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Functionaldiversityofthelateralline

systemamongpopulationsoftheWestern

Rainbowfish(Melaenoteniaaustralis)

LindseySpiller(BScHons)

NeuroecologyGroup,SchoolofAnimalBiology

ThisthesisispresentedforthedegreeofMastersofPhilosophyattheUniversityofWesternAustralia.

2016

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Summary

Sensorysystemsarevitalforanyorganisminordertoreceiveandrespondtorelevantinformationabouttheimmediateenvironment.Theabilitytoreceiveinformationviathesensescontributestofitnessrelatedbehavioursandthereforeananimal’sabilitytosurvive.Themechanosensorylaterallinesystemisauniquesensorymodalityinfishesandsomeamphibiansandisanadaptationtoaquaticenvironments.Therelationshipbetweenthemorphologyofaspecies’laterallinesystemandhabitatcharacteristics,especiallywaterflow,hasbeenextensivelyinvestigatedforarangeoffreshwaterandmarinespecies;however,therehavebeenveryfewstudiesconductedinAustralia.Mostofthesestudieshavefocussedonthediversityofthelaterallinesystemamongspeciesfromdivergenthabitattypesratherthancomparingthesamespeciesacrosshabitats.Afocusonasinglespeciesisimportantbecauseitallowsustomoreclearlydeterminethatanydifferencesthatarefoundarelikelytobeduetoexternalfactors.Thisstudyinvestigatedthemorphologyofthelaterallinesystemofafreshwaterfish,thewesternrainbowfish(Melanotaeniaaustralis),collectedfromeighthabitatsoftheinlandPilbararegionofnorthwestAustralia.Usingfluorescencelabelling,thesuperficialneuromastsystemwasmappedandthenumberandarrangementofsuperficialneuromastswasfoundtovarysignificantlyacrosspopulations,withthenumberofsuperficialneuromastsdecreasingwithincreasedratesofwaterflowunderfieldconditions.

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Thestudyalsoinvestigatedhowintraspecificvariationinthesuperficialneuromastsaffectstheabilityofthisspeciestoperformrheotaxis(orientationintowaterflows).Arheotacticstudywasdesignedtodetermineifdifferencesinthesuperficialneuromastarrangementhadaneffectontheirabilitytocorrectlyorientateatdifferingflowspeeds.Chemicalablationwasalsousedtodeterminetheroleofthelaterallineinthisbehaviour.Differencesintheabundanceofsuperficialneuromastsaffectedtheabilitytoorientateintowaterflowsandrainbowfishesshowedareducedabilitytoorientatewithouttheuseofafunctionallateralline.Furtherstudyisrequiredtofullyunderstandwhetherindividualpopulationshavethecapacitytoadapttheirlaterallinesystemtoalteredhydrologicalconditions.

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TableofContentsSummary............................................................................................................................2Acknowledgements........................................................................................................6DeclarationofCanditurecontribution...................................................................9ChapterOne.Generalintroduction.......................................................................10Aimsofthisstudy.................................................................................................................18

Chapter2.Theebbandflowofthewesternrainbowfish(Melanotaeniaaustralis):howlaterallinesystemdiversityrelatestohabitatvariability.............................................................................................................................................20Abstract....................................................................................................................................20Introduction...........................................................................................................................21Materialandmethods.........................................................................................................26Habitatcharacterisation..............................................................................................................28Fishsampling....................................................................................................................................31Neuromastcharacterisation.......................................................................................................32Scanningelectronmicroscopy(SEM).....................................................................................35Dataanalyses....................................................................................................................................36

Results......................................................................................................................................38Thelaterallinesystemofthewesternrainbowfish.........................................................38Neuromastabundanceinrelationtobodylength,populationandsex...................45Therelationshipbetweensuperficialneuromastsandwaterflow...........................46Variationincomplexityofrainbowfishhabitats...............................................................47

Discussion...............................................................................................................................48Canalsystemofthewesternrainbowfish............................................................................49Abundanceofsuperficialneuromastsofwesternrainbowfishinrelationtoflow................................................................................................................................................................52Possiblecausesofvariationinsuperficialneuromastabundance.............................55

Conclusions.............................................................................................................................56Chapter3.Laterallinemorphologyandhabitatorigindeterminerheotaxicabilitiesinthewesternrainbowfish(Melanotaeniaaustralis)57Abstract....................................................................................................................................57Introduction...........................................................................................................................58Materialsandmethods.......................................................................................................61Fishsamplingandhusbandry....................................................................................................61ExperimentalSetup........................................................................................................................64Neuromastvisualisation..............................................................................................................67Experimentalconditions..............................................................................................................68Imageanalysis..................................................................................................................................69Statisticalanalysis..........................................................................................................................70

Results......................................................................................................................................70Effectofneomycinandflowonfishorientation................................................................70Effectofhabitatorigin..................................................................................................................75

Discussion...............................................................................................................................78Effectofwaterflowonrheotaxis.............................................................................................79Theroleofthelaterallineinmediatingrheotaxis............................................................80Effectoffishhabitatoriginonrheotaxis...............................................................................84

Conclusions.............................................................................................................................87Chapter4.Generaldiscussion.................................................................................88

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Introduction...........................................................................................................................88Variationinthemorphologyofthelaterallinesystemofthewesternrainbowfish.............................................................................................................................89RoleofthelaterallinesystemofM.australisinrheotaxis....................................90Limitationsofmyresearchproject................................................................................94Concludingcommentsandfutureresearch................................................................94

References......................................................................................................................97Appendices...................................................................................................................104Appendix1:...........................................................................................................................104

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Acknowledgements

ThisresearchwassupportedbytheAustralianResearchCouncilincollaborationwithRioTintoandBHPBilliton(ARC‐LP120200002).AspecialthankyoutoSamLuccittiandSuziWild(RioTinto)forfacilitatingaccesstoWeeliWolliCreekandtheUpperFortescueCatchmentandforfeedbackduringtheprojectmeetings.Firstandforemost,Iwouldliketoacknowledgemysupervisorsfortheirincrediblesupportandguidancethroughoutmyresearch.IthankShaunCollin,PaulineGrierson,JenniferKelleyandJanHemmifortheirconstantcommitment,encouragement,supportanddedicationtomeandmythesis.TheirhelphasshapedmeintothescientistIamtoday.IacknowledgeJenniferKelleyforherconstantaccessibility,willingnesstohelpwithanythingthatIneededatanypointandalsoherguidanceonthefieldtriptothePilbara.Youweresoexceptionallypresentateverymomentthroughoutmythesis.IacknowledgeShaunCollinforhisparticularlyupbeatandpositiveattitude,especiallywhenIwasloosingmotivation,paramountguidanceandincredibleknowledgethroughoutmyproject.IacknowledgePaulineGriersonforbeinganincrediblerolemodel,supportsystemandhavingthewillingnesstohelpatanypoint.Shewasalsoanincredibleemotionalsupportthroughoutthistime.AspecialacknowledgementgoestoJanHemmi.Hisguidanceandassistanceduringthesecondchapterwasunwavering,despitenotbeinganofficialsupervisortobeginwith.Hisdedication,knowledge,understandingandhelpfulnesstohisstudentsandmyself,wasabsolutelyastonishing.Without

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him,IwouldnotbeinthepositionIamintodayandIowethecompletionofmysecondchaptertohim.IwouldliketoacknowledgethreeincredibletechniciansatUWAfortheirassistanceandguidanceaswell.CameronDuggin,CarlSchmidtandRickRoberts.Withouttheirassistanceandwillingnesstohelpcreateandfixmicroscopesandflumes,myresearchwouldhavebeenconsiderablyinhibited.AspecialmentiontoCarlSchmidtforhishandymanskills,supportandknowledgewithhelpingmecreatetheperfectarenaformysecondchapter.ThankstoSamanthaLostromforherhelpandadviceonthefieldtripinApril2014.AndreSiebersandJordanIlesalsoprovidedadditionalsitedataandassistedwithidentificationofinvertebrates.Onamorepersonalnote,IwouldliketoacknowledgemyfriendsandfamilywhohavesupportedmeoverthelastfewyearsandwithoutwhomIwouldnothavebeenabletoachievecompletion.Thankyoutomyfellowscientists,AdelaideBevilaquaandPennyBrooshooftforbeingsoundingboardswheneverIneededitandtoTanyaHevroyforprovidingadviceandguidancefromthebeginning,despitethevastdistancebetweenus.ThankyoutomyparentsRoslynandGeofffortryingsoeagerlytounderstandthetopicofmythesisandforprovidingsupportduringthehardtimes.AspecialthankyoutomysisterandbossJacquieSpiller,whowhenneededwasmorethanwillingtofindmeextratimeoff,orcoverformeintheworkplacewheneverIhadtomakethisthesismoreofapriority.

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LastbutnotleastIwouldliketothankmyincrediblysupportingpartnerJean‐SebastianCorreiaandhisverykindparentsHelenandCarlos.Carlosthankyouforyouconstantadviceontheacademicenvironment.Jean,youhavebeenmybiggestenthusiastandhavealwaysprovidedmewithlove,supportandempathy.YouhavealsoalwaysbeensoincrediblygenerouswithyourtimeandwillingtoassistmeinanythingthatIneeded.HekeptmepositiveanddriventhroughoutthisentireprocessandIwillbeeternallygrateful.

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DeclarationofCandidaturecontribution

ThisthesisdoesnotcontainworkthatIhavepublishedorworkcurrentlyunderreviewforpublication.

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ChapterOne.Generalintroduction

Sensory systems fundamentally link species to their environments, yet therehave been few studies that consider the ecological resilience of sensorysystemstoenvironmentalchange.Thisisparticularlythecaseforfreshwaterhabitatsglobally,asthesearedegradingatanalarmingrateandmorerapidlythan any other ecosystem (Kingsford, 2011). Consequently, greaterunderstanding of the responses of any given species to altered habitatconditions is necessary to determine the likelihood of its survival and hasbecomeatopicofgreatinterestforecologistsglobally.Survivalinachangingenvironmentdependsonaspecies’abilitytoadaptkeyfitness traits through phenotypic plasticity, contemporary evolution or acombination of both of these processes (Palkovacs, Kinnison et al. 2012).Whileithasbeenshownthathumanactivities,suchasthedammingofrivers,cancausechanges inmorphological,physiologicalandbehaviouraltraitsofarangeofspecies(Palkovacs,Kinnisonetal.2012),itisunclearhowthesetraitchanges affect species survivorship in the long term. It has only recentlyemergedthatsensorysystemscanvaryamongindividualsofthesamespecies(Wark & Piechel, 2010). If such within‐species variation is evident infreshwater fishes then a study of a single species across a diverse range ofhabitats couldprovideauniqueopportunity tounderstand the linkbetweenenvironmentalvariationandsensorytraitspecialisations.

