Juvenile Antarctic rockcod, Trematomus bernacchii, are physiologically robust to CO2–acidified ...

36 Pages·2016·1.12 MB·English

by  Brittany Bjelde

©2016.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
RESEARCHARTICLE
Juvenile Antarctic rockcod (Trematomus bernacchii) are
physiologically robust to CO -acidified seawater
2
BrittanyE.Davis1,2,NathanA.Miller1,3,ErinE.Flynn1andAnneE.Todgham1,*
ABSTRACT literature suggests that OA will have negative consequences for
marineorganismsacrossdifferentecosystems(Kroekeretal.,2013).
To date, numerous studies have shown negative impacts of CO -
2
In order to predict the impact of OA on marine organisms it is
acidifiedseawater(i.e.oceanacidification,OA)onmarineorganisms,
important to understand whether contemporary individuals
including calcifying invertebrates and fishes; however, limited
currently exhibit the capacity to tolerate projected OA scenarios
researchhasbeenconductedonthephysiologicaleffectsofOAon
andpossessthemechanismsnecessarytorespondtoelevatedpartial
polar fishes and even less on the impact of OA on early
pressuresofcarbondioxide(P ).
developmental stages of polar fishes. We evaluated aspects of CO2
High-latitude oceans are projected to undergo the greatest
aerobic metabolism and cardiorespiratory physiology of juvenile
decreases in pH, from 0.3 to 0.5 units by the end of the century
emerald rockcod, Trematomus bernacchii, an abundant fish in the
(Ciais et al., 2013) because of the increased solubilityof gases in
RossSea,Antarctica,toelevatedpartialpressureofcarbondioxide
(P ) [420 (ambient), 650 (moderate) and 1050 (high) μatm P ] colderwaters.Polarecosystemssupportorganismsthatareadapted
CO2 CO2 to cold, stable environments (Eastman, 1993) and as a result, are
over a 1 month period. We examined cardiorespiratory physiology,
predicted to be more sensitive than lower-latitude systems to
including heart rate, stroke volume, cardiac output and ventilation
changes in ocean conditions associated with GCC (Barnes and
rate,wholeorganismmetabolismviaoxygenconsumptionrateand
Peck, 2008; Fabryet al., 2009; Barnes et al., 2009). The physical
sub-organismalaerobiccapacitybycitratesynthaseenzymeactivity.
effectsofGCCinpolaroceanshavealreadybeendocumentedand
Juvenile fish showed an increase in ventilation rate under high
areofparticularconcern.Increasesinseaandairtemperaturesalong
P compared with ambient P , whereas cardiac performance,
CO2 CO2 the Antarctic Peninsula are occurring at the fastest rates on Earth
oxygen consumption and citrate synthase activity were not
(MeredithandKing,2005),alongsideanetdecreaseinoceanpH,
significantly affected by elevated P . Acclimation time had a
CO2 and predicted rapid changes in ocean P could create highly
significanteffectonventilationrate,strokevolume,cardiacoutputand CO2
unfavorableconditionsformarineorganismsby2050(McNeiland
citrate synthase activity, such that all metrics increased over the
Matear,2008;McNeiletal.,2010).
4weekexposureperiod.Theseresultssuggestthatjuvenileemerald
Real-timemonitoringofnear-shoreAntarctichabitatshasshown
rockcodarerobusttonear-futureincreasesinOAandmayhavethe
that P is quite variable (Kapsenberg et al., 2015), with several
capacitytoadjustforfutureincreasesinPCO2byincreasingacid-base AntarcCtOic2habitatsprojectedby2100toexperienceyearlycyclesof
compensationthroughincreasedventilation.
harmful, low pH during the Antarctic winter (Kapsenberg et al.,
KEYWORDS:Antarctica,Cardiorespiratoryphysiology, 2015).SuchresultssuggestthatinpolarregionstheeffectsofGCC
Earlylifestages,Notothenioid,Oceanacidification maymanifestingreaterP variability.Potentialbiologicaleffects
CO2
of elevatedP havealready been observed inseveral laboratory
CO2
INTRODUCTION studies on Antarctic calcifying marine organisms, including
As levels of atmospheric carbon dioxide (CO ) rise as a result of morphological changes with reduced growth (Yu et al., 2013),
2
increasedanthropogenicemissionsofCO ,theworld’soceansare developmental malformations (Byrne et al., 2013) and the
2
concurrentlyabsorbing CO andbecoming moreacidic. Increased dissolution of shells (Orr et al., 2005; McClintock et al., 2009;
2
dissolved CO in the ocean alters the chemical equilibrium, Bednaršek et al., 2012). Physiological alterations in oxygen
2
decreasing carbonate ion concentration and pH (Feely et al., consumption rates, heat shock proteins and enzymes involved in
y
2004) in a process known as ocean acidification (OA) (Sabine shell growth have also been observed (Cummings et al., 2011). g
o
et al., 2004; Caldeira and Wickett, 2005; Orr et al., 2005; Meehl Although more recent studies have expanded the list of taxa
l
o
etal.,2007;Gattusoetal.,2014).Inthepast200years,theocean vulnerable to OA to include non-calcifying organisms such as i
B
hasabsorbedover50%oftheanthropogenicallyproducedCO and fishes,theseOAstudiesinfisheshaveprimarilyfocusedontropical
2 l
open-ocean pH has decreased by 0.1 units (Sabine et al., 2004). (Dixsonetal.,2010;Nowickietal.,2012;Pimenteletal.,2014a) a
t
Global climate change (GCC) models predict that by the next andtemperatespecies(Hurstetal.,2012;Hamiltonetal.,2014;see n
e
century,oceanpHwilldeclinebyanadditional0.2–0.4units(Ciais also review by Heuer and Grosell, 2014). Few studies have been m
et al., 2013; Gattuso et al., 2014) and an accumulating body of conductedontheimpactofincreasedP onpolarfishes(Strobel i
etal.,2012,2013;Enzoretal.,2013;ECnOz2orandPlace,2014)and er
p
even fewer on early developmental stages of polar fishes (Flynn x
1DepartmentofAnimalSciences,UniversityofCaliforniaDavis,Davis,CA95616, etal.,2015). E
USA.2DepartmentofWildlife,FishandConservationBiology,Universityof f
CaliforniaDavis,Davis,CA95616,USA.3RombergTiburonCenterfor Although knowledgeoftheimpactofOAonfisheshasgrown, o
EnvironmentalStudies,SanFranciscoStateUniversity,Tiburon,CA94920,USA. large gaps remain in understanding the effects of OA on fish al
physiology.PhysiologicalandbiochemicalprocessesarelargelypH n
*Authorforcorrespondence([email protected]) r
dependent, such that changes in blood and tissue pH due to u
o
Received12October2015;Accepted15February2016 increasedenvironmentalPCO2canhavenegativeeffectsoncritical J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
reproduction), it is possible that different life-history stages may
LCiOstofsycmarbdoialcsoauntpdutabbreviations bhoavthedaicfufeterenatndsenlosnitgiveirt-iteesrmto e(1le–v4atwedeePkCs)O2e.fHfeecrtes,wofeeinxvpeosstuigreatetod
CS citratesynthase elevatedP ontheaerobicmetabolismofjuvenileT.bernacchii.
