Using Biotechnology and Bioinformatics to Track a Marine Invader

1
Using Biotechnology and Bioinformatics to Track
a Marine Invader

• Based on the work of
• Dr. Jonathan Geller, Moss Landing Marine
  Laboratories
• and
• Dr. Caren Braby, Monterey Bay Aquarium Research
  Institute

2
Outline

• Background
• Invasive species
• Cryptic invaders
• Local mussels
• Hypothesis
• Materials and methods
• The molecules
• Expected results
• Possible conclusions

Carcinus maenas, the European green crab, invader
of the Pacific west coast. Photo credit Jim
Carlton
3
Invasive species

• Non-native species are organisms introduced into
  an environment in which they did not evolve
• Invasive species are non-native species that
  significantly out-compete native species
• Mostly introduced by human
• actions
• Largest threat to biodiversity
• after habitat loss
• Estimated at least 137 billion
• in damages annually in the US
• Most marine invaders probably
• came as larvae in ballast water

Caulerpa taxifolia, an invasive marine alga, in
the Mediterranean Sea. Photo credit U. of Nice,
France
4
Ballast water
http//www.animaweb.org/images/carte_logistique.gi
f

• The coasts of the world are interconnected by
  extensively used shipping routes
• Ocean-going ships use seawater for ballast
  (balance)

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Ballast water

• The water (and plankton) are moved great
  distances and released
• Planktonic organisms after transport are alive,
  abundant, and diverse
• Reference Carlton Geller, 1993, Science
• Supplement S1 Transport of marine invaders

http//massbay.mit.edu/exoticspecies/ballast/
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Cryptic invaders
Modified from figure in Hilbish, et al. 2000

• Some invasions can be cryptic (obscure) and
  therefore difficult to track
• Many species with world-wide distribution may
  actually be successful cryptic invaders
• One example of world-wide distribution in
  temperate areas is shown above

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One cryptic marine invader
http//www.penncoveshellfish.com/PennCoveMussels.h
tm

• Before 1988 all Bay mussels were considered
  Mytilus edulis
• Thought to be distributed world-wide in temperate
  areas

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Genetic analysis

• Genetic studies uncovered three morphologically
  indistinguishable species
• Mytilus edulis
• Mytilus trossulus
• Mytilus galloprovincialis
• Only one is a successful
• invader
• Genetic markers are
• essential for species identification

Photo credit J. Geller
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World-wide distribution?

• Historic (native) ranges for each species were
  identified
• Areas of hybridization were found
• Invasion events were mapped
• Only M. galloprovincialis appears to invade, and
  it has done so repeatedly
• All done with phylogenetic analysis
• References Hilbish, et al., 2000, Marine
  Biology Riginos Cunningham, 2005, Molecular
  Ecology

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Phylogenetic trees

• A diagram showing evolutionary lineages of
  organisms
• DISCUSSION branches, clades, outgroup

A speculatively rooted tree for rRNA genes.
www.answers.com search phylogenetic tree
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Our native M. trossulus
2
1

• North Pacific M. trossulus
• Invades North Atlantic after the opening of the
  Bering Strait approx. 3.5 million years ago
• Two natural invasion events occurred (1 2)
• Reference Riginos Cunningham, 2005, Molecular
  Ecology

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Our Native
Adapted from Hilbish, et al., 2000
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Native
Adapted from Hilbish, et al., 2000
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Native
Adapted from Hilbish, et al., 2000
Our Invader
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Native
Adapted from Hilbish, et al., 2000
Invader came from Mediterranean Sea
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Native
Adapted from Hilbish, et al., 2000
Possible scenario being studied now
Invader
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Invader displaces native

• DNA from museum collections shows M. trossulus in
  southern CA
• M. galloprovincialis arrived in Southern CA in
  the 1930's
• It has progressively spread northward and
  displaced M. trossulus
• Reference Geller, 1999, Conservation Biology

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Our Location Monterey Bay

• Part of the MBNM Sanctuary
• Mixed population of M. trossulus and M.
  galloprovincialis

http//walrus.wr.usgs.gov/infobank/gazette/html/na
vigation/ncal.html
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Another California native

• Mytilus californianus
• Lives on outer coast
• Morphologically distinct
• Genetically distinct
• Will use as an outgroup

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Why do we care?

