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TheBenefitandimportanceofKleptoplastytoSacoglossanSeaSlugs

CalebBeck,UndergraduateStudent,DalhousieUniversity

Abstract

Photosynthesiswasfirstobservedbeingperformedbysacoglossanslugsinthe1960’s.However, there isstill littleknownabout thecellularprocesses thatgivesacoglossanstheiruniquephotosyntheticability.Inthepastfewdecadestheliteraturewrittenaboutphotosynthesis in sacoglossanshasbeencontradictory regardingwhetherornot theyare able to photosynthesize at all, andwhether this is an example of horizontal genetransferbetweenthesacoglossansandtheiralgalfoodsource.Thegoalofthispaperisto analyze existing literature on, and determine whether sacoglossans benefit fromphotosynthesisandifitisanexampleofhorizontalgenetransfer.Thispaperexaminesliteratureonsixsacoglossanspeciesandtheimpactsphotosynthesishasonthem.Theseimpacts include; change inweight during starvation, survival rates, yield of offspring,and the influence of non-native algal food sources. Past sacoglossan research hasfocusedon threeor fewer species at a time.Lackof comparisonbetween species andlackofdatacompilationcausedthebenefitsofphotosynthesistosacoglossansnottobeapparent. The benefits of photosynthesis become clear after data compilation, whichshowsthatthereisvariationofphotosyntheticabilitiesbetweensacoglossanspecies.Inthose sacoglossans that can photosynthesize it has been shown that they maintainplastids from their algal food source and that their photosynthetic abilities are anexampleofhorizontalgenetransfer.

1.IntroductionSacoglossan(sap-suckingseaslugs)areuniqueorganismsintheanimalkingdombecausetheyaretheonlyanimalsabletoperformphotosynthesissimilartothatfoundinplants.Sacoglossanspeciesareabletotakeinplastidsfromtheiralgalfoodsourceandincorporatethemintotheirdigestivetract,whichallowsthemtoperformakindofphotosynthesiscalledkleptoplasty.Oncetheplastidisincorporatedintothedigestivetractitisreferredtoasakleptoplast.Researchersbelievethatsacoglossansabilitytomaintainfunctioningkleptoplastsisaresultofhorizontalgenetransferbetweenthesacoglossanandtheiralgalfoodsource,whichgivesthesacoglossantheabilitytoproducetheenzymesandproteinsessentialtomaintainthekleptoplast(Pierce2015).Horizontalgenetransferisthetransferofgenesbetweenorganismsofdifferentspeciesasopposedtogenesthatareinheritedthroughthereproductionofonespecies.Manyevolutionistsbelievethathorizontalgenetransferishowearlylifeformsevolvedonearth.HorizontalgenetransferinAchaeaandBacteriahasbeenobservedinmoderntimes(Stefano2015).However,itwasbelievedthateukaryoticorganismshadlosttheabilitytoperformgenetransferbetweenorganisms,untilthediscoveryofkleptoplastyinsacoglossans(Pierce2015).Understandinghowmorecomplexformsoflifeperformhorizontalgenetransfercanimpactallbranchesofbiology.Failuretostudythebenefitofkleptoplastycouldleadscientiststomissopportunitiestobetterunderstandearlyevolutionaryhistoryonearth.

