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Zoology

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 / z o o l

Biparental immune priming in the pipeﬁsh Syngnathus typhle

Anne Beemelmanns, Olivia Roth

^∗

HelmholtzCentreforOceanResearchKiel(GEOMAR),EvolutionaryEcologyofMarineFishes,DüsternbrookerWeg20,D-24105Kiel,Germany

a r t i c l e i n f o

Articlehistory:

Received30October2015

Receivedinrevisedform8February2016 Accepted6June2016

Availableonlinexxx

Keywords:

Maternaleffects Paternaleffects

Trans-generationalimmunepriming Epigeneticeffects

Host–parasiteinteraction

a b s t r a c t

Thetransferofimmunityfromparentstooffspring(trans-generationalimmunepriming(TGIP))boosts offspringimmunedefenceandparasiteresistance.TGIPisusuallyamaternaltrait.However,iffathers haveaphysicalconnectiontotheiroffspring,andifoffspringareborninthepaternalparasiticenviron- ment,evolutionofpaternalTGIPcanbecomeadaptive.InSyngnathustyphle,asex-rolereversedpipefish withmalepregnancy,bothparentsinvestintooffspringimmunedefence.ToconnectTGIPwithparental investment,weneedtoknowhowparentssharethetaskofTGIP,whetherTGIPisasymmetricallydis- tributedbetweentheparents,andhowthematernalandpaternaleffectsinteractincaseofbiparental TGIP.Weexperimentallyinvestigatedthestrengthanddifferencesbutalsothecostsofmaternaland paternalcontribution,andtheirinteractivebiparentalinfluenceonoffspringimmunedefencethrough- outoffspringmaturation.Todisentanglematernalandpaternalinfluences,twodifferentbacteriawere usedinafullyreciprocaldesignforparentalandoffspringexposure.Inoffspring,wemeasuredgene expressionof29immunegenes,15genesassociatedwithepigeneticregulation,immunecellactivityand life-historytraits.Weidentifiedasymmetricmaternalandpaternalimmuneprimingwithadominating, long-lastingpaternaleffect.WecouldnotdetectanadditiveadaptivebiparentalTGIPimpact.However, biparentalTGIPharboursadditivecostsasshownindelayedsexualmaturity.Epigeneticregulationmay playarolebothinmaternalandpaternalTGIP.

1. Introduction

Thetransferof non-geneticinformationfromparents tooff- spring is a phenotypic plastic trait that can have fast and long-lastingeffectsindependentofgeneticinheritance(Mousseau and Fox, 1998). Such parental effects are a key source of trans-generationalphenotypicplasticitythatallowforimmediate responsestonovelenvironmentalchallenges(Bondurianskyand Day(2009);BoulinierandStaszewski(2008)).Forinstance,trans- generational immune priming(TGIP), thetransfer of immunity fromparentstoprogeny,enhancesoffspringimmunecompetence andbuffersthemagainstimpactsofpathogensformerlyexperi- encedby theirparents(Grindstaff et al.,2006; Hasselquistand Nilsson,2009;Saddetal.,2005).

Thematernaltransferofimmunologicalactivecomponentslike antibodies viablood, milk,or egg is of major importance during earlylife stages (Boulinier and Staszewski, 2008; Brambell, 1970a,b;Ehrlich,1892),whentheadaptiveimmunesystemisstill

∗Correspondingauthor.Tel.:+494316004557;fax:+494316004553.

E-mailaddress:oroth@geomar.de(O.Roth).

subjecttomaturation(Lindholmetal.,2006; Rossiter,1996).A mechanismthatenablesparentstopassontheirimmunecompo- nentstotheprogeny,andinthiswayprotecttheiryoungduringthis crucialperiod,shouldthereforebeadaptive(MarshallandUller, 2007).Particularly,iftheprevailingparasiteassemblageissimi- laracrossgenerations,persistentorevenmultigenerationalTGIP might befavoured(Lemkeet al.,1994; Reid et al.,2006).TGIP hasevolvedinvertebrates(Grindstaffetal.,2003;Hasselquistand Nilsson,2009;JiménezdeOyaetal.,2011),butcanalsobefound ininvertebrates,whereitisachievedbyothermechanismsthan antibody-mediatedimmunity(Freitaketal.,2009;Freitaketal., 2014;Littleetal.,2003;Moret,2006;Rothetal.,2010;Saddetal., 2005;Salmelaetal.,2015)

TGIPwasdeterminedasamaternaltrait,becausenon-genetic immunologicalexperiencewassupposedtobeexclusivelypassed onvia the eggor placenta, while sperm cells were considered too small to carry those (Bonduriansky and Day, 2009). How- ever,latelythepotentialofparentaltransferofepigenetictraits inﬂuencingtheoffspringphenotypehasbeendiscovered(Jablonka and Lamb, 2014,2015; Jiang et al., 2013).Epigenetics concern stimuli-triggeredchangesingeneexpressionthatdonotinvolve analterationofthenucleotidesequence,butareheritableacross http://dx.doi.org/10.1016/j.zool.2016.06.002

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generations(Bergeretal.,2009;Gómez-Díazetal.,2012).Subse- quently,factorsthataffectthegeneexpressionbutnottheDNA sequencecanbepassedontothenextgenerationandcontribute tothephenotypicplasticityoftheoffspring(JablonkaandLamb, 2014,2015; Szyf,2015; Youngsonand Whitelaw, 2008). Here, RNAmolecules(RassoulzadeganandCuzin,2015;Rassoulzadegan et al., 2006), DNA-methylation (Ci and Liu, 2015; Jiang et al., 2013) and histonemodiﬁcations (Campos et al., 2014; Gaydos et al., 2014; Jones, 2015; Ragunathan et al., 2015; Vilcinskas, 2016,thisissue), factorsassociatedwiththeregulationof gene expression,canbetrans-generationallyinheritedandmanifestas stableepigeneticmarksinthefollowinggeneration(Jablonkaand Lamb,2015;KappelerandMeaney,2010).Environmentalstress- orssuchasparasitesandpathogensmanipulatethehost’simmune systemwhile interfering withhistone acetylation/deacetylation processes (Mukherjee et al., 2012; Mukherjee et al., 2015) or DNA-methylationpatterns(Marretal.,2014).Todate,epigenetic inheritanceofimmuneprimingupon pathogenexposureacross generations based onDNA methylation changes hasonly been demonstratedtooccurinplants(Dorantes-Acostaetal.,2012;Luna etal.,2012),whereashistonemodiﬁcationsmightbeassociated withinheritableimmune memoryuponinfectionin eukaryotes (Youngbloodetal.,2010;Ragunathanetal.,2015)andinvertebrates (Vilcinskas,2016,thisissue).However,thequestionwhichmech- anismsareresponsibleformediatingimmuneprimingeffectsthat arebasedonepigeneticchangesinvertebratesisstillunresolved.

