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First published online January 30, 2009
Journal of Experimental Biology 212, 514-522 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024034
Do mitochondrial properties explain intraspecific variation in thermal tolerance?
1 Department of Zoology, University of British Columbia, 6270 University
Boulevard, Vancouver, BC V6T 1Z4, Canada
2 Department of Ecology, Evolution and Marine Biology, University of California
Santa Barbara, Santa Barbara, CA 93106, USA
* Author for correspondence (e-mail: fangue{at}lifesci.ucsb.edu)
Accepted 24 November 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: killifish, acclimation, adaptation, temperature, oxygen limited thermal tolerance, mitochondria
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Somero,
2005). Although the pervasive effects of temperature on
biochemical and physiological processes are thought to play a fundamental role
in establishing biogeographic patterns in aquatic ectotherms
(Hochachka and Somero, 2002),
the precise mechanisms involved in establishing both the upper and lower
thermal limits of organisms are still poorly understood
(Pörtner et al., 2006).
In light of evidence from polar, temperate and tropical ecosystems
demonstrating that climate change is affecting the distribution and local
abundance of many marine organisms
(Parmesan, 2006;
Pörtner and Knust, 2007;
Root et al., 2003), it is
critical to identify the precise physiological and biochemical mechanisms that
define the limits of an organism's thermal niche.
The concept of oxygen and capacity limited thermal tolerance (OLTT) has
recently been proposed as a unifying principle for understanding the
mechanistic basis of whole-organism thermal tolerance in ectotherms
(Pörtner, 2002;
Pörtner et al., 2007).
The OLTT hypothesis states that an ectotherm's thermal niche is linked to its
ability to make physiological adjustments in response to temperature in order
to maintain aerobic scope (the difference between maximum and resting
metabolic rate). At both low and high temperature extremes, the OLTT
hypothesis suggests that organismal performance is limited by the inability to
supply oxygen to the respiring mitochondria, i.e. that thermal limitations
result from a mismatch between oxygen supply and demand
(Pörtner, 2001;
Pörtner, 2002;
Pörtner et al., 2004).
Increasing temperatures result in an increase in the resting oxygen demand,
and since the ability of the circulatory and ventilatory systems to supply
oxygen has a maximum limit, aerobic scope declines as temperature increases,
causing a reduction in performance (reviewed by
Pörtner et al., 2004).
Decreasing temperatures, in contrast, are thought to cause a decline in the
ability of the mitochondria to produce ATP, compromising an organism's ability
to perform normal physiological functions including the function of the
ventilatory muscles and the circulatory pumps that are needed to supply oxygen
to the working tissues at low temperatures. Thus, acclimation or adaptation to
the cold is likely to involve increases in mitochondrial density and/or
changes in their functional properties to improve ATP production and maintain
aerobic scope in the cold. This increase in mitochondrial capacity in the
cold, however, may result in a tradeoff during warming because higher
mitochondrial capacity increases resting oxygen demand at warmer temperatures.
According to the OLTT hypothesis, this increased metabolic rate at warm
temperatures would be expected to decrease metabolic scope, thus decreasing
the upper thermal limits of the organism. Thus, Pörtner has suggested
that `adjustments of mitochondrial densities and their functional
properties appear as a critical process in defining and shifting
thermal tolerance windows'
(Pörtner, 2001).
One particularly interesting aspect of the OLTT framework is that it generates several specific predictions with respect to the relationship between mitochondrial properties and whole-organism responses to temperature: (1) cold-adapted or -acclimated organisms should have greater mitochondrial capacity (either increased mitochondrial amount or increased functional activity), (2) cold-adapted or -acclimated organisms with increased mitochondrial content or function should have higher standard (`resting') metabolic rate relative to warm-adapted organisms and (3) the increased metabolic rate in cold-adapted or -acclimated organisms should be associated with a decrease in thermal tolerance. The purpose of the current study was to test these predictions in a single species that can be acclimated to a wide range of temperatures and that has regional subspecies that are thought to be adapted to different thermal environments. By working within a single eurythermic species, we minimize the effects of phylogenetic differentiation among the organisms being compared and can simultaneously assess the effects of thermal acclimation and adaptation.
Populations of the common killifish (Fundulus heteroclitus) have
been studied extensively as a model to investigate mechanisms of local thermal
adaptation (Schulte, 2001).
These fish, which inhabit estuaries and salt marshes along the East Coast of
North America from Newfoundland to Northeastern Florida, have a broad capacity
to tolerate and acclimate to a wide range of environmental temperatures
(Fangue et al., 2006;
Powers and Schulte, 1998;
Schulte, 2001). The species
has been divided into two regional subspecies – a northern form, F.
h. macrolepidotus Walbaum, and a southern form, F. h.
heteroclitus Linnaeus (Morin and
Able, 1983) – that differ both genetically and
physiologically. We have previously shown that these subspecies differ in
thermal tolerance such that the putatively cold-adapted northern form has a
lower thermal tolerance than the southern form at all acclimation temperatures
and that in both subspecies thermal tolerance increases with increasing
acclimation temperature (Fangue et al.,
2006). In the present study, we assess whole-organism metabolic
rate and mitochondrial properties of killifish from the northern and southern
subspecies acclimated to a range of temperatures in order to test some of the
predictions of the OLTT hypothesis.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Six
replicate 75 liter tanks were used for each acclimation temperature (5°C,
15°C and 25°C), and fish were housed in a split-tank design with
northern fish on one side and southern fish on the other to minimize tank
effects. Treatment of all experimental animals was in accordance with the
University of British Columbia animal care protocol #A01-0180.
