ContentslistsavailableatScienceDirect
Applied
Animal
Behaviour
Science
jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a p p l a n i m
Dietary
fatty
acid
composition
significantly
influenced
the
proactive–reactive
behaviour
of
Senegalese
sole
(
Solea
senegalensis
)
post-larvae
Zohar
Ibarra-Zatarain,
Sofia
Morais,
Kruno
Bonacic,
Cindy
Campoverde,
Neil
Duncan
∗IRTA,SantCarlesdelaRàpita,CarreteradePobleNou,km5.5,E-43540SantCarlesdelaRàpita,Tarragona,Spain
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received14April2015
Receivedinrevisedform22July2015 Accepted10August2015
Availableonline24August2015
Keywords: Soleasenegalensis Stress
Personality Nutrition Dietarylipids Fishoil
a
b
s
t
r
a
c
t
Fewstudieshaveexaminedtheinfluenceofdietonlarvalproactive–reactivebehaviouraldimensionof stresscopingstyleresponses.Thepresentstudyevaluatedtheinfluenceofusingdifferentvegetablesoils (Linseed;Soybean;Olive)andfishoil(Codliver)forArtemiametanaupliinutritionalenrichmentonthe proactive–reactivebehaviouralresponsesofSenegalesesole(Soleasenegalensis)post-larvae(40dayspost hatch).Forty-twoSenegalesesolelarvaefromeachofthefourreplicatetankspertreatmentweretested. Twotestswereperformed:anewenvironmentindividual-basedtest,whichevaluatethelarvaelatency timetomove,totalactivitytimeandtotaldistancemoved;andariskgroup-basedtest,whichconsisted inevaluatingthelarvalcapacitytocrossfroma“comfort”zonetoa“risk”zone.Inthegroup-basedtest, proactive,intermediateandreactiveindividualswereidentifieddependingonthetimetakentocross betweentwozones.LarvaefedwithArtemiametanaupliienrichedwiththecodliveroilemulsionwere significantly(P=0.01)largerandintheindividual-basedtestpresentedsignificantlyhighertotalactivity time(P=0.08)andtotaldistancemoved(P=0.01)thanlarvaefromtheotherdietarytreatments.No sig-nificantcorrelations(P>0.05)wereobservedbetweenlarvaetotallengthandlatencytimetomove,total activitytimeortotaldistancemovedacrossalltreatmentsorwithinanydietarytreatment.Inthe group-basedtest,fishfedwithArtemiaenrichedwiththecodliveroilemulsionpresentedasignificantlyhigher proportionofproactivelarvae(P=0.02)andthelowerproportionofreactivelarvae.Thepresentstudy showedforthefirsttimethat(i)Senegalesesolepresentedadefinedproactive–reactivebehaviourfrom earlyontogenesisand(ii)dietaryfattyacidcompositionsignificantlyinfluencedtheproactive–reactive behaviouraldimensionofstresscopingstyleofsolelarvae.Thecurrentstudyhaspracticalimplications thatopenthepossibilitytoproduceorganismsthathavebehaviouralstylesthatcouldultimatelyresult inimprovedaquacultureproductivity.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Animals including fishwhen confronted with a threatening or stressful situationhave beenrecognized tohave two differ-entbehaviouralresponses,respectively namedasproactiveand reactive(Koolhaasetal.,1999;Øverlietal.,2007).So-called proac-tivefishhavebeencharacterizedtohavefight-flightbehavioural responseandwereobservedtoexploreunfamiliarenvironments andtakerisks(Bell,2005;Brelinetal.,2005;Koolhaasetal.,2007; Castanheiraetal.,2015).Bycontrast,reactivefishhavebeen char-acterizedtofreezeorhideandgenerallyhaveloweractivity,avoid
∗Correspondingauthor.
E-mailaddress:[email protected](N.Duncan).
riskandtendtostayimmobilewhensubmittedtonovel environ-ments(Brelinetal.,2005;Koolhaasetal.,2007;Tomsetal.,2010; Castanheiraetal.,2015).Physiologically,proactivefishwere char-acterized by a low hypothalamus–pituitary–adrenal/interrenal (HPA/HPI)axisactivity,leadingtolowpost-stresslevelsof gluco-corticoids,incontrasttoreactivefish,whichwerecharacterizedby ahigherHPA/HPIresponseandlevelsofglucocorticoids(Koolhaas etal.,2010;Conradetal.,2011).Togetherthebehaviouraland phys-iological dimensionscombined witha consistencyin responses over timeor contextshavebeendescribed asthestresscoping styleofanorganism(Koolhaasetal.,1999;Conradetal.,2011; Castanheiraetal.,2015).Adiverserangeofbehaviouraltestshave beenusedtoidentifythebehaviouraldimensionofstresscoping style responsesin teleostfish, suchasreaction toconfinement (Brelin et al., 2005), feedingmotivation after beingtransferred intoanovelenvironment(Mota-Silvaetal.,2010),inter-individual
aggression(Ruiz-GomezandHuntingford,2012),predatory situa-tions(Archardetal.,2012)andgroupbasedtests(Bell,2005;Wilson andGodin,2009;Castanheiraetal.,2013).
