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Molecular & Biochemical Parasitology

The ecdysone receptor (EcR) is a major regulator of tissue

development and growth in the marine salmonid ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae)

Liv Sandlund

^a,∗

, Frank Nilsen

^a

, Rune Male

^b

, Sussie Dalvin

^c

aSeaLiceResearchCentre,DepartmentofBiology,UniversityofBergen,Norway

bSeaLiceResearchCentre,DepartmentofMolecularBiology,UniversityofBergen,Norway

cSeaLiceResearchCentre,InstituteofMarineResearch,Norway

a r t i c l e i n f o

Articlehistory:

Received8January2016

Receivedinrevisedform15June2016 Accepted20June2016

Availableonlinexxx

Keywords:

Copepod Sealice Parasite RNAinterference Ecdysonereceptor Molting

a b s t r a c t

Thefunctionoftheecdysonereceptor(EcR)duringdevelopmentandmoltinghasbeenthoroughly investigatedinsomearthropodssuchasinsectsbutrarelyincrustaceancopepodssuchasthesalmon louseLepeophtheirussalmonis(L.salmonis)(Copepoda,Caligidae).Thesalmonlouseisanectoparasiteon Atlanticsalmonthathasmajoreconomicalimpactinaquacultureduetothecostofmedicaltreatment methodstoremovelicefromtheﬁsh.Handlingofsalmonlouseinfestationsisfurthercomplicatedby developmentofresistancetowardsavailablemedicines.Understandingofbasicmolecularbiologicalpro- cessesinthesalmonlouseisessentialtoenabledevelopmentofnewtoolstocontroltheparasite.Inthis study,wefoundL.salmonisEcR(LsEcR)transcripttobepresentintheneuronalsomataofthebrain,nuclei ofmuscleﬁbresandtheimmatureintestineofthesalmonlouse.Furthermore,weexploredthefunction ofLsEcRduringdevelopmentusingRNAinterferencemediatedknock-downandthroughinfectiontrials.

Ourresultsshowthatknock-downofLsEcRinthesalmonlouseisassociatedwithhypotrophyofseveral tissues,delayeddevelopmentandmortality.Inaddition,combinedknock-downofLsEcR/LsRXRresulted inmoltingarrestduringearlylarvalstages.

1. Introduction

Thesalmonlouse(Lepeophtheirussalmonis)isanectoparasitic copepodonsalmonidswithalifecyclethatincludesbothfree-living andparasiticstages.Thelousefeedsontheskin,mucosaandblood ofitshostandcancauseskinerosionandopenwoundsthatmay leadtodeathiftheparasiteinfectionbecomessevere.Duetoits highfecundity,salmonlousehasbecomeanincreasingthreatto thesalmonfarmingindustrywherehighfishdensitiesandallyear roundproductionprovidefavourableconditionsforlargenumbers oftheparasite[1,2].Thefree-livingstagesoftheparasitecanspread betweenpens,differentfarmingsitesandalsotowildsalmonid fishcausingenvironmentalconcerns.Infestationofwildfishwith

Abbreviations:L.salmonis,Lepeoptheirussalmonis;LsEcR,Lepeoptheirussalmonis ecdysonereceptor;LsRXR,LepeoptheirussalmonisretinoidXreceptor;RT-qPCR,real- timequantitativePCR;RNAi,RNAinterference;dsRNA,doublestrandedRNA.

∗Correspondingauthor.

E-mailaddress:[email protected](L.Sandlund).

salmonlouserearedonﬁshinaquaculturehasbeenarguedtocause reductioninwildpopulationsnearbyaquaculturesites[3–7].

The salmon louse life cycle consistsof eight developmental stagesnamednaupliaIandII,copepodid,chalimiIandII,pre-adult IandIIandtheadultstage[8,9]thatareseparatedbyamolt.Dur- ingmoltanewexoskeletonissynthesizedandsynchronizedwith sheddingoftheoldexoskeleton.Themoltingprocessisacyclic eventthatiscarefullyregulatedandrequirestheprecisecoordina- tionofspeciﬁchormonestobesuccessful.Thehormoneconsidered as themaindeterminant ofthemolting processin crustaceans isthesteroidhormone 20-hydroxyecdysone(20E), reviewedby (Chang et al.,2011) [10]. The effect of 20E is exertedthrough a nuclearreceptorcomplexconsistingof theecdysonereceptor (EcR)andtheretinoidXreceptor(RXR)referredtoasultraspir- acle(USP)in insects [11,12]. Thisheterodimeralsoplaysa key roleintheregulationofmanyotherphysiologicalprocessessuch asdevelopment,reproductionandlimbgeneration[13–16].Upon activation byecdysteroids, the EcR/RXRcomplex directlyregu- latesa smallsetof“early”genesthat furtherregulatesa larger setof“late”genesinagenetichierarchy[17,18].Multiplesplice

http://dx.doi.org/10.1016/j.molbiopara.2016.06.007

0/).