Estimation of mitochondrial amount
Four male and four female killifish from each subspecies and temperature
acclimation group were sampled for the estimation of mitochondrial amount
using mitochondrial enzyme activity and gene expression as proxies. Mean mass
was not significantly different between subspecies, acclimation groups or sex
(means ± s.e.m.; northern killifish, 7.39±0.72 g; southern
killifish, 7.94±0.56 g).
Total RNA was extracted from muscle and liver tissue as described in a
previous study (Fangue et al.,
2006). Gene-specific primers for citrate synthase (CS)
and cytochrome c oxidase subunit two (COXII) were designed
using Primer Express software (version 2.0.0; Applied Biosystems, Foster City,
CA, USA) from published sequences for F. heteroclitus: Accession No.
CN983139, CS forward 5' CGG CAT GAC GGA GAT GAA CT 3',
CS reverse 5' GAG GGC CCG GGA CAC A 3'; Accession No.
AY735182, COXII forward 5' AGT TTA GGA ATC AAA ATA GAC GCA GTT
3', COXII reverse 5' CGG GAG GTA ATG AAG GCT GTT
3'; qRT-PCR reactions and melt curve analyses were performed as
described previously (Fangue et al.,
2006). Results are expressed relative to total RNA used in the
reverse transcription reaction.
To assess mitochondrial enzyme activity, frozen liver or muscle tissue was
homogenized in 9 volumes of ice-cold buffer (100 mmol l–1
Hepes, 5 mmol l–1 EDTA, 1 mmol l–1 DTT and
0.05% Triton T-100, pH=7.4 at 20°C) using two low-speed passes of 10 s
each with a Polytron homogenizer (Fisher Scientific, Nepean, ON, Canada).
Cellular debris was removed by a 5 min centrifugation at 2500
g and 4°C, and preliminary tests ensured the complete
release of enzymes from the tissues using this procedure. The remaining
supernatant was diluted with buffer containing only 100 mmol
l–1 Hepes and 5 mmol l–1 EDTA, pH=7.4 at
20°C as appropriate for each assay. COX and CS activities were determined
as described previously (Moyes et al.,
1997). Protein concentrations were determined using the
bicinchoninic acid (BCA) method.
Mitochondrial respiration
Four to six fresh killifish livers were pooled for each mitochondrial
preparation (approximately 1 g total tissue) to obtain sufficient material for
5–7 respiration assays. Liver tissue was finely diced on cooled glass
plates and introduced to 9 volumes of ice-cold isolation medium (250 mmol
l–1 sucrose, 50 mmol l–1 KCl, 25 mmol
l–1 KH2PO4, 10 mmol
l–1 Hepes, 0.5 mmol l–1 EGTA, 1.5% bovine
serum albumin, fraction V `fatty acid free', pH=7.4 at 20°C)
(Bagarinao and Vetter, 1990).
Liver tissue was then homogenized on ice by three passes with a motorized
Teflon® tissue grinder. The resulting homogenate was centrifuged at 600
g for 10 min at 4°C to pellet cellular debris. The
supernatant was transferred to a new, pre-cooled tube by pouring through glass
wool to remove the majority of the fat. The defatted supernatant was then
centrifuged at 6000 g for 10 min at 4°C to pellet the
mitochondria, and any remaining fat was carefully removed from the
preparation. The pellet was washed twice with isolation media, gently
resuspended and kept on ice until all mitochondrial respiration measurements
were completed.
Oxygen consumption of isolated mitochondria was measured with an Oroboros
Oxygraph 2-k high-resolution respirometry system (Oroboros Instruments,
Innsbruck, Austria). The oxygen electrodes were calibrated daily with
air-saturated assay medium (150 mmol l–1 KCl, 25 mmol
l–1 KH2PO4 and 20 mmol
l–1 Hepes, pH=7.4 at 20°C)
(Bagarinao and Vetter, 1990) at
each experimental temperature, and zero oxygen measures were made by the
addition of sodium dithionite. Oxygen solubility in the assay medium at each
temperature was calculated as described previously
(Gnaiger and Forstner, 1983).
Oxygen consumption of approximately 0.3 mg mitochondrial protein in 1.8 ml
air-saturated assay medium was measured following the addition of 0.25 mmol
l–1 malate to spark the Krebs cycle, and 5 mmol
l–1 pyruvate was added as the carbon substrate. Pyruvate was
selected in order to obtain maximum rates of State III respiration in fish
mitochondria (Johnston et al.,
1998). State III rates of oxygen consumption were obtained by
adding saturating ADP to a concentration of 0.625 mmol l–1.
When all ADP had been phosphorylated, the rate of State IV respiration was
measured for 5 min before oligomycin, an inhibitor of mitochondrial
F0F1-ATPase, was added at 0.625 mmol
l–1 (State IVol respiration reflecting proton
leak). Respiratory control ratios (as indices of mitochondrial coupling) were
determined by dividing State III by State IV (RCR) or State III by State
IVol (RCRol)
(Estabrook, 1967).