• To identify what makes a good invader
• Best to work with closely related species
• To address major questions in natural selection
  and biodiversity
• How can an invader with limited genetic diversity
  out compete the genetically diverse native?
• How does an invader displace the native that has
  been selected for its niche?
• How does the invader move up the coast to SF bay
  when it did not invade there from ballast water?

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Quick Quiz

• A(n) _________ species is a non-native species
  that successfully out-competes the native
  species.
• a. robust
• b. invasive
• c. genetically superior
• d. cryptic

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Quick Quiz

• A(n) _________ species is a non-native species
  that successfully out-competes the native
  species.
• a. robust
• b. invasive
• c. genetically superior
• d. cryptic

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Quick Quiz

• All invasive species are non-native but not all
  non-native species are invasive.
• a. True
• b. False

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Quick Quiz

• All invasive species are non-native but not all
  non-native species are invasive.
• a. True
• b. False

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Quick Quiz

• Which of the following is an invasive mussel
  species in California?
• a. M. galloprovincialis
• b. M. trossulus
• c. M. californianus
• d. M. edulis

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Quick Quiz

• Which of the following is an invasive mussel
  species in California?
• a. M. galloprovincialis
• b. M. trossulus
• c. M. californianus
• d. M. edulis

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Outline

• Background
• Invasive species
• Cryptic invaders
• Local mussels
• Hypothesis
• Materials and methods
• The molecules
• Expected results
• Possible conclusions

Carcinus maenas, the European green crab, invader
of the Pacific west coast. Photo credit Jim
Carlton
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Hypothesis

• As Mytilus galloprovincialis spreads northward it
  hybridizes with AND displaces Mytilus trossulus

http//www.goes.noaa.gov/GIFS/WCIR.JPG
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Materials and Methods

• Materials Mussels
• "Mussel" is a common name for bivalves that
  attach using threads (byssus)
• Supplement S2 Know your mussel

Washington Dept. of Fish and Wildlife
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Materials and Methods

• Methods Biotechnology
• DNA extraction -gt PCR -gt Analysis
• Restriction digestion
• Gel electrophoresis
• DNA sequencing
• DISCUSSSION

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Materials and Methods

• Methods Bioinformatics
• DNA sequences -gt Edit -gt Align
• Search DNA database
• Build trees
• Translate DNA sequences
• View protein structure
• DISCUSSION

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The molecules

• For this study we need
• to find molecules that
• Share similarities within each species BUT
  display differences between species
• In other words, molecules that are just different
  enough!
• Also need well supported results
• Therefore we will use several (3) molecules

http//www.envirohealthtech.com/images/DNA.jpg
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The molecules

• Remember We will be working with PCR products
• Pieces of DNA
• Not whole gene
• Not just protein-
• coding regions
• Supplement S3
• Meet the Molecules

http//www.nearingzero.net/wordplay.html
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The molecules

• ITS the internal transcribed spacer of the
  nuclear ribosomal genes
• Region has restriction site polymorphism between
  species

http//fp.bio.utk.edu/mycology/Techniques/mt-what_
dna.htm
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ITS - Interpreting results
H
H
G
G
T
G
M
G
G

• Lane M
• 100 b.p. ladder lane
• Lanes G
• M. galloprovincialis
• Lanes H
• hybrid
• Lane T
• M. trossulus

Photo credit C. Braby
Easy, useful tool to differentiate species
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The molecules

• Glu Polyphenolic adhesive protein - nuclear
• Number of repeats (and therefore gene length)
  varies between species

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Glu - Interpreting results
M
G
G
G
M
T
T
T

• Lane M
• 100 b.p. ladder lane
• Lanes G
• M. galloprovincialis
• 300 500 bp bands
• Lane T
• M. trossulus
• 240 bp band
• Hybrids would have a combination of the two
  patterns.

