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18S rRNA clone 18S rRNA clone library of library of

phytoplankton in phytoplankton in the Columbia the Columbia River and its River and its coastal zonecoastal zoneBy Pete KahnBy Pete Kahn

Mentors: Lydie Herfort and Mentors: Lydie Herfort and

Peter ZuberPeter Zuber htt

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Significance of Significance of phytoplanktonphytoplankton

Major primary Major primary producers: producers:

OO22: 90% : 90%

COCO22=>C=>C33: 50%: 50% Most abundant Most abundant

eukaryotes: 10eukaryotes: 1022 to 10to 1044 cells/ mL cells/ mL

Base of aquatic Base of aquatic food chainfood chain

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Interactions with other Interactions with other DomainsDomains

Bacterioplankton Colonization :

POC=>DOC

“Competition” for

NO32- and

NH3?

http://www.fossilmuseum.net/Tree_of_Life/Domains_Archaea_Bacteria/Domains_of_life.jpg

Grossart, H. P., Czub, G. & Simon,Grossart, H. P., Czub, G. & Simon, M. (2006). Algae-bacteria interactions and their effects on aggregation and organic matter flux in the sea. M. (2006). Algae-bacteria interactions and their effects on aggregation and organic matter flux in the sea. Environmental MicrobiologyEnvironmental Microbiology 88, 1074-1084., 1074-1084.

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Traditional MethodsTraditional Methods

MicroscopyMicroscopy Flow CytometryFlow Cytometry Pigment analysis:Pigment analysis:

ChlChl a a Photosynthesis: Photosynthesis:

OO22 production production

CC1414 uptake uptake

Flow cytometryMicroscopy:

Pediastrium

Filtering for chlorophyll a

http://www.keweenawalgae.mtu.edu/ALGAL_IMAGES/chlorophyceans/Pediastrium_n16_dollartow3_402_c.jpg

http://upload.wikimedia.org/wikipedia/commons/c/c7/Picoplancton_cytometrie.jpg

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Molecular Methods for Molecular Methods for eukaryoteseukaryotes

Pitfalls of Traditional methods: Pitfalls of Traditional methods: -Microscopy: focus on numerous, -Microscopy: focus on numerous, “big” organisms; time consuming “big” organisms; time consuming -Incomplete -Incomplete understanding of diversityunderstanding of diversity

Molecular Methods: nucleic acid extraction, Molecular Methods: nucleic acid extraction, PCR, cloning, sequencingPCR, cloning, sequencing

Pitfalls of Molecular Methods: Pitfalls of Molecular Methods: biases in PCR primers & biases in PCR primers & preferential amplification of some organisms, preferential amplification of some organisms, i.e. dinoflagellates vs diatomsi.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 Problems for phytoplanktonphytoplankton

Traditional methods studies >>>> Traditional methods studies >>>> molecular methods studiesmolecular methods studies

Prokaryotic molecular studies >>>> Prokaryotic molecular studies >>>> phytoplankton molecular studiesphytoplankton molecular studies

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18S rRNA clone library for:18S rRNA clone library for: April: Wecoma & Forerunner: salinity April: Wecoma & Forerunner: salinity

gradientgradient Wecoma (CR4s, CR15s, CR30s, Wecoma (CR4s, CR15s, CR30s,

CR40s): CR40s): 20-32 psu; coastal surface samples20-32 psu; coastal surface samples Forerunner (St 1-5): estuary samplesForerunner (St 1-5): estuary samples 0-20 psu, inc. of 50-20 psu, inc. of 5 June & July: Forerunner: 0 psu & 30 psu June & July: Forerunner: 0 psu & 30 psu

from estuaryfrom estuary

The sampling planThe sampling plan

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Sample Dates & Sample Dates & LocationsLocationsWecoma April