EDV enddiastolicvolume OnthebasiCsOo2fpreviousOAstudiesonearlylifestagesoftemperate
ESV endsystolicvolume
andtropicalfishes,thesensitivityofadultAntarcticfishestoP ,
f heartrate CO2
H andthelackofanyphysiologicalstudiesonthecapacityofjuvenile
f ventilationrate
V T.bernacchiitorespondtoenvironmentalchange,wepredictedthat
GCC globalclimatechange
Ṁ metabolicrate these younger stages of fish would be more sensitive to elevated
O2
OA oceanacidification P . We hypothesized that elevated P would increase
PCO2 partialpressureofcarbondioxide phCyOs2iological costs and energetic demCOan2ds of juvenile
VS strokevolume T.bernacchii.
Environmentalstressors,suchasincreasedtemperature,hypoxia
and very high levels of P (e.g. 8000–50,000μatm P ; Cech
CO2 CO2
processes(Ishimatsuetal.,2005,2008;HeuerandGrosell,2014), andCrocker,2002;Leeetal.,2003),havebeenshowntoalterthe
and these effects may vary with acclimation time (Pörtner et al., cardiorespiratory physiology of fishes, which, in turn, can
2004). For instance, short-term acute effects of elevated P on negatively affect oxygen circulation and delivery to body tissues
CO2
physiology might include alterations in cardiorespiratory (Marketal.,2002;PörtnerandFarrell,2008).Previousresearchhas
phenotypes, acid-base balance, blood circulation and the nervous shown that adult Antarctic species, including T. bernacchii,
system,whereaslongeracclimationperiodscanreducegrowthand T. newnesi, T. hansoni and Pagothenia borchgrevinki, increase
reproduction(Ishimatsuetal.,2008;Pörtneretal.,2004;Esbaugh aerobicmetabolismwhenacclimatedtoelevatedP ,probablyto
CO2
etal.,2012;HeuerandGrosell,2014).Adultfishesaresuggestedto meetincreasedenergeticdemands(Enzoretal.,2013).Weassessed
be more tolerant to OA because of their well-developed ion- juvenile cardiorespiratory physiology by examining heart rate,
regulatorymechanisms(Melzneretal.,2009;althoughsomestudies stroke volume, cardiac output and ventilation rate in response to
have shown otherwise: e.g. Devine et al., 2012), with early life- elevatedP toprovideinsightintooxygensupplyandcirculation
CO2
history considered to be especially sensitive to environmental mechanisms.Changesinaerobicmetabolism,measuredasoxygen
change(Ishimatsuetal.,2008;Melzneretal.,2009;Pankhurstand consumptionrateandcitratesynthaseactivityinthecurrentstudy,
Munday,2011). provideinsightintothecapacityoffishestosupplyenoughenergy
Severalstudieshaveshownthattheearlydevelopmentalstagesof to support any increased physiological costs associated with OA,
fishesareindeedvulnerabletoelevatedP includingchangesin such as an increase in basic cellular maintenance mechanisms.
CO2
metabolism and swimming duration (Pimentel et al., 2014a), Investigating how juveniles of an abundant Antarctic fish species
swimming speed (Bignami et al., 2013), sensory perception and areimpacted byOA,andwhether younger stages of fishhavethe
behavior (see reviews by Munday et al., 2012; Clements and physiological strategies to compensate for increased levels of
Hunt,2015), morphologicaldeformations(Chambersetal.,2014; P byadjustmentsofcardiorespiratorymechanisms,willprovide
CO2
Pimentel et al., 2014b; Mu et al., 2015), and survival and growth much needed information on stage-specific vulnerability of polar
(Kikkawaetal.,2003;Baumannetal.,2011;Frommeletal.,2011, fishestoOA.
2014).Otherstudieshaveshownthattheearlystagesoffishesare
relatively robust, remaining unaffected by elevated P (Hurst MATERIALSANDMETHODS
CO2
etal.,2012;Frommeletal.,2013),suggestingthatsensitivitytoOA ExperimentalP systemandseawaterchemistry
CO2
isbothspeciesandlife-stagespecific.Evenfortolerantfishspecies, Three experimental P treatments were selected based on the
itremainsuncertainwhethertheadditionalenergeticcostsofcoping climate change scenaCriOo2s predicted for 2100, where 420μatm
withelevationsinP willhavenegativefitnesscosts,andhence, representsambient seawater(Matson etal., 2011;Hofmannet al.,
longer-termstudiesoCfO2theconsequencesofOAmustbeconducted. 2011), 650μatm P represents a moderate scenario, and
In the current study, we examined the effect of elevated 1050μatm P repreCsOe2nts a high, worst case scenario prediction
CO2
P (OA) on the juvenile emerald rockcod Trematomus (Meehl et al., 2007). These three P treatments were simulated
CO2 CO2
bernacchii Boulenger 1902, one of the most abundant and maintained using a gas-delivery CO system as described in
2 y
notothenioid fishes in the Ross Sea, Antarctica (Vacchi et al., Fangueetal.(2010)withmodificationsasdescribedinFlynnetal. g
o
2000). Several studies have characterized the physiological (2015). Briefly, CO2 and air (moisture and CO2 removed) were ol
responseofadultT.bernacchiitovariousenvironmentalchanges, mixed using mass-flow controller valves (Sierra Instruments, i
B
includingtemperature(Todghametal.,2007;BuckleyandSomero, Monterey, CA, USA). Mixed gas was then delivered to a venturi
l
a
2009: Jayasundara et al., 2013; Sandersfeld et al., 2015), hypoxia injectortobevigorouslybubbledwithseawatercontinuouslydrawn
t
(Davisonetal.,1994)andtoxicants(Regolietal.,2005;Borghesi fromMcMurdoSound,creatinga19lequilibratedreservoirbucket n
e
etal.,2008),withrecentworkontheeffectsofelevatedP (Enzor for each P treatment. Nine 19 l culture buckets, with three m
CO2 CO2
etal.,2013;EnzorandPlace,2014).Thereremainsnoinformation replicates per PCO2 treatment, were continuously dripped with eri
on physiological performance of earlier life-history stages in this reservoirtreatmentwater(andwaterinthereservoirsreplaced)ata
p
species. The emerald rockcod is a benthic notothen with a broad rateof16lh−1.Inaddition,appropriatemixedP gaswasdirectly x
depthrangeofroughly50–400m(Eastman,1993),withlife-history bubbledintoeachculturebucketat1.02lmin−C1.O2 E
f
stages inhabiting different mean depths (North, 1991). The Seawater chemistry was closely monitored throughout the o
juveniles in McMurdo Sound have been observed to settle into experiment. Total alkalinity was measured every 4 days in the al
n
shallower anchor ice and crevasses to avoid predation (<20m). gas-mixing reservoirs for the duration of the 28day experiment
r
Since juveniles and adults vary in the habitat use and have using open-cell titration (T50 titrator, Mettler Toledo, Columbus, u
o
different energetic demands (e.g. development and growth versus OH,USA)withacertifiedreferencematerial(CRM)standardand J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
0.1moll−1 HCl in seawater titrant (Dickson Laboratory, Scripps physiological changes and longer-term acclimatory changes.
