Chimie, Bât.B6c, Université de Liège, B-4000Liège, Belgium. hearing capacitiesfrom300to900 to theskull, volume. Bothspeciesaresensitivetosoundsup2100 theintra-neurocranium especiallycomparedwith ophidiiform species, bothspecieshavelargerotolithsthanmanynon- maximal diameters; © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2517-2525doi:10.1242/jeb.105254 Received 10March 2014;Accepted17April2014 1 ear morphologyinfishes,theyalsoraisednewquestionsconcerning studies haveinformedscientistsonthehearingcapabilitiesand inner fishes (Popperetal.,2005;LadichandFay, 2013).Althoughthese have givenincreasedattentiontothehearingcapacitiesofteleost Slabbekoorn etal.,2010).Duringthelastfewdecades,biologists crucial forfishsurvivalandreproduction(FayPopper, 2000; ability todetect,discriminateandidentifysurroundingsounds is et al.,1965;Myrberg, 1981; Ladich etal.,2006).Inthiscontext,the mammals (Fish,1964;Cato,1993)andfishesSteinberg mainly resultfrominvertebrates(Fish,1964;Cato,1993),marine 2010; LadichandSchulz-Mirbach,2013).Animalsoundsources are veryabundantinunderwaterenvironments(Slabbekoornetal., Natural abioticandbioticsoundsaswellanthropogenic KEY WORDS:Audition,Morphology, Otolith, morphology intwoophidiiformspecies: of thisstudywastoinvestigatehearingcapacitiesandinnerear totestthesizehypothesis.Themainaim good structurewithwhich occupy alargevolumeinsidetheneurocranium.Hencetheyare to increasesensitivity. Thesagittaeofmanyophidiidsandcarapids shape andsizeaffect hearingcapacities:largesagittaearethought fishes andithasbeenhypothesizedthatsagitta(saccularotolith) theprincipalhearingorganin (otolithic endorgan)isconsideredtobe of thedifferent structuresremainspoorlyunderstood.Thesaccule morphology. However, thefunctionofshape,sizeandorientation Numerous studieshavehighlightedthediversityoffishinnerear Loïc Kéver (Ophidion rochei Hearing capacitiesandotolithsizeintwoophidiiformspecies RESEARCH ARTICLE *Author ([email protected]) for correspondence Océanologique-Laboratoire Arago, B-66650Banyuls/Mer, France. Aquarium Public etdeRecherche Observatoire –ProgrammesdeConservation, 57 Rue Cuvier, CasePostale 55,75231,Paris Cedex 5,France. C.N.R.S./M.N.H.N., d’EcologieetdeGestionlaBiodiversité, Département species thatneedtoexecutepreciseandcomplexmovements. mainly forperformingvestibularfunctionsinfishes,especiallythose size. Largerotoliths(insizerelativetotheskull)mayhaveevolved capacities ofafishspeciescannotbepredictedonlybasedonsagitta 150 techniques. dissections, acus. INTRODUCTION ABSTRACT Laboratoire de Morphologie FonctionnelleLaboratoire deMorphologie etEvolutive, AFFISH-RC, Institutde Hz andfrom1200to2100 We usedamultidisciplinaryapproachthatcombines O. rochei μ Carapus acus 1 CT-scan examinationsandauditoryevokedpotential , OrphalColleye a mle aiteta h aai,butbetter smallersagittaethanthecarapid, has and O. rochei Hz. Resultsshowthathearing 1 , AnthonyHerrel and Carapusacus

Hz andsimilarsensitivitiesat Ophidion rochei sagittae havesimilar 2 3 UMR 7179 Aquariologie – and

Hz. Relative 2 , PascalRomans Carapus (Popper andFay, 2011) proposedtoillustratetherelative importance and variedbetweenspecies(Ladich andFay, 2013).Popperand Fay and Fay, 2011). Theirsensitivitytothissoundcomponentwaslow were showntorespondthepressure componentofsound(Popper However, some specieswithoutaccessoryhearingspecializations to thepressurecomponentof sound(PopperandFay, 2011). otophysic connections(‘hearing generalists’)madefishesinsensitive dichotomy wasalsobasedonthebeliefthatabsenceofspecific 1999; LechnerandLadich,2008;PopperFay, 2011). This such featuresweretermed‘hearinggeneralists’ (PopperandFay, andspecieswithout the innerearswerecalled‘hearingspecialists’, structures thatconnectorbringagasbubbleincloseproximity to and Wysocki, 2003).Historically, fishspeciesdisplayinganatomical swimbladder totheoticcapsule(BridgeandHaddon,1889;Ladich a modifiedchainofbones(Weberian ossicles)thatconnectsthe Grande, 2008;LechnerandLadich,2008).Thisstructureconsists of apparatus (Evans,1925;LadichandWysocki, 2003;Braunand have developedaspecializedhearingstructurecalledtheWeberian Wysocki, 2003).Otophysans(catfish, carp,piranhasandrelatives) bubbles totheinnerear(seePopperandFay, 1999;Ladichand Schulz-Mirbach, 2013),improvingsoundtransmissionfromgas Parmentier etal.,2011a; Parmentieretal.,2011b; Ladichand 1979; Ramcharitaretal.,2006a;BraunandGrande,2008; the swimbladdertowardoticcapsule(CoombsandPopper, auditory sensitivity:theydisplacedorextendedtheanteriorpartof Interestingly, manyfishesevolvedasimplewayforenhancingtheir highlighted inmanyteleostfamilies(Webb etal.,2008). Popper andFay, 1999;LadichandWysocki, 2003). process soundpressuregenerallyimprovesfishhearingcapacities(see to theinnerears(PopperandFay, 1999).Theabilitytoefficiently capacities ifsuchagasbubblecomesclosetoorisdirectlyconnected 1999). Thisindirectstimulationpathwaymainlyaffects hearing displacement ofagasbubble(e.g.theswimbladder)(PopperandFay, hearing becauseitisconvertedintoparticlemotionbymeansof the pressurecomponentofsoundintervenesonlyindirectlywith otoliths andsofttissues(PopperFay, 1999).Inmanyfishspecies, motion, beingexcitedbythedifferences inthedisplacementbetween called themacula(PopperandLu,2000)aresensitivetoparticle Sensory haircellsofanotolithorgan aregroupedinanepithelium considered tobethemostinvolvedinhearing(Popperetal.,2005). al., 2008).Amongthethreeotolithorgans, thesacculeisoften in theperceptionoflinearaccelerations(Popperetal.,2005;Webb et utricule andlagena)arethoughttobemainlyinvolvedinhearing angular accelerations,whereasthethreeotolithorgans (saccule, sensory epithelium(cristae)arethoughttohelpintheperceptionof Popper etal.,2005).Thesemicircularcanalsandtheirassociated three otolithorgans (PopperandFay, 1999;LadichandPopper, 2001; and Fay, 1999;Popperetal., 2005; HiggsandRadford,2013). hearing mechanismsandtheevolutionoffishinnerear(Popper Morphological specializationsthatimprovehearinghavebeen The fishinnereariscomposedofthreesemicircularcanalsand ) 3 and EricParmentier 1, * 2517

