Cardiac plasticity influences aerobic performance and thermal tolerance in a tropical, freshwater

66 Pages·2017·2.43 MB·English

by  Elizabeth Nyboer

©2018.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
RESEARCHARTICLE
Cardiac plasticity influences aerobic performance and thermal
tolerance in a tropical, freshwater fish at elevated temperatures
ElizabethA.Nyboer*andLaurenJ.Chapman
ABSTRACT optimatheyfaceperformancelimitationsatalllevelsofbiological
organization. To cope, fish must either relocate to more suitable
Fishesfacedwithnovelthermalconditionsoftenmodifyphysiological
habitatsoradapttonovelconditionsthroughgeneticchangeand/or
functioning to compensate for elevated temperatures. This
environmentallyinducedphenotypicplasticity(Schulteetal.,2011;
physiological plasticity (thermal acclimation) has been shown to
Seebacher et al., 2014). Fish species often modify physiological
improve metabolic performance and extend thermal limits in many
functioning through thermal acclimation (Angilletta, 2009;
species.Adjustmentsincardiorespiratoryfunctionareofteninvoked
Seebacher et al., 2014), a type of phenotypic plasticity that can
asmechanisms underlyingthermal plasticity becauselimitations in
alterthermaltolerancelimitsandoptimizeperformanceundernovel
oxygen supply have been predicted to define thermal optima in
temperature regimes (Schulte et al., 2011). Capacity for thermal
fishes;however, few studieshave explicitly linkedcardiorespiratory
acclimationvariesdramaticallyamongspecies,andishypothesized
plasticity to metabolic compensation. Here, we quantified thermal
tobelowerintropicalfishthatexperiencelittleseasonalvariationin
acclimationcapacityinthecommerciallyharvestedNileperch(Lates
their thermal environment (Tewksbury et al., 2008). Given the
niloticus) of East Africa, and investigated mechanisms underlying
significance of many inland tropical fishes to local and regional
observedchanges.WerearedjuvenileNileperchfor3monthsunder
food security (FAO, 2014; Lynch et al., 2016), it is of increasing
twotemperatureregimes,andthenmeasuredaseriesofmetabolic
importance to understand the capacity for and mechanisms
traits(e.g.aerobicscope)andcriticalthermalmaximum(CT )upon
max
underlyingmodificationsofthermaltoleranceinthesespecies.
acute exposure to a range of experimental temperatures. We also
For ectotherms, metabolic rate sets the pace for many
measuredmorphologicaltraitsofheartventricles,gillsandbrainsto
physiological functions, and can have important fitness
identify potential mechanisms for compensation. We found that
implications (Brown et al., 2004). Measurements of metabolic
long-term (3 month) exposure to elevated temperature induced
performance can therefore serve as comprehensive indicators of
compensation in upper thermal tolerance (CT ) and metabolic
max
physiological condition (Sibly et al., 2012), and can be used to
performance (standard and maximum metabolic rate, and aerobic
assessabilitytocopewithvariousenvironmentalstressors(Brown
scope),andinducedcardiacremodelinginNileperch.Furthermore,
et al., 2004). Key metabolic parameters often measured in this
variation in heart morphology influenced variations in metabolic
context include standard and maximum metabolic rate (SMR and
function and thermal tolerance. These results indicate that plastic
MMR,respectively),whichrepresentthelowerandupperlimitsof
changes enacted over longer exposures lead to differences in
oxygen uptake for fishes. SMR corresponds to basal oxygen
metabolic flexibility when organisms are acutely exposed to
demand, or the lowest possible metabolic rate in a resting, post-
temperature variation. Furthermore, we established functional links
absorptive fish (Fry, 1971; Brett and Groves, 1979), and MMR
between cardiac plasticity, metabolic performance and thermal
correspondstothemaximumrateofoxygenconsumption,usually
tolerance,providingevidencethatplasticityincardiaccapacitymay
measured after exhaustive exercise (Fry, 1971; Clark et al., 2011;
beonemechanismforcopingwithclimatechange.
Roche et al., 2013; Norin and Clark, 2016). Aerobic scope (AS),
KEYWORDS:Climatechange,Thermalplasticity,Nileperch, which is calculated as the difference between SMR and MMR,
Tropicalfisheries,Heartremodeling,Acclimationcapacity determines the range of oxygen-demanding processes that can be
performed simultaneously by a fish, and is thought to be a key
INTRODUCTION mechanism determining energy allocation, fitness and
y
Climate change models forecast persistent, global temperature biogeography in ectotherms (Pörtner and Farrell, 2008; Pörtner, g
increases of 1–4°C, and increases in the frequency of extreme 2010;Clarketal.,2013). o
l
o
climatic events over the next century (IPCC, 2014; Seneviratne AsfirstobservedbyFry(1947),andquantifiedinseveralrecent i
B
et al., 2014). Such drastic changes will directly affect aquatic studies (Healy and Schulte, 2012; Gräns et al., 2014; Norin et al.,
l
ecosystems,andthreatentheecologicalandphysiologicalstability 2014), SMR increases exponentially with temperature, whereas a
t
of fish species in freshwater habitats (Ficke et al., 2007). As MMRispredictedtoincreaseinitially,andthendeclineatthehighest n
e
ectotherms, fish depend on their thermal environment to regulate temperatures, ultimately bringing about a decline in AS at high m
metabolicfunctions,andwhenwatertemperaturesexceedspecific temperatures.Accordingtotheoxygen-andcapacity-limitedthermal i
r
tolerance(OCLTT)concept,declinesinperformance(i.e.AS)athigh e
p
temperatures are predicted because the cardiorespiratory system x
DepartmentofBiology,McGillUniversity,OfficeN3/11,StewartBiologyBuilding, E
cannot keep pace with oxygen demands in respiring tissues as
1205AvenueDocteurPenfield,Montreal,Quebec,Canada,H3A1B1. f
increasing temperatures elevate metabolic demands (Pörtner and o
*Authorforcorrespondence([email protected]) Knust, 2007; Pörtner et al., 2017). Mismatches between oxygen al
supplyanddemandarethereforethoughttodefinethermallimitsin n
E.A.N.,0000-0003-3004-009X r
teleost fishes, and may have negative fitness consequences (e.g. u
o
Received30January2018;Accepted4June2018 reductionsinbodymassorcondition)if,aspredicted,dropsinASare J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
physiologicalplasticityinfishes(Farrelletal.,2009;Jayasundaraand
Listofsymbolsandabbreviations Somero,2013).Changesintemperaturehaveasignificanteffecton
AGFL averagegillfilamentlength heart rate in teleosts, which influences total cardiac output (V ,
b
AS aerobicscope volume of blood pumped per minute). However, morphological
ASQ10 Q10-correctedaerobicscope changes induced by long-term exposure to different water
CTmax criticalthermalmaximum temperaturescanfacilitatemaintenanceofV acrossawidethermal
CT , adjustedcriticalthermalmaximum b
maxadj range(GamperlandFarrell,2004;Klaimanetal.,2011;Keenetal.,
DO dissolvedoxygen
2017).Forexample,fishesexposedtowarmertemperaturesgenerally
FBL filamentbaselength
FD filamentdensity havedecreasedrelativeventricularmass(RVM),leadingtoreduced
HSI hepatosomaticindex stroke volume (Keen et al., 2017); however, V can be maintained
b
K LeCren’sconditionfactor throughimprovedforceofcontraction(GamperlandFarrell,2004),
Mb bodymass which is often enabled by adjustments to the proportions of the
MMR maximummetabolicrate
myocardiallayers(i.e.spongyversuscompact)intheventricle.Gills,
MMR Q -correctedmaximummetabolicrate
Ṁ Q10 ox1y0genconsumptionrate in contrast, function primarily in oxygen uptake. Changes in the
OCO2LTT oxygen-andcapacity-limitedthermaltolerance environmentthatleadtonewrespiratoryorosmoregulatorydemands
RVM relativeventricularmass can result in alterations to gill structure over varying time scales
SL standardlength (Sollidetal.,2003,2005;Langerhansetal.,2007;Chapmanetal.,
SMR standardmetabolicrate 2008).Forexample, fish that are exposed to low dissolved oxygen
SMR Q -correctedstandardmetabolicrate
Q10 10 (DO) concentrations can increase gill size, which presumably
T averagerearingtemperature(∼25°C)
avg improves efficiency of oxygen uptake (Chapman et al., 2008). In
TFN totalfilamentnumber
TGFL totalgillfilamentlength addition, elevated temperature may affect other physiologically
THA totalhemibrancharea demandingorgansifthebulkofenergeticresourcesareallocatedto
TL totallength basal functions. For example, brain size has been observed to
Twarm warmrearingtemperature(∼29°C) decreaseunderstressfulconditions(CrispoandChapman,2010;Toli
V totalcardiacoutput(volumeofbloodpumpedperminute)
b et al., 2016). Given the high metabolic cost associated with the
%CM percentofcompactmyocardium
developmentoflargebrains,theabilitytoreducebrainsizethrough
plasticitywhenalargebrainisnotcriticalforfitnessislikelytobe
favored(CrispoandChapman,2010).
