réponses éco-éthologiques modulées par des processus physiologiques en lien avec l’environnement trophique
Recruitment is a key process in the life-cycle of benthic marine invertebrates, and most of bivalve species. It includes the larval phase, the settlement and secondary dispersal mechanisms which lead to a gradual sedentary lifestyle until the recruits begin a strict benthic life. Although the larval phase and the settlement/metamorphosis stage are well studied, post-settlement migrations remain relatively unknown or at least underestimated. These migrations are the result of hydrodynamic processes modulated by eco-ethological responses in the benthic boundary layer that can radically change primary fixation patterns and have a major impact on the life cycle of a bivalve.This project proposed to study secondary migrations mechanisms of bivalve recruits by developing an eco-etho-physiological approach in order to answer three objectives: I) estimate the potential weight of the secondary migrations in coarse sediments habitats, ii) identify environmental factors, and more specifically those of the trophic environment in relation to hydrodynamics conditions, which control these processes, iii) evaluate the potential interactions between secondary migrations and recruits physiology.Firstly, we developed an in-situ study of secondary migrations of bivalve recruits in a coarse sediment habitat in the Chausey Archipelago (Normandy, France) by using recruit traps and monitoring environmental parameters (hydrological, hydrodynamic and trophic conditions). Then, we studied in experimental conditions post-settlement dispersal potential of different exploited bivalve species as well as the influence of physiology (in terms of energy reserves) on secondary migrations with a fall velocity tube, a benthic flume and by developing the larval and post-larval rearing of the warty venus, Venus verrucosa. Our results highlight the importance of secondary migrations of bivalve recruits in coarse sediment habitats with nearly 24 different taxa of bivalves identified in migration. Some of these post-settlement migrations would correspond to ontogenic change of habitat, and so coarse sediment habitats would constitute a nursery zone for several species. For the first time, this project demonstrates, at different levels, the role of physiology and trophic environment in the control of secondary migrations. We observed synchronization between a massive active secondary migration event with a change in the composition of the phytoplankton community and more particularly a bloom of nanoeukaryotes. This response of bivalve recruits to a ‘’trophic migration trigger’’ (TMT) could be due to the additional energy cost of active migration behaviors. Indeed, we highlighted, in experimental conditions, that the ability of recruits to control their dispersal by their behavior is directly correlated with their physiological profile. Recruits with the most energy reserves are the most able to increase their dispersal potential by their behavior. Also recruits would migrate when their main food source is available in the environment in order to support the cost of active secondary migration behaviors such as bysso-pelagic drift. In addition, post-fixation migration potential appears to be strongly related to the synchronicity between the settlement period and the tidal cycle (spring tide or neap tide), but also to post-settlement growth rates which depend on trophic conditions. Patterns of secondary migrations result from a close physico-biological coupling involving hydrodynamics factors but also eco-ethological responses modulated by physiological processes related to the trophic environment

Os bivalves marinhos sÃo muitos apreciados como alimento e no aproveitamento de suas conchas para vÃrias utilidades especialmente como enfeites. A presente pesquisa teve como objetivo estudar o bivalve marinho venerÃdeo Anomalocardia brasiliana, quanto a densidade populacional e ciclo de reproduÃÃo, correlacionando este Ãltimo aos parÃmetros ambientais (salinidade, temperatura e precipitaÃÃo pluviomÃtrica). As amostragens foram realizadas mensalmente durante o perÃodo de maio/02 a abril/03, na praia do Canto da Barra, municÃpio de Fortim â CE, no estuÃrio do Rio Jaguaribe. Os indivÃduos Anomalocardia brasiliana apresentaram um padrÃo de distribuiÃÃo bem caracterÃstico, com os juvenis concentrados em Ãrea com sedimento fino e mais Ãmido e os adultos em banco arenosos com sedimento mais grosseiro. A diferenciaÃÃo sexual ocorreu provavelmente entre 12,6mm e 17,9mm de comprimento de conchas. A identificaÃÃo dos sexos foi realizada utilizando-se tÃcnicas histolÃgicas de rotina. Durante a gametogÃnese, foram reconhecidas em fÃmeas, ovogÃnias, ovÃcitos prÃ-vitelogÃnicos, ovÃcitos vitelogÃnicos e ovÃcitos maduros, e, em machos, espermatogÃnias, espermatÃcitos I, espematÃcitos II, espermÃtides e espermatozÃides. A reproduÃÃo da espÃcie à contÃnua, entretanto foram observados perÃodos de maior liberaÃÃo de gametas de julho a outubro/2002 e de fevereiro a abril/2003. O ciclo reprodutivo caracterizou-se pelas seguintes as fases do desenvolvimento gonadal: âem MaturaÃÃoâ, âMaduroâ, âem EliminaÃÃoâ, âEliminadoâ, para ambos os sexos. Dentre os fatores ambientais observados neste estudo, a precipitaÃÃo pluviomÃtrica e a salinidade foram as que apresentaram as maiores amplitudes de variaÃÃo. Entretanto, uma influÃncia significativa do efeito da diminuiÃÃo da salinidade foi observada somente em machos, provavelmente causada pela alta precipitaÃÃo pluviomÃtrica verificada em abril/03. Durante este estudo, observou-se a ocorrÃncia de parasitas do gÃnero Bucephalus sp. em 6,5% dos indivÃduos analisados. Apesar da baixa incidÃncia de organismos parasitados, a ocorrÃncia de parasitismo na populaÃÃo de Anomalocardia brasiliana, no estuÃrio do Rio Jaguaribe, alerta para necessidade de estudo sobre as condiÃÃes fisiolÃgicas desta espÃcie na regiÃo.
