Seasonal acclimatisation of muscle metabolic enzymes in a reptile (Alligator mississippiensis)
1 School of Biological Sciences A08, University of Sydney, New South Wales
2006, Australia
2 Départment de Biologie, Université Laval, Québec, PQ
G1K 7P4, Canada
3 Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife
Refuge, 5476 Grand Chenier Highway, Grand Chenier, LA 70643, USA
* Author for correspondence (e-mail: fseebach{at}bio.usyd.edu.au)
Accepted 10 January 2003
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Summary |
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Key words: thermoregulation, acclimatisation, reptile, Alligator mississippiensis, body temperature, lactate dehydrogenase, citrate synthase, cytochrome c oxidase, enzyme activity
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Introduction |
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Other than during winter dormancy, reptiles are thought not to acclimatise
biochemically but, instead, to thermoregulate behaviourally or become inactive
when environmental conditions preclude the attainment of `preferred' body
temperatures (e.g. Case, 1976;
Bartholomew, 1982
;
Grant and Dunham, 1988
;
Grant, 1990
). Many reptiles,
however, are active at seasonally varying body temperatures
(Christian et al., 1983
;
Van Damme et al., 1987
;
Seebacher and Grigg, 1997
;
Grigg et al., 1998
), and it is
conceivable that reptiles too could gain selective advantages from regulating
biochemical capacities in response to changing environmental conditions.
Semi-aquatic species, in particular, experience pronounced seasonal
fluctuations in thermal conditions
(Costanzo et al., 2000
), and
winter body temperatures, even of tropical crocodiles, for example, are
several degrees below summer averages, with the animals nonetheless remaining
active (Seebacher and Grigg,
1997
; Grigg et al.,
1998
). Hence, it was the aim of the present study to investigate
whether a reptile that experiences marked seasonal climatic variations, the
American alligator Alligator mississippiensis, shows seasonal
acclimatisation in metabolic enzyme activities to compensate for
Q10-related decreases in enzyme activity during winter.
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Materials and methods |
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In addition to tissue sampling, body temperature records were obtained from
seven animals in each season. Body temperature data and details of body
temperature data collection and analysis are given elsewhere
(F. Seebacher et al., in
press) but are summarised here to provide the ecological context.
Briefly, 20 animals were implanted with temperature loggers (iButton
thermochron; Dallas Semiconductor, Dallas, TX, USA) in each season, and seven
of the implanted animals were recaptured and had their dataloggers removed in
each season. Data were collected every 10 min or 15 min for an average of
10.3±1.3 days (mean ± S.E.M.; range 817 days) from each
recaptured animal in summer, and for 7.6±1.3 days (range 513
days) in winter, excluding the first three days of data obtained after the
release of the animals.
Biochemical assays
We measured the activities of lactate dehydrogenase (LDH), citrate synthase
(CS) and cytochrome c oxidase (CCO), which are active in anaerobic
glycolysis, the Krebs cycle and the electron transport chain, respectively
(Voet and Voet, 1995). Enzyme
activity was determined with a UV/visible spectrophotometer (Beckman DU 640 or
Pharmacia Ultrospec III) equipped with a temperature-controlled cuvette
holder. Assays were carried out in duplicate at experimental temperatures of
15°C and 30°C for summer samples and at 15°C, 22.5°C and
30°C for winter samples. Assay temperatures were chosen for their
ecological relevance indicated by body temperature measurements of animals in
the field (see Results). Calculations of enzyme activity were based on the
linear portions of the reaction rates, and enzyme activity was expressed as
units g1 wet tissue. One unit is equivalent to 1 µmol
substrate transformed min1. Saturating substrate
concentrations were determined in preliminary tests and were not limiting
reaction rates; i.e. doubling homogenate concentration in the assays doubled
activity, but doubling substrate concentrations did not alter reaction
rates.
Muscle tissue (0.050.1 g) was homogenised in nine volumes of extraction buffer (pH 7.5), consisting of 50 mmol l1 imidazole/HCl, 2 mmol l1 MgCl2, 5 mmol l1 ethylene diamine tetra-acetic acid (EDTA), 1 mmol l1 reduced glutathione and 1% Triton X-100, and tissue was kept on ice during homogenisation. For LDH assays, tissue homogenates were further diluted by a factor of 10 in summer samples and a factor of 50 in winter samples.