causallylinkedtokeyprocessessuchasgrowth(PörtnerandKnust, Alterations in cardiorespiratory function and morphology have
2007). A corollary of this hypothesis is that alterations to the been shown to improve thermal tolerance and performance in a
cardiorespiratory system that increase efficiency of oxygen uptake number of fish species (Eliason et al., 2011, 2017; Anttila et al.,
anddeliverycanimprovethermaltoleranceandaerobicperformance 2013; Keen et al., 2017). Forexample, increases in cardiac scope
in fishes exposed to elevated water temperatures. Although the correlate positively with aerobic scope within populations of
OCLTT hypothesis proposes a clear mechanistic explanation for sockeye salmon (Eliason et al., 2011), and larger RVM correlates
thermal limitation, numerous recent empirical studies have shown positivelywith upperthermal tolerancelimits (i.e.critical thermal
thatdeclinesinperformanceathightemperaturesmaybedrivenby maximum,CT )insomepopulationsofAtlanticsalmon(Anttila
max
mechanisms other than a mismatch between oxygen supply and etal.,2013).Likewise,gillscanundergosurfaceareaincreasesthat
demand, and have provided convincing evidence that optimal improve oxygen uptake upon exposure to warmer temperatures
temperaturesforASdonotnecessarilyelicitmaximalperformance (McBryanetal.,2016);however,fewstudieshaveexplicitlylinked
inallfitness-relatedmeasures(Clarketal.,2013;Gränsetal.,2014; plasticity in heart or gill morphology to metabolic compensation,
Norinetal.,2014; Wang etal., 2014; Brijsetal.,2015;Ern et al., and these patterns have, to our knowledge, never been tested in
2016), raising questions about the broad applicability of this tropicalfishes.Inaddition,littleisknownabouthowdevelopmental
framework (Jutfelt et al., 2018). In addition, there are many plastic responses will benefit fishes faced with rapidly changing
pathways for fish to respond to oxygen limitation, scaling from thermal environments (i.e. acute exposure to novel temperatures),
whole-bodytocellularlevels(Pörtner,2002;Angilletta,2009;Biro whichisrelevantwhenpredictingclimatechangeeffectswhereboth
andStamps,2010),andalterationstoanyofthesepathwayscanlead long-termincreasesandrapidchangesintemperatureareexpected.
y
to improvements in metabolic performance (e.g. reduced basal In this study, we investigated these patterns in a tropical, g
oxygendemand)andmaintenanceoffitness-relatedtraits. freshwaterteleost,theNileperch[Latesniloticus(Linnaeus1758)], lo
o
Nevertheless, cardiorespiratory function may still play an alarge,primarilypiscivorousfishthatwasintroducedtotheLake i
B
important role in metabolic compensation, and investigating VictoriabasininEastAfricainthemid-20thcentury,andcurrently
l
relationships between cardiorespiratory traits and thermal tolerance forms the basis of a lucrative 250–310 million USD yr−1 export a
t
and/ormetabolicperformancecanallowastraightforwardtestofat fishery (Indian Ocean Commission, 2015). Nile perch regularly n
e
least one prediction generated by OCLTT. If cardiorespiratory experiencetemperaturesintherangeof23–27°CinLakeVictoria, m
function can sustain performance at high temperatures, plastic and similarlystable temperatures in theirancestral habitatin Lake ri
changes in heart and gill morphology that improve oxygen uptake Albert (range: 26–31°C; Nyboer and Chapman, 2017). Despite e
p
anddeliverymaybekeytoacclimationcapacity(Sollidetal.,2005; thesenarrowthermalranges,recentstudieshavedemonstratedhigh x
E
Farrell et al., 2009; Dalziel et al., 2012; Anttila et al., 2013; levelsofacclimationcapacityinmetabolicrateandupperthermal
f
Jayasundara and Somero, 2013). Both heart and gill morphology tolerance of Nile perch under both short (3 day) and longer-term o
havebeenshowntochangewithexposuretoincreasedtemperatures (3week) exposures (Chrétien and Chapman, 2016; Nyboer and al
n
(Sollidetal.,2005;Klaimanetal.,2011;McBryanetal.,2016;Keen Chapman, 2017), making it an interesting subject for testing
r
et al., 2017). The heart is the primary power generator for the responses over longer acclimation times (i.e. time enough for u
o
circulatory system, and is often a central focus in investigations of developmental differences to take place) and for evaluating J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
mechanisms underlying thermal flexibility. The objectives of this Fishcollectionandrearing
study were to: (1) determine the acclimation capacity of upper Young (<2 months old) juvenile Nile perch [12.4±0.67 cm total
thermal tolerance limits (CT ) and metabolic rates of juvenile length(TL),mean±s.e.m.,n=600]werecollectedbyfishersinJuly
max
Nile perch reared for 3 months under elevated temperature, 2015 with small-mesh beach seines at Entebbe Bay, Uganda.
