yessotoxin production of Protoceratium reticulatum Effects of salinity, temperature and nutrients on growth, cellular characteristicsand Harmful Algae

Effects of salinity, temperature and nutrients on growth, cellular characteristics and yessotoxin production of Protoceratium reticulatum

Karin Ro¨der

^a,

*, Florian Matthias Hantzsche

^b

, Christina Gebu¨hr

^c

, Claudia Miene

^d

, Tina Helbig

^a

, Bernd Krock

^e

, Mona Hoppenrath

^f

, Bernd Luckas

^a

, Gunnar Gerdts

^c

aInstituteforNutrition,DepartmentofFoodChemistry,Friedrich-Schiller-UniversityofJena,DornburgerStraße25,D-07743Jena,Germany

bGKSSResearchCentre,Max-Planck-Straße1,21502Geesthacht,Germany

cBiologischeAnstaltHelgoland,Alfred-Wegener-InstituteforPolarandMarineResearch,POB180,D-27498Helgoland,Germany

dInstituteforNutrition,DepartmentofNutritionalToxicology,Friedrich-Schiller-UniversityofJena,DornburgerStraße25,D-07743Jena,Germany

eAlfred-Wegener-InstituteforPolarandMarineResearch,AmHandelshafen12,D-27570Bremerhaven,Germany

fGermanCentreforMarineBiodiversityResearch,ForschungsinstitutSenckenberg,Su¨dstrand44,D-26382Wilhelmshaven,Germany

1. Introduction

Yessotoxin(YTX),adisulphatedpolyethertoxinwasfirstisolated byMurataetal.(1987)fromdigestiveglandsofJapanesescallops (Patinopecten yessoensis). Three species of dinoflagellates were identified as YTX producing organisms, these are Protoceratium reticulatum (Clapare`de & Lachmann) Bu¨tschli (syn.: Gonyaulax grindleyi) (Satake et al., 1997), Lingulodinium polyedrum (Stein) Dodge(syn.:Gonyaulaxpolyedra)(Tubaroetal.,1998;Draiscietal., 1999) and Gonyaulax spinifera (Clapare`de & Lachmann) Diesing (Rhodesetal.,2006;Riccardietal.,2009).Meanwhile,theproduction ofYTXbythedinoflagellateP.reticulatumhasbeenreportedinNew

Zealand,Japan,Norway,UK,Canada,USA,Chile,Spain,Italy,and southernAfricaBay(Ciminielloetal.,2003;Finchetal.,2005;Krock etal.,2006,2009;Pazetal.,2004,2006,2007;Ramstadetal.,2001;

Samdaletal.,2004;Satakeetal.,1997,1999,2006).

Different concentrations of YTX were found in cultures of dinoflagellates around theworld. The toxicityof P.reticulatum reached from0.9to79pgYTXcell¹ (Eiki etal., 2005;Howard etal.,2008;MacKenzieetal.,1998;Mitrovicetal.,2005;Pazetal., 2004,2007;Samdaletal.,2004;Satakeetal.,1996,1999),andthe concentrationsofYTXinL.polyedrumwereupto1.5pgYTXcell¹, and inG. spiniferaupto200pgYTXcell¹ (Draisci etal., 1999;

Howardetal.,2008;Pazetal.,2004;Ramstadetal.,2001;Rhodes etal.,2006;Tubaroetal.,1998).Itseemsthattheprincipaltoxin formed by P. reticulatum is YTX, even though some strains contained homoYTX as prominent analogue (Paz et al., 2008).

Fromabout100knownYTXanaloguesonly40%arecharacterized concerning their exact chemical structure (Miles et al., 2004, 2005a,2005b,2006a,2006b;Pazetal.,2008).Suzukietal.(2007) HarmfulAlgae15(2012)59–70

ARTICLE INFO

Articlehistory:

Received8April2011

Receivedinrevisedform23November2011 Accepted23November2011

Availableonline5January2012

Keywords:

Carboxyyessotoxin Chlorophylla

Formationindependencyonnutrients Gonyaulaxgrindleyi

Ketoyessotoxin LC–MS/MSanalyses Protoceratiumreticulatum Salinityandtemperature YTX

ABSTRACT

Protoceratiumreticulatumasaproducerofyessotoxin(YTX)anditsanaloguesiscommoninseveral coastalenvironments.TheYTX-producingstrainofP.reticulatum,isolatedfromtheGermanBight(North Sea),wasanalysedtostudytoxinproductionundervariousautecologicalconditions.Experimentswere carriedouttoinvestigatetheinfluenceofN/Pratio(2.44(1/10N),24.36(f/2)and243.65(1/10P)), temperature(15and208C),salinity(5,10,15,20,25and30)andgrowthphaseonYTXformation,cell sizeandchlorophyllaconcentration.

P.reticulatumshowedthehighestgrowthat158Candhighersalinities(25and30).Inparticular, higher temperatureled to a reducedgrowth. Thetotal YTXconcentrations werehigher at lower temperature.However,aclearcorrelationbetweensalinityandYTXproductionwasnotobservedat lowertemperature.Furthermore,1/10Pandf/2culturesexhibitedthehighestcellquotaofYTXatthe endofthestationaryphase;adramaticeffectoccurredat158Cin1/10Pmedia,whenthetoxicity increasedtotenfoldhighervalues.SlightvariationsofthecompositionoftheYTXanaloguesunder nutrientlimitationwereobservable.Inaddition,theresultsindicatethatN-limitationcausealowercell size,whereasP-limitationleadstoahighercellsize;aninfluenceofthesalinityoncellsizewasalso observable.

ß2012ElsevierB.V.Allrightsreserved.

