This document summarizes research on the golden algae Prymnesium parvum. It describes the organism's characteristics, environmental impacts from toxic algal blooms, and methods to identify and detect P. parvum. Experiments analyzed changes in pigmentation and optical absorption signatures over the algae's growth cycle. Results showed pigmentation ratios varied too much to use for detection, but optical signatures remained consistent, allowing rapid detection through remote sensing of blooms.

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. Golden Algae Aquatic, primitive plant Scientific name Prymnesium parvum Photo: Dr. Carmelo Tomas UNC Wilmington

3. Kingdom: Protista Division: Haptophyta Class: Prymnesiophyceae Species: Prymnesium parvum

4. Golden Algae Family Tree Illustrations by Robert G. Howells, TPWD

5. Cell Characteristics Single-celled organism Size: 8-11 µm Two chloroplasts Two flagella Haptonema Golden Alga, Prymnesium parvum Drawing by Robert G. Howells, TPWD

6. “ Golden” Algae? Golden color comes from the carotenoid pigments Color along with other factors is characteristic of algal blooms

7. Live in brackish waters – wide range of salinities Temperate areas Eutrophic zones: high nutrient concentrations Ideal Environment for P. parvum Photo: Dave Buzan, TPWD

8. What makes this algae so important? Two toxins actively released into the water Prymnesin 1 Prymnesin 2 Toxins disrupt gill function, causes hemorrhaging then death Bivalves and all fish species affected No effect on larger wildlife species

9. Unique Characteristics Mixotroph – can obtain food through photosynthesis or is also capable of eating bacteria, other algae, or protozoans Allelopathic Effect – toxins produced hinder/inhibit the growth of competitors Photos by Urban Tillmann TIllmann, U. (1998) AME 14: 155-160

10. Where can we find golden algae? First identified in 1930’s in Holland and Denmark Israel, China, England, Norway, United States, Australia, Morocco thought to be found in Scotland, Germany, Spain, Bulgaria, and South Africa and many other areas

12. Locations found in the U.S. Texas Alabama Arkansas Georgia New Mexico North Carolina Oklahoma South Carolina Wyoming Thought to be present in Nebraska

14. History of P. parvum in Texas First discovered in Texas in 1985, suspected in fish kills since 1960’s From 1985-2003, over $7 million lost in 41 fish kill events The rate of fish kills has increased over recent years.

15. Impacts of Blooms Environmental -kills fish -toxins have been shown to inhibit growth of, and kill other phytoplankton and bacteria -discoloration, foaming of water Economic -tourism by anglers and tourists decrease because of dead fish -hatcheries Fish kill at Jackson Bend, Lake Granbury, 2003

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.

17. Current Methods of Identification Microscopic Analysis: - time consuming - requires highly skilled technicians to identify P. parvum 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.

18. Future Methods IDENTIFY Optical Signature Pigment Ratios DETECT Remote Sensing Chemical Taxonomy

19. Experiment Triplicate flasks containing growth medium inoculated with P. parvum culture (obtained from UT culture collection of algae 22181) Incubation consisted of 12/12 light /dark cycle at 19ºC Flasks were rotated in incubator and stirred daily

20. Experiment Samples were taken every 3 days up to day 24 100mL extracted for high-performance liquid chromatography 10mL extracted for spectrophotometer measurement, then preserved for microscopic analysis. Cell counts were performed to determine growth over time

22. Prymnesium parvum Growth Characteristics: Analysis of Pigmentation with Cell Growth Alexis Martinez

23. Method for Assessing Phytoplankton Assemblages Microscopic Analysis Optical Absorption bioassays using spectrophotometer, fluorometer to determine abs. / wavelength High-Performance Liquid Chromatography to determine concentration of pigments

24. Pigments -Chemical compounds which absorb specific wavelengths of light, means by which photosynthesis is carried out -Pigments are divided into classes: Chlorophylls Carotenoids (carotenes and xanthophylls)

25. Pigments Chlorophylls a -yellow/green in all photosynthetic organisms, except some bacteria b -blue/green in higher plants and algae c -green/brown algae, and a few unicellular algae including diatoms d -green, in some red algae Carotenoids (carotenes & xanthophylls) carotenes beta carotene -orange in all photosynthetic organisms except photosynthetic bacteria xanthophylls great variety -all yellow, ex. fucoxanthin *Specific algal groups contain diagnostic pigments

26. High-Performance Liquid Chromatography Fig. 1. Preparative and analytical procedures for HPLC from Millie et. al. (1993).

27. Chemical Taxonomy Initial reference pigment ratio matrix Sample pigment ratio matrix ChemTax matrix software program = Algal group abundance

28. Objective of Study -To determine if pigment ratios are conservative over the growth of P. parvum If conservative: use Chemtax to detect If not conservative : cannot use Chemtax to detect

29. Analysis of Growth Characteristics: Utilized cell counts to determine P. parvum growth. Compared pigment ratios from HPLC to cell growth.

30. Results

33. A B C

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

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

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

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 %

38. CONCLUSION Note: Zea-/Violaxanthin is not known to be present in prymnesiophytes. Further study needed to determine presence in Texas strain. 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. Ratios are not conservative throughout entire life cycle, or through individual life stages, therefore, cannot use pigment analysis for detection of P. parvum .

39. Rapid Detection through use of Optical Signatures Merissa Ludwig

40. Bio-Optical Methodologies HPLC-derived photopigment and chlorophyll a fluorescence analyses In vivo absorption assays Multi-spectral remote sensing All characterize phytoplankton biomass, composition, or physiological state

41. Finding an Optical Signature Measure absorbance of P. parvum during life and growth cycle If variance is small, optical signature can be determined Rapidly method to detect presence of P. parvum

42. Absorption Light absorption in water is attributable to: Water Colored dissolved organic matter (CDOM) Photosynthetic biota Inanimate particulate matter (tripton) Must account for these factors when calculating absorption

43. Absorption of Water Absorption of CDOM

44. Collecting Data Spectrophotometer measurements taken from Day 9 to Day 21 for values of visible spectrum – 350 nm to 750 nm Corrected data to account for water and CDOM Calculated coefficient of variation

45. Absorbance

46. Variance Values Variance values minimal Optical signature can be obtained 4.61 480 C 6.18 413 B 5.54 483 A Coefficient of Variation Wavelength (nm) Sample Maximum Variance in Absorbance

47. Optical Signature and Growth Samples A & B did not enter a growth phase optical signature of stationary phase only

48. Optical Signatures and Growth Sample C entered a second growth phase during days 9-21 Optical signature of growth and stationary phase Optical signature does not change significantly with growth!

49. Other Projects in the Lab Measuring chlorophyll-a Ash free dry weight analysis Grain-size analysis Field work at ANWR

51. Acknowledgements Advisor – Dr. Daniel Roelke Mentor – Reagan Errera Department of Wildlife and Fisheries Sciences Texas A&M University REU Faculty