High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.

Contrat de Plan État-Région Bretagne. Relance de l'huître plate. Rapport d'avancement des travaux 1988.

Un contrat de Plan État-Région Bretagne a été signé en 1983 pour financer des recherches sur la période 1984-1988 dans le but de permettre la relance de l'élevage de l'huître plate, à la suite de la deuxième épizootie due à un protozaire, Bonamia ostreae. Ce rapport présente les travaux réalisés et les résultats acquis durant l'année 1988, dans les domaines de la pathologie (étude de la maladie et des mécanismes de défense des mollusques) de la génétique (recherche de souches résistantes), de l'épidémiologie descriptive et de la zootechnie.

A importância dos ácidos fenólicos na atividade anti-inflamatória de cogumelos comestíveis

Os cogumelos comestíveis são uma fonte rica de moléculas bioativas que lhes conferem importantes atividades biológicas. Moléculas como os Polissacáridos, terpenóides e os compostos fenólicos têm sido descritos como os componentes mais importantes no que respeita á atividade anti-inflamatória dos cogumelos (1). No presente trabalho, os extratos etanólicos de cogumelos comestíveis foram obtidos por maceração e caracterizados quimicamente em termos de ácidos fenólicos por técnicas de HPLC-DAD. Além disso, derivados metilados e glucuronados dos ácidos fenólicos identificados foram também sintetizados com o objetivo de mimetizar reações de metabolização no organismo e estudar a capacidade destas moléculas de manter a bioatividade exibida inicialmente. Os extratos obtidos, os ácidos fenólicos e compostos sintetizados foram avaliados pela sua atividade anti-inflamatória. De entre as amostras analisadas, B. impolitus revelou o mais elevado conteúdo em ácidos fenólicos (675 ± 23 μg/g), seguido de C. cibarius > A. caesaria > L. deliciosus > B. aereus > M. esculenta > B. edulis; devido à contribuição do ácido cinâmico que foi encontrada em maior quantidade nesta amostra (505 ± 12 μg/g). Mais ainda, B. impolitus apresentou também maior inibição da produção de NO (EC50=166 ± 10 μg/mL) seguido das amostras A. caesaria > C. cibarius > L. deliciosus > M. esculenta > B. aereus > B. edulis. No que respeita aos compostos individuais, o ácido cinâmico (CA) revelou a atividade mais forte (EC50 = 182 ± 16 μM), seguido pelos ácidos p-hidroxibenzóico (HA) (239 ± 29 μM) e p-Cumárico (CoA) (442± 33 μM), o que realça a importância destas moléculas para a atividade anti-inflamatória dos cogumelos. Comparando a atividade exibida pelos ácidos fenólicos com os respetivos derivados, é possível verificar a seguinte ordem de atividades: ácido p-hidroxibenzóico: HA > HA-M3 > HA-M2 > HA-M1 > HA-G; ácido p-cumárico : CoA-M1 > CoA-G > CoA-M2 > CoA-M3 > CoA e ácido cinâmico: CA-G > CA > CA-M1. Perante os resultados obtidos é de realçar a importância dos ácidos fenólicos na contribuição para a bioatividade exibida pelos cogumelos em estudo. Mais ainda, foi possível concluir que as alterações das moléculas pelas reações de conjugação no organismo têm influência na bioativade das moléculas iniciais, sendo que muitas vezes esta atividade é aumentada.

Extended use of gamma irradiation in wild mushrooms conservation: validation of 2 kGy dose to preserve their chemical characteristics

Irradiation is recognized by international organizations as a conservation technology, and its application to wild mushrooms has been tested in some species. Our research group evaluated the effectiveness of gamma irradiation to conserve different samples of highly appreciated species, particularly, Lactarius deliciosus, Macrolepiota procera, Boletus edulis and Hydnum repandum. From those results and considering also international recommendations on this subject, the 2 kGy dose was chosen for further studies. Therefore, the application of gamma irradiation at 2 kGy dose was extended to Boletus pinophilus Pilát & Dermek and Clitocybe subconnexa Murrill to validate the proposed technology. Considering the obtained results, some of the analysed chemical parameters (specially sugars and fatty acids), as well as the antioxidant activity, showed significant changes after irradiation treatment, particularly in B. pinophillus, probably due to its higher water content. Nevertheless, the obtained differences did not seem to be sufficient to change the organoleptic characteristics of these mushrooms. Furthermore, the antioxidant activity was generally higher in irradiated samples. In conclusion, the detected chemical changes might be considered as acceptable, when considering the high advantages of gamma irradiation at decontamination and/or disinfestation level.