Ifepigeneticfactorsareinvolvedorifmalesovercomethemech- anisticbarrieroflimitedspermsize,TGIPmaynotbelimitedto females.Inabeetle,biparentalTGIPwasdiscoveredfirst(Rothetal., 2010;Zanchietal.,2011).Ifbothparentstransfertheirpathogen experiencetotheprogeny,offspringbenefitscouldbemorethan additive(Rothet al., 2010)resulting in an inducedphenotypic plasticity (Jokela, 2010). This could imply an enhanced adap- tivepotentialtohandleparasiticinfections (Marshall andUller, 2007).However,investmentinimmunityiscostlyandtradedoff againstotherlifehistorytraitslikefecundityand/orreproduction (Contreras-Gardu ñoetal.,2014).Asspecificcostsareassociated withmaternalandpaternalTGIP,anasymmetricinvestmentofthe parentsmayhaveevolved(Zanchietal.,2011).

Biparentalinvestmentinoffspringimmunityalsoexistsinthe sex-rolereversedpipeﬁshSyngnathustyphle(Rothetal.,2012b).

Here, females deposit theireggsinto a protective brood-pouch ofthemales,inwhichtheyarefertilisedandnurseduntilbeing releasedasindependentjuveniles(Berglundetal.,1986;Kvarnemo etal.,2011).Theevolutionofsex-rolereversalcoupledwithan internalbroodingstructuremaymechanisticallyenablea pater- naltransferofnon-geneticfactors.Ourpreviousstudy(Rothetal., 2012b)suggestedthat sex-role reversalmayopenthe doorfor biparentalinﬂuencesonoffspringimmunity.

Inthepresentstudyweaimedtoinvestigatetheconnection ofTGIPandparentalinvestmentbyunravellingthestrengthand differencesbetweenmaternalandpaternalimpactandtheirinter- activebiparentalinvestmentintooffspringimmuneprotectionin thesex-rolereversedpipeﬁshS.typhle.Wemeasuredtheeffects ofmaternal,paternalandbiparentalTGIPonoffspringimmunity andlifehistorybyutilizinganextendedsetof29immunegenes and15genesrelatedtoepigeneticmodiﬁcationprocessesaswell asimmunecellactivityandlifehistorytraits.

Wehypothesisedthat(I)thecombinationofmaternalandpater- nalimmune priming,i.e.biparentalimmunepriming,resultsin a synergisticeffect.By includingtwo differentallopatric bacte- riaspecies for maternaland paternalexposure(Vibrio spp. and Tenacibaculummaritimum)inafullyreciprocaldesign,weexplored whetherbacteria-speciﬁcbiparentalimmuneprimingmayharbour additiveimmuneprimingeffects.Wefurtherhypothesisedthat(II) parentalimmunepriminginS.typhleisasymmetricanddirectly

connectedtothereversedmatingsystemwithastronger,long- lastingpaternalimmuneprimingeffect.Toassessthemaintenance ofparentalimmuneprimingwecomparedone-weekoldandfour- month-oldjuveniles.Weexpectedthat(III)theparentalimmune primingeffectwouldceaseuponmaturation,withmaternaleffects ceasingearlierthanpaternaleffects.

Weassessedgenesassociatedwithepigeneticregulationpro- cesseslikeDNA-methylationandhistone-acetylation/methylation toexploretheconnectionbetweenepigenetic modiﬁcationand regulationofimmunegeneexpressionanditsnon-genetictrans- ferovergenerations.Wepredictedthat(IV)changesinepigenetic tracesintheoffspringgenerationwouldshowparentalsex-based differences.

Finally, we addressed the costs and beneﬁts of TGIP by monitoringkeylifehistorytraits(size,mass,conditionfactor,hepatosomaticindex)ofthejuvenilesandevaluatedtheirtimetoreach sexualmaturity.Wehypothesisedthat(V)strengthofmaternal, paternalandbiparentalimmuneprimingpredictstheseverityofa resourceallocationtrade-offandexpectedpaternalandbiparental immuneprimingtobeassociatedwithgreaterenergeticcosts.

2. Materialsandmethods

2.1. Parentalgeneration(P)treatment

Broad-nosedpipeﬁshS.typhlewerecaughtinthesouth-western BalticSea(54^◦44N;9^◦53E,Germany)inspring2013.Theywere keptunderBalticsummerconditions(salinityof15psu,18^◦C,and 14–16hlight)andfedtwiceadaywithmysids.

For theexperimentalsetupmature pipefishwere eitherleft naïve or injected with 50␮l of 10⁸ cells/ml heat-killed bacte- riasolutionaccordingto(Rothetal.,2012b).Twonovelbacteria species,Vibriospp.(Italianisolate,I2K3)(Rothetal.,2012a)and T.maritimum(DSMNo.17995)(Suzukietal.,2001)wereapplied toexcludeconfoundingeffectswithpreviouspathogenencoun- tersinthewild.Bothbacteriatypesactivatetheimmunesystem offishandcausetwodifferentdiseases:‘vibriosis‘(Alcaideetal., 2001)and‘flexibacteriosis‘(Bernardetetal.,1990;Kolygasetal., 2012),respectively.Thisallowedustoanalysethetransmissionof bacteria-specificimmunememorybymothersandfathers,respectively,andpotentialadditiveimmuneprimingeffects.Matingpairs ofimmune-challengedindividualswereformedin36cm×80cm aquariaconnectedtoasemi-flow-throughsystemwithmaternal, paternalorbiparentalimmunechallengeinafullyreciprocalmat- ingdesign:1.(MatV+PatT+),2.(MatT+PatV+),3.(MatNPatT+),4.

(MatNPatV+),5.(MatT+PatN),6.(MatV+PatN),7.(MatNPatN),with (V+) forVibrio, (T+)for Tenacibaculumand (N) fornaïve (seven parentaltreatmentswerereplicated eighttimes,resultingin56 breedingpairs(families)inseparatebreedingtanks)(Fig.S1inthe supplementaryonlineAppendix).AshamexposurewithPBSwas notimplementedasearlierstudieshadsuggestedthatTGIPisnot confoundedbythesterileneedleinjection(Rothetal.,2012b)and shaminjectionwithPBSdoesnotinduceimmunegeneexpression (Birreretal.,2012).

2.2. Filialgeneration1(F1)treatment

All56couplesmatedsuccessfullyaftertheimmunechallenge and aboutﬁve weeks later (July 2013), one-week-old offspring (8dayspostbirth)wereexposedtohomologousandheterologous heat-killedbacteria(Vibrio(V+)andTenacibaculum(T+)),orstayed naïveascontrols(N).Forthispurpose,aneedlewasdippedintoa solutionof10⁹cells/mlheat-killedbacteriaandusedtoprickthe juvenilesintraperitoneally.Subsequently,thesewerekeptinsep- aratetanksfor20h.Afterthisincubationtime,thejuveniles’body

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standardlengthsweremeasured,andwhole-bodysampleswere usedforRNAextraction.Fortheanalysisweselectedonlyfamilies withaminimumclutchsizeof15juveniles,with4replicatesper parentaltreatment(28familiesinall).Fromeachofthe28families, 15offspringwereusedanddistributedoverthethreetreatments, resultingin5specimensperoffspringtreatmentandatotalnumber of420sampledjuveniles.

