in The Netherlands bloom Characterization of multiple isolates from an Alexandrium ostenfeldii Harmful Algae

Characterization of multiple isolates from an Alexandrium ostenfeldii bloom in The Netherlands

Dedmer B. Van de Waal

^a,

*, Urban Tillmann

^b

, Helge Martens

^b

, Bernd Krock

^b

, Yvonne van Scheppingen

^c

, Uwe John

^b

aDepartmentofAquaticEcology,NetherlandsInstituteofEcology,POBox50,6700ABWageningen,TheNetherlands

bAlfredWegenerInstituteHelmholtzCentreforPolarandMarineResearch,AmHandelshafen12,27570Bremerhaven,Germany

cWaterschapScheldestromen,POBox1000,4330ZWMiddelburg,TheNetherlands

1. Introduction

Harmful algal blooms are a global threat tocoastal marine ecosystems,withconsequencesforfisheriesandshellfishproduc- tion (Anderson et al., 2002; Heisler et al., 2008; Wang, 2008).

Alexandrium is among the most common bloom forming toxic dinoflagellate genera and is generally held responsible for the outbreak of paralytic shellfish poisoning (PSP;Anderson et al., 2012).BesidesPSPtoxins,someAlexandriumspeciesareknownto produce other toxins including different spirolides (SPX) or gymnodimines (Cembella, 2003; Van Wagoner et al., 2011;

Borkmanetal.,2012;Kremp etal.,2014). Thesespeciesbelong to different phylogenetic groups of Alexandrium ostenfeldii as recentlydefined by Kremp et al. (2014),including Alexandrium peruvianum.MostA.ostenfeldiistrainshavebeenshowntoproduce SPX,somestrainsalsoproducedetectableamountsofPSPtoxins, whileonlyafewstrainswerereportedtocombinePSPtoxin,SPX and 12-methylgymnodimineproduction (Cembella, 2003;Bork- manetal.,2012;Krempetal.,2014).

PSP toxins are a group of neurotoxic compounds, including saxitoxin(STX),neosaxitoxin(NEO),gonyautoxins(GTX),andtheir N-sulfocarbamoyl variants theB- and C-toxins(Shimizu,1996;

Cembella,1998).STXishighlytoxicwithanLD50value(i.p.mice) of 8

m

gkg¹ body weight (Wiberg and Stephenson, 1960). The additionofasulfategroupattheC-11positionformsGTX,thereby reducingthetoxicitybyupto40%.Afurtheradditionofasulfonyl ARTICLE INFO

Articlehistory:

Received23December2014

Receivedinrevisedform21August2015 Accepted21August2015

Keywords:

Harmfulalgalblooms PSPtoxins

Spirolide Gymnodimine Lyticactivity Allelochemicalpotency

ABSTRACT

Alexandriumostenfeldiiisanemergingharmfulalgalbloomspeciesformingaglobalthreattocoastal marineecosystems,withconsequencesforfisheriesandshellfishproduction.TheOosterscheldeestuary isashallow,macrotidalandmesotrophicestuaryinthesouthwestofTheNetherlandswithlargestocks ofmussels,oysters,andcockles.TheseshellfishstockswerethreatenedbyarecentA.ostenfeldiibloomin theOuwerkerkseKreek,whichisabrackishwatercreekdischargingwaterintotheOosterschelde.Little isyetknownaboutthecharacteristicsoftheA.ostenfeldiipopulationinthiscreek.Wethereforeisolated 20clonesduringanA.ostenfeldiibloomin2013,andcharacterizedtheseclonesontheirgrowthandtoxin profilein theirexponential growth phase.The cyclicimines wereidentified bycomparison ofA.

ostenfeldiiextractswiththeretentiontimeandCIDspectraofstandardsolutions,orwithpublishedCID spectra.Wefurthermoreassessedtheallelochemicalpotencyandphylogenyofaselectionof10–12 clones. Morphology and molecular phylogeny showed that all clones belong to Group 1 of A.

ostenfeldii.Allclonesshowedcomparablegrowthratesofonaverage0.220.03d¹.Duringexponential growth,theyallproducedauniquecombinationofparalyticshellfishpoisoningtoxins,spirolidesand gymnodimines,ofwhichparticularlythelattershowedahighintra-specificvariability,witha25-fold differencebetweencloneswiththelowestandhighestcellquota.Furthermore,theselected12clones showedhigh allelopathic potencies with EC50 values based on lysis assays against the cryptophyte Rhodomonassalinabetween212and525A.ostenfeldiicellsmL¹.Lyticactivitieswerelowerforcellextracts, indicatinganimportantextracellularroleofthesecompounds.Ahighintra-specificvariabilitymayaddto thesuccessofgenotypicallydiverseA.ostenfeldii blooms,andmakepopulationsresilienttochangesin environmentalandclimaticconditions.

ß2015ElsevierB.V.Allrightsreserved.

* Correspondingauthor.Tel.:+31317473553.

E-mailaddress:d.vandewaal@nioo.knaw.nl(D.B.VandeWaal).

ContentslistsavailableatScienceDirect

Harmful Algae

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / h a l

http://dx.doi.org/10.1016/j.hal.2015.08.002 1568-9883/ß2015ElsevierB.V.Allrightsreserved.

groupatthecarbamoylgroupformsC-toxins,whichexhibita99%

lowertoxicityascomparedtoSTX(Wieseetal.,2010). SPXand gymnodiminesarefastactinghighlytoxicneurotoxins,withLD50

values (i.p. mice) down to 6.9 and 96

m

gkg¹ body weight, respectively(Mundayetal.,2004,2012).Besidestoxicity,various Alexandriumspecies wereshownto haveallelopathic potencies towardgrazers and otherphytoplanktonspecies (Tillmann and John,2002;Tillmannetal.,2007,2008;Johnetal.,2015).

