Foram aplicadas técnicas de análise e comparação de anatomia detalhada, através de dissecções e por análises moleculares em um grupo altamente variável como os Ostreidae com objetivo de comprovar o monofiletismo de Crassostreinae. Por ser economicamente importante em todo o mundo, a diferenciação entre as espécies é fundamental, com aplicações até na produção. Através de um estudo comparativo de morfologia detalhada, representantes de quase todos os gêneros de Ostreoidea foram analisados. Inicialmente, os representantes da subfamília Crassostreinae foram estudados isoladamente. Em cada uma foi realizada uma análise filogenética na procura de sua monofilia e de sua identidade básica. Os representantes de Ostreinae e Gryphaeidae foram utilizados como grupo-externo. Em seguida, alguns representantes de Pteriomorphia foram incorporados ao grupo-externo (Limidae, Spondylidae, Plicatulidae, Isognomonidae, Anomiidae) com enraizamento em Arcidae. Foram estudados 73 caracteres, presentemente comentados e discutidos. Uma única árvore mais parcimoniosa foi obtida: (((((((Cryptostrea permolis (Teskeyostrea weberi ((Hyotissa hyotis (Parahyotissa mcgintyi - P. numisma)) ((Eostrea puelchana - E. lurida) (Ostrea edulis (Ostreola equestris (\"Dendostrea\" sp1 ((Lopha cristagalli - Alectryonella plicatula) ((Dendostrea folium - D. sp 2) (\"Dendostrea\" frons (\"Saccostrea\" echinata (\"Saccostrea\" palmula (((Saccostrea mordax - \"Striostrea\" prismatica) (Saccostrea cucullata - S. glomerata)) ((Crassostrea virginica (C. gigas - C. bilineata)) (C. mangle (C. brasiliana - C. rhizophorae)))))))))))))))) Plicatulostrea onca) Plicatula gibbosa) Spondylus americanus) Lima sp.) (Anomia simplex - Isognomon alatus)) Barbatia cândida) tendo como índices: passos = 372, IC = 27 e IR = 58. Nota-se que a monofilia de Crassostreinae foi comprovada e é suportada por 6 sinapomorfias, Crassostrea também é monofilético, suportado por 4 sinapomorfias. Saccostrea, considerando a espécie tipo S. cucullata, é monofilético junto com S. glomerata. Dados obtidos por análises moleculares, realizadas neste e dados do GenBank®, corrobora, em parte, com os dados anatômicos; Crassostrea é considerado monofilético em ambas análises. As diferenças e semelhanças morfo-anatômicas encontradas em quase todas as estruturas foram suficientes para a separação e agrupamento entre os gêneros e respectivas espécies / Techniques of analysis and comparison of detailed anatomy through dissections and molecular analysis were applied in a highly variable group as Ostreidae in order to prove the monophyly of Crassostreinae. Being economically important worldwide, differentiation between species is essential in applications to production. Through a detailed comparative study of morphology, representatives of almost all genres of Ostreoidea were analyzed. Initially, the representatives of the subfamily Crassostreinae were studied in isolation. A phylogenetic analysis was performed in the search for your monophyly and their basic identity in each one. Representatives of Ostreinae Gryphaeidae and were used as-external. Then, some representatives of Pteriomorphia were incorporated into the group-external (Limidae, Spondylidae, Plicatulidae, Isognomonidae, Anomiidae) with rooting Arcidae. 73 characters presently commented and discussed were studied. A single most parsimonious tree was obtained: (((((((Cryptostrea permolis (Teskeyostrea weberi ((Hyotissa hyotis (Parahyotissa mcgintyi - P. numisma)) ((Eostrea puelchana - E. lurida) (Ostrea edulis (Ostreola equestris (\"Dendostrea\" sp.1 ((Lopha cristagalli - Alectryonella plicatula) ((Dendostrea folium - D. sp.2) (\"Dendostrea\" frons (\"Saccostrea\" echinata (\"Saccostrea\" palmula (((Saccostrea mordax - \"Striostrea\" prismatica) (Saccostrea cucullata - S. glomerata)) ((Crassostrea virginica (C. gigas - C. bilineata)) (C. mangle (C. brasiliana - C. rhizophorae)))))))))))))))) Plicatulostrea onca) Plicatula gibbosa) Spondylus americanus) Lima sp.) (Anomia simplex - Isognomon alatus)) Barbatia candida) while indices: = 372 steps, CI = 27 and IR = 58. We note that the monophyly of Crassostreinae been established and is supported by six synapomorphies, Crassostrea is also monophyletic, supported by four synapomorphies. Saccostrea, considering the type species S. cucullata, is monophyletic with S. glomerata. Data obtained by molecular analyzes, performed and this data GenBank® corroborates, in part, with the anatomical data; Crassostrea is considered to be monophyletic in both analyzes. Differences and morphological, anatomical similarities found in almost all structures were sufficient for separating and grouping among genera and their species

