Final Report: Linking Population and Physiological Diversity in a Toxin-producing Dinoflagellate

EPA Grant Number: R830413
Title: Linking Population and Physiological Diversity in a Toxin-producing Dinoflagellate
Investigators: Campbell, Lisa , Gold, John R.
Institution: Texas A & M University
EPA Project Officer: Hiscock, Michael
Project Period: September 1, 2002 through August 31, 2005 (Extended to February 28, 2007)
Project Amount: $464,880
RFA: Ecology and Oceanography of Harmful Algal Blooms (2002) RFA Text |  Recipients Lists
Research Category: Water Quality , Ecosystems , Water , Aquatic Ecosystems

Objective:

Blooms of Karenia brevis along the Texas coast are increasing in frequency, yet the source population and specific factors influencing bloom initiation and intensity are poorly understood. Initial results from the laboratory demonstrated genetic diversity among isolates of K. brevis from Texas waters. Such genetic diversity may serve as a repository from which populations bloom in response to appropriate environmental conditions. These results emphasized the need for new, hypervariable genetic markers that can be utilized in population- and species-level studies of harmful algal blooms (HAB). The primary objective of the research was to develop molecular tools for examining genetic diversity within and among blooms of K. brevis. Specific objectives were the following: (1) isolate, develop, and optimize a suite of hypervariable, nuclear-encoded DNA markers (microsatellites) to characterize genetic diversity among isolates of K. brevis; (2) establish clonal cultures of K. brevis during the onset, bloom, and decline of a HAB event in order to assess genetic and physiological variability within a bloom; and (3) test the following null hypotheses: (a) spatial/temporal samples from a single bloom are genetically homogeneous; and (b) geographic isolates of K. brevis from the northern Gulf are genetically homogeneous. The long-term objective of the project is to utilize the genetic information to provide a better understanding (and predictability) of the dynamics of this HAB species.

Summary/Accomplishments (Outputs/Outcomes):

A Suite of Hypervariable Nuclear-Encoded Genetic Markers Known as Microsatellites Were Developed in Order To Characterize Individual Blooms and Test Whether Spatial and/or Temporal Genetic Differences Existed Between or Among Blooms

PCR primer pairs for nine microsatellites and for one complex repeat that appears to be a satellite DNA sequence were generated from a genomic library of K. brevis DNA and evaluated via PCR amplification and sequencing of amplified products. The microsatellite markers are di- or tri-nucleotide (repeat) motifs (Table 1). Size polymorphism was found for all microsatellite markers, and the satellite DNA marker among the 28 spatial and temporal isolates of K. brevis wasexamined; the number of alleles per genetic marker ranged from 3–10 (Table 1). Primer pairs have now been multiplexed for efficient amplification and scoring of alleles (e.g., Figure 1) in cultures and for single cells.

Table 1. Summary of Microsatellite Markers Developed for Genotyping K. brevis. Sequences are given for forward (top) and reverse (bottom) PCR primers; annealing temperature for all is 52°C.

Table 1. Summary of Microsatellite Markers Developed for Genotyping K. brevis.

Figure 1. Example of a Multiplex Assay for Genotyping K. brevis.

Figure 1. Example of a Multiplex Assay for Genotyping K. brevis. Chromatograms for microsatellites Kbr6 (smaller fragment) and Kbr4 (larger fragment) are on the top row (blue = FAM dye); microsatellite Kbr3 is on the middle row (green = HEX dye); and microsatellite Kbr1 is on the bottom row (yellow/black = NED dye). Number labels represent number of base pairs for the corresponding DNA fragment (peak).

One additional microsatellite identified had characteristics of a randomly amplified polymorphic DNA (RAPD) marker. The primer pair for this marker (referred to as 2A5) generated a series of multiple bands that allowed rapid, straightforward separation of the two HAB species of Karenia (K. brevis and Karenia mikimotoi) in the Gulf of Mexico. Amplification of DNA from several isolates of K. brevis and K. mikimotoi, using the PCR primer pair for marker 2A5, revealed banding patterns that were identical for all isolates within each species but that differed markedly (and repeatedly) between the two species and among a number of other dinoflagellate genera.

