Incorporation of dietary carotenoids into the fins of yellow- and red-finned Eurasian perch Perca fluviatilis
Reiner Eckmann
∗, Romy Krammel, Dieter Spiteller
UniversityofKonstanz,FacultyofBiology,D-78457Konstanz,Germany
Keywords:
Astaxanthin Canthaxanthin Fincolour HPLCanalysis Rednessintensity
a b s t r a c t
Thecolouroftheventral,analandcaudalfinsofEurasianperchcanrangefrompaleyellowtointense red.Withinspatiallyseparatedpopulations,however,individualsareusuallyveryuniformincolour.
Wefedastaxanthin-andcanthaxanthin-enricheddryfeedtojuvenileperchfromayellow-finnedanda red-finnedpopulationtocomparetheinfluenceofdietarycarotenoidsonfincolourbetweenthesetwo populations.Inthiswaywewantedtotestwhetherfincolourinperchispredominantlyaphenotypically plastictraitorwhetherdifferentlycolouredindividualsrepresentcolourmorphs.Theventralfinsofperch fromthered-finnedpopulationalwaysexhibitedsignificantlymoreintenserednesswhentheirfeedwas supplementedwitheitheroneorbothcarotenoidadditives.Perchfrombothpopulationsdepositedmore canthaxanthinintheirventralfinsthanastaxanthin,red-finnedperchinparticular.Yellow-finnedperch probablyconvertedcanthaxanthininto-carotene,whichwasthedominantcarotenoidintheirventral fins,whereasthefinsofred-finnedperchcontainedonlytraceamountsof-carotene.Weconcludethat thesetwopopulationsrepresentcolourmorphsthatdifferfundamentallyintheirabilitytometabolise anddepositdietarycarotenoidsintotheirventralfins.Consideringthemultiplephysiologicalfunctions ofcarotenoids,thesefundamentaldifferencesincarotenoidmetabolismbetweenperchcolourmorphs mayhavefar-reachingconsequencesfortheperformanceofdifferentpopulations,forexampleintheir responsetoparasiteinfections.
1. Introduction
EurasianperchPercafluviatilisL.1758(hereafterperch)candif- fermarkedly infin colour, rangingfrompaleyellowtointense red(Pimakhin,2012).Perchwithredfinsseemtooccurmorefre- quently:inphotosuploadedbyFishbaseusers(FroeseandPauly, 2013)red-finnedperchoutnumberyellow-finnedperchby13:3 anda GooglesearchforEurasian perchimagesyieldsa ratioof 6:1.Withinpopulations,however,fincolourisoftenuniform.For example,inLakeConstanceuntilrecently,allperchhadpaleyel- lowfinswhilethoseinthesmalllakesnorthofLakeConstancehave orangeorbrightredfins.InSwedishforestlakesandintheRiver RhinenearBasel,however,yellow-andred-finnedperchco-occur (P.Hirsch,Univ.Basel,pers.comm.).Itisnotyetknownwhether fincolourinperchispredominantlyaphenotypicallyplastictrait whoseexpressionisinfluencedbyenvironmentalfactorssuchas
∗Correspondingauthor.
E-mailaddresses:reiner.eckmann@uni-konstanz.de(R.Eckmann), r.krammel1991@jahoo.de(R.Krammel),dieter.spiteller@uni-konstanz.de (D.Spiteller).
dietcompositionorwatercolourandturbidity,orwhetherdiffer- entlycolouredindividualsrepresentcolourmorphsthatdiffereven thoughtheyingestthesametypeofdietarycarotenoids.
Dietarycarotenoidscontributetotheyellow,orangeandred colourationoftheskin,finsandfilletinsalmonids (Shahidiand Brown,1998), buttheirinfluenceonthefincolourofperchhas notpreviouslybeenstudied.Watercolourorturbiditycaninflu- encethecolorationofperchasshownbyKekäläinenetal.(2010).
