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.
