Transcript M. galloprovincialis - Marine Biotechnology and Bioinformatics is a

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
Marine Biotechnology and Bioinformatics
Teacher Enhancement Program at Moss Landing Marine Labs
Outline
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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
Invasive species
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Non-native species are organisms introduced into an
environment in which they did not evolve
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Invasive species are non-native species that significantly
out-compete native species
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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
Ballast water
http://www.animaweb.org/i
mages/carte_logistique.gif
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The coasts of the world are interconnected by
extensively used shipping routes
Ocean-going ships use seawater for ballast
(balance)
Ballast water
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http://massbay.mit.edu/exoticspecies/ballast/
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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”
Cryptic invaders
Modified from
figure in Hilbish,
et al. 2000
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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
One cryptic marine invader
http://www.penncov
eshellfish.com/Pen
nCoveMussels.htm
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Before 1988 all “Bay mussels” were considered
Mytilus edulis
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Thought to be distributed world-wide in
temperate areas
Genetic analysis
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Genetic studies uncovered three morphologically
indistinguishable species
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Mytilus edulis
Mytilus trossulus
Mytilus galloprovincialis
Only one is a successful
invader
Photo credit: J. Geller
Genetic markers are
essential for species identification
World-wide distribution?
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Historic (native) ranges for each species were
identified
Areas of hybridization were found
Invasion events were mapped
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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
Phylogenetic trees
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A diagram showing evolutionary lineages of
organisms
DISCUSSION: branches, clades, outgroup
A speculatively rooted tree for rRNA genes.
www.answers.com search phylogenetic tree
Our native: M. trossulus
1
2
2
1
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North Pacific M. trossulus
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Invades North Atlantic after the opening of the Bering Strait
approx. 3.5 million years ago
Two “natural invasion” events occurred (1 & 2)
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Reference: Riginos & Cunningham, 2005, Molecular Ecology
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Our Native
Adapted from Hilbish, et al., 2000
Native
Adapted from Hilbish, et al., 2000
Native
Adapted from Hilbish, et al., 2000
Our Invader
Native
Adapted from Hilbish, et al., 2000
Invader came from Mediterranean Sea
Native
Adapted from Hilbish, et al., 2000
Invader
Possible scenario
being studied now
Invader displaces native
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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
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Reference: Geller, 1999, Conservation Biology
Our Location: Monterey Bay
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Part of the MBNM
Sanctuary
Mixed population of
M. trossulus and M.
galloprovincialis
http://walrus.wr.usgs.gov/infobank/gazette/html/navigation/ncal.html
Another California native
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Mytilus californianus
Lives on outer coast
Morphologically distinct
Genetically distinct
Will use as an outgroup
Why do we care?
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To identify what makes a good invader
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To address major questions in natural selection
and biodiversity
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Best to work with closely related species
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?
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
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
Quick Quiz
All invasive species are non-native but not
all non-native species are invasive.
a. True
b. False
Quick Quiz
All invasive species are non-native but not
all non-native species are invasive.
a. True
b. False
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
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
Outline
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