The Journal of Experimental Biology Scale bars:5 sagitta;SWB,swimbladder.correspond to(A,B) themaximaldiameteror(C,D) maximalthicknessofthesagittae. NC,neurocranium;RB,rockerbone; Sa, vertebral column(gray)andbodylimits (lightgray)isbasedonCT scans.For ophidiiform species.Thesedatashouldprovideabasistoconsider investigate hearingcapacitiesandinnerearmorphologyinthesetwo described inthesetwofamilies.Themainaimofthisstudywasto neverbeen Despite theirparticularanatomy, hearingabilitieshave (Meyer-Rochow, 1979;MatallanasandRiba,1980;Jardas,1996). and canleavetheir‘shelter’ inordertoseekfoodorsexualpartners arenocturnal 1996; MatallanasandCasadevall,1999).Bothspecies found from0to150 (Nielsen etal.,1999). commensal fromtheEasternAtlanticOceanandMediterraneanSea reconstructed anteriorpartof(A,C) 2518 Fig. rochei et al.,2012): et al.,2003;Parmentier2006b;2010;Kéver morphology remainpoorlyunderstood. 2000). However, thefactorsthatdriveevolutionoffishinnerear hearing sensitivityatlowerfrequencies(Lychakov andRebane, et al.,2005).Forexample,thegreaterotolithmass,better hearing capacities(Wilson, 1985;Nolf,1993;Paxton,2000;Popper It wasalsohypothesizedthatotolithsizeandshapemayaffect in pelagicspecies(Parmentieretal.,2001;Parmentier2002). found inbenthicorparasiticspecieswhereasthinotolithsare ecological niches.Incarapids,thethickestandheaviestsagittaeare todifferent habitatsor that sagittashapeandsizeareadaptations Lombarte andCruz,2007;etal.,2010)havesuggested Paxton, 2000;Parmentieretal.,2001;CruzandLombarte,2004; authors (Wilson, 1985;LombarteandLleonart,1993;Nolf, 1987) andOphidionrochei including someOphidiidae,e.g. saccules orsagittaewerealsoobservedinotherOphidiiformes at thelevelofoticcavity(Parmentieretal.,2001).Large cavity, pushingthebrainforward withonlyathinmyelencephalon these speciesareparticularlylarge andoccupynearlytheentireotic thesacculesof consequently, The sagittae(saccularotoliths)and, studied becauseoftheirwideoticcavity(Parmentieretal.,2001). Parmentier, 2004;Anderson,2005;Parmentier, 2012)havebeen at leasteightoutofthe33speciesCarapidae(Nielsenetal.,1999; the otolithorgans, shouldalsoimprovehearingcapacities.To date, (extensive useofthepressurecomponent). bubbles (onlyparticlemotiondetection)andotophysanfishes variations betweentwoextremesituations:fisheswithnogas of pressuredetectionmechanismsinfishhearingbyacontinuum RESEARCH ARTICLE This studyfocusesontwovocalophidiiformspecies(Parmentier Some adaptivechangesoftheinnerear, especiallyatthelevelof

.Tredmninlreconstructionofthe anteriorpartof Three-dimensional 1. Müller 1845(Ophidiidae).

mm. Carapus acus

m intheMediterraneanandBlackSeas(Jardas, Ophidion rochei (Lychakov andRebane,2000).Some (Brünnich 1768)(Carapidae)and O. rochei Acanthonus armatus Carapus acus and (B,D)C.acus. is asand-dwellingspecies is aholothurian (Fine etal., The 3Dreconstructionofsaccularotoliths (red),swimbladder(blue),neurocranium(gray), Ophidion rochei O. O. rochei,thegrowingrockerbone(orange)was alsoreconstructed.Dottedlines neurocranium of in thedorsoventralaxis. species, theywerelongeralongtheantero-posterioraxisbutlower buttheyappearedrelativelydifferent. Intheformer orientation, canaliculus (Fig. example, wereconnectedtotherestofinnerearbyalong Sagittae ofO.rochei high Eindexvaluestendtobemorecircular(versuselongate). 2003), theEindexcorrespondstooverallotolithshape:sagittaewith mean valueof23.4%. 2). in Ophidiiformesandlessthan4%otherspeciestested(Tables volume wasbetween12.7%and25.2%oftheneurocranium to theheadthaninothermeasuredspecies(Tables (including basioccipital stalk,forexample,maintainsthelagenaeclosetoa well individualizedin respectively). ca. 74%and55%, C. acus [volume ofthesagittae/intraneurocraniumvolume:(VolS/VolINC)]: volume [sagittae maximalthickness/headlength(ST/HL)]and [sagittae maximaldiameter/headlength(SD/HL)],thickness the sizesrelativetoskullshoweddifferences indiameter while theirvolumeswerehigherin thickness ofthesagittaeweresimilarbetween 1–3).Themaximaldiameterand anterior partoftheskull(Figs portion oftheoticcapsulewhilebrainismainlyrestrictedto are quitesimilarinbothspecies.Sagittaeoccupyasubstantial and sizeofthebrain,semicircularcanals,otolithsotolithorgans description andillustrationofstructuresinterest.Position,shape The combinationofdissectionsandCT scansallowedaprecise fish innerear. the possiblebenefitoffered bythepresenceoflarge sagittaeinthe are similarinbothspecies(Figs (Figs thesagittae (utricular otoliths)areextremelysmallcomparedwith Morphology RESULTS The semicircularcanalsof There areseveralbonystructuresorextensionsinsidethe According toVolpedo andEcheverría(Volpedo andEcheverría, In and Carapusacus Carapus acus C. acus 2, 3).Theirposition,shapeandlocationintheneurocranium has larger sagittaethan fowleri The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 and

3). C. acus O. rochei, asterisci(lagenarotoliths)andlapilli hwdtehgetVl/oICrto witha showed thehighestVolS/VolINC ratio, . were rounderthanthoseof (A,B) Leftlateraland(C,D)dorsalviews ofthe C. acus and ) weremorevoluminousproportionally C. acus and

O. rochei O. rochei. Theophidiiformsagittae 2, 3).Otolithorgans appearedtobe O. rochei O. rochei and (Fig. O. rochei C. acus (Fig. Tbe1).However, (Table 2G,H, Fig. C. acus