Institute,LaJolla,CA,USA)(SOP3b,Dicksonetal.,2007).Total Owing to limited previous work on the identification of juvenile
pH was measured spectrophotometrically (Shimadzu) for each T. bernacchii and the presence of juveniles with slightly different
reservoir and culture bucket every other day using m-cresol dye coloration patterns in the experiment, DNA barcoding was
(SOP6b,Dicksonetal.,2007).Temperatureofthecirculatingtank performed to confirm the juveniles in this study were
and culture buckets was recorded daily using both a handheld T. bernacchii (following Ivanova et al., 2007). The research
thermocouple (HH81A, Omega) and submerged HOBO data project was conducted in accordance with US federal animal
loggers recording tank temperature every 30min (Onset, Bourne, welfare laws by approval by the University of California Davis
MA, USA). Salinity was measured for each seawater sample Institutional Animal Care and Use Committee (protocol no.
used to measure total alkalinity (YSI 3100 conductivity meter, 18248).
Yellow Springs, OH, USA). Seawater P was calculated using
CO2
the seawater carbonate assessment package SeaCarb (Lavigne Cardiorespiratoryphysiology
etal.,2011)inR(v3.0.3,RDevelopmentCoreTeam),withinputs Video recordings of fish in their respective P treatments were
CO2
of total alkalinity, pH, temperature and salinity. Experimental usedtodetermineventilationrate(f ),heartrate(f ),strokevolume
V H
P treatmentvaluesarepresentedinTable1. (V )andcardiacoutput(CO).Foreachexperimentaltrial,threefish,
CO2 S
onefromeachP treatment,wereplacedinseparate75mlflasks.
Fishcollectionandmaintenance In an environmeCnOt2al room held at −1°C, each flask was secured
Once the experimental P treatments were stabilized, juvenile horizontallyabovethevideocameraandrecordedfromtheirventral
CO2
emerald rockcod Trematomus bernacchii (standard length=38.9± side for 15min. From the ventral side of the fish, the heart could
0.1mm, mass=541±8mg, mean±s.e.m.) were collected from 1 to easilybevisualizedthroughthetranslucentbodywall.Thisprocess
10m water depths by SCUBA in late October to early November was repeated until three fish from each triplicate bucket had been
2013 from Cape Evans Ice Wall, McMurdo Sound, Ross Sea, recorded (N=9 per P treatment, n=27 per time point). At the
Antarctica(77°38′23.8″S,166°31′09.7″E).Fishweretransferredto 4 week sampling timeC,Oa2dditional video recordings were made on
an aerated, insulated cooler (−1.8°C) and transported to the A.P. remaining fish, which were included in the cardiorespiratory
CraryScienceandEngineeringCenteratMcMurdoStationwithin measurements (28days: ambient N=10, moderate N=14, high
3h of collection (−1.2°C upon arrival). Fish were then counted, N=9). Following video recordings, fish were quickly killed in a
visually assessed for injury and placed into a flow-through lethal dose of 0.3% tricaine methanesulfonate (MS-222, Argent
circulating seawater tank at −1.0±0.2°C, and held at these ChemicalLaboratories,Redmond,WA,USA)forlengthandweight
conditions until the start of the experiment (5–9days depending measurements and DNA barcoding. Video recordings were
oncollectiondate).Fishwereassumedtoberoughlyintheirsecond analyzed using VLC media player (VideoLan, v2.0.9). Several
year of age based on a combination of previous findings that preliminarytrials wereconducted to determine: (1) ifvial volume
Antarctic fish larvae metamorphose into juveniles around 30mm andtheamountoftimeforrecordingalteredphysiologicalmetrics
from6to12monthsofage(North,1991),andweobservedasingle includingf andf ,and(2)iffishshouldbeanesthetizedduringthe
V H
ringfromthecentralnucleusoftheotolith(LaMesaetal.,1996). trial(datanotshown).Bothf andf remainedsimilar(stableand
V H
The sex of fish at this juvenile life-history stage could not be low)whenmeasuredevery5–10minovera1hperiod.Bothf and
V
determined.Thirty-ninefishwererandomlyselectedandplacedin f weresimilar(stableandlow)inthesmallerflaskscomparedwith
H
eachtriplicatebucket,N=117foreachP treatment.Fishineach alargerflasksize(75mlvs250ml)acrosstimeandsmallerflasks
P treatmentbucketwerefedfrozenbrCinOe2 shrimponcedaily(∼5 were used for all subsequent analyses. A low dose of MS-222
brCiOne2 shrimpμl−1;SanFranciscostrain,Artemiafranciscana,Brine anesthetic as in Mirkovic and Rombough (1998) continued to
ShrimpDirect,Oghen,UT,USA)asdescribedforsimilarsizedfish decreasef andf overtime,andthereforewasnotused.
V H
in Chambers et al. (2014). Every day, each culture bucket was
siphonedtoremovefecesandmaintainwaterquality.Nitrogenous Ventilation
wastelevelsinculturebucketsweremonitoredbycheckingnitrate, Ventilationrate(f )wasmeasuredasaproxyforminutevolume,a
V
nitrite and ammonia levels (Aquarium Pharmaceuticals, Mars, parameter that can provide insight into ventilatory acid-base
McLean,VA,USA)dailyduringthefirstweekoftheexperiment, compensation. f was determined by counting the number of
V
followed by monitoring ammonia levels in culture buckets every opercular movements, or breaths, per minute (breaths min−1) in
y
2daysthereafter.FishwereheldinP treatmentconditionsupto three1minvideosequences.Valueswerethenaveragedtoproduce g
CO2 o
4weeks and sub-sampled immediately before the start of the asinglefVvalueperfish.Ifa1minsectioncouldnotbeobtained ol
experiment (Pre-exp) and following 1, 7, 14 and 28days under becauseofsubtlefishmovement,a30ssequencewasanalyzedand i
B
differentP conditionstoprovideinsightintopossibleshort-term multipliedbytwoforthebreathsmin−1value.
CO2 al
t
n
Table1.Experimentalseawaterchemistryforambient,moderateandhighPCO2treatments me
Parameters Incomingseawater Ambient Moderate High
i
r
Temperature(°C) −0.99±0.21 −0.93±0.04 −0.92±0.03 −0.91±0.02 e
p
Salinity(ppt) 33.7±0.3 33.7±0.3 33.7±0.3 33.7±0.3 x
pH(totalscale) 7.962±0.004 8.010±0.003 7.844±0.007 7.641±0.006 E
P (μatm) 485±6 430±3 649±11 1065±14 f
AClkOa2linity(μmolkg−1) 2351.4±2.5 2351.1±2.8 2352.1±1.9 2352.3±2.7 o
DIC(μmolkg−1) 2265.8±3.5 2249.2±1.1 2303.0±2.0 2363.0±1.9 al
Ωaragonite(calc.) 1.16±0.01 1.28±0.01 0.91±0.01 0.58±0.01 rn
u
Temperature,salinity,pHandalkalinityweredirectlymeasured,whereasPCO2,DICandaragonitewerecalculatedusingSeaCarb(Lavigneetal.,2011).Values o
arepresentedasmean±s.d. J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
̇
Cardiacperformance were constrained to using closed-chamber M measurements.