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Inordertosurviveinaquaticenvironments,fishesandsomeamphibianshaveevolvedauniquesensoryorgancalledthemechanosensorylaterallinesystem(hereafterreferredtoasthe‘laterallinesystem’),whichallowsthemtodetectandreacttoevenminutechangesinwatermovementwithintheirimmediateenvironment(Dijkgraaf,1963).Thelaterallinesystemishighlyspecialisedtosuit theneeds of the animal in its particular habitat (Eros. etal. 2003). Forexample,predationpressure(McHenryetal.2009),developmentalconditions,micro‐habitat(Beckmannetal.2010,Vanderphametal.2013)andwaterflowspeeds(Wark&Piechel,2010)areallconsideredpotentialcausesofvariationinthelateral linesystembothwithinandamongfishspecies.Thelateral linesystem allows fishes to detect prey, avoid predators (Montgomery andMacdonald1987),communicateandshoalwithconspecifics(Partridge1980),as well as discriminate objects and orientate into water flows (known asrheotaxis) (Montgomery 1997, Baker and Montgomery 1999, Bleckmann2008).Therefore,thelaterallinesystemisthebasisofmanykeysurvivaltraitsinfishes(Engelmann,Hankeetal.2002,Bleckmann2009).The lateral line system is comprised of a series of bundles of hair cells(neuromasts)thatextendovertheheadandalongthelateralflankofthetrunkandbody (Fig.1;Webb1989,CartonandMontgomery2004,Wellenreuther,Brock et al. 2010). These bundles of hair cells may occur within pored orunpored water‐filled canals or sit on the superficial surface of the skin orscales(Bleckmann2009).Therearetwodistincttypesofspecialisedreceptorcells; superficial and canal neuromasts. Superficial neuromasts are arrangedonthesurfaceoftheskin(CartonandMontgomery2004)andaremostlyused

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todeterminethevelocityofthesurroundingwater(WarkandPeichel2010).Superficialneuromastsalsofacilitaterheotaxis(thebehaviouralorientationtowatercurrents)asthesecellsareconstantlystimulatedbywaterflow(Bakerand Montgomery 1999). Canal neuromasts usually occur in a distinct linebeneath the skin and are used for both the detection and discrimination ofobjects (Engelmann, Hanke et al. 2002). Typically, one canal neuromast isfound between two pores (Engelmann, Hanke et al. 2002). Visually‐compromisedspecies,suchastheblindMexicancavefish,(Astyanaxfasciatus)tend to have a particularly well‐developed canal and superficial system, asthey rely heavily on the lateral line sense to navigate within their darkenvironment(Windsor,Tanetal.2008,Delfinn2011).Each neuromast comprises a specialised region of epithelium fromwhich abundleofhaircellsprotrude.Thehaircellbundlecontainsonelongkinociliumandanumberofsmallerstereocilia,whichallextendintoagelatinouscupula.After theonsetofahydrodynamicevent, thecupulamoveswithin thewaterfilled canal but out of phasewith the rest of the body. The cupula (and theembedded hair cells) adopt a range of orientations but reveal a specificpolarity, whereby stimulation of the stereocilia in one direction (in thedirectionofthekinocilium)willelicitexcitation(andtherebydepolarisation),whilst stimulation in the other direction (in the direction away from thekinocilium)willelicitinhibition(andtherebyhyperpolarisation)(Engelmann,Hanke et al. 2002, Schmitz, Bleckmann et al. 2008). While the lateral linesystem has been well studied in terms of differences in structure andmorphologyacrossspecies(Wonsettler&Webb.1997,Rouse&Pickles.1991,

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Vischer, 2013), very few studies have investigated the abundance anddistributionofneuromastswithin a species, especiallya speciesoccupyingadiversityofhabitats.The function of the lateral line system and its link to water flow has beendescribed in varyingdetail throughout the years, for example Shulze (1870)wasthefirsttosuggestthelaterallinefunctionsasaflowsensor.Howeverin1963Dijkgraafproducedapioneeringstudy,“Thefunctioningandsignificanceofthelaterallinesystem”whichsolidifiedthelinkbetweenthelaterallineandflow. Since then, many studies have directly examined the relationshipbetweenthehydrodynamicenvironmentandlaterallinemorphologyandhavefoundthatlaterallinemorphologyisanadaptiontothehydrodynamicnoiseofaspecies’environment.Typically,relativelysedentaryfishesthatinhabitquietorstillenvironments,suchaslargelakesordams,wouldpossesswidecanalsor even have lost the canal structures with prolific numbers of superficialneuromasts (Beckmann, Eros, 2010). On the other hand, fishes that live in“noisy”or fast flowingenvironments, suchas streamsor rivers, are likely tohave a well developed canal system with low numbers of superficialneuromasts(Englemann,Hankeetal.2002,Beckmann,Erősetal.2010).Most studies to date have compared lateral line system diversity amongdifferentspeciesoffishes(Englemannetal.,2002,Beckmann,Erősetal.,2010,Vanderpham et al., 2013, Vischer, 1990) or have investigated lateral linesystem variation in a single species that occupies divergent habitats (WarkPiechel,2010).Forexample,in2010,WarkandPeichelconductedathorough

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study of threespine sticklebacks (Gasterosteus aculeatus), comparing thedistribution, typeand totalnumberofneuromasts found in individuals fromlake,streamandmarinepopulations.Theyfoundasignificantvariationinthetotal number of superficial neuromasts present on the body among thedifferentpopulations(marineandfreshwater),andattributedthesefindingstothewater flow characteristics found in these different habitat types. Similarfindingswere reported by Trokovich et al.(2011),who found differences inthemorphologyand the totalnumberof superficialneuromasts inninespinesticklebacks (Pungitius pungitius). However, these authors attributed theirfindingstogeneticinfluencesarisingfromhabitatdivergence(Trokovichetal.,2011). A further study by Fischer et al. (2013) on the Trinidadian guppy(Poeciliareticulata)alsoreportedhabitat‐relateddivergenceinthesuperficialneuromast system, but, unlike the two aforementioned studies, the authorswere able to attribute the observed intraspecific variation to variation inpredation pressure. Collectively, these studies suggest that the lateral linesystemishighlyspecialisedfordetectingandrespondingtovariationinwaterflowsandpredationpressure. Indeed,examiningvariation inthe lateral linesystemofasinglespeciesthatoccupiesacontinuumofhabitatsisapowerfulwayofidentifyingthesesensorysystemspecialisations.It is clear that there are many reasonable ecological explanations as to thepressures that promote intraspecific variation in the lateral line system.However,nostudyhascomparedalloftheputativeenvironmentalfactorsthataffectfishbehaviourinrelationtothefunctionalattributesoftheirlaterallinesystem in a single cohesive study. To better understand the functional

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significanceofthelateralline,itisimportanttounderstanditsinvolvementineveryday behaviours. One of the basic roles of the lateral line system isrheotaxis, the natural orientation of an animal’s body by swimming directlyintotheflow(Dijkgraaf,1963).Thisbehaviourisimportantforfishspeciesasit allows individuals to remain stationary in an otherwise dynamicenvironment.Rheotaxisisusedtoreceiveinformationfromupstreamthroughfood and olfactory signals being carried by the flow (Baker &Montgomery,1999,Montgomeryetal.,1995)andisalsousedinmigratorybehaviour.Fishestend to show stronger rheotaxis in faster flowing water than in no flowenvironments(VanTrump&McHenry,2013).Dijkgraaf(1963)wasthefirsttodemonstratethelateralline’sinvolvementinrheotaxis,althoughsomestudieshaverefutedthis.Forexample,VanTrumpandMcHenry(2013)conductedastudywiththeMexicanblindcavefish(Astyanaxfasciatus)andfoundthattheanimals were able to orientate correctly with and without their lateral linechemicallyblocked,suggestingthatthelaterallineisnotinvolvedinrheotaxisin this species. Similarly, Bak‐Coleman et al. (2013; 2014) suggests that thelateral line is either not involved in rheotaxis or only involved in sedentaryspecies.Otherstudieshavesuggestedthatthelaterallineworksinconjunctionwithothersenses,suchasvisionandthevestibularandolfactorysenses(Lyon,1904,Dijkgraaf,1963,Arnold,1974). Thesesomewhatcontradictorystudiesdemonstrate that the function of the lateral line system in relation to flowconditionsremainsunclear, inpart,becauseofthelimitednumberofspeciesandenvironmentsstudied.

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Asmany freshwater habitats worldwide are now exposed to anthropogenicinfluences such as climate change, it is important to understand howpopulations will respond to altered water flows, dependent on theirpopulation‐specific thresholds. Populations from either naturally fast ornaturallyslowflowingriversandstreamswilllikelyvaryintheirresilienceinresponsetoachangeinflowregime.Similarly,fishpopulationsfrommoreorlessvariableecosystems(e.g.perennialversusmoreintermittentrivers)mayalsorevealdifferencesintheresilienceoftheirlaterallinesystemtochangeorthe propensity for plasticity. For example, fishes that inhabit large bodies ofstillwater,suchaspondsordams thathaveneverbeenexposedtodynamicflows,maynotbeabletoadequatelyperformrheotaxisifconditionssuddenlychange to fast flows, and the reverse may be found in individuals fromdynamicenvironments.In Australia, negative anthropogenic impacts on aquatic ecosystems fromalteredwaterflowsareexpectedtobeexacerbatedbytheprojectedeffectsofclimate change (Kingsford 2011). Arid and semi‐arid lands are especiallyvulnerable owing to an expected increase in the intensity of flood eventsseparated by prolonged droughts and thus reduced surface flows (Parry,Canzianietal.,2007).Againstthisbackgroundofextremeclimaticvariability,in regions such as the Pilbara of northwest Australia, dewatering inmining(which can cause both localised drawdown but also increased flow due todischarge)cansignificantlyalterstreamflowsandconnectivityofpoolswithinindividualstreams. Incontrast,streamsmaybecome"overpowered" fromanenergetic perspective, when they are transformed from a shallow, slow

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flowing stream to a large and sustained fast flowing body of water; theseconditionsareunlikely tohavepreviouslybeenencounteredbymanynativestreamfishesduringtheirevolutionaryhistory(Baxter,1977).Whilesomefishspeciesareknowntoberesilient to theseenvironmentalvariations (Stewart2013), therehavebeennostudiesofAustralian inlandecosystems thathavetestedfordetrimentaleffectsofalteredflowsonfish andconsequentlytheirbehavioural responses. Knowledge of the fundamental physiological,morphological and behavioural adaptations of fishes to variations in waterquality and water flows underpins the development of "best practice" formanagingstreamsandriversinthefaceofincreasinghydrologicdisturbance.Theexperimentspresentedinthisthesisfocusoninvestigatingthelaterallinesystem of the western rainbowfish, Melanotaenia australis (FamilyMelanotaeniidae),afreshwaterspeciesendemictonorthwestAustralia.Theirdistribution ranges from the Pilbara region of Western Australia to theNorthernTerritory(Allenetal.,2002).Westernrainbowfishesinhabitawiderange of environments such as pools, creeks and lakes (Kelley et al., 2012),wheretheyareoftenfoundnearthewater’sedgenearvegetation.Despitethishighly dynamic and heterogeneous environment, M. australis form a largeproportion of the local fish communities of the Pilbara and are commonlylocatedinunstablepools,whicharehighlyvariableinflowvelocity,depthandotherattributesbothwithinandamongyears(Morgan&Gill,2004,Beesley&Prince,2010).M.australisexhibitsconsiderablegeographicvariation inbodyshapemorphology (Lostromet al., 2015), and this suggests that their laterallinesystemissimilarlyvariableamonghabitats.Thelaterallinesystemofthe

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western rainbowfish and its role in behaviour has not been investigatedpreviously.

Aimsofthisstudy

Thisthesisdescribesthemorphologyofthelateral linesystemofM.australisand determines whether population differences in the number andarrangementofneuromastsarerelatedtotheenvironmentalcharacteristicsofthe habitat. I also investigate the consequences of intraspecific lateral linediversity on one of the most important and innate fish behaviours i.e.rheotaxis.In this first introductory chapter, I have provided a conceptual basis formythesis.TheexperimentalcomponentofthisresearchisdescribedinChapters2and 3. These Chapters are formatted as "stand alone"manuscripts so somerepetitionisunavoidable.Chapter2characterisesthelaterallinesystemofM.australis.Specifically,Isoughttorevealthemostimportantfactorsinfluencingsensorysystemspecialisationbyinvestigatingwhetherpopulationdifferencesin thearrangementandnumberofneuromastsare related toenvironmentalvariables such aswater flow, predationpressure, prey availability, turbidity,pHandhabitatcomplexity.Lateral linemorphology isdescribed for155wild fishescaptured fromeightpopulations from the Fortescue River in the Pilbara, Western Australia.Neuromastsarevisualisedusingafluorescentvitaldyetomapthelateralline

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system and compare the number and arrangement of neuromasts amongpopulations.Chapter 3 investigates the ability of M. australis to perform rheotaxis atdifferentwaterflowspeedsinalaboratorysetting.Itestedtheextenttowhichthis species relies on the lateral line to perform rheotaxis by chemicallyblocking thesuperficialneuromastsusingneomycinsulphate. Ialsoassessedthe functional significance of the intraspecific variation in the lateral linesystem found in Chapter 2 by testing the ability of fishes from differentpopulations to orientate at varying water flows. Finally, in Chapter 4, Isummarisemymainfindings,assesssomeofthe limitationsofthestudyandsuggestpossibleareasoffuturefocus.Theimplicationsofthefindingsarealsoconsidered in regard to the management of freshwater ecosystems in thePilbarasubjecttoalteredflows.

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Chapter2.Theebbandflowofthewesternrainbowfish

(Melanotaeniaaustralis):howlaterallinesystemdiversity

relatestohabitatvariability.

