Calorespirometry reveals that goldfish prioritize aerobic metabolism over metabolic rate

32 Pages·2016·1.31 MB·English

by  Matthew Regan

©2017.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
RESEARCHARTICLE
Calorespirometry reveals that goldfish prioritize aerobic
metabolism over metabolic rate depression in all but near-anoxic
environments
MatthewD.Regan*,IvanS.GillandJeffreyG.Richards
ABSTRACT organisms inhabit and thrive in various hypoxic and even anoxic
environments (Bickler and Buck, 2007; Ramirez et al., 2007).
Metabolicratedepression(MRD)haslongbeenproposedasthekey
Fishesareparticularlyadeptatsurvivinglow-oxygenenvironments,
metabolicstrategyofhypoxicsurvival,butsurprisingly,theeffectsof
having independently evolved hypoxia tolerance numerous times
changes in hypoxic O tensions (Pw ) on MRD are largely
2 O2 (HochachkaandLutz,2001)owingtotherelativelyhighprevalence
unexplored.WesimultaneouslymeasuredtheO consumptionrate
2
̇ of hypoxia among aquatic habitats (Boesch, 2002; Diaz and
(M )andmetabolicheatofgoldfishusingcalorespirometrytotestthe
O2 Breitburg,2009;DiazandRosenberg,1995;Smithetal.,2006).
hypothesis that MRD is employed at hypoxic Pw values and
̇ O2 Metabolic rate depression (MRD) has been proposed as the
initiated just below P , the Pw below which M is forced to
crit O2 O2 hallmark response enabling hypoxic survival in hypoxia-tolerant
progressively decline as the fish oxyconforms to decreasing Pw .
O2 animals(e.g.BoutilierandSt-Pierre,2000;Hochachkaetal.,1996).
Specifically, we used closed-chamber and flow-through
MRD is achieved through reductions in whole-animal (e.g.
calorespirometry together with terminal sampling experiments to
̇ locomotion,reproduction,feeding)andcellular(e.g.growth,repair,
examine the effects of Pw and time on M , metabolic heat and
O2 O2 protein synthesis) processes (Guppy and Withers, 1999; Richards,
anaerobicmetabolism(lactateandethanolproduction).Theclosed-
2010),reducingATPdemand and rates ofanaerobicfueldepletion
chamber and flow-through experiments yielded slightly different
(glycogen)andwasteaccumulation(lactateandprotons).Although
results. Under closed-chamber conditions with a continually
MRDisawell-describedresponsetoanoxiaexposureinarangeof
decreasing Pw , goldfish showed a P of 3.0±0.3kPa and
O2 crit animalsincludingfruitflies(Callieretal.,2015),goldfish(Addink
metabolic heat production was only depressed at Pw between 0
O2 etal.,1991;vanWaversveldetal.,1989)andturtles(Jackson,1968),
and 0.67kPa. Under flow-through conditions with Pw held at a
O2 ithasbeensuggestedthatMRDwouldalsoenhancehypoxicsurvival
varietyofoxygentensionsfor1and4h,goldfishalsoinitiatedMRD
̇ (BoutilierandSt-Pierre,2000;Hochachkaetal.,1996).Indirect(i.e.
between0and0.67kPabutmaintainedM to0.67kPa,indicating
O2 non-calorimetric) measurements in common frogs (Donohoe and
thatP isatorbelowthisPw .Anaerobicmetabolismwasstrongly
activacteritdatPw ≤1.3kPa,bOu2tonlyusedwithinthefirsthourat1.3 Boutilier, 1998) and direct (i.e. calorimetric) measurements in
O2 goldfish(vanGinnekenetal.,1994,2004)andtilapia(vanGinneken
and 0.67kPa, as anaerobic end-products did not accumulate
et al., 1997) suggest that MRD may be employed at hypoxic O
between 1 and 4h exposure. Taken together, it appears that 2
tensions(Pw forwater).Indeed,goldfishreducedmetabolicheatby
goldfishreserveMRDfornear-anoxia,supportingroutinemetabolic ∼31% at ∼2O.12kPa Pw compared with normoxia (∼21kPa) (van
rateatsub-PcritPwO2valueswiththehelpofanaerobicglycolysisin Ginnekenetal.,2004),aOn2dtilapiareducedmetabolicheatby∼40%at
the closed-chamber experiments, and aerobically after an initial
∼1.1kPa Pw (van Ginneken et al., 1997). However, it is still
(<1h) activation of anaerobic metabolism in the flow-through O2
unknown how these changes in metabolic heat correspond with
experiments,evenat0.67kPaPw .
O2 changes in aerobic and anaerobic metabolism, and how this is
KEYWORDS:Calorespirometry,Criticaloxygentension, affectedbyPw .