Alexandriumostenfeldiiisgloballydistributedinbrackishand marineenvironments,andisolateshavebeencharacterizedfrom locations worldwide (Kremp et al., 2014). A. ostenfeldii is historicallyseenasbackgroundbloomer(Cembellaetal.,2000;

Johnetal.,2003),however,denseA.ostenfeldiibloomshavebeen reported recently in the Narragansett Bay and the New River EstuaryintheU.S.eastcoast(Borkmanetal.,2012;Tomasetal., 2012),theBalticSeacoastofFinland(Hakanenetal.,2012),along theAdriaticcoastofItaly(Ciminielloetal.,2006),andrecentlyina creek of the Oosterschelde estuary in the Southwest of The Netherlands(Bursonetal.,2014).ThisOosterscheldeestuaryisa shallow,macrotidalandmesotrophicestuarywithlargestocksof mussels,oysters,andcockles(Fig.1AandB;Troostetal.,2010;van Broekhovenetal., 2014). Becauseof potentialcontaminationof theseshellfishwithphytoplanktontoxins,TheNetherlandshasa regularmonitoringprogramfortoxiccompoundsinshellfish,as well as for the occurrence of harmful algal species in the Oosterscheldeestuary(vanderFels-Klerxetal.,2011).Arecent denseA. ostenfeldiibloomin acreekdischargingwater intothe Oosterscheldethreatenedtheshellfishstocks,andwasterminated bytheadditionofhydrogenperoxide (Burson etal.,2014).The bloom, however, recurred in 2013 and reached population densitiesof upto4500cellsmL¹ (Fig.1C). Littleisyet known about thecharacteristics of the A. ostenfeldii population in the creek. We therefore isolated a number of A. ostenfeldii clones duringthebloomin2013,andcharacterizedthebloompopulation interms ofgrowth, morphology,phylogeny,toxin composition, andlyticactivity.

2. Materialandmethods

2.1. Fieldsampling

TheOuwerkerkseKreekisasmallbrackishwatercreekinthe provinceofZeeland,SouthwestofTheNetherlands(Fig.1AandB;

SeealsoBursonetal.,2014).Theinletofthecreekisconnected withditchesthatdraintheagriculturallandsinthearea,andwater from the creek is regularly discharged into the Oosterschelde estuaryviaapumpingstation.Thephytoplanktonpopulationin thecreekwasmonitoredforAlexandriumostenfeldii,andweekly samplesweretakenforcellcountsduringthebloom.Anintegrated

watersamplewastakenoftheupper1mofthewatercolumnat location‘a’(Fig.1B),anda1LsubsamplewasfixedwithLugol’s iodinesolution(Lugol)toafinalconcentrationof1%.A.ostenfeldii cells were counted in a Sedgewick chamber on an inverted microscope(OlympusVanox,Hamburg,Germany).IsolatesofA.

ostenfeldiicellsweresampledattheonsetofthebloomon16July 2013fromlocations‘a’and‘b’inthecreek(Fig.1B).

2.2. Isolation

CellsofAlexandriumostenfeldiiwerepickedfromsmalldroplets in apetridish usinga Pasteurpipetteora 10

m

L mirco-pipette.

Individualcellswerecleanedfivetimesin2

m

Ldropletsofsterile mediumconsistingoffilteredanddilutedNorthSeawaterwitha salinity of about 10, or in sterilized artificial brackish water medium witha salinity of about10 (AppendixA). Bothmedia contained nutrientscorresponding to 50% of K-medium (Keller etal.,1987).A.ostenfeldiicellsweresubsequentlygrownin100

m

L mediummixedina1:1ratiowithsterilefilteredwaterfromthe creek (0.2

m

m membrane filter) in microplate wells. Clones OKNL1-10weresubsequentlyculturedinartificialbrackishwater medium(AppendixA),andclonesONNL11-22indilutedNorthSea watermedium.

2.3. Culturingofclones

Twenty of the successfully isolated clones were grown in 250mLErlenmeyerflasksat158Cunderanincidentlightintensity of100

m

molphotonsm²s¹ ata light-darkcycleof16:8.After acclimationtothesegrowthconditions(i.e.>5generations),cells were transferred and growth was monitored by cell counts performed every second day. At mid-exponential phase (6000cellsmL¹)cultureswereharvestedforanalysisoftoxins andforcellsizemeasurements.Cultureswerecountedagaintwo dayslater,confirmingthatcellsamplingtwodaysbeforewasstill duringtheexponentialphase.Fortoxinsampling,15mLsamples weretakenforextractionofSPXandgymnodimines,and50mL samples each were taken for PSP toxin and DNA extraction.

Sampleswerecentrifugedat6800gfor15min(SL16,Thermo Scientific,Waltham,USA)andafterremovalofthesupernatantthe pelletswerestoredat208C.

2.4. Cellcountsandmeasurements

Cell densities of Alexandriumostenfeldii cultures weredeter- minedbyusingsedimentationchambersforsettling0.2–1mLof culture suspension, and subareas with at least 400 cells were counted withaninvertedmicroscope(20X,ZeissAxiovert40C).

Observationanddocumentationofliveandfixedcellswascarried

Fig.1.OverviewofthesamplinglocationandthebloomdynamicsofAlexandriumostenfeldiiinRhine-Muesse-Scheldtdelta,SouthwestofTheNetherlands(A).Withthe specificsamplinglocations‘a’and‘b’intheOuwerkerkseKreek(B),andtheA.ostenfeldiipopulationdensitiesduringthe2013bloomsampledfromlocation‘a’(C).

out with eitheran inverted microscope(Axiovert 200M, Zeiss, Germany)oraZeissAxioskop2(Zeiss,Germany),bothequipped withepifluorescenceanddifferentialinterferencecontrastoptics.

Lengthandwidthofcells(n>50fromeachclone)weremeasured on freshly Lugol-fixed cells (1% final concentration) with the program AxioVs40 (v.4.8., Zeiss, Go¨ttingen, Germany) using micrographstaken with a digital camera (Axiocam MRc, Zeiss, Go¨ttingen,Germany).

Growthrates wereestimatedfor eachclonebymeansofan exponentialfunctionfittedthroughallcellcountsovertime(n=4), accordingtoNt=N0exp^mt,whereNtreferstothecellconcentra- tionsat timet,N₀ tothecellconcentrationsat thestart of the experiment,and

m

tothegrowthrate.

2.5. Cellmorphologyandmorphometry

Pattern,shapeanddimensionofthecalplateswereexamined using epifluorescence microscopy of calcofluor-stained cells accordingtothemethodofFritzandTriemer(1985).Morphomet- ricmeasurements were performedforfour randomlyselected clones (OKNL11, 12, 14, 19). Cells of exponentially growing cultureswerefixedwithneutralLugol(1%finalconcentration)for 1handthencollectedbycentrifugationat3220gfor10min (Eppendorf5810R,Hamburg,Germany).Cellswereresuspended inadropoffilteredseawaterandadropofa1mgmL¹solutionof FluorescenceBrightner28(Sigma–Aldrich,St.Louis,MO,USA)on amicroscopeslide.Cellswereinspectedat1000magnification (Zeiss,Axioskop2,Go¨ttingen,Germany)andphotographedwitha digitalcamera(AxiocamMRc,Zeiss,Go¨ttingen).Measurementsof sizeandareaofselecteddiagnosticplateswereperformedwith theprogramAxioVs40(v.4.8.,Zeiss,Go¨ttingen).Shapeofthefirst apical plate and the anterior sulcal plate was scored into categoriesgivenbyKrempetal.(2014).

