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									Detection of Karenia brevis blooms:
 A comparison of molecular, optical
and hybrid detection methodologies

               Project Update

                October 6, 2010
        Fish & Wildlife Research Institute
  EPA Site Visit Schedule
Wednesday October 6th, 2010


 9:00 - 10:30:    Presentation
 10:30 - 10:45:   Break
 10:45 - 12:00:   Presentation

 12:00 – 1:00:    Lunch

 1:00 – 1:15:     FWRI Grants Office Intro

 1:15 – 2:15:     USF - NASBA Demo
 2:15 – 3:15:     FWRI - SHA Demo
 3:15 – 5:15:     Karen Steidinger

 6:00 - TBD       Dinner
                                        Project Goals
The overall goal of this research is to evaluate the range of K. brevis detection technologies currently
available against the accepted standard and legal technique (light microscopic assessment) to determine the
appropriate uses of each technology and provide recommendations on the appropriate management and
monitoring applications of each technique.

The specific objectives to achieve this overall goal are listed below:

●To compare and intercalibrate available methods for the detection and enumeration of K. brevis against an
accepted standard method (microscopic enumeration), and determine the appropriate conditions under
which each method correlates well to the standard and each other.

● To determine the relative sensitivity and specificity of each assay and dynamic range for K. brevis detection
in reference to legal monitoring requirements (closure of commercial shellfish beds in Florida at 5000 cells L-1
K. brevis).

● Determine the effects of common procedural problems associated with sample collection (holding time for
live samples, sample volume, preservation and storage methods) for each method.

● Compare sample volume requirements through-put time for each method, and establish protocols for
minimizing sampling error and maximizing sample representativeness.

● Produce an Alliance for Coastal Technologies (ACT) report summarizing the project results and the current
state of Karenia brevis detection technologies.
                    Key Karenia Monitoring Challenges

● Detecting initiation (low concentrations offshore at depth) is still problematic
● Large geographical areas involved
● Methodological intercalibrations and appropriate applications
● Incorporate new detection methods into multiple platforms:
           Smaller, cheaper, faster, more accurate…integrated and automated for simultaneous
           measurements, Miniaturization, Single cell analyses, Monitor complex assemblages, Flow-
           through systems
● Transitioning technology from ‘research’ to ‘management’ applications & products
       ● Who transitions? Who maintains? Who funds?
       ● Who are the endusers & what are the endproducts?
● Future Uncertainties:
       ● Global Warming:
       ● Coastal Development/Landscape Changes:
 Management Issues: Multiple Karenia species


                                                  K. brevis
30



29
                                                  K. mikimotoi


28
                                                  K. papilionaceae

27

                                                 K. selliformis
26             2005
           Karenia Bloom
25                                               K. sp. #1 (‘mexican hat’)

     -87     -86   -85   -84   -83   -82   -81
  Management Issues: Multiple Emerging Detection
                  Technologies
Accepted for Human Health: Microscopic Counts
In Field Testing:
           Satellite: NOAA HAB bulletin, MODIS, Karenia/Trichodesmium algorithms
           Optical: Brevebuster, Flowcam, SIPPER, Imaging Cyto-Bot
           Compound Detection: gyroxanthin-diester, sterols
           Molecular/Genetic: Sandwich Hybridization Assay (SHA), Microsatellite
                      Markers, Carbon fixation Gene Technology, Fluorescent In
                      Situ Hybridization (FISH) Assays
           Hybrid Detection Methodologies: Electrochemical detection of nucleic
                      acids, Luminex XMAPTM
Issues:   Relationship to microscopic counts (and each other)
          Detection limits and species specificity
          Best/optimal/most efficient management use & application for each technique
          Compatibility with platform technology
          Price & Practicality
                     Project Methodologies
1. Microscope Counts – all sites
2. Sandwich Hybridization Methodology (SHA) – Fish and Wildlife Research
   Institute and Monterey Bay Aquarium and Research Institute
3. Nucleic Acid Sequence Based Amplification (NASBA) – University of South
   Florida
4. Fluorescent In Situ Hybridization Assay (FISH) – National Oceanographic and
   Atmospheric Administration
5. Luminex – University of Miami
6. High-Performance Liquid Chromatography (HPLC) – Florida Institute of
   Oceanography
7. Imaging FlowCytobot (IFCB) – Texas A&M University
8. BreveBuster (BB) – Mote Marine Laboratory
9. Satellite Products – NOAA, USF
                                           SHA Chemistry
   Anti-dig/HRP + substrate

