salinity and implications to for egg survival Egg ﬂounder, buoyancy Platichthys of ﬂesus , in the BalticSea—adaptation Fisheries Research

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Fisheries Research

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / f i s h r e s

Egg buoyancy of ﬂounder, Platichthys ﬂesus, in the Baltic Sea—adaptation to salinity and implications for egg survival

Anders Nissling

^a,∗

, Soﬁa Nyberg

^a

, Christoph Petereit

^b

aArResearchStation,DepartmentofEcologyandGenetics,UppsalaUniversity,SE-62167Visby,Sweden

bGEOMARHelmholtzCentreforOceanResearchKiel,D-24105Kiel,Germany

a r t i c l e i n f o

Articlehistory:

Received31August2016

Receivedinrevisedform31January2017 Accepted27February2017

HandledbyGeorgeA.Rose

Keywords:

Eggspeciﬁcgravity Flounderecotype Brackishwater Pelagiceggs Demersaleggs Eggsurvival

a b s t r a c t

Verticaldistributionofeggsasdeterminedbytheeggbuoyancy,i.e.thedifferenceinspecificgravity betweentheeggandtheambientwater,haveprofoundimplicationsforthereproductivesuccessand hencerecruitmentinfish.Herevariabilityineggspecificgravityofflounder,Platichthysflesus,wasstud- iedalongasalinitygradientandbycomparingtworeproductivestrategies,spawningpelagicordemersal eggs.Eggcharacteristicsof209eggbatches(coveringICESsubdivisions(SD)22–29inthebrackishwater BalticSea)wasusedtorevealthesignificanceofeggdiameterandeggdryweightforeggspecificgravity (ESG),subpopulations,andeggsurvivalprobabilitiesofpelagiceggsfollowingamajorsalinewaterinflow event.Asanadaptationtosalinity,ESG(at7^◦C)differed(p<0.001)betweenareas;threesubpopulations offlounderwithpelagiceggs:1.0152±0.0021(mean±sd)gcm⁻³inSD22,1.0116±0.0013gcm⁻³in SD24and25,and1.0096±0.0007gcm⁻³inSD26and28,contrastingtoflounderwithdemersaleggs, 1.0161±0.0008gcm⁻³.Eggdiameterdiffered(p<0.001)betweensubpopulations;from1.08±0.06mm (SD22)to1.26±0.06mm(SD26and28)forpelagiceggsand1.02±0.04mmfordemersaleggs,whereas eggdryweightwassimilar;37.9±5.0␮g(SD22)and37.2±3.9␮g(SD28)forpelagic,and36.5±6.5␮g fordemersaleggs.Botheggdiameterandeggdryweightwereidentifiedasexplanatoryvariables,explain- ing87%ofthevariationinESG.ESGchangedduringontogeny;aslightdecreaseinitiallybutanincrease priortohatching.EggsurvivalprobabilitiesjudgedbycombiningESGandhydrographicdatasuggested highereggsurvivalinSD25(26vs100%)andSD26(32vs99%)butnotinSD28(0and3%)afterthe inflowevent,i.e.highlyfluctuatinghabitatsuitability.TheresultsconfirmthesignificanceofESGforegg survivalandshowthatvariabilityinESGasandadaptationtosalinityisdeterminedmainlybywatercon- tentmanifestedasdifferencesineggdiameter;increaseindiameterwithdecreasingsalinityforpelagic eggs,anddecreaseddiameterresultingindemersaleggs.

1. Introduction

Toaccomplishsurvivalofearlylifestages,i.e.thereproductive successandthusrecruitmenttotheadultstock(cfﬁtness),different spawningstrategiesanddifferenttacticshaveevolvedinteleosts (e.g.Wootton,1990).Thismaybemanifestedase.g.theproduction ofdemersalvspelagiceggs,andasselectionofspawningarea,e.g.

incoastalareasoroffshore.Inthepresentstudythesignificanceof variabilityineggspecificgravity(ESG)forthereproductivesuccess offlounder(Platichthysflesus)wasstudiedalongasalinitygradient andbycomparingtwodifferentreproductivestrategies,spawning offshorewithpelagiceggs(eggswhichfloatfreelyinthewatercol-

∗Correspondingauthor.

E-mailaddress:anders.nissling@ebc.uu.se(A.Nissling).

umn)andincoastalareaswithdemersaleggs(eggsdevelopingon thebottom),respectively,suggestingeggspeciﬁcgravityasbeing amajorselectionprocess.

Spawningpelagiceggsisthemostcommonstrategyofmarine fishes and is acquired by uptake of water duringthe terminal growthofoocytesintheovary,oocytehydration,justpriortoovu- lation(Fulton,1898;CraikandHarvey,1987;Cerdàetal.,2007).By incorporationoffluidwithlowerosmoticpotentialthanthesea- waterenvironment,andthuscompensatingforthedenserparts oftheegg,theyolkandchorion,theoveralleggspecificgravityis lowered(CraikandHarvey,1987).Bothdemersalandpelagiceggs undergohydration,butthedegreeofwateruptake,accompanied byasignificantincreaseinoocytevolume,differbetweenpelagic anddemersaleggs.Typically,pelagiceggshavehighwatercontent, 90–92%,comparedtodemersaleggs,60–70%,andthusloweregg specificgravitythatallowsforbuoyancy(CraikandHarvey,1987).

http://dx.doi.org/10.1016/j.ﬁshres.2017.02.020

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Dependingonspeciesspecifichabitatpreferencesforeggdevel- opment,egg specificgravityand hencetheverticaldistribution varyamongspecies,andfurthermorebetweenpopulationswithin species,asadaptationstolocalenvironmentalconditions.E.g.for theextensivelystudiedcod,Gadusmorhua,eggspecificgravityvary fromca1.022–1.026gcm⁻³atmarineconditionsintheAtlanticto ca1.009–1.014gcm⁻³in thebrackishwaterBalticSea(Table in Jung,2012;TableinPetereitetal.,2014).Thisisduetodifferences inwatercontent,ca92%vsca97%,andaccordinglyineggdiameter, ca1.2–1.4mmvsca1.5–1.8mm,formarineandbrackishwatercod eggs,respectively(Thorsenetal.,1996;Jung,2012;Petereitetal., 2014).Thehigheruptakeofwaterinbrackishwatercodeggsresult inastretchingofthechorion,i.e.thinnerchorion,andachange inthechorion(aconsiderablepartofthetotaleggmass)toegg volumeratioinfluencingtheESG(Kjesbuetal.,1992).Aseggbuoy- ancyisdeterminedmainlyby salinity(Sundbyand Kristiansen, 2015)marinefishesthathavemanagedtoadapttothebrackish conditionsintheBalticSea, displaydifferenteggcharacteristics (watercontentandeggdiameter)comparedtotheirmarinecoun- terparts;forcodresultinginneutraleggbuoyancyat10–20psuvs at27–33psu(e.g.VallinandNissling,2000;Jung,2012;Petereit etal.,2014).