Theperformanceoffishwithdifferentstresscopingstyleshas beenreportedfordifferentfishspecies.Inacaptiveoraquaculture typeenvironment proactiveorganisms weregenerallyfoundto presenthighergrowth(Mas-Mu ˜nozetal.,2011),lowerdisease sus-ceptibility(MacKenzieetal.,2009),lowerlatencytimetorecover andfeedafteradisturbance(Øverlietal.,2007)andweremore dis-posedtofollowroutinesignoringnovelchangesintheenvironment (Ruiz-Gomez etal., 2011),which are characteristicsthat would favouraquacultureproduction.Thus,studyingthebehaviour dur-inglarval stagesof fishspecies rearedin aquaculturemightbe ofgreatinterest,inordertoestablishtheperiodofdevelopment ofbehaviouralcharacteristics,factorsthatinfluencethe develop-mentand theconsequences ofpresenting differentbehavioural traitsongrowth,performanceanddevelopment.Amongvarious factorsassumedtoinfluencelarvalsurvival,growthandquality, theimportanceofearlylarvalnutrition,especiallydietarylipids and essential fattyacids (EFA),have been highlighted in many studies(Izquierdoetal.,2000;Sargentetal.,2002).Diverse stud-ieshave addressed the effectof dietson fishlarvae behaviour (e.g.swimmingspeed,escapereaction,etc.)inseveralfishspecies suchasgiltheadseabream(Sparusaurata)(Benítez-Santanaetal., 2007),blackseabass(Centropristisstriata)(Rezecketal.,2010)and pikeperch(Sanderlucioperca)(Lundetal.,2013).Moreover, dif-ferentauthorshaveindicatedthatvegetableoilsand/orfishoils, usedtoimprovethenutritionoflivepreys,mayinfluencethefish growth,fattyacidbody composition, geneexpression and neu-ronalactivity(Monteroet al.,2003; Salesand Glencross,2010; Benítez-Dortaetal.,2012;Benítez-Santanaetal.,2014).Studies ontheeffectsofvegetableoilsonalllifestages(larval,juvenile: pre-ongrowingandadult:ongrowingandbreeders)ofaquaculture species are required as the replacement of fish oils with veg-etableoilsis increasingrapidlytoincreasethesustainability of theaquacultureindustry(Sargentetal.,2002;Nayloretal.,2009). Nonetheless,thequestionofhowthesedietarynutrientsandthe sourcesmighthaveanimpactonlarvaebehaviouraldimensionof stresscopingstylehas,toourknowledge,receivedlittleattention. Senegalesesole (Solea senegalensis) is a flatfishspecies that hasgreatinterestandpotentialforaquaculturediversificationin Europe.Oneoftheadvantages ofthisspeciesforaquacultureis thatcomparedtoothermarinespecieslarvalrearingisnot com-plicatedandlarvaepossesshighgrowthrateandsurvival(Morais etal.,2014).Furthermore,solehavebeenfoundtobeparticularly resilienttohandlingstressduringthelarvalandearlypost-larval stagescomparedtootherculturedspecies(Rønnestadetal.,2001). However,thisspeciespresenthighsizevariationandhigh mortal-ityrateshavebeenobservedattheweaningperiod(Moraisetal., 2014).Therefore,Senegalesesolelarvaeareaparticularly inter-estingfishmodelinwhichtostudythebehaviouraldimensionof stresscopingstylesinrelationtofattyacidnutritionsinceithas beendemonstratedthataninappropriatefattyacidprofileaffected growthandmuscleformation(Benítez-Dortaetal.,2012), diges-tivesystemmaturation(Boglinoetal.,2012)andglucocorticoids regulation(Martinsetal.,2013).Inviewofthesearguments,the presentstudyaimedtodeterminewhether(i)Senegalesesole lar-vaeexhibit proactive–reactivebehaviours instandardizednovel environmentandrisktestsand(ii)whetherdietaryfattyacid com-positionfrom vegetable oils and fish oils, used as rotifers and
Artemiaenrichments,caninfluencethebehaviourofsolelarvae. ResultswillprovidenovelinformationrelatedtoSenegalesesole larvaebehaviouratearlylifestagesandhowdietscouldinfluence physicalfitnessandbehaviour.Inaddition,resultsmaybe valu-ablefortheaquacultureindustryinordertoproducelarvaewitha specificbehaviouralcharacteristic.
2. Materialsandmethods
2.1. Ethicstatement
Alltheexperimentationonfishthatformedpartofthisstudy wereinagreementwiththeSpanishandEuropeanregulationson animalwelfare(FederationofLaboratoryAnimalScience Associ-ations,FELASA)andapprovedbytheAnimalEthicsCommitteeof IRTA.
2.2. Experimentalanimalsandhousing
Senegalesesolelarvae,suppliedbyacommercialfarm(StoltSea FarmS.A.,Galicia,Spain),werehousedinsixteen 100Ltanksat adensityof100larvaeL−1,infourreplicatetankspertreatment.
Tankswereconnectedthrougharecirculationsystem(IRTAmar®) and50%oftotalwaterwasreneweddaily.Waterparameters,such astemperature,salinityanddissolvedoxygenweremaintainedat 16.5±0.5◦C,35ppmand7.5mgL−1,respectively.Photoperiodwas
adjustedtofollowalight–darkcycleof16L:8Dh.
