Golden Alga Research Abstracts

Development of a DNA-based Assay & Partial Genome Analysis of Prymnesium parvum
Dr. John W. La Claire II, University of Texas at Austin

A sensitive and accurate method for early detection of this harmful alga is needed to determine it’s true range and to provide for early response in advance of toxic blooms. DNA-based tests offer unprecedented advantages and are being employed increasingly for the detection of micro-organisms. However, there is little published genetic information on P. parvum with which to develop species-specific DNA tests. Moreover, the absence of molecular data and the relevant tools is also hindering the study of what causes blooms to develop and what controls toxin production in this species. Therefore, our objectives are to: 1) construct and characterize a P. parvum cDNA library for developing species-specific tests for the organism and to obtain new molecular data, 2) develop a real-time PCR-based assay for accurately detecting and quantifying P. parvum in raw water samples, and 3) make the molecular data freely available to the public and researchers using publicly accessed databases. The real-time PCR method will allow accurate identification and quantification of P. parvum, even in places where it occurs below detectable levels using current morphological methodologies. A library of partial cDNA sequences will expedite the discovery of genes whose products are responsible for bloom formation and possibly those involved in toxin synthesis.

Progress Report:

We developed a set of species-specific DNA probes for P. parvum, for:

• sensitive and rapid detection of the alga from field-collected water samples, for early response in advance of toxic blooms
• quick and accurate identification of the alga from field-collected water samples to determine the alga’s geographical range
• rapid quantitation of cell numbers in field-collected water samples, eliminating the need for manual cell counts

We constructed an “expressed sequence tag” (partial mRNA sequence) database of ~3,500 genes from rapidly-growing P. parvum cultures, which:

• adds ~6,300 new P. parvum sequences to GenBank and the Plant Genome Network
• provides basic biological information on the physiological/metabolic status of bloom- forming cells
• complements the forthcoming genome project on Emiliania huxleyi (a coccolithophorid haptophyte alga)
• provides molecular tools for future studies investigating patterns of gene expression during bloom formation, collapse, and during toxin production, aimed at:
• identifying the biochemical pathways involved in toxin synthesis and secretion
• seeking pathways that might serve as targets for mitigation of bloom formation
• developing suites of gene probes to serve as biomarkers for P. parvum

Citations / Presentations:

Manning, S.R., and J.W. La Claire, 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.

La Claire, J.W., II. 2006. Analysis of expressed sequence tags from the harmful alga, Prymnesium parvum (Prymnesiophyceae, Haptophyta). Mar. Biotechnol. 8: In Press (accepted for publication March 19, 2006).

Statewide Survey for Prymnesium parvum
Loraine Fries, Texas Parks and Wildlife Department

Fish kills attributable to P. parvum have been reported in west Texas since 1985 when the organism was identified in association with a fish kill in the Pecos River. Recently, the range of P. parvum appears to be expanding, as evidenced by more frequent kills in previously unaffected areas. However, it is unknown if the range is truly increasing or if factors that contribute to blooms are becoming more widespread and common place.

It is important to know the current distribution of P. parvum in Texas in order to determine if it is spreading. While not substantiated, there is concern that TPWD Inland Fisheries stocking activities, bait minnow distribution, and boat traffic have contributed to the spread of P. parvum. While the organism is reported to inhabit brackish water worldwide, its range in Texas is unknown. A thorough understanding of the distribution of P. parvum is required in order to develop meaningful hazard analysis and planning to avoid potentially harmful incidental transport and spread of P. parvum. Additionally, it is important to understand what environmental characteristics area associated with P. parvum presence and, especially, the development of toxic blooms. Such understandings are required if meaningful mitigation methods are to be developed.