*Correspondingauthorat:DepartmentofFoodChemistry,FacultyofBiologyand Pharmacy,Friedrich-Schiller-Universityof Jena,Dornburger Straß25,D-07743 Jena,Germany.Tel.:+493641949653;fax:+493641949652.

E-mailaddress:Karin.Roeder@uni-jena.de(K.Ro¨der).

ContentslistsavailableatSciVerseScienceDirect

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

1568-9883/$–seefrontmatterß2012ElsevierB.V.Allrightsreserved.

doi:10.1016/j.hal.2011.11.006

reported on P. reticulatum strains isolated at different sites of Japanesecoasts(e.g.MutsuBayandOkiraiBay)whichproduced 45,46,47-trinoryessotoxin(trinorYTX),1-homoyessotoxin(homo- YTX), 45,46,47-trinor-1-homoyessotoxin besides YTX. In some strains 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin (norox- oYTXenone;ketoYTX)wasalsodetected,andsomeofthosestrains exhibitedapercentageofYTXanaloguesnearto50%ofthetotal YTXconcentration(Suzukietal.,2007).

Environmentalconditionsareimportantfortheproductionof toxinsbydinoflagellates.However,onlyfewstudiesarepublished howenvironmental factorsmightaffecttheYTXformationofP.

reticulatum.GallardoRodrı´guezet al.(2009) andGuerriniet al.

(2007)reported abouttherequirements ofmacronutrients; the effectofmicronutrientswasinvestigatedbyMitrovicetal.(2004) andsomedataareavailableontheinfluenceoftemperatureand salinityontheYTXproduction(Guerrinietal.,2007).

Ourinvestigationsweredirectedontheelucidationoftheeffect ofvariousgrowthfactorson formationofYTXs inastrainof P.

reticulatum from the North Sea (Helgoland Roads) for better assessmentoftheriskfortoxicdinoflagellateeventsintheNorth Seaconnectedwithclimatechangeoreutrophication.Therefore, the isolated strain of P. reticulatum was cultured at different conditionsconcerningmacronutrients,salinityandtemperature.

2. Materialsandmethods

2.1. IsolationandidentificationofP.reticulatum

NetsamplesfromsurfacewateratHelgolandRoads,German Bight,North Sea, Germany,were collected during a taxonomic phytoplanktonre-investigationproject(Hoppenrath,2004;Hop- penrath et al., 2009). P. reticulatum cells were isolated by micropipettingfroma mixednet-sampleinApril2003,washed in sterilefine-filtered seawaterand maintained in f/2 medium (Guillard,1975)inplasticPetridishes.Aftercultureestablishment tissueflaskswere used.The originalculture is available at the GermanCentreofMarineBiodiversityResearch,Wilhelmshaven, fromM.Hoppenrath.Thespecieswasidentifiedunderthelight microscope by its characteristic cell shape, size, and thecal ornamentation(Fig.1a–c).Thespeciesidentificationwasverified byscanningelectronmicroscopy(Fig.1d–j).

2.2. Mediapreparation,cultureconditionsanddeterminationofthe cellgrowth

TheP.reticulatumstrainwasmaintainedinborosilicateflasksin 500ml f/2 medium without silicate (Guillard, 1975) at 70–

90

m

mols¹m²,148Cand12:12hlight/darkregimeinnatural seawaterwithasalinityof30.

TheinfluenceofthesalinityontheYTXproductionwasestimated atsixsaltconcentrations.Therefore,thenaturalseawater ofthe NorthSea,whichhadtheoriginalsalinityof30,wasdilutedwith freshwater(drinkingwater)tothefollowingconcentrations:5,10, 15,20and25.Thesefivedilutionsandthenaturalseawaterwere preparedasf/2mediawithoutsilicate,describedbyGuillard(1975).

Fornutrientlimitationf/2mediawerepreparedcontainingthree compositions of nutrients. First nutrient sufficient f/2 medium (referredasf/2)containingtheoriginalconcentrationofnutrients, secondf/2mediumwithtentimeslessphosphorus(1/10P)and thirdf/2mediumwithtentimeslessnitrogen(1/10N)(Table1).The pH-valuewasadjustedto7.60.02usingHClandthemediawere incubatedinthermostatedroomsat158Cand208C,respectively.The light/darkregimeandthelightintensitywereretainedunchanged.The strainwasnotpre-adaptedtothechangedconditions.

Three replicatesofeachwereincubatedinErlenmeyerflasks (1l)whichwereinoculatedwith1mlofalateexponentialstock

culture of P. reticulatum and contained 11cellsml¹on day of inoculation.Inaddition,wetook10mloftheinoculumfortoxin determination (n=3). Therefore, the subsamples were filtered throughGF/Cfilters(CarlRothGmbH&Co.,Germany)underslight vacuumpressure.

Thegrowthcurvesweredeterminedbymeasurementofthein vivo fluorescence followed by successive cell counting of P.

reticulatum in 3-day intervals (n=3). 1ml of the culture was immediatelymeasuredbyafluorescencespectrophotometer(Cary Eclipse,VarianInc.,USA)usinganexcitationwavelengthof485nm and anemission wavelengthof 685nm.In addition,cells were counted using a Sedgewick-Rafter cell witha light microscope (10objective), aminimum of400cellswerecounted. Specific growthrate(

m

m[day¹]) wascalculated usingtheequationby Guillard(1973):

m

¼lnN1lnN0

t1t0

Niscelldensityatagiventime(t).

2.3. Samplingintheexponentialandthestationarygrowthphase For further analyses additional samples were taken in the exponentialand stationarygrowthphase.Chlorophyllaconcen- trationwasdeterminedusing subsamplesof25mlwhich were analysedastriplicateinvivobythemultialgalfluorimeter(BBE Moldaenke,Germany).Thisfluorometerallowsdeterminationof differentalgae classes: green (chlorophyceae), blue-green (cya- nophyceae), brown (diatoms and dinophyceae) and cryptophy- ceae.Theemissionofthepigmentsafterexcitationatcharacteristic wavelengthswasmeasured.Thus,thechlorophyllaconcentration andotherpigments(

m

gl¹)weredeterminedforalllivingcells.