1, 2).Thesagitta 3). Lagenae,for have asimilar and (E indexof O. rochei, 3). The

1,

The Journal of Experimental Biology O. rochei are highlightedingreen.The3Dreconstructionisbasedon saccules, thereisonlythemedulla oblongata(Fig. (Fig. small andmostlyrestrictedtotheanterodorsalregioninbothspecies the neurocraniumistopreserveconfigurationofinner ears. semicircular canalintheneurocraniumwasobserved Lastly, asmallgroovethatcorrespondstothepositionoflateral 4.3 and5.5 diameterofsagittais 1–6.Scaledata:maximal LA, lateralarch;NC,neurocranium;Sa,sagitta;SWB,swimbladder;SWP, swimbladderplate;V1–6,vertebrae acus. (G,H)Leftlateralviewsoftherighthalfintra-neurocranium(G) RESEARCH ARTICLE the 12O.rochei frequencies rangingfrom150to 1800 Carapus acus pterotic andiscrossedbythelateralsemicircularcanal(Figs by theposteriorsemicircularcanal.Thelateralarchisfoundon the species (Figs arches wereobservedoneachsideoftheneurocranium both theforamenmagnum (Figs ventrally to located small depressioncalledtheforamenofasteriscus, lateral viewsofthereconstructedanteriorpartafemale Fig. Hearing h eann N pcsaedvtdt h ri,whichis The remainingINCspacesaredevotedtothebrain,

.TredmninlreconstructionoftheneurocraniumandotolithsCarapusacus Three-dimensional 2. 3). Attheleveloflagenae andtheposteriorhalfof

(Fig. mm inO.rochei 2, 3).Theposteriorarchisontheepioticandcrossed 2). Thus,themainfunctionofbonystructuresinside and and nineofthe 10 O. rochei and C.acus,respectively. exhibited clearresponsestosound C. acus

z(i.4).In addition,10of Hz (Fig.

2, 3).Inaddition,twobony showed aresponse to

O. rochei.(B)Dorsal,(D)ventraland(F)leftlateralviewsofthereconstructedanteriorparta 3). μ CT scans.As,asteriscus;BS,basioccipitalstalk;DA,dorsalarch;EC,eyecavity;La,lapillus; C. acus

2, 3). and O. rochei hehls(al 3). thresholds (Table showed significantdifferences intheiroverallmeanauditory size andhearing threshold( differ significantlyfrom0,andtherewasnocorrelationbetween fish at eachfrequencytested.Theslopes ofthelinearregressionsdidnot regressions wereperformedbetween fishTL andhearingthresholds ( rochei rochei high intensitysoundsat2100 of sizeatsomefrequencies (Table significant ( effect oftotallength(TL)onoverallmeanthresholds, buta acus. Thegenerallinearmodelwithrepeatedmeasuresindicatedno from 8.1 exceeded 20 thedifference betweenthemeanhearingthresholds sensitivity), and from9.1 P <0.001) and900 Standard deviationswererelativelyhighinbothspecies(Table and (H) was moresensitivethan are sensitivetothesamefrequencyrange(Fig. dB The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 C. acus.Thebonesthathelptomaintaininnerearstructures P SPL (at 150

dB dB 005 feto Lbyfrequencysuggestedaneffect =0.045) effect ofTL and Ophidionrochei P Tbe4). SPL (Table SPL (at 2100 Hz (P Post hoc

Hz) to16.5 <0.001). At600 r 2

<0.2). Hz (Fig. et TkysHSD)revealedthat tests (Tukey’s Hz) to14.1 C. acus . (A) Dorsal,(C)ventraland(E)left

dB

3). Inbothspecies,linear 4). AlthoughC.acus SPL (at 900 at 300 Hz (O.rochei dB SPL (at 900 Hz (P

Hz) inO.rochei <0.04), 600 best hearing Hz) inC.

4), they and 2519 C.

Hz 4): O. O.

The Journal of Experimental Biology 2520 5.0 of thetwosagittae dividedbyintra-neurocraniumvolume. HL, headlength;SD,maximaldiameter ofthesagitta;ST, maximalthicknessofthesagitta; TL,totallength;VolS, sagittae;VolS/VolINC, volumeoftwo volume 17.9 151 Mean values sagittae isinagreementwiththeseobservations(Fig. benthic speciesassociatedwithsoftsubstratesandtheshapeof their associated withsoftsubstrates. with anabsentordullrostrumgenerallybelongtobenthicspecies shape andnoticedthatsagittaewitharelativelyhighEindex and Echeverría (Volpedo andEcheverría,2003)alsostudiedsagitta (Parmentier etal.,2001;Parmentier2002).Volpedo and thickest sagittae,lodgedintheproportionallylargest oticcavity et al.,2002).Incontrast,commensalandparasiticspecieshavethe Merlucciidae andMacrouridae(Parmentieretal.,2001;Parmentier organs resemblesthatoffastswimmerssuchasGadidae, andtheshapeofthese smaller labyrinthsthantheothercarapids, Carapidae, pelagicPyramodontinaehavethinnersagittaeand families sharingsimilarecologicalnichesduringtheirlifespan.In examples ofconvergence inotolithshapeamongunrelatedteleost phylogeny. Nolf(Nolf,1985;Nolf,1993)hasalsoreported related toenvironmentalandphysiologicalconstraintsthan et al.,2010),interspecificdifferences inotolithsizearemorelikely According toPaxton(Paxton,2000)andLombarteetal.(Lombarte RESEARCH ARTICLE Table C. acus C. acus C. acus Sagitta morphology: general considerations general morphology: Sagitta DISCUSSION O. rochei O. rochei O. rochei envle 6. 725.1 27.2 160.5 Mean values 1. Absoluteandrelativesizesofsagittaein 6 1255162. . 587522.8 22.1 25.2 7.5 7.5 9.2 25.8 27.1 31.4 3.4 3.3 3.2 21.6 14.4 16.9 1.6 1.3 1.4 5.5 4.7 4.8 21.2 17.3 15.3 161 143 149 C B A 6. 595.3 25.9 161.5 A 3 274.3 5.7 22.7 32.8 135 185 C B L(m L(m D(m T(m VolS (mm ST (mm) SD(mm) HL (mm) TL (mm) Carapus acus and Ophidion rochei

