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
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DNA Barcoding
DNA Metabarcoding
isolation
PCR
Mixed environmental
sample
Bulk DNA
Next generation
sequencing
Full community profile of
the sample
500K to 12 million sequences
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2012 Larval Fish Study Design
I. Probabilistic design
II. Field sampling
larval beach seine
Tucker trawl
3 cycles
- ~50 stations/cycle
- 1 sample (gear)/station
III. Morphological taxonomy
light trap
neuston net
Reconstitute
sample
IV. Molecular taxonomy
- early April (1)
- mid-May (2)
- late June (3)
Duluth Harbor Larval Fishes
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Fish species (31) detected
from larval samples through
DNA metabarcoding in 2012
• (~40 species occur)
Including 8 non-native
Only failed to find 1 species
Uncovered 4 other species
*species not native to Lake
Superior
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Common Name
Brook Silverside*
Longnose Sucker
White Sucker
Redhorse
Rock Bass
Pumpkinseed
Smallmouth Bass
Black Crappie
Common Carp*
Common Shiner
Golden Shiner
Emerald Shiner
Spottail Shiner
Mimic Shiner
Fathead Minnow
Longnose Dace
Creek Chub
Brook Stickleback
Threespine Stickleback
Round Goby*
Tubenose Goby*
White Perch*
Rainbow Smelt*
Johnny Darter
Eurasian Ruffe*
Yellow Perch
Logperch
Walleye
Trout-perch
Cisco
Freshwater Drum*
Scientific Name
Labidesthes sicculus
Catostomus catostomus
Catostomus commersonii
Moxostoma breviceps/macrolepidum
Ambloplites rupestris
Lepomis gibbosus
Micropterus dolomieu
Pomoxis nigromaculatus
Cyprinus carpio
Luxilus cornutus
Notemigonus crysoleucas
Notropis atherinoides
Notropis hudsonius
Notropis volucellus
Pimephales promelas
Rhinichthys cataractae
Semotilus atromaculatus
Culaea inconstans
Gasterosteus aculeatus
Neogobius melanostomus
Proterorhinus semilunaris
Morone americana
Osmerus mordax
Etheostoma nigrum
Gymnocephalus cernua
Perca flavescens
Percina caprodes
Sander vitreus
Percopsis omiscomaycus
Coregonus artedi
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
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27
31
% Agree
78%
75%
86%
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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)
ruffe larva
yellow perch
larva
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Incomplete larval key:
• Myomere number in ruffe mimics
centrarchids
• Overestimated sunfish and black crappy-underestimated ruffe
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- ).
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Expanded Sampling
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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)
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So, it’s 2016. Where the
(bleep) is the data?
Office of Research and Development
National Exposure Research Laboratory
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A word about technology...
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Purchased in 2010 for $750K
Provides long reads (700+ bp) Instrument discontinued
Support ended early 2016
Costs about $10K/run
Generates 300-500K sequences/run
Maximum multiplex of 160 samples
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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)
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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
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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
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Preliminary 16S Results
192 Sites
• St.Louis River/Duluth
• Chequamegon Bay
• Green Bay
• Maumee Bay
• Sandusky Bay
Alewife
Black Crappie
Bluegill
Bluntnose Minnow
Brook Silverside
Buffalo*
Burbot
Carpsucker/Quillback*
Common Carp
Common Shiner
Coregonus spp.*
Emerald Shiner
Eurasian Ruffe
Freshwater Drum
Gizzard Shad
Golden Shiner
Johnny Darter
Logperch
Longnose Sucker
Mimic Shiner
Muskellunge
Pumpkinseed Sunfish
Rainbow Smelt
Rock Bass
Silver Redhorse
Smallmouth Bass
Spot-tail Shiner
Trout-perch
Tubenose Goby
Walleye
White Bass
White Perch
White Sucker
Yellow Perch
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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
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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
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