Current surveillance methods to detect didymo presence/absence

1. Current surveillance methods to detect didymo presence/absence Maurice Duncan NIWA Presentation to MAF BNZ Didymo Science Seminar 24-07-07.

2. Outline for talk • National Delimiting Surveys and Ongoing Passive Surveillance and local surveys • Site selection • Survey frequency • Sampling protocols • Sampling methods • Disinfection • Cross-contamination • Interpretation of laboratory results • Summary of positive sites

3. Survey Types: NDS, OPS and local surveys • National Delimiting Surveys: • Planned surveys where sites are chosen for risk of infection and likelihood of didymo establishment and growth and for geographical coverage and carried out every 2-3 months. • Ongoing Passive Surveillance: • Where members of the public or interested organisations report suspected didymo • Local surveys: • Where organisations such as DOC or Regional Councils conduct surveys • Of 34 didymo +ve rivers, didymo visible at 9.

4. Site selection for NDS • First surveys - site selection based on a likely environments map and local opinion about sites with high use • More recent surveys based on ranking • Risk of establishment and growth based on the likely environment model (LEM1) • Visitor frequency (angler use surveys) • Risk activity: angling, boating, 4WD, picnic, etc • Accessibility: road type, remoteness, etc • Likelihood of transfer: proximity of infected sites • Social/cultural: iconic sites to local use. • Input on final list from, Regional Councils, F&G and DOC and then MAF BiosecurityNZ

5. Survey frequency • Oct 2004 (Southland) Oct 2005, Feb 2006, May 2006, Aug 2006, Nov 2006, Feb 2007 and May 2007. • NDS have been carried out about every 3 months • Ideal survey frequency probably about every 2 months with maybe a 4 month gap over winter • About 75% of new incursions found from planned surveys (NDS (50%) and Local surveys (25%)) and 25 % from OPS • Some OPS finds would have been picked up in the next NDS

6. Sampling protocols • To obtain a systematic and scientifically defensible sample to detect didymo even if it is rare • To disinfect equipment to prevent contamination of other sites • To prevent cross-contamination of samples and suspected false positives • The protocol is under constant revision primarily to reduce the possibility of cross-contamination that seems to continually be an issue

7. Sampling protocol – main points • Separation and labelling of materials for each site to reduce the amount of handling at each site and so reduce the possibility of cross-contamination • The separation on the duties of the two person sampling team into wet and dry work to reduce the possibility of the “wet” person contaminating other samples or unfilled sampling containers • Thorough decontamination using bleach of any piece of equipment, waders etc that have been in contact with the water • Benzalkonium chloride wet wipes to be used to decontaminate skin.

8. Sampling methods • (1) Visual inspection of the site • (2) Benthic sample: systematic pooled sample of algal scrapings from 5 stones from each of 5 transects sampling different river habitats (run, riffle, pool) • (2) Drift net sample: 40 micron mesh drift net deployed for 10 minutes • Drift net use based on scientific investigation of the best way of detecting didymo • Sub sample of drift net sample for DNA analysis

9. Gathering rocks

10. Pooling rock scrapings

11. Deploying the drift net

12. Drift net in action

13. Disinfection • Purpose: to prevent infection of the next sites to be sampled • Also to remove didymo from sampling gear so any didymo at the next site can be attributed to the that site • Survey from upstream to downstream • Bleach is best using a 2% solution with contact for 1 minute. • But a 5% solution with15 minutes contact for the drift net to denature DNA when DNA samples are being taken

14. Disinfecting

15. Cross-contamination • Cross-contamination (CC) occurs when didymo or DNA from one site occurs in a sample from another site • Much of the sampling and decontamination protocol is designed to prevent cross-contamination • CC can result in false positives • CC causes extra work when a river with a suspected false positive has to be re-sampled.

16. Laboratory protocol for microscope examination • Fixed time effort to examine samples, e.g.,10 -15 minutes per site (3 sub-samples) • Let samples settle and take sample from the bottom of the sample jar if there is only a small amount of material. • Report on the number of cells seen and their health, rather than just presence or absence to help determine whether the sample is a true positive.

17. Interpretation of lab results • Healthy cells in both net and benthic samples = true positive • Healthy cells in either net or benthic sample and dead cells in the other = true positive • Abundant dead or unhealthy cells in both samples = true positive • A few dead or unhealthy cells in either sample and none in the other = suspected false positive – resample site and perhaps sites further upstream of down stream • No cells in either sample = true negative

18. Summary of positive sites • To date 14 South Island major river systems infected. • Waiau/Mararoa, Wairaurahiri, Eglinton, Monowai • Aparima • Oreti • Mataura • Clutha, Lindis, Shotover, Kawarau, Makarora, Motatapu, Matukituki, Von, Greenstone, Dart, Wilkin, Young, Hawea. • Haast, Hollyford • Kakanui • Waitaki, Ahuriri, Tasman, Tekapo, Twizel, Maerewhenua, Ohau • North Opuha • Rangitata • Hurunui • Buller, Gowan, Matakitaki, • Motueka • Takaka • Lakes Te Anau/Manapouri, Wakatipu, Wanaka, Dunstan, Benmore

19. Extent of didymo infection in NZ shown as rivers coloured red. • = 20% of large (≥ order 5) South Island rivers • All sites in the top two categories of LEM1

20. Conclusion • Planned delimiting surveys have been successful at detecting didymo at very early stages of infection • Sites for surveys should be determined on the basis of risk of infection and likelihood of establishment and growth (LEM2) • Survey every 2 months

21. Conclusions continued • Follow the sampling and disinfection protocols to the letter to ensure didymo is found if it is rare, and to reduce cross-contamination • Laboratory analysis has to be more than just determination of presence or absence • Details of cell abundance and health help determine whether the river is clear of didymo or not.

22. Thanks • Funding: • MAF Biosecurity New Zealand • Sampling protocols: • Cathy Kilroy, Christina Vieglais, Eric Edwards, Frances Velvin. • Field sampling: • NIWA field teams, Regional Councils, Fish & Game, & DOC. • Lab analysis: • Karen Robinson, Cathy Kilroy, Karl Safi, Donna Sutherland & Laura Drummond, Nelson Boustead