Intheleasthumic(i.e.mosttransparent)of fourborealFinnish lakes,perchhadthelightestandlesscolouredbellyandperchwere morecolourfulinthelittoralthaninthepelagichabitat,suggest- ingdivergentselectioninlittoralandpelagiczones.Howeverthe mechanismbywhichdifferentfincolorationisobtainedinthedif- ferenthabitatsremainsunknown.Consistentdifferencesincaudal fincolour betweenperchfromdifferentwaterbodieshave also beenobservedbyMairesseetal.(2005)whowereabletodiscrim- inateperchfromtheRiverRhineandLakeGenevabycaudalfin colour.Howeverthisstudyencounteredaconfoundingfactorin thatcaudalfincolourattributes,suchaslightness,chromaandhue, weresignificantlyrelatedtofishsize.
Theexistenceofcolourmorphsinthisspecieswassuggested recentlyinastudybyRochetal.(2015)whocomparedage-0perch
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-389799
https://dx.doi.org/10.1016/j.limno.2016.12.001
withpaleyellowishfinsandperchwithredfinssampledfromthe littoralofLakeConstance.Duringthefewdecadespriorto2010, yellow-finnedperchwerethesolephenotypeinLakeConstance (professionalfishermenandfishery scientists,pers.comm.), but inthat yearindividuals withredfinsbegan tobecaught.Roch etal.(2015)showedbymicrosatelliteanalysisthatthered-finned fishdifferedfromtheyellow-finnedindividualsandfromfishwith mixedfincolour,whichareprobablybackcrossesbetweenred-and yellow-finnedindividuals. Theseresultssuggest thatred-finned fisharenotacolour variantofthenativeperchpopulation, but morelikelytheprogenyofindividualsthathaveinvadedthelake fromotherwaterbodiesinthedrainagebasin.
Thefirstcluethatfincolorationinperchisinfluencedbydiet camefromapreliminaryfeedingexperimentinourlaboratoryin whichjuvenilesof a red-finnedpopulationreceiveddietscom- prising either chironomid larvae or lake zooplanktonfor eight weeks(Eckmann,unpubl.data).Fishfeedingonchironomidlarvae hadpale-orangefins whereasthefinsof those feedingonzoo- planktonwereintenselyorange-red,theanalandventralfinsin particular.Followingthispreliminarystudy,twoexperimentswere designedtocomparetheinfluenceofdietarycarotenoidsonfincol- orationbetweenared-andayellow-finnedpopulationofperch.As astaxanthinandcanthaxanthinarethemainchromophorecom- poundspresentinperchfins(Czeczuga,1979b),weusedthesetwo carotenoidsasfeedadditives.Inthefirstexperiment,fishwerefed adryfeedsupplementedwithbothcarotenoids,andinthesecond experimentdifferenttreatmentgroupsreceiveddryfeedssupple- mentedwitheitherastaxanthinorcanthaxanthin.Itwastheaim ofourstudytotestthenullhypothesisthatperchfromyellow- andred-finnedpopulationsdevelopthesameventralfincoloration whenfeedingondietswithidenticalcarotenoidcontent.
2. Materialandmethods
Thered-finnedperchusedinthisexperimentoriginatedfrom LakeKarsee(Kperch),asmall(3ha)lakenorthofLakeConstance.
Theyellow-finnedperchoriginatedfromLakeConstance(Cperch), thesecond-largest(536km2)perialpinelakenorthoftheEuropean Alps.Spawnersfrombothpopulationswerekeptseparatelyinout- doormesocosmswheretheyspawnedvoluntarilyfrommid-April tomid-May.Eggstrandswerecollectedfromthemesocosmsand incubatedinthelaboratory.LarvaewererearedonArtemianauplii forfourweeksandthenweanedtoadrydiet(Inicio917,BioMar) whichtheyreceiveduntiltheexperimentsstarted.
2.1. Experiment1
In October 2013, two aquaria of 85L volume were stocked with30age-0 Kpercheach anda furthertwoaquaria with30 age-0Cpercheach. Allfourgroups received a formulated diet supplementedwith50mgeachofastaxanthinandcanthaxanthin (LucantinTMPinkandLucantinTMRed,BASF)per1000gofpellets.