Marine bivalves are very appreciated as food and its shell is used for several purposes, especially for ornament. The present research aims to study the marine bivalve Anomalocardia brasiliana in relation to its population density and reproductive cycle, correlating them with environmental parameters (salinity, temperature and precipitation). Monthly samplings were realized from May 2002 to April 2003 at Canto da Barra Beach, in the Jaguaribe River Estuary, Fortim, CearÃ. The Anomalocardia brasiliana individuals presented a quite characteristic distribution pattern, with the juvenile ones concentrated in areas with fine and moist sediment (fine sand) and the adult ones concentrated in sandy beds with coarse sediment (medium sand). Sexual differentiation probably occurred when the shell length was between 12,6mm and 17,9mm. Sex determination was realized using custom histological techniques. During gametogenesis, oogonia, previtellogenic oocytes, vitellogenic oocytes and mature oocytes were recognized in females, and spermatogonia, spermatocytes I, spermatocytes II, spermatids and spermatozoa were recognized in males. The reproductive cycle is continuous. Nevertheless, periods of intense gamete liberation were observed from July to October 2002 and form February to April 2003. For both sexes the reproduction cycle was characterized by the following stages in the development: âin Ripeningâ, âRipeâ, âin Spawningâ, âSpawnedâ. Among the environmental agents observed in this study, precipitation and salinity were the ones that presented the largest fluctuation in amplitude. However, a major influence by the salinity reduction effect was only observed in males, probably caused by the high precipitation registered in April 2003. During this study, the occurrence of parasites from the genus Bucephalus sp. was observed in 6,5% of the analyzed individuals. Despite the low influence of organisms infected with parasites, the occurrence of parasitism in the Anomalocardia brasiliana population at the Jaguaribe River Estuary alerts the necessity for further studies on the physiological conditions of this species in that area.

La producció de mol·luscs bivalves va moure als anys 2001 i 2002 més de 13 milions d'euros només a Catalunya, corresponents a la comercialització de 5 mil tones de marisc per any; en tot l'Estat Espanyol la producció del 2001 fou de més 256 mil tones. La importància econòmica dels mol·luscs bivalves creix paral·lelament al seu consum arreu del món. No obstant, el caràcter filtrador i bioacumulador d'aquest mol·luscs ha comportat que el seu consum sovint s'hagi vist associat a malalties causades per biotoxines i patògens microbians. L'aplicació de normatives dirigides a controlar aquests factors de risc ha contribuït a la disminució dels problemes sanitaris causats per biotoxines i bacteris d'origen. fecal. Tanmateix, els paràmetres establerts no han estat d'utilitat en el cas dels membres del gènere Vibrio, els quals formen part de la microbiota marina, ni del virus humans, ja que els indicadors bacterians clàssics tenen un comportament molt diferent al d'ambdós grups. En el cas dels virus humans, el problema es veu agreujat per les dificultats tècniques que la seva detecció comporta. És per això que diversos autors han proposat els bacteriòfags de bacteris entèrics com a indicadors. D'altra banda, també s'ha proposat la utilització de virus humans, en concret dels adenovirus detectats mitjançant PCR, com a índex molecular de la contaminació vírica d'origen humà. Amb tot, fins al moment la comunitat científica no ha arribat a un consens ni pel que fa a la tria de l'indicador o indicadors més apropiats ni tampoc pel que fa a un mètode de detecció dels patògens vírics en bivalves.
Estudis anteriors a la present tesi han avaluat la contaminació vírica en mol·luscs bivalves i la seva relació amb els diferents microorganismes models proposats; no obstant, aquests estudis s'han dut a terme utilitzant una gran varietat de metodologies i centrant-se en zones de producció molt concretes. En el marc de la Unió Europea, mancava un estudi comparatiu de zones de producció situades en zones geogràficament molt diverses i per tant, molt diferents a tots nivells, però que han de complir certes normatives establertes per la Unió Europea.
En aquesta tesi, s'ha validat i estandarditzat un mètode de detecció molecular de virus humans en mol·luscs bivalves molt sensible i de fàcil aplicació. També hem desenvolupat una RT-PCR Multiplex per la detecció simultània d'adenovirus, enterovirus i virus de l'hepatitis A, de forma que es redueix el cost econòmic i temporal que la detecció molecular clàssica comporta.