AbstractFishesusetheirmechanoreceptivelaterallinesystemtosensenearbyobjectsbydetectingslightfluctuationsinhydrodynamicmotionwithintheirimmediateenvironment.Specialisationsofthelaterallinesystem,inparticularthedistribution,abundanceandlocationsofsuperficialneuromasts,havebeeninvestigatedamongspeciesfromdifferenthabitats,especiallythosewithvaryingdegresofwaterflow.However,populationsofasinglespeciesoftenoccupyhighlyvariablehabitatsanditisunknownwhethersuchenvironmentalvariation,forexamplewaterbodieswithdifferentlevelsofflow,isreflectedbyintraspecificvariabilityinthelaterallinesystem.Here,Idescribethefirstinvestigationofthelaterallinesystemofthewesternrainbowfish(Melanotaeniaaustralis),awidespreadspeciesacrossfreshwatersystemsinnorthwestAustralia.Iexaminedwithinandamong‐populationvariationinthedensityandarrangementofsuperficialneuromasts.EightpopulationsweresampledfromadiversityofcatchmentsinthePilbararegionofnorthwestAustralia.Scanningelectronmicroscopy(SEM)andfluorescentdyelabellingwerebothusedtodeterminethearrangementanddensitiesofthesuperficialneuromastsandthecanalporeopenings.IfoundthatthesuperficialneuromastsystemofM.australiswashighlyvariableinthedensity,location,andarrangementofneuromastsamongindividuals,populationsand

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bodyregions,andwasparticularlyvariableinthecheekregion.Additionally,Ifoundthatfishesfromafewofthepopulationstendedtodisplaygreateramong‐individualvariabilityinsuperficialneuromastnumberthanthosefromothersites.Largeranimalspossessedmoresuperficialneuromaststhansmalleranimalsandthenumberofsuperficialneuromastsincreasedwithbenthicinvertebrateavailability.Inaccordancewithstudiesthathavelinkedinterspecificvariationsinsuperficialneuromastwithwaterflow,wealsofoundasignificantnegativecorrelationbetweenthewaterflowrateatthecollectionsiteandthetotalnumberofsuperficialneuromastsonthebody.Ourfindingthatasinglespeciescandisplaysignificantamong‐populationvariationinakeysensorysystemsuggeststhattheabilitytoacquiresensoryinformationishighlytunedtoboththeanimal’shabitatanditsbehaviour.

IntroductionAllfishesfeatureauniquesensoryorgan,thelaterallinesystem,whichallowsthemtoreceivebothphysicalandbiologicalinformationabouttheirenvironment(Mogdansetal.2004).Thelaterallinesystemformsthebasisofmanykeysurvivaltraitsinfishes(Engelmann,Hankeetal.2002,Bleckmann2009)andunderliesmanybehaviouraladaptations,includingpredatoravoidance(Montgomery&Macdonald1987),communicationwithconspecifics(Partridge1980),objectdiscrimination,andorientationtowaterflowsor‘rheotaxis’(Montgomery1997,BakerandMontgomery1999,Bleckmann2008).Whilepredationpressure(McHenryetal.2009),lifecycle,micro‐habitat(Beckmannetal.2010,Vanderphametal.2013)andwaterflow

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speed(Wark&Piechel,2010)mayallcontributetoobserveddifferencesinthelaterallinesystemamongspecies,thereisstillconsiderableuncertaintyastowhatenvironmentalfactorsmaybedrivingspecies‐specificspecialisations.Todate,studiesofthelaterallinesystemhaveprimarilyfocussedoninterspecificvariationorhavecomparedthelaterallinemorphologyofthesamespeciesinhighlydivergenthabitattypes.Forexample,WarkandPiechel(2010)comparedthesuperficialneuromastarrangementinthethreespinestickleback(Gasterosteusaculeatus)betweenindividualsoccupyingmarineandfreshwaterhabitats.Thelaterallinesystemisakeytraitinfluencingfishbehaviourandadaptability.Therefore,animprovedunderstandingofthissensorymodalityunderpinsthecapacitytobetterpredicttheresilienceoffreshwaterfishestochangingenvironmentalconditions,especiallyflowdynamics.Thisstudywillprovideevidenceessentialtoimprovingthecurrentunderstandingofthelaterallinesystemandpresentnewfindingsinonecomprehensiveanalysis.Thelaterallinesystemiscomprisedofaseriesofbundlesofhaircells(‘neuromasts’)thatextendovertheheadandthelateralflankoffishes(Webb1989,CartonandMontgomery2004,Wellenreuther,Brocketal.2010).Theseneuromastscomprisetwodistincttypesofspecialisedreceptorcells,superficialneuromastsandcanalneuromasts,whichdifferintheirperformanceandfunction,despitesimilaritiesinbasicstructure.Superficialneuromastsarelocatedonthesurfaceoftheskin(Carton&Montgomery2004)andmostlyfunctiontodeterminethevelocityofthesurroundingwater(Dijkgraaf,1963).Theydifferfromcanalneuromastsastheyareableto

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respondtoflowthatisnotorthogonaltotheirorientationaxis,whilecanalneuromastsarelimitedtothedirectionoftheaxisofthecanal(Janssen,2004),curvedcephaliclaterallinecanalsmayrespondtolaminarflow,providedthatonlysomeofthecanalporesaredirectlyexposedtoincomingflow(Bleckmann,Pers.Comm).Superficialneuromastsalsofacilitaterheotaxis(bodyorientationintocurrents)asthesecellsareconstantlystimulatedbywaterflow(Baker&Montgomery1999,Mogdans&Beckmann,2012).Thecanalneuromasts,ontheotherhand,usuallyoccurinadistinctline,sittingatthebaseofacanalrunningbeneaththeskinandextendingovertheheadandflank.Thecanalsreducethespeedoflaminarflow,andlowfrequencywatermovements,byactingashigh‐passfilters(Janssen,2004).Thecanalneuromastsrespondtohighfrequencystimuliwithinthesewatermovementsandarethereforeusedforboththedetectionanddiscriminationofobjects,suchaspredatorsandpreyinthelocalenvironment(Mogdansetal,2004).Consequently,characterisingthediversityinabundanceanddistributionofbothsuperficialandcanalneuromastswithinandamongpopulationsofanygivenspeciesmayprovidesomeinsightsintotheiradaptivecapacitytochangingflowconditions.Canalneuromastsaretypicallyarrangedsothatonecanalneuromastissituatedbetweentwocanalpores,whichactastheaccesspointsforwatertoenterthecanals.Thecanalneuromastsrespondtohydrodynamicpressureasthefluidmovesinandoutofneighbouringpores,allowingtheneuromaststodetecttheaccelerationofthewateraroundthefish’sbody(Wark&Piechel,2010,Engelmann,Hankeetal.2002).Ithaslongbeenrecognisedthat

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hydrologicalfactors,suchaswaterflowrate,canresultintheevolutionofparticularfunctionalmorphologiesofthelaterallinesystem(Dijkgraaf1963).Thissystemisdocumentedtobeanadaptationtotheecologicalrequirementsofaparticularspecies,andcanalsoexhibitsomeinterspecificvariabilityaccordingtolifehistorystageandthelocalhydrologicalconditions.Forexample,speciesthatliveinenvironmentswithslowmovingwater(or‘quiet’environments)havealargenumberofsuperficialneuromastsandareducednumber,orcompleteabsence,ofcanalneuromasts(Wark&Peichel2010,Mogdans&Bleckmann2012).Bycomparison,speciesthatliveinturbulent,fast‐flowing,‘noisy’environmentstendtodisplayfewersuperficialneuromastsandawider,moredeveloped,canalsystemwithalargenumberofcanalneuromasts(Janssen,2004,Wark&Peichel,2010).Thereisalsosomeevidencethatsuggeststhatthelaterallineislinkedwithotherenvironmentalandbehaviouralfactorssuchasfeedingbehaviour(Montgomery&Macdonald,1987,Fischer,etal.2013,Mchenry,etal.2009)andthestructuralcomplexityofthehabitat(Erosetal.2003).Forexample,anenvironmentthatisspatiallyandtemporallyvariableanddisplaysinconsistenciesalongagradient,suchasacomplexstream,wouldbehardertonavigatethanafairlyhomogeneousenvironmentsuchasalargepoolorlake(Erosetal.2003).Theaforementionedstudieshaveconsideredsinglefactorsascausesofthevariation,howevernonehaveconsideredallfactorswithinasingle,cohesivestudy,whichistheaimofthisinvestigation.Inthisstudy,Iexaminedvariationinthelaterallinesystemofthewesternrainbowfish(Melanotaeniaaustralis),ahighlyubiquitousspeciesofteleost

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foundinmanydifferentfreshwaterenvironmentsthroughoutthePilbaraandKimberleyregionsofnorthwestAustralia(Allenetal.2003,Kelleyetal.2011).Habitatsofwesternrainbowfishesincludeephemeralpools,creeksandlakes(Allen,etal.2003),wheretheyformalargeproportionofthelocalfishcommunities(Morgan&Gill,2004;Beesley&Prince,2010).DespitewesternrainbowfishesbeingcommonthroughoutnorthwestAustralia,therehavebeenveryfewecologicalstudiesofthisspeciesanditslaterallinesystemhasneverbeenformallydescribed.Mystudyfocussedonadultrainbowfishescapturedfromeightsites(i.e.fromeightpopulations)acrossthesemi‐aridPilbararegionofnorthwestAustralia.Isoughttofirstdescribethearrangementofneuromastsonthebodyusingfluorescentstaining(DASPEI)andscanningelectronmicroscopy(SEM).Ithenassessedhowmuchofthevariationinthelaterallinesystemofeachpopulationcouldbeattributedtoparticularhabitatcharacteristics,includingsurfaceandbenthicinvertebrateavailability,waterdepth,flowrate,turbidityandameasureofhabitatcomplexity.Ihypothesisedthatwaterflowratewouldbeamajordeterminantoflaterallinesystemvariation.Iexpectedthatfishcollectedfromlowornoflowpoolsandcreekswoulddisplayagreaternumberofsuperficialneuromaststhanfishcollectedfromsiteswithfasterflowingwater.

26

MaterialandmethodsStudyareaandmodelspecies

Westernrainbowfishesweresampledfromtwogeographicallydistinctsub‐catchmentsoftheFortescueRiverinthePilbararegionofnorthwestAustralia,encompassingsiteswithadiversityofwaterflowsandhabitatcomplexities.TheFortescueRivertraversesover570kmandformsacatchmentareaof480,000km2withthelowerwesternpartofthecatchmentdrainingacrosstheplainsintotheIndianOcean,whiletheuppereasternregionofthecatchmentdrainsfromtheHamersleyRangesintotheFortescueMarsh(Barrett&Commander,1985).The"mid‐Fortescue"istechnicallypartofthegreaterLowerFortescuecatchment.TheflowregimeintheFortescueRiveranditstributariesisdirectlylinkedtorainfall,withseasonaldischargeduringthewetmonthsofJanuarytoMarch(Rouillardetal.2015).Rainfallaveragesfortheregionisaround350mmperyearbutishighlyvariablebothwithinandamongyears(AustraliaBureauofMeteorology,2011;O'Donnelletal.2015).Theareareliesonthesehighrainfallperiodstosustainvariouspoolsalongthedrainagelinesandthenreconnectduringthistime.Therefore,thebiogeochemistry,hydrologyandecologyofthepoolsisinextricablylinked(Fellmanetal.2011'Siebersetal.2015).

