#97388, Waco, TX76706,USA. Biology,*Present address:Baylor University, of OneBearPlace Department Drive, Christi,TX78412,USA. Corpus Summer Street,Yarmouth MA02675,USA. Port, Hole, MA02543,USA. Alexander Costidis 1154 Received 27June 2013;Accepted19November2013 ‡ 1 alveolar compression,thismayreducetherespiratory pressure increasesduringdives.Dependingonthemechanism of compression andcollapse,resultingincessationofgasexchange as respiratory systemofmarinemammalswouldallowalveolar Scholander (Scholander, 1940)suggested thattheanatomyof respiratory airexpelledfromthecompliantalveolarspace. (Kooyman andSinnett,1979),actingasareinforcedspace for trachea mayplayaroleinalveolarcompressionandcollapse The morphologysupportsthehypothesisthatcartilaginous marine mammalshavecartilageextendingfartothealveolarsac. between species.Scholander(Scholander, 1940)notedthatsome detailed thattheextent,continuityandlengthofcartilagevaried marine mammalswasnotedbyScholander(Scholander, 1940),who The uniquedistributionofcartilageinthetracheaandbronchi Compliance, Divingphysiology, Alveolarcompression KEY WORDS:Diving,Lungcollapse,Pressure–volume, pressure–volume ( while beakedwhaleshadverystiff, lesscompliantspiralingrings.The Dolphins andporpoiseshadlessstiff, morecompliantspiralingrings the tracheaprogressedcaudally, trachealringschangedmorphology. seal (Phocavitulina more thanonecategoryalongtheirlength;forexample,theharbor compliance values(categories5A and5B).Sometracheasfellinto cartilage (categories1–4)andcetaceantracheasbyvarying categorizing pinnipedtracheasbyvaryingdegreesofcontinuity tracheas. Here,wedefineddifferent typesoftrachealstructures, structural (compliance)characteristicsofexcisedmarinemammal aim ofthisresearchwastoinvestigatetheanatomical(gross)and functional adaptationsofthemarinemammalrespiratorysystem.The mammal diving.Therearefewdataavailableonthestructuraland open andreceivedairfromhighlycompressiblealveoliduringmarine In 1940,Scholandersuggestedthatstiffened upperairwaysremained Colby Moore A comparativeanalysisofmarinemammaltracheas RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1154-1166 doi:10.1242/jeb.093146 Wilmington, NC28403,USA. BiologyandMarineBiology, of 601S.Wilmington, CollegeRoad, Department #97388, Waco, TX76706,USA. INTRODUCTION ABSTRACT dive depthorventilationratesofthespeciesinvestigated. collapse andre-inflationoflungs,perhapsinfluencingvariabilityin between species.Thesefindingslendevidenceforpressure-induced different speciesweremeasuredtoassessstructuraldifferences Author ([email protected]) for correspondence Woods HoleOceanographicInstitution,266Woods HoleRoad, MS50,Woods 1, *, MichaelMoore 2 Baylor University, Department of Biology,Baylor University, of OneBearPlace Department P ) demonstratedcompleteringscranially, andas – V ) relationshipsofisolatedtracheasfrom 5 4 Texas Christi,6300Ocean A&MUniversity-Corpus n AndreasFahlman and 3 International Fundfor AnimalWelfare,International 290 1 , StephenTrumble 4 University of North Carolina North University of 1,5,‡ 2 , MistyNiemeyer CO gas exchangeandfasterreplenishmentofO tidal volumesthatareclosetothevitalcapacity, resultinginefficient also allowsforhighventilationratesduringthesurfaceinterval,and (Denison etal.,1971;Bostrom2008).Themodifiedtrachea of thelungsmarinemammalsascomparedtoterrestrial problem inhumans(Fahlman,2008). effortless actionformarinemammals,yetasignificantclinical exchange. Uponascent,thealveoliarereinflated–anapparently into theupperairways(bronchiandtrachea),thusterminatinggas (commonly referredtoaslungcollapse),allalveolarairispushed would eventuallyresultinatelectasis,oralveolarcollapse thereby reducingthegasdiffusion rate(Bostrometal.,2008).This surface areaandincreasethethicknessofalveolarmembrane, diving. and providemoredetailon theroleoftracheaduring cetaceans andpinnipeds,inan attempttolinkformandfunction observations, encompassing both shallowanddeepdiving species ofmarinemammalsas comparedtogrossmorphological dive. Ouraimwastoassess the airwaycomplianceofseveral also dependonthevolumeof inspiredairatthebeginningofa mammal withamorerigidtrachea(Bostrometal.,2008),but will cessation ofgasexchangeshouldoccuratashallowerdepth fora collapse andgasexchangeceases.Thealveolardepth and (Bostrom etal.,2008),andtherebythedepthwherealveoli suggested toaffect theamount ofairdisplacedfromthelungs Fahlman etal.,2011). Thecomplianceofthetracheahasbeen mammal lungs(Denisonetal.,1971;Piscitelli2010; et al.,1970),withfewdatadocumentingcomplianceofmarine performed onexcisedlungsfromterrestrialmammals(Bachofen of thecompliancerespiratorytractandhasbeensuccessfully changes uncertain. al., 2005;Bagnoliet2011), makingpredictionsforrespiratory upper airwaysofmarinemammals(Sokolovetal.,1968;Cozzi system andlittlemechanicalinformationcurrentlyexistsforthe compliance estimatesforthevariousportionsofrespiratory might develop.However, thesemodelsareinfluencedby of therespiratorysystemandhowphysiologicpulmonaryshunts as tohowpressureaffects thevolumeinvariouscompartments rates, theoreticalmodels(Bostrometal.,2008)allowpredictions Sinnett, 1982).Giventhepredictedlungvolumesanddiffusion diving lungvolumeandtheambientpressure(Kooyman californianus measurements inharborsealsandCaliforniasealions( exchange atpressure(Fahlmanetal.,2009).Lungdiffusion collapse depth(Bostrometal.,2008)andsubsequentlevelsofgas compliance ofthetracheaisimportantindetermininglung volume betweentheupperandlowerrespiratorysystem, and Sinnett,1979;KooymanCornell,1981). Tracheal rigidityfacilitatestherapidandmorecompleteemptying The relationshipbetweenpressureandvolumegivesanestimate Recent theoreticalmodelssuggestthat,inadditiontotherelative 2 at thesurface(Scholander, 1940;Olsenetal.,1969;Kooyman 3 ) concurredthatthediffusion rateisrelatedtothe , BettyLentell 1 , William McLellan 2 stores andremovalof 4 , Zalophus
