Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 270-287).

The following research deals with the response of the small shore crabs, Hemigrapsus oreqonesis and Hemigrapsus nudus to paralytic shellfish toxins (PST). These shore crabs were shown to develop a remarkable seasonal resistance to administered saxitoxin (STX). No similar change in sensitivity was found after administration of tetrodotoxin (TTX), another marine neurotoxin with similar actions to the PST. Resistance to STX in the small shore crabs was linked to the presence of PST in the viscera, and this in turn was related to the presence of toxic dinoflagellate blooms in the area. Furthermore, this research provides, for the first time, evidence of a protein component (MW 145,000 daltons) which appears to be associated with acquired resistance to PST in the shore crab. In addition, this protein component was shown to appear in sensitive crab extracts after the administration of low doses of saxitoxin and tetrodotoxin in vivo. / Land and Food Systems, Faculty of / Graduate

First described by Plate (1906), Pyrodinium bahamense is a bioluminescent dinoflagellate species which forms a resting cyst as part of its life cycle.  P. bahamense forms large dense cyst beds in the flocculent layer of sediments and can remain dormant for decades before excysting and forming blooms.  The Atlantic strain has recently been discovered to produce a neurotoxin called saxitoxin.  Although saxitoxins are most commonly associated with paralytic shellfish poisoning, saxitoxin produced by P. bahamense has been implicated in a number of human illnesses following the consumption of contaminated puffer fish originating from the Indian River Lagoon, Florida.  The discovery of P. bahamense cysts on seagrass blades during an ongoing bloom raised the question of whether manatees, whose diet is composed mainly of seagrass, could be at risk of exposure to saxitoxins.  The aim of this study was to determine whether P. bahamense cysts can be digested in the manatee gastrointestinal tract or if they would pass through intact.  There are no known methods for purifying P. bahamense cysts from the contents of the manatee gastrointestinal tract, so it was necessary to develop an appropriate protocol using cysts purified from sediments in Tampa Bay, Florida.  These methods were then to be used to test whether P. bahamense cysts added to manatee digesta break down over time, and also to test archived digesta samples for presence of cysts.  A successful method for purifying cysts from manatee digesta could not be developed during the span of this study, and so no conclusions could be drawn about the likelihood of digestion.  However, archived samples were found to contain P. bahamense cysts, confirming that manatees are ingesting them while feeding.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

Aquatic microalgae produce a variety of toxic secondary metabolites, which are a concern for public health and seafood industries, while also presenting a source of pharmacologically valuable compounds. The present study deals with the physiology and molecular genetics of saxitoxin (STX), a cyanobacterial neurotoxic alkaloid. Ecological and chemical parameters have been investigated for their effects on growth and STX production in the cyanobacterium Cylindrospermopsis raciborskii T3, in order to better understand the physiological responses of this cyanobacterium to the anthropogenic eutrophication of water bodies. The results indicated that phosphate, in particular, had an incremental effect on STX production, as well as promoting the up-regulation of transcription of the STX biosynthetic gene cluster (sxt). High temperature was found to negatively affect growth and STX production in this organism. The effects of the plant hormone, jasmonic acid, were also tested, since it has previously been shown to affect plant alkaloid production. The hypothesised similarity between cyanobacterial and plant secondary metabolism in response to this plant hormone was confirmed in the neurotoxic cyanobacterium, C. raciborskii T3, as well as the non-toxic Anabaena sp. PCC7120. Furthermore, investigation of the sxt gene cluster transcriptional map in C. raciborskii T3 was carried out, with identification of three main polycistronic and one monocistronic transcripts. Promoter regions putatively involved in the regulation of STX production in C. raciborskii T3 were also identified. Transcription factor consensus motifs, the pho boxes, were identified in the main promoter region. These conserved motifs are the binding regions for the transcriptional regulator, PhoB, to the pho regulon genes, involved in phosphate uptake during conditions of its depletion in the environment. Moreover, a genomic region adjacent to the sxt gene cluster in C. raciborskii T3 was identified and characterised, putatively encoding a regulatory two-component system. This system appears to be involved in the sensing of environmental signals, in particular depleted phosphate, while activating the transcription of genes involved in its uptake and transport. The results of this study lead to a greater understanding of the complex factors associated with the regulation of STX biosynthesis and bloom-formation, by the cyanobacterium C. raciborskii T3.