SinceCoombs(1981)introducedthedensitygradientcolumn allowingforaccuratemeasurementsofeggspecificgravity,anum- berofstudieshavebeenconductedfocusingoni)themechanisms ofachievingegg buoyancyand parameterizationof variousegg componentsforeggspecificgravity(Kjesbuetal.,1992;Jungetal., 2014),ii)theecologicalsignificanceofverticaleggdistributionand variabilityineggspecificgravityforsurvivalprobabilitiesinrela- tiontoabioticambientconditions(e.g.Nisslingetal.,1994;Ouellet, 1997;MacKenzie and Mariani, 2012), and iii)opportunities for retentioninordispersaltosuitablehabitatsfordevelopment(e.g.

Ospina-Álvarezetal.,2012;Myksvolletal.,2013;Petereitetal., 2014).Hence,theverticaldistributionofeggsasdeterminedby eggspeciﬁcgravity,andambientsalinityandtemperaturecondi- tions,mayhaveprofoundimplicationsforthereproductivesuccess andtheyearclass-formationinteleosts.

ThebrackishwaterBalticSea,withrestrictedwaterexchangeby shallowstraightsintheSoundandtheBelt-Seas(ICESSD22and23;

Fig.1),ischaracterizedbyadecreaseinsalinityinthesurfacewater fromca9psuinthesouthwest(SD24)toca3psuinthenorth(SD 31),andapermanenthaloclineat50–70mdepthwithdensermore saline,ca10–20psu,deepwaterintheBalticproper(SD24–28).

Successfulreproductionofmarinefishesisrestrictedbyopportuni- tiesforfertilizationandeggdevelopmentatlowsalinities(Nissling etal.,2002,2006;Petereitetal.,2009)and/orbyopportunitiesto obtainneutraleggbuoyancyinthelessdensewater,i.e.notsinkto thebottomorbesubjectedtounfavourableoxygenconditionsin thedeeplayers(Nisslingetal.,1994;MacKenzieetal.,2000).Con- ditionsarestronglyaffectedbyhighlyirregularsalinewaterinflow events,influencingbothsalinityandoxygenconditionsandconse- quentlythereproductivesuccessandaccordinglybothabundance anddistributionofmarinefishes(Segerstråle,1969;Ojaveerand Kalejs,2005;MacKenzieetal.,2007),includingflounder(Ojaveer etal.,1985;Drews,1999;Ustupsetal.,2013).

Flounder,Platichthysﬂesus,inhabittheEasternAtlanticofWest- ernEuropefromtheWhiteSeatotheMediterraneanandtheBlack SeaincludingtheBalticSea(Nielsen,1986).IntheBalticSeaitdis- playdifferentspawningstrategies;spawningalongthecoastand onoffshorebankswithdemersaleggsinSD26–30andSD32,and off-shoreinthedeepbasinswithpelagiceggsinSD22–26andSD 28(Bagge,1981),i.e.formingtwogeneticallydistinct(Hemmer- Hansenetal.,2007;FlorinandHöglund,2008)sympatricecotypes, butsharingfeedingandwinteringareas(Nisslingetal.,2015).Fur- ther,asanadaptationtothedecreaseinsalinity;ca15–25psuin theBeltSeasandtheSound(SD22and23),ca13–20psuinthe

ArkonaBasinandBornholmBasin(SD24and25)vsca10–14psu intheGdanskDeepandGotlandBasin(SD26and28)(Fig.1),egg speciﬁcgravityoftheecotypewithpelagiceggshasbeenshownto differbetweenspawningareas(Nisslingetal.,2002;Petereitetal., 2014).

Inthepresentstudydataofeggspeciﬁcgravityfromﬁshsam- pledindifferentBalticSeaspawningareasin2011–2015,together withalreadypublisheddata(Solemdal,1971,1973;Nisslingetal., 2002;Petereitetal.,2014)werecompiledwiththeaimto:

i)Explorethesigniﬁcanceofeggdiameterandeggdryweight respectively,foreggspeciﬁcgravity,andhencefortheopportu- nitytoobtainneutraleggbuoyancyalongasalinitygradient,i.e.

adaptationtoprevailingwaterdensity.

ii)Revealdifferencesineggcharacteristicsbetweenﬁshspawn- ing in different areas to be used as a non-genetic tool in discriminationofsubpopulations(Ciannellietal.,2010;Myksvoll etal.,2013;Petereitetal.,2014).

iii)Assesstheecologicalsignificanceofeggspecificgravityand verticaleggdistributiononeggsurvivalprobabilitiesofthefloun- derecotypewithpelagiceggs.Hereweexplorehabitatsuitability foreggsurvivalatconditionsprevailingbeforeandafteramajor salinewaterinfloweventintotheBalticSeabycomparingcon- ditions in 2014 and in 2015 following themajor inflow event inDecember–January2014–2015(Mohrholzetal.,2015)causing changesinsalinityandthusinverticaleggdistribution.

iv)AssessontogenicchangesinESGuntilhatchingoftheecotype withpelagiceggsasanincreaseinESGduringdevelopment(as shownforotherspecies;seebelow)mayimplydevelopmentat lessfavourableoxygenconcentrations.

Inabroadercontextinformationaboutvariabilityineggspecific gravity(andverticaleggdistribution)offloundermaybeusedin stock-recruitmentrelationshipsbyincorporationofhydrographic conditionsandstockstructure,i.e.estimatetheeffectivespawning stockbiomass(Hinrichsenetal.,2016b),andfurther,contribute toforecasting stockdevelopmentinaccordancewithpostulated deteriorationofsalinityandoxygenconditionsinthedeepbasins asaneffectofclimatechangeaffectingtheextentofsalinewater inflowsintotheBalticSea(MacKenzieetal.,2007;Meieretal., 2012).