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Abetterunderstandingofhorizontalgenetransferandtheprocessesrequiredforoneorganismtotakeonthecellularprocessesofanothercouldalsochangethefieldsofgeneticsandcellularbiology.Althoughthescientificcommunityhaslargelyacceptedkleptoplastysincethe1960s,recentstudieshavequestionedwhethersacoglossanspeciesbenefitfromorperformkleptoplastyatall.Therehavebeenmultiplestudiesondifferentsacoglossanspeciestoseehowtheslugsbenefitfromkleptoplasty.Mostofthesestudiesfoundthatkleptoplastydoesnothelpsacoglossanspeciesmaintainweightintimesofstarvationandthereforeconcludedthatallsacoglossanspecieseitherdonotbenefitfromordonotperformkleptoplastyatall(Gregor2013).However,thesestudiesusedsmallsamplesizeswithonlyafewsacoglossanandalgalspecies.Almostallofthestudiesbasedtheirconclusionsonthebenefitofkleptoplastyinregardstoweightgain/retentioninsteadoflookingatotherbenefitssuchassurvivalorreproductivesuccess.Differentsacoglossanspeciesareseparatedgeographicallysoitisoftenhardtostudymorethanonespeciesatatime.Eachspeciesalsofeedsonadifferentalgalspeciesfromtheirnativelocation.Ithasnotbeendeterminedwhetherthespeciesofalgaecouldhaveaninfluenceonkleptoplasty.Althoughtherehavebeenmanystudiesofhowkleptoplastyeffectsindividualsacoglossanspeciestherehavebeenveryfewinstanceswheretwoormorespeciesarecompared.Therehasalsoonlybeenonestudyonasacoglossanspeciesthatspansovermultiplegenerationsandfocusesonotherbenefitsofkleptoplastysuchasbenefitonreproductivesuccess(Schmitt2014).Thepurposeofthispaperistodeterminethebenefitkleptoplastyhasonsixsacoglossanspecies,discusswhysomeofthesesacoglossanspeciesarebetteratmaintainingkleptoplaststhanothers,andhighlightareasoffutureresearch.Thiswillbedonebyreviewingthemethods,data,andconclusionsusedtoresearchtheeffectkleptoplastyhasonsurvival,weightretention,andreproductivesuccessofsixsacoglossanspecies.2.E.timida,P.ocellatus&E.trisunatagrownunderdifferentlightconditionsduringstarvationSpecimensofthesacoglossanspeciesE.timida,P.ocellatus,andE.trisunatawerecollectedforexperimentsthatmeasuredweightchangeduringtimesofstarvationunderdifferentlevelsoflight(Gregor2013&Yamamoto2012).E.timidaandP.ocellatusspecimenswerestarvedfor50dayswithtwoslugsinhigh,medium,andlowlightenvironmentsforbothspecies.Aftera50-dayperiodallspecimenshaddeclinedinweightregardlessoftheamountoflighttheyreceived(Gregor2013).However,thespecimensleftinthedarkexperiencedamorelineardescentinmasswhilethespecimenskeptinlightenvironmentsmaintainedtheirweightforfivedaysbeforeasuddendropandthenlosingmasslinearlysimilartothespecimenskeptinthedark(Gregor2013).

Gregorandassociatesconcludedthatkleptoplastsmustbeastoredfoodsourceratherthanasourceofkleptoplastyinthesespeciesbecauseallspecimenslostmassregardlessoftheirlightenvironment.Thisexperimentalsofoundthatwhenslugswerekeptinthedarktheyfixed87%lessCO2thanwhenleftinlightconditions(Gregor2013).