TheclimateofthePilbaraissemi‐aridandsub‐tropical,withrainfalloccurringintheformofcyclonesandtropicalthunderstorms,followedbyprolongedperiodsofdroughtthroughoutthewinter(AustralianBureauofMeteorology,2011).Summertemperaturesrangefrom24to40oC,whileinthewinter,temperaturesrangebetween11and26oC,suchthatannualpanevaporation

27

(2500mm)farexceedstheannualaveragerainfall(Fellmanetal.2011).Duringsummerperiodsofheavyrainfall,poolsbecomeswollenandcanconnectandspilloutontothefloodplain(Beesley&Prince,2010).Incontrast,duringthewintermonthsthewaterwayscanbecomeconstrictedthroughevaporationtoformachainofpools,alongadrainageline(Beesley&Prince,2010,Fellmanetal.2012;Siebersetal.2015).AdultrainbowfisheswerecollectedfromCoondinerCreekandWeeliWolliCreek(intheupperFortescuecatchment)andfromsixsiteswithinMillsteam‐ChichesterNationalPark(inthemidFortescuecatchment)duringApril‐May2014(Table1).CoondinerCreektypicallycomprisesaseriesofunstable,buthydrologicallyconnected,poolsthatrunalongthemaingorgeline,whicharelargelyreliantonrainfall(Fellmanetal.2011;Siebersetal.2015).WeeliWolliCreekencompassesadensenetworkoftributariesthatflowinanortherlydirectionintotheFortescueMarsh(Dogramacietal.2015).Theregionissubjecttosignificantminingactivityandconsequentlysomeofthecreeksintheareaaresubjecttoadditionalwaterflowsduetodewatering.Itisestimatedthat0.92GLofwaterisbeingpumpedintothecreekfromthedewateringoftheHopeDownIronOremineannually,whichhassignificantlychangedtheflowregimeofthecreeksincedischargebeganin2006(WRM,2010;Dogramacietal.2015).Thefreshwaterhabitatssampledfromthemid‐Fortescuearefedbyanundergroundaquiferthatcreatesalongstringofpermanent,stablepoolsover20km(Skryzpeketal.2013).Thus,flowratesinthisareatendtobeslowerandpoolsareoftendeeperthanthoseintheupperFortescue,forexamplethedepthatDeepReachreaches14.3m.

28

HabitatcharacterisationHabitatsacrossallsiteswereassessedforarangeofattributespriortofishsamplingtominimisedisturbance.Generalcharacteristicsofthesite,suchasthepresenceorabsenceofpredatorybirds(e.g.herons,cormorants),pastfloodlevels(estimatedbytheheightofdebrisfoundinnearbytrees)andthepercentageofcanopycoveroverpoolswasrecorded.Ialsomeasuredthefollowingattributes:waterflowrate(metrespersecond,ms‐1),benthichabitattype(seebelow),turbidity(measuredinNephelometricTurbidityunits,ntu)andthepredatorspeciesobserved(seebelow).Benthichabitattypewasassessedalongtransectsperpendiculartothebank(orbisectingapool)inanareawherefishweresightedfromthebank.Thelengthofeachtransectvaried,dependingonpoolwidth(min:3m,max:8m).At0.5mintervals,a20cmquadrantwasusedtodeterminethepercentagecoverofdifferentbenthichabitattypes,whichwerecategorisedaccordingtopercentagesofcoarse(>4mm)andfine(<4mm)substrateorgravel,aquaticvegetationandrocks.PhotographsofeachhabitatweretakenwithanOlympus1030SWwaterproofcameratoprovidearecordofkeyhabitatfeatures.Benthichabitatsurveysandsitephotographsweresubsequentlyusedtodevelopahabitatcomplexityrankingrangingfrom1to10.Ascoreof"1"describedsiteswithlowdiversityinaquaticbenthos,littletonoaquaticvegetationandlargelyopenwater,whileascoreof"10"wasallocatedtositeswithhighhabitatdiversity,includinghighcoverofaquaticvegetation(suchasSchoenusfalucatis,Ceratopteristhalictroides),overhangingvegetationand

29

submergeddebris.Siteswereevaluatedbytwoindependentobserversandthenaconsensusscoregiven.Followinghabitatcharacterisation,aSontek™Flowtracker,(ahandheldADV:AcousticDopplerVelocimeter)wasusedtodeterminewaterflowvelocityat0.5mintervalsacrossthetransect.Flowratewasmeasuredfromthewatersurfaceandrecordedasaproportionofthetotaldepthofthewateratreadingsof0.2,0.6and0.8,forexample,0.2refersto20%ofthedepthbelowthesurface.Thesemeasurementswereaveragedoverthemeasurementstations(min:11stations,max:16stations)togiveameanx,y,zvelocity,andvariationinvelocity(thestandarddeviationofthemeanflowmeasuredovera30secondperiod)foreachsite.Theflowtrackeralsorecordedthemeantemperatureateachdepth.Theabundanceofsurfaceinvertebratespresentateachsitewasassessedbysweepinga250mdipnetoverthesurfaceofthepoolinthree10msweeps.Thenetwasthenemptiedintoatraybyrinsingwithcleancreekwaterandtwoobserverscountedthetypeandtotalnumberofinvertebratescollectedovera5‐minuteperiod.Thespeciesthatwerecapturedincludedwatermites(orderAcarina),waterstriders(orderHemiptera,familyGerridae),mayfliesandchironomidlarvae(orderDiptera,familyChironomidae).Benthicinvertebratesweresampledusinga500mD‐netandwerecapturedbytramplingthesubstratewithina1m2areaandsweepingthenetoverthetrampledareafor30seconds.Thecontentsofthenetwerethenwashedthroughbotha2mmanda500msteelmeshsievewithcleancreekwater.Twoobserverscountedthetotalnumberofinvertebratescollectedinthesievesovera5‐minuteperiodduetotimeconstraintsateachsite.

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Predationpressurewasassessedthroughonsiteobservationofbirdsthatareknowntopreyonwesternrainbowfishes.Inaddition,recordsweremadeoftheabundanceofallfishspeciesthatwerecaughtorobservedateachsite.Fishwerecategorisedashigh‐orlow‐riskpredatorsaccordingtotheclassificationofpredationrisktoM.australisdevelopedbyYoungetal.(2011).Forexample,spangledperch(Leiopotheraponunicolor)isanomnivorouspredatorthatisconsideredlowrisk,whileFortescuegrunters(Leiopotheraponaheneus)andbarredgrunters(Amniatabapercoides)areconsideredhigh‐riskpredators.Table1.SummaryofkeyhabitatcharacteristicsforMillstreamNationalPark,CoondinerCreekandWeeliWolliCreek.Missingdataarewheresitesweretoodeeptosampleadequately.

Region

Site

ShannonW

einer

HabitatComp

lexity

0.2XF

lowVe

locity(m

‐s )

0.2XS

tErro

r

0.6XF

lowVe

locity(m

‐s )

0.6XS

tErro

r

Temp

eratur

e(o C)

Benth

icInvertebrat

es

SurfaceInv

ertebrat

es

PredationR

isk

Turbidity

Fortescu

eRive

r,Mills

tream

NationalPark

Jayawurrunha 0.224 6 0.12 0.0099 0.104 0.00812 25.4 31 1 Low 50.6

DeepReach 2 0.0054 0.0005 0.005 0.001 27.2 10 Low 43.4

OutCrossing 9 0.08 0.0307 0.057 0.0141 25.6 8 5 Low

PalmPool 0.432 6 0.02 0.0033 0.08 0.0044 23.6 11 9 Low 24.7

Jirndawurranha 0.262 8 0.305 0.0330 0.139 0.0182 28.2 20 4 Low

CrossingPool 0.394 4 0.004 0.0014 0.003 0.00114 28 4 3 Low 61.2

CoondinerCreek Coondiner 0.705 7 ‐0.002 0.00061 ‐0.005 0.00078 22.3 12 14 Low 37.6

WeeliWolli WeeliWolli 5 0.177 0.0111 0.186 0.0133 31.9

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Fishsampling

Ateachsite,20‐30adultwesternrainbowfishesofmixedsexwerecapturedusingeithera4mor10mlongseinenet(bothwith6mmmeshsize)dependingonthesizeoftheareasampled.Fisheswerehousedforuptofivedaysinthefieldinaerated,20Lplasticaquariacontainingcreekwaterandnaturalsubstratefromthecollectionsite.AllsampleswerecapturedduringafieldtripinApril2014.LivefishwerethentransportedtotheBiologicalSciencesAnimalUnitatTheUniversityofWesternAustraliabyairandplacedinaeratedaquaria(42x42.5x34)(onepopulationperaquarium)containinggravel,afilterandanartificialplant.Thetanksweremaintainedunderfluorescentlighting(12:12hlight:darkcycle)andwerefeddailyonamixeddietofcommercialflakefoodandArtemianauplii.ThreeadultswerealsocollectedfromeachoftheCoondinerCreekpools(Pool7andPool1.5)andfromCrossingPoolOutFlow(inMillstreamNationalPark)andpreservedonsiteforscanningelectronmicroscopy(SEM)tobecarriedoutatthelaboratoriesatUWA.TheseanimalswereeuthanizedusinganoverdoseofMS222(tricainemethanesulfonate;Sigma‐Aldrich,StLouis,MO,USA)(200mgl‐1)andthenplacedina50mLfalcontubefilledwithglutaraldehydefixative(25%glutaraldhyde,75%distilledwater(Proscietch,QLDAustralia)andwerekeptcoolatapproximately15oC.Bubblewrapwasslottedintothefalcontubetopreventthefishmovingaroundduringtransportandpotentiallycausingdamagetothesuperficialneuromasts.Fishthatwerefixedwereusedforassessingthenumber,locationandarrangementoftheneuromastsovertheheadandbodyusingscanningelectronmicroscopy

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(SEM).

NeuromastcharacterisationLivefishwerestainedwithafluorescentvitaldye2‐[4‐(dimethylamino)styrl]‐N‐ethylpyridiniumiodide,DASPEI(LifeTechnologies/MolecularProbes,EugeneOR,USA)tovisualisetheneuromastspresentonthesurfaceofthebody(protocoladaptedfromWarkandPiechel,2010).PreliminarytrialswereconductedatdifferentconcentrationsofDASPEIfor15minutesandthebelowconcentrationwasdeemedadequateforvisualisationofthesuperficialneuromasts.EachfishwasfirstallowedtoswimfreelyintheaeratedDASPEIsolutionataconcentrationof0.24gin1Lwaterfor15minutes.Fishwerethenanaesthetisedin200mgl‐1MS222(tricainemethanesulfonate;Sigma‐Aldrich,StLouis,MO,USA)untillightpressureonthecaudalfinyieldednoresponse.Thefishwasthenplacedrightsidedowninapetridishandexaminedusingafluorescencedissectingmicroscope(LeicaMZ75fittedwithaFITCfilterset;LeicaMicrosystemsInc.,Sydney,Australia).Images(8‐15perindividual)oftheentirebodywerecapturedatamagnificationof0.8X‐1.0X,usingadigitalcamera(LeicaDFC320).Measurementsofthelengthandsexofeachindividualwerealsorecorded.Sexwasdeterminedbasedonthefollowingfeatures:malesarebrighterincolourandhavepointeddorsalandanalfins,whilefemalesaredullerincolourandtheirdorsalandanalfinsaremorerounded.Followingflorescencephotography,fishwererevivedinfresh,aeratedaquariumwaterandreturnedtotheirhousingtank.IndividualfishfromeachpopulationunderwenttheDASPEIstainingandphotographyprocedureonlyonce.

33

Oncefishfromallpopulationswerephotographed,thecanalandsuperficialneuromastswereclassifiedintodistinctregionsonthehead,trunkandcaudalfin,basedonthemethodsofNorthcutt(1989)andWebb(1989).Thebodyregionsoccupiedbyneuromastswereclassifiedas:rostralsuperficial,nasalsuperficial,mandibularcanalandsuperficial,infraorbitalcanalandsuperficial,supraorbitalcanalandsuperficial,oticcanal,operculumsuperficial,cheeksuperficial,postoticcanal,dorsalandventralsuperficialandcaudaltailsuperficial(Figure1Aand1B).Anyphotographswherethenumberofneuromastsinaparticularsectionwereunclear(e.g.duetosuboptimallabelling)wereexcluded.Thebodywasdividedintoregionsandnotlinesofsuperficialneuromasts(Northcutt,1989),astherewassuchalargediversityinposition,abundanceanddistributionofsuperficialneuromastsoverthebodyandacrossindividualsandpopulations(Figure1B).WarkandPiechels(2010)wasusedasageneralguide,whichfollowedthelinesofNorthcutt(1989).

34

A.

B.

Figure1.ArrangementsofthesuperficialneuromastsystemoverthesurfaceofthebodyofawesternrainbowfishA)Arepresentative(43mminlength)fromCrossingpoolwithsuperficialneuromastsstainedwithDASPEIdye(photographssuperimposedtoaccountfordifferentfocalplanes).B)Diagramrepresentingtheneuromastgroupingsintobodyregions.Abbreviationsforsections:(RO)Rostral,(NO)Nasal,(MA)Mandibular,(SO)Supraorbital,(IN)Infraorbital,(CH)Cheek,(OP)Operculum,(DT)DorsalTrunk,(VT)VentralTrunk,(CT)CaudalTail.