The Texas Parks and Wildlife Department, with extensive cooperation from the Texas Commission on Environmental Quality Clean Rivers and Surface Water Quality Monitoring programs, has initiated a multi-agency cooperative effort to determine the distribution of P. parvum in Texas and the environmental characteristics associated with its presence and toxic bloom formation. Additional cooperators include the following: the Brazos River Authority, Colorado River Municipal Water District, Dallas Water Utilities, International Boundary and Water Commission, Guadalupe-Blanco River Authority, Lavaca-Navidad River Authority, Lower Colorado River Authority, North Texas Municipal Water District, Nueces River Authority, Red River Authority Sabine River Authority, Tarrant Regional Water District, Texas Institute for Applied Environmental Resources, Trinity River Authority, and the Upper Colorado River Authority. Through this cooperative effort, a total of about 120 sites representing every major drainage in Texas will be sampled quarterly for a one-year period. Sampling began in late March 2004 and through August 2004, a total of 198 samples have been collected for the project.

For each sample collected, a variety of water quality characteristics are measured. These include physicochemical measurements, nutrients, cations, and metals. Additionally, samples are examined for the presence of P. parvum. If the organism is detected, the cells are counted and bioassays are conducted to evaluate toxicity. Algal cells are harvested using centrifugation and flash frozen at ultracold temperatures for later genetic analysis. When all the data are collected, a distribution map and environmental correlates associated with P. parvum will be produced. Additionally, archived cells can be used in a variety of genetic analyses, including checking for strain differences and genetic markers associated with toxin production.

Progress Report

• 421 samples from 66 waterbodies (14 major river basins) were collected with associated parameters measured; bioassays were conducted for toxicity for all samples with P. parvum cells present; ~90% of the data from collecting partners has been received.
• Equipment for the real-time PCR assay for quantification of archived samples was transferred from the university laboratory to the hatchery laboratory; PCR quantification is pending/in-progress.
• Equipment for future genetic characterization (based on PCR application) was acquired (thermalcycler and DNA analyzer).

Citations / Presentations:

Fries, L.T., G.M. Southard, A. Barkoh, D. Buzan, D. Sager, J. Glass, and L. Singhurst. 2005. Golden Alga Prymnesium parvum: Monitoring and Research in Texas. Aquaculture America Meeting. New Orleans, USA. January 19.

Historic Data Assessment of Golden Alga Fish Kills
Liz Singhurst and members of the Kills and Spills Team, 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

• Historic fish kills due to toxic P. parvum were grouped into events based on location and time of occurrence; information relative to each event was collected and organized by river basin.
• The Colorado River Basin was the first focus for assessment, and environmental data (water quantity and flow, water quality, and meteorological data) was obtained for the time period surrounding and including the fish kills for nearby affected areas and non-affected areas (for comparison)
• A draft report for the 2001-2002 Colorado River fish kill event is in review:
• Almost 2.3 million fish (primarily small threadfin shad) were killed in seven fish kills, which included Moss Creek Lake, E.V. Spence Reservoir, Colorado City Lake, the Colorado River above O.H. Ivie Reservoir, and Wadley-Barrow Park Pond in Midland.
• Two waterbodies, E.V. Spence Reservoir and Colorado City Lake, each had two fish kills separated by a period without a fish kill. No apparent ecological differences exist between the periods with and without fish kills.
• The region was in a long-term drought before and during the fish kills.
• Hardness, conductivity, chlorides, TDS, and sulfates were higher in affected reservoirs than non-affected reservoirs.

Citations / Presentations:

Singhurst, L., J. Glass, J. Ralph, and D. Sager. 2006. Prymnesium parvum Fish Kills in the Colorado River Basin, TX (Oct. 2001 to Feb. 2002). Texas River and Reservoir Management Society Conference. Austin, TX. May 18-19.

Singhurst, L., J. Glass, J. Ralph, D. Sager, and D. Buzan. 2005. Assessment of Historical Golden Alga (Prymnesium parvum) Events. Golden Alga Researchers' Workshop. Golden Alga Task Force. Texas Parks and Wildlife Department. Austin, TX. May 15-16.

Singhurst, L. 2005. Prymnesium parvum Fish Kills in Colorado River Basin, Texas, from October 2001 to February 2002. Joint Meeting of the Oklahoma-Texas Aquatics Research Group and Texas River and Reservoir Management Society. University of Oklahoma Biological Station. Willis, OK. October 13-15.

