2005 Progress Report: The Development of a Single-cell Field Diagnostic for Nitrogen Limitation in Harmful Algae

EPA Grant Number: R830415
Title: The Development of a Single-cell Field Diagnostic for Nitrogen Limitation in Harmful Algae
Investigators: Dyhrman, Sonya , Anderson, Donald M.
Institution: Woods Hole Oceanographic Institution
EPA Project Officer: Hiscock, Michael
Project Period: November 18, 2002 through November 17, 2005 (Extended to August 17, 2007)
Project Period Covered by this Report: November 18, 2004 through November 17, 2005
Project Amount: $451,953
RFA: Ecology and Oceanography of Harmful Algal Blooms (2002) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Water Quality , Ecosystems , Water


The research proposed here will test and further refine an assay for N'-acetylglucosaminidase (NAG) activity using the enzyme labeled fluorescence (ELF)-NAG substrate. Specific objectives are to: (1) test for ELF-NAG labeling in a suite of harmful algal bloom (HAB) species, (2) pursue a method for quantifying ELF-NAG labeling on a single-cell basis in a model Alexandrium species, (3) relate the regulation of the ELF-NAG labeling to the physiological condition of the Alexandrium cells, (4) further characterize the physiological function of the NAG enzyme, (5) develop the assay for use on field populations of Alexandrium fundyense, and (6) test the assay on field populations of the toxic dinoflagellate A. fundyense during nutrient addition experiments in limno-corrals.

Progress Summary:

Nutrients play a significant regulatory role in phytoplankton growth and bloom dynamics. Nitrogen, a critical macronutrient, exists in inorganic and organic forms in the ocean. Dissolved organic forms of nitrogen (DON) comprise a nutrient pool that often requires enzymatic hydrolysis before it can be exploited by many species, such as harmful ones, for cell growth. Potential DON utilization by harmful algal species can be assessed through measures of enzymatic activity. Specifically, ELF technology provides a cell-specific method for diagnosing the nitrogen status of various HAB species. To date, 13 HAB species have been tested with ELF-NAG labeling. Over half of these species have been found to express NAG activity when grown under nitrogen deplete conditions, while those grown under nitrogen replete conditions did not express N’-acetylglucosaminidase activity. With this data in hand, this year we sought to further satisfy our main objectives and focus our work on assessing the regulation of ELF-NAG labeling to the physiological condition and characterizing the function of the N’-acetylglucosaminidase enzyme. This year, we augmented our ELF-NAG data for Aureococcus anophagefferens with a preliminary screening of the transcriptome by Serial Analysis of Gene Expression (SAGE). We have sequenced three Long-SAGE libraries to examine the A. anophagefferens transcriptome and how it changes with nutrient stress (low N and low P). In our initial screening of the SAGE libraries, we have sequenced a total of 2,500 tags for each library. After more in-depth sequencing, using available expressed sequence tags (EST) and genome sequence, we will screen the libraries for tags that map to genes (e.g., NAG) that are differentially regulated.

In addition to our SAGE work, we also continued our work to identify and characterize the function of the NAG enzyme by examining gene expression within the HAB Karenia brevis using quantitative PCR (qPCR). We assayed two K. brevis strains for three N-related genes, NAG, glutamine synthetase (GS), and NO2/NO3 transporter (NRT). We also looked at the expression of these genes for one K. brevis strain (Wilson) grown with different sources of nitrogen. In neither experiment did we see a significant difference in the gene expression, relative to the nutrient replete sample. Additional experiments are underway to investigate enzyme activity (e.g., GS) in K. brevis Wilson grown with nitrate, ammonium, and urea. Lastly, this year we collected field samples from our mesocosm experiment. Samples were assayed for bulk and cell-specific NAG activity as well as bulk and cell-specific alkaline phosphatase activity. These samples are currently being analyzed.

Future Activities:

We do not anticipate any major changes from the tasks outlined in the proposal. A particular focus will be work on Objectives 1 and 2. A postdoctoral scholar, Alena Strojsova, will continue to screen additional HAB species to satisfy Objective 1. Furthermore, we have already begun sequencing the Aureococcus SAGE libraries that we constructed. We also intend to follow up on our qPCR work with K. brevis by assaying cultures for enzyme activity (e.g., GS). This experiment is also currently underway.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 7 publications 3 publications in selected types All 2 journal articles
Type Citation Project Document Sources
Journal Article Dyhrman ST, Erdner D, La Du J, Galac M, Anderson DM. Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 2006;5(3):242-250. R830415 (2005)
  • Full-text: Science Direct Full Text
  • Abstract: Science Direct Abstract
  • Journal Article Dyhrman S. Ectoenzymes in Prorocentrum minimum. Harmful Algae 2005;4(3):619-627. R830415 (2004)
    R830415 (2005)
  • Full-text: Science Direct Full Text
  • Abstract: Science Direct Abstract
  • Supplemental Keywords:

    marine phytoplankton, nutrient limitation, enzyme labeled fluorescence, harmful algal bloom, diagnostic indicator, paralytic shellfish poisoning, ELF-NAG, N’-acetylglucosaminidase, qPCR,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Oceanography, algal blooms, Environmental Monitoring, Alexandrium Blooms, marine ecosystem, bloom dynamics, nutrient kinetics, phytoplankton, dinoflagellate, algal bloom detection

    Relevant Websites:

    http://www.whoi.edu/redtide/ Exit
    http://www.whoi.edu/sbl/liteSite.do?litesiteid=6692 Exit

    Progress and Final Reports:

    Original Abstract
  • 2003
  • 2004 Progress Report
  • 2006
  • Final