35

IestimatedthedensityofneuromastsoverthedifferentregionsofthebodyusingthefreehanddrawingtoolinthesoftwareprogramImageJ,version1.48,(NationalInstitutesofHealth,USA).Aftercalibratingtheimageforscale,theoutlineofeachbodyregion(seeninFigure1B)wastracedtocalculateitsarea.Thedensityofsuperficialneuromastswithineachareawasestimatedbycountingthetotalnumberofneuromasts,withintheareaanddividingbyitstotalarea(inmm2).

Scanningelectronmicroscopy(SEM)Portionsofthehead,bodyandtailofofeachrainbowfishwerefixedinKarnovskysfixative(10mlsof2.5%glutaraldhyde,5mlof2%paraformaldehyde,5mlof0.13MSorensonsphosphatebuffer,pH7.2),refrigeratedforthreedaysandthenusedforscanningelectronmicroscopy(SEM).Thesesamplesincludedbothfieldcollectedsamplesandlaboratoryfish.Thetissuewasthenwashedinaphosphatebufferandheatedusingamicrowaveoven(250Wfor40seconds).Sampleswerethenimmersedinincreasinglyconcentratedethanolbaths(50%,70%,90%,100%,100%)andheated(asdescribedabove)ateachconcentration.Thesampleswerethenplacedintoacriticalpointdrierfortwoandahalfhoursuntilthetissuewascompletelydry.Eachpieceoftissuewasthenmountedonastubandsputtercoatedwithgoldpalladium.AllimageswerecapturedwiththeZeiss1555VP‐FESEMatvariousmagnificationsrangingfrom78xto1,647,000x.

36

Figure2.Scanningelectronmicrographofanopercularsuperficialneuromastshowingtheaggregationofcilia.Notethatnotalloftheciliaareupright/intactduetolowlevelsofabrasionduringtransportationfromthefield.

DataanalysesTheoverallvariationintheabundanceofsuperficialneuromastswasfirstdescribedbycalculatingthecoefficientofvariation(CV)foreachpopulation(site)andforeachbodyregion.Ifirstcheckedforcorrelationsamongthetotalnumbersofdependentvariablesandexcludedbodyregionsthatweresignificantlycorrelated(Pearson’scorrelations:dorsaltrunkvs.ventraltrunk:(r=0.556,df=149,P

37

assumptionsofMANCOVAandanycorrelatedvariableswereexcluded,superficialneuromastabundanceforeachbodyregionwasusedasadependentvariable(=8dependentvariables),siteasafixedeffect(8‐levels)andstandardbodylength(SL)asacovariate.Asignificantoveralleffectonthedependentvariableswasinvestigatedfurtherbyconductingsubsequentunivariatetestsforeachregionofthebodyseparately.

AstherewasasignificanteffectofthecollectionsiteusingMANCOVA,itjustifiedfurtheranalysestodeterminetheeffectoftheenvironmentalvariablesonsuperficialneuromastabundance.Isubsequentlyperformedasecondsetofanalyses(MultivariateAnalysisofVariance:MANOVA)withsiteasarandomeffect(tocontrolforthedifferentpopulationoriginsoftheindividualssampled),andtheabundanceofneuromastsinspecificbodyregionsasthedependentvariables.ThisstudyconsideredaneffecttobesignificantatP

38

waterdepth,andthetotalnumberofsurfaceandbenthicinvertebratescaptured.SimilarityamongthesitesintermsoftheirenvironmentalcharacteristicswasvisualisedbyplottingtheresultingprinciplecomponentsusingthesoftwareprogramPrimer6.0(Primer‐Eltd,Ivybridge,UnitedKingdom).Groupscloselydisplayedontheprinciplecomponentsplotsweremoresimilarinenvironmentalvariables.

ResultsThelaterallinesystemofthewesternrainbowfishScanningelectronmicroscopyandfluorescencemicroscopyofDASPEI‐labelledsuperficialneuromastsrevealedthatalleightpopulationsofwesternrainbowfishsampledinthisstudypossessedconsistentlocationsofsuperficialneuromastsoverthetenheadregionsandthreebodyregions3(Figure1).Duringanalysis,itbecameapparentthattherewasnoonebaselineforthepositionsoftheneuromastsi.e.theirpositionwasalwaysarrangeddifferentlywithinthedesignatedregion.However,thesuperficialneuromastswereprolificacrosstheheadandbodyandwereeitherfoundinsmallclustersofvariousshapesorsingularly.Clustersofsuperficialneuromastsweremostoftenarrangedinacrescentshape,howevertheyalsoformedpatternssuchascrossesandabstractgroupings(Figure3,Figure4).

39

Table2.Univariateresultstestingfortheeffectofsite,bodylengthandwaterflowonthetotalnumberofsuperficialneuromastsforeachbodyregion.

EffectBodyregion(superficial) df F‐ratio P‐value

Site

Rostral 7,140 8.111

40

BenthicInvertebrates

Rostral 1,108 0.021

0.8855Nasal 1,112 0.145 0.7037Mandibular 1,112 8.082 0.0053Infraorbital 1,112 0.432 0.5126SupraOrbital 1,112 8.818 0.0036Operculum 1,112 4.525 0.0356Cheek 1,112 2.467 0.119PostOtic 1,111 14.168 0.0003Dorsal 1,110 7.722 0.0064Ventral 1,111 6.876 0.01CaudalTail 1,111 1.205 0.2746

41

Figure3.RepresentativeDASPEIimages:(A)MalefromCrossingPool,(B)FemalefromWeeliWolliCreek.Imagesshowdifferencesinthearrangementofsuperficialneuromastswithinthetrunkregion.

Acomparisonofthelevelofvariationinsuperficialneuromastabundanceforthedifferentbodyregionsrevealedthatthecheekregionshowedthehighestvariationinsuperficialneuromastabundance(CV=50%),whilethenumberofsuperficialneuromastsintheinfraorbitalregionwashighlyconsistent(i.e.lessvariable)acrosssamples(CV=12%).

A B

42

Figure4.Populationvariationandthenumberofsuperficialneuromastspresentonthecheek.Barsrepresentmeannumberofsuperficialneuromasts±standarderrors.TheDASPEIimagesshowthedifferentarrangementsofsuperficialneuromastsinthecheekregioninfishfrom(A)Jirndawurranha,(B)CrossingPool,(C)DeepReachand(D)OutCrossingPool

02468101214

A

B

C

DSite

43

Whencomparingthedensityofneuromastsamongthedifferentbodyregions(i.e,numberofsuperficialneuromasts(SNs)permm2ofbodysurface)ofonerepresentativeindividual,thenasalregionhadthehighestdensity(55.2SNs/mm2)followedbytherostralregions(35.7SNspermm2).RainbowfisheswiththehighestpopulationvariationinsuperficialneuromastabundanceoccurredatOutCrossing(CV=26%),whilethesitewithrainbowfishesexhibitingtheleastvariabilitywasWeeliWolliCreek(CV=13%).

44

Table3.Meanandranges(highestvalueminusthelowestvalue)forthetotalnumberofsuperficialneuromastspresentforeachbodyregionateachsite.Thecoefficientofvariationforeachsiteandbodysectionisalsogiven.Highlightedinredarethehighestsuperficialneuromastmeansforeachbodysectionandhighlightedinbluearethehighestrangesforeachbodysection.

Rostral Nasal Mandibular Infra‐orbital Supra‐orbital Operculum Cheek PostOtic Dorsal Ventral CaudalCVofSite

Jaya Mean 8.9 5.9 22.4 15.6 0.2 26.1 8.0 12.6 65.8 185.9 39.2 0.16Range 11 6 31 6 2 16 11 7 109 158 75

DeepReach Mean 8.1 4.6 33.4 15.8 0.0 32.0 10.2 14.2 75.1 185.6 39.3 0.22Range 12 5 28 5 0 23 20 15 59 195 41

OutCrossing Mean 8.4 5.6 31.1 16.2 0.0 31.9 11.9 17.3 90.1 222.3 48.7 0.27Range 8 8 34 7 0 30 18 21 88 211 61

PalmPool Mean 8.4 4.8 28.0 15.6 0.0 27.7 10.9 19.4 83.3 222.5 50.1 0.22Range 8 8 22 6 0 14 15 17 67 212 84

Jirndawurranha Mean 8.0 3.5 28.0 16.3 0.0 24.2 9.6 14.9 61.7 135.4 36.5 0.21Range 7 8 28 4 0 34 15 11 44 106 70

CrossingPool Mean 8.6 5.2 29.2 16.1 0.0 28.6 9.0 17.5 74.0 202.9 37.9 0.25Range 13 7 17 7 0 24 17 18 70 208 68

CoondinerCreek

Mean 8.2 5.1 27.1 18.0 0.0 24.1 10.0 13.5 63.1 141.6 43.1 0.16Range 16 4 22 8 0 31 19 12 62 191 43

WeeliWolliCreek

Mean 14.7 4.8 31.5 16.4 0.0 21.2 9.1 19.4 59.6 163.3 39.2 0.13Range 16 5 44 8 0 23 14 13 41 92 42

CVofBodysection

0.39 0.35 0.27 0.12 9.26 0.31 0.50 0.28 0.29 0.30 0.40

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Figure5.Themeannumberofsuperficialneuromastsacrossallsitesforeachbodyregion.Errorbarsrepresentstandarderrorsofthemean.Canalsandcanalporeswereclearlydefinedonthehead,formingfourmainlines:thesupraorbital,theotic,themandibularandinfraorbitalcanals,allwithvisibleclustersofcanalneuromastssituatedatthebaseoftheporeopenings(Figure1A).Thepositionofthesecanallineswashighlyconsistentamongindividualsandpopulations.Incontrast,nocanalporeswerevisibleonthetrunkofthebody.

Neuromastabundanceinrelationtobodylength,populationandsexTheMANCOVArevealedanoveralleffectofpopulation(F7,135,=0.62,P=

46

TherelationshipbetweensuperficialneuromastsandwaterflowTheMANOVAanalysisrevealedthattherewasasignificanteffectofwaterflowrateonsuperficialneuromastabundanceacrossthedifferentregionsofthebody(F7,137,=0.61,P

47

VariationincomplexityofrainbowfishhabitatsUnsurprisinglyforsuchalargeregionalstudy,environmentalcharacteristicswerehighlyvariableamonghabitats.Forexample,theDeepReach(midFortescue)sitewasaverylarge,deepbodyofwater(>14m),wherefishwerefoundswimmingfreelynearthesurfaceandfacedfewobstacles.Incontrast,poolsatCoondinerCreekorJirndawurranha,werequiteshallow(<2m)andhadmanyobstaclesanddebristhatwouldcreateacomplexenvironmentfornavigation,particularlyunderincreasedflow(Figure7).PrinciplecomponentsanalysisoftheeightsitesrevealedthatDeepReachandCrossingPoolwerethemostsimilarinhabitatstructure,complexity,flowratesanddepthprofiles(Figure7).Thehabitatcomplexityratingsalsosupporttheseresults,whichrevealedthatDeepReachandCrossingPoolscoredsimilarlyat2and4,respectively.

Figure7.AplotoftheprinciplecomponentsforsitesatCoondinerCreekandMilllstreamNationalPark.WeeliWolliCreekwasexcludedfromtheanalysisowingtoanincompletedataset.

48

TheJirndawurranha,JayawurranhaandOutCrossingsiteswerethemostdistinctfromeachotherandtherestofthesites.Atthetimeofsampling,flowratesvariedbetween0.000ms‐1(atPalmPool)and0.305ms‐1(atJirndawurranhachannel;Table1).AprinciplecomponentsplotofthewaterflowmeasuresrecordedateachsiterevealedthatOutCrossinghadthemostvariableflowspeedsandflowdirectionsacrossthetransect(Figure8),whileCrossingpoolandCoondinerCreekhadthemoststableflowconditions.

Figure8.Aplotoftheprinciplecomponentsfortheflowmeasurementsatthethreedifferentdepths(0.2,0.6,0.8)forallthree‐flowdirections(X,YandZ)forMillstreamNationalPark,CoondinerCreekandWeeliWolliCreek.

