Cylindrospermopsis raciborskii exudate-Cu complexes: impact on copper dynamics and bioavailability in an aquatic food chain

The increasing contamination of aquatic environments motivates studies on the interactions among natural dissolved organic matter, metals, and the biota. This investigation focused on the organic exudates of the toxic cyanobacteria Cylindrospermopsis raciborskii as a Cu carrier through a three-level aquatic trophic chain (bacteria, protozoa, and copepod). The effects of bacteria activity and growth on the metal-organic complexes were evaluated through changes in free Cu2+ ions, total dissolved, and total particulate Cu. To be sure that the added copper would be complexed to the exudates, its complexing properties were previously determined. The cyanobacteria exudate-Cu complexes were furnished to bacteria that were further used as a food source to the protozoan Paramercium caudatum. This was then furnished as food to the copepod Mesocyclops sp. The results showed that, in general, the cyanobacterial exudates decreased Cu bioavailability and toxicity to the first trophic level (bacteria), but because the heterotrophic bacteria accumulated Cu, they were responsible for the transference for the otherwise low availability metal form. Both the bacteria and protozoan organisms accumulated Cu, but no metal accumulation was detected in the copepods.

Credneramides A and B: Neuromodulatory Phenethylamine and Isopentylamine Derivatives of a Vinyl Chloride-Containing Fatty Acid from cf. Trichodesmium sp nov.

Credneramides A (1) and B (2), two vinyl chloride-containing metabolites, were isolated from a Papua New Guinea collection of cf. Trichodesmium sp. nov. and expand a recently described class of vinyl chloride-containing natural products. The precursor fatty acid, credneric acid (3), was isolated from both the aqueous and organic fractions of the parent fraction as well as from another geographically and phylogenetically distinct cyanobacterial collection (Panama). Credneramides A and B inhibited spontaneous calcium oscillations in murine cerebrocortical neurons at low micro-molar concentrations (1, IC50 4.0 mu M; 2, IC50 3.8 mu M).

Seasonal dynamics of cyanobacteria in a eutrophic reservoir (Arcoverde) in a semi-arid region of Brazil

Environmental conditions favor the predominance of dense populations of cyanobacteria in reservoirs in northeastern Brazil. The aim of this study was to understand cyanobacterial population dynamics in the rainy and dry seasons at two depths in the Arcoverde reservoir. Microalgae and cyanobacteria samples were collected during 24 hours with intervals of 4 hours (nycthemeral) at sub-surface and 10 m using a van Dorn bottle and a determined biomass. Physical and chemical variables were obtained and the data were analyzed using the principal component analysis (PCA). No nycthemeral variations in the taxonomic composition or distribution of the populations of cyanobacteria were found between the different times of day in either the rainy or dry season. In both seasons, the greatest biomass of the phytoplankton community was made up of cyanobacteria at two depths and all times of the day. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju was dominant at all times of the day on both the surface and at the bottom. In the rainy season, the differences in cyanobacterial biomass between the surface and bottom were less significant than in the dry season. The differences in cyanobacterial biomass between surface and bottom were less pronounced than those found in the dry season. We concluded that a) physical variables better explain the alterations of species in the phytoplankton community in an environment dominated by cyanobacteria throughout the year; b) seasonal climatic factors associated to periods of stratification and de-stratification are important for alterations in the community and variations in biomass and, c) the turbidity caused by rainfall favored the emergence and establishment of other cyanobacteria, especially Planktothrix agardhii (Gomont) Anagnostidis & Komarek.

Degradation of [D-Leu]-Microcystin-LR by solar heterogeneous photocatalysis (TiO2)

The present study investigates the use of solar heterogeneous photocatalyis (TiO2) for the destruction of [D-Leu]-Microcystin-LR, powerful toxin of widespread occurrence within cyanobacteria blooms. We extracted [D-Leu]-Microcystin-LR from a culture of Microcystis spp. and used a flat plate glass reactor coated with TiO2 (Degussa, P25) for the degradation studies. The irradiance was measured during the experiments with the aid of a spectroradiometer. After the degradation experiments, toxin concentrations were determined by HPLC and mineralization by TOC analyses. Acute and chronic toxicities were, quantified using mice and phosphatase inhibition in vitro assays, respectively. According to the performed experiments, 150 min were necessary to reduce the toxin concentration to the WHO's guideline for drinking water (from 10 to 1 mu g L-1) and to mineralize 90% of the initial carbon content. Another important finding is that solar heterogeneous photocatalysis was a destructive process indeed, not only for the toxin, but also for the other extract components and degradation products generated. Moreover, toxicity tests using mice have shown that the acute effect caused by the initial sample was removed. However, tests using the phosphatase enzyme indicated that it may be formed products capable of inducing chronic effects on mammals. The performed experiments indicate the feasibility of using solar heterogeneous photocatalysis for treating contaminated water with [D-Leu]-Microcystin-LR, not only due to its destruction, but also to the significant removal of organic matter and acute toxicity that can be achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Morphological and molecular studies of Sphaerospermopsis torques-reginae (Cyanobacteria, Nostocales) from South American water blooms

