Auswahl der wissenschaftlichen Literatur zum Thema „Mytilus galloprovincialis – Larves“

Les Perturbateurs Endocriniens (PE) sont une classe de contaminants environnementaux d'origine anthropique qui affectent les organismes en perturbant leur système endocrinien. Notamment, le manque de compréhension détaillée des systèmes endocriniens et leur voie de signalisation empêche la compréhension de l’effet de PEs chez les invertébrés. Conformément, cette thèse vise à aborder la question de la perturbation endocrinienne chez les invertébrés marins en exploitant le développement larvaire de la moule méditerranéenne Mytilus galloprovincialis. En appliquant la logique de l’Adverse Outcome Pathway-AOP, le travail de cette thèse vise à caractériser le mécanisme d'action de PEs modèles BPA, TBBPA et TBT chez les larves de moule et à identifier de plausibles voies neuro-endocriniennes par lesquelles ils induisent leur effet biologique délétère. Les éléments neuroendocriniens régulant la biogenèse de la coquille larvaire ont été caractérisés et le processus morphogénétique établi comme sensible à la perturbation neuroendocrinienne. Les plausibles AOPs neuro-endocriniens qui pourraient être liées aux effets biologiques de PEs modèles ont été identifiées. En plus, l'AOP possible du TBT a établi une relation plausible entre la signalisation des Récepteurs Nucléaires et le système neuroendocrinien des larves de moule. Les résultats de cette thèse représentent des preuves essentielles qui aideront et feront avancer la compréhension actuelle des mécanismes de perturbation endocrinienne chez les organismes invertébrés
Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect organisms by disrupting their endocrine system. Notably, the lack of detailed understanding of endocrine systems and their signaling pathway prevents understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms

Les écosystèmes marins côtiers sont soumis à des pressions anthropiques et naturelles qui placent les organismes aquatiques dans des situations de multi-stress.Ce travail doctoral avait pour objectif d’évaluer l’impact et de mieux comprendre les mécanismes d’adaptation des premiers stades de vie de moule exposés à deux facteurs environnementaux majeurs : la pollution métallique et l’accroissement des températures des eaux marines côtières. Les effets induits par l’exposition à deux polluants métalliques modèles (Cu et Ag) et à un stress thermique modéré seuls ou en combinaison ont été évalués sur les stades précoces de développement de deux espèces de moule : Mytilus galloprovincialis et Mytilus edulis ainsi que leurs hybrides. Nos résultats montrent une augmentation significative du pourcentage de larves D ma lformées avec l’augmentation de la température. Par ailleurs, l’Ag apparaît significativement plus toxique que le Cu pour les larves. De plus,la toxicité des métaux s’accroit avec l’augmentation de la température. La coexposition aux métaux et à une température modérée augmente les activités enzymatiques antioxydantes de la catalase (CAT), de la superoxyde dismutase (SOD)et de la glutathion-S-transférase (GST) et accroît le contenu cellulaire en métallothionéines et la peroxydation lipidique. A une température plus élevée de 22 °C,une diminution significative des activités des enzymes antioxydantes est observée.Les dommages à l’ADN chez les larves-D de moule M. gallo provincialis ont été évalués à l’aide du test des comètes avec et sans traitement par la Formamidopyrimidine ADN glycosylase. L’exposition aux métaux et/ou à la température aaugmenté de manière significative les lésions de l’ADN sur les larves de moule,avec un effet plus accentué sur les dommages oxydatifs. Il a été montré également que le Cu et l’Ag sont accumulés différentiellement dans les larves en fonction de la température d’exposition. L’étude de l’expression par RT-qPCR de 18 gènes impliqués dans les défenses antioxydantes, la réparation de l’ADN, l’apoptose,la protéolyse, la transcription, les réponses au stress thermique et la détoxification métallique a été conduite chez les larves-D de moule M. galloprovincialis.En cas d’un stress thermique modéré, une tendance à la surexpression des gènes impliqués dans les défenses cellulaires est observée. Toutefois, en cas d’une coexposition métallique et thermique, les voies d’apoptose et d’altération cellulaires sont activées.Un plugin et une macro ont été développés pour le logiciel ImageJ afin d’évaluer et de caractériser le comportement de nage des larves D de M. galloprovincialis.Une augmentation significative de la vitesse maximale des larves-Davec l’augmentation de la température est observée sans que la vitesse moyenne soit affectée. En revanche, l’exposition au Cu et à l’Ag à 22 °C augmente significativement les vitesses moyenne et maximale des larves. En condition contrôle à18 °C, les larves suivent principalement des trajectoires rectilignes (88 %). Avec l’augmentation de la température et/ou l’exposition aux métaux, les trajectoires circulaires augmentent considérablement au détriment des trajectoires rectilignes.Ces modifications de comportement pourraient être liées d’une part à l’induction de malformations mais aussi à la modulation de l’activité neuronale. Ainsi, une augmentation significative de l’activité acétylcholine estérase (AChE) des larves a été observée suite à l’exposition à un gradient de températures et/ou aux métaux.Des larves D viables et normalement développées ont été obtenues par hybridation de M. galloprovincialis et M. edulis. Les larves de M. edulis apparaissent plus sensibles à la température que les larves de M. galloprovincialis. Aucune différence significative de sensibilité n’a été observée entre les moules hybrides et pures quelque soit le métal testé. (...)