D'altra banda, durant 18 mesos s'ha estudiat de forma simultània la distribució de virus humans en mol·luscs bivalves i de diferents microorganismes proposats com a indicadors en diferents àrees de producció d'Espanya, Grècia, Regne Unit i Suècia. Durant aquest estudi he aïllat dos dels virus de major importància en l'etiologia de malalties associades al consum de marisc, virus de l'hepatitis A i norovirus, en mol·luscs bivalves destinats al consum humà i procedents de zones de producció d'Espanya, Grècia i Regne Unit. Els adenovirus humans són els virus més prevalents en totes les àrees estudiades i presentaren una relació estadísticament significativa amb la resta de virus humans estudiats, fets que aconsellen la seva utilització com a índex molecular de contaminació vírica d'origen humà. Pel que fa als bacteriòfags de bacteris entèrics que també han estat proposats com a microorgaismes model, les anàlisis mostraren que els fags F-específics d'ARN són l'únic grup de fags que presenta una relació estadísticament significativa amb tots els virus humans, tot i això, la capacitat predicitiva dels fags F-ARN és més forta amb els norovirus i en zones amb nivells d'Escherichia coli més elevats, de fet, serien útils com a model de norovirus al Regne Unit.

The transport of organic solutes from sea water by the molluscan integument occurs via a Na⁺-dependent cotransport process. Although much is known about the mechanism of this transport, little attention has been given to the influence of environmental parameters on this process. One highly variable parameter to intertidal animals is salinity. Intertidal habitats are routinely exposed to periodic changes in external salinity from ∼0 to ∼425 mM Na⁺, representing a range of ∼0 to ∼1000 mOsM osmotic concentration. This study examined the effect of salinity on organic solute transport and cell volume regulation in the gill of two representative intertidal animals, Mytilus edulis and Mytilus californianus. Integumental organic solute transport was found to adapt to reduced salinity in a 2-stage response. There was an initial, steep inhibition of transport that was far greater than predicted from the reduction of external Na⁺ alone. However, within 10 min. of acclimation to the dilute medium, transport recovered to the level predicted by the external Na⁺ concentration. Long-term acclimation to reduced salinity resulted in no upregulation of integumental transport. Thus, the degree of adaptation of integumental organic solute to reduced external salinity was always defined by the availability of external Na⁺ for the transport process. The exposure to reduced salinity also resulted in a change of cell volume. The gill preferentially lost organic solutes but spared inorganic ions following long-term acclimation to reduced salinity. Short-term acclimation of gill tissue to reduced salinity resulted in a rapid Volume Regulatory Decrease (VRD) which served to restore cell volume toward the control value. This short-term VRD did not involve the substantial loss of organic solutes, but was probably mediated by the efflux of K⁺ (and a counter anion) across the basolateral membrane. The loss of inorganic ions during brief exposures to reduced salinity conserves metabolically expensive organic compounds. The loss of organic solutes during long-term acclimation to reduced salinity may be due to the decreased Na⁺ gradient which reduces the ability of integumental transport to maintain the high solute concentrations in gill tissue of control animals.

Dissertação para obtenção do Grau de Mestre em Tecnologia e Segurança Alimentar
A cultura e consumo de moluscos bivalves em Portugal são atividades cujo início se perde no tempo e com um grande impacto na economia nacional. Contudo, o consumo destes organismos envolve problemas específicos de segurança alimentar pelo que é necessário um controlo rigoroso associado a um plano de monitorização. A amêijoa japonesa (Ruditapes philippinarum), a ameijola (Callista chione), a lambujinha (Scrobicularia plana), o mexilhão (Mytilus edulis) e a ostra portuguesa (Crassostrea angulata) são espécies apreciadas e com elevado interesse socio-económico em Portugal e que podem ser encontradas ao longo da costa portuguesa, nomeadamente nos estuários do Tejo e do Sado. Possuem as partes do corpo moles encerradas num exosqueleto sob a forma de uma concha, composta por duas valvas calcárias e alimentam-se por filtração. Os bivalves têm grande importância na dieta humana por serem fonte de nutrientes essenciais e por fornecerem proteína de elevada qualidade biológica. Contudo, os benefícios do seu consumo são contrabalançados com o perigo de exposição do consumidor a substâncias poluentes que se podem acumular nas partes edíveis, o que constitui um fator de risco para a saúde humana. O principal objetivo deste estudo foi a determinação da composição química aproximada e a avaliação da contaminação por metais pesados na parte edível de 5 espécies de moluscos bivalves capturados em Portugal. Por conseguinte, o teor de humidade, gordura, proteína, cinza e glicogénio e as concentrações de cádmio (Cd), mercúrio (Hg) e chumbo (Pb) foram determinados nos tecidos moles (em peso húmido) da amêijoa japonesa (Ruditapes philippinarum), da ameijola (Callista chione), da lambujinha (Scrobicularia plana) e da ostra portuguesa (Crassostrea angulata) do Estuário do Sado, da amêijoa japonesa (Ruditapes philippinarum) e do mexilhão (Mytilus edulis) do estuário do Tejo e do mexilhão (Mytilus edulis) da zona de Cascais. Os moluscos bivalves analisados apresentaram teores de humidade entre 79 e 85%, de proteína entre 10 e 14%, de cinza entre 2 e 4%, de gordura entre 0,3 e 1,5% e de glicogénio entre 0,5 e 2,5%. Os resultados obtidos foram semelhantes aos de outros autores. Os níveis de metais pesados encontram-se abaixo dos limites da Comissão Europeia, com exceção da ostra portuguesa capturada no estuário do Sado, que poderá representar um risco para o consumo humano. Verificou-se que apenas o chumbo apresentou diferenças entre os dois locais de amostragem, sendo que o estuário do Tejo é o que apresenta valores mais elevados.

