1

Functionaldiversityofthelateralline

systemamongpopulationsoftheWestern

Rainbowfish(Melaenoteniaaustralis)

LindseySpiller(BScHons)

NeuroecologyGroup,SchoolofAnimalBiology

ThisthesisispresentedforthedegreeofMastersofPhilosophyattheUniversityofWesternAustralia.

2016

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Summary

Sensorysystemsarevitalforanyorganisminordertoreceiveandrespondto

relevantinformationabouttheimmediateenvironment.Theabilitytoreceive

informationviathesensescontributestofitnessrelatedbehavioursand

thereforeananimal’sabilitytosurvive.Themechanosensorylateralline

systemisauniquesensorymodalityinfishesandsomeamphibiansandisan

adaptationtoaquaticenvironments.Therelationshipbetweenthemorphology

ofaspecies’laterallinesystemandhabitatcharacteristics,especiallywater

flow,hasbeenextensivelyinvestigatedforarangeoffreshwaterandmarine

species;however,therehavebeenveryfewstudiesconductedinAustralia.

Mostofthesestudieshavefocussedonthediversityofthelaterallinesystem

amongspeciesfromdivergenthabitattypesratherthancomparingthesame

speciesacrosshabitats.Afocusonasinglespeciesisimportantbecauseit

allowsustomoreclearlydeterminethatanydifferencesthatarefoundare

likelytobeduetoexternalfactors.Thisstudyinvestigatedthemorphologyof

thelaterallinesystemofafreshwaterfish,thewesternrainbowfish

(Melanotaeniaaustralis),collectedfromeighthabitatsoftheinlandPilbara

regionofnorthwestAustralia.Usingfluorescencelabelling,thesuperficial

neuromastsystemwasmappedandthenumberandarrangementof

superficialneuromastswasfoundtovarysignificantlyacrosspopulations,

withthenumberofsuperficialneuromastsdecreasingwithincreasedratesof

waterflowunderfieldconditions.

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Thestudyalsoinvestigatedhowintraspecificvariationinthesuperficial

neuromastsaffectstheabilityofthisspeciestoperformrheotaxis(orientation

intowaterflows).Arheotacticstudywasdesignedtodetermineifdifferences

inthesuperficialneuromastarrangementhadaneffectontheirabilityto

correctlyorientateatdifferingflowspeeds.Chemicalablationwasalsousedto

determinetheroleofthelaterallineinthisbehaviour.Differencesinthe

abundanceofsuperficialneuromastsaffectedtheabilitytoorientateinto

waterflowsandrainbowfishesshowedareducedabilitytoorientatewithout

theuseofafunctionallateralline.Furtherstudyisrequiredtofully

understandwhetherindividualpopulationshavethecapacitytoadapttheir

laterallinesystemtoalteredhydrologicalconditions.

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TableofContents

Summary............................................................................................................................2

Acknowledgements........................................................................................................6

DeclarationofCanditurecontribution...................................................................9

ChapterOne.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.............................................................................................................................56

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

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

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

Conclusions.............................................................................................................................87

Chapter4.Generaldiscussion.................................................................................88

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

References......................................................................................................................97

Appendices...................................................................................................................104Appendix1:...........................................................................................................................104

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Acknowledgements

ThisresearchwassupportedbytheAustralianResearchCouncilin

collaborationwithRioTintoandBHPBilliton(ARC‐LP120200002).Aspecial

thankyoutoSamLuccittiandSuziWild(RioTinto)forfacilitatingaccessto

WeeliWolliCreekandtheUpperFortescueCatchmentandforfeedbackduring

theprojectmeetings.

Firstandforemost,Iwouldliketoacknowledgemysupervisorsfortheir

incrediblesupportandguidancethroughoutmyresearch.IthankShaunCollin,

PaulineGrierson,JenniferKelleyandJanHemmifortheirconstant

commitment,encouragement,supportanddedicationtomeandmythesis.

TheirhelphasshapedmeintothescientistIamtoday.IacknowledgeJennifer

Kelleyforherconstantaccessibility,willingnesstohelpwithanythingthatI

neededatanypointandalsoherguidanceonthefieldtriptothePilbara.You

weresoexceptionallypresentateverymomentthroughoutmythesis.I

acknowledgeShaunCollinforhisparticularlyupbeatandpositiveattitude,

especiallywhenIwasloosingmotivation,paramountguidanceandincredible

knowledgethroughoutmyproject.IacknowledgePaulineGriersonforbeing

anincrediblerolemodel,supportsystemandhavingthewillingnesstohelpat

anypoint.Shewasalsoanincredibleemotionalsupportthroughoutthistime.

AspecialacknowledgementgoestoJanHemmi.Hisguidanceandassistance

duringthesecondchapterwasunwavering,despitenotbeinganofficial

supervisortobeginwith.Hisdedication,knowledge,understandingand

helpfulnesstohisstudentsandmyself,wasabsolutelyastonishing.Without

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him,IwouldnotbeinthepositionIamintodayandIowethecompletionof

mysecondchaptertohim.

IwouldliketoacknowledgethreeincredibletechniciansatUWAfortheir

assistanceandguidanceaswell.CameronDuggin,CarlSchmidtandRick

Roberts.Withouttheirassistanceandwillingnesstohelpcreateandfix

microscopesandflumes,myresearchwouldhavebeenconsiderablyinhibited.

AspecialmentiontoCarlSchmidtforhishandymanskills,supportand

knowledgewithhelpingmecreatetheperfectarenaformysecondchapter.

ThankstoSamanthaLostromforherhelpandadviceonthefieldtripinApril

2014.AndreSiebersandJordanIlesalsoprovidedadditionalsitedataand

assistedwithidentificationofinvertebrates.

Onamorepersonalnote,Iwouldliketoacknowledgemyfriendsandfamily

whohavesupportedmeoverthelastfewyearsandwithoutwhomIwouldnot

havebeenabletoachievecompletion.Thankyoutomyfellowscientists,

AdelaideBevilaquaandPennyBrooshooftforbeingsoundingboards

wheneverIneededitandtoTanyaHevroyforprovidingadviceandguidance

fromthebeginning,despitethevastdistancebetweenus.Thankyoutomy

parentsRoslynandGeofffortryingsoeagerlytounderstandthetopicofmy

thesisandforprovidingsupportduringthehardtimes.Aspecialthankyouto

mysisterandbossJacquieSpiller,whowhenneededwasmorethanwillingto

findmeextratimeoff,orcoverformeintheworkplacewheneverIhadto

makethisthesismoreofapriority.

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LastbutnotleastIwouldliketothankmyincrediblysupportingpartnerJean‐

SebastianCorreiaandhisverykindparentsHelenandCarlos.Carlosthankyou

foryouconstantadviceontheacademicenvironment.Jean,youhavebeenmy

biggestenthusiastandhavealwaysprovidedmewithlove,supportand

empathy.Youhavealsoalwaysbeensoincrediblygenerouswithyourtime

andwillingtoassistmeinanythingthatIneeded.Hekeptmepositiveand

driventhroughoutthisentireprocessandIwillbeeternallygrateful.

9

DeclarationofCandidaturecontribution

ThisthesisdoesnotcontainworkthatIhavepublishedorworkcurrently

underreviewforpublication.

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ChapterOne.Generalintroduction

Sensory systems fundamentally link species to their environments, yet there

have been few studies that consider the ecological resilience of sensory

systemstoenvironmentalchange.Thisisparticularlythecaseforfreshwater

habitatsglobally,asthesearedegradingatanalarmingrateandmorerapidly

than any other ecosystem (Kingsford, 2011). Consequently, greater

understanding of the responses of any given species to altered habitat

conditions is necessary to determine the likelihood of its survival and has

becomeatopicofgreatinterestforecologistsglobally.

Survivalinachangingenvironmentdependsonaspecies’abilitytoadaptkey

fitness traits through phenotypic plasticity, contemporary evolution or a

combination of both of these processes (Palkovacs, Kinnison et al. 2012).

Whileithasbeenshownthathumanactivities,suchasthedammingofrivers,

cancausechanges inmorphological,physiologicalandbehaviouraltraitsofa

rangeofspecies(Palkovacs,Kinnisonetal.2012),itisunclearhowthesetrait

changes affect species survivorship in the long term. It has only recently

emergedthatsensorysystemscanvaryamongindividualsofthesamespecies

(Wark & Piechel, 2010). If such within‐species variation is evident in

freshwater fishes then a study of a single species across a diverse range of

habitats couldprovideauniqueopportunity tounderstand the linkbetween

environmentalvariationandsensorytraitspecialisations.

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Inordertosurviveinaquaticenvironments,fishesandsomeamphibianshave

evolvedauniquesensoryorgancalledthemechanosensorylaterallinesystem

(hereafterreferredtoasthe‘laterallinesystem’),whichallowsthemtodetect

andreacttoevenminutechangesinwatermovementwithintheirimmediate

environment(Dijkgraaf,1963).Thelaterallinesystemishighlyspecialisedto

suit theneeds of the animal in its particular habitat (Eros. etal. 2003). For

example,predationpressure(McHenryetal.2009),developmentalconditions,

micro‐habitat(Beckmannetal.2010,Vanderphametal.2013)andwaterflow

speeds(Wark&Piechel,2010)areallconsideredpotentialcausesofvariation

inthelateral linesystembothwithinandamongfishspecies.Thelateral line

system allows fishes to detect prey, avoid predators (Montgomery and

Macdonald1987),communicateandshoalwithconspecifics(Partridge1980),

as well as discriminate objects and orientate into water flows (known as

rheotaxis) (Montgomery 1997, Baker and Montgomery 1999, Bleckmann

2008).Therefore,thelaterallinesystemisthebasisofmanykeysurvivaltraits

infishes(Engelmann,Hankeetal.2002,Bleckmann2009).

The lateral line system is comprised of a series of bundles of hair cells

(neuromasts)thatextendovertheheadandalongthelateralflankofthetrunk

andbody (Fig.1;Webb1989,CartonandMontgomery2004,Wellenreuther,

Brock et al. 2010). These bundles of hair cells may occur within pored or

unpored water‐filled canals or sit on the superficial surface of the skin or

scales(Bleckmann2009).Therearetwodistincttypesofspecialisedreceptor

cells; superficial and canal neuromasts. Superficial neuromasts are arranged

onthesurfaceoftheskin(CartonandMontgomery2004)andaremostlyused

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todeterminethevelocityofthesurroundingwater(WarkandPeichel2010).

Superficialneuromastsalsofacilitaterheotaxis(thebehaviouralorientationto

watercurrents)asthesecellsareconstantlystimulatedbywaterflow(Baker

and Montgomery 1999). Canal neuromasts usually occur in a distinct line

beneath the skin and are used for both the detection and discrimination of

objects (Engelmann, Hanke et al. 2002). Typically, one canal neuromast is

found between two pores (Engelmann, Hanke et al. 2002). Visually‐

compromisedspecies,suchastheblindMexicancavefish,(Astyanaxfasciatus)

tend to have a particularly well‐developed canal and superficial system, as

they rely heavily on the lateral line sense to navigate within their dark

environment(Windsor,Tanetal.2008,Delfinn2011).

Each neuromast comprises a specialised region of epithelium fromwhich a

bundleofhaircellsprotrude.Thehaircellbundlecontainsonelongkinocilium

andanumberofsmallerstereocilia,whichallextendintoagelatinouscupula.

After theonsetofahydrodynamicevent, thecupulamoveswithin thewater

filled canal but out of phasewith the rest of the body. The cupula (and the

embedded hair cells) adopt a range of orientations but reveal a specific

polarity, whereby stimulation of the stereocilia in one direction (in the

directionofthekinocilium)willelicitexcitation(andtherebydepolarisation),

whilst stimulation in the other direction (in the direction away from the

kinocilium)willelicitinhibition(andtherebyhyperpolarisation)(Engelmann,

Hanke et al. 2002, Schmitz, Bleckmann et al. 2008). While the lateral line

system has been well studied in terms of differences in structure and

morphologyacrossspecies(Wonsettler&Webb.1997,Rouse&Pickles.1991,

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Vischer, 2013), very few studies have investigated the abundance and

distributionofneuromastswithin a species, especiallya speciesoccupyinga

diversityofhabitats.

The function of the lateral line system and its link to water flow has been

described in varyingdetail throughout the years, for example Shulze (1870)

wasthefirsttosuggestthelaterallinefunctionsasaflowsensor.Howeverin

1963Dijkgraafproducedapioneeringstudy,“Thefunctioningandsignificance

ofthelaterallinesystem”whichsolidifiedthelinkbetweenthelaterallineand

flow. Since then, many studies have directly examined the relationship

betweenthehydrodynamicenvironmentandlaterallinemorphologyandhave

foundthatlaterallinemorphologyisanadaptiontothehydrodynamicnoiseof

aspecies’environment.Typically,relativelysedentaryfishesthatinhabitquiet

orstillenvironments,suchaslargelakesordams,wouldpossesswidecanals

or even have lost the canal structures with prolific numbers of superficial

neuromasts (Beckmann, Eros, 2010). On the other hand, fishes that live in

“noisy”or fast flowingenvironments, suchas streamsor rivers, are likely to

have a well developed canal system with low numbers of superficial

neuromasts(Englemann,Hankeetal.2002,Beckmann,Erősetal.2010).

Most studies to date have compared lateral line system diversity among

differentspeciesoffishes(Englemannetal.,2002,Beckmann,Erősetal.,2010,

Vanderpham et al., 2013, Vischer, 1990) or have investigated lateral line

system variation in a single species that occupies divergent habitats (Wark

Piechel,2010).Forexample,in2010,WarkandPeichelconductedathorough

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study of threespine sticklebacks (Gasterosteus aculeatus), comparing the

distribution, typeand totalnumberofneuromasts found in individuals from

lake,streamandmarinepopulations.Theyfoundasignificantvariationinthe

total number of superficial neuromasts present on the body among the

differentpopulations(marineandfreshwater),andattributedthesefindingsto

thewater flow characteristics found in these different habitat types. Similar

findingswere reported by Trokovich et al.(2011),who found differences in

themorphologyand the totalnumberof superficialneuromasts inninespine

sticklebacks (Pungitius pungitius). However, these authors attributed their

findingstogeneticinfluencesarisingfromhabitatdivergence(Trokovichetal.,

2011). A further study by Fischer et al. (2013) on the Trinidadian guppy

(Poeciliareticulata)alsoreportedhabitat‐relateddivergenceinthesuperficial

neuromast system, but, unlike the two aforementioned studies, the authors

were able to attribute the observed intraspecific variation to variation in

predation pressure. Collectively, these studies suggest that the lateral line

systemishighlyspecialisedfordetectingandrespondingtovariationinwater

flowsandpredationpressure. Indeed,examiningvariation inthe lateral line

systemofasinglespeciesthatoccupiesacontinuumofhabitatsisapowerful

wayofidentifyingthesesensorysystemspecialisations.

It is clear that there are many reasonable ecological explanations as to the

pressures that promote intraspecific variation in the lateral line system.

However,nostudyhascomparedalloftheputativeenvironmentalfactorsthat

affectfishbehaviourinrelationtothefunctionalattributesoftheirlateralline

system in a single cohesive study. To better understand the functional

15

significanceofthelateralline,itisimportanttounderstanditsinvolvementin

everyday behaviours. One of the basic roles of the lateral line system is

rheotaxis, the natural orientation of an animal’s body by swimming directly

intotheflow(Dijkgraaf,1963).Thisbehaviourisimportantforfishspeciesas

it allows individuals to remain stationary in an otherwise dynamic

environment.Rheotaxisisusedtoreceiveinformationfromupstreamthrough

food and olfactory signals being carried by the flow (Baker &Montgomery,

1999,Montgomeryetal.,1995)andisalsousedinmigratorybehaviour.Fishes

tend to show stronger rheotaxis in faster flowing water than in no flow

environments(VanTrump&McHenry,2013).Dijkgraaf(1963)wasthefirstto

demonstratethelateralline’sinvolvementinrheotaxis,althoughsomestudies

haverefutedthis.Forexample,VanTrumpandMcHenry(2013)conducteda

studywiththeMexicanblindcavefish(Astyanaxfasciatus)andfoundthatthe

animals were able to orientate correctly with and without their lateral line

chemicallyblocked,suggestingthatthelaterallineisnotinvolvedinrheotaxis

in this species. Similarly, Bak‐Coleman et al. (2013; 2014) suggests that the

lateral line is either not involved in rheotaxis or only involved in sedentary

species.Otherstudieshavesuggestedthatthelaterallineworksinconjunction

withothersenses,suchasvisionandthevestibularandolfactorysenses(Lyon,

1904,Dijkgraaf,1963,Arnold,1974). Thesesomewhatcontradictorystudies

demonstrate that the function of the lateral line system in relation to flow

conditionsremainsunclear, inpart,becauseofthelimitednumberofspecies

andenvironmentsstudied.

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Asmany freshwater habitats worldwide are now exposed to anthropogenic

influences such as climate change, it is important to understand how

populations will respond to altered water flows, dependent on their

population‐specific thresholds. Populations from either naturally fast or

naturallyslowflowingriversandstreamswilllikelyvaryintheirresiliencein

responsetoachangeinflowregime.Similarly,fishpopulationsfrommoreor

lessvariableecosystems(e.g.perennialversusmoreintermittentrivers)may

alsorevealdifferencesintheresilienceoftheirlaterallinesystemtochangeor

the propensity for plasticity. For example, fishes that inhabit large bodies of

stillwater,suchaspondsordams thathaveneverbeenexposedtodynamic

flows,maynotbeabletoadequatelyperformrheotaxisifconditionssuddenly

change to fast flows, and the reverse may be found in individuals from

dynamicenvironments.