Research/Studies Progressing under the Texas Legislatively-authorized funds:

Concentrated Monitoring of a Prymnesium parvum Bloom
David Buzan, Texas Parks and Wildlife Department

Goal: Describe the factors that influence the distribution of Prymnesium parvum, its toxin and toxic effects during a bloom and through the conclusion of a bloom.

Objectives:

1. Identify the physical, chemical and biological conditions that are present before blooms, during blooms and as blooms end;
2. Describe the factors influencing the distribution of Prymnesium parvum, its toxin and toxic effects during a bloom and through the conclusion of a bloom;
3. Identify of conditions that trigger blooms; and
4. Collect information supporting development of models to evaluate ways to prevent, manage or mitigate the impacts of future blooms.

Location: Brazos River at Graham downstream through Lake Whitney and downstream of Lake Whitney as necessary to follow blooms.

Progress Report:

• Blooms of golden alga are most intense in the upper reaches of Lake Whitney, particularly from Steele Creek Park to Allen's Bend.
• Even though blooms are most intense in the upper reaches of the lake, when golden alga bloom is present it can kill fish throughout the reservoir.
• Toxic blooms of golden alga seem to affect fish the worst during late winter.
• The toxic blooms affect fishes' gills, causing the gills to look like they've had chemical burns.
• Some possible patterns that we need to explore more are that golden alga blooms in the lake appear more toxic:
  • when phosphorus (an important nutrient) concentrations are lower
  • when temperatures are lower
  • when zooplankton (the microscopic animals floating in the water that might eat golden alga) numbers are lower
  • when other types of floating algae (microscopic plants) are less abundant

Citations / Presentations:

Buzan, D., M. Byrd, L. Singhurst, and J. Nelson. 2005. Marmot Trapping on Lake Whitney Spring 2004 and 2005. Texas Commission on Environmental Quality. Surface Water Quality Monitoring Annual Meeting. Bandera, TX. October.

Byrd, M. 2005. Prymnesium parvum Monitoring in Lake Whitney. Joint Meeting of the Oklahoma-Texas Aquatics Research Group and Texas River and Reservoir Management Society. University of Oklahoma Biological Station. Willis, OK. October 13-15.

Buzan, D., M. Byrd, L. Singhurst, and J. Nelson. 2005. Monitoring Prymnesium parvum Blooms in Lake Whitney (aka Marmot Trapping on Lake Whitney), TPWD Preliminary Sampling Results for 2005. Texas River and Reservoir Management Society Annual Meeting. Baylor University. Waco, TX. May 15-16.

Buzan, D. and M. Byrd. 2005. Golden Alga Monitoring. Public Meeting. Texas Parks and Wildlife Department. Lake Whitney (Lakeside Village), TX. March.

Effects of Barley Straw and Liquid Live Micro-Organisms on Density and Ichthyotoxicity of Prymnesium parvum in hatchery ponds
Dr. Aaron Barkoh, Texas Parks and Wildlife Department

Objectives:

1. Determine if decomposing barley straw inhibits P. parvum population growth and ichthyotoxicity;
2. Determine if the Liquid Live Micro-Organisms system inhibits P. parvum density and ichthyotoxicity;
3. Determine effects of decomposing barley straw on water quality, nutrient levels, phytoplankton community structure, zooplankton community structure and fish production; and
4. Determine effects of the Liquid Live Micro-Organisms system on water quality, nutrient levels, phytoplankton community structure, zooplankton community structure and fish production.

Results:

• Barley straw or LLMO did not eliminate P. parvum or inactivate its toxin.
• Barley straw or LLMO had no significant effects on fish production, zooplankton densities, and nutrient levels.

Barley Straw Final Report(PDF 202.3 KB)

Barkoh, A., J.M. Paret, D.C. Begley, D.D. Lyon, D.G. Smith, and J.Warren Schlechte. 2007. Evaluation of barley straw and Liquid Live Micro-Organisms System for controlling Prymnesium parvum in fish hatchery ponds. Final Report to Texas Parks and Wildlife Department. PWD RP T3200 1430.