                                                                              Karenia brevis

                                                                             Imaged
                                                                             array

                                      Dig-signal
                                        probe
                                       Target                                   Verification
                                       rRNA                                     by matching
                                                                                96-well bench
                                                                                run

                                         Capture
                                          probe            array spot intensity 
                                                           absorbance (450 nm)
                                   RSA/ Biotin/ linker   Direct capture of target sequence
                                                         No purification required
            solid support                                Reagents stable 2-25C
Scholin et al. 1996, 1999; Haywood et al. 2009, 2007
Karenia brevis in natural samples at 2 concentrations

                                            low abundance                                                                high abundance
                                                                                                       400000
                         100000            r2=0.81                                                                   r2=0.82
SHA equivalents (Cells.L-1)




                                                                              SHA equivalents (Cells.L-1)
                              80000
                                                                                                       300000


                              60000
                                                                                                       200000
                              40000

                                                                                                       100000
                              20000


                                  0
                                                                                                                         low abundance
                                                                                                            0

                                      0     20000 40000 60000 80000 100000                                      0     100000     200000     300000     400000

                                          Shipboard cell count (Cells.L-1)                                          Shipboard cell count (Cells.L-1)


                                                    n = 514                                                               n = 667
                                                         (From Haywood et al. 2009; & in prep)
 Summary of SHA status in Karenia spp. detection
 Since 2004, ~800 samples have been processed for 4 species

 Detects background levels to ~1000 cells L-1 of K. brevis

 Exceeds regulatory limit requirements of 5000 cells L-1

 SHA has ISO certification and is accredited for regulatory use in New Zealand
        (Ayers et al. 2005. NZ. J. Mar. & FW. Res., 39(5):1225-1231)

 SHA chemistry has been used in the laboratory, real-time on ships, and in autonomous
    deployable instruments since 2004

 Rapid, crude extraction, direct quantitation (i.e. no amplification required)

 SHA is tolerant of high biomass samples

 Can be tuned for a range of cell numbers:
    1-400K L-1 for lab assay (changeable), and to high range of cell count in situ

 Complements optical methods (Satellite, ‘Brevebusters’) & detects species
   Provides early warning of pre-bloom increase in cell numbers
                                                  NASBA (USF CMS & COT)




Figure 4. NASBA amplification pathway (upper left),
the functioning of molecular beacons (upper right), and
NASBA cell counts vs. microscopy for K. brevis bloom
samples (bottom right).



                                                                 The AMG use a syringe pump to filter water through filtration
                                                                 columns in a rotary wheel (large white drum in center of
                                                                 photo). Because each sample has a unique flow path from
                                                                 sampling to RNA extraction and then to amplification, there
                                                                 is no possibility for cross-contamination.
                                            FISH
•Fluorescent In Situ Hybridization (FISH) assays -
whole cell assays because they are used to label
molecules of intact cells.

•This method involves penetration of the probe into
chemically fixed, intact cells, hybridization to its
target sequence on the rRNA molecules, and
visualization via a fluorescent reporter.

•Algal cells labeled using FISH are examined directly
by epifluorescence microscopy.

•Cell morphology is retained, although some changes
in cellular size and shape are inevitable upon fixation.