Coastal marine ecosystems are subject to anthropogenic and natural pressures that place aquatic organisms in multi-stress situations. The aim of this doctoral work was to assess the impact and better understand the adaptation mechanisms of mussel early life stages exposed to two major environmental factors : metallic pollution and increasing temperatures in coastal marine waters. The effects induced by exposure to two model metal pollutants (Cu and Ag) and moderatethermal stress alone or in combination were evaluated on the early life stages of development of two mussel species : Mytilus galloprovincialis and Mytilus edulisas well as their hybrids. Our results show a significant increase in the percentageof malformed D-larvae with increasing temperature. Moreover, Ag is significantly more toxic than Cu for larvae. In addition, the toxicity of metals increases with increasing temperature. Co-exposure to metals and a moderate temperature increases the antioxidant activity of catalase (CAT), superoxide dismutase (SOD)and glutathione-S-transferase (GST), and increases cellular content of metallothioneinsand lipid peroxidation. At a higher temperature of 22 °C, a significant decrease in the activities of the antioxidant enzymes is observed. DNA damage in M. galloprovincialis larvae was evaluated using the comet assay with and without Formamidopyrimidine DNA glycosylase treatment. Co-exposure to metals and/ortemperature increase significantly increased DNA lesions on mussel larvae, witha more pronounced effect on oxidative damage. It has also been shown that Cuand Ag are accumulated differently in the larvae as a function of the exposure temperature. The study of the gene expression by RT-qPCR of 18 genes involvedin antioxidant defenses, DNA repair, apoptosis, proteolysis, transcription, thermal stress and metal detoxification was conducted in D-larvae of M. galloprovincialis.Under moderate thermal stress, a tendency of over expression of the cell defense pathways is observed. However, in the case of metallic and thermal co-expositions, the pathways of apoptosis and cellular alteration are rather induced. A pluginand macro were developed for the evaluation and characterization of behavior oflarvae of M. galloprovincialis. A significant increase in the maximum speed of D larvaewith increasing temperature is observed without affecting the mean velocity. In contrast, exposure to Cu and Ag at 22 °C significantly increases average and maximum larval speed. Under controlled conditions at 18 °C, larvae mainly follow rectilinear trajectories (88 %). With increasing temperature and/or exposure to metals, circular trajectories increase considerably at the expense of rectilinear trajectories. These changes in behavior may be related to the induction of malformations but also to the modulation of neuronal activity. Thus, a significant increase in the acetylcholinesterase (AChE) activity of the larvae is observed following exposure to a temperature gradient and/or metals. Viable and normally developed D-larvae were obtained by hybridization of M. galloprovincialis and M.edulis. Larvae of M. edulis are more sensitive to temperature than larvae of M. galloprovincialis.No significant sensitivity differences between species are observed whatever the metal used. Moreover, the hybrid larvae from female M. galloprovincialis are more tolerant to the increase of temperature compared to the hybrid larvae issues from rom female of M. edulis

Mestrado em Biologia Marinha
A dispersão de organismos marinhos durante a sua fase larvar é determinante para a conectividade entre populações e o estabelecimento da complexa biogeografia das espécies marinhas. A determinação dos padrões de dispersão tem sido relevante para a conservação de espécies, a gestão de stocks de pesca e mesmo para a compreensão de processos ecológicos como a extinção e a recolonização de populações. As estruturas carbonatadas de organismos marinhos representam arquivos de informação ambiental. Através da análise geoquímica de estruturas como os otólitos de peixe e os estatólitos e conchas de invertebrados é possível descobrir as condições físico-químicas ambientais sob as quais se desenvolveram estes organismos e reconstruir padrões de dispersão e locais de origem de indivíduos. No entanto, a incorporação dos elementos químicos por estas estruturas é altamente regulado por um conjunto de fatores físico-químicos ambientais e biológicos que deverão ser ponderados para uma correta utilização de assinaturas geoquímicas como meio de identificação geográfica. Com o intuito de investigar como a composição química de conchas larvares do mexilhão Mytilus galloprovincialis é influenciada por algumas condições ambientais e temporais, indivíduos desta espécie foram submetidos a diferentes temperaturas, salinidades e concentrações de elementos. Pretendia-se verificar como difere a incorporação de elementos com os diferentes parâmetros de cada fator ambiental e se a mesma é influenciada pela idade do indivíduo. Com esse objetivo foram cultivadas larvas de M. galloprovincialis por um período de 6 e 14 dias e criados tratamentos combinados de temperatura (12, 17, 20 e 25°C) e de salinidade (26, 32 and 37). Foram quantificadas as concentrações dos elementos Mg, Mn, Ba, Sr, Cu e Zn pelo rácio de Ca através de análise de ICP-MS (inductively coupled plasma mass spectometry). Paralelamente um outro conjunto de indivíduos larvares foi submetido a concentrações de Cu e Zn durante aproximadamente 3 dias. Não foi encontrada variabilidade significativa nas assinaturas geoquímicas entre as larvas com 6 dias e 14 dias de idade, sugerindo uma composição química estável, para este período, que não foi afetada pelo desenvolvimento larvar. Numa outra abordagem, foi detetada a máxima incorporação destes elementos a uma temperatura de 17°C e a diferentes combinações de temperatura e salinidade, realçando uma interacção entre estes fatores. Estes resultados realçam a possível influência da temperatura sob a disponibilidade de cada elemento. Outro aspeto a ter em conta é o facto de a 17°C serem encontradas as condições ótimas para o desenvolvimento larvar, e por isso também da secreção da concha carbonatada do mexilhão, existindo uma maior incorporação dos elementos do ambiente. A incorporação de Cu, no entanto, não mostrou ser significativamente afetada pela temperatura nem pela salinidade. Relativamente ao Zn estes efeitos foram apenas significativos quando não combinados. Para além disso, e numa terceira abordagem, a disponibilidade destes últimos na água, não se mostrou determinante para a incorporação dos mesmos nas conchas, sugerindo-se um controlo da inclusão por parte de fatores fisiológicos do próprio organismo. Os resultados obtidos permitem compreender mais detalhadamente a relação que alguns fatores ambientais exercem sob a produção de assinaturas químicas em conchas larvares desta espécie de mexilhão e poderão contribuir para uma melhor interpretação de padrões de conectividade de populações no meio marinho.
Larval dispersal is the main cause of connectivity between populations and the establishment of the complex biogeography of most marine species. The determination of dispersion patterns has been relevant to conservation studies of species, management of fisheries stocks and even for the understanding of ecological processes as the extinction and re-colonization of populations. Carbonate structures of marine organisms work as an archive of environmental information. Through geochemical analysis of fish otoliths, as well as statoliths and shells of invertebrates is possible to discover physical and chemical environmental conditions under which these organisms developed and trace dispersion patterns and natal habitats. However, the incorporation of trace elements in these structures is highly regulated by a range of physical and chemical environmental factors as well as physiological and biochemical. These factors should be considered for an accurate use of geochemical signatures as a means of geographical identification. In order to investigate how the geochemical composition of larval shells from the mussel species Mytilus galloprovincialis is influenced by environmental and temporal conditions, individuals were submitted to different temperatures, salinities and water elemental concentrations. The main objective was to understand how the elemental incorporation varies with different parameters of each environmental factor, and if the same is influenced by age. M. galloprovincialis larval were reared during 6 and 14 days under combined treatments of temperature (12, 17, 20 and 25° C) and salinity (26, 32, and 37). Concentrations of the elements Mg, Mn, Ba, Sr, Cu and Zn by the ratio of Ca were analyzed through ICP-MS (inductively wound couple plasma-mass spectrometry) analysis. Simultaneously another set of individuals were submitted to different concentrations of Cu and Zn for a period of approximately 3 days. No significant variability in chemical composition was found in larval shells between larval with 6 and 14 days of development, suggesting a stable geochemical signature, for this period, and no age effect on incorporation. On a second approach, was detected a maximum incorporation of these elements at 17° C and at different temperature-salinity combinations, suggesting an interaction between these factors. These results highlight the possible influence of temperature on each element’s availability. Another aspect is the fact that at 17° C larval seem to find optimum conditions for its development and for carbonate shell secretion, having a higher incorporation of the elements from the water. Cu incorporation, however, was not significantly affected by temperature or salinity. With regard to the Zn these effects were significant only when not interacting. Finally, the availability of the lastly mentioned elements in the water was not significant for elemental concentrations in shells, suggesting that certain elements incorporation might be specifically controlled by physiological mechanisms of the organism. Results obtained allow understanding in more detail the influence some environmental factors have on geochemical signature production in mussel larval shells of this species and may contribute to a better use of elemental fingerprinting in the interpretation of connectivity patterns.

Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect the endocrine systems of living organisms. The lack of knowledge of endocrine systems prevents the understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms.

Bivalves are some of the most studied marine organisms for ecological importance as bio-indicators and economic value in shellfish farming. They have a planktonic life stage, spending about 4 weeks as free-swimming larvae in the water column and probably this is an evolutive advantage making the most common marine molluscs. A variety of physical, biological, and genetic factors influence the initial larvae pelagic dispersion and the subsequent soil settlement. During larval stages bivalves are characterized by a very fast growth, and by morphological and metabolic changes pointed to the accomplishment of metamorphosis into adult organism. These radical modifications are probably due to the activation of different gene expression patterns associated with each developmental stage. The aim of my PhD project was to define the transcriptional signatures of 6 larval stages in Mytilus galloprovincialis to provide a comprehensive overview on the biological processes underlying the larval development. In 2009 we performed the stimulation of bivalves mating to collect samples of fertilized eggs and each larval stage. Total RNA have been extracted and each sample was fixed 4% paraformaldehyde for in situ hybridization. We set up seven stage-specific 3'-end cDNA libraries in order: i) to discover specifically expressed transcripts in larval stages, and ii) to define the first transcriptional signature of larval development in M. galloprovincialis, calculating the 3’-EST frequency at each stage. Using pyrosequencing technology (Roche 454 Titanium), we have generated a total of 751,872 high quality reads characterized by a median length of 315 bp. The assembling process (Newbler 2.5.1) of pyrosequencing reads and ESTs, previously produced by our lab (Mytibase), resulted in 14,364 unique sequences/putative transcripts and 55,493 singletons. We counted the number of reads that align with each unique sequence at each stage-specific library defining the expression pattern of each stage. After the annotation process we were able to estimate a gene discovery rate of about 45% rising to 60% in the early larval stages characterized by a lot of unknown genes. Quantitative real time PCR analysis was performed to quantify and validate the expression profile of some genes on samples collected during a new mussel mating experiment, performed at the beginning of 2011. On the basis of the high correlation values between the qRT-PCR and sequencing data, we can consider the counts reliable. A negative binomial test was performed to identify differentially expressed transcripts between at least two stages. In particular the 25% of genes results to be differentially expressed and we observed the main differences comparing early larval stages, including trocophora and D-larva, and late larval stages, including umbo stage, pediveliger, and metamorphic larva. Differentially expressed genes showing similarity with known genes or proteins were grouped into functional categories according to Gene Ontology in order to identify the biological categories involved in development such as organ development, growth control, biomineralization, and byssus secretion. Finally we are performing whole mount in situ hybridization of the most differentially expressed unknown genes to start their functional characterization defining their morphological localization. We defined the first transcriptional signatures of larval stages in M. galloprovincialis in order to better understand the molecular mechanisms involved in the process of development from larva to the adult.