2.3. Experimentalemulsions,livefeedenrichmentandfeeding protocol
Fourexperimentalemulsionswerepreparedwithdifferentoils followingthemethodologydescribedbyVillaltaetal.(2005)and usedtoenrichrotifersandArtemiametanauplii.Thefouroilsused were:codliveroilfromGadusmorhua,cod(Sigma–AldrichCo.,St Louis,MO,Germany);linseedoil,linseed(BiolasiProducts Natu-rals,S.L.,Guipùzcoa,Spain);soybeanoil,soybean(HuilerieEmile Noël,S.A.S.,PontSaintEsprit,France);andoliveoil,olive(Borges Pont,Lleida,Spain).Oilswereemulsifiedinwarmdistilledwater (50◦C)withsoylecithin(DieteticaRosa,S.A.,Barcelona,Spain)and
˛-tocopherol(Sigma–AldrichCo.,StLouis,MO,Germany),usingan Ultra-turraxT25homogenizerathighspeedfor60–90s.Emulsions werekeptrefrigeratedat4◦Cintheabsenceofairuntilusedfor enrichingtherotifersandArtemiametanauplii.
Senegalesesolelarvaewerefedtwiceaday,from2to8days post-hatching(dph)withrotifers(Brachionusplicatilis)enriched withthe oilemulsions at a density of 10rotifersmL−1. Freshly
enriched Artemia metanauplii (EG type; INVE, Belgium) were introducedin tanksat6dphuntil 24dph, inquantitiesranging from0.5to6metanaupliimL−1,adjustedbasedupontheincreaseof
weightofthelarvae,withthedailyfoodrationbeingcalculatedas describedbyCa ˜navateetal.(2006).Aslarvaemetamorphosedand becamebenthonic,liveArtemiametanaupliiweregradually substi-tutedwithfrozenArtemiametanauplii.From19to24dphandfrom 25dphlarvaewerefedexclusivelywithfrozenArtemiaatadensity of6metanaupliimL−1until30dphandthen12metanaupliimL−1
untiltheendoftheexperiment(40dph).
Rotifers (Brachionus plicatilis) were cultured as described in
Boglinoetal.(2012).Artemiametanaupliiwerehatchedinstandard conditions and metanauplii enrichment wasperformed in 20L conical containers at 150metanaupliimL−1 for 16h at 28◦C
withoxygen(≥5mgL−1), and using 0.6g of each emulsionL−1.
Subsequently, enriched Artemiametanauplii were washed with UV-treatedfilteredseawateranddisinfectedwithhydrogen per-oxide(8000ppm)for 5min. Thenwashed for 15min period in 150mplanktonnetswithUV-treatedfilteredseawater.Abatch ofArtemiawasfrozenandkeptat−20◦Cuntilbeinggivento post-metamorphosedlarvae.
2.4. Lipidandfattyacidanalysis
withMS-222,washedwithdistilledwaterandimmediatelyfrozen at−20◦C.Totallipidswereextractedinchloroform:methanol(2:1, v:v)usingthemethoddescribedbyFolchetal.(1957)and quan-tified gravimetrically after evaporation of the solvent under a nitrogenflow, followed byvacuumdesiccationovernight.Total lipidswerestoredinchloroform:methanol(2:1,20mgmL−1)
con-taining 0.01% butylated hydroxytoluene (BHT) at −20◦C prior to analysis. Methyl esters were extracted twice using isohex-ane:diethylether(1:1,v:v),purifiedonTLCplates(Silicagel60, VWR,Lutterworth, UK)and analyzed bygas–liquid chromatog-raphyonaThermoElectron-TraceGC(Winsford,UK)instrument fittedwithaBPX70capillarycolumn(30m×0.25mmid;SGE,UK). Atwo-stagethermalgradientwasused,from50◦C(injection tem-perature)to150◦C,afterrampingat40◦Cmin−1and holdingat
250◦Cafterrampingat2◦Cmin−1.Helium(1.2mLmin−1constant
flowrate)wasusedasthecarriergasandon-columninjectionand flameionizationdetectionwasperformedat250◦C.Peaksofeach fattyacidwereidentifiedbycomparisonwithknownstandards (SupelcoInc.,Spain)andawellcharacterizedfishoil,andquantified usinganinternalstandard,21:0fattyacid,addedpriorto trans-methylationusingaChrom-cardforWindows(TraceGC,Thermo Finnigan,Italy).
ThefattyacidcompositionoftheArtemiametanaupliivaried in relationtotheenrichment withthefouremulsions.Artemia
enrichedwiththecodemulsionpresentedthehighestproportions oftotal saturatedfattyacids(SFA),mainly18:0.Theamountof totalmonounsaturatedfattyacids(MUFA),mainlyoleicacid(OA, 18:1n−9),wasthehighestintheArtemiaenrichedwiththeolive emulsionandintermediaryinthoseenrichedwiththecod emul-sion.Artemiaenrichedwiththesoybeanemulsionhadthemost elevatedcontentsoftotaln−6polyunsaturatedfattyacids(n−6 PUFA),mainlylinoleicacid(LA,18:2n−6).Arachidonicacid(ARA) levelswererelativelystableamong thedietarytreatments,and rangedfrom1.2 (olivediet)to1.5 (codand soybeandiets).The contentofn−3PUFAwasthehighestintheArtemiaenrichedwith thelinseedemulsion,mainlyduetolinolenicacid(LNA,18:3n−3) contents.Finally,levelsofn−3LC-PUFA,EPAandDHA,were high-estinArtemiaenrichedwiththecodemulsion.