In Australia, negative anthropogenic impacts on aquatic ecosystems from

alteredwaterflowsareexpectedtobeexacerbatedbytheprojectedeffectsof

climate change (Kingsford 2011). Arid and semi‐arid lands are especially

vulnerable owing to an expected increase in the intensity of flood events

separated 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 to

discharge)cansignificantlyalterstreamflowsandconnectivityofpoolswithin

individualstreams. Incontrast,streamsmaybecome"overpowered" froman

energetic perspective, when they are transformed from a shallow, slow

17

flowing stream to a large and sustained fast flowing body of water; these

conditionsareunlikely tohavepreviouslybeenencounteredbymanynative

streamfishesduringtheirevolutionaryhistory(Baxter,1977).Whilesomefish

speciesareknowntoberesilient to theseenvironmentalvariations (Stewart

2013), therehavebeennostudiesofAustralian inlandecosystems thathave

testedfordetrimentaleffectsofalteredflowsonfish andconsequentlytheir

behavioural responses. Knowledge of the fundamental physiological,

morphological and behavioural adaptations of fishes to variations in water

quality and water flows underpins the development of "best practice" for

managingstreamsandriversinthefaceofincreasinghydrologicdisturbance.

Theexperimentspresentedinthisthesisfocusoninvestigatingthelateralline

system of the western rainbowfish, Melanotaenia australis (Family

Melanotaeniidae),afreshwaterspeciesendemictonorthwestAustralia.Their

distribution ranges from the Pilbara region of Western Australia to the

NorthernTerritory(Allenetal.,2002).Westernrainbowfishesinhabitawide

range of environments such as pools, creeks and lakes (Kelley et al., 2012),

wheretheyareoftenfoundnearthewater’sedgenearvegetation.Despitethis

highly dynamic and heterogeneous environment, M. australis form a large

proportion of the local fish communities of the Pilbara and are commonly

locatedinunstablepools,whicharehighlyvariableinflowvelocity,depthand

otherattributesbothwithinandamongyears(Morgan&Gill,2004,Beesley&

Prince,2010).M.australisexhibitsconsiderablegeographicvariation inbody

shapemorphology (Lostromet al., 2015), and this suggests that their lateral

linesystemissimilarlyvariableamonghabitats.Thelaterallinesystemofthe

18

western rainbowfish and its role in behaviour has not been investigated

previously.

Aimsofthisstudy

Thisthesisdescribesthemorphologyofthelateral linesystemofM.australis

and determines whether population differences in the number and

arrangementofneuromastsarerelatedtotheenvironmentalcharacteristicsof

the habitat. I also investigate the consequences of intraspecific lateral line

diversity on one of the most important and innate fish behaviours i.e.

rheotaxis.

In this first introductory chapter, I have provided a conceptual basis formy

thesis.TheexperimentalcomponentofthisresearchisdescribedinChapters2

and 3. These Chapters are formatted as "stand alone"manuscripts so some

repetitionisunavoidable.Chapter2characterisesthelaterallinesystemofM.

australis.Specifically,Isoughttorevealthemostimportantfactorsinfluencing

sensorysystemspecialisationbyinvestigatingwhetherpopulationdifferences

in thearrangementandnumberofneuromastsare related toenvironmental

variables such aswater flow, predationpressure, prey availability, turbidity,

pHandhabitatcomplexity.

Lateral linemorphology isdescribed for155wild fishescaptured fromeight

populations from the Fortescue River in the Pilbara, Western Australia.

Neuromastsarevisualisedusingafluorescentvitaldyetomapthelateralline

19

system and compare the number and arrangement of neuromasts among

populations.

Chapter 3 investigates the ability of M. australis to perform rheotaxis at

differentwaterflowspeedsinalaboratorysetting.Itestedtheextenttowhich

this species relies on the lateral line to perform rheotaxis by chemically

blocking thesuperficialneuromastsusingneomycinsulphate. Ialsoassessed

the functional significance of the intraspecific variation in the lateral line

system found in Chapter 2 by testing the ability of fishes from different

populations to orientate at varying water flows. Finally, in Chapter 4, I

summarisemymainfindings,assesssomeofthe limitationsofthestudyand

suggestpossibleareasoffuturefocus.Theimplicationsofthefindingsarealso

considered in regard to the management of freshwater ecosystems in the

Pilbarasubjecttoalteredflows.

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Chapter2.Theebbandflowofthewesternrainbowfish

(Melanotaeniaaustralis):howlaterallinesystemdiversity

relatestohabitatvariability.

AbstractFishesusetheirmechanoreceptivelaterallinesystemtosensenearbyobjects

bydetectingslightfluctuationsinhydrodynamicmotionwithintheir

immediateenvironment.Specialisationsofthelaterallinesystem,inparticular

thedistribution,abundanceandlocationsofsuperficialneuromasts,havebeen

investigatedamongspeciesfromdifferenthabitats,especiallythosewith

varyingdegresofwaterflow.However,populationsofasinglespeciesoften

occupyhighlyvariablehabitatsanditisunknownwhethersuch

environmentalvariation,forexamplewaterbodieswithdifferentlevelsof

flow,isreflectedbyintraspecificvariabilityinthelaterallinesystem.Here,I

describethefirstinvestigationofthelaterallinesystemofthewestern

rainbowfish(Melanotaeniaaustralis),awidespreadspeciesacrossfreshwater

systemsinnorthwestAustralia.Iexaminedwithinandamong‐population

variationinthedensityandarrangementofsuperficialneuromasts.Eight

populationsweresampledfromadiversityofcatchmentsinthePilbararegion

ofnorthwestAustralia.Scanningelectronmicroscopy(SEM)andfluorescent

dyelabellingwerebothusedtodeterminethearrangementanddensitiesof

thesuperficialneuromastsandthecanalporeopenings.Ifoundthatthe

superficialneuromastsystemofM.australiswashighlyvariableinthedensity,

location,andarrangementofneuromastsamongindividuals,populationsand

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bodyregions,andwasparticularlyvariableinthecheekregion.Additionally,I

foundthatfishesfromafewofthepopulationstendedtodisplaygreater

among‐individualvariabilityinsuperficialneuromastnumberthanthosefrom

othersites.Largeranimalspossessedmoresuperficialneuromaststhan

smalleranimalsandthenumberofsuperficialneuromastsincreasedwith

benthicinvertebrateavailability.Inaccordancewithstudiesthathavelinked

interspecificvariationsinsuperficialneuromastwithwaterflow,wealso

foundasignificantnegativecorrelationbetweenthewaterflowrateatthe

collectionsiteandthetotalnumberofsuperficialneuromastsonthebody.Our

findingthatasinglespeciescandisplaysignificantamong‐populationvariation

inakeysensorysystemsuggeststhattheabilitytoacquiresensory

informationishighlytunedtoboththeanimal’shabitatanditsbehaviour.

IntroductionAllfishesfeatureauniquesensoryorgan,thelaterallinesystem,whichallows

themtoreceivebothphysicalandbiologicalinformationabouttheir

environment(Mogdansetal.2004).Thelaterallinesystemformsthebasisof

manykeysurvivaltraitsinfishes(Engelmann,Hankeetal.2002,Bleckmann

2009)andunderliesmanybehaviouraladaptations,includingpredator

avoidance(Montgomery&Macdonald1987),communicationwith

conspecifics(Partridge1980),objectdiscrimination,andorientationtowater

flowsor‘rheotaxis’(Montgomery1997,BakerandMontgomery1999,

Bleckmann2008).Whilepredationpressure(McHenryetal.2009),lifecycle,

micro‐habitat(Beckmannetal.2010,Vanderphametal.2013)andwaterflow

22

speed(Wark&Piechel,2010)mayallcontributetoobserveddifferencesinthe

laterallinesystemamongspecies,thereisstillconsiderableuncertaintyasto

whatenvironmentalfactorsmaybedrivingspecies‐specificspecialisations.To

date,studiesofthelaterallinesystemhaveprimarilyfocussedoninterspecific

variationorhavecomparedthelaterallinemorphologyofthesamespeciesin

highlydivergenthabitattypes.Forexample,WarkandPiechel(2010)

comparedthesuperficialneuromastarrangementinthethreespine

stickleback(Gasterosteusaculeatus)betweenindividualsoccupyingmarine

andfreshwaterhabitats.Thelaterallinesystemisakeytraitinfluencingfish

behaviourandadaptability.Therefore,animprovedunderstandingofthis

sensorymodalityunderpinsthecapacitytobetterpredicttheresilienceof

freshwaterfishestochangingenvironmentalconditions,especiallyflow

dynamics.Thisstudywillprovideevidenceessentialtoimprovingthecurrent

understandingofthelaterallinesystemandpresentnewfindingsinone

comprehensiveanalysis.

Thelaterallinesystemiscomprisedofaseriesofbundlesofhaircells

(‘neuromasts’)thatextendovertheheadandthelateralflankoffishes(Webb

1989,CartonandMontgomery2004,Wellenreuther,Brocketal.2010).These

neuromastscomprisetwodistincttypesofspecialisedreceptorcells,

superficialneuromastsandcanalneuromasts,whichdifferintheir

performanceandfunction,despitesimilaritiesinbasicstructure.Superficial

neuromastsarelocatedonthesurfaceoftheskin(Carton&Montgomery

2004)andmostlyfunctiontodeterminethevelocityofthesurroundingwater

(Dijkgraaf,1963).Theydifferfromcanalneuromastsastheyareableto

23

respondtoflowthatisnotorthogonaltotheirorientationaxis,whilecanal

neuromastsarelimitedtothedirectionoftheaxisofthecanal(Janssen,2004),

curvedcephaliclaterallinecanalsmayrespondtolaminarflow,providedthat

onlysomeofthecanalporesaredirectlyexposedtoincomingflow

(Bleckmann,Pers.Comm).Superficialneuromastsalsofacilitaterheotaxis

(bodyorientationintocurrents)asthesecellsareconstantlystimulatedby

waterflow(Baker&Montgomery1999,Mogdans&Beckmann,2012).The

canalneuromasts,ontheotherhand,usuallyoccurinadistinctline,sittingat

thebaseofacanalrunningbeneaththeskinandextendingovertheheadand

flank.Thecanalsreducethespeedoflaminarflow,andlowfrequencywater

movements,byactingashigh‐passfilters(Janssen,2004).Thecanal

neuromastsrespondtohighfrequencystimuliwithinthesewatermovements

andarethereforeusedforboththedetectionanddiscriminationofobjects,

suchaspredatorsandpreyinthelocalenvironment(Mogdansetal,2004).

Consequently,characterisingthediversityinabundanceanddistributionof

bothsuperficialandcanalneuromastswithinandamongpopulationsofany

givenspeciesmayprovidesomeinsightsintotheiradaptivecapacityto

changingflowconditions.

Canalneuromastsaretypicallyarrangedsothatonecanalneuromastis

situatedbetweentwocanalpores,whichactastheaccesspointsforwaterto

enterthecanals.Thecanalneuromastsrespondtohydrodynamicpressureas

thefluidmovesinandoutofneighbouringpores,allowingtheneuromaststo

detecttheaccelerationofthewateraroundthefish’sbody(Wark&Piechel,

2010,Engelmann,Hankeetal.2002).Ithaslongbeenrecognisedthat

24

hydrologicalfactors,suchaswaterflowrate,canresultintheevolutionof

particularfunctionalmorphologiesofthelaterallinesystem(Dijkgraaf1963).

Thissystemisdocumentedtobeanadaptationtotheecologicalrequirements

ofaparticularspecies,andcanalsoexhibitsomeinterspecificvariability

accordingtolifehistorystageandthelocalhydrologicalconditions.For

example,speciesthatliveinenvironmentswithslowmovingwater(or‘quiet’

environments)havealargenumberofsuperficialneuromastsandareduced

number,orcompleteabsence,ofcanalneuromasts(Wark&Peichel2010,

Mogdans&Bleckmann2012).Bycomparison,speciesthatliveinturbulent,

fast‐flowing,‘noisy’environmentstendtodisplayfewersuperficial

neuromastsandawider,moredeveloped,canalsystemwithalargenumberof

canalneuromasts(Janssen,2004,Wark&Peichel,2010).Thereisalsosome

evidencethatsuggeststhatthelaterallineislinkedwithotherenvironmental

andbehaviouralfactorssuchasfeedingbehaviour(Montgomery&Macdonald,

1987,Fischer,etal.2013,Mchenry,etal.2009)andthestructuralcomplexity

ofthehabitat(Erosetal.2003).Forexample,anenvironmentthatisspatially

andtemporallyvariableanddisplaysinconsistenciesalongagradient,suchas

acomplexstream,wouldbehardertonavigatethanafairlyhomogeneous

environmentsuchasalargepoolorlake(Erosetal.2003).The

aforementionedstudieshaveconsideredsinglefactorsascausesofthe

variation,howevernonehaveconsideredallfactorswithinasingle,cohesive

study,whichistheaimofthisinvestigation.

Inthisstudy,Iexaminedvariationinthelaterallinesystemofthewestern

rainbowfish(Melanotaeniaaustralis),ahighlyubiquitousspeciesofteleost

25

foundinmanydifferentfreshwaterenvironmentsthroughoutthePilbaraand

KimberleyregionsofnorthwestAustralia(Allenetal.2003,Kelleyetal.2011).

Habitatsofwesternrainbowfishesincludeephemeralpools,creeksandlakes

(Allen,etal.2003),wheretheyformalargeproportionofthelocalfish

communities(Morgan&Gill,2004;Beesley&Prince,2010).Despitewestern

rainbowfishesbeingcommonthroughoutnorthwestAustralia,therehave

beenveryfewecologicalstudiesofthisspeciesanditslaterallinesystemhas

neverbeenformallydescribed.

Mystudyfocussedonadultrainbowfishescapturedfromeightsites(i.e.from

eightpopulations)acrossthesemi‐aridPilbararegionofnorthwestAustralia.I

soughttofirstdescribethearrangementofneuromastsonthebodyusing

fluorescentstaining(DASPEI)andscanningelectronmicroscopy(SEM).Ithen

assessedhowmuchofthevariationinthelaterallinesystemofeach

populationcouldbeattributedtoparticularhabitatcharacteristics,including

surfaceandbenthicinvertebrateavailability,waterdepth,flowrate,turbidity

andameasureofhabitatcomplexity.Ihypothesisedthatwaterflowrate

wouldbeamajordeterminantoflaterallinesystemvariation.Iexpectedthat

fishcollectedfromlowornoflowpoolsandcreekswoulddisplayagreater

numberofsuperficialneuromaststhanfishcollectedfromsiteswithfaster

flowingwater.

26

MaterialandmethodsStudyareaandmodelspecies

Westernrainbowfishesweresampledfromtwogeographicallydistinctsub‐

catchmentsoftheFortescueRiverinthePilbararegionofnorthwestAustralia,

encompassingsiteswithadiversityofwaterflowsandhabitatcomplexities.

TheFortescueRivertraversesover570kmandformsacatchmentareaof

480,000km2withthelowerwesternpartofthecatchmentdrainingacrossthe

plainsintotheIndianOcean,whiletheuppereasternregionofthecatchment

drainsfromtheHamersleyRangesintotheFortescueMarsh(Barrett&

Commander,1985).The"mid‐Fortescue"istechnicallypartofthegreater

LowerFortescuecatchment.TheflowregimeintheFortescueRiverandits

tributariesisdirectlylinkedtorainfall,withseasonaldischargeduringthewet

monthsofJanuarytoMarch(Rouillardetal.2015).Rainfallaveragesforthe

regionisaround350mmperyearbutishighlyvariablebothwithinand

amongyears(AustraliaBureauofMeteorology,2011;O'Donnelletal.2015).

Theareareliesonthesehighrainfallperiodstosustainvariouspoolsalongthe

drainagelinesandthenreconnectduringthistime.Therefore,the

biogeochemistry,hydrologyandecologyofthepoolsisinextricablylinked

(Fellmanetal.2011'Siebersetal.2015).

TheclimateofthePilbaraissemi‐aridandsub‐tropical,withrainfalloccurring

intheformofcyclonesandtropicalthunderstorms,followedbyprolonged

periodsofdroughtthroughoutthewinter(AustralianBureauofMeteorology,

2011).Summertemperaturesrangefrom24to40oC,whileinthewinter,

temperaturesrangebetween11and26oC,suchthatannualpanevaporation

27

(2500mm)farexceedstheannualaveragerainfall(Fellmanetal.2011).

Duringsummerperiodsofheavyrainfall,poolsbecomeswollenandcan

connectandspilloutontothefloodplain(Beesley&Prince,2010).Incontrast,

duringthewintermonthsthewaterwayscanbecomeconstrictedthrough

evaporationtoformachainofpools,alongadrainageline(Beesley&Prince,

2010,Fellmanetal.2012;Siebersetal.2015).