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A21-dayexperimentwasdoneonE.trisunataspecimensthatwerestarvedandkeptinthelightordarkconditions(Yamamoto2012).SimilartotheexperimentbyGregor(2013)thisexperimentshowedindividualsinthelightmaintainingtheirweightforsevendaysbeforearapiddropinmassfollowedbylineardescentsimilartoindividualskeptinthedark(Yamamoto2012).ThisresearchconcludedthatsinceE.trisunatalostmassregardlessoftheamountoflightitreceiveditdoesnotbenefitfromkleptoplasty.Althoughspecimensunderbothlightanddarkconditionsexperiencedsimilarpatternsofweightlossthesacoglossansthatwerekeptinthedarkexperiencedtheirsuddenweightdropfasterthanthoseinlightedconditions.Thissuggeststhatthesacoglossansthatwerekeptinlitenvironmentsmaintainedtheirmassbecauseoftheirkleptoplasts.WhencomparedtoothersacoglossanspecieslikeE.chloroticathatcanmaintainweightfor4monthsduringstarvationitmayseemthatE.timida,P.ocellatus,andE.trisunatadonotsharetheabilitytoperformkleptoplasty(Green2000).However,thelongevityofkleptoplastythatcanbeperformedduringstarvationandthesuddenweightlossinthesespecieswhencomparedtoE.chloroticacanbeexplainedbynaturalvariationbetweenspecies.3.SurvivalratesofE.atroviridisandE.trisinuatainlightanddarkconditionswhenfedorstarvedAkimoto(2014)comparedspecimensofbothE.atroviridisandE.trisinuataunderlightanddarkconditionsduringtimesofstarvationandnon-starvationovera16-dayperiodwithdifferentalgalfoodsources.Thisresearchfoundthatsurvivalratewasupto40%higherinbothspecieswhenfedandkeptinlightconditions,andspecimensthatwerestarvedhadsimilarsurvivalrateregardlessofthelightconditiontheywerekeptin(Akimoto2014).Overthe16dayperiodbothspecimensthatwerefedinthedarklostupto50%oftheirweightwhencomparedtospecimensthatwerefedinlightenvironments(Akimoto2014).Therewasalsonostatisticaldifferencebetweenanyofthegroupswhowerefeddifferentfoodsources.ThisexperimentconcludedthatthepresenceoflighthadpositiveeffectsonsurvivalandweightmaintenanceofbothE.trisinuataandE.atrovirdis.Thisexperimentshowsthatkleptoplastyisrequiredtomaintainsacoglossans.Sacoglossansdependequallyonalgaeandlighttosustainthem.IfkleptoplastswereonlyastoredfoodsourceassuggestedbyGregoretal(2013)thesacoglossanswouldnotrequirelighttobenefitfromthekleptoplasts.Instead,thesacoglossansrequirelightbecauselightisrequiredforkleptoplaststoperformkleptoplasty.Itisjustasbadforsacoglossanstobefedandkeptinthedarkasitisforthemtobestarvedandkeptinthelight(Akimoto2014).4.E.clarkichlorophyllconcentrationduringstarvationEvery2weekssamplesfromstarvedE.clarkispecimenswentthroughchromatography,whichisaprocessthatuseslightwavestodetectsubstanceconcentration.TheconcentrationsofchlorophyllAandBweretestedevery2weeksovera14-weekperiod.ThisresearchshowedthatE.clarkiretainedproductionofbothchlorophyllAandBfortwo

