Golden Algae

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Golden Algae

  1. 1. Prymnesium parvum : Analysis of Pigmentation Dynamics Over Growth by Alexis Martinez Identification through Bio-Optical Signatures by Merissa Ludwig REU Program Texas A&M University Summer 2005
  2. 2. Golden Algae <ul><li>Aquatic, primitive plant </li></ul><ul><li>Scientific name Prymnesium parvum </li></ul>         Photo: Dr. Carmelo Tomas UNC Wilmington
  3. 3. <ul><li>Kingdom: Protista </li></ul><ul><li>Division: Haptophyta </li></ul><ul><li>Class: Prymnesiophyceae </li></ul><ul><li>Species: Prymnesium parvum </li></ul>
  4. 4. Golden Algae Family Tree Illustrations by Robert G. Howells, TPWD
  5. 5. Cell Characteristics <ul><li>Single-celled organism </li></ul><ul><li>Size: 8-11 µm </li></ul><ul><li>Two chloroplasts </li></ul><ul><li>Two flagella </li></ul><ul><li>Haptonema </li></ul>                                Golden Alga, Prymnesium parvum Drawing by Robert G. Howells, TPWD
  6. 6. “ Golden” Algae? <ul><li>Golden color comes </li></ul><ul><li>from the carotenoid </li></ul><ul><li>pigments </li></ul><ul><li>Color along with </li></ul><ul><li>other factors is </li></ul><ul><li>characteristic of </li></ul><ul><li>algal blooms </li></ul>
  7. 7. <ul><li>Live in brackish waters – wide range of salinities </li></ul><ul><li>Temperate areas </li></ul><ul><li>Eutrophic zones: high nutrient concentrations </li></ul>Ideal Environment for P. parvum Photo: Dave Buzan, TPWD
  8. 8. What makes this algae so important? <ul><li>Two toxins actively released into the water </li></ul><ul><li>Prymnesin 1 </li></ul><ul><li>Prymnesin 2 </li></ul><ul><li>Toxins disrupt gill function, causes hemorrhaging then death </li></ul><ul><li>Bivalves and all </li></ul><ul><li>fish species affected </li></ul><ul><li>No effect on larger </li></ul><ul><li>wildlife species </li></ul>
  9. 9. Unique Characteristics <ul><li>Mixotroph – can obtain food through photosynthesis or is also capable of eating bacteria, other algae, or protozoans </li></ul><ul><li>Allelopathic Effect – toxins </li></ul><ul><li>produced hinder/inhibit the </li></ul><ul><li>growth of competitors </li></ul>Photos by Urban Tillmann TIllmann, U. (1998) AME 14: 155-160
  10. 10. Where can we find golden algae? <ul><li>First identified in 1930’s in Holland and Denmark </li></ul><ul><li>Israel, China, England, Norway, United States, Australia, Morocco </li></ul><ul><li>thought to be found in Scotland, Germany, Spain, Bulgaria, and South Africa and many other areas </li></ul>
  11. 12. Locations found in the U.S. <ul><li>Texas </li></ul><ul><li>Alabama </li></ul><ul><li>Arkansas </li></ul><ul><li>Georgia </li></ul><ul><li>New Mexico </li></ul><ul><li>North Carolina </li></ul><ul><li>Oklahoma </li></ul><ul><li>South Carolina </li></ul><ul><li>Wyoming </li></ul><ul><li>Thought to be </li></ul><ul><li>present in </li></ul><ul><li>Nebraska </li></ul>
  12. 14. History of P. parvum in Texas <ul><li>First discovered in Texas in 1985, suspected in fish kills since 1960’s </li></ul><ul><li>From 1985-2003, over $7 million lost in 41 fish kill events </li></ul><ul><li>The rate of fish kills has increased over recent years. </li></ul>
  13. 15. Impacts of Blooms <ul><li>Environmental -kills fish </li></ul><ul><li>-toxins have been shown to inhibit growth of, and kill other phytoplankton and bacteria </li></ul><ul><li>-discoloration, foaming of water </li></ul><ul><li>Economic </li></ul><ul><li>-tourism by anglers and tourists decrease because of dead fish </li></ul><ul><li>-hatcheries </li></ul>Fish kill at Jackson Bend, Lake Granbury, 2003
  14. 16. Problem Research Solution Persistent toxic algae with the ability to decimate fish, bivalve, and other phytoplankton populations. Texas economy feels effect in declining tourism and recreation due to dead fish. Knowledge about P. parvum is limited, inability to detect indicators prior to harmful algal bloom. Immediate need for simple, rapid, and more accurate method to detect P. parvum and to identify possible indicators before harmful algal bloom.
  15. 17. Current Methods of Identification <ul><li>Microscopic Analysis: </li></ul><ul><li>- time consuming </li></ul><ul><li>- requires highly skilled technicians to identify P. parvum </li></ul>Light micrograph (phase contrast) of a water sample, containing predominantly Prymnesium parvum , collected from Lake Koronia on 11th September, 2004. Inset: Motile P. parvum cell with visible flagellae and haptonema. Bars indicate 10 μm; inset 5 μm.