2. Materialsandmethods 2.1. Samplingofﬁsh

The study includes measurements of egg characteristics of ﬂounder,Platichthysﬂesus,sampledinICESSDs22–26andSD28 (Fig.1)in2011–2015.Samplingwasconductedbyeither trawl surveysintheBalticdeepbasinsbyR/VAlkor(SD22–26andSD 28)inApril,orbygillnetsurveysfromat5toat70mdepthoff easternGotland(SD28)andintheHanöBight(SD25)inmidApril- earlyMay(Fig.1).Femalesizewasmeasuredas totallengthin mmorascmlength-class,thelatterusedinfurtheranalysis.Addi- tionally,alreadypublisheddata(Solemdal,1971,1973;Nissling etal.,2002;Petereitetal.,2014)wereincluded.Intotal209egg batchesfromdifferentfemales wereused.Thesamplingproce- dureisdescribedindetailinNisslingetal.(2002)andinPetereit etal.(2014)(Rawdataavailableatthefollowinglink:https://doi.

pangaea.de/10.1594/PANGAEA.871590).

2.2. Samplingofeggs

Eggsformeasurementswereobtainedbystrippingandartiﬁ- cialfertilization,usingeggsfromonefemaleandsemenfrom2to 3males,eitherdirectlyonboard(R/VAlkor)oraftertransporta- tiontotheArResearchStationortoamobilelaboratorywhereﬁsh

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Fig.1. ICESsubdivisions(SD)intheBalticSeawithspawningareasofﬂounder,Platichthysﬂesus,withpelagiceggs:BS−BeltSea(SD22),AB−ArkonaBasin(SD24),BB− BornholmBasin(SD25),GD−GdanskDeep(SD26),GB−GotlandBasin(SD28).

werekeptintankswithrunningwateruntilstripping.Fertilization wasperformedat17–20psuatca6–10^◦C.After0.5–1hasubsam- pleoffertilizedeggswereincubatedinnewwaterat6–9^◦Cand 17–20psuuntilmeasurementsca12–24hpostfertilization.Eggs usedformeasurementsofspeciﬁcgravity,diameteranddryweight werescannedunderastereo-microscopeandonlyeggswithnor- maldevelopment(i.e.regularcellmorphology;Kjørsviketal.,1990) wereusedtoensureonlyhighqualityeggsinmeasurements.

2.3. Determinationofeggcharacteristics

Egg speciﬁc gravity (ESG) was determined using a density gradient column (Coombs,1981) using 15–30 eggsin stage IA (ThompsonandRiley,1981)ineachdetermination.Theeggswere insertedinthecolumnand,afterasettlingtimeofca45–60min,

thepositionsrecordedandcomparedwiththepositionsof4–9den- sityglassfloats(Spartel,UK;MartinInstrumentsCo,UK)ofknown specificgravity;correlationcoefficientofthedensityfloatswere

>0.99in allmeasurements.Measurementwereperformedatca 7^◦C;ifdeviatingfromthis(temperaturecheckedatthetopofthe watercolumn)ESGwasadjustedto7^◦Cusingaseawaterdensity calculator(seelinkbelow).TheprocedurefordeterminationofESG isdescribedindetailinNisslingetal.(2002)andinPetereitetal.

(2014).

Innineeggbatchesfromdifferentfemales,eggswereincubated inthedensitygradientcolumnthroughoutdevelopmentfromstage IA(12–24hafterfertilization)untilhatching,i.e.atneutralbuoy- ancy.ESGwasassessed1–2timesperdaytorecordchangesduring ontogeny;developmentstage,accordingtoThompsonandRiley (1981),determinedusingeggbatchesfromtherespectivefemale

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incubatedatsimilarconditions(at20psuand6–8^◦C).Incubation ofalimitednumberofeggsinadensitygradientcolumnimplies noeffectoneggdevelopment,resultingin82–100%viablelarvae;

seePetereitetal.(2014)formoredetails.

Egg diameter, assessed as the outside diameter set by the chorion,wasmeasuredbyonecrossdiagonalmeasurementatstage IAunderastereo-microscopeat50orat240xmagniﬁcationusing anocularmicrometrescale,with20–30eggsineachdetermina- tion.Eggdryweight(stageIA)wasassessedintwoways,either 6–8eggs,rinsedindemineralizedwater2timesforca10s,were collectedinapre-weightedaluminiumvialforfreezedrying(at

−50^◦C)andsubsequentlyweightedtothenearest0.1␮g(Sarto- riusmicrobalanceSC2),orbatchesof200eggs,rinsed2timesfor ca15sindemineralizedwater,wasincubatedat60^◦Cfor24hand weighted(0.1mg;SartoriusBP210S).

2.4. Hydrographicdataandverticaleggdistribution

Salinity,temperatureandoxygendataweremeasuredusinga CTDprovidedwithanoxygenprobe(ADM)withcontinuousmea- surementsfromthesurfaceto3mabovethebottominthemain spawningareas fortheﬂounderecotypewithpelagiceggs(see Hinrichsenetal.,2016a),theBornholmBasin(SD25),theGdansk Deep(SD26)andtheGotlandBasin(SD28).Themeasurements werecarriedoutduringsurveyswithR/VAlkorinApril2014and in April2015; data from stationBB25, GD60 and GB90(cruise ReportsAl435(DOI10.3289/CRAL435]andAl454[http://oceanrep.

geomar.de/id/eprint/28939]).

BycombiningdataofwaterdensityfromCTDcastswithmean ESGofeacheggbatch,theverticaleggdistributionwasdetermined inaccordancetotheneutraleggbuoyancy.AsnodifferenceinESG wasfoundbetweeneggsfromSD24andSD25,andbetweeneggs fromSD26andSD28,respectively,theverticaleggdistributionin theBornholmBasin(SD25)wasassessedusingeggbatchesfrom SD24andSD25pooled(n=42),andtheverticaleggdistribution intheGdanskDeep(SD26)andtheGotlandBasin(SD28)withegg batchesfromSD26andSD28pooled(n=69);seebelow.