Fordeterminationofcellsizeandmorphology,subsamplesof 50ml were analysed using the Flow CAM (Fluid Imaging Technologies,USA, further detailssee Sierackiet al.,1998). For mostof thesamples,dependingoncell density,a minimum of 400cells were measured. Particles from 5 to 100

m

m were measured using a 20 objective and a flow cellof 100

m

min depth.Consequently,informationaboutthecelldiameter,volume, lengthandwidthofeachmeasuredcellwereobtained.

The YTX concentration was determined by LC–MS/MS (see below).

2.4. ExtractionofyessotoxinsandmeasurementbyLC–MS/MS Cultures were filtered on GF/C filter (Whatman, GB) under slightvacuum,100mlintheexponentialgrowthphaseand300ml inthestationaryphase,respectively.Filterswereextractedwith methanol(MeOH)usinganultrasonicprobefor30sandanice- cooledultrasonicbath for30min,afterwards theywerecentri- fugedat14,000gfor10min.Thesupernatantwasstoredandthe filterswereextractedagainwithMeOHusingtheultrasonicbath (30min), followed by centrifugation at 14,000g for 10min.

Supernatants were combined evaporated to dryness using a heatingblock(408C)undernitrogenstream.Driedsampleswere dissolvedin1mlMeOH,admittedto2mlsingle-usesyringesand filtered through 0.45

m

m nylonfilters (Carl Roth GmbH &Co., Germany).

100ml of the culture filtrates were stored in 100ml flasks (Kautex,Germany)formeasuringofreleasedYTXintheculture media.Theflaskswerestoredat208Cuntilfurtherpurification.

Subsequently,thefiltrateswereloadedonpreparedChromabond C18ec cartridges (Macherey-Nagel GmbH & Co. KG,Germany).

Thosewereequilibratedwith3mlMeOHand3mlofdeionised

water(SynergyWaterPurificationSystem,Millipore).Afterwards theculturefiltrates(100ml)wereloadedonthecartridge,washed with3mlofdeionisedwaterandthenelutedwith3mlMeOH.The MeOH was evaporated to dryness under nitrogen stream and samples were dissolved in 1ml MeOH and filtered through 0.45

m

mnylonfilters.Allsampleswerestoredat208Cinbrown autosamplervialsuntilmeasuringbyLC–MS/MS.

Certified YTX standard solution, dissolved in MeOH, was obtained from the National Research Council (NRC) Canada (Halifax, Canada). The LC–MS/MS measurements were carried outasdescribedearlier(Ro¨deretal.,2011).Liquidchromatography was performed using Hyperclone C8 Column (3

m

m, 130A˚, 502.0mm) with security guard (Phenomenex, Germany) by gradientelutionataflowof0.3mlmin¹.Mobilephaseconsistsof eluentA:5mMammoniumformateinacetonitrile/water(10:90) andofeluentB:5mMammoniumformateinacetronitrile/water (90:10).Thegradientelutionwasdonewith100%Afor1.5min, followed by linear gradient to 40% A over 3.5min, held over 5.0min,andwithin1.0minto100%B,heldfor9.0minandatleast within1.0minbackto100%A,heldfor24min.

MS/MSanalyseswereperformedusingaTripleQuadrupoleAPI 365 LC-MS/MS (Applied Biosystems GmbH, Germany) with electrosprayionizationbyESI TurboIon sprayInterface(SCIEX, Fig.1.Light(a–c)andscanningelectron(d–j)micrographsofProtoceratiumreticulatumfromtheusedculture.(a)Leftlateralviewshowingthethecalornamentation.(b) Ventralsurfaceviewshowingthecingulumdisplacement.(c)Ventralviewindeeperfocusshowingthegeneralcellshape.(d)Rightlateraltoventralview.(e)Leftlateralto ventralview,notetheventralpore(arrow).(f)Leftlateralview.(g)Rightlateralview.(h)Apicaltodorsalviewoftheepitheca.(i)Antapicaltoventralviewofthehypotheca.(j) Antapicaltoleftlateralviewofthehypotheca.Scalebars=10mm.

Table1

Molarconcentrationandproportionofmacronutrientsinthefinalmedia.

Component Molarconcentrationinfinalmedia

Control 1/10N 1/10P

NO3 8.8210⁴M 0.88210⁴M 8.8210⁴M

PO43

3.6210⁵M 3.6210⁵M 0.36210⁵M

N/P 24.36 2.44 243.65

K.Ro¨deretal./HarmfulAlgae15(2012)59–70 61

Canada).Analyseswerecarriedoutinmultireactionmonitoring (MRM) mode with negative ionization; selected transitions ([MH]>[MHSO3]: YTX m/z 1141.5>1061.5, homoYTX m/z 1155.5>1075.5, 45-OH-YXT m/z 1157.5>1077.5, carbox- yYTXm/z1173.5>1093.5,theputative45-OH-carboxyYTXm/z 1189.8>1109.5,m/z1047.5>967.5(probablyketoYTX)andm/z 1191.5>1111.5).YTXconcentrationsweredeterminedbyathree pointcalibrationcurveusingdilutionsofacertifiedYTXstandard solution (NRC, Halifax, Canada). Concentrations of the YTX analogueswereexpressedasYTXequivalents.