Photo credit C. Kirlin
Second tool to validate ITS results
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The molecules

• CO3 Cytochrome c oxidase
• subunit III - mitochondrial
• Species level differences are observed after DNA
  sequencing
• Also, highlights an interesting phenomenon
• Mussel mitochondria dont play by the rules!
• Mitochondria are not maternally inherited as they
  are in mammals

http//www.bioeng.auckland.ac.nz/images/database/b
ioinformatics/mitochondria.gif
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Unusual mtDNA inheritance
mitochondrial type
mitochondrial type
daughter
son

• Doubly Uniparental Inheritance
• Daughters receive maternal mtDNA while sons
  receive both but only pass on paternal mtDNA
• How that works we dont know

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First seen with Bioinformatics

• Two separate mtDNA lineages
• Same sex mtDNA from different species are more
  related to each other than opposite sex mtDNA of
  their own species
• Remember the previous tree?
• Reference Geller, 1999, Conservation Biology

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Why CO3?

• CO3 gene fragments need to be
• purified and sequenced. This will
• Support mussel species identification
• Identify the mtDNA type in hybrids
• Provide novel DNA sequences to Genbank
• Provide raw material for bioinformatics work
• We will amplify the female genotype from gill
  since it is found in both sexes

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Quick Quiz

• M. galloprovincialis displaces M. trossulus by
• a. coveting the best substrates
• b. tolerating more environmental changes
• c. genetic hybridization
• d. producing more offspring

??
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Quick Quiz

• M. galloprovincialis will be distinguished from
  M. trossulus by
• a. dissection
• b. behavior
• c. shell morphology
• d. genetic analysis

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Quick Quiz

• M. galloprovincialis will be distinguished from
  M. trossulus by
• a. dissection
• b. behavior
• c. shell morphology
• d. genetic analysis

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Biotech Flowchart
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Bioinformatics Flowchart
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Outline

• Background
• Invasive species
• Cryptic invaders
• Local mussels
• Hypothesis
• Materials and methods
• The molecules
• Expected results
• Possible conclusions

Carcinus maenas, the European green crab, invader
of the Pacific west coast. Photo credit Jim
Carlton
48
Previous data
7. Santa Cruz 8. Moss Landing North 9.
Moss Landing South 10. Moss Landing ML 11.
Monterey

• Monitored the distribution of M.
  galloprovincialis along the west coast
• Reference Braby Somero, 2005, Marine Biology

49
Previous data
7. Santa Cruz 8. Moss Landing North 9.
Moss Landing South 10. Moss Landing ML 11.
Monterey

• Monitored the distribution of M.
  galloprovincialis along the west coast
• MLML the site we will be sampling as well
• Reference Braby Somero, 2005, Marine Biology

50
Previous data
7. Santa Cruz 8. Moss Landing North 9.
Moss Landing South 10. Moss Landing ML 11.
Monterey

• Monitored the distribution of M.
  galloprovincialis along the west coast
• MLML the site we will be sampling as well
• Found 2 potential refuges - PA MLML
• Reference Braby Somero, 2005, Marine Biology

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What will we learn from our data?

• Is our hypothesis supported?
• As Mytilus galloprovincialis spreads northward it
  hybridizes with AND displaces Mytilus trossulus
• Is MLML a refuge?

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Previous results

• MLML Displacement appears to be occurring
• vs.
• PA This site appears to be a stable refuge

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Sampling procedure

• Previous evidence shows that the smaller mussel
  population contains more natives
• But for an accurate comparison to previous data
  we should sample in the same random way
• Reference Braby Somero, 2005, Marine Biology

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Possible conclusions

• Displacement will continue at the MLML site
• Stable refuge will persist at the PA site

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Quick Quiz

• The data collected in this workshop will
  contribute to answering which of the following
  questions?
• a. Is MLML a refuge for M. trossulus?
• b. Is M. trossulus going extinct?
• c. Is M. galloprovincialis continuing a
  successful northward invasion?
• d. Is the M. galloprovincialis invasion
  threatening M. californianus populations?

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Quick Quiz

• The data collected in this workshop will
  contribute to answering which of the following
  questions?
• a. Is MLML a refuge for M. trossulus?
• b. Is M. trossulus going extinct?
• c. Is M. galloprovincialis continuing a
  successful northward invasion?
• d. Is the M. galloprovincialis invasion
  threatening M. californianus populations?

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END