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variantswithdifferentexpressionpatternsofEcRandRXRhave beenidentiﬁedinbothinsectandcrustaceanssuchasDrosophila melanogaster[19],Homarusamericanus[20],Daphniamagna[21]

andMarsupenaeusjaponicus[14].Differencesinspatialandtem- poralexpressionpatternsbetweentheisoformshave shownto correlatewithspeciﬁcresponsesandhavedistinctfunctionsduring development[22–24].Inthesalmonlouse,threeLsEcRtranscripts differingonlyintheir5UTRregionhasbeencharacterized[25]

whereasseveralLsRXRsplicevariantsoftheopenreadingframe wereidentiﬁedbyEichneretal.,2015[26].Duringmetamorphic reorganisationoftheinsectcentralnervoussystem,20Eregulates awide varietyof cellularresponsessuchasneuronal prolifera- tion,maturation,celldeathandremodellingoflarvalneuronsinto theiradultforms.InactivationofthetwoDrosophilaEcR-B1and EcR-B2isoformsgavedefectsintheearlystagesofneuronalremod- ellingduringmetamorphosis[22],whileheatshockinducedEcR nullmutantswerearrestedinlateembryogenesisorunabletogo throughmoltandmetamorphosisdependingontheirdevelopmen- talstage[23,27].

TheroleoftheEcRindevelopmentalprocessesandmoltingis verywellestablishedininsectsandtosomeextentindecapods, however,moleculardataregardingthespeciﬁcfunctionofEcRin copepodsisstilllimited[21].RNAinterference(RNAi)hasbeensuc- cessfullyappliedtoinvestigategenefunctioninsalmonlouseand servesasanimportanttooltounderstandthebiologyoftheparasite [28–31].Knock-downofLsEcRinadultfemalesalmonliceresulted indegenerationofvitellogeninproducingtissueandeggstringsand thereforabolishmentofreproduction,provingtheimportantroleof LsEcRinfemalereproduction[25].DuetotheimportantroleofEcR inmanyvitalbiologicalprocessesasaligandactivatedtranscription factor,itisimportanttoexplorethefunctionofLsEcRinmolting anddevelopmentandasa possibletargetforpesticides.In this study,weshowthatknock-downofLsEcRexpressioninL.salmo- nislarvaeresultsincomprehensivetissuedamageandmortalityin laterdevelopmentalstages.Inaddition,weshowthatsimultane- ousknock-downofLsEcRanditspartnerLsRXRinnaupliainterrupts themoltingprocess.Inaddition,knock-downofLsEcRindirectlyor directlyaffectstheexpressionofgenesimportantinchitinsynthe- sis.ThissuggeststhatLsEcRplaysakeyroleinmolting,growthand tissuedevelopmentofthesalmonlouse.

2. Materialsandmethods 2.1. Animals

EggsfromtheAtlanticsalmonlouseLepeophtheirussalmonis[32]

werehatchedin wellswithﬂow-throughseawaterand keptin cultureonAtlanticsalmon(Salmosalar)aspreviouslydescribed [33].Thesalmonwaskeptinsingletankswithseawater(34.5ppt) atapproximately10^◦C[34].Allexperimentswereconductedin accordancewiththeNorwegianlegislationforanimalwelfare.

2.2. Insituhybridisation

Copepodidswerefixedin4%paraformaldehydeinphosphate bufferedsalinefor24handtransferredto70%ethanolforamini- mumof24hbeforebeingembeddedinparaffinwax.Localisation ofLsEcRtranscriptincopepodidswasperformedasdescribedby Trösse et al., 2014 [31] with modifications described in Sand- lundetal.,2015[25].LsEcRspecificcDNAwasusedastemplate togenerate PCRproductwithT7promoter extensionsfollowed bysynthesisofsinglestrandeddigoxigenin-labelledRNAprobes (667bp)(PrimerslistedinTable1.).Probeconcentrationandqual- itywasdeterminedbyspectrometry,employingaNanodropND 1000(ThermoFisherScientific)and a spottest,respectively. In

brief,sectionsweredewaxedinHistoclear(NationalDiagnostics) beforerehydrationoftissueandproteinaseKtreatment(10min) followedbyﬁxationin4%formaldehydeinPBS,aceticanhydride treatmentanddehydration.100␮lhybridizationmixcontaining 20ngdigoxigenin-labelledRNAwereaddedtothetissueandincu- batedinavacuumchamberat60^◦Covernight.Chromogenesiswas carriedoutusingnitrobluetetrazolium(NBT)(RocheDiagnostics GMbH)and5-bromo-4-chloro-3-indolylphosphate(BCIP)(Roche).

SenseRNAwasusedasanegativecontrol.

2.3. Proteindetection

Rabbitpolyclonalantibodies(pAb)wereraisedagainstasyn- theticLsEcRpeptidethatcorrespondingtoauniqueregionofthe LsEcR,residues264–277(AADTTVDPKSNNNG),byGenScript(Pis- cataway,NJ,USA).ThepAbsuccessfullydetectedtheLsEcRprotein attheexpectedMwof60.3kDabyWesternblotanalysis.Several trialsofimmunoﬂuorescencestudiesusingthesameantibodywere notsuccessful.

