Golden Alga Projects

The U.S. Fish and Wildlife Service State Wildlife Grant awarded to TPWD in 2003 (T-14-P) continues and has been expanded to include other TPWD projects, and the University of Texas project previously under this grant has been completed. Additionally, the Texas Legislature has authorized an additional $225,000 for research projects addressing priority needs for the understanding and management of golden alga in Texas. This money is raised through user-based fees, specifically the licenses that fishermen pay to fish in Texas. This authorization was supplemented through matching federal aid funds under the U.S. Fish and Wildlife Service State Wildlife Grants to total $450,000 for research on golden alga in Texas from September 2005 through August 2006. Several projects will continue through August 2007. Projects funded include the following:

2005 & 2006 P. parvum Research Coordinated by TPWD

Research Progressing under the State Wildlife Grant/Legislatively authorized Funds (T-23-P/RGA):

Statewide Survey for Prymnesium parvum:

Loraine Fries (Texas Parks and Wildlife Department)
Greg Southard (Texas Parks and Wildlife Department)
Dijar Lutz-Carrillo (Texas Parks and Wildlife Department)
J. Warren Schlechte (Texas Parks and Wildlife Department)
Steven Hamby (Texas Parks and Wildlife Department)
Gary Steinmetz (Texas Parks and Wildlife Department)
John Taylor (Texas Parks and Wildlife Department)
Gary Garrett (Texas Parks and Wildlife Department)

Objective:
The goal of this research is to determine the distribution of P. parvum within the state of Texas and environmental correlates associated with prevalence and toxicity. Additionally, isolates from affected river basins will be examined for genetic differences at the first internal transcribed spacer region and compared among themselves and with available isolates from geographically disjunct locales.

Progress Report/Findings:
A total of 423 water samples from 56 water bodies representing 14 major river basins in Texas was collected for this study. Sampling was conducted from March 2004 through March 2005 with most sites sampled four times over the collection period. The vast majority of samples were collected through the Texas Commission on Environmental Quality Clean River and Surface Water Quality Monitoring programs and cooperators. For each water sample, a variety of parameters was measured including physicochemical field measurements, conventional organic and inorganic parameters, cations, and metals. Cell counts, to enumerate P. parvum cells, were accomplished using light microscopy and a hemacytometer. Bioassays, to evaluate ichthyotoxicity, were conducted whenever a sample yielded a positive cell count. Additionally, each sample was evaluated using a real-time (or quantitative PCR (qPCR)) method which has the potential to be much more sensitive than cell counts via light microscopy.

Overall, P. parvum cells were detected either by cell counts or qPCR in 49 of the 56 water bodies. This finding strongly suggests that P. parvum is widespread and could be expected to be found in virtually any water body. However, toxicity was only detected in 63 samples from seven water bodies and, in each case, these were waters where P. parvum kills have been previously reported. A thorough data analysis, which may establish correlation between environmental variables and toxicity, is planned.

To date, cell culture isolates from Texas have been obtained only from the Rio Grande, Colorado, and Brazos River Basins. Sequences from the ITS1 region of these isolates were identical to each other and more similar to those from South Carolina and Scotland than they were to isolates from Maine and England. Additional work is planned with more isolates from Texas and elsewhere to better determine the relationships between and among P. parvum populations.


See also 2003 & 2004 P. parvum Research,
Statewide Survey for Prymnesium parvum

Combined Nitrogen and Phosphorus Fertilization for Controlling Prymnesium parvum Toxicity in Fish Culture Ponds

Gerald Kurten (Texas Parks and Wildlife Department)
Dr. Aaron Barkoh (Texas Parks and Wildlife Department)
Loraine T. Fries (Texas Parks and Wildlife Department)
Drew Begley, (Texas Parks and Wildlife Department)

Objective:
The goal of this project is to determine if combined nitrogen and phosphorus fertilization of hatchery ponds will reduce the toxicity of P. parvum. Specifically, two fertilization regimens of nitrogen and phosphorus (300N:30P and 300N:60P three times weekly) will be evaluated for efficacy in reducing dominance of the phytoplankton community and toxin production by P. parvum in limnocorrals placed in a pond at the Possum Kingdom fish hatchery (PKFH).