Faisabilité biologique de la production de larves d'huîtres creuses Crassostrea gigas à partir d'eau de mer du site de Leucate. Convention de recherche Ifremer/Région Languedoc-Roussillon - Rapport final.

Afin de vérifier sur un site régional la présence d'une qualité d'eau de mer permettant la réalisation d'élevages larvaires d'huîtres creuses Crassostrea gigas (et/ou d'huîtres plates Ostrea edulis) sur une base régulière tout au long de l'année, une série d'élevages comparatifs a été réalisée à la Station IFREMER de Palavas, de mars 1995 à septembre 1995. Le matériel biologique nécessaire à cette comparaison est constitué de larves d'huîtres creuses Crassostrea gigas et en algues unicellulaires provenant de l'éc1oserie commerciale de la SATMAR. Les résultats obtenus démontrent la capacité de l'eau de mer du site étudié sur Leucate à supporter la production de larves d'huîtres creuses compétentes à se métamorphoser et à se fixer. Cette étude s'inscrit dans la démarche de la Région Languedoc-Roussillon et du CEPRALMAR visant à favoriser l'implantation d'une écloserie de production de Mollusques Bivalves sur le littoral méditerranéen.

An expert model approach to assess the potential of non-wood forest products for forest owners

Mestrado em Engenharia Florestal e dos Recursos Naturais - Instituto Superior de Agronomia - UL

Karyotype and genome size in Euterpe Mart. (Arecaceae) species.

Euterpe (Martius, 1823), a genus from Central and South America, has species with high economic importance in Brazil, because of their palm heart and fruits, known as açaí berries. Breeding programs have been conducted to increase yield and establish cultivation systems to replace the extraction of wild material. These programs need basic information about the genome of these species to better explore the available genetic variability. The aim of this study was to compare E. edulis (Martius, 1824), E. oleracea (Martius, 1824) and E. precatoria (Martius, 1842), with regard to karyotype, type of interphase nucleus and nuclear DNA amount. Metaphase chromosomes and interphase nuclei from root tip meristematic cells were obtained by the squashing technique and solid stained for microscope analysis. The DNA amount was estimated by flow cytometry. There were previous reports on the chromosome number of E. edulis and E. oleracea, but chromosome morphology of these two species and the whole karyotype of E. precatoria are reported for the first time. The species have 2n=36, a number considered as a pleisomorphic feature in Arecoideae since the modern species, according to floral morphology, have the lowest chromosome number (2n=28 and 2n=30). The three Euterpe species also have the same type of interphase nuclei, classified as semi-reticulate. The species differed on karyotypic formulas, on localization of secondary constriction and genome size. The data suggest that the main forces driving Euterpe karyotype evolution were structural rearrangements, such as inversions and translocations that alter chromosome morphology, and either deletion or amplification that led to changes in chromosome size.

Produção agroecológica de mudas de maracujá em substratos a base de húmus de minhoca e casca de arroz carbonizada.

2014

Divergência genética em genótipos de maracujazeiro azedo, com base em características físicas e químicas dos frutos.

2016

Divergência genética em genótipos de maracujazeiro azedo, com base em características físicas e químicas dos frutos.

RESUMO: Objetivou-se com o presente trabalho avaliar a divergência genética e as características físicas e químicas de frutos de duas populações do maracujazeiro azedo na região Norte do Espírito Santo, como as progênies de meio-irmãos de acesso local de um plantio comercial (genótipos: 1; 2; 3; 4; 5; 6; 7; 8; 9 e 10) e do híbrido BRS Ouro Vermelho (genótipos: 11; 12; 13; 14; 15; 16; 17; 18; 19 e 20). A divergência genética foi avaliada por procedimentos multivariados como a distância generalizada de Mahalanobis (D2) e pelos métodos de agrupamento de otimização de Tocher e UPGMA. Encontrou-se divergência genética entre as populações estudadas promovendo a formação de grupos diferentes entre o método de Tocher e do UPGMA. As características, referentes ao tamanho do fruto, diâmetro polar e equatorial, foram as que mais contribuíram na diversidade genética dos genótipos. Nas populações estudadas de maracujazeiro azedo há grande variabilidade genética quanto às características avaliadas, o que possibilita selecionar plantas com elevado potencial para fins de melhoramento genético. O híbrido BRS Ouro Vermelho apresenta boa adaptação às condições locais. ABSTRACT: The aim of the present work was to evaluate genetic divergence and physical and chemical characteristics in fruit of two populations of sour passion fruit in the northern region of the State of Espírito Santo, Brazil, these being half-sibling progenies from local accessions of a commercial crop (genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) and the hybrid BRS Ouro Vermelho (genotypes: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20). Genetic divergence was evaluated using such multivariate procedures as the generalised Mahalanobis distance (D2) and the Tocher optimisation and UPGMA clustering methods. Genetic divergence was found between the populations under study, promoting the formation of different groups between the Tocher and UPGMA methods. As characteristics for fruit size, the polar and equatorial diameters had the most impact on the genetic diversity of the genotypes. In the populations of sour passion fruit being studied, great genetic variability is seen in the evaluated characteristics, making it possible to select plants of high potential for breeding purposes. The BRS Ouro Vermelho hybrid is well adapted to the local conditions.