Aquatic microalgae produce a variety of toxic secondary metabolites, which are a concern for public health and seafood industries, while also presenting a source of pharmacologically valuable compounds. The present study deals with the physiology and molecular genetics of saxitoxin (STX), a cyanobacterial neurotoxic alkaloid. Ecological and chemical parameters have been investigated for their effects on growth and STX production in the cyanobacterium Cylindrospermopsis raciborskii T3, in order to better understand the physiological responses of this cyanobacterium to the anthropogenic eutrophication of water bodies. The results indicated that phosphate, in particular, had an incremental effect on STX production, as well as promoting the up-regulation of transcription of the STX biosynthetic gene cluster (sxt). High temperature was found to negatively affect growth and STX production in this organism. The effects of the plant hormone, jasmonic acid, were also tested, since it has previously been shown to affect plant alkaloid production. The hypothesised similarity between cyanobacterial and plant secondary metabolism in response to this plant hormone was confirmed in the neurotoxic cyanobacterium, C. raciborskii T3, as well as the non-toxic Anabaena sp. PCC7120. Furthermore, investigation of the sxt gene cluster transcriptional map in C. raciborskii T3 was carried out, with identification of three main polycistronic and one monocistronic transcripts. Promoter regions putatively involved in the regulation of STX production in C. raciborskii T3 were also identified. Transcription factor consensus motifs, the pho boxes, were identified in the main promoter region. These conserved motifs are the binding regions for the transcriptional regulator, PhoB, to the pho regulon genes, involved in phosphate uptake during conditions of its depletion in the environment. Moreover, a genomic region adjacent to the sxt gene cluster in C. raciborskii T3 was identified and characterised, putatively encoding a regulatory two-component system. This system appears to be involved in the sensing of environmental signals, in particular depleted phosphate, while activating the transcription of genes involved in its uptake and transport. The results of this study lead to a greater understanding of the complex factors associated with the regulation of STX biosynthesis and bloom-formation, by the cyanobacterium C. raciborskii T3.

O aporte artificial de nutrientes nos ambientes aquáticos tem favorecido a incidência de florações de cianobactérias, causando impactos na qualidade da água e colocando em risco a saúde humana. O gênero de cianobactéria Cylindrospermopsis, amplamente distribuído em água doce pelo mundo, é capaz de produzir diversas cianotoxinas, dentre elas saxitoxina e análogos. O objetivo deste trabalho foi elaborar um modelo de exposição referente à ingestão de água contaminada por saxitoxina e análogos através da recreação de contato primário e do consumo de água. Os dados de densidade de células de Cylindrospermopsis e de concentrações de saxitoxina e análogos na água bruta foram obtidos da Rede de Monitoramento de Qualidade de Águas Superficiais da CETESB (2015 e 2016) do reservatório Itupararanga. O modelo conceitual de exposição e o cálculo da ingestão diária crônica de saxitoxina e análogos consideraram: concentrações máxima e mínima de saxitoxina e análogos encontradas na água bruta do reservatório, vias de exposição, população exposta, taxa de ingestão de água, frequência e duração da exposição, peso corporal para adultos (maiores de 21 anos) e crianças (menores de 5 anos), para cenários de exposição relativos à recreação de contato primário e consumo de água potável. Os valores de células de cianobactérias estiveram entre 2.216 e 203.082 céls.mL-1 e as concentrações de saxitoxina e análogos estiveram entre 0,04 e 0,37 µg equivalente STX..L-1. Os resultados obtidos na análise da água bruta do reservatório, estiveram abaixo do valor estabelecido na legislação brasileira para água potável (3 µg equivalente STX.L-1), mas não atenderiam as especificações adotadas por Oregon e Ohio (0,3 µg equivalentes STX.L-1) e à proposta em discussão no Comitê Permanente para Gestão Integrada da Qualidade da Água destinada ao Consumo Humano no Estado de São Paulo (0,13 µg equivalente STX.L-1), indicando a possibilidade da ocorrência de efeito adverso à saúde em grupos