Peripheral communities of the Eastern Lau Spreading Center and Valu Fa Ridge: community composition, temporal change and comparison to near-vent communities

Western Pacific hydrothermal vents will soon be subjected to deep-sea mining and peripheral sites are considered the most practical targets. The limited information on community dynamics and temporal change in these communities makes it difficult to anticipate the impact of mining activities and recovery trajectories. We studied community composition of peripheral communities along a cline in hydrothermal chemistry on the Eastern Lau Spreading Center and Valu Fa Ridge (ELSC-VFR) and also studied patterns of temporal change. Peripheral communities located in the northern vent fields of the ELSC-VFR are significantly different from those in the southern vent fields. Higher abundances of zoanthids and anemones were found in northern peripheral sites and the symbiont-containing mussel Bathymodiolus brevior, brisingid seastars and polynoids were only present in the northern peripheral sites. By contrast, certain faunal groups were seen only in the southern peripheral sites, such as lollipop sponges, pycnogonids and ophiuroids. Taxonomic richness of the peripheral communities was similar to that of active vent communities, due to the presence of non-vent endemic species that balanced the absence of species found in areas of active venting. The communities present at waning active sites resemble those of peripheral sites, indicating that peripheral species can colonize previously active vent sites in addition to settling in the periphery of areas of venting. Growth and mortality were observed in a number of the normally slow-growing cladorhizid stick sponges, indicating that these animals may exhibit life history strategies in the vicinity of vents that differ from those previously recorded. A novel facultative association between polynoids and anemones is proposed based on their correlated distributions.

Avaliação da técnica de dispersão da matriz em fase sólida assistida por vórtex para análise de fármacos em peixes

Os fármacos classificados como contaminantes orgânicos emergentes tornaramse tópico de discussão ambiental pois estes são capazes de atingir diferentes matrizes do meio ambiente, como água, solo e organismos aquáticos, e estudos demonstrando a toxicidade tem despertado o interesse científico. Neste estudo um método analítico foi otimizado e validado para determinação de quinze fármacos em amostras de peixes empregando preparo de amostras por dispersão da matriz em fase sólida (MSPD) assistida por vórtex e determinação por cromatografia líquida acoplada a espectrometria de massas sequencial (LC-MS/MS). O estudo ainda focou na aplicação de diferentes suportes sólidos na etapa de dispersão da MSPD, sendo que três (quitina, quitosana e concha de mexilhão dourado) foram utilizados pela primeira vez para esta finalidade, O método otimizado foi validado seguindo os parâmetros do INMETRO, ANVISA, SANCO e FDA. A curva analítica e linearidade foram avaliados através da calibração externa e superposição na matriz. Os compostos demonstraram linearidade dentro da faixa recomendada, com coeficiente de correlação maior do que 0,99. Os limites de detecção do método variaram de 1 a 100 ng g -1 , e os limites de quantificação a variaram de 5 a 1000 ng g -1 . Os valores de recuperação variaram de 68% a 108%, com RSD menores que 13% para todos os compostos. O efeito de matriz foi avaliado e quatro dos quinze compostos apresentaram efeito maior que ±20%. Na aplicabilidade o método demonstrou ser eficiente para extração de fármacos de diferentes espécies de peixe, apresentando exatidão e precisão adequados. Não foram encontrados resíduos de fármacos nas amostras analisadas.

Socio-economic profile of the Philippine National Aquasilviculture Program (PNAP) beneficiaries in Jiabong, Samar, Philippines

A total of 37 beneficiaries under the Philippine National Aquasilviculture Program (PNAP) was interviewed using the structured survey questionnaire of Socioeconomic Monitoring Guidelines for Coastal Managers in Southeast Asia (SocMon SEA). Most of the members of the households are young and in-school. Household heads’ primary occupation is fishing, a shift from mussel farming- the town’s major industry in the past decades. Perceived threat by the beneficiaries is related to the environment specifically typhoon and the problems on waste disposal. It also identified law enforcement as weak leading to dwindling fish catch, mass mortality of mussel, red tide and other problems affecting their primary sources of income. However, they could not relate these phenomena to the most likely causes. The current occupation does not provide sufficient income for the family as they seek for alternative jobs. Garbage and poor implementation of laws are among the identified problems of the beneficiaries.

