Victor Galhano

Morphological, biochemical and molecular characterization of Anabaena , Aphanizomenon and Nostoc strains (Cyanobacteria, Nostocales) isolated from Portuguese freshwater habitats

Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.

Exposure of the cyanobacterium Nostoc muscorum from Portuguese rice fields to Molinate (Ordram(®)): Effects on the antioxidant system and fatty acid profile.

Herbicide contamination of aquatic ecosystems is a serious global environmental concern. Several herbicides enhance the intracellular formation of reactive oxygen species, and can lead to the damage of macromolecules and to a decrease of oxidant defenses in a wide range of non-target microorganisms including cyanobacteria. The effects of molinate (a thiocarbamate herbicide used for controlling grassy weeds in rice fields) on the activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione S-transferase were evaluated in Nostoc muscorum, a freshwater cyanobacterium with a significant spread in Portuguese rice fields. These were determined in N. muscorum cultures acutely (72h) exposed to concentrations ranging from 0.75 to 2mM of molinate. This study also analyzed the effects of molinate on: (1) the nonenzymatic antioxidant contents (reduced and oxidized glutathione, carotenoids, and proline), (2) the oxidative cell damage measured in terms of lipid peroxidation (MDA level) and electrolyte leakage (intactness of plasma membrane), and (3) the total fatty acid profile. The results showed that the activities of all antioxidant enzymes decreased dramatically with the rising concentration of molinate after 72h. Time-dependent and concentration-dependent increase in MDA and enhanced cell membrane leakage were indicative of lipid peroxidation, formation of free radicals and oxidative damage. Compared to control, 72-h herbicide exposure increased lipid peroxidation by 5.4%, 19% and 28% with 0.75, 1.5 and 2mM of molinate, respectively. Similarly, herbicide stress induced an increase in electrolyte leakage (5.8%, 29.5% and 30.2% above control, with 0.75, 1.5 and 2mM of molinate, respectively). The increased production of proline at higher molinate concentrations (the values rose above control by 45%, 95% and 156% with 0.75, 1.5 and 2mM, respectively) indicated the involvement of this osmoprotectant in a free radical scavenging mechanism. Moreover, a radical decline in both glutathione pool, carotenoids and saturated fatty acids were also observed. The results of the present study lead us to conclude that: (1) both enzymatic and nonenzymatic antioxidative defense system of N. muscorum are dramatically affected by molinate, (2) the herbicide induces peroxidation, (3) it contributes to an increase of the unsaturation level of cell membrane fatty acids. These evidences should be taken in account when using N. muscorum as an environmental indicator species in studies of herbicide biotransformation and biomarker response as well as in environmental monitoring programmes.

Comparative toxicity of bentazon and molinate on growth, photosynthetic pigments, photosynthesis, and respiration of the Portuguese ricefield cyanobacterium Nostoc muscorum

Bentazon and molinate are selective herbicides recommended for integrated weed management in rice. Their toxicity on growth and some biochemical and physiological parameters of Nostoc muscorum, an abundant cyanobacterium in Portuguese rice fields, was evaluated under laboratory conditions during time‐ and concentration‐dependent exposure for 72 h. Results showed that toxic concentrations (0.75–2 mM) of both herbicides have pleiotropic effects on the cyanobacterium. Molinate was more toxic than bentazon to growth, respiration, chlorophyll‐a, carotenoids, and phycobiliproteins contents. Protein content was increased by both herbicides although the effect was particularly evident with higher concentrations of molinate (1.5–2 mM). The herbicides had contrasting effects on carbohydrates content: molinate increased this organic fraction whereas bentazon decreased it. Photosynthesis and respiration were inhibited by both herbicides.

Bentazon triggers the promotion of oxidative damage in the Portuguese ricefield cyanobacterium Anabaena cylindrica: Response of the antioxidant system

Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75–2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide‐induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S‐transferase (GST) increased in a time‐ and herbicide dose‐response manner and were higher than those in the control samples after 72 h. A time‐ and concentration‐dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH‐dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.

