examination水星mr804

RESEARCHOpenAccessMolecularanalysisoftheSydneyrockoyster(Saccostreaglomerata)CO2stressresponseNicoleG.
Ertl1,2,WayneA.
O'Connor1,3,AaronN.
Wiegand1andAbigailElizur1*AbstractBackground:Humanactivitieshaveledtoasubstantialincreaseincarbondioxide(CO2)emission,withfurtherincreasespredicted.
ARNA-SeqstudyonadultSaccostreaglomeratawascarriedouttoexaminethemolecularresponseofthisbivalvespeciestoelevatedpCO2.
Results:Atotalof1626S.
glomeratatranscriptswerefoundtobedifferentiallyexpressedinoystersexposedtoelevatedpCO2whencomparedtocontroloysters.
Thesetranscriptscoverarangeoffunctions,fromimmunity(e.
g.
patternrecognitionreceptors,antimicrobialpeptides),torespiration(e.
g.
antioxidants,mitochondrialrespiratorychainproteins)andbiomineralisation(e.
g.
carbonicanhydrase).
Overall,elevatedlevelsofCO2appeartohaveresultedinaprimingoftheimmunesystemandinproducingcountermeasurestopotentialoxidativestress.
CO2exposurealsoseemstohaveresultedinanincreaseintheexpressionofproteinsinvolvedinproteinsynthesis,whereastranscriptsputativelycodingforproteinswitharoleinciliaandflagellafunctionweredown-regulatedinresponsetothestressor.
Inaddition,whilesomeofthetranscriptsrelatedtobiomineralisationwereup-regulated(e.
g.
carbonicanhydrase2,alkalinephosphatase),asmallgroupwasdown-regulated(e.
g.
perlucin).
Conclusions:ThisstudyhighlightedthecomplexmolecularresponseofthebivalveS.
glomeratatoexpectednear-futureoceanacidificationlevels.
Whilethereareindicationsthattheoysterattemptedtoadapttothestressor,gaugedbyimmunesystemprimingandtheincreaseinproteinsynthesis,someprocessessuchciliafunctionappeartohavebeennegativelyaffectedbytheelevatedlevelsofCO2.
Keywords:Saccostreaglomerata,Sydneyrockoyster,Molluscs,RNA-seq,Stress,Carbondioxide,Immunity,BiomineralisationBackgroundAnthropogenicactivitiessuchasdeforestationandburn-ingoffossilfuelshaveledtoa36%increaseinatmos-phericcarbondioxide(CO2)overthelastfewhundredyears.
Thisisexpectedtofurtherincreasetobetween540to970ppmby2100.
OftheanthropogenicCO2produced,aboutonethirdhasbeentakenupbyoceans,leadingtoa0.
1unitdecreaseintheocean'ssurfacepH,withpredictionsoffurtherpHreductionsof0.
14–0.
35unitsby2100.
Furthermore,CO2uptakeaffectscarbon-atechemistrythroughchangestothesaturationstateof,forinstance,aragoniteandcalcite[1–8].
DuringCO2uptake,water(H2O)interactswithCO2toproducecar-bonicacid(H2CO3)inthefirstinstance,whichthendis-sociatestoformbicarbonate(HCO3-)andcarbonateions(CO32-)that,inthepresenceofcalciumions(Ca2+),eventuallyleadstotheformationofcalciumcarbonate(CaCO3)[5,6].
Calciumcarbonateisanimportantcom-ponentofthebivalveshell,aswellascoralreefsandothermarinecalcifiers,withitsformationanddissol-utionstronglydependentonthecarbonatesaturationstateofthewatercolumn[4,6].
Oceanacidification,thetermthatencompassestheeffectsofCO2uptakebytheocean,canaffectshellformation,shellgrowthandthick-nesswithpotentialflow-oneffectsofreducedprotectionfrompredatorsandsuboptimalenvironments[3,6,9].
Inaddition,oceanacidificationcanpotentiallyaffectthe*Correspondence:AElizur@usc.
edu.
au1UniversityoftheSunshineCoast,SippyDowns,SunshineCoast,QLD,AustraliaFulllistofauthorinformationisavailableattheendofthearticle2016TheAuthor(s).
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Ertletal.
ClimateChangeResponses(2016)3:6DOI10.
1186/s40665-016-0019-ymetabolicrateofbenthicinvertebrates,proteinsynthesisandionexchange[1,3].
Anumberofstudiesexaminingtheeffectsofoceanacidificationonmolluscshavebeenundertaken,withresponsesvaryingbetweenspeciesandchangingwithontogeny[9].
Forinstance,withingeneracomparisonoflarvalshellgrowthandcalcificationinCrassostreavirgi-nicaandCrassostreaariakensisinresponsetoelevatedpCO2,showthatwhileC.
ariakensislarvaearenotaf-fected,C.
virginicalarvaedisplayedaslowershellgrowthanddecreasedcalcification[7].
C.
virginicalarvae,aswellaslarvaeoftheclamMercenariomercenariaandArgopectenirradians,alsoshoweddelayedmetamor-phosisanddecreasedlarvalsizeatelevatedCO2.
Inaddition,mortalityratesofM.
mercenariaandA.
irra-dianslarvaewerehigheratelevatedCO2thanambientCO2[10].
AstudyonthemusselMytilusgalloprovincia-lisobservednoeffectonfertilisationinresponsetoele-vatedlevelsofCO2,butfertilisedeggsexposedtoelevatedCO2showeddelayeddevelopmentintoD-veligerlarvaecomparedwiththecontrolfertilisedeggs.
Moreover,themajorityofthefertilisedeggsthateventu-allydevelopedintoD-veligerlarvaeshowedarangeofmorphologicalabnormalities(e.
g.
indentationofshellmargin)andhadalsosmallershells[11].
TheSydneyrockoyster,Saccostreaglomerata,isnativetotheeastcoastofAustralia,whereitisbothecologic-allyandeconomicallyimportant[12].
Duetoitsimport-ance,severalstudieshavealsobeencarriedouttoassessthepotentialeffectsofoceanacidificationonthisspe-cies.
Forinstance,elevatedpCO2hasbeenshowntodecreasefertilisationrateandreducethepercentageofS.
glomeratagametesthatdeveloptoD-veligerstage.
AsobservedinM.
galloprovincialis,S.
glomerataD-veligerlarvaethathadbeenexposedtoelevatedpCO2weresmallerandhadhigherpercentagesofmorphologicalabnormalitiesat24h[13].
Whilemuchoftheoceanacidificationresearchhasconcentratedontheearlylife-stagesofmolluscs,studiesindicatethatnotonlylarvae,butalsojuvenileandadultmolluscscanbeaffectedbyoceanacidification.
Observedeffectsweredecreasedshellstrength,reducedgrowth(lowerdryshellandsoft-tissuemass)andincreasedmetabolicrateinresponsetoelevatedlevelsofCO2[14,15].
Estuaries,thehabitatofmanymolluscssuchasoys-ters,arebelievedtobemorevulnerabletoincreasesinpCO2anditseffectsthantheopenoceanastheyoftenalreadyshowhighlevelsofCO2andaremuchshallower[7,10,16],whichmayaffecttherateinwhichthelocalpHchanges.
Consideringthepoten-tiallysevereandvariedimpactsoceanacidificationcanhaveonallmolluscanlife-stages,itisessentialtogainabetterunderstandingofthemechanismsunder-lyingtheobservedeffectsofoceanacidificationonestuarinebivalves.
Toaddressthisquestion,wecar-riedoutapCO2exposurestudyusingadultS.
glo-merata.
Inthisstudy,S.
glomerata,acclimatedtoelevatedtemperature(28°C)wasexposedtoelevatedtemperatureandelevatedpCO2(1000ppm)andthemo-lecularresponseoftheoysterstothesechallengesana-lysedwithRNA-Seq.
MethodsStressexposureandsamplecollectionCO2andtemperatureAdult,wildS.
glomerata,collectedfromCromartyBay(seawatertemperatureof20.
5±0.
5°Cand423.
36±17.
54μatmCO2),PortStephens(NSW,Australia)wereslowlybroughtupto28°C(1°Cchangeinwatertemperature/day),thenacclimatedtotheelevatedtemperature(28°C)for4days.
Afteracclimation,theoysterswereexposedto1)28°Candambient(385ppmor386.
9μatm)pCO2(control)or2)28°Candelevated(1000ppmor1108.
2μatm)pCO2inthreereplicate750Lheadertankspertreatmentfor4weeks.
Thistimeframewaschosenasoysterswereexpectedtobeaffectedbytheexperimentalconditionwithin4weeks.
Sixoysters(n=2perreplicate)weresampledrandomlyfromeachtreatmentattheendoftheexperiment.
AdditionalsamplesforreferencetranscriptomepreparationInaddition,adult,wildSaccostreaglomeratafromCromartyBay,PortStephens(NSW,Australia),wereexposedtoa)salinityandtemperature(n=24oysters)orb)polycyclicaromatichydrocarbons(PAH)(n=12oysters).
Fulldetailsofexperimentalset-up,oysterhus-bandryandexposureareasdescribedinAdditionalfile1.
OnlytheresultsfromtheCO2andtemperatureex-posurearediscussedinthispaper;however,sequencingreadsfromthesamplesofalltreatments(n=48oysterstotal)wereusedtobuildthereferencetranscriptome.
SamplepreparationandsequencingTissuesusedforRNA-Seqanalysisweregill,mantle,adductormuscle,gonadanddigestive(PAHandCO2experiment)orgill,mantle,adductormuscleanddigest-ive(salinity).
TotalRNAwasextractedfrom25mg(24mgforsalinitysamples)ofpooledtissue(5mgpertissueforCO2andPAHsamples,6mgpertissueforsal-initysamples)fromeachoftheoystersaccordingtothemanufacturer'sguidelines,usingtheDirect-zolRNAMiniPrepkit(ZymoResearchCorporation,USA)includ-ingtheDNaseIdigestionstep.
QualityandquantityoftheextractedRNAweretestedbygelelectrophoresis,bioanalyzer(2100Bioanalyzer,AgilentTechnologies,USA)usingtheRNA6000NanoChipkit(AgilentTech-nologies)andwiththeQuantusTMfluorometer(Promega,Australia).
ERCC(ExternalRNAControlConsortium)Ertletal.
ClimateChangeResponses(2016)3:6Page2of19RNAspike-incontrolmixes(Ambion,Australia)werediluted1:100,then2μlofthedilutedmix1andmix2addedto1μgoftotalRNAofcontrolandtreatedsamples,respectively.
RNA-Seqlibrarieswerethenpreparedfromeachofthesesamples,usingtheTruSeqRNAsampleprepkit-v2(Illumina,Australia)accordingtothekit'sprepar-ationguidelines.
Qualityandquantityofthe48cDNAlibrariesweretestedwiththe2100Bioanalyzer,usingtheHighSensitivityDNAchipkit(AgilentTechnologies)andwiththeQuantusTMfluorometer.