Statistical analyses were performed using GraphPad Prism 5.01(GraphPadSoftware,Inc.,SanDiego,CA).1-wayANOVAwas applied for analysis of variances of cell size, chlorophyll a concentration,andYTXcellquotabetweenmediawithasalinity of15,20,25,and30,aswellasbetweenf/2,1/10Nmedia,and1/10 Pmedia. 2-WayANOVA wasapplied tocompare variances of chlorophyllaconcentrationandYTXcellquotaatthedifferent salinitiesintheexponentialversusstationarygrowthphase.Both, 1-wayand2-wayANOVA includedBonferroni’smultiplecom- parison test as post test. P values<0.05 were considered as significant.

3. Results

3.1. IdentificationofP.reticulatum

Chloroplasts color thecells golden-brown. The cingulum is nearlymediananddescendingaboutonecingularwidthwithout overlap(Fig.1bandc).Cellsareslightlylaterallycompressed.P.

reticulatumis photosynthetic,hasa stronglyornamented theca (Fig. 1d–j) andthischaracteristicreticulation withoneor two poresinside eachreticulation subunit canhidethe suturesso thatitcanbedifficulttorecognizetheplateborders.Theplate pattern was discerned as Po 4⁰ 6⁰⁰ 6⁰⁰⁰ 1p 1⁰⁰⁰⁰ as has been describedbefore (Fig. 1d–j; Woloszynska,1928;Hansenet al., 1997).Somecellshadadifferentepithecalplatepatternwith3⁰ and1a,thisvariabilityis also knownfromthe literature(Von Stosch, 1969; Hansen et al., 1997). The first apical plate has a ventral pore at its right margin (Fig. 1e). The pore plate is narrow andelongated (Fig. 1f–h). For further informationsee Section2.

3.2. GrowthofP.reticulatum

P.reticulatumcellsculturedatasalinityof5and10diedinall casesshorttimeafterinoculation.Highestcellconcentrationwas observed in the f/2 media at 158C at salinities of 25 and 30 Culturingatasalinityof20orbelowdecreasedthegrowthstrongly (Fig.2a).Furthermore,1/10Ncellscultivatedat158Creachedthe stationaryphasefasterthanthoseculturedinf/2mediaandthe absolutecellnumberwasalsolower.Theinfluenceofthesalinity on growthin 1/10 N mediawas not sohigh; cells cultured at salinitiesof20,25and30hadalmostthesamemaximumofcells (Fig. 2c). P.reticulatum cultivated in 1/10 Pmedia reached the stationaryphasefasterthancellscultivatedinthef/2mediaandin the1/10Pmedia,withlowgrowthatsalinitiesof20,25and30and lowestgrowthatasalinityof15(Fig.2e).

ThesameinfluenceofthesalinityongrowthofP.reticulatum wasobservedinf/2mediaatbothtemperaturesof15and208C, butthemaximumcellcountswerehigherat158C(Fig.2aandb).

Highertemperaturecausednodifferencesingrowthbetween1/

10N cultures at 15 and 208C, except for cells cultured at a salinity of 15showed a highergrowth rate at 158C.Also the growthof1/10Pcultureswaslowat15and208C(Fig.2candf).

Growthratesintheexponentialgrowthphaserangedfrom0.21 to0.35day¹.

3.3. Variationofcellsizeandmorphology

Theinfluenceofnutrientlimitation,salinityandtemperature on cell size and morphology was investigated; therefore char- acteristics of P. reticulatum during exponential and stationary phasewerecompared.Nochangesofcellsizeormorphologywere foundinf/2mediaata salinityof30.Thus, thiscultureactsas reference to compare the influence of nutrient limitation and salinity. The strongest changes of the cell size depending on nutrientlimitationand salinitywereobservedinthestationary phase,butatrendwasalreadyvisibleintheexponentialgrowth phase(datanotshown).Ingeneralanincreaseofcellsizeduring culturingwasobservedinthef/2mediaatsalinitiesof15,20,and 25andin1/10Pmedia(allsalinities),whereasthecellsizewas highestinlowsalinityculturesinthestationaryphase;compared to cells cultured in f/2 media at a salinity of 30 significant differenceswereobserved(P<0.001).Theinfluenceofsalinityon thecellsizewashighestinthef/2media.Thelowestcellsizeswere observedincultureswith1/10Nmedia,whereasthedifferenceto cellsculturedinf/2mediumatasalinityof30wassignificantfor1/

10 N media at salinities of 20, 25, and 30 (P<0.001) and not significantatasalinityof15(P>0.05).Butcomparing1/10Ncells ofdifferentsalinitieswitheachothernostatisticsignificancewas observed for salinities of 20, 25, and 30 suggesting that the influenceofsalinityislessimportant.Fig.3showsthevolume(fl) of P. reticulatum (158C) cultured at different salinities in the stationaryphase,Pvaluesinbetweenthesalinitieswereplotted.

Statisticdifferencestocellsculturedinf/2atasalinityof30were onlyplottedfor1/10Natasalinityof15.Asimilarpatternwasalso observedat208C,evenifthedifferencesinbetweenlimitationsor withinonelimitationbetweenthesalinitieswerenotthatclear.

Statistic analyses of variances of cells cultured at 208C in the stationary growth period revealed that there were significant differencescomparingcellsculturedinf/2mediaatasalinityof30 salinityandalmostallothersamples(P<0.001);onlyellscultured atasalinityof25werenotstatisticallydifferent.Itwasevidentthat cells cultured at highertemperature showedstrong changes of morphologyinthestationaryphase.Thosecells weredeformed andshowedexcrescences.