AdultrainbowfisheswerecollectedfromCoondinerCreekandWeeliWolli

Creek(intheupperFortescuecatchment)andfromsixsiteswithinMillsteam‐

ChichesterNationalPark(inthemidFortescuecatchment)duringApril‐May

2014(Table1).CoondinerCreektypicallycomprisesaseriesofunstable,but

hydrologicallyconnected,poolsthatrunalongthemaingorgeline,whichare

largelyreliantonrainfall(Fellmanetal.2011;Siebersetal.2015).WeeliWolli

Creekencompassesadensenetworkoftributariesthatflowinanortherly

directionintotheFortescueMarsh(Dogramacietal.2015).Theregionis

subjecttosignificantminingactivityandconsequentlysomeofthecreeksin

theareaaresubjecttoadditionalwaterflowsduetodewatering.Itis

estimatedthat0.92GLofwaterisbeingpumpedintothecreekfromthe

dewateringoftheHopeDownIronOremineannually,whichhassignificantly

changedtheflowregimeofthecreeksincedischargebeganin2006(WRM,

2010;Dogramacietal.2015).Thefreshwaterhabitatssampledfromthemid‐

Fortescuearefedbyanundergroundaquiferthatcreatesalongstringof

permanent,stablepoolsover20km(Skryzpeketal.2013).Thus,flowratesin

thisareatendtobeslowerandpoolsareoftendeeperthanthoseintheupper

Fortescue,forexamplethedepthatDeepReachreaches14.3m.

28

HabitatcharacterisationHabitatsacrossallsiteswereassessedforarangeofattributespriortofish

samplingtominimisedisturbance.Generalcharacteristicsofthesite,suchas

thepresenceorabsenceofpredatorybirds(e.g.herons,cormorants),past

floodlevels(estimatedbytheheightofdebrisfoundinnearbytrees)andthe

percentageofcanopycoveroverpoolswasrecorded.Ialsomeasuredthe

followingattributes:waterflowrate(metrespersecond,ms‐1),benthichabitat

type(seebelow),turbidity(measuredinNephelometricTurbidityunits,ntu)

andthepredatorspeciesobserved(seebelow).Benthichabitattypewas

assessedalongtransectsperpendiculartothebank(orbisectingapool)inan

areawherefishweresightedfromthebank.Thelengthofeachtransectvaried,

dependingonpoolwidth(min:3m,max:8m).At0.5mintervals,a20cm

quadrantwasusedtodeterminethepercentagecoverofdifferentbenthic

habitattypes,whichwerecategorisedaccordingtopercentagesofcoarse(>

4mm)andfine(<4mm)substrateorgravel,aquaticvegetationandrocks.

PhotographsofeachhabitatweretakenwithanOlympus1030SWwaterproof

cameratoprovidearecordofkeyhabitatfeatures.

Benthichabitatsurveysandsitephotographsweresubsequentlyusedto

developahabitatcomplexityrankingrangingfrom1to10.Ascoreof"1"

describedsiteswithlowdiversityinaquaticbenthos,littletonoaquatic

vegetationandlargelyopenwater,whileascoreof"10"wasallocatedtosites

withhighhabitatdiversity,includinghighcoverofaquaticvegetation(suchas

Schoenusfalucatis,Ceratopteristhalictroides),overhangingvegetationand

29

submergeddebris.Siteswereevaluatedbytwoindependentobserversand

thenaconsensusscoregiven.Followinghabitatcharacterisation,aSontek™

Flowtracker,(ahandheldADV:AcousticDopplerVelocimeter)wasusedto

determinewaterflowvelocityat0.5mintervalsacrossthetransect.Flowrate

wasmeasuredfromthewatersurfaceandrecordedasaproportionofthetotal

depthofthewateratreadingsof0.2,0.6and0.8,forexample,0.2refersto

20%ofthedepthbelowthesurface.Thesemeasurementswereaveragedover

themeasurementstations(min:11stations,max:16stations)togiveamean

x,y,zvelocity,andvariationinvelocity(thestandarddeviationofthemean

flowmeasuredovera30secondperiod)foreachsite.Theflowtrackeralso

recordedthemeantemperatureateachdepth.

Theabundanceofsurfaceinvertebratespresentateachsitewasassessedby

sweepinga250mdipnetoverthesurfaceofthepoolinthree10msweeps.

Thenetwasthenemptiedintoatraybyrinsingwithcleancreekwaterand

twoobserverscountedthetypeandtotalnumberofinvertebratescollected

overa5‐minuteperiod.Thespeciesthatwerecapturedincludedwatermites

(orderAcarina),waterstriders(orderHemiptera,familyGerridae),mayflies

andchironomidlarvae(orderDiptera,familyChironomidae).Benthic

invertebratesweresampledusinga500mD‐netandwerecapturedby

tramplingthesubstratewithina1m2areaandsweepingthenetoverthe

trampledareafor30seconds.Thecontentsofthenetwerethenwashed

throughbotha2mmanda500msteelmeshsievewithcleancreekwater.

Twoobserverscountedthetotalnumberofinvertebratescollectedinthe

sievesovera5‐minuteperiodduetotimeconstraintsateachsite.

30

Predationpressurewasassessedthroughonsiteobservationofbirdsthatare

knowntopreyonwesternrainbowfishes.Inaddition,recordsweremadeof

theabundanceofallfishspeciesthatwerecaughtorobservedateachsite.Fish

werecategorisedashigh‐orlow‐riskpredatorsaccordingtotheclassification

ofpredationrisktoM.australisdevelopedbyYoungetal.(2011).For

example,spangledperch(Leiopotheraponunicolor)isanomnivorouspredator

thatisconsideredlowrisk,whileFortescuegrunters(Leiopotheraponaheneus)

andbarredgrunters(Amniatabapercoides)areconsideredhigh‐risk

predators.

Table1.SummaryofkeyhabitatcharacteristicsforMillstreamNationalPark,CoondinerCreekandWeeliWolliCreek.Missingdataarewheresitesweretoodeeptosampleadequately.

Region

Site

ShannonWeiner

HabitatCom

plexity

0.2XFlowVelocity(m

‐s)

0.2XStError

0.6XFlowVelocity(m

‐s)

0.6XStError

Tem

perature(o C)

BenthicInvertebrates

SurfaceInvertebrates

PredationRisk

Turbidity

FortescueRiver,M

illstream

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

Coondiner

Creek 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

31

Fishsampling

Ateachsite,20‐30adultwesternrainbowfishesofmixedsexwerecaptured

usingeithera4mor10mlongseinenet(bothwith6mmmeshsize)

dependingonthesizeoftheareasampled.Fisheswerehousedforuptofive

daysinthefieldinaerated,20Lplasticaquariacontainingcreekwaterand

naturalsubstratefromthecollectionsite.Allsampleswerecapturedduringa

fieldtripinApril2014.LivefishwerethentransportedtotheBiological

SciencesAnimalUnitatTheUniversityofWesternAustraliabyairandplaced

inaeratedaquaria(42x42.5x34)(onepopulationperaquarium)containing

gravel,afilterandanartificialplant.Thetanksweremaintainedunder

fluorescentlighting(12:12hlight:darkcycle)andwerefeddailyonamixed

dietofcommercialflakefoodandArtemianauplii.

ThreeadultswerealsocollectedfromeachoftheCoondinerCreekpools(Pool

7andPool1.5)andfromCrossingPoolOutFlow(inMillstreamNationalPark)

andpreservedonsiteforscanningelectronmicroscopy(SEM)tobecarried

outatthelaboratoriesatUWA.Theseanimalswereeuthanizedusingan

overdoseofMS222(tricainemethanesulfonate;Sigma‐Aldrich,StLouis,

MO,USA)(200mgl‐1)andthenplacedina50mLfalcontubefilledwith

glutaraldehydefixative(25%glutaraldhyde,75%distilledwater(Proscietch,

QLDAustralia)andwerekeptcoolatapproximately15oC.Bubblewrapwas

slottedintothefalcontubetopreventthefishmovingaroundduringtransport

andpotentiallycausingdamagetothesuperficialneuromasts.Fishthatwere

fixedwereusedforassessingthenumber,locationandarrangementofthe

neuromastsovertheheadandbodyusingscanningelectronmicroscopy

32

(SEM).

NeuromastcharacterisationLivefishwerestainedwithafluorescentvitaldye2‐[4‐(dimethylamino)styrl]‐

N‐ethylpyridiniumiodide,DASPEI(LifeTechnologies/MolecularProbes,

EugeneOR,USA)tovisualisetheneuromastspresentonthesurfaceofthe

body(protocoladaptedfromWarkandPiechel,2010).Preliminarytrialswere

conductedatdifferentconcentrationsofDASPEIfor15minutesandthebelow

concentrationwasdeemedadequateforvisualisationofthesuperficial

neuromasts.EachfishwasfirstallowedtoswimfreelyintheaeratedDASPEI

solutionataconcentrationof0.24gin1Lwaterfor15minutes.Fishwerethen

anaesthetisedin200mgl‐1MS222(tricainemethanesulfonate;Sigma‐

Aldrich,StLouis,MO,USA)untillightpressureonthecaudalfinyieldedno

response.Thefishwasthenplacedrightsidedowninapetridishand

examinedusingafluorescencedissectingmicroscope(LeicaMZ75fittedwitha

FITCfilterset;LeicaMicrosystemsInc.,Sydney,Australia).Images(8‐15per

individual)oftheentirebodywerecapturedatamagnificationof0.8X‐1.0X,

usingadigitalcamera(LeicaDFC320).Measurementsofthelengthandsexof

eachindividualwerealsorecorded.Sexwasdeterminedbasedonthe

followingfeatures:malesarebrighterincolourandhavepointeddorsaland

analfins,whilefemalesaredullerincolourandtheirdorsalandanalfinsare

morerounded.Followingflorescencephotography,fishwererevivedinfresh,

aeratedaquariumwaterandreturnedtotheirhousingtank.Individualfish

fromeachpopulationunderwenttheDASPEIstainingandphotography

procedureonlyonce.

33

Oncefishfromallpopulationswerephotographed,thecanalandsuperficial

neuromastswereclassifiedintodistinctregionsonthehead,trunkandcaudal

fin,basedonthemethodsofNorthcutt(1989)andWebb(1989).Thebody

regionsoccupiedbyneuromastswereclassifiedas:rostralsuperficial,nasal

superficial,mandibularcanalandsuperficial,infraorbitalcanalandsuperficial,

supraorbitalcanalandsuperficial,oticcanal,operculumsuperficial,cheek

superficial,postoticcanal,dorsalandventralsuperficialandcaudaltail

superficial(Figure1Aand1B).Anyphotographswherethenumberof

neuromastsinaparticularsectionwereunclear(e.g.duetosuboptimal

labelling)wereexcluded.Thebodywasdividedintoregionsandnotlinesof

superficialneuromasts(Northcutt,1989),astherewassuchalargediversity

inposition,abundanceanddistributionofsuperficialneuromastsoverthe

bodyandacrossindividualsandpopulations(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

Iestimatedthedensityofneuromastsoverthedifferentregionsofthebodyusingthe

freehanddrawingtoolinthesoftwareprogramImageJ,version1.48,(National

InstitutesofHealth,USA).Aftercalibratingtheimageforscale,theoutlineofeachbody

region(seeninFigure1B)wastracedtocalculateitsarea.Thedensityofsuperficial

neuromastswithineachareawasestimatedbycountingthetotalnumberof

neuromasts,withintheareaanddividingbyitstotalarea(inmm2).

Scanningelectronmicroscopy(SEM)Portionsofthehead,bodyandtailofofeachrainbowfishwerefixedinKarnovskys

fixative(10mlsof2.5%glutaraldhyde,5mlof2%paraformaldehyde,5mlof0.13M

Sorensonsphosphatebuffer,pH7.2),refrigeratedforthreedaysandthenusedfor

scanningelectronmicroscopy(SEM).Thesesamplesincludedbothfieldcollected

samplesandlaboratoryfish.Thetissuewasthenwashedinaphosphatebufferand

heatedusingamicrowaveoven(250Wfor40seconds).Sampleswerethenimmersed

inincreasinglyconcentratedethanolbaths(50%,70%,90%,100%,100%)andheated

(asdescribedabove)ateachconcentration.Thesampleswerethenplacedintoa

criticalpointdrierfortwoandahalfhoursuntilthetissuewascompletelydry.Each

pieceoftissuewasthenmountedonastubandsputtercoatedwithgoldpalladium.All

imageswerecapturedwiththeZeiss1555VP‐FESEMatvariousmagnifications

rangingfrom78xto1,647,000x.

36

Figure2.Scanningelectronmicrographofanopercularsuperficialneuromastshowingtheaggregationofcilia.Notethatnotalloftheciliaareupright/intactduetolowlevelsofabrasionduringtransportationfromthefield.

DataanalysesTheoverallvariationintheabundanceofsuperficialneuromastswasfirstdescribedby

calculatingthecoefficientofvariation(CV)foreachpopulation(site)andforeachbody

region.Ifirstcheckedforcorrelationsamongthetotalnumbersofdependentvariables

andexcludedbodyregionsthatweresignificantlycorrelated(Pearson’scorrelations:

dorsaltrunkvs.ventraltrunk:(r=0.556,df=149,P<0.001;caudalfinvs.ventraltrunk:

(r=0.298,df=151,P<0.001):cheekvs.operculum:(r=0.36,df=152,P<0.001).

MultivariateAnalysisofCovariance(MANCOVA)wasthenusedtotestfordifferences

amongcollectionsites(populations),sexandstandardbodylengthonthetotalnumber

ofsuperficialneuromastspresentindifferentregionsofthebody.Testswere

conductedtoensurethedatamettheassumptionsofMANCOVA,includingLevene’s

test,whichtestsforhomogeneityofvariances,andBox’sMtest,whichtestsforthe

equalityofcovarianceamonggroups.Afterconfirmingthatthedatametthe

37

assumptionsofMANCOVAandanycorrelatedvariableswereexcluded,superficial

neuromastabundanceforeachbodyregionwasusedasadependentvariable(=8

dependentvariables),siteasafixedeffect(8‐levels)andstandardbodylength(SL)asa

covariate.Asignificantoveralleffectonthedependentvariableswasinvestigated

furtherbyconductingsubsequentunivariatetestsforeachregionofthebody

separately.

AstherewasasignificanteffectofthecollectionsiteusingMANCOVA,itjustified

furtheranalysestodeterminetheeffectoftheenvironmentalvariablesonsuperficial

neuromastabundance.Isubsequentlyperformedasecondsetofanalyses(Multivariate

AnalysisofVariance:MANOVA)withsiteasarandomeffect(tocontrolforthedifferent

populationoriginsoftheindividualssampled),andtheabundanceofneuromastsin

specificbodyregionsasthedependentvariables.Thisstudyconsideredaneffecttobe

significantatP<0.05.Flowrate,habitatcomplexity,temperature,turbidityand

invertebrateabundancewereenteredasfixedeffects(categoricalandordinalfactors).

Giventhatrainbowfishesweremostcommonlyobservedinanestimatedtop20%of

thewatercolumn,orapproximately30cmbelowthewatersurface,webasedour

analysesofwaterflowrateonmeasurestakenat0.2(20%).Sexandbodylengthwere

includedintheunivariatetestsonlyforbodyregionsthatwerefoundtobesignificant

inapriori(MANCOVA)tests.TheMANOVAandMANCOVAtestswereperformedusing

thestatisticsprogramJMP11.0(SASltd,Cary,NC,USA).

PrincipleComponentsAnalysis(PCA)wasusedtovisualiseanyvariationamongsites

accordingtotheirenvironmentalcharacteristics,includingdepth,temperatureandthe

velocitiesateachflowdepth.Weusedthemeanwatervelocityforeachsite(0.2:

averagedacrossthetransect),habitatcomplexity,meanwatertemperature,mean

38

waterdepth,andthetotalnumberofsurfaceandbenthicinvertebratescaptured.

Similarityamongthesitesintermsoftheirenvironmentalcharacteristicswas

visualisedbyplottingtheresultingprinciplecomponentsusingthesoftwareprogram

Primer6.0(Primer‐Eltd,Ivybridge,UnitedKingdom).Groupscloselydisplayedonthe

principlecomponentsplotsweremoresimilarinenvironmentalvariables.

Results

ThelaterallinesystemofthewesternrainbowfishScanningelectronmicroscopyandfluorescencemicroscopyofDASPEI‐labelled

superficialneuromastsrevealedthatalleightpopulationsofwesternrainbowfish

sampledinthisstudypossessedconsistentlocationsofsuperficialneuromastsoverthe

tenheadregionsandthreebodyregions3(Figure1).Duringanalysis,itbecame

apparentthattherewasnoonebaselineforthepositionsoftheneuromastsi.e.their

positionwasalwaysarrangeddifferentlywithinthedesignatedregion.However,the

superficialneuromastswereprolificacrosstheheadandbodyandwereeitherfound

insmallclustersofvariousshapesorsingularly.Clustersofsuperficialneuromasts

weremostoftenarrangedinacrescentshape,howevertheyalsoformedpatternssuch

ascrossesandabstractgroupings(Figure3,Figure4).

39

Table2.Univariateresultstestingfortheeffectofsite,bodylengthandwaterflowonthetotalnumberofsuperficialneuromastsforeachbodyregion.

