animals toexploittheO terrestrial lifehistoriesbecauseinvasionoflandwould allow one ofthedrivingforcesbehindevolutionamphibious or particularly inhypoxichabitats,haveoftenbeenhypothesized tobe atmospheric air(Dejours,1988).Lowconcentrationsofaquatic O during theinvasionofland. tetrapods tocopewiththecomplexchallengesofaerialrespiration allow extantamphibiousfishes,andmayalsohaveallowedprimitive system torespondairexposureindependentofO independently, byairexposure.Theabilityofthehematological contrast, airexposureincreasedthehemoglobinO 3988 Received 4July2014; Accepted15September2014 *Author ([email protected]) for correspondence Canada. Integrative Biology, Guelph,ONN1G 2W1, of Department University of environments containonlyabout3%oftheO relatively lowinwaterandevenwell-oxygenatedaquatic these environmentsaredramaticallydifferent. Oxygensolubilityis in vertebrateevolutionbecausethephysicalconditionsbetween The transitionfromaquatictoterrestrialliferepresentsamajor step fish, Mangroverivulus Hemoglobin–oxygen affinity, Air-breathingorgan, KEY WORDS:Hemoglobin,Oxygen-carryingcapacity, that amphibiousfishesmaintainO conditions predatesthevertebrateinvasionofland,wehypothesized in hypoxemia.Giventhattheabilitytorespondlowoxygen dioxide inthebloodofamphibiousfishesduringemersionmayresult the initiallossofrespiratorysurfaceareaandaccumulationcarbon Despite theabundanceofoxygeninatmosphericairrelativetowater, Andy J.Turko*, CayleihE.Robertson,KristinBianchini,MeganFreemanandPatriciaA.Wright and airexposure strategies tomaintainoxygendeliveryduringaquatichypoxia The amphibiousfish RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3988-3995doi:10.1242/jeb.110601 suggest thatO brackish water (15‰, ~21kPa O brackish water(15‰,~21kPa remain activeforweeksinbothairandwater, for7 the amphibiousfishKryptolebiasmarmoratus,whichareableto emersed bymountingaco-optedhypoxiaresponse.We acclimated to hypoxia.AquatichypoxiaincreasedtheO pronounced inair-versuswater-acclimatedfish,butnotresponse Angiogenesis intheskinandbucco-opercularchamberwas normoxicair(~21 (~3.6kPa), capacity anddecreasedthehemoglobinO cells. AcclimationtoaerialhypoxiabothincreasedtheO increase intheaffinity ofhemoglobinforO blood viaalarge(40%)increaseinredcelldensityandsmall (decreased INTRODUCTION ABSTRACT P 50 2 ) by25%withoutaffecting thenumberofredblood transport isregulatedbothbyO 2 -rich aerialenvironment(Graham, 1997). kPa) oraerialhypoxia(~13.6kPa). 2 2 ; control),aquatichypoxia uptake andtransportwhile 2 ( 2 Kryptolebias marmoratus P affinity 2 50 2 -carrying capacityof availability andalso, decreased 11%). In
days tonormoxic 2 . 2 availability may These results available in 2 2 -carrying affinity 2 , hypoxia oftenhavebloodwithahighO 1979; SoivioandTuurala, 1981).Fishadapted oracclimatedto and morphologicaladjustmentstotheO cause amphibiousfishestobecomehypoxemicuntilphysiological surface (Bohretal.,1904).ImpairedO amphibious fishesaresurroundedbyO Farmer etal.,1979;Wells etal.,1997).Thus,despitethefactthat 2014); however, notallair-breathing fishesfollowthispattern(e.g. Accumulation ofCO coalesce, reducingthesurfaceareaavailableforrespiration. gills butduringemersionthegilllamellaetypicallycollapseand fishes. Water-breathing fishesexchangerespiratorygasesacrossthe amphibious fishessimilarlytendtopossessbloodwithahigh O Fago, 2004;BraunerandVal, 2006;Wells, 2009).Air-breathing and ability ofhemoglobin(Hb)tobindandtransportO made. the lowsolubilityofCO used tomaintainO or extantamphibiousfishes,thehypoxiaresponsecouldhave been during emersion(Farmer, 1979;Farmeret al.,1979;Graham,1997). Ultsch, 1987).HighbloodpartialpressureofCO the impairedO carrying capacity, whichhasbeenhypothesizedtocompensatefor possess HbwitharelativelyhighO high blood the Bohreffect, whichpreventsO Hb frombecomingfullyO the Rooteffect: aphenomenonfoundonlyinfishes,whichprevents in alteredprotein synthesisorhavefunctional consequences.The However, it isunknownwhetherthesetranscriptionalchangesresult strategies maybeusedtomaintain metabolism(Gracey, 2008). fish thatexperiencedaquatic hypoxia,suggestingthatsimilar Gillichthys mirabilis (Rytkӧ factor hypoxiainduciblefactor-1 duringhypoxicexposure Caenorhabditis elegans predates theinvasionoflandbyanimals,becauseeven basal emersion. Theabilitytosenseandrespondhypoxialikely hypoxia toincreasethesurfaceareaavailableforO lamellar perfusioncanbeincreasedduringexposuretoaquatic transport inresponsetohypoxiaorhypoxemia.Forexample, Root effects onO relatively pH-insensitiveHbtoreducetheconsequencesofBohrand adapted air-breathing andamphibiousfishesshouldpossess 1941; GillenandRiggs,1971).Ithasbeenhypothesizedthatwell- reduce intraerythrocyticpHandtheaffinity ofHbforO Fishes canincreasetheircapacityforO During theevolutionofamphibiouslifestylesbyearlytetrapods Taking advantageofaerialO nen et al., 2011). After 24h ofemersiontheamphibiousfish nen etal.,2011). After24h P CO 2 2 coupled withareducedrespiratorysurfaceareamay uptake andtransportcausedbyelevatedblood 2 transport (Graham,1997;ShartauandBrauner, 2 2 alters geneexpressioninasimilar patternto in thebloodofemersedfishes,resultingfrom transport inthefaceofhigh is knowntoaccumulatethetranscription 2 2 in airversuswater, canalsoimpairthe uses different saturated athigh 2 presents severalchallengesfor 2 2 -binding affinity (Weber and loading atthegas-exchange 2 transport canalsooccurvia 2 -rich airduringemersion, 2 2 transport systemcanbe -carrying capacityand 2 P uptake, bindingand CO 2 (Root andIrving, 2 2 uptake (Booth, 2 (Rahn, 1966; P ( P CO CO 2 2 during ) may P 2 CO by 2 - 2
