Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
1
Functionaldiversityofthelateralline
systemamongpopulationsoftheWestern
Rainbowfish(Melaenoteniaaustralis)
LindseySpiller(BScHons)
NeuroecologyGroup,SchoolofAnimalBiology
ThisthesisispresentedforthedegreeofMastersofPhilosophyattheUniversityofWesternAustralia.
2016
2
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.
3
Thestudyalsoinvestigatedhowintraspecificvariationinthesuperficial
neuromastsaffectstheabilityofthisspeciestoperformrheotaxis(orientation
intowaterflows).Arheotacticstudywasdesignedtodetermineifdifferences
inthesuperficialneuromastarrangementhadaneffectontheirabilityto
correctlyorientateatdifferingflowspeeds.Chemicalablationwasalsousedto
determinetheroleofthelaterallineinthisbehaviour.Differencesinthe
abundanceofsuperficialneuromastsaffectedtheabilitytoorientateinto
waterflowsandrainbowfishesshowedareducedabilitytoorientatewithout
theuseofafunctionallateralline.Furtherstudyisrequiredtofully
understandwhetherindividualpopulationshavethecapacitytoadapttheir
laterallinesystemtoalteredhydrologicalconditions.
4
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
5
Introduction...........................................................................................................................88Variationinthemorphologyofthelaterallinesystemofthewesternrainbowfish.............................................................................................................................89RoleofthelaterallinesystemofM.australisinrheotaxis....................................90Limitationsofmyresearchproject................................................................................94Concludingcommentsandfutureresearch................................................................94
References......................................................................................................................97
Appendices...................................................................................................................104Appendix1:...........................................................................................................................104
6
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
7
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.
8
LastbutnotleastIwouldliketothankmyincrediblysupportingpartnerJean‐
SebastianCorreiaandhisverykindparentsHelenandCarlos.Carlosthankyou
foryouconstantadviceontheacademicenvironment.Jean,youhavebeenmy
biggestenthusiastandhavealwaysprovidedmewithlove,supportand
empathy.Youhavealsoalwaysbeensoincrediblygenerouswithyourtime
andwillingtoassistmeinanythingthatIneeded.Hekeptmepositiveand
driventhroughoutthisentireprocessandIwillbeeternallygrateful.
9
DeclarationofCandidaturecontribution
ThisthesisdoesnotcontainworkthatIhavepublishedorworkcurrently
underreviewforpublication.
10
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.
11
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
12
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,
13
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
14
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.
16
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.
20
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
21
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.
84
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
References
Allen,G.R.,MidgleyS.H.&Allen.M.,(2002).FreshwaterFishesofAustralia.Perth:WesternAustralianMuseum.
AustralianBureauofMeteorology.(2011).UVandSunProtectionServices.CommonwealthofAustralia.Availableatwww.bom.gov.au/uv/?ref=ftr.
Bak‐Coleman,J.,Coombs,S.(2014).Sedentarybehaviorasafactorindetermininglaterallinecontributionstorheotaxis.JournalofExperimentalBiology,217,2338‐2347.
Bak‐Coleman,J.,Court,A.,Paley,D.A.andCoombs,S.(2013).Thespatiotemporaldynamicsofrheotacticbehaviordependsonflowspeedandavailablesensoryinformation.JournalofExperimentalBiology,216,4011‐4024.
Baker,C.F.andMontgomery,J.C.(1999a).ThesensorybasisofrheotaxisintheblindMexicancavefish,Astyanaxfasciatus.JournalComparativePhysiology,184,519‐527.
Baker,C.F.andMontgomery,J.C.(1999b).LaterallinemediatedrheotaxisintheAntarcticfishPagotheniaborchgrevinki,PolarBiology,21,305‐309.
Barnett,J.C.&Commander,D.P.(1985).HydrogeologyoftheWesternFortescueValley,PilbaraRegion,WesternAustralia.In‘WesternAustraliaGeologicalSurvey,Record1986/8’.(DepartmentofIndustryandResources:Perth,Australia).
Bassett,D.K.,CartonA.G.,MontgomeryJ.C.,(2006)Flowingwaterdecreaseshydrodynamicsignaldetectioninafishwithaepidermallateral‐linesystem,MarineandFreshwaterResearch,57,611‐617.