Heart rate (f ; beats min−1) was analyzed using the same three Preliminary trials were conducted showing noO2change in fish
H ̇
video sequences as in f . Heartbeats were counted foreach 1min M over2h(seeFig.S1);therefore,1htrialswereruntomaximize
V O2
video sequence and a mean was calculated from three video thenumberoffishthatcouldbeincludedateachsamplingtime.
sequences. Stroke volume (V ) was determined by slowing the Because fish activity could not be monitored during
S
videosequencesby50%andcapturingfivestillframepairsofthe measurements and fish could not be acclimated for long periods
heart at its maximal and minimal dimension, representing end- withintherespirometrychambers,quantifyingmetabolicratesthat
diastolic volume (EDV) and end-systolic volume (ESV), best approximated routine values required a unique quantile-
respectively. EDV and ESV images were converted to a tri-color analysis approach as described in Chabot et al. (2016). The first
̇
scheme to enhance contrast between tissue and blood in the heart 20minofdatashowedelevatedM comparedwiththelast40min,
O2
andanalyzedinImageJ(1.47v,Java1.6.0_65).FollowingMirkovic which is probably the result of handling stress and was therefore
andRombough(1998),theheartwasmodeledasaprolatespheroid excluded(seeFig.S1).Theremaining40minofdatawasdivided
such that V, the estimated heart volume (i.e. EDV or ESV) is into20non-overlapping2minregions,overwhichindividuallinear
calculatedusing: regressions were fit. The result was 20 individual estimates of
metabolicrateperfish.Withinthisdistributionofmetabolicrates,
4
V ¼ pab2; ð1Þ somearelikelytoincludeperiodswhenthefishwasactive(thosein
3 the right tail of the distribution) and some when the fish was
whereaisone-halfthelengthofthemajoraxis(width)ofheartand sedentary (those in the left tail). Recognizing that there is a
bisone-halftheminoraxis(length)oftheheart.Majorandminor distribution of metabolic rates for each individual fish and that
axes were determined following Mirkovic and Rombough (1998) activitywasnotmonitored,quantiles(arangeofP-valuesreferredto
and Jacob et al. (2002) with slight modifications. The outline of as q , q , q , q in this paper) were calculated (see Chabot
0.1 0.2 0.5 0.8
bloodflowwithintheheartwastraced,fittedwithanellipse,andthe etal.,2016).Withthelefttailofthedistributioncharacterizingthe
major and minor axes lengths were determined using photo lowest metabolic rates, q of the distribution (the 20th quantile)
0.2
calibrated to a micrometer standard (mm) taken during the real- wasusedastheestimateofeachindividual’sroutinemetabolicrate.
timevideorecordings.Eachofthefiveimagepairswastracedfive Thismetricwaschosenbecauseitissensitivetothedistributionof
times to reduce error and averaged. V was calculated as the metabolicratesandservesasareasonablecompromisebetweentoo
S
difference between the EDV and ESV, corrected for mass of the few data points in the tail (such as with q or q , potentially
0.05 0.1
fish, and presented as mass-specific V (μl g−1 beat−1). Cardiac assuming no spontaneous activity and/or stress) and too many
S
output (CO) was calculated as ƒ ×V and presented as mass- pointsinthetail(suchasq ,potentiallyincorporatingmetabolic
H S 0.8
specificCO(μlg−1min−1).SamplesizesforV andCOforsome ratesduringwhichthefishwasactive).Comparativequantilevalues
S
groupswerereduced(N=4–10)asaresultofthepositionoffishin have been used previously, such as q in halibut and shrimp
0.15
thevials.Whenfishwerenotrestingflatagainstthebottomofthe (Dupont-Prinet et al., 2013a,b) and q in snakes (Dorcas et al.,
vial, images of the heart were skewed, which made it difficult to 2004).CalculatedṀ ispresentedas0μ.m25olO h−1g−1wetweight.
O2 2
analyzeheartvolume.
Citratesynthaseenzymeassay
Metabolicrate At1,7,14and28days,threefishfromeachP triplicatebucket
̇ CO2
Routine mass-specific metabolic rate (M ) was indirectly (N=9 per P treatment, per sampling time) were removed and
O2 CO2
determined by measuring oxygen consumption of fish in their killed in 0.3% MS-222. Muscle tissue and the tail caudal section
respective P treatment (−1.35±0.05°C) using closed-chamber were sampled, snap frozen in liquid nitrogen, and shipped to
CO2
respirometry. The rate of oxygen consumption was measured by UniversityofCalifornia,Davis,forcitratesynthaseenzymeactivity
recordingdecreasesinoxygensaturationusingafiberopticoxygen assays.T.bernacchiimusclecontainsamixtureofmusclefibersbut
meter and oxygen sensor spots (Witrox 4, Loligo Systems, is predominantly white muscle with smaller oxidative (red) and
Denmark; accuracy of 0.4% at 20.9% O ) fixed within 150ml intermediatefibers(pink)intermixedwithintheupperandmiddle
2
chambers.At7,14and28days,fishfromeachP treatmentwere trunkandtailsectionsofmuscletissue(DavisonandMacDonald,
CO2
measuredsimultaneously,attheiracclimationP ,untilthreefish 1985). Muscle fibers could not be identified or separated in the
CO2
fromeachtriplicatebucketweremeasured(N=9perP treatment; juvenilefishandhenceasinglecross-sectionofmuscletissuefrom
CO2 y
N=27 per time point). Oxygen consumption was recorded the lower trunk areawas made for each fish for measuring citrate g
o
continuously (every 1s) for 1h and the chambers were manually synthaseenzymeactivity. l
o
mixedevery5minwithapaddleaffixedtothetopofthechamber. Citrate synthase (CS), a key enzyme in the tricarboxylic acid i
B
AcontrolchamberwasrunforeachP treatmenttoaccountfor cyclethatprovidesameasureofaerobicpotential,wasmeasuredin
CO2 ̇ al
anybackgroundbiologicalactivityintheseawater.RoutineM was whole muscle tissue as in Flynn et al. (2015) modified from
also measured for 11 fish immediately before the start oOf2 the Jayasundara et al. (2013). CS activity was monitored in a BioTek nt
̇ e
experiment(pre-exp).FollowingM trials,fishwerequicklykilled Synergy HT spectrophotometer at 25°C and measured as the m
O2
with a lethal-dose of 0.3% MS-222, measured for length and maximumrateofincreaseinabsorbanceat412nm,causedbythe ri
e
weight,andthecaudalsectionwassampledforDNAbarcodingto production of acoenzyme A-SH (sulfhydryl group) monitored by
p
confirmfishspecies.Werecognizethatourmethodsformeasuring DNTB. CS enzyme activity was calculated by subtracting the x
E
oxygen consumption do not follow standard procedures, as the backgroundactivity(negativecontrol)fromtheCSenzymeactivity
f
consensus in the field is to allow 24–48h of rest for fish in the (positivereaction)foreachsampleandquantifiedusingthemolar o
Ṁ chamberstoeliminatecaptureandhandlingstress,whichhas extinction coefficient of DTNB (14.15mlμmol−1cm−1). Protein al
O2 ̇ n
been shown to elevate M (Holeton, 1974; Clark et al., 2013). concentrations of each tissue homogenate were determined using
Unfortunately, due to Olo2gistical limitations and challenges the bicinchoninic acid method (Smith et al., 1985) with bovine ur
o
associated with the extreme cold at an isolated field station, we serumalbuminastheproteinstandard(ThermoFisherScientific). J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
CS enzyme activity was then calculated per mg of protein and and acclimation time (F =5.657, P<0.01); however, there was
2,78
expressedasμmolmin−1mgprotein−1. no interaction between P treatment and acclimation time
CO2
(F =2.337, P=0.062). Overall f was greater in the high
4,78 V
Statisticalanalyses P compared with the ambient P treatment (Tukey
CO2 CO2
StatisticalanalyseswereconductedinR(v3.0.3,RDevelopment HSD, P=0.0019) by 4 opercula beats (i.e. breaths). Ventilation
CoreTeam).Allindividualfishwerenestedwithintheirreplicate rate in the ambient (34±0.7breathsmin−1) and moderate (36±
culturebuckettocheckforaneffectof‘bucket’.Eachmodeltest 0.5breathsmin−1) P treatments did not differ significantly
showed no effect of culture bucket, and hence ‘bucket’ was not (P=0.095) nor didCOf2 between moderate and high (38±
V
keptinthestatisticalanalyses.Datawereanalyzed fornormality 0.6breathsmin−1) P treatments (P=0.266). Furthermore, f of
CO2 V
andhomogeneityofvariancesofresidualsfortheassumptionsof all fish at 28days of acclimation increased significantly (38±
an analysis of variance (ANOVA). Both visual inspections and 0.4breathsmin−1) in comparison to fish acclimated to
Shapiro–Wilks for normality tests and Levene’s test for P treatments for 14days (35±0.7breathsmin−1, P=0.017) and
variances were conducted, data were transformed (square-root 7CdOa2ys (35±0.6breathsmin−1, P=0.012). Ventilation rates were
transformation) if the assumptions were not met, and atwo-way similarat7and14days(P=0.993).
