J Comp Physiol B (2014) 184:991–1001
DOI 10.1007/s00360-014-0861-9
ORIGINAL PAPER
Thermal plasticity of skeletal muscle mitochondrial activity
and whole animal respiration in a common intertidal
triplefin fish, Forsterygion lapillum (Family: Tripterygiidae)
J. R. Khan · F. I. Iftikar · N. A. Herbert ·
Erich Gnaiger · A. J. R. Hickey
Received: 6 January 2014 / Revised: 3 September 2014 / Accepted: 9 September 2014 / Published online: 1 October
2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Oxygen demand generally increases in ecto-therms
as temperature rises in order to sustain oxidative
phosphorylation by mitochondria. The thermal plasticity of
ectotherm metabolism, such as that of fishes, dictates a
species survival and is of importance to understand within an
era of warming climates. Within this study the whole animal
O2 consumption rate of a common New Zealand intertidal
triplefin fish, Forsterygion lapillum, was investi-gated at
different acclimation temperatures (15, 18, 21, 24 or 25 °C) as
a commonly used indicator of metabolic per-formance. In
addition, the mitochondria within permeabi-lised skeletal
muscle fibres of fish acclimated to a moderate temperature
(18 °C Cool acclimation group—CA) and a warm temperature
(24 °C. Warm acclimation group—WA) were also tested at
18, 24 and 25 °C in different states of coupling and with
different substrates. These two levels of analysis were carried
out to test whether any peak in whole animal metabolism
reflected the respiratory performance of mitochondria from
skeletal muscle representing the bulk of metabolic tissue.
While standard metabolic rate (SMR- an indicator of total
maintenance metabolism) and maximal
Communicated by I. D. Hume.
J. R. Khan · N. A. Herbert
Institute of Marine Science, Leigh Marine Laboratory, University of
Auckland, P.O. Box 349, Warkworth 0941, New Zealand
F. I. Iftikar · A. J. R. Hickey ()
School of Biological Sciences, University of Auckland,
3a Symonds Street Thomas Building, Auckland, New
Zealand e-mail: a.hickey@auckland.ac.nz
E. Gnaiger
D. Swarovski Research Laboratory, Department of General and
Transplant Surgery, Medical University of Innsbruck, Innrain
52, Christoph‑Probst‑Platz, 6020 Innsbruck, Austria
˙
metabolic rate (M O2 max) both generally increased with
temperature, aerobic metabolic scope (AMS) was maximal at
24 °C, giving the impression that whole animal (meta-bolic)
performance was optimised at a surprisingly high temperature.
Mitochondrial oxygen flux also increased with increasing
assay temperature but WA fish showed a lowered response to
temperature in high flux states, such as those of oxidative
phosphorylation and in chemically uncoupled states of
respiration. The thermal stability of mitochondria from WA
fish was also noticeably greater than CA fish at 25 °C.
However, the predicted contribution of respirational flux to
ATP synthesis remained the same in both groups and WA fish
showed higher anaerobic activ-ity as a result of high muscle
lactate loads in both rested and exhausted states. CA fish had
a comparably lower level of resting lactate and took 30 %
longer to fatigue than WA fish. Despite some apparent
acclimation capacity of skel-etal muscle mitochondria, the
ATP synthesis capacity of this species is constrained at high
temperatures, and that a greater fraction of metabolism in
skeletal muscle appears to be supported anaerobically at
higher temperatures. The AMS peak at 24 °C does not
therefore represent utilisa-tion efficiency of oxygen but,
rather, the temperature where scope for oxygen flow is
greatest.
Keywords Mitochondria · Temperature acclimation ·
Electron transport system · Lactate · Anerobic metabolism
Introduction
The metabolic rates of organisms are acutely sensitive to
temperature, and for ectotherms such as intertidal fishes,
environmental temperatures have significant influence over
bio-energetic processes. The oxygen consumption rate of
13
\992
ectotherms, which serves as a common and practical meas-ure
of aerobic metabolism, typically increases with tempera-ture
to maximal limits and then collapses (Clark et al. 2013; Healy
and Schulte 2012; Pörtner 2002; Pörtner and Knust 2007;
Pörtner et al. 2004) suggesting that fish will struggle to
survive at extreme high temperatures. Understanding the basis
and the capacity to alter these thermally-mediated lim-its is
becoming increasingly important given predictions of global
warming (Clark et al. 2013; Franklin et al. 2013).
The aerobic metabolic rate of ectotherms is dependent
on activity level, and the standard metabolic rate (SMR)
reflects respiration that sustains basic physiological pro-
cesses of an unfed animal at rest under standardised con-
˙ ETS components will therefore affect ATP yields and effi-
ditions. Maximal metabolic rates (M O2max) represent an
animal’s oxygen uptake during periods of high physi-
˙ thermally acclimated animals.
ological demand. Measurement of the M O2max and SMR
˙
permits calculation of the aerobic metabolic scope (M
O2max−SMR = AMS). When determined across a range of
temperatures an inflection in the AMS can pinpoint critical
thermal limits on aerobic metabolism (Tcrit) (Farrell 2002;
Pörtner and Knust 2007; Pörtner et al. 2004).
˙ found in harbours, coastal reefs and in intertidal rock pools.
M O2max generally increase more substantially with tem-
perature than SMR, indicating disproportionate demands on
oxygen flux as temperatures increases while conse-
quently increasing the AMS (Pörtner et al. 2004). The Tcrit
˙
predicted from AMS and M O2max—temperature plots often
occur at lower temperatures compared to Tcrit values pre-
dicted from the SMR (Fry 1971).
This discrepancy suggests a loss of stability at high res-
piration rates (or O2 flux) as temperatures rise, and this
likely impacts ATP synthesis capacities of mitochondria.
This also likely coincides with changes and increased ATP
demands to support muscle contraction and recovery
immediately post exercise.
“Leak” state respiration, which represents the mitochon-
drial oxygen uptake not contributing to oxidative phospho-
rylation (OXP), likely results from hydrogen ion leak from the
mitochondrial intermembrane space back to the matrix
through uncoupling proteins the adenine nucleotide trans-
locase (ANT) and inner membrane and potential electron
slippage within the electron transport system (Pesta and
Gnaiger 2012). Leak respiration generally increases with
temperature (Hilton et al. 2010; Iftikar and Hickey 2013;
Iftikar et al. 2010, 2014; Parks et al. 1955), and likely results
from elevated proton permeability through increased
membrane fluidity with rising temperatures (Seebacher et al.
2010; Zukiene et al. 2010). This acts to depress mito-
chondrial membrane potentials as temperature rises (Zuk-iene
et al. 2010) and suppresses ATP synthesis (Iftikar and Hickey
2013). The increase in non-productive or inefficient leak
respiration may explain why whole animal metabolism is
compromised at high temperatures in ectotherms with high
