1 Ecological Exposure Research Division, Cincinnati, OH 2 Mid-Continent Ecology Division, Duluth, MN. Office of Research and Development

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1 Metagenomic apporaches to detecting invasive species in Lake Superior Erik Pilgrim 1, John Martinson 1, Joel Hoffman 2, Anett Trebitz 2, Greg Peterson 2, & Jack Kelly 2 1 Ecological Exposure Research Division, Cincinnati, OH 2 Mid-Continent Ecology Division, Duluth, MN Office of Research and Development

2 1 Goal: DNA method to detect invasive species

3 Molecular taxonomy (DNA barcoding) The use of DNA sequence data from a gene to assign species identification to an unknown specimen DNA barcoding: using a standard gene for ID For most animals, barcoding standard gene is COI 2

4 DNA Barcoding DNA barcode sequence Comparison to reference sequences Serratella micheneri 3

5 Next Generation DNA Sequencing Mixed environmental sample Bulk DNA Next generation sequencing Full community profile of the sample million bases/run (1 billion bases/day)

6 Next Generation sequencing Ability to handle bulk/batch samples Bulk extraction (water, sediment, benthos) Bulk PCR Bulk sequencing Ability to run multiple samples in a single sequencing run (multiplexing) Molecular ID tags flagging different samples 5

7 Genetic monitoring for invasives Two main investigative pathways: 1) Targeting particular invaders with developed biomarkers Advantage: sensitivity Disadvantage: works only for targeted species 2) Community profiles based on genetic data Advantage: ability to detect foreign DNA Disadvantage: foreign IDs may not be as specific as desired 6

8 Targeting specific invaders Develop list of most likely/most worrisome invaders edna Develop individual genetic markers for each invader Screen samples with invader genetic markers Determine presence/absence of targeted invaders Sample communities Bulk DNA extraction

9 Community Profiling Sample communities Foreign DNA sequences could represent new invasives Bulk DNA extraction, PCR, and DNA sequencing Test new samples against library Bioinformatic analysis to ID foreign DNA Develop DNA sequence data for identifying taxa at each community Create library of community DNA sequences *Likely to be done as part of bioassessment

10 Work in Support of GLNPO/GLRI Benthos sampling for detecting and monitoring invasives Lake Superior (Duluth Harbor, Isle Royale) Lake Huron (National Coastal Condition Assessment) Larval fish sampling Lake Superior Mock samples for sensitivity testing 9

11 Lake Superior Benthos Use identified material to enhance barcode database Zooplankton under represented currently Compare benthos of impacted area (Duluth Harbor) to less impacted sites (Isle Royale) 10

12 Lake Huron Benthos Sampled as part of NCCA Paired ponar grabs Compare metagenomic results to standard NCCA data analysis Compare results from different size fractions 500 µm 38 µm unsieved 11

13 Larval Fish Sampling in Lake Superior alewife freshwater drum ninespine stickleback smallmouth bass black crappie golden shiner northern pike spoonhead sculpin blacknose/ longnose dace johnny darter pumpkinseed spottail shiner threespine brook stickleback lake chub rainbow smelt stickleback burbot lake herring rock bass troutperch channel catfish lake sturgeon round goby tubenose goby common carp lake whitefish round whitefish walleye shorthead emerald shiner longnose sucker redhorse white perch eurasian ruffe mottled sculpin silver redhorse white sucker 40+ species from adult fin clips Database of COI and cytochrome B Bulk extractions of larval samples PCRed for COI fathead minnow muskellunge slimy sculpin yellow perch 12

14 Larval detection: Mock samples Samples prepared to test sensitivity of target detection Samples contain target and non target fish tissue Created with adult fillets to mimic biomass conditions Testing ability to find one target in a mass of non target At what point to we lose the ability to detect target species? 1 in 100? 1 in 1000? 1 in 10,000? 13

15 Where are we now? What s next? Lake Huron Benthos has been processed/sieved Bulk DNA extraction beginning next week PCR and NGS runs in the next 1 2 months Larval fish samples all extracted and PCRed NGS runs in the next month Mock samples Bulk DNA extraction beginning this week PCR and NGS runs in the next 1 2 months Lake Superior samples being processed taxonomically for barcode library development 14

16 Acknowledgments EPA Cincinnati John Darling Sara Matthews Tetra Tech Jen Stamp and field crew EPA Duluth Chelsea Hatzenbuhler Julie Barker 15