Remaining F1-offspring (approx. 420 pipefish) were pooled accordingtotheir7parentaltreatmentgroupsandtransferredinto 36cm×80cmaquariaconnectedtoasemi-flow-throughcircula- tionsystemusingthreetankreplicatesperparentaltreatmentanda densityof20pipefishpertank.TocompareTGIPbetweendifferent developmentalstages,four-month-oldjuveniles(notfullysexu- allymature)fromthe7differentparentaltreatmentgroupswere injectedwith20␮l10⁸cells/mlheat-killedVibrio(V+)orTenacibac- ulum(T+)bacteria(intraperitoneally)orstayed naïve(N),using 3replicatesperoffspringtreatmentpersamplingevent.Intotal, 9individualsofthe7parentaltreatmentgroupswererandomly collectedout of thetanks at two consecutivesampling events, resultinginatotalnumberof126sampledjuveniles.Afterincu- bation(20h),bodystandardlengthandbodymassweremeasured beforethefish weresacrificed. The liverwas weighedand the hepatosomaticindex(HSI=ratiooflivermass[g]relativetobody mass[g])wascalculatedtoevaluatetheenergystatusofthefish (Chellappaetal.,1995).Furthermore,afishbodyconditionfactor (CF)wasdeterminedafterthemethodofFrischknecht(1993).

Forcharacterizingtheimmuneresponse,andtogaininforma- tionabouttheactivityoftheadaptiveandinnateimmunesystem, wemeasuredtheabsolutenumberoflymphocytesandmonocytes intheheadkidneyandbloodaccordingtotheprotocolofRothetal.

(2011).

Remaining F1-offspring (approx. 280 pipeﬁsh) were left immunologicallyuntreatedand,tomaintainanequaldensityratio (20pipeﬁshpertank),pooledwithinthematernal,paternaland biparentaltreatmentgroupsandrandomlydistributedintothree tankreplicates.ThetimefortheF1-offspringtoreachsexualmatu- ritywasrecorded.

2.3. Quantiﬁcationofgeneexpression

WequantifiedthemRNA-levelof44pre-selectedtargetgenes ofpreviousstudies(Birreretal.,2012;Rothetal.,2012b)using quantitativerealtimepolymerasechainreaction(qPCR).Thegenes wereidentifiedandselectedbasedonanexpressed-sequence-tag (EST)libraryfromS.typhleexposedtonaturalVibrioisolates(Haase etal.,2013).Accordingly,immunegeneswereselectedamongpre- viouslyidentifiedgenesthatweredifferentiallyexpressedinthe pipefishuponinfection(Haaseetal.,2013)andpresumedtobe importanttargetgenesduringourappliedimmunechallenge.The chosengenesweregroupedintofunctionalcategories:(i)innate immune system (immediate and non-specific immune defence upon infection, i.e. phagocytosis), (ii)adaptive immune system (specific antibody-mediated immune defence), (iii) innate and adaptiveimmunegenes(genesconnectedtobothpathways),(iv) complementsystem(complementstheantibodyandphagocytic cell-mediatedimmuneresponse),and(v)epigeneticmodulators (DNAmethylation,histonede/methylation,histonede/acetylation) (TableS6).Primerefficienciesweretested,withallprimerpairs attainingstandardcurveswithslopesoflogquantityvs.threshold cycle(Ct)between−3.6and−3.1andefficienciesof90–100%(Table S7).

TheRNAextractionof420whole-bodysamplesofearly-stage juvenilepipeﬁsh(oneweekpostbirth)and126gilltissue sam- plesoflate-stagejuvenilepipeﬁsh(fourmonthspostbirth)was performedwithanRNeasy96Universal-TissueKit(Qiagen,Venlo, Netherlands)accordingtothemanufacturer’sprotocol.Extraction

yieldsweremeasuredbymeansofaspectrophotometer(NanoDrop ND-1000;Peqlab,Erlangen,Germany)toallowareversetranscrip- tionintocDNAviaaQuantiTectReverseTranscriptionKit(Qiagen) ofafixedamountof800[ng/␮l]persample.Thegeneexpression of48geneswasmeasuredusingaBioMarkHDsystem(Fluidigm, SouthSanFrancisco,CA,USA)basedon96.96dynamicarrays(GE chips).Apre-amplificationstepwasperformedbymixinga500 [nM]primerpoolofall48primerswith2.5mlTaqManPreAmp MasterMix(AppliedBiosystems,Waltham,MA,USA)and1.25␮l cDNApersample.Themixturewaspre-amplified(10minat95^◦C;

14cycles:15sat95^◦C;4minat60^◦C)andtheobtainedPCRprod- uctsdiluted 1:10with low EDTA-TEbuffer. For chiploading,a samplemixwaspreparedbycombining3.5␮l2xSsofast-EvaGreen SupermixwithLowROX(Bio-RadLaboratories,Hercules,CA,USA) with0.35␮l20xDNABindingDyeSample&AssayLoadingReagent (Fluidigm)and3.15␮lpre-ampliﬁedPCRproducts.Anassaymix waspreparedbycombining 0.7␮lof 50[␮M] primerpairmix, 3.5␮lAssayLoadingReagent(Fluidigm),and3.15␮llowEDTA-TE buffer.Finally,5␮lofeachsampleandassaymixwereﬁlledinto theGEchipsandmeasuredintheBioMarksystem,applyingthe GE-fast96.96PCRprotocolaccordingtothemanufacturer’s(Flu- idigm)instructions.Thesamplesweredistributedrandomlyacross chipsandeachoftheseincludednotemplatecontrols(NTC),con- trolsforgDNAcontamination(-Rt)andstandardsandtwotechnical replicatespersampleandgene.

2.4. Dataanalysisandstatistics

Foreachofthetwotechnicalreplicatespersample,themean cycletime(Ct),thestandarddeviation(SD),andthecoefﬁcientof variation(CV)werecalculated.SampleswithaCVlargerthan4%

wereremoved,duetopotentialmeasurementerrors(Bookoutand Mangelsdorf,2003).Thecombinationofthehousekeepinggenes ubiquitin(Ubi)andribosomeprotein(Ribop)showedthehighest stability(geNormM>0.85)(Hellemansetal.,2007)andtheirgeo- metricmeanwasusedtoquantifyrelativegeneexpressionofeach targetgene bycalculating −Ct-values.Allplots andstatistical testswereperformedinRv3.2.2(RCoreTeam,2015)andPRIMER v6(ClarkeandGorley,2006).Multivariatestatisticswereusedto inferdifferencesin theentireexpressionpattern of29immune genesand15genesassociatedwithepigeneticregulation(Table S6).

Data analysis was conducted on three different levels to address our hypotheses. First, we evaluated maternal, paternaland biparentalimmuneprimingeffectsincombinationwith thetwo pathogen species(Vibrio andTenacibaculum).Thus, we assessed the effect of seven parental treatment combinations ongene expression(29immunegenes), lifehistory parameters (body size, body mass, CF, and HSI) of one-week-old juveniles and four-month-old juvenilesandimmune cellcountmeasure- ments (lymphocyte/monocyte ratio of blood and head kidney) for the latter. On the second level, we explored the impactof maternaland paternaleffects in thebiparentaltreatmentcom- binationbyexaminingtheF0-parentalxF1-offspringinteraction terms. We elucidatedwhethermaternaland paternaleffects in a biparentalcombination hadadditiveimmune primingeffects.