3.4. Chlorophyllaconcentration

Differences of chlorophyll a cell quota were observed in P.

reticulatum.Influenceofthesalinity:Generally,theconcentrations ofchlorophyllacell¹increasedwithlowersalinityinf/2and1/10 Pcultures.Thiseffectwasstrongestat158Cintheexponential growthphase.Significantdifferenceswereobservedcomparingf/2 cells cultured at a salinity of 30 to 25 (P<0.01), and to 15 (P<0.001).Nostatisticdifferenceswereobservedcomparingf/2 cellsatacertainsalinitytothenexthighersalinity(e.g.salinityof 15 vs. salinity of 20). Furthermore, differences between f/2 culturedcellsoftheexponentialtothestationarygrowthphase were not statistically significant(Fig. 4a). The influence of the salinityonthechlorophyllaconcentration of1/10PculturedP.

reticulatum was less obvious. Significant differences only exist between1/10Pcellsculturedatasalinityof30to15.Comparing cellsofacertainsalinitytothenexthighersalinity(salinityof15 and 20) were statistically different (P<0.001, Fig. 4c). In the stationaryphasedifferencesexistbetweenf/2cellsculturedata salinityof15tosalinityof20andtosalinityof30(P<0.01,Fig.4c), respectively. No significant difference exists between 1/10 P culturedP.reticulatumcells.Nocorrelationbetweensalinityand chlorophyllaconcentrationwasobservedin1/10Pculturedcells (P>0.05).

Influenceof nutrients:Nostatisticsignificantdifferenceexists between P.reticulatumcells cultured at158Cin f/2and1/10 P

media(P>0.05).Butthedifferencesoff/2and1/10Pto1/10N cultureswereevident,especiallyinthestationaryphaseinwhichf/

2and1/10Pculturedcellsdifferfrom1/10Nculturedcells at:

salinityof15(f/2:P<0.001,1/10P:P<0.001),salinityof20(f/2:

P<0.05,1/10P:P<0.001),andsalinityof25(f/2:P<0.001).Only differencesbetween 1/10P and1/10 N ata salinity of25, and betweenf/2aswellas1/10Pand1/10Natasalinityof30werenot significant(P>0.05).

Influenceofgrowthphasewasmostevidentin1/10Nlimited culturesat158C(Fig.4b).Therewerenosignificantdifferences intheexponentialgrowthphaseat208C,neitherregardingthe different salinities nor nutrients. In the stationary phase P.

reticulatumculturedatasalinityof20containedsignificantmore chlorophyll a when cultured in f/2 medium than in 1/10 P medium (P<0.01). Furthermore cells cultured in 1/10 N mediumat a salinity of15contained significantmore chloro- phyllain comparisontosalinitiesof20,25,and30(P<0.001, respectively).

To exclude the potential influence of cell size on the chlorophyllaconcentration,theratioofdiametertochlorophyll a concentration was also calculated and the pattern did not change.

3.5. YTXconcentrationinP.reticulatumcells

YTXwasthemainanaloguewitharelativeamountofmorethan 94% of all YTXs and a total concentration of 7.22 0.20pg YTXcell¹inthelateexponentialstockcultureofP.reticulatum.In addition,lowamountsoftheputativecarboxyYTX(0.220.05pg YTX eq.cell¹) and the putative ketoYTX (0.190.05pg YTX eq.cell¹)weredetectedintheinoculum.

TheYTXcellquotasofP.reticulatumculturedat158C(inf/2 media at different salinities) were not homogenous in the exponential growth phase. The concentrations of YTX in cells cultured inf/2mediaat lowersalinitiesincreasedslightlyfrom 7.220.20pgYTXcell¹ (inoculum)to10.283.45pgYTXcell¹ (salinityof15)andto8.412.09pgYTXcell¹(salinityof20).Onthe otherhand,theconcentrationsofYTX,culturedinthesamemedium athighersalinities,decreasedduringtheexponentialgrowthphase from 7.220.20pg YTXcell¹ (inoculum) to 3.900.32pg YTXcell¹(salinityof25)andto4.020.68pgYTXcell¹(salinity of30).ComparedtotheinoculumdifferencesoftheYTXcellquota werenotstatisticallysignificant(P>0.05)(Fig.5a).

A decreaseof theYTX cellquota in theexponentialgrowth phasewithincreasingsalinitywasalsoobservedin1/10Nmedia.

Fig.2.GrowthcurvesofP.reticulatumculturedatdifferentsalinitiesinf/2-media:(a)158C,f/2media,(b)208C,f/2media,(c)158C,1/10Nmedia,(d)208C,1/10Nmedia,(e) 158C,1/10Pmedia,and(f)208C,1/10Pmedia.Theerrorbarsrepresentstandarderror(n=3).DisplayedgrowthcurvesarebasedoncellcountingbySedgwickRafter’s countingchamber.

K.Ro¨deretal./HarmfulAlgae15(2012)59–70 63

TheconcentrationsofYTXinrespectivecellsweregenerallylower comparedtotheYTXconcentrationincellsofthelateexponential inoculum.Onlycellsculturedatasalinityof15containedahigher YTXcellquota(13.512.01pgYTXcell¹).Withhighersalinitythe YTX concentrations decreased subsequently to 6.980.94pg YTXcell¹(salinityof20),6.431.21pgYTXcell¹(salinityof25) and 4.141.28pg YTXcell¹ (salinity of 30). Compared to the inoculum differences of the YTX cell quotawere not statistically significant(P>0.05)(Fig.5a).

However, the highest YTX concentrations cell¹ in the exponentialgrowthphase were observedin cultures in 1/10 P media. The YTX concentrations tended to decrease by higher salinityalsointhosecultures.TheYTXconcentrationsinthe1/10P cultivated cells were: 24.134.08pgYTXcell¹ (salinity of15), 25.296.63pgYTXcell¹(salinityof20),19.383.15pgYTXcell¹ (salinity of 25) and 17.131.28pg YTXcell¹ (salinity of 30).