Validation d’un procédé automatisé d’extraction sur phase solide de glandes digestives de moules pour l’identification et la quantification des dinophysistoxines en LC/ESI/SM2 par piégeage d’ions quadripolaire

This report presents a new extraction method of the dinophysistoxins (DTXs), confirmed by quantification using high-performance liquid chromatography coupled to mass spectrometry with an ion trap and electro spray interface (HPLC/ESI/MS2). The method originality consists on the adaptation of DTXs basic extraction procedure (liquid/ liquid) to a solid phase extraction (SPE) via a robotic station: ASPEC XLi The parameters of the automatization procedure were optimized to obtain the best DTXs recovery rate. These improvements were loaded with digestive gland mussel homogenat realized on a silica cartridge SPE, activated in hexane/chloroform (50:50), washed with hexane/chloroform (50:50) and extracted by an elution gradient (chloroform methanol (65:35) and methanol (100%)). This method was validated according to two normative referentials (linearity, detection quantification limits and accuracy…) : - The Guide of the Pharmacy industry: Analytical Validation, report of the commission SFSTP 1992 (French Corporation of the Sciences and Technical Pharmaceutical), - - The Procedure of validation of an alternative method in compare to a reference method. (AFNOR, 1998. NF V 03-110). Comparison with the classical liquid/liquid extraction and the automated method present clear advantages. In an analytical method the extraction is generally considered to be the most labor-intensive and error-prone step. This new procedure allowed us to increase throughput, to improve the reproducibility and to reduce the error risks due to the individual manual treatments.

Développement de matériaux à base de protéines extraites du byssus de la moule bleue Mytilus edulis