Baxter,R.M.(1977).Environmentaleffectsofdamsandimpoundments.AnnualReviewofEcology,EvolutionandSystematics8:255–283.
Beckmann,M.T.Erős,A.SchmitzandH.Bleckmann(2010)."NumberandDistributionofSuperficialNeuromastsinTwelveCommonEuropeanCypriniformFishandTheirRelationshiptoHabitatOccurrence."InternationalReviewofHydrobiology95(3):273‐284.
Blaxter,J.(1987).Structureanddevelopmentofthelateralline.BiologicalReviewsoftheCambridgePhilosophicalSociety62,471‐514.
BleckmannH(1994)Receptionofhydrodynamicstimuliinaquaticandsemiaquaticanimals.In:RathmayerW(ed)ProgressinZoology,41,1‐115.
Bleckmann,H.,Zelick,R.,(2009).Laterallinesysteminfish.IntergrativeZoology.4,13‐25.
98
Carton,A.G.andMontgomery,J.C.(2004).Acomparisonoflaterallinemorphologyofbluecodandtorrentfish:twosandperchesofthefamilyPinguipedidae.EnvironmentalBiologyofFish,70,123‐131.
Chagnaud,B.P.,Brucker,C.,Hofmann,M.H.,BleckmannH.,(2008)MeasuringFlowVelocityandFlowDirectionbySpatialandTemporalAnalysisofFlowFluctuations.JournalofNeuroscience,28(17)4479‐4487.
Champalbert,G.,Marchand,J.andleCampion,J.(1994).RheotaxisinjuvenilesoleSoleasolea(L.):influenceofsalinityandlightconditions.JournalSeaRestoration,32,309‐319.
Coombs,S.,Janssen,J.,andWebb,J.F.(1988).Diversityoflaterallinesystems:evolutionaryandfunctionalconsiderations.SensoryBiologyofAquaticAnimals.(EdsJ.Atema,R.R.Fay,A.N.PopperandW.N.Tavolga.)pp.553–595.(Springer‐Verlag:NewYork).
Coombs,S.andMontgomery,J.(1994).FunctionandevolutionofsuperficialneuromastsinanAntarcticnotothenioidfish.BrainBehaviouralEvolution.44,287‐298.
Dames&Moore(1984).Millstreamwatermanagementprogram.EngineeringDivision,PublicWorksDepartment,WesternAustralia.
Delfinn,T.,Patton,P.,Coombs,S.,(2011).Doblindcavefishhavebehaviouralspecializationsforactiveflow‐sensing?JournalofComparativePhysiologyA187:743‐754.
Dijkgraaf(1962).ThefunctioningandthesignificanceoftheLateralLinesystem.BiologicalReviews38(1):51‐105.
Dogramaci,S.&Skrzypek,G.,(2015).UnravellingsourcesofsolutesingroundwaterofanancientlandscapeinNWAustraliausingstableSr,HandOisotopesChemicalGeology,vol393‐394,pp.67‐78.
Engelmann,J.,HankeW.&BleckmannH.,(2002).Laterallinereceptioninstill‐andrunningwater.JournalofComparativePhysiologyA188(7):513‐526.
Eros,T.,Botta‐Dukat,Z.&GrossmanG.D.,(2003):AssemblagestructureandhabitatuseoffishinaCentralEuropeansubmontanestream:apatch‐basedapproach.EcologyofFreshwaterFish.12:141–150.
Fellman,J.B.,Petrone,K.C.&Grierson,P.F.(2012).Leaflitterage,chemicalquality,andphotodegradationcontrolthefateofleachatedissolvedorganicmatterinadrylandriver.JournalofAridEnvironments,89,30‐37.
FischerE.,K.,Soares,D.,Archer,K.,R.,GhalamborC.,K.,HokeK.,L.,(2013),GeneticallyandenvironmentallymediateddivergenceinlaterallinemorphologyintheTrinidadianguppy(PoeciliareticulataI),ThejournalofExperimentalBiology,216,3123‐3142.
