Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C, raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

Toxicidade a cianobactérias: impacte potencial na saúde pública em populações de Portugal e Brasil

Como recurso natural fundamental à vida, a água e os ecossistemas aquáticos devem ser alvo de avaliação contínua, no que se reporta à sua qualidade física, química e biológica. Segundo a Organização Mundial de Saúde cerca de 1,1 biliões de pessoas estão impossibilitadas em aceder a qualquer tipo de água potável e, as populações residentes nas proximidades de rios, lagoas, e reservatórios utilizam estas águas para as suas necessidades de consumo, aumentando o risco de transmissão de doenças. Enquanto constituintes da comunidade fitoplanctónica, as cianobactérias são microrganismos procariotas, fotossintéticos, que obtêm os nutrientes diretamente da coluna de água e, um aumento da concentração de nutrientes (principalmente azoto e fósforo), associado a condições ambientais favoráveis, pode desencadear um crescimento rápido originando fluorescências. Sob determinadas condições as cianobactérias podem produzir toxinas existindo registos que evidenciam que fluorescências toxicas são responsáveis pelo envenenamento agudo e morte de animais e humanos pelo que, a água utilizada para consumo humano deverá ser regularmente monitorizada para este elemento biológico. O objetivo deste estudo é relacionar a ocorrência de fluorescências de cianobactérias (> 2000 cel/ml) e toxicidade associada, com o impacte potencial na Saúde Pública avaliado através do consumo direto ou indireto da água. Em Portugal foram selecionados oito reservatórios situados na região Sul, pertencentes às bacias hidrográficas do Sado e Guadiana e estudados entre 2000 e 2008. No Brasil foram selecionados os reservatórios de Três Marias (Estado de Minas Gerais) e de Tucuruí (Estado do Pará) e estudados em 2005 e 2006 respetivamente. Os reservatórios foram caracterizados em termos físicos e químicos, tendo-se igualmente procedido à caracterização da comunidade fitoplanctónica através da identificação e quantificação dos principais grupos presentes em diferentes épocas do ano. Em termos fitoplanctónicos os reservatórios portugueses apresentaram maior diversidade,verificando-se contudo dominância das cianobactérias na comunidade. Associados a fluorescências, foram registados nestes reservatórios géneros produtores de hepato e neurotoxinas como Aphanizomenon sp, Microcystis aeruginosa e Oscillatoria sp. No Brasil, em situação de fluorescências, os géneros produtores de neuro e hepatotoxinas foram Microcystis (> 350.000 cels/ml) e Cylindrospermopsis. A presença destes géneros, poderá constituir um risco potencial para a saúde pública, pelo que é importante a implementação de medidas de mitigação em todos os reservatórios objeto de estudo, devendo essa atuação passar pelo controle do estado trófico no sentido de evitar o desenvolvimento de fluorescências. Assim sugere-se a implementação de um tratamento adequado para a produção de água de consumo e a organização de ações de sensibilização e aviso e informação às populações que utilizam os reservatórios em Portugal e no Brasil para diversos usos. - ABSTRACT - As a life fundamental natural resource, water and aquatic ecosystems must be continuously evaluated in their physical, chemical and biological quality. According World Health Organization, 1.1 billion people has no chance to access any kind of potable water. Populations living near rivers, lagoons or reservoirs use those waters to content their needs, increasing risks disease transmission. As members of phytoplankton community, cyanobacteria are prokaryotic, photosynthetic microorganisms and get its nutrients directly from water column. The increase of this nutrients (especially nitrogen and phosphorus) associated with favorable environment conditions, can support a sudden grow and instigate blooms. Under specific conditions cyanobacteria can produce toxins and several records have shown that toxic blooms are responsible by acute poisoning and death in animals and humans so, water for human consumption must be regularly surveyed for this biologic element. The aim of this study is to correlate Cyanobacteria blooms (>2.000cels/ml) and connected toxicity with public health impact, evaluated through water consumption. In Portugal, eight reservoirs located in the South region were selected and study between 2000 and 2008. In Brazil, Três Marias reservoir (Minas Gerais Provence) and Tucuruí (Pará Provence) were selected and study in 2005 and 2006. Reservoirs were characterized in physical and chemical aspects, as well as phytoplankton community, through identification and counting of main present groups along study period. In bloom circumstances, liver toxins and neurotoxins producers like Aphanizomenon sp, Microcystis aeruginosa and Oscillatoria sp. were founded in Portuguese reservoirs. In Brazil, cyanobacteria genera involved in toxic bloom were Microcystis (> 350.000 cels/ml) and Cylindrospermopsis. This genera presence represents a potential risk for public health, and show the requirement to implement mitigation measures in all study reservoirs. These measures can be represented by water eutrophication control to avoid blooms, by appropriate treatments of water to human consumption, and public warnings or information to dose people in Portugal and Brazil that use these reservoirs to several activities.