2.4. FunctionalstudiesusingRNAi

DoublestrandedRNA(dsRNA)fragmentsforLsEcRandLsRXR (LsEcRfragment: position782–1458, Ac.no.KP100057.1;LsRXR fragment: positionin 323–945, Ac.No. KJ361516) were gener- atedbyT7RNApolymerase(MEGAscript®RNAiKit,Ambioninc) fromPCR templateswithT7 promoter regions.An850bpgene fragmentfrom Atlanticcod (Gadus morhua)wasused as negativecontrol(PrimersarelistedinTable1.).RNAionsalmonlouse larvaewasperformedasdescribed[30].Fivebiological parallels of naupliaI(n=5×30)were soakedin either1.5␮gof control dsRNA fragment or LsEcR dsRNA alone or together with equal amountsofLsRXRdsRNA,in150␮lofseawaterandleftforincu- bationovernightat9±1^◦C.Thenextday,sampleswerechecked forthepresenceofexuvia,shedduringthemoltfromnaupliaI tonaupliaIIstage,andnaupliaIIwastransferredtoﬂowthrough incubatorsandleftforsevendaysposttreatment(d.p.t.)whenthe controlhadmoltedintocopepodids.Copepodidswereinspected underabinocularmicroscope(OlympusSZX0.5and1.6×Olym- pusobjective)andphotographed(CanonEOS600D).Theanimals werephotographed,collectedandstoredonRNAlater^TM(Ambion) orﬁxedinKarnovsky‘sreagentforembeddinginplastic.

2.5. Infectiontrial

Copepodids (100/fish) were released in tanks holding one salmon/tank.Thewater level washeld low forthe first10min oftheinfectionafterwhichwaterflowwasreturnedtonormal.

Waterfrom thetanks wasfiltered througha double filtersys- tem(20␮m)tocollectcopepodidsthatdidnotsuccessfullyattach tothefish.Threefishwereinfected withcontrol liceand three fishwereinfectedwithLsEcRdsRNAtreatedlice(knock-downof LsEcRwasverifiedbeforeinfection).Theexperimentwastermi- nated27dayspostinfectionwhenthecontrollicehaddevelopedto thepre-adult/adultstage.Thefishwasanaesthetisedinamixtureof methomidate(5mg/l)andbenzocaine(60mg/l)andlicewerecare- fullyremovedwithforceps,counted,inspectedunderamicroscope anddocumentedasdescribedinSection2.4.Bothcephalothorax length(CL)andtotal length(TL) ofthecontrolandLsEcRkdlice weremeasuredasdescribedbyEichneretal.,2015[35]inorderto determineanydifferencesinsize.

2.6. ExtractionofRNAandcDNAsynthesis

ForRNAiexperiments,30copepodidswerehomogenisedusing 1.4mmzirconiumoxidebeads(Bertin)inTRIReagent®(Sigma-

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Table.1

PrimersequencesandTaqman®assays^ausedinthisstudy.