The Journal of Experimental Biology rmtclybtentetetgop Tbe1). dramatically betweentreatmentgroups(Table precluding statisticalanalysis.Noneofthevaluesvaried calculated fromthemeanvaluesforeachtreatmentgroup, corpuscular hemoglobinconcentration(MCHC)couldonly be (MCV), meancorpuscularhemoglobin(MCH)and mean sampling wasimpossibleandthereforemeancorpuscularvolume As aresultofthesmallbloodvolumesusedinthisstudy, repeated oxygen, cells (RBCs),butairexposurehadnoeffect (two-wayANOVA: Fig. ANOVA: oxygen,P treatment groupsrelativetonormoxiccontrolfish(two-way P mangrove rivulusremainactiveandarethoughttoobtainO (Taylor,can lastupwardsof2months 1990),duringwhichtime (Taylor etal.,2008,Ellison2012;Taylor, 2012).Emersions damp leaflitterorinsiderottinglogsthatmayalsobehypoxic frequently emerseandfindrefugeinterrestrialhabitatssuchas puddles andcrabburrowsfilledwithwater, butthesefishalso condition (three-way ANOVA: medium, mangrove rivulustypicallyinhabitextremelyhypoxic( and airexposure. similar phenotypesshouldoccurinfishexposedtoaquatichypoxia (three-way ANOVA: medium, O would useaconservedsuiteofphenotypicchangestoincreasethe acclimated toeitheraquatichypoxiaoraterrestrialenvironment hypoxia response.Thishypothesispredictsthatamphibiousfishes Hb is50%saturatedwithO of highP Hb–O capacity ofblood(numberredcells,hematocritand[Hb]), significantly larger between 0.3kPa and 4.0kPa and4.0kPa significantly larger between0.3kPa to aerialhypoxicconditions (Fig. vitro availability wasfound( (two-way ANOVA: oxygen, Both hypoxiaandairexposuresignificantlyincreasedblood[Hb] response ofO RESEARCH ARTICLE i.2).A significantinteractionbetweenrespiratorymediumandO Fig. amphibious fishestomaintainO 1880) isanidealmodelforstudyingtheadaptationsusedby air exposure.Themangroverivulus fishes mayusedifferent responsestotolerateaquatichypoxiaand to extrabranchialsitesofgasexchange.Alternatively, amphibious understood. longer periods(daystoweeks)outofwaterissimilarlynotwell or aerialhypoxia( 0.3kPa and 0.6kPa P and0.6kPa 0.3kPa aquatic normoxia,hypoxia( mount ahypoxiaresponse,weexposed al., 2012;Wright, 2012).To testthehypothesisthatair-exposed fish cutaneous respiration(GrizzleandThiyagarajah,1987;Cooperet O during emersion,wehypothesizedthatamphibiousfishesmaintain Blood analysis RESULTS Hb–O post hoc 09;Fg 1A).Hematocritwassignificantlyincreasedinallthree =0.92; Fig. 2 2 Air exposuresignificantlydecreased Given thereducedcapacityfortransportofbloodO -carrying capacity, theaffinity ofHbforO uptake andtransportwhileoutofwaterbyco-optingtheir 1B). Hypoxiasignificantlyincreasedthenumberofredblood CO 2 2 affinity (reducedP affinity andextra-branchialangiogenesis,predictingthat P<0.001; medium, 2 CO tests revealedthatairexposure significantlyincreased , buttherewasnooveralleffect ofoxygenavailability 2 and thelossofrespiratorysurfaceareafromgills 2 -transport systemsinamphibiousfishesthatspend P <0.01; medium, O CO 2 P ~13.6kPa). We measuredtheO ~13.6kPa). 2 , butwasnotinfluencedbyany treatment <0.001, allotherinteractions 50 ) toagreaterextentinnormoxic relative 2 P P=0.46; interaction, and <0.001; medium, P <0.001; CO 2 uptake andtransport.Inthewild, P Kryptolebias marmoratus 50 P P O was increasedbyelevated
2). TheRooteffect was 2 =0.23; interaction, P K. marmoratus ~3.6kPa), aerialnormoxia ~3.6kPa), P 50 =0.21; O , thebloodP 2 , 2 P and theflowofblood <0.001; O P P <0.05; interaction, P=0.52; Fig. CO 2 , 2 P than between P P =0.06; CO 2 to 7 O O >0.05) and as aresult 2 2 2 2 -carrying <2.5kPa) , at which P P days of <0.05; =0.06; (Poey
1C). 2 by in 2 2 , hypoxia andhigh cooperativity (Hillcoefficient, ( epithelium, as well aswithinthebuccaland opercularchambers angiogenesis CD31wasvisible inthedorsalandventralcutaneous Under conditionsofaquaticnormoxia (control),themarkerfor P 7 and (C)redbloodcellcounts. of Kryptolebiasmarmoratus. Fig. Sample sizesaregiveninparentheses.Errorbarsrepresents.e.m. difference betweenfishin waterversusair(two-wayANOVA, was found(two-wayANOVA, significant interactionbetweenoxygenavailabilityandrespiratorymedium letters indicatesignificantdifferences betweenthefourtreatmentgroupsifa normoxia andhypoxia(two-wayANOVA, Different lowercaseletters indicatesignificantoveralldifferences between medium, water orhypoxicair. Hypoxiainwater( Angiogenesis andaerialventilation Table P
days tooneoffourtreatmentgroups:normoxicwater, normoxicair, hypoxic <0.05; allinteractions, O 2
=13.57±0.06kPa) wasachievedbymixing N =13.57±0.06kPa) .Aquatichypoxiaandairexposurecauses changesintheblood 1.
