Extreme low-level genetic detection of didymo:

a new surveillance tool

Centre for Biodiversity and Ecology Research

Department of Biological Sciences

School of Science and Engineering

University of Waikato, Hamilton

Craig Cary, Brendan Hicks, Catherine Barnett,

Chrissen Gemmill, Andreas Rueckert, Kathryn Coyne1

1University of DelawareCollege of Marine and Earth StudiesLewes, Delaware 19958, USA

2

Problem 1Current field sampling efforts rely solely on microscopy

- Lacked sensitivity at low cell concentrations - early surveillance- Effort in microscopy limits sampling capability in time and space

Need for method with increased sensitivity, and higher throughput

Would allow:

• Earliest possible detection• High frequency surveillance capability - low cost !• Integrate to ongoing incursion/mitigation response efforts

Objective: Develop a DNA detection tool (the DNA method) for didymo

that is highly specific, highly sensitive, and allows high throughput with rapid turn-around

# o

f d

idym

o c

ells

at

a s

ite

Time

Drift netDNA

method

Drift netmicro

method

Visualmicro

method

Relative Limits of Detection of Visual, Microscopicand DNA Analysis Methods for D. geminata

Assumptions: Growth of didymo in a river is exponential when flows are stable andbelow scouring velocity

Benthicmicro

method FLOODS

3

Problem 2• Origin of didymo in New Zealand• Multiple introductions from different locations?

????

Objective: Use molecular markers to reveal origin and phylogeographic history in New Zealand

Criteria for the DNA methodSpecific requirements for this new DNA amplification based

methodology:

• Robust, field compatible protocols for stabilization, extraction

• Species or strain level specificity

• Extreme sensitivity for low-level detection (single cell)

• A broad dynamic range (> 1 to 100,000 cells/mL)

• The highest possible degree of reproducibility

• Efficient, cost-effective, rapid, with high throughput capability

• Meet extremely high QC/QA standards

Collection and Stabilisation protocolsTrial procedures for environmental sampling:• Surface swabs - visible and clean surfaces• Design and testing of drift net assembly• Develop net DNA denaturation procedures• Trial different “field compatible” fixatives - stabilise

DNA

Results• Nets - concentrate didymo, increase detection chance• Swab protocols - detecting non-visible didymo• Developed simple DNA denaturation procedures • Fixation in 70% ethanol

Gomphoneis minutae var cassiae NZ TAR009

Gomphonema parvulum AJ243062

Encyonema triangulatum AJ535157

Didymosphenia geminata NZ CONTROL

Amphora montana AJ243061

Fragilaria striatula AY485474

Cymbella sp. NZ AH-26

Coscinodiscus radiatus X77705

82

89

74

54

96

0.000.020.040.060.08

Distinctiveness of didymo 18s rRNA• Nothing known about didymo genetics

• Lower Waiau sample - Target 18S rRNA

Results:• Mono-specific clone library

• 1764 bp - cloned and bi-directionally sequenced

• Sequenced related spp. In NZ - Gomphoneis sp. (NI), Cymbella sp. (SI)

• Form one clade

• NZ Cymbella not closely related to Didymosphenia

Design didymo-specific PCR primers

Objectives:

• Align all known gomphonemoid 18S rRNA gene sequences

• Look for areas of variability that will distinguish didymo

• Design primers to suit specificity and size needed for QPCR

• Amplify out the gene

Results:

Provisional alignment of 10 taxa identified 5 areas of sequence variability - designed 5 didymo-specific primers

Diatom 9F Euk 608F Didymo 602F Euk 1000 F Didymo 1565F Didymo 1659F

Didymo 753R Euk1000R Didymo 1670R

Euk B

Primers validated against related species and environmental samples

Tested 12 primers in combination with each other and universals

Lane Primer combinationsAmplicon

length (bp)