Thefattyacidcompositionofthesolelarvaefedwiththefour dif-ferentdietarytreatmentsreflectedthefattyacidpatternobserved intheenrichedArtemiametanauplii(Table1).Solelarvaefedwith thecodemulsionpresentedthehighestproportionsoftotal satu-ratedfattyacids(SFA),EPAandDHA.Respectively,EPAandDHA inlarvaefedthecoddietwas4.9and7.2comparedtorangesof 0.9–1.2and2.3–2.7inlarvaefedwiththeotherdiets.Theamount oftotalmonounsaturatedfattyacids(MUFA),mainlyoleicacid(OA, 18:1n−9),wasthehighest(35.8)inthesolelarvaefedwiththe oliveemulsionandlowerrangingfrom21.6to22.6inlarvaefed withtheotherdiets.Solelarvaefedwiththesoybeandiethadthe mostelevatedcontentsoftotaln−6polyunsaturatedfattyacids (n−6PUFA),mainlylinoleicacid(LA,18:2n−6)witha levelof 27.4comparedto11.7–19.8withotherdiets.ARAlevelswere rel-ativelystableamongthedietarytreatments,andrangedfrom2.7 (olivediet)to3.9(linseeddiet).Thecontentofn−3PUFAwasthe highestinthesolefedwiththelinseedemulsion,mainlydueto linolenicacid(LNA,18:3n−3)contents.
2.5. Proactive–reactivebehaviouralcharacterization
2.5.1. Individual-basedtest
Forty-twoSenegalesesolelarvae(aged40dph)fromeachofthe fourreplicatetankspertreatmentweretested.Larvaewere cap-turedingroupsofsixfromtheexperimentaltankandplacedin a1Lbeaker.Aftera standardized1mininthebeakertoensure alllarvaehad a similarpre-testtreatmenteach larva was indi-viduallyintroducedintoaseparateplastictransparentPetriplates
(sixdishesof9cmofdiameter,seeFig.1A)usedtoperformthe individualtest.Larvae werealwaysplaced inthesameposition (closetotheedge)inthePetriplate,whichwasgridded(squares equalto1cm2,Fig.1A).Petriplateswerefilledwith15mLofsea
waterfromtheholdingtanksandrenewedforeachsampled lar-vae.Adigitalcamera(Casio-Exilim,modelEX-ZS100,Japan)was fixed40cmabovethePetriplates(Fig.1B)tovideorecordlarval activityfor5min.Theobserverstood1mawayfromtheworking area,inordertocauseminimaldisturbancetolarvae,followingthe criteriadescribedbyØverlietal.(2002).Oncevideorecordingwere achieved,threebehaviouralresponseswereregisteredforeach lar-vae,being:(i)thelatencytimetomovedefinedasthetime (in seconds)offirstforwardmovementsincethebeginningofthetest (adaptedfromBell,2005;Archardetal.,2012);(ii)thetotalactivity timeconsideredasthetotalswimmingactivitytime(inseconds) ofthelarvaeinthenewenvironment(adaptedfromMillotetal., 2009;Benhaïmetal.,2012);and (iii)theTotalDistanceMoved determinedbycountingthetotal numberofsquarescrossedby thelarvaeduringtheexperimentaltime(adaptedfromMillotetal., 2009;Benhaïmetal.,2012).Larvaetotallengthwasmeasuredas well,withaVernierscale(0.001error;modelCD-20PK;Mitutoyo, JapanCorp).
2.5.2. Group-basedrisktest
In the group-based risk test, a differentpool of larvae than theoneusedintheindividual-basedtestwasscreenedinarisk takingexperience.Experimentalmethodologywasadaptedfrom previous studies performed in stickleback, Gasterosteus aculea-tus(Bell,2005;Ruiz-GomezandHuntingford,2012),giltheadsea bream(Castanheira et al.,2013), commoncarp,Cyprinuscarpio
(Huntingfordetal.,2010)andbluegillsunfish,Lepomismacrochirus
(WilsonandGodin, 2009).Fourgrouprisktestsweremadeper treatment,onetestpertreatmentreplica.Thetestconsistedin pla-cing30individualsinarectangulartank(80cmlength×10.5cm depth×13cmwidth)dividedintotwoareas,inordertodetermine thecapacityoflarvaetocrossfromaknownareadefinedasa “com-fortzone”toanunknownareadefinedasa“riskzone”(Fig.1C).The experimentaltankwasdividedintwoequalvolumeswithablack plasticbarrier,includinga1.5cmwidth–lengthwindowatthebase ofthebarrier,throughwhichfishwereabletopass(Fig.1C).During theacclimationperiod,eachgroupoffish(n=30)wasretainedfor 1hinthecomfortzone,bykeepingthewindowcloseduntilthe beginningofthetest.Thecomfortzonewascompletelyisolated fromlightbyablackplasticcover,whiletheriskareawas illumi-natedbyafluorescentwhitelight(OSRAMDULUX,48W,450lx insurface)placed25cmabovethewatersurface.Agentlewater flow(2Lh−1)andaerationwereprovidedduringtheperiodofthe
testandsimilarwaterparametersthanthoseinthehousingtanks (temperatureof16.5±0.5◦C,salinityof35ppmanddissolved oxy-genof7.5mgL−1)weremaintained.Adigitalcamera(Casio-Exilim,
modelEX-ZS100,Japan)wasfixedontheriskzonetovideorecord thelarvaethatsuccessfullycrossedfromcomfortzonetotherisk zone.
Table1
Fattyacidcompositionofsolepost-larvaefedwithArtemiametanaupliienrichedwithdifferentexperimentalemulsions.