ANOVA was run with significant F-values followed up with a
Tukey HSD test. The alpha value was set at 0.05. Ventilation Cardiacperformance
rate, heart rate, stroke volume, cardiac output, metabolic rate Fishheartrates(f )werenotaffectedbyP treatment (Fig.2A;
H CO2
(transformed) and citrate synthase (transformed) were the two-way ANOVA, F =0.884, P=0.417) or acclimation time
2,78
dependent parameters used in the statistical tests, whereas (F =1.812, P=0.170). In addition, there was no interaction
2,78
P treatment and acclimation time were independent between P treatment and acclimation time (F =0.898,
vaCrOia2bles. Measurements taken immediately before the start of P=0.470)onCOf2.Onaverage,f was35±2beatsmin−1(m4,e7a8n±s.d.).
H H
the experiment are presented visually in figures as a pre- Acclimationtimehadasignificanteffectonstrokevolume(V )
S
experimental(Pre-exp)value,butthesedatawerenotincludedin (Fig.2B;two-wayANOVA,F =14.69,P<0.0001),withnoeffect
2,51
statisticalanalyses.Resultsarepresentedasmeans±s.e.m.unless of P treatment (F =1.063, P=0.353) and no interaction
CO2 2,51
noted otherwise. between P treatment and time (F =0.292, P=0.882). V
significantlyCOi2ncreased from 11.6±0.7μ4l,51g−1 beat−1 at 7days toS
RESULTS 15.4±0.5μl g−1 beat−1 after 14days (Tukey HSD, P<0.001) and
Ventilation 16.1±0.6μlg−1beat−1after28days(P<0.0001),butstrokevolume
Fish ventilation rate (f ) was significantly affected by wassimilarat14and28days(P=0.689).
V
P treatment (Fig. 1; two-way ANOVA, F =6.311, P<0.01) Similar to stroke volume, mass-specific cardiac output (CO)
CO2 2,78
significantly increased over acclimation time (Fig. 2C; two-way
ANOVA, F =15.768, P<0.0001); however, fish experienced no
2,51
45 effect of P treatment (F =0.845, P=0.435) or an interaction
CO2 2,51
betweenP treatmentandtime(F =0.158,P=0.959).Cardiac
outputsignCiOfi2cantlyincreasedfrom440,551±25μlg−1min−1at7days
to549±22μlg−1min−1after14days(TukeyHSD,P<0.001).After
40 28days of acclimation, CO was 582±21μlg−1min−1, also
significantlygreaterthanat7days(P<0.0001).
1)
–
n
mi Metabolicrate
hs 35 Mass-specific metabolic rate (Ṁ ) measured by oxygen
at O2
e consumption of juvenile fish showed no effect of P treatment
br b CO2
(V (Fig. 3; two-way ANOVA, F2,72=2.462, P=0.092) or acclimation
f a,b time(F =2.503,P=0.089).Therewasalsonointeractionbetween
2,72
30
a PĊ O2 treatment and time (F4,72=0.567, P=0.687). The average
M forjuvenileT.bernacchiifromalltreatmentsandtimepoints
waOs22.6±0.1μmolO h−1g−1. gy
Time (days) 2 o
l
25 0 7 14 28 o
Citratesynthaseenzymeactivity i
A A B B
Cellularaerobic potential, measured bycitratesynthaseactivity in
l
Pre-exp Ambient PCO2 Moderate PCO2 High PCO2 muscle tissue, showed a significant effect of acclimation time ta
(Fig.4;two-wayANOVA,F =5.616,P=0.001),butnoeffectof n
Fig.1.Ventilationrate(f )inbreathsperminuteofTrematomus 3,96 e
V P treatment (F =0.876, P=0.419) or an interaction between m
breeprrneascecnhtsiithaecrmosesdidanif,fethreenbtoPxCrOe2ptrreesaetnmtsenthtes.inTtheer-qlinuearotinlethraenbgoex(IpQloRt)and PCCOO22 and acclima2t,i9o6n time (F6,96=1.583, P=0.16). In general, eri
thewhiskersextend1.5timesIQR.Pre-experiment(pre-exp,N=9),ambient CS enzyme activity significantly increased over time from p
P (N=28),moderateP (N=32)andhighP (N=27).Differentshapes 0.68±0.06μmolmin−1mgprotein−1 at day 1 compared with x
wCitOhi2ntheboxplotaremeaCOn2s±s.e.m.foreachexCpOe2rimentaldaywithineach 1.06±0.08μmolmin−1mgprotein−1 after 28days acclimation E
f
PCO2treatment;time0(N=9),7days(N=9),14days(N=9)and28days (P<0.001), with no significant differences detected at other time o
(ambient,N=10;moderate,N=14;high,N=9).Thesamplesize(N=9)was points.AlthoughtherewerenooverallsignificanteffectsofP on al
attainedbymeasuringfVofthreefishfromeachofthethreereplicatebuckets CS activity over time, fish in moderate P did inCcrOe2ase n
perPCO2treatment.Differentlettersrepresentasignificantdifferencebetween CS activity from 0.59±0.10μmol min−1 mg proCteOi2n−1 at 1day to ur
(PACNOO2tVreAa,tmP<e0n.t0s5()l,owanedrc8a7sefi)sahnidnatoctcallimwaetrieonustiemde.(uppercase)independently 1.23±0.13μmolmin−1mgprotein−1at28days. Jo
1207
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
38 A 20.0 B 700 C
f–1 (beats min)H 3333334567 PMHArmiogedhb-ee iePxranpCttOe P 2PC COO2 2 Mass-specific stroke volumeµ–1–1 beat)(l g111102577.....05055 a b b Mass-specific cardiac outputµ–1–1 min)(l g345600000000 a b b
0 7 14 28 0 7 14 28 0 7 14 28
Time (days)
Fig.2.CardiacperformancemetricsofjuvenileTrematomusbernacchiiexposedtodifferentP treatments.Dataineachpanelarepresentedas
CO2
means±s.e.m.(A)Heartrate(f )ineachP treatmentovertime[N=9perpoint,exceptat28days(ambient,N=10;moderate,N=14)]for87fish.Theminimum
H CO2
samplesize(N=9)wasattainedfrommeasurementsofthreefishfromeachofthethreereplicatebucketsperP treatment.(B)Mass-specificstroke
CO2
volume(V ).(C)Mass-specificcardiacoutput(CO)for60fish.ForBandC,samplesizeswerereducedat7,14and28days,suchthatineachP treatment
S CO2
samplesizeswereN=5,4and6(ambient),N=6,5and10(moderate)andN=8,8and8(high),respectively.Differentlettersabovelinesindicateasignificant
differencebyacclimationtime(ANOVA,P<0.05).