Thisisaconcentrationofcarotenoidscommonlyusedinfeeding studieswithsalmonids(ShahidiandBrown,1998).Asoursisthe firststudyinwhichcarotenoidsareaddedtothedietofperch,we adoptedthesevaluesasaguideline.Theadditivesweregroundina mortarwithasmallquantityofpellets,andthepowdermixedwith 1000goffeed.Themixturewaswettedwithwater,mixedthor- oughlytoapulp,passedthroughameatmincerwith2mmbore diameter,anddried.Thefishlivedat20±0.2◦C with14hlight perday.TheaquariaweresuppliedwithtapwaterfromtheCity ofConstancewaterworksat arateimplyinga theoreticalwater renewaltimeof 2–3h, and foodwasprovided adlibitumtwice perday.Afterthree months,onetankgroupofeachpopulation wasswitchedontoanon-supplementeddryfeed(codes:KCar/df
andCCar/df,respectively)whilethesecondgroupofeachpopula- tioncontinuedtoreceivethesupplementeddiet(codes:KCarand CCar,respectively).Theexperimentsendedafterfivemonths.
Samplingtookplaceatthestartoftheexperiment,afterthree monthsandattheendoftheexperiment,withtenfishremoved fromeachaquarium,euthanizedwithanoverdoseofMS222and photographedinventralaspectwithacolour referencechartin eachpicture.Photosweretakenunderawhitecanvassoftboxillu- minatedwithvideofloodlightstoeliminatesurfacereflectionsand hardshadows.Theventralfinswerecutoffatthebases,blotteddry, weighedto0.1mgandstoredat−20◦Cuntilfurtherprocessing.
2.2. Experiment2
InApril2014,asecondexperimentbeganusingfishfromthe same sourcebatches as those used in experiment 1. Four 85L aquariawere each stocked with30 Kperchand a furtherfour aquariawith30Cpercheach.Twotank-groupsfromeachpop- ulationreceivedaformulateddietsupplementedwith100mgof astaxanthinper1000gofpellets(codes:KAst1/2andCAst1/2, respectively),and theothertwogroups receivedadiet supple- mentedwith100mgofcanthaxanthinper1000g(codes:KCan1/2 andCCan1/2,respectively).Theexperimentalconditionswerethe sameasinexperiment1.Photosandfinsamplesoftenfishfrom eachaquariumweretakenatthestartoftheexperimentandafter nineweeksattheend.
2.3. Rednessintensity
PhotoswereevaluatedwithImageJ(http://rsbweb.nih.gov/ij/) usingtheRGBcolourmodel.Luminosity,i.e.theoverallbrightness ofaphoto,wascalculatedbyconvertingeach pixeltograyscale usingtheweightedRGBconversion.Thebrightnessofallphotos wasauto-adjustedsothattheaveragegreyvalue(range0–255)of thewhitereferencechartexceeded240.Anellipticalselectionof themostintenselycolouredpartofeachventralfinwasmarked, andtheaverageredandgreyvaluesofthisselectionnotedalong withthoseofthewhiteandredstandardcharts.Fincolourwas scoredaccording totheredness intensity, I,of theselected fin region,whichwascalculatedasI=100*(F-W)/(R-W)accordingto VillafuerteandNegro(1998),whereF,WandRarethequotients ofaverageredandgreyvaluesofthefinarea,thewhitestandard andtheredstandardchart,respectively.Thisgivesastandardized rednessvaluerangingfrom0to100.Inbothexperiments,right andleftventralfinsofallsampledfishmatchedintermsofredness intensity(t-testforpairedvalues,p>0.05inallcases).Therefore, meanvaluesforeachfinpairwereusedinfurtheranalyses.