2.6. Toxins

2.6.1. Toxinextraction

Freeze dried cell pellets were suspended in 500

m

L 0.03M acetic acid for PSP toxin analysis and with methanol for SPX analysis,respectively.Thesamplesweresubsequentlytransferred into a FastPrep tube containing 0.9g of lysing matrix D. The sampleswerehomogenizedbyreciprocal shakingat maximum speed(6.5ms¹)for45sinaBio101FastPrepinstrument(Thermo Savant, Illkirch, France). After homogenization, samples were centrifuged(Eppendorf5415R,Hamburg,Germany)at16,100g at48Cfor15min.Thesupernatantsweretransferredtospin-filters (pore-size0.45

m

m,MilliporeUltrafree,Eschborn,Germany)and centrifugedfor30sat800g.Thefiltratesweretransferredto HPLCvialsandstoredat208Cuntilmeasurement.Variationin cellulartoxinquotaandcompositionwastestedbygrowingfour clonesintriplicate.

2.6.2. Toxinanalysis

2.6.2.1. PSPtoxins. Theaqueous extractswereanalyzed for PSP toxins by reverse-phase ion-pair liquid chromatography with fluorescencedetection(LC-FLD) and post-columnderivatization followingminormodifications ofpreviouslypublishedmethods (Dieneretal.,2006;Krocketal.,2007).TheLC-FLDanalysiswas carried out on a LC1100 series liquid chromatography system consistingofa G1379Adegasser,aG1311Aquaternarypump,a G1229Aautosampler,andaG1321Afluorescencedetector(Agilent Technologies,Waldbronn,Germany),equippedwitha Phenom- enex Luna C18 reversed-phase column (250mm4.6mm id, 5

m

mporesize)(Phenomenex,Aschaffenburg,Germany)witha PhenomenexSecuriGuardprecolumn.Thecolumnwascoupledto

aPCX2500post-columnderivatizationsystem(PickeringLabora- tories, Mountain View, CA, USA). Eluent A contained 6mM octanesulfonicacid,6mMheptanesulfonicacid,40mMammoni- umphosphate,adjustedtopH6.95withdilutephosphoricacid, and 0.75%tetrahydrofurane. EluentBcontained13mM octane- sulfonic acid, 50mM phosphoric acid, adjusted topH 6.9 with ammoniumhydroxide,15% acetonitrile and1.5% tetrahydrofur- ane.Theflowratewas1mLmin¹withthefollowinggradient:0–

15minisocraticA,15–16minswitchtoB,16–35minisocraticB, 35–36min switch to A, 36–45min isocratic A. The injection volumewas20

m

Landtheautosamplerwascooledto48C. The eluatefromthecolumnwasoxidizedwith10mMperiodicacidin 555mMammoniumhydroxidebeforeenteringthe508Creaction coil,afterwhichitwasacidifiedwith0.75Mnitricacid.Boththe oxidizingandacidifyingreagentsenteredthesystematarateof 0.4mLmin¹.Thetoxinsweredetectedbydual-monochromator fluorescence(

l

ex333nm;

l

em395nm).Thedatawereprocessed withAgilentChemstationsoftwareandcalibratedagainstexternal standards.StandardsolutionsofPSPtoxinswerepurchasedfrom theCertifiedReferenceMaterialProgramoftheInstituteofMarine Biosciences(NationalResearchCouncil,Halifax,NS,Canada).

2.6.2.2. Lipophilictoxins.

2.6.2.2.1. Selected reaction monitoring (SRM) experiments. Mass spectralexperimentsforlipophilictoxindetectionandquantifica- tion were performed on a 4000 Q Trap (AB-SCIEX, Darmstadt, Germany),triplequadrupolemassspectrometerequippedwitha TurboSpray¹ interface coupled to an Agilent (Waldbronn, Germany)model1100LC.TheLCequipmentincludedasolvent reservoir, in-line degasser (G1379A), binary pump (G1311A), refrigerated autosampler (G1329A/G1330B), and temperature- controlledcolumnoven(G1316A).

Afterinjectionof5

m

lofsample,separationoflipophilictoxins wasperformedbyreverse-phasechromatographyonaC8column (502mm)packedwith3

m

mHypersilBDS120A˚ (Phenomenex, Aschaffenburg,Germany)andmaintainedat258C.Theflowrate was0.2mLmin¹andgradientelutionwasperformedwithtwo eluents,whereeluentAwaswaterandeluent Bwasmethanol/

water(95:5,v/v),bothcontaining2.0mMammoniumformateand 50mM formic acid. Initial conditions were elution with 5% B, followedbyalineargradientto100%Bwithin10minandisocratic elutionuntil10minwith100%B.Theprogramwasthenreturned to initial conditions within 1min followed by 9min column equilibration(totalruntime:30min).

Massspectrometricparameterswereasfollows:curtaingas:

20psi,CADgas:medium, ionsprayvoltage: 5500V, tempera- ture:6508C,nebulizergas:40psi,auxiliarygas:70psi,interface heater: on, declustering potential: 121V, entrance potential:

10V, exit potential: 22V, collision energy: 57V. SRM experi- ments were carriedout in positiveion modeby selecting the following transitions (precursor ion>fragment ion): m/z 508>490forgymnodimineA,522>504for12-methylgymno- dimine, 692>164 for 13-desmethylspirolide C (SPX-1), and 694>164 for 13-desmethylspirolide D. Dwell timesof 40ms wereusedforeachtransition.Standardsolutions ofSPX-1and gymnodimine A were purchased from the Certified Reference Material Program of the Institute of Marine Biosciences (National Research Council, Halifax, NS, Canada), and 12- methylgymnodimine was kindly provided by Kirsi Harju, VERIFIN, Department of Chemistry, University of Helsinki, Finland.

2.6.2.2.2.Productionspectra. Production spectrawererecorded in the Enhanced Product Ion (EPI) mode in the mass range fromm/z120to550.Positiveionizationandunitresolutionmode were used. The mass spectral parameters were as in SRM experiments.