Compared to the inoculum differences of YTX cell quota were statisticallysignificantfor1/10PculturedP.reticulatumatasalinityof 15(P>0.01)andasalinityof20(P>0.001).Differencesbetweenthe YTXcellquotaoftheinoculumtosalinitiesof25and30werenot significant(P>0.05)(Fig.5a).

TheinfluenceofthesalinityontheYTXcellquotawhichwas mentionedabovewasnotsignificantinallmedia(P>0.05).

IncontrasttotheYTXconcentrationsduringtheexponential growth phase, a different pattern was observed during the stationaryphase.TheYTXconcentrationinP.reticulatumcultured in f/2 mediain thestationary phase washigher than the YTX concentrationduringtheexponentialgrowthphase,withsignifi- cantdifferencescomparingsalinitiesof20(P<0.05),25(P<0.01), and30(P<0.01).P.reticulatumcellsculturedatsalinitiesof15and 20 had almost the same YTX concentration 14.231.51pg YTXcell¹ (salinity of 15), 15.392.71pg YTXcell¹ (salinity of 20) and at salinities of 25 and 30 with YTX concentrations 11.280.77pg YTXcell¹ (salinity of 25) and 11.531.81pg YTXcell¹(salinityof30).Cellsculturedin1/10Nmediacontained less YTXcell¹ compared to the inoculum of late exponential P.

reticulatum;6.273.71pgYTXcell¹(salinityof15),4.561.33pg YTXcell¹(salinityof20),5.890.42pgYTXcell¹(salinityof25), and5.121.15pgYTXcell¹(salinityof30).Differencesbetween theinoculumandf/2and1/10Nmediawerenotsignificant(P>0.05) (Fig.5b).

Otherwise, 1/10 P mediacultures containedextremely high YTX concentrations: 50.663.27pg YTXcell¹ (salinity of 15), 50.0619.49pg YTXcell¹ (salinity of 20), 56.5319.40pg YTXcell¹ (salinityof 25)and46.423.97pgYTXcell¹ (salinity of30)(Fig.5b).Allofthemweresignificantdifferentcomparedtothe

Fig.4.Chlorophyllaconcentration(pg/cell)inP.reticulatum(158C)culturedatdifferentsalinities:(a)f/2media,(b)1/10Nmedia,and(c)1/10Pmedia;theerrorbars representstandarderror(n=3).Significantresultswerelabelledwithasterisks:*P<0.05,**P<0.01,***P<0.001.

Fig.3.Volume(fl)ofP.reticulatum(158C)culturedatdifferentsalinitiesinthestationaryphase(thelinewithintheboxmarksthemedian,theboundaryoftheboxplots indicatethe25th/75thpercentile,theerrorbarsrepresentthe10th/90thpercentile,the5th/95thoutlinersareshownasdots(n=400)).Significantresultswerelabelledwith asterisks:*P<0.05,**P<0.01,***P<0.001.

YTX cell quota of the inoculum atsalinities of 15 (P>0.01), 20 (P>0.01),25(P>0.001)and30(P>0.01)(Fig.5b).

Cultivationathighertemperature(208C)ledtoanincreaseof YTX concentrations cell¹ when grown at higher salinities.

However,theYTXconcentrationswerelow whencultures were grownatsalinitiesof15and20at208C.Thiswasobviousformost ofthesamplesduringtheexponentialandstationaryphase(Fig.6a andb).Generally,1/10Nculturescontainedthelowestand1/10P culturesthehighestYTXconcentrationcell¹.Significantdiffer- encesofYTXcellquotawereobservedinthestationarygrowth phasecomparingtheinoculumandP.reticulatumcellsculturedata salinityof30inf/2medium(P<0.05),aswellasatsalinitiesof25 and30in1/10Pmedium(P<0.001).Comparingexponentialto stationary growth phase significant differences were observed between1/10Nmediaatasalinityof15(P<0.05),1/10Pmediaat salinitiesof20(P<0.05),25(P<0.001),and30(P<0.05).

The concentrations of both YTX and the YTX analogues (carboxyYTX and ketoYTX) were enhanced under P-limited conditions.Comparedtotheinoculumtheconcentrationsofthe YTX analogues decreased in the f/2 media, except that the concentration of YTX analogues increased in f/2 medium at a salinityof15.DuetothelowerconcentrationofYTXsandpartly loweramountofcellsresultinginanYTXcellquotabelowLOQ,it wasnotpossibletointerpretdetectedamountofYTXsinthecells cultured at 208C. In addition, we detected differences in the pattern of YTXs under differentlimitations independent of the salinity(Fig.7).

3.6. ExtracellularYTXconcentration

The YTX concentrations werecalculated as YTXml¹filtrate.

The results were normalized to the cell concentrations for comparisonofintracellularandextracellularquotaofYTX,which wasfoundinthefiltrateofallcultures.Generally,theconcentra- tionofYTX(normalizedtothecellcounts)inthefiltratewaslower withlowercellconcentrations,especiallyduringtheexponential growth phase. However, during the stationary phase the YTX concentration cell¹ in the filtrate increasedin all samples.In addition,aslightdecreaseoftheYTXcellquotawasobservedwith increasingsalinity.Highertemperatureduringcultivationresulted alsoinapartlydifferentexcretionofYTX(Fig.8andTable2).