The Journal of Experimental Biology compliance measurementsandwasalsousedformorphological [ descriptions only(notincludedinTable that wasfixedinformalinandthususedformorphological cetacean trachea(Gervais’ beakedwhale, Species: Pv, Phocavitulina Animal ID ( three speciesofterrestrialmammalsintotalduringthisstudy We examinedfivepinnipedspecies,fourcetaceanspeciesand RESEARCH ARTICLE *Tracheas frozenpriortocompliance measurement. p2(o)B er–J–70 8.7 140 37 12 640 66 8 190 144 – 65 5 167 – 90 53.2 100 110 36 – 185 – 13 J 220 – – A – 35 170 A 52.75 10 39 A – 21.5 43.2 – – 18 J 20 – – 18 – A 35 20 58 951 15 37.5 33 316 Terr – 35.5 46 A 18.1 I 18 Terr Terr – A 18 330 Terr 195 A 52.8 – A Od J 24 43.2 Od Bp 25 14 Od Od J 75 75 Ss Bp YOY 56 Od 34.0 J Clf Od 52 YOY Mm 21 75 24 75 YOY Bp 2(cow) Pp – 25 82.5 Pp Od Ot Bp 1(cow) Pp 53.0 24 20 Ot Ss (pig) J La 62 Ot Clf (dog) La 40 16.5 J Ot KLC112* YOY 56.6 19.5 YOY Ot DO8091Pp* Ot Dd H-0055 Zc Ot A 29.9 H-0053 J YOY Zc Ot A DO8482 Zc Ph 58.6 30.9 YOY March3 28.4 Zc Ph YOY Zc IFAW10-065 Zc CSL10328 223 Ph 26.6 Zc CSL10524 54 208 Zc CSL10518 Ph Ma 30.8 Ph Ph CSL10514 Ma 25.6 CSL9946 CSL9943 Ph Ph Pg CSL9942 Ph Ph CSL9938 Ph Hg Hg ES3208 Hg Ph ES3218 DO7435Pg* Hg Pv DO5257(Hg2) Pv Hg H-0066(Hg3) Pv DO8032* Pv DO6322(Hg1) DO7662(Hg1) DO8031* HS2171 DO5258 DO8811 Table M Age class:YOY, youngoftheyear;J,juvenile; I,immature;A,adult. Group: Ph,phocid;Ot,otariid;Od,odontocete; Terr, terrestrial mammals. primigenius Phocoena phocoena angustirostris Tracheal category types Tracheal category RESULTS N Balaenoptera physalus b , bodymass.Trachea lengthandfloodablevolumewere usedforcompliancemeasurements. HIWoods HoleOceanographicInstitution ΔV standardtemperaturepressure dry ΔP WHOI STPD P P EO NortheastFisheriesObserverProgram P International FundforAnimalWelfare generalizedlinearmodel P–V confidencelimit NEFOP IFAW GLM CL List of symbolsand abbreviations List of =32, T trach N amb 2 1. Animaldata be1). able (Bojanus 1827). (Gill 1866);Zc, tracheal pressure nitrogen tension ambient pressure change involume change inpressure pressure–volume All tracheaswerefreshwiththeexceptionofone (Linnaeus 1758);Mm, pce Group Species Linnaeus 1758;Hg, Zalophus californianus (Linnaeus 1758)]wastoolarge for Mesoplodon mirus
rFg 1 1 orFig. Halichoerus grypus Mesoplodon europaeus (Lesson 1828);Dd, ). Thefinwhale M b k)AecasTahalnt c)Trachea floodablevolume(ml) Trachea length(cm) Ageclass (kg) True 1913;Clf, (Fabricius 1791);Pg, Delphinus delphis ) Canis lupusfamiliaris opinei hssuy(i.7).Furtherhistologywouldbeneeded compliance inthisstudy(Fig. lengthwise ofrigidity, whichdeterminedtheareasofmeasured tracheal anatomy, wherethereappearedtobetwodistinctareas were groupedtogethermorphologically, astheyhadsimilarexternal The harborseal( inflation anddeflationofeachtracheawasconsideredhere(Fig. for theslope;therefore,numericalvaluecomplianceof deflation foreachspecies(Fig. this studywecomparedthecomplianceduringinflationand volume wasexpressedasapercentageofthefloodablevolume.In shown). Asthesizeoftracheavariedwithbodymass, pressure–volume ( results aredetailedinFig. The complianceestimateswereplottedforeachspeciesandthe 1–5; tracheal structuresweredesignatedinmarinemammals(categories pinniped speciesandoneforcetaceans(T gross trachealstructures,orcategories,wereobservedforthe descriptions only(notincludedinTable Harbor sealandgray pinnipeds andfunctionof Form relationships pressure–volume of Overview is2–6;Table Figs Pagophilus groenlandicus Linnaeus 1758;La, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 Linnaeus 1758;Ss, Phoca vitulina P
– 2). V ) relationshipbetweentracheas(datanot Lagenorhynchus acutus
1. Therewasgoodreproducibilityinthe Erxleben 1777;Ma,
) andgrayseal( 1). Complianceisanumericalvalue Sus scrofa
1 orFig. Linnaeus 1758;Bp, be2). able (Gray 1828);Pp, Halichoerus grypus) Mirounga 1). Fourdistinct As such,five Bos 1155
1).
The Journal of Experimental Biology 1156 RESEARCH ARTICLE progressed caudad(Fig. lengthwise changeinmorphologyandcompliance,asthetrachea caudal sectionswere‘accordion’-likeinstructure,suggestinga separating thecartilagerings(Fig. middle sectionappearedtobeastiff, rigidarea,withminimaltissue differing flexibilitybetweenthetworegions.Thecranialendto lengthwise (versuscross-sectionally),thuspotentiallyindicating to determinethetypeandamountoftissuepresentbetweenrings in volumeover a (Figs revealed basedonmacroscopicdifferences incartilagecontinuity caudal regions(Figs category 1andchangedmorphologically tocategory2inmiddleand a slip(Figs trachea progressedtowardsthe lungs,specificallydemonstratedby cartilage ringscraniallywithadistinct changeinmorphologyasthe lungs (Fig. becoming moreaccordion-likeinstructuremovingtowards the (external lengthwise)adistinctcranialareaofrelativerigidity, seal tracheaconformedtocategory2morphologically(Fig. well asa‘slip’ (category3;Fig. sections oftheharborsealtracheawereincomplete,formingagap as this cranialsectionofcompleteringsascategory1(Fig. cartilage withnoconnectivetissuebridges(Fig. throughout theircircumference,thusformingacompletecircle of cartilaginous ringsoftheharborsealtracheawerecomplete (Table differing rigiditywereconfirmedbycompliancemeasurements –1 For boththeharborsealandgray seal,theslipallowedforvariation Specific totheharborseal,threedifferent trachealcategorieswere As previouslymentioned,thegraysealtracheaalsodisplayed Compliance (% kPa ) 10 15 20 25 30 35 10 15 20 25 30 35 0 5 0 5 2–4, 7;Table B A
3). A cross-sectionofthecranialportionrevealedthat 1,2,3 Pv Hg Phocid Pv Hg Phocid 1,2 7). Cross-sectionalexaminationrevealedcomplete 2, 7).Thus,thegraysealtrachea begancraniallyas
Hg1 Hg2 Hg1 Hg2 Δ P
2) andtwodistinctregions(lengthwise)of 2, 7;Table
in thephysiological range(upto4 Hg3 Hg3 ;Tbe3). 7; Table
Pg MaZc Pg MaZc 2
4). Caudalcross-sectionsoftheharbor
4 2).
