Pseudo-nitzschia copepods by calanoid seriata Induction acid production of domoic in the toxic diatom Aquatic Toxicology

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Aquatic Toxicology

jou rn al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

Induction of domoic acid production in the toxic diatom Pseudo-nitzschia seriata by calanoid copepods

Anna Tammilehto

, Torkel Gissel Nielsen

, Bernd Krock

, Eva Friis Møller

, Nina Lundholm

aNaturalHistoryMuseumofDenmark,UniversityofCopenhagen,Sølvgade83S,DK-1307CopenhagenK,Denmark

bNationalInstituteofAquaticResources,DTUAqua,SectionforOceanecologyandClimate,TechnicalUniversityofDenmark,DTU,Kavalergården6, DK-2920Charlottenlund,Denmark

cGreenlandClimateResearchCentre,GreenlandInstituteofNaturalResources,Nuuk,Greenland

dAlfredWegenerInstitut-HelmholtzZentrumfürPolar-undMeeresforschung,ÖkologischeChemie,AmHandelshafen12,27570Bremerhaven,Germany

eDepartmentofBioscience,Roskilde,AarhusUniversity,Frederiksborgvej399,POBox358,4000Roskilde,Denmark

a r t i c l e i n f o

Articlehistory:

Received16August2014 Receivedinrevisedform 26November2014 Accepted28November2014 Availableonline5December2014

Keywords:

Domoicacid Pseudo-nitzschiaseriata Calanus

Diatom Toxinproduction Grazing

a b s t r a c t

ThetoxicdiatomPseudo-nitzschiaseriatawasexposeddirectlyandindirectly(separatedbyamembrane) tocopepods,CalanushyperboreusandC.finmarchicus,toevaluatetheeffectsofthecopepodsondomoic acidproductionandchainformationinP.seriata.ThetoxicityofP.seriataincreasedinthepresenceof thecopepods.Thisresponsewaschemicallymediatedwithoutphysicalcontactbetweentheorganisms suggestingthatitwasinducedbypotentialwaterbornecuesfromthecopepodsorchangesinwater chemistry.DomoicacidproductionmayberelatedtodefenseagainstgrazinginP.seriataalthoughitwas notshowninthepresentstudy.Toevaluateiftheinductionofdomoicacidproductionwasmediated bythechemicalcuesfromdamagedP.seriatacells,liveP.seriatacellswereexposedtoaP.seriatacell homogenate,butnoeffectwasobserved.ChainformationinP.seriatawasaffectedonlywhenindirect contactwiththecopepods.Thisstudysuggeststhatthepresenceofzooplanktonmaybeoneofthefactors affectingthetoxicityofPseudo-nitzschiabloomsinthefield.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Manyphytoplanktonspeciesproducetoxicsecondarymetabo- lites, whose functions are still unknown in most groups of microalgae.Thesecondarymetabolitedomoicacid(DA)isapotent neurotoxin produced by several species in the diatom genus Pseudo-nitzschiaPeragallo(reviewedinLelongetal.,2012;Trainer etal.,2012).DAmayaccumulateinorganisms grazingontoxic Pseudo-nitzschiaspeciesandafterwardsbetransferredtohigher trophiclevels(e.g.Scholinetal.,2000;McHuronetal.,2013)where itmayleadtoamnesicshellfishpoisoning(ASP).Thesymptomsof ASPincludee.g.nausea,diarrhea,short-termmemoryloss,paraly- sisandinextremecases,death.

ThefirstrecordedASPincident,whichaffectedmorethan100 humanstookplaceinPrinceEdwardIsland,Canada,in1987,and

∗Correspondingauthor.Tel.:+4535330854.

E-mailaddresses:atammilehto@snm.ku.dk(A.Tammilehto),tgin@aqua.dtu.dk (T.G. Nielsen), Bernd.Krock@awi.de (B. Krock), efm@bios.au.dk (E.F. Møller), nlundholm@snm.ku.dk(N.Lundholm).

itwascausedbyconsumptionofDA-contaminatedbluemussels (Batesetal.,1989).TheeffectsofDAonseabirds,sealionsandfish havebeenstudied(Fritzetal.,1992;Scholinetal.,2000;Lefebvre etal., 2012), whereas there isnot muchknowledge ofpossible effectsofDAon,e.g.planktonicgrazerssuchascopepodsthatfeed onDA-producingPseudo-nitzschia.Shawetal.(1997)studiedthe effectsofdissolvedDAonthecopepodTigriopuscalifornicusgrazing onthenon-toxicdiatomThalassiosirapseudonana.Reducedgraz- ingduetoDAwasnotobserved,butDAwastoxictothecopepods andcausedmortalityatrelativelylowconcentrations(Shawetal., 1997).InmostgrazingstudiesontoxicPseudo-nitzschiathecope- pods haveaccumulatedDA,but noapparentadverse effects on grazers,e.g.reducedgrazing,weredetected(Lincolnetal.,2001;

Tester et al., 2001; Maneiroet al., 2005; Leandro et al., 2010).

Hence,thepotentialroleofDAasagrazingdeterrenthasnotbeen supported.However,Bargu etal.(2003)foundkrilltofeeddis- continuouslyontoxicPseudo-nitzschiamultiseries,andthesame observationwasmadebyTammilehtoetal. (2012)whenfeed- ingcopepods,CalanusfinmarchicusandC.hyperboreus,withtoxic P.seriata.Bothstudiessuggestedthatthegrazerssufferedfrom physiological incapacitation. The indication that DA mayaffect http://dx.doi.org/10.1016/j.aquatox.2014.11.026

0166-445X/©2014ElsevierB.V.Allrightsreserved.

grazingbycopepodshasimplicationsforthephytoplankton,and maygivetoxicspeciesanadvantageovernon-toxicones(Huntley etal.,1986).ToinvestigatewhetherDAdeterscopepodgrazingon thetoxicPseudo-nitzschiaspecies,andwhatthepotentialmodeof actionis,furtherstudiesarestillneeded.