Onthethirdlevel,weanalyzedmaternal,paternalandbiparental effects on genes associated with epigenetic regulation processes (DNA-methylation and histone-acetylation/deacetylation andmethylation/demethylation).Parentalbacteria-speciﬁcgroups werecombined,merging theparental Vibrioand Tenacibaculum treatmentsintofourparentaltreatments.

ThePERMANOVAmodel(‘vegan’package −‘adonis’function in R) was based on a Bray-Curtis matrix of non-transformed

−Ct-values,applying‘F0-parents’ and‘F1-offspring’ treatment as ﬁxed factors and including ‘family’ as nested factor within

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the‘F0-parents’treatmentsand ‘size’ascovariatetocorrect for size-relevantinﬂuences.Theanalysisofsimilarity(ANOSIM)was performedwiththesoftwarePRIMERv6(Clarke,1993;Clarkeand Gorley,2006)basedonaBray-Curtisdistancematrixand4th-root transformation,whichallowedapairwisecomparisonbetweenthe levelsofparentalandoffspringtreatment.Weappliedaprincipal componentanalysis(PCA)fortheevaluationofdifferentialgene expressionproﬁlescausedbytheparentaltreatments.Further,we useda between-classanalysis(BCA) toillustratetheinteraction betweenF0-parentaland F1-offspring treatment(Chesselet al., 2004;DolédecandChessel,1987;Thioulouseetal.,1995).Inaddi- tion,a correspondingscatterplotwasaddedtodemonstratethe contributionofeachsinglegene.Here,thedirectionofthearrows determinesthecorrelationbetweenvariables(genes)andprinci- palcomponents.Thelengthofthearrowisdirectlyproportionalto thecontributionofeachgenetothetotalvariability(Oksanenetal., 2007).

Statisticalunivariateapproaches wereappliedforlifehistory parametersandimmunecellcounts.Assumptionsofnormalityfor eachresponsevariableweregraphicallyexaminedandtestedwith theShapiro–WilkandLevinetestsafterBox–Coxtransformation usingJmp9(SASInstituteInc.,Cary,NC,USA).Alinearmodelwas fittedusingthefixedfactors‘F0-parents’and ‘F1-offspring’,the non-independentfactor‘family’or‘samplingday’or‘tank’nested into‘F0-parents’whiletaking‘size’ascovariate.Forthebodysize analysisthesamemodelwithoutcovariatewasused.Allsignificant ANOVASforF0-parentalandF1-offspringeffectswerefollowedby post-hocTukey’sHSDtest.

3. Results

3.1. Maternal,paternalandbiparentalimmuneprimingeffectsin one-week-vs.four-month-oldjuveniles

3.1.1. One-week-oldF1-juveniles−immunegeneexpression 29 candidate genes connected to pathways of the innate immune system, the adaptive immune system and the com- plement component system were signiﬁcantly affected upon the parental bacteria treatments (PERMANOVA-immune:

F_6,377=12.44, p<0.001 seeFig. 1A and TableS1 in the supple- mentaryonline Appendix). ThePCA revealed both a difference betweenthetwoappliedparentalbacteriatreatments(Vibrioand Tenacibaculum)andaparentalsex-basedeffect(Fig.1AandTable S2).F1-offspring of mothersand fatherschallenged withVibrio orTenacibaculumdisplayedasignificantlydifferentialexpression profilealongthesecondprincipalcomponent(PC2:18.9%oftotal variation) clustering according to the specific bacteria species (ANOSIM-immune:MatV+vs.MatT+, p=0.001;PatV+ vs.PatT+, p=0.001;Fig.1AandTableS2).However,bothbiparentaltreat- mentgroupsclusteredtogetherwiththematernal-Tenacibaculum aswellasthepaternal-Tenacibaculumtreatments,whichincluded theirrespectivesingleparentalTenacibaculumtreatment(Fig.1A).

Hence,thebiparentaltreatmentsweremoresimilartothemater- nalandpaternalTenacibaculumtreatmentsincomparisontothe parentalVibriotreatment.Particularly,thepaternalTenacibaculum treatmentwasnotsigniﬁcantlydifferentfromthebiparentaltreat- mentcomprisingtherespectivepaternalTenacibaculumtreatment (ANOSIM-immune:PatT+vs.Bi-(MatV+PatT+),p>0.05;Fig.1Aand TableS2).

3.1.2. Four-month-oldF1-juveniles–immunegeneexpression In four-month-old juveniles the identical29 immune genes weresigniﬁcantlyinﬂuencedbytheparentalbacteriatreatments (PERMANOVA-immune:F_6,98=3.21,p<0.001;Fig.2AandTableS1).

InthemultivariatePCAanalysisweidentiﬁedadifferencebetween

thetwoparentalbacteriatreatments(VibrioandTenacibaculum) independentoftheparentalsexeffectclusteredinopposeddirec- tionsalongtheﬁrstprincipalcomponent(PC1:27.8%ofthetotal variation)(ANOSIM-immune:MatV+vs.MatT+,p=0.001;PatV+vs.

PatT+,p<0.028;Fig.2AandTableS3).Juvenilesfrombothpater- naltreatmentswerenotsigniﬁcantlydifferentfromthoseofthe respectivebiparentaltreatments(ANOSIM-immune:PatT+vs.Bi- (MatV+PatT+),p>0.05;PatV+vs.Bi-(MatT+PatV+),p>0.05;Fig.2A andTableS3)indicatingalong-lastingpaternalimmunepriming effect.

3.1.3. Four-month-oldF1-juveniles–immunecellcounts

Theparentalimmunechallengeoffour-month-oldF1-offspring signiﬁcantlyaffectedthenumberoflymphocytesandmonocytesin theheadkidney(PERMANOVA-cellshk:F6,97=7.29,p<0.001;Table S1) and blood (PERMANOVA-cells blood: F_6,97=4.66, p<0.001;

TableS1).Thelymphocyte/monocyte-ratioofF1-offspringinthe head kidneywas strongly elevatedby the maternal as wellas the paternal Vibrio treatment (ANOVA-LM-ratio hk: F_6,73=6.4, p<0.001,TukeyHSD:C<MatV+,C<PatV+;Fig.4AandTableS5).

Incontrast,solelythepaternalTenacibaculumtreatmentelevated the L/M-ratio in F1-offspring (TukeyHSD: C=MatT+, C<PatT+;

Fig. 4A and Table S5). When parents received maternal-Vibrio andpaternal-Tenacibaculum treatmentin a biparentalcombination, the L/M-ratio was significantly raised in contrast to the controlgroup(TukeyHSD: C<Bi-(MatV+PatT+);Fig.4 andTable S5) but showed a similar pattern as the maternal or paternal combinations.In contrasttothat, theL/M-ratioin theblood of F1-offspring waspositivelyinfluenced upon the paternalVibrio treatment only (ANOVA-LM-ratio blood/F0-parents: F_6,73=5.15, p<0.001,TukeyHSD:C<PatV+;Fig.4BandTableS5)andfurther revealed a Vibrio-specific offspring treatment effect (ANOVA- LM-ratio blood/F1-offspring: F_2,73=7.89, p<0.001, TukeyHSD:

F1-N<F1-V+;TableS5).