Distinguishing the two Karenia species (K. brevis and K. mikimotoi) by characteristic sizes (base pairs; bp) of fragments generated by the primers for marker 2A5 may prove useful as a rapid taxonomic assay, as HAB blooms in the Gulf of Mexico are rarely composed of a single species.

Recent blooms observed in Florida, for example, have included several species that, in some cases, have been dominated by K. mikimotoi (C. Heil, personal communication). Amplification of genomic DNA from other dinoflagellate species—including representatives of Amphidinium, Lingulodinium, Prorocentrum, Gymnodinium, Symbiodinium, and Pyrocystis—with the 2A5 primer pair has revealed species-specific patterns, which indicate that marker 2A5 may have broad application as a species-specific diagnostic tool.

Assess Genetic and Physiological Variability Within a Bloom

Over the initial 3-year period of this project, there were no blooms of K. brevis in waters offshore of Texas, and difficulties were encountered in transporting samples from Florida. Because of this, alternative protocols were developed in order to provide sufficient material for analysis. A simple and effective protocol was developed for a multiplex PCR amplification of single cells of K. brevis from bloom samples that had been stored in Lugol’s preservative. Development of this protocol permitted us to address the question of genetic variability within a bloom population and among bloom populations over historical time.

Multiplex PCR Amplification of Single Cells of K. brevis. The protocol is a two-step process in which the first multiplex PCR includes six primer pairs; subsequently, the product is then amplified in six individual reactions with a single primer pair. This procedure requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol’s iodine preservative. Destaining of Lugol’s-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years. The capability to work with preserved material permits examination of archived samples (when stored refrigerated) for analysis of allele distributions within prior blooms. This approach will minimize the need to obtain large cultures for DNA extraction.

Genetic Diversity Among Established Clonal Cultures of K. brevis. All available cultures of K. brevis were genotyped at each of the 10 microsatellites; allele distributions are shown in Figures 2 and 3. Distinct genotypes were observed among each of the Texas clones isolated from the 1999 bloom (TX001, TX002, TX003, TX004, CCFWC266, and CCFWC267). Analysis of the “toxic” (TX003) and “nontoxic” strains (TX004) by liquid chromatography-mass spectrometry (LC-MS) revealed the “nontoxic” strain to contain brevenal, the nontoxic polyether compound that acts as a brevetoxin antagonist. Growth rate and toxicity measurements also support variability among clones of K. brevis (see below).

Figure 2. Allele Distributions at Each of the 10 Microsatellites Among 28 Clonal Cultures of K. brevis Isolated From Blooms Offshore of Texas and Florida

Figure 2. Allele Distributions at Each of the 10 Microsatellites Among 28 Clonal Cultures of K. brevis Isolated From Blooms Offshore of Texas and Florida

Figure 3. Gel Image for Genotypes of Isolates of K. brevis From Waters of Florida and Texas.

Figure 3. Gel Image for Genotypes of Isolates of K. brevis From Waters of Florida and Texas. Upper portion of figure: Multiplex reaction I (Kbr 8, 9, 11, 1, 4); Texas: Lane 1: TX001, 2: TX002, 3: TX003, 4: TX004, 5: CCFWC266, 6: CCFWC267, 7: TX005, 8: CCFWC269, 9: C2, 10: C5; Florida: Lane 11: CCMP718-2004, 12: CMP2228, 13: CCMP2229, 14: CCMP2281, 15: NOAA-1, 16: C4, 17: CCFWC254, 18: CCFWC253, 19: CCFFWC263, 20: CCFWC265, 21: CCFWC250, 22: CCFWC251, 23: CCFWC252, 24: CCFWC258, 25: CCFWC259, 26: CCFWC260, 27: CCFWC261, 28: CCFWC262, 29: CCFWC256, 30: CCFWC257. Lower portion of figure: Multiplex reaction II (Kbr 10, 5, 6, 3, 7): Lanes as in Upper portion of figure.