Thepresence of grazersmayaffectphytoplankton morphol- ogy,suchasthelengthofchainsandcolonysize(HessenandVan Donk,1993;JakobsenandTang,2002;Jiangetal.,2010;Selander et al.,2011; Bergkvist et al., 2012)and physiology (Janget al., 2003).Theseresponsesmayreducetheriskofbeinggrazed,i.e.

thedefensemechanismisinducible.Selanderetal.(2006)found thatwaterbornecuesfromthecopepodAcartiatonsainducedpara- lyticshellfishpoisoningtoxin(PST)productioninthedinoflagellate Alexandriumminutum,whichledtoreducedgrazingofA.tonsaon A.minutum.Ithasalsobeenshownthatthechemicalandmorpho- logicalresponsesmaytakeplacesimultaneously(Selanderetal., 2012).Such inducible defensesmay bebeneficial in conditions wheretheriskofbeinggrazedisunpredictable,ase.g.inmarine environments,giventhatdefensivetraitsarecostly(Karbanand Baldwin,1997).Inadditiontothepotentialcuesreleasedbythe grazers,mechanicaldamage ofthealgaemaysignaltherisk of grazing,especiallywhenfeedingis“sloppy”,evenifthemessage maynotalwaysbereliablesincealgaealsolyse,e.g.duetoattack byviruses(VanDonketal.,2011).Theeffectofmechanicaldam- ageonthepotentialinducibledefensesinphytoplanktonhasbeen rarelystudied(reviewedinVanDonketal.,2011;seeLampertetal., 1994;Lürling,1998).Itispossiblethatthechemicalsreleasedby thegrazersandthedamagedcellsinteracttoproducethepotential signalthateventuallyinducesthedefensereactioninthephyto- plankton(VanDonketal.,2011).Indicationsthatthepresenceof thecopepodsmayenhanceDAproductioninP.seriatawerefound byTammilehtoetal.(2012),wheretheDAcellquotainP.seriata increasedafter12hinthepresenceofC.hyperboreus.Inthesame studynoincreaseinDAwasfoundinthepresenceofC.glacialisor C.finmarchicus.

Inthepresentstudyweexploredthefollowingquestions:

1)DoesthepresenceofthetwocopepodspeciesCalanushyper- boreusandC.finmarchicusinducephysiologicalormorphological responses,i.e.enhancedDAproductionorchangesinchainfor- mationinPseudo-nitzschiaseriata?

2)Arethepotentialcuesinvolvedintheinducedresponsechemical orisphysicalcontactrequiredtoelicitthem?

3)CanthepotentialtriggersbereleasedfromdamagedP.seriata cellsonly,withoutthepresenceofthecopepods?

2. Materialsandmethods

2.1. Studyorganisms

The copepods Calanus finmarchicus and C. hyperboreus were collectedfromDisko Bay(69^◦14N,53^◦23W) ata 300-m-deep monitoringstation(Madsenetal.,2001;Hansenetal.,2012)during thespringbloominApril2012.DiskoBayislocatedonthewestern coastofGreenland.Thecopepodsweresampledfromtheupper 100musingaWP-2net(200␮m),transportedtothelaboratory inthermoboxes,pickedindividuallyusingastereomicroscopein ice-chilledpetridishesandkeptinthedarkattheinsitutemper- atureforamaximumof1weekin0.45-␮mfilteredseawaterand fedwithThalassiosirasp.priortotheexperiments.Thereafter,the copepodswerestarvedfor24h.TheDA-producingP.seriatastrain P5G3wasisolatedasdescribedinTammilehtoetal.(2012)and grownin50%silica-reducedL1-medium(GuillardandHargraves, 1993)at12:12light:darkcycle.ThelengthandwidthofP.seriata cellswere64.8±3.9(SD)␮mand5.5±0.8(SD)␮m,respectively.

Fig.1.Schematicdiagramoftheincubatorwithtwoflasksconnectedviatwo2-␮m polycarbonatemembranes(diameter4.5cm).FlaskA:Pseudo-nitzschiaseriataplus copepods(exceptinthecontrol);flaskB:P.seriataonly.

2.2. Inductionexperimentswithcopepods

Theinductionexperimentswereconductedinincubatorscon- sistingoftwopolystyrenetissuecultureflasks(Sarstedt)(volume 710mlflask⁻¹)thatwereconnectedthroughtwoholes(4.5cmin diameter)coveredby2.0-␮mpolycarbonatemembranestoallow waterexchangebetweenflasks(AandB)buttopreventexchange ofcopepodsandalgalcellsbetweentheflasks(modifiedfromTang, 2003;Fig.1).Non-toxicaquariumsilicone(DanaLimaquariumsil- icone579)wasusedtogluethefiltersontothepolystyreneflasks andtoconnecttheflasks.Thepermeabilityofthe2-␮mpolycarbo- natemembranewastestedvisuallybyobservingthetransportof fooddyefromoneflasktotheother.Tang(2003)usedsimilarmem- branesandreportedreachingthediffusionequilibriumof∼50%and 90%after3and5days,respectively.Theincubators(flasksAand B)bothcontainedP.seriataculture.ThecopepodsandP.seriata cellswereaddedtoflaskAseparatedbythe2.0-␮mpolycarbonate membranefromtheP.seriatacellsinflaskB(Fig.1;Table1).

Theseawaterusedfortheexperimentswascollectedatleast 1weekbeforestartingtheexperimentstominimizethepotential cuesoriginatingfromthefield.Thesalinity35waterwasfiltered (0.45-␮mporesize)andstoredat4±2^◦C.Bothflasks(AandB) oftheincubatorswereinoculatedwithP.seriataataconcentra- tionof1000cellsml⁻¹,correspondingtoanaverageof162␮gCl⁻¹ (forcalculationsseeTammilehtoetal.,2012).Thiscellconcentra- tionwasachievedbypoolingtogetherfourP.seriatabatchcultures (forgrowthconditionsseeSection2.1,thebatchcultureshadbeen grownfor9days)inearlystationaryphase(16,000cellsml⁻¹ in 1.1l)anddilutingitwith16.9lof0.45-␮mfilteredseawater(=a pooledculture)onthedaywhentheexperimentwasstarted.To measuretheDAconcentrationofP.seriatabeforetheexperiments, triplicatesubsamples(100ml)ofP.seriataweretakenfromthe

Table1

Experimentalsetupoftheincubationexperimentswithcopepodsandhomogenized Pseudo-nitzschiaseriatacells,showingthecontentsofflaskA,containingcopepods orhomogenizedP.seriata(exceptinthecontrol)pluslivealgae,andB,containing onlylivealgae.

Experiment Treatment Flask

A B

Copepods Control Algae Algae

C.hyperboreus Copepodsand algae

Algae C.finmarchicus Copepodsand

algae

Algae

Damagedcells Control Algae Algae

Cellhomogenate addition

Homogenized algaeandalgae

Algae

pooledcultureusinga100-mlsyringeandfilteredontoGF/Ffil- tersusinggentlevacuum.Thesampleswerestoredat−20^◦Cuntil analysis.Afewhoursbeforetheexperiments,activelyswimming copepodswere addedtotheflasks filledwith0.45-␮mfiltered seawaterandkeptat4^◦C.