Progress Report/Findings:
Continued refinement of nutrient application rates and concentrations has proceeded at the TPW Dundee fish hatchery in three additional studies. The most recent study was transitioned from 220 liter containers to ponds to determine if findings in controlled studies were consistent with “real world” applications. Although numerous application rates and concentrations have been tested, the most effective nutrient (nitrogen and phosphorus) applications continue to be the original tested rates of 30 µg/L P and 300 µg/L N. Lower application rates have positive effects in reducing golden alga toxicity and cell densities, but response times are much slower.

Future studies will examine the use of these most successful nutrient application rates combined with techniques for reducing the one negative consequence of the existing successful nutrient application rates, which is the eventual elevation of pH to levels that can be harmful to pH sensitive species such as striped bass.

A progress report outlining the findings of additional nutrient manipulation work should be completed by the Fall of 2007.

Citations / Presentations:
Kurten, G. L, A. Barkoh, L.T. Fries, D. Begley. 2006. Combined Nitrogen and Phosphorus Fertilization for Controlling Golden alga Prymnesium parvum in Fish Culture Ponds. Southern Division of American Fisheries Society. Spring Meeting. San Antonio, TX. February 9-12.

Kurten, G. L., A. Barkoh, L.T. Fries and D. Begley. 2007. Combined nitrogen and phosphorus fertilization for controlling the toxigenic alga Prymnesium parvum. North American Journal of Aquaculture 69:214-222.

Abstract:

Combined Nitrogen and Phosphorus Fertilization for Controlling Golden alga Prymnesium parvum in Fish Culture Ponds.

Kurten, G. L. Texas Parks and Wildlife Department, Inland Fisheries Division, Possum Kingdom State Fish Hatchery, 600 South Highway 16, Graford, Texas 76449, USA

Barkoh, A. Texas Parks and Wildlife Department, Inland Fisheries Division, Heart of the Hills Fisheries Science Center, 5103 Junction Hwy, Ingram, Texas 78025, USA

Fries, L. T. Texas Parks and Wildlife Department, Inland Fisheries Division, A. E. Wood Fish Hatchery, 507 Staples Road, San Marcos, Texas 78666, USA

Begley,* D. Texas Parks and Wildlife Department, Inland Fisheries Division, Dundee State Fish Hatchery, Route 1, Box 123A Electra, Texas 776360, USA

Prymnesium parvum has caused significant fish kills at Texas hatcheries since 2001. Copper sulfate and ammonium sulfate can control P. parvum but they provide short-term relief and have undesirable side effects. Copper sulfate can kill desirable algae and invertebrates, and ammonium sulfate can be toxic to fish. Because dominance of the phytoplankton community and toxin production by P. parvum appears to be nutrient-related, we evaluated two fertilization regimens of nitrogen (N) and phosphorus (P) for their efficacy in controlling P. parvum populations and toxin production. The experiment was conducted with three treatments (30P:300N and 60P:300N three times weekly and no fertilization) in limnocorrals at the Texas Parks and Wildlife Department’s Dundee fish hatchery. In unfertilized treatments P. parvum cell densities and toxicity persisted throughout the 40 d experiment. In both fertilized treatments P. parvum cell densities and toxicity declined below detectable levels within two weeks and remained undetectable during the duration of the study.