2.7. Rhodomonassalinabioassays

Clonal isolates of Alexandrium ostenfeldii (OKNL11-22) were growninbatchculturesin100mLErlenmeyerflasksatstandard culture conditions described above. Growth was followed by countingLugol-fixed(1%finalconcentration)sampleseverysecond orthirddayusinganinvertedmicroscope.Atlateexponentialphase, at a population density of 8–1210³cellsmL¹, cells were harvestedforestimatingbothlyticcapacityofwholecellcultures andofcellextracts.Asubsampleof20mLlivesamplewastakenfor wholecelllyticactivity.Asubsampleof50mLwas collectedby centrifugation(Eppendorf5810R,Hamburg,Germanyat3220g for10min).Thecellpelletwastransferredtoa 1mLmicrotube, centrifugedagain(Eppendorf5415,16,000g,5min),andstored frozen(208C)untiluse.

Lyticactivityofwholecellcultureandcellextractwereboth estimatedbyaRhodomonassalinalysisassay(Tillmannetal.,2009).

Forthisbioassay,R.salina(strainKAC30,Kalmarculturecollection, Sweden)waspre-adaptedtoandsubsequently grownin10PSU mediumforatleastfourweeks.Fortestingthe variationinlytic activity,afulldose-response-curveoflyticactivityofwholeculture andcellextractwasrecordedfortriplicateculturesofcloneOKNL19.

Here,ninedifferentdilutionsrangingfrom4000to50cellsmL¹and from30,000to100cellsmL¹forwholecultureandcellextract, respectively,werepreparedintriplicateandprocessedasdescribed below.Lyticactivityofallothercloneswasestimatedwithareduced numberof dilutions.For wholecellculture lysisestimates, four differentdilutionsoftheAlexandriumostenfeldiilivesampleswere prepared(undiluted,3000,1000,and250cellsmL¹;finalconcen- trationintheassay)intriplicatevolumesof3.9mLin6mLglass vials.Eachsamplewasspikedwith0.1mLofapre-adjustedculture ofR.salinayieldingafinalconcentrationof1010⁴cellsmL¹.The firstoftwonegativecontrolscontainedonlyK-medium,whereasthe secondnegativecontrolwasperformedwithAlexandriumtamar- ense,strainAlex5,astrainwhichpreviouslywasshowntobenon- lytic(TillmannandHansen,2009).Vials werethenincubatedat 158Cinthedark.After24h,sampleswerefixedwithLugol(2%final concentration),andconcentrationsofintactcellsofbothspecies weredeterminedunderaninvertedmicroscope.

Forestimatinglyticactivityofcellextracts,cellpelletswere thawed and re-suspended in 1mL filtered seawater. Cellular extractswerepreparedbysonicatingthecellsuspensionwitha SonoplusHD70disintegratorequippedwithaMS73sonotrodefor 1minon iceusing thefollowingsettings: 50%pulsecycle,70%

amplitude.Basedontheknownnumberofcellsinthepellet,the extractwasusedtopreparefourconcentrationsof‘‘Alexandrium ostenfeldiicells’’inthe4mLvolumeusedinthebioassays(15,000, 7000, 3000, and 1500cellsmL¹). The bioassays were then performed as described above except that just intact cells of Rhodomonassalinawerecounted.

AllresultswerereportedasfinalconcentrationofRhodomonas salinaexpressedaspercentoftheseawatercontrol.Thedatafrom eachclonewereplottedaslog-transformedcellconcentrationsof AlexandriumostenfeldiiandcorrespondingpercentagesurvivalofR.

salina(meanofthreereplicates).Data,evenifalimitednumberof dilutions were performed, followed a sigmoid curve and were fittedasadose–responsecurvewithEq.(1)inordertoestimatethe EC50ofA.ostenfeldii,definedastheconcentrationofA.ostenfeldii cellsresultinginamortalityof50%oftheR.salinapopulation.

Y¼ 100

1þ _EC^X

50

k (1)

Here,YistheconcentrationofintactRhodomonassalinacells(as percentageofthecontrol),Xislog-transformedcellconcentration ofAlexandriumostenfeldii,andEC50andkarefittedparameters.

ForthecellextractofcloneOKNL18,EC50calculationbycurve fittingfailedasthehighestdose(15,000cellsmL¹)yieldedjust 41%celllysis.EC50herewasdefinedas‘‘>15,000cellsmL¹’’.

2.8. Phylogeny

RibosomalDNA sequencedata forthephylogeneticanalyses were obtained from Alexandrium ostenfeldii clones OKNL11-22.

Initially, 50mL samples of exponentially growing cells were collected by centrifugation at 3220g for 15min at room temperature (Eppendorf 5810R, Hamburg, Germany). The cell pelletswerefrozenat208Cfor20minbeforeextractionoftotal DNA with the DNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and quantity of the DNA was assessed by UV-spectroscopy witha NanoDropND-1000system(Peqlab,Erlangen,Germany)andthe integrityofDNAwasconfirmedusing1%agarosegelelectropho- resis wherea majorityoftheextractedgenomicDNAexceeded 20kilobases.

The D1/D2regionsofthe28S largesubunit(LSU) ribosomal DNAandtheinternaltranscribedspacerregion,includingtheITS1, 5.8Ssubunit,and ITS2sequenceslociwereamplifiedfromeach totalDNAextractbypolymerasechainreaction(PCR).Theforward and reverseprimers forLSUamplification were:D1R-F (5⁰-ACC CGCTGAATTTAAGCATA-3⁰)andD2C-R(5⁰-CCTTGGTCCGTGTTT CAAGA-3⁰),respectively.TheforwardandreverseprimersforITS/

5.8amplificationwere:ITSa(5⁰-CCAAGCTTCTAGATCGTAACA AGG (ACT)TC CGT AGG T-3⁰)and ITS b (5⁰-CCT GCA GTC GAC A(GT)ATGCTTAA(AG)TTCAGC(AG)GG-3⁰),respectively.Reaction conditions wereas follows: HotMasterTaq¹ (5Prime,Hamburg, Germany)buffer1X,0.1mMofdNTPs,0.1mMofeachforwardand reverseprimerand1.25unitsofTaqpolymerasewereaddedto10–

30ngoftheextractedgenomicDNAintotalreactionvolumesof 50

m

L.For28SrDNAamplifications,thereactionsweresubjected tothefollowingthermocyclingconditions:onecycleat958Cfor 7min,35cyclesat948Cfor45s,at548Cfor2min,andat708Cfor 1.5min, and a final extensionat 708C for 5min. The thermal cyclingconditionsfortheITS/5.8amplificationswere:onecycleat 948Cfor4min,9cyclesat948Cfor50s,at608Cfor40sandat 708Cfor1min,and29cyclesat948Cfor45s,at508Cfor45s,and at 708C for 1min, and a final 5min extension step at 708C.