(2) examine links between variation in metabolic function, body Specimens were transported to the Aquaculture Research and
mass and condition, and plastic responses in oxygen uptake and DevelopmentCenterinKajjansi,Uganda,wheretheywerestocked
delivery capacities of cardiorespiratory organs and (3) assess intosixlarge(6×10×1m)outdoorconcreteaquaculturepondsand
whether plastic changes associated with rearing are beneficial for rearedfor3months(2Julyto5October2015).Thisrearingperiod
coping with rapid temperature changes (i.e. acute exposures to a waschosenasweanticipatedthistimeframewouldallowforsome
rangeoftemperatures). morphologicalanddevelopmentalresponsestooccurbasedonNile
We predicted that wewould detect plasticity in metabolic traits perch growth rates (Nkalubo, 2012), and because it spanned a
andCT leadingtometaboliccompensationinfishrearedunder portion of one dry and one rainy season, capturing some natural
max
warmerconditions.Wealsoanticipatedfindingdifferencesinheart seasonal variation. The ponds were organized into three blocks,
and/or gill morphology among rearing regimes; specifically, we eachwithonereplicateofthetwothermalregimesdescribedabove
expectedsmallerRVM,thickercompactmyocardiumandlargergill (Fig. S1A). These rearing temperatures shall be referred to as the
sizes in warm-reared fish, as these are possible mechanisms for averagerearingtemperature,orT (∼25°C),andthewarmrearing
avg
copingwiththeincreasedoxygendemandlikelytobeexperienced temperature, or T (∼29°C). To create these divergent thermal
warm
under elevated temperatures. If these morphological changes regimes, wooden frames were constructed over each pond and
improve thermal tolerance and metabolic performance, we would coveredwitheithergreenhouseplastic(TeraMax®,Teris,Montréal,
expecttofindrelationshipsamongthesetraitswithinrearinggroups. QC, Canada) or agricultural shade-cloth (GreenTek Shade-Rite®,
Teris,Montréal, Québec;Fig.S1B).Priortostocking,pondwater
qualityparametersincludingtemperature(°C),luminosity(i.e.lux,
MATERIALSANDMETHODS expressedinlmm–2),DO,turbidity(Secchidepth,expressedincm)
Overview andpHwerecarefullymonitoredfor4monthstoensurethatwater
Wild-caughtjuvenileNileperchwerecapturedinJulyandrearedfor temperatures and physicochemical conditions were stable. Each
3months under two temperature regimes, one resembling annual pondwasequippedwithflow-throughinletandoutletmechanisms
average temperatures in Lake Victoria (∼25°C maximum; control) sothatwaterinsidethepondscouldbeflusheddirectlywith∼25°C
and one resembling temperatures predicted under climate change localriverwater(seeFig.S1Bforaschematicdiagramofthepond
(∼29°Cmaximum).Fishfromtheserearingtemperatureswerethen set-up).
acutelyexposedinthelaboratory(3days)toarangeofexperimental During stocking, all ponds were filled with river water and
temperatures encompassing both rearing temperatures and a higher maintained at the natural ∼25°C. Experimental fish (n=100 per
temperature (33°C) not previously experienced in either regime, rearingpond)wereweighedtothenearest0.01g,andmeasuredfor
therebytestingtheirabilitytocopewithrapidchangesintemperature. TL and standard length (SL) to the nearest 0.1cm to ensure
SMR, MMR, AS and CT were measured, and organs (livers, uniformity of initial stocking size (Table S1). After 3days, T
max warm
hearts,gills,brains)wereexaminedtoidentifypotentialmechanisms pondswereheatedto∼29°Cover48h.Thiswasaccomplishedby
for metabolic compensation. This research was conducted under temporarily cutting inflow of cool river water to the T ponds,
warm
McGillUniversityAnimalCareProtocol5029. whichallowedthewaterinthepondstowarmunderthegreenhouse
Table1.Resultsoftwo-wayANOVAonwaterqualityvariables
Variable Rearingtemperature Block Rearingtemperature×Block
Maximumtemperature(°C) F 1832.797 5.866 9.337
1,511
P <0.001 0.003 <0.001
η2 0.782 0.022 0.035
Minimumtemperature(°C) F 1766.408 18.962 10.882
1,511 y
P <0.001 <0.001 <0.001 g
η2 0.776 0.069 0.041 o
l
Lux(lmm–2) F1,511 29.049 0.848 9.249 Bio
P <0.001 0.429 <0.001
η2 0.048 0.003 0.031 al
t
AverageDO(mgl−1) F 0.428 0.694 n
1,377 e
P 0.513 0.500 m
η2 0.001 0.004 i
r
e
pH F1,232 1.819 0.59 p
P 0.179 0.555 x
E
η2 0.008 0.005
f
o
Secchi F 1.081 1.279
1,372 l
P 0.299 0.28 a
η2 0.003 0.007 rn
u
Variablesincludemaximumandminimumwatertemperature,lux(luminosity),dissolvedoxygen(DO),pHandturbidity(Secchidepth),acrossrearing o
temperatures(T ,T )andamongblocks.BoldP-valuesindicatesignificanceatα<0.05. J
avg warm
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
covers. During this time, DO concentrations were monitored to were no differences in any other water quality variables (DO,
ensuremaintenanceofadequateoxygenlevels.AfterT ponds turbidity, pH) between rearing temperatures or among blocks
warm
hadwarmedtothedesiredtemperature(∼29°C),allponds(T and (Table1,TableS2,Fig.S2B–E).
avg
T )wereflushedeachdayforthesameamountoftimewithfresh During rearing, Nile perch were fed daily on live, aquaculture-
warm
riverwater.Thelargevolume(60m3)ofthepondsensuredthatthe rearedNiletilapiafry,whichweregradedtoensureappropriateprey
T ponds stayed within 3°C of the desired temperature, even sizes. Daily feeding rations were calculated to equal ∼15% of the
warm
during periods of flushing. Throughout the experiment, water biomassofNileperchstockedineachpondtoensureadequatefood
qualityparameters(temperature,luminosity,DO,turbidityandpH) supply at rates similar to natural conditions as estimated by Nile
wererecordedtwicedaily,onceinearlymorningandonceinmid- perchbioenergeticanalyses(Kitchelletal.,1997)andfromprevious
afternoontocapturedailyfluctuations.Twenty-four-hourHOBO™ trials of Nile perch rearing. The levels were adjusted based on
light and temperature loggers (Onset®, Bourne, MA, USA) took estimatesofmortalitiesandestimatedgrowthratesofNileperchin
hourlytemperatureandluminosityreadingsforthedurationofthe theponds.
rearing period (see Table S2, Fig. S2 for a summary of all water
quality parameters). To determine how water quality parameters Laboratoryexperiments
varied between rearing temperatures (T and T ) and among After completing 3months in the rearing ponds, Nile perch were
avg warm
rearing pond replicates (blocks 1, 2 and 3), data from the HOBO transportedfrompondstothelaboratory,wheretheywereplacedin
loggers(temperatureandlux)andfromdailywatersampling(DO, temperature-controlled tanks (n=4 per tank) containing filtered,
turbidityandpH)wereplottedovertimeforvisualassessment(Fig. oxygenated well water (see Nyboer and Chapman, 2017 for a
S2A–E) and analyzed with two-way ANOVA, with rearing description of the laboratory system). All fish were individually
temperature and block as fixed effects. Water temperatures in the marked using Visible Implant Elastomer (Northwest Marine
T rearingpondshadhighermaximum(28.6°C)andminimum Technology Inc., Shaw Island, WA, USA). We employed a fully
warm
(27.1°C) daily temperature than those in the T regime crossedexperimentaldesignforlaboratorytestssothatindividuals
avg
(maximum=24.5°C; minimum=23°C; Table 1; Table S2, Fig. from each rearing temperature (T and T ) and block (3
avg warm
S2A). There were significant effects of block and block×rearing replicates) were subjected to each experimental laboratory
temperature;however,theeffectsize(η2)ofthesefactorswassmall temperature for metabolic rate experiments (Fig. S1A, Table 2).