Le byssus est un amas de fibres que les moules produisent afin de s’ancrer aux surfaces immergées sous l’eau. Ces fibres sont pourvues de propriétés mécaniques impressionnantes combinant rigidité, élasticité et ténacité élevées. De plus, elles possèdent un comportement d’auto-guérison de leurs propriétés mécaniques en fonction du temps lorsque la contrainte initialement appliquée est retirée. Les propriétés mécaniques de ces fibres sont le résultat de l’agencement hiérarchique de protéines de type copolymère blocs riches en collagène et de la présence de métaux formant des liens sacrificiels réversibles avec certains acides aminés comme les DOPA et les histidines. Bien que cette fibre soit très intéressante pour la production de matériaux grâce à son contenu élevé en collagène potentiellement biocompatible, cette ressource naturelle est traitée comme un déchet par les mytiliculteurs. L’objectif de cette thèse était de valoriser cette fibre en extrayant les protéines pour générer une nouvelle classe de matériaux biomimétiques. Un hydrolysat de protéines de byssus (BPH) riche en acides aminés chargés, i.e. ~30 % mol, et permettant de former des films a pu être généré. Lorsque solubilisé à pH 10.5, le BPH forme un hydrogel contenant des structures en triple hélice de collagène et des feuillets β anti-parallèles intra- et inter-moléculaires. Suite à l’évaporation de l’eau, le film de BPH résultant est insoluble en milieu aqueux à cause des structures secondaires très stables agissant comme points de réticulation effectifs. Les propriétés mécaniques des films de BPH sont modulables en fonction du pH. Au point isoélectrique (pI = 4.5), les interactions électrostatiques entre les charges opposées agissent comme points de réticulation et augmentent la rigidité des films et leur contrainte à la rupture sans affecter la déformation à la rupture. À pH plus élevé ou plus bas que le pI, les performances mécaniques des films sont plus faibles à cause de la répulsion entre les groupements fonctionnels de même charge qui interagissent plutôt avec les molécules d’eau et causent le gonflement de la matrice protéique des films. Le BPH contenant un nombre élevé d’acides aminés chargés et réactifs, nous avons pu réticuler les films de manière covalente à l’aide d’EDC ou de glutaraldéhyde. Les propriétés mécaniques des films sont modulables en fonction de la concentration d’EDC utilisée lors de la réticulation ou en employant du glutaraldéhyde comme agent réticulant. Les films sont à la fois plus rigides et plus forts avec un degré de réticulation élevé, mais perdent leur extensibilité à mesure que les segments libres de s’étirer lors d’une traction deviennent entravés par les points de réticulation. La réticulation augmente également la résistance à la dégradation enzymatique par la collagénase, les films les plus fortement réticulés lui étant pratiquement insensibles. La spectroscopie infrarouge montre enfin que la réticulation entraîne une transition de feuillets β anti-parallèles inter-moléculaires vers des structures de type hélices de collagène/PPII hydratées. Des liens sacrificiels ont été formés dans les films de BPH par traitement au pI et/ou avec différents métaux, i.e. Na+, Ca2+, Fe3+, afin de moduler les propriétés mécaniques statiques et d’évaluer le rôle de ces traitements sur le comportement d’auto-guérison lors de tests mécaniques cycliques avec différents temps de repos. Plus la valence des ions métalliques ajoutés augmente, plus les propriétés mécaniques statiques affichent un module, une contrainte à la rupture et une ténacité élevés sans toutefois affecter la déformation à la rupture, confirmant la formation de liens sacrificiels. Les tests mécaniques cycliques montrent que les traitements au pI ou avec Ca2+ créent des liens sacrificiels ioniques réversibles qui mènent à un processus d’auto-guérison des performances mécaniques dépendant du pH. L’ajout de Fe3+ à différentes concentrations module les performances mécaniques sur un plus large intervalle et la nature plus covalente de son interaction avec les acides aminés permet d’atteindre des valeurs nettement plus élevées que les autres traitements étudiés. Le Fe3+ permet aussi la formation de liens sacrificiels réversibles menant à l’auto-guérison des propriétés mécaniques. Les spectroscopies Raman et infrarouge confirment que le fer crée des liaisons avec plusieurs acides aminés, dont les histidines et les DOPA. Les résultats dans leur ensemble démontrent que les films de BPH sont des hydrogels biomimétiques du byssus qui peuvent être traités ou réticulés de différentes façons afin de moduler leurs performances mécaniques. Ils pourraient ainsi servir de matrices pour des applications potentielles dans le domaine pharmaceutique ou en ingénierie tissulaire.

Hydrodynamism and its influence on the density of the decorator crab Microphrys bicornutus (Mithracidae) on intertidal rocky shores from a subtropical region

Hydrodynamism is an environmental stressor on marine communities with effects on populations of intertidal organisms. Microphrys bicornutus is a crab species associated with secondary substrates on rocky shores and little is known about its population dynamics and its relation to the shore hydrodynamics. The aim of this study is to describe the population structure of Microphrys bicornutus on intertidal rocky shores from the north coast of the São Paulo State, Brazil, and the influence of wave exposure on the density of this decorator crab. There was a greater density of M. bicornutus on sheltered than on exposed rocky shores, which is probably due to the hydrodynamic influence on M. bicornutus populations on rocky shores: directly ( by the physical stress caused by wave impact) and indirectly ( by determining the dominance of the secondary substrate) acting on the density of these crabs. Our results also suggest a vertical distribution of juveniles and adults, where the juveniles might be more abundant on the infralittoral fringe and, thus, more influenced by hydrodynamism than adults in the infralittoral.

The role of biological processes in affecting the dynamics and fate of microplastics in coastal marine systems

