Estudo de dinoflagelados bênticos da Ria de Aveiro (NW Portugal)

A Ria de Aveiro apresenta ao longo das margens pequenos charcos com características muito peculiares, que oferecem condições para a proliferaçãode um grande número de algas, especialmente algas bênticas pertencentes avários grupos, com a dominância de diatomáceas, dinoflagelados emacroalgas verdes. Verificou-se que a comunidade de dinoflagelados é dominada essencialmentepor espécies pertencentes ao género Prorocentrum (P. lima, P. micans, P. cassubicum, P. rhathymume duas prováveis novas espécies de Prorocentrum), mas também por outras espécies como Coolia monotis, Amphidinium massartii, Bysmatrum subsalsum, Scrippsiella cf. trochoidea, Kryptoperidinium foliaceum, Oblea rotunda, Akashiwo sanguineae, embora menos frequentes, espécies dos géneros Protoperidinium e Peridinium. Na costa portuguesa a maioria destas espécies já tinham sido anteriormenteregistadas, excepto A. massartii, O. rotunda, B. subsalsum, P. cassubicum e P. rhathymum, mas para a Ria de Aveiro apenas S. trochoidea, Peridinium quinquecorne, P. lima, P. minimum e P. micans já tinham sido referenciados(Moita & Vilarinho 1999). O teste de Artemiademonstrou a toxicidade de apenas três espécies:Prorocentrum lima, P. cassubicum e Coolia monotis. As culturas de P. limaforam de longe as mais tóxicas para a larva de Artemia salina. A ocorrência de espécies tóxicas na Ria deve ser tida em conta uma vez que nesta região háprodução de moluscos, que podem acumular estas toxinas afectando espéciesem níveis tróficos superiores, incluindo o Homem. Os charcos estudados foram bastante semelhantes quanto à variação dos parâmetros ambientais e comunidades biológicas. Apesar das variaçõesclimatéricas provocarem alterações acentuadas na salinidade e temperaturada água, há um leque de espécies que está sempre presente, enquanto outrassó ocorrem em determinadas alturas, podendo atingir elevadas densidades. Neste estudo da comunidade bêntica de dinoflagelados da Ria de Aveiropretendeu-se incluir a correcta identificação das espécies, usando técnicasavançadas de microscopia, o estabelecimento de culturas em laboratório e a realização de ensaios de toxicologia (com Artemia e com Hydrobia) com o objectivo de descobrir o papel que estas espécies desempenham na cadeiaalimentar. O conhecimento adquirido através do estudo de organismos tóxicos é importante para aumentar o conhecimento de quais as espécies, quando ecomo podem surgir nestas áreas como um surto nefasto. Outro aspectointeressante acerca dos dinoflagelados reside na sua filogenia; a análisedetalhada de sequências genéticas como SSU-rDNA e LSU-rDNA vêm clarificar estas relações, podendo ainda ajudar em eventuais identificaçõesambíguas. ABSTRACT: The Ria de Aveiro contain numerous ponds and sheltered areas, which, offerthe appropriate conditions for the proliferation of a very diverse algal community, especially benthic algae, in which diatoms, dinoflagellates andgreen macroalgae predominate. Some of the benthic dinoflagellates are known to produce toxic substances, theepibenthic dinoflagellate community is therefore the focus of this work. The dominant species of dinoflagellates in the ponds were from the genusProrocentrum (P. lima, P. micans, P. cassubicum, P. rhathymumand probably two new species), but also other frequent species like Coolia monotis, Amphidinium massartii, Bysmatrum subsalsum, Scrippsiella cf. trochoidea, Kryptoperidinium foliaceum, Oblea rotunda, Akashiwo sanguineaand some species of the genera Protoperidinium and Peridinium were present. Most of this species had been previously referred for Portuguese waters, except A. massartii, O. rotunda, B. subsalsum, P. cassubicum and P. rhathymum, although most of them had not been reported from the Ria deAveiro (only S. trochoidea, Peridinium quinquecorne, P. lima, P. minimumand P. micans had already been cited from this area) (Moita & Vilarinho 1999). Prorocentrum lima, P. cassubicum and Coolia monotis were found to be toxic to the brine shrimp, Artemia salina, but with different intensities, being P. limathe most toxic and P. cassubicumthe less toxic. The occurrence of toxic species should be taken in consideration, since they can affect species inhigher trophic levels, including humans that feed on organisms that accumulatethe toxins, like molluscs. Ria de Aveiro is one of the major shellfish productionsareas. In relation to the environmental changes, most of the benthic dinoflagellateswere found to be very tolerant to these modifications, although some speciesonly appeared in some periods of the year, sometimes in large numbers. The analysis of the benthic dinoflagellates from Ria de Aveiro includes theidentification of the organisms, using light and electron microscopy (highresolution scanning and transmission electron microscopy), the establishmentof laboratory cultures and the study of the toxicity of the species by bioassays(using Artemia and Hydrobia), with the aim of better understanding the role of these organisms in marine food webs. This study also included a genetic approach, based on the sequence of theLSU-rDNA of the cultured species, for future incorporation in a phylogenetictree, but the only sequences obtained were from A. massartii and P. micans