99
FranssenN.R.,(2011).Anthropogenichabitatalterationsinducesrapidmorphologicaldivergenceinnativestreamfish.EvolutionaryImplications.4,791‐804.
HarrisJ.A.,ChengA.G.,CunninghamL.L.,MacDonaldG.,RaibleD.W.,RubelE.W.,(2003),Neomycin‐InducedhaircelldeathandrapidregenerationintheLateralLineofZebrafish(Daniorerio),JournaloftheAssociationfortheResearchinOtolaryngology,4,219‐234.
Hemmi,J.M.,PfeilA.,(2010)Amulti‐stageanti‐predatorresponseincreaseinformationonpredationrisk,JournalofExperimentalBiology,213,1484‐1489.
Hofer,B.(1908).StudienuberdieHautsinnesorganederFische.I.DieFunktionderSeitenorganebeidenFischen.BerKglBayerBiolVersuchsstationMunchen1:115–64.
Janssen,J.(2004)Laterallinesensoryecology.In:vonderEmdeG,MogdansJ,KapoorBG(eds)Thesensesoffishes:adaptationsforthereceptionofnaturalstimuli.NarosaPublishingHouse,NewDelhi,231–264.
Kanter,M.J.andCoombs,S.(2003).RheotaxisandpreydetectioninuniformcurrentsbyLakeMichiganmottledsculpin(Cottusbairdi).JournalExperimentalBiology,206,59‐70.
Kelley,J.L.,Phillips,B.,Cummins,G.H.,Shand,J.(2012).Changesinthevisualenvironmentaffectcoloursignalbrightnessandshoalingbehaviourinafreshwaterfish.JournalofAnimalBehaviour,83,783‐789.
KendrickP.,(2001)Pilbara3(PIL3–Hamersleysubregion),ABiodiversityAuditofWesternAustralia’s53BiogeographicalSubregions,DepartmentofConservationandLandManagement.
Kingsford,R.T.(2011).Conservationmanagementofriversandwetlandsunderclimatechange‐asynthesis.MarineandFreshwaterResearch62(3),217‐222.
Kulpa,M.,Bak‐Coleman,J.,CoombsS.,(2015)Thelaterallineisnecessaryforblindcavefishrheotaxisinnon‐uniformflow.JournalofExperimentalBiology,218,1603‐1612.
Lostrom,S.,EvansJ,GriersonPF,CollinSP,DaviesPE,KelleyJL.(2015).Linkingstreamecologywithmorphologicalvariabilityinanativefreshwaterfishfromsemi‐aridAustralia.EcologyandEvolution5,3272‐3287.
Lyon,E.P.(1904)Onrheotropism.I.Rheotropisminfishes.AmericanJournalPhysiology,12,149–161.
Marshall,N.J.,(1996)Thelaterallinesystemsofthreedeep‐seafish.JournaloftheMarineBiologicalAssociationoftheUnitedKingdom,66,2,323‐333
100
McGuigan,K.,Franklin,C.E.,Mortiz,C.&Blows,M.W.(2003).Adaptationofrainbowfishtolakeandstreamhabitats.Evolution1,104‐118.
McHenry.M.J.,Feitl,K.E.,Strother.J.A.,VanTrumpJ.A.,(2009),Zebrafishrapidlysensethewaterflowofapredatorsstrike.BiologyLetters:AnimalBehaviour.5,477‐479.
MillerR,P.J.,(1973).TheosteologyandadaptivefeaturesofRhyacichthysaspro(Teleostei:Gobioidei)andtheclassificationofgobioidfish.JournalofZoology,171,397‐434.
Miller,P.J.(1986).Affinities,originandadaptivefeaturesoftheAustraliandesertgobyChlamydogobiuseremius(Zietz,1896)Teleostei:Gobiidae.JournalNaturalHistory.21,687–705.
Mogdans,J.&BleckmannH.(2012).Copingwithflow:behavior,neurophysiologyandmodelingofthefishlaterallinesystem.BiologicalCybernetics106(11‐12),627‐642.
Mogdans,J.,Kröther,S.,Engelmann,J,(2004).NeurobiologyofthefishLateralLine:AdaptationsfortheDetectionsofhyrdronamicStimuliinRunningWater,SpringerNedlands,265‐287.