Mestrado em Engenharia Agronómica - Produção Animal - Instituto Superior de Agronomia / Faculdade de Medicina Veterinária
A cultura de moluscos bivalves (moluscicultura) é uma atividade com elevada expressão na aquacultura portuguesa, representando 55% da produção total e a produção de amêijoas representa cerca de 24% do total produzido. A amêijoa-boa, Ruditapes decussatus, é a espécie de maior importância económica, mas, nos últimos anos, a sua produção diminuiu associada a diversos fatores, como a sobre-exploração e doenças como a Perkinsiose. Para enfrentar a diminuição de R. decussatus, esta foi substituída pela amêijoa-japonesa, Ruditapes philippinarum, que aparentava ter maior resistência a doenças e maiores taxas de crescimento. Apesar da rápida expansão desta espécie em águas portuguesas, a amêijoa nativa continua a ser comercialmente mais importante e apreciada do que a amêijoa-japonesa pela sua qualidade. O objetivo deste estudo foi avaliar os métodos de diagnóstico da Perkinsiose em duas espécies de amêijoas de locais distintos. Utilizaram-se como métodos o teste RFTM (teste referência) e a histopatologia (método complementar). As amostras foram coletadas em Outubro de 2017 e Abril de 2018 em 6 locais diferentes, com n=50 para cada local (exceto no Estuário do Tejo (2017) com n=47) correspondendo a um total de 297 espécimes examinados. Os resultados mostraram que a amêijoa-boa tinha maior prevalência da doença, principalmente a produzida em Olhão. A amêijoa-japonesa também teve casos positivos, embora com menor nível de infeção. Este estudo sugeriu que para diagnosticar a Perkinsiose, o teste RFTM é eficaz, menos dispendioso e mais rápido. Mas deve ser complementado por análises histopatológicas especialmente em animais com baixa infeção. Tendo em conta as condições ambientais nos diferentes locais, é notório que o stress ambiental causado por altas salinidades e temperaturas da água, a baixa concentração de oxigénio nos sedimentos, altas densidades e stress fisiológico devido à época de desova contribuem para aumentar a prevalência e propagação da doença
N/A

The ability of a species to show plasticity throughout its range is suggested to be significant to the survival, maintenance, and expansion of populations. In the Southern Ocean, plastic traits may have enabled resilience since the onset of cooling, and given species the capacity to exploit empty niches after the retreat of ice in interglacial periods. Phenotypic plasticity has rarely been investigated in Southern Ocean invertebrates however, and the cold stenothermal environment, which prevails, has often been considered homogenous in its selection on fauna. Previous ecological studies have often pooled together material collected within predetermined biogeographic regions to overcome the limitations of sampling difficulties. Subtle differences between environments may however, be forcing ecological divergence in species, with possible implications for speciation processes. This thesis investigates the phenotypic plasticity and reproduction among populations of the small shallow-water brooding bivalve Lissarca miliaris over its Antarctic range, and of deep-sea protobranch bivalves Yoldiella ecaudata, Y. sabrina, and Y. valettei from contrasting benthic regions. The reproductive studies of L. miliaris revealed a previously unknown hermaphrodite trait, maximising the reproductive efficiency in a short-lived species where the female’s capacity to brood its young is limited. Reproduction is also described for the first time in deep-sea Antarctic protobranch bivalves and demonstrates lecithotrophic larval development. Additionally, Y. valettei shows evidence of simultaneous hermaphroditism, which may increase the likelihood of successful reproduction in low population densities. Phenotypic plasticity is observed among populations of bivalves, irrespective of geographical proximity, and with no latitudinal trends, but subtle differences in the environment. Significant differences in morphology and growth rates are identified among populations, and reproductive plasticity identified in L. miliaris and Y. sabrina. Increasing atmospheric temperature is also measured to show an effect on the ecophysiology of intertidal populations of L. miliaris at Signy Island over the past 40 years, with increasing growth rates at the cost of smaller offspring and pressure from endolithic algal decay.