LDH activity was determined by following the absorbance of NADH at 340 nm. The assay medium was 100 mmol l1 potassium phosphate (KH2PO4/K2PO4) buffer (pH 7.0), 0.16 mmol l1 NADH and 0.4 mmol l1 pyruvate. The millimolar extinction coefficient of NADH is 6.22. CS activity was measured as the reduction of DTNB [5,5' dithiobis-(2-nitrobenzoic) acid] at 412 nm. The assay was conducted in 100 mmol l1 Tris/HCl, pH 8.0, 0.1 mmol l1 DTNB, 0.1 mmol l1 acetyl CoA and 0.15 mmol l1 oxaloacetate. Control assays (in which oxaloacetate was omitted) were performed to quantify any transfer of sulfhydryl groups to DTNB other than that caused by CS activity. The millimolar extinction coefficient of DTNB is 14.1. The oxidation of reduced cytochrome c by CCO was measured at 550 nm against a reference of 0.05 mmol l1 cytochrome c oxidised with 50 µmol l1 K2F(CN)6. The assays were performed in 100 mmol l1 KH2PO4/K2PO4, pH 7.5 and 0.05 mmol l1 cytochrome c reduced with sodium hydrosulphide (Na2S2O4). Excess sodium hydrosulphide was removed by bubbling air through the solution. The millimolar extinction coefficient of cytochrome c is 19.1.
Thermal sensitivities of enzyme were expressed as Q10 values that were calculated as: Q10=(k2/k1)10/T2T1, where k = reaction rate at temperatures 1 and 2, and T = temperature.
Statistical analysis
Enzyme activities were compared by a three-factor analysis of variance
(ANOVA) with season (summer and winter), sex (male and female) and assay
temperature (15°C and 30°C) as factors. Individual means were compared
by Tukey's post-hoc tests. Linear model 1 regressions were performed
to test for significant relationships between enzyme activities and body mass.
Values are given as means ± S.E.M.
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Results |
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As expected, all enzyme activities were significantly greater at an assay temperature of 30°C compared with 15°C (LDH, F1,116=133.72, P<0.0001; CS, F1,116=97.68, P<0.0001; CCO, F1,116=47.34, P<0.0001; Fig. 3). Activities in winter samples were significantly greater than in summer samples for LDH (F1,116=1195.21, P<0.0001) and CCO (F1,116=63.82, P<0.0001) but not for CS (F1,116=0.84, P=0.36). The interaction between assay temperature and season was, however, significant for CS (F1,116=6.00, P<0.02), and activity at 15°C was significantly greater in winter compared with summer but did not vary between seasons at 30°C. Interestingly, activity at 15°C in winter alligators was not significantly different from activity at 30°C in summer alligators for LDH and CCO (Fig. 3), suggesting complete thermal compensation.
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The thermal sensitivity of enzyme activity, expressed as Q10 values calculated between 15°C and 30°C, changed with season in all enzymes (Fig. 4). Q10 values were significantly lower in winter compared with in summer for the mitochondrial enzymes (two sample t-test; CS, t=7.57, P<0.0001; CCO, t=3.26, P<0.002). By contrast, Q10 values for LDH were greater in winter than in summer (t=2.57, P<0.02; Fig. 4). In winter samples, Q10 values were similar between 1522.5°C (LDH, 1.62±0.047; CS, 1.52±0.055; CCO, 1.41±0.12) and 22.530°C (LDH, 1.55±0.091; CS, 1.41±0.11; CCO, 1.36±0.092).
|
The activity of the mitochondrial enzymes was significantly greater in males than in females (CS, F1,116=9.14, P<0.01; CCO, F1,116=8.89, P<0.01; Fig. 5), but sex did not interact with either season or assay temperature (all F1,116<2.50, all P>0.1), indicating that, although absolute activities varied, the seasonal and thermal responses were similar in males and females (Fig. 5). There was no difference between the sexes in LDH activity (F1,116=1.56, P=0.21).
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Enzyme activities did not change with body mass at any season or assay temperature (linear regression: all F1,30<4.0, all P>0.05, all r2<0.1; Fig. 6).
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Discussion |
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These data may have important implications for reptilian thermal
physiology, because acclimatisation of enzyme activity indicates that
performance in reptiles may be less dependent on the animals attaining a
`preferred' body temperature range than previously thought. The notion of
`preferred' or `selected' body temperatures should be employed with caution,
because there may not be a single species-specific optimal body temperature
(e.g. Hertz et al., 1993;
Christian and Weavers, 1996
;
Andrews et al., 1999
). On the
contrary, optimal body temperatures may be plastic and change with
acclimatisation, reflecting a shift in the thermal dependency of physiological
processes, so that, as demonstrated for alligators in this study, the a
priori assumption that `warm is always better' may not always be true.
Thermoregulation in reptiles is often interpreted as the ability of animals to
behaviourally maintain near-constant body temperatures in the face of biotic
and abiotic constraints (Christian and
Tracy, 1981
; Angiletta, 2001;
Grbac and Bauwens, 2001
;
Seebacher and Grigg, 2001
).
Our data indicate, however, that it may not be sufficient to base conclusions
about thermoregulatory ability of reptiles entirely on behavioural patterns
and that comparisons of field body temperatures with single `selected' body
temperatures may be temporally confounded because biochemical acclimatisation
may change thermal optima.