Primername^b Sequence(5‘-3‘) Method

LsEcRspeciﬁcP1 CCGATTTGCCATTACGTAGGCTTGTAGAGC insitu/dsRNA

LsEcRspeciﬁcP2 CCGCAGCTGCAGCCGACACAACTGTAGAT insitu/dsRNA

LsEcRspeciﬁcP3 CGAGCGTTTCCACTTACTTGCCAT dsRNA

LsEcRspeciﬁcP4 CGCCAACAACGACGACCCTCCACCAACAGCACT dsRNA

LsRXRspeciﬁcP1 CGGAATTGGGATGTCTACGAGCCATCATA dsRNA

LsRXRspeciﬁcP2 CTTCCTCTGACTCACTATAGAAGCATA dsRNA

CodspeciﬁcT7f ATAGGGCGAATTGGGTACCG dsRNA

Cod speciﬁc T7r AAAGGGAACAAAAGCTGGAGC dsRNA

LsEF1␣f CATCGCCTGCAAGTTTAACCAAATT RTq-PCR

LsEF1␣r CCGGCATCACCAGACTTGA RTq-PCR

LsEF1␣TaqMan^® ACGTACTGGTAAATCCAC RTq-PCR

LsEcRtotalf TCGGGAGAAAGTCCCTCTTCT RTq-PCR

LsEcRtotalr ACAGCTCCAGTAGGTGTTAAAGGA RTq-PCR

LsEcRtotalTaqMan^® TCGCAGTCCATTCTC RTq-PCR

LsRXRtotalf CCTAGTTGAACTCATCGCCAAAATG RTq-PCR

LsRXRtotalr TGAAGAGTATGATGGCTCGTAGACA RTq-PCR

LsRXRtotalTaqMan^® CCGCTTTGTCCATTTGCAT RTq-PCR

LsHR38f CCGTTCTCACAACTTCCTTTACCAT RTq-PCR

LsHR38r CGTCGAAATCGATGTCAGTTTTGC RTq-PCR

LsHR38TaqMan^® CCCACGGCAAGACAT RTq-PCR

LsE74f GGTCACGTTAAAGATGGGTCAATTT RTq-PCR

LsE74r CCAACAGGAGTACTAACACAACTGAT RTq-PCR

LsE74TaqMan^® CAGCGCCTCGTTCAC RTq-PCR

LsE75f CCTTGACCAATTTTCAGAACGGTTT RTq-PCR

LsE75r AATCCAGGGATCCGCTTGG RTq-PCR

LsE75TaqMan^® CACGTTCGCCAAGTTT RTq-PCR

LsCPDf TGTCAAATAAGCGTGAGGTTAAGGAA RTq-PCR

LsCPDf GCGCGTGAATACCACAATCC RTq-PCR

LsCPD TaqMan^® ACCCAAACGATCTTCC RTq-PCR

LsChit1f TCCATTCATTTGTACACATGTGGCTTA RTq-PCR

LsChit1r CATTGTAAGGGTCAAGGAGTCGAAT RTq-PCR

LsChit1TaqMan^® CAGACCAGCAAATCCA RTq-PCR

LsChit2f GTTAATTTCTTAAACAAGTATAACTTCGACGGTT RTq-PCR

LsChiti2r GGAACACCACCTCGTTTAGCA RTq-PCR

LsChit2TaqMan^® TTCCCAGTCAATATCC RTq-PCR

LsChs1f TTTCGAGGTAAAGCACTTATGGATGAT RTq-PCR

LsChs1R AGCCATCTATCTTCTCCTTGATCGT RTq-PCR

LsChs1TaqMan^® ATGCCTTGGTTCTTCC RTq-PCR

LsChs2f AGCAGTGACTATGCTTTTAGATTTGATGA RTq-PCR

LsChs2r CTGAGCCTAAAGGATGAATTCTTCCA RTq-PCR

LsChs2TaqMan^® CACTGCGCCGGTTTT RTq-PCR

RACE,rapidampliﬁcationofcDNAends;TOPO,DNAtopoisomeraseI;dsRNA,double-strandedRNA;RTq-PCR,real-timequantitativePCR.

aTaqman®assayswereprovidedbyAppliedBiosystems,Branchurg,NJ,USA.

bAllgeneralprimerswerepurchasedfromSigma-Aldrich,StLouis,MO,USA.

Aldrich), according to the manufacturers protocol. RNA was extractedfromthewaterphaseusingRNeasymicrokit(Qiagen) asdescribedbythemanufacturer.Sampleswerestoredat−80^◦C.

Concentrationsof RNA wasdetermined by NanoDropND-1000 spectrophotometerat260nm(NanoDropTechnologiesInc.)Two parallelreactionsofcDNAsynthesiswereachievedusingAfﬁni- tyScriptqPCRcDNAsynthesisKit(AgilentTechnologies)according tothemanufacturer‘sprotocol.AﬁvefolddilutionofcDNAwas storedat−20^◦C.

2.7. DetectionoftranscriptlevelindsRNAtreatedlicebyreal timequantitative-PCR(RT-qPCR)

RT-qPCRwascarriedoutusingAppliedBiosystems7500 Real- TimePCRSystem(AppliedBiosystems^®)understandardconditions (initiation:50^◦C2min,holding:95^◦C 10min,40cyclesof95^◦C 15s followed by 60^◦C 1min) aspreviously described in Sand- lundetal.,2015[25].Transcriptionlevelsofallgenesinvestigated werenormalisedtotheestablishedsalmonlousestandard gene eEF1␣ [36]. Eightdilutionsof RNA wereused tocreate a stan- dardcurveandallRT-qPCRswerecarriedoutinduplicate.Samples wereanalysedindependentlybutsimultaneouslyaseEF1␣using

thesamecDNAandmastermix(TaqMan®UniversalPCRMaster Mix;AppliedBiosystems).Fivebiologicalparallelsofcopepodids (n=5×30)wererunforeachdsRNAtreatmentgroup.Resultswere analysedbythe2ˆ⁻^Ct approachandpresentedwith95%con- ﬁdenceintervalcalculatedfromthe2ˆ^−Ctvalues.T-testswere usedtodeterminedifferencesinexpressionbetweenthecontrol groupanddsRNA treatedanimals.Ap-value of0.05 wassetas threshold.

2.8. Histology

CopepodidsforlightmicroscopywereﬁxedinKarnovsky‘sﬁx- ative,rinsedinPBSanddehydratedinaseriesofethanolsolutions (50%,70%and 96%),beforeembeddinginTechnovit7100(Her- aeusKulzerTeqhnique)asdescribed by[35].Sections(1–2␮m) werestainedwithToluidineblue(1%in2%borax).Sectionswere mountedusingHistomount(Invitrogen).

MicroscopyandimagingwereacquiredwithanAxioScope.A1 lightmicroscopeconnectedtoAxiocam105colorcamera(Zeiss International)andprocessedusingAdobePhotoshopCS6(Adobe Systems).

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Fig.1.LocalisationofLsEcRtranscriptinLepeophtheirussalmoniscopepodidlarvae.A)Lightmicroscopeimageofacopepodidlarvae(sevendays)toshowtheoutlineofthe animal.B)InsituhybridisationusingLsEcR-speciﬁcprobeswasusedfordetectionoftranscript.Negativecontrol(senseRNA)isshown(insert).Transcriptwasdetectedin theneuronalsomataofthebrain(whiteframe)andmyonucleithroughoutthecopepodid(asterisk).Aweakpositivestainingwasalsoobservedintheimmatureintestine (blackframe).Weakunspeciﬁccolouringoftheoutercuticularlayerwasseenusingboththesenseandtheanti-senseprobes.C)Ahistologicalsectionofacopepodidwas shownforbettervisualisationofselectivetissues.Whiteandblackframesareusedtobettervisualizethelocalisationofneuronalandintestinaltissue,respectivelyand asterisksareusedtomarkmuscletissue.Scalebars=200␮m(A–C).