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weeksbeforeasignificantdrop.Afterthisdropchlorophyllproductionsloweduntiltherewasnochlorophyllproductionbyweek14(Middlebrooks2012).MiddlebrooksandassociatesconcludedthatE.clarkiisabletomaintainchloroplastsforlongerthanothersacoglossanspecies,andthatchlorophyllproductionwouldnothavestoppediftheE.clarkihadaconstantsourceofalgaetoeatandperformhorizontalgenetransferwith(Middlebrooks2012).TheresearchbyMiddlebrooksetal(2012)showsthataconstantintakeofalgaeisneededtomaintainfunctioningkleptoplasts.Duringstarvationsacoglossansaredeprivedoftheenzymesandproteinsfromtheiralgalfoodsourcethatarerequiredforkleptoplastmaintenance.WithoutalgaetheproductionofchlorophyllAandBismaintainedforashortamountoftimebeforeasuddendrop.AlthoughtheamountoftimevariesbetweenspeciesthesuddendropinchlorophyllproductionIssimilartothesacoglossanssuddenlossofweightintheresearchdonebyGregoretal(2013)andYamamotoetal(2012).5.E.chloroticaandhorizontalgenetransferAnine-monthstudylookedattherelationshipbetweenE.chloroticasabilitytomaintainkleptoplastyanditsalgalfoodsourceV.litorea.JuvenileE.chloroticaspecimenswerekeptinalaboratorycultureandfedV.litoreafor1month,whichwasthenfollowedbyan8-monthstarvationperioduntiltheyreachedmaturity.EverymonthaspecimenwasfrozenandsamplesfromthesespecimenswereusedtoperformDNAspectroscopy.TheDNAspectroscopyfoundthatjuvenileE.chloroticasharednosimilargeneticsequenceswithV.litoreaandthereforedidnotcontainthegenesrequiredtoproducetheenzymesorproteinsrequiredtomaintainkleptoplasts.However,afterthejuvenileE.chloroticafedonV.litoreaforamonththetwospeciesshowedsimilarDNAsequences.AstheE.chloroticaateV.litoreatheygainedtheDNAsequencesforkleptoplastmaintenancethroughhorizontalgenetransfer(Green2000).HorizontalgenetransferallowedE.chloroticatomaintainfullyfunctionalkleptoplastsfor4monthsandpartiallyfunctioningkleptoplastsforupto9months.AfterninemonthstherewasmassmortalityintheE.chloroticasuggestingthattheylosttheirabilitytomaintaintheirkleptoplasts.TheconclusionofthisstudywasthatgenesweretransferredfromV.litoreatoE.chloroticaafteringestionandthatiswhyE.chloroticaisabletomaintainfunctioningkleptoplastsovera9-monthperiod(Green2000).Thisresearchshowsthataconstantsourceofalgaeisrequiredforhorizontalgenetransfer,whichisusedtomaintainkleptoplasts.AlthroughE.chloroticacanmaintainkleptoplastsformanymonthsduetohorizontalgenetransferwithV.litoreaothersacoglossanssuchasE.timida,P.ocellatus,andE.trisunatamaintainkleptoplastsforshorteramountsoftime.Thiscouldbeduetospeciesvariation,orthedifferenceinnaturalfoodsources.6.ReproductivesuccessofE.timidaduetodifferentfoodsourcesOver250daysSchmittetalraisedthreegenerationsofE.timidathatwereseparatedintofourgroupswithdifferentfeedingconditions.Figure1outlinesthefulllifecycleofonegenerationofE.timidafeedingontheirnaturalfoodsource,A.acetabulum.Thesecond

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groupofE.timidawasstarved,andthelasttwogroupsfedonV.litoreaandC.verticillatarespectively.V.litoreaandC.verticillatawerechosenbecausetheyarethenaturalfoodsourceofE.chloroticaandE.clarkiwhomaintainfunctionalkleptoplastsfrom14weeksupto9months,whichislongerthanmostsacoglossanspecies(Schmitt2014).AlthoughV.litoreaandC.verticillataareresponsibleforincreasedkleptoplastfunctioninothersacoglossanspeciesitwasfoundthatE.timidathatwerefedA.acetabulumasjuvenileshadahighersurvivalratewhencomparedtoindividualsthatfedontheirnon-nativefoodsource(Schmitt2014).WhengiventhechoicebetweentwoalgalspeciesjuvenileE.timidafedonA.acetabulum.OverthreegenerationstheE.timidawhofedoffofA.acetabulumhadfasterratesofmaturation,highersurvivalratesduringstarvation,andanincreaseinoffspringyieldfromeggs(Schmitt2014).ThehigheroverallhealthofE.timidawhenfedonA.acetabulumshowsthatthealgalfoodsourcehasaneffectonthesurvivalofthesacoglossansandcouldimpactthelongevityofkleptoplastmaintenanceandthereforekleptoplasty.AlthoughE.timidashowedmanypositivebenefitsfromfeedingonA.acetabulum,therewasnonoticeablechangeinthelifespanofE.timidaasaresultoffoodsourceexceptforduringstarvation(Schmitt2014).Schmittandcolleaguesconcludedthatalthoughfoodsourcedoesnotimpactthelifespan,E.timidadoesbenefitfromperformingkleptoplasty.Thesebenefitsincludebutarenotlimitedto;increasedreproductivesuccess,survivalduringtimeswithlowfoodavailability,andfastermaturation.

