edna: Implications for Aquaculture Wisconsin Aquaculture Association, 2013

Transcription

1 edna: Implications for Aquaculture Wisconsin Aquaculture Association, 2013 Nathan Stone Aquaculture/Fisheries Center University of Arkansas at Pine Bluff

2 DNA

3 What is edna? Environmental DNA DNA that can be extracted from environmental samples (soil, water, air). Taberlet et al. (2012) Within cells or dissolved. Complex mixture of DNA from many different organisms, may be degraded.

4 DNA in the Environment Extracellular DNA is common in freshwater, seawater and benthic environments. Freshwater environments and oceans constitute a great reservoir of extracellular DNA. Pote et al (2009) Persistence is highly variable. 1-3 weeks? Dejean et al. (2011); Degraded or utilized by organisms. Total DNA in surface water ranges from 2-90 µg/l. Pote et al (2009)

5 Lake Erie Water: bacteria cells and virus particles Matteson et al. (2011), qpcr : Summer bacterial abundances: 3.3 to 16 million per milliliter (ml) Summer virus abundances: million per ml (About 237 ml per cup)

6 Releases of fish DNA Scales Urine Feces

7 edna Detection Variety of methods used for: Sample collection Laboratory analysis Concentration filtration / centrifugation Extract / amplify specific segments of DNA using species specific primers Use PCR (or qpcr) Gel electrophoresis of PCR product Processing time up to 2 weeks Photo by Biosurplus.com, real-time PCR, Cepheid SmartCycler II Model SC1000-2

8 Uses for edna? Research Monitoring Enforcement

9 Uses Under Research Invasive species detection Endangered species detection Food habit studies Pathogens in the water Biodiversity

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11 Monitoring Dan Egan

12 Possible Sources of Asian Carp edna

13 ECAL Report February 2013 Fisheries Gear Vessel hulls have considerable amounts of adhering DNA, which can persist for days and is not removed by overland transport. Adhering DNA also does not appear to be completely or quickly washed off of boats moving through the water. Nets appear to be sources of very large amounts of edna but require confirmation...

14 ECAL Report February 2013 Bird Transport and Deposition of edna Piscivorous birds have the capacity to be a direct vector of Asian carp DNA or to contaminate boats with Asian carp DNA. Silver carp DNA could be amplified from bird fecal samples collected up to 1 week following consumption of a silver carp meal. Silver carp DNA in fecal material deposited on metal sheets persisted for 30 days at temperatures exceeding 60 C (140 F).

15 ECAL Report February 2013 Bird Transport and Deposition of edna Satellite-tagged double-crested cormorants exhibited large variation in daily and seasonal bird movement. Throat and cloacal swabs found silver carp DNA in 13 of 15 cormorants from a rookery near Peoria, IL and 7 of 15 cormorants from a rookery near Baker s Lake (within the CAWS). [CAWS : Chicago Area Waterway System]

16 Fish Farming News (6):2. edna & fish meal

17 Photo by Juanmonino/iStockphoto, Would anchovy DNA show up?

18 Asian Carp & Baitfish?

19 Asian carp reproduction in the wild Photos: Kansas Department of Wildlife and Parks Concern over use as bait and possible transfer above barriers like dams.

20 Michigan Draft Asian Carp Plan Prevent transport and release or improper disposal of Asian carps that may be intermixed with baitfish baitfish could be certified as containing no Asian carps and testing could be accomplished at aquaculture facilities using edna surveillance.

21 Illinois, Ohio, Michigan

22 52 bait shops No Asian carp Successful at detecting goldfish visual inspections, and edna screenings found no evidence that the Chicago area bait trade is currently a pathway for Asian carp introduction.

23 edna for Enforcement of Regulations?

24 Concerns with regulatory testing of aquaculture facilities or hauling trucks Validity of tests unknown Quality of labs unknown Sources of DNA other than live Asian Carp Unknown how long DNA can persist If a sample is positive?

25 Spiked samples sent to 11 labs for PCR detection of zebra & quagga mussel larvae: 75.8% accuracy, 7.1% false positives Dr. S. Jerrine Nichols, U.S. Geological Survey Great Lakes Research Center

26 Invasive Species Advisory Committee ISAC is a group of non-federal experts and stakeholders mandated to provide advice to the National Invasive Species Council (NISC) on invasive species-related issues.

27 Main Point of White Paper: The establishment of well regulated sample collection, sample custody, and analyses protocols will allow for: 1) Authoritative public announcements. 2) Appropriate regulatory decisions. 3) Avoid wasteful use of regulatory resources, unnecessary public confusion or unrest, national and international commercial damage, and legal remediation.

28 Advisory Committee Recommendations: 1. National Academy of Sciences review of the reliability and effectiveness of DNA-based methods. 2. Independent performance testing of laboratories. 3. Develop validation & accreditation programs for edna methods and labs. Validation is the Bridge between Research and Regulatory Decisions!

29 Standard protocols, proficiency testing

30 EPA molecular ecologist John Darling: "As technology moves forward, we will see improvements... But the usefulness of all of this is contingent on understanding the likelihood of error and recognizing and communicating the implications for management accordingly.

31 KEY: Understanding Sources of Error In a perfect world, edna would be ideal for detecting rare, illegal, or unwanted organisms. False positive paradox: when used for detecting very rare organisms, even highly specific DNA-based methods could give misleading results when the detection rate is near or below the false positive rate (Darling and Mahon 2011).

32 Contamination is a Crucial Concern (van Oorschot et al. 2010) From Wikipedia, the free encyclopedia: The Phantom of Heilbronn a hypothesized female serial killer whose existence was inferred from DNA evidence found at numerous crime scenes in Austria, France and Germany from 1993 to In late March 2009, investigators concluded that the "Phantom" criminal did not exist, and the DNA recovered at the crime scenes had already been present on the cotton swabs used for collecting DNA samples.

33 Although edna methods show great potential for inventory and monitoring aquatic species, there are still details to resolve. Before adoption of standard procedures for edna sampling and analysis, further development and comparative testing of protocols is necessary. January 2013

34 Summary edna tremendous potential. Extremely sensitive. Potential for positive results but no live fish. Potential for false negatives and false positives. Before used for regulatory purposes: Need for standardization of methods. Validation of tests. Accreditation of laboratories. Performance testing of laboratories.

35 Questions?