3. Results

3.1. LsEcRtranscriptexpressioninL.salmoniscopepodids

Sectionsoffree-livingcopepodids(7daysold)wereusedforin situhybridisationinordertoidentifythelocalisationofLsEcRtran- scriptsincopepodids.ExpressionofLsEcRtranscriptwasdetected intheneuronalsomataofthebrain(Fig.1B;whiteframe)aswell asinmyonucleithroughouttheanimal(Fig.1B;muscleﬁbresare markedwithasterisks).Inaddition,aweakstainingoftheimma- tureintestinecouldalsobeobserved(Fig.1B;blackframe).

3.2. Knock-downofLsEcRbyRNAididnotinhibitmoltingfrom naupliaIItocopepodidlarvae

ToassessthefunctionofLsEcRduringearlydevelopmentand molting,RNAiexperimentswereperformedbysoakingnaupliaI

larvaeindsRNAfragmentspecifictoLsEcR.Theexperiment was terminatedsevend.p.t.whencontrolanimalshadmoltedtothe copepodidstage.Twoseparateexperimentswererunshowingsim- ilarresults.ThedsLsEcRtreatedgroupdevelopednormallyfrom nauplia II to the copepodid stage and did not show any loss- of-functionphenotype(Fig.2B)comparedtothecontrolanimals (Fig. 2A) sevend.p.t. RTq-PCR analysiswas performedin order todeterminethedegreeofLsEcRknock-down (LsEcR)and con- firmedasignificantdecreaseofLsEcRtranscriptbyanaverageof 57%(Fig.3A).HistologicalsectionsofthedsLsEcRtreatedlicewere examinedtoidentifyanydifferencesininternalmorphologycom- paredto thecontrol, however noabnormalities wereobserved (resultsnotshown).

Thebinding of ecdysone hormonesto theEcR/USP complex isknowntodirectlyand indirectlyregulateseveralothergenes includingthetranscriptionfactorsLsE75andLsE74andthehor- monereceptor HR38.Consequently RT-qPCR wasperformed in

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Fig.2.SelectionofrepresentativeanimalsobtainedfromtheRNAiexperimentusingmoltingnaupliusIlarvae.A)Controlgroup.B)Larvaetreatedwithdouble-strandedRNA (dsRNA)targetedtoLsEcRalonedevelopedintocopepodidsanddidnotshowanyabnormalphenotypecomparedtothecontrolgroup(A).C)Larvaetreatedwithfragments targetingbothLsEcRandLsRXR,wasobservedtohavedevelopedcopepodidlikefeatures(markedwithwhitearrow)insidetheexuvia(markedwithblackarrow)andwas viableatdaysevenpostsoaking.However,themoltingfromnaupliaIItocopepodidwasarrestedandtheanimalsstartedtodieafter12days.Morphologyofanormally developednaupliaIIlarvaeisshowninD(forreferenceonly).Scalebars=200␮m(A–D).

Fig.3.Relativeexpressionoftheecdysonereceptor(EcR)andtheretinoidXrecep- tor(RXR)togetherwithpossibletargetgenesregulatedbytheLsEcR/LsRXRcomplex, aftertreatmentwithdouble-strandedRNA(dsRNA).LepeophtheirussalmonisEcR (LsEcR)andLsEcR/LsRXRtreatedcopepodidswerecollected7daysposttreatment.

Quantitativereal-timePCR(RTq-PCR)ofLsEcR,LsRXRandthepossibletargetgenes LsE75,LsE74,LsChi1,LsChi2,LsCHS1,LsCHS2,LsCP1andLsHR38wereevaluated.Gen- BankAccessionNumbers:LsEcR:KP100057,LsRXR:KJ361516,LsChi1:AJD87505.1, LsChi2:AIE45495.1,LsCHS1:KX349436,LsCHS2:KX349435,LsE75:KX260137LsE74:

KX349434,LsCP1:BT078319.1,LsHR38:KX349433.Thegraphsarerepresentativesof twoindividualexperiments.Theexpressionlevelsoftherespectivegenesinthecon- trolgroupsweresetto1.Valuesareexpressedasmean±confidenceintervalsoffive biologicalreplicates(n=30copepodids/sample).*Statisticallysignificant(P<0.05).

StatisticalanalysiswasperformedusingaT-test.

dsRNA treated lice on these genes and several genes directly involvedinthemoltingprocess:LsChitinase1(LsChi1),LsChitinase2 (LsChi2),LsChitinsynthase1(LsCHS1),LsChitinsynthase2(LsCHS2), LsZinc carboxypeptidase (LsCP1) transcripts were measured in dsRNAtreated lice.Asigniﬁcantdown-regulationwasobserved for LsCP1, LsCHS2 and LsHR38 while a signiﬁcantup-regulation wasdetectedforLsChi1intheLsEcRkdanimals(Fig.3A)(T-test, P<0.05).