  16. 18. Future Methods <ul><li>IDENTIFY </li></ul><ul><li>Optical Signature Pigment Ratios </li></ul><ul><li>DETECT </li></ul><ul><li>Remote Sensing Chemical Taxonomy </li></ul>
  17. 19. Experiment <ul><li>Triplicate flasks containing growth medium inoculated with P. parvum culture (obtained from UT culture collection of algae 22181) </li></ul><ul><li>Incubation consisted of 12/12 light /dark cycle at 19ºC </li></ul><ul><li>Flasks were rotated in incubator and stirred daily </li></ul>
  18. 20. Experiment <ul><li>Samples were taken every </li></ul><ul><li>3 days up to day 24 </li></ul><ul><li>100mL extracted for high-performance liquid chromatography </li></ul><ul><li>10mL extracted for spectrophotometer measurement, then preserved for microscopic analysis. </li></ul><ul><li>Cell counts were performed to determine growth over time </li></ul>
  19. 22. Prymnesium parvum Growth Characteristics: Analysis of Pigmentation with Cell Growth <ul><li>Alexis Martinez </li></ul>
  20. 23. Method for Assessing Phytoplankton Assemblages <ul><li>Microscopic Analysis </li></ul><ul><li>Optical Absorption </li></ul><ul><ul><li>bioassays using spectrophotometer, fluorometer to determine abs. / wavelength </li></ul></ul><ul><ul><li>High-Performance Liquid Chromatography to determine concentration of pigments </li></ul></ul>
  21. 24. Pigments <ul><li>-Chemical compounds which absorb specific wavelengths of light, means by which photosynthesis is carried out </li></ul><ul><li>-Pigments are divided into classes: </li></ul><ul><li>Chlorophylls </li></ul><ul><li>Carotenoids (carotenes and xanthophylls) </li></ul>
  22. 25. Pigments <ul><li>Chlorophylls </li></ul><ul><li>a </li></ul><ul><li>-yellow/green in all photosynthetic organisms, except some bacteria </li></ul><ul><li>b </li></ul><ul><li>-blue/green in higher plants and algae </li></ul><ul><li>c </li></ul><ul><li>-green/brown algae, and a few unicellular algae including diatoms </li></ul><ul><li>d </li></ul><ul><li>-green, in some red algae </li></ul><ul><li>Carotenoids (carotenes & xanthophylls) </li></ul><ul><li>carotenes </li></ul><ul><li>beta carotene </li></ul><ul><li>-orange in all photosynthetic organisms except photosynthetic bacteria </li></ul><ul><li>xanthophylls </li></ul><ul><li>great variety </li></ul><ul><li>-all yellow, ex. fucoxanthin </li></ul>*Specific algal groups contain diagnostic pigments
  23. 26. High-Performance Liquid Chromatography Fig. 1. Preparative and analytical procedures for HPLC from Millie et. al. (1993).
  24. 27. Chemical Taxonomy <ul><li>Initial reference pigment ratio matrix </li></ul><ul><li>Sample pigment ratio matrix </li></ul>ChemTax matrix software program = Algal group abundance
  25. 28. Objective of Study <ul><li>-To determine if pigment ratios are conservative over the growth of P. parvum </li></ul><ul><li>If conservative: use Chemtax to detect </li></ul><ul><li>If not conservative : cannot use Chemtax to detect </li></ul>
  26. 29. Analysis of Growth Characteristics: <ul><li>Utilized cell counts to determine P. parvum growth. </li></ul><ul><li>Compared pigment ratios from HPLC to cell growth. </li></ul>
  27. 30. Results
  28. 33. A B C
  29. 34. Pigment ratio statistics of entire sampling period (Day 3 – 24) 57 30 38 18 24 15 COV %   0.01 0.06 0.01 0.09 0.04 0.01 STDV   0.03 0.20 0.01 0.48 0.15 0.05 AVG C     39 24 53 21 23 27 COV %   0.01 0.05 0.01 0.11 0.04 0.02 STDV   0.03 0.23 0.01 0.53 0.18 0.06 AVG B     56 13 57 12 21 33 COV %   0.01 0.03 0.01 0.06 0.03 0.02 STDV   0.02 0.19 0.01 0.49 0.16 0.05 AVG A Zeax- Diado-/Diato Viola- Fuco- Chl c1c2 Chl c3 TOTAL  
  30. 35. Growth Phase Pigmentation Dynamics (highlighted areas display very high correlation of coefficient values) 86 23 40 14 21 16 COV %   0.01 0.04 0.01 0.07 0.03 0.01 STDV   0.02 0.16 0.01 0.47 0.13 0.05 AVG C     56 28 82 25 31 39 COV %   0.01 0.06 0.01 0.14 0.05 0.02 STDV   0.02 0.21 0.01 0.55 0.16 0.06 AVG B     74 10 90 10 30 42 COV %   0.01 0.02 0.01 0.05 0.05 0.02 STDV   0.01 0.17 0.01 0.50 0.15 0.06 AVG A Zeax- Diado-/Diato Viola- Fuco- Chl c1c2 Chl c3 GROWTH  