Field Results. In September 2005, a bloom of K. brevis occurred along the South Texas coast. With the cooperation of Texas Parks and Wildlife, bloom samples were collected weekly, and in some cases daily, at a number of locations from Brownsville north to Corpus Christi, TX. Genotyping of these samples will provide data to assess the spatial and temporal variation in population structure. Results for one station are shown in Figure 4. A comparison of allele distributions for the existing culture collections and the 2005 bloom sample revealed a greater number of alleles present in the field sample (Figure 5). This observation suggests that our culture collection does not represent fully the diversity among bloom populations of K. brevis.

Figure 4. Gel Image for Genotypes at the Kbr 10 Locus of 96 Single Cells From Corpus Christi Bay, TX, Collected and Preserved in October 2005

Figure 4. Gel Image for Genotypes at the Kbr 10 Locus of 96 Single Cells From Corpus Christi Bay, TX, Collected and Preserved in October 2005

Figure 5. Distribution of Alleles at Six Microsatellites Among Established Cultures of K. brevis

Figure 5. Distribution of Alleles at Six Microsatellites Among Established Cultures of K. brevis (as in Figure 3) and for Single Cells (n > 85) From a Bloom in Corpus Christi Bay in October 2005. The number of alleles at most microsatellites in the October 2005 bloom sample (green bars) exceeded the number present in established cultures (blue).

Physiological Diversity Among K. brevis Isolates. For genetically distinct clones of K. brevis cultured under varying salinity and temperature, growth rates, toxicity, and toxin production were determined to assess physiological variability within and among haplotypes of K. brevis. Experiments were conducted using eight existing clonal isolates of K. brevis (five isolates from the 1999 bloom off South Padre Island, near Brownsville, TX, and three isolates from the western Florida region) grown under a range of environmentally relevant temperatures and salinities. Average growth rates ranged from 0.15 to 0.37 d-1, with highest growth rates observed for cultures grown at salinities of 30 practical salinity units (PSUs). Considerable variability in growth rate was observed among clonal cultures grown under similar conditions. Variation in maximum growth rate appeared to be limited by salinity (Figure 6).

Figure 6. Specific Growth Rate (d[-1]) for Clones of K. brevis Isolated From a 1999 Bloom off Brownsville, TX (Left) and Florida (Right).

Figure 6. Specific Growth Rate (d-1) for Clones of K. brevis Isolated From a 1999 Bloom off Brownsville, TX (Left) and Florida (Right). Growth rates were calculated from chlorophyll a fluorescence measured on a Turner fluorometer for cultures at 25°C, 70 μEinst m-2s-1 at salinities from 20 to 35.

Development of a New Assay To Determine Toxicity Due to Hemolytic Activity.A modified assay for hemolysis was developed using red drum (Sciaenops ocellatus), an environmentally relevant fish species, as the source of erythrocytes in order to quantify differences in hemolytic activity among clonal isolates of Karenia (Neely and Campbell, 2006). Assays were conducted with eight clones of K. brevis (five from Texas waters and three from Florida waters) and four clones of K. mikimotoi grown under the same range of temperatures and salinities as in growth rate experiments above. Average hemolytic activity for the clones of K. mikimotoi ranged from 30–80% of the saponin standard; average hemolytic activity for the K. brevis clones ranged from 23–75% of the saponin standard (Figure 7). The highest hemolytic activity was observed in K. mikimotoi clone B1, suggesting that K. mikimotoi may play a previously unrecognized role in fish kills in the Gulf of Mexico. We hypothesize that the finding of variable toxicity among strains of these two species of Karenia may explain part of the variability in toxicity and fish kills among HAB events.

Figure 7. Hemolytic Activity

Figure 7. (A) Hemolytic Activity (As % of Saponin Standard) Varied Approximately 3-Fold Among K. brevis and K. mikimotoi Clonal Isolates Grown Under Identical Conditions of 25°C, 70 μEinst m-2s-1, and a Salinity of 30. (B) Hemolytic activity varied among K. brevis clones when grown at three salinities, 25°C, and 70 μEinst m-2s-1. Brevetoxin (PbTx-3) showed no activity in this assay.