LibrariesweresenttoAGRF(Australia)for100bppaired-endsequencing,wherethe48librarieswererandomlydistributedacrossfourlanesandrunonanIlluminaHiSeq2000sequencer.
ReadprocessinganddenovoreferencetranscriptomeassemblyPaired-endreadqualitywasexaminedpre-andpost-cleanupwithFastQC(http://www.
bioinformatics.
babraham.
ac.
uk/projects/fastqc/).
ReadsweretrimmedforqualitywithTrimmomatic[17],andadaptersequencesandreadswithasizebelow40bpaftercleanupre-moved.
Inaddition,readswereassessedforartefactslikeE.
coli,phiX,humansequences,aswellasforthelevelofribosomalsequences.
Processedreads,alongwithS.
glomerataprocessednon-strandspecificandnon-normalisedreadsfromapreviousIlluminaHiSeq2000sequencingrun(Additionalfile1)wereassembledintotranscripts,usingTrinity[18]withtheinsilicoreadnor-malisationoption.
AssembledtranscriptswerealignedtotheERCCandphiXreferencesequences,usingBLAT[19]andtranscriptsmappingtoeitherreferencesre-movedfromtheS.
glomeratareferencetranscriptomeassemblywithanin-housescript.
TheremovedERCCtranscriptsfunctionedasindicatorsofhowsuccessfultheassemblyofthereferencetranscriptomewasandwerecloselyexaminedintheCLCworkbench(CLCBio,USA,version7.
5).
Inaddition,redundancywasremovedwithcd-hit-est[20],usinga99%cut-off,andcomplete-nessoftheassemblywasexaminedwithCEGMA(CoreEukaryoticGenesMappingApproach)[21].
Inordertoeliminateanypotentialbiasoftheanalysis,ERCCrefer-encesequenceswereaddedtothecleanS.
glomerataref-erencetranscriptomethatwasthenusedasareferenceforthedifferentialgeneexpressionanalysis.
DifferentialgeneexpressionanalysisofCO2samplesTranscriptssignificantlydifferentiallyexpressedbetweencontrolandelevatedCO2samplesweredeterminedwithRSEM[22]andEBSeq[23].
Post-processedreadsofthe12CO2sampleswerealignedtotheS.
glomeratarefer-encetranscriptomewithBowtie[24](Additionalfile2:TableS1),usingaRSEMinternalscript.
ReadcountswereestimatedwithRSEM,afterwhichreadcountdatafortranscriptsthathadreceivedazeroestimatedcountforall12sampleswasremovedfromtheanalysis.
Tran-scriptswithmeancountsof>0acrossallsampleswerethenfurtheranalysedwithEBSeqinR(version3.
1.
1).
Asadenovoassembledreferencetranscriptomewasusedfordifferentialgeneexpressionanalysiswherethegene-isoformrelationshipwasnotknown,mappingambiguityclusterswereproducedwiththeRSEMscriptrsem-generate-ngvector.
Mediannormalisationoftheesti-matedcountdatawascarriedoutinEBSeq,afterwhichEBTestwasrunfor12iterationsuntilconvergencehadbeenreached.
Afalsediscoveryrate(FDR)thresholdof0.
05wasappliedtodetermineisoforms/transcriptssig-nificantlydifferentiallyexpressedbetweencontrolandelevatedCO2samples.
Foldchangevaluesusedthrough-outthetextwerebasedontheposteriorfoldchangevalues.
FunctionalannotationofdifferentiallyexpressedtranscriptsBlastxsimilaritysearcheswerecarriedoutontranscriptsfoundtobesignificantlydifferentiallyexpressedagainsttheNCBInon-redundant(nr)database(downloaded08.
09.
14),usingane-valuecut-offof1e-5withahitnumberthresholdof25.
Mappingandfunctionalanno-tationofthetranscriptswascarriedoutwithBlast2GO[25],usingstandardparameters(hitadjustedto25).
Inaddition,InterProScansearcheswererunthroughBlas-t2GOwiththeresultsmergedwiththealreadyexistingannotations.
Wheredomain/familyinformationwasavailablefortranscriptswithasequencedescriptionof"—NA—"or"hypotheticalprotein/uncharacterisedpro-tein",andthisinformationofferedanindicationastothepotentialidentityofthetranscript,itwasaddedtotherespectivetranscript.
Furthermore,transcriptsofinterestweremappedtothetissuespecificS.
glomeratatranscripts(Additionalfile1),usingtheCLCGenomicsWorkbenchversion7.
5(CLCBio,USA)withdefaultpa-rameters(minimumlengthfraction:0.
7,similarity:0.
8),todeterminetheirputativetissuedistributioninS.
glomerata.
QPCRanalysisQPCRanalysiswascarriedouttovalidatethedifferentialtranscriptexpressionanalysismethodusedinthisstudy.
Forthis,totalRNAwasextractedfrom25mgofpooledtissue(5mgofeach,gill,mantle,adductormuscle,gonadanddigestive)fromthesixcontrol(28°Cand385ppmpCO2)andsixtreated(28°Cand1000ppmpCO2)oystersusedtopreparethe12CO2RNA-Seqlibrariesofthisstudy.
Inaddition,totalRNAwasalsoisolatedfromawild,non-stressedS.
glomerata(using6mgeachofgill,mantle,adductormuscle,gonadanddigestivetissueoftheoyster)andusedasareferencesampleintheqPCRanalysis.
TheDirect-zolRNAErtletal.
ClimateChangeResponses(2016)3:6Page3of19MiniPrepkit,includingtheDNaseIdigestionstep,wasusedforRNAisolationaccordingtothemanufacturer'sguidelines.
QualityoftheextractedRNAwastestedwiththe2100Bioanalyzer,usingtheRNA6000NanoChipkit,andquantitywiththeQuantusTMfluorometer.
cDNAsynthesiswascarriedoutonthereferencesample(500ngoftotalRNA),aswellasonthecontrolandtreatedsamples(1000ngoftotalRNAeach),usingtheQuantiTectreversetranscriptionkit(Qiagen,Australia)asdescribedinthekit'sguidelines.
Furthermore,nega-tivereversetranscription(-RT)reactionswereper-formedonthreeoutof12samples,whereRNasefreewaterwasusedinsteadofthereversetranscriptasetotestforgenomiccontaminationofthesamplesduringqPCRanalysis.
PrimerdesignandtestingOfthetranscriptsfoundtobesignificantlydifferentiallyexpressedbetweenthesixcontrolandsixCO2treatedS.
glomerata,twotranscripts,showinganestimatedreadcountof>100forthelowestsamplewererandomlychosenforqPCRanalysis.
FourtranscriptspecificprimerpairspertranscriptweredeterminedwithPrimer3Plus(http://primer3plus.
com/cgi-bin/dev/primer3plus.
cgi),withaprimersizeof19–23bp,melttemperatureof60–61°Candlowtonoself-annealing.
PotentialprimerpairsweremappedbacktotheS.
glomeratareferencetranscrip-tomewiththeCLCGenomicsWorkbenchversion7.
5(CLCBio,USA)todetermineprimerpairsthateithera)onlymappedtothetranscriptofinterest,orb)mappedtothetargettranscriptandaverysmallnumberofothertranscriptswithaminimumoftwonucleotidemismatchatthe3′end.
Next,.
bammappingfiles,producedduringthedifferentialtranscriptexpressionanalysiswithBowtie[24]andRSEM[22]werevisualisedintheIntegrativeGenomicsViewer(IGV;https://www.
broadinstitute.
org/igv/)andusedtoexaminepotentialprimerpairsfornu-cleotidemismatchesintheprimersequenceacrosscontrolandCO2treatedsamples.
Primerpairswitha)nonucleo-tidemismatchesacrosseitheroftheprimersequencesinall12samples,orb)amaximumoftwonon-crucial(no3′)nucleotidemismatchesacrosseitheroftheprimersequencesinall12samplesweresynthesisedbySigmaAldrich(Australia)forqPCRanalysis(Table1).
AtemperaturegradientPCRwasperformedtodeter-minethebestprimerannealingtemperatureforeachoftheeightprimerpairs.
TheprimerschosenforuseinthisstudyarepresentedinTable1.
TriplicatePCRreac-tionswerepreparedforeachoftheprimerpairs,using0.
6μLofreferencesampleastemplate,addedto14.
4μLofmastermix,containing1.
5μLof10xPCRreactionbuffer,2mMofMgCl2,200μMofdNTPs,1UofTaq(allreagentsfromFisherScientific,Australia),200nMeachoftherespectiveforwardandreverseprimer,and10.
74μLofRNasefreewater.
Cyclingconditionswereaninitialdenaturationfor1minat95°C,followedby35cyclesofdenaturationfor30sat94°C,annealingfor30sateithera)58.
1°C,b)60.
6°Corc)61.
9°Cwiththetriplicatesdistributedacrossthethreetemperatures,thenextensionfor45sat72°C.
Finalextensionoc-curredat72°Cfor2min,afterwhichgelelectrophoresiswascarriedouton3μLofeachPCRproduct.
FragmentpreparationforqPCRstandardcurvesPCRproductsofaknownlengthwereproducedforthetwotranscriptschosenforqPCRanalysis.
Tektin-2andtektin-4-likePCRproductswereobtainedbyPCRamplifi-cation,usingprimerpairsT2F_2andT2R_4,andT4F_1andT4R_2,respectively.
FourreplicatePCRreactionsweresetupforbothtranscripts,using1μLofreferencesampleastemplate,0.
2μMeachoftherespectiveforwardandreverseprimer,12.
5μLofMyTaqmix(Bioline,Australia)and10.
5μLRNasefreewaterforatotalreac-tionvolumeof25μL.
PCRamplificationconditionswere:a)initialdenaturationfor2minat95°C,followedby35cyclesofb)denaturationfor30sat95°C,c)annealingfor30sat60°C,d)extensionfor2minat72°C,withafinalextensionfor2minat72°C.
GelelectrophoresiswasusedtodeterminesinglebandingofthePCRproducts,afterwhichproductsfortektin-2andtektin-4-likewerepurifiedwiththeQIAquickPCRpurificationkit(Qiagen,Australia)accordingtothekit'sguidelines.
Purifiedproductsofthetwotranscriptswereassessedwithgelelectrophoresisandthefourreplicatesofeachprimerpairpooledtoobtainasinglecleanproductperprimerpair.
ThetwocleanproductswereanalysedontheNanoDrop2000spectrophotometer(ThermoFisherScientific,USA)threetimesandthemeanconcentrationvalueofeachproductusedtodeterminecopynumbersofeachproductwiththecopynumbercalculator(http://cels.
uri.
edu/gsc/cndna.
html).
Table1PrimerpairsforqPCRanalysis.