comparedwithrearingtemperaturealone(Table1).Posthoctests Laboratorytankswereinitiallyheldattheaveragemaximumrearing
revealed that differences among blocks were only apparent in the temperature(29or25°C)andthenslowlyraisedorloweredtothe
T rearingtanks,withreplicate1ofT significantlywarmer desired experimental temperature (25, 29 or 33°C) over a 12 h
warm warm
thanreplicates2(P=0.002)and3(P<0.001).Averagelux(lmm−2) period, and then held for 3days. Fish were brought into the
alsodifferedamongrearingtemperatures,withT havinghigher laboratory in a staggered fashion so that each batch was held for
warm
luminosity than T ; however, the effect size was small, and thesame12h+3dayperiodbeforeexperimentaltrials.Onthefirst
avg
differenceswereapparentonlyin blocks1and3(Table1).There day of acclimation, Nile perch were fed two tilapia fry each, and
Table2.Samplesizes(N)andmeans±s.e.m.ofbodymass(M ),standardlength(SL),totallength(TL)andLeCren’sconditionfactor(K)ofjuvenile
b
Nileperchusedinrespirometryexperiments
Experimentaltemperature
Rearingtemperature andblock N M (g) TL(cm) SL(cm) K
b
T 25 7 49.11±4.45 16.98±0.17 14.2±0.24 1.01±0.015
avg
1 2 34.12±2.01 15.55±0.04 13±0 0.97±0.023
2 2 51.4±2.18 17.3±0.02 14.35±0.09 1.03±0.007
3 3 57.57±4.83 17.73±0.14 14.9±0.21 1.01±0.017
29 9 30.95±3.26 14.66±0.24 12.26±0.3 1.04±0.026
1 2 25.56±3.34 13.95±0.1 11.65±0.06 1.06±0.04
2 3 34.19±1.87 15.1±0.11 12.6±0.15 1.11±0.027
3 4 30.68±7.16 14.7±0.35 12.32±0.44 0.99±0.011
y
33 9 23.44±2.27 13.94±0.24 11.67±0.26 0.95±0.013 g
1 2 18.77±1.43 12.75±0.24 10.75±0.22 1.04±0.046 o
l
2 3 27.45±3.6 14.7±0.13 12.4±0.18 0.91±0.037 o
i
3 4 22.77±4.04 13.97±0.24 11.6±0.26 0.93±0.019 B
Twarm 25 9 42.9±5.26 15.91±0.27 13.31±0.36 1.09±0.023 al
1 3 36.52±3.26 15.46±0.15 12.96±0.15 1.04±0.018 t
n
2 3 56.24±12.77 17.26±0.33 14.33±0.5 1.11±0.035 e
3 3 35.95±5.02 15±0.17 12.63±0.25 1.11±0.01 m
29 10 33.33±4.8 15.31±0.32 12.69±0.41 0.98±0.038 ri
1 3 20.69±0.28 13.6±0.19 11.13±0.16 1.02±0.054 e
p
2 4 48.16±6.38 17.1±0.21 14.17±0.3 1±0.04 x
3 3 26.22±3.45 14.63±0.14 12.26±0.17 0.9±0.012 E
33 7 30.9±4.9 15.17±0.27 12.65±0.36 0.91±0.023 f
o
1 2 19.94±0.67 14.1±0.03 11.55±0.06 0.86±0.028
l
2 3 42.67±5.19 16.6±0.16 13.93±0.24 0.94±0.011 a
n
3 2 24.2±7.02 14.1±0.2 11.85±0.24 0.92±0.04 r
u
Eachrearingtemperature×experimentaltemperature×blockcombinationisrepresented.Samplesizeswereselectedtoensureadequatepowerbasedonknown o
levelsofvariabilityinrespirometrydata,andaccountingforavailabilityofspecimensinponds. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
thereafter(48h)foodwaswithheldtoensureapost-absorptivestate closedportiontoensurethewaterinthechamberhadfullymixed.
duringtrials.Intotal,therespirometryexperimentswerecompleted Oxygen concentrations were converted to metabolic rate
overa3-weekperiod(5–26October2015),sofishbroughtintothe (mg O min−1) after accounting for respirometer volume and fish
2
laboratorylasthadanextra17daysintherearingponds. mass.Backgroundrespiration(onaverage9.9%ofSMRand3.6%
of MMR) was subtracted from the metabolic rates by assuming a
Metabolictraits linearchange between the two 20-min ‘empty’ runs. MMR isthe
Metabolic traits (SMR, MMR, AS) were estimated for Nile perch highest Ṁ measurement recorded over any 3-min period
from each rearing temperature×block×experimental temperature throughoutOt2he trial. To calculate SMR, Ṁ measurements were
O2
combination (Table 2) by measuring oxygen consumption rate plotted against time to detect the point where the recovery curve
(Ṁ ) using intermittent-flow respirometry. The experimental set- leveled off (on average 4.4±1.6 h, mean±s.d.). SMR was then
upOis2describedindetailinNyboerandChapman(2017),butbriefly, calculatedasthemeanofthelowest10%ofallṀ measurements
fourpolypropylenerespirometers(volume=1.57–2.80l),eachfitted afterthispoint.Outliers±2s.d.fromthemeanwerOe2excludedfrom
withaFiresting®temperatureprobeandfiberopticcablefocusedon thiscalculation(Clarketal.,2013).Wefollowedmethodsoutlined
acontactlessoxygensensorspot(PyroScienceSensorTechnology, inNyboerandChapman(2017)toensurethatcalculationsofSMR
Bremen, Germany), were submerged in a temperature-controlled did not over- or under-estimate this trait. We detected allometric
water bath held at the desired experimental temperature. Oxygen effects of body mass on metabolic rate, and therefore used mass
and temperature were recorded every 2s, and outputs adjustments (following Ultsch, 1995) instead of classic mass
were monitored and recorded by Firesting® Profix software corrections(mgmin−1kg−1)toaccountfortheeffectsofbodysize
(PyroScience) for the duration of each trial. Respirometers were on metabolic rate. Mass-adjusted AS was calculated as the
setonautomated10-minloopscomprisinga5-minclosedportion differencebetweenmass-adjustedMMRandSMR(notethatfrom
duringwhichDOlevelsweremeasured,anda5-minopenportion hereon,mass-adjustedmetabolictraitswillbereferredtoasMMR,
during which fresh water was flushed through the respirometry SMRandAS).
chambers.
For each trial, MMR was measured first using an established Bodysize,conditionandorganmeasurements
3-minexhaustivechaseprotocol(Rocheetal.,2013).Inshort,this To estimate fish condition (K), fish were weighed for M to the
b
protocolinvolvedhand-chasingindividualNileperchtoexhaustion nearest 0.01g and measured for TL and SL to the nearest 0.1cm
andthentransferringthemtotherespirometer,wheretheywereheld before respirometry trials. K was calculated using LeCren’s
for an average of 10h after the chase, allowing ample time for equation as detailed in Froese (2006). Because there was
recovery and to derive estimates of SMR (Nyboer and Chapman, homogeneity of slopes in the bi-logarithmic M ×SL relationship
b
2017), with oxygen consumption measured throughout the 10-h among rearing temperatures and blocks, coefficients were derived
period. Twenty-minute ‘empty’ runs were conducted before and bypoolingdataforallindividuals.
after each trial to quantify background respiration. Prior to this Attheendofallexperimentaltrials,fishwereeuthanizedwitha
study, a number of exploratory trials were conducted on recovery sharp blow to the head, and livers, hearts, gills and brains were
times of Nile perch to validate this protocol. Results from these removed. Livers were weighed fresh to the nearest 0.001g, and
experimentsshowedthatreliableSMRmeasurescouldbeobtained hepatosomaticindex(HSI)wascalculatedasapercentageofM .All
b
<10 h after employing exhaustive chase (Nyboer and Chapman, othertissueswerepreservedin10%formalinuntilprocessing.