Sphaerospermopsis torques-reginae (Komarek) Werner, Laughinghouse IV, Fiore & Sant'Anna comb. nov. was originally described as Anabaena torques-reginae Komarek from planktonic populations of Cuban eutrophic environments, characterized by twisted trichomes with spherical akinetes adjacent to the heterocytes. Recently, using molecular analyses, all planktonic Anabaena Bory ex Bornet & Flahault morphospecies were transferred into the genus Dolichospermum (Ralfs ex Bornet & Flahault) Wacklin el al., including Dolichospermum torques-reginae (Komarek) Wacklin et al. However, by a polyphasic characterization of strains of Anabaena reniformis Lemmermann and Aphanizomenon aphanizomenoides (Forti) Horecka & Komarek (=Anabaena aphanizomenoides Forti), these planktonic species were reclassified into Sphaerospermopsis Zapomelova et al. Our study's main objective was to characterize morphologically and molecularly cyanobacterial populations identified as Dolichospermum torques-reginae, observed in different aquatic ecosystems in South America. The 16S rRNA gene of two Dolichospermum torques-reginae strains (ITEP-024 and ITEP-026) was sequenced and phylogenetically analyzed for the first time. The morphological and phylogenetic analyses demonstrated the affiliation of the studied populations with the genus Sphaerospermopsis and, consequently, were denominated as Sphaerospermopsis torques-reginae. Furthermore, geographic distribution, ecology, and toxicity of the species are discussed. It was observed in different aquatic environments, natural and artificial, tropical and subtropical in Brazil, temperate in Argentina, and tropical in Colombia, suggesting a wide distribution in South America. It normally occurred in dense freshwater blooms, although it was also found in water with low salinity. Sphaerospermopsis torques-reginae toxic blooms have been reported in tropical water bodies in northeastern Brazil.

Biomonitoring genotoxicity and cytotoxicity of Microcystis aeruginosa (Chroococcales, Cyanobacteria) using the Allium cepa test

Water pollution caused by toxic cyanobacteria is a problem worldwide, increasing with eutrophication. Due to its biological significance, genotoxicity should be a focus for biomonitoring pollution owing to the increasing complexity of the toxicological environment in which organisms are exposed. Cyanobacteria produce a large number of bioactive compounds, most of which lack toxicological data. Microcystins comprise a class of potent cyclic heptapeptide toxins produced mainly by Microcystis aeruginosa. Other natural products can also be synthesized by cyanobacteria, such as the protease inhibitor, aeruginosin. The hepatotoxicity of microcystins has been well documented, but information on the genotoxic effects of aeruginosins is relatively scarce. In this study, the genotoxicity and ecotoxicity of methanolic extracts from two strains of M. aeruginosa NPLJ-4, containing high levels of microcystin, and M. aeruginosa NPCD-1, with high levels of aeruginosin, were evaluated. Four endpoints, using plant assays in Allium cepa were applied: rootlet growth inhibition, chromosomal aberrations, mitotic divisions, and micronucleus assays. The microcystin content of M. aeruginosa NPLJ-4 was confirmed through ELISA, while M. aeruginosa NPCD-1 did not produce microcystins. The extracts of M. aeruginosa NPLJ-4 were diluted at 0.01, 0.1, 1 and 10 ppb of microcystins: the same procedure was used to dilute M. aeruginosa NPCD-1 used as a parameter for comparison, and water was used as the control. The results demonstrated that both strains inhibited root growth and induced rootlet abnormalities. The strain rich in aeruginosin was more genotoxic, altering the cell cycle, while microcystins were more mitogenic. These findings indicate the need for future research on non-microcystin producing cyanobacterial strains. Understanding the genotoxicity of M. aeruginosa extracts can help determine a possible link between contamination by aquatic cyanobacteria and high risk of primary liver cancer found in some areas as well as establish water level limits for compounds not yet studied. (C) 2012 Elsevier B.V. All rights reserved.

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including conesnails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.