Key Words: toxic alga, fish kills, harmful algal blooms, golden alga, fertilization

Isolation of Prymnesium parvum Analytical Standards for Research on Golden Alga Toxicity

Dr. Kevin Schug (University of Texas at Arlington)
Dr. James P. Grover (University of Texas at Arlington)

Objective:
The goal of this research is to reproduce and optimize the current protocol for the isolation of the molecules believed to be responsible for Prymnesium parvum toxicity with respect to analytical figures of merit in order to unequivocally obtain high purity standards; as well as to devise improvements towards a more streamlined procedure for extraction and purification. Once isolated, these standards will be available for further assay development; as well as in limited quantities to members of the TPWD for other research purposes.

Progress Report/Findings (April 2007):
We have been working for approximately a year to reproduce the literature method of Igarishi et al.1,2 for isolation of analytical standards of prymnesin-1 and prymnesin-2, the major toxic constituents released by Golden Alga. The first batch of the “Texas strain” of Prymnesium parvum was cultured (on a 60 L scale) in the laboratory of Dr. Grover. Culture conditions had been previously optimized for high toxicity through the use of a modified nutrient protocol with reduced phosphate and nitrate levels. Unfortunately, these nutrient levels appeared to limit algal biomass in the cultured samples, compared to levels reported in the literature. It is therefore anticipated that the reduced biomass will result in a lower amount of toxin isolated; and subsequent batches of culture with a modified nutrient protocol are necessary to reach the targeted isolation yield (milligrams).

The algae produced in the initial batch have been processed in accordance with the multi-step literature method for isolating prymnesins in the laboratory of Dr. Schug. At this point, we have not yet completed the full procedure for this first batch. This procedure features multiple column chromatography steps and is complicated by the poor solubility of the prymnesins. Furthermore, we have found the need to develop supporting technology along the way to track the fractions containing prymnesins, as well as to identify other potential toxic constituents. These include electrospray ionization – mass spectrometry for directly monitoring prymnesins in isolated fractions, as well as the development of an erythrocyte lysis assay to monitor the toxicity of various fractions;3 to direct subsequent extraction steps.

We will continue to work toward completion of the extraction procedure for the first algal “test” batch. In the meantime, we have made preparations for subsequent batches to be grown (~ 120 L) for extraction. Air will be bubbled through the cultures and a greater concentration of nitrates will be employed relative to the first batch. We expect to provide the promised quantity of prymnesins by the end of this grant contract (end of summer 2007). We will also have completed preliminary set-up of the lysis assay. Repetition of the extraction procedure for subsequent batches should proceed much more smoothly with all supporting technology in place. This work is expected to be continued (with renewal of funding) for an additional year or more.

1. Igarishi, T.; Satake, M.; Yasumoto, T., “Prymnesin-2: A Potent Ichthyotoxic and Hemolytic Glycoside Isolated from the Red Tide Alga Prymnesium parvum,” J. Am. Chem. Soc. 1996, 118, 479.
2. Igarishi, T.; Satake, M.; Yasumoto, T., “Structures and Partial Stereochemical Assignments for Prymnesin-1 and Prymnesin-2: Potential Hemolytic and Ichthyotoxic Glycosides Isolated from the Red Tide Alga Prymnesium parvum,” J. Am. Chem. Soc. 1999, 121, 8499.
3. Eschbach, E.; Scharsack, J.P.; John, U.; Medlin, L.K., “Improved Erythrocyte Lysis Assay in Microtitre Plates for Sensitive Detection and Efficient Measurement of Haemolytic Compounds from Ichthyotoxic Algae,” J. Appl. Toxicol. 2001, 21, 513-519.

Historic Data Assessment of Golden Alga Fish Kills

Liz Singhurst (Texas Parks and Wildlife Department)
Joan Glass (Texas Parks and Wildlife Department)

Kip Portis (Texas Parks and Wildlife Department)
Jack Ralph (Texas Parks and Wildlife Department)
Dr. David Sager (Texas Parks and Wildlife Department)

Goal: To obtain, organize, and analyze data related to historic fish kills due to golden alga in Texas.