1). B A C P 6 –3 Hematocrit (%) Blood [Hb] (g dl–1)
=0.52; O Red blood cells (10 ) mm 10 20 30 40 10 12 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110601 0 1 2 3 4 5 6 7 8 0 0 2 4 6 8 ae Air Water ae Air Water ae Air Water P (15) 2 (9) (7) A CO , Normoxia omxaHypoxia Normoxia omxaHypoxia Normoxia P 2 <0.01; CO , butnotairexposure(three-wayANOVA: a a P Kryptolebias marmoratus <0.05). Asterisksdenoteasignificantoverall (A) Hbproteinconcentration,(B)hematocrit (14) Air (9) 8 (5) (8) Air P Air B * >0.05; Table n H 2 P , ) wassignificantlyincreasedby O P P 2 =3.59±0.06kPa) andair =3.59±0.06kPa) <0.01; allinteractions, <0.05). Different uppercase Water Water Water (16) (7) (5) B 2 Hypoxia gas withatmosphericair.
1). Hb–O b b were acclimatedfor (9) (6) B * P <0.05). 2 P binding >0.05; 3989
The Journal of Experimental Biology Mean corpuscularhemoglobinconcentration(gdl hypoxia. Asterisksdenoteasignificantoveralldifference ( encruclrvlm f)5.752 36±.46.042 50.68±5.47 63.10±4.24 73.68±5.84 59.07±5.22 Mean corpuscularhemoglobin(pgcell Mean corpuscularvolume(fl) air-exposed fish(body 1 1 and ‘gulping’ airwhenoutofwater. Ratesofaerialventilationafter Fig. body regionsmeasured(three-wayANOVA: region, was observedintheventralepitheliumcomparedwithother interactions, rus omxcwtr omxcar yoi ae rhpxcar Hypoxiainwater( groups: normoxicwater, normoxicair, hypoxicwater orhypoxicair. the hypoxiaresponse.Instead,wefoundthatK.marmoratus sizes aregivenin parentheses. Errorbarsrepresents.e.m. P mixing N 3990 RESEARCH ARTICLE Fig. maintain O Our resultsdonotsupportthehypothesisthatamphibiousfishes availability (three-wayANOVA: medium,P significantly increasedafterairexposure,independentofO Across allfourbodyregions,CD31fluorescentintensitywas in fluorescenceofthebuccalandopercularchambers(Fig. 3A).After7 (Fig. Hypoxia inwater( P movements by more than 10min (Fig. movements bymorethan10min movement, althoughsomeventilationswereseparatedfrombody ofabody majority ofaerialventilationsoccurredwithin30s ( towards decreasedmovementafter1day air-exposure period,althoughtherewasatrend over the7day s.e.m. Samplesizesaregiveninparentheses.Superscriptletterswithinarowrepresentsignificantoveralldifferences ( Table1. HematologicalpropertiesofKryptolebiasmarmoratus Table1. one P Root effect (%) DISCUSSION Hill coefficient
<0.05), buttherewasnosignificant interaction betweenCO <0.05). P h ofair-breathing weremorethandoubletheratesobservedafter day or7 3.7kPa P 3.7kPa 4.0kPa CO 4.0kPa CO 0.6kPa 0.3kPa P 0.3kPa 0.3kPa CO 0.3kPa We observedair-exposed mangroverivulusopeningtheirmouths
.Air exposureincreasestheaffinityofhemoglobinforO 2. 3C). CO 2 2 ) between the four treatment groups when a significant interaction between oxygen availability and respiratory medium was found (three-wayANOVA,) betweenthefourtreatmentgroups whenasignificantinteractionbetweenoxygenavailabilityandrespiratory mediumwasfound gas withatmosphericair. Different uppercaselettersindicatesignificantoveralldifferences in 50 CO CO days ofairexposure(P is presented in Torr; 1Torr=133Pa. Therewasasignificantoveralldifferenceis presentedinTorr; in 1Torr=133Pa. 2 2 2 2 2 2 uptake andtransportwhileoutofwaterbyco-opting P shift shift >0.05; Fig. P O days ofairexposure,therewasastrikingincrease 2 =3.59±0.06kPa) andair( =3.59±0.06kPa) movementsh
P 3C). Significantlyreducedfluorescence 50 (Torr) 10 20 30 40 0 − omxaHypoxia Normoxia 1 ) 9 (10) (9) 00;Fg 4A).Theactivityof <0.05; Fig. A − 1 P ) didnotsignificantlychange
CO 4C). 9 (13) (9) AirWater B P 2 =0.3 kPa O − 2 1 =13.57±0.06kPa) wasachievedbymixingN =13.57±0.06kPa) <0.001; O Water ) P C =0.06; Fig. C Air P <0.05) fromallotherCO 2 2 and oxygenavailability( , 114.60±12.07 19.40±2.21 Treatment qai omxaAra omxaAutchpxaAerialhypoxia 1.58±0.07 (9) Aquatichypoxia 1.39±0.06 (9) 1.35±0.03 (9) Aerialnormoxia Aquatic normoxia 1.830 9*–95±.2() 97±.0(0*–7.82±3.61(8)* –9.78±4.60(10)* –19.54±6.92(9)* –14.18±3.09 (9)* –7.07±3.72 (9) P 2 =0.94; all P 4B). The in Kryptolebiasmarmoratus. <0.001;
have acclimated for 7days todifferentO acclimated for7days 3B). omxaHypoxia Normoxia 9 (9) (9) A 2 P a, a a CO 1.45±0.07(9) * 9 (8) (9) AirWater B 2 =0.6 kPa RBCs, withoutanymajoreffect onHb–O carrying capacityofthebloodviaanincreaseindensity exposed toairandhypoxia.AirexposureincreasedHb–O contrast toourpredictions,different responseswereobservedinfish flexibility inresponsetochangestherespiratoryenvironment.In to thecutaneousepithelium.Thehematologicalsystemalsodisplays opercular chamberprovideasupplementarygasexchangesurface data suggestthat,duringairexposure,themouthandbucco- the respiratoryenvironment.Behavioralandimmunohistochemical compared withwaterispredictedtoresultinrelativelylowerHb–O flexible O uptake anddelivery, theabundanceofO might, forexample,respondtochangesinbloodCO pathway inthecaseofaerialhypoxia.Air-exposed mangroverivulus that canbeactivatedconcurrentlywiththehypoxia-activated availability, butinsteadmaybecontrolledbyasecondarypathway both aquaticandaerialenvironmentssubstantiallyincreasedtheO increasing