O2
Environmentalhypoxia,Fish,Metabolicdepression,Metabolicheat MRDwouldbeparticularlyimportantatPw valuesbelowan
animal’scriticalPw forO consumptionrate(OM2̇ ),referredtoas
O2 2 ̇ O2 y
INTRODUCTION P , which is the Pw at which M becomes dependent on g
Aerobic pathways of ATP production yield ∼15 times more ATP encrvitironmental P . P O2is largely deteOrm2 ined by the O binding o
O2 crit 2 ol
than anaerobic pathways (Hochachka and Somero, 2002). affinity of hemoglobin (Hb) (Mandic et al., 2009), and at i
B
Consequently,environmentalhypoxiaandthecorresponding shift Pw values below P the ability to extract environmental O to
to anaerobic metabolism seriously threaten energy balance by satuOr2ate Hb is constrcariitned and thus unable to support rout2ine al
t
reducing an animal’s ability to generate sufficient ATP to meet metabolicrate(MR)aerobically.Theanimalcanattempttosustain n
e
metabolic demands. Despite the critical importance of aerobic ATP production at routine levels through an upregulation of m
respiration to the maintenance of metabolic function, many anaerobic glycolysis, but this comes with the depletion of i
r
carbohydrate reserves and the accumulation of deleterious e
p
anaerobic end-products (Richards, 2009), ultimately limiting x
DepartmentofZoology,UniversityofBritishColumbia,6270UniversityBlvd., E
hypoxic survival time (Lague et al., 2012; Speers-Roesch et al.,
Vancouver,BC,CanadaV6T1Z4. f
2013). However, if the animal is capable of reducing its energy- o
*Authorforcorrespondence([email protected]) consumingprocessesthroughacontrolled,hypoxia-inducedMRD, al
then it could simultaneously mitigate the negative consequences n
M.D.R.,0000-0001-9341-5747 r
of reduced ATP production and increased rates of fuel depletion u
o
Received11July2016;Accepted21November2016 and waste accumulation. We therefore hypothesized that MRD is J
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RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
replaced weekly. We fed the fish to satiation daily (Nutrafin Max
Listofsymbolsandabbreviations GoldfishFlakes)exceptfor24hbeforetransfertotheexperimental
C oxygencontent apparatus, when feeding ceased. The University of British
FO2 flowrate Columbia’sAnimalCareCommitteeapprovedallprocedures.
Hb hemoglobin
M mass
Ṁ oxygenconsumptionrate Calorespirometer
O2 Weusedadifferentialcalorespirometertosimultaneouslymeasure
MR metabolicrate ̇
metabolicheatandM underclosedandflow-throughconditions.
MRD metabolicratedepression O2
P P atwhichHbis50%saturatedwithoxygen The design and operation of the calorespirometerare described in
P50 crOit2icalpartialpressureofoxygenforṀ detailinReganetal.(2013).Briefly,themetabolicheatofafishis
crit O2
PO2 partialpressureofoxygen detected as a voltage by a collection of Peltier units (Custom
PwO2 partialpressureofoxygeninwater Thermoelectric Peltier module 12711-5L31- 03CQ, Bishopville,
T time
MD,USA)viatheSeebeckeffectandconvertedtowattageusingan
V volume
empirically determined calibration coefficient (see Regan et al.,
2013). The design of the calorespirometer allows for the
̇
simultaneous measurements of metabolic heat and M using
ewmhperleoyethdeatnheygpaotixviecPimwpOa2cvtsaluoefsraendducisediniatieartoebdicjusctabpealcoiwtyPacnridt, tPhOe2oinpftloodweisng(OacnedanoOutpftliocwsiOngR1w2a5t,erDulinneedsina,sFwLe,lUl SasA)inpOlta2hceedfioshn
increasedanaerobicreliancebegintoaccrue. chamber. To determine Ṁ under closed-chamber conditions, a
Wetestedthishypothesisusingclosed-chamberandflow-through P optode within the fisOh2 chamber measured the change in
cMalRoDres(pviiraommeettraybtoolicsimheualtt)anaenodusalnyaemroebaiscurgelyOc2olcyosnissu(vmiaptieoxncrreattieosn, PchOwa2Om2boevrevroslueqmueenantidalfi5shmminasinstaecrvcoalrsdianngdtow:as then corrected for
rates of the anaerobic end-product ethanol) in goldfish held at M_ ¼ðDC (cid:2)DT(cid:3)1(cid:2)VÞM(cid:3)1; ð1Þ
tPewrmOi2nvaallusaems rpalninggingexfproermimneonrtmsooxniagtooldafnisohxiae.xpWoeseadlstooptehrefosrmamede whereC istheO Oco2ntentofOt2hewaterconvertedtoμmoll−1from
Pw values as used in the calorespirometry experiments to fully Pw usOi2ng the s2olubility factor of 1.9312μmol l−1 mmHg−1
O2 O2
quantify whole-body anaerobic metabolism. Goldfish were chosen (Boutilieretal.,1984),Tisthetimeperiodoverwhichthechangein
owing to theirexceptional hypoxiatolerance and well-documented C is calculated (5min), V is the fish chamber volume (32ml)
O2
ability to induce MRD (e.g. Addink et al., 1991; van Waversveld minusthevolumedisplacedbythefishitself,andMisthemassof
̇
etal.,1989;vanGinnekenetal.,2004),somethingnotallfishspecies the fish. To determine M under flow-through conditions, the
O2
are capable of (Stangl and Wegener, 1996). We used difference in Pw between inflowing and outflowing water lines
calorespirometry because it is the ‘gold standard’ of MR supplying the fOis2h chamber was measured using the same
measurements and the only way to accurately measure MR on P optodeandcorrectedforflowrateandfishmassaccordingto:
O2
hypoxemic animals in real time (see Kaiyala and Ramsay, 2011;
Nelson,2016).Despitethesuperiorityofcalorespirometry,onlyafew M_ O2 ¼½ðCiO2 (cid:3)CoO2Þ(cid:2)F(cid:4)M(cid:3)1; ð2Þ
studies have measured the metabolic heat of fishes (Addink et al.,
1991;Reganetal.,2013;StanglandWegener,1996;vanGinneken where Ci and Co are O content of inflowing and outflowing
O2 O2 2
etal.,1994,1997,2004;vanWaversveldetal.,1989),andonlythree water,respectively,convertedfromPw asdescribed above,Fis
of these (van Ginneken et al., 1994, 1997, 2004) have measured water flow rate (22mlh−1) and M is thOe2 mass of the fish. Under
metabolicheatatPw otherthannormoxiaandanoxia.Whilethe flow-throughconditions,thechamberPw couldbeheldconstant
O2 O2 ̇
data from these studies suggest that MRD is employed at hypoxic for extended time periods, allowing us to measure M and
O2
Pw values, they exposed their organisms to progressive hypoxia metabolicheatatdifferenttimepointsatanydesiredPw .