Vibrio parahaemolyticus (Vp) é uma bactéria naturalmente presente em regiões estuarinas, sendo a principal causa de gastrenterite de origem bacteriana associada a pescados, principalmente ostras cruas. Nesta pesquisa, foi desenvolvida uma avaliação quantitativa de risco para avaliar a probabilidade de Vp causar doença após o consumo de ostra crua, produzida e comercializada no Estado de São Paulo. O estudo incluiu a identificação e caracterização do perigo, a avaliação da exposição e a caracterização do risco. Um modelo matemático foi desenvolvido. Este modelo leva em consideração o comportamento de Vp em ostras na cadeia produtiva, em cada estação do ano, além da relação entre a dose de Vp ingerida e a probabilidade de desenvolver a doença. A avaliação da exposição foi desenvolvida em três etapas: cultivo, pós-coleta e consumo. Na etapa de cultivo foram considerados os fatores que influenciam a prevalência e o número de Vp em ostras no momento da coleta. Na etapa pós-coleta, foram descritas as práticas da indústria e foram considerados os fatores associados ao processamento, transporte e manipulação. Já na etapa de consumo foram considerados os fatores como a quantidade de ostras consumidas por porção, o peso médio por ostra consumida e a população de Vp patogênico no momento do consumo. O resultado do modelo quantitativo da avaliação da exposição foi, então, integrado ao modelo dose-resposta, Beta-Poisson, para se obter uma estimativa do risco. Esta estimativa expressa o impacto da exposição humana a Vp, sobre a saúde pública, associada ao consumo de ostras. A simulação de Monte Carlo foi utilizada para avaliar o efeito da variabilidade e incerteza das variáveis do modelo sobre a estimativa do risco. O modelo prediz uma probabilidade de ocorrência de doença de 4,6x10-4, por porção de ostra, consumida ao longo do ano. As variáveis que possuem maior influência sobre o risco de ocorrência de doença são a população de Vp em ostras no cultivo, a temperatura de transporte das ostras até o varejo e a porcentagem de Vp patogênico em ostra, no momento do seu consumo. O modelo evidencia que uma das maneiras de reduzir o risco de ocorrência de doença seria intervir nas condições de transporte de ostras até o varejo por meio da sua refrigeração. Com o modelo é possível identificar fatores e simular cenários para avaliar o comportamento de V. parahaemolytícus como um perigo microbiológico, ao longo da cadeia produtiva de ostra até o momento do seu consumo. Também é possível avaliar o impacto de medidas de intervenção na cadeia produtiva. As suposições adotadas limitam a aplicabilidade do modelo. Portanto, é necessário que o modelo seja validado, particularmente com relação ao número de casos de doença causados por Vp, cujos dados de vigilância epidemiológica inexistem no Brasil. / Vibrio parahaemolyticus (Vp) is naturally present in estuarine regions and is the main cause of gastroenteritis associated with the consumption of bivalve molluscan shellfish, specially raw oysters. In this research, a quantitative risk assessment was developed to evaluate the probability of Vp causing disease after consumption of raw oyster, produced and commercialized in the state of Sao Paulo. The study included the identification and characterization of the hazard, exposure assessment and risk characterization. A mathematical model was developed. This model takes into account the behavior of Vp in oysters in the productive chain, for each season of the year, besides the relationship between the number of cells of Vp ingested and the probability of developing the disease. The exposure assessment was done in three steps: farming, after harvesting and consumption. At the farming