thenumberofcirculatingRBCs.Incontrast,hypoxiain and increasedtheconcentrationofHbinbloodwithout for O indicate thatthechangesinangiogenesisandbindingaffinity ofHb 2010). than O 2 If thebindingaffinity ofHbforO concentrations. P C P O 2 2 =0.71) orbetweenCO =3.59±0.06kPa) andair( =3.59±0.06kPa) 2 observed duringairexposurearenottheresultofalteredO 146.96±20.72 1.136 9 49±.2(0 –6.55±1.69(8) –4.96±2.72(10) –11.41±3.66 (9) 19.95±2.62 1.45±0.06 (9) 1.35±0.03 (9) (Putnam etal.,2004;Casey2010;Tresguerres etal., C AirWater P 2 50 2 uptake anddeliverystrategiesthatrespondtochangesin The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110601 gas withatmosphericair. Values areexpressedasmeans± between eachconcentrationofCO Fish wereacclimatedfor7daystoone offourtreatment P a, a a 50 1.49±0.05(10) * omxaHypoxia Normoxia (i.e. acrossallconcentrationsofCO 9 (8) (9) A 2 P and respiratorymedium( CO 2 9 (10) (9) P AirWater B conditions 2 O =4.0 kPa 2 134.33±15.50 21.29±2.39 1.40±0.09 (10) 1.44±0.07 (10) =13.57±0.06kPa) wasachievedby =13.57±0.06kPa) P Water C <0.05) betweennormoxiaand 2 reflects abalancebetweenO C Air 2 b, b b 2 used (three-wayANOVA, 1.70±0.17(8) * affinity. Theseresults 2 in atmosphericair P =0.82). Sample 135.72±21.72 26.78±3.95 1.50±0.05 (8) 1.48±0.09 (8) 2 2 , notwithin or pHrather 2 affinity b, b b 2 * 2 2 2 -
The Journal of Experimental Biology exposure? Thelungfish Neochanna burrowsius outofwater, theamphibiousCanterburymudfish After 3weeks shift frombreathinginwatertoair(Graham,1997). this patternandincreaseaffinity ofHbforO (Graham, 1997;Wells, 2009).Whydomangroverivuluscontradict aestivating lungfish. affinity, suggestingthatthesefishareusingadifferent strategyto and Clariaslazera air-breathing fishes affinity inair-breathing animals.Consistentwiththishypothesis,the RESEARCH ARTICLE however, therewasnosignificanteffect ofO (Johansen etal.,1976).Inthenon-aestivatingmangroverivulus, tidal volumeswhileinhabitinghypoxicsubterraneanenvironments air-breathing duringaestivation,probablytoenablereducedlung characterized byhighHb–O 1984). Conversely, hypoxia-tolerantorganisms aregenerally C A C Frequency Aerial breaths h–1 A 10 20 30 40 50 60 70 80 10 15 20 25 30 0 0 5
Fluorescent intensity (a.u.) Mouth 06 01010102020203030303040404050505060>600 600 570 540 510 480 450 420 390 360 330 300 270 240 210 180 150 120 90 60 30 10 20 30 40 50 60 70 80 0 8 8 (8) (8) (8) 1 h a Ventral epithelium Dorsal epithelium Duration ofairexposure (4) (4) Branchial cavity Mouth Water all reduceHb–O a (4) Protopterus annectens 1 d b Branchial cavity P. amphibius also decreasesHb–O (4) Normoxia * 1 hairexposure 2 Time between breathandmovement(s) (5) affinities tomaximizeO 7 d Ventral Dorsal b 2 (5) increases Hb–O affinity duringtheontogenetic Air b epithelium epithelium (5) (5) , 2 * 2 Polypterus senegalus availability onHb–O
B affinity (Wells etal., 2 after 7 B 1 mm
Movements h–1 (5) 1 dairexposure 10 12 14 2 0 2 4 6 8 affinity while (5)
Water days ofair a 2 (4) 8 8 (8) (8) (8) 1 h Duration ofairexposure uptake (4) * Hypoxia 2 (5) 1 d effect inemersedfishresultingfromincreasedblood acclimated fishata tropical fishes–wasapproximatelyequaltothatofbloodfromair- 1 environmental conditions,regardlessofwidelyvariablebloodCO may bemaintainedin decreased bloodpH.The mangrove rivulusata Oncorhynchus mykiss of alternativetroponinisoformshelptomaintaincardiacfunctionin environmental conditions(Somero,1995).Forexample,expression animals isoftenhighlyconservedduringacclimationtochanging levels. LikeHbinmangroverivulus,proteinfunctionother typical levelforemersedamphibiousfishes.Thus,Hb–O effective exposed mangroverivulusservestomaintainarelativelystable
Torr=133Pa) –atypicalpartialpressureinvenousbloodofaquatic Torr=133Pa) We proposethattheelevatedaffinity ofHbforO (4) 7 dairexposure Air b P=0.06 (5) P 7 d (5) 50 * The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110601 across respiratorymediabycounterbalancingtheBohr P body movements(B)wasmeasuredover7 Frequency ofaerialventilation(A)andnumber Kryptolebias marmoratus Fig. 7 Fish (N 1 ventilations inrelationtowholebodymovements parentheses. Errorbarsrepresents.e.m. by different letters.Sample sizesaregivenin ANOVA, P air exposure.Significantdifferences (one-way CO represent s.e.m. epithelium andallotherbodyregions.Errorbars (three-way ANOVA, Asterisks denoteasignificantoveralldifference in waterversusair(three-wayANOVA, indicate significantoveralldifferences betweenfish water, normoxicairorhypoxicair. Different letters of fourtreatmentgroups:normoxicwater, hypoxic fluorescence (arbitraryunits;a.u.)infishfromone using AdobePhotoshop.(C)QuantificationofCD31 photomicrographs stitchedtogetherandoverlaid fluorescent anddifferential interferencecontrast images werecreatedfromlow-magnification(×4) normoxic water(A)orair(B)for7 the headof marker CD31(green)inasagittalsectionthrough immunohistochemical stainingfortheangiogenesis Representative compositeimagesarepresentedof chamber ofKryptolebiasmarmoratus. cutaneous epitheliumandbucco-opercular Fig.
