Files
Abstract
The toxic dinoflagellate Karlodinium veneficum forms blooms in estuaries worldwide. These blooms are often associated with severe fish mortalities that are largely caused by production of karlotoxins. The toxicity of these blooms can vary between bloom events and within blooms over time. Laboratory experiments also indicate production of these toxins is inversely related to growth rate, and is light dependent. Additionally, cell cycle studies have found G1 phase of the cell cycle to occur during daylight hours and growth limited cells to arrest in G1 phase. These data suggest a model of karlotoxin biosynthesis that closely corresponds to the cell cycle, wherein rapidly dividing cells have relatively low cellular karlotoxin content while slowly dividing cells arrest in G1 phase proceed through multiple light/dark cycles and light dependent karlotoxin synthesis cycles leading to increased cellular toxicity. Application of this model to field populations of K. veneficum may explain the variable toxicity of natural blooms. However, this requires optimization of methods for measuring in situ growth rates. The goals of this dissertation were to 1) optimize methodologies for measuring in situ growth rates of K. veneficum, 2) to evaluate the synchrony of karlotoxin synthesis and the cell cycle, and 3) evaluate the influence of mixotrophic nutrition on cell cycle synchrony. To achieve these goals we optimized image cytometry, a quantitative fluorescence microscopy technique, for cell cycle analysis of K. veneficum and demonstrated its feasibility as a technique for estimating in situ growth rates following the cell cycle method. We also determined the duration of cytokinesis, an important component of determining in situ growth rates using the mitotic index technique that in the case of K. veneficum must be determined in the laboratory. Lastly, we evaluated the synchrony between karlotoxin synthesis and the cell cycle and the effects of mixotrophic nutrition on cell cycle synchrony in laboratory cultures. This work established a set of tools for measuring in situ growth rates in K. veneficum blooms, demonstrated synchrony between karlotoxin synthesis and G1 phase of the cell cycle, and determined that the K. veneficum cell cycle remains synchronous under mixotrophic nutrition. The work described in this dissertation sets the ground work for future field studies that will seek to explain the source of karlotoxin variability observed in natural blooms.