LSOb CLOa SBOc OOd
Formulation(mgg−1)
Codliveroila 0 528 0 0
Linseedoilb 528 0 0 0
Soybeanoilc 0 0 528 0
Oliveoild 0 0 0 528
Supplementse 52 52 52 52
Distilledwater 420 420 420 420
Fattyacidcomposition(%TFA)
Totalsaturated 16.5±2.2 19.7±1.2 17.9±0.7 15.6±0.7
18:1n−9(OA) 22.3±0.7 22.6±0.4 21.2±0.5 35.8±0.8
Totalmonounsaturated(MUFA) 30.0±1.0 35.4±0.6 29.0±0.7 45.6±1.3
18:2n−6(LA) 14.9±0.2 7.8±0.7 22.9±0.5 10.1±0.3
20:4n−6(ARA) 3.9±0.3 3.0±0.1 3.2±0.1 2.7±0.4
Totaln−6PUFA 19.8±0.7 11.7±1.0 27.4±0.3 14.2±0.7
18:3n−3(LNA) 25.2±3.6 13.1±1.1 15.1±1.5 14.3±0.8
20:5n−3(EPA) 0.9±0.2 4.9±0.2 1.0±0.2 1.2±0.2
22:6n−3(DHA) 2.3±0.6 7.2±0.2 2.7±0.5 3.1±1.2
Totaln−3PUFA 31.6±3.5 30.7±1.2 22.4±0.7 22.4±0.9
TotalPUFA 51.4±3.5 42.4±1.9 49.8±1.0 36.6±1.2
MUFA/PUFA 0.6 0.8 0.6 1.2
n−3/n−6 1.6 2.6 0.8 1.6
DHA/EPA 2.6 1.5 2.7 2.6
ARA/DHA 1.7 0.4 1.2 0.9
ARA/EPA 4.3 0.6 3.2 2.3
aCLO:codliveroil.
b LSO:linseedoil.
c SBO:soybeanoil.
d OO:oliveoil.
eSupplements:soylecithin,4g;vitaminE1,2g.
Thevideoanalysiswascorroboratedwithrealtimecounts that weremadeevery10minoftheriskzones.
2.6. Statistics
Resultswereexpressedasmeans±standarderrorS.E.(n=168 fortheindividualtests andTL).Alldatawerechecked for nor-mality(Shapiro–Wilktest)andhomogeneityofvariance(Bartlett’s test).Individual-basedtestmeans(n=42)fromeachreplicawithin eachtreatmentwerecomparedwithaone-wayAnalysisof Vari-ance(ANOVA)andasnodifferenceswereobservedthedatafrom replicas was combined. Two Multivariate Analysis of Variance (MANOVA)wereperformed,firstamongtreatmentsonlarval activ-ityparameters(n=42×4=168)oftheindividual-basedtest and theposthocTukeyHSDtestwasperformedwhensignificant dif-ferenceswerefoundandsecondtodetectpossibleinterferences amongthepositionof thePetriplates (Fig.1A)onthe individ-ualsresponsesofsolelarvae(length,latencytomove,totalactivity time and total distancemoved). No statisticaldifferences were observedamongthelocationofthePetriplates(Fig.1B)forthe lar-vaelength(F5=1.29,P=0.30),thelatencytimetomove(F5=0.65,
P=0.65),totalactivitytime(F5=0.88,P=0.49)andtotaldistance
moved (F5=0.63, P=0.67) to discount that there wasan effect
onbehaviourofPetridishpositionoftheexperimentalsetup.A Pearsoncorrelationwasperformedinordertodetermine possi-blecorrelationsbetweenvariablesincludingtotallength.Arank analysis,withN-tilesfrequenciessubcommand,wasperformedto examinethepossibleeffectofsizeoflarvaeonthelatencytime tomove,thetotalactivityandthetotaldistancemoved. Individ-ualsfromalldietarytreatmentstakentogetherwereseparatedinto sizeranges:smallfrom0.5to0.8mm,mediumfrom0.9to1.0mm andlargefrom1.1to1.9mm.Thenumbersofindividualsinthe mediumsizerangewere49,64,67and76respectivelyforcod, lin-seed,oliveandsoybeandietarytreatments.Aone-wayANOVAwas performedtocomparethebehaviouralparametersamongstthe treatmentsfortheselectedlarvaethatwereallinthesamemedium sizerange.Groupsof30larvae(n=30)fromeachreplica(4 repli-cas)ineachtreatment(4treatments)wereusedineachgrouptest (total4×4=16grouptests).AChisquare(2)testwasperformed
ondatafromthegrouped-basedtests,inordertodeterminethe dif-ferencesintheproportionsofproactive,intermediateandreactive larvae.First,aChisquaredtestwasmadetocomparethedatafrom
Fig.2.SizeandbehaviouralperformanceofSenegalesesole(Soleasenegalensis)larvaefedwithArtemiametanaupliienrichedwithoneoffourdifferentoilemulsions:linseed oil;codliveroil;oliveoilandsoybeanoil.(A)Totallength;(B)latencytimetomove;(C)totalactivitytimeand(D)totaldistancemoved.n=168pertreatment.Different lettersindicatesignificantdifferencesbetweentreatments.
replicaswithineachtreatment(3×4matrix).Nodifferenceswere observedamongstreplicaswithintreatments.Thereplicatedata wascombined(n=30×4=120)anddietarytreatmentswere com-pared(3×4matrix).AvalueofP<0.05wasacceptedasstatistically significantforallstatisticaltestsrealizedondatafromthe individ-ualandgroupingtests.Allthestatisticalanalysiswasconducted usingSPSSstatisticsV.17(IBMInc.,Chicago,Illinois,USA).
