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

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)

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

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

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)

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

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

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

9

Key

- Gave a PCR signal

- Gave no PCR signal

(Surface – LeftMiddle – CenterBottom – Right)

Results, continued

Eukaryotes M. rubra

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)

11Sampling Experience

• Went water sampling in Astoria and Ilwaco Harbor with Sheedra Futrell and Dr. Lydie Herfort

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

13Thank You!

• My frontline mentor, Dr. Lydie Herfort

• My senior mentor, Dr. Peter Zuber

• Vikki Campbell

• Karen Wegner

• Dr. Antonio Baptista