demands on ATP synthesis (Iftikar and Hickey 2013;
13
J Comp Physiol B (2014) 184:991–1001
Iftikar et al. 2014; Seebacher et al. 2010). While thermal
acclimation of mitochondria has been explored and dis-
cussed (Bouchard and Guderley 2003; Guderley and St-
Pierre 2002; Seebacher et al. 2010; St-Pierre et al. 1998),
little of the previous work has focused on the relative con-
tributions of components of the electron transport system
and relative proportions of flux derived from the OXP and
the ETS when flux is measured in the presence of a chemi-
cal uncoupler (ETSu) at different temperatures (Iftikar et
al. 2014). For example Complex II of the ETS, which does
not directly pump protons contributes less to ATP syn-
thesis that Complex I. Changes in flux contributions from
ciency, and this has yet to be explored in skeletal muscle of
Here we test how animal respiration and skeletal mus-cle
mitochondrial function at different temperatures using an
intertidal New Zealand triplefin fish, Forsterygion lapil-lum.
We chose skeletal muscle because in unfed states this
represents the bulk of a fish’s metabolically active tissue.
Forsterygion lapillum, is a small (20–50 mm) benthic fish
Lower latitude populations inhabit waters with winter and
summer sea surface temperatures of approximately 15 and 23
°C respectively, and rockpool habitats occasionally, albeit
briefly, reach 28 °C (Hilton et al. 2010). This species
therefore provides a useful experimental model to explore
thermo-tolerance and acclimation metabolism, which is
becoming increasingly relevant in the contexts of climate
change (Iftikar and Hickey 2013).
Fish were acclimated to a range of temperatures to
determine the SMR, MMR and AMS. Then mitochondria
within permeabilised skeletal muscle fibres were com-
pared between fish acclimated to 18 °C (a cool acclimation
group—CA) and 24 °C (a warm acclimation group—WA),
the latter representing a maximal but chronically toler-able
temperature. These fish then had their mitochondrial
parameters tested at different 18, 24 and 25 °C, with 25 °C
providing insight to the effects of temperature immedi-
ately above the maximum tolerable temperature. Multiple
substrates were used in assays to activate the two princi-pal
electron chains centred around Complexes I(CI) and
II (CII), both independently and in combination. Respira-
tion was measured in the leak, OXP and uncoupled state to
resolve potential limits on skeletal muscle mitochondria
and how their function alters with acclimation. We also
measured tissue lactate loads at rest and following exhaus-
tive exercise at relevant acclimation temperatures. Overall
we aimed to test whether any peak in the AMS tempera-
ture relationship was mirrored by changes in mitochondrial
function with subtle elevations in temperature. We show
that while the combination of substrates to fuel both CI and
CII simultaneously elevates flux in both acclimation
J Comp Physiol B (2014) 184:991–1001\
groups, acclimation impacts the stability of mitochondrial
function, with greater stabilities yet lower overall
capacities being apparent in WA fish.
Materials and methods
Specimens and acclimation
Forsterygion lapillum (2.08 ± 0.087 g, 69 ± 2.98 mm SL)
were collected using baited minnow traps and also using
custom-built slurp-guns on snorkel at Ti Point and Mathe-
son’s Bay around Leigh, New Zealand. Upon capture they
were immediately transported to the Leigh Marine Labora-
tory, University of Auckland. Specimens were acclimated
to one of five different temperature treatments (15, 18, 21,
24 and 25 ± 0.5 °C) for a minimum of 4 weeks in temper-
ature-controlled tanks. The responses of whole animal per-
formance traits by acclimation in fish can be observed by
4–6 weeks of exposure to the new external condition (Con-
don et al. 2010; Hammill et al. 2004; Johnston and
Lucking 1978; Wilson et al. 2007). Mitochondrial
oxidative capaci-ties are known to alter in fish within 2
weeks of warm and cold acclimation (Bouchard and
Guderley 2003). We contend that the 4 week steady state
exposure used in this study would have led to complete
acclimation of F. lapil-lum. Three insulated 36 L tanks
were used to house fish at an approximate low density of 6
3 specimen) were taken to test for bacterial respiration. This
g/m at each temperature. Temperatures did not vary more
than ±0.5 °C during the 4 week acclimation period in any
tank and water which was pumped from the ocean was
−1
heated and then pumped a 200 mL min (replacing each
tanks’ volume every 3 h). All specimens were exposed to a
constant 12L: 12D light cycle and were fed daily on a
mixture of freeze-dried mysis shrimp and fresh mussel.
Respirometry
24 h prior to experimentation, fish were housed individu-ally
and fasted. Metabolic rate was determined as the mass-
˙ ratus for several days. M O2max were induced by remov-
specific rate of oxygen consumption (M O2) per hour per
gram. Measurements were carried out with an automated,
intermittent-flow respirometer (Khan and Herbert 2012). A
100 mL acrylic tube respirometry chamber housed individ-ual
fish and was placed in an insulated 36 L reservoir tub filled
with filtered, UV sterilised seawater. The entire appa-ratus
was maintained at the specimens’ acclimation temper-ature
using freshwater within coils of PVC that was heated/ cooled
® oxygen debt repayment as a proxy for M O2max is often used in
by a Julabo F12 heater/chiller unit. The reservoir was
aerated rigorously using a compressor connected to an air
stone and a “flush” pump (Sicce® MiMouse) was used to
flush aerated water (from the reservoir) through the chamber.
The apparatus was connected to a custom-built
993
data acquisition system (DAQ) and a computer was used to
control the measuring/flush phase of the respirometry protocol
(see below). Adequate mixing in the chamber was achieved
using a Gilson® Minipuls 2 peristaltic pump that continuously
˙
circulated water. M O2 was measured repeat-edly by
switching the flush pump off for 20 min and meas-uring the
decline in [O2] within the chamber in a sealed state. During
respiration measurements, [O2] was main-tained above 85 %
saturation, and water was re-oxygenated to 100 % between
measurement phases in 5 min pulses with the flush pump. The
change in oxygen saturation in the chamber was recorded by a
fibre-optic O2 sensor (NTH-PSt1-L5-NS-40x1, 20-YOP.
Presens, Germany) connected to a Microx® TX3 meter
(Presens, Germany) operating at a frequency of 1 Hz. The
Microx® oxygen measurements were integrated using custom
software, which also coor-dinated the flush pump control. The
decrease in chamber
˙
[O2] was calculated by the slope α ( O2sat/ t) and M O2 (mgO2
−1 −1
g h ) was subsequently calculated using the fol-
lowing formula:
−1
MO2 = α × VRESP × β × M
where Vresp is the volume of the respirometer less the vol-
ume of the fish (mL), β is the oxygen solubility constant
and M is the mass of the fish (g). After each experiment
was complete, background oxygen consumption readings