DiscussionThisstudyhasshownthatthereisasignificantrelationshipbetweentheflowrateoftheenvironmentandthestructureandabundanceofthesuperficialneuromastsinthisspecies.Italsosupportspreviousstudiesthathaveconcludedthatlimnophilicfishlivinginquieter,slowerenvironmentshavemoresuperficialneuromaststhanrheophilicfishthatlivein“noisier”,fast‐

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pacedenvironments(Jakubouski,1967,Vischer,2013,Bleckmann,1994,Coombsetal.1998,Tyke,1990,Dijkgraaf,1963,Englemannetal.2002,Beckmann&Eros,2010,Tan,2011,Janssen,2004,Teyke,1990).Thisstudyshowedthattheabundanceofsuperficialneuromastsvariedoverspecificregionsofthebody,andalsovariedamongindividualsandpopulations.Thelevelofvariabilitythatwefoundinthesuperficialneuromastswithinaparticularbodyregionhasnotbeenreportedinanyotherspeciestomyknowledge.Ialsoconductedabriefinvestigationofthecanalstructurebutwasunabletolocateanycanalporesonthetrunk.However,furtherSEManalysesofmorefishacrossagreaterrangeofsitesislikelyrequiredtodefinitivelyconcludethatthisspecieslacksacanalsystemonthetrunk.Collectively,thefindingsfromthisexperimentsuggestthattheplacementofsuperficialneuromastsandthecanalsystemcanbeexplainedbysensoryadaptationstodifferentenvironments.

CanalsystemofthewesternrainbowfishThisinvestigationintothecanalstructureofthewesternrainbowfishrevealedfourcanalsoverthehead;themandibular,theotic,thesupraorbitalandtheinfraorbitalcanals.However,ourbriefinvestigationswereunabletofindanyevidenceofacanalsystemonthetrunkofM.australis.Thisrequiresamuchmorethoroughinvestigation,whichisoutsidethescopeofthecurrentstudy.IfM.australisdoesindeedlackatrunkcanalsystemitwillundoubtedlyhaveaneffectontheanimal’smechanoreceptiveabilities.Severalofthepopulationsinvestigatedinthisstudyareexposedtorelativelyhighflowrateswithhighbackgroundnoiseandwouldbenefitfromawell‐developedcanalsystem.Thiswouldadequatelyallowthemtosenseaspectsoftheirenvironment,suchas

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predators,preyanddisturbanceswithsuchahighbackgroundnoise(Webb,1989).Numerouspreviousstudieshaveindeedfoundthatafishfroma‘noisier,’fasterflowingenvironmentwillhaveamuchmoredevelopedcanalsystemthananimalsfrom‘quieter’environments(Tyke,1990;Englemannetal.2002;Wark&Piechel,2010).Theabsenceoftrunkneuromastsappearstobecharacteristicofbenthic,planktivorousorschoolingspecies(Webb,1988).Asthewesternrainbowfishisashoalingspecies,onepossibleexplanationfortheproposedabsenceofthecanaltrunksystemisthatitistheresultofabehaviouraladaptation.Thisstudyhasalreadyrevealedthattherearedifferencesinthearrangementofthesuperficialneuromastsystem,butitremainsunclearifthecanalsystemofthewesternrainbowfishsimilarlydiffersamongpopulations.However,other,closelyrelated,speciesalsoshowdifferencesintheircanalsystemarrangement.Forexample,Vanderphametal.(2013)foundthattwospeciesofcommonbully(GobiomorphuscotidianusandGobiomorphushuttoni)haddifferingnumbersofcanalporesontheheadandattributedthistodifferencesinthehabitatthatthesefishexperiencedasadults(Vanderpham,etal.2013).Thissuggeststhatawell‐developedcanalstructureinturbulentflowingenvironmentsmayprovideaselectiveadvantageatcertainlifestages.Allthewesternrainbowfishinthisstudywerematureadultfishesandthereforesubsequentanalysisofjuvenilesmightrevealdifferencesinthecanalstructures.Incomparisonwithotherspecies,suchasthecardinalfishApogoncyanosoma,thebodyofthewesternrainbowfishhasaconsiderablenumberofsuperficialneuromasts;wewouldexpectthatspecieswiththispatternaremoresensitive

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topreymovementandhavehighercaptureefficiencythanfisheswithfewerneuromasts(Janssen,2004).WhiletheexactdietofwesternrainbowfishesinthePilbarahasnotyetbeendocumented,itisplausiblethatitsdietissimilartothatoftheeasternrainbowfish(Melanotaeniasplendidasplendida)astheyoccupyasimilarecologicalniche(McGuiganetal.2003).Thedietoftheeasternrainbowfishincludesmacroalgae(42.5%),aquaticinvertebrates(19.2%)andterrestrialinvertebrates(12.3%)(Puseyetal.2004).Therefore,itislikelythatinvertebrates(bothsurfaceandbenthic)comprisealargeportionoftheirdiet.Thedietoftherainbowfishmayexplainthesignificantrelationshipbetweensuperficialneuromastnumberandbenthicinvertebratenumbersfoundinthisstudy,andthusfoodavailabilitymaybeanotherexampleofthelaterallineshowingspecialisationforaparticularenvironmentorbehaviouraltask.Investigatingthefeedinghabitsinourmodelspeciescouldprovideanotherexplanationfortheobserveddifferencesintheabundanceofcanalandsuperficialneuromastsamongpopulations,astherearemanyothercompetingspeciesforfoodsourcesthatwereobservedduringfieldworkexpeditions.Previousstudieshavelinkedthecanalsystemtootherenvironmentalpressuressuchaspredatorandpreyrelationships,asthiscomponentofthelaterallinesystemisusedinthedetectionanddiscriminationofobjects.Torrentfish(Cheimarrichthysfesteri),aspeciesthatresidesinturbulentfastflowinghabitats,hasprolificnumbersofsuperficialneuromastsandasimplebranchedcanalsystem(CartonandMontgomery2004),whichistheoppositeofwhatmightbepredictedifthelaterallinesystemdevelopmentwasdrivenbywaterflowrates.Alternatively,theauthorsconcludedthatotherfactorssuchasthe

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nocturnalfeedinghabitsoftorrentfishmighthaveastrongerinfluenceonthehypertrophyofthetwolaterallinesubsystems(Carton&Montgomery,2004).Thisexampleprovidesevidencethatsuperficialneuromastandcanalneuromastpatternscannotalwaysbegeneralisedforspecificrolesandmaybespecie‐sorhabitat‐specific.Here,Ifoundthatrainbowfishmightalsoshowanadaptationofitslaterallinetofeedingbehavioursaswellasflowrates.

AbundanceofsuperficialneuromastsofwesternrainbowfishinrelationtoflowMyobservationsforthewesternrainbowfishesareconsistentwithstudiesofotherfishspeciesthathaveshownsuperficialneuromastsaremorevariableintheirnumberandlocationthancanalneuromasts(Fischer2013,Webb1989,WebbandNoden,1993,WebbandShirey,2003).Thepatternobservedinthisstudy(moreneuromastsinslow‐flowenvironments)isalsoconsistentwithmostoftheliteratureanditsdocumentedrelationshiptoflow(Dijkgraaf1963,Wark&Piechel,2010,Engelmann,etal.2002,BleckmannandZelick,2009,Bassettetal.2006).Myinvestigationintothewesternrainbowfish,supportsthenotionthatincreasedhydrodynamicactivity(i.e.a“noisy”environment)willproduceadecreasednumberofsuperficialneuromasts,whileslowordecreasedflowrates(i.e.quieterenvironments)willproduceahighernumberofsuperficialneuromasts(Dijkgraaf,1963,Wark&Piechel,2010,MogdansandBleckmann2012,Miller1986,Puzdrowski,1989andVischer,1990).Thisstudyalsoshowedthatsuperficialneuromastnumberishighlyvariablebothwithinbodyregionsandwithinpopulations.Inrainbowfishes,themostdenselypackedareasonthebodywerethenasalandrostralregions.Asthesearetheareasthatmakefirstcontactwithoncomingflow,itislikelythatthisis

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anadaptationfortheearlyassessmentofhydrodynamicflow.Onestudyhaspreviouslylinkedthedistributionofsuperficialneuromastsoverthebodywithparticularbehaviouralfunctions.Yoshizawaandcolleagues(2010)usedVibrationAttractionBehaviour(VAB)andaregion‐specificsuperficialneuromastablationtreatment(usingVetbondTM,anon‐toxictissueglue)todeterminetheareasthatareusedinthisbehaviourtoavibrationstimulus.Theyfoundthattherewasasignificantdecreaseintheanimal’sabilitytoperformVABwhentheSO‐3(Supraorbital3region)anddorsaltrunkareawereablated,comparedwithcontrolanimals,wherefunctionoftheseregionsremainedintact.Thisarea(SO‐3)coverspartoftheinfraorbital,operculumandcheekregionsdefinedinthecurrentstudyandsuggeststhattheseregionsarelargelyresponsiblefordetectingvibrations(Yoshizawa,etal.2010).Consequently,thisopensupquestionswhethertheseregionsareequallyimportantintherainbowfish,orifthenasalandrostralregionsareutilisedmoreforthediscriminationofobjects.ThisstudyshowedM.australisdisplayedhighersuperficialneuromastvariation(27%)acrossallpopulations,withtheintra‐populationvariabilityvaryingbetween13%and28%.Forexample,thesevariationsaremuchhigherthanthevariationsSchmitzfoundin2008.Heexaminedthesuperficialneuromastsystemofthecommongoldfishandfoundonly9%variationinthenumberofsuperficialneuromastsacrossthebody(Schmitz,etal.2008).Asalltheanimalswerewildcaught,Iwasunfortunatelyunabletodeterminewhetherthelargeamountofvariationobservedisduetoenvironmentalselectivepressures,sincehighlevelsofvariationcouldsignificantlyaffectsurvival.Fishes,inparticular,arehighlysensitivetoflowalteration,showingconsistentdeclinesinabundance

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anddiversity,regardlessofwhetherflowshaveincreasedordecreasedrelativetothenaturalregime(Poff&Zimmerman2010).Furthermore,theresponsetoalteredflowscanbespecifictoeachspecies(Haxton&Findlay2008),suggestingthatitmaybecriticaltoassesseachspecies’resiliencetovariableflowstopredictresponsesatthepopulationlevel.Ifthevariationiscausedbyenvironmentalfactors,itthereforechangesanindividual’ssensoryboundaryandpotentiallyitsabilitytoadequatelysenseitssurroundings.Furthermorethismayaffectthedetectionofexternalstimuli.Thehighlevelofinter‐individualvariationinsuperficialneuromastabundancefoundinthisstudysuggeststhatindividualswillhavevariablesensoryabilities.Thesedifferencescouldberesponsibleforsubsequentvariationinbehaviouraltraitsandshapeanindividual’sperception(Wark&Piechel,2010).If,however,thevariationisduetogeneticfactorsitislikelythatthiswillbeinstrumentalinnaturalselectionaswehavealreadydiscoveredthatthelaterallineisresponsibleformanyfitness‐relatedbehaviourssuchasfeeding,avoidingpredationandmating(Montgomery&Macdonald,1987).Incontrast,manystudieshavefoundtheoppositepatternofvariabilityinthesuperficialneuromastsinspeciesinhabitingdifferenthydrodynamicconditions,suchasfishesfromnoisierenvironmentsdisplayingmoresuperficialneuromasts.CartonandMontgomery(2004)insteadlinkedthearrangementofthelaterallinesystemineachspeciestotheirpredatorytacticsanddiurnalandnocturnalhuntingbehaviours.Theyexplainthatthetorrentfishisanocturnalfeederandthattheprolificnumberofsuperficialneuromastspresentinthisspeciesisresponsibleforfindingpreywithoutvisualcues,abehaviourtheyhaveadaptedthroughnaturalselection.