2007 Forerunner April 2007

Forerunner July 2007Forerunner June 2007

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Goals within CMOPGoals within CMOP Short term: Short term: -Good clone library of phytoplankton-Good clone library of phytoplankton -Determining good primers for isolating 18S rRNA-Determining good primers for isolating 18S rRNA Long term: Long term: -Understand changes in populations between -Understand changes in populations between

seasons and locationsseasons and locations -Better understand unexpected changes in -Better understand unexpected changes in community: monitor ecosystem healthcommunity: monitor ecosystem health -Bacteria & Archaea relationships/ interactions -Bacteria & Archaea relationships/ interactions

(Dan Murphy)(Dan Murphy) -Better understanding of microbial ecosystem as -Better understanding of microbial ecosystem as a wholea whole

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What we know about What we know about Columbia River Columbia River phytoplanktonphytoplankton

Anderson GC. 1972. Aspects of marine Anderson GC. 1972. Aspects of marine phytoplankon studies near the Columbia River phytoplankon studies near the Columbia River with special reference to subsurface with special reference to subsurface chlorophyll maximum. The Columbia River chlorophyll maximum. The Columbia River estuary and adjacent ocean waters, University estuary and adjacent ocean waters, University of Washington Press, Seattle, WA, p. 219-240.of Washington Press, Seattle, WA, p. 219-240.

Contaminant ecology of fish and wildlife of Contaminant ecology of fish and wildlife of the lower columbia river-final report. April the lower columbia river-final report. April 1996. Lower Columbia River Bi-State 1996. Lower Columbia River Bi-State Program.Program.

Frey BE, R Lara-Lara, LF Small. 1984. Frey BE, R Lara-Lara, LF Small. 1984. Primary production in the Columbia River Primary production in the Columbia River Estuary water column. Columbia River Estuary water column. Columbia River Estuary Data Development Program.Estuary Data Development Program.

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What we know about What we know about Columbia River Columbia River phytoplanktonphytoplankton

Estuary: Estuary: >50 species of diatoms; >50 species of diatoms; Asterionella formosaAsterionella formosa;; Asterionella karianaAsterionella kariana; ; Skeletonema CostatumSkeletonema Costatum; ; ThallasiosiraThallasiosira; ; Stephanodiscus Stephanodiscus hatzschiihatzschii

Coastal: Coastal: Chaetoceros Chaetoceros convolutusconvolutus;; Dactyliosolen Dactyliosolen mediterraneusmediterraneus;; Thalassionema Thalassionema nitzschioidesnitzschioides

Asterionella formosa: most

abundant diatom

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Thalassionema nitzschioides

http://thalassa.gso.uri.edu/flora/imagesfl/tnema1.jpg

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DNA

RNA

Variations in nucleic acid in Variations in nucleic acid in environmental samplesenvironmental samples

Oct 2006 Between seasonsBetween seasons

Between locationsBetween locations

April Wecoma & April Wecoma & Forerunner: Forerunner: Freshwater << Freshwater << Surface CoastalSurface Coastal

Feb 2007

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Methods OverviewMethods OverviewWater sample => Sterivex

on site

Total nucleic acid extraction from sterivex in lab

Amplification through PCR

DNA removal?

TOPO cloning/ transformation

Plasmid isolation (Minipre

p)

96 well plates?

Sequences

BLAST=> Clone library

Water samples pass through sterivex filter

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Extraction/ DNA removalExtraction/ DNA removal

Extraction with Extraction with LETS buffer + LETS buffer + Phenol Phenol ChloroformChloroform

DNA removal DNA removal with TURBO with TURBO DNA free kit or DNA free kit or Ribopure kitRibopure kit

Sterivex

Gel after extraction

& DNA removal

DNA remov

ed

DNA not

removed

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PCRPCR

Primers: Euk A: Primers: Euk A: AACCTGGTTGATCCTGCC AACCTGGTTGATCCTGCC Euk B: Euk B: TGATCCTTCTGCAGGTTCACCTACTGATCCTTCTGCAGGTTCACCTAC Specific for Specific for eukaryotes, Examined eukaryotes, Examined with BLAST: highly with BLAST: highly conservedconserved