O2 O2
over sometimes prolonged periods of time and they did not relate
their measurements to Pcrit nor directly assess the contributions of Hypoxicexposures
anaerobicmetabolismatvarioushypoxicPw values.Furthermore, Individualfishweretransferredtoaflow-throughcalorespirometer
O2
other studies that have attempted to examine the role of MRD heldat17°Candaflowrateof22mlh−1,andinthisapparatuswe
y
and other metabolic and respiratory responsesto hypoxia have not performedbothclosed-chamberandflow-throughcalorespirometry g
o
attempted, to our knowledge, to directly assess the relative experimentsfollowinga16hnormoxichabituationperiod.Forthe
l
o
contributions of MRD, aerobic respiration and anaerobic closed-chamber experiments (n=8), the trial began by stopping i
B
metabolism at different hypoxic Pw values over time. waterflowandallowingthefishtoreducePw fromnormoxiato
Consequently, we still do not have a compOr2ehensive picture of the anoxia over 60–90min. The experiment waOs2 ended when the al
t
hypoxicsurvivalstrategiesoffishes. chamberPw reachedanoxia,atwhichpointweintroducedalethal n
O2 e
doseofanaesthetic(bufferedMS-222,finalchamberconcentration m
MATERIALSANDMETHODS of 150mgl−1) to determine the calorespirometer’s baseline heat i
r
Studyorganisms signature. For the flow-through experiments, inflowing Pw was e
O2 p
We obtained adult goldfish [Carassius auratus auratus (Linnaeus manuallyadjustedtoyieldoneoffourchamberPw valuesovera x
1758); 2.06±0.39g wet mass; n=264; sex unknown] from a ∼60min period (20, 1.3, 0.67 or 0kPa; n=3–6 foOr2each) and the E
f
commercial supplier (Delta Aquatics, Burnaby, BC, Canada) and animalsweremaintainedatoneofthesePw valuesforupto4h o
held them under a 12 h:12h light:dark cycle in a 76litre (referred to as the experimental period). WOe2measured metabolic al
recirculating system of aerated, dechlorinated, 17°C water at the heat over the full 21h period (16h normoxia habituation, 1h n
r
UniversityofBritishColumbia(Vancouver,BC,Canada).Stocking transitiontoexposurePw ,4hexperimentalperiod)andcollected u
density was <0.4gl−1 and water in the recirculating system was effluentwatersampleseitOh2erbefore(time0)orat1and4hduring Jo
565
RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
theexperimentalperiodformeasurementsofethanol(aglycolytic matchtheexperimentalperiodsofthecalorespirometryexperiments
end-product excreted across goldfish gills). Following the (n=8 per time point). We sampled fish by inconspicuously
experiment, we introduced a lethal dose of anaesthetic (buffered introducing a lethal dose of anaesthetic (buffered MS-222, see
MS-222, see above) to determine the calorespirometer’s baseline above),weighingtheindividualfish,freezingthemimmediatelyin
heat signature.At the end of each experiment, werecalibrated the liquid N , and then storing them at −80°C for later metabolite
2
P optodestodetermineanydriftthathadoccurredoverthecourse analyses.Weensuredthattheconditionsbetweentheseexperiments
ofOt2heexperiment(upto∼10%)forthepurposeoflatercorrection, and the calorespirometry experiments were similar by including a
and then washed the calorespirometer and its water lines with a 16h habituation period followed by a 1h transition period to the
10% bleach solution. The flow-through and closed-chamber desiredPw ,conductingexposuresinthedark,andbypreventing
calorespirometry experiments were performed in fall 2014 and the fish inOth2e tank from accessing the air–water interface (which
winter2015,respectively. was not available to the calorespirometry fish). Thus, the main
difference between this experiment and the calorespirometer
Comparisonofclosed-chamberandflow-through experiment was vessel size (calorespirometer chamber was 32ml
calorespirometry andexposuretankswere10litres),whichcouldaffecttheabilityof
To more directly compare the results of the closed-chamber and thefishtomoveduringourhypoxiaexposuresandyielddifferent
flow-throughcalorespirometryexperiments,weconductedaback- levels of lactate and ethanol accumulation. We are, however,
to-backcomparisonofthetwotechniquesusingthesamefish.This confident that fish movement is minimal in the calorespirometer
wasrequiredbecauseourfirstexperiments(presentedinFigs1and based on the relatively smooth heat traces observed over the
2)usingthesetechniqueswereconductedatdifferenttimesofyear habituation and experimental periods, and periodic visual
̇
and yielded different routine normoxic M values, which could inspection of the fish in the 10litre tanks revealed little to no
O2 ̇
affect our determination of P . We measured routine M using movement,especiallyduringthehypoxiaexposures.Thus,despite
crit O2
both techniques and determined P during closed-chamber the differences in exposure regimes, the fish from both the
crit
respirometry (P was not determined via flow-through calorespirometry and the terminal sampling experiments likely
crit
calorespirometry because it would require the fish to undergo respondedtohypoxiainasimilarmanner.