3.3. Simultaneousknock-downofLsEcRandLsRXRisoforms resultedinmoltingarrestofnaupliaIIlarvae

Anadditionalexperimentwassetuptoinvestigatetheeffect ofsimultaneousknock-downofLsEcRandLsRXRinsalmonlouse larvae,asdescribedabove.IncontrasttothedsLsEcRtreatedlice, moltingoftheLsEcR/LsRXRknock-downlicewasinterruptedand the treated lice did not molt from nauplia II (Fig. 2D) to the copepodid stage(Fig.2A). ThedsLsEcR/dsLsRXR treatedlice had developedsomefeaturessimilartothatofthecontrolcopepodid suchassegmentationoftheabdomen(Fig.2C;markedbywhite arrow)but theywerenot abletoemergefromtheoldcuticula (Fig.2C;edge ofexuviamarkedby blackarrow).RT-qPCR con- firmedasignificantknock-downofbothLsEcRandLsRXRby46%and 57%,respectively(Fig.3B)(T-test,P<0.05).Inaddition,thesame genesmentionedinSection3.2wereanalysedbyRT-qPCRanal- ysisinthedsLsEcR/dsLsRXRtreatedanimals.SimilartotheLsEcR knock-downanimals,LsCP1,LsCHS2andLsHR38weresignificantly down-regulatedinthedsLsEcR/dsLsRXRtreatedanimals(Fig.3B) (T-test,P<0.05).However,incontrasttothedsLsEcRtreatedlice nosignificantup-ordown-regulationwasobservedfortheLsChi1 orLsChi2inthedsLsEcR/dsLsRXRtreatedanimals(Fig.3B)(T-test, P<0.05).

3.4. InfectiontrialusingLsEcRknock-downanimals

An infection trial with LsEcR knock-down copepodids was performed.Atlantic salmonwasinfected witheither controlor LsEcRkdcopepodidsandcultivatedonthehostﬁshuntilthecon- trol group had developed into pre-adult I and II females and pre-adultIIandadultmales(27d.p.i.).Attermination,noabnor-

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Fig.4.RepresentativeanimalsobtainedafterinfectiontrialusingLsEcRknock-down(LsEcRKd)copepodids.A,D)showspre-adultIIfemale(A)andmale(D)controlanimals 27daysafterinfection.B,E)representsLsEcRkdfemale(B)andmale(E)animals.C)ThefemaleLsEcRkdlicecouldbedividedintotwogroupsthatwereeithersigniﬁcantly smallerorthesamesizeasthecontrolgroup.F)AllmaleLsEcRkdliceweresigniﬁcantlysmallercomparedtothecontrol.StatisticalanalysiswasperformedusingaT-test;

P<0.05.Scalebar=1mmA–B,D–E.

malities couldbe observed by light microscopy in thedsLsEcR lice. However, high mortality was evident and a significantly lowernumber ofLsEcRkd lice(n=29)were recoveredfromthe hostcompared tothecontrolgroup(n=96).TheLsEcRkdmales weresignificantlyshorterinCLandTL(Fig.4E,F;T-test,P<0.05) comparedtothecontrolgroup(Fig.4D,F)withafewindividu- alsthat wereevenshorter thannormallydevelopedpre-adultI males[9].In contrast,thefemaleLsEcRkd licecouldbedivided intotwogroups.OnegroupthathadsignificantlysmallerCLand TL(Fig.4B,C;T-test,P<0.05)comparedtothecontroland one groupwithCLandTLsizesimilartothecontrolgroup(Fig.4C;T- test,P>0.05).HistologicalsectionsfromfemaleandmaledsLsEcR treatedlice(Fig.5F–J)bothrevealedabnormalmorphologycom- pared tothe control (Fig.5A–E). Most strikingwas the severe reductionofmuscletissueofthedsLsEcRtreatedanimals(Fig.5F).

The intestine of the dsLsEcR treated animals (Fig. 5H) resem- bledtheintestinaltissueobservedincopepodidsratherthenthe anatomydescribed foradultanimals(Fig.5C)[37].Hypotrophy ofthecellularstructureofthesub-cuticula(Fig.5G),theneuro- logicaltissue (Fig.5Jframed),femaleovaries (Fig.5I) and male testes(Fig.5Jmarkedwithasterisks)wasapparentintheknock- downanimalscompared tothecontrol(Fig.5B–E).Histological examinationsofLsEcRkdfemalesthatwerethesamesizeasthe

controlgroupshowedsimilarmorphologyastheotherLsEcRkd animals.

4. Discussion

While extensiveknowledge of theEcR functionin a variety ofdevelopmentalandphysiologicalprocessesininsectshasbeen obtained,thefunctionalimportanceofthisreceptorincopepods remainslargelyunstudied.Herewereportonthetranscriptional distributionandfunctionofthenewlycharacterizedL.salmonisEcR (LsEcR)[25]duringmoltinganddevelopmentofthesalmonlouse.