PCR cleanup with PCR cleanup with MO-BIO ultra clean MO-BIO ultra clean kitkit

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/ TransformationCloning/ Transformation TOPO TA Cloning KitTOPO TA Cloning Kit Transformation with Top 10 chemical Transformation with Top 10 chemical

competent cells => competent cells => E. coliE. coli Plated onto LB XGAL-AMP platesPlated onto LB XGAL-AMP plates -Selective: cells gain ampicillin -Selective: cells gain ampicillin

resistance from resistance from plasmidplasmid -Differential: cells w/plasmid insert turn -Differential: cells w/plasmid insert turn

whitewhite cells w/ no insert turn bluecells w/ no insert turn blue

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Plasmid isolation Plasmid isolation (miniprep)(miniprep)

For 96 well plate, no For 96 well plate, no miniprep. Send to miniprep. Send to Washington Washington UniversityUniversity

Recover plasmid from Recover plasmid from E. coliE. coli host host

Clean plasmid and Clean plasmid and prepare to prepare to concentration of 100 concentration of 100 ng/ ulng/ ul

Take to primate Take to primate center for sequencingcenter for sequencing

Gel from miniprep

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Sequences from AprilSequences from April St 1 (0 psu): St 1 (0 psu):

RhizophydiumRhizophydium St 4 (15 psu): St 4 (15 psu):

Pseudopedinella Pseudopedinella elasticaelastica; ; Protperidinium leonisProtperidinium leonis; ; Katablepharis Katablepharis japonicajaponica

CR4s (27 psu): CR4s (27 psu): Asterionella kariana*Asterionella kariana*; ; Thalassiosira Thalassiosira aestivalis*aestivalis*

Pirsonia guinardiaePirsonia guinardiae Gyrodinium rubrumGyrodinium rubrum CR 15s (20 psu): CR 15s (20 psu):

Thalassiosira Thalassiosira pseudonana*pseudonana*::

http://genome.jgi-psf.org/Thaps3/Thaps3.home.html

http://www4.fimr.fi/project/algaline/GALLERY/0709NBP.JPGhttp://www.bsu.edu/classes/ruch/msa/barr/4-1.jpg

Fungus Dinoflagellate

Diatom: 1st to have genome

sequenced

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Sequences from Station 1 Sequences from Station 1 (0 psu) June(0 psu) June

Skeletonema costatum*Skeletonema costatum* --Diatom; extremely Diatom; extremely

abundant in temperate abundant in temperate areasareas

Aulacoseira ambiguaAulacoseira ambigua --river diatomriver diatom Stephanodiscus Stephanodiscus

hantzschii*hantzschii* --eutrophic freshwater eutrophic freshwater

diatomdiatom Cyclotella meneghinianaCyclotella meneghiniana -river diatom; areas of -river diatom; areas of

high conductivityhigh conductivity

http://thalassa.gso.uri.edu/ESphyto/list/taxa/skel/skelcos.htm

http://craticula.ncl.ac.uk/EADiatomKey/html/taxon11.html

http://www.lancs.ac.uk/staff/kingl/telford/stephhant.htm

http://craticula.ncl.ac.uk/Eddi/jsp/taxon.jsp?TaxonId=AU002A

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Next stepNext step

Use multiple primer sets to decrease Use multiple primer sets to decrease PCR biasesPCR biases

Traditional methods: ChlTraditional methods: Chl a a analysis; analysis; Flow cytometry; Primary Flow cytometry; Primary productivity ratesproductivity rates

Apply protocol to ETM on future Apply protocol to ETM on future cruisescruises

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AcknowledgementsAcknowledgements

Lydie and PeterLydie and Peter Michiko NakanoMichiko Nakano Dan MurphyDan Murphy Everyone in Peter & Michiko’s labsEveryone in Peter & Michiko’s labs Michael Wilkin & the crew of the Michael Wilkin & the crew of the

ForerunnerForerunner Antonio BaptistaAntonio Baptista NSFNSF All of youAll of you