step, the factors that influence the prevalence and the population of Vp at the time of harvesting were considered. At the after harvesting step, the factors associated with transportation, handling and processing were considered. At the consumption step, factors related to the amount of oysters and the average weight per oyster consumed and the density of pathogenic Vp at the time of consumption were considered. Then, the quantitative model of exposure assessment was integrated to the dose-response model, BetaPoisson, in order to obtain a risk estimate. This calculation expresses the impact of the human exposure to Vp associated with the consumption of oysters on public health. The Monte Carlo simulation was used to evaluate the effect of variability and uncertainty of variables of the model in the risk estimation. The model predicts a probability of occurrence of the disease of 4,6x10-4 per serving of oyster consumed during one year. The variables showing the greatest influence on the risk of occurrence of disease are the density of Vp in oyster in the farming step, the temperature during transportation of oysters to the retail market and the percentage of pathogenic Vp strains in oysters,\' at the moment of consumption. The model indicates that the use of refrigeration during transportation of oysters to retail could reduce the risk of disease. The model allows the identification of factors and the simulation of scenarios in order to evaluate the behavior of V. parahaemolyticus, as a microbiologícal hazard, in the productive chain of oyster to the consumption. It is also possible to evaluate the impact of intervention measures in the productive chain. The assumptions Iimit the application of the model. Therefore, it is necessary to validate the model, particularly in relation to the number of cases of dísease caused by V. parahaemolyticus of which the data on epidemiologic surveillance do not exist in Brazil.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Crassostrea (Sacco, 1897) é o gênero mais importante do mundo de ostras de cultivo e consiste de 34 espécies distribuídas pelas regiões tropicais e temperadas do globo. C. gasar e C. rhizophorae são as duas espécies nativas que estão distribuídas ao longo de toda a costa do Brasil até o Caribe. C. gasar também ocorre na costa da Africa. Ainda que sua distribuição seja extensa e com disponibilidade abundante, o cultivo de ostras nativas no Brasil ainda é incipiente e a delimitação correta dos estoques mantém-se incerta. O sucesso do desenvolvimento da malacocultura, que é recomendada internacionalmente como forma sustentável de aquicultura, depende da resolução desses problemas. Assim, com o objetivo de determinar geneticamente seus estoques no Atlântico como também estimar sua história demográfica, dois diferentes marcadores moleculares foram empregados: sequências de DNA da região controle mitocondrial e loci de microssatélites espécie-especifícos, desenvolvidos no presente estudo. Foram sequenciados fragmentos da região controle de um total de 930 indivíduos de C. gasar e C. rhizophorae coletados em 32 localidades que incluíram o Caribe, a Guiana Francesa, a costa brasileira e a África. Também foram realizadas genotipagens de 1178 indivíduos, e ambas as espécies, com 9 e 11 loci de microssatélites para C. gasar e C. rhizophorae, respectivamente. Os dados genéticos foram analisados através de diferentes abordagens (índices de estruturação (FST) e de (Jost D), análise molecular de variância (AMOVA), análise espacial molecular de variância (SAMOVA), Bayesian Skyline Plots (BSP), análise fatorial de correspondência (AFC) e análise de atribuição Bayesiana (STRUCTURE)). Os resultados indicaram um padrão geral de estruturação, onde dois diferentes estoques foram detectados para ambas as espécies: grupos do norte e do sul, onde o Rio de Janeiro seria a região limitante entre os dois estoques. Os maiores valores dos índices de estruturação foram encontrados para C. gasar, indicando que esta espécie estaria mais estruturada do que C. rhizophorae. As análises demográficas indicaram uma provável expansão das populações durante o ultimo período glacial e uma possível origem americana