Grantee Research Project Results
Final 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 Delaware , Old Dominion University
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 2003 through August 31, 2006
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:
Goals
The original general goal of this project was to carry out a multifaceted molecular, ecological and physiological analysis of Delaware Inland Bays Raphidophyte communities and isolated strains, with an emphasis on population dynamics.
The specific original goals of this project were:
- To gain a better understanding of the effects of environmental perturbations and grazing pressure on Raphidophyte community dynamics.
- To identify environmental factors that stimulate the growth of Raphidophytes relative to other algal species.
- To investigate the potential of Raphidophyte cyst distributions as an indicator of seasonal bloom “hot spots”.
Summary/Accomplishments (Outputs/Outcomes):
Accomplishments
We have now completed all of the major goals of the grant, with the addition of several accomplishments that were not envisioned in the original proposal. Our accomplishments include:
- Development of quantitative PCR (QPCR) probes for all the known species of raphidophytes in the Delaware Inland Bays (DIB) , including Chattonella subsalsa, C. cf. verruculosa, Heterosigma akashiwo, Fibrocapsa japonica, and the newly described species and genus Veiridita minima (see below). Using these species-specific primers and probes in conjunction with an internal standard for QPCR, we now have a new set of molecular enumeration tools to identify and accurately count these species in environmental water and sediments samples. In combination with funding from other sources (MERHAB), we also developed new multiplexing and multi-probing methods to simultaneously enumerate more than one HAB species in the same QPCR reaction, offering a major advance in HAB monitoring methodology.
- Environmental work using QPCR to understand factors controlling the abundance of DIB raphidophytes. The results of QPCR analyses in conjunction with environmental surveys suggests that Heterosigma can tolerate a wider range of temperatures and salinities compared to the other raphidophyte species, validating the results of our complementary laboratory culture experiments (see below). In addition, using Genescan, we demonstrated the existence of several distinct strains of Heterosigma akashiwo in the Inland Bays. The distribution of each of these strains appears to be regulated by salinity.
- Laboratory culture work on responses of raphidotphytes to varying nutrients, temperature, salinity and light. Results of laboratory culture experiments reported in year 1 suggest that Heterosigma akashiwo can grow to higher density in nitrate compared to ammonium. Heterosigma also appears to be a better competitor for nutrients and light and tolerates wider variations in salinity of temperature than the sympatric species Chattonella subsalsa. Laboratory culture experiments were also paired with environmental sampling using QPCR methods to evaluate competitive interactions between DIB raphidophytes and other co-existing species such as HAB dinoflagellates.
- Assessment of grazing impacts on DIB raphidophytes. Grazing experiments with collected mesozooplankton grazers (copepods) were 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. On the other hand, micrograzing experiments using a novel species-specific QPCR dilution method show that protozoans can exert a strong top-down control on Heterosigma akashiwo, but not on the larger sympatric raphidophyte species. Thus, micrograzers may offset some of the advantages Heterosigma enjoys with respect to bottom-up control factors, allowing co-existence of the entire multi-species raphidophyte consortium.
- Evaluation of benthic cysts as a recruitment mechanism for raphidophyte blooms. Field and laboratory experiments examined excystment as a factor that could contribute to bloom development in the DIB. Results suggest that excystment of different raphidophyte species is regulated in very different manners, and that gradual supplies of vegetative cells from hatching cysts over the course of the season may be an important recruitment mechanism.
- Investigations of the regulation of nitrate assimilation in Heterosigma. Species-specific primers for nitrate reductase were designed and a series of experiments were 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 constitutively, even in the absence of nitrogen in the culture media. 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 other microalgal species examined to date and suggests that Heterosigma is well adapted to take advantage of pulses of nitrate within the natural environment.
We have also begun to evaluate nitrate reductase enzyme activity in Heterosigma and Chattonella subsalsa using a novel fluorescent substrate for NR, CNOB. Traditional methods used to determine in vitro NR activity only measure Vmax, while in vivo methods require harsh treatments and are difficult to replicate. The CNOB method does not require harsh treatment of the cells and can be performed on live cultures using a flow cytometer to measure changes in fluorescence. Although this method is still being evaluated in our lab, preliminary experiments suggest that NR activity in Heterosigma is not inhibited by the presence of NH4+ in the culture media. For Chattonella subsalsa, however, the presence of NH4+ results in a ~90% inhibition in NR enzyme activity. These results suggest that Heterosigma akashiwo and Chattonella subsalsa do not compete for the same nitrogen source when both ammonium and nitrate are present, and may explain why we see mixed blooms of these species in the Delaware Inland Bays.