Objectives:

  1. To obtain and organize data on historic blooms and fish kills due to golden alga in Texas;
  2. To obtain and organize meteorological and aquatic physico-chemical data for time periods and locations relevant to blooms and historic fish kills due to golden alga in Texas;
  3. To analyze available data to determine if contributing factors can be identified with blooms and fish kills due to golden alga in Texas; and
  4. To report on the potential contributing factors and current data gaps, while summarizing historic fish kills due to golden alga in Texas.

Progress Report/Findings:

Citations/ Presentations:
Singhurst, E., J. Glass, K. Portis, J. Ralph, and D. Sager. 2007. Historical assessment of golden alga (Prymnesium parvum) fish kills in Texas: 2001 Colorado River fish kills. Report to Texas Parks and Wildlife Department. PWD RP T3200-1422. Austin.

See also 2003 & 2004 P. parvum Research,
Historic Data Assessment of Golden Alga Fish Kills

Efficacy of Oxidation Compounds to Control P. parvum Cells and Toxicity

Greg Southard (Texas Parks and Wildlife Department)

Objective:
The objective of this research is to refine pond control and management methods for P. parvum by investigating new means of algal and toxin controls using oxidative compounds that are EPA-approved algaecides. These compounds will be evaluated for use in public ponds and larger waterbodies for management options to control golden alga blooms and protect public aquatic ecosystems. The evaluation of compounds is scheduled to begin in June 2007.

Prymnesium parvum Sampling along the Texas Coast

Meridith Byrd (Texas Parks and Wildlife Department)
Janet Nelson (Texas Parks and Wildl.ife Department)

Goal:
Although Prymnesium parvum is known world-wide as an estuarine algae, in Texas P. parvum blooms and fish kills had been limited primarily to the western and northern portions of the state. In the springs of 2006 and 2007, however, blooms and fish kills occurred in waters close to Galveston Bay estuaries. These occurrences delimited the need for a baseline sampling of Texas coastal waters for P. parvum to gain an understanding of its distribution and possible areas to focus response efforts. The tidal estuaries of Texas are scheduled to be sampled beginning winter 2008.

Monitoring Blooms of Prymnesium parvum in Lake Whitney

David Buzan (Texas Parks and Wildlife Department)
Dr. Ayal Anis (Texas A&M University at Galveston)

Objective:
The objective of this proposed project is to describe the physical, chemical and biological conditions before, during and following a bloom of Prymnesium parvum in a Texas reservoir. This objective addresses directly or indirectly the following research priority goals identified by the TPWD Golden Alga Task Force.

Research Priority Goals:

  1. Monitor affected systems for specific blooms: Concentrating monitoring and experiments on Lake Whitney might increase the effectiveness of the research due to existing data and studies on golden alga at this location.
  2. Explore bloom triggers and ending points for P. parvum; understand the importance of grazing and nutrient limitation (including carbon dioxide availability) for P. parvum growth.
  3. Investigate the conditions and triggers necessary for toxin production and fish kills; include the roles/dependencies of obligate needs for a dark cycle, nutrients and their interactions, conflicting temperature/salinity evidence, fish stimulated production, and P. parvum densities.
  4. Examine differences between areas with P. parvum fish kills, areas with P. parvum but no fish kills, and areas without P. parvum. Possibly examine multiple waterbodies for differences. Possibly conduct laboratory/mesocosm invasion studies based on the differences.
  5. Examine potential impacts to other organisms (e.g., Daphnia, coots & other birds, etc.).
  6. Understand the physical response of Lake Whitney and P. parvum blooms to external physical forcing factors. Measure effects of wind-stress, surface buoyancy flux, inflow and outflow, variations in stratification and mixing processes, and the temporal and spatial variability (e.g. lateral boundaries vs. lake center) of lake dynamics.

Progress Report/Findings:
Analysis is pending.

Citation:
Anis, A. 2007. Hydrology and meteorology of Lake Whitney, Texas. Final REport to Texas Parks and Wildlife Department.