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7.ConclusionAftercompilingandcomparingdifferentstudiesitisclearthatdeterminingthebenefitthatkleptoplastyhasonthesacoglossanspeciesreviewedinthispaperrequiresamoreindepthunderstandingofeachspeciesabilitytomaintainkleptoplasts.Initiallysacoglossanweightlossduringstarvationregardlessoflightconditionsmakesitdifficulttounderstandhowsacoglossansbenefitfromkleptoplasty.Althoughthereisvariabilitybetweentheamountsoftimeeachspeciesisabletomaintainkleptoplastsitislikelythateachspeciesdecreaseinsurvivalrateduringtimesofstarvationcanbeattributedtothelossoftheiralgalfood-source.Withoutthealgae,whicharetheirsourceofhorizontalgenetransfer,sacoglossanscannotmaintainkleptoplastsand

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thereforelosetheirabilitytoperformkleptoplasty(Middlebrooks2012,Akinmoto2014,Gregor2013,Yamamoto2012,Green2000). Sacoglossanspeciesthatareabletomaintainfunctionalkleptoplastsforlongperiodsoftimeproduceessentialproteinsandenzymesforkleptoplastmaintenance,whichmakeshorizontalgenetransferanessentialaspectofkleptoplasty(Green2000).Althoughrequiringfurtherresearchitappearsthatalgalspecieshasaneffectonthelongevityofkleptoplastyaswellasimpactingdevelopmentandyieldofoffspring(Schmitt2014).Whenstudiedindividuallythebenefitofkleptoplastyonsacoglossansisnotimmediatelyapparentduetothevariationintheamountoftimethesacoglossanscanmaintainfunctionalkleptoplasts.However,whentheresultsfromeachstudyarecompileditisclearthat;horizontalgenetransferisrequiredtomaintainkleptoplasts(Pierce2015),differentspeciesarebetteratperforminghorizontalgenetransferresultingingreater/lesserabilitytomaintainkleptoplastsindifferentspecies(Green2000),thereisadecreaseinsurvivalrateswhensacoglossansarepreventedfromacquiringkleptoplasts(Middlebrooks2012),andthereisapositiveinfluenceonreproductivesuccesswhensacoglossanshavekleptoplastsandareperformingkleptoplasty(Schmitt2014).Inconclusionkleptoplastyisbeneficialtosacoglossanspeciessurvival,andrequireshorizontalgenetransfer.TobetterunderstandthephenomenonofkleptoplastyandhorizontalgenetransferfuturestudiesshouldfocusonthegeneticdifferencesbetweenspeciessuchasE.chlorotica,whichcanmaintainkleptoplastsformonths(Green2000),andE.trisinuata,whichcanmaintainkleptoplastsforonlyafewdays(Yamamoto2012).Pairingupsacoglossanspecieswithnon-nativealgalfoodsourcescouldalsohelpresearchersunderstandwhysomespeciesareabletomaintainchloroplastsforsomuchlongerthanothers(Schmitt2014).Understandingwhatgeneticallymakesonespeciesmoresuccessfulinperforminghorizontalgenetransferastomaintainproductionofessentialproteinsandenzymesformaintenanceofforeignorganellescouldgiveresearchersabetterunderstandingofcellularprocess,genetics,andtheearlyevolutionarytree.Researchersfocusingonecologyshouldtendawayfrommeasuringtheweightchangeofsacoglossansinregardtothebenefitthatkleptoplastyhasandshouldinsteadlookintothesocialbehaviorsthatkleptoplastyaffectsaswellasreproductivesuccess.Astudyofthebenefitsofkleptoplastywouldalsobeimprovedbyconductingexperimentsinthefieldratherthaninthelaboratory.Researchfocusingonhowkleptoplastyaffectssacoglossansinteractionwithotherorganismsintheirecosystemcouldprovidemoreevidenceoftheevolutionarybenefitsthatkleptoplastyandhorizontalgenetransferhaveonsurvival.ReferencesAkimotoA,HiranoYM,SakaiA,YusaY.2014Relativeimportanceandinteractiveeffectsof

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