EffectBodyregion(superficial) df F‐ratio P‐value

Site

Rostral 7,140 8.111 <0.0001Nasal 7,145 4.236 0.0003Mandibular 7,144 2.487 0.0193Infraorbital 7,145 3.01 0.0056SupraOrbital 7,145 2.439 0.0216Operculum 7,145 7.609 <0.0001Cheek 7,145 1.699 0.1134PostOtic 7,144 4.791 <0.0001Dorsal 7,143 7.212 <0.0001Ventral 7,144 11.547 <0.0001

CaudalTail 7,144 1.853 0.0815

Length

Rostral 1,140 0.025 0.8723Nasal 1,145 2.731 0.1006Mandibular 1,144 0.206 0.6506Infraorbital 1,145 0.049 0.8253SupraOrbital 1,145 0.854 0.357Operculum 1,145 7.312 0.0077Cheek 1,145 12.348 0.0006PostOticSN 1,144 2.248 0.136Dorsal 1,143 7.855 0.0058Ventral 1,144 24.101 <0.0001

CaudalTail 1,144 0.001 0.9721

Flow

Rostral 7,141 11.33 <0.0001Nasal 7,146 3.859 0.0007Mandibular 7,145 3.045 0.0051Infraorbital 7,146 3.047 0.0051SupraOrbital 7,146 2.797 0.0092Operculum 7,146 8.859 <0.0001Cheek 7,146 3.606 0.1134PostOticSN 7,145 7.976 <0.0001Dorsal 7,144 7.212 <0.0001Ventral 7,145 11.548 <0.0001

CaudalTail 7,145 1.853 0.0815

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 0.01

CaudalTail 1,111 1.205 0.2746

41

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

Acomparisonofthelevelofvariationinsuperficialneuromastabundanceforthe

differentbodyregionsrevealedthatthecheekregionshowedthehighestvariationin

superficialneuromastabundance(CV=50%),whilethenumberofsuperficial

neuromastsintheinfraorbitalregionwashighlyconsistent(i.e.lessvariable)across

samples(CV=12%).

A B

42

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

0

2

4

6

8

10

12

14

A

B

C

DSite

43

Whencomparingthedensityofneuromastsamongthedifferentbodyregions

(i.e,numberofsuperficialneuromasts(SNs)permm2ofbodysurface)ofone

representativeindividual,thenasalregionhadthehighestdensity(55.2

SNs/mm2)followedbytherostralregions(35.7SNspermm2).Rainbowfishes

withthehighestpopulationvariationinsuperficialneuromastabundance

occurredatOutCrossing(CV=26%),whilethesitewithrainbowfishes

exhibitingtheleastvariabilitywasWeeliWolliCreek(CV=13%).

44

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

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

CVofSite

JayaMean 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

DeepReachMean 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

OutCrossingMean 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

PalmPoolMean 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

JirndawurranhaMean 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

CrossingPoolMean 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 43.10.16

Range 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 39.20.13

Range 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

45

Figure5.Themeannumberofsuperficialneuromastsacrossallsitesforeachbodyregion.Errorbarsrepresentstandarderrorsofthemean.

Canalsandcanalporeswereclearlydefinedonthehead,formingfourmain

lines:thesupraorbital,theotic,themandibularandinfraorbitalcanals,allwith

visibleclustersofcanalneuromastssituatedatthebaseoftheporeopenings

(Figure1A).Thepositionofthesecanallineswashighlyconsistentamong

individualsandpopulations.Incontrast,nocanalporeswerevisibleonthe

trunkofthebody.

Neuromastabundanceinrelationtobodylength,populationandsexTheMANCOVArevealedanoveralleffectofpopulation(F7,135,=0.62,P=<0.001)

andbodylength(F1,135,=0.10,P<0.001),butnoeffectofsex(F1,135,=0.01,P

=0.20)ontheabundanceofsuperficialneuromastsacrossthebodyof

rainbowfish.Thesubsequentunivariatetestsrevealedthatsitehadasignificant

effectonsuperficialneuromastabundanceforallbodyregionsexceptthecheek

areaandthecaudaltailarea(Table2).

0

20

40

60

80

100

120

140

160

180

200

Meannumberofsuperficialneuromasts

Bodyregion

46

TherelationshipbetweensuperficialneuromastsandwaterflowTheMANOVAanalysisrevealedthattherewasasignificanteffectofwaterflow

rateonsuperficialneuromastabundanceacrossthedifferentregionsofthe

body(F7,137,=0.61,P<0.001),andasignificanteffectofbenthicinvertebrate

abundanceonsuperficialneuromastnumber(F1,104,=0.12,P=0.0006).In

contrast,therewasnooveralleffectofhabitatcomplexity(F1,143,=0.01,

P=0.892),temperature,(F1,123,=0.00,P=0.930),turbidity(F1,87,=0.10,P=0.74),or

invertebrateabundance(F1,123,=1.93,P=0.16)onsuperficialneuromast

abundance.Theeffectofwaterflowonsuperficialneuromastnumberwas

significantforallregionsofthebodyexcludingthecheekandoticcanalareas

(Table2).Thetotalnumberofsuperficialneuromasts(summedacrossallbody

regionsforeachanimal)wasnegativelycorrelatedwithwaterflowrater155=‐

0.35,P<0.0001),revealingthatfishfromstill,orslow‐waterhabitatshavemore

superficialneuromasts,whilethosefromsiteswithfast‐flowingwaterhavea

lowerabundanceofsuperficialneuromasts(Table2).

Figure6.Scattergraphofthetotalnumberofsuperficialneuromastsfoundoneachindividualagainstflowratesfortheeightstudysitesdisplayedbyasingledatapoint.Negativevaluesofflowspeedrepresentflowthatmovedintheoppositedirection.Lineofbestfit(r155=‐0.35,P<0.0001).

0200

400

600

800

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Numberofsuperficialneuromasts

Flowspeed(m‐s)

47

VariationincomplexityofrainbowfishhabitatsUnsurprisinglyforsuchalargeregionalstudy,environmentalcharacteristics

werehighlyvariableamonghabitats.Forexample,theDeepReach(mid

Fortescue)sitewasaverylarge,deepbodyofwater(>14m),wherefishwere

foundswimmingfreelynearthesurfaceandfacedfewobstacles.Incontrast,

poolsatCoondinerCreekorJirndawurranha,werequiteshallow(<2m)and

hadmanyobstaclesanddebristhatwouldcreateacomplexenvironmentfor

navigation,particularlyunderincreasedflow(Figure7).

PrinciplecomponentsanalysisoftheeightsitesrevealedthatDeepReachand

CrossingPoolwerethemostsimilarinhabitatstructure,complexity,flowrates

anddepthprofiles(Figure7).Thehabitatcomplexityratingsalsosupportthese

results,whichrevealedthatDeepReachandCrossingPoolscoredsimilarlyat2

and4,respectively.

Figure7.AplotoftheprinciplecomponentsforsitesatCoondinerCreekandMilllstreamNationalPark.WeeliWolliCreekwasexcludedfromtheanalysisowingtoanincompletedataset.

48

TheJirndawurranha,JayawurranhaandOutCrossingsiteswerethemost

distinctfromeachotherandtherestofthesites.Atthetimeofsampling,flow

ratesvariedbetween0.000ms‐1(atPalmPool)and0.305ms‐1(at

Jirndawurranhachannel;Table1).Aprinciplecomponentsplotofthewater

flowmeasuresrecordedateachsiterevealedthatOutCrossinghadthemost

variableflowspeedsandflowdirectionsacrossthetransect(Figure8),while

CrossingpoolandCoondinerCreekhadthemoststableflowconditions.

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

DiscussionThisstudyhasshownthatthereisasignificantrelationshipbetweentheflow

rateoftheenvironmentandthestructureandabundanceofthesuperficial

neuromastsinthisspecies.Italsosupportspreviousstudiesthathave

concludedthatlimnophilicfishlivinginquieter,slowerenvironmentshave

moresuperficialneuromaststhanrheophilicfishthatlivein“noisier”,fast‐

49

pacedenvironments(Jakubouski,1967,Vischer,2013,Bleckmann,1994,

Coombsetal.1998,Tyke,1990,Dijkgraaf,1963,Englemannetal.2002,

Beckmann&Eros,2010,Tan,2011,Janssen,2004,Teyke,1990).Thisstudy

showedthattheabundanceofsuperficialneuromastsvariedoverspecific

regionsofthebody,andalsovariedamongindividualsandpopulations.The

levelofvariabilitythatwefoundinthesuperficialneuromastswithina

particularbodyregionhasnotbeenreportedinanyotherspeciestomy

knowledge.Ialsoconductedabriefinvestigationofthecanalstructurebutwas

unabletolocateanycanalporesonthetrunk.However,furtherSEManalysesof

morefishacrossagreaterrangeofsitesislikelyrequiredtodefinitively

concludethatthisspecieslacksacanalsystemonthetrunk.Collectively,the

findingsfromthisexperimentsuggestthattheplacementofsuperficial

neuromastsandthecanalsystemcanbeexplainedbysensoryadaptationsto

differentenvironments.

CanalsystemofthewesternrainbowfishThisinvestigationintothecanalstructureofthewesternrainbowfishrevealed

fourcanalsoverthehead;themandibular,theotic,thesupraorbitalandthe

infraorbitalcanals.However,ourbriefinvestigationswereunabletofindany

evidenceofacanalsystemonthetrunkofM.australis.Thisrequiresamuch

morethoroughinvestigation,whichisoutsidethescopeofthecurrentstudy.If

M.australisdoesindeedlackatrunkcanalsystemitwillundoubtedlyhavean

effectontheanimal’smechanoreceptiveabilities.Severalofthepopulations

investigatedinthisstudyareexposedtorelativelyhighflowrateswithhigh

backgroundnoiseandwouldbenefitfromawell‐developedcanalsystem.This

wouldadequatelyallowthemtosenseaspectsoftheirenvironment,suchas

50

predators,preyanddisturbanceswithsuchahighbackgroundnoise(Webb,

1989).Numerouspreviousstudieshaveindeedfoundthatafishfroma‘noisier,’

fasterflowingenvironmentwillhaveamuchmoredevelopedcanalsystemthan

animalsfrom‘quieter’environments(Tyke,1990;Englemannetal.2002;Wark

&Piechel,2010).Theabsenceoftrunkneuromastsappearstobecharacteristic

ofbenthic,planktivorousorschoolingspecies(Webb,1988).Asthewestern

rainbowfishisashoalingspecies,onepossibleexplanationfortheproposed

absenceofthecanaltrunksystemisthatitistheresultofabehavioural

adaptation.

Thisstudyhasalreadyrevealedthattherearedifferencesinthearrangementof

thesuperficialneuromastsystem,butitremainsunclearifthecanalsystemof

thewesternrainbowfishsimilarlydiffersamongpopulations.However,other,

closelyrelated,speciesalsoshowdifferencesintheircanalsystemarrangement.

Forexample,Vanderphametal.(2013)foundthattwospeciesofcommonbully

(GobiomorphuscotidianusandGobiomorphushuttoni)haddifferingnumbersof

canalporesontheheadandattributedthistodifferencesinthehabitatthat

thesefishexperiencedasadults(Vanderpham,etal.2013).Thissuggeststhata

well‐developedcanalstructureinturbulentflowingenvironmentsmayprovide

aselectiveadvantageatcertainlifestages.Allthewesternrainbowfishinthis

studywerematureadultfishesandthereforesubsequentanalysisofjuveniles

mightrevealdifferencesinthecanalstructures.

Incomparisonwithotherspecies,suchasthecardinalfishApogoncyanosoma,

thebodyofthewesternrainbowfishhasaconsiderablenumberofsuperficial

neuromasts;wewouldexpectthatspecieswiththispatternaremoresensitive

51

topreymovementandhavehighercaptureefficiencythanfisheswithfewer

neuromasts(Janssen,2004).Whiletheexactdietofwesternrainbowfishesin

thePilbarahasnotyetbeendocumented,itisplausiblethatitsdietissimilarto

thatoftheeasternrainbowfish(Melanotaeniasplendidasplendida)asthey

occupyasimilarecologicalniche(McGuiganetal.2003).Thedietoftheeastern

rainbowfishincludesmacroalgae(42.5%),aquaticinvertebrates(19.2%)and

terrestrialinvertebrates(12.3%)(Puseyetal.2004).Therefore,itislikelythat

invertebrates(bothsurfaceandbenthic)comprisealargeportionoftheirdiet.

Thedietoftherainbowfishmayexplainthesignificantrelationshipbetween

superficialneuromastnumberandbenthicinvertebratenumbersfoundinthis

study,andthusfoodavailabilitymaybeanotherexampleofthelateralline

showingspecialisationforaparticularenvironmentorbehaviouraltask.

Investigatingthefeedinghabitsinourmodelspeciescouldprovideanother

explanationfortheobserveddifferencesintheabundanceofcanaland

superficialneuromastsamongpopulations,astherearemanyothercompeting

speciesforfoodsourcesthatwereobservedduringfieldworkexpeditions.

Previousstudieshavelinkedthecanalsystemtootherenvironmentalpressures

suchaspredatorandpreyrelationships,asthiscomponentofthelateralline

systemisusedinthedetectionanddiscriminationofobjects.Torrentfish

(Cheimarrichthysfesteri),aspeciesthatresidesinturbulentfastflowing

habitats,hasprolificnumbersofsuperficialneuromastsandasimplebranched

canalsystem(CartonandMontgomery2004),whichistheoppositeofwhat

mightbepredictedifthelaterallinesystemdevelopmentwasdrivenbywater

flowrates.Alternatively,theauthorsconcludedthatotherfactorssuchasthe

52

nocturnalfeedinghabitsoftorrentfishmighthaveastrongerinfluenceonthe

hypertrophyofthetwolaterallinesubsystems(Carton&Montgomery,2004).

Thisexampleprovidesevidencethatsuperficialneuromastandcanal

neuromastpatternscannotalwaysbegeneralisedforspecificrolesandmaybe

specie‐sorhabitat‐specific.Here,Ifoundthatrainbowfishmightalsoshowan

adaptationofitslaterallinetofeedingbehavioursaswellasflowrates.

AbundanceofsuperficialneuromastsofwesternrainbowfishinrelationtoflowMyobservationsforthewesternrainbowfishesareconsistentwithstudiesof

otherfishspeciesthathaveshownsuperficialneuromastsaremorevariablein

theirnumberandlocationthancanalneuromasts(Fischer2013,Webb1989,

WebbandNoden,1993,WebbandShirey,2003).Thepatternobservedinthis

study(moreneuromastsinslow‐flowenvironments)isalsoconsistentwith

mostoftheliteratureanditsdocumentedrelationshiptoflow(Dijkgraaf1963,

Wark&Piechel,2010,Engelmann,etal.2002,BleckmannandZelick,2009,

Bassettetal.2006).Myinvestigationintothewesternrainbowfish,supportsthe

notionthatincreasedhydrodynamicactivity(i.e.a“noisy”environment)will

produceadecreasednumberofsuperficialneuromasts,whileslowordecreased

flowrates(i.e.quieterenvironments)willproduceahighernumberof

superficialneuromasts(Dijkgraaf,1963,Wark&Piechel,2010,Mogdansand

Bleckmann2012,Miller1986,Puzdrowski,1989andVischer,1990).

Thisstudyalsoshowedthatsuperficialneuromastnumberishighlyvariable

bothwithinbodyregionsandwithinpopulations.Inrainbowfishes,themost

denselypackedareasonthebodywerethenasalandrostralregions.Asthese

aretheareasthatmakefirstcontactwithoncomingflow,itislikelythatthisis

53

anadaptationfortheearlyassessmentofhydrodynamicflow.Onestudyhas

previouslylinkedthedistributionofsuperficialneuromastsoverthebodywith

particularbehaviouralfunctions.Yoshizawaandcolleagues(2010)used

VibrationAttractionBehaviour(VAB)andaregion‐specificsuperficial

neuromastablationtreatment(usingVetbondTM,anon‐toxictissueglue)to

determinetheareasthatareusedinthisbehaviourtoavibrationstimulus.They

foundthattherewasasignificantdecreaseintheanimal’sabilitytoperform

VABwhentheSO‐3(Supraorbital3region)anddorsaltrunkareawere

ablated,comparedwithcontrolanimals,wherefunctionoftheseregions

remainedintact.Thisarea(SO‐3)coverspartoftheinfraorbital,operculumand

cheekregionsdefinedinthecurrentstudyandsuggeststhattheseregionsare

largelyresponsiblefordetectingvibrations(Yoshizawa,etal.2010).

Consequently,thisopensupquestionswhethertheseregionsareequally

importantintherainbowfish,orifthenasalandrostralregionsareutilised

moreforthediscriminationofobjects.

ThisstudyshowedM.australisdisplayedhighersuperficialneuromastvariation

(27%)acrossallpopulations,withtheintra‐populationvariabilityvarying

between13%and28%.Forexample,thesevariationsaremuchhigherthanthe

variationsSchmitzfoundin2008.Heexaminedthesuperficialneuromast

systemofthecommongoldfishandfoundonly9%variationinthenumberof

superficialneuromastsacrossthebody(Schmitz,etal.2008).Asalltheanimals

werewildcaught,Iwasunfortunatelyunabletodeterminewhetherthelarge

amountofvariationobservedisduetoenvironmentalselectivepressures,since

highlevelsofvariationcouldsignificantlyaffectsurvival.Fishes,inparticular,

arehighlysensitivetoflowalteration,showingconsistentdeclinesinabundance

54

anddiversity,regardlessofwhetherflowshaveincreasedordecreasedrelative

tothenaturalregime(Poff&Zimmerman2010).Furthermore,theresponseto

alteredflowscanbespecifictoeachspecies(Haxton&Findlay2008),

suggestingthatitmaybecriticaltoassesseachspecies’resiliencetovariable

flowstopredictresponsesatthepopulationlevel.Ifthevariationiscausedby

environmentalfactors,itthereforechangesanindividual’ssensoryboundary

andpotentiallyitsabilitytoadequatelysenseitssurroundings.Furthermore

thismayaffectthedetectionofexternalstimuli.Thehighlevelofinter‐

individualvariationinsuperficialneuromastabundancefoundinthisstudy

suggeststhatindividualswillhavevariablesensoryabilities.Thesedifferences

couldberesponsibleforsubsequentvariationinbehaviouraltraitsandshapean

individual’sperception(Wark&Piechel,2010).If,however,thevariationisdue

togeneticfactorsitislikelythatthiswillbeinstrumentalinnaturalselectionas

wehavealreadydiscoveredthatthelaterallineisresponsibleformanyfitness‐

relatedbehaviourssuchasfeeding,avoidingpredationandmating

(Montgomery&Macdonald,1987).

