Results

ConclusionsThisstudyfoundthatthereweredifferencesinproteinexpressionbetweenlarvaerearedinalowsalinityenvironmentcomparedtothoserearedinafluctua:ngsalinityenvironment.Thesodium/potassiumATPasewasupregulatedinFSlarvae,whiletheLStreatmentwassimilartothecontrol(C).(Figure1A).AproteinbelievedtobeamechanosensoryproteinwasupregulatedinFSlarvae.(Figure1B).Larvalposterolateralarmlengthdifferedsignificantlybetweentreatments.(Figure2).

References&AcknowledgementsPia,T.S.,Johnson,T.,andGeorge,S.B."Salinity-inducedMorphologicalChangesinBashevkin,S.M.,Lee,D.,Driver,P.,Carrington,E.,George,S.B.(inpress)."PriorWewouldliketothanktheBlinks-NSFREU-BEACONprogramatFridayHarborLaboratories(NSFgrantnumberDBI1262239).WewouldliketothankDr.RichardStrathmannforprovidingalgaeands:rringapparatus

IntroducIon

•  GelswerestainedusingLifeTechnologiesColloidalBlueStainingKit

•  GelimagesweretakenusingaKodakImager

•  Duringeachlow-salinityevent,10larvaefromeachjarweremeasuredusingimageJtodetermineaverageposterolateralarmforeachtreatment

EarlyGastrulaBipinnariaBipinnariaBrachioloaria

•  LarvaewerefedamixeddietofIsochrysisgalbana,Dunaliellater3olecta,andRhodomonasspp.every2-3days

•  Fourlowsalinityeventsweresimulated.Duringeachlow-salinityevent,~1000larvaewereobtainedfromeachjarforproteinanalysis

•  Sampledlarvaewerespundown,seawaterremoved,andlysisbufferadded

•  ABradfordassaywasusedtodeterminelysateproteincontentto

MethodsII

5jarspertreatment,4000larvaeperjar

Figure2,lea:Posterolateralarmlengthoflarvae34and42daysold.Differencesinarmlengthweresignificant.Valuesaremeans±standarddevia:on(p<0.05,n=120).Right:Photosof42dayoldlarvaefromtheControls(C),Fluctua:ngsalinity(FS)andlowsalinity(LS).

•  Thisrunoffcancreatesurfacewaterswithsalinityaslowas21‰,andcanpersistfordays

•  SinceP.ochraceuslarvaecantakeover200daystodevelop,theyareboundtoexperienceatleastonelow-salinityeventduringtheirdevelopment(below)

•  Previousstudieshaveshownthatlowsalinitycancauselarvaetoundergomorphologicalchanges,includingthedevelopmentofashorterandwidershape,aswellasreducedarmlengthduringthebrachiolariastageofdevelopment,affec:ngswimmingability(Piaetal,2012).

•  Throughtheuseofmassspectrometry,previousresearchhasalsoshownthatalow-salinityenvironmentcausesachange

•  Thepresentstudyisthefirsttoexamineeffectsofaconstantlow-salinityenvironmentversusanenvironmentthatexperiencesfluctua:onsbetweenlowandnormalsalinityseawater

ThisstudysettoanswerthefollowingquesIons:

Na/KATPaseProteinExpression MechanosensoryProteinExpression

Figure1A,lea:Sodium-PotassiumATPaseproteinexpressionin34dayoldlarvae.Figure1B,right:PossibleMechanosensoryproteinexpressionin24dayoldlarvae.

RiversfeedingintotheSalishSea.ManycarryfreshwaterfromtheArIc.