3. Results
3.1. Individualbehaviourcharacteristics
Feeding Senegalese sole larvae with Artemia metanauplii enrichedwiththefourdifferentemulsionssignificantlyaffected growthandindividualactivityperformancewhenintroducedinto a newenvironment (Fig. 2).Larvae fed withArtemia metanau-pliienrichedwiththecodemulsionpresentedasignificanthigher totallength(Fig.2A;F1.24=8.6,P=0.01;1.13±0.03cm)thanlarvae
fedArtemiaenrichedwithlinseed,oliveandsoybean emulsions (1.01±0.03cm;0.92±0.03cm and 0.93±0.02cm, respectively). TheactivityoflarvaeinthenewPetridishenvironmentinall treat-mentsrangedfromlarvaethatthat didnotmovetolarvaethat movedimmediately(lowlatencytime)withahighlevelofactivity (distancemovedandtimeofactivity)duringthe5min.No signif-icantdifference(F1.24=2.3,P=0.079)wasfoundinlatencytimeto
moveamonglarvaefedthedifferentdietarytreatments(Fig.2B), althoughlarvaefedwithArtemiaenrichedwiththecodemulsion tendedtohave a lowerlatencytime tomove (36.6±3.4s) and thoseontheolivetreatmenthada slightly higherlatencytime (47.5±2.4s).Larvaefromthecodgrouphadsignificantlyhigher total activitytime(Fig.2C;F1.24=4.1, P=0.08;50.4±3.7s)than
thosefromthelinseedandolivegroups(36.3±3sand37.6±2.7s, respectively), while larvae fed the soybean diet displayed an intermediate and non-significantly different total activity time value(39.6±3.6s).Similarly, larvae fedwithArtemia metanau-pliienrichedwiththecodemulsionshowedasignificantlyhigher total distance moved (Fig. 2D; F1.24=12.8, P=0.01; 51.47±3.4
squares)thanthosefedthethreeotherdietsandlarvaefedwith
Artemiaenrichedwiththesoybeanemulsionpresenteda signif-icantlylowertotaldistancemoved(F1.24=4.4,P=0.06;25.3±2.3
squares) than theolive group (39.9±3.2 squares), while those fed thelinseeddiet showedanintermediate valueof total dis-tance moved (32.4±3 squares). Whenlarvae were selectedby mediumsize(0.9–1.0mm,mediumofalltreatments)fromeach treatmentgroupandcompared thesamepattern ofdifferences wasfoundamongsttreatmentgroups.The0.9–1.0mmlarvaefrom theCodtreatmentpresentedsignificantlylowerlatencytime to move (F252=3.603, P=0.014), significantly higher total activity
time(F252=5.926,P=0.001)andsignificantlyhighertotaldistance
moved(F252=20.414,P<0.001)than0.9–1.0mmlarvaefromsome
or alloftheothertreatments. In addition,nosignificant corre-lations(P>0.05)wereobservedbetweenlarvaltotallengthand latencytimetomove,totalactivitytimeortotaldistancemoved acrossalltreatmentsorwithinanydietarytreatment.The corre-lationcoefficientswerelowrangingfromthehighestofR2=0.27, P=0.08observedbetweentotallengthandtotalactivitytimeinthe codtreatmenttothelowestbetweentotallengthandtotaldistance movedinthelinseedtreatment(R2=0.08,P=0.62).
3.2. Risktakingbygroup
Alltreatmentshadlarvaethatpassedthroughthedoortothe riskzonesoonafteropening(proactive)andlarvaethatdidnot cross fromthecomfort zone totherisk zone (reactive).Larvae fromthecodtreatmentpresentedasignificanthigherproportion (2=24.27,df=6,P=0.02)ofproactiveandintermediateorganisms
Fig.3.Numberofproactive,intermediateandreactiveSenegalesesole(Solea sene-galensis)larvaefedwithArtemiametanaupliienrichedwithoneoffourdifferentoil emulsions:linseedoil;codliveroil;oliveoilandsoybeanoil(2,P=0.001).n=120 pertreatment.*Indicatesproportionsweresignificantlydifferentfromexpected values.
4. Discussion
Thisisoneofthefirststudiesthatinvestigatedtheeffectofdiets ontheproactive–reactivebehaviouraldimensionofstresscoping styleduringthelarvaldevelopmentperiod.Thetestsemployedin thepresentstudyenabledthecharacterization,forthefirsttime, ofproactive–reactivebehaviourofSenegalesesolepost-larvaeat 40dph.Theactivityofsolepost-larvaeinthenewenvironment individual-basedtestsandlatencytoenteranunknownriskarea intheriskgroup-basedtestenabledtheidentificationofproactive andreactivebehaviouraltraits.Inagreementwiththe classifica-tionused in otherstudies, proactivelarvae had low latencyto moveintoariskzone orinanewenvironmentandweremore activein a newenvironment,whilst reactivelarvaehad higher latencytomoveintoa riskzone orin a newenvironment and wereless active in a new environment. Alldietary treatments had this range of behaviours, although thelevelsof activity in a newenvironment and proportionsof proactive, intermediate andreactivelarvaesignificantlychangeddependingondiet.The identifiedproactive–reactivebehavioursasdeterminedby activ-ityintheindividual-basedtestsdidnotappear toberelatedto thesize of the larvae as the same pattern of differences were foundin thebehaviours of similarly sized (0.9–1.0mm) larvae thatwereselectedfromeachtreatmentgroupandtherewasno correlationbetweenlarvaelengthandindividual-basedtest activ-ityvariableslatencytimetomove,totalactivityortotaldistance moved.Theactivityinnewenvironmentandtherisktakingtests usedinthepresentstudytodifferentiatetheproactiveand reac-tivebehaviours have beenbothpreviously reportedasrelevant measurementstodetermineproactiveandreactivetraitsinawide rangeoffishspecies(Tomsetal.,2010;Castanheiraetal.,2013) includingSenegalesesole(Mota-Silvaetal.,2010;Martinsetal., 2011).This behaviouraldimensionof stress coping styleswere describedin thedamselflyLestes congener(Brodin, 2008), com-moncarp(Huntingfordetal.,2010),rainbowtroutOncorhynchus mykiss (Ruiz-Gomez et al., 2011) and three-spined stickleback (Ruiz-Gomez and Huntingford,2012).Altogether,thesestudies, whichareinlinewiththepresentresults,haveprovidedasolid baseofevidencefortheexistenceofproactive–reactivecopingstyle responsesinthosefishspeciesanddemonstratedthatproactive individuals,when innovelenvironments,resumed activity ear-lier,tendedtotakerisksandspentmoretimeinmovementthan reactiveindividuals.