See also 2003 & 2004 P. parvum Research,
Concentrated Monitoring of a Prymnesium parvum Bloom

Advancing the Predictive Understanding of Bloom Formation and Toxicity
in Prymnesium parvum

Dr. James P. Grover (University of Texas at Arlington),
Dr. Daniel L. Roelke (Texas A&M University),
Dr. Bryan W. Brooks (Baylor University),
Dr. Richard L. Kiesling (University of Austin & US Geological Survey)

Scope of Research:
We propose to carry out a research program to advance our predictive understanding of P. parvum toxic bloom formation and toxicity. Our investigation will address open questions remaining from our work to date using focused laboratory and field experiments to examine population growth and toxicity in relation to limiting factors of salinity, temperature, and nutrients, and to examine population dynamics and toxicity of P. parvum in the presence of competitors and grazers, and other natural enemies. The experiments proposed here build from our results to date. We intend to resolve remaining uncertainties in our understanding of the relevant ecophysiology, using laboratory experiments, and move towards understanding the role of the larger planktonic community, using additional laboratory and field experiments.

Laboratory Research Questions:
1. What is the growth potential of P. parvum at low salinity typical of Texas inland waters (< about 5 psu)?
2. What is the influence of temperature and other environmental factors on nutrient-dependent growth kinetics?
3. What values for yield coefficients are appropriate to model P. parvum growing under differing environmental conditions?
4. What is the coupling (or lack of it) between abundance, cell composition, and toxicity in P. parvum growing under differing environmental conditions?
5. Do competing algae suppress bloom formation or toxicity of P. parvum?
6. Do grazers suppress bloom formation or toxicity of P. parvum?
7. What is the coupling (or lack of it) between growth, abundance, and toxicity of P. parvum when in a setting with competitors and grazers present?

Field Research Questions:

  1. Will characteristics of filtered water from Lake Waco (dissolved constituents and very small particles, e.g. viruses) prevent initiation of toxic P. parvum blooms in Lake Whitney by suppressing proliferation or toxicity?
  2. Will characteristics of the competitor and grazer assemblage from Lake Waco prevent initiation of toxic P. parvum blooms in Lake Whitney by suppressing proliferation or toxicity?
  3. Do losses from zooplankton grazing or mortality due to pathogens or both terminate P. parvum blooms in Lake Whitney?

Major Findings:

Publications:
Baker, J.W., J.P. Grover, B.W. Brooks, F. Ureña-Boeck, D.L. Roelke, R. Errera, and R.L. Kiesling. 2007. Growth and toxicity of Prymnesium parvum (Haptophyta) as a function of salinity, light and temperature. J. Phycol. 43: 219-227.

Errera, R.M., D.L. Roelke, R. Kiesling, B.W. Brooks, J.P. Grover, L. Schwierzke, F. Ureña-Boeck, J.W. Baker, J.L. Pinckney. 2007. The effect of nitrogen and phosphorus availability, barley straw extract, and immigration on Prymnesium parvum community dominance and toxicity: Results from in-lake microcosm experiments, Texas, USA. Aquat. Microb. Ecol. (In Review).

Grover, J.P., J.W. Baker, F. Ureña-Boeck, B.W. Brooks, R.M. Errera, D.L. Roelke, R.L. Kiesling. 2007. Laboratory tests of ammonium and barley straw extract as agents to suppress abundance of the harmful alga Prymnesium parvum and its toxicity to fish. Water Res. (in press).

Roelke, D. L., R. M. Errera, R. Kiesling, B. W. Brooks, J. P. Grover, L. Schwierzke, F. Ureña-Boeck, J. Baker, and J. L. Pinckney. 2007. Effects of nutrient enrichment on Prymnesium parvum population dynamics and toxicity: Results from field experiments, Lake Possum Kingdom, USA. Aquat. Microb. Ecol. 46: 125-140.