3.2. Interactionofmaternal,paternalandbiparentalimmune primingeffectsandbacteriaspeciﬁcity

3.2.1. One-week-oldF1-juveniles–immunegeneexpression Basedonthe29immunegenesasignificantinteractionbetween theF0-parentalandtheF1-offspringtreatmentintheone-week- oldjuvenileswasdetected(PERMANOVA-immune:F_12,377=1.75, p<0.001;Fig.1BandTableS1).Theappliedpairwisecomparison ofinteractiontermsshowedthatthebiparentalimmunepriming effect was significantly influenced by the paternal Tenacibac- ulum immune challenge. Tenacibaculum-exposed F1-offspring of the biparental treatment Bi-(MatV+PatT+) were not significantly differentfromTenacibaculum-challenged offspring ofthe singlepaternal Tenacibaculumtreatment (ANOSIM-immune:Bi- (MatV+PatT+)/F1-T+ vs. PatT+/F1-T+, p>0.05; Table S2). With paternalVibriotreatmentthetwogroupsrevealedasignificantly different immune gene expression pattern (ANOSIM-immune:

Bi-(MatT+PatV+)/F1-V+ vs. PatV+/F1-V+, p=0.004; Table S2).

In contrast, both maternal treatment interaction terms were signiﬁcantly different from both biparental treatment interac- tions(ANOSIM-immune:Bi-(MatV+PatT+)/F1-V+vs.MatV+/F1-V+, p=0023;Bi-(MatT+PatV+)/F1-T+vs.MatT+/F1-T+,p=0.001;Table S2).

Tosimplifythevisualizationofthecomplexinteractionterms, the two respective parental and offspring bacteria treatment groups(VibrioandTenacibaculum)werecombinedintoonesingle bacteriatreatmentgroup(bac)tofocusonparentaleffectsonly.

Asdemonstrated in thebetween-class analysis (BCA), a robust F1-offspring treatmenteffectis explained by theﬁrst principal component (52.8% of total variation) displaying a clustering of the parental bacteria treatments apart from the parental con-

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Fig.1.Immunegeneexpressionofone-week-oldSyngnathustyphleF1-juveniles(N=420).(A)PCAplotbasedon29immunegenesvisualizingtheexpressionproﬁlesof 7F0-parentaltreatmentgroups(parentalcontrol(Control),paternalVibrio(PatV+)andTenacibaculum(PatT+),maternalVibrio(MatV+)andTenacibaculum(MatT+),and thebiparentaltreatmentgroups(Bi-MV+xPT+andBi-MT+xPV+)).(B)BCAplotbasedon29immunegenesdisplayingtheinteractionoffourF0-parentaltreatmentgroups (parentalcontrol(Control),paternal(Pat),maternal(Mat),andbiparental(Bi))withF1-offspringtreatment(Vibrio,Tenacibaculumcombinedinbacterialchallenge(Bac) versusnochallengeornaïve(N)).Colorsrepresentdifferentparentalbacterialchallenges,withVibrioinred,Tenacibaculuminblueandthejointbiparentalgroupsinpurple.

Thecorrespondingarrow-scatterplotsontherighthandsiderepresentthecontributionofeachvariable(immunegene)tothetotalvariability.Thecontributionofeachgene issymbolisedbythelengthofthearrow,whichisdirectlyproportionaltothecontributionofeachvariable.Thescaleofthegraphsisgivenbygridsizedintheupperright handcornerofeachpanel.TheEigenvaluesarerepresentedbyabarchartinthelowerrighthandcorner,withthetwoblackbarsequivalenttothetwoaxesusedtodraw thePCAorBCAplotandtheircorrespondingscatterplots(A:PC1=24.7%,PC2=18.9%;B:PC1=52.8%,PC2=20.1%).

trol(PERMANOVA-immune: F_2,377=18.36,p<0.001;Fig. 1Band TableS1).Moreover,F1-offspringofthebiparentalbacteriatreat- ment,whichwerechallengedwithbacteria(F0-biparental/F1-bac) assembled together with the F1-offspring of the correspond- ing paternal bacteria treatment (F0-paternal/F1-bac), whereas F1-offspringofthematernaltreatmentexposedtobacteria(F0- maternal/F1-bac) were similar to F1-offspring of the control (F0-control/F1-bac)(Fig.1B).

3.2.2. Four-month-oldF1-juveniles–geneexpressionand immunecellcounts

Infour-month-oldoffspringtherewasnointeractionbetween F1-parental treatment and F0-offspring treatment on the 29 immune genes (PERMANOVA-immune: F_12,377=0.83, p>0.05;

Fig. 2B and Table S1). The lymphocyte/monocyte ratio in the head kidney of four-month-old juveniles revealed a signiﬁcant F1-parental treatment and F0-offspring treatment interaction (PERMANOVA-cellshk:F_12,97=2.75,p<0.005;TableS1).TheL/M- ratio in the head kidney of F1-offspring with paternal Vibrio challengewasmorepronounceduponhomologousVibrioexposure compared to the heterologous Tenacibaculum treatment, indi- cating a paternal Vibrio speciﬁcity effect on the cellular level

(ANOVA-LM-ratiohk:F_12,73=2.9,p<0.002,TukeyHSD:F0-PatV+x F1-T+<F0-PatV+xF1-T+;Fig.S2andTableS5).

3.3. Maternal,paternalandbiparentalimmuneprimingeffects connectedtoepigeneticregulationmechanisms

3.3.1. One-week-oldF1-juveniles–geneexpressionofepigenetic regulationgenes

Inone-week-oldF1-offspring15genesresponsibleforepige- neticregulationmechanismslikeDNA-methylationandhistone- modiﬁcations displayed a signiﬁcant parental treatment effect (PERMANOVA-epigen:F6,377=6.6,p=0.001;Fig.3andTableS1).

Toreducecomplexityandfocusonparentaltreatmenteffects,we appliedanANOSIMforpairwisecomparisonsbetweencombined maternal,paternalandbiparentaleffectsindependentofthetwo parentalbacteriatreatments(TableS4).TheANOSIMindicatedthat theeffectwasnotdrivenbytheparentaltreatment,sincetherewas nodifferentialexpressioncomparedtothecontrolgroup(ANOSIM- epigen:Patvs.C,p>0.05;Matvs.C,p>0.05;Bivs.C,p>0.05;Fig.3A andTableS4).Itwasratherduetodifferenceswithinthematernal, paternalandbiparentaltreatments(ANOSIM-epigen:Patvs.Mat, p=0.019;Bivs.Mat,p=0.002,Bivs.Pat,p=0.004;Fig.3AandTable S4).