2.4. High-performanceliquidchromatography(HPLC)
FortheanalysisofcarotenoidsinventralfinsbyHPLC,deep- frozenfinsweregroundinliquidnitrogeninamortar.Carotenoids were extracted withacetone,dried over Na2SO4 and the sam- pleswerecentrifugedfor6–12minat13000rpm.Thesupernatants weretransferredintoHPLCvials,evaporatedunderanitrogenflow, andthevialswerestoredat−20◦C.ForHPLCanalysiscarotenoids werere-dissolvedin0.2mLacetoneandseparatedonanAgilent ZorbaxEclipse XDB-C8column (150mm×4,6mm, 5m) using anAgilent1100HPLCsystemwithadiodearraydetector.HPLC conditionswere:solventA:H2O0.1%aceticacid,solventB:ace- tonitrile;HPLCprogramme:gradientelutionfrom70%Bto100%B in10min,100%Bfor15min;flowrate:1mL/min,diodedetector range280–550nm;theUVtraceof462nmwasusedforquantita- tiveanalysis.Injectionvolumesrangedfrom5to50L.Astaxanthin (retentiontime:8.3min),canthaxanthin(retentiontime:11.5min) and -carotene(retentiontime: 21.5min) (BASF)were usedas
Table1
Totallength(cm)andredness(cf.Materialsandmethodsfordefinitionofredness)of theventralfinsofjuvenileEurasianperchfromLakeConstance(C)andKarsee(K) afterthreeandfivemonthsofadlibitumfeedingondryfeedenrichedwith50mg/kg ofastaxanthinandcanthaxanthin(10fishanalysedpertreatment).Thefishreceived thecarotenoid-enrichedfeedforfivemonths(Car)orforthreemonthsfollowedby twomonthsonnon-enricheddryfeed(Car/df).Valueswiththesamesuperscript indicatenon-significantdifferenceswithincolumns.Significancelevelsweresetat P<0.05inallcomparisons.
Treatment Totallength(cm) Redness
3mo 5mo 3mo 5mo
C Car 10.1±0.7b 13.2±0.7bc 14.5±5.9b 23.1±5.1bc CCar/df 10.3±0.8b 13.1±1.2c 12.5±2.9b 13.9±2.1c KCar 11.4±1.3a 14.5±1.2a 57.8±22.1a 74.5±14.6a KCar/df 11.1±1.3ab 14.1±1.3ab 60.5±17.1a 55.1±12.2ab
standardsandtogeneratecalibrationcurvesforthequantitative analysisofextractedcarotenoids.
2.5. Dataanalysis
Start values of total length and of redness intensity were comparedbetweenpopulationsbyt-test.Treatmentmeanswere compared with ANOVA when test assumptions of normality (Shapiro-WilkWtest)and homoscedasticity(Bartletttest)were fulfilled,otherwiseKruskal-Wallis-ANOVA(KW-ANOVA)wasused.
Multiplecomparisons ofmeanswere conductedwithStudent’s tonthedifferenceacrosseverypairoflevelsorwithNemenyi- test,respectively.Resultswereconsideredstatisticallysignificant atP<0.05.TestswerecalculatedusingJMP(SASInstituteInc.Cary, NorthCarolina,USA)version7.0.1.orSTATeasy(Wissenschaftliche Auswertungen,Hamburg,Germany),version2009.
3. Results 3.1. Experiment1
Atthestartoftheexperiment,Kperchweresignificantlylarger thanCperch(7.0±0.7vs.6.4±0.6cmtotallength;t-test,P<0.05) probablybecauseKperchspawnedandhatchedaboutthreeweeks earlier.AfterthreemonthsKperchcontinuedtobelargerthan Cperch,andthesamewastrueafterfivemonthswhenonetreat- mentgrouppermorphhadbeenswitchedtoanon-enrichedfeed (Table 1).Thesedifferenceshowever, couldonlypartly becon- firmedstatistically(ANOVA,F3,34=3.02,P=0.043andF3,36=3.67, P=0.021,respectively).
RednessintensitywasalreadysignificantlygreaterforKperch thanforCperchatthebeginningoftheexperiment(4.3±1.4vs.
3.3±0.8;t-test,P<0.05).Afterthreemonths,rednessintensitywas significantly greater for Kperchthan for Cperch (KW-ANOVA, H=27.5>2=7.81;Table1).Afterfivemonths,rednessintensity hadincreasedfurtherinthetwogroupsreceivingthecarotenoid- enrichedfeedbutnotinthegroupsthatfedonnon-enrichedfeed forthelasttwomonths.Evenso,Kperchstillexhibitedsignificantly moreintenserednessthanCperchthatreceivedthesametypeof feed(KW-ANOVA,H=33.6>2=7.81;Table1;Fig.1a+b).