Phytoplankton survey of “Barrinha”lagoon (Mira – Portugal)

This work reports a one year survey (October 2008 to October 2009) where limnology and plankton data from Barrinha lagoon (Mira-Portugal - 40° 27′ 22″ N, 8° 48′ 7″ W) were analyzed. This lagoon shows peculiar geochemical features being one of the few places in Portugal where the freshwater bivalve Anodonta cygnea can be found. This study consisted on monthly collections of water samples, with identification and quantification of the planktonic communities found in the water. Physical-chemical parameters of the water were also analyzed (temperature, dissolved oxygen, conductivity, pH, nitrites, nitrates, ammonia) as well as organic and inorganic particles and bacteria in the water. The importance of plankton and of phytoplankton in particular is unquestionable; it is the base of the food chain, source of oxygen and can also be used as bio-indicator of water quality. A total of 75 taxa of plankton were identified in the water samples, being 71 of phytoplankton and 4 of zooplankton. The most abundant taxon was Chlorophyceae with 30 taxa (abundances vary from 25% to 75%), then by Bacillariophyceae with 20 taxa (abundances vary from 10% to 75%), followed by the Cryptophyceae represented dominantly by only one genus the Crypotmonas (abundances vary from 10% to 35%) and Cyanophyceae 10 taxa (which reach up a maximum of 7.5% of total abundances of phytoplankton). Among the zooplankton, that was always very scarce, the Protozoa were the main group found. In term of physical-chemical parameters of the water the data were quite uniform except in the end of summer, in this period the lagoon begins to show some signs of eutrophication: decay of nutrients concentration, organic and inorganic particles, followed by oxygen depletion, which favours a simple algae to growth (Cryptomonas) in detriment of all other species of plankton, that in this period reach its minimums. Escherischia coli, Enterococci and total aerobic mesophilic (TAM) microorganisms were indentified and quantified in the water, their abundances change throughout the year, their occurrence are human correlated and they give a good scenario about the water quality in the habitat

Antimicrobial and antibiofilm activity of unionid mussels from the north of Portugal

The populations of freshwater mussels belonging to the family Unionidae have been facing drastic changes in terms of diversity and numbers caused by constant aggressions on their natural habitat. Nevertheless, bivalves are capable of developing strategies of defense to overcome potential aggressors. The present work aimed to assess the potential antibacterial capacity of different species of unionid mussels from the north of Portugal. For this purpose, circulating cells (hemocytes), fluids, and mucus were obtained by nonlethal methods from the species Anodonta anatina (Linnaeus), Anodonta cygnea (Linnaeus), Potomida littoralis (Cuvier), and Unio delphinus (Spengler), and tested against bacterial reference strains and multidrug-resistant isolates. The cellular fraction of A. anatina, A. cygnea, and P. littoralis showed antibacterial activity, detected by the agar disc diffusion method, against Bacillus subtilis ATCC 6683, Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606. Circulating cells from P. littoralis and A. anatina also inhibited Listeria monocytogenes ATCC 19111 and A. cygnea has also inhibited a multidrug-resistant isolate of Pseudomonas putida. The plasma of all mentioned freshwater mussels, used directly or diluted, showed great ability to hamper or inhibit the biofilm formation of Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. Anodonta cygnea hampered the biofilm formation by Enterococcus faecalis ATCC 29212 as well. Overall, these results showed that not only cells play a relevant role in the immune system of these species but also the plasma, which likely contains antibiofilm substances. Anodonta cygnea stood out by presenting the best antibacterial inhibition potential.This work was supported by the Portuguese Foundation for Science and Technology (FCT) with a research grant to M. H. (SFRH/BD/76265/2011). This article was also supported by the project INNOVMAR-Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035, within Research Line NOVELMAR), by North Portugal Regional Operational Programme( NORTE2020), under the PORTUGAL2020 Partnership Agreement, and through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Estudos preliminares de populações de náiades nos sectores terminais dos rios Tua e Sabor (bacia do Douro): análise do habitat e da qualidade da água e sedimentos