Mitochondrial preparations were first assayed at the fish's acclimation temperature (5, 15 or 25°C), and the RCR was calculated to determine the coupling of the preparation. Preparations with RCR values substantially less than 4 were not used for further analysis. Samples of the mitochondrial preparation were assayed at temperatures between 2 and 37°C. The order of assay temperature was randomized, and the final assay of each mitochondrial preparation was repeated at the fish's acclimation temperature to ensure that the preparation had not become progressively uncoupled over the duration of the experiment. All assays for each mitochondrial preparation were completed within 8 h from the start of the mitochondrial isolation, but preparations were often stable for more than 12 h. Within each acclimation temperature group and killifish subspecies, mitochondrial respiration measurements were conducted at each assay temperature for 4–6 independent mitochondrial preparations. Protein concentrations were determined using the BCA method.
Whole-animal respirometry
Mass-specific oxygen consumption (µmol g–1
h–1) was determined for northern and southern killifish
acclimated to and tested at one of three temperatures (5, 15 or 25°C) or
acclimated to 5°C and acutely challenged with increasing measurement
temperatures. Mean mass did not differ between subspecies (northern killifish,
5.98±0.25 g, N=22; southern killifish, 6.71±0.34 g,
N=21). Fish were placed in 250 ml flow-through respirometers at their
acclimation temperature overnight (12–18 h) to recover from handling
stress and were not fed during this time. Following the recovery period, an
oxygen probe (FOXY-R, Ocean Optics Ltd, Dunedin, FL, USA) was introduced and
the respirometer sealed. The decline of oxygen was recorded, and the
mass-specific oxygen consumption was calculated as previously described
(Sloman et al., 2008). In the
acute thermal challenge experiment, six northern 5.49±0.27 g
(mean ± s.e.m.) and six southern 6.64±0.82 g killifish
were held in respirometers overnight at 5°C, and metabolic rate was
measured as previously described. The respirometer was then opened and flushed
with well-oxygenated water while the temperature was increased by 5°C at a
rate of 0.3°C min–1. The respirometer was then closed and
metabolic rate was measured over the course of 30 min. This procedure was
repeated such that metabolic rate was measured at temperatures of 5, 10, 15,
20 and 25°C. Oxygen consumption measurements at water temperatures of
30°C could not be determined because, at these temperatures, killifish
began to lose equilibrium, leading to heat death.
Statistical analyses
Killifish metabolic rates, mitochondrial respiration measurements, enzyme
activities and mRNA data sets were analyzed by multiple analysis of variance
(ANOVA) with subspecies, acclimation group and/or assay temperature as
factors. Metabolic rate data from the acute thermal challenge experiment were
analyzed with a two-factor, repeated-measures ANOVA. All data met the
assumptions of normality, and data were log transformed where necessary to
meet assumptions of homogeneity of variance. When interaction terms were not
significant, post-hoc comparisons were performed among the groups
with the Student-Newman-Keuls multiple range test (SNK MRT). If the
interaction terms were significant, the data were separated and analyzed
independently using one-way ANOVA followed by SNK MRT. For all analyses,
was set at 0.05. For the analysis of proton leak (IVol),
slope discontinuities were determined using the Regress algorithm developed by
Yeager and Ultsch (Yeager and Ultsch,
1989) for statistical determination of critical points in
continuous data sets by determining the intersection of two best fit
lines.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Lucassen et al.,
2006; Moyes et al.,
1997). Liver CS activity differed significantly between subspecies
and with acclimation temperature (two-way ANOVA P=0.013,
P<0.001 respectively), and there was no interaction
(Table 1). Cold acclimation
increased CS activity in both subspecies, and northern fish had significantly
higher CS activity compared with southern fish (significant in
post-hoc tests at 5°C; Table
1). Similar results were observed in muscle (two-way ANOVA,
P=0.033 for subspecies and P=0.022 for acclimation
temperature), with increased CS activity in the cold and in northern fish
(Table 1). In both liver and
muscle, CS mRNA levels showed a similar pattern, as did protein
activity, with highest expression observed in cold-acclimated northern fish
(two-way ANOVA, liver P
0.003 for acclimation temperature and
subspecies, muscle P<0.001 for acclimation temperature and
subspecies) (Table 1).
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In the liver, COX activity was greater in the northern subspecies (two-way ANOVA, P=0.003), and there was no significant effect of acclimation temperature or an interaction (Table 1). This pattern was not observed in muscle, however, where there was no significant difference in COX activity between subspecies or acclimation groups (Table 1). Levels of COXII mRNA did not correspond with protein activity in either muscle or liver. In liver, COXII mRNA levels increased significantly with decreasing acclimation temperature (two-way ANOVA, P=0.001), and there was no significant difference between subspecies and no significant interaction (Table 1). In muscle, COXII mRNA levels differed significantly between subspecies, with northern fish having higher mRNA levels (two-way ANOVA, P=0.003; Table 1), and there was no significant effect of acclimation temperature or an interaction.
These data provide mixed evidence for an increase in mitochondrial amount in northern fish, or with cold acclimation. However, there is a consistent pattern that cold-acclimated northern fish tend to have greater mitochondrial enzyme activity and mRNA levels in most tissues than do southern fish. There is less support for differences in mitochondrial enzyme activity between subspecies when the fish are acclimated to warm temperatures.
