Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Development of an FIA system with amperometric detection for determination of bentazone in estuarine waters

On the basis of its electrochemical behaviour a new flow-injection analysis (FIA) method with amperometric detection has been developed for quantification of the herbicide bentazone (BTZ) in estuarine waters. Standard solutions and samples (200 µL) were injected into a water carrier stream and both pH and ionic strength were automatically adjusted inside the manifold. Optimization of critical FIA conditions indicated that the best analytical results were obtained at an oxidation potential of 1.10 V, pH 4.5, and an overall flow-rate of 2.4 mL min–1. Analysis of real samples was performed by means of calibration curves over the concentration range 2.5x10–6 to 5.0x10–5 mol L–1, and results were compared with those obtained by use of an independent method (HPLC). The accuracy of the amperometric determinations was ascertained; errors relative to the comparison method were below 4% and sampling rates were approximately 100 samples h–1. The repeatability of the proposed method was calculated by assessing the relative standard deviation (%) of ten consecutive determinations of one sample; the value obtained was 2.1%.

Exploring bioactive properties of marine cyanobacteria Isolated from the Portuguese coast: high potential as a source of anticancer compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

Functional role of macrobenthos in estuarine sediment dynamics

Thesis submitted to the Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia for the degree of Doctor of Philosophy in Environmental Engineering

Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: an overview

Microbiology (2009), 155, 3476–3490

Integrated biomarker responses of an estuarine invertebrate to high abiotic stress and decreased metal contamination

An integrated chemical-biological effects monitoring was performed in 2010 and 2012 in two NW Iberian estuaries under different anthropogenic pressure. One is low impacted and the other is contaminated by metals. The aim was to verify the usefulness of a multibiomarker approach, using Carcinus maenas as bioindicator species, to reflect diminishing environmental contamination and improved health status under abiotic variation. Sampling sites were assessed for metal levels in sediments and C. maenas, water abiotic factors and biomarkers (neurotoxicity, energy metabolism, biotransformation, anti-oxidant defences, oxidative damage). High inter-annual and seasonal abiotic variation was observed. Metal levels in sediments and crab tissues were markedly higher in 2010 than in 2012 in the contaminated estuary. Biomarkers indicated differences between the study sites and seasons and an improvement of effects measured in C. maenas from the polluted estuary in 2012. Integrated Biomarker Response (IBR) index depicted sites with higher stress levels whereas Principal Component Analysis (PCA) showed associations between biomarker responses and environmental variables. The multibiomarker approach and integrated assessments proved to be useful to the early diagnosis of remediation measures in impacted sites.

Joint effects of salinity and the antidepressant sertraline on the estuarine decapod Carcinus maenas

Concurrent exposure of estuarine organisms to man-made and natural stressors has become a common occurrence. Numerous interactions of multiple stressors causing synergistic or antagonistic effects have been described. However, limited information is available on combined effects of emerging pharmaceuticals and natural stressors. This study investigated the joint effects of the antidepressant sertraline and salinity on Carcinus maenas. To improve knowledge about interactive effects and potential vulnerability,experiments were performed with organisms from two estuaries with differing histories of exposure to environmental contamination. Biomarkers related to mode of action of sertraline were employed to assess effects of environmentally realistic concentrations of sertraline at two salinity levels. Synergism and antagonism were identified for biomarkers of cholinergic neurotransmission, energy production,anti-oxidant defences and oxidative damage. Different interactions were found for the two study sites highlighting the need to account for differences in tolerance of local ecological receptors in risk evaluations.

Assessing the balance between biodiversity conservation and coastal and estuarine management of the Sado Estuary Natural Reserve, Portugal, using BIO-SAFE

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia do Ambiente, perfil de Engenharia Ecológica

Biomarkers in Solea Senegalensis Kaup, 1858 exposed to contaminated estuarine sediments: a multi-level approach

Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia for the degree of Doctor of Philosophy in Environmental Sciences

Sertraline accumulation and effects in the estuarine decapod Carcinusmaenas: Importance of the history of exposure to chemical stress

Sertraline is widely prescribed worldwide and frequently detected in aquatic systems. There is, however, a remarkable gap of information on its potential impact on estuarine and coastal invertebrates. This study investigated sertraline accumulation and effects in Carcinus maenas. Crabs from a moderately contaminated (Lima) and a low-impacted (Minho) estuary were exposed to environmental and high levels of sertraline (0.05, 5, 500 μg L−1). A battery of biomarkers related to sertraline mode of action was employed to assess neurotransmission, energy metabolism, biotransformation and oxidative stress pathways. After a seven-day exposure, sertraline accumulation in crabs’ soft tissues was found in Lima (5 μg L−1: 15.3 ng L−1 ww; 500 μg L−1: 1010 ng L−1 ww) and Minho (500 μg L−1: 605 ng L−1 ww) animals. Lima crabs were also more sensitive to sertraline than those from Minho, exhibiting decreased acetylcholinesterase activity, indicative of ventilatory and locomotory dysfunction, inhibition of anti-oxidant enzymes and increased oxidative damage at ≥0.05 μg L−1. The Integrated Biomarker Response (IBR) index indicated their low health status. In addition, Minho crabs showed non-monotonic responses of acetylcholinesterase suggestive of hormesis. The results pointed an influence of the exposure history on differential sensitivity to sertraline and the need to perform evaluations with site-specific ecological receptors to increase relevance of risk estimations when extrapolating from laboratory to field conditions.

