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

1. 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

2. 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

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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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)

10. 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)

11. 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

12. 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

13. 1. Gel electrophoresis 2. High resolution melt 3. Sequence analysis Three-fold validation process: Validation of positives Most critical step - confidence in results

14. 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

15. Sites sampled with the DNA method No didymo Didymo Rivers Samples 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

16. 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

17. Risk assessment Maintains a high negative predictive value

18. 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

19. 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

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

21. 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

22. 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

23. 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

24. 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

25. ? 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

26. 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.

27. 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

28. 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

29. 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