Hypothesis Testing

Spatial/Temporal Samples from a Single Bloom are Genetically Homogeneous. The range in alleles is greater among the samples from Corpus Christi than among the laboratory cultures (Figure 5). This suggests, again, that diversity of natural populations may not be well represented by culture collections.

Geographic Isolates of K. brevis from the Northern Gulf are Genetically Homogeneous. Results to date suggest that allele distributions are not correlated with bloom (year) or location (Florida vs. Texas) and lead to rejection of our hypothesis that geographic isolates are homogeneous. Note that the dominant allele for both TX and FL isolates is the same (Figure 2).

Conclusions:

Hypervariable genetic markers that are useful for cataloguing clones of K. brevis using both live material and Lugol’s-preserved material have been developed. The capability to distinguish genetically among strains of K. brevis is now possible in both real time and from archived samples. Successful PCR amplification of single Karenia cells from Lugol’s-preserved material will be extremely useful for population-genetic studies, as it is possible to routinely genotype 50–100 cells per sample; this would not be feasible if 50-100 individual clonal cultures per sample were required. Results based on the existing clonal cultures available to date suggest that allele distributions are not correlated with bloom (year) or location (Florida vs. Texas).

A new assay to determine hemolytic activity of toxins from species of Karenia has been developed (Neely and Campbell, 2006). Hemolytic activity observed in extracts of K. mikimotoi, was, in some cases, greater than in K. brevis. We hypothesize that this finding of variable toxicity among strains of these two species of Karenia may explain part of the variability in toxicity and fish kills among HAB events.

With these data, a genetic database for K. brevis on both spatial and temporal scales has been established for the Gulf of Mexico. The diversity in growth rate, hemolytic activity, and brevetoxin and brevenal production among clones examined from this project provides the basis to link allelic profiles and toxicity. Ultimately, this information will provide the capability to predict how environmental factors influence toxicity or potency of blooms of Karenia.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 21 publications 6 publications in selected types All 5 journal articles
Type Citation Project Document Sources
Journal Article Henrichs DW, Renshaw MA, Santamaria CA, Richardson B, Gold JR, Campbell L. PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol’s iodine solution. Marine Biotechnology 2008;10(2):122-127. R830413 (Final)
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  • Other: Texas A&M
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  • Journal Article Mikulski CM, Morton SL, Doucette GJ. Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA. Harmful Algae 2005;4(1):49-60. R830413 (2004)
    R830413 (Final)
  • Full-text: Science Direct
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  • Abstract: Science Direct
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  • Other: Science Direct
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  • Journal Article Neely T, Campbell L. A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico. Harmful Algae 2006;5(5):592-598. R830413 (2005)
    R830413 (Final)
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  • Abstract: Science Direct
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  • Journal Article Renshaw MA, Soltysiak K, Arreola D, Loret P, Patton JC, Gold JR, Campbell L. Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis. Molecular Ecology Notes 2006;6(4):1157-1159. R830413 (Final)
  • Full-text: Molecular Ecology Notes
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  • Abstract: Blackwell Synergy Abstract
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  • Journal Article Van Dolah FM, Lidie KB, Monroe EA, Bhattacharya D, Campbell L, Doucette GJ, Kamykowski D. The Florida red tide dinoflagellate Karenia brevis: new insights into cellular and molecular processes underlying bloom dynamics. Harmful Algae 2009;8(4):562-572. R830413 (Final)
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  • Abstract: ScienceDirect
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  • Supplemental Keywords:

    RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Oceanography, algal blooms, Biochemistry, Ecological Risk Assessment, Ecology and Ecosystems, marine ecosystem, toxin monitoring programs, marine biotoxins, bloom dynamics, brevetoxins, phytoplankton, gene sequences, dinoflagellate, algal bloom detection, dinoflagellate blooms, K. brevis, Karenia brevis

    Progress and Final Reports:

    Original Abstract
  • 2003 Progress Report
  • 2004 Progress Report
  • 2005 Progress Report
  • 2006