Microplastics (MP) are omnipresent contaminants in the marine environment. Ingestion of MP has been reported for a wide range of marine biota, but to what extent the uptake by organisms affects the dynamics and fate of MP in the marine system has received little attention. My thesis explored this topic by integrating laboratory tests and experiments, field quantitative surveys of MP distribution and dynamics, and the use of specialised analytical techniques such as Attenuated-Total-Reflectance- (ATR) and imaging- Fourier Transformed Infrared Spectroscopy (FTIR). I compared different methodologies to extract MP from wild invertebrate specimens, and selected the use of potassium hydroxide (KOH) as the most cost-effective approach. I used this approach to analyse the MP contamination in various invertebrate species with different ecological traits from European salt marshes. I found that 96% of the analysed specimens (330) did not contain any MP. As preliminary environmental analyses showed high levels of environmental MP contamination, I hypothesised that most MP do not accumulate into organisms but are rather fast egested. I subsequently used laboratory multi-trophic experiments and a long-term field experiment using the filter-feeding mussel Mytilus galloprovincialis and the detritus feeding polychaete Hediste diversicolor to test the aforementioned hypothesis. Overall, results showed that MP are ingested but rapidly egested by marine invertebrates, which may limit MP transfer via predator-prey interactions but at the same time enhance their transfer via detrital pathways in the sediments. These processes seem to be extremely variable over time, with potential unexplored environmental consequences. This rapid dynamics also limits the conclusions that can be derived from static observations of MP contents in marine organisms, not fully capturing the real levels of potential contaminations by marine species. This emphasises the need to consider such dynamics in future work to measure the uptake rates by organisms in natural systems.

Molecular mechanisms controlling physiological plasticity in marine mussels under the influence of natural and anthropogenic stress factors

Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

Characterization of microplastics ingested by marine benthos - a methodological and field-experimental study

Microplastics have become ubiquitous pollutants in the marine environment. Ingestion of microplastics by a wide range of marine organisms has been recorded both in laboratory and field studies. Despite growing concern for microplastics, few studies have evaluated their concentrations and distribution in wild populations. Further, there is a need to identify cost-effective standardized methodologies for microplastics extraction and analysis in organisms. In this thesis I present: (i) the results of a multi-scale field sampling to quantify and characterize microplastics occurrence and distribution in 4 benthic marine invertebrates from saltmarshes along the North Adriatic Italian coastal lagoons; (ii) a comparison of the effects and cost-effectiveness of two extraction protocols for microplastics isolation on microfibers and on wild collected organisms; (iii) the development of a novel field- based technique to quantify and characterize the microplastic uptake rates of wild and farmed populations of mussels (Mytilus galloprovincialis) through the analysis of their biodeposits. I found very low and patchy amounts of microplastics in the gastrointestinal tracts of sampled organisms. The omnivorous crab Carcinus aestuarii was the species with the highest amounts of microplastics, but there was a notable variation among individuals. There were no substantial differences between enzymatic and alkaline extraction methods. However, the alkaline extraction was quicker and cheaper. Biodeposit traps proved to be an effective method to estimate mussel ingestion rates. However their performance differed significantly among sites, suggesting that the method, as currently designed, is sensible to local environmental conditions. There were no differences in the ingestion rates of microplastics between farmed and wild mussels. The estimates of microplastic ingestion and the validated procedures for their extraction provide a strong basis for future work on microplastic pollution.

Ions and Small Molecules as Modulators of F1FO-ATPase, Mitochondrial Bioenergetics and Cell Metabolism

The properties of the mitochondrial F1FO-ATPase activated by the natural cofactor Mg2+ or by Ca2+, were studied, mainly on heart mitochondria from swine, widely used in translational medicine. The Ca2+ driven conformational changes in the F1FO-ATPase form the mitochondrial permeability transition pore (mPTP), which triggers regulated cell death and is involved in severe pathologies. The Ca2+-activated F1FO-ATPase hydrolyzes ATP with kinetics slightly different from those of the Mg2+-ATPase. Known F1-ATPase inhibitors inhibit both the Ca2+-activated F1FO-ATPase and the mPTP formation strengthening the molecular link between them. The different Gd3+ effects on the Ca2+- and Mg2+-activated F1FO-ATPases confirm their difference as also phenylglyoxal which preferentially inhibits the Ca2+-activated F1FO-ATPase. The effects of phenylarsine and dibromobimane, which interact with differently distant Cys thiols, show that mPTP opening is ruled by nearby or distant dithiols. Bergamot polyphenols and melatonin inhibit the mPTP and ROS formation. H2S, a known cardiovascular protector, unaffects the F1FO-ATPase, but inhibits Ca2+ absorption and indirectly the mPTP, both in swine heart and mussel midgut gland mitochondria. New generation triazoles inhibit the Ca2+-activated F1FO-ATPase and the mPTP, but unaffect the Mg2+-activated F1FOATPase. In parallel, the energy metabolism was investigated in mammalian cells. In boar sperm ATP is mainly produced by mitochondrial oxidative phosphorylation (OXPHOS), even if it decreases over time because of less active mitochondria. Insufficient ATP may induce sperm dysfunction. Also, canine mesenchymal stem cells rely on OXPHOS; those from umbilical cord which produce more ATP than those from adipose tissue, seem preferable for transplant studies. The intestinal porcine enterocyte cell line IPEC-J2, used for human gut research, responds to different fetal bovine serum concentrations by remodeling OXPHOS without altering the bioenergetic parameters. The IPEC-J2 bioenergetics is modulated by Vitamin K vitamers. These data shoulder cell bioenergetics as precious tool for medical research.