(rate of oxygen decline in the respirometer without the
was always negligible.
˙ ˙
M O2, SMR, M O 2max, and subsequently AMS, were
measured in 10 fish at each of the five acclimation tem-
˙
peratures (15, 18, 21, 24 and 25 °C). M O2 was recorded
from the point fish were first introduced to the chamber
and for the following 19 h. SMR was estimated by aver-
˙
aging the lowest 10 % of M O 2 values recorded from the
point fish were introduced to the respirometry chamber.
˙ 2
Only M O2 values with R values >0.85 for α were used.
Pilot experiments showed that SMR estimates were simi-
lar to those of specimens that had been left in the appa-
˙
ing fish from the respirometer and forcing the fish to the
point of exhaustion by lightly tapping its tail in aerated
seawater at the appropriate temperature for a period of 5
min. The specimen was then returned to the respirom-eter
˙
and the highest observed M O2 value resulting from three
short (10 min) measuring periods (Reidy et al. 1995)
˙
were assumed to be the M O2max. This method of meas-uring
˙
species that are not necessarily suited to
Brett-type swim flume respirometers (Clark et al. 2013;
Dupont-Prinet et al. 2013; Khan and Herbert 2012). AMS
was then estimated as the difference between the SMR and
˙
M O2max for each specimen.
13
\994
Mitochondrial experiments
Given the small size of F. lapillum mitochondrial isola-
tion was not feasible. Therefore, we used a permeabilised
fibre method used elsewhere (Hilton et al. 2010), which
preserves the arrangements and structure of mitochondria
within cells (Picard et al. 2011). The caudal peduncle, or
trunk musculature, is predominantly fast-glycolytic, with
minimal superficial slow-oxidative fibres (Hickey and
Clements 2003). Skeletal muscle fibres were dissected by
filleting each side of a fish. The myotomes were rap-idly
teased into 0.5 × 1 mm fibre bundles after placement in 1
mL ice-cold high-energy relaxing solution (in mM unless
stated, BIOPS: 2.77 CaK2EGTA, 7.23 K2EGTA, 5.77
Na2ATP, 6.56 MgCl2·6H2O, 20 taurine, 20 imidazole,
0.5 dithiothreitol, 50 K-MES, 15 Na-phosphocreatine and
50 Sucrose, pH 7.1). Fibres were then permeabilised, fol-
lowing the transfer into 2 mL fresh high-energy relaxing
solution with saponin (50 µg), and agitated for 30 min on
ice, as described previously (Veksler et al. 1987). Saponin
perforates the sarcolemma of muscle fibres by targeting
cholesterol but leaves intracellular structures such as mito-
chondria intact due to their lower cholesterol content. Sap-
onin and cytosolic constituents were washed from fibres
following three 10 min washes in ice-cold assay medium
(0.5 EGTA, 3 MgCl2·6H2O, 60 K-lactobionate, 20 taurine,
−1
10 KH2PO4, 20 HEPES, 160 sucrose and 1 g. L BSA,
essentially free fatty acid, pH 7.24 at 20 °C). Fibre bundles
were rapidly blot-dried on lint free filter paper and
approxi-mately 20–30 mg of skeletal muscle was weighed
for respi-ration assays.
A multiple substrate-inhibitor titration protocol was
employed to test respirational flux in vivo and to explore
the relative capacity of the ETS and OXP components
®
(Gnaiger 2008). Three OROBOROS Oxygraph-2Ks
(Oroboros Instruments, Innsbruck, Austria) were used, and
respiratory measurements of fibre bundles were performed
at 18, 24 and 25 °C in 2 mL incubation assay medium.
Respiration was measured as weight-specific oxygen flux
−1
[pmol O2 (s · mg wet weight) ], calculated as the time
derivative of oxy-gen concentration using DatLab 4.3.2.7
®
software, OROB-OROS (Innsbruck, Austria).
To avoid O2-limitation due to the diffusion constraints
across muscle fibres, the chamber stoppers were raised and
gaseous O2 was added to the header space left within the
chambers to super saturate the assay medium to ~450 nmol
−1
mL . Once achieved the stoppers were lowered expelling
remaining gas. Chambers were re-oxygenated to maintain
−1
oxygen above 280 nmol mL (Gnaiger 2008). The titra-
tion protocol maximised respirational flux through the
ETS. Firstly, malate (5 mM), glutamate (10 mM) and pyru-
vate (10 mM) were added to determine the leak state res-
piration [synonymous with state 2 respiration (Pesta and
13
J Comp Physiol B (2014) 184:991–1001
Gnaiger 2012)]. ADP (1.5 mM) was added to stimulate
oxidative phosphorylation (OXP), followed by cytochrome
c (10 µM), to test mitochondrial integrities, and then fol-
lowed 2 mM ADP to ensure Complex I(CI) mediated OXP
was maximised. Succinate was added to maximise OXP
flux with combined electron inputs from Complexes I and
II (CII). Carbonyl cyanide p-(trifluoromethoxy) phenyl-
hydrazone was then titrated (3 × 1 µM FCCP) to uncou-ple
respiration and to determine the maximal uncoupled flux
capacity through the electron transport system (ETS).
Complex I, II and III were selectively inhibited by addition
of rotenone (1 µM), malonate (15 mM), and antimycin a (1
µM) respectively. The residual non-mitochondrial oxy-gen
flux was subtracted from other measures of flux. Three
flux-control ratios were calculated. (1) The relative con-
tribution of leak respiration to OXP, (2) the ratio between
maximal ETS flux (uncoupled)/OXP, and the additive
effect of succinate (CII substrate) was determined by
subtracting the flux attributable to CI.
Concentrations of lactate, and aerobic and glycolytic
potential indicators (CS, LDH) in rested and exhausted
CA and WA fish
Fish were acclimated to either 18 or 24 °C for a period of 4
weeks prior to experimentation (n = 16 at each, mass 1.27
g ± 0.95 g, FL 49.56 mm ± 1.4 mm). 8 each of the 18 and
24 °C acclimated specimens were placed individually in 10
L buckets of 50 µm filtered seawater at their acclima-tion
temperature and chased to exhaustion by light physi-cal
stimulation applied to the tail. The specimens were
considered to be exhausted when fish failed to react to the
physical stimulus and the time to exhaustion for each indi-
vidual was recorded. Once exhausted each individual was
euthanized immediately and placed into liquid nitrogen.
The remaining 8 specimens at each of the acclimation tem-
peratures were transferred individually to 10 L buckets of
seawater at their acclimation temperature but were imme-
diately euthanized and placed in liquid nitrogen without
being chased to exhaustion.
A portion of peduncle muscle was cut from semi-
thawed frozen specimens and homogenised in a 1:10 w/v 4
% ice-cold per-chloric acid and neutralised with NaOH.
Lac-tate was measured in the muscle by the reversible L-
LDH method and all measurements were performed using
a Molecular Devices Spectramax-340 plate reading
spectro-photometer at 25 °C.
Additional muscle (~40–60 mg) was cut and equili-
brated within a 1:20 volume (w/v) of ice-cold homogenisa-
tion buffer (25 mM Tris–HCl pH 7.8, 1 mM EDTA, 2 mM
MgCl2, 50 mM KCl, 0.5 % Triton X100) in 2 mL centri-
fuge tubes containing metal ball bearings and homogenised
with a tissue lyser (Qiagen, New Zealand). Homogenates
J Comp Physiol B (2014) 184:991–1001\
were centrifuged at 14,000g and the resulting supernatant