Sampleswerekeptat 108Cuntil analysison 1%agarosegel,in ordertoensuretheexpectedamplificationproductswerepresent.

After PCR amplificationand subsequentcloning intothevector provided withthe TOPOTA Cloning¹ kit (Invitrogen, Carlsbad, California,USA),3–8clonesperampliconweresequencedusing the M13vector primerssupplied withthekit. Sequencingwas conducted witha standard cycle sequencingchemistry ABI3.1 (Applied Biosystems, Darmstadt, Germany). Cycle sequencing products wereanalyzedon anABI3130XL capillary sequencer (Applied Biosystems, Darmstadt, Germany) and the generated sequenceswereassembledwithCLCmainworkbenchversion6.0 (www.CLCbio.com). The resultingsequences weresubmittedto GenBank(AppendixB).

TheLSUandITS/5.8Ssequenceswerethencombinedwiththose availableinGenBank(AppendixB)andacombinedalignmentsof LSU and ITS sequences was constructed in order to conduct phylogeneticanalyses(AppendixC).Thedatasetwasalignedwith MAFFT usingtheq-insioption (Katohet al.,2005).The fullML analysis(GTRGAMMA,1000bootstrapreplicates)wasconducted withRAxML(Stamatakisetal.,2005).

2.9. Statisticalanalysis

Normalityofcellsizeparameters(length,width,length/width ratio,andvolume),toxins(PSPtoxins,SPXandgymnodimines)and

lyticactivitywastestedusingtheShapiro–Wilktest.Significance ofdifferencesbetweencloneswastestedusingaone-wayANOVA ifthedatawasnormaldistributed,andaKruskal–Wallistestifthe distribution was non-normal. The associations between the cellularquotaof thedifferenttoxins weretestedusingPearson product-momentcorrelation.

3. Results

3.1. Growth,morphology,morphometry,andphylogeny

Growth rates for all isolates were comparable and ranged between 0.16¹ and 0.26¹ with an average of 0.220.03d¹

(n=20).Generally,cellswereroundtoellipsoidinshape(Fig.2A–

F).Theepithecawasslightlyvariableinshapewithanoutlineranging from sigmoidto dome-shapeor round (Fig. 2B–F). The generally round hypotheca occasionally was slightly asymmetric (Fig. 2K).

Withinandamongclonestherewasalsoaconsiderablevariabilityin size(Fig.2G–K,Table1).Celllengthofallmeasuredcellsrangedfrom 26.3to53.4

m

m,thewidthfrom30.6to40.5

m

m,andthelength/

widthratiofrom0.9to1.2withsignificantdifferencesamongclones inmeancelllength(Kruskal–Wallistest;H=259;df=19;P<0.001), width(Kruskal–Wallistest;H=322;df=19;P<0.001),andlength/

widthratios(Kruskal–Wallistest;H=175;df=19;P<0.001).

Theplate patternwitha poreplate,4 apical,6 precingular, 5 postcingular, 2 antapical, 6 cingular, and 8 sulcal plates

Fig.2.Alexandriumostenfeldiimorphology.(A–K)Brightfieldimagesofalivingcell(A),andLugol-stainedcells(B–K)toshowvariabilityincellshape(A–F)andcellsize(G–K).

(L–Ad)Epifluorescenceimagesofcalcofluorstainedcellsshowingtheplatepattern(L–R),anddetailsofthe6⁰⁰place(SandT),the1⁰plateandventralpore(U–X),andthes.a plate(Y–Ad).PlatelabelsaccordingtotheKofoideansystem.Sulcalplatesabbreviations:s.p.=posteriorsulcalplate.s.s.p.=leftposteriorsulcalplate.s.d.p.=rightposterior sulcalplate.s.m.p.=medianposteriorsulcalplate.s.d.a.=rightanteriorsulcalplate.s.s.a.=leftanteriorsulcalplate.s.ac.a.=anterioraccessorysulcalplate.s.m.a.=median anteriorsulcalplate.s.a.=anteriorsulcalplate.

(Fig.2L–R)wastypicalforthegenusAlexandriumandthetypical shapeofthefirstapicalplateandthelargeventralpore(Fig.2L–N) clearly identified all isolatesas Alexandrium ostenfeldii. Detailed morphometricanalysesoffourisolatesindicatedsomevariabilityin shapeofthediagnosticplates1⁰,6⁰⁰ands.a.(Table2).Formostofthe cellstherightanteriormarginofthefirstapicalplate1⁰wasstraight (Fig.2LandM),howeverforallfourclonestherewerecells(upto 27%forcloneOKNL11)withcurvedorirregularmargins(Fig.2Uand V).Asignificantnumberofcellsrangingfrom22%to43%hadan anteriorlyextended1⁰plate(Fig.2WandX),whichcanexplainsome ofthevariabilityofthe1⁰area(Table2).Themeanventralporearea rangedfrom2.3to4.6

m

m² andwasdifferentbetweenthe four analyzedclones(Table2).Theshapeoftheanteriorsulcalplates.a.

wasfoundtobevariablewithinallfouranalyzedclones.Forthe majorityofcellstheshapewasclassifiedas‘‘A-shaped’’(Fig.2Aaand Ab),butabout20%and30%ofthecellsofeachisolatehada‘‘door- latch’’shaped(Fig.2YandZ)anda‘‘horse-shoe’’shaped(Fig.2Acand Ad)s.a.plate,respectively(Table2).Thes.a.platewasmorewide thanhighwiththewidth/heightratioofabout1.3beingquitesimilar forthefourclones(Table2).Theterminalprecingularplate6⁰was foundtobequitevariableinitswidth/heightratio(Fig.2SandT), withameanbeingevenbelowoneforcloneOKNL12(Table2).Based on the ITS/LSU phylogenetic analyses (Fig.3), the tested clones belongedtoGroup1ofA.ostenfeldii.

