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Expanding Larval Fish DNA Metabarcoding to All the Great Lakes

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Expanding Larval Fish DNA Metabarcoding to All the Great Lakes Expanding Larval Fish DNA Metabarcoding to All the Great Lakes Erik Pilgrim, Sara Okum, John Martinson, Joel Hoffman, Greg Peterson, Julie Lietz & Chelsea Hatzenbuhler U.S. EPA-Cincinnati/U.S. EPA-Duluth Office of Research and Development National Exposure Research Laboratory Genetic Monitoring for Invasives • Two main investigative pathways: – 1) Targeting particular invaders with developed biomarkers (most eDNA work) • Advantage: sensitivity • Disadvantage: works only for targeted species – 2) Community profiles based on genetic data (DNA metabarcoding) • Advantage: ability to detect ‘foreign’ DNA • Disadvantage: IDs are only as good as the reference database 2 DNA Barcoding DNA Metabarcoding isolation PCR Mixed environmental sample Next generation Bulk DNA sequencing Full community profile of the sample 500K to 12 million sequences 4 2012 Larval Fish Study Design I. Probabilistic design II. Field sampling III. Morphological taxonomy larval beach seine light trap Tucker trawl neuston net Reconstitute 3 cycles sample - ~50 stations/cycle - 1 sample (gear)/station IV. Molecular taxonomy - early April (1) - mid-May (2) - late June (3) Duluth Harbor Larval Fishes Common Name Scientific Name Brook Silverside* Labidesthes sicculus Longnose Sucker Catostomus catostomus White Sucker Catostomus commersonii Fish species (31) detected Redhorse Moxostoma breviceps/macrolepidum • Rock Bass Ambloplites rupestris from larval samples through Pumpkinseed Lepomis gibbosus Smallmouth Bass Micropterus dolomieu DNA metabarcoding in 2012 Black Crappie Pomoxis nigromaculatus • (~40 species occur) Common Carp* Cyprinus carpio Common Shiner Luxilus cornutus • Including 8 non-native Golden Shiner Notemigonus crysoleucas • Only failed to find 1 species Emerald Shiner Notropis atherinoides Spottail Shiner Notropis hudsonius • Uncovered 4 other species Mimic Shiner Notropis volucellus Fathead Minnow Pimephales promelas Longnose Dace Rhinichthys cataractae Creek Chub Semotilus atromaculatus Brook Stickleback Culaea inconstans Threespine Stickleback Gasterosteus aculeatus Round Goby* Neogobius melanostomus Tubenose Goby* Proterorhinus semilunaris White Perch* Morone americana Rainbow Smelt* Osmerus mordax Johnny Darter Etheostoma nigrum Eurasian Ruffe* Gymnocephalus cernua *species not native to Lake Yellow Perch Perca flavescens Logperch Percina caprodes Superior Walleye Sander vitreus Trout-perch Percopsis omiscomaycus Cisco Coregonus artedi 6 Freshwater Drum* Aplodinotus grunniens Morphological- vs. NGS-based ID Morph vs R1 Morph vs R2 R1 vs R2 Richness 32 vs 32* 32 vs 31** 32 vs 31 Common Species 28 27 31 % Agree 78% 75% 86% • NGS found species morph ID did not! – Freshwater Drum (likely as eggs: 2/2 R1 w/eggs; 7/9 R2 w/eggs) – Brook Stickleback (one hit; morph confused with non-native Threespine Stickleback) – Walleye (likely as eggs: 2/2 w/eggs and no unmatched percids) – Creek Chub (morph ID one as hornyhead chub, one blacknose dace) • NGS missed Bluegill and Pumpkinseed – why? The ruffe conundrum 57 sites (DNA) vs. 3 sites (morph) Eurasian ruffe (Gymnocephalus cernua) Incomplete larval key: • Myomere number in ruffe mimics ruffe larva centrarchids • Overestimated sunfish and black crappy-- underestimated ruffe yellow perch larva 8 Total length = 5.0 mm Ruffe Black Crappie Take Home Message (in 2013) • DNA metabarcoding applied to larval fish communities provides a new measure of invasive fish propagules. • Next step: Expand sampling to more of the Great Lakes (2014- ). 10 Expanded Sampling 11 Updated Design/Workflow • Most samples collected as tow • Pick/sort larvae from tow sample • Approximately 25 samples per site undergoing morphological ID • Enter molecular workflow (DNA extraction -> PCR -> DNA Sequencing -> Analysis) 12 So, it’s 2016. Where the (bleep) is the data? Office of Research and Development National Exposure Research Laboratory 13 A word about technology... • Purchased in 2010 for $750K • Provides long reads (700+ bp) Instrument discontinued • Costs about $10K/run Support ended early 2016 • Generates 300-500K sequences/run • Maximum multiplex of 160 samples • Purchased in 2015 for $100K • Provides shorter reads (400-500 bp) • Costs $1-1.5K/run • Generates 12-20 million sequences/run • Currently multiplexing up to 384 samples • (can expand to approx. 1000 samples) 14 Technological “Advancement” • Positives: – New instrument costs less to purchase and run – Generates at least 40X the amount of sequence data as the previous instrument – Very responsive tech support • Challenges: – Create and optimize new markers for the new instrument 15 Where are we now? • Designed, optimized, and processed all samples for new 16S marker (300 bp) – Lots of bioinformatics and analyses to complete in the coming weeks • Designed and optimized new COI marker (about half the standard barcode) – Starting to process these samples 16 Preliminary 16S Results Alewife Logperch Black Crappie Longnose Sucker Bluegill Mimic Shiner Bluntnose Minnow Muskellunge 192 Sites Brook Silverside Pumpkinseed Sunfish • St.Louis River/Duluth Buffalo* Rainbow Smelt • Chequamegon Bay Burbot Rock Bass • Green Bay Carpsucker/Quillback* Silver Redhorse • Maumee Bay Common Carp Smallmouth Bass • Sandusky Bay Common Shiner Spot-tail Shiner Coregonus spp.* Trout-perch Emerald Shiner Tubenose Goby Eurasian Ruffe Walleye Freshwater Drum White Bass Gizzard Shad White Perch Golden Shiner White Sucker Johnny Darter Yellow Perch 17 Upcoming Work • Comparisons, comparisons, and more comparisons – 16S against COI • Possible comparison against old instrument – Molecular results against morphological IDs – Diversity across and between lakes – Sampling period • Share data and results with FWS and USGS collaborators 18 Acknowledgements EPA Cincinnati: Sara Okum, John Martinson EPA Duluth: Joel Hoffman, Anett Trebitz, Chelsea Hatzenbuhler, Julie Lietz, Greg Peterson, Christy Meredith EPA GLNPO/Chicago: Jamie Schardt USFWS: Stephen Hensler, Anjie Bowen , Tim Strakosh, Henry Quinlan Dynamac: Barry Wiechman, Ana Braam, Xiao Song 19.
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