Biosynthesis-Assisted Structural Elucidation of the Bartolosides, Chlorinated Aromatic Glycolipids from Cyanobacteria

The isolation of the bartolosides, unprecedented cyanobacterial glycolipids featuring aliphatic chains with chlorine substituents and C-glycosyl moieties, is reported. Their chlorinated dialkylresorcinol (DAR) core presented a major structural-elucidation challenge. To overcome this, we discovered the bartoloside (brt) biosynthetic gene cluster and linked it to the natural products through in vitro characterization of the DAR-forming ketosynthase and aromatase. Bioinformatic analysis also revealed a novel potential halogenase. Knowledge of the bartoloside biosynthesis constrained the DAR core structure by defining key pathway intermediates, ultimately allowing us to determine the full structures of the bartolosides. This work illustrates the power of genomics to enable the use of biosynthetic information for structure elucidation.

Marine Cyanobacteria Compounds with Anticancer Properties: Implication of Apoptosis

Marine cyanobacteria have been proved to be an important source of potential anticancer drugs. Although several compounds were found to be cytotoxic to cancer cells in culture, the pathways by which cells are affected are still poorly elucidated. For some compounds, cancer cell death was attributed to an implication of apoptosis through morphological apoptotic features, implication of caspases and proteins of the Bcl-2 family, and other mechanisms such as interference with microtubules dynamics, cell cycle arrest and inhibition of proteases other than caspases.

Mangrove sedimentary characteristics and implications for crab Ucides cordatus (Crustacea, Decapoda, Ucididae) distribution in an estuarine area of the Amazonian region

At the coastal zone sediments, water and organisms interact intensely. At equatorial tidal-dominated coast mangroves are abundant. These areas are well-known for their ecological importance. Considering the mangroves of Atlantic South America, the mangrove crab, Ucides cordatus has ecological and economic prominence. High densities of this crab are found on the Amazon coast. This study investigates the sediment distribution of the Bragança mangrove area (Amazon coast, Brazil) and its correlations with vegetation and mangrove crab distribution. Sediments of 47 sites, as well as crabs from sites with different sediment and vegetation, were sampled. Results show that surface sediment of the area is mainly composed by silt (59%), with 21% sand and 20% clay. Variations in sorting and skewness are the product of local variations in clay and sand content. The vegetation type was significantly correlated to mangrove crab characteristics, abundance and weight/size. Sediment characteristics are also substantially different according to the vegetation type. Areas where Avicennia germinans prevails have more sand and clay than areas of Rizophora mangle, in which silt is dominant and crabs were significantly heavier. The present results have showed that the distribution of sediments, crabs and vegetation at mangrove areas are strongly correlated. Thus, they should be studied in conjunction.

Reproductive biology of the sesarmid crab Armases rubripes (Decapoda, Brachyura) from an estuarine area of the Sahy River, Sepetiba Bay, Rio de Janeiro, Brazil

The aim of this work was to gain knowledge about reproductive biology of the crab Armases rubripes (Rathbun, 1897) from an estuarine area of the Sepetiba Bay. Samples were taken monthly from February 2003 to January 2004 in the Sahy River estuary (22º56'S; 44º01'W), Rio de Janeiro, Brazil. The crabs were collected by hand during 15-minute catch-effort sessions conducted by two people. In the laboratory, the specimens were separated by sex, carapace width was measured and gonadal stage was checked macroscopically. A total of 830 individuals were caught - 304 males, 373 females (60 ovigerous females) and 153 juveniles. The ovigerous females were found almost year-round, except in November and April, showing a continuous reproductive period. They presented a size range from 8.2 to 15.0 mm carapace width (12.1 ± 1.7 mm). Color and macroscopical aspects determined five gonadal stages for males and females (immature, rudimentary, intermediary, developed and resting). First sexual maturity was estimated at 6.5 mm of carapace width for males and 8.1 mm for females. Individual fecundity varied from 200 to 11,460 eggs (4,458 ± 2,739 eggs). Mean egg size was 0.248 ± 0.026 mm, varying from 0.213 to 0.333 mm, while the volume ranged from 0.0051 to 0.0188 mm³ (0.0082 ± 0.0029 mm³).

Ostracodes (Crustacea) from Cananéia-Iguape estuarine/lagoon system and geographical distribution of the mixohaline assemblages in southern and southeastern Brazil

The ostracode assemblages from Cananéia-Iguape estuarine/lagoon system (southernmost State of São Paulo) are here discussed in detail for the first time. Thirty-four sites, approximately 1 km equidistant, were sampled along the system, including the Cananéia Sea, Pequeno Sea, Cubatão Sea, Ribeira de Iguape River and Itapitangui River. The ostracodes throughout this area have poor assemblages, with a total of 662 specimens of dead and living organisms. The majority of the ostracode fauna is composed of euryhaline species, as follows: Cyprideis multidentata Hartmann, 1955 (174 specimens), Minicythere heinii Ornellas, 1974 (54 specimens), Tanella gracilis Kingma, 1948 (96 specimens) and Whatleyella sanguinettiae Coimbra, Carreño & Ferron, 1994 (226 specimens). Although there are few studies on the Brazilian mixohaline ostracode faunas, including the euryhaline marginal marine taxa, the published data show that the group is best known in the south and southeast regions. Based on this review and with the new data presented in this paper, the geographical distribution of eight mixohaline key species in southern and southeastern Brazil is also discussed.