Montgomery,J.C.&MacdonaldJ.A.,(1987).SensoryTuningofLateralLineReceptorsinAntarcticFishtotheMovementsofPlanktonicPrey.Science235(4785),195‐196.
MontgomeryJC,CoombsS,Halstead,M.D.B.(1995)Biologyofthemechanosensorylaterallineinfishes.ReviewsFishBiology,5,399‐416.
Montgomery,J.C.,Baker,C.F.andCarton,A.G.(1997).Thelaterallinecanmediaterheotaxisinfish.Nature,389,960‐963.
Morgan,D.L.&Gill,H.S.(2004).FishfaunaininlandwatersofthePilbara(IndianOcean)DrainageDivisionofWesternAustralia—evidenceforthreesubprovinces.Zootaxa,636,1‐43.
MünzH.,(1985).SingleunitactivityintheperipherallaterallinesystemofthecichlidfishSarotherodonniloticusL.JournalofComparativeandPhysiologyA‐SensoryNeuralandBehaviouralPhysiology157,555–68.
Northcutt,R.G.(1989).Thephylogeneticdistributionandinnervationofcraniatemechanoreceptivelaterallines.InTheMechanosensoryLateralLine(ed.S.Coombs,P.GörnerandH.Münz),pp.17‐78.
Palkovacs,E.,P.,KinnisonM.T.,CorreaC.,DaltonC.M.&HendryA.P.,(2012).Fatesbeyondtraits:ecologicalconsequencesofhuman‐inducedtraitchange.EvolutionaryApplications5(2),183‐191.
101
Parry,M.L.,CanzianiO.F.,PalutikofJ.P.,vanderLindenP.J.,&HansonC.E.,(2007).ContributionofWorkingGroupIItotheFourthAssessmentReportoftheIntergovernmentalPanelonClimateChange,2007.Cambridge,UKandNewYork,USA,IntergovernmentalPanelonClimateChange.
Partridge,B.L.P.,Pitcher,T.J.(1980).Thesensorybasisoffishschools;relativerolesofalteallineandvision.TheJournalofComparativePhysiology.135,315‐325.
Poff,N.L.,Allan,J.D.,Palmer,M.A.,Hart,D.D.,Richter,B.D.,(2003).Riverflowsandwaterwars:emergingscienceforenvironmentaldecisionmaking.FrontiersinEcologyandtheEnvironment1,298–306.
Puzdrowski,R.L.(1989).Peripheraldistributionandcentralprojec‐tionsofthelateral‐linenervesingoldfish,Carassiusauratus.BrainBehavouralEvolution34,110–131.
RouillardA.,SkrzypekG.,DogramaciS.,TurneyC.,GriersonPF.(2015)ImpactsofachangingclimateonacenturyofextremefloodregimeofnorthwestAustralia,HydrologicalandEarthSystemSciences.19,2057–2078.
Schmitz,A.,Bleckmann,H.andMogdans,J.(2008).Organizationofthesuperficialneuromastsystemingoldfish,Carassiusauratus.JournalofMorphology,269,751‐761.
SchulzeF.(1861.)UberdieNervenendigungindensogenann‐tenSchleim‐KanalenderFischeunduberentsprechendeOrganederdurchKiemenatmendenAmphibien.ArchAnatuPhys,759–69.
SiebersA.R.,PettitN.E.,SkrzypekG.D.,FellmanJ.B.,DogramaciS.,GriersonP.F.,(2015).Alluvialgroundwaterinfluencesdissolvedorganicmatterbiogeochemistryofpoolswithinintermittentdrylandstreams.FreshwaterBiologyDOI:10.1111.
Skrzypek,G.,Dogramaci,S.&Grierson,P.F.(2013)GeochemicalandhydrologicalprocessescontrollinggroundwatersalinityofalargeinlandwetlandofnorthwestAustralia.ChemicalGeology,357,164–177.
Stewart,W.,Cardenas,G.,McHenry,M.(2013).Zebrafishlarvaeevadepredatorsbysensingwaterflow.TheJournalofExperimentalBiology,216,388‐398.