2.5. Habitatsuitability

Habitatsuitability foregg survival (Hinrichsenet al., 2016c) wasassessedasthewater column atwhich eggswouldobtain neutralbuoyancyabovecriticallevelsfordevelopment,i.e.1ml oxygenl⁻¹(Vitinsh,1980;Grauman,1981)and>2^◦C(Hinrichsen etal.,2016b;IsaWallin,personalcommunication).Accordingly, eggsurvivalprobabilitiesandcauseofmortalitywereevaluatedin relationtotemperatureandoxygenconditionsatthedepthwhere therespectiveeggbatchachieveneutralbuoyancy(atstageIA), orasdeathfromsedimentationofnon-buoyanteggbatches;no eggsoftheecotypewithpelagiceggsareexpectedtosurvivecon- ditionsprevailingatthebottom(seeHinrichsenetal.,2016c;and referencestherein).

3. Results

3.1. Differencesineggspeciﬁcgravitybetweenareas

Eggspecific gravity (ESG)at stage IA (Thompsonand Riley, 1981), measured at 7^◦C ca 12–24h post fertilization, varied between areas. The highest values occurred in SD 23, 1.02038±0.00062(mean±sd)gcm⁻³andthelowestinSD26and SD 28, 1.00955±0.00074gcm⁻³ and 1.00955±0.00068gcm⁻³, respectively(Table1;Fig.2).AGLM(generallinearmodel;SPSS ver.22)withESGasdependentvariable,area(ICESSD)asfixed factorandfemalelength-classascovariateresultedina signifi- canteffectofarea,df=5,F=16.58,p<0.001(datafromSD23and

fordemersaleggsinSD25wereexcludedinanalysisduetofew measurements).Pairwisecomparisonsrevealedthreegroupsfor ﬂounderwithpelagiceggs,SD22,SD24and25,andSD26and 28,respectivelyseparatedfromﬂounderwithdemersaleggsinSD 28(Table2).Aweakpositiveeffect(df=1,F=4.18, p=0.042)of length-classonESGoccurred.Thiseffectwashoweverrelatedto measurementsinSD22only.ExcludingSD22inanalysisresulted innoeffect(df=1,F=0.410,p=0.523)withp=0.245-0.645forthe respectivegroup,SD24and25,SD26and28andSD28withdemer- saleggs,i.e.noevidenteffectoffemalesizeonESG.Apartfromthat measurementsinSD22,incontrasttootherareas,yieldedaposi- tiverelationshipbetweenlength-classandESG,variabilityinESG washigh,standarddeviationof0.0021gcm⁻³comparedto0.0007- 0.0013gcm⁻³fortheotherareas(Table1).TheresultsfromSD22 mayberelatedtosamplingoccasion(trawlstationand/ordate) potentiallyandeffectofmixedpopulationsinthearea(Petereit etal.,2014;discussionbelow).

3.2. Signiﬁcanceofeggdiameterandeggdryweight

Regressionanalysisrevealedastrongeffectofeggdiameteron ESG,df=1,F=582.1,p<0.001,butnoeffectofeggdryweighton ESG,df=1,F=0.00,p=0.98(Fig.3aand3b)suggestingthatvari- ability inESG among subpopulations/areasis related mainlyto differencesinwatercontent,manifestedasdifferencesineggdiam- eter,butwithsimilardryweightsamongareas(Table1).Thiswas supportedbyanANOVAanalysiswitheggdiameterasdependent variableandSDasfixedfactor,resultinginasignificantdifference betweenSDs,df=4,F=63.57,p<0.001.AcorrespondingANOVA witheggdryweightasdependentvariableyieldedasignificantdif- ferencebetweenareas,df=4,F=3.19,p=0.017.However,thiswas duetoasignificantdifferencebetweenSD25(N.B.n=9)andSD26 (p=0.015;pairwisecomparisons)whereasnodifferencesoccurred betweene.g.eggsfromSD22andpelagiceggsinSD28(p=1.00) orbetweenpelagicanddemersaleggsinSD28(p=1.00).Hence, variabilityinESGamongsubpopulations/areasisrelatedtodiffer- encesineggdiameter(i.e.watercontent)withnodifferenceinegg dryweight.

Amultipleregressionmodelsuggestedinclusion ofbothegg diameter(df=1,t=25.53,p<0.001;negativerelationship)andegg dryweight(df=1,t=8.15,p<0.001;positiverelationship)explain- ing87%ofthevariationinESG(df=2,F=326.0,p<0.001)when measurementsfromallSDswerepooled,i.e.largereggswithlower dryweightresultinmorebuoyanteggs.ThiswasvalidalsoforSD 22andSD26and28,i.e.withpelagiceggs,andclosetosigniﬁcant forSD28withdemersaleggs(Table3),i.e.withintherespective sub-population/areavariabilityinESGisdeterminedbybothegg diameter(i.e.watercontent)anddryweight.

3.3. Ontogenicchangesineggspeciﬁcgravity

ESGduringdevelopmentinnineeggbatchesfromearlystage 1A to stage IV and hatching is presented in Fig. 4. In general, ESGdecreasedsomewhatduringdevelopment(Fig.4aandc),or remainedmoreorlessstable(Fig.4b)untilmid/latestageIIIfol- lowed by an increase in ESG during stage IV until hatching. A decreaseinESGduringdevelopmentresultinanupwardmove- mentoftheegg(untilstageIII)followedbysinkingduringstage IVuntilhatching.ThehighestdecreaseinESGuptostageIIIwas approximately0.0015–0.0020gcm⁻³corresponding,roughly,toca 50%ofthevariabilityinESGofeggbatchesinstageearlyIAwithin therespectivearea(basedonthevariabilitybetweeneggbatchesin SD24and25,andSD26and28,respectively)andmaythusincrease opportunitiesforeggsurvivalasevaluatedbelow.Similarly,the highestincreaseinESGduringstageIV,ca0.0015gcm⁻³compared

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Table1

Eggcharacteristicsofflounder,Platichthysflesus,indifferentICESsubdivisions(SD)intheBalticSea(mean±sd)togetherwithfemalesizeofanalyzedfish.InSD25andSD 28,wherebothecotypesoccur,PindicatespelagiceggsandDdemersaleggs.Numberofmeasurements(eggbatchesfromdifferentfemales)withinbrackets.

