Grantee Research Project Results
2005 Progress Report: Investigation of Toxic Raphidophyte Population Dynamics Using Molecular and Physiological Tools
EPA Grant Number: R831041Title: Investigation of Toxic Raphidophyte Population Dynamics Using Molecular and Physiological Tools
Investigators: Hutchins, David A. , Cary, S. Craig , Coyne, Kathryn J. , Doblin, Martina
Institution: University of Southern California , Old Dominion University , University of Delaware
Current Institution: University of Delaware , Old Dominion University
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 2003 through August 31, 2006
Project Period Covered by this Report: September 1, 2004 through August 31, 2005
Project Amount: $567,331
RFA: Ecology and Oceanography of Harmful Algal Blooms (2002) RFA Text | Recipients Lists
Research Category: Water Quality , Aquatic Ecosystems , Water
Objective:
The goals of this project are to:
- Gain a better understanding of the effects of environmental perturbations and grazing pressure on raphidophyte community dynamics.
- Identify environmental factors that stimulate the growth of raphidophytes relative to other algal species.
- Investigate the potential of raphidophyte cyst distributions as an indicator of seasonal bloom “hot spots.”
Progress Summary:
The second year of this project has seen progress toward all of the major goals of the grant. We now have quantitative polymerase chain reaction (qPCR) probes for all four species of raphidophytes in the Delaware Inland Bays (DIB): Chattonella subsalsa, C. cf. verruculosa, Heterosigma akashiwo, and Fibrocapsa japonica. Using the species-specific primers and probes in conjunction with an internal standard for qPCR, we are now able to identify and accurately enumerate these species in environmental water and sediment samples, and correlate abundances of each species with environmental characteristics. These results suggest that Heterosigma can tolerate a wider range of temperatures and salinities compared to the other raphidophyte species, a finding that has been validated by laboratory culture experiments. Using Genescan, however, we demonstrated the existence of several distinct strains of H. akashiwo in the DIB. The distribution of each of these strains appears to be regulated by salinity.
Results of laboratory culture experiments reported in year 1 suggest that H. akashiwo can grow to higher density in nitrate compared to ammonium. We used molecular methods to investigate the regulation of nitrate assimilation in this species. Species-specific primers for nitrate reductase were designed and a series of experiments was conducted to evaluate expression of nitrate reductase after addition of nitrate to nitrate-starved cultures and after addition of ammonium to nitrate-replete cultures. Our results demonstrate that nitrate reductase is expressed in the absence of nitrogen in the culture media but is repressed by addition of ammonium. Addition of nitrate to nitrogen-starved cultures resulted in a ~ 100-fold increase in expression of nitrate reductase within 30 minutes. This induction of gene transcription is much higher than in other microalgal species examined to date and suggests that H. akashiwois well adapted to take advantage of pulses of nitrate within the natural environment.
We conducted a series of experiments to examine microzooplankton grazing on H. akashiwo, C. subsalsa, C. cf. verruculosa, and F. japonica. Dilution experiments were carried out using natural water samples collected during blooms of each of these species. In contrast to previous dilution experiments where grazing on the whole community was evaluated, however, we used a species-specific approach. qPCR was used to assess changes in raphidophyte abundances in these experiments to evaluate grazing pressure on each species. This was then compared to grazing on the total community. Our results clearly indicate grazing on H. akashiwo by microzooplankton, but not on the other raphidophyte species.
Two sets of grazing experiments with collected mesozooplankton grazers (copepods) were also carried out using natural bloom samples. Our results suggest that copepods are not exerting a major grazing pressure on raphidophytes. Copepod abundance in the DIB is highly variable, being affected by time of day and salinity (influenced by tide), as well as other factors. Consequently, grazing pressure by copepods is likely to be highly variable.
Perhaps the most exciting result of this year’s work is our identification of a new species (and potentially a new genus) of raphidophyte in the DIB. A bloom of this species, which superficially resembles H. akashiwo, occurred at near zero salinity in Little Assawoman Bay during the summer of 2005. This bloom was notable in that the abundance of the new species was estimated at 200 million cells per liter. We have been able to isolate the new species from environmental bloom samples and we are currently characterizing it in comparison to H. akashiwo.
The work is now considerably ahead of our original schedule, and we anticipate that the project will continue as planned over the next 12 months. Two papers have also been published and six are in advanced stages of preparation and should be submitted in the near future. The following publications and meeting presentations have credited support from this grant.
Future Activities:
In 2006, we will continue the application of our qPCR methods to enumerate all five raphidophyte species (including the new raphidophyte, Veridita minima) in natural blooms and in further mesocosm experiments, including experiments with manipulations such as nutrient enrichments. We will continue with cyst excystment, nitrate reductase, and zooplankton grazing studies, and will synthesize our extensive data set on relative environmental tolerances within the raphidophyte consortium and compared to other taxa with the aim of making predictive models. Finally, we are now putting considerable effort into writing papers from our research, and publication will be a priority in the third year of the grant.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 32 publications | 13 publications in selected types | All 13 journal articles |
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Coyne KJ, Cary SC. Molecular approaches to the investigation of viable dinoflagellate cysts in natural sediments from estuarine environments. The Journal of Eukaryotic Microbiology 2005;52(2):90-94. |
R831041 (2005) R831041 (Final) |
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Coyne KJ, Handy SM, Demir E, Whereat EB, Hutchins DA, Portune KJ, Doblin MA, Cary SC. Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnology and Oceanography: Methods 2005;3(Sep):381-391. |
R831041 (2005) R831041 (Final) |
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Handy SM, Coyne KJ, Portune KJ, Demir E, Doblin MA, Hare CE, Cary SC, Hutchins DA. Evaluating vertical migration behavior of harmful raphidophytes in the Delaware Inland Bays utilizing quantitative real-time PCR. Aquatic Microbial Ecology 2005;40(2):121-132. |
R831041 (2005) R831041 (Final) |
Exit Exit |
Supplemental Keywords:
water, watersheds, marine, estuary, ecological effects, bioavailability, cellular, population, ecosystem, aquatic, habitat, public policy, decision-making, environmental assets, environmental chemistry, biology, ecology, genetics, limnology, analytical, surveys, measurement methods, northeast, Atlantic coast, mid-Atlantic, Delaware, DE,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Oceanography, Monitoring/Modeling, algal blooms, Environmental Monitoring, Ecological Risk Assessment, Ecology and Ecosystems, marine ecosystem, aquatic ecosystem, bloom dynamics, pigment analysis, nutrient kinetics, algal growth, aquatic toxins, marine biogeochemistry, Raphidophyte blooms, algal pigments, Delaware, ChattonellaProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.