das populações africanas. Todos os resultados sugeriram a existência de uma barreira geográfica próxima ao Rio de Janeiro, que poderia ser a cadeia de Vitória-Trindade e o fenômeno de ressurgência que ocorre em Cabo Frio (RJ). Esses resultados serão de grande utilidade para estabelecer critérios para seleção de sementes para cultivo ao longo da costa do Brasil que permitirá o manejo adequado dos estoques ostreícolas, prevenindo seu desaparecimento como já ocorrido em outros recifes no mundo. / Crassostrea (Sacco, 1897) is the most important genus of cultivated oysters in the world and consisting of 34 species distributed by tropical and temperate regions of the globe. C. gasar and C. rhizophorae are the two native species which have wide distribution along the entire Brazilian coast up to the Caribbean. C. gasar also occurs on coast of Africa. Despite its extensive distribution and abundant availability, cultivation of those oysters in Brazil is incipient, and the correct delimitation of the existing stocks is still uncertain. The successful development of malacoculture which is recommended internationally as an environmentally sustainable form of aquaculture depends on the resolution of these issues. Thus, in order to genetically determinate their stocks in the Atlantic and to estimate their demographic history, two different molecular markers were employed: sequences of the mitochondrial DNA control region and species-specific microsatellite loci, developed in the present study. We have sequenced a fragment of the mitochondrial control region from a total of 930 individuals of C. gasar and C. rhizophorae collected in 32 localities including the Caribbean, French Guyana, Brazilian coast and Africa. We have also genotyped 1178 individuals of both species with 9 and 11 loci of microsatellites for C. gasar and C. rhizophorae, respectively. Genetic data were analyzed with different approaches (fixation (FST) and differentiation (Jost D) indices, analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), Bayesian Skyline Plots (BSP), factorial correspondence analysis (AFC) and Bayesian attribution analysis (STRUCTURE)). The results indicated a general structure pattern, where two different stocks were detected for both species: north and south groups, where Rio de Janeiro would be the limited region between them. Higher values of fixation indices were found for C. gasar, indicating that this species would be more structured than C. rhizophorae. Demographic analyses showed a probable expansion of populations during the last glacial period and a probable American origin of African populations. All results suggested the existence of a barrier next to Rio de Janeiro, which could be Vitoria-Trindade chain and the upwelling in the region of Cabo Frio (RJ). These results will be useful to establish criteria for the selection of seeds for cultivation along the Brazilian coast which will allow proper management of stocks of oysters preventing its disappearance as in other reefs around the world.

As bactérias patogênicas em moluscos bivalves podem ser agentes causadores de doenças como a gastroenterite e responsáveis por vários surtos de origem alimentar, representado um risco para os consumidores. Os vírus entéricos são a causa mais comum de surtos de gastroenterites não bacteriana em humanos no mundo e podem ser encontrados nas águas utilizadas no cultivo de moluscos bivalves. Este estudo teve como objetivo avaliar a contaminação de mexilhões (Mytella falcata) e ostras (Crassostrea brasiliana) provenientes do Complexo Estuarino Lagunar de Cananéia-Iguape, Estado de São Paulo, por bactérias (coliformes totais, coliformes termotolerantes, patotipos de Escherichia coli), por astrovírus e norovírus humanos. Um total de 150 amostras de moluscos bivalves (75 ostras e 75 mexilhões) foram coletadas de junho de 2016 a fevereiro de 2017. A estimativa de coliformes totais nos tecidos das ostras variou de 14,1 a 154,5 número mais provável (NMP)/g e de coliformes termotolerantes de 3,0 a 48,6 NMP/g, enquanto que para as amostras de mexilhões, os coliformes totais variaram de 97,4 a 1300 NMP/g e coliformes termotolerantes de 3,6 a 927 NMP/g. E. coli foi