SD Eggspeciﬁcgravity(gcm⁻³) Eggdiameter(mm) Eggdryweight(␮g) Femalesize(cm)

SD22 1.01520±0.00206(33) 1.08±0.06(33) 37.9±5.0(32) 35.5±6.3

SD23 1.02038±0.00062(4) 30.0±4.1

SD24 1.01220±0.00122(13) 30.8±2.8

SD25P 1.01129±0.00123(29) 1.17±0.07(18) 34.5±2.5(9) 31.3±4.4

SD25D 1.01663±0.00057(3) 1.06(1) 30.0±3.8

SD26 1.00955±0.00074(17) 1.26±0.06(17) 41.1±5.9(16) 32.2±5.6

SD28P 1.00955±0.00068(52) 1.26±0.05(36) 37.2±3.9(33) 30.0±3.8

SD28D 1.01601±0.00076(58) 1.02±0.04(35) 36.5±6.5(11) 29.7±4.9

7.7 10.5 13.3 16.1 18.9 21.7 24.5 27.3 1.0060

1.0080 1.0100 1.0120 1.0140 1.0160 1.0180 1.0200 1.0220

20 25 30 35 40 45 50

Salinity of neutral buoyancy (psu)

Egg specific gravity (g/cm3)

Length-class (cm)

SD 22 SD 22 SD 23 SD 24 SD 25P SD 25D SD 26 SD 28P SD 28D

Fig.2. Therelationshipbetweeneggspecificgravity(gcm⁻³)andfemalesize(cmlengthclass)forflounder,Platichthysflesus,indifferentICESsubdivisions(SD).Corresponding salinityofneutralbuoyancyat7^◦Cshownonsecondverticalaxis.

Table2

Pairwisecomparisonsofeggspecificgravityofflounder,Platichthysflesus,indifferentICESsubdivisions(SD)byGLM-analysis(evaluatedatlengthclass31.25cm).InSD25 andSD28wherebothecotypesoccurPindicatespelagiceggsandDdemersaleggs.

SD mean 95%lower 95%upper SD22 SD24 SD25P SD26 SD28P SD28D

SD22 1.0143 1.01389 1.01474 <0.001 <0.001 <0.001 <0.001 <0.001

SD24 1.0122 1.01165 1.01278 <0.001 0.100 <0.001 <0.001 <0.001

SD25P 1.0113 1.01091 1.01166 <0.001 0.100 <0.001 <0.001 <0.001

SD26 1.0096 1.00911 1.01010 <0.001 <0.001 <0.001 1.000 <0.001

SD28P 1.0096 1.00935 1.00994 <0.001 <0.001 <0.001 1.000 <0.001

SD28D 1.0160 1.01573 1.01628 <0.001 <0.001 <0.001 <0.001 <0.001

Table3

Resultsfrommultiple-regressionanalysisbetweeneggspecificgravity(dependentvariable)andeggdiameterandeggdryweight,forflounder,Platichthysflesus,indifferent ICESsubdivisions(SD);forallareaspooledandfortherespectivesub-population.P=pelagiceggs.D=demersaleggs.n=numberofeggbatches.

Eggdiameter Eggdryweight Modelsummary

t p t p n F adjustedr² p

SDspooled −25.5 <0.001 8.15 <0.001 100 326.0 0.868 <0.001

SD22 −16.49 <0.001 12.53 <0.001 32 151.6 0.907 <0.001

SD25P −1.56 0.181 −0.218 0.836 8 1.90 0.205 0.243

SD26and28P −5.24 <0.001 3.21 <0.01 49 15.30 0.373 <0.001

SD28D −2.65 <0.05 1.81 0.108 11 3.61 0.343 0.077

totheinitialESG,involve sinkingand potentiallyincreasedegg mortality.

3.4. Eggsurvivalprobabilities

InFig.5verticalproﬁlesoftemperature,salinityandoxygencon- centrationinApril2014andinApril2015,areshownfortheBaltic Seadeepbasins,theBornholmBasin(SD25),theGdanskDeep(SD 26)andtheGotlandBasin(SD28).Themajorsalinewaterinﬂow eventinthewinter2014–2015resultedinimprovedconditions

foreggdevelopmentbelowthehalocline,i.e.inthemainspawn- ingareasforﬂounderwithpelagiceggs.IntheBornholmBasinand theGdanskdeepbothsalinityandoxygenconcentrationsincreased whereas in theGotlandbasin there wasan increasein salinity while oxygenconditions remainedlow.The onaveragesalinity belowthehaloclineincreasedfrom13.7±2.0(mean±sd)psuin April2014to15.5±2.6psuinApril2015intheBornholmBasin, from10.6±1.3psuto13.8±1.6psuintheGdanskDeep,andfrom 9.4±1.0psuto11.0±1.3psuintheGotlandBasin.Highersalini- ties(denserwater)resultedinasigniﬁcantchangeindepthrange

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7.7 10.2 12.7 15.2 17.7 20.2 22.7 25.2 1.0060

1.0080 1.0100 1.0120 1.0140 1.0160 1.0180 1.0200

0.90 1.00 1.10 1.20 1.30 1.40 1.50

Egg diameter (mm)

SD 22 SD 25P SD 26 SD 28P SD 28D

a)

7.7 10.2 12.7 15.2 17.7 20.2 22.7 25.2 1.0060

1.0080 1.0100 1.0120 1.0140 1.0160 1.0180 1.0200

25.0 30.0 35.0 40.0 45.0 50.0 55.0

Egg dry weight (µg)

SD 22 SD 25P SD 26 SD 28P SD 28D

b)

Fig.3. Therelationshipbetweeneggspecificgravity(gcm⁻³)anda)eggdiameter(mm)andb)eggdryweight(␮g)forflounder,Platichthysflesus,indifferentICESsubdivisions (SD).Correspondingsalinityofneutralbuoyancyat7^◦Cshownonsecondverticalaxis.

Table4

Averagedepth(m)anddepthrangeforstudiedﬂounder,Platichthysﬂesus,eggbatchesobtainingneutralbuoyancywithinthewatercolumnin2014and2015,andtheaverage temperature(^◦C),salinity(psu)andoxygenconcentration(mll⁻¹)experiencedbytheseeggbatchesinICESsubdivisions(SD)25,26and28respectively.Mean±standard deviation.nreferstothenumberofeggbatchesobtainingneutralbuoyancyinthewatercolumn,withthetotalnumberoftestedbatcheswithinbrackets.

