The application of qPCR assays for the early detection of toxic Alexandrium in eastern australian waters

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Harmful algal blooms that produce Paralytic Shellfish Toxins (PSTs) are prevalent and affect shellfish harvesting areas worldwide. PSTs have caused shellfish harvesting closures and product recalls, resulting in economic losses, as well as brand damage and damage to the wider economy including the tourism industry. In Tasmania, it is known that four PST producing species co-occur, comprising ๐˜ˆ๐˜ญ๐˜ฆ๐˜น๐˜ข๐˜ฏ๐˜ฅ๐˜ณ๐˜ช๐˜ถ๐˜ฎ ๐˜ค๐˜ข๐˜ต๐˜ฆ๐˜ฏ๐˜ฆ๐˜ญ๐˜ญ๐˜ข, ๐˜ˆ. ๐˜ฑ๐˜ข๐˜ค๐˜ช๐˜ง๐˜ช๐˜ค๐˜ถ๐˜ฎ, ๐˜ˆ. ๐˜ข๐˜ถ๐˜ด๐˜ต๐˜ณ๐˜ข๐˜ญ๐˜ช๐˜ฆ๐˜ฏ๐˜ด๐˜ฆ and ๐˜Ž๐˜บ๐˜ฎ๐˜ฏ๐˜ฐ๐˜ฅ๐˜ช๐˜ฏ๐˜ช๐˜ถ๐˜ฎ ๐˜ค๐˜ข๐˜ต๐˜ฆ๐˜ฏ๐˜ข๐˜ต๐˜ถ๐˜ฎ. In particular, of these, three species are morphologically almost identical, the species of the former ๐˜ˆ๐˜ญ๐˜ฆ๐˜น๐˜ข๐˜ฏ๐˜ฅ๐˜ณ๐˜ช๐˜ถ๐˜ฎ ๐˜ต๐˜ข๐˜ฎ๐˜ข๐˜ณ๐˜ฆ๐˜ฏ๐˜ด๐˜ฆ species complex (๐˜ˆ. ๐˜ค๐˜ข๐˜ต๐˜ฆ๐˜ฏ๐˜ฆ๐˜ญ๐˜ญ๐˜ข, ๐˜ˆ. ๐˜ฑ๐˜ข๐˜ค๐˜ช๐˜ง๐˜ช๐˜ค๐˜ถ๐˜ฎ, and ๐˜ˆ. ๐˜ข๐˜ถ๐˜ด๐˜ต๐˜ณ๐˜ข๐˜ญ๐˜ช๐˜ฆ๐˜ฏ๐˜ด๐˜ฆ), which cannot be differentiated using light microscopy. Therefore, phytoplankton monitoring using light microscopy and total PST in shellfish using High Performance Liquid Chromatography (HPLC) may not be sufficient to allow for an early warning with enough time to take appropriate shellfish harvesting management decisions. In this thesis, quantitative Polymerase Chain Reaction (qPCR) assays are investigated as an in-field early warning system, as well as a tool for long-term risk assessment of PST-associated harmful algal blooms. A commercial on-farm pipeline based on the collection and filtration of water samples using a custom designed gravity filter, a cell lysis, and a qPCR assay based on ๐˜ด๐˜น๐˜ต๐˜ˆ4 was also developed and validated. QPCR assays based on ribosomal DNA (rDNA) โ€˜barcodingโ€™ regions and an assay based on a gene associated with PST biosynthesis (๐˜ด๐˜น๐˜ต๐˜ˆ4) were found to be generally specific, sensitive and efficient. The efficacy of an rDNA-based assay for cyst quantification was demonstrated, showing potential for its use as a long-term risk assessment tool for a new harvest area. However, qPCR assays based on rDNA gene regions were found to overestimate cell abundances. An analysis of rDNA copy number variation among strains of species of ๐˜ˆ๐˜ญ๐˜ฆ๐˜น๐˜ข๐˜ฏ๐˜ฅ๐˜ณ๐˜ช๐˜ถ๐˜ฎ showed a variation of up to 3-5 orders of magnitude within a species, and was correlated significantly with genome size, which also varied within a species. An analysis of the variation in genomic copies of ๐˜ด๐˜น๐˜ต๐˜ˆ4 genes showed variation as well, however this was of a lesser degree, of up to one order of magnitude. A positive correlation was found between ๐˜ด๐˜น๐˜ต๐˜ˆ4 copies per cell and the total PST produced per cell, showing that the dosage effect may contribute to the regulation of PST biosynthesis.
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