Rapid tests for the detection of Prymnesium parvum and its toxins

Linda Medlin, Gundula Ellers, Kerstin Toebe, & Katja Kerkmann Bremerhaven, Germany Why rRNA probes?

* universally found

* high target numbers per cell

* variable and conserved regions (can make nested probes for quantification) MakeMake hierarchicalhierarchical rRNArRNA probesprobes

How to design and test a probe

* Amass data bases from rRNA sequences

* use ARB program to design probe

* check probe for possible matches in RDP and Genbank

* test specificity in dot blot (DIG-labelled probe) and in situ (FITC or CY3 labelled probe) tests

* final check with flow cytometer EUK CHLO PRYM PELA PELA 1209R 02 02 01 02

ChangeChange fromfrom PFAPFA toto ETOETOHH SalineSaline

ChChaannggee frfromom SDSSDS toto NNoonniidedett P-P-4400

WithoutWithout DiDimethmethylyl ForFormmaammideide

WWiithth DDiimemethylthyl FoFormamidermamide DoubleDouble StainingStaining ofof CellsCells toto differentiatedifferentiate cellscells hierarchicallyhierarchically

252500

202000 y y t t i i s s

ten 150

ten 150 n n I I ve ve 101000 ti ti a a l l e e R R 5050

00 05051100115522002255 Distance[µm] Pr yy mGllA FFLUOLUOS NoPrroobbee Eukk11220099 CY55 Distance[µm]

TheThe cellscells werewere hybridisedhybridised withwith thethe universaluniversal eukaryoticeukaryotic probeprobe (labelled(labelled bblue)lue) andand thethe genusgenus specificspecific probeprobe forfor PrymnesiumPrymnesium PrymGl01APrymGl01A (labelled(labelled green).green). MixtureMixture ofof E.huxleyiE.huxleyi andand PrymnesiumPrymnesium parvumparvum AnalysisAnalysis ofof AlexandriumAlexandrium strainsstrains fromfrom EuropeanEuropean waterswaters usingusing hierarchicalhierarchical probesprobes

EUK1209EUK1209 DINODINO BB ATAM01ATAM01 ATNA02ATNA02 ATWE03ATWE03 ATME04ATME04 ATME05ATME05 ATME06ATME06

AlAlex.ex. tamarensetamarense (English(English Channel)Channel)

AlAlex.ex. cf.cf. tamarensetamarense (Italy)(Italy) Tetrahymena thermophila (Tetr.the) Gonyaulax spinefera (Gony.spi) Pyrocystis noctiluca (Pyrocyst) PhylogeneticPhylogenetic treetree Alexandrium affine (CCMP112) Alexandrium tamarense (Alex.tam) ofof AlexandriumAlexandrium Alexandrium tamarense (Alextama) Alexandrium tamarense (31/4) (18S(18S rRNA)rRNA) 100/10 Alexandrium tamarense (Aletamar) 0/100 Alexandrium catenella (BAHME217) 100/9 Alexandrium tamarense (SZN19) 8/100 Alexandrium tamarense (SZN01) Alexandrium fundeynse (Alexfund) 55/ Alexandrium fundeynse (Alex.fun) 66/ 65/6 76 Alexandrium tamiyavanichii (Atamiy) 69/8 5/72 Alexandrium minutum 8/78 (Alexminu) Alexandrium minutum (Alexmin1) 92/6 8/66 Alexandrium minutum (SZN30) Alexandrium minutum (AL3T)

70/9 Alexandrium tamanutum (8/1) 9/96/5699/ Alexandrium tamanutum (AT5) 8795/73 Alexandrium tamanutum (SZN29) /92 63/5 Alexandrium tamanutum (SZN28) 8/68 95/8 Alexandrium ostenfeldii (Alexostf) 2/92 65/7 Alexandrium ostenfeldii (K0324) 6/66 Alexandrium ostenfeldii (K0287) 100/10 Alexandrium ostenfeldii (AOSH1) 0/100 Alexandrium ostenfeldii (BAHME136) Alexandrium margalefii (Alexmarg) Alexandrium taylori (AY2T) Alexandrium taylori (AY4T) 100/10 0/100 Alexandrium taylori (AY1T) 0.01 substitutions/site