2017).Fishwereweighedforbodymass(M )andmeasuredforSL Preserved heart ventricles for a subset of fish (Table S3) were
b
andTLafterthetrials. isolatedfromtherestofthehearttissues(Fig.1A),blottedtoremove
excessliquid,andweighedfivetimestothenearest0.01g.Themean
Criticalthermalmaximum wasusedtocalculateRVMasapercentageofM .Ventricleswere
b
After respirometry trials, fish were returned to their rearing ponds thenembeddedhorizontally inparaffin,sectioned(4μm thickness)
for ∼10daysto recover, after which asubset were returned to the along the dorso-ventral axis (Fig. 1A,B), stained with hematoxylin
laboratoryandre-acclimatedtothesameexperimentaltemperature andeosin,andphotographedat4.5×magnificationwithaLeica6SD
for 3days to measure CT as an estimate of thermal tolerance. zoom microscope with a Lumenera Scientific Infinity camera
max
Owing to stresses incurred during this transfer process, a smaller (Ottawa,ON,Canada).ImageJv.1.50i (https://imagej.nih.gov)was
samplesizewasusedforCT trials(TableS3);however,boththis used to quantify the areas of the spongy and compact myocardial
max y
current work and previous studies showed low variance in CT layers(Fig.1B).Theareaofcompactmyocardiumwasdividedbythe g
max o
values,thusevenwithlowsamplesizesthesefindingsarelikelyto total area (compact+spongy) of the heart section to calculate the l
o
beaccurate.CTmaxwasmeasuredfollowingChenetal.(2013).In percentofcompactmyocardium(%CM). Bi
these trials, groups of two to four Nile perch from each rearing To quantify gill size, four gill arches from the left side of the
l
temperature×experimental temperature combination were branchial basket of 9–10 fish (Table S3) from each rearing a
t
transferred into a water-filled cooler, held overnight (∼8 h) and temperature were extracted, laid flat and photographed at 6.3× n
e
then subjected to aconstant temperature increase of 0.3°C min−1. magnification on both hemibranchs using the same microscope m
Thisheatingratedoesnotallowforacclimationduringthetrial,but setup described above. A series of six gill traits were selected to ri
e
also does not lethally shock the animal (Beitinger et al., 2000). represent the overall size and area of the hemibranchs. These
p
CT was determined as the temperature at which each fish lost measuresweretakentothenearest0.1mmor1mm2andincluded: x
max E
equilibriumfor10s. totalfilamentnumber(TFN),totalhemibrancharea(THA),totalgill
f
filament length (TGFL), average gill filament length (AGFL), o
Calculationsofmetabolictraits filament base length (FBL) and filament density (FD) (Chapman al
n
Metabolic rates (SMR and MMR) were estimated by calculating etal.,2008;CrispoandChapman,2010).TFNwascalculatedasthe
r
linear regressions between oxygen concentration and time for the numberoffilamentsalongthefilamentbaseonbothhemibranchs. u
o
closedperiodofeachloop,omittingthefirstandlast30sofeach THAwasthesumoftheareaofallfilamentsonbothhemibranchs. J
5
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
A B
Bulbus arteriosus
Ventricle
Atrium
Sinus venosus
C
Total hemibranch area
Filament base length
Length of gill filament
D
Spongy
Cerebellum
Optic lobe layer
Cerebrum
Compact layer
Inferior lobe
Fig.1.IllustrationsdepictingmorphologyanddissectionpointsforNileperchorgans.(A)Dissectionpoints(dashedredlines)andthelocationofthecross-
section(dashedblackline)fortheheartventricles.(B)Alow-magnificationimageofanH&E-stainedcross-sectionofaheartventriclewiththespongyand
compactmyocardiumlayersindicated.(C)Onesideofagillarchwithmeasurementsusedforanalysisofgillsize(dashedredlines).(D)Dissectionpointsfor
brains(dashedredlines).
TGFL was quantified by measuring the length of every 10th allblockswererepresentedinallrearingtemperature×experimental
filament, and using the average of the two outer measures to temperature combinations; block was therefore not included as a
estimate the lengths of the nine filaments in between (Fig. 1C). fixed effect. For tests where the rearing temperature×experimental
AGFLwascalculatedastheTGFL/TFNratiooneachhemibranch. temperatureinteractiontermwassignificant,andoveralltrendsacross
FBL was calculated as the length of the filament base of each experimentaltemperaturewerevisuallysimilar,posthoctestsforthe
hemibranch(Fig.1C).FDwascalculatedastheFBL/TFNratioon effect of experimental temperature within rearing temperature and
eachhemibranch.Toobtainestimatesforthewholegill,valuesfor rearingtemperaturewithinexperimentaltemperaturewereconducted
TFN,THA, TGFLandFBLweresummed acrossthefourarches, to detect differences among levels. Equality of variances and
andthenmultipliedby2toaccountforbothsidesofthebranchial normality of residuals were assessed with diagnostic residual plots
basket. Values for FD and AGFL were averaged across the four andnormalQ–Qplots.
arches(Chapmanetal.,2008;CrispoandChapman,2010).Wedid Effects ofrearing temperatureon K, M ,SL,TL,RVM,%CM,
b
not measure total gill surface area; however, previous inter- RBMandHSIwereanalyzedwithtwo-wayANOVA,withrearing
population and rearing studies have found that populations or temperatureandblockasfixedeffects.Non-significantinteraction
treatmentsoffishwithalargerTGFLarecharacterizedbyalarger termswereremovedfrommodels.PotentialinteractiveeffectsofM
b
TGSA (Chapman and Hulen, 2001; Chapman, 2007; Chapman and rearing temperature on organ masses were tested using
etal.,2008).WethereforeassumethatalargerTGFLpergramof ANCOVA with raw organ masses as response variables, rearing
y
Nileperchreflectsgreateroxygenuptakecapacity. temperatureandblockasfixedeffects,andM asacovariate.Raw g
b o
Preservedbrainsforasubsetoffish(TableS3)wereprocessedby livermasswaslog10-transformedtomeetassumptionsofstatistical ol
trimmingexcesstissueandensuringallbrainswereseveredfromthe tests.ResultsofANCOVAsrevealedsimilaroutcomesasthetwo- i
B
spinalcord1mmpastthecerebellum(Fig.1D).Blottedbrainswere way ANOVAs (except for a significant M ×rearing temperature
b l
a
weighedfivetimestothenearest0.001g,andthemeanwasusedto interactionforventriclemass),soonlyANOVAresultsarereported
t
calculaterelativebrainmass(RBM)asapercentageofM . (butseeTableS4forcomparisonwithTable3).Gilltraitswerefirst n
b e
analyzedwitheachmetricseparatelyusingANCOVAwithrearing m
Statisticalanalysis temperature and block as fixed effects, and Mb as a covariate. ri
e
EffectsofrearingandexperimentaltemperatureonSMR,MMRand Second,principalcomponentsanalyses(PCA)wereusedtoreduce
p
AS were tested with three-way ANOVA with rearing temperature, gill metricsto majoraxes, and PCA scores wereused as response x
E
blockandexperimentaltemperatureasfixedfactors.Mass-adjusted variablesinANOVAmodelswithrearingtemperatureandblockas
f
metabolic traits were log -transformed to meet assumptions of fixedeffects.ToperformPCA,wefirststandardizedgilltraitstoa o
10
statisticaltests.Interactionsamongfixedeffectswereremovedfrom commonbodymassfollowingCrispoandChapman(2010). al
n
the model if not significant. Effects of rearing and experimental ANCOVAwasusedtotestthehypothesisthatvariationinRVM,%
r
temperature on CT were tested with a two-way ANOVA with CM and gill size had an effect on metabolic performance (SMR, u
max o
rearingandexperimentaltemperatureasfixedfactors.ForCT ,not MMRandAS)andthermaltolerance(CT ).Beforethesetests,Q J
max max 10
6
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
A B1.10 Table3.Resultsoftwo-wayANVOAtestingfordifferencesinbodysize
40.0 andorgantraitsacrossrearingtemperaturesandblocks
g) 38.0 1.05 Trait Factor F d.f. P η2
y mass ( 3364..00 ondition 1.00 TMLb((cgm)) RBRleeoaacrrkiinnggtteemmppeerraattuurree 600...498454232 11,,4477 000...530060932 000...002012988
d 32.0 C
o Block 7.589 0.001 0.244
B
30.0 0.95 SL(cm) Rearingtemperature 0.606 1,47 0.44 0.013
Block 7.473 0.002 0.241
28.0
K Rearingtemperature 0.031 1,47 0.861 0.001
26.0 0.90 Block 0.536 0.589 0.022
C D RBM(%M ) Rearingtemperature 0.013 1,20 0.911 0.001
b
a 9.5 Block 4.459 0.025 0.308
0.060 %) b RVM(%Mb) Rearingtemperature 9.219 1,33 0.005 0.218
00..005586 um ( 89..50 %CM BRleoacrkingtemperature 121..292676 1,32 00..102042 00..121792
0.054 di Block 0.404 0.671 0.025
RVM00..005520 b myocar 78..50 HSI(%Mb) BRleoacrkingtemperature 107..087265 1,28 <00..900217 00..030859
00..004486 act 7.0 a PC1gills RBleoacrkingtemperature 00..191864 1,15 00..733988 00..010186
p
0.044 m PC2gills Rearingtemperature 0.002 1,15 0.969 0.000
o 6.5
0.042 C Block 0.043 0.958 0.006
0.040 6.0 Traitsincludebodymass(M ),totalandstandardlength(TLandSL),LeCren’s
b
E F conditionfactor(K),relativebrainmass(RBM),relativeventricularmass
(RVM),percentcompactmyocardium(%CM),hepatosomaticindex(HSI)and
0.6 0.6 twoprincipalcomponentsforgilltraits(PC1andPC2).Rearingtemperatures
0.4 0.4 includeTavgandTwarm,andeachregimewasrepresentedbythreeblocks.Bold
P-valuesindicatesignificanceatα<0.05.