Mitochondrial respiration
We measured maximum ADP stimulated (State III) oxygen consumption rates of
mitochondria isolated from liver as an index of mitochondrial functional
capacity. Respiratory control ratios, RCR (III:IV) as well as RCRol
(III:IVol; the respiratory control ratio calculated using the
respiratory rate of mitochondria inhibited by oligomycin), were measured as an
indicator of mitochondrial coupling (Table S1 in supplementary material).
Estimates were always greater than 4.0 (RCR) and 10.4 (RCRol) when
assayed at the fish's acclimation temperature. Thus, liver mitochondria from
both northern and southern fish were generally highly coupled, with only
modest (and non-statistically significant) declines in coupling at the highest
assay temperatures at which accurate State III rates could be determined.
Thermal acclimation affected the upper temperature sensitivity of mitochondrial oxidative phosphorylation. State III rates of mitochondria from both northern and southern fish acclimated to 5°C and assayed at 37°C could not be measured, as these mitochondria were insensitive to ADP at this assay temperature whereas mitochondria from 15°C- and 25°C-acclimated fish remained coupled and responsive to ADP up to 37°C, uncoupling only at higher temperatures.
Thermal acclimation had different effects on the response of mitochondria to acute temperature challenge in each killifish subspecies (Fig. 1). Mitochondria from both northern and southern fish acclimated to 25°C showed the expected exponential increase in State III respiratory rates with increasing test temperature. Surprisingly, this pattern was not maintained in mitochondria isolated from fish acclimated at 15°C or 5°C. The acute thermal response curves deviated from the standard exponential form as acclimation temperature decreased, and the extent and nature of this deviation differed between northern and southern fish. As a result, the relationship between the respiratory rates of northern and southern mitochondria at any given test temperature differed, depending on the acclimation temperature of the fish from which the mitochondria were derived. At low test temperatures (2 and 5°C), mitochondria from northern fish had greater State III respiratory rates than those from southern fish when the mitochondria were isolated from fish acclimated to 5 and 25°C but not when mitochondria were isolated from fish acclimated to 15°C. In contrast, at higher test temperatures (25 and 30°C), mitochondria isolated from southern fish had higher State III respiratory rates than those isolated from northern fish when the fish were acclimated to either 5 or 15°C but not when the fish were acclimated to higher temperatures.
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Warm acclimation came at a cost in terms of mitochondrial function in the cold. Warm-acclimated mitochondria (25°C) from both northern and southern fish exhibited State III respiratory rates one-third lower than rates for 5°C- or 15°C-acclimated mitochondria in both subspecies when assayed at low temperatures (compare Fig. 1C with Fig. 1A,B). Interestingly, mitochondria isolated from warm-acclimated southern fish tended to have reduced performance compared with mitochondria from cold-acclimated southern fish, even when assayed at the warm acclimation temperature (25°C). At 25°C, warm-acclimated mitochondria from southern fish respired at approximately one-half the rate of mitochondria isolated from cold-acclimated southern fish (compare Fig. 1C with Fig. 1A). In contrast, mitochondria from northern killifish, regardless of acclimation temperature, had equivalent State III rates between acclimation groups when assayed at 15 and 25°C.
We determined the rates of oxygen consumption for mitochondria inhibited by oligomycin (State IVol) as an indicator of proton leak. Assay temperature had a significant effect on State IVol, with rates increasing exponentially with assay temperature in all acclimation groups and for both subspecies (Fig. 2). However the State IVol rates were always low compared with the ADP-stimulated (State III) rates and demonstrate no evidence of the extreme non-linearities observed in the State III rates, and thus these data cannot explain the unusual shape of the State III oxygen consumption curves, particularly those observed for mitochondria from cold-acclimated fish (Fig. 1A).
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Whole-organism metabolic rate
To determine whether the differences in mitochondrial properties between
subspecies and acclimation groups had any effect on properties at higher
levels of organization, we measured whole-organism oxygen consumption of fish
from both subspecies acclimated to 5, 15 and 25°C. There was a significant
effect of subspecies and acclimation temperature on metabolic rate, with no
significant interaction (two-way ANOVA, P<0.001 for subspecies and
acclimation temperature) (Fig.
3A). As expected, metabolic rate increased with increasing
acclimation temperature. Across the whole temperature range, Q10
for metabolic rate was approximately 2.3 and was similar between the two
subspecies. Q10 was slightly greater between 5 and 15°C (2.8)
and slightly lower (1.9) at higher temperatures in both killifish subspecies.
At all acclimation temperatures, northern fish had a higher metabolic rate
than did southern fish.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The OLTT hypothesis predicts that both cold acclimation and adaptation
should cause an increase in mitochondrial amount. Although there is ample
evidence for an increase in mitochondrial amount with cold acclimation in a
variety of species (Guderley,
2004; Johnston et al.,
1998; Pörtner et al.,
2007), we observed only modest, if any, increases in mitochondrial
enzyme activity in response to cold acclimation, and the responses differed
between enzymes and tissues. It is possible that mitochondrial enzyme activity
is simply a poor proxy for mitochondrial amount, as other studies have also
observed disconnections between the effects of acclimation on the activities
of various mitochondrial enzymes (Lannig
et al., 2003; Lucassen et al.,
2003; Lucassen et al.,
2006). To account for this possibility, we also examined mRNA
levels for these genes as an alternative index of mitochondrial amount.