The decrease in thermal sensitivity of citrate synthase activity in winter
may explain the greater activity of this enzyme in winter compared with in
summer at 15°C but not at 30°C. By contrast, cytochrome c
oxidase activity was significantly elevated in winter animals at all test
temperatures, as well as being less thermally sensitive in winter compared to
in summer. The mechanisms responsible for the acclimatisation response in
mitochondrial enzymes appear, therefore, to differ for citrate synthase and
cytochrome c oxidase. In so far as changes in Q10 values
reflect protein characteristics, it is possible that modifications in citrate
synthase in muscle explain the seasonal changes in activity. On the other
hand, cytochrome c oxidase is a membrane-bound enzyme, so seasonal
changes in membrane properties could explain the modifications in activity.
Marked modifications of membrane lipids are known to occur during seasonal
acclimatisation of ectotherms (Hazel,
1995), and such changes are likely to modify the activity and
thermal sensitivity of membrane-bound enzymes
(St Pierre et al., 1998
;
Guderley and St Pierre, 2002
).
An increase in enzyme concentration
(Pierce and Crawford, 1997
) or
changes in mitochondrial density and/or characteristics
(St Pierre et al., 1998
;
Guderley and St Pierre, 2002
)
could also intervene. As for cytochrome c oxidase, the activity of
lactate dehydrogenase was significantly elevated in winter animals, but
lactate dehydrogenase also had a greater Q10 value in winter than
in summer. The latter finding is somewhat baffling because it would be
expected that a decrease in thermal sensitivity would be advantageous at a
time when the animals experienced significantly greater fluctuations in body
temperature as well as significantly lower body temperatures.
The fact that male alligators had significantly greater aerobic enzyme
activities is interesting in the context of ecological differences between
male and female crocodilians. Male crocodiles travel significantly further
than females during periods of dispersal
(Tucker et al., 1998), and
males must establish territories in preparation for courtship and breeding
(Vliet, 2001
) in spring
(Seebacher and Grigg, 2001
).
Both dispersal and territoriality require sustained activity likely to be
fuelled by aerobic metabolism (Elsworth et
al., in press
), so selection pressures may favour higher aerobic
metabolic capacity in males compared with females. Hence, although the
phenotypic responses of enzyme activities to seasonal climatic changes were
similar in males and females (no interaction between sex and other variables),
the seasonal phenotypic differences appear to be superimposed on genotypic
gender-based differences.
The lack of a scaling relationship in metabolic enzyme activity does not
reflect the typical mass-related decrease in oxygen consumption observed in
crocodilians (Grigg, 1978;
Wright, 1986
;
Emshwiller and Gleeson, 1997
).
It may be that scaling of oxygen consumption is caused by oxygen transport
constraints rather than by mass-specific changes in oxygen demand
(Goolish, 1991
;
Bejan, 1997
). The lack of
constant scaling of metabolic enzyme activity is not uncommon among ectotherms
(Baldwin et al., 1995
;
Norton et al., 2000
), as
scaling of enzyme activity may be a function of several biotic factors such as
developmental stage (Garenc et al.,
1999
) and size-specific demands for locomotory performance
(Somero and Childress, 1980
).
Hence, more detailed experimental studies are needed to determine the nature
of the scaling relationship, or lack thereof, of metabolic enzyme activity in
alligators, particularly considering the relatively narrow body mass range of
our study animals in winter. Moreover, it would be useful to assay more
metabolically active organs, such as heart and liver, in addition to
muscle.
Many aquatic ectotherms change biochemical capacities with seasonal
acclimatisation or thermal acclimation (e.g. see
Guderley and St Pierre, 2002),
and this ability may be the result of their inability to compensate
behaviourally for environmental variation in homogeneous marine environments.
Body temperatures of aquatic and semi-aquatic ectotherms are often closely
tied to water temperature fluctuations, particularly to long-term, seasonal
fluctuations (Seebacher and Grigg,
1997
), as a result of the high rates of convective heat exchange
in water. Hence, aquatic or semi-aquatic habits may provide the context within
which acclimatisation is advantageous. The notion that acclimatisation is
restricted to aquatic species (Wilson and
Franklin, 2000
), however, may be a little simplistic because the
proximate cause for biochemical/physiological acclimatisation is body
temperature, but body temperature is determined by a complex suite of
parameters such as behaviour, heat transfer characteristics and body mass, as
well as environmental conditions. It is conceivable, therefore, that
terrestrial species experience similar seasonal fluctuations in body
temperature and could gain similar advantages from biochemical acclimatisation
as aquatic animals despite the fact that they are able to thermoregulate on a
daily basis. Acclimatisation may be less pronounced, however, in animals that
experience large daily fluctuation in body temperature, because selection
would decrease the thermal sensitivity of biochemical traits
(Wilson and Franklin, 2000
).
In addition, metabolic acclimatisation may be energetically expensive, for
example with respect to ATP used during increased rates of transcription, so
that the benefits of maintaining biochemical/physiological performance may by
outweighed by the increased energetic costs, and dormancy becomes the more
advantageous response, particularly in extreme climates (e.g.
St Pierre and Boutilier,
2001
).
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Acknowledgments |
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