removed and immediately assayed for citrate synthase (CS)
and lactate dehydrogenase (LDH), with modifications from
previous workers (Hickey and Clements 2003; Newsholme
and Crabtree 1986).
Citrate synthase (CS) was determined at 412 nm with
5 μL tissue homogenate in 100 mM Tris–HCl pH 8.0, 0.1
mM acetyl coenzyme A, and 0.2 mM 5,5′-dithiobis-(2-
nitrobenzoic acid) (DTNB), and reactions were started
following addition of 5 mM oxaloacetate. LDH was meas-
ured at 340 nm with 5 μL tissue homogenate using a reac-
tion mixture containing 100 mM Tris–HCl pH 7.0, 1 mM
EDTA, 2 mM MgCl2, 1 mM dithiothrietol (DTT) and 0.15
mM NADH, and reactions were started following the
addition of 1.5 mM pyruvate. Measurements were made at
340 nm by observing the disappearance of NADH. All
assays were conducted at 25 °C.
Statistical analyses
AMS estimates were subjected to ANOVA and Tukey post
hoc pairwise comparisons to identify any significant differ-
ences between acclimation temperatures. Comparisons of
enzyme activities were made using students’ T-tests. Where
Fig. 1 The standard metabolic rate (SMR, dashed line) and maxi-
˙ slopes of linear regressions are theoretically proportional to activa-
mal metabolic rate (M O2max solid grey line) of F. lapillum specimens
acclimated to 15, 18, 21, 24 or 25 °C and measured as the rate of oxy-
gen consumption (mgO2·g− ·h− , n = 10 at each temperature). The
1 1
aerobic metabolic scope (AMS, black solid line) was determined as
˙ significant differences between temperatures within each measure of
the difference between SMR and M O2max for each individual. Inset
995
normality was not met data were log transformed. T-tests
were also used to make comparisons of respirational fluxes
between treatment groups at respective assay temperatures.
Differences in skeletal muscle lactate were measured in
rested and exhausted F. lapillum following log transforma-
tion of data and a two way ANOVA (assay versus acclima-
tion temperature), followed by Holms-Sidak test for spe-
cific post hoc differences.
Results
Whole animal respirometry
SMR increased linearly with temperature in F. lapil-lum
between 15 and 24 °C but increased at a greater rate
˙
between 24 and 25 °C. M O2max were essentially stable over
the entire investigated temperature range except for a dis-
proportionate increase at 24 °C which decreased at 25 °C
(Fig. 1). AMS was therefore unchanged at all investigated
temperatures except for at 24 °C where it showed a signifi-
cantly higher peak than at 15 and 21 °C (Fig. 1). While the
AMS plot indicates the potential for a modest increase in
scope at the elevated temperature of 24 °C, slopes from the
˙
shows Arrhenius plots for SMR (open circles) and M O2max where the
tion energy of a system. Therefore, the more negative the slope the
greater the sensitivity to increases in temperature. Letters represent
metabolism. All values are shown with ± sem
13
\996
Fig. 2 Representative trace respiration titration protocol measuring
oxygen concentration (nmol. mL− , blue line, left y-axis) over time
1
(mins) and mitochondrial respirational flux (pmol O2. (s. mg)− , red
1 and Complex II (CII). Carbonyl cyanide p-(trifluoromethoxy) phenyl-
line, right y-axis). Titrations of mitochondrial substrates, poisons and
inhibitors and their time of addition are shown with arrows: Respi-
ration states are indicated. Leak respiration (state 2) represents res-
piration with Complex I (CI) substrates malate (M), pyruvate (P) and
glutamate (G) in the absence of ADP. Oxidative phosphoryla-tion
(OXP-CI) was then initiated with ADP addition (1.5 mM) and
cytochrome c (c) added to test mitochondrial integrity (no significant
effect was detected across all samples), and further followed by addi-
˙ addition of excess ADP, flux increased with CI substrates and
Arrhenius plots of M O2max and SMR differ (P < 0.05,
inset Fig. 1 inset), suggesting that SMR is potentially more
˙ substrates (OXP-CI, Fig. 3b). By the subtraction of leak
sen-sitive to temperature increase than M O2max.
Mitochondrial function
Mitochondrial outer membrane integrity remained intact at
all assay temperatures with no significant increase in flux
observed with the addition of cytochrome c (Fig. 2). We
note a 25–30 % increase in respirational flux on addition of
succinate, showing the added contribution of CII to
respira-tion. This contribution was also mirrored in
measurements following the addition of rotenone in the
FCCP uncoupled state. Data for this state were not
presented as the time till maximal inhibition was variable.
Respiration was generally highest in all respiration
states for the 18 °C acclimated fish relative to 24 °C accli-
mated fish at equivalent assay temperatures. Oxygen flux
generally increased with increasing temperature, and flux
rates of muscle fibres from fish acclimated to 18 °C and
measured at 18 °C were similar to 24 °C acclimated fish
assayed at 24 °C. This was most evident for leak state
respiration (Fig. 3a) with similar rates when groups were
assayed at their acclimation temperatures. Following
13
J Comp Physiol B (2014) 184:991–1001
tional ADP (2 mM) to ensure OXP was maximised. Succinate was
added to maximise OXP flux with combined electron inputs from CI
hydrazone was then titrated (3 × 1 µM FCCP) to chemically
uncouple respiration to determine the maximal uncoupled flux capac-
ity through the electron transport system (ETSu). Complex I was then
inhibited with rotenone and Complex III with antimycin. The residual
non-mitochondrial oxygen flux (ROX) was determined. Respiration
states discussed in the text have been highlighted. Note the increase
in O2 concentration during the assay represents reoxygenation, to
ensure O2 was not limiting (colour figure online)
this provided information on OXP capacities with CI
respiration from OXP-CI it is apparent that similar frac-tions
of the net flux contribute to phosphorylation (Fig. 3b). This
residual fraction of OXP relative to leak was greater for 24 °C
acclimated fish at all temperatures indicating a relative
increase in the apparent efficiency in OXP-CI in this group
(Fig. 3c). The fraction of OXP-CI contributing to
phosphorylating respiration was between 5 to 10 % greater in
the 24 °C acclimated group for all temperatures meas-ured.
When assayed at respective acclimation temperatures the
acceptor control ratios ACRs were 5.73 ± 0.22 at 18 °C and
5.35 ± 1.99 at 24 °C (Fig. 3e).
In both CA and WA groups the addition of succinate
activated CII and increased flux above that of CI mediated
respiration (Fig. 3d). With succinate addition the fractional
flux increase was greatest for 18 °C acclimated fish
assayed at 18 and 24 °C with 35 and 30 % increases
respectively (Fig. 4b). However, when fibres from CA fish
were assayed at 25 °C, the fractional increase in OXP-CI
to OXP-CI,CII was equivalent to that for the WA fish.
While chemical uncoupling with FCCP also ele-vated
flux in both acclimation groups, FCCP had the
J Comp Physiol B (2014) 184:991–1001\ 997

