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The Global Invasion of the Exotic Alga, Didymosphenia Geminata (Didymo

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The Global Invasion of the Exotic Alga, Didymosphenia Geminata (Didymo Extreme low-level genetic detection of didymo: a surveillance tool Craig Cary, Brendan Hicks, Catherine Barnett, Andreas Rueckert, Kathryn Coyne1 Centre for Biodiversity and Ecology Research 1University of Delaware Department of Biological Sciences College of Marine and Earth School of Science and Engineering Studies Lewes, Delaware 19958, USA University of Waikato, Hamilton Didymo distribution in NZ – 2011 Epidemiology: First discovered Oct. 2004, Waiau River, Southland 209 infected sites – Mar. 9, 2009 Broke to Buller in 2005 Spread to Central Otago 2006/07 Spread with in Nelson area 2007 Spread to Fiordland 2007 2004 in 2 rivers 2005 in 11 rivers 1,801 rivers 2006 in 29 Rivers 2007 in 55 rivers and 4 lakes by 2024 2009 in 132 rivers and 5 lakes 2011 in 243 rivers and 7 lakes What will stop a North Island Incursion? • Continued targeted public outreach • Interior border protection - anglers, 4WD, kayakers • Early warning - high risk area surveillance in NI • Rapid and decisive mitigation • Continued high frequency surveillance Develop a new early warning method with high negative predictive value Developing a long-term didymo surveillance capability for New Zealand New didymo specific approaches were needed: • Sampling tools and strict procedures • New highly sensitive analysis tools • Database development and management 4 Passed sampling efforts relied 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 5 Standardized collection, stabilisation, and denaturation 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 • Swab protocols - detecting non-visible attached didymo • Developed simple DNA denaturation procedures • Fixation in 70% ethanol or Vodka ! • Developed complete didymo sampling kit The Didymo Sampling Kit Designed to enable reproducible contamination free sampling Didymo kits ready to ship to NZ agencies 17 Dec 2007 Field Guide for Didymo Sampling • Didymo drift net sampling preparations • Sampling kit contents • Drift net sampling procedures • Sampling handling and preservation • Decontamination and denaturation • Sample mailing to Waikato 9 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 Polymerase Chain Reaction Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated ….. And then what Distinctiveness of didymo 18s rRNA • Nothing known about didymo genetics • Lower Waiau sample - Target gene 18S ribosomal RNA (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 Ladder (known bp) Long primer set 1670-602 = 968 bp Short primer set 753-602 = 151 bp Use for QRT PCR Quantitative Real Time-PCR (QRT-PCR) Fluorescent dye Quencher Primers and TaqMan probe anneal to DNA – fluorescence quenched Polymerase progresses along Rotor Gene 6000 gene (Corbett) Primer knocks off fluorescent dye Primer knocks off fluorescent dye – no longer quenchedCt http://www.appliedbiosystems.com 16 Ct: cycle threshold value. Lower Ct = greater abundance of target Sensitivity of QPCR reaction Calibrator concentration 10 ng 1 ng 100 pg 1 pg (~ 1 cell) - threshold 10 pg Negative controls 100 fg - below threshold (BT) Normalised fluorescence Cycle 39 R2 = 0.9974 34 Sensitivity = 68 copies of target gene (~1 cell) 29 Linear over 6 orders of magnitude R 2 = 0.9974 24 (R2=0.997) Threshold cyclenumber 19 14 Threshold cycle cycle Threshold number 14 1.E- 04 -04 1.E- 03 -03 1.E- 02 -02 1.E- 01 -01 1.E+00 1.E+01 1.E+02 CalibratorCalibrator DNA DNA [ng reaction (ng- 1 ] reaction-1) 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 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 18 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 Detection and enumeration N in a natural system 0 10 km • Buller, Gowan, and Owen Rivers Owen River 66 33 7 5 7 2 • 8 locations (Oct. 2006) 4 Gowan 4 8 River 1 Buller • Localise populations River Lake Lake Rotoiti Rotoroa • Owen River - didymo free 20 QPCR validation for New Zealand Rivers Samples Sites sampled with the DNA method NI 56 75 SI 56 134 Rivers found positive NI 0 (May 2007 delimiting survey) No didymo SI 50 Didymo 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 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), Iran (1) Rivers found positive International 12 All positive samples validated as didymo by QPCR method 22 Proposed rapid response for North Island incursion N • Immediate resampling • Sequence validation 0 10 km • Microscopy Owen River 6 3 7 5 2 4 • Delimiting survey (5km) Gowan 8 River 1 Buller • Quantitation River Lake Lake Rotoiti • Mitigation Rotoroa • Controlled access • GemX treatment (?) 23 Cost of DNA analyses $100 NZ 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 – 96 per week, ~400 samples per month • Robotic operation, investment in equipment • Cost savings for volume likely 24 Current didymo surveillence program in the North Island • Samples collected by regional councils, DOC, Fish and Game • Processing at Univ. of Waikato – 4day turn around • Field data entered by sample source • Didymo results entered at Waikato into NZ Didymo Samples Database • 25-30 samples per month • To date over 2000 samples have been run • NI still negative for didymo 25 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. 26 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 • Christina Vieglais – Biosecurity New Zealand • 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 27 .
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