2). Thesagittae O. rochei . 17.6 1.4 . 27 1.5 . 26.7 17.4 35.7 1.6 1.3 1.8 are and Carapusacus oe h opooia ytmflil inthenaturalenvironmentof roles themorphologicalsystemfulfills systems requiresfirstlyacompleteknowledgeofallthebiological of aspeciesandtheparticularitiesoneitsmorphological Schulz-Mirbach etal.,2012). al., 2007;PopperandFay, 2011; Schulz-Mirbachetal.,2011; Yan etal.,2000;Lovell2005;Webb etal.,2006;Maruska (e.g. Wilson, 1985;PopperandLu,2000;Yan andCurtsinger, 2000; together withtheneurocraniumseemtoreinforceourobservations comparisons withotherteleostinnerearsphotographedordrawn ratio hasneverbeenreportedinpreviousstudies.However, 2001) speciesinvestigatedtodate.Unfortunately, theVolS/VolINC (Marshall, 1971;Fineetal.,1987)andcarapid(Parmentier highlighted thatsacculesarelarge relativetotheskullinallophidiid study, thevolumeofsagittae( inner earstructuresandbrain(Table neurocranium volume,limitingthespacedevotedtoremaining headlengthand of bothspeciesareunusuallylarge comparedwith sls hn4 fteICvlm (Tables is lessthan4%oftheINCvolume whereassagittaevolumeoftheotherspecies in ophidiiformspecies, occupied atleast12.7%(upto25.2%in dal,tesuyo h eainhp betweenthecharacteristics Ideally, thestudyofrelationships 3 DT % DH % TH % VolS/VolINC (%) ST/HL (%) SD/HL (%) SD/TL (%) ) 3.3 3.3 3.2 3.2 3.1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 28.1 20.5 18.9 19 17.3 neurocranium of(A) brain andtheleftinnerearin the morphologyandpositionof U, utriculus. canals; T, tectum;Te, telencephalon; S, saccule;Sa,sagitta;SC,semicircular LA, lateralarch;MO,medullaoblongata; EC, eyecavity;L,lagena;La,lapillus; stalk; C,cerebellum;DA,dorsalarch; rochei Ophidion rochei brain andskullof Fig. O. rochei,

.Morphologyoftheinnerear, 3. . As,asteriscus;BS,basioccipital

7.8 5.8 5.9 5.7 5.5 1, Figs C. acus)oftheINCvolume

C. acus 1, 2).Otherauthorsalso . 1–3). Inthepresent Left lateralviewsshow Carapus acus C. acus and 23.4 16.1 14.9 14.5 14.1 and (B)O. O. fowleri) and

The Journal of Experimental Biology circles), RESEARCH ARTICLE 2009). al., 2009)andCarassiusauratus Fig. Ophidion rochei rochei, suggeststhatthesagittaeof significance forthefishes.This considerations thatlarge sacculesareexpectedtohaveafunctional environmental factors(Parmentieretal.,2002).Itresultsfromthese appears asanassemblyoffunctionalcharactersinteractingwith ecological conditions.Themorphologyofaspeciesconsequently interacting toimprovethefitnessofindividualingiven single morphologicalcomplex,butofmanysuchcomplexes, the studiedspecies.Moreover, anorganism doesnotconsistofone ND, notdetermined. of thetwosagittaedividedbyintra-neurocraniumvolume. HL, headlength;SD,maximaldiameterofthesagitta;ST, maximalthicknessofthesagitta;TL,totallength;VolS, sagittae;VolS/VolINC, volumeoftwo volume Dashed linesandopensymbolswere usedforfrequenciesover1800 A. clarkii P. lacrymatus C. chromis E. marginatus E. marginatus O. fowleri O. fowleri (Pomacentridae). Table argenteus the hearingthresholds(between150 and2100 because noteveryfishresponded.The areabetweenthegreylinescontains Sagitta size andhearingcapacities Sagitta considered whenfunctionalmorphologyofotolithsisdebated. functions (sounddetectionandvestibularcues),bothshouldbe similar selectivepressures.Becauseotolithorgans havetwomajor swimbladder horn-likeextensionscorrespondingtoaccessoryhearing almost matchthoseof O. rochei Wysocki etal.,2009;PopperandFay, 2011). Thebestsensitivityfor structures (Ramcharitaretal.,2006a;Vasconcelos andLadich,2008; (Fig. (Serranidae), represented. Wysocki, 2009).Forgreaterclarity, standarddeviationswerenot by severalauthorsusingdifferent techniques(adaptedfromLadich and Sound pressure level (dB re. 1 µPa)

.AudiogramsofCarapusacus 4. 100 120 140 60 80 4), whichisquitehighforfisheslackingaccessoryhearing

2. Absoluteandrelativesizesofsagittaeinfiveteleostspecies: Mean hearingthresholdsof E. argenteus C. acus 993180523231. . 12.7 17.4 5.4 6.7 19.4 20.1 2.3 2.7 2.3 1.3 0.5 0.7 1.8 2.1 9.3 10.4 79 79 B A was at600 (Parmentier etal.,2011a), 150 300 7 8. . . 8 . 3.5 2.6 2 1.5 5 6.2 1.6 2.1 489 195 3.7 2.1 9.2 8.9 184.2 143.9 574 B 420 A Chromis chromis and L(m L(m D(m T(m VolS (mm ST (mm) SD(mm) HL (mm) TL (mm) 42. . . . . 31281.6 3.6 3.1 2.8 4.7 3.8 13.1 18.2 15.3 3.8 ND ND 4.6 5.3 7.9 0.7 0.8 0.9 3.2 3.1 3.6 24.7 16.9 23.6 84 ND ND and (black diamonds)and O. fowleri Hz. Athigherfrequencies,thehearingthresholds C. acus

600 Eucinostomus argenteus were abletodetectsoundsup2100

900 Frequency (Hz) C. acus (adapted fromLadichandWysocki, evolved understrongandpotentially (Pomacentridae), , Ophidionrochei Amphiprion clarkii

1200 1500 (green squares), A. clarkii

Hz) obtainedfor (black triangles). (Fig. Plectroglyphidodon lacrymatus , Eucinostomus