2 Deflation 7). Conversely, themiddleand Inflation Species
Species Otariid Otariid
Dd Dd 5a Odontocete Odontocete
La La 5a
Pp Pp 5a
2). We categorized
Mm Mm 5b
Clf Clf
4 Terrestrial mammal Terrestrial mammal
Ss Ss 3). kPa). At
2 2). Mid
Bp Bp1Bp2 Bp Bp1Bp2 2 along itsentire length. Theoverlappingtracheal structureallowed reducing thecircumferenceof the airway. compression, cartilagesideswere abletoslipovereachother, the entirelengthoftrachea (category2;Table were discontinuousaroundthe circumferenceofthetracheaalong have provedmoredifficult with smallertrachealpiecestomeasure. slightly different compliance measurements;however, thiscouldalso harbor seal,asopposedtothetwoareasmeasured,mayhaverevealed compliance measurementsinallthreecross-sectionalareasof the developing abreakincartilagemorecaudalregions(Fig. lungs, beginningcraniallywithacompletecartilagering and demonstrated achangeinmorphologyasthetracheaapproached the in trachealcompliance(Fig. intervals mayaffect theresults. did notaffect thevalidityofdata,althoughlongerfreezing (DO6322), indicatingthatfreezingthetracheaspriortomeasurements were identicaltothoseoftheothergraysealinitsageclass measurements weretakenpre-andpost-freezing.Complianceresults One grayseal(DO7662)tracheawasfrozenandcompliance The cartilaginousringsoftheharp seal( deflation, ascomparedwithyoungergrayseals(Hg2andHg3:Fig. Fig. groups (Hg1,Hg2andHg3).Interestingly, oldergrayseals(Hg1: changes (Fig. varied withinflationanddeflationalsodemonstratedage-related deflated (datanotshown).Thecomplianceinthegraysealtrachea higher differential pressures,thetracheastiffened, specifically when Harp seal The harpsealshowednodistinct variationintrachealcompliance Overall, theharborsealandgraydemonstratedsimilarrigidity 1) demonstratedhighercompliancevaluesonbothinflationand from theinternalvolumewhentransmuralpressurewas0 absolute transmuralpressureandvolumeasthepercentagechange individual animalasarandomfactor. Pressurewasexpressedasthe volume byfittingthedatawithagenerallinearizedmodel(GLM)using determined astheslopeofrelationshipbetweenpressureand tracheas duringdeflation(A)andinflation(B). Fig. value. seals analyzed.Bp1isanadultcow, Bp2acalfandBptheaverage (YOY) isH-0066Hg(Table DO7662Hg andDO6322Hg,Hg2(juvenile)isDO5257HgHg3 Table Numbers abovetheerrorbarsindicatetrachealcategoryasdefinedby Phocoena phocoena californianus Pagophilus groenlandicus familiaris
1. Elasticproperties(compliance)ofmarinemammal
2. Species:Pv, Phocavitulina The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146
Ss,Susscrofa;Bp,Bosprimigenius ; 1). Therefore,thegraysealsweredividedintothreeage ; Dd,Delphinusdelphis ; Mm,Mesoplodonmirus ; Ma,Miroungaangustirostris
1). Hgrepresentsanaverageofallgray
1). Inaddition,bothspeciesalso ; Hg,Halichoerusgrypus ; La,Lagenorhynchusacutus Pagophilus groenlandicus ; Clf,Canislupus . Hg1(adult)includes The compliancewas ; Zc,
2). During
7). Further Zalophus ; Pg,
kPa. ; Pp,
1). )
The Journal of Experimental Biology incomplete cartilagecircumferencestretched(Fig. inflations, theconnectivetissueseparatingendsof alternatively asonesidewasabletoslipovertheother. During expansion orcompressionastheconnectivetissuestretched,and RESEARCH ARTICLE 3 2 1 Category 4 5b 5a Table 2. Marinemammaltrachealcategories 2. Table
Morphology
‘Slip’ and‘gap’:lessstiff/compliant ‘Slip’ feature:lessstiff/compliant Complete ring:stiff/lesscompliant Description ag gp:ls tf/opin Dog lessstiff/compliant ‘gap’: Large prln ig:rgdls opan True’sand Spiraling rings:rigid/lesscomplaint Spiraling rings:compliant
1). Inother
Harbor porpoise, white-sided dolphin, Harbor porpoise,white-sided Harbor seal Cow Pig California sealion Harp seal Gray seal Harbor seal Ribbon seal(Sokolovetal.,1968) Bearded seal(Sokolovetal.,1968) Walrus (Sokolovetal.,1968) Gray seal Harbor seal Species Ribbon seal(Sokolovetal.,1968) Bearded seal(Sokolovetal.,1968) Northern elephantseal Human (Netter,2011) <4 deflations, butatverylowvolumes,ordifferential pressures when inflated.Similarly, thetracheawasalsoverycompliantduring words, theharpsealtracheawascomparativelyverycompliant common dolphin kPa, itbecamerigidandnon-compliant(datanotshown,outof
Gervais’ beakedwhales
The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146
and harborseals. indicates cross-sectionalareataken)inbothgray cartilage ringswerefoundatthecranialcut(arrow Fig.
.Category1trachealmodel. 2.
Entire tracheallength tracheallength Entire Entire tracheallength Mid andcaudalsections Mid Mid andcaudalsections Mid Entire tracheallength Entire tracheallength Entire tracheallength Entire tracheallength Mid andcaudal Caudal Cranial Cranial Entire tracheallength Cranial Cranial Location Entire tracheallength Entire Entire tracheallength (with dorsalridge)
Complete
1157
The Journal of Experimental Biology 4 (Table 1158 RESEARCH ARTICLE changes. the harpsealactedasanelasticbuffer, dampeningthepressure kept thediscontinuousringstogether. Thus,theexcisedtracheaof probably duetotheelasticstructureofharpsealtracheathat Elephant seals( where stable valuewithin10–15 Δ physiological range).Duringaninflationoftheharpsealtrachea, Northern elephantseal P initially increasedsharplybutthengraduallydeclinedtoa Δ
2). Thetracheawashorseshoeshaped,wherecartilagerings P stabilized immediatelyfollowinginflation.Thiswas Mirounga angustirostris ) wereclassifiedascategory
s. Thiswasnotseeninthegrayseal, discontinuous cartilageringsfusedbyconnectivetissue,progressing California sealionsdemonstratedacylinderofoverlapping among pinnipeds(Fig. seal wereneitherthehighest(harpseal)norlowest(harbor pinniped species.Inotherwords,compliancevaluesfortheelephant tracheal compliancedatavalues(Fig. caudally. morphology continuedthelengthoftracheaasitprogressed were incompletebutnotoverlapping.Inallcases,this California sealion During bothinflationanddeflationexperiments,theelephantseal The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 cut (arrowindicatescross-sectionalareataken). and gapwerepresentincartilageringsatthemid- Fig. (arrow indicatescross-sectionalareataken). was presentincartilageringsatthecaudalcut Fig.
1).
.Category3trachealmodel. 4. .Category2trachealmodel. 3.
1) werebetweenthoseofother A slipfeature A slipfeature
The Journal of Experimental Biology porpoise. Subcategory B(dorsalview)referstotheless compliant/rigidspiralingcartilage ringsfoundinbeakedwhalespecies. (Odontocete andMysticete).Subcategory A dolphinspecies andtheharbor (ventralview)referstothecompliant/less rigidspiralingcartilageringsfoundin Fig. overlapped oneanother(Table discontinuous cartilaginousringsformedaslipwheresides The sealiontrachealmorphologywasclassifiedascategory2, 8;Table from thelarynxtofirstbronchialbifurcation(Fig. RESEARCH ARTICLE
.Category 5cetaceantrachealmodel 6.
2). . A blue arrowindicatesthelocationofrightaccessorybronchusfoundinall cetaceanspeciesexamined
2). section untilthecaudalregionswheretracheaseparatedintotwo exhibited tissueinvolutioncranially, whichcontinuedinthemid- overlapped throughoutthetracheallength.Thesealiontrachea During compression,thelateralsidesofsealiontrachea The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 throughout thetracheallength. shaped cartilagerings;thesewerepresent connective tissue/musculature,i.e.horseshoe- Fig.