Fig. 3 Respirational fluxes were

determined at 18, 24 and 25 °C a 0.7 Leak b 3.0 OXP CI *

(s.mg)-1

.(s.m
for skeletal peduncle muscle from
0.6 2.5

g)-1
F. lapillum moder-ate acclimation
temperature of 18 °C (open 0.5
2.0
circles, MA) and warm
0.4

2
acclimation temperature of 24 °C 1.5

.2
(closed circles, WA). Leak 0.3

m
ol
O
pmol

p
respiration (a) was higher in CA 1.0
0.1

O
OXP CI-Leak
fish (open circles) than WA fish
0.5

0.2
at all assay tempera-tures. b
Complex I(CI) medi-ated
oxidative phosphorylation (CI 0.0 0.0
Oxp flux, grey represents CI leak 17 19 21 23 25 17 19 21 23 25
flux prior to ADP addition).
While similar flux rates were
apparent for fish assayed at their c 0.7 Residual OXP (OXP-Leak) d 4.5
respective acclimation tempera- OXP CI,CII
0.6 4.0
.(s.mg)-

m
tures, flux rates are highest for in

g
s

1
(

)
.

-
CA fish at each temperature. c 0.4 3.5
1

2.5

0.5
Subtraction of the leak flux from 3.0

2
OXP-CI predicts that

.
2

the flux contributing to ATP

O
p

o
l
pmol

synthesis remains similar 0.3 2.0

O

1.0

across temperatures within 1.5

0.2
groups, but was lowest in the
CA group at 23 °C. d Addition
of succinate further elevated 0.1 0.5 OXP CI
respiration flux (black) in CA 0.0 0.0
and WA fish. The fractional 17 19 21 23 25 17 19 21 23 25
increase in suc-cinate mediated
flux is greater in CA fish than
in WA fish when assayed at 24 e 10 f 0.40 Leak/OXP-CI
ACR
°C, yet this increase was not
sustained with a further 1 °C 8
0.30
increase in assay temperature
(n = 4 duplicates, error bars = 6
SEM, *P > 0.05, ***P > 0.005) 0.20
4
0.10
2

0 0.00
17 19 21 23 25 17 19 21 23 25
Temperature (°C) Temperature (°C)

greatest effect when assayed at 18 °C (Fig. 4c). This chemi-
cal uncoupling response with FCCP diminished from a 15– 20
% flux increase to only 5 % flux increase as temperature was
increased to 25 °C in both groups. While the overall ETS flux
was highest in the 18 °C acclimated fish, the frac-tional
increase was similar in both groups (Fig. 4c).
To make comparisons between groups in terms of their
responses to temperature, we compared the change in
oxygen flux per °C ( O2 flux/°C) was calculated for each
group in different flux states (Fig. 4d). We note here that
traditional Q10 relationships are extremely sensitive over
small temperature increments and resulted in highly
variable estimates, moreover they do not represent direct
changes in flux. It was apparent that between 18 and 24 °C
acclimated groups, the 18 °C acclimated fish are more
responsive to increasing temperatures in all high flux states
(i.e. excluding leak). However, above 24 °C OXP-CI,CII
and ETS decreased with a change of only 1 °C in the 18 °C
acclimated fish while the 24 °C acclimated fish showed a
greater stability at higher temperatures.
Marker enzyme activities, resistance to exercise
fatigue and tissue lactate‑loading
Citrate synthase activities were almost 40 % higher in CA
fish WA fish, while LDH activities remained simi-lar
between treatments (Table 1). LDH/CS ratios further
reinforced a shift to increase aerobic capacities in CA fish
relative to WA fish (Table 1). CA fish took almost 30 %
longer to fatigue than WA fish (Table 1). Muscle lactate

13
\998 J Comp Physiol B (2014) 184:991–1001

Fig. 4 a Respiration flux a 0.4

increased from the coupled 5.00 ETS b 1.40 Flux increase in OXP CII

.(s.m
OXP flux (grey) state on addi-

g)-1
pmol O2. (s.mg)-1
1.20
tion of the uncoupling agent 4.00 0.80 0.35
0.3

1.00
FCCP (black). b The absolute
3.00
increase in flux declined with

ol O2
increasing temperature. c 0.60
2.00
When presented as a fractional

m
p
increase from the OXP flux OXP CI,CII 0.40 0.25
1.00 Fractional increase
state, the apparent “reserve” 0.20 with CII
ETS capacity decreases 0.00 0.00 0.2
similarly in both CA (open 17 19 21 23 25 17 19 21 23 25
circles) and WA (closed circles)
acclimated fish. d Comparison Temperature oC Temperature oC
o
of the changes in flux/ C in leak c 1.20 ETSu/OXP CI,CII 0.25 d 2.00
(squares), OXP-CI (diamonds),
Fractional increase
pmol O2. (s.mg)-1
OXP-CI,CII (triangles) and

Co
1.00 0.20 1.50
uncoupled respiration-ETS

(s.mg /-
1.00

1
(circles). In both groups of fish 0.80
0.15

)
OXP-CI,CII and ETS show a 0.50
0.60

2
lack of capacity to increase 0.10 0.00

O
0.40
flux between 24 and 25 °C. In -0.50

∇
0.05
CA animals a general loss of 0.20 ETS-OXP
-1.00
capacity in the 24-25 °C
0.00 0.00 18-24, 24-25, 18-24, 24-25,
occurs (n = 4 duplicates, error 17 19 21 23 25 18 Accl 18 Accl 24 Accl 24 Accl
bars = SEM, *P > 0.05)
Temperature o C 18oC 24oC
acclimated acclimated