  31. 36. Stationary Phase Pigmentation Dynamics 20 0 47 12 27 0 COV %   0.01 0.00 0.01 0.06 0.04 0.00 STDV   0.04 0.23 0.02 0.48 0.16 0.05 AVG C     16 23 0 12 13 17 COV %   0.01 0.06 0.00 0.07 0.03 0.01 STDV   0.04 0.25 0.01 0.55 0.20 0.06 AVG B     29 5 11 13 7 15 COV %   0.01 0.01 0.00 0.07 0.01 0.01 STDV   0.03 0.21 0.01 0.50 0.18 0.05 AVG A Zeax- Diado-/Diato Viola- Fuco- Chl c1c2 Chl c3 STATIONARY  
  32. 37. Comparison of separating analysis over phases versus using entire sampling period 57 30 38 18 24 15 TOTAL   20 0 47 12 27 0 STATIONARY   86 23 40 14 21 16 GROWTH C       39 24 53 21 23 27 TOTAL   16 23 0 12 13 17 STATIONARY   56 28 82 25 31 39 GROWTH B       56 13 57 12 21 33 TOTAL   29 5 11 13 7 15 STATIONARY   74 10 90 10 30 42 GROWTH A Zeax- Diado-/Diato Viola- Fuco- Chl c1c2 Chl c3 COV %  
  33. 38. CONCLUSION <ul><li>Note: </li></ul><ul><ul><li>Zea-/Violaxanthin is not known to be present in prymnesiophytes. Further study needed to determine presence in Texas strain. </li></ul></ul><ul><ul><li>Even though ratios for pigments were statistically variable, for the stationary growth phase for Flask B, all values were less inconsistent than when analyzed over the entire growth period. Needs further study. </li></ul></ul><ul><li>Ratios are not conservative throughout entire life cycle, or through individual life stages, therefore, cannot use pigment analysis for detection of P. parvum . </li></ul>
  34. 39. Rapid Detection through use of Optical Signatures Merissa Ludwig
  35. 40. Bio-Optical Methodologies <ul><li>HPLC-derived photopigment and chlorophyll a fluorescence analyses </li></ul><ul><li>In vivo absorption assays </li></ul><ul><li>Multi-spectral remote sensing </li></ul><ul><li>All characterize phytoplankton biomass, composition, or physiological state </li></ul>
  36. 41. Finding an Optical Signature <ul><li>Measure absorbance of P. parvum during life and growth cycle </li></ul><ul><li>If variance is small, optical signature can be determined </li></ul><ul><li>Rapidly method to detect presence of </li></ul><ul><li>P. parvum </li></ul>
  37. 42. Absorption <ul><li>Light absorption in water is attributable to: </li></ul><ul><ul><li>Water </li></ul></ul><ul><ul><li>Colored dissolved organic matter (CDOM) </li></ul></ul><ul><ul><li>Photosynthetic biota </li></ul></ul><ul><ul><li>Inanimate particulate matter (tripton) </li></ul></ul><ul><ul><li>Must account for these factors when </li></ul></ul><ul><ul><li>calculating absorption </li></ul></ul>
  38. 43. <ul><li>Absorption of Water </li></ul><ul><li>Absorption of CDOM </li></ul>
  39. 44. Collecting Data <ul><li>Spectrophotometer measurements taken from Day 9 to Day 21 for values of visible spectrum – 350 nm to 750 nm </li></ul><ul><li>Corrected data to account for water and CDOM </li></ul><ul><li>Calculated coefficient </li></ul><ul><li>of variation </li></ul>
  40. 45. Absorbance
  41. 46. Variance Values <ul><li>Variance values minimal </li></ul><ul><li>Optical signature can be obtained </li></ul>4.61 480 C 6.18 413 B 5.54 483 A Coefficient of Variation Wavelength (nm) Sample Maximum Variance in Absorbance
  42. 47. Optical Signature and Growth <ul><li>Samples A & B did not enter a growth phase </li></ul><ul><ul><li>optical signature of stationary phase only </li></ul></ul>
  43. 48. Optical Signatures and Growth <ul><li>Sample C entered a second growth phase during days 9-21 </li></ul><ul><ul><li>Optical signature of growth and stationary phase </li></ul></ul><ul><li>Optical signature does not change </li></ul><ul><li>significantly with growth! </li></ul>
  44. 49. Other Projects in the Lab <ul><li>Measuring chlorophyll-a </li></ul><ul><li>Ash free dry weight analysis </li></ul><ul><li>Grain-size analysis </li></ul><ul><li>Field work at ANWR </li></ul>
  45. 51. Acknowledgements <ul><li>Advisor – Dr. Daniel Roelke </li></ul><ul><li>Mentor – Reagan Errera </li></ul><ul><li>Department of Wildlife and Fisheries Sciences </li></ul><ul><li>Texas A&M University </li></ul><ul><li>REU Faculty </li></ul>

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