COXII mRNA has been suggested as a useful measure of the
transcription rate of the mitochondrial genome as the entire mitochondrial
genome is transcribed simultaneously
(Fernandez-Silva et al., 2007).
Our results show that cold significantly increases COXII mRNA
expression in the liver in both northern and southern fish but that this was
not the case in muscle. Taken together, these data clearly indicate that there
is no simple and uniform response of all tissues to increase mitochondrial
amount in response to cold acclimation in F. heteroclitus.
Similarly, our data only provide equivocal support for the prediction of
the OLTT hypothesis that cold adaptation is associated with increases in
mitochondrial amount, as there are few consistent differences in mitochondrial
amount between the subspecies across all acclimation temperatures. However,
our data do suggest that there may be an interaction between cold adaptation
and cold acclimation in this eurythermal species, in that differences between
the subspecies in both mitochondrial enzyme activity and mRNA levels were much
more pronounced in fish acclimated to low temperatures than in fish acclimated
to warm temperatures. Six of the eight variables tested differed significantly
between subspecies in cold-acclimated fish while only two of these variables
differed between subspecies in warm-acclimated fish. Thus, Fundulus
heteroclitus macrolepidotus (the northern subspecies) may have somewhat
greater capacity to acclimate to the cold by increasing mitochondrial amount
than does Fundulus heteroclitus heteroclitus (the southern
subspecies). A similar pattern has been seen in cod populations distributed
across a latitudinal cline. Lucassen et al. showed that, in both muscle and
liver, differences between cod populations in mRNA expression and enzyme
activities of CS and COX were more significant when fish were acclimated to
colder temperatures (Lucassen et al.,
2006). Together, these similar observations in two
phylogenetically distant fish species suggest that there may be a common
pattern of increased plasticity in mitochondrial amount in cold-adapted
eurythermal fishes.
The OLTT hypothesis also predicts that mitochondrial function (as measured
by State III respiratory rates) should increase with cold acclimation, and
substantial previous data in other species support this idea
(Bouchard and Guderley, 2003;
Guderley and Johnston, 1996;
Kraffe et al., 2007;
Sommer and Pörtner,
2004). In contrast, our data do not show a strong signal of
increased mitochondrial respiratory rates in mitochondria isolated from fish
acclimated at lower temperatures, when compared at equivalent assay
temperatures. Warm acclimation did, however, result in a shift in the upper
thermal sensitivity of mitochondria to acute temperature challenge such that
mitochondria from northern and southern fish acclimated to 15 and 25°C
remained responsive to ADP at assay temperatures up to 37°C
(Fig. 1B,C) whereas the
respiratory rates of 5°C-acclimated mitochondria could not be measured at
temperatures greater than 35°C (Fig.
1A). Similarly, Dahlhoff and Somero found that the Arrhenius break
temperature for mitochondrial function increased with acclimation temperature
in multiple eurythermal species of abalone
(Dahlhoff and Somero, 1993).
This idea is consistent with the OLTT hypothesis, in that the more thermally
sensitive mitochondria from cold-acclimated fish cease to function at a lower
temperature upon acute warming, potentially limiting the aerobic scope of
cold-acclimated fish at warm temperatures. In addition, we observed that the
inflection point of the response of State IV respiration to acute temperature
challenge was somewhat different between different acclimation groups
(Fig. 2), with mitochondria
from warm-acclimated fish having a higher inflection temperature than
mitochondria from cold-acclimated fish. Although at present no data are
available that bear on the possible mechanistic basis for these differences, a
possible explanation could be changes in mitochondrial membrane lipid
composition with acclimation, resulting in different fluidity and phase change
behaviour. Such changes have been observed in other fish species (e.g.
Kraffe et al., 2007) and have
been associated with changes in mitochondrial function.
One additional critical issue that must also be considered is that, at present, complete data on the thermal windows of F. heteroclitus are lacking. CTMax and CTMin provide an index of the maximum and minimum temperatures at which a fish can survive and thus provide an index of one aspect of the thermal window. The thermal windows for growth and reproduction are, however, likely to be much narrower, and it is this narrower index of the thermal window that is perhaps most relevant to considerations of the OLTT hypothesis. Measurements of the pejus temperature (i.e. the temperature at which aerobic scope begins to decline) may be particularly revealing with respect to the relationship between mitochondrial properties and the thermal window at the organismal level and thus provide a fruitful avenue for further study in this system.
The OLTT hypothesis also predicts that mitochondrial respiratory rate should increase with cold adaptation. Our data provide some support for this idea in that mitochondria from both cold-acclimated and warm-acclimated northern killifish have higher State III respiratory rates than do mitochondria from southern fish when assayed at low temperatures. However, this was not the case for mitochondria from fish acclimated to intermediate temperatures nor for mitochondria assayed at higher temperatures. In fact, at high assay temperatures, mitochondria isolated from intermediate and cold-acclimated southern killifish had higher respiratory rates than did northern mitochondria. This peculiar observation is the result of the complex shape of the acute response to temperature of mitochondria from cold-acclimated fish and the fact that these patterns differ between killifish subspecies.