multiplerunsatdifferentPw values).Briefly,fishwereintroduced
O2
to the calorespirometer and allowed to habituate under the same Lactateandethanolanalyses
conditions as the calorespirometry experiments described above. Tolinkourwhole-bodycalorespirometrymeasurementsofMRto
Following the habituation period, we first measured the fish’s the activation of anaerobic metabolism, we measured whole-body
̇
routine M under normoxia using flow-through respirometry (as concentrations of lactate and ethanol. Entire goldfish from the
O2
described above), then immediately closed off the respirometer 10litretankexposuresweregroundintoafinepowderusingaliquid
̇
chamberandmeasuredM usingclosed-chamberrespirometry(as N -chilledmortarandpestle.Toextractthemetabolites(lactateand
O2 2
describedabove).Werepeatedthisthreetimesforeachofsixfish, ethanol)fromthepowder,an aliquotof powderwas weighed and
and the Pw was not allowed to drop below 16kPa during these transferred to a 2ml centrifuge tube containing 1ml of ice-cold
O2
closed-chambermeasurements.Followingthefinalclosed-chamber 30%HClO andimmediatelyhomogenizedat0°CusingaPolytron
4
measurement, we allowed the fish to deplete the chamber’s O homogenizer set to the highest setting for 30s. The resulting
2
contentsoastodetermineitsP .Metabolicheatwasnotmeasured homogenatewasthencentrifugedat20,000gfor5minat4°Cand
crit
duringtheseback-to-backexperiments. thesupernatantwastransferredtoanew1.5mlcentrifugetubeand
neutralizedusing3moll−1Trisbasetoavoidthevolatilizationof
Terminalsamplingexperiments ethanol that occurs in association with vigorous CO production
2
TobetterestimatetheeffectsofPw onanaerobicmetabolism,we when HClO is neutralized with K CO . We confirmed that
O2 4 2 3
ran parallel hypoxia exposures where we euthanized animals to neutralizationwithTrisbasedoesnotaffectourenzymaticanalysis.
measurewhole-bodyconcentrationsoflactateandethanol.Foreach Wemeasuredethanolimmediatelyfollowingneutralizationusinga
Pw ,weexposed24goldfishspreadacrosssixtanks(fourfishper commercialkitdesignedforbiologicalethanolanalysis(Diagnostic
O2
tank) and sampled two replicate tanks at each of 0, 1 and 4h to ChemicalLtd.,PEI,Canada),andthenfrozetheunusedportionof
8 4 Fig.1.Closed-chambercalorespirometry y
6 b b b b d,e d d d d d,e d,e 3 m6mw0aeettateoasrb9u(oP0relwimcmOihn2e)enbwatesatcsioanfruegosdoexulydocgfefiedtshnhefr.cofPoimsanhrsn’tsiuoamrOlmpproetcxisooiasnnustorrueamatoenpfot(oiMxoẋinOay.g2o)Devanaentriadn Biolog
µ–1–1mol h g) 4 b,c b,c,ec,d,eb b b b b b b 2 –1)olic heat (J g asirgenmifiecaannstl±ysd.eiff.emre,nn=t8(o.nDea-twaapyoAinNtsOsV2hAar,inPg>0a.l0e5tt)e.rarenot erimental
M. (O2 2 a b 1 Metab Exp
f
o
a al
0 0 n
0 1 2 3 4 5 6 20 r
u
Pw (kPa) o
O2 J
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RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
3.0 A 1.2 15 1.0 Fig.2.Flow-throughcalorespirometry
a a measurementsofṀ O2,metabolicheatandglycolytic
end-productsingoldfishheldatdifferent
12 0.8 Pw valuesfordifferentperiodsoftime.(A)1h;
2.0 a a 0.8 (B)O42h.Dataaremeans±s.e.m,n=3–6,anddatapoints
areoffsetslightlyonthex-axisforclarity.Datapoints
a 9 0.6
c a sharingaletterarenotsignificantlydifferent(one-way
ANOVA,P>0.05).
Mµ.–1–1 (mol h g) O2 231...0000 Bbcb aba a ba a aa 0001...482–1Metabolic heat (J g) 91103625µ–1ole-body lactate + ethanol (mol g) 000001.....24680 µ–1–1 g) Ethanol excretion rate (mol h
h
W
b
6 0.4
b
1.0 0.4
3 0.2
b
a
0 b 0 0 0
0 1 2 3 19 20 21
Pw (kPa)
O2
the sample extract for later lactate analysis. Lactate concentration timesthestandarddeviationofanindividual’saverageṀ between
O2
was measured using the LDH reaction according to the protocols 13and21kPaPw .
O2
outlinedinBergmeyer(1983).
Dataanalysisandstatistics
̇
O equilibriumcurves M andethanolproductionrateswerecalculatedateachtimepoint,
To2understandhowthegoldfish’sHb–O affinityisrelatedtoP whOi2lemetabolicheatwasrepresentedbyaveragingthecontinualheat
2 crit
and MRD, we constructed O equilibrium curves for the whole measurementsmadeoverthe20minstraddlingthetimepoint (e.g.
