Browsing by Author "Manning, Schonna Rachelle"
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Item Molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum Carter (Haptophyta)(2010-08) Manning, Schonna Rachelle; La Claire, John W., 1951-; Brand, Jerry J.; Herrin, David L.; Jansen, Robert K.; Kitto, George B.This dissertation includes molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum, including analysis of known polyketide metabolites as a function of salinity and growth. Initially, the development of molecular and phytochemical tools was necessary for the detection and quantification of P. parvum and its associated toxins. Suites of oligonucleotides and molecular beacons were designed for conventional and quantitative multiplex PCR to amplify four species- and gene-specific products simultaneously that were used for the detection and quantitation of P. parvum. This built-in redundancy provided increased confidence in reactions with the positive confirmation of four discrete products. Techniques were also developed for the chemical enrichment of toxins produced by P. parvum. Until now, isolation of “prymnesins” has never been reproduced. Polyketide prymnesins possess unique spectral properties that were used to generate an LC-MS fingerprint that comprised 13 ion species. Preliminary investigations using chemifluorimetric methods were also capable of detecting prymnesins in the pico- and nano-molar range. Environmental samples were tested as an independent assessment of these methods. Lastly, the roles of polyketide prymnesins were analyzed with respect to total hemolytic activity (HA) as a function of culture age and salinity. Variation in HA of supernatants was statistically significant relative to both variables (p << 0.05). Salinity was inversely related to HA wherein cultures growing in 5-25 psu were 150-200% more hemolytic. Total HA was inversely related to culture age during the first three weeks, but positively related to it during the next three weeks. Interestingly, no hemolysis was detected in fractions containing prymnesins from culture supernatants and the majority of hemolysins remained in the aqueous phase. Prymnesins extracted from cells varied significantly over the 6-week observation period (p << 0.05); HA was positively correlated during the first half and inversely related during the last half of the study. Salinity was directly related to HA from cell extracts, but these effects were not significantly different until the last three weeks. These investigations suggest that polyketide prymnesins are present at much lower quantities than previously believed, and they may not be the key compounds associated with hemolysis due to P. parvum.Item Multiplex polymerase chain reaction (PCR) method for the rapid and sensitive species-specific detection of the harmful alga, Prymnesium parvum Carter (Haptophyta)(2006-08) Manning, Schonna Rachelle; La Claire, John W., 1951-Harmful algal blooms have caused much concern during the past century due to an increasing frequency and severity of events. The toxin-forming alga, Prymnesium parvum Carter (Haptophyta), has been a nuisance since the early 1900's, causing massive fish fatalities around the globe. Due to the widespread impact of P. parvum, it is evident that sensitive methods for early detection and quantification are needed to aid the conservation of species and ecosystems affected by this ichthyotoxic alga. In this context, a multiplex polymerase chain reaction (PCR) method was developed for the rapid, species-specific detection of P. parvum. Poly-adenylated [poly-A(+)] mRNA was selected from isolated total RNA and used for cDNA library construction. Selected cDNA sequences were examined for oligonucleotide design, and primer sets were tested using PCR to establish amplicon product quality and specificity. For multiplex reactions, four primer sets with the required parameters were collectively tested in single reaction tubes. All reactions simultaneously produced four species- and gene-specific amplicons, as determined by gel electrophoresis and nucleotide sequencing. Multiplex PCR reactions were performed with isolated DNA and whole cells from P. parvum isolates from various geographic regions, including Texas, South Carolina, Maine and the United Kingdom. Isolated DNAs from related species, including other haptophytes, and distant outgroups were utilized as negative controls. The multiplex reactions resolved the same unique banding pattern for all P. parvum isolates examined and this pattern was not found for any other organisms studied so far. In electrophoresis gels, products were detectable from as few as [varies with, or, similar to]1600 cells. Incorporating a second set of re-amplification (50 cycles total) increased the level of detection at least 100-fold. Using this assay, researchers should be able to determine the presence of this toxin-forming alga in environmental samples within a time frame suitable for determining appropriate responses.