.Category4trachealmodel. 5. A gapof 1159
The Journal of Experimental Biology deflation measurements(Fig. addition, similarcompliancevalueswerefoundfortheinflation and trachea wasshortened,affecting overallcompliance(Fig. In otherwords,eitherthetwobronchiwereelongatedortrue perhaps duetotheprimarybifurcationbeinglocatedmorecranially. 1160 RESEARCH ARTICLE The trachea was cut in the middle and the upper (from pharynx to the middle) and lower (from the middle to the bifurcation) portions testedseparately.The tracheawascut inthemiddleandupper(from pharynxtothemiddle)andlower(from themiddletobifurcation)portions HS2171 Animal ID the maintrachealbifurcation,andjoinedtocranialsection of the in thisstudy. Anaccessoryrightbronchuswasobservedcranialto 9),similartootherodontocetesinvestigated conical andwide(Fig. The tracheaoftheharborporpoise( medium compliancerelativetootherspeciesinthisstudy(Fig. separately oraspartofthetruetrachea. tube wasalsoconsideredtobeincategory2,althoughnotmeasured allowing thetwotubestocompressseparately. Thus,eachbronchial bronchi displayedoverlayingcartilage,orslipfeaturesaswell, was indicativeofthethoracicinletregioninotariids.Bothsealion fused primarybronchithattraveledtowardsthelungs(Fig. both harborandgrayseals. (cranial, middleandcaudal)intheharborsealtracheatwograytrachea.Verticalof differing arrowsindicatetworegions rigiditylengthwisefor Fig. Table Form and function of odontocetes andfunctionof Form CSL10328 DO8032 The discontinuouscartilaginousringsofthesealionallowedfor
.Lengthwisechangeinmorphologyof harborsealandgraytracheas. 7. 3. Complianceestimatealongthelengthoftracheainthree differentspecies
1). Species P. vitulina Z. californianus H. grypus Phocoena phocoena Cranial 1.9 Inflation (% 1.9 8.4
8). This
) was 1). In
1),
kPa − 1 ( observed inonebeakedwhalespecies:True’s beakedwhale complaint spiralingrings(Fig. cetaceans: (A)oneswithcompliantand(B)rigid/less structure. Compliancevaluesrevealedtwosubcategoriesfor classified ascategory5becauseoftheiroverallsimilarityingross baleen whale,thefinwhale.Allcetaceanspeciesexaminedwere right lunginallodontocetespeciesstudied(Fig. for furtherstudy. compliance differences between subcategories.Thisisafutureaim to confirmanymicroscopicdiscontinuitysuggestedbytheobserved were notdistinguishedhere,andfurtherhistologywouldbeneeded microscopic discontinuitiesinthecartilageofcetaceantracheas morphology, asitwasinafixedstateofpreservation(Fig. beaked whaletracheawasnotanalyzedforcompliance, just were lessrigidoverall(category5A;Fig. porpoise anddolphinspeciesdemonstratedgreatercompliance,or dolphin (Lagenorhynchus acutus ) tracheasweremorecompliant ) Mesoplodon mirus)(category5B;Table The commondolphin( Horizontal arrowsindicatethreedifferent cross-sectionalmorphologies Caudal 6.3 6.8 14.4 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 Upper Deflation (% 4.7 1.9 1.4 Delphinus delphis
6; Table
kPa − 1 )
2). Tracheal rigiditywas
6; Table Lower 7.9 3.6 4.7
2), whereasharbor ) andwhite-sided
6) aswellone
2). TheGervais’
6). Any
The Journal of Experimental Biology other odontocetespeciesinvestigated(Fig. lower compliancevaluesoninflationanddeflationthananyofthe inflation thandeflation(Fig. porpoise demonstratedtheopposite,beingmorecomplianton bifurcation. The tracheawascutcraniallyatthe larynxandcaudallyafterthefirst Fig. (Table present betweenendsofthecartilage,formingahorseshoeshape category 4ascartilaginousringswereincompleteandagapwas The dog(Canislupusfamiliaris)tracheawasdeterminedtobe during deflation(Fig. RESEARCH ARTICLE Form and function of terrestrial animals terrestrial andfunction of Form
.Ventral viewoftheexcisedharborporpoise (DO8803)trachea. 9.
2). Thegapwaspresentthroughtheentirelengthof
1A) thanwheninflated(Fig.
1A,B). TheTrue’s beakedwhalehad
1A,B).
1B). Theharbor similar compliancevaluesforthedogandcows(Fig. compliant tracheaforterrestrialmammalswasthepigfollowed by similar compliancevaluesoninflationanddeflation(Fig. group forthismorphology. Compliancevaluesforcattleindicated although deflationdatawerenotmeasured(Fig. demonstrated thehighestvaluesofcomplianceoninflation, entire lengthofthesealionandharpsealtracheas.Thepigtrachea this categoryweredistalportionsoftheharborsealtracheaand feature seeninsomepinnipedspecies.Examinedspeciessharing overlapping ofcartilaginoussides,formingthecharacteristicslip Table were groupedincategory2(Table common dorsalridgeuniquetothisspeciesinstudy. Thus,cattle as number, distributionand patternoffusionspiraling cartilages rings andanaccessorybronchus. Itshouldbenotedthatdetailssuch For example,allcetaceansdemonstrated similarspiralingcartilage odontocete trachea,evengiven their equallydiversedivingbehavior. compliance dataandmorphology wasdemonstratedinthe rings throughoutthetrachea. However, lessvariationbasedon were foundtohavecomplete,incomplete and/orchangingcartilage physical propertiesamongpinnipedspecies.Forexample,tracheas suggested thatthereisgreatvarietyintrachealmorphology and properties ofseveralpinnipedandcetaceantracheas.Theresults lineages. Specifically, wehavedescribedgrossandstructural marine mammalspecieswithdifferent divingbehaviorsand categorically classifiedthevariedrespiratoryanatomyof some In thisstudy, wehaveprovideddetaileddescriptionsand not varymuchamongstthemselves(Fig. Compliance valuesforbothinflationanddeflationthedogsdid showed similartrachealmorphologytotheelephantseal(Table trachea. Dogs,althoughnottheclosestlandrelativetopinnipeds, DISCUSSION The cattle( The pig(
2), astherewereincompletecartilaginousringswithdistinct The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 Sus scrofa) tracheawasgroupedincategory2(Fig. Bos primigenius)tracheashadincompleteringswitha fused bronchialtubes. the locationoftracheaandelongated representative graphic. Fig.
2), asitwastheclosestcategorical
.Californiasealiontracheaand 8.
1).
1).
1). Arrows indicate
1). Themost 1161
2).
3;
The Journal of Experimental Biology 1162 RESEARCH ARTICLE harp sealandelephantseal),thecompliancematchedwell to manner. Forallphocidsealsinthis study(harborseal,gray correlating formandfunctioninamorecompletedescriptive circumference ofthetrachealringsandcompliancevalues,thus categories dependingonthecontinuityofcartilagethroughout the variousstructuralcharacteristicsobservedintofivedistinct Bagnoli etal.,2011; Cozzietal.,2005).Inthisstudy, weseparated Slijper, 1962;Sokolovetal.,1968;KooymanandSinnett,1979; characteristics ofthetracheainmarinemammals(Scholander, 1940; Previous studieshavepublishedaccountsoftheanatomical mammals. growing fieldofknowledgeinrespiratoryadaptationsformarine respiratory behavior, thisworkcanbeconsideredanadditiontothe descriptions. Giventhelimiteddatapertainingtomarinemammal revealed withfuturehistology, whichmayhelptoimprovecategory microscopic differences inpinnipedandcetaceantracheasmaybe categorized tracheasbasedonvisualobservation.Undiscovered samples, especiallywhenmatchedtogrossanatomy. Inaddition,we proved tobeusefulforindicatingfunctionaldifferences intracheal from different individualswithinthesamespeciesandourmethod limitations, therewasgoodreproducibilitybetween both compressionandexpansionofthetrachea.Despitethese coeruleoalba compliance ofthetracheainstripeddolphin( al. (Cozzietal.,2005),wherestraincurveswereusedtoassessthe otherwise. Ourstudywasdifferent fromtheapproachbyCozziet further histologyandgreatersamplesizemayhaveindicated specific variationintrachealringsapartfromage-relatedchanges, the thoracicwall,visceralmass,etc.Althoughwedidnotfindinter- situ prove tobedifferent. We suggestthatfutureexperimentsattempt Rommel, 2012).Thus,thetrachealcomplianceinliveanimalsmay venous plexus)wasnotinvestigated(Cozzietal.,2005;Costidisand and endotrachealtissueproperties(e.g.engorgement ofendotracheal difficult toquantify. Additionally, thecontributionofperitracheal in tracheallengthduringthecompliancemeasurementsthatproved decomposition ofthecollectedtissuesandlongitudinalchange involved. evolutionary pressuresotherthandivingadaptationcouldbe cetacean grossmorphologywassimilar. Thisfindingsuggestedthat were notassessed.Althoughmicroscopicdifferences mayexist, Tracheal andfunction form Table Z. californianus angustirostris M. P. groenlandius H. grypus P. vitulina Species Our experimentalapproachhadlimitations,specificallyregarding inflations anddeflationsinordertoaccountforthedynamicsof 4. Approximate pinniped dive depths and citations from the literature theliterature pinnipeddivedepthsandcitationsfrom 4. Approximate