Thistableonlyshowsthehighestefficiencyprimerpairs,aswellastheprimersusedtoproducethepurifiedPCRproductsforthestandardcurvesTranscriptPrimerIDSequence(5′to3′)Length(bp)tektin-2T2F_1CCACACCCTTCAGCAGTGT19T2R_1GCGATCTTTGCGCGGATTT19T2F_2AGTTCGCCAGGAGAGTCGA19T2R_4CTCCTCTAGAGCCCTCTTCGT21tektin-4-likeT4F_1ACAATGGGGTTCAGGGCTG19T4R_2CGGACGCTGACACACTTGT19T4F_3TGAGAGAATTCGCCACGAG19T4R_3TGTTGCACGGAGTCCATTT19Ertletal.
ClimateChangeResponses(2016)3:6Page4of19PrimervalidationandqPCRanalysisofCO2samplesPrimerefficiencyandspecificityweredeterminedwithqPCR(Table1).
A1in10,10pointserialdilutionwaspreparedfromthe108copynumberstocksolutionofeachofthetwopurifiedPCRproducts.
Theserialdilu-tionswerethenusedasqPCRtemplatestodetermineprimerefficiencyandspecificityforallprimerpairs.
Forthis,200nMeachperforwardandreverseprimerwereaddedto1μLoftemplate,5μLofPlatinumSYBRGreenqPCRSuperMix-UDG(Invitrogen,Australia)and3.
6μLofRNasefreewater.
Reactionswereperformedintriplicate,includingtriplicatenotemplatecontrols(NTCs),usingtheRotor-Gene6000thermalcycler(CorbettResearch,Australia).
Cyclingconditionswereasfollows:initialholdingstepat50°Cfor2min,holdat95°Cfor2min,then40cyclesof95°Cfor15s,60°Cfor15sand72°Cfor25s,withthelaststepsettoacquiretoGreen.
Meltcurveanalysiswasperformedbyincreasingthemeltingtemperatureby1°Cincrementsfrom72°Cto95°C.
TheRotor-Gene6000software,ver-sion1.
7.
87(CorbettResearch,Australia),wasusedforquantificationandmeltcurveanalysis.
Reactioneffi-ciency(E)wascalculatedforeachprimerpairbytheRotor-Gene6000software,usingthefollowingequation:E=[10(-1/M)]-1,whereMstandsfortheslopeofthecurve.
Absolutetranscriptexpressionlevelsoftektin-2andtektin-4-likeweredeterminedwithqPCRinthesixcon-trolandsixCO2treatedS.
glomeratasamples,withreac-tionvolumesandcyclingconditionsasdescribedforprimervalidation.
Reactionswerecarriedoutindupli-cate,includingduplicateNTCsand–RTs,aswellasonepointofthestandardcurveandapositivecontrolintriplicate.
Primerpairsusedfortheindividualtranscriptswere:a)T2F_1andT2R_1(efficiency:0.
9855)andb)T4F_3andT4R_3(efficiency:0.
9698).
StatisticalanalysisofqPCRdataAtwo-samplet-testassumingunequalvariances(Micro-softExcelSoftware)wasusedtodeterminesignificantdifferencesbetweenthetranscriptexpressionlevelsofcontrolandCO2stressedS.
glomerata.
InordertoallowforadirectcomparisonbetweenRNA-SeqandqPCRresults,thesignificancelevelforthet-testwassetatp100)andKOBAS(e-valueof1e-5)usingDIAMOND(v0.
8.
5)[26],andagainsttheHMMER/Pfamproteindatabase(v28.
0,DomainScore>20)[27]usingHMMER3.
1b[28].
Wheremultipleanno-tationswereavailableforasingleopenreadingframe(ORF)ormultipleORFswerepredictedforagiventran-script,thebestmatchingannotation(highestBitScore,loweste-value)orORFwiththebestannotationwerechosen,respectively.
GOenrichmentanalysisUsingTrinotate(v3.
0.
1,https://trinotate.
github.
io/)scripts,theannotationanddifferentialexpressionfileswereloadedintoaSQLitedatabase(v3.
9.
2,http://www.
sqlite.
org/)andaGOenrichmentanalysiscarriedoutusingthe'goseq'Rpackage[29].
Takingthelengthofeachtranscriptintoaccount,GOseqdeterminedGOandCOG/eggNOG(ClustersofOrthologousGroups/evolutionarygenealogyofgenes:Non-supervisedOrtho-logousGroups)termsthatareover-representedamongthetranscriptsdifferentiallyexpressedbetweencontrolS.
glomerataandoystersexposedtoelevatedCO2.
ResultsanddiscussionS.
glomeratareferencetranscriptomeForthisstudy,aS.
glomeratareferencetranscriptomewasproducedandthenusedtoanalysethemolecularresponseofadult,wildS.
glomerataexposedtoelevatedpCO2(1000ppm)andtemperature(28°C),whencom-paredtocontrolS.
glomeratathatwereexposedtoele-vatedtemperatureandambientpCO2(385ppm).
ApCO2concentrationof1000ppmwaschosenbasedonpredictionsfortheyear2100[8].
Inordertoobtainacomprehensivereferencetranscriptome,tissue(gill,mantle,adductormuscle,gonadanddigestive)ofS.
glo-merataexposedtoCO2andtemperature,salinityandtemperature,andPAHwasextractedandnon-strandspecificandnon-normalisedlibraries(n=48)foreachindividualoysterprepared.
LibrariesweresequencedwithIllumina,resultinginatotalof818,834,356paired-endreadswithaGCcontentof42–44%.
AsimilarGCcontenthasalsobeenfoundinothermolluscs[30,31].
Rawreadswereprocessedandtheresultingprocessedreads(98.
7%),alongwiththeprocessednon-strandspe-cificandnon-normalisedreadsofourpriorS.
glomeratastudy(Additionalfile1),assembledintoareferencetranscriptome.
Assemblystatisticsbeforeandafterre-dundancyremovalaresummarisedinTable2.
Com-pletenessofthereferencetranscriptomeassemblywasassessedwithCEGMA[21],aswellasbythesuccessfulassemblyofERCCandphiXsequences.
EventhoughtheCEGMAsoftwarewasoriginallydevelopedtoassessthecompletenessofgenomes,variousstudies[32,33]havealsousedthissoftwaretodeterminethecompletenessoftranscriptomes.
Oftheoriginal92ERCCreferencese-quencesandonephiXsequence,89ERCC'sandoneErtletal.
ClimateChangeResponses(2016)3:6Page5of19phiXsequencewerefoundintheS.
glomeratareferencetranscriptome,withallsequencesclosetofulllength.
TheN50value(836bp)ofthenon-redundantreferencetranscriptomeiscomparabletotheN50'sobservedforothermolluscandenovotranscriptomes[34–36].
Basedontheseresults,theassemblywasconsideredtobeofsuitablequalityforthedifferentialgeneexpressionanalysis.
DifferentialtranscriptexpressionanalysisofCO2samplesEBSeq,anRbasedprogramwasusedtodetermineS.
glomeratatranscriptsdifferentiallyexpressedbetweencontroloysters(exposedtopCO2of385ppmand28°C)andoystersexposedtoelevatedpCO2(1000ppmand28°C).
EBSequsesanempiricalBayesianapproachandwaschoseninthisstudyasittakestheestimationuncer-taintyinherentinisoformexpressionanalysisintocon-sideration[23].
Graphicalresultsofthestandarddiagnosticsonthedifferentialtranscriptexpressionana-lysiswithEBSeqarepresentedinAdditionalfile3:Fig-ureS1,Additionalfile4:FigureS2,Additionalfile5:FigureS3andAdditionalfile6:FigureS4.
Atotalof1626S.
glomeratatranscriptswerefoundtobedifferen-tiallyexpressed(DE)betweencontrolandelevatedpCO2S.
glomerata,usingafalsediscoveryrate(FDR)thresh-oldof0.
05.
FunctionalannotationoftheDEtranscriptswithBlast2GOagainstNCBI'snon-redundantdatabasewithane-valuecut-offof1e-5resultedintheannotationof75.
2%oftheDEtranscripts.
Annotationinformationfor73.
9%ofDEtranscriptscouldbeobtainedwhentranscriptsweresearchedagainsttheInterProScandata-basethroughBlast2GO.
OfthefunctionallyannotatedDEtranscripts,GO-termsassociatedwithcellularandmetabolicprocesses,cellandmembrane,andcatalyticactivityandbindingcontainedthemostDEtranscripts(Fig.
1a,bandc).
WhiletheDEtranscriptspresentonlyasmallsectionoftheS.
glomeratareferencetranscrip-tome,thispatternofGO-termsiscomparabletothemostcommonGO-termsfoundinthetranscriptomesofothermolluscs[37,38].
InadditiontothemainGO-terms,termsassociatedwithresponsetostimulus,im-munesystemprocess,biologicalregulation,signallingandtransporteractivitywerealsoobservedfortheDEtranscripts(Fig.
1a,bandc),indicatingthattheDEtran-scriptsarepotentiallyinvolvedinawiderangeofpro-cessesandfunctions.
GOenrichmentanalysisGOenrichmentanalysisidentifiedarangeofGOandCOG/eggNOGtermsthatwereover-representedineithercontrolorCO2stressedS.
glomerata(Table3).
OftheGOtermsfoundtobeover-representedinthecon-trolgroup,'oxidation-reductionprocess'(biologicalprocess)and'oxidoreductaseactivity'(molecularfunc-tion)werethemostenrichedGOtermintheirrespect-ivecategory.
OtherenrichedGOtermswere'hydrolaseactivity'(8)and'carbohydratemetabolicprocess'(8).
Incomparison,'calciumionbinding'(molecularfunction)and'regulationofapoptoticprocess'(biologicalprocess)werethemostenrichedGOtermsfoundtobeover-rep-resentedinCO2stressedS.
glomerata.
Apoptosisisanim-portantcomponentoftheinnateimmunesystemofmolluscs[39]andanover-representationof'regulationofapoptoticprocess',alongwithanover-representationof'alternativeoxidaseactivity'inCO2stressedoysterscouldsuggestthatpotentiallyprotectivemechanismswerein-ducedinresponsetothestressor.
AnalysisoftheenrichedCOG/eggNOGtermsindicatedanover-representationof,forexample,'collectinsub-familymember12(1),'glutathi-oneS-transferase(2)or'RNAbindingmotifprotein'(2)incontrolS.
glomerata,whereas'alternativeoxidase'(2),'sol-utecarrierfamily17'(3)or'alkalinephosphatase'(2)wereover-representedinCO2stressedoysters.
ImmunityCloserexaminationofthe1626S.
glomeratatranscriptsdifferentiallyexpressedbetweencontrolandCO2stressedoysters,showedmultipletranscripts,potentiallyinvolvedininnateimmunity.
Innateimmuneresponsesaretriggeredbytherecognitionofpathogen-associatedmolecularpatterns(e.
g.
lipopolysaccharides)ordamage-associatedmolecularpatterns(DAMPs)throughpatternrecognitionreceptors(PRRs)[40,41].
DAMPsaremole-culessuchasheatshockproteins,RNA,DNA,galectins,Table2Assemblystatistics.
Statisticsof"totaltranscripts"includesassembledERCCandphiXtranscripts.