I bivalvi sono la classe di organismi marini più studiata, sia per il loro ruolo ecologico di bioindicatori del grado di inquinamento costiero, sia per la loro rilevanza economica nel settore alimentare. Il loro sviluppo larvale è caratterizzato da una rapida crescita e da profondi cambiamenti morfologici e metabolici volti al raggiungimento dello stadio adulto. I radicali mutamenti a cui l’organismo va incontro sono riconducibili all’espressione coordinata di specifici geni. Lo scopo del mio progetto di dottorato è stato quello di definire i profili trascrizionali associati a ciascuno dei 6 stadi di sviluppo larvale di Mytilus galloprovincialis per far luce sui processi biologici alla base dell sviluppo larvale. Nel 2009 abbiamo riprodotto in laboratorio il naturale processo di fecondazione tra 8 femmine e 20 maschi inducendo l’emissione dei gameti. Abbiamo quindi raccolto campioni di RNA totale dell’uovo fecondato e di ciascuno stadio larvale e abbiamo inoltre conservato in paraformaldeide alcune larve per effettuare esperimenti di ibridazione in situ. A partire dall’RNA totale estratto abbiamo allestito sette librerie stadio specifiche costituite da frammenti 3’ terminali di cDNA. Queste sono state sequenziale con le moderne tecnologie di sequenziamento massivo così da ottenere sia le sequenza di trascritti espressi durante gli stadi larvali, sia una stima del loro profilo di espressione nei vari stadi larvali calcolando la loro frequenza di rappresentazione in ciascuna libreria. Per il sequenziamento ci siamo avvalsi della tecnologia 454 (Roche) che ci ha permesso di ottenere 751,872 reads caratterizzate da una lunghezza mediana di 315 bp. Le reads ottenute sono state assemblate con sequenze prodotte precedntemente dal nostro laboratorio e depositate su MytiBase ottenendo 14,364 sequenze consensus e 55,493 singletons. Quindi abbiamo calcolato il numero di reads che si allineano con ciascun trascritto in ciascuna libreria stadio specifica così da ottenere una stima dei profili trascrizionali associati a ciascuno stadio di sviluppo larvale. Sfruttando un algoritmo da noi sviluppato siamo riusciti ad annotare il 55% dei trascritti. Per verificare l’attendibilità delle conte come mezzo per stimare l’espressione genica abbiamo eseguito una serie di esperimenti di PCR quantitativa su un nuovo campionamento biologico eseguito all’inizio del 2011. Sulla base dell’elevata correlazione tra i dati di espressione stimati con le due tecnologie indipendenti possiamo giudicare affidabile il nostro approccio. Tramite un test binomiale negativo abbiamo determinato che il 25% dei trascritti risulta differenzialmente espresso in almeno due stadi di sviluppo. Inoltre le maggiori differenze di espressione si rilevano confrontando gli stadi precoci (troco fora e larva D) con quelli tardivi (larva umbonata, pediveliger e larva metamorfica). Mediante analisi di Gene Ontology abbiamo individuato i processi biologici maggiormente implicati nello sviluppo larvale quali ad esempio il controllo della crescita, lo sviluppo degli organi e i processi di biomineralizzazone e sintesi del bisso. Infine stiamo eseguendo degli esperimenti di ibridazione in situ per incominciare la caratterizzazione di alcuni dei geni, differenzialmente espressi ma non annotati, definendo la loro localizzazione. Concludendo, abbiamo ottenuto la prima stima dei profili trascrizionali associati a ciascuno stadio larvale di M. galloprovincialis per comprendere i meccanismi molecolari su cui fonda il processo di sviluppo larvale.

Mestrado em Meteorologia e Oceanografia Física
A cultura de mexilhão na costa NW da Península Ibérica, explorada desde o início do século XX, tem vindo a crescer na economia desta região. Desta forma, torna-se necessário conhecer os detalhes do ciclo de vida das larvas de mexilhão, durante a sua fase planctónica. Com o objetivo de estudar a dispersão, a conectividade larvar, e o recrutamento de larvas de mexilhão nas diferentes regiões da costa NW da Península Ibérica, foi implementado um modelo lagrangiano, ROFF. Este permite simular as trajetórias, o crescimento nas diferentes fases larvares, a taxa de mortalidade abiótica e por advecção para o ano particular, 2009. Os resultados obtidos com este modelo foram validados através da comparação com um conjunto de observações em estações amostradas nas Rias Baixas Galegas. Através da análise das matrizes de conectividade e do balanço de líquido, observou-se que a Ria de Arousa é a maior fornecedora de larvas. Para além disto, a Ria de Muros é a que apresenta um balanço emissãoreceção mais favorável. E ainda uma tendência na conectividade larvar em direção a N ao logo do ano, observando um fornecimento liquido de larvas nas regiões a N das principais Rias.
The mussel culture in the NW coast of the Iberian Peninsula, is exploited since the beginning of the XX Century, has been growing in the economy of this region. Thus, it is important to understand the details of the life cycle of mussel larvae, during its planktonic stage. In order to study a dispersion, the larvar connectivity, and the recruitment of mussel larvae in different regions of the NW coast of the Iberian Peninsula, it was implemented a Lagrangian model, ROFF. This allows the simulation of trajectories, growth in different larvar stages, the abiotic mortality rate and the advection for the year 2009. The results obtained with this model were validated through comparison with a set of observations at stations sampled in the four main Rias. Through the analysis of connectivity matrices and the net supply, it was observed that the Arousa’s Ria is the largest supplier of larvae. Furthermore, the Ria de Muros is the one presenting a more favorable emission - reception balance. And yet a trend in larvar connectivity towards the N throughout the year, noting a liquid supply of larvae in the regions N of the main Rias.