). Ourapproachallowedasimultaneousassessmentof
Sea lion Elephant seal Harp seal Gray seal Harbor seal Common name
274 1735 Female 90 Female 122 122 Max. depth m m m m
m
P – V Male: 312 Male: 89 Pre-breeding: 51 33 456 Female: 106 Female: Post-molt: 48 Female: 12–40 Mean depth Stenella curves m
in m m
m m m compared withtheharpseal’s thinconnectivetissuebridge,may connective tissueinthegapbetweencartilaginousrings,as and grayseals.Inaddition,thegreaterthicknessprominenceof aided inthehighercompliance,comparedwiththatofharbor cartilage throughoutthecircumferenceoftracheamayhave study (Table morphology wasdifferent fromthatofotherpinnipedsinthecurrent horseshoe shapedthroughoutthelengthoftrachea(Table and Kovacs,1993;Folkowetal.,2004)(Table greater depthascomparedwiththeharborandgrayseals(Lydersen majority ofdivesinshallowwater, theyarecapableofdivestoeven category 2(Table with thatoftheharborandgrayseal(Fig. The tracheaoftheharpsealhadevengreatercompliancecompared species (Fig. stiff tracheaduringdeflation(compression)inyounganimalsofboth tracheal anatomyandcompliance.Thecomplianceindicatedarather physiology, wewouldexpectthesespeciestoexpresssimilar 4).Ifmorphologicaltraitslimit Moore etal.,2009)(Table 1996; Lesageetal.,1999;Gjertz2001;Vincent etal.,2002; (Thompson andFedak,1993;Bonessetal.,1994;Thompson deep dives(meandepth:12–40and48–51 species arerelativelyshallow;however, theysometimesperform range andreportshaveindicatedthataveragedivedepthsforboth thus allowingasmalldegreeofdeeperdivingbehavior. caudal incompleteringsaddcompliancetoanoverallrigidtrachea, stiffer regioncraniallyandamorecompliantcaudally. The changing morphologymaybethatslipspresentcaudallyresultina morphology theentirelengthoftheirtrachea.Onehypothesisfora species (Californiasealion,elephantandharpseal)hadaconsistent al., 2009;Robinsonet2012)(Table (Fig. Despite themorphologicaldifferences, thetracheawascompliant (Sokolov etal.,1968),thedogandhuman(Netter, 2011). progressed towardsthelungs(Table seals alldemonstratedchangingtrachealmorphologyasthetrachea 1968) andribbon(Histriophocafasciata The harbor, gray, bearded( structural properties(compliance)whendescribingmorphology. observable morphology, and illustratedtheefficiency ofthe Pinnipeds –Phocidae m The trachealmorphologyoftheNorthernelephantsealwas Harbor andgraysealsareoftensympatricintheirNorthAtlantic m
1) inthisdeepdivingspecies(LeBoeufetal.,2000;Kuhn The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 >100 80 Female: 304.9–614 100–400 80–100 50% below40 45% below4 Depth range
1). Inthegrayseal,complianceincreasedwithage. 2), butsimilartothatofthebeardedandribbonseals m
m
m 2). Althoughharpsealstendtomaintainthe m
m m
Erignathus barbatus)(Sokolovetal., m
2). However, otherpinniped Weise etal.,2006 Weise Feldkamp etal.,1989 Kuhn etal.,2009 LeBoeuf etal.,2000 Robinson etal., 2012 Folkow etal.,2004 Lydersen andKovacs,1993 Thompson andFedak,1993 Vincent etal.,2002 Beck etal.,2000 Moore etal.,2009 Gjertz etal.,2001 Lesage etal.,1999 Bonessl etal.,1994 Moore etal.,2009 Reference
) (Sokolovetal.,1968) 4). Therelativelackof
1) andwasclassifiedin
4).
m, respectively)
2). The
The Journal of Experimental Biology (Table of theelephantseal(Feldkampetal.,1989;Weise etal.,2006) greater thanthatoftheharborandgraysealsbutshallower of thephocids(Fig. inflation anddeflation,hadpropertiesthatfellinbetweenthose The Californiasealiontracheawasequallycompliantduring was correlatedwithlongerbronchialtubes(Sokolovetal.,1968). in lengthregardlessofnecklength,whereasthelonger described (Sokolovetal.,1968)andwasshowntoremainconsistent category wasusedforcetaceansinthispaper(Fig. tracheas examinedshowedthistypeofmorphology, asingle discontinuous trachealringsseeninothermammals.Asallcetacean seemingly morerobuststructurethanthegenericgaporsliptypeof form acompletering,thefirmfusiontoadjacentringsforms more locations.Althoughthetwoendsofeachringmaynotmeetto rings, whichfusewithadjacentcranialandcaudalringsatoneor cylinder oftightlycompact,spiraling,irregularlyshapedtracheal sampled appearedtobecomposedofarelativelyshort,single the upperrightlobeoflung(Fig. cetacean tracheasalsodisplayedanaccessorybronchusleadingto similar. Tracheas maintainedthesamegeneralshapeandall Gross anatomicalfeaturesofallthecetaceantracheaswerevery The sealion( exchange helpstolowertheend-diveN unexpected result.Theoreticalmodelssuggestthatcontinuedgas deep divingspecies.Thereareseveralpossibleexplanationsforthis that thealveolarcollapsedepthshouldoccurathigherpressurein collapse (Bostrometal.,2008),theresultsinphocidssuggest (Fig. explain itslowercompliancevaluesversusthoseoftheharpseal RESEARCH ARTICLE accurately describethecompliance Additionally, thecompliance oftheexcisedtracheamaynot bubbles forming(Fahlmanetal.,2009;Hooker2009). and therebyreduceexcessivesupersaturationthelikelihoodof Odontocete trachealcategories Pinnipeds –Otariidae ascent (Hooker et al.,2009). 1940; Kooymanetal.,1970;Hooker etal.,2005)orexhaleduring dive witheitherapartiallyor completely filledlung(Scholander collapse depthbyvaryingtherespiratory volumeduringdiving,and rigid tracheas.However, marinemammalsmayalsoalteralveolar compliant upperairwayandshallow diverstendedtohavemore the pinnipedtracheas,where deeper divinganimalshadamore (Bostrom etal.,2008).Thiswastheoppositeofwhatseen in shallower depthandreducegasexchangeduringdeeper dives tracheas. Thestiffer trachea would causethealveolitocollapseata Baird etal.,2006;Tyack et al.,2006),yethaverelativelyrigid claim. compliance values.However, histologyis requiredtosupportthis compression underpressureandmayexplainthedifferences in discontinuities incartilageringslikelyaidedtheease of continuity, thicknessandlongitudinalareaofcartilage.Any microscopic differences in trachealmorphology, specifically relationship (A andB).Variation incompliancemay bedueto the cetaceantracheasintotwosubcategoriesbasedon especially theTrue’s beakedwhale(Fig. the white-sideddolphinfromthoseofotherodontocetes, If trachealcompliancehasimportantconsequencesforalveolar The trachealcompliance Beaked whalesareknowndeepdivers(HookerandBaird,1999; 1).
4). Eumetopias jubatus 1). ThedivedepthoftheCaliforniasealionis P – V relationships wereverydifferent for
in situ ) tracheahasbeenpreviously 6). Thetracheasofallcetaceans 2
1). Therefore,wedivided levels duringdeepdives, in aliveanimal.