2
blood of five normoxia-acclimated goldfish using the thin film 50–70min for 1h time point). All data are presented as means±
spectrophotometric technique (Lilly et al., 2013). Blood was s.e.m.TheeffectsofPw oneachvariableweredeterminedusing
collected from the caudal artery of anaesthetized fish using 60μl one-wayANOVA(SigmaOS2tat11.0).
heparinized capillary tubes. We then centrifuged the tubes and
resuspendedtheredbloodcellsinHEPESbuffer(pH7.8)toensure RESULTS
a consistent blood pH across all samples. AWöstoff gas mixing Closed-chambercalorespirometryexperiments
pump (H. Wösthoff Messtechnik GmbH, Bochum, Germany) We used closed-chambercalorespirometry to measure P and to
crit
mixed compressed O and N to each of seven P values for the characterize the effects of a progressive reduction in Pw on
2 2 O2 ̇ O2
construction of the O equilibrium curves, and Hb P values M and metabolic heat.Pw in the closed-chamberexperiments
(the P at which Hb 2is 50% saturated with O ) were c5a0lculated waOs2decreasedfromnormoxiaO2toanoxiabythefish’sownṀ over gy
using Oth2e equation of each sigmoidal curve2as calculated by 60–90min(dependingonthefish’sṀO2).PcritwascalculateOd2tobe olo
SigmaStat11.0. 3.0±0.3kPa (Fig. 1). At PwO2 values above Pcrit, there werėno Bi
significanteffectsofchangesinPw ontheaverageroutineM ,
PPcritcisalcduelfainteiodnas the Pw at which an organism’s routine MẇhilepraotgPrewsOsi2vvealylufeesllbteolozweroPcarsitt(haetwOgo2hlidcfhisthhedefipslhetsepdenthte∼a3v0aimlaibnOl)2e, ntal
ċrit O2 O2 e
M transitionsfrombeingindependentoftobeingdependentupon oxygen. Metabolic heat was maintained at routine levels at all m
O2
PchwaOm2.beWr cealdoeretesrpmirionmedetrPycerixtpfeorrimeeancths uinsdinigvidthuealBiAnSIthCepcrologsreadm- uPpwoOn2rveaalcuheisngbeatwnoexeina,2e0veanntdua0l.l5yksPtaabi(lFizigin.g1)atbu∼t2w1%asodfeproreustsiende eri
p
(Yeager and Ultsch, 1989), which uses a two-segment linear normoxicvalues(anMRDof79%;Fig.1)after∼20min. x
E
regression model to determine P as the Pw at which the two
lineartrendlinesintersectonagrcaripthplottingMȮ2 asafunctionof Flow-throughcalorespirometryexperiments of
Pw . Some individuals’ Ṁ values increasOed2 above routine Weusedflow-throughcalorespirometry to characterize the effects al
̇ O2 O2 ̇ n
M levelsathypoxicPw valuesclosetoP ,andincludingthese ofPw andtimeonM ,metabolicheatandexcretedethanol.We
ṀO2valueswouldoveresOti2mateP .Toprecvreitntthis,weexcluded heldinOd2ividualsatoneOof2fourPw values(20,1.3,0.67or0kPa) ur
O2 ̇ crit ̇ O2 ̇ o
from our routine M estimation any M value that exceeded 1.5 for 1 and 4h. For 1h exposures, M and metabolic heat were J
O2 O2 O2
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RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
maintained at routine lėvels to PwO2 of 0.67kPa, while at 5 A
Pw valuesbelowthis,M felltozeroandmetabolicheatfellto
∼32O%2 ofroutinelevels(anOM2 RDof68%;Fig.2A).Similarly,for –1g)
4lteovhzeeelsrxotpoaonPsudwrmeOs2e,otMafḃ0Oo.26lia7cnkhdPemaat,ewfteahlbliolteoliac∼tPh2e0wa%Ot2wovefarrleuoeumstiabnieenltolaewivneetlhdsis(a,atMnrȯOMu2tRfienDlel µ–1mol h 4
of80%;Fig.2B). . (O2
M
Ethanolexcretionrateswereundetectablefollowing1hexposure
h
at all Pw values (Fig. 2A). These rates increased following 4h g 3
exposure,O2and higher rates were generally detected at lower hrou
PsiwgnOi2ficvaanlute(sFi(gF.ig2.B2).B), but these increases were not statistically Flow-t
2
Whole-bodyanaerobicend-productconcentrations 2 3 . 4 5
Closed-chamber M (µmol h–1 g–1)
Whole-body concentrations of lactate significantly increased over O2
time0valuesfollowing1and4hat1.3kPa,0.67kPaandanoxia
(Table 1). Whole-body concentrations of ethanol significantly 1) 4 B
–
g
increasedovertime0valuesfollowing1and4hofanoxiaexposure 1
–
(Table1).Thetotalanaerobicend-productconcentrationsat1.3and ol h 3
0.67kPa following 4h were similar to those following 1h, m
µ
suggesting that the rate of anaerobic end-product accumulation .(O2
fell to near-zero levels after 1h (Fig. 2). A similar result was M
observedfortheanoxia-exposedfish,thoughtoalesserextent,with xic 2
anaerobic end-product concentrations being ∼1.8-fold higher mo
following4hexposurethanfollowing1hexposure(Fig.2). or
n 1
e
n
Closed-chamberversusflow-throughcalorespirometry uti
̇ o
IndividualM valuesdeterminedinthesamefishinaback-to-back R
O2 0
comparisonofclosed-chamberandflow-throughrespirometrywere Closed-chamber Flow-through
positively correlated (n=18, r=0.925, P<0.0001; Fig. 3A) and
yieldedsimilarmeanṀO2values(t=0.423,P=0.678;Fig.3B).The cFliogs.e3d.A-chcaommbpearriasnodnfolofwṀ-Oth2rmoueagshucreamloreenstpsiorofmgoeltdryfi.sBhomthardeespuirsoimngetric
closed-chamberportionoftheseexperimentsyieldedaP of2.7±
crit techniqueswereperformedinthesameapparatusfollowinga≥16h
0.2kPa(n=6). habituationperiod,atthesametimeofday(∼10:00hPST),andat
Pw ≥16kPa.(A)Ṁ valuesresultingfromclosedandflow-through
Hb–O equilibriumcurves techOn2iques,withmeaOs2urementsmadeback-to-backonthesamefish(n=18,
2
TheHbofgoldfishdisplayedaveryhighaffinityforO2,resultingin r=0.925,P<0.0001).(B)AverageṀ O2measurementsforeachtechnique,with
a steep O equilibrium curve and an average whole-blood P of errorbarsrepresentings.e.m.(n=18,t=0.423,P=0.678).
