1. 1 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 www.stccmop.org Aerial photograph of M. rubra bloom by A. Derr

2. 2 What 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)

3. 3 M. 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

4. 4 M. 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

5. 5 M. 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)

6. 6 Question & 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

7. 7 Data 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

8. 8 Results 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. 9 Key - Gave a PCR signal - Gave no PCR signal (Surface – Left Middle – Center Bottom – Right) Results, continued EukaryotesM. rubra

10. 10 Conclusions 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)

11. 11 Sampling Experience Went water sampling in Astoria and Ilwaco Harbor with Sheedra Futrell and Dr. Lydie Herfort

12. 12 Future 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

13. 13 Thank You! My frontline mentor, Dr. Lydie Herfort My senior mentor, Dr. Peter Zuber Vikki Campbell Karen Wegner Dr. Antonio Baptista