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Fig.2.Immunegeneexpressionoffour-month-oldjuvenilesofS.typhle(N=126).(A)PCAplotbasedon29immunegenesvisualizingtheexpressionproﬁlesof7F0-parental treatmentgroups(parentalcontrol(Control),paternalVibrio(PatV+)andTenacibaculum(PatT+),maternalVibrio(MatV+)andTenacibaculum(MatT+),andthebiparental treatmentgroups(Bi-MV+xPT+/Bi-MT+xPV+).(B)BCAplotbasedon29immunegenesdisplayingtheinteractionoffourF0-parentaltreatmentgroups(parentalcontrol (Control),paternal(Pat),maternal(Mat),andbiparental(Bi))withF1-offspringtreatment(Vibrio,Tenacibaculumcombinedinbacterialchallenge(Bac)versusnochallenge (N)).ColorsrepresentdifferentparentalbacterialchallengeswithVibrioinred,Tenacibaculuminblueandthejointbiparentalgroupsinpurple.Thecorrespondingarrow- scatterplotsontherighthandsiderepresentthecontributionofeachvariable(immunegene)tothetotalvariability.Thecontributionofeachgeneissymbolisedbythe lengthofthearrow,whichisdirectlyproportionaltothecontributionofeachvariable.Thescaleofthegraphsisgivenbygridsizedintheupperrighthandcornerofeach panel.TheEigenvaluesarerepresentedbyabarchartinthelowerrighthandcorner,withthetwoblackbarsequivalenttothetwoaxesusedtodrawthePCAorBCAplot andtheircorrespondingscatterplots(A:PC1=27.8%,PC2=14.7%;B:PC1=41.6%,PC2=17.3%).

3.3.2. Four-month-oldF1-juveniles–expressionofepigenetic regulationgenes

In contrast to the effects we found for one-week-old juveniles,epigeneticregulationgenesoffour-month-oldjuvenileswere affected by the parental bacteria challenges in comparison to thecontrolgroup(PERMANOVA-epigen:F_6,93=6.6,p=0.001,Table S1; ANOSIM-epigen: Pat vs. Control, p=0.001; Matvs. Control, p=0.006; Bivs.Control, p=0.001;Fig.3BandTableS4). Thisis additionallysupportedinthegraphicalvisualizationofthePCA,in whichparentaltreatmentsarefoundindistinctclustersalongthe firstprincipalcomponent(45.8%oftotalvariation).Nosignificant differencesbetweenmaternal,paternalandbiparentaltreatment couldbeidentified(Fig.3BandTableS4).

3.4. Costsofimmunepriming–lifehistorytraits 3.4.1. One-week-oldF1-juveniles–lifehistory(size)

One-week-oldF1-offspringwithparentalbacteriatreatments hadalargerbodysizethanthecontrolgroup(ANOVA-size.one- week: F2,378=30.04, p<0.001, C<MatV+, C<MatT+, C<PatV+, C<PatT+;Fig.S3AandTableS5).F1-offspringfrommotherschal- lengedwithbacteriaevenrevealedalargerbodysizethanoffspring withpaternaltreatment(TukeyHSD:PatV+<MatV+,PatT+<MatT+;

Fig.S3AandTable7).Inaddition,F1-offspringofmotherswithVib- rioexposurewerebiggerinsizethanoffspringofmotherswith Tenacibaculumtreatment(TukeyHSD:MatT+<MatV+;Fig.S3Aand TableS5).

3.4.2. Four-month-oldF1-juveniles–lifehistory (size/mass/CF/HSI)

Inamultivariateapproach,bodysize,mass,CFandHSIoffour- month-oldjuvenilepipefishweresignificantlyinfluencedbythe parentalimmune challenge(PERMANOVA-life-size/mass/CF/HSI:

F6,98=2.56,p<0.004;TableS1).However,body lengthandmass of four-month-old juvenileswere only increased if exposed to a maternal Vibrio immune challenge. Offspring with maternal Vibrioexposurewere1.35±0.32cmlargerand0.27±0.071gheav- ier than the control group (ANOVA-size: F_6,98=2.86, p=0.013, TukeyHSD: C<MatV+; Table S5 and Fig. S3B; ANOVA-mass:

F_6,98=4.43,p=0.001,TukeyHSD:C<MatV+,PatT+<MatV+,C<Bi- (MatV+PatT+); Table S5 and Fig. S3C). Further, the paternal Tenacibaculum treatment positively inﬂuenced the HSI of F1- offspring,whichhadalargerliver(1.24±0.24mg)thanoffspringof theparentalcontroltreatment(ANOVA-HSI:F6,74=2.39,p=0.034, TukeyHSD:C<T+;TableS1andS5andFig.S3D).

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Fig.3. Expressionof15epigeneticgenesfor(A)one-week-oldand(B)four-month-oldjuvenilesofS.typhle.ThePCAplotsarebasedon15genesassociatedwithepigenetic regulationprocesses(DNA-methylationandhistonemodiﬁcations)andshowtheexpressionproﬁlesofcombinedfourF0-parentaltreatmentgroups(parentalcontrol(black), paternal(red),maternal(blue),andbiparental(purple).Thecorrespondingarrow-scatterplotsontherighthandsiderepresentthecontributionofeachvariable(epigenetic gene)tothetotalvariability.Thecontributionofeachgeneissymbolisedbythelengthofthearrow,whichisdirectlyproportionaltothecontributionofeachvariable.The scaleofthegraphsisgivenbygridsizedintheupperrighthandcornerofeachpanel.TheEigenvaluesarerepresentedbyabarchartinthelowerrighthandcorner,with thetwoblackbarsequivalenttothetwoaxesusedtodrawthePCAplotandthecorrespondingscatterplots(A:PC1=44.2%,PC2=28.3%;B:PC1=45.8%,PC2=22.7%).

3.4.3. Six-month-oldF1-offspring–lifehistory(sexualmaturity) Upon a parental immune challenge, juvenile males devel- oped a brood pouch (i.e. sexualmaturity) one month (average 38.6±2days)laterthanoffspringofunexposedparents(ANOVA- maturitymales:F_3,168=158.8,p<0.001,TukeyHSD:C<Mat,C<Pat, C<Bi;Fig.S4andTableS5).Ifbothparentsreceivedthebacterial treatment,malesreachedsexualmaturityevenlater(6±1days) (TukeyHSD:Mat<Bi,Pat<Bi;Fig.S4andTableS5).

4. Discussion

4.1. Doescombinedmaternalandpaternalimmunepriming causeamorethanadditiveeffect?

Weexploredthesynergisticeffectsofmaternalandpaternal immuneprimingbyapplyingtwodifferentbacteriaspecies(Vibrio spp.andT.maritimum).Contrarytoourexpectations,thebiparental effectsprettymuchcompensatedeachotheranddidnotpileup to an additiveimpact. In contrast, biparental immune priming withthesamebacteriaphylotypesforbothparentsinducedsyn- ergisticbeneﬁcialeffects(Rothetal.,2012b).Thisdiscrepancymay havearisenduetostrongbacteria-speciﬁcimmuneprimingeffects (BeemelmannsandRoth,2016,unpublisheddata).Theimpactof biparental treatment in one-week-old juveniles was driven by Tenacibaculumbacteria,asoffspringreceivingbiparentaltreatment

combinationsrevealedasimilarimmunegeneexpressionproﬁleas theirsiblingswiththerespectivesinglematernalandsinglepater- nalTenacibaculumtreatments.Incontrast,thefactthattheoffspring treatmentgroupwithsingleparentalVibriotreatmentsclustered farapartfromthecontrolgroupandinanoppositedirectionto theparentalTenacibaculumgroupspointstoanantagonisticVibrio- mediatedparentaleffect.Ourresultsthussuggestthatthebacteria type determinesthestrengthand degreeofbiparentalimmune priming.