Theresults fromthe present studyhave clearly shown that diets differing in fatty acid profile significantly influenced the proactive–reactivebehaviouraldimensionofstresscopingstyleof Senegalesesolelarvae.Toourknowledge,thereisnoinformation ontheearlyinteractionsbetweendietaryfattyacidcomposition andthestresscopingstyleofSenegalesesolelarvae,oranyother fishlarvae.SolelarvaefedwithArtemiametanaupliienrichedwith the fish oilemulsion showedsignificantly higher activity time andswamlongerdistancesinthenewenvironment individual-basedtestandasignificantlyhigherproportionoflarvaetookthe risktocrosstoandexploreanunknownenvironmentintherisk group-basedtest comparedtolarvaefedwithArtemiaenriched withthevegetableoilemulsions(linseedoil,oliveoilandsoybean oil).Incontrast,larvaefromthelattergroupswerelessactivein theindividual-basedtest,morecautiousinthereactiontonovel situations and mostly stayed shelteredin thesafe zone witha significantlylowerproportioncrossingtoanunknown environ-ment(riskgroup-basedtest).Therefore,thefishoilenrichment(cod liveroil)appearedtopromoteaproactiveexplorativebehaviour, whilevegetableoils(linseedoil,oliveoilandsoybeanoil)resulted inareactivebehaviourinSenegalesesolelarvae.Possible expla-nationsforthesesignificantdietaryeffectsonproactive–reactive behaviourcouldbeadirecteffectofnutritionalstatuson physiol-ogy,metabolicrateand/ordevelopmentthatresultedinretarded growthordifferencesinphysicalconditionthataffectactivityand personalityofthelarvae.
CodliveroildietcontainedhigheramountsofSFAandofthe EFAs,EPA and DHA (15.2; 4.7 and 2.6% TFA, respectively)than the otherdiets. In addition, it had the second highest level of MUFA,aftertheolivetreatment.Therefore,overall,thisdiet pos-sibly supplied higher levels of important energy substrates,as wellasof criticalstructuralcomponentsofbio-membranesand precursorsofessentialmetabolites,resultinginamorebalanced dietforfishlarvalandmetamorphosingstageswithfastgrowth associatedwithhighrequirementsfordevelopmentand organo-genesis(Conceic¸ão, 1997;Moraisetal.,2005).Thisdifferencein nutritionmayalsoexplainthehighergrowthofsolefedthecod diet.OtherstudieshaveshownthatredseabreamPagrusmajor
(Nakayamaetal.,2003),giltheadseabream(Benítez-Santanaetal., 2007)andpikeperch(Lundetal.,2012)fedwithhigherinclusions ofPUFA,particularlyDHA,indietsimprovedfishgrowth, activ-ity,swimmingspeed,escapeabilityandreducedmortalityaftera stressconfinementsituation.Similarly,Liuetal.(2002)andAtalah etal.(2011)suggestedthatincreasingthedietaryamountofEPA and ARAsupplied togilthead seabreamand Europeanseabass improvedresistancetohandlingstressincomparisontolarvaefed withdietscontainingpoorlevelsoftheseingredients.However,in thepresentstudy,thepoorcorrelationbetweenlarvallengthand activityandthesamepatternofbehaviouraldifferencesamongst fishofthesamesizefromthedifferenttreatmentsprovides com-pellingevidencethatthedieteffectonthebehaviouraldimension of stress coping style was more profound than just increasing energyreservesandgrowthtoincreaseactivityandhencethe pro-portionofproactivelarvae.
observedthatlarvaefedwithhigherinclusionsofDHAandEPA showedhigher ability toswim, formedschooling patterns and improvedantipredatorescapecapacity.Therefore,higherdietary levelsofLC-PUFA,particularlyofDHAandEPA,asprovidedbythe coddietinthepresentstudy,couldhavehadapositiveeffectonthe ontogenicdevelopmentoftheneuralsystemandsensorialorgans whichinturninfluencedthefishbraindevelopmentor neurogene-sis,cognition,swimmingactivity,learningcapacityandexplorative behaviouroflarvae’s.Theseassumptionsareinagreementwiththe studiesperformedbyØverlietal.(2007),Sørensenetal.(2007)and
Koolhaasetal.(2010)whomconfirmedthatbehaviouralstress cop-ingstylesoffishatdifferentagesareassociatedwithneurogenesis andneuralplasticity.Inreferencetothevegetableemulsions,these threedietsledtosimilarcopingstyleresponsesinsolelarvae.A possibleexplanationofthisisthatvegetableoilspresentedlower DHA/EPAratio,higher ARA/DHAandARA/EPAratiosand unbal-ancedratiosof MUFA/PUFAand n−3/n−6 PUFAs.Therefore, it ispossibletosuggestthatenergy,physicalfitnessandenzymatic materialoflarvaewassimilarwithinthethreevegetable emul-sions,butlowerincomparisonwithlarvaefedfishoilemulsion (Sargentetal.,2002;Villaltaetal.,2005;Benítez-Dortaetal.,2012; Boglinoetal.,2012).However,furthersystematicstudiesshould beperformedinordertoexploretherelationbetweennutrition, neurogenesisandcopingstyles,sinceitisnotentirelyclearhow theseaspectscanbeinvolvedinfishlarvalbehaviour.