TheexperimentswerestartedbyaddingC.hyperboreusorC.

finmarchicustoflaskA,twooreightcopepods, respectively,per flask.Thecontrolincubator(flaskA)containednocopepods.Forcell counts,a3-mlsamplewastakenfromeachflaskandfixedwith1%

(finalconcentration)acidicLugol’ssolution.Thegrazingtreatments andthecontrolswererunsimultaneouslyinquadruplicateandin triplicate,respectively.Theincubatorsweremountedonaplankton wheelrotatingat1.3rpm.Theexperimentwasrunfor8daysat 4±2^◦Cusinga 12:12light:darkcycleundera lightintensityof 100␮molphotonsm⁻²s⁻¹.

CellandDAconcentrations werequantifiedagainondays2, 5 and 8. Both flasks (A and B) were sampled simultaneously, andtheflaskswererefilledfilteredseawater.Onday2,thecell concentrationsampleswerefixedwith10%glutaraldehyde(final concentration)topreservethechains.AcidicLugol’ssolution(1%

finalconcentration)wasusedforfixationontheotherdaysandthe sampleswerecountedwithinthreemonthsaftertheexperiments.

Onday3,19mlofP.seriatabatchculture(350,000cells,culture grownfor12days)wasaddeddirectlytotheflaskswithcopepods (flaskA)toensurethatthecopepodshadenoughP.seriatacellsfor grazing.ThesamevolumeofP.seriatawasaddedtothecontrolflask Atoensuresimilartreatmentofthegrazingandthecontrolflasks.

Thedilutionsduetosamplingandaddingcellsonday3werecon- sideredincalculatingthegrowthandingestionrates.Thegrowth ratewascalculatedaccordingtoFrost(1972)andtheingestionrate accordingtoHarrisetal.(2005)(forformulasseeTammilehtoetal., 2012).

Attheendoftheexperiment(day8),samplesweretakenfor cellcounts(4ml),DA(200ml)andchlorophyll-aconcentrationof P.seriatacells.InadditiontomeasuringtheparticulateDAconcen- trationofthecellscollectedonGF/Ffilters,a3-mlsampleofthe filtratewastakentomeasuretheconcentrationofdissolvedDA.

Thefiltratesampleswerestoredat−20^◦Cpriortoanalysis.TheDA cellquotawascalculatedbydividingtheamountofDAcollectedon theGF/Ffilter(P.seriatacells)bythecellnumber.Inaddition,the ratiobetweendissolvedDAandcellnumberswascalculated.To measurechlorophyll-aconcentration,a100-mlsamplefromeach flaskwasfilteredontoGF/Ffiltersusinggentlevacuum.Thefilters werethenextractedin5mlof96%ethanolfor24h(Jespersenand Christoffersen,1987)and measuredfluorometricallybeforeand afterHCladdition,usingaTurnerfluorometer(TD-700)calibrated againstachlorophyll-astandard.Thenumberofcopepodsperflask wasrecordedattheend oftheexperiment and theircondition checkedvisually.

2.3. InductionexperimentwithdamagedP.seriatacells

FlaskAcontainedliveandhomogenizedP.seriatacellsandflask BonlyliveP.seriatacells(Table1).TheP.seriatahomogenatewas preparedbyfilteringaknownvolumeofcultureontoa2.0-␮m polycarbonatefilterusinggentlevacuum,afterwhichthecellson thefilterwerehomogenizedusinganautoclavedmortar.Inthe controls, both flasks containedonly live P.seriata cells. Allthe incubatorswerefilledwithaged0.2-␮mfilteredseawaterwitha salinityof30andP.seriataculture(15mlof50,000cellsml⁻¹)was addedtoeachflasktoreachafinalconcentrationof1000cellsml⁻¹. ThehomogenateofP.seriataculture,correspondingtotheconcen- trationof1000cellsml⁻¹ in710mlwasaddedintoflaskA.The filterwasrinsedwith a knownvolumeof 0.2-␮mfilteredsea- watertocollectthehomogenate.Toensurethatthecells were crushed,thehomogenatewascheckedvisuallyusinganinverted

microscope(OlympusCKX31)at200×magnificationbeforeuse.

ThehomogenatewasaddedtoflaskAdirectlyafterhomogeniza- tionandcarewastakentokeepthehomogenatecoolatalltimes.

Afterfillingtheincubatorswith0.2-␮mfilteredseawaterandtheP.

seriataculture,1.65mlofthehomogenatewasaddedtothetreat- mentflaskAandthesamevolumeof0.2-␮mfilteredseawater intothecontrolflaskA.Boththehomogenateandcontroltreat- mentswereruninquadruplicate.Theincubationexperimentwas run ona plankton wheel (1.1rpm)for 8 days at 3±1^◦C using 12:12light:darkcycleunder alight intensityof100␮molpho- tonsm⁻²s⁻¹.

Samples for cell and DA concentrations were collected and treatedasdescribedinSection2.2.Thehomogenateofdamaged P.seriatacells wasprepared andadded tothetreatmentflasks dailyduringtheexperiment (days0–7)toprovidefreshpoten- tialcues,andatthesametimeanequalvolumeof0.2-␮mfiltered seawaterwasaddedtothecontrolflasks.TheP.seriatacultures usedforinoculatingtheexperimentalincubatorsandformaking cellhomogenateweregrownat4^◦Cusing12:12light:darkcycle underalightintensityof100␮molphotonsm⁻²s⁻¹andwerehar- vestedatlateexponentialtolatestationaryphase(culturesgrown for16–23days).

Subsamples(50ml)formeasuringdissolvedinorganicnitrogen (ammonium,nitrateandnitrite),dissolvedinorganicphosphorus (phosphate)and dissolvedsilica atthebeginningoftheexperi- mentweretakenfromtwoextraflaskssimilartothecontrolflasks andthesameflaskswereusedformeasuringpH.Attheendof theexperiment(day8)pHwasmeasuredfromalltheBflasks.pH wasmeasuredusinganalyticalpH-meter(Radiometer).Thenutri- entsamplesattheendoftheexperiment(day8)werecollected fromonecontrolflaskBandonetreatmentflaskB.Thesamplefor dissolvedsilicaanalysiswaspassedthrougha5.0-␮mpolycarbo- natefilterandthosefornitrogenandphosphorusanalysesthrough GF/Cfilters.Thenutrientfiltrateswerefrozenat−20^◦Cimmedi- atelyaftersamplingandlateranalyzedattheAarhusUniversity (Denmark)onaflowinjectionautoanalyzer,followingHansenand Koroleff(1999).DAanalysesfortheincubationexperimentswere conductedasdescribedinTammilehtoetal.(2012).Thedetection limitforDAwas10ngsample⁻¹.

2.4. Microscopy

Pseudo-nitzschiacellconcentrationsandchainlength(number ofcellsinperchain)wereenumeratedinaSedgewick-Raftercham- berusinganinvertedmicroscope(NikonTMSandOlympusCKX31) at100×magnification.Aminimumof400cellswascountedineach sampleandifasamplecontainedfewerthanthis,atleasthalfof thecellsinthecountingchamberwerecounted.Thelengthand widthofthecellsweremeasuredusingOlympusBX53microscope at400×magnification.