•Used to monitor for HAB species is in New Zealand
(Rhodes et al. 1998, 2000, 2001, 2004), where WC-
formatted probes have been integrated into two-
tiered biotoxin monitoring programs for industry and
public health.                                             K. brevis cells with K. brevis probe
                                                  LUMINEX
                         Luminex xMAP Detection System

                                                                             Species specific
                                                                             microsphere-probe A
 Red fluorescent
  633 nm laser




                                                                                  Intensity of reporter
 Green fluorescent




                                                                                   fluorescence
   532 nm laser




                                                                                                  *Wave length of microsphere
                                                                                                  fluorescence
                                                                            *Bead classification spectral address
                                                                            is within 658 nm/712 nm emission ratio

Illustration of xMap Luminex detection system. Species specific microsphere-probes are interrogated individually in
a fast flowing fluid as they pass by two separate laser beams. A high digital signal processing, classifies individual
microspheres based on their unique fluorescence spectral address. Quantitation of the reaction is based on the
emission of a red fluorochrome.
xMAP: DIVERSITY OF APPLICATIONS FROM
         INDIVIDUAL SAMPLES

 1.   Specific Species Detection
 2.   Functional Genes
 3.   Toxins
 4.   Relate to Environmental Conditions

               Cost Effective: ~10 Cents/Probe

               Speed: 20,000 Beads/minute

               Flexible: In contrast to chip arrays
Pigment Chromatogram (HPLC) – Karenia brevis
                                              444                                    1) Chl c3
                                                     469                         8
                                                                                     2) Chl c1 & c2
                                                                                     3) 19’-butanoyloxyfucoxanthin
 Relative Absorbance (440 nm)



                                                                                     4) Fucoxanthin
                                                                                     5) 19’-hexanoyloxyfucoxanthin
                                    Gyroxanthin-diester                              6) Diadinoxanthin (Diatoxanthin)
                                                                                     7) Gyroxanthin-diester
                                    400        450          500       550
                                              Wavelength
                                                                                     8) Chl a
                                                                                     9) b-y carotene

                                                           6                                   Key:
                                               2 4                                              Green - chlorophylls
                                                                                                Brown – xanthophylls
                                                35                7                             Orange - carotenoids

                                          1
                                                                                           9

                                0     10                   20               30       40         50          60
                                                           Retention Time (minutes)
Pigment Chromatogram (HPLC) – Karenia brevis
Optical Phytoplankton Discriminator (BreveBuster)
              Sample                               Sample
       Particle Absorbance                     4th Derivative   Library of
                                                                Standards
                                                                                  Cyanophyceae




                                                                Prymnesiophyceae Raphidophyceae
                               Cyano-
                              Cyano-
                              phyceae
                              phyceae
        Karenia sp.
            Karenia sp.




    Prymnesio-
       Prymnesio-
                                                Multiple          Karenia sp.    Bacillariophyceae
        phyceae
     phyceae
                                     Chloro-
                                     Chloro-
                                                Regression
                                     phyceae
                                     phyceae

           Bacillario-
                                                Analysis
        Bacillario-
             phyceae      Raphido-
         phyceae          Raphido-
                           phyceae
                          phyceae


       Community Structure                                       Chlorophyceae    Cryptophyceae
     Karenia Similarity Index (SI) from BreveBuster overlain on MODIS
Fluorescence Line Height (FLH) image of Karenia bloom in November 2005
       Satellite Detection
NOAA Gulf of Mexico HAB Bulletin




                         Produced biweekly
                         for environmental
                              managers
                       Satellite Detection


             Center for Prediction
              of Red Tides (CPRT)


•   What is CPRT? A cooperative effort between USF and FWC/FWRI

•   Goal: To develop and implement a routine predictive capacity for Florida
    red tides and their potential impacts.

•   How: The CPRT will synthesize data produced by the USF College of
    Marine Science, FWC Fish and Wildlife Research Institute and others to
    provide continuing red tide forecasts for the purpose of managing and
    mitigating red tide occurrences.

•   Where: Facility is housed at the USF’s College of Marine Science in St.
    Petersburg
   USF-FWRI Center for Red Tide Prediction (CPRT)
    What does it do? - The 2007 Red Tide Example
In 2007 there were three                                 Were these Red Tides related?
 simultaneous red tides                 Based on USF/FWRI CPRT models, both east coast and
Panhandle, east coast and               panhandle red tides probably originated from
       SW Florida                       transport of the SW Florida red tide


                                           Path of drifters                                 Path of drifters
                                           released at 30 ft                                released at 90 ft




                                         The paths of surface drifters over 70 days, representing water parcels with red tides of
                                         K. brevis, released on 6 September 2007. Color changes depict days after release.