Mytilus galloprovincialis is invasive on rocky shores on the west coast of South Africa where it has become the dominant intertidal mussel. The success of this species on the west coast and its superior competitive abilities, have led to concern that it may become invasive on the south coast at the expense of the indigenous mussel Perna perna. On shores where these species co-occur, there appears to be habitat segregation among zones occupied by mussels. M.galloprovincialis dominates the high-shore and P.perna the low-shore, with a mixed zone at mid-shore level. This study examined the factors responsible for these differences in distribution and abundance. The study was conducted in Plettenberg Bay and Tsitsikamma (70km apart) on the south coast of South Africa. Each site included two randomly selected locations (300-400m apart). A third mussel species, Choromytilus meridionalis, is found in large numbers at the sand/rock interface at one location in Plettenberg Bay. Aspects of settlement, recruitment, growth and mortality of juvenile and adult mussels were examined at different tidal heights at each site. Quantitative analysis of mussel population structure at these sites supported the initial observation of vertical habitat segregation. Post-larvae were identified to species and this was confirmed using hinge morphology and mitochondrial DNA analysis. Size at settlement was determined for each species to differentiate between primary and secondary settlement. Adult distribution of C.meridionalis was primarily determined by settlement, which was highly selective in this species. Settlement, recruitment and growth of P.perna decreased with increasing tidal height, while post-settlement mortality and adult mortality increased higher upshore. Thus all aspects of P.perna’s life history contribute to the adult distribution of this species. Presumably, the abundance of P.perna on the high-shore is initially limited by recruitment while those that survive remain prone to elimination throughout adulthood. M.galloprovincialis displayed the same patterns of settlement and recruitment as P.perna. However, post-settlement mortality in this species was consistently low in the low and high zones. Juvenile growth also decreased upshore, suggesting that M.galloprovincialis may be able to maintain high densities on the high-shore through the persistence of successive settlements of slow-growing individuals. The low cover of M.galloprovincialis on the lowshore appeared to be determined by adult interactions. M.galloprovincialis experienced significantly higher adult mortality rates than P.perna in this zone. There were seasonal variations in the competitive advantages enjoyed by each species through growth, recruitment or mortality on the low-shore. In summer, P.perna had higher recruitment rates, faster growth and lower mortality rates, while M.galloprovincialis had slightly higher recruitment rates and faster growth rates in winter. P.perna is a warm water species while M.galloprovincialis thrives on the cold-temperate west coast of South Africa. Therefore both species appear to be at the edge of their optimal temperature regimes on the south coast, which may explain the seasonal advantages of each. Nevertheless, P.perna has maintained spatial dominance on the low-shore suggesting that it may ultimately be the winner in competition between these species. M.galloprovincialis appears to have a refuge from competition with P.perna on the high-shore due to its greater tolerance of desiccation stress, while being competitively excluded from the low-shore. Warm water temperatures coupled with poor recruitment rates at most sites may limit the success of M.galloprovincialis on this coast.

Protecting water and marine resources - and ensuring their environmental status - is one of the pillars of EU environmental policy. The acceleration of the process of loss of marine biodiversity or of essential goods to the ecosystem has required the adoption of special measures on nature and biodiversity (Sixth Community Environment Action Program). The process of recovering the natural bio filter from the coastal area of the sea coast requires the development of ecological methods designed to increase populations of epibiotic organisms that are filters capable of accelerating the restoration of marine quality in tourist areas - leisure on the coast. The modular system designed and developed by our specialists is intended both for the extension of the natural epibiont bio filter in traditional coastal areas, and for the cultivation for food purposes of mussels - Mytillus Galloprovincialis - the largest bivalve in the Black Sea and oysters - Crassostrea gigas. The location of the modular system has the following specific hydro meteorological characteristics: the winds in the northern sector have the highest dominance and intensity, the orientation of the Romanian Black Sea coast (from north to south) favors the dominance of currents parallel to the shoreline, and the structure of the substrate is composed of an alternation of submerged rocks and stony platforms covered with terry mud. The system has been successfully tested until the third stage of development of the epibiont organisms, the tests continuing at present. The presented solutions will contribute to the restoration of the sea filters, by ensuring an optimal breeding place for a variety of marine organisms.