2.5. Statisticalanalyses

Changesover timewithin eachtreatmentwere testedusing repeatedmeasuresANOVA(RMANOVA),andtheFriedmantestwas usedasanon-parametricalternative.Pairedsamplesweretested usingthepairedt-testorthenon-parametricMann–WhitneyU- test.Thedifferencesbetweenmeansweretestedusingthet-test (twosamples)andone-wayANOVA(threeormoresamples)or thenon-parametricMann–WhitneyU-testandtheKruskall–Wallis test.NormaldistributionwastestedusingtheShapiro–Wilktest andhomogeneityofvariancesusingLevene’stest.Ifthedataset violatedtheassumptionofnormaldistributionorhomogeneityof variances,itwaslntransformed.Iftheassumptionsofnormality orvariancehomogeneitywerenotfulfilled,evenafterthetrans- formation,anon-parametrictestwasused.TheX²-testwasused

tocomparetheproportionsofcellsinsinglecellsandchains(three categories:singlecells,2-cellchainsand3-ormorecellchains) usingtheproportionsinthecontrolastheexpectedfrequency.A commonsignificancelevelof0.05wasapplied.

3. Results

TheDAcellquota(toxicity)ofP.seriataincreasedduringthe experimentwithbothC.hyperboreusandC.finmarchicus,incon- trasttothecontrolwheretheDAcellquotaofP.seriataremained stable(Fig.2).ThetoxicityofP.seriataincreasedinbothsidesofthe incubators,i.e.bothwhencellswereindirectphysicalcontactwith thecopepods(flaskA)andalsowhenthecopepodsandthealgae werephysicallyseparatedintodifferentcompartmentsandonly waterexchangebetweenthecompartmentswasallowedthrough a2-␮mpolycarbonatemembrane(flaskB).Onday0,theDAcell quotaofP.seriatawasbelowthelimitofdetection.Thegreatest toxicityincreasewasrecordedwhenP.seriatawasindirectphys- icalcontactwithC.hyperboreus,risingfromundetectedto13.1pg DAcell⁻¹(day8mean)inflaskA(Fig.2A).ThetoxicityofP.seriatain flaskAwithC.hyperboreusincreasedsignificantlyduringtheexper- iment(RMANOVA,F_2,6=58.4,p<0.01),andpost-hocanalysiswith BonferroniadjustmentshowedthetoxicityofP.seriatatobesignif- icantlyhigherondays5and8thanonday2(p<0.05andp<0.01, respectively)(Fig.2A).InflaskBwithcuesfromC.hyperboreus,a

Fig.2. Domoicacid(DA)cellquota(pgDAcell⁻¹,mean±SD)ofPseudo-nitzschia seriataindifferenttreatmentsondays0,2,5and8.(A)FlaskAwithcopepods (exceptcontrol);(B)flaskBcontainingonlyalgaewithcuesfromthecopepods (exceptcontrol).Onday0,DAcellquotameasuredfromthepooledculture.Inthe control,n=3andinthecopepodtreatments,n=4.

Table2

Domoicacid(DA)cellquotaofPseudo-nitzschiaseriata(pgDAcell⁻¹)anddissolved DAconcentrationintheculturefiltrate(ngDAml⁻¹)onday8(mean±SD)inflaskA, containingcopepods(exceptcontrol)andflaskB,withcuesfromcopepods(except control).

Cellular(pgDAcell⁻¹) Filtrate^a(ngDAml⁻¹)

A Control 0.10±0.04 bd

C.hyperboreus 13.1±1.98 17.0±5.55

C.finmarchicus 4.15±1.52 bd

B Control^b 0.01±0.00 bd

C.hyperboreus 0.51±0.25 bd

C.finmarchicus 2.62±0.89 bd

abd=measurementbelowthelimitofdetection.

bOnemeasurementofcellularDAbelowthelimitofdetection.

significantincreaseintoxicity(RMANOVA,F_2,6=5.6,p<0.05)was alsoseen,butpost-hocanalysiswithBonferroniadjustmentcould notrevealsignificantpairwisedifferences(Fig.2B).Thetoxicityof P.seriataalsochangedsignificantlywheninphysicalcontactwith C.finmarchicus(RMANOVA,F2,6=30.3,p<0.01),reachingthehigh- estlevelonday5(7.3pgDAcell⁻¹,mean)(Fig.2A).Thepost-hoc analysiswithBonferroniadjustmentshowedthatthetoxicitywas significantlyhigheronday5thanonday2(p<0.05).InflaskBwith cuesfromC.finmarchicus,thetoxicityofP.seriatachangedsignif- icantly(RMANOVA,F_2,6=36.3,p<0.001)andwashigherthanin flaskBwithcuesfromC.hyperboreus,butlowerthaninflaskA withC.finmarchicus(Fig.2).Thepost-hocanalysiswithBonferroni adjustmentrevealedthatthetoxicityofP.seriatainflaskBwith cuesfromC.finmarchicuswassignificantlyhigherondays5and8 thanonday2(p<0.01andp<0.05,respectively).Inthecontrols, thetoxicityofP.seriatadidnotchangeinflaskA(Friedman’stest, X²₃=4.2,p=0.24)norinflaskB(Friedman’stest,X₃²=3.4,p=0.33) duringtheexperiment(Fig.2).

TheDAcellquotaandtheconcentrationofdissolvedDAinthe mediumonday8werecomparedbetweentreatments(Table2).

CellularDAwasdetectedinallthetreatments,althoughsubstan- tially less in thecontrols compared to the grazing treatments, especiallyinflaskA(Table2).DissolvedDAwasobservedonlywith C.hyperboreusinflaskA,whereasintheotherflask,themeasure- mentswerebelowthedetectionlimit(bd)(Table2).TheDAcell quotawassignificantlyhigherintheflaskswiththecopepodsthan inthecontrolandalsohigherwithC.hyperboreusincomparison to C. finmarchicus (one-way ANOVA, F2,8=68.6, p<0.001, post- hocanalyseswithBonferroniadjustment:control-C.hyperboreus:

p<0.001,control-C.finmarchicus:p=0.025,C.hyperboreus–C.fin- marchicus:p<0.001)(Fig.2;Table2).InflaskBonday8,theDA cellquota(lntransformed)wassignificantlyhigherwithcuesfrom C. finmarchicuscompared tothe control and withcuesfromC.

hyperboreus;C.hyperboreuswasalsosignificantlyhigherthanthe control(one-way ANOVA,F2,8=171.2,p<0.001,post-hocanaly- seswithBonferroniadjustment:control-C.finmarchicus:p<0.001, C.hyperboreus–C. finmarchicus: p=0.001,control-C.hyperboreus:

p<0.001)(Fig.2;Table2).TheconcentrationofdissolvedDA(exter- nalDAinthemedium)onday8wassignificantlyhigherinflaskA withC.hyperboreusthaninthecontrolflaskA(bd)(Kruskall–Wallis test,X²₂=8.9,p=0.012,post-hocpairwisecomparison,p=0.009), showingthattheP.seriatacellsalsoexcretedorleakedthetoxin (Table2).TheconcentrationofdissolvedDAwithC.finmarchicus wasnondetectableinflaskA,however,andit didnotdiffersig- nificantlyfromC.hyperboreusorthecontrol(non-censoredvalues wereused).InflaskB,theconcentrationsofdissolvedDAwithcues fromC.hyperboreusandC.finmarchicusandinthecontrolwereall belowthelimitofdetection(Table2).TheamountofdissolvedDA perP.seriatacellinflaskAwithC.hyperboreuswas21.3±19.9pg DAcell⁻¹.