Barkoh, A., J.M. Paret, D.C. Begley, D.D. Lyon, D.G. Smith, and J.W. Schlechte. 2007. Evaluation of barley straw and a commercial probiotic for controlling Prymnesium parvum in fish production ponds. accepted by North American Journal of Aquaculture.

Research/Studies Progressing under the Texas Legislatively-authorized funds:

Developing a Predictive Understanding of Prymnesium parvum Toxic Bloom Formation and its Control
Dr. Richard L. Kiesling (US Geological Survey),
Dr. Bryan W. Brooks (Baylor University),
Dr. James P. Grover (University of Texas at Arlington),
Dr. Daniel L. Roelke (Texas A&M University)

Scope of Research:
We propose to carry out an integrated research program focused on developing a predictive understanding of P. parvum toxic bloom formation and its control. Our investigation will assess P. parvum bloom dynamics by measuring the in-reservoir response of the phytoplankton community to experimental manipulation of each reservoir's planktonic foodweb, by assessing the population growth physiology of P. parvum under the influence of potential limiting factors, and by assessing the toxicity response of P. parvum under these changing conditions.

Our results will provide an understanding of how P. parvum populations respond to direct intervention (i.e., additions of barley straw) as well as to ecological controls more suitable to entire reservoir systems (i.e., nutrient supply, trophic dynamics). Relative toxicity tests will establish linkages between toxicity and physiological status of P. parvum populations, and comparisons of various toxicity endpoints will assess the potential of using faster, more economical tests of toxicity in assessing system risk. The research will provide a predictive model of P. parvum growth capable of application to a number of reservoir systems.

Objectives:

1. Assess the in-field population dynamics of a toxic P. parvum bloom in Possum Kingdom Reservoir using in-reservoir enclosure experiments;
2. Perform laboratory chemostat and semi-continuous experiments designed to elucidate physiological and toxicological characterizations of P. parvum cultures;
3. Perform bioassays with samples from field experiments and laboratory cultures to identify toxic conditions caused by P. parvum; and
4. Develop and validate a predictive model of P. parvum population demographics.

Major Findings

• Because direct chemical analysis of P. parvum toxins remains infeasible we compared toxicity assays using three model organisms: bacteria (Vibrio fischeri), zooplankton (Daphnia magna), and juvenile fathead minnows (Pimephales promelas). Acute toxicity was strong only for fathead minnows, though sublethal effects on D. magna were occasionally noted. Acute toxicity assays with fathead minnows became our routine method for further studies.
• Three factors were tested in field experiments for their impact on P. parvum abundance and toxicity: nutrient additions, barley straw extract, and enhanced initial densities of P. parvum. Only nutrient addition had a consistent and significant effect, increasing the abundance of P. parvum and other algae, but reducing the toxicity of P. parvum to fish.
• Laboratory experiments demonstrated that the growth rate of P. parvum increases from about zero at 5° C to an optimum at about 28° C, and declines to zero at 35° C. Growth is also optimal at light and salinity levels higher than is typical in Texas inland waters. Under the suboptimal growth conditions in the cooler seasons when P. parvum blooms in Texas lakes toxicity to fish is higher than under optimal growth conditions.
• Laboratory experiments show that P. parvum growth is saturated by nitrate and phosphate at very low concentrations. P. parvum is likely to be a good competitor for these nutrients compared to other algae common in Texas inland waters.
• In laboratory experiments, growth of P. parvum is not inhibited by the addition of barley straw extract, but is inhibited by sufficiently large additions of ammonium.
• Equations for a provisional mathematical model of P. parvum dynamics have been constructed and parameterized. The model is currently under investigation for further refinement of structure and parameters.

Citations / Presentations:

Baker, J., J. Grover, B. Brooks, F. Urena-Montoya, R. Kiesling, D. Roelke. 2005. Niche Definition, Nutrient Kinetics and Possible Bloom Control of Prymnesium parvum (“Golden Algae”). Oklahoma-Texas Aquatics Research Group/Texas River and Reservoir Management Society, Oklahoma Biological Station OK, USA. October 13-15.