SD25 SD26 SD28

2014 2015 t p 2014 2015 t p 2014 2015 t p

Depth 61.4±6.9 58.1±4.3 2.31 <0.05 91.8±3.3 83.2±2.9 14.58 <0.001 108.6±9.6 94.6±10.6 3.13 <0.001

Depthrange 51–72 52–69 80–98 79–91 94–118 75–110

Temperature 6.5±1.0 7.4±0.5 5.9±0.4 6.5±0.5 5.1±0.2 5.8±0.3

Salinity 14.2±1.4 15.1±1.5 11.7±0.6 12.2±0.8 10.6±0.4 11.1±0.5

Oxygen 3.3±1.4 4.0±0.9 2.20 <0.05 1.3±0.8 2.8±0.6 10.94 <0.001 0.3±0.2 0.5±0.3 2.33 <0.05

Batches(n) 31(42) 42(42) 46(69) 68(69) 7(69) 24(69)

atwhicheggsobtainedneutralbuoyancy(Fig.5),fromonaverage 61.4mto58.1mintheBornholmBasin,91.8mto83.2minthe Gdanskdeep,and108.6mto94.6mintheGotlandBasin,andthus atmorefavourableoxygenconditions(Table4).

Thechange(uplift)indepthrangeforeggdevelopmentbetween 2014and2015affectedeggsurvivalprobabilitiessigniﬁcantly.In theBornholmBasinandtheGdanskDeep opportunitiesforegg survivalincreasedfrom26%to100%(p<0.001;Fisher´ısexacttest) andfrom32%to99%(p<0.001)respectively,whereasprobabili- tiesforeggsurvivalintheGotlandBasinwaslowinbothyears,0%

and3%(p=0.496),respectively(Fig.6).Thehabitatsuitabilitywas improvedduetobothmorefavourableoxygenconditionsandto

increasedopportunitiesforremainingneutrallybuoyantwithinthe watercolumn(Fig.6;Table4).Temperaturehadnoimpactonegg survivalprobabilitiesastemperatureswerewithinpreferredpref- erencesforeggdevelopment(4–10^◦C;Hinrichsenetal.,2016b;Isa Wallin,personalcommunication)inallbasinsinbothyears.

4. Discussion

Inprinciple,eggspeciﬁcgravityofﬁsheggsisdeterminedbythe fractionalcontributionsofthemaincomponents,yolk+embryo, chorion and the perivitelline space between the chorion and vitellinemembrane(CraikandHarvey,1987;Kjesbuetal.,1992;

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1.0095 1.0105 1.0115 1.0125 1.0135 1.0145 1.0155 1.0165 1.0175 1.0185

0.0 10.0 20.0 30.0 40.0 50.0 60.0

KB_DK1_2015 KB_DK2_2015 KB_DK3_2015 1.0095

1.0105 1.0115 1.0125 1.0135 1.0145 1.0155 1.0165 1.0175 1.0185

0.0 10.0 20.0 30.0 40.0 50.0 60.0

KB_DK1_2011 KB_DK2_2011 KB_DK3_2011 1.0095

1.0105 1.0115 1.0125 1.0135 1.0145 1.0155 1.0165 1.0175 1.0185

0 10 20 30 40 50 60

BB_DK1_2011 BB_DK2_2011 BB_DK3_2014

IA IB II III IV

mean egg density (g cm-3) during ontogenc egg development unl ﬁrst hatch

Degree-days post ferlizaon

SD 25

b)

c) a)

SD 22

Fig.4. Meanvaluesofeggspecificgravityofflounder,Platichthysflesus,duringonto- genesisfromstageIAtostageIVandhatchingfromtwoICESsubdivisions(SD),25 (a)and22(bandc).DatainPanelBaretakenfromPetereitetal.(2014)andare fromFebruary/March.DatashowninPanelAandCarefromexperimentsinApril.

GovoniandForward,2008;SundbyandKristiansen,2015)withthe highwatercontentoftheyolkbeingakeyfactorcompensatingfor theheavycomponents,proteins,inthechorionandembryo;seee.g.

Jungetal.(2014)orSundbyandKristiansen(2015)forparameteri- zationoftherespectivecomponentfortheoverallESG.Inaddition tothehighwatercontentoftheyolk,alsooildroplets andyolk lipids,withlowerdensitythanseawater,maycontributetoegg buoyancyinsomespecies(Cerdàetal.,2007;GovoniandForward, 2008).

Forflounder,Platichthysflesus,intheBalticSea,ESG atearly stageIAvariedsignificantlybetweenareasinaccordancewiththe decreaseinsalinity,i.e. thehighest values(1.0152±0.0021and 1.0204±0.0006gcm⁻³;mean±sd)wereobtainedinthewestern parts,SD22andSD23,andthelowest(1.0096±0.0007gcm⁻³) intheeastern,SD26adSD28,i.e.allowingeggstoobtainneu- tralbuoyancy intherespectivespawningarea. ESG offlounder spawningdemersaleggs,sampledinSD25andSD28,isanexcep- tion withESG of 1.0161±0.0008gcm⁻³, a strategy adopted as spawningatlowsalinities(ca6–8psu)preventseggstoachieve buoyancy.SimilarvaluesofESG,1.0151–1.0157gcm⁻³,forfloun- derspawningdemersaleggshavebeenobtainin SD27and SD

29/32(Solemdal,1971,1973).Hence,obtainedresultssuggestthat theecotypewithpelagiceggsmaybeseparatedintothreesub- populations(SD22,SD24and25,andSD26and28,respectively), withdifferenteggcharacteristics(ESG)inaccordancewithsalin- ityconditionsintherespectivespawningarea,separatedfromthe ecotypewithdemersaleggs.N.B.datafromSD23wereexcluded in comparison due to few measurements.In SD 22 ESG varied between1.01116and1.01887gcm⁻³correspondingtoasalinityof ca14.3–24.2psu,i.e.thevariabilityinESGwassubstantial.Salin- ityintheareavariesbetweenca14and25psubutaccordingto datainPetereitetal.(2014)about60%ofﬂoundereggbatcheshad ahigherESGcomparedtothewaterdensity(measuredbyCTD- casts)in3outof6samplingareas.Similarresultswereobtained byvonWesternhagenetal.(1988),38of60eggbatches(65%)did notcorrespondtotheambientsalinity.TheareasSD22andSD23 aremixingareasbetweentheBalticandtheKattegat,withpoten- tiallymorethanonepopulationutilizingtheareaasdiscussedby Petereitetal.(2014),potentiallycausingthehighvariabilityinESG.

