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Faculty of Science, Medicine and Health
2012
High muscle mitochondrial volume and aerobic
capacity in a small marsupial (Sminthopsis
crassicaudata) reveals flexible links between energyuse levels in mammals
Terence Dawson
University of Wollongong
Koa Webster
University of New South Wales
Enhua Lee
University of New South Wales
William A. Buttemer
University of Wollongong, buttemer@deakin.edu.au
Publication Details
Dawson, T., Webster, K., Lee, E. & Buttemer, W. A. (2013). High muscle mitochondrial volume and aerobic capacity in a small
marsupial (Sminthopsis crassicaudata) reveals flexible links between energy-use levels in mammals. The Journal of Experimental
Biology, 216 (7), 1330-1337.
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research-pubs@uow.edu.au
High muscle mitochondrial volume and aerobic capacity in a small
marsupial (Sminthopsis crassicaudata) reveals flexible links between
energy-use levels in mammals
Abstract
We investigated the muscle structure–function relationships that underlie the aerobic capacity of an
insectivorous, small (~15 g) marsupial, Sminthopsis crassicaudata (Family: Dasyuridae), to obtain further
insight into energy use patterns in marsupials relative to those in placentals, their sister clade within the Theria
(advanced mammals). Disparate hopping marsupials (Suborder Macropodiformes), a kangaroo (Macropus
rufus) and a rat-kangaroo (Bettongia penicillata), show aerobic capabilities as high as those of ‘athletic’
placentals. Equivalent muscle mitochondrial volumes and cardiovascular features support these capabilities.
We examined S. crassicaudata to determine whether highly developed aerobic capabilities occur elsewhere in
marsupials, rather than being restricted to the more recently evolved Macropodiformes. This was the case.
Treadmill-trained S. crassicaudata attained a maximal aerobic metabolic rate ( or MMR) of 272 ml O2 min−1
kg−1 (N=8), similar to that reported for a small (~20 g), ‘athletic’ placental, Apodemus sylvaticus, 264 ml O2
min−1 kg−1. Hopping marsupials have comparable aerobic levels when body mass variation is considered.
Sminthopsis crassicaudata has a basal metabolic rate (BMR) about 75% of placental values but it has a notably
large factorial aerobic scope (fAS) of 13; elevated fAS also features in hopping marsupials. The of S.
crassicaudata was supported by an elevated total muscle mitochondrial volume, which was largely achieved
through high muscle mitochondrial volume densities, Vv(mt,f), the mean value being 14.0±1.33%. These data
were considered in relation to energy use levels in mammals, particularly field metabolic rate (FMR). BMR is
consistently lower in marsupials, but this is balanced by a high fAS, such that marsupial MMR matches that of
placentals. However, FMR shows different mass relationships in the two clades, with the FMR of small
(<125>g) marsupials, such as S. crassicaudata, being higher than that in comparably sized placentals, with the
reverse applying for larger marsupials. The flexibility of energy output in marsupials provides explanations for
this pattern. Overall, our data refute widely held notions of mechanistically closely linked relationships
between body mass, BMR, FMR and MMR in mammals generally.
Keywords
mitochondrial, volume, aerobic, capacity, small, high, marsupial, muscle, sminthopsis, crassicaudata, reveals,
flexible, links, between, energy, levels, mammals
Disciplines
Medicine and Health Sciences | Social and Behavioral Sciences
Publication Details
Dawson, T., Webster, K., Lee, E. & Buttemer, W. A. (2013). High muscle mitochondrial volume and aerobic
capacity in a small marsupial (Sminthopsis crassicaudata) reveals flexible links between energy-use levels in
mammals. The Journal of Experimental Biology, 216 (7), 1330-1337.
