Determination and characterization of genes involved in toxic mechanisms of the prymnesiophyte Prymnesium parvum

Abstract

This thesis represents a study of the ecophysiology and toxicity of the prymnesiophyte Prymnesium parvum. The first aim was to investigate changes in the relative toxicity of P. parvum following a series of physiological shock treatments, meant to simulate environmental conditions under which harmful blooms of this species have been observed. As blooms of this haptophyte often occur in dynamic coastal brackish water systems, Prymnesium parvum is noted for its physiological flexibility, which may contribute to providing a competitive advantage over other coexisting species. Due to the unconfirmed nature of the compounds involved in toxigenic processes, two bioassays were employed to characterize changes in lytic capacity (extracellular vs. intracellular). These bioassays are considered physiologically relevant, as observed icthyotoxicity occurs through lysis of the gill cell membranes, rendering the fish unable to perform gas-exchange processes and obtain oxygen. Additionally, the gene expression of three polyketide synthase genes (PKS) were analyzed via quantitative PCR (qPCR), based on current chemical characterizations of toxic compounds produced by P. parvum. Low salinity and high irradiance were observed to alter the lytic effects of P. parvum on the sensitive cryptophyte Rhodomonas salina and erythrocytes. Furthermore, these two shock treatments were found to increase the transcript copy number in selected PKS genes, suggesting a possible correlation between toxicity and the PKS biosynthetic pathway. Allelochemical mediation has been suggested to affect competition and predatory relationships associated with formation of P. parvum blooms. As interactions between species are an integral part of understanding plankton ecology, interspecific interactions between P. parvum and three coexisting species were accordingly investigated. Combining bioassays with a functional genomic approach allowed differential characterization of cell-cell contact vs. waterborne cues depending on the organism with which incubated. A unique response on both the levels of toxicity, gene expression profile as well as PKS transcript copy number to the potential predator Oxhyrris marina suggest a fundamentally different type of interaction between the two species. Additionally, a dose-response time series experiment showed that changes in gene expression and toxicity did not occur immediately in P. parvum, rather after 60-90 minutes. Such a response by P. parvum may in fact signify a co-evolutionarily adaptive defense. Finally, examination of the effects of phosphorous limitation and low salinity stress on the gene expression profile and lytic capacity showed that the combination of these two stressors induces secretion or extracellular transport of toxic substances to a much higher degree than either stressor individually. Whether this observation is due to changes in membrane integrity due to homeostatic processes needs further research. The pattern of gene expression, however, revealed regulation of among others genes associated with active cellular transport processes, suggesting that maintenance of intracellular-extracellular homeostasis may play a role in the observed toxicity. In summary, these studies integrate the concepts of ecophysiology and functional genomics, providing a useful platform for further research regarding environmental factors associated with the toxicity of P. parvum. As functional genomic methods become more accessible, such approaches illustrate their potential application within the field of harmful algal research.