Thisbacteria-specificeffectwasalsofoundinfour-month-old juvenilesas indicated bysimilar immunegene expression pro- files.Olderjuvenilesofbiparentaltreatmentgroupsweresolely influenced by the paternal bacteria treatment, independent of theappliedbacteriaspecies.Hence,offspringoffatherswhohad beenchallengedwithVibrioorTenacibaculumbacteriashoweda similarimmunegeneexpressionprofileasoffspringofthecorre- spondingbiparentaltreatmentcombinations,whilethematernal groupsweresignificantlydifferentfromtherespectivebiparental groups. Neither was any synergistic parental immune priming effect detected in four-month-old juveniles, but we did find a dominatingpaternalimmuneprimingeffect.Inlinewiththis,the immunecellactivityintheheadkidneyandbloodoffour-month- old juveniles was upregulated upon the paternal treatmentas comparedtothecontrol.

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A

0.0 0.2 0.4

Control MatT+ MatV+ PatT+ PatV+

MatT+PatV+

MatV+PatT+

Lympho cyte/mono cyt e ratio of head kidney (4 −month)

B

0.0 0.4 0.8 1.2

Control MatT+ MatV+ PatT+ PatV+

MatT+PatV+

MatV+PatT+

Lymphocytes:Monocytes[ratio]

Lymphocyte/mono cyt e rati o of bloo d (4 −month)

Lymphocytes:Monocytes[ratio]

Fig.4. Parentaltreatmenteffectsbasedonimmunecellcountmeasurementsof four-month-oldjuvenilesofS.typhle(N=126).(A)Lymphocyte/monocyteratioof headkidney,(B)lymphocyte/monocyteratioofblood.Barplotsaregroupedaccord- ingtoF0-parentaltreatments(parentalcontrol(Control),maternalTenacibaculum (MatT+)andVibrio(MatV+),paternalTenacibaculum(PatT+)andVibrio(PatV+),and biparental(MatT+PatV/MatV+PatT+).Errorbarsrepresentthestandarderrorofthe mean(±sem).

4.2. Doweﬁndasymmetricparentalimmunepriming?

Consistentwithourhypothesis (II)we identified differences betweenmaternalandpaternalimmunepriming.Ourdataindi- cateasymmetricparentalinvestmentintooffspringimmunitywith astrongerpaternalthanmaternalcontribution.One-week-oldF1- offspringoffathersexposedtoTenacibaculumrevealedanimmune geneexpressionprofilethatcouldnotbedifferentiatedfromthat ofF1-offspringofparents exposedtobiparentaltreatment,also includingthepaternalTenacibaculumchallenge(Bi-(MatV+PatT+) identicaltoPatT+). In thiscombination, we furtheridentified a significantinteractiontermindicating a paternalTenacibaculum specificityeffect.Thissuggeststhatfathersmediatedthebiparental immuneprimingeffect.Four-month-oldjuvenilesevenrevealed stronger paternal immune priming effects independent of the appliedbacteriaspecies. Whenfocussingonlyonthematernal, paternalandbiparentalinteractionterm,wefounda stableand strongerimpactofthepaternaltreatmentonimmunegeneexpres- sionthanthatofthematernaltreatmentforbothone-week-oldand four-month-oldjuveniles.

F1-offspringoffathersexposedtoVibrioorTenacibaculumbac- teriainducedtheirimmunecellproliferationintheheadkidney, themajor immune organ of teleosts, earlier than those of the controlgroup. In addition, a homologous paternalVibrio expo- sure increased the cellular immune defense compared to the heterologouscombination,whichindicatesastrongpaternalVib- riospeciﬁcityeffect.Thatthelymphocyte/monocyte-ratiointhe bloodofF1-offspringwasincreasedonlyuponthepaternalVibrio treatmentsuggeststhatthepaternalVibriotreatmentcausedafast

inductionofcellularimmunityinthenextgeneration.Inthecase ofthematernaltreatment,onlytheexposuretoVibrioinducedcell proliferationinthehead-kidneyofF1-offspring,whichhighlights thatmothersonlyprovidedcellularprotectionagainstpotentially familiarVibriobacteria.Theparentalgenerationwaswild-caught intheKielFjordwherepipefishareinconstantcontactwithvari- ousVibriophylotypes(Rothetal.,2012a).Duringthepresentstudy, weusedaVibriophylotypeisolatedfromanItalianpipefish(Roth etal.,2012a),whichwesupposedtobeallopatricandmostlikely noveltotheimmunesystemofBalticpipefish.However,poten- tiallytheepitopestructureofItalianandBalticVibriophylotypesof theirnaturalhabitatwasnotdifferentiableforS.typhleeitherdue tostrongsimilaritiesorduetoimmunologicalcross-reactivity.In contrast,theTenacibaculumbacteriawereisolatesfromadifferent fishspeciesinthePacific(Suzukietal.,2001).Mostlikely,theshort maternalcontacttoTenacibaculumbacteriawasnotsufficientto provideimmunologicallong-termprotectiononthecellularlevel viatheegg.Thecloseconnectiontothefathersduringmalepreg- nancy,however,waslongenoughtoboost thecellularimmune defenseagainstbothexperiencedbacteria.

Theseresultssupportourhypothesis(II)thatinthissex-role reversedmatingsystemchallengedfathershaveastrongerinﬂu- enceonoffspringimmunity.Sincethefathersarecloselyconnected totheprogenyduringmalepregnancytheyprobablytransferred immunityforalongertimeperiod.In teleosts,femalesproduce costlyeggs,inwhichtheydepositnutritionproteinsandimmune componentsovertheeggyolk(Blyetal.,1986;BobeandLabbé, 2010; Swain et al., 2006). In the sex-role reversed pipeﬁsh S.

typhle females oviposit theireggsinto the male’sbrood pouch wheretheyarefertilisedandremainduringpregnancy(4weeks) (Berglund etal.,1986).Freshlyhatchedjuvenilesconsumepro- teinsofthematernalyolksac,butarealsosuppliedwithadditional nutrition and oxygen via the paternal brood pouch (Azzarello, 1991;Carcupinoetal.,1997,2002;Kvarnemoetal.,2011;Ripley and Foran, 2006;Ripley andForan, 2009).Consequently,males and females have different possibilities totransfer non-genetic componentstostimulatetheoffspring’simmunity.Infemales,a depositionintotheeggyolkismostlikely,whilemalesmaytransfer substancesthroughthespermﬂuidandtheplacenta-likestruc- tureinthebroodpouch.Malesandfemalesseemtohaveevolved differentmechanismstoinvestintotheimmunedefenceoftheir progeny.