6; Table
2). P – V and terminationofgasexchange at70 Howard (RidgwayandHoward, 1979)estimatedalveolarcollapse depth ofalveolarcollapsefor marinemammals.Ridgwayand 2008; Fahlmanetal.,2009). alveolar collapse,andgasdynamicsduringdiving(Bostrom et al., may affect compressionof the respiratorysystem,depthof properties oftheconductingairwaysbetweenspecies.Thisvariation that thereisgreatvariationinthemorphologyandstructural the odontocete–mysticeteevolutionarytree. in cetaceanevolutionaryhistory, andhaspersistedonbothsidesof mysticete. Thus,theaccessorybronchusislikelyaconservedtrait shared thesamebasicgrosstrachealstructureasfinwhale,a porpoise, white-sideddolphinandcommondolphin)inthisstudy conserved evolutionarytrait. in alveolarcompression,itspresencemayhavepersistedasa function oftheaccessorybronchus.Ifthisanatomicalfeatureaids work lookingattheairwaydynamicsmayhelpelucidate rates whileatthesurface(Kooymanetal.,1975).Futureimaging Kooyman andSinnett,1982)thatallowextremerespiratoryflow musculature intheconductingairways(Denisonetal.,1971; (Kooyman andSinnett,1979)aswelladditionalelasticfibers cetaceans havecartilageextendingdowntothealveolarsac bronchus mayaidthegenerationoftheseflowrates.Inaddition, (Kooyman andCornell,1981;Cottenetal.,2008).Theaccessory rectus abdominustopowerrespiratoryflowratesup160 have strongmusculature,amongthemthescalenus,intercostalsand important function.Forexample,ithasbeenshownthatcetaceans bronchus incetaceanspecies,thisanatomicalfeaturemayhavean (Berta etal.,2006).Giventheuniversalprevalenceofaccessory although cetaceanslacklunglobes,whicharedistinctinhumans The accessorybronchusalsoinveststherightlungincetaceans, bronchus isassociatedwiththerightlobe(BaratandKonrad,1987). 2010). Thereisanincreasedprevalenceofdiseasewhenthetracheal and isassociatedwithrespiratorydisease(ManjunathaGupta, during inhalationandexhalation. flow rates,and/oralterationsininternaltrachealandlungpressures function, asanadditionalbronchusmayallowforalteredventilatory accessory bronchus.Itspresencecouldbesignificanttolung relative, arerelatedtocetaceansandsimilarlydemonstratedan (Nickel etal.,1979).Pigs,althoughnottheirclosestterrestrial found inthepinniped,buthasbeendescribedArtiodactylspecies characteristic: anaccessorybronchus.Thisanatomicalfeatureisnot All cetaceansexaminedinthisstudysharedacommon weddelli has beenshowninlivesubmerged Weddell ( incompressible. Thatthetracheaisinfactcompressedduringdiving suggest thatnotalltracheasofmarinemammalscanbeconsidered (which isinthebeakedwhalefamily),haverigidupperairways, we ampullatus). Whilethegrayseal,andpossiblybottlenosewhale based onthegraysealandbottlenosewhale( marine mammalswasarigidstructure,hisobservationsweremainly While Scholander(Scholander, 1940)suggestedthatthetracheaof (Fahlman etal., 2006).Pulmonaryshuntmeasurements inthe the alveolarcollapsemightoccur atdepthsgreaterthan70 calculation, whichaccountedfor thediveresponse,suggestedthat The roleofanaccessorybronchusindiving adaptation trachea’s inlungfunctionandasadiving role The There areinconsistentdatawith regardstohypotheticalmodeled In anevolutionarycontext,thesmallerodontocetespecies(harbor In humans,thepresenceofthisadditionalbronchusisananomaly ) andelephantseals(Kooymanetal.,1970).We propose The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146
m, whereasamorerecent Leptonychotes Hyperoodon 1163
l
s
m − 1
The Journal of Experimental Biology 1164 RESEARCH ARTICLE tension (P changes inarterialpartialpressureofO (Kooyman andSinnett,1982).Morerecentstudies,measuring diving lungvolumesaslow~20%ofthetotalcapacity collapse maynotoccuruntildepthsgreaterthan150 harbor sealandCaliforniasealionimplythatcompletealveolar differently. This isalimitationofthisstudy thatcouldbefurther Furthermore, different tissuetypesmayaffect compliancevalues in measurementstaken associated tissuesaswellengorgement wouldaffect compliance al., 2005;CostidisandRommel, 2012).Itwouldmakesensethat which mayfillanddisplacethe intra-trachealairvolume(Cozziet example, connectivetissueor theendotrachealvenousplexus, anatomical featuresofthetracheamayaffect compliance;for demonstrated vastlydifferent divebehavior. Inaddition,other cetaceans (beakedwhales)maynotbeappropriate,asthey have between shallowdivingspeciesofcetaceananddeep behavior, sizeandage.Simplemorphological comparisons individuals andmaybedependentonparticularrange,animal morphology, asdivedatamayshowgreatvariabilityamong (Fahlman etal.,2009). as afasteralveolarcollapsecouldlimitN al., 2008).Thismayseemcounterintuitiveasanadaptiveadvantage, reducing theavailablealveolaraircirculationvolume(Bostromet concurrently withthelowersectionsofrespiratorysystem, deeper. Thereasonisthattheupperrespiratorysystemcompresses variables equal,amorecomplianttracheapushesthecollapsedepth depth inmarinemammals(Bostrometal.,2008).With allother respiratory tractareimportantindeterminingthealveolarcollapse and betweenspecies,alsoindividuals. depth ofalveolarcollapsemaysimilarlyvaryconsiderablywithin considerably betweenpinnipedsandcetaceans;consequently, the degree ofpulmonaryfillingattheonsetadivemightvary 225 suggested thatthealveolarcollapsedepthcouldbeasdeep collapse depthduringdeepdivesreducesend-divevenous N different. However, theoreticalresultssuggestthatadeeperalveolar in vivo result representstrachealcomplianceofjuvenileanimals,orthatthe may bedeeperintheelephantseal.Itispossiblethatthisunexpected harbor seals(Fig. seal ascomparedwiththecomparativelyshallowdivinggrayand (compression) wassignificantlyhigherinthedeeperdivingelephant et al.,2008).Forexample,thetrachealcomplianceduringdeflation under pressure,thiswillalterthealveolarcollapsedepth(Bostrom pushed upintothetrachea,butiftracheaisalsocompressed during adive.Asananimaldives,airfromcollapsingalveoliis morphology, anatomymayplayanimportantroleinlungfunction that althoughdivingabilityisnotentirelydictatedbytracheal diving species(elephantseal),lendingmoreevidencetothenotion shallow divingpinnipedspecies(harborandgrayseal)versusdeeper structure maycorrelatewithlifehistoryanddivingability. while theanimalisdiving.Thus,anatomicaldifferences intracheal determining thecollapsedepthaswellbehavioraladjustments (trachea andbronchi)alveolarspaceareimportantin diving lungvolumeandtherelativesizeofconductingairways the gasfromtissues(Fahlmanetal.,2009).Regardless, during ascentcouldactuallybebeneficial,asthishelpstoremove there mayalsobeoccasionswheregasexchangeatadeeperdepth Caution shouldbetakenwhencorrelatingdiveabilitytotracheal Recent studiessuggestthatthestructuralcharacteristicsof There wereimportantdifferences intrachealcompliancebetween m (McDonaldandPonganis,2012).Itshouldbenotedthatthe compliance, whenthetracheaissurroundedbytissue, N 2 ) andtherebytheriskofbubblesformingduringascent 1). Thissuggeststhatthealveolarcollapsedepth in situ rather thaninexcisedtracheas. 2 2 absorption. However, in divingsealions