Notsurprisingly,analysisofthespatialdistributionofLsEcRincope- podidsusinginsituhybridizationdemonstratedawideexpression patterntheintestine,neuronalsomataofthebrainandinthenuclei ofthemusclecells(Fig.1B).A similarwidedistributionpattern ofLsEcRtranscriptwasobservedintheadultfemalesalmonlice [25]andthecopepodMarsupenaeusjaponicus[14].Theextensive expressionofLsEcRisinaccordancewiththebroadrangeofbio- logicalprocessesinvolvingthisnuclearreceptorsuchasmolting, remodellingofneurons[38,39]andmuscletissueduringmetamor- phosis[40].

Knock-downstudiesofLsEcRusingRNAicausednear60%down- regulation(Fig.3A)ofLsEcRtranscriptinthesalmonlicelarvae,

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Fig.5.HistologicalexaminationofLepeophtheirussalmonisEcRknock-down(LsEcRkd)animals.ToluidinestainedcontrolsectionsandsectionsfromLsEcRkdanimalsare showninA–EandF–J,respectively.AandF)showssectionsofthewholeanimalwithlettersasguidestothecorrespondingphotosofindividualtissues.EandJ)aresections ofmaleswheretestesaremarkedwithasterisksandbraintissueisframedwithablackcircle.Testesandovariesarelocatedatthesamepositionintheirrespectivegender (A,Fmarkedwithasterisk).Muscletissuewasobservedthroughoutthecontrolanimal(A)incontrasttotheLsEcRkdanimalswhereaseverereductionofmuscletissuewas evident(F).Thesub-cuticulartissue(G)ovaries(I),testes(J*)andneuronaltissue(Jframed)werehypotrophiccomparedtothecontrol(B,D,E*,Eframed)whiletheintestinal tissueoftheLsEcRkdanimalsappearedtoresemblethatfoundincoepodidlarva(H)ratherthenthecontrol(C).Scalebar=300␮m(A,F)and100␮M(B–E,G–H).

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butdidnotresultinmoltingarrestoranyobservablemorpholog- icalabnormalitiesinthecopepodids(7d.p.t.).Similarresultshave beenreportedinLsRXRknock-downcopepodidsafterdsRNAbath treatment[30].Thenormalphenotypewassurprisingasdeple- tionofbothEcRandRXRtranscriptsinlarvaefromseveralinsect andcrustaceanspeciesbothcauseseveredevelopmentaldefects and moltingarrest that eventually lead todeathof the animal [27,41–43].Studieshaveshown thatbothEcRandUSPareable tointeractwithDNAeitherashomodimersormonomers[44,45].

In addition, USP alone has shown to bind ligandand exert its functionwithout EcR[46].These resultsfrom ourexperiments indicatethatLsEcRandLsRXRhaveotherpartnersormanageto retainits function alone. The apparentnormal development of theLsEcRkdcopepodidscouldbeduetoinsufﬁcientknock-down ofLsEcRorthepresenceresidualLsEcRprotein.However,dueto unsuccessfuldetectionofLsEcRprotein,wecannotdetermineif theextentofproteindepletioncorrespondstothedecreaseinLsEcR transcript.Moltingarrestwashoweverachievedbysimultaneous knock-downofbothLsEcRandLsRXR.Toourknowledge,notmany knock-downstudiesofthenuclearreceptorcomplexhavebeen performedincrustaceans.StudiesofEcR/RXRdepletionintheﬁd- dlercrabUcapugilatorshowedsimilarresultswitharrestofgrowth duringearlyblastemaldevelopmentandduringlimbgeneration.In addition,thedsEcR/dsRXRinjectedcrabsfailedtomoltandsubse- quentlydied[43].

Despite the lack of observable abnormalities in thedsLsEcR treatedcopepodids(7d.p.t),theinfectiontrialusingthesamecope- podidsresultedin70%mortalityanddevelopmentaldefectsthat obstructedthesurvivinglicefromnormaldevelopment.Whether highmortalityiscausedbydisruptionofthemoltingprocessor otherphysiologicalmechanismsisuncertain.Inadditiontothehigh mortality,theCLandTLoftherecoveredanimalsweresignificantly shorterforthemalesandonegroupofthefemales(T-test:P<0.05) comparedtothecontrol.Thiscouldeitherreflect adecrease in sizeand/oradelayindevelopment.Similarresultswereobserved inthecottonmiridbug,Apolyguslucorum,wherethedurationof developmentincreasedinthethird,fourthandfifthinstarafter siRNAtreatmenttargetedtoEcR.InterferenceofAlEcRaddition- allycausedalethalphenotypewheremortalityincreasedfromthe fourthnymphinstartoadultanimals[47].Asignificantincreasein developmentaltimeswasalsoobservedforlarvaandpupalstages ofSpodoptera exiguaafterinjectionof dsRNA inthefifth instar [48].Duringmetamorphosisofholometabolousinsects,neurons undergoacrucialremodellingprocessdependentonEcR(reviewed inBoulangeretal.,2015)[49].Theecdysonetiterandthepatternof EcRisoformexpressionhavealsoshowntobeafundamentalfactor duringregrowthofmuscleduringmetamorphosisinManduca[40]

