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You are here: UMCOHH Home > Research Projects: HAB Functional Genomics

Functional Genomics of a Subtropical Harmful Algal Bloom Species: Karenia brevis Davis (HAB Functional Genomics)

Principal Investigators: Douglas L. Crawford Ph.D. and Gary Hitchcock Ph.D.
Co-Investigators: Kelly Rein Ph. D., Peter Minnett Ph. D.
Participating Facilities Cores: Remote Sensing, Toxic Algal Culture and Genomics

Using Functional Genomic tools (see infrastructure description), plankton ecology and satellite imagery to investigate the distribution and molecular mechanisms affecting Harmful Algal Blooms (HABs). This research is to provide better predictive ability about HAB effects on human health. Phytoplankton that make up HABs regulate gene expression by altering mRNA levels (Pichard et al., 1993; Pichard et al., 1997; Wyman et al., 1998; Amaro et al., 2000; Paul et al., 2000; Taroncher- Oldenburg and Anderson, 2000). This proposal seeks to create tens of thousands of expressed sequence tags (EST), which are full-length or near full-length cDNA in which only 1-70% or 400-600 base pairs have been sequenced, for Karenia brevis . These short sequences (ESTs) are used to define or tag each gene . The ESTs are used to manufacture microarrays; the microarrays will be used to quantify patterns of mRNA expression and thus lead to the discovery of genes that reflect changes in population growth, environmental change or toxin production. Microarrays of K. brevis genes will be used to achieve four overall goals: (1) quantify variation in mRNA expression among populations in bloom and non-bloom conditions; (2) quantify patterns of gene expression among cultures of K. brevis exposed to different environmental conditions; (3) use genetic markers to discern population divergence among blooms, and (4) examine variation in gene expression among different populations. To interpret these data, microarray studies will be combined with analyses of toxin production, ecological and physiological measurements, and satellite imagery (see Remote Sensing Core description). These diverse approaches will allow us to interrogate patterns of gene expression and thus discover genes involved in HABs. These studies will provide linkages between population genetics, oceanography, and functional genomics to better inform scientific and public concerns about the ocean’s effects on human health.

Undergraduate, graduate and post-doctoral training is an integral part of this project. Educating students about integrative science (molecules to oceanography) increases general scientific knowledge, understanding of diverse scientific fields, and creation of human resources for future discovery. Student and post-doctoral scholars will present data at National and International meetings and will publish in peer-reviewed journals. Training in molecular, physiological, ecological and oceanographic techniques will be enhanced by the Center grant through institutional forums for students and researchers to exchange ideas.

There are six specific aims that integrate ocean studies with genomics to better inform us about HAB biology and its impact on human health.

  • Isolation, sequencing and production of microarrays. A cDNA library will be created from mRNA isolated from clonal (single-cell isolate) cultures of K. brevis . ESTs will be isolated from normalized and subtracted libraries, and sequenced using a high-throughput system (ABI 3730 with 96 capillaries) located in the genomics facility at RSMAS. Annotation of ESTs will use a serial search of Unigene databases and results will be publicly available. Approximately 10,000 unique ESTs will be printed using facilities at RSMAS. This aim creates tools and bioinformatics that will enhance the study of HABs by us and other interested groups.
  • Creation of molecular markers to investigate genetic variation and population dynamics. Genetic diversity in K. brevis is not well studied, yet the population dynamics and genetic variation among populations should affect the impact of this HAB organism on human health.
  • Determination of gene expression from bloom and non-bloom K. brevis populations. Approximately, 10 6 to 10 7 K. brevis cells (free of other dinoflagellates) can be isolated using flow cytometry (Algal Core Facility; see Cores descriptions). RNA & DNA will be isolated and then used for microarray and genetic analyses (respectively). Pre-bloom populations on the West Florida mid-shelf will be identified by satellite imagery (e.g., Tester and Stumpf, 1998). Intense temporal ship-board sampling of K. brevis and environmental features will be used to identify temporal, environmental, and bloom patterns of gene expression.
  • Determine growth rates as an index of the physiological state of bloom and nonbloom populations. Investigating the distribution and growth rate of K. brevis provides information to better understand the variation in gene expression from field samples. This integration of ocean science and functional genomics will provide information on critical molecular markers that distinguish important ecological conditions that influence growth rate.
  • Determine patterns of gene expression of K. brevis cultured in the laboratory under different environmental conditions. Based on the study of natural populations (Aim 3), environmental conditions associated with patterns of induced genes can be experimentally tested. Laboratory cultures of K. brevis provide the means to test experimentally how environmental and population differences affect patterns of gene expression.
  • Determine distribution and genetic variation in K. brevis populations and variation in gene expression. The goal of this research is to integrate measures of genetic variation with remote sensing and ship-based sampling to determine the distribution and transport of K. brevis. These data will address the hypothesis that transport of high cell concentrations at the shoreward edge of the Florida Current occurs primarily in frontal features such as filaments, the Tortugas Gyre, and mesoscale and sub-mesoscale eddies. Variation in gene expression will be measured among samples collected in waters off south Florida and in the Gulf of Mexico. Determine patterns of gene expression associated with genetically different populations of K. brevis.

Aims 1-5 can be thought of as building a foundation so we can interpret data from the molecular and genomic tools. Aim 6 uses this knowledge to investigate how ocean transport influences the population dynamics of K. brevis and how this interaction affects patterns of gene expression.

This is an ambitious proposal to create genomic and molecular tools to investigate patterns of gene expression and genetic divergence in order to understand the biology of harmful algal blooms. This research integrates an understanding of phytoplankton ecology and oceanographic processes to define the spatial and temporal patterns, processes, and timing that affect the production of toxins that affect the health of humans. These tools will be made available to the larger oceanographic community and thus provide a broader impact on the scientific community.