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PossiblecausesofvariationinsuperficialneuromastabundanceThefindingsofthecurrentstudyraiseaninterestingquestionasmanyofthepopulationsfoundinMillstreamNationalParkareconnectedduringthewetseasonandwouldhaveaccesstogeneticmixing.Therefore,itwouldbelikelythatthereisastrongergeneticbasistotheobservedvariation.Manystudieshaveattemptedtodeterminewhetherthelaterallinevariationfoundamongpopulationsisduetoenvironmentalorgeneticdrivers,(oravariationofboth)andmosthaveconcludedthatitisindeedacombinationofboth(Fischer,2013,Trokovich2011).However,certainregionsofthelaterallineappeartobemoreinfluencedbyonedriverthananother.Fischer’s(2013)investigationoftheTrinidadianguppy(Poeciliareticulata)concludedthatpredationpressureisanenvironmentalfactorthatcanresultinamong‐populationdivergenceinlaterallinemorphology,specificallysuperficialneuromasts,ashefoundnodifferencesinthecanalsystem.Hisstudyrevealedthatguppiesoccurringinhighpredationpressureareashadmoreneuromastsinthedorsalandtrunkregionsthananimals,whichinhabitedlowriskenvironments.Theyarguethatahigherneuromastnumberinthesebodyregionsallowsguppiestoshoaltightlyandthereforeavoidpredationmorethanguppiesthatshoallooselyandoccurinlow‐riskhabitats(Fischer.etal,203).Incontrast,astudybyTrokovich(2011)investigatedthenumberandarrangementofsuperficialneuromastsinthethreespinemarinesticklebacks(Pungitiuspungitius)andtheirrelatedfreshwaterpondinhabitants.Theyfoundthatsevenofthethirteenbodyregionsshowedsignificantdifferencesbetweenthetwohabitatsandthatthesedifferencesweremaintainedwhenasecondgenerationwasrearedinthelaboratory.They,therefore,concludedthatitis

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likelythatthepatternsfoundinnaturehaveageneticbaseandthatnaturalselectionplaysanimportantrole(Trokovich2011).Geneticandlaboratoryrearingstudiesareneededtoconcludeiftheseexplanationsalsoexplainifthevariationfoundwithinthedifferentregionsareenvironmentally‐orgenetically‐determined.

ConclusionsThisinvestigationintothelaterallinesystemofthewesternrainbowfishhasprovidedthefirstdescriptionofthestructureofthecanalandsuperficialneuromastsystems.Thisstudyhasinvestigatedanddescribedthelocationsofthesuperficialneuromastsystemandthevariabilitybetweenindividualsandpopulationsfromdifferenthabitats.Thisincludedthenumberandtheplacementofthesuperficialneuromaststotheextentthathadnotbeendocumentedpreviously.Wehavealsocompletedapreliminaryinvestigationintothecanalstructureofthelaterallinesystem.Thisstudyisthefirsttoconsidertheeffectofmultipleenvironmentalfactorsonlaterallinesystemdiversityinasinglespecies.Theresultsshowthatthereisasignificanteffectofflowontheamountofsuperficialneuromasts,howevertherewasalsoasignificanteffectofsite,lengthandbenthicinvertebratenumbers,suggestingthatthecompositionofthelaterallinemaybemultifactorial.Ithasthereforeestablishedafoundationforfurtherstudieshighlightingtheimportanceofthelaterallinesystemthatisvitalforthebehaviourandsurvivaloffishes.

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Chapter3.Laterallinemorphologyandhabitatorigin

determinerheotaxicabilitiesinthewesternrainbowfish

(Melanotaeniaaustralis)

AbstractRheotaxisisamultisensorybehaviourthatallowsfishestoorientthemselvesinthedirectionofwaterflowtoresistbeingsweptdownstream.Visual,olfactory,vestibularandlaterallinesystemshavesomeinvolvementinrheotacticbehaviourbuttherelativecontributionsofdifferentsensesundervaryingconditionsarepoorlyunderstood.Furthermore,thebehaviouralconsequencesofthisvariationareunknown.Inthisstudy,Iinvestigatedtheroleofthelaterallinesysteminmediatingrheotaxisinpopulationsofthewesternrainbowfish(Melanoteaniaaustralis)fromfreshwaterhabitatswithdiversityofflowregimes.Neomycinsulphatewasusedtochemicallyablatethelaterallineandevaluateitsroleinfacilitatingrheotaxisinfishexposedtothreedifferentwaterflowspeeds.Overall,meanorientationdirectionoffishdidnotchangewithwaterflowspeed.However,meandirectionallengthincreasedwithflowspeed,implyingthatfishspentmoretimeorientatedwithrespecttoflowasthespeedincreased.Itwasfoundthatneomycintreatedfishhadasmallermeandirectionallengthandspentlesstimeorientatingwithrespecttoflow.ThisresultsuggeststhatthelaterallineisrequiredforrheotaxisinM.australisandisparticularlyimportantinslowwaterenvironments.Thisindicatesalinkbetweenintraspecificlaterallinesystemdiversityandswimmingbehaviourinthewesternrainbowfish.Thisstudyalsonotedthatpopulationdifferencesin

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thenumberofsuperficialneuromastsofthelaterallinesystemcanaffecttheabilityofM.australistoorientwithrespecttothedirectionofwaterflow.

IntroductionWaterflow,includingitsdirectionandspeed,isadominantfactorinanyaquaticenvironment.Navigatingandutilisingthesecurrentsiscriticalforthespeciesthatinhabitdynamicenvironments.Rheotaxisdescribeshowfishesandotheraquaticspeciesorienttheirbodydirectlyintotheflowandmaintaintheirpositionrelativetothesubstratumbyswimmingdirectlyintothecurrent.Rheotaxisisknowntoreduceenergycosts,providingthefishwitholfactoryinformationcarriedwiththecurrentandalsoprovidingdirectionalguidanceforexample,duringtheupstreammigrationofsalmonduringspawning(Montgomeryetal.1995).Fishesuserheotaxisacrossarangeofdifferenthabitattypes,includingbenthic,freshwaterspecies(Montgomery,1997)andspeciesthatliveinhabitatswithvariableflowspeedssuchasinlakes(Kanter,Coombs,2003)andoceans(Champalbert,1994).Rheotaxisalsooccursinstreamdwellingspeciessuchasthegiantdanio(Devarioaequipinnatus),(Bak‐Colemanetal,2013)andtheblindMexicancavefish(Astyanaxfasciatus)thatliveinlightlesssubterraneancavepools(VanTrump&McHenry,2013).Thus,regardlessofhabitattypeandsensoryability,rheotaxisisintegralforallfishspecies’survivability.

Mostresearchtodatehasinvestigatedwhichofthesensescontributetorheotacticbehaviours.Itappearsthatthelateralline,vision,vestibularandolfactorysensesareallinvolved(Lyon,1904,Dijkgraaf,1963,Arnold,1974),but

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therelativeimportanceorinputofeachsenseisstillpoorlyunderstood.Forexample,afishcanperformrheotaxisevenintheabsenceofafunctionallaterallinesystem(Montgomery1997,BakerandMontgomery1999,VanTrumpandMcHenry2013)orwhenvisionisimpeded(Sulietal.2012).Thelaterallinesystemhasbeenthoroughlyinvestigatedformorphologicalvariationwithinandamongspecies,aswellasitsrelationshiptoaquaticenvironments,sincetheconnectionwasfirstmadebySchulzein1861(Schulze,1861).Thesystemiscomprisedoftwodistincttypesofspecialisedreceptorcells;superficialandcanalneuromasts.Superficialneuromastsarearrangedonthesurfaceoftheskin(CartonandMontgomery,2004;Chapter2)andareconsideredtobemostlyusedtodeterminethevelocityofthesurroundingwater(WarkandPeichel,2010),aswellasfacilitaterheotaxis(BakerandMontgomery,1999).Incomparison,moststudieshavesuggestedthatthecanalsystemiseithernotinvolvedinrheotaxisorhasaveryminorrole(VanTrumpandMcHenry,2013).Thewesternrainbowfishhasavaryingnumberofsuperficialneuromastsandshowssignificantdifferenceinthearrangementandnumberofsuperficialneuromastsamongcloselylocatedpopulations(Chapter2).Thisfindingraisesthepossibilitythatthelaterallinesystemdiversityobservedinindividualsfromthesamehabitatandfromdifferenthabitatsmayallowsomepopulationstoorientateintoflowsmoreaccuratelythanothers.Onewaytodemonstratethecontributionofthelaterallinesysteminfacilitatingrheotaxisistoablatetheneuromastsandexaminethecorrespondingchangeinorientationbehaviour.Forexample,Montgomeryandcolleagues(1997)foundthattherewasasubstantialreductionintheabilityofthreefishspeciesto

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orientatewithanablatedlaterallinesystem.However,eachofthesespecies(torrentfishCheimarrichthysfosteri,Antarcticfish:Pagotheniaborch‐grevinki,blindmexicancavefish:Astyanaxfasciatus)haddifferingrheotacticthresholdsatflowspeedslessthan0.5cm‐s,2cmsand3cms,respectively(Montgomeryetal.,1997).Athigherflowspeeds,nodifferencesinrheotaxiswereobservedamongthespecies(Montgomeryetal.,1997).However,thisstudycomparedphylogeneticallydistinctspeciesthatoccupyextremelydifferenthabitatsandecologicalniches.Itisthereforeunclearwhetherdifferencesintheirrheotacticthresholdsweresolelyexplainedbyspecies’variationinlaterallinemorphology.Examiningrheotacticresponsesinasinglespeciesthatoccupiesavarietyofhabitatsmayelucidatetheoverallimportanceofthelaterallinesysteminrheotaxiswithrespecttootherenvironmentalfactors.Inthisstudy,Iexaminedwhetherwithin‐speciesvariationinneuromastarrangementcorrespondswithdifferencesinrheotacticresponsesoffishtowaterflows.Thefocalspeciesforthisstudyisthewesternrainbowfish(Melanotaeniaaustralis),anativefreshwaterfishspeciesfoundthroughoutthePilbaraandKimberleyregionsofWesternAustralia.Thewesternrainbowfishflourishesinavarietyofhabitatsrangingfromlarge,stagnantpoolstofastflowingriversandstreams.Previousinvestigationsintothisspecieshavedemonstratedthatfishfromfasterflowingenvironmentsgenerallyhavefewerneuromastsincomparisonwithindividualsfromslow‐flowhabitats(Chapter2).TheobjectiveofthestudyistodetermineifthelaterallinesystemisactivelyinvolvedintheabilityofM.australistoperformrheotaxis.Inaddition,thisstudyinvestigateshowdifferentarrangementsofsuperficialneuromastsinfluencetheabilityofwesternrainbowfishtoorientinflowsthataredifferenttothoseof

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theirnaturalenvironments.Thiswasinvestigatedbyseparatingthepopulationsaccordingtotheirhabitatorigin(e.g.fromlargestagnantlakescomparedtofastflowingchannels,thesewerecalledlowandhighflowhabitats,respectively)asthepreviouschapterhasshownthatthesehabitatoriginshavesignificanteffectsonthenumbersofsuperficialneuromasts.

MaterialsandmethodsFishsamplingandhusbandryRainbowfisheswerecollectedfromtwoareasofthePilbararegioninthenorthwestofWesternAustralia.ThefirstareawastheupperFortescuecatchment,whichincludesCoondinerCreekandWeeliWolliCreek.ThesecondareawasthemidFortescuecatchment,whichincludessitesinMillstream‐ChichesterNationalPark(seeChapter2forfurtherdetails).Thetwocatchmentsdifferintheirhydrology;sitesintheuppercatchmenttypicallycompriseaseriesofunstableintermittentpoolsthatrunalongthemaingorgelineandarelargelyreliantonrainfall(Fellmanetal.,2011).TheuppercatchmentalsoincludedWeeliWolliCreek,whichislocatedEastoftheGoodiadarrieHillsandactsasadischargepointfordewateringoperationsfromanumberofmines(WRM,2010).ThestreamcomprisesadensenetworkoftributariesthatflowinanortherlydirectionintotheFortescueMarsh(Kendrick2001,WRM2010,Dogramacietal.,2015).ThestreamishydrologicallysignificantasitisfedbyWeeliWollispringandisapermanentsourceofwaterinacharacteristicallyaridenvironment.Around1GLofwaterispumpedintothecreekannuallyfromcontinuousdischarge(Dogramacietal.2015),withsomesectionsofthecreeksubjecttobothcontinuousand,attimes,muchfaster

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flowsthanwouldoccurnaturally(Dogramacietal.2015).Incontrast,MillstreamNationalParkisfedbyanundergroundaquiferthatcreatesalongstringofpermanent,stablepoolsoveradistanceof20km.However,thepoolsvaryinhydrologyfromlargepermanent,stagnantpoolssuchasDeepReachandCrossingPooltofastflowingchannelssuchasOutCrossing,JirndawurranhaandJayawurrunha.