2 50
0.49±0.12kPa(Fig.4).
Pw values just below P , metabolic heat was maintained at
O2 crit
DISCUSSION routinenormoxiclevelstoaPw of0.67kPa,andMRDwasonly
O2
We hypothesized that goldfish employ MRD at hypoxic evidentingoldfishexposedtoanoxia.Themagnitudeoftheanoxia-
Pw values and initiate it at Pw values just below P . This induced MRD [79% depression in closed-chamber experiments;
O2 O2 crit
hypothesis predicted that metabolic heat would decrease from 68% (1h) and 80% (4h) in flow-through experiments; Figs 1, 2]
routinelevelsataPw belowP ,whenthefish’sabilitytotakeup was very similar to what has been shown previously for anoxia-
O2 crit ̇
environmentalO tosupportaroutineM wascompromised.Our exposedgoldfishusingcalorimetry(Addinketal.,1991;Stangland
2 O2
closed-chambercalorespirometryexperimentsyieldedaP of3.0± Wegener,1996;vanGinnekenetal.,1994).
crit y
0.3kPa (Fig. 1), consistent with the P values reported in other g
crit o
studiesongoldfish(FryandHart,1948;Fuetal.,2011).However, Metabolicresponsestohypoxia l
o
contrary to our hypothesis that MRD is initiated at hypoxic Goldfishmaintainedroutine MR atseverelyhypoxic PwO2 values Bi
underbothclosed-chamberandflow-throughconditions(Figs1,2),
l
a
Table1.Whole-bodyconcentrations(μmolg−1)oflactateandethanolin butappeartohaveuseddifferentstrategiestodoso.Intheclosed- t
goldfishexposedtodifferentPwO2(partialpressureofoxygeninwater) chamber experiments, metabolic heat was maintaiṅed at routine en
valuesfor1and4hfollowinga16hnormoxichabituationperiod(0h) normoxic levels to 0.67kPa despite a decrease in M at 3.0kPa m
O2
0kPa 0.67kPa 1.3kPa 20kPa (Fig. 1), suggesting that anaerobic glycolysis was upregulated to eri
supportMR(thoughlactateandethanolcouldnotbemeasuredin
p
[Lactate] 0h 1.20±0.40a 0.22±0.18a 0.53±0.47a 0.21±0.20 closed-chamber experiments as a function of Pw ). In the flow- x
1h 6.42±0.54b 4.21±0.34b 3.11±0.48b 0.15±0.14 O2 E
through experiments, metabolic heat was similarly maintained at
4h 10.6±0.93c 4.89±0.86b 4.52±0.72b 0.11±0.15 f
[Ethanol] 0h 0.61±0.11a 0.74±0.14 0.87±0.20 – routinenormoxiclevelsto0.67kPaatboth1and4h,butunlikethe o
14hh 12..2204±±00..1270bc 11..0027±±00..1100 00..7475±±00..2018 –– slceulvopespelosdra-tctehdaallmahebyroeprboiexcxiacpllePyrwimeOve2ennvtasal,utMesṡeOtv2eeswrteealdys.mThyhapiisnotsxauiicgngePdewstasttnhveaaatrlMu-reoRsuwtainnades urnal
Differentlettersindicatesignificantdifferencesbetweentimepointswithina O2 o
Pw exposure(P<0.05).[Ethanol]measurementsat20kPawerenottaken. that MRD is reserved for all but severely hypoxic (<0.67kPa) or J
O2
568
RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
100 in normoxia to ∼17 and 15µmol h−1 g−1 at 1.3 and 0.67kPa,
respectively (assuming P:O of 6 and ATP:lactate/ethanol of 1),
2
%) 80 while heat production does not change. These inconsistencies are
n ( likely a consequence of not being able to temporally match our
atio 60 measurements of anaerobic metabolism (taken as the delta
ur accumulation of lactate and ethanol over the entire hour plus the
at Pw adjustment period) with those of Ṁ and heat, which were
O s2 40 takeOn2 attheendofthe1h(between50anOd270min exposure).As
Hb– 20 Hb P = 0.49±0.12 kPa such,itispossibletherearetemporalshiftsinfuelselectionwithin
50 the first hour of hypoxia exposure, with lactate and/or ethanol
accumulatingduringtheinitialdescenttowardsthetargetPw as
0 ̇ O2
0 1 2 3 4 5 6 22 MO2-sustainingmechanismsareupregulated.Finer-scalestudiesare
P (kPa) neededtoconfirmthisidea.
O2
TheP valuesderivedfromtheclosed-chamberandflow-through
crit
Fig.4.O2equilibriumcurveforthewholebloodoffivenormoxia- calorespirometryexperimentsdifferedsubstantially,withPcritshifting
acclimatedgoldfish.Redbloodcellswereseparatedfromplasmaand from 3.0kPa in the closed-chamber experiments to somewhere
resuspendedinHEPESbuffer(pH7.8).O levelswereachievedusinga
2 between 0 and 0.67kPa in theflow-through experiments (the exact
WösthoffgasmixingpumpattachedtocylindersofcompressedO andN .