Astaxanthin was only foundsporadically in the ventralfins ofbothmorphs,exceptKperchthathadfedforfivemonthson carotenoid-enrichedfeed,whosefinsconsistentlycontainedlow amountsofastaxanthin.CanthaxanthinwasdetectedinCperch inverysmallamounts,butwasthedominantcarotenoidinven- tralfinsofKperch(Table2).Afterswitchingtonon-enrichedfeed, theconcentrationofcanthaxanthininthefinsofKperchdidnot changesignificantlybutinthegroupthatcontinuedtofeedona carotenoid-enricheddiet,theconcentrationincreasedaboutthree- fold (ANOVA, F3,16=3.64, P=0.036; Table 2).Beta-carotene was
Fig.1.VentralviewsofjuvenilePercafluviatilisfromtwospatiallyseparatedpopula- tionsfedwithdryfeedssupplementedwithcarotenoids.a)Karseeperchfedforfive monthswith50mg/kgastaxanthin-and50mg/kgcanthaxanthin-supplemented feed.b)LakeConstanceperchreceivingthesametreatment.c)Karseeperchfedfor nineweekswith100mg/kgastaxanthin-supplementedfeed.d)Karseeperchfedfor nineweekswith100mg/kgcanthaxanthin-supplementedfeed.e)LakeConstance perchfedfornineweekswith100mg/kgastaxanthin-supplementedfeed.f)Lake Constanceperchfedfornineweekswith100mg/kgcanthaxanthin-supplemented feed.Bars:10mm.
Table2
Thecontentofastaxanthin,canthaxanthinand-caroteneintheventralfinsof Eurasianperchafterthreeandfivemonthsofadlibitumfeedingonacarotenoid- enricheddryfeed.Valuesaregiveningcarotenoidpergfreshweightandare presentedasmeans±S.D.forfivefish.WhenavaluewithoutS.D.isgiveninatable cell,thenonlyoneoutoffivefishcontainedthatparticularcarotenoid.Valueswith thesamesuperscriptindicatenon-significantdifferencesbetweencanthaxanthin valuesforKperchafterthreeandfivemonths,andbetween-carotenevaluesfor Cperchafterthreeandfivemonths.SignificancelevelsweresetatP<0.05inboth comparisons.FortreatmentcodesrefertoTable1.
Treatment Astaxanthin Canthaxanthin -carotene
3mo 5mo 3mo 5mo 3mo 5mo
CCar 0 0 0.1±0.1 0.1±0.1 2.2±0.8a 4.7±2.7a
CCar/df 0.3 0.2 0 3.0±1.5a 1.2±0.5a
KCar 0.8 0.4±0.3 4.8±6.1b 12.1±6.8a 0 0 KCar/df 0.4 0.2 4.1±2.9b 2.9±1.7b 0 0
notfoundinKperchfinsbutwasthedominantcarotenoidinthe ventralfinsofCperch.InbothCperchgroupstheconcentrations of-carotenefoundafterfivemonthsdidnotdiffersignificantly fromthosemeasuredafterthreemonths(Table2),eventhoughthe concentrationinthegroupthatcontinuedtofeedonacarotenoid- enricheddietwasaboutfourfoldhigherthaninthegroupthatfed thenon-enricheddietduringthelasttwomonths(KW-ANOVA, H=8.42>2=7.81).
Table3
Totallength(cm)andredness(cf.Materialsandmethodsfordefinitionofredness)of theventralfinsofjuvenileEurasianperchfromLakeConstance(C)andKarsee(K) afternineweeksofadlibitumfeedingoncarotenoid-enricheddryfeeds.Thedryfeed wassupplementedwitheither100mg/kgofastaxanthin(Ast)orcanthaxanthin (Can).Thereweretworeplicatespertreatment(10fishanalysedperreplicate)and theresultsaregivenasmeans±S.D.Valueswiththesamesuperscriptindicatenon- significantdifferences.Foreachvariablethegroupsreceivingthecanthaxanthin- enrichedfeedarecomparedinthefirstcolumnbelowthevariablename,those receivingtheastaxanthin-enrichedfeedarecomparedinthesecondcolumn,and thefourgroupsofLakeConstanceorKarseepercharecomparedwitheachotherin thethirdcolumn.SignificancelevelsweresetatP<0.05inallcomparisons.
