a molecular investigation of m. rubra pre-bloom distribution in the columbia river estuary
DESCRIPTION
A Molecular Investigation of M. rubra pre-bloom Distribution in the Columbia River Estuary. Deirdre Dr. Lydie Herfort, Frontline Mentor Dr. Peter Zuber, Senior Mentor. Observation ● Prediction ● Analysis ● Collaboration. Aerial photograph of M. rubra bloom by A. Derr. - PowerPoint PPT PresentationTRANSCRIPT
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1
A Molecular Investigation of M. rubra pre-bloom
Distribution in the Columbia River Estuary
Deirdre
Dr. Lydie Herfort, Frontline MentorDr. Peter Zuber, Senior Mentor
Observation ● Prediction ● Analysis ● Collaboration
www.stccmop.org Aerial photograph of M. rubra bloom by A. Derr
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2What is Myrionecta rubra?
• Mixotrophic ciliate, most likely of marine origin• Forms non-toxic red tides in estuaries, fjords, and other
coastal margin environments• Photosynthesizes through use of acquired chloroplasts of
cryptophyte prey– Karoklepty (predation)– Symbiotic co-evolution
M. rubra under transmitted light (left) & epoflourescence microscopy (right) by D. Stoecker, University of Maryland
Microscope image of cryptophyte prey(T. Peterson)
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3M. rubra blooms in the Columbia River estuary
• Blooms from late July to October• Based on ‘18S-28S’ rRNA gene analysis, a single variant leads
to blooms each year (variant B)• Only one cryptophyte, Teleaulax amphioxea, is associated
with M. rubra variant B
Aerial photography of M. rubra bloom by A. Derr, 2008
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4M. rubra in Oceanic Waters
• At least five different variants detected in coastal waters based on ‘18S-28S’ rRNA gene analysis
Sites of sequence polymorphisms of M. rubra partial ‘18S-28S’ rRNA gene sequences
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5M. rubra in Oceanic Waters, continued
• Water samples collected during CMOP May-June cruise 2010• FlowCAM analysis of 15 mL of water showed M. rubra in only
two locations
FlowCAM images of M. rubra among phytoplankton assemblages (T. Peterson)
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6Question & Research Goal
Question:• FlowCAM – 15 mL of sample water• Molecular analysis – 1-4 L of sample water
• Is molecular analysis a more sensitive approach?
Goal:• Determine M. rubra presence in coastal water samples using
molecular identification methods• Polymerase Chain Reaction (PCR) DNA amplification
with M. rubra specific primers• Agarose gel electrophoresis to visualize PCR products
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7Data Collection Methods
• Analyze 18 samples taken from the CMOP May-June cruise 2010 at varying depths and locations
• Extract nucleic acid from filtered water samples using a phenol/chloroform extraction method
• Use 18S rRNA gene PCR primers (EukA & EukB) for general identification of microbial eukaryotes
• Use ‘18S-28S’ rRNA gene PCR primers (MR18Sf & MR28Sr) specific to M. rubra – Amplifies M. rubra Internal Transcribed Spacer gene region (below)
• Run all PCR products on agarose electrophoresis gel to visualize PCR products
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8Results
• Nucleic acid successfully extracted from filtered water samples
• Microbial eukaryotes detected in 14 / 18 samples
• M. rubra detected in 17 / 18 samples, even when not detected by FlowCAM
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9
Key
- Gave a PCR signal
- Gave no PCR signal
(Surface – LeftMiddle – CenterBottom – Right)
Results, continued
Eukaryotes M. rubra
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10Conclusions
• M. rubra present in most coastal samples during pre-bloom season
• M. rubra not detected by FlowCAM analysis because it is likely present in low abundance
Preparing PCR products for an agarose electrophoresis
gel (J. Schilling)
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11Sampling Experience
• Went water sampling in Astoria and Ilwaco Harbor with Sheedra Futrell and Dr. Lydie Herfort
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12Future Work
• Continue monitoring M. rubra presence during non-blooming periods (Nov. – Jun.)
• Identify which variant of M. rubra is most common in oceanic samples
• Develop an alternative method to gene sequencing for identification of M. rubra variants
• Culture the five known variants of M. rubra to use as positives for PCRs
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13Thank You!
• My frontline mentor, Dr. Lydie Herfort
• My senior mentor, Dr. Peter Zuber
• Vikki Campbell
• Karen Wegner
• Dr. Antonio Baptista