0.2 0.2
C1 0 C2 0
P P temperature ofthe individual (e.g.adjusted to 29°C for T fish).
warm
–0.2 –0.2 Q -correctedmetabolictraitsaredenotedasSMR ,MMR and
10 Q10 Q10
–0.4 –0.4 AS .CT wasadjustedusingtheslopeforthelinearequationof
Q10 max
CT ×experimental temperature within each rearing temperature.
–0.6 –0.6 max
AdjustedCT isdenotedasCT .ANCOVAswithCT or
G H max max,adj max,adj
SMR andMMR asresponsevariables,rearingtemperatureand
Q10 Q10
0.60 0.9 a blockasfixedeffects,andorgantraits(e.g.RVM,%CM,gillPCs)as
0.8 covariates were applied to detect whether these organ traits had an
0.58
overall effect on metabolic traits or CT . Significant effects were
max
0.56 0.7 confirmed with Pearson’s correlation of CT or SMR and
SI max,adj Q10
M 0.54 H 0.6 b MMRQ10withorgantraits.Alphavaluesweresetto0.05toconfirm
B
R 0.5 significance for all statistical tests. All analyses were performed in
0.52 IBMSPSSStatistics,version22.0(IBMCorp.,Armonk,NY,USA).
0.50 0.4
0.48 0.3 RESULTS
Bodysizeandcondition
Tavg Twarm Tavg Twarm NileperchfrombothrearingconditionsachievedthesameMb,SL,
Rearing temperature TLandKoverthe3-monthrearingperiod(Fig.2A,B).Becausethe y
stocking size and feeding regime were the same among rearing g
Fig.2.ResultsofANOVAexaminingtheeffectsofrearingtemperatureon o
temperatures, fish were assumed to have similar growth rates l
fitness-relatedtraitsandorgantraits.Fitness-relatedtraitsinclude(A)body o
massand(B)LeCren’sconditionfactor.Organtraitsinclude(C)relative betweenrearingconditions,althoughwecouldnotquantifyprecise Bi
ventricularmass(RVM),(D)percentcompactmyocardium,(E)PC1ofgills feedingratesoractivitylevels.Therewere,however,strongeffects l
representinggillarea,(F)PC2ofgillsrepresentinggillwidth,(G)relativebrain of block for all body size traits (Table 3). Post hoc pairwise ta
mass(RBM)and(H)hepatosomaticindex(HSI).Valuesarepresentedas comparisons revealed that fish from block 2 were significantly n
means±s.e.m.Differentlettersindicatesignificantdifferences(α=0.05)among largerforallmeasuresthanthosefromblock1(M :P=0.002,TL: me
rreeaarriinnggtteemmppeerraattuurrees(T[awvaermra),g∼e2r9e°aCri]n.gBtleomckpweraastuinrecl(uTdaevgd),a∼s2a5f°iCxe;dwfaarcmtorin Pth=a0n.0t0h1osaendfroSmL:bPl=oc0k.0031)(Mand: Psi=g0n.i0fi6c4a,ntTlyLo:rPm=ba0r.g0i4n3allayndlarSgLer: eri
ANOVAmodels. b p
P=0.0064). K was stable across rearing temperature and block x
E
(Table 3). Mortality rates of Nile perch in the rearing ponds
f
corrections were applied to SMR, MMR and AS to adjust for averaged65%,andweresimilaracrossthermalregimes(64.3%in o
the effects of experimental temperature on metabolic traits. Q T ,66%inT ),withmost (73%)ofthemortalityoccurringin al
10 avg warm
temperaturecoefficientswerecalculatedusingthevan‘tHoffequation thefirst3weeksoftherearingtreatments.Althoughthesourceof n
r
(McNab, 2002); these values were subsequently used to adjust mortalitiescouldnotbedetermined,webelievethattheinitialstress u
o
metabolic rates at a given experimental temperature to the rearing ofcaptureandtransferwaskey,inadditiontocompetitionforfood J
7
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
A B b Fig.3.ResultsofANOVAScomparing
5.5 Tavg c 14 metabolictraitsandthermaltolerance
–1g) 5.0 Twarm –1g) 13 ltiemmitpseirnatNuirleespaenrcdhbaemtwoenegnerexapreirnigmental
k k
–1 4.5 –1 12 temperatures.(A–C)Resultsofthree-way
min min ANOVAcomparingmeans±s.e.m.for
2 4.0 b 2 11 (A)standardmetabolicrate(SMR),
O O a a (B)maximummetabolicrate(MMR)and
mg 3.5 a y mg 10 (C)aerobicscope(AS)betweenrearing
R ( 3.0 R ( 9 temperaturesacrossarangeof
M M experimentaltemperatures.(D)Resultsof
S 2.5 x x M 8 two-wayANOVAcomparingmeans±s.e.m.
2.0 7 ofcriticalthermalmaximum(CTmax)
betweenrearingtemperaturesandacross
C D b experimentaltemperatures.Differentletters
b inAindicatesignificantdifferencesamong
b
40 experimentaltemperatureswithinarearing
9
1) temperature,andasterisksindicate
–1–min kg 87 a (°C)ax 3398 a stteeigmmnppifeeicrraaanttuutrrdeeis.ffDewirfietfhenircneenastnbleeetxttewpreesreiimnnerBen–atDarilng
m indicateoveralldifferencesinmetabolic
O 2 a CT 37 traitsamongexperimentaltemperatures.
mg 6 Significanceofresultsarepresentedin
S ( 5 36 Table4.Samplesizesarepresentedin
A Table1.