sensíveis que utilizem água do reservatório, dependendo da eficiência de remoção dessas cianotoxinas no processo de tratamento. Os valores de ingestão diária crônica estimados, para recreação, variaram de 2,55 x 10-6 a 1,08 x 10-4 µg equivalente STX. kg-1.dia-1 (adultos) e 2,78 x 10-9 a 4,35 x 10-4 µg equivalente STX. kg-1.dia-1 (crianças). Para ingestão de água potável, esses valores variaram de 2,5 x 10-4 a 1,47 x 10-2 µg equivalente STX. kg-1.dia-1 (adultos) e 5,36 x 10-5 a 2,55 x 10-2 µg equivalente STX. kg-1.dia-1 (crianças). Esses valores indicaram que as crianças estão mais expostas que os adultos, fato preocupante, pois elas são mais sensíveis aos efeitos de substâncias tóxicas, e apesar de estudos indicarem efeitos da saxitoxina em cultura de células neurais, sua capacidade de atravessar a barreira hematoencefálica e de causar danos cognitivos em ratos, os efeitos da exposição crônica em humanos ainda são desconhecidos. Não foi possível estimar os riscos à saúde da população exposta à saxitoxina e análogos, pela falta de dados na literatura, porém o modelo de exposição proposto é um avanço para a identificação de lacunas para a condução da avaliação de risco à saúde humana considerando a exposição crônica. / The artificial enrichment of nutrients in aquatic environments has favored the incidence of cyanobacteria blooms, causing impacts on water quality and putting human health at risk. The genus of cyanobacteria Cylindrospermopsis, widely distributed throughout the world in freshwater environments, is able to produce cianotoxin, among them saxitoxin and its analogues. The objective of this work was to elaborate a model of exposure referring to the ingestion of water contaminated by saxitoxin and analogues through recreation of primary contact and water consumption. Cylindrospermopsis cell density and saxitoxin and analogues concentrations in surface water were obtained from the CETESB Surface Water Quality Monitoring Network for the Itupararanga reservoir in 2015 and 2016. The development of the conceptual model of exposure and the calculation of the chronic daily intake of saxitoxin and analogues, considered: maximum and minimum concentrations of saxitoxin and analogues found in the raw water of the reservoir, route of exposure, exposed population, rate of water intake, frequency and duration of exposure, body weight for adults (over 21 years) and children (under 5 years), for exposures scenarios concerning recreation of primary contact and consumption of drinking water. The values of cyanobacteria cells were between 2,216 and 203,082 cells.L-1 and the concentrations of saxitoxin and analogues were between 0.04 and 0.37 µg STX equivalent. L-1. The comparison of these variables indicated a significant positive correlation between them. The results obtained in the raw water analysis of the reservoir were below the value established in Brazilian legislation for drinking water (3 µg equivalent STX.L-1), but would not meet the specifications adopted by US states Oregon and Ohio (0.3 µg equivalents.L-1) and the proposed value for the Standing Committee for Integrated Water Quality Management for Human Consumption in the State of São Paulo (0,13 µg equivalent STX.L-1), indicating the possibility of adverse health effects in sensitive groups using water from the reservoir. Estimated chronic daily intake values, for recreation, ranged from 2.55 x 10-6 to 1.08 x 10-4 µg STX equivalent. kg-1.day-1 (adults) and 2.78 x 10-9 to 4.35 x 10-4 µg STX equivalent. kg-1.day-1 (children). For water intake, these values ranged from 2.5 x 10-4 to 1.47 x 10-2 µg STX equivalent. kg-1 day-1 (adults) and 5.36 x 10-5 to 2.55 x 10-2 µg STX equivalent. kg-1. day-1 (children). These values indicated that children are more exposed than adults, a worrying fact because they are generally more sensitive to the effects of toxic substances than adults, and although studies indicate that saxitoxin was able to cross the blood brain barrier and to cause cognitive damage in rats, as well as changes in neural cell cultures, the effects of chronic exposure in humans are still unknown. It was not possible to estimate the health risks of the population exposed to saxitoxin and the like, due to the lack of data in the literature, but the proposed exposure model is an advance for the identification of gaps for conducting the human health risk assessment considering the exposure chronic.