4. Discussion

P.reticulatumiscommonindifferentgeographicalareasallover theworldandmanyreportsexistaboutaccumulationofYTXin several molluscan shellfish species (e.g. Aasen et al., 2005;

Ciminiello et al., 1997, 2003; Finch et al., 2005; Krock et al., 2006;MacKenzieetal.,1998;Pazetal.,2004,2006,2007,2008;

Ramstadetal.,2001;Samdaletal.,2004;Satakeetal.,1997,1999, 2006;Suzukietal.,2007;YasumotoandTakizawa,1997).TheP.

reticulatumstrainusedinthisstudywasisolatedintheNorthSea andidentifiedasYTXproducer(Hoppenrath,2004).Uptonowno seriousaccumulationofYTXsinshellfishspecieswasreportedin theGermanBight.However,thepresenceofP.reticulatuminthe Fig.5.ConcentrationofYTX(m/z1141.4>1061.5)inP.reticulatum(158C)in(a)the

exponentialand(b)thestationaryphase;theerrorbarsrepresentstandarderror (n=3). Significant results were labelled with asterisks: *P<0.05, **P<0.01,

***P<0.001.

Fig.6.ConcentrationofYTX(m/z1141.4>1061.5)inP.reticulatum(208C)in(a)the exponentialand(b)thestationaryphase;theerrorbarsrepresentstandarderror (n=3). Significant results were labelled with asterisks: *P<0.05, **P<0.01,

***P<0.001.

K.Ro¨deretal./HarmfulAlgae15(2012)59–70 65

Fig.7.PercentageofYTXanditsanalogues(COOHYTYandketoYTX)inthestationaryphase(158C)atdifferentsalinitiesindependenceofthenutrientlimitation.

Fig.8.ConcentrationofYTX(m/z1141.4>1061.5)intheculturefiltrate(158Cand208C)intheexponentialandthestationaryphase.Concentrationsinthefiltratewere relatedtothecells/ml(asanindicator).

NorthSeaplanktonnecessitatesinvestigationswithfocusonthe potentialriskofbloomsofthisdinoflagellate.

P.reticulatum,isolatedfromtheNorth Seadisplaysagrowth optimumat158Cinf/2medium(Fig.2a).Incontrast,athigher temperature(inthis case 208C)growthdecreasedconsiderably (Fig.2b).Hence,aP.reticulatumbloomwithhighcelldensitiesis morelikelytooccurduringthespringorearlysummermonths.Up tonow only low amounts of P.reticulatum were foundin the Germanbight.

DinoflagellatesarecapableofstoringNandPinintracellular poolsforuseduringtimesofdeficiency(Cembellaetal.,1984;

Dortchetal.,1984).Thelowchlorophyllaconcentration(Fig.4), whichwasdetectableinallN-deficientcultures, indicatedthat thosecellswereindeedNdeficient(Boyeretal.,1987).Beside temperatureandnutrient-availabilitysalinity is a factorinflu- encingthegrowthofP.reticulatum,whichisknowntogrowina widerangeofsalinities(Guerrinietal.,2007;Koikeetal.,2006).In our experiments the growth of P. reticulatum decreased with lower salinity and is close to zero at salinities underneath a salinityof15.

Eutrophicationadherentwithalterationofthecompositionof nutrientscould enhancethe chances for accelerated growthof harmfulalgalbloom(HAB)species(Andersonetal.,2002).Inthis context,risinganthropogeniceffectstogetherwithsimultaneous climatechange have beenreported worldwide and also in the NorthSea(Edwardsetal.,2006;Wiltshireetal.,2008).NandPare important nutrientsfor the growthof microalgae, where upon microalgaeareknowntorequireNinahigherquantitycompared toP. N is mostly limiting factor withregard to phytoplankton growthin marineand estuarywaters albeit P can also be the limiting factor under large N input (Anderson et al., 2002).

Therefore,changesofnutrientcompositionortemperatureofthe NorthSeaduringtheseasonsleadtocharacteristicdifferencesin growth of the phytoplankton species within HABs. However, increasingtemperatureofthewateroftheNorthSeacausedby climatechangewouldnotleadtoahigherriskforabloomofP.

reticulatum.IntheNorthSeaphosphorous(P)seemstobethefirst limiting nutrient in spring and nitrogen (N) in summertime (Peetersetal.,1991).Ahigherinputofnutrientsduringthespring causedbyeutrophicationcouldresultinincreasedphytoplankton growth and a possible P-limitation at the end of the bloom

stopping cell division can cause very high YTX concentrations cell¹.

4.1. YTXconcentrationinP.reticulatumcells

FromourexperimentsitisevidentthatforP.reticulatumaclear correlationbetweenthetotalconcentrationofYTXsandnutrient limitationcanbeobserved(Fig.5).WhereasthecellquotaofYTX generallyincreasedinthefollowingorder:1/10N<f/2<1/10P media.Unfavourablegrowthconditionscaninducechangesofcell size,morphologyandearlierentranceintothestationarygrowth phase.Thiseffectcancorrelatewiththeformationoftemporary restingstages(hypnocygotes)andsuchcellswerefoundinthef/2 media (at lower salinities) and in 1/10 P media, whereby the occurrencewaspartlycorrelatedwithanincreasedproductionof YTXs. Adeformationofcells wasnoticeableat highertempera- tures,especiallyinthef/2mediaathighersalinities.

Recently,itwasreportedthattheYTXconcentrationincreased withincreasingsalinity(Guerrinietal.,2007).Afternormalization of theYTX cellquota tothecell size,this YTX profilewas not observedduringtheexponentialgrowthphasebutitwasfound duringthestationaryphase(Fig.9aandb).Generally,cellquotasof YTXsculturedat208Cwerelowercomparedto158C.At208Cthe cellquotasofYTXsincreasedwithincreasingsalinityduringthe stationary phase, whereby theinfluence oflimitations was not obvious(Fig.9candd).Possiblyhighertemperaturesinhibitedthe toxinproductionwhichisnotinagreementwithobservationsby Guerrinietal.(2007)andPazetal.(2006).Buttheconditionsofthe cultureswerenotoptimalat208Candtherefore,wesupposethat YTX production is associated to a functioning metabolism.