detectada em 24 amostras (16%), em concentrações variando entre <3 e >927 NMP/g. Quatro amostras (17%) foram identificadas com Escherichia coli enteropatogênica (EPEC), apresentando o gene eae por PCR (Reação em Cadeia da Polimerase) e RFLP (Polimorfismo no Comprimento de Fragmentos de Restrição), e os amplicons positivos foram sequenciados. As porcentagens de similaridade relativas ao gene phoA de E. coli, para as cinco amostragens realizadas no estudo, apresentaram valores iguais ou superiores a 88,6%. As sequências de EPEC agruparam-se em diferentes clados com outras sequências do Brasil, Suíça e Uruguai, exibindo similaridade de 57,7 e 97,1% quando comparadas umas as outras. Quando comparadas a outras sequências de referência depositadas no GenBank, a similaridade variou entre 56,2 e 95,4%. Estes resultados são os primeiros a indicar a presença de EPEC em moluscos bivalves no Brasil. Astrovírus não foram identificados nas amostras de moluscos analisadas neste estudo. Norovírus (NoV) foi identificado em 21 (14%) das amostras, sendo 38% de mexilhões e 62% de ostras. As amostras de NoV genogrupo II (GII) foram agrupadas num clado único, juntamente com outras sequências de NoV GII, sendo mais próximas filogeneticamente de sequências originárias do Brasil, Japão e México, com similaridade de 93,8 a 96,6% do que com as outras sequências homólogas. A triagem de moluscos bivalves para coliformes, E. coli e presença de vírus entéricos significativos para a saúde pode ajudar na prevenção de surtos entre os consumidores e contribuir para a melhoria do ambiente estuarino. / The pathogenic bacteria in bivalve molluscs are causative agents of diseases such as gastroenteritis and responsible for several food-borne outbreaks, representing a risk to consumers. Enteric viruses are the most common cause of outbreaks of non-bacterial gastroenteritis in humans in the world and can be found in waters used in the cultivation of bivalve molluscs. The objective of this study was to evaluate the contamination of mussels (Mytella falcate) and oysters (Crassostrea brasiliana) from the estuarine complex Lagunar of Cananéia-Iguape, State of São Paulo, by bacteria (total coliforms, thermotolerant coliforms, Escherichia coli) and by human astroviruses and noroviruses. A total of 150 samples of bivalve molluscs (75 oysters and 75 mussels) were collected from June 2016 to February 2017. The total coliform estimate in oyster tissues varied from 14.1 to 154.5 most probable number (MPN)/g and thermotolerant coliforms from 3.0 to 48.6 MPN/g, whereas for mussel samples, total coliforms ranged from 97.4 to 1300 MPN/g and thermotolerant coliforms from 3.6 to 927 MPN/g. E. coli was detected in 24 samples (16%) at concentrations ranging from <3 to >927 NMP/g. Four (17%) were identified with enteropathogenic Escherichia coli (EPEC), presenting the gene eae by PCR (Polymerase Chain Reaction) and RFLP (Restriction fragment length polymorphism), and the positive amplicons were sequenced. The percentages of similarity relative to the phoA gene of E. coli, for the five samplings carried out in the study, presented values equal or superior to 88.6%. The EPEC sequences were grouped in different clades with other sequences from Brazil, Switzerland and Uruguay, exhibiting similarity of 57.7 and 97.1% when compared to each other. When compared to other reference sequences deposited in GenBank, the similarity ranged from 56.2 to 95.4%. These results are the first to indicate the presence of EPEC in bivalve molluscs in Brazil. Astroviruses were not identified in the mollusk samples analyzed in this study. Norovirus (NoV) was identified in 21 (14%) of the samples, representing 38% of mussels and 62% of oysters. NoV genogroup II (GII) samples were clustered in a single clade, along with other NoV GII sequences, keeping phylogenetically closest to sequences originating in Brazil, Japan and Mexico, with similarity of 93.8 to 96.6% than with the other homologous sequences. The screening of bivalve molluscs for coliforms, E. coli and the presence of enteric viruses significant to health can help preventing outbreaks among consumers and contribute to the improvement of the estuarine environment.