1800 (Parmentier et O. rochei ) whichhas 4), C. auratus

2100

Hz (blue

Hz O. Onuxodon fowleri td,Lychakov andRebane(Lychakov andRebane,2000)showed study, demonstrated. Althoughthemasswasnotconsideredinpresent neverbeenexperimentally mass. Thisassumptionhas,however, displacement ismaximalshiftstowardlowfrequencieswithotolith increases andthefrequencyatwhichotolithamplitude low frequencies(Lychakov and Rebane, 2000):thesensitivity 2000; CruzandLombarte,2004;LombarteCruz,2007). improve hearingcapacities(Lychakov andRebane,2000;Paxton, otoliths (absoluteorrelativesize)arecommonlyconsideredto in ordertodeterminethefunctionofotolithsizefishhearing.Larger Lombarte andCruz,2007)sizeswerebothconsideredintheliterature 2000; Schulz-Mirbachetal.,2010)andrelative(e.g.Paxton, 2004; Ramcharitaretal.,2004).Absolute(e.g.Lychakov andRebane, to respondacousticstimuli(Paxton,2000;CruzandLombarte, Echeverría, 2003;Popperetal.,2005)thatcouldinfluencetheability shows animportantdiversityinshapeandsize(Volpedo and thesagitta 2005; SisnerosandBass,2005).Amongfishspecies, structure implicatedinfishhearing(LuandXu,2002;Popperetal., (extracted from O. rochei, thisdistancewas2.9±0.2and5.3±2.5 (Ramcharitar etal.,2006b;Ramcharitar2006a).In swimbladder waslessthan3 response tosoundover1500 et al.(Ramcharitaral.,2006b;Ramcharitar2006a)showeda al., 2012).Two outofthefivesciaenidspeciestestedbyRamcharitar sciaenids (Ramcharitaretal.,2006b)andcichlids(Schulz-Mirbach asshownforholocentrids(CoombsandPopper, 1979), detection, between theswimbladderandoticcapsulecanincreaseindirectsound structures (Parmentieretal.,2011a). A relativelyshortdistance Significant dependent variable andspeciestotallength(TL) as fixedfactors. wascalculatedwithauditorythreshold(soundpressurelevel)as a The model Within-subjects effects MS Between-subjects effects Source d.f. repeated measurements(Rm) Table ro 1 80.76 0.378 0.304 112 1.085 1.129 2.134 23.573 421.006 4.601 475.201 87.65 9924.425 172.31 16 371.54 1 7 1 7 7 Error Rm (Frequency)×Species Rm (Frequency)×TL Rm (Frequency) Error Species TL Heavier sagittaeshouldimprovehearingcapacities,especiallyat Among innerearstructures,thesacculeisthoughttobemain 3 DT % DH % TH % VolS/VolINC (%) ST/HL (%) SD/HL (%) SD/TL (%) )

.Gnrllna oe sn feuny 1020 z as 3. Generallinearmodelusing‘frequency’ (150–2100Hz) P-values areinbold. (Pomacentridae) and The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 μ CT scansofsixfishes). (Carapidae), Epinephelus marginatus Amphiprion clarkii

mm awayfromtheoticcavity z nteetoseis the Hz. Inthesetwospecies,

m respectively mm, FP C. acus <0.001 0.046 <0.001 2521 and

The Journal of Experimental Biology larger sagittaethan from atheoreticalview. Relativetotheneurocranium, way relativesagittasizeaffects hearinghasneverbeenaddressed improve hearingcapacities.Unfortunately, toourknowledge,the relatively (generallycomparedwithbodylength)larger sagittae Lombarte, 2004;LombarteandCruz,2007)alsosuggestedthat the neurocranium.Severalotherauthors(Paxton,2000;Cruzand frequency soundsbecauseofitslarge saccularotolithsrelativeto armatus lower bestfrequencyofsensitivity. to statethatlarge sagittaeinabsolutesizearenotsynonymouswith P. mexicana A. clarkii higher hearingthresholdfrom300to900 threshold ishighly affected bythechoice ofthecalibration studies. Inaddition,theestimated valueoftheparticlemotion fornumerousother thatsimilarobservations canbemade concluded shapes foreachacousticmodality (pressureandparticlemotion) whichobservedsimilaraudiogram (Anderson andMann,2011), setup usedinthisstudywasthe sameasinAndersonandMann hearing capacities(Radfordetal., 2012).However, theexperimental ofsoundwhenaspeakerisusedtotest separate bothcomponents pressure andparticlemotionofsoundbecauseitisnotpossible to wouldideallydeterminehearingthreshold using pressure audiograms.Futurestudiesonhearingcapacities in capacities offishes. study alsofoundnodirectlinkbetweenotolithsizeandhearing least intermsofauditorythresholds.Resultsfromthepresent that theseotolithdifferences donotaffect hearingsensitivities,at between bothecotypesbutnotinhearingcapacities,concluding mexicana. Theyfoundnumerousdifferences inotolithmorphology cave- andsurface-dwellingecotypesoftheAtlanticmolly, investigated themorphologyofotolithsandhearingabilitiesin significant. Schulz-Mirbachetal.(Schulz-Mirbachal.,2010) while thedifferences observedfortheotherfrequencieswerenot mean areavaluesvariedfrom1.11 to1.24 investigated inthestudy. 2522 acus 1and2showthat sagittae arealsoheavier. Tables lapillus)butnotonfishspecies.Larger or (sagittae, asteriscus that otolithmassisrelatedtoitsareaanddependsontype relativeto1 SPL, soundpressurelevelindecibels RESEARCH ARTICLE chromis higher frequencyofbestsensitivity(600 from Table 9.2±1.9 populations (Schulz-Mirbachetal.,2010).Thisareawas 150 300 600 900 2100 1800 1500 1200 Fine etal.(Fineal.,1987)speculatedthat Comparisons performedinthisstudywereonlybasedonsound

z144811 1. 2710 12.7 114.5 12 8.1 104.4 Hz z1661. 21311. 10 10.6 123.1 12 13.0 106.6 Hz z9. 421 2. 3610 13.6 126.8 12 14.2 99.8 Hz z1081. 21201. 10 14.1 122.0 12 16.5 100.8 Hz

z1991. 0110919 10 10 9.1 10 13.3 11.1 10.1 151.0 136.3 10 125.0 12 123.9 11 12 15.9 13.4 12.7 15.7 139.9 129.3 Hz 116.4 Hz 115.5 Hz Hz have larger sagittae(14.4to35.7 4. HearingthresholdsofOphidionrochei μ CT scans).Despitetheirlarger sagittae, mm (Ophidiiformes) wouldhaveagoodsensitivitytolow- (150 (7.9 en(P)s.d.(SPL) Mean (SPL) O. rochei 2 [200 in mm Hz), C. acus Hz (Schulz-Mirbachetal.,2010)].Itisreasonable 3 ) andthe O. rochei C. chromis Poecilia mexicana and 13.2±3 (Table 1), butitshowsasignificantly [200 Amphiprion clarkii N Hz (Wysocki etal.,2009)]and mm