Database of diazotrophs in global ocean: abundances, biomass and nitrogen fixation rates

[EN] Marine N2 fixing microorganisms, termed diazotrophs, are a key functional group in marine pelagic ecosystems. The biological fixation of dinitrogen (N2) to bioavailable nitrogen provides an important new source of nitrogen for pelagic marine ecosystems 5 and influences primary productivity and organic matter export to the deep ocean. As one of a series of efforts to collect biomass and rates specific to different phytoplankton functional groups, we have constructed a database on diazotrophic organisms in the global pelagic upper ocean by compiling about 12 000 direct field measurements of cyanobacterial diazotroph abundances (based on microscopic cell counts or qPCR 10 assays targeting the nifH genes) and N2 fixation rates. Biomass conversion factors are estimated based on cell sizes to convert  abundance data to diazotrophic biomass. The database is limited spatially, lacking large regions of the ocean especially in the Indian Ocean. The data are approximately log-normal distributed, and large variances exist in most sub-databases with non-zero values differing 5 to 8 orders of magnitude. 15 Lower mean N2 fixation rate was found in the North Atlantic Ocean than the Pacific Ocean. Reporting the geometric mean and the range of one geometric standard error below and above the geometric mean, the pelagic N2 fixation rate in the global ocean is estimated to be 62 (53–73) TgNyr−1 and the pelagic diazotrophic biomass in the global ocean is estimated to be 4.7 (2.3–9.6) TgC from cell counts and to 89 (40–20 200) TgC from nifH-based abundances. Uncertainties related to biomass conversion factors can change the estimate of geometric mean pelagic diazotrophic biomass in the global ocean by about ±70 %. This evolving database can be used to study spatial and temporal distributions and variations of marine N2 fixation, to validate geochemical estimates and to parameterize and validate biogeochemical models. The database is 25 stored in PANGAEA (http://doi.pangaea.de/10.1594/PANGAEA.774851).

Adaptación de la Directiva Europea de Gestión de la Calidad de Aguas de Baño a las playas de Canarias

[ES] Hemos realizado una serie de consideraciones con respecto a la Directiva 2006/7/CE para lograr su correcta aplicación en las playas de baño de Canarias. En nuestras aguas se producen algunos fenómenos que no se producen en el resto de la Unión Europea. Por esta razón hemos establecido una serie de conceptos que no están previstos de forma explicita en los artículos de la Directiva y que, con algunas matizaciones, podremos adaptarlos a la generalidad de la Directiva, o ?sensu contrario?, adecuar la Directiva a nuestra realidad ambiental. En este sentido, hemos adaptado conceptos como ?contaminación de corta duración?, ?tendencia a la proliferación de macroalgas?, ?tendencia a la proliferación de fitoplancton marino?, ?proliferación de cianobacterias? y otros a las especificidades que se producen en nuestras costas y que podrían alterar la calidad de las aguas de baño

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

Microbial community analysis of Lake Chillisquaque, a small water system in Central Pennsylvania

Cyanobacteria are photosynthetic organisms that require the absorption of light for the completion of photosynthesis. Cyanobacteria can use a variety of wavelengths of light within thevisible light spectrum in order to harvest energy for this process. Many species of cyanobacteria have light-harvesting proteins that specialize in the absorption of a small range of wavelengths oflight along the visual light spectrum; others can undergo complementary chromatic adaptation and alter these light-harvesting proteins in order to absorb the wavelengths of light that are mostavailable in a given environment. This variation in light-harvesting phenotype across cyanobacteria leads to the utilization of environmental niches based on light wavelength availability. Furthermore, light attenuation along the water column in an aquatic system also leads to the formation of environmental niches throughout the vertical water column. In order to better understand these niches based on light wavelength availability, we studied the compositionof cyanobacterial genera at the surface and depth of Lake Chillisquaque at three time points throughout the year: September 2009, May 2010, and July 2010. We found that cyanobacterialgenera composition changes throughout the year as well as with physical location in the water column. Additionally, given the light attenuation noted throughout the Lake Chillisquaque, we are able to conclude that light is a major selective factor in the community composition of Lake Chillisquaque.

Evolution of the Southern Kenya Rift from Miocene to present with a focus on the Magadi area