Fig.3.CellconcentrationsofPseudo-nitzschiaseriata(cellsml⁻¹,mean±SD)ondays0,2,5and8in(A)thecontrol(flasksAandB,containingnocopepods,n=3);(B)flaskA, containingCalanushyperboreus(n=4);(C)flaskA,containingC.finmarchicus(n=4),incomparisonwithflaskBthatcontainednocopepodsbutonlyalgae.Thearrowsshow whenextraculturewasaddedtotheflaskAonday3.

P.seriata grewin allexperimentalincubators(Fig.3)and at thestartoftheexperimentcelldensitieswerethesameinflask A (Kruskall–Wallistest, X₃²=0.33, p=0.85) for each treatment.

The algae reachedthe highest cell density in thecontrol flask A,wheretherewerenograzersandextraculturewasaddedon day3.InflaskA,wherethealgaewereindirectcontactwiththe copepods,thecelldensitiesdecreasedduetograzing(Fig.3).The decreasewassignificant(one-way ANOVA,F2,8=55.0, p<0.001) and morepronounced withC. finmarchicusthan withC. hyper- boreus.Thepost-hocanalyseswithBonferroniadjustmentrevealed thatthecellconcentrationsweresignificantlylowerinflaskAwith C.hyperboreusandC.finmarchicusthaninthecontrolflaskA(both p-values<0.001)at theend ofthe experiment,confirming that thecopepods weregrazing onP. seriata.In flaskB, there were nodifferences incellconcentrationsbetweentreatmentsatthe start(one-wayANOVA,F_2,8=0.75,p=0.50)norattheendofthe experiment(one-wayANOVA,F_2,8=0.08,p=0.93).Theoverallnet growthrate(betweendays0and8)wassimilarinallBflasks(one- wayANOVA,F_2,8=0.38,p=0.70),indicatinghomogeneousgrowth conditions(datanotshown).

BothC. hyperboreus and C. finmarchicus grazed onP. seriata during the 8-day incubation (Figs. 3 and 4) and no copepod mortality was detected. The mean ingestion rates during the

Fig.4. Ingestionrates(cellscopepod⁻¹h⁻¹,mean±SD,n=4)ofCalanushyperboreus andC.finmarchicusondays0–2,2–5,5–8,and0–8.

experimentforC.hyperboreusandC.finmarchicuswere3219and 542cellscopepod⁻¹h⁻¹,respectively(Fig.4), andC.hyperboreus ingestedsignificantlymorecellsthandidC.finmarchicus(t-test;

variancesnotequal;t_3.06=9.0,p=0.003).Themeaningestionrate ofC.hyperboreuswashighestfromdays5to8buttherewerenosig- nificantdifferencesiningestionratesofC.hyperboreusbetweenthe differenttimepoints(RMANOVA,F_2,6=1.0,p=0.42).C.finmarchicus ingestedP.seriataatsignificantlydifferentratesduringtheexperi- ment(RMANOVA,F_2,6=27.6,p=0.001)andpost-hocanalysiswith Bonferronicorrectionshowedthattheingestionratewassignifi- cantlylowerondays5to8thanondays0to2and2to5(p=0.017 andp=0.005,respectively)(Fig.4).

AddinghomogenateofP.seriatatotheP.seriataculturedidnot affectthetoxicity ofthecultureduringtheexperiment(Fig.5).

Thereweresignificantdifferencesintoxicitybetweendaysinflask Awithhomogenate(RMANOVA,F_2,6=8.1,p=0.020)andpost-hoc pairwisecomparisonswithoutadjustmentshowedtoxicitytobe significantlyhigheronday2thanonday8(p=0.043)(withBon- ferroniadjustmentnodifferencescouldbedetected).However,the samesignificantdifferencebetweendayswasalsodetectedinthe controlflaskA(Friedmantest,X₂²=8.0,p=0.018,post-hocpairwise comparisonsp=0.014).Hence,theminorchangeintoxicityonday 2inflasksAwasduetofactorsotherthanaddingthehomogenate and therewasnosignificantdifferencein toxicitybetweenthe treatments inflaskA onday2 (t-test, t₆=−0.51, p=0.63). In B flasks (control and treatment), toxicity did not change signifi- cantlyduring theexperiment (Friedman test, X₂²=1.5, p=0.47, andRMANOVA,F1.02,3.06=0.042,p=0.86,respectively).Becausethe assumptionofsphericitywasnotmet(assessedbyMauchly’stest), theGreenhouse–GeisercorrectionwasappliedfortheRMANOVA onthetreatmentflaskB.Cellnumbersdecreasedinalltheflasksto around∼500cellsml⁻¹untilday5,afterwhichtheyremainedsta- bleorincreasedslightlyuntilday8(datanotshown).Netgrowth ratesdidnotdifferbetweenthecontrolandthetreatmentinflask A(t-test,t₆=−2.3,p=0.057)norinflaskB(Mann–Whitney,U=4.0, z=−1.2,p=0.34),confirminguniformgrowthconditions(datanot shown).Dissolvedinorganicnitrogenandsilicalevelsdecreased bothinthecontrolandtreatmentincubators(flaskB)duringthe experimentandwereatthesamelevel(datanotshown).There waslittlephosphateintheseawaterused(mean1.3␮moll⁻¹)and itsleveldidnotchangeinthecontrolbutdecreasedslightlyinthe treatmentincubator(flask B)(datanotshown).pHvalueswere 7.9inboththecontrolandtreatmentBflasksattheendofthe experiment(t-test,t6=−1.1,p=0.33).