35
4
25 29 33 25 29 33
Experimental temperature
becauseoftheemaciatedappearanceofnon-survivingfishandthe 33°C,whereasT fishshowedsignificantincreasesinSMRateach
avg
excellent condition of survivors. Although selection imposed by experimental temperature (Fig. 3A). Overall, SMR of T fish
avg
these mortality rates may bias results, similar rates across rearing increased 37% across experimental temperatures compared with a
temperaturesvalidatecomparisons. lower 22% increase in T fish. Rearing and experimental
warm
temperature both had significant effects on MMR (Table 4). T
warm
Nile perch had a 10–19% lower MMR than T fish at all
avg
Metabolictraitsandcriticalthermalmaximum experimental temperatures (Table 4, Fig. 3B). Patterns of increase
NileperchrearedunderwarmconditionshadlowSMRscompared across experimental temperatures were remarkably similar between
with T fish (Fig. 3A, Table 4), a difference that was especially rearing temperatures, with MMR remaining constant from 25 to
avg
pronouncedat29°Cand33°C,whereT fishshoweda37%and 29°C, and showinganexponential 31% increaseat33°C(Table 4,
27% reduction in Ṁ , respectively. Atw2ar5m°C, SMR values did not Fig. 3B). T Nile perch showed reduced AS overall, with lower
O2 warm
differ between fish from the two rearing temperatures (Fig. 3A). values at both the coolest and warmest experimental temperatures
There was a significant rearing temperature×experimental (Table4,Fig.3C).At29°C,however,ASofT fishequaledthatof
warm
temperature interaction, and post hoc tests revealed that T fish the T fish. AS showed a general increase with experimental
warm avg
performedequallywellat29°Casat25°C,butthatSMRincreasedat temperatureinfishfrombothrearingtemperatures(Table4,Fig.3C).
Table4.Resultsoftwo-andthree-wayANOVAsexaminingtheeffectsofrearingtemperatureandexperimentaltemperatureoncriticalthermal
maximum(CTmax)andmetabolictraits y
g
Rearing Experimental Rearingtemperature× o
Variable temperature temperature Block Experimentaltemperature ol
i
CT (°C) F 87.974 14.02 0.199 B
max 2,13
P <0.001 0.001 0.822 al
η2 0.871 0.683 0.03 t
n
SMR(mgO2kg−1min−1) F2,43 23.623 33.148 0.171 4.929 e
P <0.001 <0.001 0.843 0.012 m
η2 0.355 0.607 0.008 0.187 i
r
MMR(mgO kg−1min−1) F 12.914 32.288 0.174 0.366 e
2 2,43 p
P 0.001 <0.001 0.841 0.696 x
η2 0.231 0.6 0.008 0.017 E
AS(mgO kg−1min−1) F 4.749 16.545 0.13 1.949 f
2 2,43 o
P 0.035 <0.001 0.878 0.155
η2 0.099 0.435 0.006 0.083 al
n
Thetwo-wayANOVAtestedtheeffectsofrearingtemperature(T ,T ),experimentaltemperature(25,29and33°C)andtheirinteraction(R×E)onCT .The r
avg warm max u
three-wayANOVAtestedtheeffectsofrearingtemperature,experimentaltemperature,blockandR×Eonstandardandmaximummetabolicrate(SMRandMMR) o
andaerobicscope(AS).BoldP-valuesindicatesignificanceatα<0.05. J
8
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
A Organdevelopmentandplasticity
3.8 Tavg: R=–0.32, P=0.185 Cardiac remodeling was apparent in Nile perch, with T fish
T : R=–0.55, P=0.021 warm
1) warm having reductions in RVM and increases in %CM (Fig. 2CD,
–
min 3.3 Table 3), as predicted. The average RVM of Twarm fish (0.046±
1 0.002g,mean±s.e.m.)wasapproximately20%smallerthanthatof
–
O kg2 2.8 mTaavsgsfisshho(w0.e0d57th±a0t.0v0e2ntgr)i.clLeinseizaerrdegivreersgsieodnsatoflaMrgbewr ibthodvyenstirzicelse,
mg indicating possible growth-related changes in the onset of cardiac
(0 remodeling(Fig.S3A).WetestedthisinteractionwithANCOVAs
Q1 2.3 withventriclemassasaresponsevariableandM asacovariate,and
R b
M thesetestsrevealedasignificantM ×rearingtemperatureinteraction
S b
for raw ventricle mass. However, differences between rearing
1.8
3.0 5.0 7.0 9.0 11.0 13.0 temperatureweresimilartotheANOVAresults(TableS4,Table4).
Compact myocardium (%) Forsimplicity,onlyANOVAresultswereretainedinthemaintext
(butseeTableS4forcomparison).For%CM,ANOVArevealedthat
T fish had a higher %CM compared with T fish (Fig. 2D,
B warm avg
11 T : R=0.59, P=0.007 Table3).Fortestsofhowvariabilityinventriclemorphologyaffects
–1) 10 Tawvagrm: R=0.39, P=0.125 oQr1%0-cCoMrreacstecdomvaertiaabteoslircervaetaelstahnadtCvaTrmiaatxio,andj,inAN%CCOMVhAaSdwainthegRaVtivMe
n
mi 9 effect on SMR and a positive effect on AS , but did not
–1g 8 influenceMMRQQ1100orCTmax,adj(Fig.4,Table5),aQn1d0thatvariation
O k2 7 in RVM had a positive effect on CTmax,adj (Table 5). Pearson’s
mg 6 creolrarteiolantisohnipsbceotwnfeiremneRdVMtheasnedpAatSteirnnsT, anfdishre(vFeigal.e4d).a positive
(10 5 Gill traits did not differ between rearianvgg temperatures (Table 6).
Q
S 4 PCAonmass-standardizedgilltraitsextractedtwocomponentswith
A
3 eigenvalues >1, with PC1 explaining 66.5% and PC2 explaining
3.0 5.0 7.0 9.0 11.0 13.0 26.9% of the variance. Gill metrics related to filament and
Compact myocardium (%) hemibranch size (AGFL, TGFL and THA) loaded on PC1, and
thoserelatedtogillwidth(AFNandFBL)loadedonPC2(TableS5).
FDwasnotincludedinthePCAasitdidnotcorrelatewiththeother
C traits. Results of ANOVA using PC scores as response variables
11 Tavg: R=0.64, P=0.003 confirmed the lack of divergence in gill size among rearing
T : R=–0.12, P=0.639
1) 10 warm temperatures (Fig. 2E,F, Table 3). Rearing temperature had a
–n significanteffectonHSI(Fig.2G,Table3),withT fishhaving
–1 mi 98 41% smaller HSI values (0.47±0.03, mean±s.e.m.w)atrhman Tavg fish
g (0.81±0.05). Rearing temperature had no effect on RBM (Fig. 2H,
k
O 2 7 Table 3), with fish from both Tavg and Twarm regimes having
g 6 remarkably similar values (0.54±0.034 and 0.531±0.045,
m
(0 5 respectively).Noneofthegilltraits,HSIorRBMhadaneffecton
Q1 anyQ -adjustedmetabolictraitsorCT .