One interesting aspect of the unusual shape of the mitochondrial
temperature curves for cold-acclimated fish is that mitochondria from northern
fish have a large zone across which the mitochondria demonstrate limited
thermal sensitivity (Q10=1.1, 20–30°C assay temperatures)
whereas the State III respiratory rates of southern mitochondria increased
with increasing assay temperature across this range (Q10=2.1) and
instead had a zone of limited temperature sensitivity (Q10=1.2)
over a higher temperature range. Studies comparing acute effects of
temperature on mitochondrial respiration with thermal acclimation and
adaptation are rare, and most recent studies have found simple linear or
exponential increases in State III rates with increasing assay temperatures,
with Q10s of 2–3 across a broad range of temperatures (e.g.
Johnston et al., 1998;
Sommer and Pörtner,
2004). However, early studies of eurythermal marine invertebrates
(Newell and Pye, 1970;
Newell and Pye, 1971) have
provided evidence for a large zone of thermal independence of mitochondrial
respiration across environmentally relevant temperature ranges, similar to
that observed here for cold-acclimated F. heteroclitus. The
observation that this region of relative thermal independence is at a higher
temperature in mitochondria from cold-acclimated southern fish compared with
those for mitochondria from cold-acclimated northern fish is consistent with
the observed difference between these subspecies in the position of their
thermal windows, which are shifted upwards by approximately 1.5°C in
southern fish relative to northern fish
(Fangue et al., 2006). However,
the observed shift in the thermal window between subspecies is present
regardless of acclimation temperature, whereas differences in the shape of the
acute response of the mitochondria to temperature change are not present in
warm-acclimated fish. This observation suggests that additional mechanisms
must be involved in specifying thermal windows in F.
heteroclitus.
A critical aspect of the OLTT hypothesis is that increases in mitochondrial
properties and function as a result of cold adaptation cause increases in
whole-organism metabolic rate, which in turn result in decreases in thermal
tolerance. To test this hypothesis, we assessed the metabolic rate of northern
and southern F. heteroclitus acclimated to a range of temperatures.
Consistent with the OLTT hypothesis, metabolic rate was greater in northern
fish than in southern fish at all acclimation temperatures. As expected under
the OLTT framework, this increased metabolic rate is associated with the
decreased thermal tolerance that we have observed in the northern subspecies
(Fangue et al., 2006).
Differences in the metabolic rate of killifish embryos
(DiMichele and Westerman, 1997)
as well as isolated heart tissue at warm acclimation temperatures
(Podrabsky et al., 2000) have
also been recorded, with northern fish having higher metabolic rates than
southern fish. However, these differences in whole-organism metabolic rate do
not relate in any simple way to the differences in mitochondrial properties
that we have observed between these subspecies; differences in mitochondrial
properties were more evident at low acclimation temperatures whereas the
differences in whole-organism metabolic rate between subspecies were present
at all acclimation temperatures.
In addition, measurements of metabolic rate at a single acclimation
temperature did not capture the effects of acclimation on the acute response
of the mitochondria to temperature that we have observed in the mitochondria
of this species. To determine whether these mitochondrial properties were
manifested at higher levels of organization, we assessed the effects of acute
thermal challenge on whole-organism metabolic rate in 5°C-acclimated fish,
which demonstrate a non-linear response of mitochondrial respiration to acute
thermal challenge. As was the case for mitochondrial respiratory rates,
whole-organism metabolic rates differed between subspecies at low temperatures
and then converged at intermediate temperatures. At high temperatures
(>20°C), however, the differences between subspecies in mitochondrial
respiratory rate were much greater than those at the whole-organism level.
Thus, the effects observed at the mitochondrial level are partially, but not
fully, reflected at the whole-organism level. Taken together, these data
support the idea that the relationship between maximum oxygen consumption
rates of mitochondria measured in vitro and mitochondrial metabolic
rates in vivo is not a simple one. In vivo rates of
mitochondrial respiration are influenced by a host of factors including
intracellular pH (Moyes et al.,
1988), availability and affinity for ADP and NADH
(Brand and Murphy, 1987;
Guderley and St Pierre, 1999),
membrane properties (Kraffe et al.,
2007) and delivery of oxygen and fuels by the circulation
(Mathieu-Costello, 1992). It
is possible that the oxidative performance of killifish mitochondria in
vivo is regulated at one or several of these levels.
Implications for whole-organism performance
Unifying principles relating the mechanisms of aerobic capacity modulation
to seasonal acclimatization and latitudinal thermal adaptation remain elusive.