m evenat 2 collection (dog,cattleandpig). terrestrial mammalswereutilizedincomplianceandmorphologydata were recoveredatadecompositioncodeof2.Inaddition,threespecies caught andstrandedmarinemammalsusedforcompliancemeasurements transported totheNorthCarolinaStateVeterinary SchoolinRaleigh.By- Gervais’ beakedwhalewerestrandedinnorthernNorthCarolina,and chilled toWHOIforcompliancestudies.BoththeTrue’s beakedwhale and Mammal Center(Sausalito,CA,USA)andtheexcisedtracheaswereshipped necropsy. TheCaliforniasealionsinthisstudywerecollectedbytheMarine transported toWHOIassoonpossible,andalsostoredat4°Cprior by theInternationalFundforAnimalWelfare (IFAW) staff andvolunteers were storedina4°Cchillerpriortonecropsy. Strandedanimalswerecollected Institution (WHOI),MarineResearchFacility, within24 on icewhenavailableandtransportedtotheWoods HoleOceanographic from theNortheastFisheriesObserverProgram(NEFOP).Animalswerekept By-caught marinemammalswererecoveredinassociationwithfishinggear possible thatthisreflectstheneedtolimitN exhalation versusinhalationbeforeadive(Snyder, 1983).Itis Differences couldalsobelinkedtodiverging behaviorssuchas different strategiesforpinnipedsascomparedwithcetaceans. cetacean. Asfarasgasexchangeisconcerned,thissuggests a morecomplianttrachea,whiletheoppositewastruefor carefully examiningtheassociatedtissues. addressed infutureworkbyrepeatingmeasurements values forcetaceans. were determinedbycontinuityof cartilage forpinnipedsandcompliance differences ingrossmorphology(Figs caudal areas.Fivetrachealcategories wereassignedtodescribemajor length ofeachtracheaaswellcross-sectional viewsatcranial,middleand airway wasexcisedclosertothesecondary bronchialbifurcation(Fig. and wasfoundinassociationwith anaccessorybronchus,theconducting length oftheodontocetetracheaismuchshorterthanthatpinniped larynx andjustcranialtothefirstbronchialbifurcation.Becauserelative accounted forinthisstudy. Thetracheaincludedalltissuecaudaltothe Removal ofassociatedtissuecouldaffect compliancedata,andwasnot was separatedfromthelungsandexcessassociatedtissueremoved. been discussedinaseparatepublication(Fahlmanetal.,2011). Thetrachea each carcass.Thestructuralpropertiesofthelungsweremeasuredand have The entirerespiratorysystemwasexcised(lungs,bronchiandtrachea) from removed asdetailedbelow. porpoise) orhindflipperlength(seals).Duringthenecropsy, thetracheawas included standardlength,girth,flipperdorsalfin(dolphinand (Table using aTri-coastal, classIIIscale(modelno.LPC-4,Snohomish,WA, USA) Upon arrival,sexwasdeterminedandeachanimalweighed(±0.2 mechanics duringdeepdives. in thesespecies.Futureresultsmaylendmoreinsightintolung degree oftrachealcomplianceasrelatedtotheontogenydiving wider rangeofageswilllikelyrevealmoredetailpertainingtothe the visualobservationspresentedincurrentstudy. Inaddition,a species ofmarinemammalsandincludehistologicaldatatoconfirm work isaimedatexpandingthesedatatoencompassmanymore marine mammalsispoorlyunderstood(Bagnolietal.,2011). Future pinnipeds, whichcommonlyexhalebeforediving(Snyder, 1983). diving cetaceansoninspirationascomparedwithdeep-diving Animals MATERIALS ANDMETHODS Protocol Morphometrics Tracheal grossmorphologywas determinedbyexaminationoftheentire Our datasuggestedthatinthepinnipedmodel,deeperdivershad Unlike mostterrestrialspecies,therespiratoryphysiologyof 1). Routinemorphometricmeasurementswerealsocompleted.These The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146
2–5; Table
2 2). Tracheal categories absorption indeep-
h oflanding.Animals in situ
9). and kg)
The Journal of Experimental Biology between and deflationexperiments, internal (floodable)volumeoftherelaxedtrachea(i.e. removed fromthetracheatoreacha trachea horizontallyonatrayinroomair. Thetotalairvolumeinjectedor at 2 card (USB1208LS,MeasurementComputing,Norton,MA,USA)sampling A, HarvardApparatus)andthedatawerecollectedonalaptopusinganA/D Harvard Apparatus,Holliston,MA,USA)connectedtoanamplifier(Tam- was measuredusingadifferential pressuretransducer(MPXtype339/2, flask. Floodable volume wasmeasuredintriplicatebyweighing afilledvolumetric trachea floodedwithwater, andthetotalvolume ofwatermeasured. or changeinlength(Table immersing thetracheahorizontally inawaterbathtoavoidtissueexpansion trauma (BrownandButler, 2000). physiological range,andmuchhigher differential pressureswould resultin tracheal ( RESEARCH ARTICLE structural propertiesbymeasuringthe balance betweenthesepressures.Inthecurrentstudy, weinvestigatedthe changes intheinternalvolumeofrespiratorysystemwilldepend on the properties ofthetracheaandalveolarspace(Bostrometal.,2008). Thus, P negative duringdeflationtrials.Astheanimaldives,twoforceswillbalance inflation ( between theinsideoftrachea( pressure difference ( was estimatedbydeterminingthe measurements. measuring additionalvolumeandsubtractingthatfromtheend floodable volumebyassumingthatthetwoinstrumentsarenotcollapsible, measurement slightly. Thiswasaccountedforduringmeasurementsofthe well astheadditionofendotrachealtube,affected thevolume USA). ThecompressionattheendoftracheabyTwixit clip,as were sealedshutwithaTwixit clip(LindenSweden,Inc.,Edina,MN, a volumetricsyringeofsuitablesize(3–60 size. Theendotrachealtubewasattachedtoasystemof3-wayvalveswith Jorgensen Laboratories,Loveland,CO,USA)endotrachealtubeofsuitable Triangle Park,NC,USA)orveterinary(EquineNasotrachealTube, intubated usingahuman(HudsonRCI,Teleflex MedicalInc.,Research and Bwasrigid). morphology withonlycompliancedifferences (A waslessstiff/complaint, subcategories, A andB(Fig. of cetaceantracheas,comprisedspiralingringsdividedintotwo cartilage ringsseparatedbyone‘gap’.Tracheal category5,consistedsolely category (Fig. large gapincartilage,itwasdefinedascategory3(Fig. trachea wascategorizedas2(Fig. cartilage caneasilyslideovertheother, reducinginternalvolume,the slip, waspresentincross-sections,whereduringcompressiononesideof trachea wasdesignatedascategory1(Fig. of thetrachea(Table tracheal compliancewasseparatelymeasuredattheupperandlowersection length ofthetracheainsomespecies,e.g.harborseal.Inthose tracheal volume.Thecomplianceappearedtochangealongthe constant untilthepressurestabilizedbeforenextstep-wisechangein sample. Afteraninjectionorremovalofabolusair, thevolumewasheld minimum ofthreeleak-freeinflation/deflationcurveswererecordedforeach then inflatedordeflatedwiththesevolumesusingavolumetricsyringe.A respectively, wasdividedintofourtofiveequalincrements.Thetrachea exceed 30 trachea. Thetrans-pulmonarypressuresinthemammalianlungseldom amb The volumeoftherelaxedtrachea (floodablevolume)wasmeasuredby The complianceofthetrachea,orinsomecasessections To estimatecompliance,excisedtracheaswereplacedonatrayand For pinnipedsspecifically, ifcompleteringswerepresent,aportionofthe The volumechange( : (1)thepressureinairways(e.g. Hz. Compliancemeasurementswereperformedthroughlayingthe Δ P Δ