S 4 10 max,adj
A
3 DISCUSSION
0.03 0.04 0.05 0.06 0.07 0.08 0.09
Thethreemostimportantfindingsofthisstudywerethat:(1)Nile
RVM perch reared under elevated temperatures showed evidence for
compensation in upper thermal tolerance and metabolic rate, and
Fig.4.Linearrelationshipsbetweenmetabolictraitsandhearttraits. y
(A–C)Q10-correctedmetabolictraits(SMRQ10andASQ10)andhearttraits attainedsimilarbodysizeandconditionbetweenrearingtreatments; og
includingrelativeventricularmass(RVM)andpercentcompactmyocardium. (2) Nile perch are capable of cardiac remodeling, and variation in l
o
Colorsindicatedifferentrearingtemperatures(Tavg,blue;Twarm,red). heart morphology correlated with metabolic function in fish from Bi
CorrelationsforCTmaxwithRVM(notshown)hadsimilarpatternsasASQ10 bothrearingtreatments;and(3)plasticchangesenactedoverlonger l
(C),withamarginallysignificantpositivecorrelationintheTavggroup(R=0.756, exposureseffecteddifferencesinmetabolicflexibilitywhenacutely ta
P=0.082). exposed to temperature change. This work has important n
e
implicationsforclimatechangeresilienceinthisspecies,whichis m
Nile perch from both rearing temperatures showed asignificant ofmajoreconomicimportancetoinlandfisheriesinEastAfrica. ri
increase of 1.89°C (T ) and 1.95°C (T ) in CT over an e
warm avg max p
experimental temperature range of 7°C (Table 4, Fig. 3D), with a Developmentalplasticityinupperthermaltoleranceand x
slope relating CTmax to temperature of 0.218±0.060 for the Twarm metabolictraits fE
fishand0.244±0.067fortheT fish.T fishhadhigheroverall Rearing Nile perch under different temperature regimes for o
avg warm
CTmax (Table 4, Fig. 3D), with values 2.4–2.8°C higher at all 3months led to adjustments in upper thermal tolerance (CTmax). al
experimentaltemperatures.Theseindividualswereabletoachieve GiventhestenothermalevolutionaryhistoryoftheNileperchused n
r
CT of nearly 40°C after only 3days of acclimation to 33°C, in this study, one might expect low thermal plasticity in this u
max o
whereasTavgfishreachedonly37.5°C. population (Tewksbury et al., 2008). However, Nile perch J
9
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb178087.doi:10.1242/jeb.178087
Table5.Resultsoftwo-wayANCOVAtestingfortheinfluenceofcardiactraitsonthermaltoleranceandmetabolicperformanceofNileperchfrom
differentrearingtemperatures(T ,T )
avg warm
Variable
Rearing Covariate×
Outcome Covariate temperature Block Covariate Rearingtemperature
CT SMR F 139.898 9.737
max Q10 1,11
P <0.001 0.01
η2 0.927 0.47
RVM F 22.644 10.728 7.233
1,11
P 0.001 0.008 0.023
η2 0.694 0.518 0.42
SMR %CM F 8.031 0.456 6.184
Q10 1,31
P 0.008 0.638 0.018
η2 0.206 0.029 0.166
RVM F 13.89 0.17 0.724
1,31
P 0.001 0.844 0.401
η2 0.303 0.011 0.022
MMR %CM F 8.597 0.412 2.464
Q10 1,31
P 0.006 0.666 0.127
η2 0.217 0.026 0.074
RVM F 3.484 0.336 0.236
1,31
P 0.071 0.717 0.63
η2 0.098 0.021 0.007
AS %CM F 0.168 1.009 9.471
Q10 1,31
P 0.684 0.376 0.004
η2 0.005 0.061 0.234
RVM F 14.244 2.584 1.935 11.958
1,31
P 0.001 0.092 0.174 0.002
η2 0.315 0.143 0.059 0.278
Outcomevariablesincludedadjustedcriticalthermalmaximum(CT ),andQ -correctedstandardandmaximummetabolicratesandaerobicscope
max,adj 10
(SMR ,MMR andAS ).Cardiactraitcovariatesincludedrelativeventricularmass(RVM),andpercentcompactmyocardium(%CM).Blockandthe
Q10 Q10 Q10
covariate×rearingtemperatureinteractionwereincludedasfactorsinthemodel.BoldP-valuesindicatesignificanceatα<0.05.
experiencing chronically elevated temperatures were able to with experimental temperature, which indicates comparable levels
increase thermal tolerance without having restrictions on their of plasticity in thermal tolerance between rearing temperatures.
ability to further modulate thermal limits upon acute temperature TheseresultsconfirmpreviouslydocumentedtrendsinNileperch
increases.ThisisevidencedbythesimilarlinearincreasesinCT thermal plasticity over shorter exposures (Nyboer and Chapman,
max
2017),andareinlinewithfindingsinrecentmeta-analysesthatdo
not find evidence for predicted latitudinal trends in ectotherm
Table6.Resultsoftwo-wayANOVAtestingtheeffectsofrearing
temperature(T ,T )onaseriesofgilltraits thermalplasticity(Seebacheretal.,2014;GundersonandStillman,
avg warm
2015).
Rearing
Rearingtemperatureaffectedmetabolicperformance,withwarm-
Gilltrait temperature Block M
b reared fish showing overall reductions in SMR, MMR and AS
TFN F1,14 0.199 0.154 10.385 relative to T fish at most experimental temperatures, indicating
P 0.662 0.858 0.006 avg
that compensatory mechanisms underlying basal metabolic
η2 0.014 0.022 0.426
reductions are effective during rapid transfer to temperatures both
THA(mm2) F 0.094 0.234 30.933
1,14
P 0.764 0.794 <0.001 aboveandbelowtheirrearingtemperature(asopposedtoincurring y
η2 0.007 0.032 0.688 costsofthermalspecialization).DecreasesinSMRareverylikely g
o
TGFL(mm) F1,14 0.57 0.719 30.065 indicativeofthermalcompensationbecauselowerself-maintenance ol
P 0.463 0.504 <0.001 costs presumably allow more energy allocation to growth and i
η2 0.039 0.093 0.682 B
reproduction(Priede,1985;Sandblometal.,2014).Reductionsin
FBL(mm) F1,14 0.000 0.307 23.030 SMR in warm-reared fish were accompanied by even greater al
P 0.986 0.741 <0.001 t
η2 0.000 0.042 0.622 reductions in MMR, which ultimately resulted in reduced AS en
AGFL(mm) F1,14 0.202 0.483 17.26 relativetotheTavg-rearedfish.AlthoughdecreasesinMMRandAS m
P 0.66 0.627 0.001 are not necessarily predicted outcomes of thermal compensation, ri
η2 0.014 0.065 0.552 suchpatternshavebeenreportedinapreviousstudyonacclimated e
p
FD(TFN/FBL) F1,14 0.402 1.762 6.634 Nile perch (Nyboer and Chapman, 2017), and may indicate that x
P 0.536 0.208 0.022 E
long-termexposureinvokesmechanismsthatreduceSMRbutalso
η2 0.028 0.201 0.322 f
leadtoreductionsinpeakperformance(MMR).Similartrendshave o
Gilltraitsincludetotalfilamentnumber(TFN),totalhemibrancharea(THA), been found in a congener of Nile perch, the tropical barramundi al
totalgillfilamentlength(TGFL),filamentbaselength(FBL),averagegill n
(Norin et al., 2014), and in northern populations of shorthorn
filamentlength(AGFL)andfilamentdensity(FD).Bodymass(M )was r
includedasacovariate,andblockasafixedfactorinthemodel.BobldP-values sculpin (Sandblom et al., 2014), and suggest that there may be ou
indicatesignificanceatα<0.05. energetic costs associated with maintenance of high MMR. J
10