Using Biotechnology and Bioinformatics to Track a Marine Invader Based on the work of Dr. Jonathan...

Using Biotechnology and Using Biotechnology and Bioinformatics Bioinformatics

to Track a Marine Invaderto 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

OutlineOutline

BackgroundBackground Invasive speciesInvasive species Cryptic invadersCryptic invaders Local musselsLocal mussels

HypothesisHypothesis Materials and methodsMaterials and methods

The moleculesThe molecules Expected resultsExpected results Possible conclusionsPossible conclusions

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

Invasive speciesInvasive species

Non-native species are organisms introduced into Non-native species are organisms introduced into an environment in which they did not evolvean environment in which they did not evolve

Invasive species are non-native species that Invasive species are non-native species that significantly out-compete native speciessignificantly out-compete native species Mostly introduced by human Mostly introduced by human

actionsactions Largest threat to biodiversity Largest threat to biodiversity

after habitat lossafter habitat loss Estimated at least $137 billion Estimated at least $137 billion

in damages annually in the USin damages annually in the US Most marine invaders probably Most marine invaders probably

came as larvae in ballast watercame as larvae in ballast waterCaulerpa taxifolia, an invasive marine alga, in the Mediterranean Sea. Photo credit: U. of Nice, France

The coasts of the world are interconnected The coasts of the world are interconnected by extensively used shipping routes by extensively used shipping routes

Ocean-going ships use seawater for ballast Ocean-going ships use seawater for ballast (balance)(balance)

Ballast waterBallast water

http://www.animaweb.org/images/carte_logistique.gif

Ballast waterBallast water

The water (and The water (and plankton) are moved plankton) are moved great distances and great distances and released released

Planktonic organisms Planktonic organisms after transport are alive, after transport are alive, abundant, and diverseabundant, and diverse

Reference: Carlton & Geller, Reference: Carlton & Geller, 1993, Science1993, Science

Supplement: S1 “Transport of Supplement: S1 “Transport of marine invaders”marine invaders”

http://massbay.mit.edu/exoticspecies/ballast/

Cryptic invadersCryptic invaders

Some invasions can be cryptic (obscure) and Some invasions can be cryptic (obscure) and therefore difficult to tracktherefore difficult to track

Many species with world-wide distribution may Many species with world-wide distribution may actually be successful cryptic invadersactually be successful cryptic invaders

One example of world-wide distribution in One example of world-wide distribution in temperate areas is shown abovetemperate areas is shown above

Modified from figure in Hilbish, et al. 2000

One cryptic marine invaderOne cryptic marine invader

Before 1988 all “Bay mussels” were Before 1988 all “Bay mussels” were considered considered Mytilus edulisMytilus edulis

Thought to be distributed world-wide in Thought to be distributed world-wide in temperate areastemperate areas

http://www.penncoveshellfish.com/PennCoveMussels.htm

Genetic analysisGenetic analysis

Genetic studies uncovered three Genetic studies uncovered three morphologically indistinguishable species morphologically indistinguishable species Mytilus edulisMytilus edulis Mytilus trossulusMytilus trossulus Mytilus galloprovincialisMytilus galloprovincialis

Only one is a successful Only one is a successful invaderinvader

Genetic markers are Genetic markers are essential for species identificationessential for species identification

Photo credit: J. Geller

World-wide distribution?World-wide distribution?

Historic (native) ranges for each species Historic (native) ranges for each species were identifiedwere identified

Areas of hybridization were foundAreas of hybridization were found Invasion events were mappedInvasion events were mapped

Only Only M. galloprovincialis M. galloprovincialis appears to invade, appears to invade, and it has done so repeatedlyand it has done so repeatedly

All done with phylogenetic analysisAll done with phylogenetic analysis

References: Hilbish, et al., 2000, Marine Biology; Riginos & References: Hilbish, et al., 2000, Marine Biology; Riginos & Cunningham, 2005, Molecular EcologyCunningham, 2005, Molecular Ecology

Phylogenetic treesPhylogenetic trees

A diagram showing evolutionary lineages A diagram showing evolutionary lineages of organismsof organisms

DISCUSSION: branches, clades, outgroupDISCUSSION: branches, clades, outgroup

A speculatively rooted tree for rRNA genes. www.answers.com search phylogenetic tree

Our native: Our native: M. trossulusM. trossulus

North Pacific North Pacific M. trossulusM. trossulus Invades North Atlantic after the opening of the Bering Invades North Atlantic after the opening of the Bering

Strait approx. 3.5 million years agoStrait approx. 3.5 million years ago Two “natural invasion” events occurred (1 & 2)Two “natural invasion” events occurred (1 & 2)