IdentificationIdentification ofof A.A. tamarensetamarense inin LabLab CulturesCultures andand FieldField SamplesSamples byby Species-Species- && Strain-specificStrain-specific PProbesrobes

nono probeprobe

ATAM01ATAM01 (species(species specific)specific)

ATNA02ATNA02 (strain(strain specific)specific) J.J. Brenner,Brenner, unpubl.unpubl... resultsresults

A.A. tamarensetamarense (WE(WE strain)strain) FieldField samplesample (Ork(Orkneyney Islands)Islands) Harmful Algal Blooms

Noctiluca

Karenia brevis AlexandriumAlexandrium tamarensetamarense A.ostenfeldiiA.ostenfeldii

V.p . V.p .

Alexandrium ostenfeldii 10 µm 10 µm (AOSH1)

20 µm Coccolithus pelagicus Cruciplacolithus neohelis Reticulosphaera japonensis Coccolithus sp. CCMP 300 Pleurochrysis carterae Emiliania huxleyi Prymnesium parvum Prymnesium patelliferum Prymnesium calathiferum Chrysochromulina polylepis Chrysochromulina kappa Chrysochromulina hirta Chrysochromulina campanulifera Chrysochromulina strombilis Chrysochromulina simplex Chrysochromulina leadbeateri Chrysochromulina cf. ericina Chrysochromulina throndsenii Chrysochromulina throndsenii Chrysochromulina sp. P16 Chrysochromulina sp. TH2 Phaeocystis antarctica Phaeocystis pouchetii Phaeocystis globosa Pavlova sp. CCMP1416 Pavlova gyrans Pavlova sp. CCMP 625 Pavlova salina Coccolithus pelagicus Cruciplacolithus neohelis Reticulosphaera japonensis Coccolithus sp. CCMP 300 Pleurochrysis carterae Emiliania huxleyi Prymnesium parvum Prymnesium patelliferum Prymnesium calathiferum Chrysochromulina polylepis Chrysochromulina kappa Chrysochromulina hirta Chrysochromulina campanulifera Chrysochromulina strombilis Chrysochromulina simplex Chrysochromulina leadbeateri Chrysochromulina cf. ericina Chrysochromulina throndsenii Chrysochromulina throndsenii Chrysochromulina sp. P16 Chrysochromulina sp. TH2 Phaeocystis antarctica Phaeocystis pouchetii Phaeocystis globosa Pavlova sp. CCMP1416 Pavlova gyrans Pavlova sp. CCMP 625 Pavlova salina Application & detection methods for rRNA probes

•• DNADNA dotdot blotsblots

•• InIn situsitu hybridizationhybridization // FluorescenceFluorescence MicroscopyMicroscopy

•• InIn situsitu hybridizationhybridization // FlowFlow CytometryCytometry lliquidiquid && solidsolid

•• DDNANA microchipsmicrochips EukEuk 12091209

PrymGenusPrymGenus ProbeProbe 18S18S rRNArRNA

PrymPrym SpeciesSpecies ProbeProbe 28S28S rRNArRNA ConclusionsConclusions

•• SSpecificpecific probesprobes couldcould bebe mademade forfor differentdifferent groupsgroups ofof phytoplanktonphytoplankton (from(from higherhigher groupgroup downdown toto speciesspecies andand strainstrain level)level) •• MMoreore thanthan aa dozendozen probesprobes forfor toxictoxic algaealgae areare availableavailable oror underunder developmentdevelopment •• IItt isis possiblepossible toto useuse thethe probesprobes withwith lablab culturescultures andand withwith fieldfield samplessamples •• TThehe probesprobes couldcould bebe usedused withwith differentdifferent kindskinds ofof techniquestechniques (dot(dot blots,blots, fluorescencefluorescence microscopy,microscopy, flowflow cytometry,cytometry, DNADNA chips,chips, etc.)etc.) PlatformsPlatforms forfor thethe detectiondetection ofof toxictoxic algaealgae