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
Hypothesis

As Mytilus
galloprovincialis
spreads northward it
hybridizes with AND
displaces Mytilus
trossulus
http://www.goes.noaa.gov/GIFS/WCIR.JPG
Materials and Methods
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Materials: Mussels
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"Mussel" is a common
name for bivalves that
attach using threads
(byssus)
Supplement: S2 “Know your
mussel”
Washington Dept. of Fish and Wildlife
Materials and Methods
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Methods:
Biotechnology:
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DNA extraction -> PCR
-> Analysis
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Restriction digestion
Gel electrophoresis
DNA sequencing
DISCUSSSION
Materials and Methods
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Methods:
Bioinformatics:
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DNA sequences ->
Edit -> Align
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Search DNA database
Build trees
Translate DNA
sequences
View protein structure
DISCUSSION
The molecules
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For this study we need
to find molecules that:
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http://www.envirohealthtech.com/images/DNA.jpg
Share similarities within each species BUT
display differences between species
In other words, molecules that are just
different enough!
Also need well supported results
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Therefore we will use several (3) molecules
The molecules
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Remember: We will be working with PCR
products
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Pieces of DNA
Not whole gene
Not just proteincoding regions
Supplement: S3
“Meet the Molecules”
http://www.nearingzero.net/wordplay.html
The molecules
1. ITS = the internal transcribed spacer of the
nuclear ribosomal genes
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Region has restriction site polymorphism between
species
DNA
RNA
http://fp.bio.utk.edu/my
cology/Techniques/mtwhat_dna.htm
Ribosome
(RNA + proteins)
ITS - Interpreting results
M G G G H H G G T
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
The molecules
2. Glu = Polyphenolic adhesive protein nuclear
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Number of repeats (and therefore gene
length) varies between species
= repeat
protein
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
Photo credit: C. Kirlin
Hybrids would have a combination
of the two patterns.
Second tool to validate ITS results
The molecules
3. CO3 = Cytochrome c oxidase
subunit III - mitochondrial
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Species level differences are observed after
DNA sequencing
http://www.bioeng.auckland.ac.nz/images/
database/bioinformatics/mitochondria.gif
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Also, highlights an interesting phenomenon:
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Mussel mitochondria don’t play by the rules!
Mitochondria are not maternally inherited as they
are in mammals
Unusual mtDNA inheritance
mitochondrial type:
mitochondrial type:
daughter
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son
“Doubly Uniparental Inheritance”
Daughters receive maternal mtDNA while sons
receive both but only pass on paternal mtDNA
How that works we don’t know
First seen with Bioinformatics
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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
Why CO3?
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CO3 gene fragments need to be
purified and sequenced. This will…
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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
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
Quick Quiz
M. galloprovincialis will be distinguished from
M. trossulus by:
a. dissection
b. behavior
c. shell morphology
d. genetic analysis
Quick Quiz
M. galloprovincialis will be distinguished from
M. trossulus by:
a. dissection
b. behavior
c. shell morphology
d. genetic analysis
Biotech Flowchart
Collect mussels
Extract DNA
Check on gel
ITS PCR
CO3 PCR
Glu PCR
Digest product
Check on gel
Analyze on gel
Analyze on gel
Purify product
DNA sequencing
Analyze
Bioinformatics Flowchart
DNA sequence files
Blast
Is it Mytilus CO3?
YES
NO - Discard
View 4-color trace files
Edit trace files
Align both orientations
Resolve all anomalies
Align with:
Rest of class
Seq's from Genbank
Translate
Align with:
Proteins from other species
Build tree
Determine species
View 3D structure
Outline
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Background
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Hypothesis
Materials and methods



Invasive species
Cryptic invaders
Local mussels
The molecules
Expected results
Possible conclusions
Carcinus maenas, the European green crab, invader of the
Pacific west coast. Photo credit: Jim Carlton
Previous data
7.
8.
9.
10.
11.
Santa Cruz
Moss Landing – North
Moss Landing – South
Moss Landing ML
Monterey
 Monitored the distribution of M. galloprovincialis along
the west coast

Reference: Braby & Somero, 2005, Marine Biology
Previous data
7.
8.
9.
10.
11.
Santa Cruz
Moss Landing – North
Moss Landing – South
Moss Landing ML
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
Previous data
7.
8.
9.
10.
11.
Santa Cruz
Moss Landing – North
Moss Landing – South
Moss Landing ML
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
What will we learn from our data?
1. Is our hypothesis
supported?

As Mytilus
galloprovincialis
spreads northward it
hybridizes with AND
displaces Mytilus
trossulus
2. Is MLML a refuge?
100%
90%
80%
70%
60%
M.tross
hybrid
M.gal
50%
40%
30%
20%
10%
0%
2002
Previous results
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100%
80%
60%
M.tross
hybrid
M.gal
40%
vs.
20%
0%
2002
MLML
2006
MLML
2007
MLML
2002
PA
2007
PA
MLML:
Displacement
appears to be
occurring
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PA: This site
appears to be a
stable refuge
Sampling procedure
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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
Possible conclusions
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Displacement will
continue at the MLML
site
Stable refuge will
persist at the PA site
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?
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?
END