Lastly,itshouldbementionedthatstresscopingstyleisdefined asacoherentsetofbehaviouralandphysiologicalstressresponses that is characteristicto a certaingroupof individual (Koolhaas etal.,1999).Inthepresentstudy,dietsinfluencedthebehaviour of40DAHSenegalesesolelarvaeinacoherentway.Thedietary treatments had a coherent influenceon larvalbehaviour as no differences werefoundin thebehavioural parameters amongst thefour replicas within each dietarytreatment indicating that thebehavioural patternsin thereplicas were highlyconsistent betweenindividualsthathadreceivedthesametreatment.Stress copingstylehasbeenshowntobedeterminedorperhaps influ-enced by genetic and environmental factors (Dingemanse and Réale,2005;Koolhaasetal.,2007).Ithasalsobeenhypothesized thatindividual’sbehaviourmaychangeinaccordancewith devel-opmentalstageorage(GroothuisandTrillmich,2011).Froman environmentalpointofviewfishbehaviourmaydependon envi-ronmentalstimuli(i.e.predators,density,etc.),foodavailability, social structure or motivational state and negative experiences (Koolhaasetal.,2007;Ruiz-Gomezetal.,2011;Frostetal.,2013). The present studyhighlighted that nutrition wasan important environmentalfactor indetermining orinfluencing behavioural developmentoftheproactive–reactivebehaviouraldimensionof stresscopingstyleofsolelarvaeindifferentcontexts.
Theimplication of understandingfishstress coping stylesis ofmajorimportance,notonlyfromanevolutionaryperspective butalsoinpracticaldisciplines,suchasneurosciences( Benítez-Santana et al., 2014), disease susceptibility (MacKenzie et al., 2009)andespeciallyaquacultureproduction(Øverlietal.,2002). In this context, the characterization of Senegalese sole larvae proactive–reactivebehaviouraldimensionof stresscoping style may have important practical applications for the aquaculture industry for theselection or production of larvae withspecific behaviouralcharacteristicthatmayhavebenefitsforwelfare pro-tocols, selective genetic programmes, improved growth, stress resistanceand/orsurvival. For instance,Øverliet al.(2002)and
Pottinger(2006) foundthatselectedlines ofproactiverainbow troutjuvenilespresentedhigherlevelsoflocomotoractivityand growthrates.MacKenzieetal.(2009)indicatedthatcopingstyle responsesincommoncarpwererelatedtosusceptibilityto dis-easesin inflammatory challenges.Hansen etal. (2009)showed thatproactiveAtlanticcod,Gadusmorhua,individualspresented
ahigherlearningabilitycomparedtoreactivefish.Inthepresent study,proactivelarvae(fedwithcodoil)weremoreactive,showed higherlengthandtakemoreriskthanreactivelarvae(fedwith vegetableoils).
5. Conclusion
Thepresentstudyindicatedthatproactive–reactivebehavioural dimension of stress coping style developed early during onto-genesis and, furthermore, that the proportion of the different proactive–reactivebehaviourwasmodulatedbydiets.These find-ingsareofgreatinteresttocertainsectorssuchtheaquaculture, sinceitmayofferthepossibilitytoproduceahigherproportion oforganismsthathavesimilarbehaviouralstylesthatmayresult in improvedgrowth,welfare, stressresistance, fitnessandthus incrementaquacultureproductivity.
Conflictofinterest
Theauthorsconfirmthattherearenoknownconflictsofinterest associatedwiththispublication.
Acknowledgments
WewouldliketothankGloriaMaciaandMagdaMonllaóand othertechnicalstafffromIRTA,SantCarlesdelaRápita,for main-tainingthelarvaeandassistancewiththeinstallationofequipment. We aregratefultoMartaSastre,NoeliaGrasand AliciaEstevez, PhD,forperformingthefattyacidsanalysisinthepresentstudy. We thank Anaïs Boglino, PhD, for her valuables comments on this manuscript.ThisworkwassupportedbytheSpanish Min-istryof Economyand Competitiveness (projectAGL2011-23502 coordinatedbyS.M.,whoholdsaRamónyCajalpost-doctoral con-tractalsoawardedbyMINECO),InstitutoNacionaldeInvestigación y Tecnología Agraria y Alimentaria, Spain (project INIA-FEDER RTD2011-00050coordinatedbyN.D.)andbyPhDgrantsawarded byConsejoNacionalde CienciayTecnología,CONACYT,México (Z.I.Z.),Secretaría Nacionalde EducaciónSuperior,Ciencia, Tec-nología eInnovación, SENESCYT, Ecuador(C.C.) and Agènciade Gestiód’AjutsUniversitaris ideRecerca, AGAUR,Generalitatde Catalunya,Spain(K.B.).
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