Innovative microbiome-based system for the improvement of productivity, safety and sustainability of mussels farming

Northwestern Adriatic Sea Mediterranean mussels are exposed to fluctuating environmental parameters and to natural and anthropogenic stressors. Today is well known that mussels can be defined as holobiont, even if remains a lot to elucidate about how an organism and its microbial component response to environmental stress. This PhD dissertation aims to investigate microbiome possible adaptive patters exploiting the organism physiology response to stress, using the NGS sequencing method. The experimental approach consisted of two phases to first determine (i) the microbiome at a tissue scale level, (ii) the microbiome and physiological response to natural and anthropogenic stress environment and the chemical assessment of the microecosystem the Northwestern Adriatic Sea Mediterranean Mussel lives in. Results revealed firstly a robust microbiome well differentiated from seawater microecosystem, with compositional variations at the organ level. Thanks to those findings, digestive gland, the organ in which digestive and detoxification processes allow animal to tolerate and accumulate xenobiotics of natural and anthropogenic origin, was the selected tissue for the second phase of the project. The second phase of the project evaluated the putative physiological variations and the compositional changes in microbiome of digestive gland. I then manage to assess microbiome region trends across the north Adriatic, with each sampling site well differentiated from the others. Finally, a chemical method able to a powerful tool for the analytical detection of the major pollutants in mussels were validated. These first results may provide baseline information for future studies approaches of seasonal and region trends of microbiota profiles and physiological responses in terms of metabolism.

Monitoring of macro- and microlitter in the Venice lagoon and coastal area

Marine litter and plastics are a significant and growing marine contaminant that has become a global problem. Macrolitter is subject to fragmentation and degradation due to physical, chemical and biological processes, leading to the formation of micro-litter, the so-called microplastics. The purpose of this research is to assess marine litter pollution by using remote sensing tools to identify areas of macrolitter accumulation and to evaluate the concentrations of microplastics in different environmental matrices: water, sediment and biota (i.e. mussels and fish) and to contribute to the European project MAELSTROM (Smart technology for MArinE Litter SusTainable RemOval and Management). The aim is to monitor the presence of macro- and microlitter at two sites of the Venice coastal area: an abandoned mussel farm at sea and a lagoon site near the artificial Island of Sacca Fisola; The results showed that both study areas are characterised by high amounts of marine litter, but the type of observed litter is different. In fact, in the mussel farm area, most of the litter is linked to aquaculture activities (ropes, nets, mooring blocks and floating buoys). In the Venice lagoon site, the litter comes more from urban activities and from the city of Venice (car tyres, crates, wrecks, etc.). Microplastics is present in both sites and in all the analysed matrices. Generally, higher microplastics concentrations were found at Sacca Fisola (i.e., in surface waters, mussels and fish). Moreover, some differences were also observed in shapes and colours comparing the two sites. At Sacca Fisola, white irregular fragments predominate in water samples, blue filaments in sediment and mussels, and transparent irregular fragments in fish. At the Mussel Farm, blue filaments predominate in water, sediment and mussels, while flat black fragments predominate in fish. These differences are related to the different types of macrolitter that characterised the two areas.