Notre etude se situe dans le cadre d'un programme de recherches mene en partenariat dans le but de mettre au point des cultures cellulaires chez les bivalves marins. Les travaux ont eu pour objectif l'etablissement de cultures primaires de cellules fonctionnelles de coquille st-jacques pecten maximus et d'huitre crassostrea gigas. Ils ont necessite la selection et l'optimisation d'un protocole de dissociation cellulaire et la mise en place d'un procede de decontamination microbienne de certains tissus pour eviter la presence de microorganismes parmi les suspensions cellulaires (branchies notamment). Les essais ont ete realises a partir de cur et de branchies de coquille st-jacques et a partir d'embryons d'huitre. La technique d'isolement des cellules a par la suite ete validee par les resultats des cultures realisees dans un milieu de base que nous avons defini. Certaines cellules adherent en effet systematiquement au support apres deux ou trois jours de culture. Deux types cellulaires sont differenciables, des cellules de type epithelial, peu nombreuses, et des cellules fibroblastiques, largement majoritaires. Les cellules fusiformes adherentes en culture, de type fibroblaste, ont ete caracterisees, dans le cas du cur, par microscopie electronique (presence de myofibrilles) et immunomarquage (reaction positive a l'anti-myosine et anti- tropomyosine) comme etant des myocytes. Cette conclusion est d'ailleurs corroboree par le fait qu'apres environ une semaine de culture, lorsque les cellules sont quasiment confluentes, elles reconstituent des formations plurinucleees de type myotubes qui se contractent spontanement in vitro. Les resultats des cultures, reproductibles dans le cas du cur de coquille st-jacques restent plus variables dans le cas des branchies et des cellules embryonnaires. Ces donnees nous ont donc conduit peu a peu a selectionner les cellules de cur pour les experimentations ulterieures. Diverses techniques analytiques (histoautoradiographie, comptage en scintillation, electrophorese et autoradiographie d'electrophorese, chromatographie sur couche mince et autoradiographie de chromatographie sur couche mince), mises en uvre a differents temps de culture, apres incubation des cellules en presence de precurseurs marques ont montre que les cellules de cur en culture dans le milieu de base neosynthetisent des proteines, de l'adn et des lipides pendant au moins 20 jours (c'est aussi le cas des branchies). Ces activites de biosynthese peuvent d'ailleurs etre stimulees par addition au milieu de base de certains facteurs d'origine marine notamment et des lipides en particulier ce qui prouve que le milieu de culture peut etre encore ameliore. Un protocole de cryopreservation des cellules de cur a pu etre mis au point, permettant d'obtenir des cultures a partir des cellules decongelees. L'activite de biosynthese de ces cellules en culture, evaluee apres sept jours, apparait equivalente, au moins, a celle des cellules temoins non congelees et le profil des proteines et des lipides neosynthetises par les cellules en culture apres congelation-decongelation est comparable a celui obtenu pour les cellules fraiches. De plus, comme pour les cellules non congelees, une differenciation terminale des myoblastes en myotubes est observee en culture. A titre preliminaire, pour une etude de faisabilite, ces modeles experimentaux ont aussi ete utilises comme bioessais pour des tests de cytotoxicite aigue (effet de sels de metaux lourds) et de genotoxicite (effet d'un produit mutagene de reference, l'aflatoxine b1), l'objectif etant de pouvoir proposer a terme, des biotests et biomarqueurs d'effets cytotoxique et genotoxique de xenobiotiques presents dans le milieu marin, completant ainsi la panoplie des tests deja disponibles pour la surveillance de la qualite de l'eau de mer (algues, bacteries, embryons). En conclusion, les donnees acquises au cours de ce travail et les techniques analytiques mises au point ont permis pour la premiere fois: - d'etablir des cultures primaires de cellules de cur de coquille st-jacques dont l'activite de biosynthese a ete verifiee avec rigueur au cours du temps par l'etude de certaines fonctions du metabolisme de base (en l'absence de biomarqueurs specifiques connus), - de congeler des cellules de cur de coquille st-jacques permettant, apres decongelation, d'obtenir des cultures dont l'activite de biosynthese in vitro est conservee. Ces resultats, qui ouvrent sur de larges perspectives vont, en outre, faciliter la mise en place de nouveaux modeles cellulaires chez des invertebres marins

Mestrado em Toxicologia e Ecotoxicologia
Carbamazepine (CBZ), an antiepileptic drug, is one of the most commonly detected pharmaceutical drugs in aquatic ecosystems, being used as a marker of anthropogenic pollution. Since CBZ is designed to exert a biological effect, when it reaches aquatic environment high probability exists for toxic effects on non-target organisms. In this way, the present study evaluated the acute (96 h) and chronic toxicity (28 d) of environmentally relevant concentrations of CBZ (0.00, 0.03, 0.30, 3.00, 9.00 μg/L) in the edible clams Venerupis decussata (a native species) and Venerupis philippinarum (an invasive species) collected from the Ria de Aveiro lagoon. The effects on both species were assessed through the use of a battery of biomarkers mainly related with health status and oxidative stress in the organisms. Furthermore, it was applied a promising alternative technique, the immunoassay ELISA for the direct CBZ quantification in clams’ tissues. The results obtained for the acute test showed that CBZ levels in clams’ tissues increased along the exposure concentration range, where V. decussata accumulated more CBZ (except for CBZ 9.00 μg/L) than V. philippinarum. After an exposure of 28 d V. decussata accumulated a higher concentration of CBZ with exception of the highest concentration of exposure, where the levels of CBZ accumulated were similar to the acute test. Although the clams accumulated lower levels of CBZ than the concentration of exposure, these concentrations were enough to impair the health status of the species under study and induce oxidative stress. A different response to CBZ was observed for both species exposed to the acute test. V. philippinarum increased the lipid peroxidation levels at the highest CBZ concentration, whereas V. decussata presented a significant decrease in this parameter. Glutathione S-transferase activity was stimulated for V. decussata and decreased for V. philippinarum. Nevertheless, after exposure to CBZ, for both species it was found an induction of glutathione reductase and superoxide dismutase. The results indicated that, probably, V. philippinarum have a deficient antioxidant defense system compared with V. decussata, being less capable to neutralize reactive oxygen species and thus appeared to be the most sensitive species to the CBZ effects. A 28 d exposure to CBZ resulted in a higher toxicity in V. decussata compared with the same species exposed to the acute test. This was mainly reflected by a lower activity/content in the mechanisms involved in the antioxidant defense system and thus, a lower capability to lead with oxidative stress induced by CBZ. The risk quotient determined for the Ria de Aveiro was higher than 1 indicating that an ecotoxicolgical risk is suspected. Furthermore, the bioaccumulation of CBZ in clams should be taken into consideration since it might be transferred along the food chain, ultimately affecting humans.