AllotherstatisticsareexcludingERCCandphiXtranscriptsReferencetranscriptomeTotaltranscripts(#)718,804N50length(bp)913Meantranscriptlength(bp)620Mintranscriptlength(bp)201Maxtranscriptlength(bp)36,260Non-redundanttranscripts(#)708,463N50length(bp)836Meantranscriptlength(bp)595Mintranscriptlength(bp)201Maxtranscriptlength(bp)36,260ntranscripts1000bp93,805CEGMACompleteproteins(%)93.
15Partialproteins(%)98.
79Ertletal.
ClimateChangeResponses(2016)3:6Page6of19defensinsandannexins,thatarereleasedfromstressed(e.
g.
hypoxia),injuredornecroticcells[42,43].
ManyoftheseDAMPshavebeenfoundtobedifferentiallyexpressedinS.
glomerataexposedtoelevatedCO2(Additionalfile7:TableS2).
Forinstance,S.
glomerataDEtranscriptsoftheheatshockHsp20familywereup-regulatedinresponsetoelevatedCO2,whilesixoutofeightHsp70transcriptswere4-foldandhigherdown-regulated.
Asidefromheatshockproteins,twooutoffourannexinswerealsoup-regulated(2-foldandhigher)Fig.
1GOanalysisofS.
glomerataDEtranscripts.
GO-termsweredeterminedwithBlast2GO,usingdefaultparameters.
Level2GO-termsareassociatedwith(a)biologicalprocess,(b)cellularcomponentand(c)molecularfunctionaredepictedErtletal.
ClimateChangeResponses(2016)3:6Page7of19inCO2challengedoysters.
Thiscorrelateswiththeresultsoftheenrichmentanalysis,whereannexinA7wasshowntobeover-representedinS.
glomerataexposedtoelevatedCO2(Table3).
Inaddition,theanti-microbialpeptidemantledefensinwas4-foldandhigherup-regulatedandgalectins,whichalsofunctionasaPRR,hadonetranscript4-foldandhigherupandonetranscript4-foldandhigherdown-regulatedinchallengedoysters.
OtherPRRsfoundtobedifferentiallyexpressedinS.
glomerataweregram-negativebacteriabindingproteins(GNBPs),scavengerreceptors(SRs),fibrinogen-relatedproteins(tenascins,fibrinogencdomain-containingproteinsandfibroleukins),c-typelectins,collec-tins,peptidoglycanrecognitionproteins(PGRPs),c-typemannosereceptors/macrophagemannosereceptorsandC1qdomaincontainingproteins(Additionalfile7:Table3GOtermssignificantlyover-representedamongthetranscriptsdifferentiallyexpressedbetweencontrolandCO2stressedoysters.
ThistableincludesGO(GO_BP=Biologicalprocess,GO_MF=Molecularfunction),COG(ClustersofOrthologousGroups)andeggNOG(evolutionarygenealogyofgenes:Non-supervisedOrthologousGroups)termsthatwerefoundtobesignificantlyover-representedamongthetranscriptsdifferentiallyexpressedinS.
glomerataexposedtoelevatedlevelsofCO2whencomparedtocontroloystersCategoryTermCountp-valueover-representedinGO_BPOxidation-reductionprocess150.
0013controlCarbohydratemetabolicprocess80.
0019controlRegulationofapoptoticprocess50.
0077elevatedCO2GO_MFOxidoreductaseactivity70.
0096controlCopperionbinding61.
00E-04controlO-glycosylhydrolaseactivity50.
0024controlCarbon-nitrogenhydrolaseactivity30.
0065controlPeptidyl-dipeptidaseactivity20.
001controlCationbinding20.
0017controlCalciumionbinding149.
00E-04elevatedCO2Alternativeoxidaseactivity22.
00E-04elevatedCO2Calcium-dependentphospholipidbinding20.
0056elevatedCO2COGEndonucleaseIVplaysaroleinDNArepair10.
0084controlAlkalinephosphatase20.
0044elevatedCO2Homocysteines-methyltransferase10.
0049elevatedCO2eggNOGGlutathioneS-transferase23.
00E-04controlActivatingsignalcointegrator1complexsubunit25.
00E-04controlLeucine-richrepeatextensin-likeprotein27.
00E-04controlNiemann-Pickdisease27.
00E-04controlAngiotensinIconvertingenzymepeptidyl-dipeptidaseA20.
001controlRNAbindingmotifprotein20.
0019controlWDrepeatdomain83oppositestrand10.
0044controlChromosome5openreadingframe6310.
0053controlKiaa152410.
0056controlCollectinsub-familymember1210.
0085controlSolutecarrierfamily1730.
0013elevatedCO2Alternativeoxidase21.
00E-04elevatedCO2K06254agrin22.
00E-04elevatedCO2Hydrolasefamily20.
0015elevatedCO2AnnexinA720.
0019elevatedCO2Ribosomalproteinl2110.
0092elevatedCO2Familywithsequencesimilarity21010.
0092elevatedCO2Follistatin10.
0096elevatedCO2Ertletal.
ClimateChangeResponses(2016)3:6Page8of19TableS2).
Overall,slightlymorePRRs(53.
8%)wereup-regulatedinresponsetoelevatedCO2thandown-regulated,with,forinstance,GNBPtranscripts2-foldandhigherdown-regulatedandfouroutofsixcollectins4-foldandhigherup-regulated.
ThissuggeststhatprolongedCO2exposureinducedtheinnateimmunedefencesystemofS.
glomerata,likelyduetotheactionofthemostlyup-regulatedDAMPsthatwerealsodetectedinthisstudy.
Inaddition,SRswhichwerefoundtobe4-foldandhigherup-regulatedinresponsetoCO2,arealsolinkedtophago-cytosis[44].
AsidefromSRs,othermoleculesassociatedwithphagocytosis,suchasantimicrobialpeptides(bacteri-cidalpermeabilityincreasingproteinandmantledefensin)andlysozymewerealsoobservedtobe4-foldandhigherup-regulatedinS.
glomerataexposedtoelevatedCO2(Additionalfile7:TableS2).
Phagocytosisisamechanismoftheinnateimmunesystemthatrecognisesandremovesdeadcells,foreignbodies(e.
g.
bacteria)andenvironmentaldebris[44,45],anditsinductioninCO2stressedS.
glo-meratamightbeapreventivemechanismoraresponsetodamageatthecellularlevel.
Asimilarexpressionpatternhasalsobeenobservedininvertebratesexposedtodiffer-enttypesofstress.
Forinstance,theimpactofinjuryontheimmuneresponsesofHydrawasanalysedandshowedanup-regulationofantimicrobialpeptides(hydramacinandarminin),alectin(L-rhamnosebindinglectinCSL3)thatfunctionsasaPRRandsmallheatshockproteinsinresponsetoinjury[46].
TheauthorssuggestedthatthesmallheatshockproteinsmightactascytoprotectorsagainstROS(reactiveoxygenspecies)andinjurystress,whiletheHydraantimicrobialpeptidescouldhaveadd-itionalrolesinfunctionssuchasregeneration[46].
Expos-ureofC.
virginicatoelevatedlevelsofCO2(800and2000μatm)for4weeksinturnshoweddecreasedHsp70mRNAlevelsandanincreaseinhaemocytelysozymeac-tivityinresponsetothestressor[47].
Incontrast,Hsp70geneexpressionofthecoralDesmophyllumdianthustoelevatedpCO2(997μatm)for8monthswasstronglyup-regulated,alongwiththegeneexpressionofmannose-bindingC-typelectin[48].
Allograftinflammatoryfactor1-like,anotherS.
glomerataDEtranscriptthatwasfoundtobe2-foldandhigherup-regulatedinCO2stressedoys-ters(Additionalfile7:TableS2)hasbeenshowntobein-ducedbybacterialchallenge,tissueinjuryandshelldamageinthepearloysterPinctadamartensii,suggestingaroleininnateimmunity[49].
TheseresultsindicatethatstressorssuchaspCO2andtissueinjuryappeartoresultinasimilarinductionoftheaffectedani-mal'simmunesystem,ashasbeenseenintheS.
glo-merataexposedtoelevatedpCO2ofthisstudy.
Thismightbeapre-emptivestrategytoprotecttheoysterfrompotentialinfectionduringstressexposureoratissueprotectivemeasureinresponsetopotentialdamageatthecellularleveldueto4weeksofcontinuouspCO2challenge.
Whilenoapparenttissuedamageorinfectionwasobservedthroughouttheex-perimentandduringsamplecollection,damageatthecellularlevelcouldstillhaveoccurred,ashasbeenimpliedbytheincreaseinDAMPsinCO2challengedS.
glomerata.
Furthermore,consideringthepresenceofpotentiallyopportunisticbacteriaintheoyster'snaturalhabitat[50,51],non-specificprimingoftheinnateimmunesystemmightprotectstressedS.
glo-meratafromsuchinvadingopportunisticbacteria.
ThatstresscanaffectthecompositionofthebacterialcommunityinoystershasalreadybeenshowninastudyinS.
glomerata,whereindividualsinfectedwiththeprotozoanparamyxeanparasite,Marteiliasydneyi,showedadifferentbacterialcommunityintheirdi-gestiveglandthannon-infectedS.
glomerata[51].
Interestingly,onetranscriptputativelycodingforamacrophage-expressedgene1(MPEG1)proteinandmultiplelaccasetranscriptswerefoundtobe2-foldandhigherdown-regulatedinthepCO2stressedS.
glomerata(Additionalfile7:TableS2).
MPEG1ofthediskabaloneHaliotisdiscusdiscusshowedantibacterialactivityagainstGram-positiveand–negativebacteriaandwasup-regulatedinresponsetobacteriaandviralhemorrhagicsepticaemiavirus[52].
Similarly,alaccaseofthespongeSuberitesdomunculawasup-regulatedinresponsetobacteriallipopolysaccharideandalsoshowedantibacterialactivitywhenthelaccasemedi-atorABTS[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)]waspresent[53].
Consideringthefunc-tionofMPEG1andlaccaseintheseinvertebratesandtheirdown-regulationintheS.
glomerataexposedtoelevatedpCO2,itispossiblethata)bothareonlyin-ducedasadirectresponsetospecificinvadingpatho-gens,orb)protectionfrompotentialinfectionmightnotbetheprimaryreasonfortheup-regulationoftheotherimmunefactors(e.
g.
antimicrobialpeptidesandPRRs)inS.
glomerata.
OtherDEtranscriptsobservedtobedifferentiallyexpressedinCO2challengedS.
glomerataareputativelyinvolvedinapoptosis.
Caspase,whichhasaroleincelldeathsignalling[54],was2-foldandhigherdown-regulatedinS.
glomerataexposedtoelevatedpCO2,whileanti-apoptotictranscriptssuchasBcl-2,Fasapop-toticinhibitorymolecule(FAIM)andsometranscriptsoftheinhibitorofapoptosisprotein(IAP)familywereup-regulatedinresponsetoelevatedpCO2(Additionalfile7:TableS2).