Table 1 Mass length ratios showed no significant differences the predicted respirational flux that can contribute to ATP
between treatment groups pro-duction remains similar for both CA and WA groups at
Acclimation temperature 18 °C 24 °C their respective acclimation temperatures. While this indicates
an efficiency gain in ATP synthesis with warm acclimation,
Mass length ratio (g/mm) 0.033 ± 0.003 0.028 ± 0.001 mus-cle lactate loads in resting animals were high in WA
***
CS (U/g wet wgt) 3.33 ± 0.240 2.019 ± 0.18 (t 366) rested fish, demonstrating a relative deficiency in aerobic
LDH (U/g wet wgt) 177.7 ± 15.9 155.7 ± 10.2 (t −1.163) capacity even with acclimation to warmer temperatures.
LDH/CS 54.3 ± 3.9 83.9 ± 7.8*** (t −8.082)
**
An apparent maximisation of aerobic performance, or
Time till exhaustion (s) 213.6 ± 10.6 165.0 ± 12.01 (t 3.03) scope following acclimation to 24 °C could be inferred by
Citrate synthase (CS) activity differed while lactate dehydroge-nase the AMS to be maximal at 24 °C (Fig. 1) but these contrast
(LDH) did not, which was reflected by the ratio of LDH to CS with the species’ general habitat temperature of around 20
activity. Time till exhaustion was determined by stimulating fish till
exhaustion
°C. While temperatures as high as 28 °C are reached in
rock pools inhabited by F. lapillum (Hilton et al. 2010),
Mean ± sem, ** P < 0.01, *** P < 0.001, n = 16
this is limited to short periods at low tide. This species also
suffered from high mortality with chronic acclimation at
concentrations at rest and following exhaustive exercise 25 °C (55 % after 2 weeks) in this present work. The maxi-
detected a significant effect of acclimation temperature misation of AMS at temperatures towards the upper ther-
(ANOVA, F = 4.506, P = 0.04) and exercise stress (Fig. 5, mal limit of a species follows the alternative model of the
F = 10.68, P = 0.003). Most relevant for this study was the oxygen- and capacity-limited thermal tolerance (OCLTT)
lack of significant difference between rested WA fish and hypothesis (Clark et al. 2011, 2013). While high tempera-
exercised CA fish, indicating that relatively high baseline tures are encountered by wild fish populations, reproduc-
lactate loads are present in rested WA fish. tive success is greater at cooler temperatures (Clark et al.
2013; Richter and Kolmes 2005). Moreover, decreased
growth rates occur in northern killifish (Fundulus hetero-
Discussion clitus) acclimated to temperatures maximising the AMS
(Healy and Schulte 2012).
In this study, we demonstrate that while the apparent aerobic Although temperature-dependent growth rates have not
scope was maximal in WA fish, skeletal muscle mitochondrial been investigated in F. lapillum, this species appears to
capacities were lower relative to CA fish (Fig. 3a). However, preferentially select temperatures (21 °C) below those