                                       What does CPRT do?
• The Center provides FWRI with ‘state of the art’ tools (satellite image interpretation, models) to help
track and predict red tides.
• In 2007, CPRT models demonstrated that both panhandle and east coast red tides probably
originated in SW Florida
     USF-FWRI Center for Red Tide Prediction (CPRT)
                   What does it do?
              The 2007 Red Tide Example
   Satellite Image Interpretation                                          Forecasting Red Tide

9/29/2007                    10/15/2007




                             3 red
                             tides




                          MODIS satellite image showing
MODIS satellite image                                         (LEFT) Particle Trajectories forecasted from physical oceanography
                          3 red tides in white
showing blooms of
                          corresponding to the monitoring     measurements and USF Model, (RIGHT) Forecast of red tide
Trichodesmium in blue.
                          map (ABOVE)                         trajectories based on model and FWRI monitoring data. Note: green
                          .                                   dots indicate that no red tide is present at the time of the forecast.
Trichodesmium, a
                          Blooms of Karenia brevis and
cyanobacteria that can
                          Trichodesmium are identified by
provide N, is often a
                          the unique ways the cells reflect
precursor to red tides.
                          light.

                                                    Current USF Tools: http://ocgweb.marine.usf.edu
Role of ACT in HAB Detection Methodologies

    A third-party testbed for evaluating coastal technologies

    ACT does not have a “horse in the race” and can remain
     impartial

    Over 10 years experience in testing and evaluating sensors for
     research and coastal management
                    Project Sampling Design


                         Combined                Dedicated
    Lab
                            Field                 Bloom
Experiments
                         Bloom/Lab               Sampling

    Cultures:             Sanibel Bloom:       RV Weatherbird Cruise:
                                                      Bloom

    Methods                  Methods          Using on site sampling for
       vs                       vs           Intercalibration/Comparison
Microscope Counts        Microscope Counts
                     LAB EXPERIMENT SUMMARY

●May, 2009 – Shipping Test & Initial Comparison: Initial shipping test
conducted to see if shipping live samples overnight resulted in viable
samples

●July, 2009 – Concentration Test: K. brevis (C2) culture with different
cell concentrations (5,000 c L-1, 50,000 c L-1, and 500,000 c L-1)

●October, 2009 – ‘Wild’ Multiple Species Test: Wild bloom sample
from SW Florida Coast (Sanibel Island) with multiple Karenia species
present (K. brevis, K. mikimotoi, K. selliformis)

●December, 2009 – Karenia Species Differentiation Test: Test
conducted to test methodology differentiation between K. brevis and
other Karenia species (K. mikimotoi).

●May, 2010 – K. brevis Strain Differentiation Test: Test conducted to
test methodology differentiation between different strains of K. brevis
(C2 from Charlotte Harbor, B5 from Apalachicola, B4 from Texas)
                 Aliquot Flow Chart
Shipping
Test & Initial
Comparison
Concentration Test
‘Wild’ Multiple          Wild Bloom/Lab Experiment
 Species Test




   ● Water samples were collected at three stations, however Karenia cells were only detected at
   Station 1 [26o 28.592’, 82o 23.617’]

   ● Surface samples were collected with 13 L bucket and immediately poured through 153
   micron filter into 3 4-Liter brown bottles.
Karenia Species
 Differentiation
      Test
K. brevis Strain
Differentiation
      Test
 Day 2 Microscope Counts vs SHA ‘Shipped’ Microscope Count Results




                                 Dotted line is 1 to 1
                                 Solid line is best linear fit




Note: All samples for local PI’s (Microscope, HPLC, NASBA, SHA) were held overnight at FWRI
under simulated shipping conditions (wrapped in wet newspaper in cooler) to simulate offsite PI
conditions. SHA example above demonstrates inherent variability between flasks on Day 2
Dotted line is 1 to 1
Solid line is best linear fit
                 Sandwich Hybridization Assay
• How cells are counted DOES matter - Live counts differ from preserved
  counts for the same sample

• Sample volume counted IS important - Live counts were performed on
  100ul sample, a potential error source when multiplied to get L-1
  concentrations. PI counts were performed on 1ml samples and therefore
  the error is reduced by a factor of 10.