Baker, J., J. P. Grover, B. Brooks, R. Kiesling, D. Roelke. 2005. Growth of Prymnesium parvum (“Golden Algae”) in Relation to Temperature, Light and Salinity. Texas River and Reservoir Management Society. Waco, TX. May 15-16.

Brooks, B.W., F. Ureña-Boeck, D.L. Roelke, R. Errera, R.L. Kiesling, J.P. Grover. 2006. Prymnesium parvum Impacts on Freshwater Zooplankton: Laboratory and Field Studies. Aquatic Sciences Meeting, ASLO. Victoria, Canada. June 4-9.

Brooks, B., F. Boeck, J. Baker, R. Errerra, J. Grover, R. Kiesling, D. Roelke. 2005. Prymnesium parvum Impacts to Freshwater Organisms: Developing a Predictive Understanding of Toxicity Using a Weight-of-Evidence Approach in Laboratory and Field Investigations. Society of Environmental Toxicology and Chemistry. Baltimore, USA. November 13-17.

Brooks, B.W., F. Urena-Montoya, J.K. Stanley, M. Alvarez, R.L. Kiesling, D.L. Roelke, R. Errerra, J. Baker, J.P. Grover. 2005. A Predictive Understanding of Prymnesium parvum Toxicity to Freshwater Organisms: Initial Findings from Laboratory and Field Studies. Texas River and Reservoir Management Society. Waco, TX. May 15-16.

Errera, R.M., D.L. Roelke, R.L. Kiesling, B.W. Brooks, J.P. Grover, F. Ureña-Boeck, J. Pinckney. 2006. The Role of Inorganic Nutrients and Barley Straw Extract in the Invasion and Inhibition of Prymnesium parvum. Aquatic Sciences Meeting, ASLO. Victoria, Canada. June 4-9.

Errera, R., D.L. Roelke, J. Pinckney. 2005. Invasion Success of Prymnesium parvum in a Deep, Semitropical Reservoir: The Role of Inorganic Nutrients. Aquatic Sciences Meeting, ASLO. Salt Lake City, USA. February 20-25.

Errera, R., D. Roelke, R. Kiesling, B. Brooks, F. Ureña-Montoya, J. Grover, J. Pinckney. 2005. The Role of Inorganic Nutrient, Barley Straw Extract, and Initial Population Densities in the Invasion Success of Prymnesium parvum in Filtered Assemblages. Oklahoma-Texas Aquatics Research Group/Texas River and Reservoir Management Society, Oklahoma Biological Station OK, USA. October 13-15.

Errera, R., D. Roelke, J. Pinckney, R. Kiesling, J. Grover, and B. Brooks. 2005. Optimization of ChemTax for Prymnesium parvum Nutrient Enrichment Experiments in Lake Whitney, TX. Texas River and Reservoir Management Society. Waco, TX. May 15-16.

Grover, J.P., J. Baker, F. Urena-Montoya, B. Brooks, R. Kiesling and D. Roelke. 2005. Growth, Toxicity, and Composition of Prymnesium parvum in Relation to Temperature, Light, and Salinity. 3rd Symposium on Harmful Algae in the U.S. Monterey, CA. October 2-7. Abstract on-line (p. 105).

Roelke, D.L., R. Errera, R. Kiesling, B. Brooks, F. Ureña-Montoya, J. Grover, J. Pinckney. 2005. Prymnesium parvum Demographics in Limnocorral Experiments using Natural Plankton Assemblages from Possum Kingdom Reservoir: The Role of Nutrients, Barley Straw Extract, and Initial Population Density. Oklahoma-Texas Aquatics Research Group/Texas River and Reservoir Management Society, Oklahoma Biological Station OK, USA. October 13-15.

Roelke, D., R. Kiesling, R. Errera, B. Brooks, F. Ureña-Montoya, and J. Grover. 2005. The Influence of Barley Straw Extract on Prymnesium parvum in Natural Plankton Assemblages from Possum Kingdom Reservoir under Ambient and Nutrient Enriched Conditions. Texas River and Reservoir Management Society. Waco, TX. May 15-16.