The data presented here suggest that these two processes may be linked and
that thermal adaptation can proceed via changes in the ability to
acclimate to local temperatures. The potential for complex interactions
between acclimation (plasticity) and adaptation may be a critical component of
conceptual frameworks such as the OLTT (particularly for temperate zone
eurytherms), and consideration of these issues requires careful examination of
thermal niche size and shape. The size of the thermal niche as estimated using
CTMax and CTMin is similar between
killifish subspecies but shifted to slightly higher temperatures in southern
fish; killifish populations differ in their thermal tolerance such that
southern fish are 
1.5°C more tolerant of high temperatures than
northern fish at all acclimation temperatures
(Fangue et al., 2006). Swimming
performance studies in killifish, regardless of population of origin, have
shown that these fish maintain consistent swimming performance across a wide
thermal acclimation temperature range of 7–34°C
(Q101), with performance declining at temperatures outside of
this range. These data suggest that killifish have a wide temperature zone
over which aerobic scope is maintained and that the boundaries of the thermal
niche across which activity can be maintained may be quite similar between the
subspecies if sufficient time is allowed for acclimation
(Fangue et al., 2008). However,
we have also shown that there may be differences between the subspecies in
swimming performance in response to acute thermal challenge
(Fangue et al., 2008). In the
current study, we observed that northern fish have a higher metabolic rate
than do southern fish at all acclimation temperatures and that the acute
response of metabolic rate to temperature differs between the subspecies in
cold-acclimated fish. At the level of the mitochondria, we observed no
differences in mitochondrial amount or function between subspecies at warm
acclimation temperatures, despite the substantial differences in
whole-organism standard metabolic rate in these fish. We also observed
differences between populations in mitochondrial amount and function with cold
acclimation. Collectively, these data suggest that while northern fish are
slightly less thermally tolerant than southern fish, they may be able to
maintain high activity across a wider range of temperatures. It has been
suggested that high-latitude, cold-adapted eurythermal species exposed to more
thermally variable environments may be under strong selection for the
maintenance of plasticity, high metabolic rate, large metabolic scope and
broad tolerance (Angilletta et al., 2006). Our data support this suggestion
and clearly point to the use of contrasting strategies between killifish
subspecies and between acclimation temperatures. Thus, we conclude that
mitochondrial function and content modulation may differ between subspecies of
eurythermal killifish, leading us to suggest the possibility that variation in
plasticity may be an important component of local adaptation to a seasonally
variable environment in this species.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Bagarinao, T. and Vetter, R. D. (1990). Oxidative detoxification of sulphide by mitochondria of the California killifish Fundulus parvipinnis and the speckled sanddab Citharichthys stigmaeus. J. Comp. Physiol. 160,519 -527.
Bouchard, P. and Guderley, H. (2003). Time
course of the response of mitochondria from oxidative muscle during thermal
acclimation of rainbow trout, Oncorhynchus mykiss. J. Exp.
Biol. 206,3455
-3465.
Brand, M. D. and Murphy, M. P. (1987). Control of electron flux through the respiratory chain in mitochondria and cells. Biol. Rev. Camb. Philos. Soc. 62,141 -193.[Medline]
Dahlhoff, E. and Somero, G. N. (1993). Effects of temperature on mitochondria from abalone (Genus Haliotis): adaptive plasticity and its limits. J. Exp. Biol. 185,151 -168.[Abstract]
DiMichele, L. and Westerman, M. E. (1997). Geographic variation in development rate between populations of the teleost F. heteroclitus. Mar. Biol. 128, 1-7.[CrossRef]
Estabrook, R. W. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP/O ratios. Meth. Enzymol. 10,41 -47.[Medline]
Fangue, N. A., Hofmeister, M. and Schulte, P. M.
(2006). Intraspecific variation in thermatolerance and heat shock
protein gene expression in common killifish, Fundulus heteroclitus.J. Exp. Biol. 209,2859
-2872.
Fangue, N. A., Mandic, M., Richards, J. G. and Schulte, P. M. (2008). Swimming performance and energetics as a function of temperature in killifish, Fundulus heteroclitus. Physiol. Biochem. Zool. 81,389 -401.[CrossRef][Medline]
Fernandez-Silva, P., Enriquez, J. A. and Montoya, J. (2007). Replication and transcription of mammalian mitochondrial DNA. Exp. Physiol. 88,1 .
Gaston, K. J. (2003). The Structure and Dynamics of Geographic Ranges. New York: Oxford University Press.
Gnaiger, E. and Forstner, H. (1983). Polargraphic Oxygen Sensors: Aquatic and Physiological Applications. New York: Springer-Verlag.
Guderley, H. (2004). Locomotor performance and muscle metabolic capacities: impact of temperature and energetic status. Comp. Biochem. Physiol. 139,371 -382.[CrossRef][Medline]
Guderley, H. and Johnston, I. A. (1996). Plasticity of fish muscle mitochondria with thermal acclimation. J. Exp. Biol. 199,1311 -1317.[Abstract]
Guderley, H. and St Pierre, J. (1999). Seasonal cycles of mitochondrial ADP sensitivity and oxidative capacities in trout oxidative muscle. J. Comp. Physiol. 169,474 -480.
Hochachka, P. W. and Somero, G. N. (2002). Biochemical Adaptation: Mechanism and Process in Physiological Evolution. New York: Oxford University Press.
Johnston, I. A., Calvo, J., Guderley, H., Fernandez, D. and
Palmer, L. (1998). Latitudinal variation in the abundance and
oxidative capacities of muscle mitochondria in perciform fishes. J.
Exp. Biol. 201,1
-12.
Kraffe, E., Marty, Y. and Guderley, H. (2007).
Changes in mitochondrial oxidative capacities during thermal acclimation of
rainbow trout Oncorhynchus mykiss: roles of membrane protein
phospholipids and their fatty acid compositions. J. Exp.
Biol. 210,149
-165.
Lannig, G., Eckerle, L., Serendero, I., Sartoris, F. J., Fischer, T., Knust, R., Johansen, T. and Pörtner, H. O. (2003). Temperature adaptation in eurythermal cod (Gadus morhua): acomparison of mitochondrial enzyme capacities in boreal and Arctic populations. Mar. Biol. 142,589 -599.