Treatment Totallength(cm) Redness
CAst1 10.4±0.7a b 17.4±3.6b b
CAst2 10.6±0.7a b 16.4±2.5b b
C Can1 12.2±0.5a a 25.9±1.9b a
C Can2 10.6±0.7b b 23.3±3.7b a
KAst1 10.5±0.8a b 57.9±8.8a a
KAst2 9.6±0.6b c 42.7±9.0a b
KCan1 10.9±0.5b b 33.8±8.6a c
K Can2 12.4±0.6a a 33.4±7.9a c
3.2. Experiment2
At the start of the experiment, total body length did not differ between Kperch and Cperch (7.3±0.5 vs. 7.2±0.9cm;
t-test, P>0.05). Afternine weeks total body length showedno consistentpattern betweenperch morphs feeding oncanthax- anthin(ANOVA,F3,36=22.68,P<0.0001)orastaxanthin(ANOVA, F3,36=4.13, P=0.0129) aswell asamong perchmorphs feeding ontwo different enriched feeds (Cperch: ANOVA,F3,36=15.45, P<0.0001; Kperch: ANOVA, F3,36=33.55, P<0.0001). For both perchmorphs,however,thelargestfishwerefoundingroupsfeed- ingoncanthaxanthin-enricheddiets(cf.secondcolumninTable3).
Redness intensity (I) was significantly higher for Kperch than for Cperch at the beginning of the experiment (6.5±2.2 vs. 4.6±1.4; t-test, P<0.05). After nine weeks, Kperch exhib- ited significantly greater I values than Cperch feeding on bothastaxanthin-andcanthaxanthin-enrichedfeed(KW-ANOVA, H=31.60>2=7.81andH=15.11>2=7.81,respectively;Table3).
Among Cperch, fish feeding on canthaxanthin-enriched feed exhibitedsignificantlygreaterrednessintensitythanthosefeed- ingonastaxanthin-enrichedfeed(ANOVA,F3,36=23.34,P<0.0001) whereasinKperchfinswereredderinfishfeedingonastaxanthin- enrichedfeed,(ANOVA,F3,36=17.93,P<0.0001;Fig.1,c–f).
AstaxanthinwasfoundintheventralfinsofallanalysedKperch receivingtheastaxanthin-enrichedfeed,whereasinallothertreat- mentgroupsthiscarotenoidwasonlyfoundoccasionally(Table4).
Canthaxanthinwasfoundconsistentlyinallgroupsreceivingthe canthaxanthin-enricheddiet,andtheaveragecontentofKperch ventralfinswasnearlytenfoldgreaterthaninCperch(KW-ANOVA, H=15.46>2=7.81).Inthegroupsreceivingastaxanthin-enriched feed,canthaxanthinwasfoundinonlyafewfish(Table4).Beta- carotenewasnotdetectedineitherCAstorinKAstgroups,nor wasit foundin sevenoutoftenKCan fish.Cperchfeedingon canthaxanthin-enrichedfeed,however,hadconsistentlyhighcon- centrationsof-caroteneintheirventralfins(Table4).
4. Discussion
Theperchpopulationscomparedinthesetwoexperimentsdif- feredmarkedlyinventralfincolorationwhenfeedingonthesame carotenoid-enricheddry feeds,inthatKperchalwaysexhibited moreintenserednessthanCperch.Thenullhypothesisthatperch fromyellow-andred-finnedpopulationsdevelopthesameven- tralfincolorationwhenfeedingondietswithidenticalcarotenoid contentmustthereforeberejected.Thisresultsuggeststhatthedif- ferenceinventralfincolorationandcarotenoiddepositionbetween
yellow-andred-finnedperchhasageneticbasis,perhapssimilar tothatpreviouslyshowntoinfluencevariablepigmentationand colorationinsalmonids(Bjerkeng,2000;Choubertetal.,1995;Rye andGjerde,1996;Withler,1986;WithlerandBeacham,1994).In contrasttosalmonids,dietarycarotenoidsapparentlydonotinflu- encefleshcolourinperch.Whenadultyellow-finnedperchwere fedonbroodstockfeedfortroutwithahighcarotenoidcontentfor severalmonths,wedidnotobserveanychangeinthefish’spale- whitefleshcolour(Eckmann,unpublisheddata),andinthepresent studywedidnotobservebygrossinspectionanydifferenceinflesh colourbetweenperchfromdifferenttreatmentseither.