2 2 value cannot be determined). These technique-specific differences
in P are consistent with a recent study comparing closed and
crit
near-anoxicenvironments.Thisisdifferentthantheresultsofvan intermittent-flowrespirometry(Snyderetal.,2016),whichattributed
Ginnekenandcolleagues(1994,2004),whoshowedmoderate27% the higher P in closed respirometry to metabolic waste
̇ crit
and 33% decreases in heat production along with lower M in accumulationandafasterdeclineinPw .Similarfactorsmaybeat
O2 O2
goldfish exposed to 3.5 and 2.1kPa, respectively. These playinourclosed-chambercalorespirometryexperiments,resultingin
incongruent results are likely due to differences in experimental anoverestimationP .Anotherpossibleexplanationmightbethatthe
ċrit
designandstudygoals.vanGinnekenetal.(1994,2004)exposed routinenormoxicM (andheat)intheclosed-chamberexperiments
O2
each fish in their studies to progressive hypoxia over prolonged was approximately twofold higher than in the flow-through
periodsoftime(e.g.8.4,4.2,2.1andfinally0.63kPaovera16h experiments (cf. Figs 1 and 2). All else being equal, this would
period in van Ginneken et al., 2004), which does not allow the necessitatethefishfromtheclosed-chamberexperimentsadoptingan
authors to disentangle the effects of Pw and time on metabolic oxyconforming strategy at a higher Pw , yielding a higher P .
̇ O2 O2 crit
heat and M . In contrast, our flow-through calorespirometry However, our back-to-back comparison of calorespirometry
O2
experimentsexposedgoldfishtoonlyasinglehypoxicPw (aftera techniques suggests that technique per se does not explain the
O2 ̇
1hadjustmentperiod)forupto4handweassessedtheeffectsof twofoldchangeinM andheatproduction,whichmightinsteadbe
O2 ̇
varyinghypoxicPw valuesusingdifferentindividuals,allowing explainedbythetimeofyear.Inanycase,thedifferencesinM do
O2 O2
ustoindependentlyassesstheeffectsofPw andtimeonmetabolic not appear to affect P and therefore do not explain why P is
O2 crit crit
responses.Usingthisapproach,weclearlyshowthatwithin1hof higher in the closed-chamber experiments than in the flow-through
exposure,goldfisharecapableofmaintainingoxygenuptakeunder experiments.
severely hypoxic conditions (0.67kPa), obviating the need for Another factor possibly contributing to the lower P values
crit
hypoxia-inducedMRD. obtained from the flow-through experiments versus those from
ElevatedlevelsoflactateandethanolatPw ≤1.3kPaat1and the closed-chamber experiments is time. Fishes possess many
O2
4h indicate that anaerobic glycolysis also contributed to mechanisms that enhance O uptake with decreasing Pw ,
2 O2
maintaining MR, though in slightly different ways in anoxia and including increases to gill surface area (Sollid et al., 2003), Hb
hypoxia.Inanoxia,lactateandethanollevelscontinuedtoincrease synthesis (Gracey et al., 2001) and concentration in the blood
throughout the 4h exposure, but their rate of accumulation (Affonso et al., 2002), hematocrit (Lai et al., 2006; Turko et al.,
decreased from 5.81µmol h−1 g−1 during the first hour to 2014), Hb–O affinity (Turko et al., 2014), ventilation frequency
2
1.73µmol h−1 g−1 during the subsequent 3h. These results are and amplitude (Holeton and Randall, 1967; Itazawa and Takeda,
consistent with those observed in tissues from anoxia-exposed 1978;Tzanevaetal.,2011;VulesevicandPerry,2006),aswellas
y
turtles(Trachemysscriptaelegans),wherelactateproductionrates redistributed blood supply to critical tissues (Sundin et al., 1995). g
o
were elevated during the first hour of anoxia exposure and While these mechanisms effectively enhance the uptake of l
o
subsequently decreased between 1 and 5h anoxia in brain, liver environmental O2 and its distribution throughout the body, their Bi
andwhitemuscle(KellyandStorey,1988).Combined,theseresults inductiontakestime,varyingfromminutestodaysdependingonthe
l
a
suggest that there is an initial reliance on anaerobic metabolism physiological response examined. Because the fish in the flow-
t
uponanoxiaexposurethatmaycompensatefortheanoxia-induced through experiments had spent 1 or 4h at each Pw when their n
limitations on aerobic ATP production while MRD is initiated. In Ṁ was measured (in addition to the ∼1h requiredOto2 reduce the me
O2
hypoxia,theearlyrelianceonanaerobicmetabolismwastemporally PwO2 from normoxia to the target PwO2), they may have had eri
even more profound than in anoxia. Lactate and ethanol additional time to initiate some of these mechanisms of enhanced
p
accumulation was confined entirely to the first hour of hypoxia O uptake compared with the closed-chamber fish that saw only x
exposure at 1.3 and 0.67kPa, while Ṁ was concurrently ∼320minofcontinuallydecreasingsub-P hypoxicconditions.If E
maintained at routine normoxic levels throOu2ghout the hypoxic P is in fact influenced by the rate acnritd duration of hypoxia of
crit
exposures. Taken together, these data suggest that total ATP induction over relatively short time scales, then it becomes al
n
turnover is higher over the first hourof hypoxia exposure than in important to apply similar methodology both within and between
r
normoxia. Indeed, whole-body estimates of total ATP turnover studies (something that is not currently done; see Rogers et al., u
o
duringthisperiodindicatethatitincreasesfrom∼10µmolh−1g−1 2016)toensureP valuesarecomparable.Thisisespeciallytrue J
crit
569
RESEARCHARTICLE JournalofExperimentalBiology(2017)220,564-572doi:10.1242/jeb.145169
whenP isusedasareferencepointformodelsthat,forexample, respiration is therefore the ideal strategy for surviving long-term
crit
predict how climate changewill reshape the distribution of fishes hypoxicboutsbecauseitminimizesthetimethefishisforcedtorely
aroundtheworld(Deutschetal.,2015). onMRDandtheassociatedphysiologicalcosts.