ObtainedESGforflounderintheBalticSeadiffersignificantlyfrom ESGofflounderatmarineconditions,ca1.0246gcm⁻³(Solemdal, 1971).Asevidentfromreciprocalexperiments,eggcharacteristics ofmarineandbrackishwatereggsremainessentiallyunchanged whenfisharetransferredfrombrackishtoamarineenvironment andviceversa;Solemdal(1971,1973)forflounderandNisslingand Westin(1997)forcod.Hence,withrespecttoeggcharacteristics fishintheBalticSeaformspecificpopulations.Asimilaradaptive patternisalsoassumedforspeciesspawninginthebrackishwater BlackSea,specificallybluefintuna,Thunnusthynnus(MacKenzie andMariani,2012).

AlthoughmultipleregressionanalysisshowedthatESGinBaltic ﬂounder wasrelated toboth eggdiameter (i.e. water content;

Thorsen et al.,1996)and eggdry weight (massofmainly proteins)withintherespectivesubpopulation/area,differencesinESG betweenareas/subpopulationswere relatedonly toegg diameter,withlargereggsintheeasternpartsoftheBalticSeathanin thewestern,whileeggdryweightwasthesameirrespectiveof area/subpopulation,includingdemersaleggs.Thisindicatesthat watercontentacquiredintheovaryduringoocytehydrationbya breakdownofyolkproteinstoFAAasosmoticeffectorsandacor- respondinginflowofwater(Thorsenetal.,1996;FinnandFyhn, 2010)mediatedbyaselectiveinfluxofwaterbychannelmem- braneproteins,aquaporins(Fabraetal.,2005;Cerdà,2009)isthe keymechanism.Thisprocessisconsideredhighlyregulatedand potentiallyspeciesspecific(Cerdà,2009)and,asshownbyThorsen etal.(1996),differencesinthedegreeofbreakdownofyolkpro- teinandthusintheosmoticpotential,resultinahigherinflowof waterduringhydrolysisincodeggsatbrackishwaterconditions comparedtoatmarineconditions.Concomitanttothis,theresults suggestthatthesameamountofmaterialisdepositedintheegg irrespectiveofareaand spawningstrategy, andthatdifferences inESGbetweensubpopulationsaredeterminedbydifferencesin thewatercontent,potentiallyduetodifferencesinthedegreeof degradationofyolkproteinsintoFAAduringhydrolysis.

Withintherespectivearea/subpopulationhowever,bothvari- abilityineggdiameterandineggdryweightdetermineESG,and resultindifferencesinverticaleggdistributionwithimplications for eggsurvival probabilities.Thisisin agreementwithstudies onotherspecies,e.g.forcod,intra-populationvariabilityinESGis causedbydifferencesinboththeamountofmaterialdepositedinto thechorion,andtothedegreeofuptakeofwaterduringhydration priortoovulation;i.e.tothewatercontent/eggdiameter(Kjesbu etal.,1992;Jungetal.,2014).

Incubation of eggs from early stage IA to stage IV showed that ESG maychangeduring ontogenyupto hatching; aslight decreaseinESGduringdevelopmentwasnotedfor eggbatches sampled in SD 22 and SD 25 in April, with an initial ESG of

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Fig.5. Depthprofilesshowingtemperature(^◦C;dottedline),oxygen(mll⁻¹;solidline)andsalinity(psu;dashedline)inICESsubdivisionsa)25,b)26andc)28(Fig.1)in April2014(totheleft)andApril2015(totheright),anddepthrange(greyhorizontalfield)andmeandepth(horizontalline)offlounder,Platichthysflesus,eggbatchesthat wouldobtainneutralbuoyancy(seeTable4);basedoneggspecificgravityandhydrographicdatafromCTD-castsintherespectivearea.

1.0104–1.0125gcm⁻³, whereas ESG for egg batches from SD 22 sampled in February–March, withan initial ESG of 1.0138- 1.0166gcm⁻³,remainedstableuntilstageIV.Theresultscontrast tothegeneralpictureofchangesinESGduringontogenyofmarine ﬁshes(Jungetal.,2014),i.e. aslightincreasein ESGuntilmid- gastrulation (stage II), followed by a gradual decrease in ESG, and ﬁnally an increase in ESG just prior to hatching. As con- cludedbyJungetal.(2014),changesinESGduringdevelopment areassociatedwithchangesintheyolk+embryofractionwitha correspondingchangein the perivitellinespace. Asopposed to thechorionwhichispermeabletowater,theyolkmembraneis impermeabletowaterfollowingthecorticalreactionatfertiliza- tionbutwithanincreasingpermeabilityduringeggdevelopment (Mangor-Jensen,1987),i.e.densityofthepervitellinespaceisin equilibriumwiththesurroundingenvironmentwhereasdensityof theyolk+embryochangesduringontogeny.Priortothegastrula- tionthereisadecreaseinthevolumeofyolk+embryo(i.e.water

content)andacorrespondingincreaseinvolumeofthepervitelline space,whichisreversedfollowinggastrulationasosmoregulation organsstarttofunction(Riis-Vestergaard,1987).Apartfromdif- ferencesbetweenspeciesinsizeofthepervitellinespace(e.g.in sardine,Sardinapilchardus,thepervitellinespaceoccupy>80%of theeggvolume,andca74%inEuropeaneel,Anguillaanguilla,comparedto<10%forthemajorityofpelagicﬁsheggs;Coombsetal., 2004;Sørensenetal.,2015), ambientsalinityconditions canbe expectedtoinvolvedifferencesin therelationbetweenvolume ofyolk+embryoandvolumeofthepervitellinespace.ForBaltic cod eggsincubatedat differentsalinities,ESG decreasedduring developmentinbatchesincubatedat10psubutincreasedwhen incubatedat17psuandinparticularat30psu(NisslingandVallin, 1996).Thus,changesinESGduringdevelopmentmayberelatedto initialESG.Beingneutrallybuoyantatlowsalinitiesmayinvolve lowerloss ofwater initiallyas thedifferencebetweenexternal and internal osmolality is low;internal osmolality correspond-