days. (C)Cumulativefrequencyofaerial h recordingperiodsafter1 2 and
.Aerialventilationandbodymovements in 4. of 4.0kPa (P of 4.0kPa
.Airexposureinducesangiogenesisinthe 3. K. marmoratus P =8) wereacclimatedtoairfor1 CO Danio rerio P <0.05) betweentimepointsareindicated 50 2 Kryptolebias marmoratus of 0.6kPa (P of 0.6kPa of control(aquaticnormoxia) 50 P =24.3±1.5Torr), whichisa =24.3±1.5Torr), <0.05) betweentheventral acclimated tovarying under avarietyof exposed toair.
h, 1 50 day and7 2
=24.7±1.3Torr; days. Composite observed inair-
acclimated to h, 1 P P CO <0.05). 2
day or days of
2 days. affinity and/or 3991 2
The Journal of Experimental Biology Hb–O a moreefficient strategyfor copingwithhypoxiathanadjusting without compromisingunloadingatthetissuesandmaytherefore be increasing Hb–O Perhaps hypoxia-acclimatedmangroverivulusareconstrainedfrom Hb–O at highP buffering capacityandthuslessenthemagnitudeofBohreffect significantly increased[Hb],whichwouldprovideextrapH- 20.2±1.3Torr, comparedwith21.0±0.7 McLeod etal.,1978;Routley2002).IncreasedO Soldatov, 1996;Laietal.,2006),butthereareexceptions(e.g. fishes (e.g.Tetens andLykkeboe, 1981;Muradetal.,1990; via erythropoiesisorsplenicrelease,isastrategyusedbymany optimizing O mean reductioninmaximumO aerial gasexchange(BaldwinandWells, 1990). and impedeflowthroughtheepidermalcapillarybedsused for increased RBCdensity. ExtraRBCsmayincreasebloodviscosity requiring modificationstoO facilitate O during behavioralorontogeneticshiftstoair-breathing inorderto fishes increaseorganic phosphateconcentrationsrelativeto[Hb] Weber, 2000;Rutjesetal.,2007).Mostcommonly, air-breathing (Grigg, 1969;Marinskyetal.,1990;Jensen,1991;Val, 2000; extent. AtaP why fishacclimatedtohypoxiadidnotincreaseaffinity tothesame O mangrove rivulusHbiswelladapted tomaintainingnear-maximal with otherteleosts(Pelsterand Randall,1998).Thissuggeststhat the magnitudeofthisshift,whichwasgenerallysmallcompared maximize O Val, 2000).Conversely, hypoxia-tolerantfishspeciesoften expression ofHbisoformswithdifferent O the RBCintracellularenvironmentorbychangingrelative CO water-breathing fish,the maintain consistentO 3992 RESEARCH ARTICLE air exposurealoneincreasedHb–O Conservation ofHb–O temperatures (Aldermanetal.,2012;Genge2013). aquatic hypoxia. Insteadofrecruitingadditional sitesofgas region ofair-exposed mangroverivulus,butnotinfishexposedto leaving water. under normoxicconditions; muscle (HoppelerandVogt, 2001;Fraseretal.,2006). capacity allowsfishtoincreaseO phosphate concentrationstoincreaseHb–O 2009). Ifair-exposed mangroverivulusareabletodecreaseorganic compared withfishesthatinhabitwell-oxygenatedwater(Wells, At highpartialpressuresofCO surface area(Richards,2011; Turko etal.,2012;Cook2013). as increasingthedensityofredcellsinbloodorenlarging gill tissues andinsteadrelyonotherhypoxia-tolerancestrategiessuch environmental O circulating RBCsinresponsetohypoxiaenhanceO but notduringairexposurealone.Increasingthenumberof increased RBCdensity, byhypoxicacclimationineitherwaterorair 2 Overall, weobservedarelativelyminorRootshift(12.2±2.1% Angiogenesis wasobservedin theskinandbucco-opercular Fishes areabletomodifyHb–O Oxygen-carrying capacitywassignificantlyincreased,via saturation athigh 2 gradient. Neitherair-breathing norhypoxiaexposure affected 2 2 affinity inhypoxia-acclimatedfish;thismayresultfromthe affinity (BraunerandWang, 1997).Itiscurious,then,why CO 2 2 unloading atthetissues(Babiker, 1984;Graham,1997; 2 (Weber, 2000;Wells, 2009). 2 CO uptake usinglowratiosoforganic phosphatetoHb uptake toRBCsandO 2 2 2 of 0.3kPa, whichistypicalofarterialbloodina of 0.3kPa, affinity bytheneedtofacilitateO is limitedthereoftenatrade-off between P CO 2 2 affinity mightallowmangroverivulusto 2 delivery inbothairandwaterwithout during emersion,evenimmediately after P P 50 2 2 -accepting tissuessuchasskeletal O 2 there wasatrendtowardsincreased 2 of hypoxia-acclimatedfishwas =22kPa) acrossalarge (3.7kPa) =22kPa) saturation inalltreatmentgroups 2 2 affinity primarilybymodifying uptake fromtheenvironment 2 affinity butdidnotinduce