A carbamazepina (CBZ), uma droga antiepilética, é uma das drogas farmacêuticas frequentemente detetadas em ecossistemas aquáticos, sendo utilizada como um marcador da poluição antropogénica. Uma vez que a CBZ foi desenhada para exercer um efeito biológico, quando alcança o ambiente aquático existe uma elevada probabilidade para provocar efeitos tóxicos em organismos não-alvo. Desta forma, no presente estudo foi avaliada a toxicidade aguda (96 h) e crónica (28 d) de concentrações ambientalmente relevantes de CBZ (0.00, 0.03, 0.30, 3.00, 9.00 μg/L) em amêijoas comestíveis Venerupis decussata (nativa) e Venerupis philippinarum (invasora) capturadas na Ria de Aveiro. Os efeitos em ambas as espécies foram estudados utilizando uma bateria de biomarcadores principalmente relacionados com o estado de saúde e stress oxidativo nos organismos. Foi aplicada também uma técnica alternativa promissora, o imunoensaio ELISA para a quantificação direta de CBZ nos tecidos das amêijoas. Os resultados obtidos para o teste agudo mostraram um aumento da concentração de CBZ nos tecidos das amêijoas ao longo do intervalo de exposição, tendo V. decussata acumulado mais CBZ (exceto para CBZ 9.00 μg/L) do que V. philippinarum. Após uma exposição de 28 d a V. decussata acumulou uma concentração superior de CBZ, exceto na concentração mais elevada, onde os níveis concentrados foram similares aos do teste agudo. Apesar das amêijoas acumularem baixos níveis de CBZ comparando com a concentração de exposição, estes foram suficientes para comprometer o estado de saúde das espécies em estudo levando a uma condição de stress oxidativo. As duas espécies apresentaram uma resposta diferente à CBZ quando submetidas ao teste agudo. V. philippinarum aumentou os níveis de peroxidação lipídica na concentração mais alta de exposição, ao passo que V. decussata apresentou uma diminuição significativa deste parâmetro. A atividade da glutationa S-transferase foi estimulada no caso da V. decussata e diminuída para V. philippinarum. Não obstante, após a exposição a CBZ, em ambas as espécies ocorreu uma indução das atividades da glutationa reductase e superóxido dismutase. Os resultados indicaram que, provavelmente, V. philippinarum possui um sistema de defesa antioxidante deficiente quando comparada com V. decussata, sendo menos apta na neutralização das espécies reativas de oxigénio e portanto apresentou-se como a espécie mais sensível aos efeitos da CBZ. Uma exposição de 28 d à CBZ resultou numa toxicidade superior na espécie V. decussata por comparação com o teste agudo. Tal foi principalmente refletido na reduzida atividade/conteúdo nos mecanismos envolvidos no sistema de defesa antioxidante, traduzindo-se numa baixa capacidade para combater o stress oxidativo provocado pela CBZ. O quociente de risco determinado para a Ria de Aveiro foi superior a 1 indicando que se suspeita de um risco ecotoxicológico. A bioacumulação de CBZ pelas amêijoas torna evidente a possibilidade de ser transferida ao longo da cadeia alimentar, afetando em último caso o ser humano.

