Extreme low level genetic detection of didymo a new surveillance tool
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Extreme low-level genetic detection of didymo: a new surveillance tool. Craig Cary, Brendan Hicks, Catherine Barnett, Chrissen Gemmill, Andreas Rueckert, Kathryn Coyne 1. Centre for Biodiversity and Ecology Research Department of Biological Sciences School of Science and Engineering

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Extreme low-level genetic detection of didymo: a new surveillance tool

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Extreme low level genetic detection of didymo a new surveillance tool

Extreme low-level genetic detection of didymo: a new surveillance tool

Craig Cary, Brendan Hicks, Catherine Barnett,

Chrissen Gemmill, Andreas Rueckert, Kathryn Coyne1

Centre for Biodiversity and Ecology Research

Department of Biological Sciences

School of Science and Engineering

University of Waikato, Hamilton

1University of Delaware

College of Marine and Earth Studies

Lewes, Delaware 19958, USA


Problem 1

Problem 1

Current 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


Problem 2

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


Extreme low level genetic detection of didymo a new surveillance tool

Criteria for the DNA method

  • Specific 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


Extreme low level genetic detection of didymo a new surveillance tool

Collection and Stabilisation protocols

  • Trial 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


Extreme low level genetic detection of didymo a new surveillance tool

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


Extreme low level genetic detection of didymo a new surveillance tool

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

Didymo 753R Euk1000R Didymo 1670R Euk B

  • 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


Extreme low level genetic detection of didymo a new surveillance tool

Diatom9F and D753R

Diatom9F and D1670R

Euk608F and D753R

D1659F and EukB

D602F and D1670R

D602F and Euk1000R

Euk1000F and D1670R

D602F and D753R

D1565F and EukB

D1565F and D1670R

Euk608F and D1670R

Long primer set

Short primer set

Use for QRT PCR

Primers validated against related species and environmental samples

Tested 12 primers in combination with each other and universals

Ladder

(known bp)

1670-602 = 968 bp

753-602 = 151 bp


Quantitative real time pcr qrt pcr

Quencher

Fluorescent dye

Primers and TaqMan probe anneal to DNA – fluorescence quenched

Polymerase progresses along gene

Rotor Gene 6000

(Corbett)

Primer knocks off fluorescent dye

Primer knocks off fluorescent dye – no longer quenched

Ct

http://www.appliedbiosystems.com

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

Quantitative Real Time-PCR (QRT-PCR)


Extreme low level genetic detection of didymo a new surveillance tool

39

39

1 pg (~ 1 cell) - threshold

R2 = 0.9974

34

34

100 fg - below threshold (BT)

29

29

Threshold cycle number

Threshold cycle number

2

R

= 0.9974

24

24

19

19

14

14

1.E

1.E

-

04

-04

1.E

1.E

-

03

-03

1.E

1.E

-

02

-02

1.E

1.E

-

01

-01

1.E+00

1.E+00

1.E+01

1.E+01

1.E+02

1.E+02

Calibrator DNA (ng reaction-1)

-

1

Calibrator DNA [

ng

reaction

]

Sensitivity of QPCR reaction

Calibrator concentration

10 ng

1 ng

100 pg

Normalised fluorescence

10 pg

Negative controls

Cycle

Sensitivity = 68 copies of target gene (~1 cell)

Linear over 6 orders of magnitude (R2=0.997)


Reproducibility of qpcr

  • Calculated cell abundance for triplicate samples :

  • Sample 1: 20.6 cells/L

  • Sample 2: 19.7 cells/L

  • Sample 3: 21.4 cells/L

  • Average: 20.6 cells/L (+/-0.85)

Ten-fold dilution of each sample confirms absence of inhibitors

Reproducibility of QPCR

Is a single sample representative of didymo abundance from 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


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

Validation of specificity

  • 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

1. Gel electrophoresis

2. High resolution melt

3. Sequence analysis

Three-fold validation process:

Validation of positives

Most critical step - confidence in results


Detection and enumeration in a natural system

N

N

0

0

10 km

10 km

Owen

Owen

River

River

6

3

6

3

5

5

7

7

2

2

4

4

Gowan

Gowan

8

1

8

1

River

River

Buller

Buller

Lake

Lake

River

River

Rotoiti

Rotoiti

Lake

Lake

Rotoroa

Rotoroa

Detection and enumeration in a natural system

  • Buller, Gowan, and Owen Rivers

  • 8 locations (Oct. 2006)

  • Localise populations

  • Owen River - didymo free


Qpcr validation for new zealand

Sites sampled with the DNA method

No didymo

Didymo

RiversSamples

NI 56 75

SI 56 134

QPCR validation for New Zealand

Rivers found positive

NI 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

Manganui-a-te-ao, NI

- still negative


Extreme low level genetic detection of didymo a new surveillance tool

QPCR validation - on-going international survey

Sampled sites

Sites with didymo

Rivers Sampled

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

Rivers found positive

International 12

All positive samples validated as didymo by QPCR method


Risk assessment

Risk assessment

Maintains a high negative predictive value


Cost of dna analyses

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

Taqman analysis pipeline and response strategy

Developed to support high frequency surveillance

  • If positive

  • - notify BNZ and end users

  • - rapid response

  • 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 remains unvalidated - targeted surveillance


Extreme low level genetic detection of didymo a new surveillance tool

Where did didymo come from?Phylogeography of didymo:using molecular markers to reveal its origins and geographic history in New Zealand

?

?


Preliminary phylogeography of didymo

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


Internal transcribed spacers of the 18s rrna

Hypervariable piece of DNA - population level distinction

Internal Transcribed Spacersof the 18S rRNA

D602F

D1659F

ITS3F

ITS1

ITS2

5.8S

18S

28S

D753R

D1670R

ITS4R

ITS2


Phylogeny of didymo based on partial 18s rdna

Phylogeny of didymobased on partial 18S rDNA

(relatedness to similar taxa)

Amphora montana AJ243061

Eolimna minima AJ243063

Anomoeoneis shpaerophora AJ535153

72

Didymospenia geminata BC5 (Montana USA)

Didymosphenia geminata Lower Waiau LW1 (NZ)

100

Didymosphenia geminata N2 (Norway)

Didymosphenia geminata Oreti OR3 (NZ)

67

Didymosphenia geminata UKC1

Cymbella sp. (NZ)

68

79

98

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


Phylogeography of didymo

USA, Boulder Creek, MO (BC5)

Phylogeography of didymo

USA, Lee Vining Creek, CA (LV1)

Maximum Parsimony analysis of 703 bp ITS data

Canada, Vancouver Island (VI2)

USA, Rapid Creek, SD (RC1)

USA, Wenatchee River, WA (WE1)

100% bootstrap support

NZ, Oreti (OR2)

2 bp

NZ, Upper Oreti (UO2)

NZ, Upper Waiau(UW1)

NZ, Upper Waiau (UW2)

Iceland (IC1)

NZ, Lower Waiau (LW2)

NZ, Lower Waiau (LW3)

United Kingdom (UK1)

3 bp

Norway (N2)

5 bp


Conclusions and future directions for phylogeography

?

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


Key outcomes

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.


Critical future research opportunities

Critical future research opportunities

  • Extensive multi-loci phylogeography study

    • Identify 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


Recommendations for ni surveillance

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

  • Ultimate result:

    • Negative predictive value

    • Earliest possible detection

    • Possibility of mitigation or containment

  • Critical to increase frequency and range of sampling in NI

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

Take 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.

  • Matching support given to get more samples analysed faster


Acknowledgements

Acknowledgements

We 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


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