C

FS

LSMethodsI•  P.ochraceusadultswerecollectedfromCan:leverPoint,FridayHarbor,WA

andinjectedthroughthemouthwith4mLof100μM1-MethylAdeninetoinducespawning

•  Eggswerefer:lizedwith8-10dropsofdilutesperm•  Onceinthegastrulastage,larvaeweredividedinto15jars,fivejarsthatwere

keptconstantlyat30‰(control,C),fivejarskeptconstantlyat21‰(lowsalinity,LS),andfivejarsthatweredroppedto21‰for24honceperweek,butotherwiseweremaintainedat31‰(fluctua:ngsalinity,FS)

Sodium/potassiumATPasesareusedbycellstohelpregulatetheirvolume,thusup-regula:onbyFSlarvaeproducedmoresodium/potassiumATPaseinresponsetothelowsalinitycondi:ons.However,LSlarvaedidnotshowthissameup-regula:ondespitebeingexposedtoaconstantlowsalinityenvironment.Theselarvaehavebeeninthelowsalinityenvironmentforalongeramountof:me,andmighthaveadjustedtothisenvironment.

Previousresearchhasshownthatplanktotrophicechinodermlarvaecansensefoodbymechanicals:mula:onofthecilia(Hart&Strathmann1987)Thus,up-regula:onofmechanosensoryproteinsinFSlarvaecouldhelpthemsensechangesinfoodpar:clesinthewaterandosmo:cpressure.However,thelackofup-regulatedmechanosensoryproteinsintheLStreatedlarvaecouldindicatethattheselarvaemighthaveproblemssensingtheirenvironment,whichcouldcausethemtohavereducedfoodintakeandmakethemmorelikelytobekilledbypredators.Ontheotherhand,reducedexpressioncouldindicatethatLStreatedlarvaehaveadjustedtotheirsurroundingsandnolongerneedtoup-regulatemechanosensoryproteinexpressiontosuccessfullysensetheirenvironment.

FutureWork• WesternBlottoquanIfychangesinmechanosensoryandosmoregulatoryproteinexpression•  Immunolabelingtodeterminewhereinthebodytheseproteinsareexpressed•  LookathowP.ochraceusjuvenilesareimpactedbylarvaldevelopmentinalow-salinityenvironment

Figure3:Percentageoflarvaethatsurvivedeachtreatment.Datatakenat34daysaaerfourlowsalinityevents.Nosignificantdifferencesbetweentreatments(P>0.05)despitechangesinproteinexpressionandposterolateralarmlength.

•  PisasterochraceuslarvaedevelopinsurfacewatersoftheSalishSeafromMaythroughSeptember

•  Duringthis:me,freshwaterrunofffrommel:ngarc:ciceentersriversystems,andisul:matelydispelledintheSalishSea

ThereducedarmlengthsinLStreatedlarvaeindicatethatthelow-salinitytreatmentstuntedlarvalgrowthandmightaffectswimmingasshowninrecentstudyBashevkinetal.inpress.

inlarvalproteinexpression,includingproteinsinvolvedinosmoregula:on,metabolism,andmotorabili:es

determinehowmuchvolumeshouldbeusedtoloadgels•  LysatesampleswerefixedwithSDSsamplebufferandrunonBis-Trisgels

CiliatedbandinPisasterochraceuslarvae.Ifthisbandisdamaged,larvaemighthaveproblemssensingfood,thehalocline,orthesurfaceinthewatercolumn.

112 kDA 75 kDA

PisasterOchraceus(Echinodermata:Asteroidea)Larvae."JournalofPlanktonResearch34.7(2012):590-601.

•  Doesproteinexpressionvarybetweenlarvaerearedinnormalsalinity(30‰),fluctuaIngsalinity(21‰/30‰),andconstantlow-salinity(21‰)?

•  Iftherearedifferences,whichproteinsareup-anddown-regulatedinthegivenenvironments?

•  Dothesedifferentsalinitytreatmentsaffectposterolateralarmlength?•  Dothesedifferentsalinitytreatmentscausedifferencesinsurvival?

exposuretolowsalinityaffectsthever:caldistribu:onofPisasterochraceus(Echinodermata:Asteroidea)larvaeinhaloclines."MarineEcologyProgressSeries.