Hz) than en(P)s.d.(SPL) Mean (SPL) C. acus

2 mm

in µPa. has verysmallsagittae:

mm

3 Hz (Table O. rochei ) thanthe and Carapusacus 2 C. acus O. rochei O. rochei in threedistinct Acanthonus C. acus

(4.6 (extracted 3, Fig. (150 Chromis and had a mm Hz), had N C. 4) P. 3 ) 2002). Because equilibrium maintenanceandorientation(AnkenRahmann, Fishes generallyusevisualandvestibularsensesforpostural 60 accelerometer),differences inthresholdsreached or shaker table calibration; dependingonthetechniqueemployed(Eulerequation, tested byasingleteamusingdifferent techniquesforparticlemotion were hearing capacitiesbasedonauditoryevokedpotentials(AEP) technique (Radfordetal.,2012).Inapreviousstudy, goldfish higher thanthe fundamentalfrequency(ca. 250 than thefirstpeakfrequency(ca. 200 capacities wereobtainedbetween 300and1200 detect intheirnaturalhabitat. In determine whatkindofsounds thedifferent speciesare able to One ofthemainpurposesstudies onanimalhearingabilityisto implications Sagitta morphology andvestibular function morphology Sagitta Hearing capacities of of Hearing capacities Ophidion scrippsae personal observation).A similar behavior wasalsodescribedin first (Codinaetal.,2012)inanalmostverticalposture(L.K., head (Codinaetal.,2012).However, theyalsoenterthesandtail barbel-like pelvicfinsandkeeptheirtailslightlyhigherthanthe al., 2012).Ophidionrochei Vandewalle, 2003;ParmentierandVandewalle, 2005;Schwarzet tail down(almostvertical)positiontoenterintoit(Parmentierand swim headdownwhentheyaresearchingforhostbutswitchtoa and theprecisemovementstheyhavetoexecute. sense. Theyneedveryacutevestibularcues,mainlyfortheirposture thesefunctionswillmainlyrelyonthevestibular environments, acoustic constraints. in Parmentier etal.,2002).Large relativesacculethicknessandlength thin otolithsarefoundinpelagicspecies(Parmentieretal.,2001; whereas heaviest sagittaearefoundinbenthicorparasiticspecies, inner earwouldbeadvantageous.Incarapids,thethickest and whereanacutesenseofbalancebasedonthe in cavepopulations, Consequently, theysuggestedthatthickerotolithsmayhaveevolved mexicana no difference inhearingcapacities ofcave-andsurface-dwelling 2000). Schulz-Mirbachetal.(Schulz-Mirbachal.,2011) observed otoliths leadstoalossofresolvingpower(Lychakov andRebane, wide perceptibleaccelerationrangeintheotolithicorgans withsmall accelerations towhichtheotolithorgan issensitive.However, the 2000), thesmallermassofotolith,widerrange al., 2001). about thegravityvectorwouldhavebiggerotoliths(Parmentieret slow swimmingpatternsandfishesthatneedpreciseinformation excitation bygravity. Thus,itisconceivablethatfishesexhibiting consider thatotolithmass,andconsequentlyvolume,affects haircell in theforceexertedonunderlyinghaircells.Thesedataleadusto the earthatcontrolsotolithsizeinordertomaintainanequilibrium micro-gravity) andproposedafeedbackloopbetweenthebrain experiments onotolithgrowthunderalteredgravity(hyper- and perception oftheirbodypositioning. strongly suggeststhat layer ofsand(L.K.,personalobservation).Thissetobservations are alsoabletotravelmorethan1 According toLychakov andRebane (Lychakov andRebane, Anken etal.(Ankenal.,1998;2000)conducted C. acus dB atsomefrequencies(Radfordetal.,2012). despitethattheformer populationhadthickerotoliths. , and The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 O. rochei O. rochei (Greenfield, 1968).Onceinthesand, C. acus O. rochei O. might haveevolvedtorespondnon- generally followtheseafloorwiththeir and and m horizontallyina10 and C. acus

Hz) ofmanymalesoundsand O. rochei, thebesthearing O. rochei C. acus C. are activeindark : ecological

Hz. Thisishigher need anaccurate

Hz) offemale Carapus acus O. rochei cm deep P.

The Journal of Experimental Biology 300 detected bytheirconspecifics. where allthesoundfrequenciesoftheircallshouldbe 1996), O. rochei conspecific soundstobedetectedfromfurtheraway. Additionally, relatively goodhearingcapacitiesaround600 sounds (Parmentieretal.,2006b). seems possiblebecauseholothuriantissuesarealmosttransparentto acoustic communicationbetweenconspecificsthroughthehost fish wereintheirhostholothurians(Parmentieretal.,2006b), conspecifics. AlthoughsoundsofC.acus survival todetectsoundsfromothersourcesratherthan Ladich, 1999).Insuchcases,itmaybemoreimportantforfish observations wereperformedforotherspecies(Myrberg, 1981; C. acus was relativelyflatbetween300and1500 auditory sensitivitydonotmatchperfectly. However, thethreshold (Parmentier etal.,2006b).Thismeansthatsoundcharacteristicsand pcmn eecuh namxmmo 10 specimens werecaughtinamaximumof attenuated fasterthanthesecondharmonics.Giventhat in veryshallowwaterandobservedthatthefundamentalfrequency Lenhardt, 1983)investigatedthepropagationof frequency’ isca.11 attenuation (Mann,2006).In100 sandy bottomand filledwithseawater. sandy bottom,soundsofca.215 1000 8°43W; Corsica, France). in frontofthemarineresearchcenter STA.RE.SO. inCalviBay(42°34N, collected bySCUBA diving(40 RESEARCH ARTICLE 21:30 and02:00 (May 2010,July2010andSeptember2011), fishweretrappedbetween Croatia) andBanyuls-sur-mer (42°28N,3°08E;France).InDùlce-Glàva took placeintwodifferent locations:Dùlce-Glàva(43°26N,16°40E; Specimens of Ophidion rochei low-frequency sounds.Forexample,in5 frequency soundsbecausetheirhabitatpreventsthepropagationof advantageous forshallow-waterfishestohaveadaptationshigh- Rogers andCox(RogersCox,1988)statedthatitwouldbe more sensitivetothehigher-frequency componentsofthesesounds. O. rochei sounds (Parmentieretal.,2010;Kéver2012).Consequently, second harmonics(ca.500andca.750 second peakfrequency(ca.380 sounds (Kéveretal.,2012).However, itmatchesprettywellthe with handnets. SCUBA diving(5to10 In Banyuls-sur-mer (August2010and2012),fishwerecollectedby outer wingand2 Fish collection MATERIALS ANDMETHODS Conclusions of theirparticularwaylife. whichisneededinbothspeciesbecause acute senseofequilibrium, fish hearingabilities.Relativesagittasizecouldberelatedtoan absolute orboth)doesnotseemtobeasufficient criteriontopredict manynon-ophidiiformspecies.Sagittasize(relative, compared with At thelaboratory(Universityof Liège, Belgium),fishwerekeptina Ten In z hl on nryrne rm250to1600 whilesoundenergy rangesfrom Hz, l tank(14 C. acus,thebesthearingcapacitiesarebetween150and C. acus to detectsoundfromcloselylocatedconspecifics.Similar r bet rcs onso hi oseiis butseem are abletoprocesssoundsoftheirconspecifics, is alsoknowntoliveat150 were extractedfromca.250 O. rochei

h:10 h withabeachseine(22

mm atthecentralpart)inshallowwaters(<2 and h light:darkcycle, internalfilter)witha0.1

