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Developing Genetic Barcoding for use in Water Quality Studies. Charles S. Spooner U.S. Environmental Protection Agency National Water Quality Monitoring Council December 10, 2006. Perspectives. Biology is of increasing interest in defining water quality

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Developing genetic barcoding for use in water quality studies

Developing Genetic Barcoding for use in Water Quality Studies

Charles S. Spooner

U.S. Environmental Protection Agency

National Water Quality Monitoring Council

December 10, 2006


Perspectives
Perspectives Studies

  • Biology is of increasing interest in defining water quality

  • Taxonomy has been a part of EPA, USGS water data systems

    • NODC Dataset

    • Integrated Taxonomic Data System (I T I S)

      • Council’s Data Elements ( WQDE)

      • EPA Substance Registry System

      • WQX Schema described yesterday

  • Data systems only want clear records of what was counted


Bioassesments
Bioassesments Studies

State Water Use Designations: Aquatic Life Support

Uses correlations of species to water quality through species tolerance values and trophic guides

Evaluate the incidence and abundance of aquatic fauna as indicators of aquatic ecosystem condition

Involves field collection, subsampling, sorting, and identification by morphological traits by a taxonomist in a lab


Inherent problems with morphology
Inherent Problems With StudiesMorphology

  • Reliance on a small and dwindling pool of taxonomic expertise in morphological taxonomy

  • Concerns about morphological assessments

    • Consistency (accuracy)

    • Taxonomic resolution (precision)

  • Time and cost associated with manual sampling, sorting, and identifying organisms

    • Roughly 7 labor hours and $250 in supply costs per 100 specimens


The research basis
The Research Basis Studies

  • Research in the 1980 established species tolerance values and trophic guides to allow the use of fish and macro-invertabrates as water quality indicators

  • EPT Taxa are three sensitive genera

  • In 2003, researchers at the University of Guelph in Ontario, Canada, proposed “DNA barcoding” as a way to identify species. Barcoding uses a very short genetic (but standard) sequence from a standard part of the genome

  • EPA used DNA to identify species in ballast water and to understand environmental determinants of fish population structure


The ami project
The AMI Project Studies

EPA’s Advanced Monitoring Initiative began in 2007, using Maryland as the test state

(1) determine whether DNA barcoding can improve efficiency of bioassessment and

(2) to use this information to determine how to most efficiently incorporate DNA barcoding into state bioassessment programs.


Project design
Project Design Studies

Reference

Barcode

Database

EPA

Maryland

DNR

If taxonomic agreement

Smithsonian

+

=

Adult Voucher Specimens

EPA-contract

Lab

Guelph

If taxonomic disagreement

Taxon Experts

Morphology

DNA

Repeatability

Accuracy

Precision

Cost

Repeatability

Precision

Cost

Species

Description


Process
Process Studies

  • A leg is sub sampled from the voucher and placed into a 96 well extraction block

  • DNA is extracted from the subsample

    • A robot that can process 384 samples in six hours

  • Amplification of the barcode gene

    • Polymerase Chain Reaction; PCR

      • Partitions subunit 1 of the cytochrome oxidase (CO1) gene

  • Sequencing

    • Determining the composition and order of the nucleotide bases

  • Enter the sequences and metadata into the Barcode of Life Database

  • In the future: into GenBank


  • Bugs in the system
    Bugs in the System Studies

    • ~10% COI amplification failure

    • ~25% unacceptable sequence


    BOLD Studies


    BOLD Studies


    Morphological results
    Morphological Results Studies

    From 2007 MBSS collection 803 specimens were sent to independent lab for identification to the lowest possible level

    Small percentage only identified to order or family

    73.2% to genus, 22.5% to species

    - Reidentification in a separated lab:

    6.8% disagreement in genus level identifications

    Main offenders: Ephemerella and Serratella, Prostoia and Ostrocerca, and Cheumatopsyche and Diplectrona


    Can DNA barcodes Studiesadd resolution to bioassessments?

    Isoperla sp. 1 n=4

    Isoperla sp. 2 n=3

    Isoperla sp. 3 n=3

    Isoperla sp. 4 n=7

    Isoperla sp. 5 n=4

    Isoperla sp. n=27

    Isoperla sp. 6 n=6


    4 Studies

    1

    Genetic groups within Isoperla

    4

    7

    7

    7

    4

    1

    116

    54

    1% sequence divergence

    1


    Conclusions
    Conclusions Studies

    Great potential to enhance bioassessments, after a reference library is formed

    Larger centralized effort is needed to is needed to create an EPT reference library

    Field and lab protocols need to be standardized


    Acknowledgements
    Acknowledgements Studies

    US EPA

    Mark Bagley, Stefanie Swensen, contractor, John Martinson, S.A. Jackson, A.C. Roth, Steve Young

    Smithsonian Institution, Laboratories of Analytical Biology

    Jeffery Hunt, Andrea Ormos, Genelle Harrison, Amy Driskell, Lee Weigt

    Smithsonian Institution, National Museum of Natural History

    Terry Erwin, Oliver S. Flint Jr., David Furth

    Maryland Department of Natural Resources

    Ron Klauda, Ellen Friedman, Istvan Turcsanyi

    Consortium for the Barcode of Life

    David Schindel

    Canadian Barcoding Network

    Pia Marquardt

    Dynamac Corporation

    Ana Braam, Carrie Drake, Mandy Schable, Mike Sergeant,

    Ian Struewing, Charles Watson


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