18S rRNA clone library of phytoplankton in the Columbia River and its coastal zone

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18S rRNA clone library of phytoplankton in the
Columbia River and its coastal zone
http//www.mapwatch.com/news-blog/images/phytoplan
kton.jpg

• By Pete Kahn
• Mentors Lydie Herfort and
• Peter Zuber

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Significance of phytoplankton

• Major primary producers
• O2 90
• CO2gtC3 50
• Most abundant eukaryotes 102 to 104 cells/ mL
• Base of aquatic food chain

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Interactions with other Domains
http//www.fossilmuseum.net/Tree_of_Life/Domains_A
rchaea_Bacteria/Domains_of_life.jpg
Bacterioplankton Colonization POCgtDOC
Competition for NO32- and NH3?

• Grossart, H. P., Czub, G. Simon, M. (2006).
  Algae-bacteria interactions and their effects on
  aggregation and organic matter flux in the sea.
  Environmental Microbiology 8, 1074-1084.

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Traditional Methods
Flow cytometry
Microscopy Pediastrium

• Microscopy
• Flow Cytometry
• Pigment analysis
• Chl a
• Photosynthesis
• O2 production
• C14 uptake

http//upload.wikimedia.org/wikipedia/commons/c/c7
/Picoplancton_cytometrie.jpg
http//www.keweenawalgae.mtu.edu/ALGAL_IMAGES/chlo
rophyceans/Pediastrium_n16_dollartow3_402_c.jpg
Filtering for chlorophyll a
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Molecular Methods for eukaryotes

• Pitfalls of Traditional methods
  -Microscopy focus on
  numerous, big organisms time consuming
  
  -Incomplete understanding of diversity
• Molecular Methods nucleic acid extraction, PCR,
  cloning, sequencing
• Pitfalls of Molecular Methods
  biases in PCR primers
  preferential amplification of some organisms,
  i.e. dinoflagellates vs diatoms

Savin, M. C., Martin, J. L., LeGresley, M.,
Giewat, M. Rooney-Varga, J. (2004). Plankton
diversity in the Bay of Fundy as measured by
morphological and molecular methods. Microbial
Ecology 48, 51-65.
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Problems for phytoplankton

• Traditional methods studies gtgtgtgt molecular
  methods studies
• Prokaryotic molecular studies gtgtgtgt phytoplankton
  molecular studies

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The sampling plan

• 18S rRNA clone library for
• April Wecoma Forerunner salinity gradient
• Wecoma (CR4s, CR15s, CR30s, CR40s)
• 20-32 psu coastal surface samples
• Forerunner (St 1-5) estuary samples
• 0-20 psu, inc. of 5
• June July Forerunner 0 psu 30 psu from
  estuary

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Sample Dates Locations
Wecoma April 2007
Forerunner April 2007
Forerunner June 2007
Forerunner July 2007
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Goals within CMOP

• Short term
• -Good clone library of phytoplankton
• -Determining good primers for isolating 18S
  rRNA
• Long term
• -Understand changes in populations between
  seasons and locations
• -Better understand unexpected changes in
• community monitor ecosystem health
• -Bacteria Archaea relationships/
  interactions (Dan Murphy)
• -Better understanding of microbial ecosystem
  as
• a whole

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What we know about Columbia River phytoplankton

• Anderson GC. 1972. Aspects of marine
  phytoplankon studies near the Columbia River with
  special reference to subsurface chlorophyll
  maximum. The Columbia River estuary and adjacent
  ocean waters, University of Washington Press,
  Seattle, WA, p. 219-240.
• Contaminant ecology of fish and wildlife of the
  lower columbia river-final report. April 1996.
  Lower Columbia River Bi-State Program.
• Frey BE, R Lara-Lara, LF Small. 1984. Primary
  production in the Columbia River Estuary water
  column. Columbia River Estuary Data Development
  Program.