O exíguo conhecimento das condições ecológicas presentes nos rios Tua e Sabor, que permitem a existência de populações viáveis das náiades Unio delphinus (anteriormente designado por Unio cf. pictorum), Anodonta anatina e Potomida littoralis, é condição básica para a monitorização destes ecossistemas. Neste sentido, no Verão de 2009 foram feitos estudos preliminares de caracterização do habitat e microhabitat usado pelas espécies de náiades bem como análises à qualidade da água e sedimentos. No que concerne ao habitat, foi aplicada a metodologia RHS (River Habitat Survey) complementada com uma análise do microhabitat baseada em transeptos por cada troço de rio seleccionado. Foram, por isso, determinadas as variáveis profundidade, substrato dominante e sub-dominante, velocidade da corrente medida na coluna de água e no leito, e cobertura em cada área amostrada (0,25 m2). As náiades apresentaram uma distribuição espacial característica, concentrando-se principalmente em zonas ensombradas e de substrato fino. Relativamente à qualidade da água detectaram-se valores de condutividade >100 µS.cm-1, nutrientes, Amónia 0,18 +/- 0,02 mg/L, Nitritos e Nitratos 4cm) 7,0 x 103 ufc/ml e biofilme 5,0 x 104 ufc/ml e especialmente de E.coli (água 115 ufc/ml, sedimento superior (4cm) 12 ufc/ml e biofilme 400 ufc/ml) indicadoras de perturbação de origem antrópica. Como medida de conservação das diferentes espécies encontradas e destes ecossistemas aquáticos (rios Tua e Sabor), considera-se essencial a monitorização que vise a manutenção/melhoria da qualidade da água e do habitat, controlo das espécies exóticas e minimização dos efeitos da regularização e sobrepesca com o intuito de evitar a regressão assinalada noutros rios de Portugal

Estudos preliminares de populações de mexilhão-de-rio (Margaritifera margaritifera l.) nos rios Rabaçal e Tuela Nordeste de Portugal): análise do habitat e da qualidade da água e sedimentos

Permanecem pouco estudadas as condições ecológicas dos rios Rabaçal e Tuela que permitem a existência de populações viáveis de mexilhão-de-rio (Margaritifera margaritifera L.). Estudos preliminares realizados no Verão de 2009 permitiram caracterizar o habitat e microhabitat usado pela espécie assim como a qualidade da água e sedimentos. Relativamente ao habitat foi aplicada a metodologia RHS (River Habitat Survey) complementada com uma análise do microhabitat. Realizaram-se 30 transectos por cada troço de rio seleccionado e determinadas as variáveis da profundidade, substrato dominante e sub-dominante, velocidade da corrente, medida na coluna de água e no leito, e cobertura em cada área amostrada (0.25 cm2). A M. margaritifera apresentou uma distribuição espacial não aleatória, concentrando-se em zonas específicas. As curvas de preferência permitiram detectar diferenças entre os juvenis, presentes maioritariamente em habitats com corrente, menor profundidade e substrato grosseiro (pedras e seixos), e os adultos, de distribuição mais ampla, capazes de colonizar zonas lênticas, com maior profundidade, ausência de corrente e substrato de granulometria fina (areias). No que respeita à qualidade da água detectou-se baixo teor sais dissolvidos (condutividade < 50 µScm-1) e nutrientes (N-Total <0,1 mg/L) (POM <3 mg/L PIM < 0,01mg/L). Apesar da qualidade da água ser elevada foi detectada uma baixa concentração de coliformes totais na água e sedimento indicando alguma influência antropogénica. Como medida de conservação da espécie nestes rios afigura-se essencial a monitorização das descargas de efluentes domésticos, dos efeitos da regularização e sobrepesca com o intuito de evitar a regressão assinalada noutros rios de Portugal

Ionic regulation and shell mineralization in the bivalve Anodonta cygnea following heavy metal exposure