FAIMwasshowntoprotectavarietyofcelltypessuchashepatocytesfromcelldeath[55],andBcl-2suppressedirradiation-inducedapoptosisintrans-geniczebrafish[56].
SimilartoFAIM,IAPscanprotectdifferentcelltypes(e.
g.
neurons,macrophages)fromstressinducedapoptosis[57].
Inourstudy,twooutoffiveIAPtranscriptswere4-foldandhigherup-regulatedErtletal.
ClimateChangeResponses(2016)3:6Page9of19inCO2stressedS.
glomerata,indicatingthata)someapoptoticactivitywasstilloccurringorb)asdifferenttypesofIAPscanhavearangeoffunctionsindifferentcells(reviewedinDubrez-Dalozetal.
[57]),itispossiblethatthedifferentexpressionpatternseenislinkedtoseparateanti-apoptoticfunctionsoftheindividualIAPs.
AdiscordantexpressionpatternofIAPswasalsoobservedintheclamRuditapesphilippinarumexposedtoibuprofenforsevendays[58].
Asidefromanti-apoptotictranscripts,thepro-apoptoticinterferonalpha-inducibleprotein27(IFI27)andprogrammedcelldeathprotein7,alongwithTNF(tumornecrosisfactor)ligandsuperfamilymemberandTNFreceptorsuperfamilymemberproteinswerealsodifferentiallyexpressedinS.
glomerataexposedtoelevatedpCO2(Additionalfile7:TableS2).
IFI27thatwas4-foldandhigherup-regulatedinCO2stressedS.
glomerata,haspro-apoptoticfunc-tionsinvertebrates,whichmightbeblockedbythesim-ultaneousexpressionofBcl-2[59].
WhilesparseresearchisavailableonIFI27,itcouldactsimilarlyinoysters,suggestingthattheconcomitantlyup-regulatedBcl-2couldfunctionasanIFI27mediatorinS.
glomer-ata.
Anotherpro-apoptoticS.
glomerataDEtranscriptwasprogrammedcelldeathprotein7(lessthan4-folddown-regulated),whichwasshowntocauseincreasedcellapoptosisinmicewhenover-expressed[60].
TNFandTNFreceptorsuperfamilymembersthatwerealsoob-servedintheS.
glomerataDEtranscripts(both4-foldandhigherup-regulated),havediversefunctions,suchasrolesinapoptosisandacuteinflammation[61].
TNF-mediatedcelldeathsignallingiscomplex,activatingdifferentcom-ponentsthroughvariedpathwaysandresultingeitherincelldeath(dependentonROSandJNK[JunNH2-terminalkinase]signalling)orcellprotection(activationofnuclearfactorkB)[54].
Similartoourstudy,aTNFreceptorwasfoundtobeup-regulatedinregeneratingtissueofHydraafterinjury[46].
WhileCO2exposedS.
glomeratahadnoobviousinjuries,itispossiblethattheincreasedTNFandTNFreceptorexpressionintheoystersalsohadacellpro-tectivefunction.
Overall,itappearsthatapoptosiswasde-creasedinS.
glomeratasubjectedto1000ppmpCO2for4weeks,potentiallytoprotectitscellsfromtheCO2apop-toticstimuli.
Comparabletoourstudy,brineshrimps(Artemiasinica)exposedtoelevatedpCO2for14daysup-regulatedtheanti-apoptoticfactorApoptosisinhibitor5,withtheauthorssuggestingthatthismightbeamechan-ismtodealwiththetoxiceffectofthisstressor[62].
An-otherstudy,assessingmoleculardifferencesbetweendroughtintolerant(Pyganodongrandis)andtolerant(Uni-omerustetralasmus)freshwatermusselsshowedoverallaninductionofapoptosisinintolerantmusselsandaninhib-itionofapoptosisindroughttolerantmussels,suggestingthatapoptosisinhibitionmightbeanimportantmecha-nismsindroughttoleranceofU.
tetralasmus[63].
Insummary,primingoftheinnateimmunesystemandsup-pressingcellapoptosisappeartobetwomechanismsbywhichS.
glomerataexposedtoelevatedpCO2copewithpotentiallydetrimentaleffectsofoceanacidification.
RespirationandantioxidantdefenceS.
glomeratatranscriptspotentiallyinvolvedinantioxi-dantdefence,ROSproductionandrespirationwerealsoobservedamongtheDEtranscripts(Additionalfile7:TableS2).
Dualoxidase,whichislinkedtophagocytosisandtheproductionofROS[64],wasfoundtobe4-foldandhigherdown-regulatedintheelevatedtreatmentwhencomparedtocontroloysters(Additionalfile7:TableS2),suggestingthatCO2stressedoystersaimtolimittheirROSproductiontoprotectthemselvesfromoxidativestress.
ThisdiffersfromtheexpressionpatternseenforS.
glomeratatranscriptswhicharepartofthemitochondrialrespiratorychain,whereNADHdehydro-genase[ubiquinone]iron-sulfurprotein2(complexIprotein),cytochromeb-c1complexsubunit7(complexIIIprotein)andalternativeoxidasewere4-foldandhigherup-regulatedandcytochromecoxidasesubunit5Amitochondrial(complexIVprotein)4-foldandhigherdown-regulatedintheelevatedtreatmentwhencomparedtocontroloysters(Additionalfile7:TableS2).
Both,complexIandcomplexIIIareconsideredtobethemajorsourcesofROSproductioninthemitochon-drialrespiratorychain[65,66],suggestingthatup-regulationofmitochondrialrespirationinCO2chal-lengedS.
glomeratawouldleadtoanincreaseinROSproduction,elevatingtheriskofoxidativedamagetothetissuesoftheoysters.
However,thisappearstobecoun-teractedbytwomeasuresintheS.
glomerataexposedtotheelevatedCO2treatment.
Firstly,alternativeoxidase(Additionalfile7:TableS2),locatedintheinnermito-chondrialmembrane,allowstheanimaltocircumventcomplexIIIbytransferringelectronsfromcoenzymeQtooxygenandthereforelimitstheamountofROSpro-ducedduringcellularrespiration[66,67].
Secondly,ROSoriginatingfromcomplexIarethoughttoberemovedbymitochondrialmatrixantioxidantssuchasglutathi-oneperoxidaseandcatalase[65].
InS.
glomerataex-posedtoelevatedpCO2,bothcatalase(4-foldandhigher)andglutathioneperoxidase(2-fold)wereup-regulated(Additionalfile7:TableS2)whencomparedtocontroloysters,potentiallyprotectingtheanimalfromROSproducedbycomplexI.
Whiletranscriptscodingfortheantioxidantsperoxiredoxinandglutatredoxinwere4-foldandhigherdown-regulatedinCO2chal-lengedS.
glomerata,nearlyhalf(twooutoffive)ofthetranscriptscodingforglutathioneS-transferasewere2-foldandhigherup-regulatedinS.
glomerataexposedtoelevatedpCO2for4weeks(Additionalfile7:TableS2).
OftheantioxidantsfoundintheDEtranscriptsofS.
Ertletal.
ClimateChangeResponses(2016)3:6Page10of19glomerata,catalase(CAT),glutathioneperoxidase(GPX),peroxiredoxinandglutaredoxinareabletore-movetheROShydrogenperoxide(H2O2),withGPXalsofunctioningintheremovaloforganichydroperox-ides(e.
g.
fattyacidandphospholipidhydroperoxides)[68,69].
GlutathioneS-transferases,ontheotherhand,areactiveagainstsecondarymetabolites(e.
g.
epoxides,hydroperoxidesandunsaturatedaldehydes),thusoffer-ingprotectionfromtheeffectsofoxidativestress[69,70].
Ferritin,whichisinvolvedinhydroxylradicalscav-enging[71],wasalsofoundtobe2to4-foldup-regulatedinS.
glomerataexposedtoelevatedpCO2(Additionalfile7:TableS2).
Up-regulationofCAT,GPX,ferritinandglutathione-S-transferasetranscripts,aswellastranscriptsinvolvedincellularrespirationintheCO2challengedS.
glomeratasuggestthattheele-vatedCO2treatmentledtooxidativestress,whichS.
glo-merataattemptedtocounteractbyup-regulatingarangeofantioxidantsthatactonavarietyofsubstrates.
Furthermore,S.
glomerataincreasedtheexpressionofalternativeoxidasetopotentiallyreduceproductionofROSfromcellularrespiration(mainlycomplexIII),anddecreasedexpressionofdualoxidasewhosemainfunc-tionistheproductionofROS.
Asimilarpatternofex-pressionhasbeenobservedinCrassostreagigasexposedtohypoxiaandinC.
virginicaexposedtoelevatedpCO2[67,71,72].
Alternativeoxidaseandpyruvatekinase,an-otherS.
glomeratatranscriptfoundtobe4-foldandhigherdown-regulatedinpCO2exposedS.
glomerata(Additionalfile7:TableS2)ofourstudy,weremeasuredinC.
gigasremovedfromwaterfor3h,thenre-immersedintoeithernormoxicorhypoxicwater[71].
TheyshowedanincreaseinalternativeoxidasemRNAlevelsinC.
gigasre-immersedintonormoxicwater,andahigherlevelofpyruvatekinasemRNAlevelsinoystersundernormoxicconditionswhencomparedtooystersinhypoxicwater[71].
Theauthorssuggestedthatthein-creaseinalternativeoxidasewaslinkedtoincreasedoxy-genconsumptionandwouldprotecttheoysterfromtheresultingROSproduction[71].
Similarly,alternativeoxi-dasemRNAlevelsinthegillsanddigestiveglandofC.
gigasexposedto12hand24hofhypoxiawereshowntobeup-regulatedwhencomparedtonormoxiccondi-tions,suggestingthathypoxiacouldcausechangestotherespiratoryfunctionofmitochondriainC.
gigas[67].
Furthermore,comparabletotheS.
glomerataDEtran-scripts,avariedantioxidantdefenceresponsewasob-servedinC.
virginica(oneperoxiredoxinproteindown-regulated,threeperoxiredoxinsup-regulated)inre-sponseto2weeksofelevatedCO2exposure[72],andthespidercrabHyasaraneusinresponsetodifferentconcentrations(390μatm,1120μatmand1960μatm)ofCO2for10weeks,whereperoxiredoxinandascorbateperoxidasewereup-regulatedandthioredoxinandthioredoxinperoxidasedown-regulatedincrabsexposedto1120μatmofCO2[73].
Moreover,GPXwasup-regulatedinH.
araneusexposedto1960μatmofCO2,whilesuperoxidedismutaseandthioredoxinweredown-regulatedunderthesameCO2conditions[73].
Thesere-sultsindicatethatstressorslikehypoxiaandpCO2canaffectarangeofgenesthatfunctioninROSprotection,withthespecificantioxidantresponsepotentiallyinflu-encedbytheconcentrationofCO2thattheinvertebratesareexposedto.
ThishasalsobeenobservedintheS.
glo-merataofourstudy,showingthatS.
glomerataexposedto1000ppmofpCO2appeartoemployarangeofmechanisms(e.
g.
antioxidants,alternativeoxidase)tocopewithoxidativestress,potentiallycausedbyexpos-uretoelevatedCO2.
