Karenia brevis is a planktonic dinoflagellate that is responsible for the occurrence of harmful algae blooms (or “red tides”) along the coast of the southeastern United States, primarily in the Gulf of Mexico. During these blooms toxins produced by K. brevis, called brevetoxins, lead not only to massive fish mortalities, but also the mortality of marine mammals, sea birds and other marine life, the contamination of shellfish stocks, and adverse effects to human health. For K. brevis, little is known about its physiology--even less about what environmental factors are responsible for or contribute to its ability to form blooms or produce toxins. The physiology of an organism and how it responds to its environment are determined by the organism's genes and how the expression of those genes is controlled. One of my aims is to use a functional genomics approach to investigate and understand the correlation between gene expression of K. brevis and the physiological and ecological nature of the organism. To this end, I have created an expressed sequence tag (EST) library of K. brevis. Currently, I have over 18,000 high-quality ESTs isolated from cultures of K. brevis. From these sequences I have create K. brevis microarrays containing almost 6,000 unique elements. In collaboration with researchers at the University of Miami and Florida International University (as part of the Ocean and Human Health Center located at the University of Miami), different measures of physiology, such as growth rate and rate of carbon fixation, or toxin production are measured for cultures of K. brevis grown under different environmental conditions. Then, I harvest mRNA from these cultures and use it with my microarrays to determine what genes are differentially regulated in response to these different culture conditions, e.g. varying levels of light, nitrogen, phosphorus, or iron.
A second aim is to determine what is the population structure of K. brevis within a bloom and to compare the structure(s) between blooms, i.e. to determine how genetically diverse individual cells are from each other within a bloom and from cells in subsequent blooms. There are two major lines of reasoning as to the level of genetic diversity that may exist for K. brevis, as is true for the majority of single-celled planktonic organisms: 1) because K. brevis primarily (exclusively?) reproduces asexually, the population of K. brevis in the Gulf of Mexico may be a single population of highly-related, if not genetically-identical, cells, 2) conversely, differing mini-environments throughout the Gulf of Mexico may provide selective pressures that help to maintain genetically diverse populations of K. brevis. Understanding which of these hypotheses is true will significantly boost our understanding of K. brevis ecology. If genetically identical, then the cells are likely responding to a specific factor, or defined suite of factors, that lead to bloom formation, e.g. introduction of a nitrogen source. If genetically diverse, then some sub-population of cells, optimized to take advantage of prevailing conditions, may bloom, and the factor(s) responsible for bloom formation may be different at different times or locations.
Genotyping of individual cells within a bloom and between blooms of K. brevis will allow us to determine how genetically diverse this organism is. Recently, we and others have shown that gene sequences from nuclear, mitochondrial, and chloroplast genomes in K. brevis are virtually identical between cultures of K. brevis that were initiated from cells collected at different locales and dates. A more sensitive method of genotyping is microsatellite analysis. This type of analysis is indeed showing that many planktonic protists are more genetically diverse than we could have imagined. What isn’t clear from this analysis is whether the genetic diversity translates into phenotypic diversity, i.e. are the genetic differences neutral or do they provide differences to the cell’s physiology? I would like to utilize microarrays as a means of determining the genetic profile of individual cells, and then compare the profiles to assess levels of genetic diversity. If successful, the profiles will not only produce a more global view as to the level of variation, but also potentially provide clues as to how the diversity might impinge upon the physiology of the cell, e.g. one individual may show higher expression of genes A, B, and C which are known to be involved in O2 evolution.