Earlierwork hassuggested thatgrowth rate,as a proxy for uptakeofdietarycarotenoids,couldexplaindifferencesinventral fincoloration.Mairesseetal.(2005)reportedthatcaudalfincolour inEurasianperchwassignificantlyrelatedtofishsize,andasimilar positive relationshipbetweenbody sizeand carotenoiddeposi- tionwasreportedforsalmonidsbyStorebakkenandNo(1992).At theendofourexperiments,however,bodylengthsofKperchand Cperchdifferedbyonlyaround1cminexperiment1(Table1),and showednoconsistentpatterninexperiment2(Table3),whereas inthestudyofMairesseetal.(2005),fishdifferedbyupto20cmin standardlength.Furthermore,inexperiment1afterfivemonthsof feedingCperchwerearound2cmlongerthanKperchafterthree months,yetthesmallerKperchhadhigherrednessintensitythan thelargerCperch(Table1).Fishsize,therefore,isnotaconfound- ingfactorinourstudy,suggestingthatouryellow-andred-finned populationsrepresentcolourmorphswithmarkeddifferencesin carotenoidmetabolismand/orincorporationintotheirfins.
The analysis of fin carotenoid content revealed pronounced differencesbetweenthetwopopulations.Whenthedryfeedcon- tainedcanthaxanthin,Kperchdepositedconsiderableamountsof thiscarotenoidintheirventralfins,betweenone(experiment2) andtwo(experiment1)ordersofmagnitudemorethanCperch.
Supplementaryastaxanthinwasincorporatedconsistently,albeit insmallquantitiesbyKperch,afternineweeksinexperiment2 andafterfivemonthsinexperiment1.InCperchfins,astaxanthin wasfoundonlysporadically.Atlanticsalmonand rainbowtrout areknowntoutilizeastaxanthin moreefficientlythan canthax- anthinforflesh pigmentation(StorebakkenandNo,1992).Both colourmorphsofperch,however,utilizethesecarotenoidsforfin colorationinadifferentway,favouringcanthaxanthinoverastax- anthin.
Themoststrikingdifferencebetweenthetwopopulationsisthe
-carotenecontentoftheventralfins.Whilethiscarotenoidwas completelyabsentinKperch,itwaspresentinlargeamountsin Cperchconsumingdryfeedwithcanthaxanthin.Hence,Cperch likely converted canthaxanthinin -carotene, probablyusing a metabolicpathwaythathaspreviouslybeendetectedintheskin andliverofrainbowtrout(Guillouetal.,1989).Feedinglabelled carotenoidstoperchmighthelptosubstantiatethesuggestedcon- versionofcanthaxanthinin-carotene.
Whenthefishwereswitchedfromcarotenoid-enrichedtonon- enrichedfeedinexperiment 1,therewasnochangeinredness intensity in either K or Cperch. By contrast, carotenoid con- tentdecreasedaftertwomonths,mostnotablyforcanthaxanthin and-carotene,butunfortunatelythesedifferencescouldnotbe confirmedstatisticallyasvarianceswereratherhigh.Inrainbow trout musclecarotenoid concentrationhad not decreased after 85days of starvation (Choubert, 1985). The marginal decrease of carotenoid concentration in perch ventralfins suggests that carotenoidsdepositedintoperchventralfinsarenotpermanently incorporatedandmightbeatleastpartlyremobilizedataslow rate.Longerperiodsofcarotenoiddeprivationareneededtogain betterinformationonthepossibleremobilizationofcarotenoids fromperchventralfins.