Doutoramento em Biologia
De forma a reduzir a transmissão de agentes patogénicos através do consumo de bivalves, devem ser desenvolvidas novas tecnologias a associar à depuração, tal como a terapia fágica. A eficácia da utilização de fagos para controlar infeções bacterianas tem sido relatada em diversos estudos. No entanto, relativamente à utilização da terapia fágica para inativar bactérias patogénicas durante a depuração de bivalves, existe apenas um estudo preliminar, feito sem circulação de água (contrariamente ao que acontece na depuração industrial). O sucesso da combinação destas duas tecnologias depende da compreensão detalhada da dinâmica sazonal e espacial da comunidade bacteriana total, incluindo bactérias associadas a doenças devido ao consumo de bivalves e indicadores de qualidade microbiológica da água, nas zonas de produção de bivalves. Durante o Verão, a comunidade bacteriana total apresentou uma maior complexidade e aumento da abundância dos principais grupos de bactérias patogénicas, indicando que deste pode ser um período crítico onde a terapia fágica deve ser aplicada. No entanto, devido ao impacto das fontes de poluição antropogénicas e outras fontes de contaminação, a terapia fágica poderá ter que ser aplicada também durante a estação fria. Cinco novos fagos foram isolados e caracterizados para controlar Salmonella Typhimurium (phSE-1, phSE-2 e phSE-5) e Escherichia coli (phT4A e EC2A), a fim de avaliar a sua potencial aplicação durante a depuração de bivalves. Os ensaios in vitro demonstraram que o uso de fagos individuais (phT4A e EC2A), pode ser uma alternativa eficaz para o controlo de E. coli, em particular, quando combinados em cocktail. A depuração num sistema estático com MOI 1 usando os fagos phT4A e EC2A, foi a condição em que se obteve melhores resultados (diminuição ~2.0 log CFU/g) em berbigões artificialmente contaminados. Quando berbigões naturalmente contaminados foram tratados em sistema estático com as suspensões de fagos individuais e cocktails de fagos, foram obtidas reduções semelhantes na concentração de E. coli (diminuição ~0.7 log UFC/g). Quando os berbigões naturalmente contaminados foram depurados com o fago phT4A com circulação de água, a concentração de bactéria foi reduzida mais rapidamente que na ausência de fagos. Os resultados dos ensaios in vitro mostraram igualmente que o controlo de S. Typhimurium com fagos phSE-5 e phSE-2 e o cocktail destes dois fagos foi eficiente. A aplicação do fago phSE-5 e cocktail (phSE-2/phSE-5) inativou eficazmente S. Typhimurium em berbigões contaminados artificialmente (redução de 1.7 UFC/g com o fago phSE-5 e 0.7 log UFC/g com o cocktail phSE-2/phSE-5) e em berbigões naturalmente contaminados (redução de 0.9 log UFC/g para ambos para a suspensão simples e para o cocktail) durante a depuração em sistema estático, especialmente quando são usadas suspensões do fago phSE-5 a uma MOI baixa. Os berbigões artificialmente contaminados também foram mais eficazmente descontaminados durante a depuração com circulação de água na presença do fago phSE-5 que quando foi usada apenas depuração sem adição de fagos (i.e. o processo convencional de depuração). Este é o primeiro trabalho em que foi testado o uso de fagos durante a depuração de berbigões naturalmente contaminados e contaminados artificialmente em sistemas com circulação de água, tal como acontece na depuração industrial de bivalves, provando assim que esta tecnologia poderá ser transposta para a indústria. Os resultados obtidos usando fagos de E. coli e de S. Typhimurium mostraram que a combinação da terapia fágica e depuração melhora a segurança microbiana dos bivalves para consumo humano, melhorando a eficiência de descontaminação. Além disso, esta abordagem também permite reduzir o tempo necessário para a depuração e consequentemente, os custos a ela associados. No entanto, a seleção e caracterização dos fagos e determinação da MOI mais adequado para ser utilizado durante a terapia fágica, é essencial para o sucesso da terapia fágica no controlo de bactérias patogénicas.
In order to reduce the infections by microbial pathogens through the consumption of bivalves, it is essential to develop alternative approaches to the conventional depuration practices. One new promising approaches is to combine the depuration of bivalves with phage therapy. The use of phages to control bacterial infections has been reported across numerous fields by many researchers. However, relatively the combination of depuration and phage therapy to eliminate pathogenic bacteria in bivalves there is only one study, and this study did not replicate industrial depuration procedures. The successful combination of those two technologies depends on a detailed understanding of the seasonal and spatial dynamics of the overall bacterial communities, including the bacteria implicated in bivalves-related illness and the indicators of microbiological water quality, in the harvesting areas. During the summer, the total bacterial community presented high complexity and an increase of abundance of the main pathogenic bacteria, indicating that this season is the critical time frame when phage therapy should be applied. However, due to the impact anthropogenic and other sources of contamination, phage therapy could be necessary also during the cold season. Five new phages were isolated and characterized to control Escherichia coli (phT4A and ECA2) and Salmonella Typhimurium (phSE-1, phSE-2 and phSE- 5) in order to evaluate their potential application during depuration. The in vitro assays indicated that the use of phages individually (phT4A and EC2A) or combined in cocktails (phT4A/EC2A), can be an effective alternative to control of E. coli, particularly if combined in a phage cocktail. Depuration in static seawater at MOI 1 with phage phT4A and ECA2 revealed to be the best condition (decreased of the 2.0 log CFU/g) in artificially contaminated cockles. When naturally contaminated cockles were treated in static seawater with single phage suspensions and the phage cocktail, similar decreases in the concentration of E. coli (decreased of the 0.7 log CFU/g) were achieved. When naturally contaminated cockles were depurated using conventional practices with phage phT4A, bacterial concentration was reduced sooner. The assays in vitro, demonstrated that the control S. Typhimurium with phages phSE-5 and phSE-2 and with these two phages combined in a cocktail was efficient, paving way for the in vivo studies. The efficiency of bacterial inactivation with single phage suspensions of phSE-5 and phSE-2 leaded to further in vivo studies to control of Salmonella in bivalves. The application of phage phSE-5 and phage cocktail phSE-2/phSE-5 can be successfully employed to inactivate S. Typhimurium (reduction of 1.7 log CFU/g for phSE-5 and 0.7 log CFU/g for phSE-2/phSE-5 in artificially contaminated cockles and reduction of 0.9 log CFU/g for both in naturally contaminated cockles) during depuration in static system, especially if phSE-5 phage is used individually and if a low MOI is employed. Using conventional depuration practices in the presence of phage phSE-5, bacterial concentration is more quickly and efficiently reduced in artificially contaminated cockles. To our knowledge, this is the first report of a depuration trial using phages in the artificially and naturally contaminated cockles using industrial depuration procedures, proving that this technology can be ported into the bivalves industry. The obtained results using phages of E. coli and S. Typhimurium indicated that combining phage therapy with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs. However, the selection and characterization of appropriate phages and the most adequate multiplicity of infection to be used in phage therapy is a critical stage to achieve a successful phage-mediated control of pathogenic bacteria.