3.2. Toxinsandlyticactivity

All20isolatesofAlexandriumostenfeldiiproducedPSPtoxins with cellular quota ranging between 9.5 and 51pgcell¹

(Fig.4A).ThetotalPSPcontentwassignificantlydifferent(one- wayANOVA;F3,11=21.3;P<0.001)betweenthefourreplicated clones(OKNL11,12,15and19),withanaveragerelativestandard deviation(i.e.thestandarddeviationrelativetotheaveragePSP contentofeachclone)of105%.TheGTXandC1/C2toxinswerethe predominant analogs in all clones, the latter comprising around 845%ofthetotalPSPcontent(Fig.4B).Therelativecontributionof GTX2/3wasalsolargelycomparablebetweenallclonescontributing toaround9.62%ofthetotalPSPcontent,whileSTXshowedamuch largervariabilitycontributingbetween2.4%and18%ofthetotalPSP content (Fig. 4B). Among the cyclic imine toxin group, 13- desmethylspirolideC(SPX-1)andgymnodimineA(datanotshown), and12-methylgymnodiminecouldbe identifiedbycomparisonof retention time (Fig. 5)andCIDspectra (Fig. 6) fromA.ostenfeldii extractswiththosefromstandardsolutions.13-desmethylspirolideD was identified by comparison with a previously published CID spectrum(Slenoetal.,2004).

Thetotalgymnodiminecontent(i.e.gymnodimineAand12- methylgymnodimine) showed a large variation between clones witha25-folddifferencebetweenthelowestandhighestvalues (Fig. 7A). The total gymnodimine content differed significantly between the four tested clones (one-way ANOVA; F3,11=98.2;

P<0.001),withanaveragerelativestandarddeviationof73%.

All clones predominantly produced gymnodimine A, while most clonesalsoproduced12-methylgymnodiminewithrelativecontri- butionsrangingbetween0.2%and37%(Fig.7A).Thecellularquotaof totalSPX(expressedasSPX-1equivalents)generallyrangedbetween 2.4 and 4.4pgcell¹, with three clones containing less than 1.8pgcell¹ (Fig. 7B). The SPX included 13-desmethylspirolide C (76%to86%)and13-desmethylspirolideD(14%to24%,Fig.7B).All clones also produced trace levels of several unknown SPX-like compounds (data not shown). The totalSPX contentsignificantly differed between the four tested clones (one-way ANOVA;

F3,11=37.1;P<0.001),withanaveragerelativestandarddeviation of82%.Weobservedasignificantcorrelationbetweenthecellular quotaoftotalPSPtoxinsandtotalSPX(r=0.568;n=20;P=0.009), totalPSPtoxinsandtotalgymnodimines(r=0.472;n=20;P=0.031), andtotalSPXandtotalgymnodimines(r=0.526;n=20;P=0.017).

All12selectedclonesinducedlysisofRhodomonassalina,with EC50valuesrangingbetween212and525Alexandriumostenfeldii cellsmL¹ (Fig. 8A and B). The lytic response toward cellular extractwerelesspronouncedandcorrespondedto1222to5839A.

ostenfeldiicellsmL¹,andwasevenbeyondthehighesttestedcell concentration of 15,000cellmL¹ for stainOKNL18 (Fig. 8C). A quantitativecomparisonofcelllysisbetweenwholecellculture andcellfreesupernatantofstrainOKNL21showedthatabout70%

of thetotallytic activityis present withoutthecells (datanot shown).Wedidnotobserveasignificantcorrelationbetweenthe EC50valuesandthedifferentcellulartoxinquota.

4. Discussion

The morphological characteristics show a high variability withinthepopulation,supportingearlierdescriptionsofmembers Table1

SizemeasurementsofalltestedOKNLcloneswithlength(L),width(W),andL/W ratio.ValuesshowmeanSDwiththenumberofanalyzedcells(n).

Clone L(mm) W(mm) L/Wratio n

OKNL1 41.74.1 38.43.4 1.090.06 50

OKNL2 41.12.8 38.03.2 1.080.06 50

OKNL3 43.13.9 39.33.1 1.100.05 50

OKNL4 39.93.2 37.73.4 1.060.05 42

OKNL7 40.13.1 37.82.9 1.060.06 40

OKNL8 40.93.0 39.03.0 1.050.04 40

OKNL9 38.63.5 33.62.7 1.150.07 50

OKNL10 41.93.8 37.92.5 1.110.07 50

OKNL11 42.12.9 40.52.3 1.040.03 56

OKNL12 39.14.5 37.04.3 1.060.05 56

OKNL13 37.96.1 35.35.4 1.070.05 56

OKNL14 40.64.2 38.83.7 1.040.04 55

OKNL15 42.74.4 40.43.5 1.060.05 53

OKNL16 41.33.8 38.83.1 1.070.05 55

OKNL17 40.23.9 38.93.1 1.030.04 55

OKNL18 34.43.8 32.63.4 1.060.05 56

OKNL19 42.34.1 40.13.2 1.060.05 55

OKNL20 39.84.4 37.34.2 1.070.05 55

OKNL21 32.54.2 30.64.1 1.060.04 54

OKNL22 40.83.3 39.32.6 1.040.05 56

Overallmean 40.12.6 37.62.6 1.070.03 20

Table2

DiagnosticplatefeaturesoffourOKNLclones.Valuesshowmean,andmeanSD,withthenumberofanalyzedcells(n).

Clone Shape1⁰plate 1⁰extension Shapes.a Areavp(mm²) Area1⁰(mm²) 6⁰⁰ratiow/h s.aratiow/h

%Straight(n) %Yes(n) %Door-latch %A-shaped %Horseshoe- shaped

n

OKNL11 72.6(62) 21.9(64) 16.1 55.4 28.6 56 4.61.6(44) 76.616.2(36) 1.30.2(45) 1.30.2(38)

OKNL12 86.5(37) 22.2(45) 24.4 40.0 35.6 45 3.00.8(21) 52.613.5(16) 0.90.1(37) 1.20.2(21)

OKNL14 92.1(63) 42.9(63) 17.9 53.8 28.2 78 2.31.0(58) 70.116.2(48) 1.10.2(64) 1.20.2(68)

OKNL19 93.8(96) 33.3(99) 14.4 58.9 26.7 90 3.31.0(115) 76.316.7(90) 1.10.1(97) 1.30.2(88)

fromGroup1ofAlexandriumostenfeldii.Thehighvariabilityincell sizewithineachclone,incombinationwitharelativelyconsistent length/widthratioof1.1,isinlinewithearlierfindingsforclones belonging to Group 1 (Kremp et al., 2014). About 30% of the investigatedcellsshowedananteriorlyextended1⁰ plate,more common for Group 2. Previously considered as an important diagnosticplate,ouranalysisshowsavarietyofshapesforthes.a.

platewithintheclones,dominatedbyA-shapedplatesgenerally accountingfor52%,butalsowithupto36%ofthecellscontaininga horseshoe-shapeds.a. plate,previously ascribedtoAlexandrium peruvianum. We thus confirm the conclusions by Kremp et al.