cmH P amb V ) pressuresuptoabout4 ) wereexpressedaspositivevalueswhileboth
5) consistedofahorseshoe-shapedtrachealring:incomplete and 2 O (2.93 P
trach 3). Δ P Δ kPa). Thus,weonlyexposedthetracheastotrans- . Asaconvention,Δ = V P ) wasexpressedasafractionalchangefromthe
1). Oneendwassealedwithatwixit clip,the trach Δ P –
P 6; Table was expressedasthepressurechange amb
3). Whenaslipwasaccompaniedby P
kPa, asthosepressureswerewithinthe trach P , cmH P – – V Δ ) andtheambientpressure( V P relationship. Thetrans-luminal
2), becauseofsimilargross 2). Ifanoverlayofcartilage,or relationship (compliance)ofthe 2 of 50
O), thedifferential pressure ml). Theendsofthetrachea P P trach and thevolumeusedfor cmH ) and(2)thestructural Δ
2 4). The4thtracheal P O or− =0). Forinflation Δ P and 50 Δ cmH V P were amb 2 O, ), mean values±s.d.unlessotherwisestated. Foundation forStatisticalComputing,version2.5.1,2007). using R(R:A LanguageandEnvironmentforStatisticalComputing,R determining whichsampleshadoverlappingCL.Parameterswerefitted determine the95%CL anddifferences amongspecieswereassessedby estimate ofcompliance).Thes.e.m.foreachparameterwasusedto species, the95%confidencelimit(CL)wasestimatedforslope(the slope representingamorecomplianttissue.To comparecomplianceamong relationship representedtheelasticpropertiesoftrachea,withincreasing ID asarandomvariable(Littelletal.,1998).Theslopeofeach experiments. to AmberPfeifer, NicolePoulsonandErinEschenbrennerforhelpwithcompliance collection andsupport.ThankyoutoHaleyPfaff forgraphics,andalsothankyou Rebel Sanders,AnnPabstandUNCWGeraldKooymanforsamples,sample Baylor UniversityDepartmentofBiology, DrSeanTodd andCollegeoftheAtlantic, Fund forAnimalWelfare, NOAA ObserverProgram,TheMarineMammalCenter, We wouldliketothanktheWoods HoleOceanographic Institution,International variable, generalized linearmodel(GLM),withrelativevolumeasthedependent used toinflatetheexcisedtracheawascompletelysaturatedwithwatervapor. volumes tostandardtemperaturepressuredry(STPD).Itwasassumedthatair The roomtemperatureandtheambientpressurewereusedtoconvertall number N00014-10-1-0059]. This projectwassupportedbyagrantfromtheOffice ofNavalResearch[grant sample collectionanddraftingeditingofthemanuscript. manuscript draftingandediting.M.N.,B.L.,W.M. andA.C.wereresponsiblefor interpretation. M.M.andS.T. assistedinexperimentdesignandexecution, C.M. andA.F. wereresponsiblefordevelopmentofconcepts,approachand The authorsdeclarenocompetingfinancialinterests. ahfn . idbad,J n ahfn M. Bachofen, and J. Hildebrandt, H., Bachofen, Acknowledgements Data andstatistical processing analysis References Funding Author contributions Competing interests anl,P,Czi . afr,A,Aoel,F,Fmr,R n otnio M.L. Costantino, and R. Fumero, F., Acocella, A., Zaffora, B., Cozzi, P., Bagnoli, ar,R . ese,D . cwee,D . io,A . cor .S and G.S. Schorr, A.D., Ligon, D.J., McSweeney, D.L., Webster, R.W., Baird, eio,D . arl,D .adWs,J.B. West, and D.A. Warrell, D.M., M.L. Denison, Costantino, and F. Acocella, P., Bagnoli, B., and Cozzi, J.L. Dearolf, S.A., Rommel, W. A., McLellan, M.A., Piscitelli, P. B., Cotten, ek .A,Bwn .D n vro,S.J. Iverson, and W. D. Bowen, C.A., Beck, H.R. Konrad, and M. Barat, ala,A. Fahlman, S.A. Rommel, A., Costidis, oto,B . ala,A n oe,D.R. Jones, and A. Fahlman, B.L., Bostrom, K.M. Kovacs, and J.L. Sumich, A., Berta, rw,R .adBte,J.P. Butler, and R.E. Brown, oes,D . oe,W .adOtdl O.T. Oftedal, and W. D. Bowen, D.J., Bonessl, and liquid-filledexcisedlungs-surfacetension Biomech. tracheo-bronchial treeofthebottlenosedolphin( (2011). Experimentalandcomputationalbiomechanicalcharacterisationofthe alw J. Barlow, ( emptying inthelungsofsealionsanddogs. evidence forevolutionaryadaptationsto diving. biomechanical propertiesofthetrachea of thestripeddolphin muscles inbottlenosedolphins,Tursiops truncatus. D.A. Pabst, and divingbehaviourofadultgreyseals. and itseffect onlungfunction. on physiology. in theheadofbottlenosedolphin( during divinginbreath-holdmammals. 161 alveolar collapseduringdeepdivinginmarinemammals. Behav. Ecol.Sociobiol. foraging cycleresemblingthatofotariidsealsinasmallphocid,theharbor seal. Biology In thisstudy, P The relationshipbetweenpressureandvolumewasdeterminedusinga eoldndensirostris Mesoplodon , 298-305. . Burlington,MA:Elsevier, Inc. Δ 44, 1040-1045. P (2008). Thepressure tounderstandthemechanismoflung compression (2006). Diving behaviour of Cuvier’s ( (2006). DivingbehaviourofCuvier’s , bodymassandspeciesasindependentvariables,animal The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093146 Front. Physiol (2008). Thegrossmorphologyandhistochemistry ofrespiratory ≤ 0.05 wasconsideredsignificant.Dataarepresentedas 34, 95-104. ) beakedwhalesinHawai’i. (1987). Tracheal bronchus. (2012). Vascularization oftheairsinusesandfatbodies . 3 (2000). Theabsolutenecessityofchest-wallcollapse J. Appl.Physiol. , 243. usostruncatus Tursiops J. Exp.Biol. Respir. Physiol. in situ.J.Appl.Physiol. (2006). (2000). Seasonalchangesinbuoyancy Aquat. Mamm. (1970). Pressure-volumecurvesofair- 104 (1971). Airwaystructureandalveolar (2008). Tracheal compressiondelays Anat. Rec. usostruncatus Tursiops ihu cavirostris Ziphius , 907-908. J. Morphol. (1994). Evidenceofamaternal Marine Mammals:Evolutionary Can. J.Zool. 203 Am. J.Otolaryngol. ): morphologicalimplications , 2323-2330. Respir. Physiol.Neurobiol. 284A 13, 253-260. 26, 26-32. tnlacoeruleoalba Stenella (2005). Structureand 269 , 500-510. 84, 1120-1128. ) andBlainsville’s , 1520-1538. ) duringdiving. 29, 422-431. 8 , 118-122. 1165 P – V J. :
The Journal of Experimental Biology itl,R . er,P .adAmra,C.B. Ammerman, and P. R. Henry, R.C., Littell, 1166 RESEARCH ARTICLE un .E,Cokr .E,Teba,Y n ot,D.P. Costa, and Y. Tremblay, D.E., Crocker, C.E., Kuhn, eae . O’ V., Lesage, ala,A,Hoe,S . lzwa . oto,B .adJns D.R. Jones, and B.L. Bostrom, A., Olszowka, S.K., Hooker, A., Fahlman, ala,A,Osok,A,Bsrm .adJns D.R. Jones, and B. Bostrom, A., Olszowka, A., Fahlman, eBef .J,Cokr .E,Csa .P,Bakel .B,Wb,P .and P. M. Webb, S.B., Blackwell, D.P., Costa, D.E., Crocker, B.J., Le Boeuf, oya,G .adCrel L.H. Cornell, and G.L. Kooyman, E. Sinnett, and G.L. Kooyman, R.L. Gentry, and K.S. Norris, G.L., Kooyman, oya,G .adSnet E. Sinnett, and G.L. Kooyman, J.P. Schroeder, and D. Hammond, G.L., Kooyman, ala,A,Lrn,S . ergo . or,C,Ery . imyr M., Niemeyer, G., Early, C., Moore, M., Ferrigno, S.H., Loring, A., Fahlman, okr .K,Bid .W n ala,A. Fahlman, and R.W. Baird, S.K., Hooker, okr .K,Mle,P .O,Jhsn .P,Cx .P n od I.L. Boyd, and O.P. Cox, M.P., Johnson, P. 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