Incontrast,manystudieshavefoundtheoppositepatternofvariabilityinthe

superficialneuromastsinspeciesinhabitingdifferenthydrodynamicconditions,

suchasfishesfromnoisierenvironmentsdisplayingmoresuperficial

neuromasts.CartonandMontgomery(2004)insteadlinkedthearrangementof

thelaterallinesystemineachspeciestotheirpredatorytacticsanddiurnaland

nocturnalhuntingbehaviours.Theyexplainthatthetorrentfishisanocturnal

feederandthattheprolificnumberofsuperficialneuromastspresentinthis

speciesisresponsibleforfindingpreywithoutvisualcues,abehaviourthey

haveadaptedthroughnaturalselection.

55

PossiblecausesofvariationinsuperficialneuromastabundanceThefindingsofthecurrentstudyraiseaninterestingquestionasmanyofthe

populationsfoundinMillstreamNationalParkareconnectedduringthewet

seasonandwouldhaveaccesstogeneticmixing.Therefore,itwouldbelikely

thatthereisastrongergeneticbasistotheobservedvariation.Manystudies

haveattemptedtodeterminewhetherthelaterallinevariationfoundamong

populationsisduetoenvironmentalorgeneticdrivers,(oravariationofboth)

andmosthaveconcludedthatitisindeedacombinationofboth(Fischer,2013,

Trokovich2011).However,certainregionsofthelaterallineappeartobemore

influencedbyonedriverthananother.Fischer’s(2013)investigationofthe

Trinidadianguppy(Poeciliareticulata)concludedthatpredationpressureisan

environmentalfactorthatcanresultinamong‐populationdivergenceinlateral

linemorphology,specificallysuperficialneuromasts,ashefoundnodifferences

inthecanalsystem.Hisstudyrevealedthatguppiesoccurringinhighpredation

pressureareashadmoreneuromastsinthedorsalandtrunkregionsthan

animals,whichinhabitedlowriskenvironments.Theyarguethatahigher

neuromastnumberinthesebodyregionsallowsguppiestoshoaltightlyand

thereforeavoidpredationmorethanguppiesthatshoallooselyandoccurin

low‐riskhabitats(Fischer.etal,203).

Incontrast,astudybyTrokovich(2011)investigatedthenumberand

arrangementofsuperficialneuromastsinthethreespinemarinesticklebacks

(Pungitiuspungitius)andtheirrelatedfreshwaterpondinhabitants.Theyfound

thatsevenofthethirteenbodyregionsshowedsignificantdifferencesbetween

thetwohabitatsandthatthesedifferencesweremaintainedwhenasecond

generationwasrearedinthelaboratory.They,therefore,concludedthatitis

56

likelythatthepatternsfoundinnaturehaveageneticbaseandthatnatural

selectionplaysanimportantrole(Trokovich2011).Geneticandlaboratory

rearingstudiesareneededtoconcludeiftheseexplanationsalsoexplainifthe

variationfoundwithinthedifferentregionsareenvironmentally‐orgenetically‐

determined.

ConclusionsThisinvestigationintothelaterallinesystemofthewesternrainbowfishhas

providedthefirstdescriptionofthestructureofthecanalandsuperficial

neuromastsystems.Thisstudyhasinvestigatedanddescribedthelocationsof

thesuperficialneuromastsystemandthevariabilitybetweenindividualsand

populationsfromdifferenthabitats.Thisincludedthenumberandthe

placementofthesuperficialneuromaststotheextentthathadnotbeen

documentedpreviously.Wehavealsocompletedapreliminaryinvestigation

intothecanalstructureofthelaterallinesystem.Thisstudyisthefirstto

considertheeffectofmultipleenvironmentalfactorsonlaterallinesystem

diversityinasinglespecies.Theresultsshowthatthereisasignificanteffectof

flowontheamountofsuperficialneuromasts,howevertherewasalsoa

significanteffectofsite,lengthandbenthicinvertebratenumbers,suggesting

thatthecompositionofthelaterallinemaybemultifactorial.Ithastherefore

establishedafoundationforfurtherstudieshighlightingtheimportanceofthe

laterallinesystemthatisvitalforthebehaviourandsurvivaloffishes.

57

Chapter3.Laterallinemorphologyandhabitatorigin

determinerheotaxicabilitiesinthewesternrainbowfish

(Melanotaeniaaustralis)

AbstractRheotaxisisamultisensorybehaviourthatallowsfishestoorientthemselvesin

thedirectionofwaterflowtoresistbeingsweptdownstream.Visual,olfactory,

vestibularandlaterallinesystemshavesomeinvolvementinrheotactic

behaviourbuttherelativecontributionsofdifferentsensesundervarying

conditionsarepoorlyunderstood.Furthermore,thebehaviouralconsequences

ofthisvariationareunknown.Inthisstudy,Iinvestigatedtheroleofthelateral

linesysteminmediatingrheotaxisinpopulationsofthewesternrainbowfish

(Melanoteaniaaustralis)fromfreshwaterhabitatswithdiversityofflow

regimes.Neomycinsulphatewasusedtochemicallyablatethelaterallineand

evaluateitsroleinfacilitatingrheotaxisinfishexposedtothreedifferentwater

flowspeeds.Overall,meanorientationdirectionoffishdidnotchangewith

waterflowspeed.However,meandirectionallengthincreasedwithflowspeed,

implyingthatfishspentmoretimeorientatedwithrespecttoflowasthespeed

increased.Itwasfoundthatneomycintreatedfishhadasmallermean

directionallengthandspentlesstimeorientatingwithrespecttoflow.This

resultsuggeststhatthelaterallineisrequiredforrheotaxisinM.australisandis

particularlyimportantinslowwaterenvironments.Thisindicatesalink

betweenintraspecificlaterallinesystemdiversityandswimmingbehaviourin

thewesternrainbowfish.Thisstudyalsonotedthatpopulationdifferencesin

58

thenumberofsuperficialneuromastsofthelaterallinesystemcanaffectthe

abilityofM.australistoorientwithrespecttothedirectionofwaterflow.

IntroductionWaterflow,includingitsdirectionandspeed,isadominantfactorinanyaquatic

environment.Navigatingandutilisingthesecurrentsiscriticalforthespecies

thatinhabitdynamicenvironments.Rheotaxisdescribeshowfishesandother

aquaticspeciesorienttheirbodydirectlyintotheflowandmaintaintheir

positionrelativetothesubstratumbyswimmingdirectlyintothecurrent.

Rheotaxisisknowntoreduceenergycosts,providingthefishwitholfactory

informationcarriedwiththecurrentandalsoprovidingdirectionalguidancefor

example,duringtheupstreammigrationofsalmonduringspawning

(Montgomeryetal.1995).Fishesuserheotaxisacrossarangeofdifferent

habitattypes,includingbenthic,freshwaterspecies(Montgomery,1997)and

speciesthatliveinhabitatswithvariableflowspeedssuchasinlakes(Kanter,

Coombs,2003)andoceans(Champalbert,1994).Rheotaxisalsooccursin

streamdwellingspeciessuchasthegiantdanio(Devarioaequipinnatus),(Bak‐

Colemanetal,2013)andtheblindMexicancavefish(Astyanaxfasciatus)that

liveinlightlesssubterraneancavepools(VanTrump&McHenry,2013).Thus,

regardlessofhabitattypeandsensoryability,rheotaxisisintegralforallfish

species’survivability.

Mostresearchtodatehasinvestigatedwhichofthesensescontributeto

rheotacticbehaviours.Itappearsthatthelateralline,vision,vestibularand

olfactorysensesareallinvolved(Lyon,1904,Dijkgraaf,1963,Arnold,1974),but

59

therelativeimportanceorinputofeachsenseisstillpoorlyunderstood.For

example,afishcanperformrheotaxisevenintheabsenceofafunctionallateral

linesystem(Montgomery1997,BakerandMontgomery1999,VanTrumpand

McHenry2013)orwhenvisionisimpeded(Sulietal.2012).

Thelaterallinesystemhasbeenthoroughlyinvestigatedformorphological

variationwithinandamongspecies,aswellasitsrelationshiptoaquatic

environments,sincetheconnectionwasfirstmadebySchulzein1861(Schulze,

1861).Thesystemiscomprisedoftwodistincttypesofspecialisedreceptor

cells;superficialandcanalneuromasts.Superficialneuromastsarearrangedon

thesurfaceoftheskin(CartonandMontgomery,2004;Chapter2)andare

consideredtobemostlyusedtodeterminethevelocityofthesurrounding

water(WarkandPeichel,2010),aswellasfacilitaterheotaxis(Bakerand

Montgomery,1999).Incomparison,moststudieshavesuggestedthatthecanal

systemiseithernotinvolvedinrheotaxisorhasaveryminorrole(VanTrump

andMcHenry,2013).Thewesternrainbowfishhasavaryingnumberof

superficialneuromastsandshowssignificantdifferenceinthearrangementand

numberofsuperficialneuromastsamongcloselylocatedpopulations(Chapter

2).Thisfindingraisesthepossibilitythatthelaterallinesystemdiversity

observedinindividualsfromthesamehabitatandfromdifferenthabitatsmay

allowsomepopulationstoorientateintoflowsmoreaccuratelythanothers.

Onewaytodemonstratethecontributionofthelaterallinesysteminfacilitating

rheotaxisistoablatetheneuromastsandexaminethecorrespondingchangein

orientationbehaviour.Forexample,Montgomeryandcolleagues(1997)found

thattherewasasubstantialreductionintheabilityofthreefishspeciesto

60

orientatewithanablatedlaterallinesystem.However,eachofthesespecies

(torrentfishCheimarrichthysfosteri,Antarcticfish:Pagotheniaborch‐grevinki,

blindmexicancavefish:Astyanaxfasciatus)haddifferingrheotacticthresholds

atflowspeedslessthan0.5cm‐s,2cmsand3cms,respectively(Montgomeryet

al.,1997).Athigherflowspeeds,nodifferencesinrheotaxiswereobserved

amongthespecies(Montgomeryetal.,1997).However,thisstudycompared

phylogeneticallydistinctspeciesthatoccupyextremelydifferenthabitatsand

ecologicalniches.Itisthereforeunclearwhetherdifferencesintheirrheotactic

thresholdsweresolelyexplainedbyspecies’variationinlateralline

morphology.Examiningrheotacticresponsesinasinglespeciesthatoccupiesa

varietyofhabitatsmayelucidatetheoverallimportanceofthelateralline

systeminrheotaxiswithrespecttootherenvironmentalfactors.

Inthisstudy,Iexaminedwhetherwithin‐speciesvariationinneuromast

arrangementcorrespondswithdifferencesinrheotacticresponsesoffishto

waterflows.Thefocalspeciesforthisstudyisthewesternrainbowfish

(Melanotaeniaaustralis),anativefreshwaterfishspeciesfoundthroughoutthe

PilbaraandKimberleyregionsofWesternAustralia.Thewesternrainbowfish

flourishesinavarietyofhabitatsrangingfromlarge,stagnantpoolstofast

flowingriversandstreams.Previousinvestigationsintothisspecieshave

demonstratedthatfishfromfasterflowingenvironmentsgenerallyhavefewer

neuromastsincomparisonwithindividualsfromslow‐flowhabitats(Chapter

2).Theobjectiveofthestudyistodetermineifthelaterallinesystemisactively

involvedintheabilityofM.australistoperformrheotaxis.Inaddition,thisstudy

investigateshowdifferentarrangementsofsuperficialneuromastsinfluencethe

abilityofwesternrainbowfishtoorientinflowsthataredifferenttothoseof

61

theirnaturalenvironments.Thiswasinvestigatedbyseparatingthepopulations

accordingtotheirhabitatorigin(e.g.fromlargestagnantlakescomparedtofast

flowingchannels,thesewerecalledlowandhighflowhabitats,respectively)as

thepreviouschapterhasshownthatthesehabitatoriginshavesignificant

effectsonthenumbersofsuperficialneuromasts.

Materialsandmethods

FishsamplingandhusbandryRainbowfisheswerecollectedfromtwoareasofthePilbararegioninthe

northwestofWesternAustralia.ThefirstareawastheupperFortescue

catchment,whichincludesCoondinerCreekandWeeliWolliCreek.Thesecond

areawasthemidFortescuecatchment,whichincludessitesinMillstream‐

ChichesterNationalPark(seeChapter2forfurtherdetails).Thetwo

catchmentsdifferintheirhydrology;sitesintheuppercatchmenttypically

compriseaseriesofunstableintermittentpoolsthatrunalongthemaingorge

lineandarelargelyreliantonrainfall(Fellmanetal.,2011).Theupper

catchmentalsoincludedWeeliWolliCreek,whichislocatedEastofthe

GoodiadarrieHillsandactsasadischargepointfordewateringoperationsfrom

anumberofmines(WRM,2010).Thestreamcomprisesadensenetworkof

tributariesthatflowinanortherlydirectionintotheFortescueMarsh(Kendrick

2001,WRM2010,Dogramacietal.,2015).Thestreamishydrologically

significantasitisfedbyWeeliWollispringandisapermanentsourceofwater

inacharacteristicallyaridenvironment.Around1GLofwaterispumpedinto

thecreekannuallyfromcontinuousdischarge(Dogramacietal.2015),with

somesectionsofthecreeksubjecttobothcontinuousand,attimes,muchfaster

62

flowsthanwouldoccurnaturally(Dogramacietal.2015).Incontrast,

MillstreamNationalParkisfedbyanundergroundaquiferthatcreatesalong

stringofpermanent,stablepoolsoveradistanceof20km.However,thepools

varyinhydrologyfromlargepermanent,stagnantpoolssuchasDeepReachand

CrossingPooltofastflowingchannelssuchasOutCrossing,Jirndawurranhaand

Jayawurrunha.

Between15and20maleandfemalerainbowfisheswerecapturedusingeithera

4mor10mnet(witha6mmmeshsize),dependingonthesizeofthepool.

Individualswerehousedforupto5daysinthefieldinaerated20Lplastic

aquariacontainingcreekwaterandnaturalsubstratebeforebeingtransported

backtotheBiologicalSciencesAnimalUnitatTheUniversityofWestern

Australia.Oncebackatthelaboratory,mixed‐sexpopulationswereplacedin

aeratedaquaria(42x42.5x34cm)containinggravel,afilterandanartificialplant

andwerehousedundernormallight/darkconditions(12:12hlight:darkcycle)

at26oC±1oC.Fisheswerefedadailymixeddietofcommercialflakefoodand

Artemianauplii.

63

Table1.SummaryofhabitatresultsforMillstreamNationalPark,CoondinerCreekandWeeliWolliCreek.

Site

0.2Flow

Velocity(m

‐s)

0.2StError

0.6Flow

Velocity(m

‐s)

0.6StError

Tem

perature(o C)

FortescueRiver,M

illstream

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)was

usedtodeterminethewaterflowvelocityateachsiteandwasmeasuredat

0.5mintervalsalongatransect.Therangeofflowspeedthatcanbemeasured

withtheflowtrackeris±0.001to4.0m/s. Thelocationofthetransectwas

selectedbyobservingthepresenceofMelanotaeniaaustralisfromthebank.

Waterflowmeasurementswerecapturedinthreedimensions(0.2,0.6and0.8

asaproportionofthetotaldepthofthewater)atthreedifferentdepthsforeach

intervalonthetransect,andwereaveragedforeachstationovera10speriod.

Themeasurementswerethenaveragedoverthestations(minimum:11

stations,maximum:16stations)toobtainthemeanflowandflowstandard

deviationsforeachsamplesite.Atallsites,M.australiswereobserved

inhabitingthetop20%ofthewatercolumn(approx.30cmbelowthewater’s

surface),thusonlythe0.2flowvelocityreadingwasusedforthisexperiment.

64

Accordingtotheflowratemeasuresobtainedinthefield,andtoreducethe

numberoffishrequiredfortheexperiment,populationsofM.australiswere

characterisedasoriginatingfromeitherslowfloworfastflowhabitats.

CoondinerCreek(Meanflowspeedrange=‐0.002+‐0.00061m‐s),Deepreach

(0.0054+‐0.0005m‐s)andCrossingpool(‐0.004+‐0.0014m‐s)wereassignedas

originatingfromslowflowhabitats,whileJirndawurranha(0.305+‐0.0330m‐

s),Jayawurrunha(0.12+‐0.0099m‐s)andWeeliWolliCreek(0.177,+‐0.0111

m‐s)weregroupedintothefastflowgroup.