LindaLinda Medlin,Medlin, G.G. Eller,Eller, K.K. Toebe,Toebe, R.Groben,R.Groben, M.M. Lange,Lange, && K.K. KerkmannKerkmann Bremerhaven,Bremerhaven, GernmanyGernmany 1.1. ChemChemScanRDIScanRDI,, aa laserlaser basedbased systemsystem toto quicklyquickly analyseanalyse FISH-experimentsFISH-experiments

2.2. ElectrochemicalElectrochemical detectiondetection ofof toxictoxic algaealgae viavia aa handholdhandhold devicedevice

3.3. DevelopmentDevelopment ofof DNA-microarraysDNA-microarrays forfor monitoringmonitoring phytoplanktonphytoplankton compositioncomposition FISHFISH detectiondetection ofof toxictoxic algaealgae viavia thethe ChemChemScan,Scan, solidsolid phasephase cytometercytometer ChemScanRDI combines fluorescent cell labelling with laser scanning

1. Membrane Filtration

2. Target cells by using fluorescently labled molecular probes or antibodies

3. Automated analysis of the fluorescent cells by ChemScan RDI (Chemunex Inc.; Maisons-Alford, France)

Electrochemical detection of toxic Algae via a handheld device A Handheld Device for the Detection of Harmful Algae

ƒ Alexandrium tamarense ƒ Alexandrium ostenfeldii

RNA Enzyme Detection Oligo catalyzed transfeEnzymer ocoupledf electronsto an antibody Helper Electrode Immobilized Oligo

Work Electrode Reference Electrode

Disposable Sensorchip Electrochemical Detection of rRNA from Alexandrium Species

2500

2000 ] A 1500

t [n A.ostenfeldii n e

r A. tamarense

r 1000 u C 500

0 rRNA Negative Control Positive Control

ƒ ~ 500 ng rRNA have been hybridized to probes ƒ The probes were directed against ribosomal RNA of A. tamarense, respectively A. ostenfeldii Concentration Series of Decreasing Amounts of Target

A. Decreasing amounts of 2500 A. tamarense rRNA

] 2000 A 1500

t [n hybridized n e r 1000 r u to a A. tamarense probe C 500

0 600 ng RNA 300 ng RNA 150 ng RNA 75 ng RNA Negative Pos tiv e Control Control

B. Decreasing amounts of

2500 a 70 bp Oligonucleotide

] 2000 A hybridized to a 1500 t [n n

e 1000 r

r A.ostenfeldii u 500 C 0 probe M M M M M M M n n n n n n n 14 ,4 ,7 14 57 71 0 7 71 35 7, 3, 0, Ta rg et C oncen trat ion Comparison of cell counts with electrode readings

y = 40,919 + 0,032024x R= 0,91791 Data 1 500 cell counts/L 400

300

200

100

0 Alexandrium tamarense 0 200 400 600 800 1 103 1,2 103 Microchip electrode readings 250

200

Rep 1 150 Rep 2 Rep 3 Rep 4 100 Rep 5

50 0 0 0 5 oren 1-5 oren 6-10 oren 16-2 ns oren 26-3 oren 11-1 e ns ns e ns ns S e e e S S Sensoren 21-25 S S Sensoren 31-35

C. Detection of rRNA in natural samples from the Orkneys islands (column sets 1-4) as compared to rRNA from Prorocentrum mexicanum (column sets 5&6) and hybidized with Alexandrium tamarense probe. Long term stability of treated sensors

1800 1600 Avidin PC 1400 DNA PC A] 1200 n [

t 1000 Frisch PC n

e 800 r Avidin NC r 600

Cu DNA NC 400 200 Frisch NC 0

s h h h ys k t t isch a e n n r d e o o ont F 3 2 w 1 m 2 m 3 m

ƒ Sensors have been treated with Avidin (Avidin) and Avidin/Probe (DNA) ƒ The coated Sensors were then stored at 4oC over the indicated times ƒ Freshly prepared sensors have been prepared before each hybridization as positve controls for the experimental conditions (Frisch) ƒ To control the stability of the coated sensors, a hybridization was carried out with a 70 bp oligonucleotide (PC) ƒ For the negative control (NC) a hybridization was carried out without target-DNA Alexandrium ostenfeldii K0324 RNA per Cell in Log-, Lag- and Stationary Phase