Hz (Mann,2006).FineandLenhardt(Fine m depth)aftersunset(between21:30and00:00 C. acus were collectedduringfivefieldcampaignsthat

m depth)inOctober2011 andMarch2012 have larger oticcavitiesandsagittae

Hz) ofmalesoundandthefirst

Hz orbelowsuffer verystrong

ae et,this‘cut-off m waterdepth,

m long,meshsizeof4 Holothuria tubulosa

z respectively)offemale Hz,

m ofwaterdepth(Jardas, Hz, whichshouldenable eentrecordedwhile were not

m waterdepthwitha Opsanus tau

Hz couldallow m waterdepth, , whichwere O. rochei

mm atthe m depth).

sounds m high

Hz h) Two (Scanner SiemensSomatonSensation16-slice,AG,München).(2) by themaximalresolution(isotropicvoxelsize:600 Veterinary InstituteoftheUniversityLiège.Structuredetailswerelimited oriented vertically, closedat thebottomwithasquaresteelplate (40×40 2013): twoO.fowleri,Epinephelusmarginatus et al.,2002;Parmentier2006a;2007;Bertucci scans fromdifferent vocalspecies(CourtenayandMcKittrick,1970;Picciulin unknown intheliterature,samemeasurementswereperformedonfishCT order tomakecomparisonsandbecausethiskindofdataarecompletely sagittae totheoverallintra-neurocranium(INC)volumewascalculated.In aswellheadlength.Inaddition,theratioofvolumetwo TL with similarlength.Inthepresentstudy, wecomparedsagittalength but areofa otherfishspeciesthathavebiggerskulls have smallotolithscomparedwith length. However, thismayleadtoabiasandwouldsuggestanguilliformfishes comparing itslargest diameter(usuallylength,rarelyheight)tofishstandard authors (e.g.Paxton,2000)haveexpressedtherelativesizeofsagittaby Company). StructuremeasurementswerealsoperformedinAMIRA.Several Volume andsurfacerenderingwasperformedwithAMIRA 5.4.0(VSG,FEI 10 isotropic voxelsizebetween22.4and60.4 imaging systemwassetat42or70 whichallowedgreaterprecision.Dependingonfishsize,the phoenix|x-ray), with aμ in TL)werescannedattheNationalMuseumofNaturalHistoryParis rochei investigated withcomputedtomography(CT)imagingsystems.(1)An The internalmorphologyofthree M10) coupledtoacameralucida. Dissections wereperformedunderastereoscopicmicroscope(Leica,Wild in theirsonicapparatus(Emery, 1880;CourtenayandMcKittrick,1970). but species, measured 149,169and177 TL) and10females(from151to217 [87 of Kéveretal.(Kéveral.,2012),thissampleincludedtwojuveniles investigate innerearandbrainmorphologies.Accordingtothenomenclature The skullsof15 being transferredto70%ethanol. Aldrich, StLouis,MO,USA)andfixedinformalin(7%)for15 All fishinvestigatedwereeuthanizedwithanoverdoseofMS-222(Sigma- Twelve 22 suspend thefish10 respiration. Theharnesswasclosed dorsallywithsmallpliersandusedto cellophane. Theywerethenplaced inacustom-madeharnessallowing whichallowedustowraptheposteriortwo-thirdsoffishbody in 222, mucus production.Thus,fisheswere anesthetizedwith150 anguilliform shape,theabsenceof hardspinesinfins,andthesubstantial However, Ophidiiformeswerehardertoimmobilizebecauseoftheir that ofParmentieretal.(Parmentieral.,2009;2011a). determination ofhearingthresholds.Ourexperimentalsetupwassimilar to response forsoundsatdifferent intensitiesandfrequenciesthusallowsthe sounds wererecordednearthebrainstem.Thepresenceorabsence ofa (Corwin etal.,1982;Kenyon1998),bulkneuralresponseselicited by were usedtomeasuretheirhearingcapacity. FollowingtheAEP technique al., 2009;Wysocki etal., 2009). capacities ofthelasttwospeciesareknownfromliterature(Parmentier et lacrymatus, oneChromis chromis CT scans Dissections Inner earmorphology Hearing capacities Specimens weresubmerged ina5%phosphomolybdicacidsolutionfor The innerearsandbrainsofthree days beforescanningtoimprovecontrastbetweenthedifferent tissues. cm diameter, 0.7 mm and114 O. rochei (161.5 O. rochei CT scan(v|tome|x240L,GESensing&InspectionTechnologies Carapus The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105254 mm TL)anda (135 and185 mm totallength(TL)],threemales(169,184and201 O. rochei (106 to210 cm belowthewatersurfaceinasteel tube(115

cm thick).Thistube wasplacedinasoundproofbooth, species generallydonothaveintersexualdifferences were openeddorsallyanddissectedinorderto mm inTL)andtwo

mm TL)and10 mm TL.Sexwasnotdeterminedforthis C. acus

and oneAmphiprionclarkii kV andspecimenswerescannedatan

mm TL). O. rochei (149 C. acus μm. mm TL)werescannedatthe C. acus were alsodissected.Fish C. acus and three , onePlectroglyphidodon (127 to177 μm) ofthescanner (143 and161

mg C. acus days before l − . Hearing

1 cm high, mm TL) of MS- 2523

cm),

was mm mm O.