andmidgutmorphogenesisduringembryogenesis in Drosophila [50].Histologicalexaminationsshowedalackofmuscletissueand hypotrophyofneuronal-,sub-cuticularandgonadaltissueinthe LsEcRkdlice(Fig.5F–J).OurresultsindicatethatLsEcRplayasimilar roleinthesalmonlouseandisrequiredeitherdirectlyorindirectly inthedevelopmentofdifferenttissues.Knock-downofLsEcRin pre-adultIIfemalesresultedinfailureinnormaleggdevelopment andoffspringgeneration[25].Thehypotrophicgonadsfoundin bothfemaleandmaledsLsEcRtreatedliceinthisstudysuggests thattheanimalswouldnotbeabletosuccessfullyreproduceand thatknock-downofLsEcRtranscriptinlarvalstagesaffectsmat- urationofthegonadaltissueofthetreatedanimalsandthereby theabilitytoreproduce.Similardestructionofthegonadaltissues wasobservedinadultfemaleLsRXRknock-downlice[26].Dueto thesigniﬁcantreduceddevelopmentofinternaltissues,reduced growthorevenmortalityisexpected.Thisisinaccordancewith presentresults.

Incrustaceans,chitinsynthases,chitinasesandcarboxypepti- dasesareimportantenzymesinchitinmetabolismthatiscrucial

for reorganisation and exchange of the exoskeleton [51,52]. A significantdecreaseofLsCP1andLsCHS2transcript(Fig.3)were evidentintheLsEcRandLsEcR/LsRXRknock-downanimalssuggest- ingthemtoplayaroleinmolting.Theresultsaresupportedby previousexpressionstudiesinL.salmonislarvalstages [30]and RNA-seqdata(unpublished;Licebase.org)thatconfirms expres- sionofLsCP1andLsCHS2inallmoltingstages.Nodown-regulation wasobserved for LsCHS1 that is mainlyexpressedin the adult femaleintestine(unpublished;Licebase.org).Asimilardifference inCHStranscriptexpressionwasdemonstratedintheshrimpPan- dalopsisjaponica[53].OntogeneticanalysisofLsChi1and LsChi2 showedthatexpressionvarieswithintheintermoltandpostmolt stagesandpeaksbetweenpremoltnaupliusIandpostmoltnauplius II [35]. LsChi1 were significantly up-regulated (Fig. 3.), respec- tivelyintheLsEcRkdanimals.PreviousRNAistudiesofLsChitinases demonstratedthemtoaffectremodellingoftheexoskeleton[35], supportingourresultsthatregulationoftheLsChi1geneinLsEcRkd copepodidsmaycontributetothehighmortalityrateandpheno- typeobservedinanimalsfromtheinfectiontrial.Noregulationwas observedfortheLsChigenesintheLsEcRkd/LsRXRkdgroup.How- ever,duetothephysiologicaldifferencesbetweentheLsEcRkdand theLsEcRkd/LsRXRkdgroups(Fig.2B–C)adirectcomparisonoftran- scriptexpressionofdown-streamgenesisnotpossible.Oftheearly responsegenesE74,E75andHR38(Fig.3.)onlythelatterwassig- nificantlydown-regulatedinbothtreatmentgroups.Thiscouldbe becauseofsecondaryresponsescausedbydepletionofLsEcRand LsEcR/LsRXRanddisruptionofseveralbiologicalprocesses.Com- parison of expression of the response genes betweendifferent LsEcR/LsRXRknock-downexperimentsshowthattheexpressionof thesegenesaredependentonthedegreeofLsEcR/LsRXRknock- downindicatingthattheresponsegenesaretightlyregulatedby thenuclearreceptorcomplex.

Insummary,wefoundthatLsEcRtranscriptisexpressedinsev- eraltissuesintheL.salmoniscopepods.Thisisinaccordancewithits expectedroleinmanymolecularbiologicalprocesses.Functional studiesusingRNAishowedthatknock-downofLsEcRinL.salmonis larvaresultsinincreasedmortalityandseverehistologicalchanges.

Incontrast,simultaneousknock-downofLsEcR/LsRXRresultedin moltingarrest.ThisshowsthatEcRclearlyhasanimportantfunc- tioninmoltinganddevelopmentofthesalmonlouseL.salmonis.

Thelastfewyears,developmentofincreasedtoleranceandresis- tancetoavailableanti-sealicedrugshasmadeitcrucialtodevelop novelmethodstocontrolsalmonliceinfestations.Ininsects,the EcRhaslongbeenusedasatargetforecdysoneagonistssuchas thebisacylhydrazines.Thesechemicalcompoundsattaingreattar- getspeciﬁcityduetotheirvariationsinbindingafﬁnitybetween differentecdysozoans[54,55]hence,thereisapotentialfornovel medicinestargetingtheecdysonehormonesysteminL.salmonis.

However,furtherinvestigationsofthestructureoftheligandbind- ingpocketofLsEcRarenecessaryinordertoreducethepotential endocrinedisruptionofotheraquaticinvertebrates.Theﬁndings identifyLsEcRasapossibletargetforchemicalsdisruptingdevel- opmentalprocessesandmaybevaluableinthedevelopmentof newtoolstoﬁghttheparasite.

Funding

This research has been funded by the Research Council of Norway,SFI-SeaLiceResearchCentre,GrantNumber203513/O30.

Acknowledgements

WewouldliketothankHeidiKongshaug,PerGunnarEspedal, LarsHamreandTeresaCieplinskaforexcellenthelpinthelabora- tory.