Abstract for a paper in publication:
Field experiments on a harmful alga: effects of nutrient enrichment, barley straw extract, and Prymnesium parvum immigration on phytoplankton dynamics and toxicity to fish
Daniel Roelke, Reagan Errera, Richard Kiesling, Bryan W. Brooks, James P. Grover, Fabiola Ureña-Boeck, Jason Baker, James L. Pinckney

Toxic Prymnesium parvum blooms occur worldwide and result in large fish kills. In the past five-years, incidences of P. parvum blooms in inland water bodies of Texas (USA) have increased dramatically. In this research, we conducted in-lake experiments (Lake Possum Kingdom) during the fall and early-spring of 28-day duration using twenty-four 1.57 m3 enclosures. The experiments focused on the impact of nutrient enrichment, immigration of P. parvum, and addition of barley straw extract on phytoplankton biomass and assemblage structure, P. parvum population density, and toxicity to fish. Nutrient enrichment stimulated P. parvum population growth beyond bloom proportions. But P. parvum did not dominate the assemblage under these conditions, as it does during natural blooms. Instead, euglenophytes and chlorophytes dominated. Toxicity to fish, which is linked to P. parvum’s allelopathic and mixotrophic effectiveness, was greatly reduced (eliminated in many cases) under conditions of nutrient enrichment. Outlier phytoplankton assemblage dynamics (divergent trajectories) were not observed with the addition of nutrients as they were in treatments that received no nutrient additions. This further suggests an important role of competition for limiting nutrients as a nonlinear process that could lead to chaos in plankton communities. Immigration of P. parvum into natural assemblages had little impact on phytoplankton dynamics. This suggested that local interactions, such as grazing processes, were stronger than the destabilizing effect of immigration. Finally, addition of barley straw extract had no effect on phytoplankton dynamics, which suggested that degradation of oxidized polyphenolics occurred over times-scales shorter than our 7-day sampling period.

Estimating the Economic Impacts of Golden Algae (Prymnesium parvum) on Recreational Fishing at Possum Kingdom Lake
Dr. Robert B. Ditton, Texas A&M University

Study Purpose:
The purpose of this study is to estimate the extent of economic impact of the outbreak and continued presence of golden algae at Possum Kingdom Lake (PKL) on the local three-county area.

Objectives:

1. To expand county-level time series state sales tax data (pre and post golden algae outbreak) to total expenditures to understand changes in expenditures over time;
2. To complete personal interviews with operators/ owners of area businesses to assess the direct economic impacts of various golden algae events at PKL;
3. To complete a case study of the golden algae outbreak at PKL and to understand the extent of direct economic impacts on the study area;
4. To use an input-output model to estimate the extent of indirect and induced economic effects of the golden algae outbreak on the study area by economic sector and to demonstrate interrelationships among sectors; and
5. To suggest a protocol for monitoring recreational fishing activity and related economic impacts at PKL in the future.

Citations/Presentations:

Oh, Chi-Ok, and R.B. Ditton. 2005. Estimating the Economic Impacts of Golden Alga (Prymnesium parvum) on Recreational Fishing at Possum Kingdom Lake, Texas. Final Report to Texas Parks and Wildlife Department.

Economic Impacts Final Report (PDF 328 KB)

Testing and Evaluation of Clays and Chemical Flocculants for Management of Prymnesium parvum Blooms in Texas
Dr. Donald M. Anderson (Woods Hole Oceanographic Institution)
Dr. Mario R. Sengco (Woods Hole Oceanographic Institution)

Goal: Evaluate the use of clay minerals to potentially address, flocculate, and sediment Prymnesium parvum blooms in Texas.

Objectives:

1. To select the most effective clay minerals against Prymnesium parvum;
2. To test whether chemical flocculants alone can remove cells and/or improve cell removal in combination with clay;
3. To measure the ability of selected clays to remove Prymnesium parvum toxins from suspension; and
4. To test the clay flocculation concept in tank mesocosms in Texas.

Citations/Presentations:

Sengco, M.R., and D. M. Anderson. 2005. Removal of Prymnesium parvum through clay and chemical flocculation. Final Report to Texas Parks and Wildlife Department.

Clay Floculants Final Report (PDF 458.3 KB)

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