2 Torr incontrolfish.When unloading atthetissues. 2 affinity, itispuzzling 2 -binding affinities 2 delivery tothe 2 2 transport, -carrying cutaneous bloodflowincreasesO in bloodflowtoextrabranchialrespiratorysurfaces.Augmented 1 effort tomaintainastable other amphibiousfishes,weproposethatthischangerepresents an P 2007; Cooperetal.,2012).Instead,aerialO branchial regionwaspresumedtobenon-functional(Ongetal., considering gillsurfaceareaisreducedduringairexposureandthe ventilation (Ongetal.,2007). area, whichmayfurtherlimitwaterlossduringbucco-opercular of theinter-lamellar cellmass,reducingtheeffective gillsurface Burggren andMoalli,1984).Airexposurealsoinducesenlargement aerial ventilationfrequency(Burggren andMwalukoma,1983; mangrove rivulustoreduceevaporativewaterlossbydecreasing novel adaptationstotheO opted byfishesduringtheinvasionoflandandinsteadsuggest that results contradictthehypothesisthathypoxiaresponsewas co- chamber, whichmangroverivulusventilate by‘gulping’ air. These that airexposurepromotesangiogenesisinthebucco-opercular movements, suggestingthattransientincreasesinO These aerialventilationswereusuallyassociatedwithbody rivulus, stronglyimplicatingthisregionasasiteofgasexchange. observed, forthefirsttime,‘gulping’ activitybyemersedmangrove chamber (Zhangetal.,2000;Gonzales2011). We also capillary densityonthetongueandwithinbucco-opercular with anincreasedrelianceonair-breathing possessincreased 1966). Phylogeneticanalysesofmudskipperssuggestthatspecies ofemersion(Todd andEbeling, are filledwithbloodafter10–20min 1997). Forexample,bucco-pharyngealbloodvesselsin surfaces (Johansen,1970;Satchell,1976;Olson,1994;Graham, organs suchaslungs,buccalpouchesorcapillary-richcutaneous During emersion,amphibiousfishesoftenrelyonair-breathing by reductionoftheinter-lamellar cellmass(Turko etal.,2012). their capacityforbranchialO exchange totoleratehypoxia,mangroverivulusappearincrease individually in 120ml semi-transparent plasticcontainers (FisherBrand individually in120ml maintained intheHagenAqualab, UniversityofGuelph.Fishwerereared strain) (Tatarenkov etal.,2010) wereobtainedfromthebreedingcolony Kryptolebias marmoratus appears tobeindependentlyregulatedbybothO rivulus haveaphenotypicallyflexiblerespiratorysystem that ventilatory activity(A.T., unpublishedobservation). disturbances typicallycausefishinbothairandwatertocease behaviour hasbeenoverlooked,especiallyconsideringthat itisnotsurprisingthat this ‘gulping’ (1 breath every~12min), (~100 exposed fish(Cooperetal.,2012).However, giventhesmallsize our laboratorydemonstratedangiogenesisinthecaudalfinofair- capillaries (GrizzleandThiyagarajah,1987)arecentpaperfrom been knowntobecoveredwithadensenetworkofepithelial (Wright, 2012).Thedorsalsurfaceofmangroverivulushaslong was thoughttobeachievedentirelyacrossthecutaneousepithelium Aerial ventilationfrequencysignificantlydecreasedbetween1 provide astimulustorefreshtheairinbucco-opercularchamber. exposed fishincreasetheaffinity ofHbforO the respiratorymedium.Althoughitinitiallyappearsthat air- Experimental animals MATERIALS ANDMETHODS
CO day ofairexposureandwasprobablycorrelatedwithanincrease Observing bucco-opercularrespirationwashighlyunexpected, Overall, theseresultsprovidethefirstevidencethatmangrove 2 or pHwhenoutofwater. We alsohaveshownforthefirst time mg) ofmangroverivulusandtheinfrequentnature The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110601 hermaphrodites atleast6 2 P transport systemhaveevolved. 50 2 uptake byincreasinggillsurfacearea in thefaceoflarge changes in blood 2 uptake andprobablyallowed 2 uptake inthisspecies 2 , incontrasttomost
months ofage( 2 availability and 2 demand might G. mirabilis
h and Slc8E
The Journal of Experimental Biology saturation curvesateach inairoverthecourseofexperiments.The 13.57±0.06kPa (mean±s.e.m.; hereandthroughout)inwater and averaged3.59±0.06kPa connected toHachHQ30dmeter, HachCompany, Missisauga,ON,Canada) 2011). Oxygenlevelsweremeasureddaily (HachLDO101electrode nitrogen intoclosedchamberscontainingexperimentalfish(Reganetal., both waterandairwasachievedbyintroducingafinelyregulatedflowof 2013). ThelowerCO volumes (Reeves,1980;Henrikssonetal.,2008;BianchiniandWright, Trobe University, Melbourne,Australia) designed toutilizesmallblood fish acclimatedtonormoxicair. didnotshowanyhematologicalresponsethatdiffered relativeto (~17kPa) hypoxia waslethal,whereasfishacclimatedtolesssevereaerial was selectedafterpreliminaryexperimentsindicatedthatmoresevereaerial between 0 and 22kPa in 1kPa increments.EachO in1kPa between 0and22kPa was mixedfromcompressed O partial pressures (0.3, 0.6, 4.0kPa) usingaspectrophotometer (P partial pressures(0.3,0.6,4.0kPa) situation. TheredbloodcellcountsatO measurements maybeconsistentoverestimatescomparedwiththe have causedtheacutesplenicreleaseofredbloodcellsandthusour should benotedthatstressinducedbythismethodofbloodsamplingmay tubes (VWR,Missisauga,ON,Canada)(BianchiniandWright, 2013).It blood wascollectedbycaudalseveranceusingheparinisedmicrohematocrit To measurehematocrit,hemoglobinconcentrationandredbloodcellcounts, tension ofmangroverivulus(Turko etal.,2012).TheaerialO 10G008). University ofGuelphAnimalCareCommittee(AnimalUtilizationProtocol times perweek.Theexperimentsinthisstudywereapprovedbythe with reverseosmosiswater. Fishwerefed Marinemix; MarineEnterprisesInternational,Baltimore,MA,USA)made changes