Toxicity of seven priority hazardous and noxious substances (HNSs) to marine organisms: Current status, knowledge gaps and recommendations for future research

Shipping industry and seaborne trade have rapidly increased over the last fifty years, mainly due to the continuous increasing demand for chemicals and fuels. Consequently, despite current regulations, the occurrence of accidental spills poses an important risk. Hazardous and noxious substances (HNSs) have been raising major concern among environmental managers and scientific community for their heterogeneity, hazardous potential towards aquatic organisms and associated social-economic impacts. A literature review on ecotoxicological hazards to aquatic organisms was conducted for seven HNSs: acrylonitrile, n-butyl acrylate, cyclohexylbenzene, hexane, isononanol, trichloroethylene and xylene. Information on the mechanisms of action of the selected HNS was also reviewed. The main purpose was to identify: i) knowledge gaps in need of being addressed in future research; and ii) a set of possible biomarkers suitable for ecotoxicological assessment and monitoring in both estuarine and marine systems. Main gaps found concern the scarcity of information available on ecotoxicological effects of HNS towards marine species and their poorly understood mode of action in wildlife. Differences were found between the sensitivity of freshwater and seawater organisms, so endpoints produced in the former may not be straightforwardly employed in evaluations for the marine environment. The relationship between sub-individual effects and higher level detrimental alterations (e.g. behavioural, morphological, reproductive effects and mortality) are not fully understood. In this context, a set of biomarkers associated to neurotoxicity, detoxification and anti-oxidant defences is suggested as potential indicators of toxic exposure/effects of HNS in marine organisms. Overall, to support the development of contingency plans and the establishment of environmental safety thresholds, it will be necessary to undertake targeted research on HNS ecotoxicity in the marine environment. Research should address these issues under more realistic exposure scenarios reflecting the prevailing spatial and temporal variability in ecological and environmental conditions.

Impact of Herbicides on Non-Target Organisms in Sustainable Irrigated Rice Production Systems: State of Knowledge and Future Prospects

Victor Galhano1, Jose Gomes-Laranjo1, Eduardo Fernandez-Valiente2, Romeu Videira3 and Francisco Peixoto3 1CITAB – Centre for Research and Technology of Agro-Environment and Biological Sciences, University of Tras-os-Montes and Alto Douro (UTAD), Vila Real 2Department of Biology, Faculty of Sciences, Autonomous University of Madrid 3CECAV – Centre of Animal Sciences and Veterinary, UTAD, Vila Real 1,3Portugal 2Spain

Differences in photosynthetic apparatus of leaves from different sides of the chestnut canopy

In crowns of chestnut trees the absorption of radiant energy is not homogeneous; leaves from the south (S) side are the most irradiated, but leaves from the east (E) and west (W) sides receive around 70 % and those from north (N) face less than 20 % of the S irradiation. Compared to the S leaves, those from the N side were 10 % smaller, their stomata density was 14 % smaller, and their laminae were 21 % thinner. N leaves had 0.63 g(Chl) m−2, corresponding to 93 % of total chlorophyll (Chl) amount in leaves of S side. The ratios of Chl a/b were 2.9 and 3.1 and of Chl/carotenoids (Car) 5.2 and 4.8, respectively, in N and S leaves. Net photosynthetic rate (PN) was 3.9 µmol(CO2) m−2 s−1 in S leaves, in the E, W, and N leaves 81, 77, and 38 % of that value, respectively. Morning time (10:00 h) was the period of highest PN in the whole crown, followed by 13:00 h (85 % of S) and 16:00 h with 59 %. Below 500 µmol m−2 s−1 of photosynthetic photon flux density (PPFD), N leaves produced the highest PN, while at higher PPFD, the S leaves were most active. In addition, the fruits from S side were 10 % larger than those from the N side.

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs

Hot, dry summers and mild to cool, wet winters are the characters of the Mediterranean climate. Drought, extreme temperatures and extreme irradiation (UVs) often concomitantly in some cases also together with salinity, significantly affect the growth, yield and quality of the Mediterranean crops. Olive (Olea europaea L), grapevine (Vitis vinifera L) and sweet chestnut (Castanea sativa) are the most important woody crops in the Mediterranean among others. The olive tree and vineyard are familiar features of the Mediterranean landscape. In some mountain regions, these features are accompanied by the orchards of chestnut. Olive oil and wine are important products in that region. In some regions, such as Italy, Turkey, Spain, Portugal, and Greece, chestnut is one of the most important fruit products as well. Olive oil, grape and wine are a traditional icon of the Mediterranean diet. Enjoying the plentiful indigenous plant products, especially wine, olive oil and chestnut, is part of the Mediterranean civilization. Olive oil is the main source of fat in the Mediterranean diet and one of those basic ingredients essential to life in the Mediterranean. It may also protect against heart disease, stroke, and certain cancers. The vine and wine are among the most important symbols of

Searching for Algaecide or Algaestatic Effects of Several Plant Extracts on Phytoplankton: Preliminary Results

Aims: Evaluate the in vitro effects of essential oils and water extracts of Laurus nobilis , Rosmarinus officinalis , Mentha suaveolens and Fraxinus angustifolia on the growth of Anabaena cylindrica and Chlorella vulgaris.