Over the last years, there has been an increasing demand of shellfish for consumption. The ability of traditional capture fisheries to supply bivalves is unlikely to increase significantly because of the drops in natural recruitments of seed, which are mainly due to overexploitation of natural stocks. Production of bivalve seed in hatcheries is a relatively new industry for which empirical approaches were developed, adapting methods across species and measuring the resulting effect in terms of growth and survival. To date, only a few bivalve species of major aquacultural importance in Europe have benefited from newly developed genomic resources (e.g. microarrays and RNA sequencing) and gene silencing approaches, which are both expected to significantly improve the biological knowledge on these commercially important species in the coming years. The present PhD thesis aimed firstly at increasing, through Next Generation Sequencing, genomic information available for two emerging species, Venerupis decussata and Pecten maximus. The second aim was to investigate two main bottlenecks hampering the bivalve production in hatchery: efficiency of reproduction and susceptibility to pathogens. By means of microarray analysis, a gene expression study on V .decussata oocytes at different maturation stages was performed and the major biological processes involved in gamete maturation were identified. A RNA sequencing experiment was conducted on pathogen-challenged hemocytes and unchallenged controls to study the P. maximus immune transcriptome. mRNAs encoding proteins with a known immune function were detected and a global analysis of differential expression comparing gene-expression levels in stimulated hemocytes against controls provided evidence on a large set of transcripts involved in P. maximus immune response. Finally, reverse genetic and real-time PCR were implemented to investigate the role of IkB2 in the Crassostrea gigas immune response against Ostreid herpesvirus type 1. Following the injection of a dsRNA targeting IkB2, juveniles were infected with OsHV-1 and mRNA levels of four immune genes (IkB1, IkB2, Rel, SOCS) were evaluated in gonads and gills demonstrating their implication in the Pacific oyster antiviral response.
Nel corso degli ultimi anni, si assistito ad una crescita della domanda di molluschi destinati al consumo alimentare, alla quale però la pesca tradizionale non ha saputo far fronte, principalmente a causa dell’impoverimento degli stock naturali e alla conseguente scarsità di seme. La produzione di seme di bivalvi in schiuditoio è un’attività relativamente recente e le attuali metodiche di allevamento non sono altro che protocolli già usati in altre specie, adattati e verificati in termini di crescita e sopravvivenza. Ad oggi, ad aver beneficiato delle recenti risorse genomiche, come microarry ed RNA sequencing, e delle sofisticate strategie di silenziamento genico sono state solo alcune specie di bivalvi di notevole interesse alimentare a livello europeo. Tuttavia, si pensa che, nel corso dei prossimi anni, questi approcci molecolari possano costituire una risorsa importante per lo studio della biologia di numerose specie di bivalvi non ancora allevate su larga scala ma di crescente interesse commerciale. Il primo obiettivo della presente tesi di dottorato è stato quello di incrementare, mediante tecniche di Next Generation Sequencing, le risorse genomiche attualmente disponibili per due specie emergenti: la vongola verace Venerupis decussata e la cappasanta atlantica Pecten maximus. In secondo luogo, si sono considerati due importanti fattori che ostacolano l’allevamento dei bivalvi in schiuditoio: l’efficienza riproduttiva e la suscettibilità ai patogeni. Utilizzando una piattaforma microarray, sono stati valutati i livelli di espressione genica di oociti di V. decussata a due diversi stadi di maturazione. L’analisi dei dati ha quindi permesso di identificare i principali processi biologici coinvolti nella maturazione dei gameti femminili. Inoltre, al fine di studiare il trascrittoma immunitario di P. maximus, è stato effettuato un esperimento di RNA sequencing su emociti immuno-stimolati e di controllo. Questo studio ha permesso di identificare trascritti di mRNA che codificano per importanti proteine del sistema immunitario. In aggiunta l’individuazione di geni differenzialmente espressi in emociti stimolati e controlli ha messo in evidenza un set di trascritti potenzialmente implicati nella risposta immunitaria di P. maximus. Infine, allo scopo di valutare il ruolo di IkB2 nella risposta immunitaria di Crassostrea gigas all’infezione da herpesvirus di tipo 1, è stato effettuato un esperimento di RNA interference. In seguito all’iniezione di un RNA a doppio filamento codificante una porzione del trascritto IkB2, individui giovani di C. gigas sono stati infettati con un omogenato contenente il virus HV-1. Infine, per valutare l’importanza di quattro geni della risposta immunitaria (IkB1, IkB2, Rel, SOCS), i loro livelli di espressione sono stati valutati in due diversi tessuti: le gonadi e le branchie.