(2014)thatthediagnosticcharactersoriginallydefinedtoseparate A.ostenfeldiiandA.peruvianumaremorevariablethanpreviously assumed and can show considerable intra- and inter-strain variability. Furthermore, Kremp et al. (2014) showed that A.

ostenfeldii and A. peruvianum morphotypes often had nearly identicalrDNAsequencesindicatingthattheyrepresentextreme endsinacontinuumofA.ostenfeldiimorphotypes(Krempetal., 2014).Nevertheless,Krempetal.(2014)alsoshowedthatselected platefeatures,whentestedstatistically,aswellastoxinprofiles, differedsignificantlyamongphylogeneticclusters.Our morpho- metric analysis thus indicate a designation of the Dutch A.

ostenfeldiipopulationasmembersoftheGroup1(seealsoKremp etal.,2014),aconclusionclearlysupportedbyboththemolecular dataandthepresenceofPSPtoxins.

The phylogeneticanalysis reveals a closerelationshipof the DutchAlexandriumostenfeldiipopulationwithisolatescoveringa widegeographicaldistribution,rangingfromthecoastalareasin theBalticSeatoembaymentsintheNorth-EastcoastoftheUS (Fig.3).Thisgeographicallywidedistributionmayindicaterecent

anthropogenic-driven dispersal, or that the phylogenetic clade comprisesgloballydistributedpopulationswithabroadtraitrange similartotheGroups(I-V) oftheformerAlexandriumtamarense speciescomplex(Johnetal.,2014).TheemergenceofA.ostenfeldii intheOuwerkerkseKreekseemstohaveoccurredonlyrecently, althoughsomesporadicobservationsofAlexandriumsp.inthearea were made in the last decade during monitoring programs (unpublisheddata).However, onlyindepthpopulation genetics will help to evaluate a common origin and/or the potential dispersalroutesoftheA.ostenfeldiiGroup1populations.

Interestingly, within Alexandrium ostenfeldii Group 1, toxin profilesoftheisolatesfromTheNetherlandsaremostcomparable to two isolates from North America, which are the only representativeswhichalsoproducePSPtoxins,SPX,andgymno- dimines(VanWagoneretal.,2011;Borkmanetal.,2012;Tomas et al., 2012). The Dutch isolates, however, differ in their gymnodiminecomposition. Specifically, theisolatesfromNorth Americawerereportedtoonlyproduce12-methylgymnodimine, whiletheOKNLisolatesproduceboth12-methylgymnodimineas well as gymnodimine A, a compound previously known from Kareniaonly(Sekietal.,1995;MacKenzieetal.,1996;Milesetal., 2000).Itthusseemsthatthecloneswereportherearethefirst recordofgymnodimineAforA.ostenfeldii,andpossessaunique combinationoftoxins.WhethertheisolatesfromNorthAmerica are not producing gymnodimine A, or if the levels are below detectionlimit requiresfurther analyses. Generally,taking into accountthatadvancinganalyticaltechniqueswilllowerthelimit ofdetectionforamultitudeoftoxins,futurestudiesmayshowthat morecomplextoxinprofiles mightgenerallybecommon forA.

ostenfeldiipopulations.

AlexandriumminutumCCMP113 Alexandrium insuetum CCMP2082

Alexandrium tamutumAL2T

AP0704-3,New River, USA AOVA0917, Gotland, Sweden AOF0901, Åland, Finland

AP0411, New River,USA AP0905, Narrangansett Bay, USA

AOKAL0909, Kalmar,Sweden OKNL17, Ouwerkerk, The Netherlands

OKNL13, Ouwerkerk, The Netherlands OKNL14, Ouwerkerk, The Netherlands OKNL11, Ouwerkerk, The Netherlands OKNL12, Ouwerkerk, The Netherlands OKNL21,Ouwerkerk, The Netherlands OKNL18, Ouwerkerk, The Netherlands OKNL22, Ouwerkerk, The Netherlands OKNL16, Ouwerkerk, The Netherlands OKNL19, Ouwerkerk, The Netherlands AOPL0917, Hel, Poland

K1354, Öresund, Denmark AP0704-2, New River, USA ASBH01, BohaiSea, China

Group 1

IEO-VGOAM10C, Palamos,Spain IEO-VGOAMD12, Palamos, Spain WW516, Fal River, UK WW517, Fal River, UK LSA06, Lough Swilly,Ireland LSE05, Lough Swilly, Ireland

Group 2

AOPC1, Saanich,Canada IMPLBA033, Callao, Peru

CCMP1773, Limfjord,Denmark BYK04, North Sea,Ireland CCAP111947, NorthSea, Scotland CCAP111945, North Sea, Scotland S0601301, NorthSea, Scotland NCH85, North Sea, Norway AONOR4, Oslofjord, Norway S6P12E11, North Sea, Scotland K287, Limfjord, Denmark

Group 6

LKE6, Gulf ofMaine, USA IMRV062007, North Sea, Norway HT120B7, Gulf of Maine, USA AOIS4, Breidafjord, Iceland F301, Gulf of Maine, USA HT120D6, Gulf of Maine, USA

Group 5

CAWD135, New Zealand

CAWD136, New Zealand Group 4 AOFUN0801, Hokkaido,Japan

AOFUN0901, Hokkaido,Japan Group 3 69

87 85

100 80

100

90

100 100 100

100 99

100 99

85 99 77

0.002

Alexandrium ostenfeldii

Fig.3.PhylogenictreeofthecombinedD1-D2LSUandITS1-5.8S-IST2rDNAofAlexandriumostenfeldiiandotherAlexandriumspecies,whereAlexandriumminutum, Alexandriumtamutum,andAlexandriuminsuetumwereusedasoutgroups,withaselectionof10OKNLclonesindicatedinbold.NodelabelswerederivedusingRandomized AxeleratedMaximumLikelihoodanalyses,RAxML(Stamatakis,2014),inGTRGAMMAwith1000bootstrapsupport.