Reference: Riginos & Cunningham, 2005, Molecular EcologyReference: Riginos & Cunningham, 2005, Molecular Ecology

1

2

1

2

Our NativeOur Native

Adapted from Hilbish, et al., 2000

NativeNative


NativeNative

Our InvaderOur Invader


NativeNative

Invader came from Mediterranean SeaInvader came from Mediterranean Sea


NativeNative

InvaderInvader Possible scenario Possible scenario being studied being studied

nownow


Invader displaces nativeInvader displaces native

DNA from museum DNA from museum collections shows collections shows M. M. trossulustrossulus in southern CA in southern CA

M. galloprovincialisM. galloprovincialis arrived in arrived in Southern CA in the 1930'sSouthern CA in the 1930's

It has progressively spread It has progressively spread northward and displaced northward and displaced M. M. trossulustrossulus

Reference: Geller, 1999, Conservation Reference: Geller, 1999, Conservation BiologyBiology

Our Location: Monterey BayOur Location: Monterey Bay

Part of the MBNM Part of the MBNM SanctuarySanctuary

Mixed population Mixed population of of M. trossulusM. trossulus and and M. galloprovincialisM. galloprovincialis

http://walrus.wr.usgs.gov/infobank/gazette/html/navigation/ncal.html

Another California nativeAnother California native

Mytilus californianusMytilus californianus Lives on outer coast Lives on outer coast Morphologically Morphologically

distinct distinct Genetically distinctGenetically distinct Will use as an Will use as an

outgroupoutgroup

Why do we care?Why do we care?

To identify what makes a good invaderTo identify what makes a good invader Best to work with closely related speciesBest to work with closely related species

To address major questions in natural To address major questions in natural selection and biodiversityselection and biodiversity How can an invader with limited genetic How can an invader with limited genetic

diversity out compete the genetically diverse diversity out compete the genetically diverse native?native?

How does an invader displace the native that How does an invader displace the native that has been selected for its niche?has been selected for its niche?

How does the invader move up the coast How does the invader move up the coast to SF bay when it did not invade there from to SF bay when it did not invade there from ballast water?ballast water?

Quick QuizQuick Quiz

A(n) _________ species is a non-native A(n) _________ species is a non-native species that successfully out-species that successfully out-competes the native species. competes the native species.

a. robusta. robustb. invasiveb. invasivec. genetically superiorc. genetically superiord. crypticd. cryptic


A(n) _________ species is a non-native A(n) _________ species is a non-native species that successfully out-species that successfully out-competes the native species. competes the native species.

a. robusta. robustb. b. invasiveinvasivec. genetically superiorc. genetically superiord. crypticd. cryptic


All invasive species are non-native but All invasive species are non-native but not all non-native species are not all non-native species are invasive. invasive.

a. Truea. Trueb. Falseb. False


All invasive species are non-native but All invasive species are non-native but not all non-native species are not all non-native species are invasive. invasive.

a. a. TrueTrueb. Falseb. False


Which of the following is an invasive Which of the following is an invasive mussel species in California? mussel species in California?

a. a. M. galloprovincialisM. galloprovincialisb. b. M. trossulusM. trossulusc. c. M. californianusM. californianusd. d. M. edulisM. edulis

OutlineOutline



The moleculesThe molecules Expected resultsExpected results Possible conclusionsPossible conclusions

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

HypothesisHypothesis

AsAs Mytilus Mytilus galloprovincialisgalloprovincialis spreads northward spreads northward it hybridizes with it hybridizes with AND displaces AND displaces Mytilus trossulusMytilus trossulus

http://www.goes.noaa.gov/GIFS/WCIR.JPG

Materials and MethodsMaterials and Methods

Materials: MusselsMaterials: Mussels

"Mussel" is a "Mussel" is a common name for common name for bivalves that attach bivalves that attach using threads using threads (byssus) (byssus)

Supplement: S2 “Know Supplement: S2 “Know your mussel”your mussel”

Washington Dept. of Fish and Wildlife


Methods: Methods: Biotechnology:Biotechnology: DNA extraction -> DNA extraction ->

PCR -> AnalysisPCR -> Analysis Restriction digestionRestriction digestion Gel electrophoresisGel electrophoresis DNA sequencingDNA sequencing

DISCUSSSIONDISCUSSSION


Methods: Methods: Bioinformatics:Bioinformatics: DNA sequences -> DNA sequences ->

Edit -> Align Edit -> Align Search DNA databaseSearch DNA database Build treesBuild trees Translate DNA Translate DNA

sequencessequences View protein View protein

structurestructure

DISCUSSIONDISCUSSION

The moleculesThe molecules

For this study we need For this study we need

to find molecules that:to find molecules that: Share similarities within each species Share similarities within each species