Ecofisiologia de morfotipos reto e espiralado de Cylindrospermopsis raciborskii (Cyanobacteria) em condições controladas

Pós-graduação em Ciências Biológicas (Biologia Vegetal) - IBRC

Effects of Lyngbya majuscula (Cyanophycea) blooms on sediment nutrients and meiofaunal assemblages in seagrass beds in Moreton Bay, Australia

Blooms of Lyngbya majuscula have been increasingly recorded in the waters of Moreton Bay, on the south-east coast of Queensland, Australia. The influences of these blooms on sediment infauna and the implications for sediment biogeochemical processes was studied. Sediment samples were taken from Moreton Bay banks during and after the bloom season. The deposition of L. majuscula seems to be responsible for the higher total Kjedahl nitrogen (TKN) concentrations measured during the bloom period. Total organic carbon (TOC) concentrations did not change. Lyngbya majuscula blooms had a marked influence on the meiobenthos. Nematodes, copepods and polychaetes were the most abundant groups of meiofauna, and the bloom produced a decrease in the abundance and a change in the sediment depth distribution of these organisms. The distribution of nematodes, copepods and polychaetes in sediment became shallower. Further, the bloom did not affect the abundance and distribution of polychaetes as strongly as it did copepods and nematodes. The changes observed in the distribution of meiofauna in the sediment during the bloom period indicate that L. majuscula produces oxygen depletion in sediments, and that different fauna seem to be affected to different degrees.

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.

A bloom of Lyngbya majuscula in Shoalwater Bay, Queensland, Australia: An important feeding ground for the green turtle (Chelonia mydas)

Lyngbya majuscula, a toxic cyanobacterium, was observed blooming during June-July (winter) 2002 in Shoalwater Bay, Queensland, Australia, an important feeding area for a large population of green turtles (Chelonia mydas). The bloom was mapped and extensive mats of L majuscula were observed overgrowing seagrass beds along at least 18 km of coast, and covering a surface area of more than I I km(2). Higher than average rainfall preceded the bloom and high water temperatures in the preceding summer may have contributed to the bloom. In bloom samples, lyngbyatoxin A (LA) was found to be present in low concentration (26 mu g kg(-1) (dry weight)), but debromoaplysiatoxin (DAT) was not detected. The diet of 46 green turtles was assessed during the bloom and L. majuscula was found in 51% of the samples, however, overall it contributed only 2% of the animals' diets. L. majuscula contribution to turtle diet was found to increase as the availability of the cyanobacterium increased. The bloom appeared to have no immediate impact on turtle body condition, however, the presence of a greater proportion of damaged seagrass leaves in diet in conjunction with decreases in plasma concentrations of sodium and glucose could suggest that the turtles may have been exposed to a Substandard diet as a result of the bloom. This is the first confirmed report of L. majuscula blooming in winter in Shoalwater Bay, Queensland, Australia and demonstrates that turtles consume the toxic cyanobacterium in the wild, and that they are potentially exposed to tumour promoting compounds produced by this organism. (c) 2005 Elsevier B.V. All rights reserved.

Florida Bay: Water quality status and trends, historic and emerging algal bloom problems

Florida Bay is a unique subtropical estuary that while historically oligotrophic, has been subjected to both natural and anthropogenic stressors, including hurricanes, coastal eutrophication and other impacts. These stressors have resulted in degradation of water quality in the past several decades, most evidenced by reoccurring blooms of the picocyanobacterium Synechococcus spp. Major nutrient inputs consist of freshwater flows to the eastern region from runoff and regulated canal releases, inputs from the Everglades to the central region via Taylor Slough, exchanges with the Gulf of Mexico, which include intermittent Shark River inputs to the western region, stormwater and wastewater from the Florida Keys, and atmospheric deposition. These nutrient inputs have resulted in a transition from strong phosphorus (P) limitation of phytoplankton in the eastern bay to nitrogen (N) limitation in the western bay. Large blooms of Synechococcus were most pronounced in the central bay region, in the area of transition between P and N limitation, in the mid-1990s. Although non-toxic, these blooms, which have continued intermittently through the early 2000s, resulted in significant sea-grass and benthic organism mortalities. A new suite of stressors in 2005, including the passages of Hurricanes Katrina, Rita, and Wilma, additional canal releases, and the initiation of road construction to widen the main roadway leading to the Keys, were correlated with a large Synechococcus bloom in the previously clear, strongly P- limited, northeastern region of the bay. Sustained for 3 years, this bloom was accompanied by a shift from P limitation to N limitation during its course. Nutrient bioassay experiments suggest that this bloom persisted due to the ability of Synechococcus to access organic N and P sources, microbial and geochemical cycling of organic and inorganic nutrients in the water column and between the water column and sediments (both suspended particles and benthos), and decreased grazing by benthic fauna due to their die-off.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system

Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB) is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed.

Toxic Epidermal Necrolysis-like Cutaneous Lupus Erythematosus: A Series of Three Patients

Toxic epidermal necrolysis-like lesions have been described in the setting of lupus erythematosus, and have been considered as a specific hyperacute variant of cutaneous lupus erythematosus, with features different from classical drug-related toxic epidermal necrolysis. We report here a series of three patients with lupus erythematosus who presented with severe worsening of their cutaneous disease in a toxic epidermal necrolysis-like fashion. We compared these cases with cases reported previously. Based on this discussion, we speculate that some of these patients may have classical drug-related toxic epidermal necrolysis rather than lupus erythematosus-related toxic epidermal necrolysis.