BiomineralisationandcytoskeletonBivalveshellsconsistofanouterlayer(periostracum)madeupofconchiolin,aprismaticlayer(ostracum)andalamellarlayer(hypostracum)whichisfoundclosesttothebivalvebody.
Thelayersbelowtheperiostracumconsistofcalciumcarbonatecrystalsdepositedinanor-ganicmatrix(proteins,glycoproteins,polysaccharidesandlipids),withthelamellarlayercontainingaragoniteandcalcite[74–78].
SeveralstudieshavebeencarriedoutinmolluscssuchasthemusselMytilusedulis,thepearloystersPinctadamargaritiferaandPinctadamax-ima,theclamLaternulaellipticaandtheseasnailPa-tellavulgatatodeterminegenespotentiallyassociatedwithshellformation[78–82].
Someofthemanygenessuggestedtobeinvolvedinbiomineralisationaretyro-sinase,chitinase,chitinsynthase,calponin,carbonicanhydrase,perlucin,nacrein-like,silk-like,perlustrin,lustrin,follistatin,sarcoplasmiccalciumbindingandcal-modulin[79–81].
Inourstudy,threeS.
glomeratacar-bonicanhydrasetranscriptswerefoundtobedifferentiallyexpressed,withtwocarbonicanhydrase2transcripts4-foldandhigherup-regulatedandcarbonicanhydrase15-like4-foldandhigherdown-regulated(Fig.
2).
Carbonicanhydrase,whichcatalysesthereac-tionofCO2tobicarbonate(HCO3-)[81],hasbeenexam-inedindifferentinvertebratesandshowedvaryingresponsestoelevatedlevelsofCO2.
InM.
edulis(2monthsexposure)carbonicanhydrasewasshowntobenotsignificantlyaffectedbyelevatedpCO2[83],whileanovelcarbonicanhydraseofHyriopsiscumingii(2weeksexposure)wassignificantlydown-regulatedinelevatedpCO2[84].
Incontrast,carbonicanhydrasewasfoundtobeup-regulatedinjuvenileC.
gigas(28daysexposure)[85],inthecoralD.
dianthus(8monthsexposure)[48]andinthecrabHyasaraneus(10weeksexposure)[73]exposedtoelevatedpCO2.
TheseresultssuggestthatcarbonicanhydraseexpressioninresponsetoCO2stressisnotconsistentacrossmarinespeciesandexposureErtletal.
ClimateChangeResponses(2016)3:6Page11of19times,emphasisingthenecessitytodeterminethere-sponseofindividualmarineanimalstopotentialfuturechangesinpCO2.
Furthermore,thedifferentexpressionpatternobservedforthetwotypesofcarbonicanhydraseintheS.
glomerataofthisstudyindicatesthattheremightalsobesomevariationwithinaspecies.
Ascar-bonicanhydrasesalsohaverolesinprocessessuchasrespiration,iontransportorpHhomeostasis[84,86],itispossiblethatthedifferenceinexpressionseeninourS.
glomeratastudywasduetodifferentrolesofthetwotypesofcarbonicanhydrase.
Metabolismofchitin,abiopolymerthathasbeende-tectedinmolluscanshells,involveschitinsynthase,chi-tindeacetylaseandchitinase[79,87,88],whichhaveallbeenfoundtobedifferentiallyexpressedintheS.
glo-merataofthisstudy(Fig.
2).
Chitinsynthase(synthesiseschitin)andchitinase(degradeschitin)were2-foldandhigherdown-regulatedinresponsetoelevatedCO2,whereaschitindeacetylase(modifieschitin)waslessthan2-foldup-regulated,suggestingthatoystersexposedto1000ppmpCO2protectthechitinshellcomponentfromdegradationandconcentrateonchitinmodificationinsteadofchitinsynthesis.
Additionalworkisneededtodeterminetheeffect(detrimentalorbeneficial)ofchitinmodificationonS.
glomeratashells.
AsimilarpatternofexpressionhasbeenshowninM.
edulis,whereelevatedlevelsofCO2exposureresultedinadecreaseinchiti-nase;however,chitinsynthaseshowednoresponsetohighCO2levels[83].
DEtranscriptsputativelycodingforfollistatin,silklikeprotein,perlucin,alkalinephosphatase,sarcoplasmiccalcium-bindingprotein(onetranscript2-foldandhigherup-,onetranscript4-foldandhigherdown-regulated),calmodulinandcalmodulin-like,whichhavealsobeenassociatedwithbiomineralisationinmol-luscs[79–81,89],werealsofoundinS.
glomerataexposedtoelevatedpCO2(Fig.
2).
Whileperlucinandcalmodu-linwere4-foldandhigherdown-regulated,silklikeprotein,follistatin,calmodulin-likeandalkalinephos-phatasewereupto4-foldandhigherup-regulatedinCO2stressedS.
glomerata.
Inaddition,theenrich-mentanalysisalsoshowedanover-representationoftheCOG/eggNOGterms'follistatin'and'alkalinephosphatase'inCO2challengedS.
glomerata(Table3),furtherhighlightingtheeffectCO2exposurecouldpotentiallyhaveonbiomineralisation.
Contrarytoourresults,sarcoplasmiccalcium-bindingproteinandsilklikeproteinexpressioninM.
edulisdidnotchangeunderelevatedpCO2[83].
Alkalinephosphataseontheotherhand,wasalsofoundtobeup-regulatedinA.
sinicaexposedtotwolevelsofCO2for7days,butshowedadecreaseinexpressionlevelsatday14[62].
AstudyinPinctadafucataexaminedcalmodulin(roleincalciummetabolism[90])andcalmodulin-likeandtheirpotentialfunctionsinbiomineralisationandfoundthatbothhadaroleinbiomineralisationbutweredifferentlydistributedinthemantletissueoftheoyster[91].
Ingeneral,theseresultssuggestthatele-vatedpCO2levelsmighthaveaffectedtheshellFig.
2HeatmapofS.
glomerataDEtranscriptsinvolvedinbiomineralisation.
HeatmapshowstheposteriorfoldchangevaluesoftheDEtranscriptsinvolvedinbiomineralisation,withtranscriptsdepictedinredup-regulatedandtranscriptsingreendown-regulatedinCO2treatedoysterswhencomparedtocontroloysters.
Fulllistof1626DEtranscripts,whichincludetheabovebiomineralisationtranscripts,wasdeterminedwithBowtie-RSEM-EBSeq,usingaFDRthresholdof0.
05Ertletal.
ClimateChangeResponses(2016)3:6Page12of19compositionofS.
glomeratabycausingadown-regulationofsomeofthebiomineralisationgenes(e.
g.
perlucin),andup-regulationofothers(e.
g.
chitindea-cetylase,calmodulin-like),whichcouldhaveconse-quencesinregardstoshellstrength.
ThiscouldpotentiallyexplainthedecreaseinshellstrengththathasbeenobservedinP.
fucataexposedtolowpH[14],aswellasthesignificantchangesinshellstrengthobservedinC.
gigasexposedtothesameel-evatedCO2conditionsusedwithinourstudy[92].
Also,inaccordwiththemolecularresultsofourstudyinS.
glomerata,researchanalysingtheshellultrastructureofthemusselM.
edulisexposedtopro-jectednear-futureoceanacidificationlevels(550,750and1000μatmpCO2)showedchangesinshellcom-positionandcrystalformation[93].
Whileshellgrowthdidnotappeartobeaffectedbyexposureto1000μatmpCO2,asignificantreductioninaragonitethicknessandasignificantincreaseincalcitethick-nesswasobservedunderthistreatmentcondition[93].
Inaddition,newcalcitecrystalsformedintheshellofM.
edulisexposedtoelevatedoceanacidifica-tionlevelsweredisorientatedandcouldpotentiallyresultinreducedshellstrengthintheexposedmus-sels[93].
OtherS.
glomerataDEtranscriptsfoundareputativeelementsofthecytoskeletonandextracellularmatrix(ECM).
Theeukaryoticcytoskeletonconsistsofmicrotu-bules(α-andβ-tubulin),microfilaments(actin)andintermediatefilaments(proteinsofthekeratinfamily),maintainscellshapeandhasaroleinmotility(e.
g.
movementoforganelles)[94,95].
AmongtheS.
glomer-ataDEtranscripts,onebetatubulinandoneactintran-scriptwerelessthan2-foldup-regulated,whilefouractintranscriptswere4-foldandhigherdown-regulatedinS.
glomerataexposedtoelevatedpCO2(Additionalfile7:TableS2).
SimilartranscriptexpressionhasbeenobservedinH.
araneusinresponsetodifferentlevelsofelevatedpCO2,wherealphaandbetatubulintranscripts,aswellasactintranscriptswereup-regulatedinre-sponsetotheCO2treatment[73].
Inaddition,aβ-actintranscriptanddifferentactinproteinsofD.
dianthusandC.
virginica,respectively,werealsofoundtobeup-regulatedinresponsetoelevatedpCO2[48,72].
Whilethemajorityofactintranscriptsweredown-regulatedinourstudy,onetubulinandoneactintranscriptwereup-regulated,indicatingthattheincreaseinpCO2for4weeksmighthavehadsomeimpactonthecytoskeleton,forcingtheoysterstocompensatebyslightlyincreasingtheexpressionofcytoskeletaltranscripts.
Ithasbeensuggestedthatoxidativestresscouldaffectthecytoskel-eton[73],which,consideringtheincreaseincomplexIandcomplexIIItranscriptsalongwiththeup-regulationofantioxidantdefencemechanismsappearstobesupportedbytheresultsofourS.
glomeratastudy.
Integ-rin,atransmembranereceptorthatconnectstheECMwiththeactincytoskeleton[96,97]was4-foldandhigherup-regulatedinCO2exposedS.
glomerata(Additionalfile7:TableS2).
Asintegrinsarealsoinvolvedintransmittingmechanicalandchemicalinformationtothecytoskeleton[96],itispossiblethattheup-regulationoftheS.
glomer-ataintegrintranscriptisacopingmechanismoftheoystertotheCO2stresstoprotectthecytoskeleton.
Inadditiontocytoskeletalcomponents,transcriptsputativelycodingforECMcomponentswerealsofoundtobedifferentiallyexpressedinS.
glomerataexposedtoelevatedpCO2for4weeks.
TheECMineukaryotes,whichiscomposedofthemacromoleculesglycoproteinsandfibrousproteins,pro-videsaphysicalscaffoldforcellsandhasotherrolessuchastransmittingmechanicalcues[97–99].
Collagens,whicharethemostabundantproteinsoftheECM[97,99],andanaggrecancoreproteinthatisalsoacomponentoftheECM[99]weredifferentiallyexpressedintheS.
glomerataofourstudy(Additionalfile7:TableS2).