RNA/Cell (pg) 10,0

8,0

6,0

4,0

2,0

0,0 Lag-Phase Log-Phase Stat.-Phase Development of DNA-microarrays for monitoring phytoplankton composition Scheme of a DNA-Chip Experiment

Fluorescently labeled ssDNA

Imobilized DNA (Oligonucleotides, PCR-Fragmentes, cDNA)

Glass-Slide Scheme of a DNA-Chip Experiment

Fluorescently labeled ssDNA

Imobilized DNA (Oligonucleotides, PCR-Fragmentes, cDNA)

Glass-Slide Low Density Chips

ƒ ~ 625 Spots per cm2 ƒ Spotdiameter: ~ 200 µm ƒ Spotting with needles (A) or piezotechnology (B)

Pipette Piezo- element 500 µm A. B. Monitoring Phytoplankton composition with DNA-Chip Technology

Phytoplankton samples

Isolation of genomic DNA from the sample

Amplification of the 18S- PCR products 18S rDNA

Hybridization of the 18S PCR-products with a DNA-Chip that contains probes initially designed for FISH Probes Probes and Targets used for preliminary Chip-Experiments

Class Probe Species Dinophyceae DINO B ƒ Alexandrium tamarense DINO E-12 ƒ Prorocentrum minimum Prymnesiophyceae PRYM01 ƒ Prymnesium patelliferum PRYM02 PRYM03 Chlorophyceae CHLO01 ƒ Dunaniella salina CHLO02 ƒ Pyramimonas obovata Pelagophyceae PELA01 ƒ Coccoid pelagophyte ƒ Pulvinaria spec. Bolidophyceae BOLI01 ƒ Clone. No. 151 PICODIV BOLI02 Localization of the Class-level probes in the 18S-Sequence

2 9

1 0

3

- 1

1

2 2 0

B E 0 0

1

0

M

A

O O

L

Y

NO L

NO UK

E

R

H E

P

DI

P DI CHL

PRYM01 PRYM02 C

0 bp 900 bp 1800 bp

18S-DNA ~ 1800 bp Preliminary results of a DNA-Chip with Class-level Probes

1. Dunaniella salina PELA 01 2. Prymnesium patelliferum 3. Coccoid pelagophyte BOLIO 02 4. Alexandrium tamarense BOLI 01 DINO E-12 DINO B PRYM 01 PRYM 02

s PRYM 03

t

o

p

S EUK 1209

5

2 CHLO 02 CHLO 01 PC 1 2 3 4 25 Spots Identification of Phytoplankton on Class-level in a Mix of Laboratory Strains

ii d a e m l g 1 2 3 4 5 6 a u fe n rk v n lo a r e o p a st r A . p o p P . . . P A P B C D

A1- PC C1- Pela 02 A2- Euk 328 C2- Dino B A3- Euk 1209 C3- Dino E-12 Genomic A4- Chlo 01 C4- NS 03 DNA A5- Chlo 02 C5- NS 04 A6- Hetero 01 C6- Pras 04 B1- Boli 01 D1- Bathy 01 18S-PCR B2- Boli 02 D2- Micro 01 B3- Prym 01 D3- Ostreo 01 B4- Prym 02 D4- Crypto B Hybridisation B5- Prym 03 D5- NC to DNA-Chip B6- Pela 01 D6- PC 1 2 3 4 5 6

A -- PrymnesPrymnesiumium parvumparvum -- AlexandriumAlexandrium ostenfeldiiostenfeldii B -- TThresholdhreshold forfor aa positivepositive signal:signal: C signal/noisesignal/noise ratioratio >> 22 D

A1- PC C1- Pela 02 12 A2- Euk 328 C2- Dino B A3- Euk 1209 C3- Dino E-12 10 e s A4- Chlo 01 C4- NS 03 i 8 o N /