The Kenya (a.k.a., Gregory) Rift is a geologically active area located within the eastern branch of the larger East African Rift System (EARS). The study area is located in the southern Kenya Rift between 1° South and the Kenya-Tanzania border (covering approximately 1.5 square degrees, semi-centered on Lake Magadi) and is predominantly filled with extrusive igneous rocks (mostly basalts, phonolites and trachytes) of Miocene age or younger. Sediments are thin, less than 1.5Ma, and are confined to small grabens. The EARS can serve both as an analogue for ancient continental rifting and as a modern laboratory to observe the geologic processes responsible for rifting. This study demonstrates that vintage (as in older, quality maps published by the Kenya Geological Survey, that may be outdated based on newer findings) quarter-degree maps can be successfully combined with recently published data, and used to interpret satellite (mainly Landsat 7) images to produce versatile, updated digital maps. The study area has been remapped using this procedure and although it covers a large area, the mapping retains a quadrangle level of detail. Additionally, all geologic mapping elements (formations, faults, etc.) have been correlated across older map boundaries so that geologic units don't end artificially at degree boundaries within the study area. These elements have also been saved as individual digital files to facilitate future analysis. A series of maps showing the evolution of the southern Kenya rift from the Miocene to the present was created by combining the updated geologic map with age dates for geologic formations and fault displacements. Over 200 age dates covering the entire length of the Kenya Rift have been compiled for this study, and 6 paleo-maps were constructed to demonstrate the evolution of the area, starting with the eruption of the Kishalduga and Lisudwa melanephelinites onto the metamorphic basement around 15Ma. These eruptions occurred before the initial rift faulting and were followed by a massive eruption of phonolites between 13-10 Ma that covered most of the Kenya dome. This was followed by a period of relative quiescence, until the initial faulting defined the western boundary of the rift around 7Ma. The resulting graben was asymmetrical until corresponding faults to the east developed around 3Ma. The rift valley was flooded by basalts and trachytes between 3Ma and 700ka, after which the volcanic activity slowed to a near halt. Since 700ka most of the deposition has been comprised of sediments, mainly from lakes occupying the various basins in the area. The main results of this study are, in addition to a detailed interpretation of the rift development, a new geologic map that correlates dozens of formations across old map boundaries and a compilation of over 300 age dates. Specific products include paleomaps, tables of fault timing and displacement, and volume estimates of volcanic formations. The study concludes with a generalization of the present environment at Magadi including discussions of lagoon chemistry, mantle gases in relation to the trona deposit, and biology of the hot springs. Several biologic samples were collected during the 2006 field season in an attempt to characterize the organisms that are commonly seen in the present Lake Magadi environment. Samples were selected to represent the different, distinctive forms that are found in the hotsprings. Each sample had it own distinctive growth habit, and analysis showed that each was formed by a different cyanobacterial. Actual algae was rare in the collected samples, and represented by a few scattered diatoms.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

The Cytotoxic Mode of Action of the Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei (Fig.16.1) is rich in components which belong to different functional groups. Besides low molecular mass compounds, the venom contains several disulphide-rich peptides, also called mini-proteins, which act as neurotoxins on ion channels or as enhancers of neurotoxins. Likewise, a variety of small cytolytic peptides, which destroy membranes very efficiently, and enzymes are present in the venom. Neurotoxins with cytolytic activity, cytolytic a-helical small cationic peptides and enzymes most probably attacking connective tissue and phospholipid membranes cause the overall cytotoxic effect of this venom. Synergistic and enhancing interactions between components enable the spider to achieve a maximum of toxicity with a minimum of venom quantity.

STUDIES ON CHOLINERGIC FUNCTION AND MEMORY IN MICE (CHOLINOTOXINS, WORKING, REFERENCE MEMORY, RADIAL ARM MAZE)

Considerable evidence suggests that central cholinergic neurons participate in either acquisition, storage or retrieval of information. Experiments were designed to evaluate information processing in mice following either reversible or irreversible impairment in central cholinergic activity. The cholinergic receptor antagonists, atropine and methylatropine were used to reversibly inhibit cholinergic transmission. Irreversible impairment in central cholinergic function was achieved by central administration of the cholinergic-specific neurotoxins, N-ethyl-choline aziridinium (ECA) and N-ethyl-acetylcholine aziridinium (EACA).^ ECA and EACA appear to act by irreversible inhibition of high affinity choline uptake (proposed rate-limiting step in acetylcholine synthesis). Intraventricular administration of ECA or EACA produced persistent reduction in hippocampal choline acetyltransferase activity. Other neuronal systems and brain regions showed no evidence of toxicity.^ Mice treated with either ECA or EACA showed behavioral deficits associated with cholinergic dysfunction. Passive avoidance behavior was significantly impaired by cholinotoxin treatment. Radial arm maze performance was also significantly impaired in cholinotoxin-treated animals. Deficits in radial arm maze performance were transient, however, such that rapid and apparent complete behavioral recovery was seen during retention testing. The centrally active cholinergic receptor antagonist atropine also caused significant impairment in radial arm maze behavior, while equivalent doses of methylatropine were without effect.^ The relative effects of cholinotoxin and receptor antagonist treatment on short-term (working) memory and long-term (reference) memory in radial arm maze behavior were examined. Maze rotation studies indicated that there were at least two different response strategies which could result in accurate maze performance. One strategy involved the use of response algorithms and was considered to be a function of reference memory. Another strategy appeared to be primarily dependent on spatial working memory. However, all behavioral paradigms with multiple trails have reference memory requirements (i.e. information useful over all trials). Performance was similarly affected following either cholinotoxin or anticholinergic treatment, regardless of the response strategy utilized. In addition, rates of behavioral recovery following cholinotoxin treatment were similar between response groups. It was concluded that both cholinotoxin and anticholinergic treatment primarily resulted in impaired reference memory processes. ^