Development of a whole-cell immunolabeling protocol for evaluation of toxin production in Microcystis sp.

摘要

The ubiquity and predominance of toxic microcystin-producing bloom-forming Microcystis sp. in water supplies and recreational water bodies jeopardizes ecosystem integrity and the public welfare of exposed populations. Microcystin is a hepatotoxin and suspected carcinogen that has been implicated in human and animal poisonings worldwide. Although various analytical methods for microcystin detection have been developed, no efficient, accurate means of assessing intracellular toxins in situ has been available to help characterize toxin production in cells, to discriminate morphologically identical toxic and nontoxic colonies of Microcystis sp. and to aid in rapid diagnosis of toxic blooms. The objectives of this thesis included development and validation of a protocol for fluorescence-based whole-cell immunolocalization of microcystin in toxin-producing Microcystis sp., optimization of this method for detection of naturally-derived toxic cyanobacterial colonies and blooms and application of this method in studies of toxin regulation at the organismal level of microcystin-producing cyanobacteria. The immunolabeling method was developed and validated using flow cytometric assessment of fluorochrome intensity and ELISA-based Qmcyst as independent measures of culture toxin content. A semi-quantitative relationship was established between microcystin-specific, antibody-conferred immunofluorescence intensity and intracellular toxin levels. The immunolabeling protocol was adapted for use in low and high phytoplankton density water samples containing Microcystis sp. colonies. The protocol adapted for field use enabled microscopic visualization of toxic colonies within hours of sample collection. The immunolabeling method was also incorporated into studies designed to help characterize regulation of toxin production in Microcystis cells. Studies of the effects of light stress on toxin production and investigations linking quorum sensing and toxin production provided evidence of enhanced intracellular toxin content during atypical laboratory growth conditions (high light stress and darkness) and evidence of intercellular signaling via the detection of N-acylhomoserine lactone production by toxic Microcystis aeruginosa. Overall, these studies offer a new technique to both researchers interested in differential microcystin synthesis within and among cells and water management officials interested in rapid and simple analysis of toxicity in Microcystis sp. blooms.