BUT display differences between speciesBUT display differences between species In other words, molecules that are just In other words, molecules that are just

different enough!different enough! Also need well supported resultsAlso need well supported results

Therefore we will use several (3) Therefore we will use several (3) moleculesmolecules

http://www.envirohealthtech.com/images/DNA.jpg


Remember: We will be working with PCR productsRemember: We will be working with PCR products Pieces of DNAPieces of DNA Not whole geneNot whole gene Not just protein-Not just protein-

coding regionscoding regions

Supplement: S3 Supplement: S3 ““Meet the Molecules”Meet the Molecules”

http://www.nearingzero.net/wordplay.html


1.1. ITSITS = the internal transcribed spacer of = the internal transcribed spacer of the the nuclearnuclear ribosomal genes ribosomal genes

Region has restriction site polymorphism Region has restriction site polymorphism between speciesbetween species

http://fp.bio.utk.edu/

mycology/Techniques/mt-what_dna.htm

DNADNARNARNA

RibosomeRibosome

(RNA + proteins)(RNA + proteins)

H H G G T

ITS - Interpreting resultsITS - Interpreting results

GMLane M: Lane M:

100 b.p. ladder lane100 b.p. ladder lane Lanes Lanes GG: : M. galloprovincialisM. galloprovincialisLanes Lanes HH: :

hybridhybrid Lane Lane TT: :

M. trossulusM. trossulus

Easy, useful tool to differentiate speciesEasy, useful tool to differentiate speciesPhoto credit: C. Braby

G G


2.2. Glu Glu == Polyphenolic adhesive Polyphenolic adhesive protein - protein - nuclearnuclear

Number of repeats (and therefore gene Number of repeats (and therefore gene length) varies between specieslength) varies between species

protein

= repeat

M G

Glu - Interpreting resultsGlu - Interpreting results

Second tool to validate ITS resultsSecond tool to validate ITS results

Photo credit: C. Kirlin

Lane M: Lane M: 100 b.p. ladder lane100 b.p. ladder lane

Lanes Lanes GG: : M. galloprovincialisM. galloprovincialis

300 & 500 bp bands300 & 500 bp bands

Lane Lane TT: : M. trossulusM. trossulus 240 bp band240 bp band

HybridsHybrids would have a would have a combination of the two combination of the two patterns.patterns.

G G M T T T


3.3. CO3CO3 = Cytochrome c oxidase = Cytochrome c oxidase subunit III - subunit III - mitochondrial mitochondrial

Species level differences are observed Species level differences are observed after DNA sequencing after DNA sequencing

Also, highlights an interesting Also, highlights an interesting phenomenon:phenomenon:

Mussel mitochondria don’t play by the Mussel mitochondria don’t play by the rules!rules!

Mitochondria are not maternally inherited Mitochondria are not maternally inherited as they are in mammalsas they are in mammals

http://www.bioeng.auckland.ac.nz/images/database/bioinformatics/

mitochondria.gif

Unusual mtDNA inheritanceUnusual mtDNA inheritance

““Doubly Uniparental Inheritance”Doubly Uniparental Inheritance” Daughters receive maternal mtDNA while sons Daughters receive maternal mtDNA while sons

receive both but only pass on paternal mtDNAreceive both but only pass on paternal mtDNA How that works we don’t knowHow that works we don’t know

mitochondrial type:

mitochondrial type:

daughter son

Two separate mtDNA Two separate mtDNA lineageslineages

Same sex mtDNA Same sex mtDNA from different species from different species are more related to are more related to each other than each other than opposite sex mtDNA opposite sex mtDNA of their own species of their own species

Remember the Remember the previous tree?previous tree?

Reference: Geller, 1999, Reference: Geller, 1999, Conservation BiologyConservation Biology

First seen with First seen with BioinformaticsBioinformatics

Why CO3?Why CO3?

CO3 gene fragments need to be CO3 gene fragments need to be purified and sequenced. This will…purified and sequenced. This will… Support mussel species identificationSupport mussel species identification Identify the mtDNA type in hybridsIdentify the mtDNA type in hybrids Provide novel DNA sequences to GenbankProvide novel DNA sequences to Genbank Provide raw material for bioinformatics workProvide raw material for bioinformatics work

We will amplify the female genotype from We will amplify the female genotype from gill since it is found in both sexesgill since it is found in both sexes


M. galloprovincialisM. galloprovincialis displaces displaces M. trossulusM. trossulus by: by:

a. coveting the best substratesa. coveting the best substrates

b. tolerating more environmental b. tolerating more environmental changeschanges

c. genetic hybridizationc. genetic hybridization

d. producing more offspringd. producing more offspring

??