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0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

2014 2015 2014 2015 2014 2015

8 2 D S 6

2 D S 5

2 D S

Cumulave probability of survival and causes of death

Oxygen deﬁciency Sedimentaon Surviving

n=42 n=69 n=69

Fig.6.Assessedcumulativecausesofmortality(duetooxygendeficiencyorsedimentation)andsurvivalprobabilitiesofflounder,Platichthysflesus,eggsinICESsubdivisions (SD)25,26and28(Fig.1)inApril2014andApril2015,respectively.nrefertonumberofeggbatches.

ingtoasalinityofca11psu(SundbyandKristiansen,2015),i.e.

onlyaminordecreaseinthevolumeofyolk+embryo.Moreover, lowambientsalinitymayfacilitatewater uptakeaftergastrula- tion,i.e.anincreaseinthevolumeofyolk+embryo,influencing ESG.Furthermore,lowambientsalinityresultinlowerdensityof theperivitellinespacecomparedtoatmarineconditions(asthe chorionispermeabletoseawater,densityoftheperivitellinespace isequaltothedensityofambientwater)influencingtheoverall ESG.Despitethatthecontributionoftherespectiveeggcompo- nentwasnot studied,resultsofthepresent studysuggest that ambientsalinitymaybeofsignificanceforchangesinESGduring ontogeny;developmentatlowsalinities,ca13.5–16psu(Fig.4a andc)involvedadecreaseinESG,andineggbatcheswithaninitial ESG,correspondingto17.7–21.3psu(Fig.4b),ESGremainedmore orlessstableuntilstageIV(incontrasttowhatisknownforstudied speciesatmarineconditions;Jungetal.,2014).

MajorsalinewaterinﬂowsintotheBalticSearesultinimproved salinity and oxygen conditions (Matthäus and Franck, 1992;

Matthäusand Lass,1995)andthus increasedhabitatsuitability (increasedopportunitiestokeepbuoyantinthewatercolumnand decreasedriskofbeingsubjectedtolethaloxygenconditions)for fishwithpelagiceggs,as shownin earlierstudiesfor both cod (Nisslingetal.,1994;MacKenzieetal.,2000;Kösteretal.,2005) andflatfishes(Nisslingetal.,2002;Ustupsetal.,2013),aprereq- uisitefortheformationofstrongyearclasses.Ingeneral,themost favourableconditionsoccurintheBornholmBasincomparedto intheGdanskDeepandGotlandBasingiventherelativedistance fromtheshallowsillsinSD22andSD23.TheBalticSeaisheav- ilyaffectedbyeutrophicationand,asaconsequence,largepartsof thedeepareasareoxygendepletedwithregularlynegativeoxy- genvalues(Conleyetal.,2002;DiazandRosenberg,2008).This impliesthattheeffectofinfloweventsonhabitatsuitabilityfor fishreproductionnowadaysislimitedandtransitory, withsoon deterioratedconditions(seeMacKenzieetal.,2007).Thesituation isfurtheraggravatedbylessfrequentinfloweventsduringthelast decades,potentiallyaneffectofclimatechange(Meieretal.,2012).

Theinfloweventin2014improvedhabitatsuitabilityforflounder eggssignificantlyintheBornholmBasin(SD25)andtheGdansk Deep(SD26)butwasnotpotenttosignificantlyimproveconditions intheGotlandBasin(SD28).

Highly fluctuating conditions for egg survival of flounder (Hinrichsen etal., 2016b; present study) mayaffectboth stock abundanceanddistribution,althoughstockdevelopmentaddition- allyisinfluencedbye.g.retention/dispersalofearlystages(Petereit et al.,2014Hinrichsen et al., 2016a), conditionsin thenursery areas(e.g.IlesandBeverton,1998,2000),aswellasfishingmor- tality.PoorerconditionsforeggsurvivalintheGotlandBasinat present are in accordance withthe current stock development (seeHinrichsenetal.,2016b);decreasingCPUEinSD28butsta- ble CPUEin SD25 andSD 26.Evaluationof stockdevelopment ishoweverhamperedbytheoccurrenceofthetwofloundereco- typeswithdifferentspawningstrategiesandrequirementsforegg survival(Nisslingetal.,2002)andthusrecruitment.Accordingto ICES(2015)theecotypewithpelagiceggsconsistofthreediffer- entsubpopulationsoccurringinSD22–23,SD24–25andinSD26 andSD28,respectively,whereastheecotypewithdemersaleggs isconsideredasonestock,presentinSD27andinSD29–32.With respecttoeggcharacteristicsthepresentstudyconfirmsthesug- gestionoftwo populationsofflounderwithpelagiceggsin the BalticProper(oneinSD24andSD25,andanotherinSD26and SD28)separatedfromflounderintheBelt-Seas(SD22).Concern- ingtheecotypewithdemersaleggs, howeverthepresent study shows,incontrasttoassuggested(ICES,2015)thatthistypeof flounderoccursinbothSD25andSD28.Thattheecotypewith demersaleggsinhabitnot onlythenorthernparts oftheBaltic Proper;e.g.ESGofca1.0151–1.0157gcm⁻³inSD27andSD29/32 (Solemdal,1971,1973),i.e.withESGequaltotheresultsinpresent investigation(suggestingonehomogenouspopulation),butoccur alsointhesouthernBalticisconsistentwithearlierobservations;

demersalfloundereggshavebeenfoundattheOderBankandat theAdlergrundinSD24(e.g.MielckandKünne,1932).Thestock abundanceoftheecotypewithdemersaleggsinthesouthernparts is,however,poorlyknown.Anyway,thesuggestedstockstructure (ICES,2015)shouldberevisedtodisentanglestockdevelopment of therespective flounderecotype and forevaluation of stock- recruitment relationshipsof subpopulations. Further,presented dataofeggspecificgravity,andthusinverticaleggdistribution andeggsurvival,maybeusedtoestimatevariabilityintheviable eggproductionindifferentareas/subpopulations,i.e.contributeto forecast stockdevelopmentofBalticSeaflounderatpotentially