Taken together, the goldfish’s overall hypoxiatolerance strategy
Hb–O affinityandinitiationofMRD appearsfinelytunedtoitsparticularhypoxicenvironment,whichis
2
Our results show that MRD is initiated in goldfish at a characterizedbylong,protracteddescentsintoeventualanoxia.This
Pw somewhere between 0 and 0.67kPa. Interestingly, our maybethecasewithotherspeciestoo;becausehypoxicenvironments
anaOly2sisofwhole-bloodHb–O affinityrevealsaHbP valueof varygreatlyinseverity,durationandrateofhypoxicinduction,the
2 50
0.49±0.12kPa(Fig.4;consistentwithBurggren,1982),withinthe hypoxiatolerancestrategiesemployedbyorganismsnativetothese
̇
Pw rangethatgoldfishappeartoreduceM intheflow-through differentenvironmentsarelikelytobejustasvariable.
O2 O2
experimentsandinitiateMRD.Itisthereforetemptingtothinkofa
causallinkbetweenthesupplyofO tothetissuesandtheinitiation Conclusions
2
ofMRD.ConsiderabledebateexistsregardingthesignalforMRD, BydemonstratingthatgoldfishprioritizeO uptakeoverMRDinall
2
withsomedatasupportingsignalsresidingontheenergyproduction but nearly anoxic environments, our results suggest two things.
sideofthecellularenergyfluxpathways(deZwaanandWijsman, First,theexceptionalhypoxiatoleranceofgoldfishowesmoretoits
1976;Hochachka,1982,1985;PlaxtonandStorey,1984;Reesand O extraction abilities than to MRD. Second, MRD is not
2
Hand,1991;BishopandBrand,2000;Bishopetal.,2002)andsome necessarily a key mechanism of hypoxic survival, as has been
datasupportingsignalsontheenergyconsumptionside(Caligiuri hypothesized (Hochachka et al., 1996), but of anoxic survival.
etal.,1981;FlaniganandWithers,1991;Robinetal.,1979;Sick WhileMRDisaneffectivemeansofbalancingenergysupplyand
etal.,1982;seereviewsbyGuppy,2004;GuppyandWithers,1999; demand, the potential costs associated with reducing cellular and
StoreyandStorey,1990).IfHb–O affinitywereinfactasignalfor whole-bodyprocessesmaythreatenorganismalfitnessandpreclude
2
MRD, this would place the signal on the energy production side, itsselectioninallbutthemostextremeenvironments.
consistent with some of the more recent views in the field (see
Guppy, 2004). Similarly, Coulson (1977) postulated that MR was Acknowledgements
directlyproportionaltothecirculatorysystem’sabilitytosupplythe Wethankthereviewersfortheirinsightfulcomments,aswellasMilicaMandic,
BruceGillespieandJoeVeneri.WededicatethispapertoJohnM.Gosline
tissues with O2, and this idea gained empirical support when van (1943–2016),afriendandmentorwhoalwaysfoundthejoyinscience.
Ginneken et al. (2004) showed a correlation between hypoxia-
induced decreases in MR and heart rate. All told, it is not Competinginterests
unreasonableto speculatethatasignal for hypoxia-induced MRD Theauthorsdeclarenocompetingorfinancialinterests.
involvesthesupplyofO tothetissue.TheassociationbetweenHb
2
P andthePw ofMRDinitiationisthereforeenticingandworth Authorcontributions
50 O2 M.D.R.andJ.G.R.designedthestudy.M.D.R.performedallexperimentsanddata
furtherinvestigation.
analyses.I.S.G.assistedwithmetaboliteassays.M.D.R.draftedthemanuscriptand
allauthorscommented.
EcologicalimplicationsofMRD
ThefactthatgoldfishappeartoinitiateMRDonlynearanoxiaand Funding
maintain Ṁ without a long-term activation of anaerobic ThisworkwassupportedbyaNaturalSciencesandEngineeringResearchCouncil
metabolismiOs2wellsuitedtothegoldfish’s(andthecloselyrelated ofCanada(NSERC)DiscoveryGranttoJ.G.R.M.D.R.wassupportedbyanNSERC
postgraduatescholarshipandaUniversityofBritishColumbiaZoologyGraduate
crucian carp’s, Carassius carassius) natural lake habitat. While Fellowship.
these lakes become ice-covered in winter and eventually anoxic,
theyareseverelyhypoxic(Vornanen,2004)formostofthewinterat Dataavailability
Pw values at which our study reveals goldfish remain aerobic. DatahavebeendepositedintheDryadDigitalRepository(Reganetal.,2017):http://
O2 dx.doi.org/10.5061/dryad.719bt
GoldfishcanthereforemaintainroutineMRformostofthewinter
without relying on anaerobic glycolysis and/or MRD until it is
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