• FWRI counts are higher for December experiment than SHA counts. SHA
  PI counted samples several days after fixation and FWRI counts were fixed
  and counted on the same day. Temporal lag between fixing with Lugols
  and counting DOES matter.

• Counts and SHA data are consistent (DOF=18), r2=0.95.

• Future field testing on cruise will provide almost real-time analysis and
  may minimize the above sources of error and elucidate others
(actual shipped samples)




                           Results of optical experiment




                                 (simulated shipped samples)
r-squared w/o outlier is 0.83
                   Imaging Flowcam/Cytobot

• Small sample size effect on counts comparison – PI did not always include
  microscope counts.

• FWRI counts are higher than PI counts for large concentrations.

• Counts and IFCB data are consistent (DOF=20), r2=0.83 with outlier
  omission.

• IFCB may have difficult time in regions of low cell concentration.

• Future field testing on cruise will provide almost real-time analysis and
  may eradicate sources of error.
                                     Cruise Summary
                                              OBJECTIVES

•Locate and track a bloom of Karenia brevis using CTD and flow-through equipment. Obtain samples
and test instrumentation inside and outside of bloom.

•Field test of various K. brevis detection instruments (both optical based and molecular based
detection instruments) for presence of Karenia brevis and cross-reaction to other phytoplankton
species in water samples.

•Field-based techniques evaluation comparing the results of various K. brevis detection instruments vs.
the gold standard of K. brevis detection, microscope cell counts. This is to be conducted inside and
outside of a bloom of K. brevis.

•Determine the relative sensitivity and specificity of each assay and range of detection of K. brevis in
field conditions.
                                         STATION POSITIONS

•Starting point will be determined @ a later date, prior to departure date (projected region of focus is
offshore Ft. Myers, Charlotte Harbor, and Sanibel Island.

•Station positions are very much dependant on the finding of a Karenia brevis bloom
(none known to exist yet in the research area).
ACT Priorities
 Transition emerging technologies to operational use
  rapidly and effectively
 Maintain a dialogue among technology users, developers,
  and providers
 Identify technology needs and novel technologies
 Document technology performance and potential
 Provide the IOOS with information required for the
  deployment of reliable and cost-effective networks
ACT Services
 A third-party testbed for evaluating coastal technologies

 A forum for capacity and consensus building

 An information clearinghouse for coastal technologies
                              ACT Partner Institutions

                                          University of Michigan
                                          Cooperative Institute for
                                          Limnology & Ecosystems Research
                                                                                        Gulf of Maine
                                                                                    Ocean Observing System




  Moss Landing
Marine Laboratories




          Monterey Bay Aquarium
            Research Institute
                                                                            Skidaway Institute
                                                                             of Oceanography
                            Deliverable – ACT Report
          ACT staff currently working with FWRI to synthesize and interpret results
        Draft one will be completed by cruise on RV Weatherbird II (October 18, 2010)
                      Outline (similar in organization to this presentation)
I.     Introduction
      A. Background                            E.    HPLC
       B. Goals and Objectives                                1. Summary of Method
II. Sampling Design                                           2. Results
       A. Shipping Tests                                      3. Management Implications
      B. Field Tests                           F.    Cytobot
III. Technologies and Methodologies                           1. Summary of Method
      A. Microscope Counts                                    2. Results
               1. Summary of Method                           3. Management Implications
               2. Results                      G.    Brevebuster
               3. Management Implications                     1. Summary of Method
     B. SHA                                                   2. Results
               1. Summary of Method                           3. Management Implications
               2. Results                      H.    Satellite
               3. Management Implications
    C. FISH                                    IV.   Summary and Conclusions
               1. Summary of Method
               2. Results                      This section will summarize findings but will
               3. Management Implications            NOT pick a “winner”.
    D. Luminex
               1. Summary of Method
               2. Results
               3. Management Implications
               Final Products

• ACT Report: Summarize results of each method
vs microscope ‘gold standard’

• Grant Final Report to EPA: Will include methods
comparison and management recommendations

• Peer Reviewed Publication (for Harmful Algae)

								
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