Whiletheaggre-cancoreproteintranscriptwas4-foldandhigherdown-regulatedinCO2challengedS.
glomerata,fivecollagentranscriptswere4-foldandhigherup-regulatedandeightwere4-foldandhigherdown-regulatedinresponsetoele-vatedpCO2.
Inaddition,oneADAMTS(adisintegrinandmetalloproteinasewiththrombospondinmotifs)transcriptwasfoundtobelessthan2-folddown-regulatedandtwoTIMPs(metalloproteinaseinhibitors)were4-foldandhigherup-regulatedinS.
glomerataexposedtoelevatedpCO2(Additionalfile7:TableS2).
ADAMTSareafamilyofproteinasesthatfunctioninECMdegradation[98],whereasTIMPsareregulatorsofADAMTSandhavearoleinthecontinuousremodellingoftheECM,whichisimportantinmaintaininghomeostasisduring,forin-stance,injury[97–100].
ThedifferentialexpressionpatternofS.
glomerataECMtranscriptssuggeststhatECMdeg-radationisinhibited,whileasmallnumberofcollagentranscriptsareup-regulatedtopotentiallycounteractanydetrimentaleffectscausedbytheelevatedCO2stressex-posure.
AslightlydifferentresponsewasobservedinC.
virginicaexposedtoelevatedpCO2for2weeks,wherethecytoskeletalcomponentactinwasup-regulatedbutonecollagenproteindown-regulated[72],indicatingthathypercapnanegativelyaffectedtheECMbutinducedactintopotentiallyprotectthecytoskeletonagainstROSinthisoysterspecies.
Interestingly,studiesonTIMPfunctioninoysterssurmisedthatitcouldhaveadditionalroles.
Forinstance,aTIMPinP.
martensiiwasshowntohaveapu-tativeroleinnacreformationassuppressionofTIMPex-pressionresultedindisorderedgrowthoftheoyster'snacre[101].
Similarly,TIMPexpressioninC.
gigaswasup-regulatedinoysterswithdamagedshells[102].
Basedonthesestudies,itispossiblethattheup-regulationofS.
glomerataTIMPsofthisstudycouldalsohavebeeninErtletal.
ClimateChangeResponses(2016)3:6Page13of19responsetoputativedamagecausedbytheprolongedCO2exposureoftheoysters.
ProteinsynthesisNuclearrespiratoryfactor1(NRF-1),whichisanu-cleartranscriptionfactorthatisinvolvedinthetran-scriptionalexpressionofmitochondrialrespiratorychaincomponentsandothermitochondrion-relatedgenes[103],hasbeenfoundtobeup-regulated(lessthan2-fold)inS.
glomeratainresponsetoelevatedconcentrationsofCO2(Additionalfile7:TableS2).
BasedonthefunctionofNRF-1invertebrates,itsup-regulationinS.
glomeratacouldpotentiallybelinkedtotheup-regulationofmitochondrialrespira-torychaincomponents,whichwasalsoobservedinthisstudy,indicatingthatmaintenanceoftherespira-toryapparatuswasofimportancetotheCO2chal-lengedS.
glomerata.
ComparabletoNRF-1,transcripts,putativelycodingforribosomalproteins,werefoundtobe2-foldandhigherup-regulated(sevenoutofeighttranscripts)inS.
glomerataafterCO2exposure(Additionalfile7:TableS2).
Ribosomalproteinshavearoleinproteinsynthesis[104]andsimilartoourstudy,wereup-regulatedinD.
dianthusandC.
virginicafollowingCO2exposure[48,72],showingthatproteinsynthesiswasnotonlyaffectedbyCO2inourS.
glomeratastudybutalsoinothermarineorganisms.
AsidefromribosomalproteinsandNRF-1,transcriptsbelongingtothefamilyofmRNAhelicasesandRNA-bindingproteinswerealsodiffer-entiallyexpressedinCO2challengedS.
glomerata(Additionalfile7:TableS2).
Ofthesetranscripts,threeareputativelycodingforeukaryotictranslationinitiationfactors(eIFs),witheIF4A2,eIF4BandeIF3hlessthan2-foldup-regulatedintheelevatedCO2treatment.
Ineukaryotes,eIF4andeIF3areinvolvedintranslationinitiation,wheretheyunwindsecondarystructuresinthemRNA5′untranslatedregion[105].
Similarly,heterogeneousnuclearribonucleoproteins(hnRNPs)areRNA-bindingproteinswithfunctionsinthenucleusandcytoplasmofthecells[106]thatweremostlylessthan2-foldup-regulatedintheS.
glomerataofthisstudy.
TherolesofhnRNPsrangefromtranscriptiontomRNAtransport,splicing,3′-endprocessingandmRNAstability[106],showingthathnRNPsareanimportantgroupoftranscrip-tionalregulationproteins.
InP.
fucata,ahnRNPwasclonedandshowntobeexpressedinthegonad,gillandvisceraoftheoyster,withitslocalisationinsidethecellrestrictedtothenucleus[107].
Increasingtheexpressionoftranscriptsputativelycodingforpro-teinsinvolvedinproteinsynthesiswouldallowCO2stressedS.
glomeratatoexpressmoleculesthatmightmakethemmoreresilienttothestressor.
Inaddition,aslightlyhigherpercentageofthetotal1626DEtranscriptswereup-regulated(53.
3%)thandown-regulated,whichmightmakeitnecessarytoincreasethenumberofproteinsinvolvedintranslationandpost-translationalprocessing.
CiliaryfunctionInbivalves,ciliaarefoundonavarietyoftissuessuchasgill,mantleorstomach,withimportantfunctionsin,forinstance,filtration,respirationandpseudofecesexpul-sion[108–111].
MultipleS.
glomerataDEtranscriptspu-tativelyinvolvedinciliaformationorfunction(e.
g.
tektin,CCDC65,CCDC176)werefoundtobelessthan4-folddown-regulatedinoystersexposedtoelevatedpCO2for4weeks(Fig.
3).
TheseRNA-SeqresultswereconfirmedwithqPCR,whichshowedthatboth,tektin-2andtektin-4-likeweresignificantlydown-regulatedinS.
glomerataexposedtoelevatedpCO2for4weeks(Fig.
4).
Tektinsareafamilyofproteinsthathavebeenobservedineukaryoticorganismssuchasmammals,insectsandseaurchinsandinawiderangeoftissues(e.
g.
testis,brain),andarevitalcomponentsofciliaandflagella[112].
Similarly,analysisoftheputativetissuedistribu-tionofthetektintranscriptsfoundtobedifferentiallyexpressedinourstudyshowedthattheywereexpressedinthehaemolymph,gill,mantle,adductormuscle,di-gestivesystemandgonadofS.
glomerata(mappingdatanotshown).
Comparabletotheresultsofourstudy,tek-tinhasalsobeenfoundtobedown-regulatedinC.
gigaslarvaeexposedtoapHofabout7.
5for6days[113].
Inaddition,anotherstudyinC.
gigasshowedthattwoformsoftektinwerepresentinthespermatozoaoftheoyster[114].
Correspondingly,CCDC135(coiled-coildomain-containingproteinlobohomolog),aflagellarprotein,hasbeenshowntolocalizeinthetestisandalongthespermflagellumoftheflyDrosophilamelano-gaster,withsomespermmotilitydefectsseeninthesin-gleanddoublemutants[115].
Anothercoiled-coildomaincontainingprotein(CCDC65)wasdetectedinthehumanspermtailandtheciliaofairwayepithelialcells,withamutationorsilencingoftheproteinnega-tivelyaffectingciliamotility[116].
Basedonthesestud-iesandtheimportanceoftheflagellumforspermmovement,down-regulationoftektin,CCDC135andCCDC65intheS.
glomerataofourstudycouldmeanapotentialimpairmentofspermmotilityintheaffectedoysters.
Whilesomeresearchhasexaminedspermmo-tility,contradictoryresultshavebeenobtainedinthesestudies,withC.
gigasspermexposedtoelevatedCO2for48hshowingadecreaseinmotilityandStrongylocentro-tusnudussperm(20minexposure)notaffectedbyele-vatedCO2[117,118].
AlthoughknowledgeregardingtheeffectofincreasedpCO2ongametesisimportant,exposureoftheadultbeforeitreachesgravidstagecouldErtletal.
ClimateChangeResponses(2016)3:6Page14of19alreadypotentiallyimpactonthegametesbeforetheyarereleasedintothewatercolumn.
OtherS.
glomerataDEtranscriptsputativelyinvolvedinciliafunctionandformationareCCDC176(coiled-coildomain-containingprotein176),CC2D2A(coiled-coilandc2domain-containingprotein2A),CEP131(5-azacytidine-inducedprotein1)andbardet-biedlsyn-dromeproteins(Fig.
3).
AstudyofCCDC176inXen-opuslaevisshowedanup-regulationofthegeneduringtheformationofmotileciliaandaninvolvementinthealignmentandmaintenanceofciliaorientation[119].
TheauthorsalsoobservedthatmisalignmentoftheciliainX.
laevisappearedtohavenegativelyaffectedtheflowgeneratedbythebeatingcilia[119].
Bardet-biedlsyn-drome(BBS)genesplayaroleinintraflagellartrans-portandciliaryfunction,withBBS2,BBS4andBBS6observedtoaffectflagellaformationofsperminmiceandBBS2andBBS4-BBS8lossinzebrafishwasasso-ciatedwithmaintenanceorsurvivalofciliaofKupf-fer'svesicle[120].
CEP131andCC2D2A,ontheotherFig.
3HeatmapofS.
glomerataDEtranscriptsinvolvedinciliaformationandfunction.
HeatmapshowstheposteriorfoldchangevaluesoftheDEtranscriptsinvolvedinciliaformationandfunction,withtranscriptsdepictedinredup-regulatedandtranscriptsingreendown-regulatedinCO2treatedoysterswhencomparedtocontroloysters.
Fulllistof1626DEtranscripts,whichincludetheabovebiomineralisationtranscripts,wasdeterminedwithBowtie-RSEM-EBSeq,usingaFDRthresholdof0.
05Fig.
4QPCRresultsofdifferentialtranscriptexpressionanalysis.
Transcriptexpressionlevels(mean±SD)oftektin-2andtektin-4-likewereanalysedwithqPCRincontrol(n=6)andCO2stressed(n=6)S.
glomerata,andsignificantdifferencesexaminedwithatwo-samplet-testassumingunequalvariances(p<0.
05)Ertletal.
ClimateChangeResponses(2016)3:6Page15of19hand,havebothbeenimplicatedinciliogenesisinvertebrates[121,122].
Whileresearchontheeffectsofstressonbivalveciliarymotilityappearstobesparse,studiesinM.
edulisshowedadecreaseincil-iaryactivityinresponsetogammaradiationandlossofciliainmusselsexposedtoenvironmentalpollution[123,124].
Overall,themolecularresponseofCO2stressedS.
glomerataofourstudysuggestthatelevatedpCO2couldalsonegativelyaffectciliafunctioninoysters,potentiallyresultinginimpairmentstotheoyster'sabilitytogenerateflowforfiltrationandspermmovement.