ExperimentalSetup.Rheotacticexperimentswereconductedinacustom‐builtindoorflume(5.1m

lengthx0.8mheightx1.4mwidth)witharecirculatingflowchamberandvalve

thatcouldbeusedtocontrolthewaterflowspeed(Figure9).Tocreatelaminar

flow,lengthsofstackedPVCpiping(3.5cmdiameter)wereplacedbehindthe

flowoutletvalve.Anobservationarena(67cmlongx32cmhighx37cmwidth)

madeofopaqueplasticwasplaced1mbehindtheoutletvalveandraised10cm

abovethebaseoftheflume.Laminarflowsweresubsequentlydirectedintothe

observationarenausingsectionsofcoreflute™,sothatallflowpassedthrough

observationarena.Diffuseinfraredlighting(940nm;obtainedfrom

LEDlightinghut.com)wasprovidedtotheobservationarenabyattachingrows

ofinfraredLEDlights(embeddedinresinforincreasedwaterresistance)tothe

baseofthearenaandplacingtwolayersofTeflondiffuseroverthetop(at0.25

mmand4cm).PreviousinvestigationsbyKelley,etal.(unpublished)

determinedthatthisspecieshasavisualsystemsensitivityrangefrom

approximately360nm(ultraviolet)to575nm(red)andthereforecouldnotsee

theinfraredlightingusedinthisexperiment.Theobservationarenawasdivided

65

intotwoequal‐sizedsectionswithopaque(white)coreflute™toallowtwofish

tobetestedatonetime.Bothendsofthearenawerecoveredwithaplastic0.5

cmmesh,allowingwatertopassthroughwhilepreventingthefishfrom

escaping(Figure9).Observationswereconductedatflowspeedsof0.003m‐s,

0.18m‐s,0.48m‐s,(hereafterreferredtoasnoflow,mediumflowandfastflow),

whichwasmeasuredwiththesameflowtracker(Sontek™)usedinthefield.

Eightmeasuresforeachspeedweretakenfromwithintheobservationarena

andthenaveragedtoproducethemeanandstandarddeviationflowspeedsfor

eachexperimentalflowcondition.

66

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

67

NeuromastvisualisationTotesttheeffectivenessofneomycininablatingthelaterallinesystem,we

comparedthesuperficialneuromastsoffishfromtheablationtreatmentto

thoseofcontrolfish.Thiswasperformedforonetreatmentandonecontrol

fishfromeachpopulation,todeterminewhethertherewasanypopulation

variationintheeffectivenessofthechemicalablation.DASPEIstainingwas

thusonlyconductedontwoindividuals(treatedandcontrol)becauseallfish

usedinthisexperimenthadbeenpreviouslytestedwithDASPEIinthe

previousexperiment(Chapter2).Theprocessofanaesthesia,DASPEIstaining

andneuromastvisualisationwassimilartothatdescribedintheprevious

chapter.Tovisualisethesuperficialneuromasts,fisheswereexposedto

fluorescentvitaldye2‐[4‐(dimethylamino)styrl]‐N‐ethylpyridiniumiodide,

DASPEI(LifeTechnologies/MolecularProbes,EugeneOR,USA)for15minutes

ataconcentrationof0.24gin1Lwater,(1200mM).Fisheswerethen

anaesthetisedwith200mgl‐1MS222(tricainemethanesulfonate;Sigma‐

Aldrich,StLouis,MO,USA)untillightpressureonthecaudalfinyieldedno

response.Eachindividualwasthenplacedrightsidedowninapetridishand

placedonthestageofafluorescence‐dissectingmicroscope(LeicaMZ75fitted

withaFITCfilterset;LeicaMicrosystemsInc.,Sydney,Australia).Imageswere

takenusingadigitalcamera(LeicaDFC320)toillustratechemicalblockingof

thelateralline.Neuromaststhatweresuccessfullyblockedshowedweak

fluorescence(i.e.werenotverybright)orshowednofluorescenceatall(see

Appendix1).

68

ExperimentalconditionsFishwereexposedtoahighconcentrationofneomycinsulphate(1200uM)

(FischerScientific,Pittsburgh,PAUS)fortwohoursbeforeobservations

commencedtochemicallyblockthelateralline.Thisagentwaschosenforthis

studybecauseittargetsonlythesuperficialneuromasts(notthecanal

neuromasts)(Kulpaetal.,2015).Preliminarytrialswereconductedtotestthe

effectivenessofneomycinsulphateonM.australis(seeAppendix1).The

concentrationthatwastrialledandfoundtobeeffectivewas1200uM.

Individualfishweretestedonlyonceandallexperimentalprocedureswere

completedinthedarktopreventtheanimalsrelyingonvisualcues.Priorto

observations,eachtestfishwasisolatedina10Ltank(20cmheightx28.5cm

lengthx13.5cmwidth)containingeitherthelaterallineblockingagent,

neomycinsulphate(ablationtreatment;seebelow)orconditionedaquarium

water(control)foraperiodoftwohours.Fishweremaintainedinvisual

isolationandaerationwasprovidedusingabatteryoperatedaquariumpump.

Afterthisperiod,pairsoffishwerethenplacedintothearena(oneineach

observationchannel)andwereacclimatisedtothedarkunderconditionsofno

flowforaperiodof10minutes.Wethenrecordedthebehaviourofthefish

underconditionsofnoflow(0.003cm‐s),mediumflow(0.18cm‐s)andfast

flow(4.8cm‐s),witheachobservationperiodlastingforfourminutes.The

behaviourofthefishwascapturedininfraredusingaSonyHandycamHDR‐

CX550positioneddirectlyabovetheobservationarena.Followingprevious

studiesofrheotaxis(VanTrumpandMcHenry,2013),fishweresubjectedto

successivelyincreasingflowspeedsandwerenotrandomisedwithrespectto

flowtoavoidcarryovereffectsassociatedwithfishbecomingfatigued.

69

ImageanalysisAllvideorecordingswereanalysedusingacustommadeMatlab(The

MathworksInc.,programversionR2014a)program(Hemmi&Pfiel,2010),

thattrackedthefish’sheadpositionandorientationintotheflowdirectionfor

eachframe.Whileimageswerecapturedat25frames/second,aresolutionof

1frame/secondwasfoundtobesufficientforgeneratinganaccurate

representationoffishorientation.Videoclipsthatcouldnotbeautomatically

trackedbecauseoflightingissues(e.g.poorcontrastattheedgesofthearena)

orairbubblesonthewater’ssurfacewerecapturedmanuallyusingthe‘point

data’option.Foreachfishandflowspeed,themeandirection(i.e.orientation

ofthefish’sbodywithrespecttoflowdirection)andmeanlength(calculated

astheamountoftimespentorientatinginagivendirection)wascalculated.

Therewerenoinstanceswheretheanimalswereobservedmakingcontact

withthebottomofthearenaandweexcludedframesinwhichfishwere

stationaryandpositionedatthesideofthearena.Datawascollatedovera

numberofframesandthisrangedfrom3000to6000framesusing10oangular

bins.Toensurethatdirectorientationintoflow(i.e.0o)formedthemidway

pointofthefirstbin,binsweregeneratedinincrementsof‐5Oto+5O.The

meandirectionandmeanvectorlengthwerecalculatedfromtheoriginal

orientationdataandwerecalculatedusingthefourquadrantinversetangent

function.AllcircularstatisticswerecalculatedusingMatlabCircularStatistics

Toolbox(Berrens,2009).Theresultingmeanvectorlengthanddirectionfor

fishwithineachtreatment/habitattype/flowspeedisascoreofbetween0

and1(highervaluesrepresentconsistentorientationataparticularangle).

70

Statisticalanalysis.PermutationalAnalysesofVariance(PERMANOVAs)wereusedtoanalysethe

data,thustherewerenounderlyingassumptionsaboutthedistributionofthe

data.EachPERMANOVAwasrunwith10,000permutationsbutthelevelof

permutationvarieddependingontheanalysisconducted.Toinvestigatethe

effectofwaterflowspeed(noflow,mediumfloworfastflow)onmeanvector

lengthandmeandirection,permutationswererandomisedamongall

individuals.Fortheanalysisoftreatmenteffects(controlandneomycin)and

habitatorigin(i.e.stillwaterpoolorfastflowingstream)permutationswere

randomisedbetweenthetreatment/habitatgroups.Asignificancelevelof

p<0.05wasusedforallanalysestoevaluatetheprobabilitythatthemeasured

effectwasduetochancealone(Hemmi&Pfiel,2010).

Results

EffectofneomycinandflowonfishorientationThepermutationanalysisoverallflowsrevealedthatthattherewasnoeffect

oftreatment(neomycinorcontrol)onmeandirection(p=0.8880)butthere

wasasignificanteffectoftreatmentonmeanvectorlength(p=0.0294)

Thereforeoverallcontrolfishhadahigherdirectionallengththanthe

neomycintreatedfish,whichimpliesthatthecontrolfishspentmoretime

orientatedwithrespecttoflowthanneomycintreatedfish.Whenconsidering

theeffectoftreatmentateachflowspeed,theeffectwasstrongestatmedium

speed(0.18cm‐s),(Figure10).

71

Figure10:Meanvectorlengthsandstandarderrorsoforientationoverthewholedataset,acrossallthreeflowspeedscomparingtherheotacticresponsesofbothneomycinandcontrolfish.Neomycinfish(n=26),controlfish(n=28).

Toinvestigateiftherewasadifferenceintheeffectivenessoftheneomycin

treatmentonfishcollectedfromdifferentsites,wedidfurtheranalysistesting

forfishsiteoriginonmeandirectionallength.Thesetestsrevealedthatfish

fromJirndawurranhashowedastrongestresponsetotheneomycintreatment

(p=0.001)andthetreatmentwhileweakeffectofneomycintreatmentwas

observedinsitessuchasWeeliWolliCreek(p=0.7964),Deepreachand

Jayawurranha.

TheresultswereconfirmedduringtheDASPEItrialsandrevealedthat

neomycinhadvaryinglevelsofeffectivenessinablatingthesuperficial

neuromastsofindividualsfromdifferentpopulations(whenadministeredat

thesameconcentration).Thiswasdeterminedthroughthefluorescentfading,

whichrevealedthatsomesuperficialneuromastsappearedbrighterthan

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

NoFlow(0.003cm‐s) MediumFlow(0.18cm‐s)

FastFlow(4.8cm‐s)

Meanlength

Flowspeed

Control

Neomycin

72

others.Thesedifferencesinbrightnesswerenoted,howeverformost

populations,onlyonefishwastestedandthereforewecannotruleoutthat

thesedifferenceswerenotonlyatanindividuallevel.Interestingly,

Jirndawurranhawasthepopulationthatwasusedduringthetrialandwasthe

populationthatdeemedthetreatmentsuccessful.Itwasalsothepopulation

thatshowedtheleastfluorescentlabellingofthesuperficialneuromasts

(Figure12andAppendix1).

Figure11:Meanvectorlengthsandstandarderrorsoforientationcomparingtherheotacticresponsesofneomycinandcontrolfishateachsite.Thesitesthatrepresentlargestagnantpools(SP)areCrossingPool,DeepreachandCoondiner.Thesitesthatrepresentfastflowing(FF)streamsareWeeliWolli,JindaandJaya.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

WeeliWolli

Jinda CrossingPool

Coondiner DeepReach

Jaya

Vectormeanlength

(FF)(FF)(SP)(SP)(SP)(FF)Population

Control

Neomycin

73

Figure12.DASPEIneuromaststainingoftheheadofM.australisshowingtheeffectivechemicalablationofsuperficialneuromasts:(A)Neomycinsulphate(female)treatedand(B)control(male)fishfromWeeliWolliCreekandneomycinsulphate(Female)treated(C)andcontrolfish(male)(D)fromCoondinerCreek.ArrowspointtoInfraorbitalneuromaststhathavebeenstainedwithDASPEIunderthesameconditions,howeverthelackoffluorescenceseenonthetreatedfishindicatesthelevelofablation.

Duringbehaviouralobservationswhilstreviewingthevideofootage,I

observedadifferenceinthebehaviourofthefishatdifferentflowrates.

Duringthenoflowobservations,themajorityofthefishspentmoretime

exploringthecentreofthearenaaswellastheouteredgeswithandwithout

theirlaterallinesablated.Thiswasclearlyseenintheangularhistograms

depictedinfigure13A.Somefishthathadbeentreatedwithneomycin

sulphatewereseenswimmingtowardsthearena’sedges,knockingupagainst

thembeforeswimmingtowardsanotheredge.Duringthemediumflowand

fastflowrecordings,thefishwereobservedspendingmoretimeateitherend

A B

C D

74

ofthearenadirectedinto,orslightlyatanangle,totheflowdirectionand

continuouslyswimmingatasteadypace(i.e.exhibitingarheotacticresponse

toflow).Isubsequentlyinvestigatedtheeffectofwaterflowonrheotaxisfor

allfish(bothcontrolandneomycintreatedfish).Ifoundthattherewasno

significanteffectofwaterflowspeedonthemeandirectionoffish,when

measuredovertheentiredataset(Table5)(p=0.427).However,asignificant

effectofflowonmeandirectionallength(p<0.0001)wasrevealed,whichis

depictedinthehistogramofthepermutateddatacomparedtotheobserved

effect(Figure14).Themeanvectorlengthfortheoveralltrialrangedbetween

0.2and0.7andshowedthatrheotacticstrengthincreasedwiththeflowrate

withinthechamber(Table5).Whentestingseparatelyacrossthetwo

treatments,thisfindingwasconsistentforboththecontrol(p=0.0002),and

theneomycintreatedfish(p=0.0014)(Table5andFigure11).

Figure13A,BandC:Rawdataangularhistogramsofthecontrolfishonlyorientationindicatingthefrequencyoforientationatthethreedifferentspeeds.(A.Noflow,B.mediumflow,C.fastflow).Datashownforcontrolfishonly.PleasenotedifferentscalesonFigure13A.

Theangularhistogramsshowthatevenatthemediumandhighflowsthe

responseofindividualfishisnotalwayspositivelyrheotactic,withsomefish

pointingintheoppositedirectiontoflow,thereforeperformingnegative

rheotaxis.Howevercontrolfishweremoreconsistently(shownbymean

A. B. C.

75

directionallength)orientatedthanneomycintreatedfish(Table5).Negative

rheotaxiswasalsoobservedwhilstwatchingthevideorecordings.Itislikely

thatthisnegativerheotaxiswouldbereplacedbypositiverheotaxisat

increasedflowspeeds(13cm‐s),similartothoseusedinotherstudies(e.g.Van

Trump&McHenry.2013).

Figure14.Histogramshowing10000permutations(n=54fish)ofthevectorlength,permutedstrictlywithinindividualfish.Thebluelineontherightshowstheobservedmeanvectorlength.Thep‐valuecomparingthepermutedvaluestotheobservedmeanvectorlengthisgivenabove.

EffectofhabitatoriginWhentestingforaneffectofhabitatoriginonrheotacticresponse,Ifirstused

theentiredatasetandfoundtherewasnoeffectoffishhabitatoriginonmean

directionormeanvectorlength(p=0.481andp=0.2278,respectively).As

populationdifferencesareonlyexpectedtobeobservedincontrolfish,further

analyseswereconductedusingthereduceddatasetofonlythecontroltreated

fishandneomycintreatedonlyfish.Thistestrevealedthattherewasno

significanteffectofhabitatoriginonvectorlengthoncontroltreatedfish

(p=0.5763),orneomycintreatedfish(p=0.2172).

76

Figure15:Meanvectorlengthsandstandarderrorsoforientationofcontrolfishfromeachhabitatoverthethreeflowspeeds.

TodetermineiffishfromWeeliWolliCreek,asitethathasanaltered

hydrologicalenvironmentduetodewateringofanearbyminesite,displayed

rheotacticbehaviourthatwastypicalofotherfastflowhabitats,weconducted

ananalysisformediumflowspeedonly.Theresultsshowed(meanlength=

0.4406,standarddeviation=0.2514)thattherheotacticresponseoffishes

fromthissiteweremidwaybetweenthemeanresponsesoffishesfromlow

flowhabitatsandhighflowhabitats(Figure11).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

NoFlow(0.003cm‐s)

SlowFlow(0.18cm‐s)

FastFlow(4.8cm‐s)

MeanVectorLength

Flowspeeds(Arena)

Pool(lowflow)

Stream(highflow)

77

Table5:ResultsobtainedfromPERMANOVAsrepresentingtheoverallaffectsofflow,treatmentandhabitatonthemeanvectorlengths.(A)

EffectofFlowonOverallModel Meanlength

StandardDeviation

pvalue=0.0001 Noflow 0.2696 0.2126Mediumflow 0.4621 0.2831Highflow 0.604 0.2791NeomycinTreated pvalue=0.0014 Noflow 0.2465 0.1858Mediumflow 0.384 0.2476Highflow 0.5427 0.2737ControlTreatment pvalue=0.0002 Noflow 0.2909 0.2362Mediumflow 0.5434 0.2993Highflow 0.6653 0.3038

(B)

OverallEffectofTreatmentOverallModel Mean

StandardDeviation

pvalue=0.0294 Control 0.4966 0.3038Neomycin 0.3929 0.2737NoFlow pvalue=0.4572 Control 0.2909 0.2362Neomycin 0.2465 0.1858MediumFlow pvalue=0.0421 Control 0.5434 0.2993Neomycin 0.384 0.2476FastFlow pvalue=0.1145 Control 0.6653 0.2507Neomycin 0.5427 0.2973

78

(C)

OverallEffectofHabitatOriginonFishrheotaxis

Meanlength

StandardDeviation

pvalue=0.2278 LowFlowHabitat 0.4701 0.3075HighFlowHabitat 0.4115 0.2709NoFlow pvalue=0.4599 LowFlowHabitat 0.2503 0.1921HighFlowHabitat 0.2958 0.2399MediumFlow pvalue=0.0357 LowFlowHabitat 0.533 0.304HighFlowHabitat 0.3688 0.2269FastFlow pvalue=0.4509 LowFlowHabitat 0.629 0.2849HighFlowHabitat 0.5699 0.2754

DiscussionThefindingspresentedhereareconsistentwithpriorstudiesshowingthatthe

laterallineplaysanimportantroleindeterminingrheotacticbehaviour.