Freshwater mussels are one of the most imperiled faunistic groups in the world and environmental exposure to toxic heavy metals, which result in deregulation of calcium absorption and deposition in the laboratory, may be a contributing factor in their decline. To address potential effects of heavy-metal exposure on calcium transport and metabolism in freshwater bivalves, adult Anodonta cygnea (L., 1758) were exposed to a sublethal concentration (1.0 × 10−6 mol/L) of essential (Zn2+ and Cu2+) or nonessential (Pb2+ and Cr3+) metal for 30 days in the laboratory. Inorganic composition of extrapallial, haemolymph, heart, and pericardium fluids, and kidney tissue, as well as shell morphology by scanning electron microscopy, were compared in treated and untreated mussels. Calcium levels in fluids varied after exposure to any of the metals investigated, although the magnitude and threshold of effect were metal- and compartment-specific. Ca2+ levels increased robustly in all fluids following exposure to Zn2+, Cu2+, or Cr3+, whereas levels decreased significantly in heart fluid alone following Pb2+ exposure (p < 0.05). In constrast to exposure to the other metals, Cu2+ revealed an interesting reverse-accumulation pattern, decreasing in the fluids but not in the kidney, where it clearly accumulates for excretion. In addition, whereas essential Cu2+ and Zn2+ are closely regulated, the nonessential metals Pb2+ and Cr3+ increase to very high levels. Drastic alterations in shell morphology, specifically the structure of border and inner pallial regions of the nacreous layer, were observed after Cu2+ or Cr3+ exposure. Collectively, data suggest that prolonged exposure to a sublethal concentration of these heavy metals can adversely affect compartmental calcium availability and shell composition in A. cygnea

Biomineralization studies on cellulose membrane exposed to biological fluids of Anodonta cygnea

The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half ‘Ussing chambers’ with different composition solutions in order to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity, occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape, mainly on the phosphate surface. When organic fluids of A. cygnea were included, these crystal forms presented a high tendency to aggregate under rosaceous shapes, also predominantly in the phosphate side. The cellulose membrane was permeable to small organic molecules that diffused from the calcium towards the phosphate side. In the calcium side, very few similar crystals were observed. The presence of organic matrix from A. cygnea fluids induced a preliminary apatite–brushite crystal polymorphism. So, the present results suggest that cellulose membranes can be used as surrogates of biological epithelia with preferential ionic diffusion from the calcium to the phosphate side where the main mineral precipitation events occurred. Additionally, the organic fluids from freshwater bivalves should be also thoroughly researched in the applied biomedical field, as mineral nucleators and crystal modulators on biosynthetic systems

Conbiomics: the missing approach for the conservation of freshwater bivalves

The decline of freshwater biodiversity hás reached alarming proportions. The extinction rate of freshwater biodiversity is predicted to be five times faster than ali other groups of species. The Unionidae, being the largest of the freshwater bivalve families, is among the most endangered group in the world. They are important providers of aquatic ecosystem services and are characterized by an unusual pattem of mtDNA inheritance. However, phylogeny, population genetic structure and species-level diversity remains unclear for much of the group. With an experienced and well-qualified team, the present proposal aims to define the most important freshwater mussel taxa and most criticai áreas for conservation, at European and Global leveis, to inform the most relevant policy niakers. This will be achieved by integrating a genomics approach with available distribution data, in order to determine and map global species richness, phylogenetic diversity, and weighted endemism. This complementary approach will not only help conserve this vulnerable group but will offer wider benefits to freshwater ecosystems whose billions of people depend globally. Additionally, as these animals have an extraordinary unusual pattern of mtDNA inheritance, they will be used as model-taxa to study the evolution of mtDNA and life in general.This research was developed under Project Nº NORTE-01-0145-FEDER-030286 (ConBiomics: the missing approach for the Conservation of freshwater Bivalves), co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds.info:eu-repo/semantics/publishedVersio

Extracellular matrix in Staphylococcus epidermidis biofilms: a consequence of bacterial production or cell wall degradation?

Staphylococcus epidermidis is a leading pathogen accounting for nosocomial infections. The ability to form biofilms is considered the major virulence factor of this bacterium. The hallmark of this type of infection is the presence of an extracellular polymeric matrix that, in the case of S. epidermidis biofilms, is mainly constituted by an N-acetylglucosamine polymer. We have identified a subpopulation of bacteria that we believe to be the responsible for the extracellular matrix accumulation in S. epidermidis biofilms as they have comparative significant higher amount of surface N-acetylglucosamine. Flow cytometric evaluation of cell wall permeability and transmission electronic microscopy are highly suggestive of primary wall degradation in these bacteria. In overall, these results suggest that the extracellular matrix in S. epidermidis biofilms is a consequence of the degradation of the bacteria cell wall and that propide iodium should be used with care when used as a marker for bacteria dead in biofilms