4.3. Aretheredifferencesintermsofstabilitybetweenmaternal, paternalandbiparentalimmunepriming?

Parentaleffectsareofshortduration(Lindholmetal.,2006).Yet, inlinewithpreviousfindings(Rothetal.,2012b)ourdataindicate thattheimpactofparentalTGIPpersistedtofour-month-oldoff- spring.TheTGIPeffectsdidnotceaseafterthematurationofthe adaptiveimmunesystem,whichtakesapproximately4–8weeks infish(Magnadóttiretal.,2005).ThecontinuityofTGIPwasinflu- encedbythesexoftheparent.Inaccordancewithourhypothesis (III),paternaleffectswerefoundtobelonger-lastingthanmaternal effects.Infour-month-oldjuveniles,theimpactofpaternalimmune primingonimmunegeneexpressioncouldnotbedifferentiated fromthebiparentaltreatmentcombinations.Thisimpliesthatthe biparentalimpactwasdrivenbythepaternaltreatment.Moreover, immunecellactivityintheheadkidneycamewithspecificityinthe caseofpaternalVibrioexposure,andthepaternalimmunepriming effectwasevenpreservedintheblood.Ourresultsthusindicate thatpaternalimmuneprimingwasdrivingthebiparentaleffectsin four-month-oldjuveniles.

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4.4. Canchangesinepigeneticregulationgenesbeattributedto parentalsex-baseddifferences?

In one-week-old F1-juveniles genes involved in epigenetic regulation processes like DNA-methylation and histone acetylation/methylationrevealednodifferentialexpressionaccording to the parental treatment. Yet, we found paternal effects on histone-acetylation/deacetylationgenes.Throughanalteredtran- scriptional regulation,paternal immune priming mayinﬂuence offspringimmunegeneexpression,potentiallyfacilitatedviainher- itable immune memorycarried acrossgenerations by histones (Ragunathanetal.,2015).

In four-month-old juveniles, the same genes were affected bymaternal,paternalandbiparentalbacterialchallenge.During developmentandmaturation,DNA-methylationpatternsandepi- geneticmarksareunderconstantchangeandhighlyﬂexible(Monk etal.,1987;RazinandShemer,1995).Aconsistentpatternofepi- genetictracescanthuslikelyonlybeestablishedaftermaturation whenprogrammingoftheparentalDNAmethylomeiscomplete(Ci andLiu,2015).Recentdataevensuggestthatlong-lastingparental immuneprimingeffectscanbemaintainedviaepigeneticmarks andarepreserveduntilthesecondgeneration(Beemelmannsand Roth, 2016, unpublished data). In contrast to the opinion that TGIPceasesafterthematurationoftheadaptiveimmunesystem, long-lastingTGIPinfour-month-oldjuvenilesandeveninthesec- ondgeneration(BeemelmannsandRoth,2016,unpublisheddata) aremediatedthroughinheritedmaternalandpaternalepigenetic marks.

4.5. Arethereanyallocationtrade-offsandcostsassociatedwith maternal,paternalandbiparentalimmunepriming?

Themaintenance ofinducedimmunityisenergeticallycostly and,therefore,enhancedlevelsofimmunedefenceinprimedoff- springimplytrade-offswithotherﬁtness-relatedtraits(Lochmiller andDeerenberg,2000;Schmid-Hempel,2011;Zanchietal.,2011).

Inlinewiththis,TGIPexhibitedcostsintermsofprolongedtimeto sexualmaturity.F1-malesofthebiparentaltreatmentneededsig- nificantlylongertoproducebroodpouchtissueand,hence,reach sexualmaturitythanindividualsofuni-maternaloruni-paternal treatments.Thisimplieshighcostsofbiparentalimmuneprim- ing.Aslong-lastingpaternalimmune primingbearsequalcosts asmaternalimmuneprimingin termsofdelayedmaturity,this suggeststhatthepaternalmechanismofimmunetransferismore effectiveand less costly.However, maternally primedoffspring revealedadditionalbenefitsthroughanimprovedbodycondition, asreflectedbylargeroffspringsize,indicatingthatexposedmoth- ersinvestmoreintothecurrentclutchduetothepossibleriskof notsurvivingthenextreproductiveevent(Landisetal.,2015).

5. Conclusion

Our study highlights the difference between maternal and paternalimmunepriminginpipeﬁsh.Itsupportsourpredictions ofasymmetricparentalinvestmentintooffspringimmunityand of dominatinglong-lastingpaternal immunepriming. Enhance- mentofimmunityuponpaternalimmunechallengealteredgene expressionandimprovedpathogen-speciﬁcimmunecellactivity.

Asoffspringarebornintheirfather’senvironmentandmostlikely experienceasimilarpathogenassembly,transferofasolidlong- termprotection against current pathogens should befavoured.

WhilematernalTGIPhadastrongimpactonyoungjuveniles,it diminishedduringthecourseofoffspringmaturationand came withlowspeciﬁcity.Eventhoughmaternaleffectswereweakerand oflowerstability,prolongedreproductiontimewascausedequally

bybothparents.However,thesedetrimentaleffectsmaybecom- pensatedbyapositivematernalimpactonoffspringsizeandbody mass.

Itseemsthatparentalpathogenexperienceinduceddifferen- tialand sometimesevenantagonistic geneexpression patterns.

Hence,ina biparentalcombinationwithtwo differentbacterial species,thespecifictypeofbacteriumdeterminedthestrengthand degreeofbiparentalimmunepriming.Thisledtoacompensated biparentalimpactinsteadofsynergisticadditivebiparentaleffects ashasbeenshowninapreviousstudythatusedidenticalpathogens forbothparents(Rothetal.,2012b).Asthebiparentaltreatment didnotleadtoanadditivelong-termeffectandalsocausedasevere delayinoffspringmaturation,biparentalimmuneprimingseemsa costlystrategy(Contreras-Gardu ñoetal.,2014).Ourresultsindi- catethat TGIPhasimportant consequencesontheevolution of lifehistory,host–pathogencoevolutionandparentalinvestment strategies.Wefoundthefirstevidencefortheinvolvementofepi- genetic regulationprocesses like DNA-methylation and histone acetylation,whichbothparentsaffectedmorewithincreasingage oftheiroffspring.Wesupposethatmaternalandpaternalepige- neticmarksmightmediatelong-termprotection.Furtherresearch shouldfocusonparentalDNA-methylationandhistoneacetyla- tionpatternsuponpathogenexposuretounravelthemechanisms behindTGIP.TostudywhetheritholdstruethatTGIPcorrelates withparentalinvestment,matingsystemswithvaryingmaternal andpaternalcontributionsshouldbeexamined.

Acknowledgments

WethankMaudePoirier,SophiaWegner,MartinGrimm,Verena Klein,FabianWendt,JoaoGladeck,IsabelKeller,SusanneLandis, JoannaMiest,DianaGill,FranziskaBrunnerandAndreaFrankefor supportduringexperimentalﬁeldandlabwork.Thisstudywas ﬁnancedbygrantstoO.R.fromtheGermanResearchFoundation (DFG,project4628/1-1,associatedwiththePriorityProgramme SPP1399,Host–ParasiteCoevolution)andtheVolkswagenstiftung.

A.B.wassupportedbyastipendfromtheInternationalMaxPlanck ResearchSchoolforEvolutionaryBiology(IMPRS).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.zool.2016.06.002.

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