Specifically,M.australisdisplayedstrongerrheotaxis(i.e.,increased

orientationwithrespecttoflow)atfasterwaterflowspeedscomparedwith

lowerwaterflowspeeds.Ialsofoundthatblockingthelaterallinewith

neomycinsulphatealterstherheotacticresponseinthisspecies,supporting

thenotionthatthelaterallineisinsomewayutilisedinrheotaxis.Thisstudy

showedapatterninwhichpopulationvariationinlaterallinemorphologymay

belinkedtobehaviouraldifferencesamongpopulations,asfishfromslowflow

habitats(poolsandlakes)showedstrongerrheotacticresponsesthanthose

fromstreamsunderfastflowconditions.Itwasalsorevealedthatrheotactic

directionisnotsignificant,suggestingthattheneomycintreatmentdoesnot

directlyaffectthedirectioninwhichtheyswim.Howeverinneomycintreated

79

fishweobservedadecreaseintheaccuracyandtheamountoftimetheyspent

orientatingwiththedirectionofflow.Thisprovidesevidencethatthelateral

line,andthebehavioursthatareinfluencedbythissensorysystem,are

optimisedtothefish’shydrologicalenvironment.

EffectofwaterflowonrheotaxisThesefindingsaresimilartothosereportedinotherstudiesandshowthatfish

positionthemselvesinthegeneraldirectionoftheflowandwillincreasethe

precisionofthisorientationwithanincreaseinwaterflowspeed(Kanter&

Coombs,2003).Therewasasignificanteffectofflowonfishmeanorientation

accuracyatallthreeflowspeeds;aresponsethatwasalsoobservedincontrol

fish,andthosewiththelaterallineablated.Thissuggeststhatalthoughthe

laterallineisanintegralpartofrheotacticbehaviours,thefishcanstill

performrheotaxisintheabsenceofvisionandthesuperficialneuromastsof

thelaterallinesystem,althoughatparticularflowsrheotaxisissignificantly

impaired.Thiscouldindicatethatthecanalsystemofthelaterallinealsoplays

arole,orcanbeutilisedforrheotaxisintheabsenceofasuperficialsystem.

Otherstudiesthatusedneomycinandstreptomycinsulphatesupportthis

assertion(Sulietal.,2012,Bak‐Colemanetal.,2013).

Thereareseveralgeneralexplanationsfortheanimalsspendingmoretime

orientatedintotheflowathighwaterflowspeeds.Firstly,thereisan

increasedamountofhydrodynamicinformationthatiscarriedwithafaster

flowrate,whichprovidesmorestimulitothesensorysystem,suchasvortices,

andturbulence,aswellasolfactoryinformation.Therefore,highflowspeeds

80

offertheindividualmoreinformationontheflowdirection.Bothvisualcues

andvestibularinformationrelyontheindividualmaintainingadownstream

position,whichismorereadilyattainedatincreasedflowspeedsorhigh

turbulenceconditions(Bak‐Colemanetal.,2013,Kanter&Coombs,2003).If

thefishexperienceslittletonodisplacementviathesurroundingwater

current,therewillalsobelittletonoinformationcapturedbythelateralline.

Thesecondexplanationforastrongerrheotacticresponseatfasterwater

flowsisnon‐sensoryandexplainedbythegeneralmotivationbehindthis

behaviourinfishes.Withalowwaterflowspeed,individualsmaybelesslikely

toperformrheotaxisintheinterestofsavingenergy,sincethecostofthelift

anddragforcesthatcarryafishdownstreamaremuchlowerinslowflows

thaninfastflows(Bak‐Colemanetal.,2013).Athirdlikelyexplanationisthat

vigorousswimmingisknowntoactivatetheoctavolateralisefferentsystem,

whichisutilisedinhabitatswithfastflows.Thissystemisknowntoreducethe

sensitivityofthelaterallineandthereforereduceself‐generatednoise,while

increasingsensitivitytowardsrelevantstimuli(Bak‐Colemanetal.,2013,Bak‐

ColemanandCoombs,2014).Theadditionofthisefferentsystemmayincrease

theanimal’sabilitytosenseitssurroundingsandthereforeincreaseits

rheotacticcapacity.Alloftheseexplanationshavebeenreadilyinvestigated

(Montgomeryetal.,1997,BakerandMontgomery,1999a,Sulietal.,2012)and

togethersupportthisstudy’sfindings.

Theroleofthelaterallineinmediatingrheotaxis

81

Ourstudyconfirmsthefindingsofpreviousresearch(BakerandMontgomery

etal,1999a,BakerandMontgomery,1999b,Sulietal,2012)thathas

demonstratedtheinvolvementofthelaterallineinrheotaxis.More

specifically,thisstudyprovidesevidencetosupportthenotionthatthe

superficialneuromastsplayanimportantpartinmediatingthisbehaviourand

arerequiredforfishtoorientatewithrespecttoflow.Superficialneuromasts

arewellknowntoplayabiggerroleinrheotaxisthanthecanalneuromast

system.Neomycinsulphateisknowntoblockthesuperficialneuromastsby

destroyingthehaircellsthatmakeupthebasicciliastructureofthelateral

line(Harrisetal.,2003).Thisagentwaschosenforthisstudybecauseit

targetsonlythesuperficialneuromasts(notthecanalneuromasts)and

previousinvestigationsofspecieshasshownthattheydisplayawidediversity

ofmorphologies,despitebeingcollectedfromsitesthatarerelativelyclosein

proximity(Chapter2).Fishthathadtheirlaterallineblockedshoweda

reducedabilitytoorientateintotheflow.However,duringthisexperimentthe

animalsexperiencedmulti‐sensoryblockingastheirvisionwasalso

compromised,whichislikelytoaffecttheirbehaviour.Bak‐Colemanetal.,

(2013)alsofoundthattherewasadecreaseinorientatingpotentialwhenboth

thelaterallineandvisualsystemswerecompromisedinthegiantdanio

(Devarioaequipinnatus).However,thisstudyfoundnosignificantdifference

betweentreatmentandcontrolfishwhenonlythelaterallineorvisionwas

blockedsingularly.Furtherinvestigationwouldneedtobeconductedtofully

understandtheeffectsofboththesesensesinM.australis.

82

Previousstudiesthathavefoundthatthelaterallineisnotinvolvedwith

rheotaxisdifferfromthecurrentstudywithrespecttothechoiceofchemical

orphysicalblockingagents.Themostrecentstudies,conductedbyBak‐

Colemanetal.(2013),Bak‐ColemanandCoombs(2014)andVanTrumpand

McHenry(2013),alluseddifferentchemicalssuchasstreptomycinsulphate

andgentamicinsulphate(VanTrump&McHenry,2013),respectively.In

comparison,gentamicinsulphateistoxictoallhaircellsinthelateralline(Van

Trumpetal.,2010)andstreptomycinsulphateblocksboththesuperficialand

canalneuromasts(Bak‐ColemanandCoombs,2014).Collectively,these

findingsprovideadditionalevidencethatsuperficialneuromastsaremore

importantforrheotacticbehavioursthanthecanalneuromasts.Trumpand

McHenry(2013)foundthatthebehaviouralresponsetoflowwas

indistinguishablewhencanalneuromastswerecompromisedcomparedwith

whentheywerefunctional.Thesedifferencescouldbeexplainedbythe

effectivenessofthesechemicalmethodstoblockall,orcertainpartsofthe

lateralline.Furthertotheseinvestigations,amorerecentstudybyKulpaetal.

(2015)testedbothneomycinandstreptomycinforitseffectivenessinablation

andfoundthatstreptomycincausedonlypartialblockingofthelateralline

system,whileneomycinresultedincomplete,ornearcomplete,blockingofthe

superficialneuromasts.

Allsiteswhereourfishwerecollectedwerehighlyspatiallyandtemporally

variableandallthesiteswerenon‐uniformintheirflowcharacteristics.A

recentstudyillustratedthatthesuperficialneuromastsofblindcavefish

(Astyanaxmexicanus)respondmoretoanon‐uniformflowasitprovidesmore

83

informationaboutthesurroundings(Kulpaetal.,2015).Manystudiesare

basedontheflowarenapresentedbyVogelandLaBarbera(1978),whichwas

designedtoreduceflowheterogeneitiesandcreatealaminarflowthroughout

theentirearena.Theirdesignwassimilartothisstudyinthatithada

recirculatingflowchamberwithPVCpipingstackedtoreduceturbulenceand

providelaminarflow.However,thecurrentstudy’sexperimentalsetup

featuredonedifference:thesizeofthepumpoutlet.Ouroutletwassmaller

thanthatusedbyVanTrumpandMcHenry(2013)andbyBak‐Colemanand

Coombs(2014)andthereforewouldhavecreatedaflowthatwaslessuniform.

CorefluteTMandothertechniqueswereutilisedtocreateuniformity,however

itislikelythattheflowinsidethearenawasnotcompletelyconsistent.This

lackofuniformitymayexplainwhywefoundasignificantdifferenceinthe

meanlengthinorientationwithrespecttoflowbetweenourtreatedand

controlfish,becausethelaterallinerequiresnon‐uniformityforthemajority

superficialneuromaststobestimulated(Kulpaetal.2015).Furthermore,non‐

uniformflowsrepresenttheflowenvironmentthatfisharemostlikelyto

experienceintheirnaturalhabitat.Forexample,iftheflowpathofany

environmentwasspatiallystablewithnoturbulence,thewaterwoulddisplace

theindividualdownstreamandtherewouldbenonetmotionbetweenthefish

andthewater,thuscreatinganinefficientstimulustothelateralline(Kulpaet

al.,2015).Thiscouldbeanotherexampleofhowhabitatcanbeanimportant

determiningfactorinaspecies’abilitytoperformrheotacticbehaviours.

Howeversomestudies(Chagnuadetal.,2008)havefoundtheretobe

microdisturbancesinlaminarflowandhowtheseeffectthesuperficial

neuromastscouldbeanareaforinvestigationinfuturestudies.

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EffectoffishhabitatoriginonrheotaxisPreviousinvestigationswiththewesternrainbowfish(Chapter2)have

revealedsignificantvariationinlaterallinemorphologyamongpopulations,

particularlyinthearrangementandnumberofsuperficialneuromasts.

Furthermore,Ihaveshownthatthismorphologicalvariationislinkedtothe

hydrologicalcharacteristicsofthefish’snaturalenvironment.Itistherefore

integraltoourunderstandingofthelateralline’sinvolvementinsensory

adaptationtodetermineifthesemorphologicaldifferencesaresufficientto

affectthefish’srheotacticcapabilities.Ourfindingthatthefish’shabitatorigin

affectsrheotaxis,butonlyundermediumflowconditions,suggeststhatthe

superficialneuromastsaremostlyutilisedduringwaterflowspeedsof

approximately1.8cms‐1.Thisisconsistentwithstudiesthathaveexamined

therheotacticthresholdsofthesuperficialandcanalneuromasts.Specifically,

anumberofstudieshaveshownthatthevelocitythresholdofsuperficial

neuromastsislowerthancanalneuromasts,meaningthatsuperficial

neuromastsaremorelikelytobestimulatedatslowerwaterflows(McHenry

etal.,2009).Thisisaspecialisationofthesurfaceneuromastsastheycan

detectweakwatermotions,however,theyareonlyeffectiveinan

environmentthathaslittletonobackground‘noise’createdbythe

surroundingwatermovements(Wark&Piechel,2010).

Thedifferentrheotacticthresholdsthatwereobservedatslow,medium,and

85

fastwaterflowspeeds(discussedabove)areconsistentwithastudy

conductedonthecommonbully(Gobiomorphuscotidianus)byBassettand

Carton(2006).Thisspecieshasaprolificnumberofsuperficialneuromasts

andalmostcompletelylacksacranialcanalsystem.Bassett&Carton(2006)

investigatedthevelocitythresholdofbulliesbycomparingtheirsensitivityto

avibratingsphere,astimulusthatmimicsthehydrodynamicprofileof

invertebratepreyspecies(zooplankton),inquietandnoisyflow

environments.Theyfoundthatthecommonbullywasmostsensitiveatwater

speedsof0‐1.5cms‐1andanyfurtherincreasesinbackgroundwaterflow

velocityresultedinaten‐folddecreaseinsensitivitytothesphere.Asthis

thresholdisclosetothemediumwaterflowspeedusedinthecurrent

experiment(1.8cms‐1),itislikelythatatthesespeeds,thesuperficial

neuromastsofrainbowfishareabletomorecorrectlyrespondto

hydrodynamicinformationandallowfishtoorientateintoflow.

Mypreviousinvestigationswiththewesternrainbowfishrevealedthatthereis

asignificantdifferenceinthenumberofsuperficialneuromastsinfish

capturedfromlowflowenvironments,suchaspoolsandlakes,comparedwith

thoseoriginatingfromhighflowhabitats,suchasstreams(Chapter2).A

numberofpreviousstudieshaveshownasignificantrelationshipbetweenthe

numberofsuperficialneuromastsandtheenvironmentinwhichaspecies

lives,withfishfromslower,“quieter”hydrologicalenvironments

characteristicallyhavingmoresuperficialneuromaststhanthosefromfaster,

“noisierenvironments”(Janssen,2004,WarkandPeichel,2010,Mogdansand

Bleckmann,2012).Thereforeitwouldbelikelythatindividualsfromslowflow

habitatsarebetteratorientatingintoslowflowingwatersthanthosefromthe

86

highflowpopulations.Tofurtherinvestigatethis,afullmappingofthecanal

systemwouldclarifyoveralldifferencesinthemorphologyofthelateralline

system(i.e.forbothsuperficialandcanalneuromasts)amongfishfromhigh

andlowflowenvironments.

Tomyknowledge,thisisthefirststudythathasinvestigatedhowafish’s

habitatoriginaffectsitsabilitytoperformrheotaxis.Theseresults(discussed

above)openupthepossibilityoffurtherinvestigationsintootherbehaviours

thatutilisethelateralline.Itisclearthatfromthecurrentstudythatthe

superficialneuromastsareutilisedmostduringmediumflowspeeds(1.8cms‐

1)andblockingtheseneuromastsleadstoasignificantdecreaseinrheotactic

ability.Itwouldbeinterestingtodetermineifatthiswaterflowspeed,animals

fromahabitatwithfasterflows(i.e.withreducedrheotacticcapabilitiesat

mediumflows)arelessabletosensepredatorsandcapturepreythanfish

fromsiteswithlittletonoflow.Previousstudieshaveinvestigatedthe

rheotacticabilitiesoflaboratory‐rearedfishwithimpulsechambers(McHenry

etal.,2009),vibratingspheresandlivepredatorspecies(Stewartetal.,2013).

Usingsimilarablationtechniquestothecurrentstudy,they(Stewartetal.,

2013)revealedthatthelaterallineplaysacrucialroleinallowingpreyto

sensepredatorsanddetectpreyitems.Importantly,thefindingspresented

hereraisequestionsastohowananthropogenicchangeinthehydrological

environment,suchastheeffectsofminingatWeeliWolliCreek,couldaffectall

ofthesefitness‐relatedbehavioursthatutilisethelateralline.WeeliWolli

creekwasoriginallyamuchslowerflowingenvironment,butforthepast12

yearsthecreekhasbeenexposedtoomuchhigherwaterflowspeeds(0.177

cm‐s).Therheotacticresponseoffishfromthispopulationfallsinbetweenthat

87

offishfromlowflowandfastflowhabitats,suggestingthatthispopulation

maybeadaptingtoitsalteredhydrologicalenvironmentthroughsensory

plasticityand/ormicro‐evolutionarychange.

ConclusionsThroughtheuseofchemicalablation,thisstudyhasestablishedthatthe

superficialneuromastsplayasignificantroleinrheotaxisinthisspeciesand

arelikelytobecriticalforthisbehaviour.Thestudyhasshownthatneomycin

sulphatecanadequatelyablateanddestroythehaircellsoftheneuromast

systemifexposedforsignificantperiodsoftime.Inaddition,thisstudyhas

builtonmorerecentfindingsbyinvestigatingthelinkbetweenthevarying

arrangementsofthesuperficialneuromastsandrheotacticbehaviour.The

dataalsoconfirmstheimportanceofaspecies’originalhabitatandhow

alteringtheflowrateoftheenvironmentmayhavedetrimentaleffectsonthe

abilitytoperformrheotaxis,whichmayultimatelyaffectsurvivalrates.The

studyprovidesaclearlinkbetweenthesuperficialneuromastsystemand

behaviour.