Especially in 1/10P medium cells at 158Ca longer cultivation timecausedhigherYTXcellquota,whichwasalsoobservedwhen thesizewasincluded.BecauseP-limitationisknowntostopthe celldivisioninotherdinoflagellateswithoutdiebackofthecells, anaccumulationofYTXunderongoingmetabolismislikely,since it is established for other species that P-limitation can cause accumulationofseveralfattyacidsandthoseareknowntohavea similarprecursoraspolyketides.

Specificenvironmentalconditionsaschangesofthesalinityor nutrient limitation can lead to an altered toxin production or altered toxinpatterns indinoflagellates (Andersonet al., 1990;

Table2

PercentageofYTX(%)incellsofP.reticulatumandinthemediafiltrateintheexponentialandthestationaryphase.

Temperature(8C) Salinity Percentage(%)ofYTXcellquotainthefiltrateandYTXcellquotaincellsofP.reticulatum

C 1/10N 1/10P

Filtrate Cells Filtrate Cells Filtrate Cells

15 Exponentialphase

15 8 92

20 7 93

25 4 96

30 7 93 4 96 3 97

Stationaryphase

15 25 75

20 24 76

25 19 81

30 13 87 43 57 4 96

20 Exponentialphase

15 10 90

20 10 90

25 2 98

30 3 97 3 97 3 97

Stationaryphase

15 24 76

20 33 67

25 20 80

30 7 93 12 88 16 84

K.Ro¨deretal./HarmfulAlgae15(2012)59–70 67

Beanietal.,2000;Boyeretal.,1987;Grzebyketal.,2003;Maier Brownetal.,2006;Roederetal.,2010).Hence,theformationof Brevetoxins(PbTx)wasinvestigatedundervarioussalinitiesand thedata revealedthattheinfluenceofsuchparameters isvery complexanditwasevidentthatPbTxconcentrationswerealways higherin the stationary phase (Maier Brownet al., 2006). The productionof paralytic shellfish poisoning(PSP) toxins (e.g.by Alexandrium spp.) is higher at low phosphorus concentrations (Anderson et al., 1990; Beani et al., 2000; Boyer et al., 1987;

Grzebyketal.,2003)andlowsalinityvalues(HwangandLu,2000) whereasN-limitationcancauseadecreaseoftoxinconcentration (Boyeretal.,1987).Thesameeffectontoxicitycausedbylower salinity wasreported in a work about toxinproduction of the raphidophycean flagellate Heterosigma akashiwo (Haque and Onoue,2002).Thoseexamplesarein largepartconsistentwith ourexperimentand illustratetheneed fortheelucidation of a potentialriskfortoxicdinoflagellateeventsintheNorthSeaand othermarineenvironments.

It is still unknown why polyether toxins are produced by severaldinoflagellatespeciesandthequestionremainswhyin onestrainnutrientlimitationleadstoanincreasedtoxinquota percellbutnotintheother.Hence,otherstrainsfromdifferent locations should be studied concerning the influence of nutrientson the toxin formation. In addition more investiga- tions are needed to discover the dynamics and mechanisms concerning toxin formation in marine dinoflagellates during HABs.

4.2. ExtracellularYTXconcentration

InseveralstudiesitwasfoundthatYTXwasreleasedintothe mediumandseveralreasonshavebeensuggested(Mitrovicetal., 2005;Pazetal.,2004,2006,2007). DatapublishedbyPazetal.

(2004,2006,2007)showedthatthetotaltoxinamountofYTXin someculturefiltratesreachesaconsiderableamountupto38%of total YTX concentrations. During our experiments YTX was

detected in the filtrate in low concentrations during the exponential growth period. It was evident, that the measured YTXfoundintheculturemediaincreasedsignificantlyduringthe stationary phase. Obviously, the release of YTX in the media dependsonthesalinityandonnutrientavailability.Guerrinietal.

(2007)foundthatthereleaseofYTXintotheculturemediaseems tobehigherunderN-limitation,whichwasobservableinour1/10 Nculturesat158C.

WesupposethatthedetectedYTXinthefiltrateiscausedby leakagefromdisintegratedcells.Thishypothesisissupportedby increasingYTXconcentrationsinthestationaryphaseandbythe higherconcentrations incultures withunfavourableconditions.

The percentage of intracellular to extracellular YTX in 1/10 P culturesdidnotchangeduringculturing.Therefore,itisevident thatless YTXwasreleasedintothemediumin1/10Pcultures.

Furthermore,1/10Pcellsculturedat158Ccontainedahighcell quotaofYTXinthelatestationaryphasecausedbytheputative entrance into a ‘‘temporary resting stage’’. In addition the assumption that lower YTX concentrations in the filtrates are causedbylessdisintegratedcells,supportsthetheorythatresting stageswereformedandhighYTXcellquotascouldbeaproductof ongoingmetabolisminthoseP-limitedcellsofP.reticulatum(see Figs.5and9andTable2).

5. Conclusion

ThestrainofP.reticulatumunderinvestigationduringthisstudy showedagoodgrowthatsalinitiesintherangeofsalinityof20–30, with strong influence of water temperature and of nutrient limitationontheformationofYTXs.Generally,N-limitedcultures displayedthelowestandP-limitedculturesthehighestYTXcell quota. Lower salinities caused a higher volume of the cell accompaniedbyanincreaseofYTXconcentration.Summarizing itcanbestatedthatthehigherriskfortoxicP.reticulatumblooms intheNorthSeaexistsduringabloominspringwithP-limitation attheendofthebloom.

Fig.9.RatioofYTXconcentrationtovolume,dependingonnutrientlimitationandsalinity,forP.reticulatumculturedat158Cin:(a)theexponentialgrowthphaseand(b)the stationaryphaseandat208Cin:(c)theexponentialgrowthphaseand(d)thestationaryphase.