wereperformedweeklyusingartificialseawater(CrystalSea 15–18‰, pH Collection Containers;FisherScientific)underconstantconditions(25°C, Control fish were maintained under standard rearing conditions (~21kPa O Control fishweremaintainedunderstandardrearingconditions(~21kPa hypoxia. Allfishwerefastedforthedurationofacclimationperiod. treatment groups:brackishwater(control),air, aquatichypoxiaoraerial of deoxygenatedHb(Iuchi,1973; Hoxter, 1979), atO respectivelytheisosbestic pointandpeakabsorbance and435nm, 390nm RESEARCH ARTICLE aquatic hypoxiahasbeenpreviouslyfoundtobeslightlyabovethecriticalO (Heisler, 1982;Perry, 1986,Tufts andPerry, 1998),whilethehigher respectively. calculated fromHct/RBCcount,([Hb]/RBCcount)×10and[Hb]/Hct, cellular Hbcontent(MCH)andmean[Hb](MCHC), were Trypan Bluesolution.TheRBCindices,meanvolume(MCV), blood: Cortland’s isotonicsaline(Wolf, 1963)furtherdiluted1:1with0.4% were countedinastandardhemocytometerusing1:400dilutionofwhole using thecyanmethemoglobinmethod(BlaxhallandDaisley, 1973).RBCs (Bianchini andWright, 2013).Hemoglobinconcentrationwasquantified were takenunderadissectingmicroscopeandanalyzedusingImageJ USA). To accuratelymeasurehematocritofsmallbloodvolumes,images Clinical Centrifuge(ModelCL,InternationalEquipment,Needham,MA, sealed microhematocrittubesat7000 collection. Hematocrit(Hct)wasdeterminedbycentrifugingsamplesin small sizeof (e.g. KlitzmanandDuling,1979;Hudetzetal.,1999).However, giventhe or epithelialcapillaries)mayalsodiffer fromtheaorticbloodwegathered arterial (0.3kPa CO arterial (0.3kPa water, ~21kPa O water, ~21kPa Air-acclimated fishweremaintainedonmoistfilterpaper(15‰brackish circular sampleholderusinganO-ring andinsertedintotheP Go-Between FreezerFilm,Hobart, Tasmania, Australia)heldinplaceona immediately spreadthinlybetween twogas-permeablemembranes(Glad Graham, 1997).Bloodsampleswereobtainedviacaudalseveranceand were typical oftropicalamphibiousfishduringairexposure(Johansen, 1970; Blood analysis Experimental fishwereacclimatedfor7 Hb–O 2 equilibrium curvesweredetermined(25°C)atthreedifferent CO 8.1, 12h:12h light:darkcycle)(FrickandWright, 2002).Water 8.1, 12h:12h K. marmoratus,thiswastheonlypracticalmethodofblood 2 ) inplasticrearingcontainers(Ongetal.,2007).Hypoxia 2 ) and venous (0.6kPa CO ) andvenous(0.6kPa 2 partial pressuresapproximatedrestingvaluesfor P CO 2 2 , CO were createdbymeasuringabsorbance at 2 and N
r.p.m. for 2min inanInternational r.p.m. for2min 2 -exchanging surfaces(e.g.thegills 2 Artemia nauplii
by theP days (25°C)tooneoffour 2 ) bloodintropicalfishes 2 and CO wee 50, humidifiedand 2 partial pressures 2 P wee 2 concentration three tofour O combination 2 50. Oxygen chosen for wee P in vivo 50, La CO 2 2 is ). 2 2 considered measuringthepHofwholebloodateach effect wouldbemoreorlessconsistentacrossalltreatmentgroups.We method ofcollectingblood.Ifstressimpactedbloodparameters,thenthe respiratory mediumandO with the durationofairexposureinfluencedventilationrate.Two-way ANOVA overall effects ofrespiratorymedium,O way ANOVAs withposthoc Hb–O necessary tomeet theassumptionsofnormaldistribution andequalvariance. on angiogenesis(CD31fluorescence). Datawerelogtransformedwhen have overestimatedHb–O a manufacturer-supplied spreadsheet.Thebloodsamplingmethodusedmay and theintegrateddensitywascalculatedusingImageJ. regions (themouth,branchialcavity, dorsalepitheliumorventralepithelium) (Cooper etal.,2012).A boxwasthendrawnaroundoneoffourbody Bethesda, MD,USA)andthenthesameintensitythresholdwasapplied the ‘rollingball’ subtractiontoolinImageJ(USNationalInstitutesofHealth, (NIS Elements,Nikon).Thebackgroundofeachimagewassubtractedusing NY, USA)andimageswerecaptured(×40)usingidenticalcamerasettings epifluorescent microscope(NikonEclipse90imicroscope,Nikon,Melville, and sealedwithnailpolish. washed fivetimeswithPBS,mountedFluoromount(Sigma-Aldrich) (1:100 goatanti-ratIgG:PBS;Invitrogen)inPBS.Sampleswerethen incubated overnightwithAlexa-Fluor-488-labelled secondaryantibody PECAM/CD31:PBS; BDPharmingen),rinsedbrieflywithPBSand temperature inprimaryantibody(1:400Ratanti-Mouse atroom were processedatthesametime.Sectionsincubatedfor1h and rehydratedwithagradedethanolseriespriortostaining.Allsamples in paraffin, seriallysectionedin5 were obtainedfromtheleftside.Dissectedtissuesroutinelyembedded 1 h at20°Candstoredin70%ethanol(4°C)untilembedding.Gillarches decalcified(Surgipath DecalcifierII,Winnipeg, Canada)for 4°C for24h, al., 2012).Euthanizedfishwerefixedin10%neutralbuffered formalinat been usedtomeasureangiogenesisin (Albelda etal.,1991;BalukandMcDonald,2008),whichhaspreviously also knownasplateletendothelialcelladhesionmolecule(PECAM-1) endothelial integralmembraneproteinclusterofdifferentiation 31(CD31), Angiogenesis