- Identification of a new genus and species of HAB raphidphyte from the DIB. One of the most exciting results of this project is our identification of a new species (and potentially a new genus) of raphidophyte in Delaware’s Inland Bays. A bloom of this species, which superficially resembles Heterosigma 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 characterized it in comparison to Heterosigma akashiwo.
- Preliminary work on global change impacts on DIB raphidophyte growth and dominance. We began a series of laboratory and field experiments to examine how rising CO2 and temperature may interact with eutrophication to affect HAB blooms in the DIB in the future. Results of these early experiments showed suggestive differences in the responses of major HAB groups to global change variables, and were used to obtain further grant support from ECOHAB for a new global change-oriented project that is currently beginning.
Education and human resources development.
This project provided support for the graduate work of University of Delaware students Sara Handy (Ph.D. 2007), Elif Demir (Ph.D. 2007) and Kevin Portune (Ph.D. expected in 2008)
Conclusions:
Raphidophytes are readily eaten (at rates which match their growth) when provided as a sole food source to copepods. The other major conclusion--that when Raphidophytes are present in a mixture of prey types (either as laboratory cultures or in natural populations), grazing rates are low--remains unchanged.
Journal Articles on this Report : 13 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) |
Exit |
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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) |
Exit Exit |
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Coyne KJ, Hare CE, Popels LC, Hutchins DA, Cary SC. Distribution of Pfiesteria piscicida cyst populations in sediments of the Delaware Inland Bays, USA. Harmful Algae 2006;5(4):363-373. |
R831041 (Final) |
Exit Exit Exit |
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Demir E, Coyne KJ, Doblin MA, Handy SM, Hutchins DA. Assessment of microzooplankton grazing on Heterosigma akashiwo using a species-specific approach combining quantitative real-time PCR (QPCR) and dilution methods. Microbial Ecology 2008;55(4):583-594. |
R831041 (Final) R833221 (2007) R833221 (Final) |
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Doblin MA, Coyne KJ, Rinta-Kanto JM, Wilhelm SW, Dobbs FC. Dynamics and short-term survival of toxic cyanobacteria species in ballast water from NOBOB vessels transiting the Great Lakes—implications for HAB invasions. Harmful Algae 2007;6(4):519-530. |
R831041 (Final) |
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Fu F-X, Zhang Y, Leblanc K, Sanudo-Wilhelmy SA, Hutchins DA. The biological and biogeochemical consequences of phosphate scavenging onto phytoplankton cell surfaces. Limnology and Oceanography 2005;50(5):1459-1472. |
R831041 (Final) |
Exit Exit |
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Fu F-X, Zhang Y, Warner ME, Feng Y, Sun J, Hutchins DA. A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum. Harmful Algae 2008;7(1):76-90. |
R831041 (Final) R833221 (Final) |
Exit Exit Exit |
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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) |
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Handy SM, Hutchins DA, Cary SC, Coyne KJ. Simultaneous enumeration of multiple raphidophyte species by quantitative real-time PCR: capabilities and limitations. Limnology and Oceanography: Methods 2006;4(Jun):193-204. |
R831041 (Final) |
Exit Exit |
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Handy SM, Demir E, Hutchins DA, Portune KJ, Whereat EB, Hare CE, Rose JM, Warner M, Farestad M, Cary SC, Coyne KJ. Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies. Harmful Algae 2008;7(5):599-613. |
R831041 (Final) |
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Popels LC, MacIntyre HL, Warner ME, Zhang Y, Hutchins DA. Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae). Journal of Phycology 2007;43(1):32-42. |
R831041 (Final) |
Exit |
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Sun J, Feng Y, Zhang Y, Hutchins DA. Fast microzooplankton grazing on fast-growing, low-biomass phytoplankton: a case study in spring in Chesapeake Bay, Delaware Inland Bays and Delaware Bay. Hydrobiologia 2007;589(1):127-139, DOI10.1007/s10750-007-0730-6. |
R831041 (Final) |
Exit Exit |
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Zhang Y, Fu F-X, Whereat E, Coyne KJ, Hutchins DA. Bottom-up controls on a mixed-species HAB assemblage: a comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA. Harmful Algae 2006;5(3):310-320. |
R831041 (Final) |
Exit Exit Exit |
Supplemental Keywords:
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.