88

Chapter4.Generaldiscussion

IntroductionTheresearchpresentedinthisthesisinvestigatedthemechanosensorylateral

linesystemofthewesternrainbowfish(Melanotaeniaaustralis)inorderto

understandhownativefreshwaterfishessurviveandflourishunderthehighly

dynamichydrologicconditionsthatcharacterisethearidPilbararegionof

inlandnorthwestAustralia.Thefindingsofthisstudyconsiderablyenhance

ourcurrentunderstandingoftherelationshipbetweenthelaterallinesystem

andwaterflowinwesternrainbowfish,demonstratingconsiderablevariation

bothamongandwithinpopulations,andhabitats.Thesefindingsthushave

relevancetounderstandingadaptationstohigh‐oftenextreme‐variationin

localconditionsandhowtheseadaptationsmayberelevanttopredicting

responsesofthewesternrainbowfishtobothman‐madeandclimatedriven

changesinstreamhydrology.

Itiswelldocumentedthatariver’sflowregimeisintegraltotheecological

integrityoftheecosystem(Poffetal.,2010).Thus,alteringthemagnitude,

frequencyortimingofflowscanhavemultipleecologicaleffectsonfreshwater

fishpopulations.Whetherthesealterationsarenaturaloranthropogenic,the

speciesthatsurviveintheseecosystemsposeaninterestingmodelto

investigatepopulationresponsestorapidenvironmentaldisturbances

(Franssen,2011).Theresearchpresentedinthisthesisemphasizesthe

importanceofwithinandamongpopulationvariationinthelaterallinesystem

andthustherolethatthelaterallinesystemlikelyplaysinbehavioural

89

adaptationsofthewesternrainbowfishtoitsdynamicenvironment.Inthis

finaldiscussionchapter,Ireviewandcompilethefindingsoftheresearchand

presentanoverallpictureregardingthesignificanceoftheseresultsinterms

ofsensoryandbehaviouraladaptationstodynamichydrological

environments.Finally,Iconsidersomeofthelimitationsofthestudyand

presenttopicsforpotentialfutureresearch.

VariationinthemorphologyofthelaterallinesystemofthewesternrainbowfishMyresearchhasaddedtoonlyahandfulofstudiesthathavemappedlateral

linesystemdiversitywithinonespecificspecies.Oneofthemajorfindings

frommyresearchisthatthewesternrainbowfish(M.australis)hasahighly

variablenumberandarrangementofsuperficialneuromastsbothwithinand

amongpopulations,whichhasnotbeendescribedinanyotherfishspecies.In

M.australis,theneuromastswerealwaysfoundinparticularlocationsoverthe

body,includingregionsoverthehead,bodyandcaudaltail(Figure1,Chapter

2).Ialsofoundthepatterningofneuromastswashighlyinconsistentacross

bodyregions.Themostvariablewasthecheekarea(withthehighest

coefficientofvariation),whilethenasalregionhadthehighestdensityof

superficialneuromasts(Chapter2).Inconjunctionwiththisvariability,Ialso

foundasignificantlinkbetweenthehydrologicalconditionsofthefish’s

naturalhabitatandthetotalnumberofsuperficialneuromastspresentonthe

body.Thisfindingisconsistentwithmanypreviousstudies,wherelimnophilic

fisheslivinginquieter,slowerenvironmentswillhavemoresuperficial

neuromaststhanrheophilicfishthatliveinnoisier,fast‐pacedenvironments

90

(Dijkgraaf,1963,Jakubouski,1967,Teyke,1990,Bleckmann,1994,Coombset

al.,1998,Englemannetal.,2002,Janssen,2004,Tan,2011,Vischer,2013).

Collectively,theseresultssuggestthatthesuperficialneuromastsarea

specialisedsensorytraitinM.australisthataretightlycoupledtothe

hydrologicalenvironmentinwhichthefishlive.Thestudyalsoreveals

interestingresultsforWeeliWolliCreek,asthisisthesitewherehydrological

conditionshavebeenalteredsignificantlyduetominingdischarge(Dogramaci

etal.,2015).Interestingly,WeeliWolliCreekwasthesitethatshowedtheleast

amountofvariationintotalsuperficialneuromastabundancecomparedtoall

othersites.Thislowlevelofvariationsuggeststhatthelaterallinesystemis

finelytunedtoaparticularenvironmentandthatselectiveprocessescouldbe

operatingtomaintainthisstate.Theseobservationsposequestionsastowhat

isthegeneticbasisofthelaterallinesysteminthispopulation(andother

populationsinothersites)andwhataffectsthechangesinhydrologyhavehad

ontheirsensorysystems?Alteredhydrologicalconditions,particularlywhen

theyoccuroverashortperiodoftimemaydisruptananimal’sabilityto

performbehavioursreliantonthelaterallinesystem.

RoleofthelaterallinesystemofM.australisinrheotaxis

MyresearchrevealedthattheabilityofM.australistoorientintoflowwas

significantlyreducedwhentheirlaterallinewaschemicallyblocked,

confirmingthatthissenseisanintegralpartofrheotaxis(Chapter3).These

rheotacticexperimentsindicatethatdrasticallyalteringflowratesislikelyto

influencethefish’sbehaviouralresponses.Forexample,particularsectionsof

theWeeliWolliCreekandthepopulationofwesternrainbowfishthatinhabit

91

thisareahavebeensubjecttoconsistentandfasterflowingwaterfor

approximately10years,whichisduetominewaterdischarge(Dogramaciet

al.,2015).Thefindingsfromthisstudyaswellascontinuedpopulation

monitoringclearlydemonstratethatM.australishasbeenabletopersistand

adapttothehydrologicalchangesinthisecosystem;thefindingsofmy

researchsuggestthatbehaviouraladaptationhasplayedaroleinthissuccess,

asevidencedbythedifferencesintheirsuperficialneuromastsystem

comparedtootherpopulations.

However,long‐termpopulationviabilityatbothWeeliWolliaswellassites

notimpactedbyartificialchangesinflowsrequiremonitoringofpopulations

overasustainedperiodofstudyinordertoassessthepersistenceofadaptive

capacityandhowwidespreadthiscapacityisacrossthefullrangeofthe

species.

Anthropogenicalterationstoenvironmentscancauserapidchangesinthe

phenotypictraitsofwildpopulations(Palkovacs,2011)andthesechangescan

occurasaresultofeithercontemporaryevolution,orphenotypicplasticity,or

acombinationofthetwo.Itislikelythattheobserveddiversityofthelateral

linesystem,andspecificallythesuperficialneuromasts,areaproductofboth

theseprocesses.However,wedonotknowtowhatextentthissensorysystem

diversitywillaffectotheraspectsoftheirbehaviourbesidesrheotaxis.The

laterallinehasbeenlinkedtonumerousothercrucialbehaviourssuchas

foraging,avoidingpredation,preycaptureandschooling,anditislikelythat

thesetooareaffectedbypopulationvariationinlaterallinemorphology.Many

92

studieshavedocumentedthatthelaterallineisimportantforpredator

avoidanceandpreycapture(McHenryetal.2009,Stewartetal.2013).In

particular,Stewartetal.(2013)usedvideorecordingsofpredator‐prey

interactionstodeterminethelateralline’sinvolvementinpredatoravoidance.

Theytoo,chemicallyablatedthelaterallineandfoundthatthepreyanimals

wererarelyabletoevadeapredator’sstrikeincomparisontothosewitha

functionallaterallinethatwereabletoevadepredation70%ofthetime.

Althoughchangingthehydrodynamicenvironmentisanextremeexampleof

whattheanimalsmightexperiencewhentheirlaterallineisablated,itmay

hindertheirabilitytoperformallthebehavioursdiscussedabove.

WhilethedirecteffectsofartificialwaterflowsatWeeliWolliCreekonfish

populationsappearoverallpositive,thereremainsthepossibilityof

concurrentchangesinthepH,turbidityanddissolvedoxygenlevelsofthe

water.Thesealterationscouldhaveconsiderablecascadingeffectsonthe

microfaunaandfloraoftheecosystemthatpotentiallytranslateintoother

importantecologicaleffects.Forexample,theremaybealteredavailabilityof

foodforrainbowfishesandtheotherspeciesinhabitingtheecosystem,which

willconsequentlyaltertheenergyflowthroughfoodwebs(Palkovacs,2011).

Furtherinvestigationsarealsorequiredintotheadaptivecapacityofotherfish

speciesthatinhabitWeeliWollicreek.Similarly,cessationofdewateringatthe

terminationoftheminingprojectwilleventuallyshiftafastflowing

permanentsystembacktoamoreintermittentanddisconnectedstream.

Giventheresponsivenessofrainbowfishesatleasttoadiversityofhabitats,

reductionofwaterflowshouldbegradualandoveralongperiodoftime

93

(years)toallowtheanimalsthatinhabitthecreektoadapttothechanges,

ratherthanadrasticchangeinflow.

Manystudieshaveinvestigatedmethodsforconservingfreshwater

ecosystems,yettodatethesesystemshavebeenlargelyoverlookedfor

conservation(Kingsford,2011).Thisisparticularlytruefortheremotenorth

ofWesternAustralia.Nonetheless,conservationeffortsareimportantandwill

contributetocurrentknowledgeofthislittle‐studiedregion.Itisalso

importanttomanageanyalteredflowsduetoanthropogenicactivitiessuchas

miningand/orwaterallocationstonewagricultureinlightofprojectedeffects

ofclimatechange.Climatechangeisexpectedtohavethefollowing

detrimentaleffectsonfreshwaterecosystems:increasedtemperatures,

decreasedorincreasedintensityofrainfall,alteredflowratesandanalteration

inthetimingandvariabilityofflowregimes(Kingsford,2011)ThePilbara

regionisalreadyahostileandharshenvironmentandmoresusceptibleto

climatechangeduetotheincreasedintensityoffloodeventsseparatedby

prolongedperiodsofdroughtandthusreducedsurfaceflows(Parryetal.

2007).Thisareashouldbeconsideredapriorityinconservationwithin

Australia,althoughitisgenerallyunderstoodthatcurrently,most

environmentalflowsareinadequatetomeettheneedsofdownstream

ecosystems.Therefore,suggestionsformanagementwouldincludeadoptinga

methodtomaintaintheecosystems’naturalflowratesinthefaceofclimate

change.Modellingtoolshavebeendevelopedtoprovidelargespatialandlong‐

termtemporalresolutionthatwillaidinthedecision‐makingprocessforbest

practicemanagementalthoughgenerallythesemodelsareconstrainedbynot

94

includingvaluableinformationregardingtheimpactsofclimatechangeonkey

fishspecies(Aldousetal.2011).

LimitationsofmyresearchprojectMystudyhighlightssomeofthechallengesofundertakingfish‐focussed

researchinhot,aridandremoteenvironments.Mystudywasrestrictedto

onlyeightpopulations,whichisarelativelysmallsample,yetreflectsthe

limitednumberofsiteswithwateracrossaridlandscapes(seealsoLostromet

al.,2014).ThePilbaraisaregionofclimaticextremes,remoteandforthemost

partundisturbed,whichmeansaccesstostreamsandcreeksislimited.For

example,atCoondinerCreek,Westernrainbowfishweresightedatsomeofthe

poolsandlocationsthatotherresearchershavesampledpreviously

(approximately7kmupstream),butwhichareonlyaccessiblebylongtrekson

footandthusnotfeasibletosafelytransportviablefishforfurtherstudyunder

controlledconditionsinPerth,some1500kmaway.Whileideally,Iwould

havelikedtosamplefromafewpopulationsupstreamatCoondinerCreekas

wellasinthemidFortescuearea,fromareassuchasKarijiniNationalPark,

thiswasfeasiblegivenlogisticsandlimitedbudgets.Helicopteraccessand

speciallydesignedfishtransportsystemsmayovercomethissampling

challenginginthefuture.

ConcludingcommentsandfutureresearchThisthesishasprovidedsomeofthefirstinvestigationsofthelateralline

systeminnativeAustralianfreshwaterfishes.However,muchmoreworkis

requiredtounderstandifthelaterallinevariationshownbyM.australisis

95

determinedbygeneticdivergenceamongpopulations,contemporary(rapid)

evolutionarychangeorphenotypicplasticity(oracombinationofthese

differentprocesses).Indeed,therehavebeenrelativelyfewstudiesofthis

natureandthesehavebeenfocusedononlyahandfulofspecies.Forexample,

Trokovichandcolleagues(2011)reportedasignificantdifferencein

neuromastnumberinpopulationsofnine‐spinedstickleback(Pungitius

pungitius)capturedfrommarineandfreshwaterpondhabitats.Theyused

"commongarden"experiments,wherefishwerebornandrearedinthe

laboratoryforoneyear.Theyfoundthatthelaboratory‐rearedfishhadsimilar

numbersofneuromaststothosecollectedfromwildpopulations,suggesting

thatneuromastnumberhasageneticbasisinthisspecies(Trokovicetal,

2011).Theyalsofoundthatwildpondpopulationsshowedgreatervariationin

thenumberofsuperficialneuromastspresentthanmarinepopulations.

However,populationsrearedinthelaboratorydisplayedareducedlevelof

neuromastnumbervariabilityafteronegeneration(Trokovicetal,2011),

therebyindicatingthatpopulationsrearedinanenvironmentwithcontrolled

andconsistentwaterflowswillhavelessneedforvariability.A“common

garden”experimentaugmentedbyageneticanalysisforthelociassociated

withlaterallinedevelopmentinM.australismaythusbeausefulnextstepto

determinehowgeneticallydistinctareeachofthepopulationsassessedinthis

study.

Theresearchpresentedwasprimarilymotivatedbytheneedtoinvestigatethe

responseofthelaterallinesystemofM.australisinrelationtoflow.Further

researchisrequiredtodetermineifdifferencesinthesuperficialsystemalso

affectotherbehavioursthataredependentonthelateralline.Forexample,it

96

hasrecentlybeenreportedthatsuperficialneuromastsmayplayasignificant

roleinpredatoravoidance(Stewartetal.2013);afindingthatcontradicts

previousstudiesthathavesuggestedthatcanalneuromastsarespecialisedto

determineanddiscriminateobjects(BleckmannandZelick.2009).Further

experimentsonrainbowfishesapplyingthemethodsofthispreviousstudy

(Stewartetal.2013),butchangingtheflowrateswithinthechambersothat

theywerefaster/slowthanthoseofthefish’snaturalhabitat,wouldreveal

whetherthelaterallinesystemisadaptedforbehavioursinanykindof

hydrologicalconditions.Iftheresultsshowedthatfishwerelessabletosense

apredator’sstrike,itwouldaugmentmystudyonrheotaxisandagain

illustratethedetrimentaleffectsthatalteringahydrologicalenvironmentcan

haveonaspecies’behaviour.

Inconclusion,thisstudyhaslaidthefoundationforassessingthebehavioural

ecologyandadaptivecapacityofnativefreshwaterfishesofinlandAustralia.

97

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Appendices

Appendix1:

Initialtrialswereconductedonthewesternrainbowfishwithneomycin

sulphatetodeterminetheconcentrationthatmosteffectivelyblocksthelateral

line.Concentrationswerefirsttrialedonlaboratoryfishat500uM,750uM,

800uM,1000umandfinallyat1200uM.Afterbeingimmersedinneomycin

sulphatefor1hour(ormore–seebelow)atthegivenconcentration,animals

werestainedtovisualisethesuperficialneuromasts.Stainingwasperformed

byexposingfishtofluorescentvitaldye2‐[4‐(dimethylamino)styrl]‐N‐

ethylpyridiniumiodide,DASPEI(LifeTechnologies/MolecularProbes,Eugene

OR,USA)for15minutesataconcentrationof0.24gin1Lwater,(1200mM).

Fisheswerethenanaesthetisedwith200mgl‐1MS222(tricaine

methanesulfonate;Sigma‐Aldrich,StLouis,MO,USA)untillightpressure

onthecaudalfinyieldednoresponse.Theindividualwasthenplacedright

sidedowninapetridishandplacedonthestageofafluorescencedissecting

microscope(LeicaMZ75fittedwithaFITCfilterset;LeicaMicrosystemsInc.,

Sydney,Australia).Imagesweretakenusingadigitalcamera(LeicaDFC320)

toillustratetheblockingofthelateralline.

Initially,thefishwereexposedtotheblockingagentforonehour,butareview

oftheliteratureshowedthatotherexperimentersexposedanimalsforlonger

periodsoftime.I,therefore,increasedtheimmersiontimeinneomycin

sulphatetotwohours.Thetreatmentwasdeemedeffectivewhenthe

superficialneuromastsappeareddullorabsentunderthefluorescentscope,

105

relativetocontrolsthatwerereadilyvisible.Thetrialsresultswereviewedby

twoindividualsandaconsensuswasdetermined.Thisrevealedthatthemost

effectiveconcentrationofneomycinsulphatewas1200umusinganexposure

timeoftwohours.Shownbelowareimagesoftrialrainbowfishes,stainedwith

differentconcentrationsofneomycinsulphate.

Figure16.LightmicrographsshowingDASPEI‐stainedsuperficialneuromasts

overtheheadandbodyofM.australisduringpreliminaryneomycinsulphate

trials.Picturesdepicttheleveloffluorescenceafteranimalswereexposedto

concentrationsof500um,700um,800um,andthefinalconcentrationusedfor

theexperiment(1200um),incomparisontothecontrolfish.

500um 700um 800um

1200um

Control