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Using Biotechnology and Bioinformatics to Track a Marine Invader

Marine Biotechnology and Bioinformatics Teacher Enhancement Program at Moss Landing Marine Labs. Using Biotechnology and Bioinformatics to Track a Marine Invader. Based on the work of Dr. Jonathan Geller, Moss Landing Marine Laboratories and

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Using Biotechnology and Bioinformatics to Track a Marine Invader

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  1. MarineBiotechnologyandBioinformatics Teacher Enhancement Program at Moss Landing Marine Labs 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.gif • The coasts of the world are interconnected by extensively used shipping routes • Ocean-going ships use seawater for ballast (balance)

  5. 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/

  6. 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

  7. One cryptic marine invader http://www.penncoveshellfish.com/PennCoveMussels.htm • Before 1988 all “Bay mussels” were considered Mytilus edulis • Thought to be distributed world-wide in temperate areas

  8. 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

  9. 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

  10. 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

  11. 1 2 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

  12. Our Native Adapted from Hilbish, et al., 2000

  13. Native Adapted from Hilbish, et al., 2000

  14. Native Adapted from Hilbish, et al., 2000 Our Invader

  15. Native Adapted from Hilbish, et al., 2000 Invader came from Mediterranean Sea

  16. Native Adapted from Hilbish, et al., 2000 Possible scenario being studied now Invader

  17. 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

  18. 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/navigation/ncal.html

  19. Another California native • Mytilus californianus • Lives on outer coast • Morphologically distinct • Genetically distinct • Will use as an outgroup

  20. 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?

  21. 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

  22. 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

  23. Quick Quiz All invasive species are non-native but not all non-native species are invasive. a. True b. False

  24. Quick Quiz All invasive species are non-native but not all non-native species are invasive. a. True b. False

  25. 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

  26. 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

  27. 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

  28. Hypothesis • As Mytilus galloprovincialis spreads northward it hybridizes with AND displaces Mytilus trossulus http://www.goes.noaa.gov/GIFS/WCIR.JPG

  29. 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

  30. Materials and Methods • Methods: Biotechnology: • DNA extraction -> PCR -> Analysis • Restriction digestion • Gel electrophoresis • DNA sequencing • DISCUSSSION

  31. Materials and Methods • Methods: Bioinformatics: • DNA sequences -> Edit -> Align • Search DNA database • Build trees • Translate DNA sequences • View protein structure • DISCUSSION

  32. 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

  33. 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

  34. DNA RNA Ribosome (RNA + proteins) 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

  35. 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

  36. = repeat protein The molecules • Glu =Polyphenolic adhesive protein - nuclear • Number of repeats (and therefore gene length) varies between species

  37. 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 Hybridswould have a combination of the two patterns. Photo credit: C. Kirlin Second tool to validate ITS results

  38. The molecules • CO3 = Cytochrome c oxidase subunit III - mitochondrial • Species level differences are observed after DNA sequencing • Also, highlights an interesting phenomenon: • Mussel mitochondria don’t play by the rules! • Mitochondria are not maternally inherited as they are in mammals http://www.bioeng.auckland.ac.nz/images/database/bioinformatics/mitochondria.gif

  39. 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 don’t know

  40. 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

  41. 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

  42. 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 ??

  43. Quick Quiz M. galloprovincialis will be distinguished from M. trossulus by: a. dissection b. behavior c. shell morphology d. genetic analysis

  44. Quick Quiz M. galloprovincialis will be distinguished from M. trossulus by: a. dissection b. behavior c. shell morphology d. genetic analysis

  45. Biotech Flowchart

  46. Bioinformatics Flowchart

  47. 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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