wasdeterminedbyimmunohistochemicalstainingforthe but becauseofthesmallsize quickly aspossibletominimizetheeffects ofacatecholaminergic response, (Nikinmaa andBoutilier, 1995;Reidetal.,1998).Bloodwascollectedas spectrophotometer. Hb-O introduced tothesamplewithintemperature-controlled One-way ANOVA and used onlyonce. mouth and/oropercula)andbodymovementswererecorded.Eachfish was microscope for30 (Logitech QuickcamPro,Fremont,CA,USA)underadissecting above. After1 aerial ventilation.Mangroverivuluswereacclimatedtoairasdescribed gas exchangewhileemersed,weperformedastudytomeasurerates of rivulus. To confirmthatmangroverivulusareabletousethese surfacesfor air exposureincreasedbloodflowtothebucco-opercularcavityofmangrove Contrary toourpredictions,theimmunohistochemicaldatasuggestedthat unfeasible. but thesmallbloodvolumesandtendencyoftoclotmadethis which activateNHE,increaseredbloodcellpHandultimatelyreduce determine theeffect ofrespiratorymedium,O magnitude oftheRooteffect. A three-wayANOVA wasalsousedto coefficient, Angiogenesis Statistical analysis Aerial ventilation All slideswereviewedonthesameafternoonusingaNikon post hoc 2 affinity measurementstocalculatethemagnitudeofBohreffect, n H The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110601 Holm–Sidak testswereusedtodetermine theoveralleffects of ) wereautomaticallycalculatedfromthesaturationcurvesby
h, 1 min andventilatoryactivity(definedasanopeningofthe day or 7days ofairexposure, fishwerevideorecorded day or7days post hoc 2 2 2 affinity ( affinity bycausingthereleaseofcatecholamines, availability on[Hb],HctandRBCcounts. Three- Holm–Sidak testswereusedtodetermine the Holm–Sidak testswereusedtotestwhether K. marmoratus μm increments,deparaffinised inxylene P 50 Kryptolebias marmoratus ), Rooteffect andcooperativity(Hill 2 availability andCO 2 availability andbodyregion , thiswastheonlypractical P CO 2 2 on used forthe P (Cooper et 50 and the 3993 P 50
The Journal of Experimental Biology or . aslac,K n rg,A. Krogh, and K. Hasselbalch, C., Bohr, K.W. Daisley, and P. C. Blaxhall, P. A. Wright, and K. Bianchini, au,P n coad D.M. McDonald, and P. Baluk, M. Babiker, rue,C .adVl A.L. Val, and C.J. Brauner, ugrn .adMal,R. Moalli, and W. T. Burggren, Wang, and C.J. Brauner, adi,J n el,R.M.G. Wells, and J. Baldwin, ot,J.H. Booth, T. E. Gillis, and C.A. Deck, J.M., Klaiman, S.L., Alderman, ugrn .adMauoa A. Mwalukoma, and W. Burggren, ae,J . rnti,S n rosi J. Orlowski, and S. Grinstein, J.R., Casey, amr M. Farmer, lio,A,Wih,P,Tyo,D . opr . ea,K,Cri,S and S. Currie, K., Regan, C., Cooper, D.S., Taylor, P., Wright, A., Ellison, P. Dejours, rsr . eMlo .V,Wr,D,Re,H . ilas .R,Fn,Y,Brass, Y., Fang, D.R., Williams, H.H., Rees, D., Ward, L.V., deMello, J., Fraser, 3994 volunteers forhelpwithfishmaintenance. regarding thedata.ThankyoutoB.Frank,M.Cornish,Daviesandseveral also thankDrsG.Scott,K.GamperlandGilmourforthoughtfuldiscussions RESEARCH ARTICLE A.J.T., C.R.andK.B. Sciences andEngineeringResearchCouncilofCanadagraduatescholarshipsto of CanadaDiscoverygrantsprogramtoP.A.W. (RGPIN-120513)andNatural Funding wasprovidedbytheNaturalSciencesandEngineeringResearchCouncil draft manuscript.A.J.T., C.R.,K.B.,M.F. andP.A.W. revisedthemanuscript. C.R., K.B.,M.F. executedtheexperiments.A.J.T. analyzedthedataandwrote A.J.T., C.R.,K.B.,M.F. andP.A.W. conceivedanddesignedtheproject.A.J.T., The authorsdeclarenocompetingfinancialinterests. hemocytometer andDrG.Tattersall forhelpoptimizingtheP The authorswithtothankDrD.Fudgeforuseofthemicroscopeand (critical SigmaPlot 11 (SystatSoftware,SanJose,CA, USA) wasusedforallanalyses led,S . ulr .A,Bc,C .adNwa,P. J. Newman, and C.A. Buck, W. A., Muller, S.M., Albelda, ok .G,Itkr .I,Bkr .W,Hce,A .R n ebr,N.A. Herbert, and A.J.R. Hickey, D.W., Baker, F. I., Iftikar, D.G., Cook, opr .A,Ltilr .L,Mrat .L n rgt P. A. Wright, and C.L. Murrant, S.L., Litwiller, C.A., Cooper, amr . yn .J,Fh,U .H n ol,R.W. Noble, and U.E.H. Fyhn, H.J., Fyhn, M., Farmer, Acknowledgements References Funding Author contributions Competing interests with fishblood. mykiss, duringchronichypoxiaexposure. embryonic hemoglobinanderythrocytesinlarvalrainbowtrout,Oncorhynchus binding. relationship –theinfluenceofcarbondioxidecontentbloodonitsoxygen lymphangiogenesis andangiogenesis. blood viscosity. elasmobranchs fromtheGreatBarrierReef:relationshipbetweenhaematologyand GTP) inair-breathingfishes. the distributionofbloodflowintroutgills. J. Physiol. acclimation ontroponinIisoformexpressioninstriatedmuscleofrainbowtrout. skin respiration. capillary recruitmentinamphibians:experimentalevidenceandarevisedmodelfor comparison betweenenvironmentalhypoxiaandanemia. Randall), pp277-306.NewYork, NY: AcademicPress. 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