Lucassen, M., Schmidt, A., Eckerle, L. G. and Pörtner, H. O. (2003). Mitochondrial proliferation in the permanent versus temporary cold: enzyme activities and mRNA levels in Antarctic and temperature zoarcid fish. Am. J. Physiol. 258,1410 -1420.
Lucassen, M., Koschnick, N., Eckerle, L. G. and Pörtner, H.
O. (2006). Mitochondrial mechanisms of cold adaptation in cod
(Gadus morhua L.) populations from different climatic zones.
J. Exp. Biol. 209,2462
-2471.
Mathieu-Costello, O. (1992). Capillary-fiber geometrical relationships in tuna red muscle. Can. J. Zool. 70,1218 -1229.[CrossRef]
Morin, R. P. and Able, K. W. (1983). Patterns of geographic variation in the egg morphology of the fundulid fish, F. heteroclitus. Copeia 1983,726 -740.[CrossRef]
Moyes, C. D., Buck, L. T. and Hochachka, P. W. (1988). Temperature effects of pH of mitochondria isolated from carp red muscle. Am. J. Physiol. 254,611 -615.
Moyes, C. D., Mathieu-Costello, O. A., Tsuchiya, N., Filburn, C. and Hansford, R. G. (1997). Mitochondrial biogenesis during cellular differentiation. Am. J. Physiol. 272,1345 -1351.
Newell, R. C. and Pye, V. I. (1970). The influence of thermal acclimation on the relation between oxygen consumption and temperature in Littorina littorea (L.) and Mytilus edulis L. Comp. Biochem. Physiol. 34,385 -397.
Newell, R. C. and Pye, V. I. (1971). Temperature-induced variations in the respiration of mitochondria from the winkle, Littorina littorea (L.). Comp. Biochem. Physiol. 40,249 -261.[CrossRef][Medline]
Parmesan, C. (2006). Ecological and evolutionary responses to recent climate change. Annu. Rev. Ecol. Evol. Syst. 37,637 -669.[CrossRef]
Podrabsky, J. E., Javillonar, C., Hand, S. C. and Crawford, D. L. (2000). Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles. Am. J. Physiol. 279,2344 -2348.
Pörtner, H. O. (2001). Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. Naturwissenschaften 88,137 -146.[CrossRef][Medline]
Pörtner, H. O. (2002). Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. 132,739 -761.[CrossRef][Medline]
Pörtner, H. O. and Knust, R. (2007).
Climate change affects marine fishes through the oxygen limitation of thermal
tolerance. Science 315,95
-97.
Pörtner, H. O., Mark, F. C. and Bock, C. (2004). Oxygen limited thermal tolerance in fish? Answers obtained by nuclear magnetic resonance techniques. Respir. Physiol. Neurobiol. 141,243 -260.[CrossRef][Medline]
Pörtner, H. O., Bennett, A. F., Bozinovic, F., Clarke, A., Lardies, M. A., Lucassen, M., Pelster, B., Schiemer, F. and Stillman, J. H. (2006). Trade-offs in thermal adaptation: the need for a molecular to ecological integration. Physiol. Biochem. Zool. 79,295 -313.[CrossRef][Medline]
Pörtner, H. O., Peck, L. and Somero, G. N.
(2007). Thermal limits and adaptation in marine Antarctic
ectotherms: an integrative view. Philos. Trans. R. Soc. Lond. B,
Biol. Sci. 362,2233
-2258.
Powers, D. A. and Schulte, P. M. (1998). Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: a multidisciplinary approach to address the million-year saga of a small fish. J. Exp. Zool. 282,71 -94.[CrossRef][Medline]
Root, T. L., Price, J. T., Hall, K. R., Schneider, S. H., Rosenzweig, C. and Pounds, J. A. (2003). Fingerprints of global warming on wild animals and plants. Nature 421, 57-60.[CrossRef][Medline]
Schulte, P. M. (2001). Environmental adaptations as windows on molecular evolution. Comp. Biochem. Physiol. 128,597 -611.[CrossRef][Medline]
Sloman, K. A., Mandic, M., Todgham, A. E., Fangue, N. A., Subrt, P. and Richards, J. G. (2008). The response of the tidepool sculpin, Oligocottus maculosus, to hypoxia in laboratory, mesocosm and field environments. Comp. Biochem. Physiol. 149,284 -292.
Somero, G. N. (2005). Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Front. Zool. 2,1 -9.[CrossRef][Medline]
Sommer, A. M. and Pörtner, H. O. (2004). Mitochondrial function in seasonal acclimatized versus latitudinal adaptation to cold in the lugworm Arenicola marina (L.) Physiol. Biochem. Zool. 77,174 -186.[CrossRef][Medline]
Yeager, D. P. and Ultsch, G. R. (1989). Physiological regulation and conformation: a BASIC program for the determination of critical points. Physiol. Zool. 62,888 -907.
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indicates a significant difference
between the 25°C acclimation group and the 5 and 15°C acclimation
groups when assayed at the same temperature (indicated on 25°C acclimation
data). Data are expressed as means ± s.e.m. (N=4–6), and
P
) indicates
the temperature of inflection (the assay temperature where the largest change
in slope was observed) within each acclimation temperature group. Data are
expressed as means ± s.e.m. (N=4–6), and
P
O2) values are
expressed as means ± s.e.m. (N=6–8 per population and
temperature treatment). Different letters indicate significant differences
(P