Molecular Detection of Karenia spp. in the

Mid-AtlanticKathryn J. Coyne1, Edward Whereat1, Muns Farestad1, Jennifer Torora1, Lauren Salvitti1 and Carmelo Tomas2

1University of Delaware College of Earth, Ocean, and Environment

2University of North Carolina, Center for Marine Science

Interstate Seafood Seminar, Sept. 16, 2010

OverviewOverview

• Molecular approach – Description– Rationale– Development– Validation

• Karenia spp. in Mid-Atlantic coastal waters– Timeline– Molecular (presence/absence) results– Validation and results of quantitative approach for

enumeration– Morphological variability– Distributions of Karenia spp. in DE and NJ

What are Molecular Methods?What are Molecular Methods?

C C C CCT AA GG G GT T T T T T TT AA

C C CCT AA GG GT T T T T T TT AAA A

C C C CCT AA GG GT T T T TT AAA AG

Molecular MethodsMolecular Methods• Polymerase chain reaction (PCR)

– Specific amplification of DNA fragment from a specific gene

DNA primers bracket the area to be amplified

Polymerase enzyme copies DNA fragment bracketed by primers to produce a product of a single size.

Why do we use Molecular Methods for HAB Research?

To determine presence/absence or abundance of HAB speciesAdvantages over microscopy:

– Increased sensitivity– Increased accuracy in detection– Applicable to mixed assemblages– Higher throughput

Species are identified and discriminated based on variations in DNA sequence.

rRNA genes - 10s to 100s of copies

Highly conserved regions

-Group or genus-specific primers

Variable regions

-Species or strain-specific primers

18S small subunit ITS1 5.8S ITS2 28S large subunit

Quantitative Real-Time PCR

Wide dynamic range: Linear over 8 orders of magnitude

Sensitive: Detection of less than 4 copies of target DNA is possible

Ct

Yes

How do we validate PCR methods?

Design primers for target species or

group

Use primers to amplify DNA from several samples

Sequence DNA to make sure primers

are specific

STEP 1: Primer design

YesNo

STEP 2: QPCR assay development

Optimize primers for QPCR

Evaluate sensitivity and range of detection

Determine accuracy

Cell counts

QPCR results

Research and

Monitoring

No

2008June - Nov., 2008: Water samples (n=33) from 4 sites in DE processed for molecular analysis. 2009Mar. - Nov., 2009: Water samples (n=30) from 4 sites in DE processed for molecular analysis. Aug. 26-27: Water samples (n=7) from NJ coastal bays processed for molecular analysis.2010March - present, 2010: Water samples from 4 sites in DE processed for molecular analysis.

2007August 30, 2007: Initial observation of K. papilionaceae in water samples.Aug. - Sept., 2007: Microscopic identification of K. pap in off-shore waters and ocean beaches.Sept. 6 - 7, 2007: Smaller “K. brevis -like” cells observed in water samples.

Karenia Karenia spp. in Mid-Atlanticspp. in Mid-Atlantic

Primer design and evaluation (Kpap)Primer design and evaluation (Kpap)

Primers for K. pap were initially provided by Miguel de Salas (UTasmania)

•Targets the 28S rRNA gene.

•DNA was amplified by PCR from environmental samples collected Aug.-Sept., 2007.

•Sequenced PCR products from 3 env. samples collected on 2 different days.

•Comparison of DE strain of Kpap with Kpap sequences in GenBank from Hong Kong, NZ and Australia

Results: Primers are specific to Kpap, but sequence is slightly different

Later confirmed by sequence analysis of pure culture of NZ and DE strains provided by Dr. Carm Tomas (UNC)

M 1 2 3 N

When did When did K. papilionaceae K. papilionaceae first appear first appear in DE coastal waters?in DE coastal waters?

• DNA from inlet and Fenwick beach water samples collected in June and July, 2007 were tested.

• K. pap was not present on June 25, 2007.• Samples collected on July 11, 2007 through the

end of August were positive.

Results suggest that K. pap was present early in the summer

K. pap K. pap PCR Screening (presence/absence) PCR Screening (presence/absence) for DE coastal waters 2008-2009for DE coastal waters 2008-2009

• 2008: 33 samples from 4 sites. – 13 samples collected before Aug. 14th were negative for K.

papilionacea– All 20 samples collected between Aug. 14th and Nov. 13th were

positive for K. pap.

• 2009: 33 samples from 4 sites. – 16 samples collected before Aug. 17th were negative for K.

papilionacea– 1 sample collected on Aug. 3rd was positive (IR39)– All 14 samples collected between Aug. 17th and Nov. 4th were

positive for K. papilionacea

• 2010: Samples collected Mar. through present– Samples collected before Aug. were negative for K. pap

DE isolate genomic DNA

DE isolate positive control

NZ isolate positive control

DE isolate pos cont and genomic DNA

NZ isolate positive control

QPCR assay for QPCR assay for K. papilionaceaeK. papilionaceae

New primers and QPCR probes were designed based on sequence data

- Specific to NZ and DE strains

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QPCR

cell count

QPCR Results for QPCR Results for K. papilionaceaeK. papilionaceae 2008 Samples 2008 Samples

Cell Count Detection limit = 167 cells/L

• QPCR results are comparable to cell counts when above limit of detection

• QPCR is more sensitive than cell counts

Calibrator sample

M 1 2 3 4 5 6 7 P N

Distribution of Distribution of K. pap K. pap north of Delawarenorth of Delaware

Samples collected along NJ on Aug. 26-27, 2009

K. pap was present from Cape May to Ocean City

Molecular detection of Molecular detection of K. brevisK. brevis

• Culture obtained from CCMP to use as positive control• 2 Different primer sets designed targeting 28S rRNA gene• Primers for K. brevis RUBISCO (Gray et al. 2003)• PCR optimized using DNA from K. brevis culture

Results: Environmental samples do not amplify with the 3 different K. brevis primer sets

- Weak, non-specific amplification

M 1 2 3 N 1 2 3 N Kpap Kbrev

Morphological Variability Morphological Variability • Unialgal cultures of the DE strain of K. papilionaceae

were established by Dr. Carm Tomas.• Small “K. brevis –like” cells were observed in these

cultures.• Small and large cells were picked into separate tubes

and sent for DNA analysis.

Results: Both large and small cells amplified with K. pap primers but not with K. brevis primers.

It’s possible that the “K. brevis –like” cells in environmental water samples were actually K. papilionaceae.

Another possibility is that the “K. brevis –like” cells in environmental water samples were an unknown Karenia strain or species.

More work needs to be done

Summary and ImplicationsSummary and Implications

• K. papilionacea has been detected in DE coastal waters each year since 2007– Endemic population or imported each year?

• K. brevis-like cells have been observed but have not been detected using molecular methods– Morphological variability of K. papilionaceae

or new strain/species of Karenia?

Questions?Questions?

Funding provided by DNREC EPA Beach Grant