Table4
Thecontentofastaxanthin,canthaxanthinand-caroteneintheventralfinsofEurasianperchafternineweeksofadlibitumfeedingoncarotenoid-enricheddryfeed.Values aregiveningcarotenoidpergfreshweightandarepresentedasmeans±S.D.forfivefishperreplicate.Whenlessthanfivefishperreplicatecontainedtherespective carotenoid,replicatesweremergedandthecombinednumberoffishcontainingthecarotenoidisindicated.Thesetreatmentswereomittedfromstatisticalcomparisons.
Valueswiththesamesuperscriptindicatenon-significantdifferenceswithincolumns.SignificancelevelsweresetatP<0.05inallcomparisons.Fortreatmentcodesreferto Table3.
Treatment Astaxanthin Canthaxanthin -carotene
CAst1 0.2±0.1(n=3/10) 0.1±0.2(n=4/10) 0
CAst2 0
CCan1 0.1±0.1(n=5/10) 0.7±0.2b 4.1±1.6a
C Can2 1.2±0.5b 5.8±2.2a
KAst1 0.5±0.1a 0.1±0.1(n=3/10) 0
KAst2 0.2±0.1b 0
KCan1 0.1±0.1(n=4/10) 12.2±5.3a 0.8±2.1(n=3/10)
KCan2 6.4±4.3ab
Astaxanthinseemstoprovidemoreintenserednessthancan- thaxanthininKperch.Inexperiment2,astaxanthin-supplemented feed led to greater redness intensity than canthaxanthin- supplemented feed (Table 3) even though the canthaxanthin contentofKperchventralfinswasmorethananorderofmag- nitudegreaterthantheircontentofastaxanthin(Table4).Similar resultshavebeenobtainedintrout,whereastaxanthinledtomore intenserednessofthefleshthancanthaxanthinatcomparablecon- centrations(SkredeandStorebakken,1986;Skredeetal.,1989).
Beta-carotene hasfull vitamin A activityin fish, while both astaxanthin andcanthaxanthin arevitamin Aprecursors (Gross and Budowski, 1966). Howeverdetails ofthe furtherbiological rolesofcarotenoidsinfishareonlypartlyunderstood(Shahidiand Brown,1998).Otherphysiologicalfunctionscommonlyattributed tocarotenoidsincludetheirabilitytoabsorbpotentiallydamaging radiationandtoquenchsingletoxygenandthusserveasantiox- idants(Tacon,1981).Highlevelsofcarotenoidshavebeenlinked tobetterresistancetobacterialandfungaldiseasesinsalmonids (Czeczuga,1979a),andtoincubation-survivalanddiseaseresis- tanceineggsofOncorhynchustshawytscha(Tyndaleetal.,2008).
Giventhesepotentiallybeneficialeffectsofcarotenoids,theques- tionariseswhetheryellowandredcolourmorphsofperchdifferin physiologicalperformanceaswellasfincolour.Inarecentstudy, Rochetal.(2015)showedthatthetwocolourmorphsnowresident inLakeConstancediffer intheirsusceptibilitytomacroparasite infection.Red-finned specimens andfish withmixedfin colour showed better resilience to the pike tapeworm Triaenophorus nodulosusandthegillwormAncyrocephaluspercae.Theseresults suggestthatparasitesusceptibilitymayberelatedtodifferencesin carotenoidmetabolismbetweenperchcolourmorphs.
Inconclusion,fincolourisnotsimplyaphenotypicallyplastic traitthatisinfluencedbyenvironmentalfactorssuchasdietcom- positionorwatercolourandturbidity,butyellow-andred-finned Eurasianperchrepresentcolourmorphsthatdifferfundamentally intheirabilitytometaboliseanddepositdietarycarotenoidsinto theirventralfins.Differencesincarotenoidmetabolismmayhave unexploredbutfar-reachingconsequencesforperchperformance, forexampleinresistingparasiteinfection.
Acknowledgements
Dr.W.Pelletier,BASF,kindlyprovidedthefeedcarotenoids.H.
Thieletookcareoftheperchspawnersandsuccessfullyweaned perchfry todry feed.M.Schmidtookcare ofthe juvenilefish stockandhelpedduringtheentirecourseoftheexperiments.We thankA.-J.Beerforlanguagecorrectionandimprovementofthe manuscriptand twoanonymous refereesfortheirhelpfulcom- ments.
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