A5- Chlo 02 C5- NS 04 l 6

A6- Hetero 01 C6- Pras 04 gna

i 4 B1- Boli 01 D1- Bathy 01 S 2 B2- Boli 02 D2- Micro 01 B3- Prym 01 D3- Ostreo 01 0

1 2 2 2 2 3 4 4 1 B4- Prym 02 D4- Crypto B 9 1 1 3 01 B 0 1 1 B 0 0 0 0 i 01 i 0 02 0 0 o -1 0 0 0 0 NK 2 o o o l a a s 0 o to l ol m 0 m m l n E a y p 1 hl er B Bo y el Di o NS NS th icr reo y k Ch C ry ry Pe P Pr t B5- Prym 03 D5- NC et P P Pr n M s Cr Eu H Di Ba O B6- Pela 01 D6- PC Probes DNA CHIP Clone librairy Dec 2000

Prasinophyceae Cryptophyceae 1 Prymnesiophyceae 2 3 Stramenopiles 4 Dinophyceae 5 Ciliates Rosko I (Telonema) Rosko II Choanoflagellates Cercozoa

April 2001

Prasinophyceae

1 Cryptophyceae 2 Prymnesiophyceae 3 Stramenopiles 4 all dinos 5 Ciliates Rosko I (Telonema) Rosko II Choanoflagellates Cercozoa Summary

ƒ The ChemScanRDI is a laserbased system that reduces the time required for FISH due to an automatic analysis, visual recovery of cells with positive signals

ƒ It is possible to detect toxic Alexandrium species via a handheld device

ƒ DNA-Chip technology provides the possibility to analyse numerous hybridzations in parallel Research Needs

ƒ Monitoring of toxic phytoplankton populations is an important scientific issue

ƒ Efficient monitoring requieres quick and reliable techniques

ƒ Currently the identification of species is done mainly by light or electron microscopy

ƒ New tools are needed to be developed which cut down the time necessary for toxic phytoplankton classification

ƒ Methods that involve oligonucleotide probes have the potential to fulfill these needs People involved in the projects

Dr. Gundula Eller Dr. Kerstin Toebe FISH/ ChemScanRDI

Susanne Huljic Handheld device/ A.ostenfeldii

Dr. Katja Kerkmann DNA-Chip Technology

Dr. Martin Lange Handheld device/ A. tamarense

Dr, Rene Groben Probe Hybridisation optimisation

Dr. Linda Medlin Principal investigator Rapid Tests for the Detection of Haemolytic Compounds 2.4 414 2.0 lysed erythrocytes 1.6 algal extract

1.2 abs. units 0.8 540 575

0.4

0.0 350 400 450 500 550 600 650 700 wavelength [nm] 1.0 414 nm: 0.8 y = 0.9829x - 0.0024 R2 = 0.9994 0.6

abs. units 0.4 y = 0.1051x - 0.0007 R2 = 0.9994 0.2 540 nm: 0.0 0.0 0.2 0.4 0.6 0.8 1.0 cell concentration [5 x 106] Prymnesiophytes

4 h 100 12 h 80 20 h

60 % lysis 40

20

0 NK C1 C2 C3 C4 P1 P2 P3 Dinophytes

4 h 100 12 h 80 20 h

60 % lysis 40

20

0 NK G1 G2 G3 G4 A1 A2 A3 A4 Prymnesium parvumRL10

100 24 h

80 48 h 60

% lysis 40

20

0 60 30 15 7.5 3.8 1.9 0.9 0.5 NK

number of cells *103 / well Alexandrium tamarense CCMP115

100 24 h 80 48 h sis

y 60

% l 40 20 0

.5 .8 .9 9 5 5 3 6 3 5 8 K 7 3 1 0. 0. 1 0 04 02 0.2 0.1 0.0 0.0 0 N 0.0 0.0 0.0 0.

number of cells *102 / well AllelochemicalAllelochemical effectseffects ELAELA TestsTests provideprovide rapidrapid meansmeans forfor detectingdetecting hhaemolyticaemolytic compoundscompounds butbut thesethese mustmust bebe coupledcoupled withwith speciesspecies teststests forfor 100%100% ReliabilityReliability