Between15and20maleandfemalerainbowfisheswerecapturedusingeithera4mor10mnet(witha6mmmeshsize),dependingonthesizeofthepool.Individualswerehousedforupto5daysinthefieldinaerated20LplasticaquariacontainingcreekwaterandnaturalsubstratebeforebeingtransportedbacktotheBiologicalSciencesAnimalUnitatTheUniversityofWesternAustralia.Oncebackatthelaboratory,mixed‐sexpopulationswereplacedinaeratedaquaria(42x42.5x34cm)containinggravel,afilterandanartificialplantandwerehousedundernormallight/darkconditions(12:12hlight:darkcycle)at26oC±1oC.FisheswerefedadailymixeddietofcommercialflakefoodandArtemianauplii.

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Table1.SummaryofhabitatresultsforMillstreamNationalPark,CoondinerCreekandWeeliWolliCreek.

Site

0.2Flow

Veloc

ity(m

‐s )

0.2StError

0.6Flow

Veloc

ity(m

‐s )

0.6StError

Temp

eratur

e(o C)

Fortescu

eRive

r,Mills

tream

NationalPark

Jayawurrunha 0.12 0.0099 0.104 0.00812 25.4 DeepReach 0.0054 0.0005 0.005 0.001 27.2 OutCrossing 0.08 0.0307 0.057 0.0141 25.6 PalmPool ‐0.02 0.0033 0.08 0.0044 23.6

Jirndawurranha 0.305 0.0330 0.139 0.0182 28.2 CrossingPool ‐0.004 0.0014 0.003 0.00114 28

CoondinerCreek Coondiner ‐0.002 0.00061 ‐0.005 0.00078 22.3 WeeliWolli WeeliWolli 0.177 0.0111 0.186 0.0133 31.9

Waterflowmeasurements

ASontek™Flowtracker,(ahandheldADV:AcousticDopplerVelocimeter)wasusedtodeterminethewaterflowvelocityateachsiteandwasmeasuredat0.5mintervalsalongatransect.Therangeofflowspeedthatcanbemeasuredwiththeflowtrackeris±0.001to4.0m/s. ThelocationofthetransectwasselectedbyobservingthepresenceofMelanotaeniaaustralisfromthebank.Waterflowmeasurementswerecapturedinthreedimensions(0.2,0.6and0.8asaproportionofthetotaldepthofthewater)atthreedifferentdepthsforeachintervalonthetransect,andwereaveragedforeachstationovera10speriod.Themeasurementswerethenaveragedoverthestations(minimum:11stations,maximum:16stations)toobtainthemeanflowandflowstandarddeviationsforeachsamplesite.Atallsites,M.australiswereobservedinhabitingthetop20%ofthewatercolumn(approx.30cmbelowthewater’ssurface),thusonlythe0.2flowvelocityreadingwasusedforthisexperiment.

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Accordingtotheflowratemeasuresobtainedinthefield,andtoreducethenumberoffishrequiredfortheexperiment,populationsofM.australiswerecharacterisedasoriginatingfromeitherslowfloworfastflowhabitats.CoondinerCreek(Meanflowspeedrange=‐0.002+‐0.00061m‐s),Deepreach(0.0054+‐0.0005m‐s)andCrossingpool(‐0.004+‐0.0014m‐s)wereassignedasoriginatingfromslowflowhabitats,whileJirndawurranha(0.305+‐0.0330m‐s),Jayawurrunha(0.12+‐0.0099m‐s)andWeeliWolliCreek(0.177,+‐0.0111m‐s)weregroupedintothefastflowgroup.

ExperimentalSetup.Rheotacticexperimentswereconductedinacustom‐builtindoorflume(5.1mlengthx0.8mheightx1.4mwidth)witharecirculatingflowchamberandvalvethatcouldbeusedtocontrolthewaterflowspeed(Figure9).Tocreatelaminarflow,lengthsofstackedPVCpiping(3.5cmdiameter)wereplacedbehindtheflowoutletvalve.Anobservationarena(67cmlongx32cmhighx37cmwidth)madeofopaqueplasticwasplaced1mbehindtheoutletvalveandraised10cmabovethebaseoftheflume.Laminarflowsweresubsequentlydirectedintotheobservationarenausingsectionsofcoreflute™,sothatallflowpassedthroughobservationarena.Diffuseinfraredlighting(940nm;obtainedfromLEDlightinghut.com)wasprovidedtotheobservationarenabyattachingrowsofinfraredLEDlights(embeddedinresinforincreasedwaterresistance)tothebaseofthearenaandplacingtwolayersofTeflondiffuseroverthetop(at0.25mmand4cm).PreviousinvestigationsbyKelley,etal.(unpublished)determinedthatthisspecieshasavisualsystemsensitivityrangefromapproximately360nm(ultraviolet)to575nm(red)andthereforecouldnotseetheinfraredlightingusedinthisexperiment.Theobservationarenawasdivided

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intotwoequal‐sizedsectionswithopaque(white)coreflute™toallowtwofishtobetestedatonetime.Bothendsofthearenawerecoveredwithaplastic0.5cmmesh,allowingwatertopassthroughwhilepreventingthefishfromescaping(Figure9).Observationswereconductedatflowspeedsof0.003m‐s,0.18m‐s,0.48m‐s,(hereafterreferredtoasnoflow,mediumflowandfastflow),whichwasmeasuredwiththesameflowtracker(Sontek™)usedinthefield.Eightmeasuresforeachspeedweretakenfromwithintheobservationarenaandthenaveragedtoproducethemeanandstandarddeviationflowspeedsforeachexperimentalflowcondition.

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Figure9.Schematicdrawingoftheflowtank(above)andobservationarenafromtheviewpointoftheoverheadcamera.Anenlargeddiagramoftheobservationarenaisalsoprovided(below).Thecamerawaspositioneddirectlyabovetheobservationarena.

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NeuromastvisualisationTotesttheeffectivenessofneomycininablatingthelaterallinesystem,wecomparedthesuperficialneuromastsoffishfromtheablationtreatmenttothoseofcontrolfish.Thiswasperformedforonetreatmentandonecontrolfishfromeachpopulation,todeterminewhethertherewasanypopulationvariationintheeffectivenessofthechemicalablation.DASPEIstainingwasthusonlyconductedontwoindividuals(treatedandcontrol)becauseallfishusedinthisexperimenthadbeenpreviouslytestedwithDASPEIinthepreviousexperiment(Chapter2).Theprocessofanaesthesia,DASPEIstainingandneuromastvisualisationwassimilartothatdescribedinthepreviouschapter.Tovisualisethesuperficialneuromasts,fisheswereexposedtofluorescentvitaldye2‐[4‐(dimethylamino)styrl]‐N‐ethylpyridiniumiodide,DASPEI(LifeTechnologies/MolecularProbes,EugeneOR,USA)for15minutesataconcentrationof0.24gin1Lwater,(1200mM).Fisheswerethenanaesthetisedwith200mgl‐1MS222(tricainemethanesulfonate;Sigma‐Aldrich,StLouis,MO,USA)untillightpressureonthecaudalfinyieldednoresponse.Eachindividualwasthenplacedrightsidedowninapetridishandplacedonthestageofafluorescence‐dissectingmicroscope(LeicaMZ75fittedwithaFITCfilterset;LeicaMicrosystemsInc.,Sydney,Australia).Imagesweretakenusingadigitalcamera(LeicaDFC320)toillustratechemicalblockingofthelateralline.Neuromaststhatweresuccessfullyblockedshowedweakfluorescence(i.e.werenotverybright)orshowednofluorescenceatall(seeAppendix1).

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ExperimentalconditionsFishwereexposedtoahighconcentrationofneomycinsulphate(1200uM)(FischerScientific,Pittsburgh,PAUS)fortwohoursbeforeobservationscommencedtochemicallyblockthelateralline.Thisagentwaschosenforthisstudybecauseittargetsonlythesuperficialneuromasts(notthecanalneuromasts)(Kulpaetal.,2015).PreliminarytrialswereconductedtotesttheeffectivenessofneomycinsulphateonM.australis(seeAppendix1).Theconcentrationthatwastrialledandfoundtobeeffectivewas1200uM.Individualfishweretestedonlyonceandallexperimentalprocedureswerecompletedinthedarktopreventtheanimalsrelyingonvisualcues.Priortoobservations,eachtestfishwasisolatedina10Ltank(20cmheightx28.5cmlengthx13.5cmwidth)containingeitherthelaterallineblockingagent,neomycinsulphate(ablationtreatment;seebelow)orconditionedaquariumwater(control)foraperiodoftwohours.Fishweremaintainedinvisualisolationandaerationwasprovidedusingabatteryoperatedaquariumpump.Afterthisperiod,pairsoffishwerethenplacedintothearena(oneineachobservationchannel)andwereacclimatisedtothedarkunderconditionsofnoflowforaperiodof10minutes.Wethenrecordedthebehaviourofthefishunderconditionsofnoflow(0.003cm‐s),mediumflow(0.18cm‐s)andfastflow(4.8cm‐s),witheachobservationperiodlastingforfourminutes.ThebehaviourofthefishwascapturedininfraredusingaSonyHandycamHDR‐CX550positioneddirectlyabovetheobservationarena.Followingpreviousstudiesofrheotaxis(VanTrumpandMcHenry,2013),fishweresubjectedtosuccessivelyincreasingflowspeedsandwerenotrandomisedwithrespecttoflowtoavoidcarryovereffectsassociatedwithfishbecomingfatigued.

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ImageanalysisAllvideorecordingswereanalysedusingacustommadeMatlab(TheMathworksInc.,programversionR2014a)program(Hemmi&Pfiel,2010),thattrackedthefish’sheadpositionandorientationintotheflowdirectionforeachframe.Whileimageswerecapturedat25frames/second,aresolutionof1frame/secondwasfoundtobesufficientforgeneratinganaccuraterepresentationoffishorientation.Videoclipsthatcouldnotbeautomaticallytrackedbecauseoflightingissues(e.g.poorcontrastattheedgesofthearena)orairbubblesonthewater’ssurfacewerecapturedmanuallyusingthe‘pointdata’option.Foreachfishandflowspeed,themeandirection(i.e.orientationofthefish’sbodywithrespecttoflowdirection)andmeanlength(calculatedastheamountoftimespentorientatinginagivendirection)wascalculated.Therewerenoinstanceswheretheanimalswereobservedmakingcontactwiththebottomofthearenaandweexcludedframesinwhichfishwerestationaryandpositionedatthesideofthearena.Datawascollatedoveranumberofframesandthisrangedfrom3000to6000framesusing10oangularbins.Toensurethatdirectorientationintoflow(i.e.0o)formedthemidwaypointofthefirstbin,binsweregeneratedinincrementsof‐5Oto+5O.Themeandirectionandmeanvectorlengthwerecalculatedfromtheoriginalorientationdataandwerecalculatedusingthefourquadrantinversetangentfunction.AllcircularstatisticswerecalculatedusingMatlabCircularStatisticsToolbox(Berrens,2009).Theresultingmeanvectorlengthanddirectionforfishwithineachtreatment/habitattype/flowspeedisascoreofbetween0and1(highervaluesrepresentconsistentorientationataparticularangle).

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Statisticalanalysis.PermutationalAnalysesofVariance(PERMANOVAs)wereusedtoanalysethedata,thustherewerenounderlyingassumptionsaboutthedistributionofthedata.EachPERMANOVAwasrunwith10,000permutationsbutthelevelofpermutationvarieddependingontheanalysisconducted.Toinvestigatetheeffectofwaterflowspeed(noflow,mediumfloworfastflow)onmeanvectorlengthandmeandirection,permutationswererandomisedamongallindividuals.Fortheanalysisoftreatmenteffects(controlandneomycin)andhabitatorigin(i.e.stillwaterpoolorfastflowingstream)permutationswererandomisedbetweenthetreatment/habitatgroups.Asignificancelevelofp

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Figure10:Meanvectorlengthsandstandarderrorsoforientationoverthewholedataset,acrossallthreeflowspeedscomparingtherheotacticresponsesofbothneomycinandcontrolfish.Neomycinfish(n=26),controlfish(n=28).Toinvestigateiftherewasadifferenceintheeffectivenessoftheneomycintreatmentonfishcollectedfromdifferentsites,wedidfurtheranalysistestingforfishsiteoriginonmeandirectionallength.ThesetestsrevealedthatfishfromJirndawurranhashowedastrongestresponsetotheneomycintreatment(p=0.001)andthetreatmentwhileweakeffectofneomycintreatmentwasobservedinsitessuchasWeeliWolliCreek(