ThecellularquotaofPSPtoxinsfellwithintherangeofearlier reportedAlexandriumostenfeldiiandAlexandriumtamarenseclones (Tillmannetal.,2009;Suikkanenetal.,2013).Thepredominanceof C1/C2hasbeenearlierreportedforA.ostenfeldiiclones,thoughthe PSP toxin composition differs between isolates (Kremp et al., 2014).B1toxinswerenotfoundinclonesOKNL4,12,13,and14, whichmayindicatethatthesecloneslacktheabilitytoproduceB1, or that the values were below the level of detection (<0.1pgcell¹).BoththecellularquotaofSPXandgymnodimines arerelativelylowascomparedtoanearlierreportedA.ostenfeldii clone(thendesignatedtoAlexandriumperuvianum)fromtheUS EastCoast(Tattersetal.,2012),whileSPXquotafellwithinthe range of several A. peruvianum clones from the NE Atlantic (Suikkanenetal.,2013).SPXconsistedmainlyof13-desmethyl- spirolide C, which was largely consistent with other clones in Group1and2oftheA.ostenfeldiispeciescomplex(Krempetal., 2014),thoughallOKNLclonesalsocontainedsome13-desmethyl- spirolide D. Clones OKNL13 and OKNL22 did not produce 12- methylgymnodimine,oronlyverylowamountsthatfellbelowthe limit of detection (<0.1pgcell¹). We furthermore observed differencesbetweencloneswiththelowestandhighestdetected toxin quota of about 5-foldfor PSP toxins, 4-fold for SPX and

25-foldforgymnodimines.Thus,ourresultsdemonstrateahigh intraspecificvariabilityintoxinquota,whichisinlinewithearlier reports on Alexandrium sp. populations (Gribble et al., 2005;

Tillmannetal.,2009;Alpermannetal.,2010;Tillmannetal.,2014).

9 8 7 21 2 10 16 *11 18 1 13 *12 *14 4 17 20 *19 22 3 15

PST content (pg cell

-1

)

0 10 20 30 40 50

A

60

B

Total

A. ostenfeldii clone numbe r

9 8 7 21 2 10 16 *11 18 1 13 *12 *14 4 17 20 *19 22 3 15

PSP composit ion (%)

0 20 40 60 80 100

GTX2/3 B1 C1/C2 STX

Fig.4.VariationincellularPSPtoxinscontent(A)andcomposition(B)inallisolated A.ostenfeldiiOKNLclones.Barsshowmeanoftechnicalreplicates,exceptfor OKNL11,OKNL12,OKNL14andOKNL19,whichindicatemeanSD(n=3),also denotedbyanasterisk.

Fig. 5. Ion chromatograms (m/z 522>406)of (A) a 12-methylgymnodimine standardand(B)anA.ostenfeldiiextract(cloneOKNL3).

Fig.6.CIDspectra(m/z522)of(A)a12-methylgymnodiminestandardand(B)an Alexandriumostenfeldiiextract(cloneOKNL3).

This variability cannot be explained by differences in growth conditionsorgrowthphase,asallculturesweregrownunderthe same conditions and harvested during the mid-exponential growthphase.Theobservedvariabilityintoxinquotathusseems theresultofgeneticdifferencesbetweenclones.

In addition to the production of PSP toxins, spirolides, and gymnodimines, all isolates produced alleochemicals with the capacitytolysecellsofthetargetspeciesRhodomonassalina.Lytic activity of extracellular secondary metabolites is rather wide- spreadinthegenusAlexandrium(TillmannandJohn,2002)andhas beenshown for various Alexandrium ostenfeldii clones (Hansen etal.,1992;Tillmann etal.,2007;Tomasetal.,2012; Hakanen etal.,2014;Tillmannetal.,2014).Ourcorrelationanalysesconfirm earliercomparativeapproachesthathaveshownthatlyticactivity isunrelatedtoPSPtoxin(TillmannandJohn,2002)andspirolide content (Tillmann et al., 2007), and also a comparison of gymnodimine cell quota and lytic activity gave no obvious evidencethat gymnodimines might contributeto theobserved celllysisofR.salina.

WithEC50valuesfrom0.18to0.510³cellsmL¹theDutch populationseemtopossessacomparable lyticactivityasother Alexandriumostenfeldiiisolatesofotherphylogeneticgroups.Their EC50 values have been shown to range from 0.2 to 1.910³cellsmL¹(Tillmannetal.,2007;Hakanenetal.,2014)

A. ostenfeldi i clone numbe r

9 8 7 21 2 10 16 11* 18 1 13 12* 14* 4 17 20 19* 22 3 15

SPX c o ntent (pg cell

-1

)

0 1 2 3 4 5 6

13-Desmethylspirolide C 13-Desmethylspirolide D

9 8 7 21 2 10 16 11* 18 1 13 12* 14* 4 17 20 19* 22 3 15

Gymnodimine content (pg c e ll

-1

)

0 2 4 6 8 10 12 14

Gymnodimine A 12-methylgymnodimine

A

B

Fig.7.Variationincellulargymnodiminecontentandcomposition(A),andSPX contentandcomposition(B)inallisolatedAlexandriumostenfeldiiOKNLclones.Bars showmeanoftechnicalreplicates,exceptforOKNL11,OKNL12,OKNL14 and OKNL19,whichindicatemeanSD(n=3),alsodenotedbyanasterisk.

21 16 11 18 13 12 14 17 20 *19 22 15

EC

50

A.ostenfeldii cells (cells mL

-1

)

0 100 200 300 400 500 600

A. ostenfe ldii OKNL19 (cel ls mL

^-1

)

10¹ 10² 10³ 10⁴ 10⁵

Rhodomonas s u rv iv al (% )

0 20 40 60 80 100

A. ostenfeldi i OK NL clone numbe r

21 16 11 18 13 12 14 17 20 *19 22 15

EC

50

A.ostenfeldii cell ex tr acts (cells mL

-1

)

0 1000 2000 3000 4000 5000 6000

A

B

C

>15,000

Fig.8.VariationinallelopathicactivitiesinaselectionofA.ostenfeldiiOKNLclones expressedasEC50valuesofwholecellculturesbasedonRhodomonassalinasurvival (%),withtypicaldose-responsecurves(A;circles,squaresandtrianglesrepresent triplicatecultureswithmeanSD(n=3),withopensymbolsforwholecultureand grayfilledsymbolsforcellextract),andanoverviewoftheEC50valuesofthetested clonesbasedonwholecellcultures(B),andbasedoncellextracts(C).Barsshowmean oftechnicalreplicates(n=3),exceptforOKNL19,whichindicatesmeanSD(n=3) basedonthetriplicateculturesshownin(A),alsodenotedbyanasterisk.