DISCUSSION etal.,2015).Thismarkeddevelopmentofthejuvenilelife-history
Althoughearlystagesoffishesarepredictedtobemorevulnerableto stagemightexplainwhyheartrate(f ),strokevolume(V ),cardiac
Ḣ S
the physical drivers of GCC compared with adults (Pörtneret al., output (CO),oxygenconsumption(M )andcitratesynthase(CS)
O2
2004; Ishimatsu et al., 2004), juvenile T. bernacchii appear to be enzyme activityall remained largely unaffected byelevated P .
CO2
physiologicallyrobusttoincreasedP predictedbyGCCemission Most studies characterizing the effects of elevated P or OAon
CO2 CO2
scenarios(Meehletal.,2007).Smallchangesatthewhole-organism earlylife-historystagesoffishessuchaslarvaeandjuvenileshave
level in terms of increased ventilation rate (f ) suggest increased been conducted in temperate (Hurst et al., 2012; Hamilton et al.,
V
minute volume may contribute to acid-base compensation and is 2014)andtropicalspecies(Mundayetal.,2009;Dixsonetal.,2010;
likely to be a sufficient strategy for dealing with shifts in Nowickietal.,2012;Pimenteletal.,2014a).Thisisthefirststudy
environmental P . Thejuvenile rockcod collected for this study to characterize the impact of OA on a juvenile Antarctic fish and
CO2
wereestimatedtobeintheirsecondyearofage(atleast>1year)with to describe the physiology of juvenile rockcod, Trematomus
functionalgillsandkidneys,importantforacid-baseregulationand bernacchii, a dominant fish (by biomass) in the Ross Sea,
gas-exchangeinteleosts(seereviewbyEvansetal.,2005;Lawrence Antarctica(Vacchietal.,2000).
6 Pre-exp
Ambient PCO2
MHiogdhe PraCtOe 2P CO2 –1otein) 1.5 PMArmoedb-eeiexranptte P PCCOO2 2 b
–1g) 4 mg pr High PCO2 a,b
1 1 a,b
–O h2 –min 1.0 a
ol mol
m
µ
Mµ.(O2 2 ctivity ( ogy
a
e 0.5 ol
s
ha Bi
ynt al
s
0 0 7 14 28 Citrate ment
Time (days) 0 i
0 1 7 14 28 er
Fcoign.s3u.mMpatsiosn-spoefcjuifvicenmileeteamboelricalrdatreosck(MċoOd2)imneeaascuhrPedbytroexaytmgeennt.Pre- Time (days) xp
experimental(N=10)dataserveasareferenceattime0;CṀO2 valuesareshown Fig.4.Citratesynthaseenzymeactivityinmuscletissueofjuvenile E
forambientP (N=9,exceptat28days,whereN=8),moOd2erateP (N=9) Trematomusbernacchii.Barsindicatethemeanenzymeactivity±s.e.m.for of
CO2 CO2
andhighPCO2(N=9)treatments(N=90fishintotal).Theopencirclewithinthe fishinambientPCO2(N=9),moderatePCO2(N=9)andhighPCO2(N=9,exceptat al
boxplotsrepresentsthemeanandthelinerepresentsthemedian.Thebox 28days,whereN=8)overa28dayperiod.Pre-experimentalCSactivityis n
representstheinter-quartilerange(IQR),withthewhiskersextendingto1.5 plottedasareference(N=9)foratotalof116fishusedforcitratesynthase r
u
timestheIQR.Pointsbeyondthewhiskersareoutliersbutwereincludedinthe analyses.Statisticallettersindicateasignificantdifferenceincitratesynthase o
dataanalyses. activitybyacclimationtime(ANOVA,P<0.05). J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
̇
ElevatedP hadasignificanteffectonf inemeraldrockcod AlthoughtheM ofjuvenileswasunaffectedbyP ,elevated
juvenilessuchCOth2atfishunderhighP hadoVverallgreaterf than P ofsimilarmaOg2nitudestothecurrentstudy(∼950C–O12025μatm;
CO2 V CO2 ̇
fishin currentambientP conditions(Fig.1).Itiscommon for Enzoretal.,2013)havepreviouslybeenshowntoincreaseM in
CO2 O2
fishestoalterf inresponsetoenvironmentalchanges(Janssenand adultT.bernacchiifollowinga1weekexposure.Following2weeks
V ̇
Randall, 1975; Randall et al., 1976; Smith and Jones, 1982), and of acclimation to elevated P , the M of adult T. bernacchii
CO2 O2
fish primarily use metabolic adjustments to cope with acid-base returned to basal levels and remained stable for up to 4weeks
disturbances and changing oxygen levels (Ishimatsu et al., 2004, (Enzoretal.,2013).Othercloselyrelatedadultnotothenioidspecies
2005; Heuer and Grosell, 2014). Fish have specialized including T. hansoni and P. borchgrevinki, however, showed no
̇
neuroepithelial cells (NECs) in their gill arches, positioned to effect of elevated P on M after acclimation for 1 or 4weeks
CO2 O2
sensebothpartialpressureofgases(e.g.P andP )intheblood (Enzor et al., 2013). It is possible that juvenile T. bernacchii
CO2 O2 ̇
andintheexternalenvironment(Baillyetal.,1992;Zacconeetal., increasedtheirM intheshortterm(i.e.withindays)butwereable
O2
1994;Perryetal.,2009).IncreasesinexternalP stimulatethese to compensate more quickly than adults, such that by 1week of
CO2 ̇
gillchemoreceptors and initiateacardiorespiratory response, such exposuretoelevatedP ,M wasalreadyreducedtobasallevels.