Suli,A.,Watson,G.M.,Rubel,E.W.andRaible,D.W.(2012).Rheotaxisinlarvalzebrafishismediatedbylaterallinemechanosensoryhaircells.PLoSONE7,e29727.
Tan,D.,Patton,P.,Coombs,S.,(2011).Doblindcavefishhavebehaviouralspecializationsforactiveflow‐sensing.JournalofComparativePhysiologyA,197,743‐754.
102
Trokovic,N.,Herczeg,G.,McCairns,R.J.,AbGhani,N.I.andMerilä,J.(2011).Intraspecificdivergenceinthelaterallinesysteminthenine‐spinedstickleback(Pungitiuspungitius).JournalofEvolutionaryBiology,24,1546‐1558.
TykeT.,(1990)MorphologicaldifferencesinneuromastsinblindcavefishAstyanaxhubbsiandthesightedriverfish,BrainBehaviouralEvolution,35,23–30.
UnmackP.J.,(2001)BiogeographyofAustralianfreshwaterfish.JournalofBiogeography,28,1053‐1089.
VanderphamJ.P.,NakagawaS.,ClossG.P.,(2013),Habitat‐relatedpatternsinphenotypicvariationinaNewZealandfreshwatergeneralistfish,andcomparisonswithacloselyrelatedspecialist,FreshwaterBiology,58,396‐408.
VanTrumpW.J.,CoombsS.,DuncanK.,McHenryM.J.(2010).Gentamicinisototoxictoallhaircellsinthefishlaterallinesystem.HearingResearch,261,42–50.
VanTrump,W.J.,McHenry,M.J.(2013).ThelaterallinesystemisnotnecessaryforrheotaxisintheMexicanblindcavefish(Astyanaxfasciatus).IntegrativeandComparativeBiology,53,799‐809.
Vischer,H.A.,(1989),Themorphologyofthelaterallinesystemin3speciesofPacificcottoidfishesoccupyingdisparatehabitats.Experientia.46,244‐250.
Vogel,S.,LaBarbera,M.(1978).Simpleflowtanksforresearchandteaching.Bioscience28,638‐643.
Wark,A.R.,&Peichel,C.L.(2010).Laterallinediversityamongecologicallydivergentthreespinesticklebackpopulations.JournalofExperimentalBiology,213(1),108‐117.
Webb,J.F.(1989a).Grossmorphologyandevolutionofthemechanoreceptivelateral‐linesysteminteleostfish.BrainBehaviouralEvolution.33,34‐53.
Webb,J.F.(1989b).Grossmorphologyandevolutionofthemechanoreceptivelateral‐linesysteminteleostfish.BrainBehaviouralEvolution.33,34‐53.
Webb,J.F.andNoden,D.M.(1993).Ectodermalplacodes:contributionstothedevelopmentofthevertebratehead.AnimalZoology.33,434‐447.
Webb,J.F.andShirey,J.E.,(2003).Postembryonicdevelopmentofthecraniallaterallinecanalsandneuromastsinzebrafish.DevelopmentalDynamics228,370‐385.
Wellenreuther,M.,BrockM.,MontgomeryJ.&ClementsK.D.,(2010).ComparativeMorphologyoftheMechanosensoryLateralLineSystemina
103
CladeofNewZealandTriplefinFishes.Brain,BehaviorandEvolution.75(4),292‐308.
Windsor,S.P.,TanD.,&MontgomeryJ.C.,(2008).SwimmingkinematicsandhydrodynamicimagingintheblindMexicancavefish(Astyanaxfasciatus).JournalofExperimentalBiology211(18),2950‐2959.
WRM(2010)CumulativeimpactsofRTIOminingontheWeeliWolliCreekSystem,Dry08andWet09Sampling.UnpublishedreportbyWetlandResearch&ManagementtoRioTintoHamersleyHopeManagementServices.July2010.
Yoshizawa,M.,Goricki,S.,SoaresD.,Jeffery,W.R.,(2010),EvolutionofabehaviouralshiftMediatedbytheSuperficialNeuromastsHelpsCavefishFindFoodinDarkness,CurrentBiology,20,1631‐1636.
104
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