Acúmulo de biomassa e nutrientes de duas leguminosas arbóreas introduzidas em sistema de pousio na Amazônia.

A vegetação de pousio é um dos principais componentes dos sistemas agroflorestais sequenciais de derrubada e queima, praticados tradicionalmente pela agricultura familiar na Amazônia. A remoção progressiva de nutrientes do solo por essa prática implica em reduções contínuas nos estoques de carbono e nutrientes, causando declínio da produtividade do solo, perda da capacidade regeneração e diminuição da diversidade de espécies da vegetação. O melhoramento de pousio com espécies leguminosas fixadoras do nitrogênio atmosférico pode contribuir para uma maior produção de biomassa e acúmulo de nutrientes em comparação com a vegetação espontânea, atendendo à demanda nutricional das culturas subsequentes, podendo ser considerada como uma tecnologia de produção sustentável. Esse trabalho avaliou o efeito da adubação fosfatada de baixa solubilidade no acúmulo de biomassa e nutrientes da parte aérea de leguminosas arbóreas utilizadas em enriquecimento de vegetação de pousio. O experimento foi conduzido por 23 meses, em um sistema agroflorestal de ?corte e trituração? em Marapanim, Pará. Foram utilizadas as espécies tachi-branco - Sclerolobium paniculatum Vogel e ingá - Inga edulis Mart e foram estimados biomassa, teor e estoque de nutrientes dos compartimentos folha, galho e tronco. Houve acúmulo de biomassa, estoques de P, Ca e Mg nas folhas, estoque de P nos galhos e o teor de cálcio nas folhas das leguminosas é limitado por fósforo, segundo os resultados encontrados. A espécie Inga edulis apresentou maiores teores de potássio, cálcio e magnésio nos compartimentos folha, galho e tronco, enquanto que a espécie Sclerolobium paniculatum apresentou maiores acúmulos de biomassa e estoques de nitrogênio, fósforo, potássio, cálcio e magnésio nos compartimentos folha, galho e tronco.

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.

Distribuzione spaziale e temporale delle specie macrobentoniche aliene e native in ambienti portuali

La maggior parte degli insediamenti urbani si trova in prossimità delle coste e più del 50% della popolazione vive entro 100 km dal mare. Questo significa che l’ambiente marino è fortemente influenzato dalle attività antropiche e, per questo, è necessario creare un piano di gestione degli ambienti costieri, in quanto la resilienza dell’habitat marino dipende dall’uso sostenibile degli ecosistemi marini urbani. Lo scopo dello studio è quello di analizzare la distribuzione spaziale e temporale delle specie native e aliene, presenti sulle banchine del porto-canale di Ravenna. Sono stati analizzati i popolamenti intertidali di 6 siti lungo il canale Candiano dal 2016 al 2018 nei mesi di maggio. I risultati hanno illustrato un gradiente terra-mare a partire dai siti più interni lungo il porto-canale. Le specie più abbondanti identificate sono i due mitili Mytilus Galloprovincialis (specie nativa) e Xenostrobus securis (specie invasiva); queste specie si avvicendano nelle stazioni centrali del Candiano e si cedono il posto a vicenda: la specie nativa rimane nelle acque esterne e meno impattate mentre quella invasiva resta nei siti interni più soggetti a disturbi di vario tipo, come quello antropico. La terza specie più numerosa è Sphaeroma serratum, isopode nativo del Mediterraneo, presente da sempre nelle zone lagunari del ravennate; in questo studio è stato rinvenuto nelle stazioni più interne, che potrebbe sfruttare come rifugio da possibili competitori. I risultati hanno mostrato l’abbondanza e la diversità delle specie, indigene e non ,del porto di Ravenna: su un totale di 71 taxa diversi, le specie aliene rappresentavano il 17%; queste sono state rinvenute soprattutto nei siti di campionamento più interni, dove sono presenti condizioni più limitanti e sfavorevoli per le specie native. Inoltre, è stato osservato come la diversità specifica aumenti lungo il gradiente mare-terra, considerando le zone con maggiore diversità anche quelle con maggiore specie aliene.