1 Didymo 602F and Didymo 753R 170

2 Didymo 1565F and Didymo 1670R 110

3 Didymo 602F and Didymo 1670R 1073

4 Diatom 9F and Didymo 753R 774

5 Euk 608 F and Didymo 753R 228

6 Didymo 602F and Euk 1000R 367

7 Diatom 9F and Didymo 1670R 1079

8 Euk 608F and Didymo 1670R 1129

9 Euk 1000F and Didymo 1670R 741

10 Didymo 1565F and Euk B 235

11 Didymo 1659F and Euk B 1141

D1565F and D1670R

D602F and D1670R

Diatom9F and D753R

Euk608F and D753R

D602F and Euk1000R

Diatom9F and D1670R

Euk608F and D1670R

Long primer set

Use for QRT PCR

D602F and D

753R

Short primer set

Euk1000F and D1670R

D1565F and EukB

D1659F and EukBLadder(known bp)

753-602 = 151 bp

1670-602 = 968 bp

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Quantitative Real Time-PCR (QRT-PCR)

Primers and TaqMan probe anneal to DNA – fluorescence quenched

Polymerase progresses along gene

Primer knocks off fluorescent dye

Primer knocks off fluorescent dye – no longer quenched

http://www.appliedbiosystems.com

Fluorescent dye Quencher

Ct

Ct: cycle threshold value. Lower Ct = greater abundance of target

Rotor Gene 6000 (Corbett)

Sensitivity of QPCR reaction

Sensitivity = 68 copies of target gene (~1 cell)Linear over 6 orders of magnitude (R2=0.997)

10 ng

1 ng

100 pg

10 pg1 pg (~ 1 cell) - threshold

100 fg - below threshold (BT)Negative controls

Cycle

No

rmal

ised

flu

ore

scen

ce

Calibrator concentration

R2 = 0.9974

14

19

24

29

34

39

1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02

Calibrator DNA [ng reaction-1]

Th

resh

old

cycl

e n

um

ber

R2 = 0.9974

14

19

24

29

34

39

1.E-04 1.E -03 1.E -02 1.E -01 1.E+00 1.E+01 1.E+02

Th

resh

old

cyc

le n

um

ber

Calibrator DNA (ng reaction-1)

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Reproducibility of QPCR

Triplicate samples collected from Buller River (NZ) - same site• 2-minute drift net collection, 0.69 m/s water velocity (~3,750 L filtered)• Heavily controlled process

Is a single sample representative of didymo abundance from QPCR?

Calculated cell abundance for triplicate samples :

Sample 1: 20.6 cells/LSample 2: 19.7 cells/LSample 3: 21.4 cells/LAverage: 20.6 cells/L (+/-0.85)

Ten-fold dilution of each sample confirms absence of inhibitors

Validation of specificity

A robust, highly controlled QC/QA pipeline • Every sample is run in duplicate

• Internal standard controls processing efficiency and environmental inhibitors

• All reagents used (extraction, QPCR) are tested with QPCR daily

• Full set of QPCR controls (negative, positive, calibrator) run daily

Strong validation protocol

• Every positive or BT sample is 3X validated (gel, HRM, seq.) for didymo

• Risk assessment established on all positive or BT samples

Assures unprecedented negative predictive value

Validation of positives

Most critical step - confidence in results

Three-fold validation process:

151 bp151 bp

1. Gel electrophoresis

2. High resolution melt

Didymo (602-753) Tm

pGEM standard Tm

84.25°C

86.5°C

Temperature (oC)d

F/d

T

Didymo (602-753) Tm

pGEM standard Tm

84.25°C

86.5°C

Temperature (oC)d

F/d

T

3. Sequence analysis

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0 10 km

N

18

76

5

4

3

2

Lake RotoitiLake

Rotoroa

BullerRiver

GowanRiver

Owen River

0 10 km

N

18

7 5

4

32

Lake RotoitiLake

Rotoroa

BullerRiver

GowanRiver

Owen River 6

Detection and enumeration in a natural system

• Buller, Gowan, and Owen Rivers

• 8 locations (Oct. 2006)

• Localise populations

• Owen River - didymo free

Site Location Water velocity (m/s)

Volume filtered

(m3)