Oil spills in the marine environment can threaten vulnerable ecosystems that support ecologically and economically significant organisms, such as the eastern oyster (Crassostrea virginica), in coastal habitats. The use of chemical dispersant (Corexit 9500) was applied as a cleanup effort in response to the Deepwater Horizon blowout to minimize crude oil slicks, but also resulted in increased concentrations of polycyclic aromatic hydrocarbons in the water column. The effects of increased soluble fractions of crude oil and dispersant components may be harmful to marine organisms. This study aimed to investigate possible sublethal impacts to the eastern oyster at multiple life history stages in order to understand potential implications on performance at an organismal, population, and ecosystem levels. Specifically, this study addressed 1) veliger swimming, 2) pediveliger settlement rates, 3) pollutant induced larval inactivity and 4) adult clearance rates after acute exposures to relevant concentrations (10 – 100 µL L-1) of water accommodated fractions of crude oil (WAF) and with a combination of chemical dispersant (CEWAF). No significant differences were observed in any tested swimming kinematics between controls and WAF or CEWAF treatments after 24 hour exposures for early staged veligers at concentrations up to 100 µL L-1 WAF and CEWAF. However, settlements rates of competent pediveligers were significant decreased compared to control (52.1 % s.d. 1.66) rates at concentrations of 50 µL L-1 WAF (30.9% s.d. 6.16) and 10 (41.2 % s.d. 0.857) and 50 (22.0% s.d. 1.23) µL L-1 CEWAF. Later staged larvae also showed increased vulnerability to oil pollution given that a higher percentage of organisms were inactive (48.3% s.d. 4.80) compared to early staged larvae (12.7% s.d. 7.68 ) after initial exposure at 50 µL L-1 CEWAF. Based on this result, we assumed effects of oil pollution were not manifested until the later larval life history stage evident by metamorphosis failure during the complex settlement transformation that results in reduced spat and eventually reduced adult oysters. Adult oysters were also exposed to increasing concentrations of WAF and CEWAF for 24 hours and feeding experiments were conducted in both clean seawater and the same oiled seawater conditions as their initial exposure. Oysters fed in oiled seawater had decreased clearance rates, but oysters fed in clean water had increased clearance rates, suggesting feeding efficiency can be returned to control rates when moved to the presence of clean water. However, our long term study conducted in clean seawater suggested of the oysters exposed to crude oil only (9.31 L h-1 g-1 s.d. 2.04) are able to return to clearance rates comparable to controls (7.69 L h-1 g-1 s.d. 1.89) after the 33 day time period but oysters exposed to crude oil with a combination of chemical dispersant (2.12 L h-1 g-1 s.d. 1.08) were not. Decreased feeding efficiency can have negative impacts on water quality in estuarine ecosystems that support productive habitats. Understanding the impacts of crude oil, and crude oil with a combination of chemical dispersant on ecologically significant organisms can aid in future oil spill response decisions in order to minimize environmental impacts.

Estuarine acidification, caused by disturbance of acid sulfate soils (ASS), is a recurrent problem in eastern Australia. Affected waters are characterised by low pH and elevated concentrations of metals, principally aluminium and iron. The effects of acid and elevated metal concentrations associated with ASS, on adult Sydney rock oysters, have not been previously investigated. This study tested links between ASS-affected drainage, subsequent estuarine acidification and Sydney rock oyster production problems on the Hastings and Manning Rivers, mid north coast New South Wales. The primary objective of this thesis was to establish if estuarine acidification causes mortality and slow growth in individual Sydney rock oysters by exposing oysters to low pH, iron and aluminium using field and laboratory experiments. Water quality data showed that estuarine acidification was spatially extensive in the Hastings and Manning Rivers following heavy rainfall and was due to mineral acids originating from drained or excavated ASS. Estuarine acidification regularly affected areas used for Sydney rock oyster production following heavy rainfall. Field experiments showed that Sydney rock oyster mortality rates were significantly higher at sites exposed to ASS-affected waters compared to locations that were isolated from ASS-affected waters. Oyster mortality increased with the time of exposure and smaller oysters (mean weight = 5 g) experienced significantly higher mortality relative to larger oysters (mean weight = 29 g). This was caused by acid-induced shell degradation resulting in perforation of the smaller oysters??? under-developed shells. Additionally, Sydney rock oyster growth rates were dramatically reduced at sites exposed to ASS-affected waters and the overall mean condition index of oysters at ASS-affected field sites was significantly lower than the overall mean condition index of oysters at non-impacted sites. Findings from laboratory experiments showed that ASS-affected water alters oyster valve movements and significantly reduces oyster feeding rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg L-1 of aluminium or ASS-affected water caused changes in the mantle and gill soft tissues following short-term exposure. Degenerative effects described in oysters in this study were also due to iron contained in ASS-affected waters. Iron precipitates accumulated on the shell, gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. This study concluded that Sydney rock oysters are unable to tolerate acidic conditions caused by ASS outflows and cannot be viably cultivated in acid-prone areas of the estuary.