CO2 O2
as hyperventilation (Burleson and Smatresk, 2000; Perry and Aswedidnotmeasureimmediate,short-termresponses(f ,f and
̇ H V
Abdallah,2012;HeuerandGrosell,2014)tominimizetheeffectsof M )toelevatedP ,itisnotpossibletoknowwhetherjuvenile
O2 CO2
elevated P on blood pH (Gilmour, 2001; Perry and Abdallah, rockcod make acute adjustments to elevated P . Alternatively,
CO2 CO2
2012). Numerous studies on teleost fishes have shown somestudieshaveshowntolerancedecreaseswithontogeneticstage
hyperventilation in response to hypercapnia (Sundin et al., 2000; because of increasing physiological performance capacity of
PerryandMcKendry,2001;PerryandReid,2002,alsoseereview juveniles and sub-adults at a smaller size, whereas adults may
by Gilmour, 2001); but typically these studies exposed fishes to become oxygen limited with increasing demand and insufficient
substantiallyhigherlevelsofCO (e.g.10,500–48,000μatm). supply (for review, see Pörtner and Peck, 2010). For example,
2
Commonly, hyperventilation is coupled with other temperaturetoleranceofjuvenilesastheNorthSeasole(Rijnsdorp
physiological adjustments (Perry and Abdallah, 2012); however, et al., 2009), California anchovy (Brewer, 1976) and delta smelt
adjustments in aerobic metabolism and cardiac output were not (Komoroskeetal.,2014)isgreatercomparedwiththeadultofeach
̇
observed in this study, because only f was significantly affected species.ItisofnotethattherewashighvariabilityinM ofjuvenile
V O2
by increased P . Increased ventilation can be a mechanism to T.bernacchiiunderallP treatments,whichwasnotasapparent
CO2 CO2
increase oxygen supply to offset metabolic costs or oxygen intheadultT.bernacchiiexposedtoelevatedP inEnzoretal.
CO2
demand associated with coping with environmental change (2013). Steffensen (2002) showed that over the course of asingle
̇
(Frederich and Pörtner, 2000; Mark et al., 2002). M in the day, oxygen consumption rates of adult T. bernacchii held under
current study was not increased in response toO2elevated ambienttemperaturesof−1°Cwerehighlyvariable,rangingfrom
P (Fig. 3), suggesting fish did not have an increased oxygen 17to85mgO kg−1h−1andsodifferencesmightreflectthetimeof
CO2 ̇ 2
demandunderconditionsofOA.ThemismatchbetweenM and day measurements were made. It is noteworthy that the current
O2
f couldbeexplainedbydifferencesingassolubilityanddiffusion environmental CO levels of the shallow, benthic habitats where
V 2
ofCO andO inseawater.At0°C,CO isabout∼38timesmore boththeadults(Enzoretal.,2013)andjuvenileswerecollectedare
2 2 2
soluble in seawater than O and diffuses roughly 1.2 times faster similar and relatively benign compared with experimental CO
2 2
(Denny,1993).Thedifferencesingassolubilityanddiffusionrate levels (Matson et al., 2014). Therefore, it is not likely that
between CO and O coupled with no evidence of increased differences in environmental P exposure have led to
2 2 CO2
aerobic metabolism suggests hyperventilation or an increase in differencesinsensitivitybetweenadultsandjuveniles.
ventilatory minute volume is probably a sufficient mechanism to In addition to whole-organism measurements of aerobic
off-load/excrete CO quickly, contributing to acid-base metabolism, sub-organismal aerobic potential, measured as citrate
2
compensation without increasing uptake of O . Respiratory synthase(CS)enzymeactivityinmuscletissue,wasalsounaffected
2
plasticity in response to OA (1000μatm) has been described in byelevatedP (Fig.4).Thisfindingprovidesadditionalevidence
̇ CO2
the estuarine red drum Sciaenops ocellatus: although M was that juvenile emerald rockcod are not adjusting their capacity for
O2
unaffected by acclimation to elevated P , the capacity for CO aerobicmetabolisminresponsetoelevatedP .Itisimportantto
CO2 2 CO2
excretion increased as marked by an increase in CO channel notethatCSactivityistissuespecificinseveralfishes(Michaelidis
2
proteins and a significant reduction in the branchial distance over etal.,2007;Strobeletal.,2013)andwasonlymeasuredinmuscle
y
which diffusion can occur (Esbaugh et al., 2015). The respiratory tissueinthecurrentstudy.IntheAntarcticnotothenioidNotothenia g
o
physiology of fishes does facilitate excretion of CO2 from the rossii from the western Antarctic peninsula, CS activity was ol
countercurrent exchange from water to blood and unidirectional unaffected by PCO2 (0.2kPa or ∼1975μatm) in cardiac, liver and Bi
waterflowacrossthegillstomaintainstablebloodpH(Heuerand white muscle tissue, whereas CS activity in red muscle increased
l
a
Grosell,2014).However,morecommonmechanismsforacid-base (Strobeletal.,2013).IntheMediterraneanseabreamSparusaurata
t
balance in fish include the secretion of H+ and the retention and acclimationtoincreasedP significantlydecreasedCSactivityin n
absorptionofHCO−throughdifferentionexchangesandde novo red and white muscle butCiOn2creased in cardiac tissue (Michaelidis me
3
synthesis of HCO3− by the kidney (Wright, 1995; Wood et al., etal.,2007).Concurrently,althoughaerobicpotentialdecreasedin ri
e
1999; Lawrence et al., 2015), facilitated by carbonic anhydrase the sea bream, activity of the anaerobic enzymes pyruvate kinase
p
(GilmourandPerry,2009;Perryetal.,2010;Esbaughetal.,2012; and lactate dehydrogenase increased, demonstrating a switch in x
E
Tseng et al., 2013). Future studies could quantify plasma HCO− metabolicpathwaysinresponsetoelevatedP (Michaelidisetal.,
and P and analyze the activity or mRNA expression of thes3e 2007). Although anaerobic metabolites wereCOn2ot measured in this of
enzymCeOs2and transporters to provide additional insight into any study, aerobic metrics of Ṁ , f , and CS in response to elevated al
O2 H n
acid-base imbalances that might have occurred in response to P provideindirectevidencetosuggestthatthesejuvenileswere
elevated P (Melzner et al., 2009; Perry et al., 2010; Esbaugh noCtOr2elyingheavilyonanaerobicpathwaystofuelenergeticdemands ur
CO2 o
et al., 2012; Lawrence et al., 2015). ofexposuretoelevatedP .Antarcticfisheshavebeenshownto J
CO2
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
express20–50%moremitochondrialproteinsthantemperatefishes whilefeeding,suchastailbitingorchasingandhence,suggestive
(Johnston et al., 1998). High mitochondrial densities might allow that juvenile emerald rockcod in low densities might experience
for sustained aerobic capacity in response to changing comparablealterationsinbehaviorasdemonstratedbyzebrafishand
environmental parameters such as P . Furthermore, O is not Arcticchar.Ifgreaterstressandenergydemandsincreasedfrom2to
CO2 2
limitingintheAntarcticanditisthereforeunlikelythatthesefishes 4weeks, changes in f (O extraction) and CO marked by stroke
V 2
needtorelyonanaerobicpathways. volume (O circulation) might represent mechanisms to maintain
2
Pörtnerand colleagues (2004) have suggested that hypercapnic organismalandsub-organismaloxygendemands.Asthisisthefirst
limitations might be defined by cardiac and circulatory system laboratorystudyofjuvenileT.bernacchii,moreresearchisneeded
failure at extremely high levels of CO , rather than ventilation to understand how laboratory factors affect fish behavior and
2
capacity being the limiting mechanism (Ishimatsu et al., 2004); physiology.