M. galloprovincialis M. galloprovincialis will be distinguished will be distinguished fromfrom M. trossulus M. trossulus by:by:

a. dissection a. dissection

b. behaviorb. behavior

c. shell morphologyc. shell morphology

d. genetic analysisd. genetic analysis


M. galloprovincialis M. galloprovincialis will be distinguished will be distinguished fromfrom M. trossulus M. trossulus by:by:

a. dissection a. dissection

b. behaviorb. behavior

c. shell morphologyc. shell morphology

d. d. genetic analysisgenetic analysis

Biotech FlowchartBiotech Flowchart

Check on gel

Analyze on gel

Digest product

ITS PCR

Analyze

DNA sequencing

Purify product

Check on gel

CO3 PCR

Analyze on gel

Glu PCR

Collect musselsExtract DNA

Bioinformatics FlowchartBioinformatics Flowchart

Build treeDetermine species

Align with:Rest of class

Seq's from Genbank

View 3D structure

TranslateAlign with:

Proteins from other species

Edit trace filesAlign both orientationsResolve all anomalies

View 4-color trace files

YES NO - Discard

BlastIs it Mytilus CO3?

DNA sequence files

OutlineOutline



The moleculesThe molecules Expected resultsExpected results Possible conclusionsPossible conclusions Carcinus maenas, the European green crab, invader of the

Pacific west coast. Photo credit: Jim Carlton

Previous dataPrevious data

Monitored the distribution of Monitored the distribution of M. galloprovincialisM. galloprovincialis along the west coastalong the west coast

Reference: Braby & Somero, 2005, Marine BiologyReference: Braby & Somero, 2005, Marine Biology

7. Santa Cruz 8. Moss Landing – North 9. Moss Landing – South10. Moss Landing ML 11. Monterey



MLMLMLML = the site we will be sampling as well = the site we will be sampling as well





MLMLMLML = the site we will be sampling as well = the site we will be sampling as well Found 2 potential refuges - Found 2 potential refuges - PA & MLMLPA & MLML



What will we learn from our What will we learn from our data?data?

1.1. Is our hypothesis Is our hypothesis supported?supported?

AsAs Mytilus Mytilus galloprovincialisgalloprovincialis spreads northward spreads northward it hybridizes with it hybridizes with AND displaces AND displaces Mytilus trossulusMytilus trossulus

2.2. Is MLML a refuge?Is MLML a refuge?

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

2002

M.trosshybridM.gal

Previous resultsPrevious results

MLML: MLML: Displacement Displacement appears to be appears to be occurring occurring

vs.vs.

PA: This site PA: This site appears to be appears to be a stable a stable refuge refuge

0%

20%

40%

60%

80%

100%

2002MLML

2006MLML

2007MLML

2002PA

2007PA

M.trosshybridM.gal

Sampling procedureSampling procedure

Previous evidence Previous evidence shows that the smaller shows that the smaller mussel population mussel population contains more nativescontains more natives

But for an accurate But for an accurate comparison to comparison to previous data we previous data we should sample in the should sample in the same random waysame random way

Reference: Braby & Somero, Reference: Braby & Somero, 2005, Marine Biology2005, Marine Biology

Possible conclusionsPossible conclusions

Displacement will Displacement will continue at the continue at the MLML site MLML site

Stable refuge will Stable refuge will persist at the PA persist at the PA sitesite


The data collected in this workshop will The data collected in this workshop will contribute to answering which of the contribute to answering which of the following questions? following questions?

a. Is MLML a refuge for a. Is MLML a refuge for M. trossulusM. trossulus? ? b. Is b. Is M. trossulusM. trossulus going extinct? going extinct? c. Is c. Is M. galloprovincialisM. galloprovincialis continuing a continuing a successful northward invasion? successful northward invasion? d. Is the d. Is the M. galloprovincialisM. galloprovincialis invasion invasion threatening threatening M. californianusM. californianus populations? populations?


The data collected in this workshop will The data collected in this workshop will contribute to answering which of the contribute to answering which of the following questions? following questions?

a. a. Is MLML a refuge for Is MLML a refuge for M. trossulusM. trossulus? ? b. Is b. Is M. trossulusM. trossulus going extinct? going extinct? c. c. Is Is M. galloprovincialisM. galloprovincialis continuing a continuing a successful northward invasion? successful northward invasion? d. Is the d. Is the M. galloprovincialisM. galloprovincialis invasion invasion threatening threatening M. californianusM. californianus populations? populations?

ENDEND

Using Biotechnology and Bioinformatics to Track a Marine Invader Based on the work of Dr. Jonathan...

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Transcript of Using Biotechnology and Bioinformatics to Track a Marine Invader Based on the work of Dr. Jonathan...