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What we know about Columbia River phytoplankton
Asterionella formosa most abundant diatom

• Estuary gt50 species of
  diatoms Asterionella formosa Asterionella
  kariana Skeletonema Costatum Thallasiosira
  Stephanodiscus hatzschii
• Coastal Chaetoceros convolutus Dactyliosolen
  mediterraneus Thalassionema nitzschioides

http//www.serc.si.edu/labs/phytoplankton/guide/di
atoms/images/Asterionella/Asterionella-formosa-PA.
jpg
Thalassionema nitzschioides
http//thalassa.gso.uri.edu/flora/imagesfl/tnema1.
jpg
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Variations in nucleic acid in environmental
samples
Oct 2006
Feb 2007

• Between seasons
• Between locations
• April Wecoma Forerunner Freshwater ltlt
  Surface Coastal

DNA
RNA
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Methods Overview
Water sample gt Sterivex on site
Total nucleic acid extraction from sterivex in lab
DNA removal?
Amplification through PCR
TOPO cloning/ transformation
96 well plates?
Plasmid isolation (Miniprep)
Water samples pass through sterivex filter
Sequences
BLASTgt Clone library
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Extraction/ DNA removal

• Extraction with LETS buffer Phenol Chloroform
• DNA removal with TURBO DNA free kit or Ribopure
  kit

DNA removed
DNA not removed
Gel after extraction DNA removal
Sterivex
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PCR

• Primers Euk A AACCTGGTTGATCCTGCC
  Euk B TGATCCTTCTGCAGGTTCACCTAC Specific
  for eukaryotes, Examined with BLAST highly
  conserved
• PCR cleanup with MO-BIO ultra clean kit

Gel after PCR
Diez, B., Pedros-Alio, C. Massana, R. (2001).
Study of genetic diversity of eukaryotic
picoplankton in different oceanic regions by
small-subunit rRNA gene cloning and sequencing.
Applied and Environmental Microbiology 67,
2932-2941.
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Cloning/ Transformation

• TOPO TA Cloning Kit
• Transformation with Top 10 chemical competent
  cells gt E. coli
• Plated onto LB XGAL-AMP plates
• -Selective cells gain ampicillin resistance
  from
• plasmid
• -Differential cells w/plasmid insert turn
  white
• cells w/ no insert turn
  blue

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Plasmid isolation (miniprep)

• For 96 well plate, no miniprep. Send to
  Washington University
• Recover plasmid from E. coli host
• Clean plasmid and prepare to concentration of 100
  ng/ ul
• Take to primate center for sequencing

Gel from miniprep
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Sequences from April

• St 1 (0 psu) Rhizophydium
• St 4 (15 psu) Pseudopedinella elastica
  Protperidinium leonis Katablepharis japonica

• CR4s (27 psu) Asterionella kariana
  Thalassiosira aestivalis
• Pirsonia guinardiae
• Gyrodinium rubrum
• CR 15s (20 psu) Thalassiosira pseudonana

Diatom 1st to have genome sequenced
Dinoflagellate
Fungus
http//genome.jgi-psf.org/Thaps3/Thaps3.home.html
http//www.bsu.edu/classes/ruch/msa/barr/4-1.jpg
http//www4.fimr.fi/project/algaline/GALLERY/0709N
BP.JPG
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Sequences from Station 1 (0 psu) June

• Skeletonema costatum
• -Diatom extremely abundant in temperate
  areas
• Aulacoseira ambigua
• -river diatom
• Stephanodiscus hantzschii
• -eutrophic freshwater diatom
• Cyclotella meneghiniana
• -river diatom areas of high conductivity

http//thalassa.gso.uri.edu/ESphyto/list/taxa/skel
/skelcos.htm
http//craticula.ncl.ac.uk/Eddi/jsp/taxon.jsp?Taxo
nIdAU002A
http//www.lancs.ac.uk/staff/kingl/telford/stephha
nt.htm
http//craticula.ncl.ac.uk/EADiatomKey/html/taxon1
1.html
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Next step

• Use multiple primer sets to decrease PCR biases
• Traditional methods Chl a analysis
  Flow cytometry Primary productivity rates
• Apply protocol to ETM on future cruises

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Acknowledgements

• Lydie and Peter
• Michiko Nakano
• Dan Murphy
• Everyone in Peter Michikos labs
• Michael Wilkin the crew of the Forerunner
• Antonio Baptista
• NSF
• All of you