Cells in total volume

Cells/L

1 Buller R -St Arnaud's 0.55 2.99 187 0.06

2 Buller R - Howard R 0.69 3.75 77,143 21

3 Buller R - Howard Junction 1.25 6.79 1,203,501 177

4 Gowan R - holiday camp 0.76 4.13 83,661 20

5 Gowan R - Buller R confl 1.45 7.87 324,500 41

6 Owen R upstream site 0.15 0.81 0 0.00

7 Buller R at Owen R Rec Res 0.21 1.14 467,098 410

8 Buller R at Murchison 0.16 0.87 132,748 153

35

40

45

170 175

0 200 km

N

Sites sampled with the DNA method

QPCR validation for New ZealandRivers Samples

NI 56 75

SI 56 134

Rivers found positiveNI 0 (May 2007 delimiting survey)

SI 50

All positive samples validated to be didymo

A proportion of the samples shown positive by the DNA method were negative by microscopy

No didymoDidymo

Manganui-a-te-ao, NI- still negative

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Sampled sitesSites with didymo

QPCR validation - on-going international survey

Rivers SampledInternational 14 (Canada (2), Norway (4), Iceland (1), Poland (1), UK (1), USA (5)

Rivers found positiveInternational 12

All positive samples validated as didymo by QPCR method

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Risk assessmentOrigin of

contaminationControls implemented

Sample collection

Dead cells Wildfowl, upstream samplers, sampling gear, or recreational users.

Field personnelTraining for field personnel, disposable plastic ware for each

sample, denaturation procedures.

DNA Extraction

Extraction supplies Isolated samples, use new sterile plastic ware with barriers

Extraction reagents “No-sample” controls

QPCR

QPCR reagents No-template controls for each QPCR experiment performed

QPCR instrument Wiped out before each experiment, QPCR negative controls

Environmental inhibitors

Include 2 pGEM internal standard controls for each sample run.

Maintains a high negative predictive value

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Cost of DNA analyses $70 per sample

1. DNA extraction

• Consumables

• Tech time – 1 day

2. Quantitative PCR

• Consumables (4 reactions to control for efficiency)

• Tech time – 2 days

3. Throughput and turn-around time (max)

• Single sample – 30 per week, 120 per month

• High thoughput – 90 per week, 400 samples per month

• Robotic operation, investment in equipment

• Cost savings for volume likely

Taqman analysis pipeline and response strategy

• If at or below the BT (early detection)

– 3 x validation - 48hrs - notify end user

– re-extract sample - repeat - 48 hrs

– re-sample - ASAP

• If positive

- notify BNZ and end users

- rapid response

Developed to support high frequency surveillanceSample in 70%

ethanol

Mix and split

Extraction buffer + Proteinase K + pGem

DNA extraction Reagent control

–20oC

–20oC

–20oC

Archive

Quantify

2 X pGEM QPCR2 X Didymosphenia geminata QPCR

Detection and enumeration

2.5 ml

2.5 ml

Validation

Sample in 70% ethanol

Mix and split

Extraction buffer + Proteinase K + pGem

DNA extraction Reagent control

–20oC

–20oC

–20oC

Archive

Quantify

2 X pGEM QPCR2 X Didymosphenia geminata QPCR

Detection and enumeration

2.5 ml

2.5 ml

Validation

• If remains unvalidated - targeted surveillance

Where did didymo come from?Phylogeography of didymo:

using molecular markers to reveal its origins and geographic history in New Zealand

????

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Preliminary phylogeography of didymo

• Phylogeography– Using molecular markers to reveal geographic history of species

and populations• Questions

– What are the origin(s) of didymo in New Zealand?– Have there been multiple introductions from different locations?