ConclusionsRNA-SeqexaminationofthemolecularresponseofadultS.
glomerataexposedtoexpectednear-futureoceanacidificationlevelsfor4weeksshowedthatoverallslightlymoreoftheDEtranscriptswereup-regulatedthandown-regulated.
TheexpressionpatternssuggestthatS.
glomerataprotectsitselffromthestressorbyprim-ingitsimmunesystem,suppressingcellapoptosisandadjustingantioxidantdefencemechanismstocompensateforwhatappearstobeanincreaseinmitochondrialres-pirationanditsrespectiveleakageofROS.
Asidefromthisprotectivemechanism,thatissimilartowhathasbeenseeninresponsetoinjuryandpathogensinothermarineorganisms,transcriptsassociatedwithmaintenanceandrepairwerealsoup-regulatedinS.
glomerata.
Thesetran-scriptshaveputativerolesintranslationandpost-translationalprocessingandwerelikelyincreasedtocovertheelevatedexpressionofprotectivemoleculessuchasantioxidantsandotherimmunerelatedproteins.
Contrarytothis,prolongedexposuretoelevatedCO2seemstohavenegativelyaffectedstructuralproteins(e.
g.
actin)andpro-teinsputativelyinvolvedinciliaandflagellafunction.
Ciliaareimportantstructureswithmanyfunctions,suchasfa-cilitatingfilteringandparticletransportintothestomach.
ThedecreaseintheexpressionoftranscriptsimplicatedinciliaandflagellafunctionsuggeststhatincreasedpCO2mightimpairprocessesintheoysterthatrelyontheirop-timalaction,suchasfeedingorspermmotility,whichinthelong-termcouldbelife-threatening.
Whilesomeoceanacidificationstudieshavebeencarriedoutinregardstoitseffectonspermmotility,thesestudiesonlyassessedhowaveryshortexposuretoelevatedpCO2impactsthereleasedsperm,ignoringthatprojectednear-futureoceanacidifica-tionlevelscouldalsopotentiallyaffectspermproductionandmaturation,whichcouldhavedownstreameffectsonitsmotilityandsuccessinfertilizinganegg.
Expressionpatternsoftranscriptsputativelyinvolvedinbiominerali-sation,suggestthatcontinuousexposureofS.
glomeratatoelevatedCO2resultedinachangeoftheshellcompos-itionwithpotentialdownstreameffectsonshellstrength.
Incontrasttolarvaethatquitelikelyhavetoextendmoreenergyintotheinitialformationoftheshell,adultsonlyneedtomaintainandslowlygrowtheirshells.
AsourstudyassessedtheeffectsofelevatedCO2onadultS.
glo-merata,theincreaseinbiomineralisationtranscriptex-pressionseenmightbesufficienttomaintainanadequatelevelofbiomineralisation.
Insummary,thisstudydetailedthecomplexmolecularresponseofS.
glomeratatopro-jectednear-futurelevelsofoceanacidification.
However,tofullyelucidatethemolecularandphysiologicalresponseofbivalvestofutureoceanacidificationlevels,long-termstudiesneedtobecarriedoutthatincluderecoverype-riodstoassessthepotentialofbivalvestoreverseanydet-rimentaleffectsofoceanacidification.
AdditionalfilesAdditionalfile1:Detailedmethodsofexperimentalexposuretrialsandprevioussequencing.
(DOCX23kb)Additionalfile2:TableS1.
Bowtiealignmentstatistics.
Tableshowsthenumberofrawreadsandreadssurvivingtheprocessing,withthetotalalignmentpercentagebasedonthepost-processedreadsaligningtotheS.
glomeratareferencetranscriptome,usingBowtie.
(DOCX12kb)Additionalfile3:FigureS1.
VarianceversusmeanplotforeachNggroup(C1).
Thisplotshowsthemean-variancerelationship(usingpoly-nomialregression)foreachisoform(Ng)groupofcondition1(controlsam-ples).
MappingambiguityclusterswereproducedwithRSEM(rsem-generate-ngvector),whiletheplotwasvisualisedinRusingEBSeq'sPolyFitPlotfunction.
(PDF260kb)Additionalfile4:FigureS2.
VarianceversusmeanplotforeachNggroup(C2).
Thisplotshowsthemean-variancerelationship(usingpolynomialregression)foreachisoform(Ng)groupofcondition2(elevatedCO2samples).
MappingambiguityclusterswereproducedwithRSEM(rsem-generate-ngvector),whiletheplotwasvisualisedinRusingEBSeq'sPolyFitPlotfunction.
(PDF268kb)Additionalfile5:FigureS3.
Quantile-quantileplot.
QQ-plotsshowthefittedBetapriordistributionswithineachconditionandeachIggroup(uncertaintygroup)andwerevisualisedinRusingEBSeq'sQQPfunction.
(PDF337kb)Additionalfile6:FigureS4.
Densityplot.
PlotshowsthepriordistributionfitwithineachconditionandeachIggroup,visualisedinRusingEBSeq'sDenNHistfunction.
(PDF38kb)Additionalfile7:TableS2.
S.
glomerataDEtranscripts.
Listof1626DEtranscriptsdeterminedwithBowtie-RSEM-EBSeq,usingaFDRthresholdof0.
05.
SequencedescriptionsarebasedonblasthomologysearchesagainsttheNCBInrdatabase(e-valuecut-off:10-5,hitnumberthreshold:25),andonInterProScandomain/familyinformation.
Posteriorfoldchange(FC)wasbasedonthenormaliseddata,whereasrealFCwasbasedontherawdata.
C1standsforcontrol,C2fortreatmentcondition.
(XLSX124kb)AbbreviationsA.
irradians:Argopectenirradians;A.
sinica:Artemiasinica;ABTS2:2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid);ADAMTS:Adisintegrinandmetalloproteinasewiththrombospondinmotifs;AGRF:AustralianGenomeResearchFacility;BBS:Bardet-biedlsyndrome;C.
ariakensis:Crassostreaariakensis;C.
gigas:Crassostreagigas;C.
virginica:Crassostreavirginica;CAT:Catalase;CC2D2A:Coiled-coilandc2domain-containingprotein2A;CCDC:Coiled-coildomaincontainingprotein;CCDC135:Coiled-coildomain-containingproteinlobohomolog;CEGMA:CoreEukaryoticGenesMappingApproach;CEP131:5-azacytidine-inducedprotein1;D.
dianthus:Desmophyllumdianthus;DAMPs:Damage-associatedmolecularpatterns;DE:Differentiallyexpressed;ECM:Extracellularmatrix;eIFs:Eukaryotictranslationinitiationfactors;ERCC:ExternalRNAControlConsortium;FAIM:Fasapoptoticinhibitorymolecule;FDR:Falsediscoveryrate;GNBPs:Gram-negativebacteriabindingproteins;GPX:GlutathioneErtletal.
ClimateChangeResponses(2016)3:6Page16of19peroxidase;H.
araneus:Hyasaraneus;hnRNPs:Heterogeneousnuclearribonucleoproteins;Hsp:Heatshockprotein;IAP:Inhibitorofapoptosisprotein;IFI27:Interferonalpha-inducibleprotein27;IGV:Integrativegenomicsviewer;JNK:JunNH2-terminalkinase;M.
edulis:Mytilusedulis;M.
galloprovincialis:Mytilusgalloprovincialis;M.
mercenaria:Mercenariomercenaria;MPEG1:Macrophage-expressedgene1;NCBI:NationalCenterforBiotechnologyInformation;nr:Non-redundant;NRF-1:Nuclearrespiratoryfactor1;NSW:NewSouthWales;NTCs:Notemplatecontrols;P.
fucata:Pinctadafucata;PAH:Polycyclicaromatichydrocarbon;PGRPs:Peptidoglycanrecognitionproteins;PRRs:Patternrecognitionreceptors;qPCR:Quantitativepolymerasechainreaction;ROS:Reactiveoxygenspecies;-RTs:Negativereversetranscriptions;S.
glomerata:Saccostreaglomerata;SRA:Sequencereadarchive;SRs:Scavengerreceptors;TIMPs:Metalloproteinaseinhibitors;TNF:Tumornecrosisfactor;U.
tetralasmus:Uniomerustetralasmus;X.
laevis:XenopuslaevisAcknowledgementsWewouldliketothankthefollowingpeopleandorganisations:MrJohnWrightforgivingusaccesstohisCO2andtemperatureexperimentsforthesamples;MrStephanO'Connor,MrKyleJohnstonandtherestoftheDPIhatcheryteamfortheirhelpandadvicewithoysterhusbandry;DrDavidSchoemanforstatisticaladviceandwritingtheRbasedscriptusedtoproducetheheatmaps;DrIdoBarforcarryingouttheGOenrichmentanalysis;CSIROforapostgraduatestudentshipforNGE;theNationalComputationalInfrastructureSpecialisedfacilityinBioinformatics(Barrine@UQ)forBarrinesupport.
FundingTheprojectwasfundedbytheFisheriesResearch&DevelopmentCorporation(FRDC–http://frdc.
com.
au/),theAustralianSeafoodCooperativeResearchCentre(CRC–http://www.
seafoodcrc.
com/)(2011/718toNGE,AEandWAO),andtheUniversityoftheSunshineCoast,Australia.
NGEwassupportedbyanAustralianSeafoodCRCandUniversityoftheSunshineCoastscholarship,aswellasascholarshipfromQueenslandEducationandTrainingInternational(QETI).
Thefundingpartieshadnoroleinstudydesign,datacollection,analysisandinterpretationofdata,decisiontopublish,orpreparationofthemanuscript.
AvailabilityofdataandmaterialsTherawCO2readssupportingtheconclusionsofthisarticleareavailablefromthesequencereadarchiveandcanbeaccessedundertheSRAstudyaccessionnumberSRP055052.
Authors'contributionsNGE:designedthestudy,collectedsamplesfromthestressexperiments,carriedoutallrelevantlaboratoryandbioinformaticswork,aswellasthedifferentialgeneexpressionanalysis,draftedmanuscript.
WAO:designedstudy,advisedonallexperiments,providedoystersandnecessaryequipmentforexperiments,reviewedmanuscript.
ANW:wrotethein-housescriptusedtoremoveERCCandphiXtranscriptsfromthetranscriptomeandadvisedonlinuxbasederrors.
AE:designedstudy,reviewedmanuscript.
Allauthorshavereadandacceptedthemanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authordetails1UniversityoftheSunshineCoast,SippyDowns,SunshineCoast,QLD,Australia.
2AustralianSeafoodCooperativeResearchCentre,BedfordPark,SA,Australia.
3DepartmentofPrimaryIndustries,TaylorsBeach,NSW,Australia.
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Weacceptpre-submissioninquiriesOurselectortoolhelpsyoutondthemostrelevantjournalWeprovideroundtheclockcustomersupportConvenientonlinesubmissionThoroughpeerreviewInclusioninPubMedandallmajorindexingservicesMaximumvisibilityforyourresearchSubmityourmanuscriptatwww.
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