however, we found no evidence of adjustments in heart rate (f , ThisstudydemonstratesthatjuvenileT.bernacchiiappeartobe
H
Fig. 2A), stroke volume (V , Fig. 2B) or cardiac output (CO, robust to elevated P levels that are predicted to occur over the
S CO2
Fig. 2C) in juvenile emerald rockcod in response to predicted next century. However, as the current experiment only lasted
P levels(650and1050μatm).Previousstudieshaveshownthat 4weeks,researchwithmorechronicexposurestoelevatedP are
fisChOe2sadjustf ,V andCOinresponsetoelevatedP ,withthe needed to thoroughly assess the vulnerability of these fishCeOs2– a
H S CO2
responsevaryingacrossspecies(Perryetal.,1999;Crockeretal., difficult undertaking with field seasons limited to the austral
2000;McKendryetal.,2001;Leeetal.,2003).Mostofthesestudies summersinMcMurdoSound,Antarctica.Resultsfromthecurrent
characterizedresponsesafteranacuteexposuretoelevatedP of studysuggestthatthephysiologicalresponsesofjuvenileemerald
much higher magnitudes (e.g. 8000–26,000μatm), whereaCsO2we rockcoddodifferfromtheadultT.bernacchiisuchthatjuvenilefish
assessed cardiac performance over 1–4 weeks of acclimation and appeartohaveagreatercapacitytobufferelevatedP asseenby
CO2
foundnoeffectsofelevatedP predictedforOAscenarios.While relativelystablecardiovascularphysiologyandaerobicmetabolism.
CO2
itismorecommonfortemperatefishestoregulateCObymodifying InadditiontoelevatedP ,temperatureispredictedtoincreaseby
V , rather than f ,Antarctic fishes havebeen foundto increasef 2–4°C. While several CstOu2dies have demonstrated that adult T.
S H H
withincreasedoxygenuptake,notV (Farrell,1991;Axelssonetal., bernacchiihavethecapacityforthermalacclimationandtheability
S
1992). Egginton et al. (2006) have suggested that f is a good toincreasethermaltolerancelimits(BilykandDeVries,2011;Bilyk
̇ H
predictor of M in adult T. bernacchii and P. borchgrevinki, et al., 2012), few studies have investigated how both elevated
O2
because both exhibit a linear relationship between f and P and temperature will impact Antarctic fishes (although see
̇ H CO2
M (Egginton et al., 2006; Campbell et al., 2009). In the current Enzoretal.,2013;EnzorandPlace,2014;Strobeletal.,2012,2013)
O2 ̇
study, there was no change in either f or M in juvenile and with only a single study on early developmental stages of
H O2
T.bernacchiiinresponsetoelevatedP . Antarctic dragonfish (Flynn et al., 2015). Stressors can interact in
CO2
Of interesting notein this study wasthatjuvenile T. bernacchii non-linearways,makingitdifficulttopredicthowfishwillrespond
showedasignificantresponsetoacclimationtime,independentof tomultiplestressorsfromsinglestressorexperiments(Todghamand
P treatment. As time held under laboratory conditions (i.e. Stillman, 2013). More research is warranted to understand the
CO2
captivity)increased,aerobicperformanceparametersincludingf , potential combined and synergistic effects of elevated P and
V CO2
V ,COandCSactivityalsoincreased(Fig.1,Fig.2B,CandFig.4). temperatureonphysiologicalperformanceandbehaviorofjuvenile
S
Acclimationtimeeffectsmaybeconfoundedbysubtlechangesin fishes.
developmentoverthe4weekexperimentalperiod;however,growth
appeared to be minimal. On average, fish length increased ∼1% Acknowledgements
whereaswetmassincreased∼3%overthe4weekperiod(datanot WewouldliketothanktheUnitedStatesAntarcticProgramandLockheedMartin
shown). Although it is challenging to unravel the specific cause formakingthisprojectpossible,includinglogisticalandfieldsupportwhileat
McMurdoStation.Particularly,weappreciatetheimmenseeffortsprovidedbythe
underlyingacclimationtimeeffects,wildfishduringdevelopment
ASCSCUBADivers,RobRobbinsandSteveRupp,forallcollectionsofjuvenile
may undergo confinement stress with an increase in the cortisol fishandtheCraryLabstaffformaintainingtheaquariumroomandsupportingthe
stress hormone, a well-documented occurrence in teleost fishes laboratoryscience.Inaddition,wethankDrAmandaKelleyforheradviceand
(Barton and Iwama, 1991; Bonga, 1997). Furthermore, previous assistanceduringthe2013AntarcticfieldseasonandDrNannFangueforher
expertiseandadvicewiththeCO system,discussionofresultsandreviewofthe
studies have shown that laboratory conditions, such as tank color 2
manuscript.
and densities of fish held together, can affect behavior and
y
physiology (Brown et al., 1992; Pavlidis et al., 2013; Hasenbein g
Competinginterests o
etal.,2016).Forexample,whenheldinlowerdensities,Arcticchar Theauthorsdeclarenocompetingorfinancialinterests. ol
(Salvalinusalpinus)exhibitedalteredswimmingbehaviorandmore i
B
aggressive interactions (Brown et al., 1992). Whereas crowding Authorcontributions l
a
increased stress in zebrafish, lower densities altered the social B.E.D.,N.A.M.andA.E.T.designedthestudy.B.E.D.,E.E.F.andN.A.M.maintained t
environment as hierarchies were formed (Pavlidis et al., 2013). theCO2systemandmeasuredseawaterchemistry.B.E.D.,N.A.M.andA.E.T. en
carriedoutthephysiologicalmeasurementsoffishduringexperimentationat
More negative interactions between fish are suggested to increase m
McMurdostation.B.E.D.carriedoutallanalysesatUCDavisanddraftedthe
energetic demands (Brown et al., 1992). Stress markers were also manuscript.B.E.D.andA.E.T.revisedthemanuscriptcriticallywitheditsfromE.E.F. ri
e
significantly greater in late larval delta smelt (Hypomesus andN.A.M. p
transpacificus) held at lower densities of 7 and 14 individuals x
E
compared with higher densities of 28 and 49 (Hasenbein et al., Funding
f
2016). Here, 39 fish were initially placed in each P triplicate ThisprojectwasfundedbytheNationalScienceFoundation[NSFANT-1142122to o
bucket, after which nine fish were sequentially rCeOm2oved for A.E.T.]. al
n
sampling metrics. After 1 and 2weeks of sampling therewere 24
and15fishremainingineachbucket,respectively,fortheduration Supplementaryinformation ur
Supplementaryinformationavailableonlineat o
of the 4 week period. We did observe some dominance behavior http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.133173/-/DC1 J
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RESEARCHARTICLE JournalofExperimentalBiology(2016)219,1203-1213doi:10.1242/jeb.133173
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Axelsson, M., Davison, W., Forster, M. E. and Farrell, A. P. (1992). Cardiorespiratory responses of white sturgeon to environmental hypercapnia.
Cardiovascular responses of the red-blooded Antarctic fishes Pagothenia Am.J.Physiol.279,617-628.
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