The Journal of Experimental Biology by electrodeswascausedthefishbrainbeingstimulatedsound. specimens ofbothspecieswerealsotestedtoconfirmthatactivityrecorded audibilitythreshold.Dead the such apeakwasapparentdefinedas were atleast3 analyzed forthepresenceofpeaksattwicefrequencystimulusthat were calculatedusinga4096-pointfastFouriertransform.Thesespectra averagedandpowerspectra pressure levelofsound.Evokedresponseswere connected toacalibratedBrüelandKjær2610amplifierthatgavetheabsolute Denmark; sensitivity stimuli werecalibratedwithaBrüelandKjær8101hydrophone(Nærum, and 90 500 times.Foreachstimuluspresentation,signalphasewasalternated( software. Foreachsoundlevelandatfrequency, thesignalwaspresented BioSig real-time processor, routedintothecomputer, andaveragedusing connectedtoanRP2.1enhanced (TDT HS4-DB4amplifier;10,000gain), dB 2524 Recherche dansl’Industrie etl’Agriculture(F.R.S.-FNRS). This studywassupported bygrantsfromtheFondspour la formationà analyses, andwrotethepaper;O.C.,P.R., A.H.andE.P. revisedthepaper. CT scansand3Dreconstructions;L.K.performedthedissections andthe field campaigns;L.K.andO.C.tested hearing abilities;L.K.andA.H.performed L.K. andE.P. conceivedanddesignedtheexperiments;L.K.,O.C. P.R. didthe The authorsdeclarenocompetingfinancialinterests. F. Bertucci fortheirfruitfulcommentsduringthewritingofthismanuscript. K.Boyle and the Veterinary InstituteofUniversity ofLiège.We are gratefultoDrs campaigns. G.BolenandL.Van BossuytkindlyhelpedforCT scanscarriedoutat Dr G.Lepointandthestaff oftheSTA.RE.S.O. stationkindlyhelpedduringfield beach seineduringfieldcampaignsconductedinSplit. Ministry ofScienceandEducationdeliveredanauthorizationforusing a required tocatchspecimensofthesespecieswithhandnets.TheCroatian they werenotcaughtinprotectedareas.Nospecificauthorization was Ophidion rochei Institutional AnimalCareandUseCommittee(protocolno.LA1610430). All experimentalprocedureswereapprovedbytheUniversityofLiège HSD selected asfixedfactors.Tukey’s were different frequenciestested(repeatedmeasures).SpeciesandTL compare soundpressurethresholds(dependentvariable)obtainedatthe tests. A generallinearmodelwithrepeatedmeasureswasperformedto The normalityofthevariableswasinvestigatedusingKolmogorov–Smirnov 179 al., 2011a). Ateachfrequency, soundlevelswerepresentedatupto seeParmentieretal.(Parmentier description ofsoundstimuligeneration, 900, 1200,1500,1800,2100,2400,2700and3000 performed withtheanimalbeingawake. were placedwhilefishesstillanesthetizedbutrecordings musculature whilethegroundelectrodewasinwater. Theseelectrodes the oticregion.Thereferenceelectrodewasplacedwithinfishepaxial 1 or recordingelectrodewasinsertedapproximately Medical, Lutz,FL,USA)wereusedforrecordingelectricalsignals.Thefirst tube. was placedonthebottomofsteel 22°C. A UW-30 underwaterspeaker(LubellLabs,Colombus,OH,USA) and filledtoaconstantheight(105 RESEARCH ARTICLE Funding Author contributions Competing interests Acknowledgements Ethics statement Statistical tests (StatSoft Inc.,Tulsa, OK,USA). using linearregressions.AllstatisticswereperformedinSTATISTICA 10 fish sizeandauditorysensitivityatthedifferent frequencieswasexamined comparisons betweenspeciesateachfrequency. Therelationshipbetween Eleven different frequencieswerepresentedtoeachfish:150,300,600, Three subdermalstainlesssteelneedleelectrodes(RochesterElectro- re. dB 1 e)t euesiuu riat.Sudpesr eesoftheacoustic deg) toreducestimulusartifacts.Soundpressurelevels re. μPa). Evokedpotentialsrecordedbytheelectrodewereamplified 1

V

μPa dB abovebackgroundlevels.Thelowestsoundlevelatwhich and − 1 − and weredecreasedin6 C. acus 184 dB are notendangeredorprotectedspeciesand re. 1

cm) withsaltwaterkeptbetween20and V μPa − 1 ; bandwidth0.1 post hoc

dB steps(dBvaluesare

mm intothehead,over Hz. Foradetailed tests allowedfor Hz to200 kHz) − 90 ie .L,Hr,M .adCx B. Cox, and M.H. Horn, M.L., Fine, M.L. Lenhardt, and M.L. Fine, A.N. Popper, and R. Fay, ih M.P. Fish, H.M. Evans, nesn E. Anderson, ehe,W n aih F. Ladich, B.G. and Kapoor, W. and Lechner, P. Moller, S.P., Colin, F., Ladich, L.E. Wysocki, and F. Ladich, References ig,D .adRdod C.A. Radford, and D.M. Higgs, D.W. Greenfield, Ladich, T. Schulz-Mirbach, and F. Ladich, ne,R . apl .adRhan H. Rahmann, and T. Kappel, R.H., Anken, H. Rahmann, and R.H. Anken, D.A. Mann, and P. A. Anderson, obre .adCu,A. Cruz, and A. Lombarte, ée,L,Bye .S,Dragič K.S., Boyle, L., Kéver, H.Y. Yan, and F. Ladich, T. N., Kenyon, I. Jardas, aih .adPpe,A.N. Popper, and F. Ladich, R. Fay, and F. Ladich, ne,R . enr . rur .adRhan H. Rahmann, and J. Breuer, K., Werner, R.H., Anken, obre .adLenr,J. Lleonart, and A. Lombarte, aih F. Ladich, rde .W n adn A.C. Haddon, and T. W. Bridge, etci . een,P,Pyo,J n amnir E. Parmentier, and J. 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The Journal of Experimental Biology amnir . adwle . rdrc,B n ie M.L. Fine, and B. Frédérich, P., Vandewalle, E., Parmentier, amnir . oly,O n an D. Mann, and O. Colleye, E., Parmentier, J.-P. Lagardère, and J.-J. Ducamp, P., Vandewalle, M.L., Fine, E., Parmentier, amnir . oilc . Dragič G., Bouillac, E., Parmentier, P. Vandewalle, and G. Goffinet, B., Focant, V., Gennotte, E., Parmentier, u .adX,Z. Xu, and Z. Lu, amnir . oly,O,Fn,M . rdrc,B,Vneal,P n Herrel, and P. Vandewalle, B., Frédérich, M.L., Fine, O., Colleye, E., Parmentier, Y. T. Rebane, and D.V. Lychakov, amnir . an .adMn,D. Mann, and K. Mann, E., Parmentier, P. Vandewalle, and M. Chardon, E., Parmentier, F. Lagardère, and P. Vandewalle, E., Parmentier, P. Vandewalle, and E. Parmentier, RESEARCH ARTICLE aalns .adCsdvl,M. Casadevall, and J. Matallanas, D. Mann, M.A. Pegg, and J.R. Nedwell, R.M., Moate, M.M., Findlay, J.M., Lovell, amnir .adVneal,P. Vandewalle, and E. Parmentier, T. C. Tricas, and L.R. 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