ChangesinthechainlengthofP.seriatawereobservedinthe experimentwithcopepodsbutnotintheexperimentwiththeP.

seriatahomogenate.Theproportionofsinglecellsversuscellsin

Fig.5. Domoicacid(DA)cellquota(pgDAcell⁻¹,mean±SD)ofPseudo-nitzschia seriatainthecontrol(n=4)andthehomogenatetreatment(n=4)ondays0,2,5and 8in(A)flaskA,withhomogenateadded(exceptcontrol),and(B)flaskB,containing onlyalgae.

chainsduringthe experiment wasmoreor less stablewhen C.

hyperboreusandC.finmarchicusgrazed onP.seriata,butonlyin theflaskswherethecopepodswereinphysicalcontactwiththe cells(flaskA),whereasinthecontrols,theproportionofcellsin chainsincreasedduringtheexperiment(Fig.6).Intheexperiment withtheP.seriatahomogenate,theproportionofsinglecellswas

∼60%throughouttheexperimentinboththecontrolandtreatment flasks(datanotshown).TheX²-testdidnotrevealrelevantdiffer- encesbetweenthecontrolandthegrazingtreatment/homogenate additionbecausesignificantdifferencesbetweenthecontroland thetreatmentsalreadyexistedatthestartoftheexperiment.

4. Discussion

WeshowthatthetoxicityofP.seriatacellsincreasedsignif- icantlyinthepresenceofcopepods. Theresponseischemically mediated,i.e.itwasalsofoundwhentheorganismshadnophysi- calcontact.ChemicalcuesfromthedamagedP.seriatacellsalone didnotenhanceDAproductioninliveP.seriatacells.

ThesignificantincreaseintoxicityofP.seriatainthepresenceof copepods,butwithoutphysicalcontact,suggeststhatwaterborne cuesorchangesinwaterchemistryinducedthecellularresponse.

ThetoxicityofP.seriataincreasedmoreinthepresenceofcuesfrom C.finmarchicusthanwithC.hyperboreus,whenthecopepodswere notinphysicalcontactwiththediatomcells.However,thetoxicity ofP.seriatapeakedwhenthecellswereindirectcontactwithboth

CalanusspeciesandthehighestDAcellquotawasrecordedafter8 dayswithC.hyperboreus.Wecannottotallyruleoutthepossibility thatthetoxicityofP.seriataindirectcontactwiththecopepods increasedbecauseofselectivegrazingofthecopepodsonlesstoxic P.seriatacells.However,wefinditunlikelybecauseC.finmarchicus isnotknowntodiscriminatebetweentoxicandnon-toxicPseudo- nitzschiaspecies(Leandroetal.,2010).Moreover,therewasalarge differenceintoxicitybetweentheP.seriatacellsindirectcontact withthecopepodsandthecontrol.Instead,wewouldexpectto seeamorepronouncedchemicallymediatedresponsewhenthe copepodsandthealgaeareinphysicalcontact,sincetheresponse islikelytobeattenuatedwhenthesignalmustfirstpassthroughthe 2.0-␮mpolycarbonatemembrane.Anexplanationforthiscouldbe thatthecueshavelipophiliccomponents,whichpartiallyadsorbto thepolycarbonatemembraneanddonotentirelypassthroughit.A similarattenuationinresponsewasobservedinananalogousstudy onthePST-producingdinoflagellateA.minutuminthepresenceof threecalanoidcopepods(Bergkvistetal.,2008).

TriggersforDAproductioninPseudo-nitzschiaspecieshavebeen attributedtoenvironmentalfactors,e.g.nutrientsin relationto thegrowthphase(reviewedinBates,1998;Lelongetal.,2012), pH(e.g.Lundholmetal.,2004),andtracemetallimitationortox- icity(Maldonadoetal.,2002).Evidenceofallelopathiceffectsof DA,however,hasnotbeenfound(Lundholmetal.,2005).DAis anaminoacidandthusnitrogenisneededforitssynthesis.Since dilutedL1-medium(1/16)withreducedsilicatewasusedinthe presentstudy,withanextraadditionofmediumcontainingP.seri- ataonday3, we assumethat noothernutrient exceptsilicate (whichwasintentional)waslimitingthegrowthofthealgaein anyexperimentalflask.Thisissupportedbytheequalgrowthrates observedinflaskBofthecontrolandtreatments.InflaskAwhere thecopepodscouldgrazeonthealgae,thegrowthofP.seriatawas assumedtobesimilartotheotherexperimentalflasks,sothatthe observeddifferenceincell concentrationwasdue onlytograz- ing.Thecopepodsexcreteorleaknutrients,suchasammonium, whichmayalterthegrowthconditionsofthealgaeintheexper- imentalflaskscontainingthecopepodswithrespecttoavailable nutrientsand theirratios. Highlevelsof ammonium(>200␮M) mayleadtoincreasedcellularDAquotasinPseudo-nitzschiabut, atthesametime,mayalsoresultinloweredgrowthrates(Bates etal.,1993).Nochangeingrowthwasobserved,evenwhenthe P.seriatacellswerenotindirectcontactwiththecopepodsinB flasks,wheretheDAcellquotaincreasedsignificantly.Therefore, weassumethatfactorsotherthannutrientscausedtheincrease inDAproduction.Levelsofmajorinorganicnutrientshavebeen measuredin onlya fewstudies,which haveshownthatchemi- calsreleasedbythezooplanktoninducedeitherphysiologicalor morphologicalresponsesinphytoplankton(Selanderetal.,2006;

Longetal.,2007;LundgrenandGranéli,2010);however,thenutri- entsthemselveshavenotbeenfoundtobetheinductivefactor in thesestudies.Selanderetal. (2006)assumedthat thepossi- bleeffectofexcretions(ammonium)fromA.tonsatobenegligible at saturatingnutrient conditionsin comparison totheeffectof chemicalcuesfromA.tonsainenhancingPSTproductionA.min- utum.Thiswaslatersupportedbytheobservationthatammonium concentrationsdidnotdifferbetweenthegrazingtreatmentsand thecontrols,althoughthecell-specificPSTcontentofA.minutum increasedsignificantlyinthepresenceofA.tonsainnitrate-rich butnot inlow-nitrateconditions(Selanderet al.,2008).Colony formationinthegreenalgaeScenedesmusalsoseemstobeinduced byspecialgrazer-releasedinfochemicalsratherthanbyexcretory productsofthegrazers,orothernutrients(Lürling,2001).pHis knowntoaffectDAproductioninPseudo-nitzschiaspeciesbutthe resultsfromstudiesarecontradictory.Lundholmetal.(2004)and Trimbornetal.(2008)foundthatelevatedpHincreasedDAcell quotainP.multiseries,whereasSunetal.(2011)showedthatthe

Fig.6. PercentageofPseudo-nitzschiaseriataassinglecellsandascellsinchains(mean±SD)ondays0,2,5and8.(A)FlaskAcontrol,withoutanycopepods;(B)flaskA, containingCalanushyperboreus;(C)flaskA,containingC.finmarchicus;(D)flaskBcontrol,withoutanycopepodcuesfromflaskA;(E)flaskBwithcuesfromC.hyperboreus inflaskA;(F)flaskBwithcuesfromC.finmarchicusinflaskA.Allflaskscontainedalgae.Inthecontrol,n=3andinthecopepodtreatments,n=4.