13
 J Comp Physiol B (2014) 184:991–1001\
14
1
12
mas
wet s-
10
µmol
.gra
8
m
6 namic effects (Q10) on enzyme function, and also as a result
lact
ate
4 2
0 at 24 °C, as OXP flux rates of fibres from WA fish are less
18oC Rest 18oC Exhausted 24oC Rest
Fig. 5 Lactate is elevated in resting skeletal muscle of WA F. lapil-
lum. Skeletal muscle lactate was measured in rested and exhausted
WA and CA fish. Lactate was lowest in rested CA fish, which
elevated following exhaustive exercise (P = 0.02). Resting mus-
cle contained more lactate in WA (n = 8) fish than those CA fish
(n = 8, P = 0.017), and had similar lactate loads to exhausted CA
fish (P = 0.12) and exhausted WA fish (P = 0.36). Symbols represent
similar groups
where the AMS is maximal (Khan and Herbert 2012). Sur-
vival at 25 °C was limited, (55 % mortality after 2 weeks)
and not impacted at all at 18 or 24 °C (0 %).
We also provide a paradoxical observation in that while
˙
SMR does not increase with temperature as much as M
O2max in terms of absolute rates, SMR increases propor-
tionally faster with elevation in acclimation temperature
(inset Fig. 1), i.e. that it is more sensitive to temperature.
Arrhenius plots show that SMR has an approximately
˙ different crab species (Iftikar and Hickey 2013; Iftikar et al.
66 % greater sensitivity to temperature than MO2max.
As F. lapillum is a benthic species it will likely gener-
˙
ally function at levels closer to the SMR than MO2max.
One view is that this should increase resource utilisation
at higher temperatures. An alternative perspective is that
the ˙ MO may be under tighter constraints for increas-
ing capacity with rising temperature relative to the SMR,
in particular at temperatures above 24 °C, as species
must survive environmental extremes, not environmental
means.
As a cautionary note, we highlight that our measure-
ments of MO2max are not measured during sustained swim-
ming, as it is not possible to measure respiration rate while
swimming. We therefore make the assumption, as have oth-
ers (e.g. Clark et al. 2013; Dupont-Prinet et al. 2013; Khan
and Herbert 2012), that post-exhaustive exercise respiration
closely represents ˙ MO and in turn reflects maximum
rates of mobilization and disposal of lactate.
The AMS peak has been suggested to present a critical
point, beyond which organismal function is compromised
999
(Pörtner et al. 2004). For F. lapillum survival was impaired
above 24 °C and mitochondrial function (discussed below)
reflected this as it was less robust above 25 °C, or ceased to
increase regardless of acclimation. Therefore, AMS likely
represents for the maximal oxygen flow, but not the most
efficient use of oxygen. Temperature-mediated increases
in mitochondrial oxygen flux occur because of thermody-
of increased mitochondrial inner membrane proton leak,
the latter of which results in a loss of efficiency (Brand The s keletal mus cle fibre res pi ration data do not mir ror
1990).
the profiles for the whole animal AMS data, in particular
24oC Exhausted than those of CA fish. The increase in AMS at 24 °C may
therefore mirror higher order processes such as hepatic lac-
tate clearance or increased cardiovascular demands may be
elevate. Notably mortality for F. lapillum was not impacted
at 18 or 24 °C (0 %), but at 25 °C was 55 % mortality after
2 weeks. This also means that the AMS measurements for
25 °C acclimated fish warrant caution as it was performed
on the survivors, and therefore these fish do not represent
those of a general population.
Mitochondrial flux rates were also tested at saturating
substrate concentrations to mimic substrate supplies to
˙
skeletal muscle at MO2max. High sub strate concentrations
should elevate mitochondrial potentials, and therefore leak
respiration rates (Brand 1990, 2005), which are further
exacerbated by high temperatures (Fig. 3b, c). An increase
in mitochondrial leak respiration with increasing tempera-
tures is well described in ectothermic and endothermic
mitochondria (Seebacher et al. 2010; Zukiene et al. 2010),
and has been observed in permeabilised cardiac fibres of
2010), and triplefin fish heart fibres including F. lapillum
(Hilton et al. 2010).
Leak respiration flux was also similar for CA and WA
groups when measured and assayed at their respective
2max acclimation temperatures, indicating adaption of this vari-
able. Assuming that the subtraction of leak from OXP-CI
provides a measure of the residual flux that contributes to
ATP synthesis, it is apparent that this value remains simi-
lar across assay temperatures and acclimation regimes
(Fig. 3b). These data indicate that despite higher absolute
oxygen fluxes in OXP for CA fish, no more ATP is prob-
ably formed aerobically, but oxygen demand to support
leak respiration increases. The ACR, or ratio of leak res-
piration to OXP was higher in CA fish than WA fish at 18
and 24 °C, but both indices were similar at 25 °C (Fig. 3e,
2max f). This will place greater demands on the cardio-branchial
systems of CA fish acutely exposed to warmer tempera-
tures as more oxygen is required to maintain equivalent
ATP supplies.
13
\1000
The multiple substrate approach of this study also pro-
vides insight to dynamics of the ETS. Mitochondrial Com-
plexes I and II did not respond uniformly at each tempera-
ture, or with acclimation. Historically most mitochondrial
analyses employed simpler titration protocols with isolated
substrates (Galli and Richards 2012). Mitochondria in vivo
are fuelled by multiple substrates (e.g. CI and CII are
simultaneously active) and in combination these increase
oxygen flux and place additional demands on ubiquinone
pools and downstream respiratory complexes (Gnaiger
2011). While succinate addition should increase the respi-
rational flux in both acclimation groups, OXP-CI,CII flux
increased more in absolute terms and as a fractional meas-
ure (Fig. 4b) in CA fish. This increase was not uniform, as
while there was a 35 % increase at 18 °C in the CA group,
this increase dropped to ~30 % at 24 °C, and then 26 % at
25 °C. When measured at 18 °C the CA fish also showed
the greatest fractional increase in flux when transitioning
from the OXP-CI state to the OXP-CI,CII state (112 %).
This difference was less when assayed at 24 °C, and was
abolished (i.e. it was similar to WA fish) at 25 °C assay
temperatures (Fig. 4b). WA fish maintained similar frac-
tional increases regardless of temperature. These data indi-
cate that temperature acclimation influences the dynamics
within the ETS and OXP system. We also note that we
have observed different contributions to from CI and CII to
OXP in heart mitochondria from tropical and temperate
wrasse species (Iftikar et al. 2014).
The increase in flux with chemical uncoupling with
FCCP provided a measure of ETS reserve capacity (ETSu).
While the meaning of this apparent reserve capacity
remains unresolved, this too showed temperature sensi-
tivity and the flux enhancement with FCCP was greatest at
cooler temperatures. The CA fish appeared to show the
greatest increases with chemical uncoupling. However,
when presented as a percentage increase from the OXP
state to the uncoupled state, both acclimation temperatures
showed similar responses to temperature. This indicates
similar controlling, or limiting effects on the OXP system
at cooler temperatures, or of the ETS at higher tempera-
tures. The loss of excess capacity ranged from 15 to 20 %
when assayed at 18 and 24 °C, with only a 5 % increase
when assayed at 25 °C.
The OXP system (F1/F0 ATP synthase, phosphate
transporter and/or adenine nucleotide transporter (ANT))
may be limiting at cooler temperatures, and/or the ETS
may become limited at warmer temperatures. The greater
chemical uncoupling effect at cooler temperatures has been
observed in permeabilised teleost heart fibres (Iftikar and
Hickey 2013; Iftikar et al. 2014). A loss of the apparent
excess ETSu may impair the capacity, or plasticity of mito-
chondria to adjust to increased flux demands, or endog-
enous ETS inhibitors such as nitric oxide (Brown 1999).
13
J Comp Physiol B (2014) 184:991–1001
Given that F. lapillum survives brief exposure above this
temperature, anaerobic metabolism likely meets temporary
metabolic demands.
We further assayed fibres from both CA and WA groups at
25 °C. It was generally apparent that for the CA group, a drop
in OXP-CI, and OXP-CI,CII was observed. While for WA
fish 25 °C appeared to be better tolerated. These data indicate
that 24 °C is probably very close to the maximal temperature
that this species can chronically tolerate.
When fibres from CA fish were measured at 18 °C, they
showed 38 and 42 % higher OXP-CI,CII and ETSu fluxes
respectively relative to WA fish. When measured at a stand-
ard temperature of 24 °C, CS was also 40 % higher in CA
fish. The similarity between CS and mitochondrial flux
measures indicates a tight linkage between citric acid cycle
flux, OXP and the ETS, and this indicates a depression in
mitochondrial mass at 24 °C. LDH activities however dif-
fered less with acclimation temperature, and this indicates that
within these acclimation temperatures F. lapillum adjusts