Oral presentations:
Brooks, B.W., Ureña-Boeck, F., Roelke, D.L., Errera, R., Kiesling, R.L. and Grover, J.P. Prymnesium parvum impacts on freshwater zooplankton: Laboratory and field studies. Meeting of the American Society of Limnology and Oceanography, Victoria, Canada, 2006.

Errera, R.M., Roelke, D.L., Kiesling, R.L., Brooks, B.W., Grover, J.P., Ureña-Boeck, F. and Pinckney, J. The role of inorganic nutrients and barley straw extract in the invasion and inhibition of Prymnesium parvum. Meeting of the American Society of Limnology and Oceanography, Victoria, Canada, 2006.

See also 2003 & 2004 P. parvum Research,
Developing a Predictive Understanding of Prymnesium parvum Toxic Bloom Formation and its Control

Development of Molecular Biomarkers for Determining Prymnesium parvum
Bloom Status and Ichthyotoxicity

Dr. John W. La Claire II (University of Texas at Austin)
Dr. David L. Herrin (University of Texas at Austin)

Objective:
The primary objective of this study is to identify specific genes and corresponding DNA sequences that will serve as biological markers (“biomarkers”) for different stages of P. parvum bloom development and toxin production. This work will utilize DNA microarray technology and the 6,286 P. parvum expressed sequence tags (ESTs) that we developed with previous TPWD/USFW funding. [The database of expressed gene sequences from late log-phase P. parvum cells is publicly available.] Collectively, these ESTs comprise 3,415 unique genes that are to be probed for suitable biomarkers. The selected biomarkers will provide the resources and groundwork necessary for developing hand-held tools for identifying the organism, and moreover, for potentially determining bloom growth and toxicity status.

Progress Report:

Publications/Presentations:
La Claire, J.W., II. 2006. Analysis of expressed sequence tags from the harmful alga Prymnesium parvum (Prymnesiophyceae, Haptophyta). Marine Biotechnology 8:534-546.

La Claire, J.W. 2006. Functional genomics of the harmful alga, Prymnesium parvum (Haptophyta). Talk presented at the 60th Annual Meeting of the Phycological Society of America, at the University of Alaska Southeast, Juneau, July, 9, 2006. Abstract published online at: http://www.psaalgae.org/ops/PSA2006Program.pdf (pp. 34-35)

Manning, S.R. 2006. Multiplex polymerase chain reaction (PCR) method for the rapid and sensitive species-specific detection of the harmful alga, Prymnesium parvum Carter (Haptophyta). Thesis submitted in partial fulfillment of the Masters of Arts Degree in Plant Biology, The University of Texas at Austin (Supervisor: John W. La Claire II) [THESIS 2006 M3165 Electronic Information Center PCL 2.200].

Manning, S.R., and J.W. La Claire II. 2005. A multiplex PCR method for species-specific detection and quantification of the harmful alga, Prymnesium parvum (Haptophyta). Abstract of a poster presented at the Botany 2005 Meetings (Botanical Society of America) in Austin, TX, August 16, 2005. Abstract published online at: http://www.2005.botanyconference.org/engine/search/index.php?func=detail&aid=51

See also 2003 & 2004 P. parvum Research,
Development of a DNA-based Assay & Partial Genome Analysis of Prymnesium parvum

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Aditional Information:

Would you like to know more?
The Biology of Golden Alga summarizes what we know about the alga and its toxins.

Where does golden alga fit compared to other single-celled organisms?
The Golden Alga Family Tree gives examples of and information about golden alga and other protists.

What does golden alga look like?
TPWD Golden Alga Images has photos of fish kills, golden algal cells, and short videos of live golden alga. These images may be used for noncommercial/educational purposes as long as TPWD is given credit and other site policies are followed.

Golden Alga Information Card: TPWD has collaborated with TCEQ and other entities to produce a golden alga information card(pdf document). The purpose of this card is to educate the public on golden alga blooms and answer some common questions. Hard copies of this card are available for free by contacting 512 389-8750.

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