DAlevelsofP.multiseriesincreasedwithloweredpH.Inthepresent study,pHis expectedtoincrease asCO₂ is used forphotosyn- thesis,butitislikelytobecounterbalancedbytherespirationof thecopepodsthatproduceCO₂.BecausepHwasnotmeasuredin thepresentstudyintheflasksthatcontainedthecopepods and becauseoftheobviously complexmechanismsbywhichitmay affectDAproduction,wecannottotallyexcludethepossibilitythat pH,atleastinpart,hadanimpactontheDAlevels.Longetal.

(2007)andLundgrenandGranéli(2010)foundnodifferencesin pHbetweenthegrazingtreatments(A.tonsaandP.globosa)and controls(onlyP.globosa).Inothersimilarinductionexperiments withcopepoditesandP.seriata,nosignificantchangesinpHwere found(N.Lundholmunpubl.).Intheexperimentwithhomogenate added,nodifferencesinpHwereobservedbetweenthecontroland treatment.

Inthepresentstudy,DAwasproducedduringtheexponential growthphase.TheDAcellquotaofP.seriataincreasedatthesame timethatcellnumbersincreaseduntilday5withcuesfromC.fin- marchicusinflaskB.Aswellonday8,P.seriatawasmoretoxic inflaskBwithcuesfromC.hyperboreusthaninthecontrol.With physicalcontactbetweenthecopepodsandthealgae(flaskA),the DAcellquotaofP.seriataincreaseduntilday5withC.finmarchicus, anduntiltheendoftheexperiment(day8)withC.hyperboreus.

Thisindicatesthatactivelydividingcellswereproducingincreas- ingamountsofDA.Thisiscontrarytothefindingsofmostearlier studieswhichshowedthatthehighestDAlevelsoccuredduring thestationarygrowthphase,whencelldivisionceased(reviewed inBates,1998;Fehlingetal.,2004;Thessenetal.,2009).Garrison etal.(1992)reportedthatP.australisproducedDAduringtheexpo- nentialphase,butconcludedthattheresultscouldbebiaseddue

totheverydensestartinginoculum,whichmighthavestressed thealgaeand,hence,enhancedDAproduction.P.australisgrown onureaproducedDAwhengrowingexponentially,buttheexpo- nentialgrowthrateof P.australiswassignificantly lowerwhen grownonureacomparedtonitrateandammonium(Howardetal., 2007).Pseudo-nitzschia pseudodelicatissima(identitynot certain) producedDAthroughouttheexponentialphase,buttheproduc- tionwassubstantiallyhigherinthelatestationaryphase(Adams etal.,2000).

Theexperimentwithcopepodsinthepresentstudywastermi- natedwhenthecultureswerestillintheexponentialgrowthphase.

Thus,higherDAcellquotasthanrecordedintheexponentialphase mighthavebeenobservedinthestationaryphase.However,with C.finmarchicusthetoxicityofP.seriatadecreasedafterday5,sug- gestingthatthepotentialchemicalsignalmayberelatedtothe activelygrazing copepods.Thisissupportedbythefindingthat theabundanceofP.seriatadecreasedtoverylowlevelsduringthe experiment,meaningthatC.finmarchicuspracticallystoppedgraz- ingbeforetheendoftheexperiment.Selanderetal.(2006)found thatcuesreleasedbythegrazingA.tonsaincreasedthePSTcon- tentinthedinoflagellateA.minutumsignificantlymorethancues releasedfromthestarvingA.tonsa.Inductionofcolonyformation inScenedesmusacutuswasalsostrongerwhenexposedtoDaphnia exudatesfromfeedingcomparedtostarvedgrazers(Lampertetal., 1994).Similarly,Lürling(1998)observedthatonlyexudatesfrom DaphniagrazingondigestiblefoodinducedcolonyformationinS.

acutus.Cuesrelatedtoactivefeedingmaythusbethemostreliable indicationoftheriskofherbivoryforphytoplankton(VanDonk etal.,2011),explainingwhycuesfromactivelygrazingconsumers mayproduceastrongerinduction.

TheproportionofP.seriatacellsinchainsindirectcontactwith thecopepodsinflaskAwasrelativelystableduringtheexperiment.

Ondays5and8withC.finmarchicusandonday8withC.hyper- boreus,theproportionofthechainswaslowercomparedtothe control.Thisislikelyduetoahigheringestionrateofchains,which maybeeasierforthecopepodstodetectduetotheirlargersize comparedtothesinglecells.Onthecontrary,intheBflaskswith- outphysicalcontactwiththecopepods,aswellasinthecontrol flasks,theproportionofchainsincreasedduringtheexperiment.

Bergkvistetal.(2012)showedthatthediatomSkeletonemamari- noireducedchainlengthinthepresenceofthreecopepodspecies;

theresponseinS.marinoiwasalsoinducedwithoutphysicalcon- tactwithA. tonsa.Grazingwassignificantlyreduced onshorter chainscomparedtolongerchains.Theauthorssuggestedthatthe suppressionofchainformationinS.marinoiwasadefensemecha- nismagainstcopepodgrazing(Bergkvistetal.,2012).Inthepresent study,noeffectsofpotentialwaterbornecuesfromthecopepods onchainformationofP.seriatawereobserved.Indeed,inalltheB flaskswithcuesfromthecopepodstheproportionofcellsinchains increasedsimilartothecontrols.InflaskA,withdirectphysicalcon- tactwiththecopepods,possiblecuesmayhavesuppressedchain formationinP.seriatabutsincethecopepodswerealsograzingthe cells,wecannotdistinguishbetweentheeffectsofthesetwofac- tors.ThisisparticularlyrelevantforC.finmarchicus,whichalmost exhaustedtheP.seriatacellsbyday5.Theincreasedproportionof cellsinchainscomparedtosinglecellsduringtheexperimentin thecontrolsshowsconvincinglythetendencyofP.seriatatoform chainsduringexponentialgrowth.

OurresultssuggestthatpotentialchemicalcuesfromP.seriata, producedbymechanicaldamage,donotinducethedefensemech- anismsaddressedinthepresentstudy(increaseinDAproduction, chainformation).Similarly,Lampertetal.(1994)andLürling(1998) didnotobserveinductionofcolonyformationinS.acutuswhenthe algawastreatedonlywiththecellhomogenateofS.acutusinstead ofDaphniaexudates.Nordidwefindanyeffectsofpossiblecues fromdamagedcellsonchainformationofP.seriata.However,we cannotexcludethepossibilitythatanychemicalcuesreleasedfrom damagedcellscouldinteractwithchemicalsfromthegrazerand thusinduceastrongersignaloftheriskofbeinggrazedupon,than ifthechemicalisreleasedonlybythegrazer.