O aporte artificial de nutrientes nos ambientes aquáticos tem favorecido a incidência de florações de cianobactérias, causando impactos na qualidade da água e colocando em risco a saúde humana. O gênero de cianobactéria Cylindrospermopsis, amplamente distribuído em água doce pelo mundo, é capaz de produzir diversas cianotoxinas, dentre elas saxitoxina e análogos. O objetivo deste trabalho foi elaborar um modelo de exposição referente à ingestão de água contaminada por saxitoxina e análogos através da recreação de contato primário e do consumo de água. Os dados de densidade de células de Cylindrospermopsis e de concentrações de saxitoxina e análogos na água bruta foram obtidos da Rede de Monitoramento de Qualidade de Águas Superficiais da CETESB (2015 e 2016) do reservatório Itupararanga. O modelo conceitual de exposição e o cálculo da ingestão diária crônica de saxitoxina e análogos consideraram: concentrações máxima e mínima de saxitoxina e análogos encontradas na água bruta do reservatório, vias de exposição, população exposta, taxa de ingestão de água, frequência e duração da exposição, peso corporal para adultos (maiores de 21 anos) e crianças (menores de 5 anos), para cenários de exposição relativos à recreação de contato primário e consumo de água potável. Os valores de células de cianobactérias estiveram entre 2.216 e 203.082 céls.mL-1 e as concentrações de saxitoxina e análogos estiveram entre 0,04 e 0,37 µg equivalente STX..L-1. Os resultados obtidos na análise da água bruta do reservatório, estiveram abaixo do valor estabelecido na legislação brasileira para água potável (3 µg equivalente STX.L-1), mas não atenderiam as especificações adotadas por Oregon e Ohio (0,3 µg equivalentes STX.L-1) e à proposta em discussão no Comitê Permanente para Gestão Integrada da Qualidade da Água destinada ao Consumo Humano no Estado de São Paulo (0,13 µg equivalente STX.L-1), indicando a possibilidade da ocorrência de efeito adverso à saúde em grupos sensíveis que utilizem água do reservatório, dependendo da eficiência de remoção dessas cianotoxinas no processo de tratamento. Os valores de ingestão diária crônica estimados, para recreação, variaram de 2,55 x 10-6 a 1,08 x 10-4 µg equivalente STX. kg-1.dia-1 (adultos) e 2,78 x 10-9 a 4,35 x 10-4 µg equivalente STX. kg-1.dia-1 (crianças). Para ingestão de água potável, esses valores variaram de 2,5 x 10-4 a 1,47 x 10-2 µg equivalente STX. kg-1.dia-1 (adultos) e 5,36 x 10-5 a 2,55 x 10-2 µg equivalente STX. kg-1.dia-1 (crianças). Esses valores indicaram que as crianças estão mais expostas que os adultos, fato preocupante, pois elas são mais sensíveis aos efeitos de substâncias tóxicas, e apesar de estudos indicarem efeitos da saxitoxina em cultura de células neurais, sua capacidade de atravessar a barreira hematoencefálica e de causar danos cognitivos em ratos, os efeitos da exposição crônica em humanos ainda são desconhecidos. Não foi possível estimar os riscos à saúde da população exposta à saxitoxina e análogos, pela falta de dados na literatura, porém o modelo de exposição proposto é um avanço para a identificação de lacunas para a condução da avaliação de risco à saúde humana considerando a exposição crônica. / The artificial enrichment of nutrients in aquatic environments has favored the incidence of cyanobacteria blooms, causing impacts on water quality and putting human health at risk. The genus of cyanobacteria Cylindrospermopsis, widely distributed throughout the world in freshwater environments, is able to produce cianotoxin, among them saxitoxin and its analogues. The objective of this work was to elaborate a model of exposure referring to the ingestion of water contaminated by saxitoxin and analogues through recreation of primary contact and water consumption. Cylindrospermopsis cell density and saxitoxin and analogues concentrations in surface water were obtained from the CETESB Surface Water Quality Monitoring Network for the Itupararanga reservoir in 2015 and 2016. The development of the conceptual model of exposure and the calculation of the chronic daily intake of saxitoxin and analogues, considered: maximum and minimum concentrations of saxitoxin and analogues found in the raw water of the reservoir, route of exposure, exposed population, rate of water intake, frequency and duration of exposure, body weight for adults (over 21 years) and children (under 5 years), for exposures scenarios concerning recreation of primary contact and consumption of drinking water. The values of cyanobacteria cells were between 2,216 and 203,082 cells.L-1 and the concentrations of saxitoxin and analogues were between 0.04 and 0.37 µg STX equivalent. L-1. The comparison of these variables indicated a significant positive correlation between them. The results obtained in the raw water analysis of the reservoir were below the value established in Brazilian legislation for drinking water (3 µg equivalent STX.L-1), but would not meet the specifications adopted by US states Oregon and Ohio (0.3 µg equivalents.L-1) and the proposed value for the Standing Committee for Integrated Water Quality Management for Human Consumption in the State of São Paulo (0,13 µg equivalent STX.L-1), indicating the possibility of adverse health effects in sensitive groups using water from the reservoir. Estimated chronic daily intake values, for recreation, ranged from 2.55 x 10-6 to 1.08 x 10-4 µg STX equivalent. kg-1.day-1 (adults) and 2.78 x 10-9 to 4.35 x 10-4 µg STX equivalent. kg-1.day-1 (children). For water intake, these values ranged from 2.5 x 10-4 to 1.47 x 10-2 µg STX equivalent. kg-1 day-1 (adults) and 5.36 x 10-5 to 2.55 x 10-2 µg STX equivalent. kg-1. day-1 (children). These values indicated that children are more exposed than adults, a worrying fact because they are generally more sensitive to the effects of toxic substances than adults, and although studies indicate that saxitoxin was able to cross the blood brain barrier and to cause cognitive damage in rats, as well as changes in neural cell cultures, the effects of chronic exposure in humans are still unknown. It was not possible to estimate the health risks of the population exposed to saxitoxin and the like, due to the lack of data in the literature, but the proposed exposure model is an advance for the identification of gaps for conducting the human health risk assessment considering the exposure chronic.

Thesis (M. Sc.), Memorial University of Newfoundland, 1998. / Restricted until June 1999. Bibliography: leaves 84-95.