An evanescent wave fiber-optic biosensor was used in an effort to develop an assay for the rapid detection of two biological toxins: cholera toxin and botulinum toxin. The Analyte 2000 fiber-optic biosensor utilizes a sandwich immunoassay format. Gangliosides or liposomes are directly adsorbed to the surface of the fiber-optic waveguide through hydrophobic interactions. The waveguide is exposed to a sample containing the toxin of interest, then subsequently exposed to a polyclonal detection antibody conjugated to the fluorophore cyanine 5. Excitation light from a 635nm laser diode is propagated through the waveguide and fluorescent molecules within approximately 100nm of the waveguide are excited. The emission light from the excited cyanine 5 molecules reverberates into the waveguide and is quantitated in pico Amperes and displayed on a computer. The exotoxins of Vibrio cholerae and Clostridium botulinum, cholera and botulinum toxin, respectively, were used for pote ntial assay development. Assay development utilizing the biosensor was attempted for the detection of botulinum toxin in buffer. The limit of detection remained too high to generate a positive signal for the detection of botulinum toxin. Biosensor assays were developed to detect cholera toxin in buffer, oyster homogenate, pure culture and induction media. A cholera toxin standard curve was generated with a limit of detection of 1 ng/ml. The values were normalized by setting 100 ng/ml of cholera toxin to a value of 100. Signals were detected in oyster homogenate spiked at 5 ug/ml as well as unspiked oyster homogenate. A Western blot showed that there were cross reactive proteins in the oyster matrix at molecular weights different from those of the cholera toxin. Cholera toxin production by three strains of Vibrio cholerae with values estimated to range from 100 pg -- 100 ng was detected with the biosensor. Additionally, oysters were harvested from Tampa Bay and placed in a 10 gallon tank filled with different types of induction media. The tank was inoculated with Vibrio cholerae and the oysters and induction medium were analyzed at varying times for the presence of cholera toxin. Vibrio cholerae cells were viable through 24 hours but no toxin was detectable.

Estuarine acidification, caused by disturbance of acid sulfate soils (ASS), is a recurrent problem in eastern Australia. Affected waters are characterised by low pH and elevated concentrations of metals, principally aluminium and iron. The effects of acid and elevated metal concentrations associated with ASS, on adult Sydney rock oysters, have not been previously investigated. This study tested links between ASS-affected drainage, subsequent estuarine acidification and Sydney rock oyster production problems on the Hastings and Manning Rivers, mid north coast New South Wales. The primary objective of this thesis was to establish if estuarine acidification causes mortality and slow growth in individual Sydney rock oysters by exposing oysters to low pH, iron and aluminium using field and laboratory experiments. Water quality data showed that estuarine acidification was spatially extensive in the Hastings and Manning Rivers following heavy rainfall and was due to mineral acids originating from drained or excavated ASS. Estuarine acidification regularly affected areas used for Sydney rock oyster production following heavy rainfall. Field experiments showed that Sydney rock oyster mortality rates were significantly higher at sites exposed to ASS-affected waters compared to locations that were isolated from ASS-affected waters. Oyster mortality increased with the time of exposure and smaller oysters (mean weight = 5 g) experienced significantly higher mortality relative to larger oysters (mean weight = 29 g). This was caused by acid-induced shell degradation resulting in perforation of the smaller oysters??? under-developed shells. Additionally, Sydney rock oyster growth rates were dramatically reduced at sites exposed to ASS-affected waters and the overall mean condition index of oysters at ASS-affected field sites was significantly lower than the overall mean condition index of oysters at non-impacted sites. Findings from laboratory experiments showed that ASS-affected water alters oyster valve movements and significantly reduces oyster feeding rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg L-1 of aluminium or ASS-affected water caused changes in the mantle and gill soft tissues following short-term exposure. Degenerative effects described in oysters in this study were also due to iron contained in ASS-affected waters. Iron precipitates accumulated on the shell, gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. This study concluded that Sydney rock oysters are unable to tolerate acidic conditions caused by ASS outflows and cannot be viably cultivated in acid-prone areas of the estuary.

Thesis (M.S.)--University of North Carolina at Wilmington, 2006. / Includes bibliographical references (leaves: 53-59)

Thesis (M.S.)--University of North Carolina at Wilmington, 2006.