ThehighestDAcellquotaofP.seriataobservedinthisstudy (13.1pgDAcell⁻¹)isin therangeofthehighestDA cellquotas recordedfor P.seriata, (33.6pg DAcell⁻¹; duringthestationary phase)(Lundholmetal.,1994).PriortoexposingP.seriata(P5G3)to thecopepodsinthisstudy,themeanDAcellquotaofP.seriatawas belowthelimitofdetection.Thepreviousyear,theaveragelevel was0.55pgDAcell⁻¹(Tammilehtoetal.,2012)andasubstantially higherDAcellquota(47pgDAcell⁻¹,singlemeasurement)forthe strainwasmeasuredjust2monthsbeforethat.Thisillustratesthe decreaseinDAproductionovertimeinstrainskeptinculturefor longerperiods,evenyears(e.g.Batesetal.,1999).However,our studyshowsthattheP.seriatastrain(P5G3)usedhadnotcom- pletelylostitsabilitytoproduceDA,becauseitthenregainedit wheninducedbythecopepods.ThedecreaseinDAproductionin unialgallaboratoryculturesmaythusillustratetherelaxationofan inducibledefense.

DissolvedDAdetectedinflaskAwithC.hyperboreusisinthe rangeofthatpreviouslyreportedintheculturemediumofP.seri- ata,butthepercellproductionofdissolvedDAwasonaverage 23–250timeshigher (Fehling etal.,2004;Hansenet al.,2011).

Maldonadoetal.(2002)foundthatP.multiseriesandP.australis producedDAduringtheexponentialgrowthphaseiniron-deficient ortoxic copperconcentrations. Almostall(95%) oftheDApro- ducedwasreleasedintothemediumandtheauthorsconcluded thistobeastressreactionagainstunfavorablemicronutrientlev- els(Maldonadoetal.,2002).Inthepresentstudy,∼60%ofthetotal

DAproduced(cellularanddissolved)byP.seriatainphysicalcon- tactwithC.hyperboreuswasreleasedintothemedium.Thismay havebeenanactivestressresponsetothepresenceofagrazer, aninvoluntaryleakageofDAduetothehighintracellularDAcon- centrationoraconsequenceof“sloppy”feedingbythecopepods (Mølleretal.,2003),leadingtoreleaseofDAintothemediumfrom thedamagedcells.

In thepresent study,wedidnotseeDAtodetergrazing on P.seriatabythecopepods.However,ifDAproduction inP.seri- ataisinducedbychemicalcuesreleasedbycalanoidcopepods,it may,becauseofitstoxicity,beassumedtobeadefensemechanism againstgrazing.Inthiscase,theriskofbeinggrazedshouldbelower inthe‘induced’cellsincomparisonto‘non-induced’cells.Aspre- viouslymentioned,Selanderetal.(2006)showedthattheinduced dinoflagellateA.minutumcontainedahigherPSTcellquotathan non-inducedcellsandthattherewaslessgrazingbythecopepod A.tonsaoninducedA.minutumcomparedtonon-inducedcells.

Copepodsmayindeedstopgrazingonthepreybecauseitistoxic orotherwiseunpalatable(behavioralselection)(Teegarden,1999).

Inaddition,grazingbycopepodsmaybepreventedbyphysiologi- calincapacitationcausedbytoxicfood(Huntleyetal.,1986;Ives, 1987).Indications of C.finmarchicusand C. hyperboreusto stop grazingontoxicP.seriataafter6hduetopossiblephysiological incapacitationwerefoundbyTammilehtoetal.(2012).Barguetal.

(2003)foundasimilareffectwhenstudyingkrill.Suchatempo- ralgrazingeffectwasnotobservedinthepresentstudy,probably becauseofthelongersamplingintervals(2–3days)comparedto the3hintervalusedbyTammilehtoetal.(2012).IfDAdetersgraz- ingbycopepodsduetophysiologicalincapacitation,thecopepods areexpectedtoreducefurthergrazinguntiltheDAlevelsinthe grazerarebelowacertainthresholdlevel.Inductionofhigherpro- ductionofDAinP.seriataduetothepresenceofgrazersistherefore expectedtoresultinafasterreductionofgrazing.Withregardto grazing,this wouldgivetoxicP.seriataanadvantageatleastin bloomconditions,becausegrazingonP.seriatawouldbeimpaired.

ProvingthatthecopepodsarephysiologicallyincapacitatedbyDA requires furtherstudies.Thehypothesis thatDA causesphysio- logicalincapacitationincopepodsdoesnotconflictwithfindings showingthatcopepodsdonotselectagainsttoxicPseudo-nitzschia species,evenwhennontoxicalternativefoodisoffered(Maneiro etal.,2005;Olsonetal.,2006;Leandroetal.,2010).Thereasonis thatthenegativeeffectsofDAongrazersareexpectedtooccuronly ifDAintakebythegrazersishighenough,whichmaynothavebeen thecaseinthestudiesbyManeiroetal.(2005),Olsonetal.(2006) andLeandroetal.(2010),orthattheeffectsofpossiblephysiolog- icalincapacitationbyDAareleftunnoticed.DAmaynevertheless haveadverseeffectsonthefecundityofthegrazers,whichmaylead tosubsequentreducedgrazingpressure.However,onlytwostud- ieshaveaddressedthisquestionandfoundthatfeedingontoxic P.multiseriesdidnotaffecteggproductionoregghatchingsuccess ofthecopepodsA.tonsa(Lincolnetal.,2001)orA.clausi(Maneiro etal.,2005).Mostcopepodgrazingexperimentsontoxicphyto- planktonspecies,however,havenotlastedmorethan24h,which maybetooshortatimetodetectthepotentialadverseeffectsof phycotoxinsongrazers(Turner,2014).

AssumingthattheproductionofDAasasecondarymetaboliteis costly,itismorelikelytobemaintainedbynaturalselectionifithas severalfunctions(Wink,2003).Inthepresentstudy,nocostsofpro- ducingDAwereobserved,astherewerenodifferencesingrowth ratesbetweenthehighlytoxicandthelesstoxiccells.However, theremaybeindirectcoststhatcouldnotbedetectedinthebatch cultures,assuggestedbyBergkvistetal.(2008)andSelanderetal.

(2012)forthePST-producersA.minutumandA.tamarense,respec- tively.Inthefield,DAproductionislikelytobecontrolledbyseveral interactingfactors,whichmakesitdifficulttopredicttheformation ofatoxicbloom.ThiswasemphasizedbyMarchettietal.(2004),