aerobic pathways but not anaerobic glycolytic capac-ities. The
lack of shift in LDH and CS/LDH ratios means that CA fish
have greater aerobic capacities, yet have simi-lar anaerobic
capacities to the WA group. On that basis it is not surprising
that resting levels of lactate were higher in WA fish than
rested CA fish, and lactate levels within rested WA fish did
not differ significantly from fish exposed to exhaus-tive
stress. Exhaustion also occurred 30 % more rapidly in WA
fish than CA fish tested at 18 °C. These data indicate that WA
fish have less endurance than CA fish when stressed at their
acclimation temperatures, and this contrasts with the AMS
data predicting optimal aerobic performance at 24 °C.
In summary, we have shown that for this species the AMS
does not mirror mitochondrial function directly, but the peak
at 24 °C is consistent with a failure in metabolism
immediately above this point. Mitochondria within skeletal
muscle fibres showed an expected increase in leak respira-
tion, and this increased disproportionately relative to OXP-CI
with increasing temperature. With warm acclimation the
fraction of leak respiration could be decreased indicating that
species can adjust this parameter. The dynamics with multiple
electron inputs also show that the two major sites of electron
input (CI and CII) respond differently to tem-perature and
acclimation. Further research should explore the relative ATP
production rates at different temperatures in the presence of
different substrates (e.g. Iftikar and Hickey 2013). The limits
or control of the ETS and OXP systems also appear to change
with temperature and, while a rising leak respiration
contribution may depress OXP effi-ciencies, loss of the
reserve ETS capacity may also become limiting, and the
uncoupled ETS state presents less evi-dence for acclimation.
The decreased ETS reserve capacity and mitochondrial mass
may explain the high lactate loads in rested and WA F.
lapillum.
J Comp Physiol B (2014) 184:991–1001\
References
Bouchard P, Guderley H (2003) Time course of the response of mito-
chondria from oxidative muscle during thermal acclimation of
rainbow trout, Oncorhynchus mykiss. J Exp Biol 206:3455–3465
Brand MD (1990) The contribution of the leak of protons across the
mitochondrial inner membrane to standard metabolic rate. J
Theor Biol 145:267–286
Brand MD (2005) The efficiency and plasticity of mitochondrial
energy transduction. Biochem Soc Trans 33:897–904
Brown GC (1999) Nitric oxide and mitochondrial respiration. Bio-
chim Biophys Acta 1411:351–369
Clark TD, Jeffries KM, Hinch SG, Farrell AP (2011) Exceptional
aerobic scope and cardiovascular performance of pink salmon
(Oncorhynchus gorbuscha) may underlie resilience in a warming
climate. J Exp Biol 214:3074–3081
Clark TD, Sandblom E, Jutfelt F (2013) Aerobic scope measurements
of fishes in an era of climate change: respirometry, relevance and
recommendations. J Exp Biol 216:2771–2782
Condon CH, Chenoweth SF, Wilson RS (2010) Zebrafish take their
cue from temperature but not photoperiod for the seasonal plas-
ticity of thermal performance. J Exp Biol 213:3705–3709
Dupont-Prinet A, Vagner M, Chabot D, Audet C (2013) Impact of
hypoxia on the metabolism of Greenland halibut (Reinhardtius
hippoglossoides). Can J Fisheries Aquat Sci 70:461–469
Farrell AP (2002) Cardiorespiratory performance in salmonids during
exercise at high temperature: insights into cardiovascular design
limitations in fishes. Comp Biochem Physiol 132A:797–810
Franklin CE, Farrell AP, Altimiras J, Axelsson M (2013) Thermal
dependence of cardiac function in arctic fish: implications of a
warming world. J Exp Biol 216:4251–4255
Fry FEG (1971) The effect on environmental factors on the physiol-
ogy of fish. In: Hoar WS, Randall DJ (eds) Fish physiology, 4th
edn. Academic Press, New York, pp 1–98
Galli GLJ, Richards JG (2012) The effect of temperature onmitochon-
drial respirationin permeabilized cardiac fibres from the fresh water
turtle, Trachemys scripta. J Therm Biol 37:195–200
Gnaiger E (2008) Mitochondrial pathways through complexes I + II:
convergent electron transport at the Q-junction and additive
effect of substrate combinations. In: Gnaiger E (ed)
Mitochondrial pathways and respiratory control, 2nd edn.
MiPNet Publications, Innsbruck, pp 21–37
Gnaiger E (2011) Mitochondrial pathways through Complexes I + II:
convergent electron transport at the Q-junction and additive
effect of substrate combinations. Oroboros Instruments,
Mitochondrial Physiology Network
Guderley H, St-Pierre J (2002) Going with the flow or life in the fast
lane: contrasting mitochondrial responses to thermal change. J
Exp Biol 205:2237–2249
Hammill E, Wilson RS, Johnston IA (2004) Sustained swimming
per-formance and muscle structure are altered by thermal
acclimation in male mosquitofish. J Therm Biol 29:251–257
Healy TM, Schulte PM (2012) Thermal acclimation is not neces-sary
to maintain a wide thermal breadth of aerobic scope in the
common killifish (Fundulus heteroclitus). Physiol Biochem Zool
85:107–119
Hickey AJ, Clements KD (2003) Key metabolic enzymes and muscle
structure in triplefin fishes (Tripterygiidae): a phylogenetic com-
parison. J Comp Physiol B 173:113–123
Hilton Z, Clements K, Hickey A (2010) Temperature sensitivity of
cardiac mitochondria in intertidal and subtidal triplefin fishes. J
Comp Physiol B 180:1–12
Iftikar FI, Hickey AJR (2013) Do mitochondria limit hot fish hearts?
Understanding the role of mitochondrial function with heat stress
in Notolabrus celidotus. PLoS ONE 8:e64120
1001
Iftikar FI, MacDonald J, Hickey AJR (2010) Thermal limits of por-
tunid crab heart mitochondria: Could more thermo-stable mito-
chondria advantage invasive species? J Exp Mar Biol Ecol
395:232–239
Iftikar FI, MacDonald JR, Baker DW, Renshaw GMC, Hickey AJR
(2014a) Could thermal sensitivity of mitochondria deter-mine
species distributions in a changing climate? J Exp Biol
217:2348–2357
Iftikar FI, MacDonald JR, Baker DW, Renshaw GMC, Hickey AJR
(2014) Could thermal sensitivity of mitochondria determine spe-
cies distributions in a changing climate? J Exp Biol (in press)
Johnston I, Lucking M (1978) Temperature induced variation in the
distribution of different types of muscle fibre in the goldfish
(Carassius auratus). J Comp Physiol 124:111–116
Khan JR, Herbert NA (2012) The behavioural thermal preference of
the common triplefin (Forsterygion lapillum) tracks aerobic
scope optima at the upper thermal limit of its distribution. J
Therm Biol 37:118–124
Newsholme EA, Crabtree B (1986) Maximum catalytic activity of
some key enzymes in provision of physiological useful informa-
tion about metabolic fluxes. J Exp Zool 239: 159–163
Parks RE, Adler J, Copenhaver JH (1955) The efficiency of oxidative
phosphorylation in mitochondria from diabetic rats. J Biol Chem
214:693–698
Pesta D, Gnaiger E (2012) High-resolution respirometry: OXPHOS
protocols for human cells and permeabilized fibers from small
biopsies of human muscle. In: Palmeira CM, Moreno AJ (eds)
Mitochondrial bioenergetics. Humana Press, New York, pp 25–58
Picard M, Taivassalo T, Gouspillou G, Hepple RT (2011) Mitochon-
dria: isolation, structure and function. J Physiol
Pörtner HO (2002) Climate variations and the physiological basis of
temperature dependent biogeography: systemic to molecular
hier-archy of thermal tolerance in animals. Comp Biochem
Physiol 132A:739–761
Pörtner HO, Knust R (2007) Climate change affects marine fishes
through the oxygen limitation of thermal tolerance. Science 315
Pörtner HO, Mark FC, Bock C (2004) Oxygen limited thermal tol-
erance in fish? Answers obtained by nuclear magnetic resonance
techniques. Resp Physiol Neurobiol 141:243–260
Reidy SP, Nelson JA, Tang Y, Kerr SR (1995) Post-exercise meta-
bolic rate in Atlantic cod and its dependence upon the method of
exhaustion. J Fish Biol 477:377–386
Richter A, Kolmes SA (2005) Maximum temperature limits for chi-
nook, coho, and chum salmon, and steelhead trout in the Pacific
northwest. Rev Fisheries Sci 13:23–49
Seebacher F, Brand MD, Else PL, Guderley H, Hulbert AJ, Moyes CD
(2010) Plasticity of oxidative metabolism in variable climates:
molecular mechanisms. Physiol Biochem Zool 83:721–732
St-Pierre J, Charest PM, Guderley H (1998) Relative contribution of
quantitative and qualitative changes in mitochondria to metabolic
compensation during seasonal acclimatisation of rainbow trout
Oncorhynchus mykiss. J Exp Biol 201:2961–2970
Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA
(1987) Mitochondrial respiratory parameters in cardiac tissue: a
novel method of assessment by using saponin-skinned fibers.
Biochim Biophys Acta 892:191–196
Wilson RS, Condon CHL, Johnston IA (2007) Consequences of
thermal acclimation for the mating behaviour and swimming
performance of female mosquito fish. Philos Trans R Soc B
362:2131–2139
Zukiene R, Nauciene Z, Ciapaite J, Mildaziene V (2010) Acute tem-
perature resistance threshold in heart mitochondria: febrile tem-
perature activates function but exceeding it collapses the mem-
brane barrier. Int J Hyperthermia 26:56–66
13