• Approaches/Challenges– Use rapidly evolving molecular markers to trace the routes of

introduction and subsequent patterns of dispersal within New Zealand

– Didymo cells are generally contaminated with other microorganism species

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Internal Transcribed Spacersof the 18S rRNA

28S5.8S18S

D602F ITS3FD1659F

D1670RD753R ITS4RITS2

ITS1 ITS2

Hypervariable piece of DNA - population level distinction

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Phylogeny of didymo based on partial 18S rDNA

Amphora montana AJ243061

Eolimna minima AJ243063

Anomoeoneis shpaerophora AJ535153

Didymospenia geminata BC5 (Montana USA)

Didymosphenia geminata Lower Waiau LW1 (NZ)

Didymosphenia geminata N2 (Norway)

Didymosphenia geminata Oreti OR3 (NZ)

Didymosphenia geminata UKC1

Cymbella sp. (NZ)

Gomphoneis minutae var. cassiae TAR009 (NZ)

Gomphonema parvulum AJ243062

Encyonema triangulatum AJ535157

Dickieia ulvacea AY485462

Eolimna subminuscula AJ243064

Fragilaria striatula AY485474

Navicula cryptocephala AJ297724

0.01 substitutions/site

72

100

98

68

67

79

(relatedness to similar taxa)

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Phylogeography of didymo

USA, Boulder Creek, MO (BC5)

USA, Lee Vining Creek, CA (LV1)

NZ, Lower Waiau (LW2)

NZ, Lower Waiau (LW3)

NZ, Oreti (OR2)

USA, Rapid Creek, SD (RC1)

NZ, Upper Oreti (UO2)

NZ, Upper Waiau (UW1)

NZ, Upper Waiau (UW2)

Canada, Vancouver Island (VI2)

USA, Wenatchee River, WA (WE1)

Iceland (IC1)

United Kingdom (UK1)

Norway (N2)5 bp

2 bp

3 bp

100% bootstrap support

Maximum Parsimony analysis of 703 bp ITS data

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??

Conclusions and future directionsfor phylogeography

• 18S gene provides resolution at the generic level

• ITS provides adequate variation at the species and population level to reveal geographic history of didymo

– A possible N. American invasion ???

• Future analyses will focus on:– More samples within and between river systems

in New Zealand - type each river– Acquisition of multiple samples for each global

location– Search for more variable population-level marker

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Key outcomes

We have:

• Developed robust field compatible protocols for the collection, stabilization and extraction of didymo DNA

• Demonstrated genus level specificity that has been environmentally validated

• Shown extreme sensitivity for low-level detection (< 1 cell per ml) with a broad dynamic range (> 6 orders of magnitude)

• Demonstrated a high degree of reproducibility

• DNA Method can now be implemented for monitoring and surveillance of didymo nationally and internationally.

• Phylogeography studies may soon reveal the origin and number of different didymo introductions to New Zealand.

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Critical future research opportunities

Extensive multi-loci phylogeography studyIdentify origin, movement, vectors, multiple invasions?

Didymo biology - search for the Achilles heel Understanding effects of water quality and chemistry Critical links to possible symbiosis (?)

Biocontrol - search for nature's magic bullet International and local Bacterial Viral

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• Ultimate result:– Negative predictive value– Earliest possible detection– Possibility of mitigation or containment

Recommendations for NI surveillance

Dan Simberloff, Leading invasion biologist, U. Tennessee:Control at the earliest possible stage is much cheaper and easier than

at any later stage

• Critical to increase frequency and range of sampling in NI

• Strong relationships between Regional Councils, DOC, Fish & Game NZ, and MAF BNZ

• Matching support given to get more samples analysed fasterTake home message:

The extensive research conducted by MAF Biosecurity New Zealand and contractors in the South Island has been critical in defining the strategy to keep the NI free of didymo.

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AcknowledgementsWe thank: • NIWA programme collaborators• DOC collaborators – Emily Atkinson, Eric Edwards• Susie Wood, Cawthron Institute• Cathy Kilroy and other NZ collectors for samples• Sarah Spaulding, EPA, USA - samples• NZ Fish & Game staff - samples and field guides• Naomi Crawford, Tanya Chubb – technical assistance• International colleagues for supplying samples• MAF Biosecurity NZ – funding and logistic support

• Especially - close attention and fantastic support from Christina Vieglais, Biosecurity NZ