This journal article is available at Research Online: http://ro.uow.edu.au/smhpapers/506
1330
The Journal of Experimental Biology 216, 1330-1337
© 2013. Published by The Company of Biologists Ltd
doi:10.1242/jeb.079087
RESEARCH ARTICLE
High muscle mitochondrial volume and aerobic capacity in a small marsupial
(Sminthopsis crassicaudata) reveals flexible links between energy-use levels
in mammals
Terence J. Dawson1,2, Koa N. Webster1,3,*, Enhua Lee1 and William A. Buttemer2,4
1
School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW 2052, Australia,
School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia, 3Department of Biological Sciences,
Faculty of Science, Macquarie University, Sydney, NSW 2109, Australia and 4Centre for Integrative Ecology, Deakin University,
Geelong, VIC 3217, Australia
2
*Author for correspondence (koa.webster@mq.edu.au)
SUMMARY
We investigated the muscle structure–function relationships that underlie the aerobic capacity of an insectivorous, small (~15g)
marsupial, Sminthopsis crassicaudata (Family: Dasyuridae), to obtain further insight into energy use patterns in marsupials
relative to those in placentals, their sister clade within the Theria (advanced mammals). Disparate hopping marsupials (Suborder
Macropodiformes), a kangaroo (Macropus rufus) and a rat-kangaroo (Bettongia penicillata), show aerobic capabilities as high as
those of ʻathleticʼ placentals. Equivalent muscle mitochondrial volumes and cardiovascular features support these capabilities.
We examined S. crassicaudata to determine whether highly developed aerobic capabilities occur elsewhere in marsupials, rather
than being restricted to the more recently evolved Macropodiformes. This was the case. Treadmill-trained S. crassicaudata
attained a maximal aerobic metabolic rate (VO2,max or MMR) of 272ml O2min–1kg–1 (N=8), similar to that reported for a small (~20g),
ʻathleticʼ placental, Apodemus sylvaticus, 264ml O2min–1kg–1. Hopping marsupials have comparable aerobic levels when body
mass variation is considered. Sminthopsis crassicaudata has a basal metabolic rate (BMR) about 75% of placental values but it
has a notably large factorial aerobic scope (fAS) of 13; elevated fAS also features in hopping marsupials. The VO2,max of S.
crassicaudata was supported by an elevated total muscle mitochondrial volume, which was largely achieved through high muscle
mitochondrial volume densities, Vv(mt,f), the mean value being 14.0±1.33%. These data were considered in relation to energy use
levels in mammals, particularly field metabolic rate (FMR). BMR is consistently lower in marsupials, but this is balanced by a high
fAS, such that marsupial MMR matches that of placentals. However, FMR shows different mass relationships in the two clades,
with the FMR of small (<125g) marsupials, such as S. crassicaudata, being higher than that in comparably sized placentals, with
the reverse applying for larger marsupials. The flexibility of energy output in marsupials provides explanations for this pattern.
Overall, our data refute widely held notions of mechanistically closely linked relationships between body mass, BMR, FMR and
MMR in mammals generally.
Key words: aerobic scope, basal metabolism, exercise, field metabolism, maximum metabolism, mitochondria.
Received 6 September 2012; Accepted 3 December 2012
INTRODUCTION
Marsupials (Metatheria) are the sister clade of placentals (Eutheria).
Together they comprise the Theria or advanced mammals and they
have many anatomical and physiological characteristics in common,
which largely reflect ancestral traits that evolved prior to the
divergence of these two groups about 148 million years ago
(Bininda-Emonds et al., 2007). Differences between the two groups,
such as their distinct reproductive features, reflect divergent
evolutionary trajectories during their long, separate histories.
Another area where differences seem to have occurred concerns
metabolic patterns. Marsupials have relatively low basal metabolic
rates (BMRs), generally about 75% of placental values, and
historically were seen as being less competent at thermoregulation
and also as ‘low energy’ mammals (Martin, 1902; Dawson, 1973).
While distinctions regarding the thermal biology of these clades
have been long discarded (Dawson, 1973; Dawson, 1989), debate
about differences in metabolic capabilities of marsupials has
persisted in some disciplines (e.g. McNab, 1980; McNab, 2005).
Here, we expand on investigations of the aerobic capacities of
marsupials and also focus on the functional structures that underlie
the capacity for oxygen use in their muscles. Our aim was to put
these into perspective relative to the features that have recently been
established for the structure–function relationships underlying the
aerobic capacities of the placental mammals (Weibel et al., 2004).
It has become apparent that some marsupials have substantial
aerobic capabilities that result from a relatively large factorial aerobic
scope (fAS), such that they achieve levels of maximum oxygen
consumption (VO2,max) similar to those seen in placentals (Dawson
and Dawson, 1982; Hinds et al., 1993). Recent data (Kram and
Dawson, 1998; Dawson et al., 2004) for Macropus rufus (Family
Macropodidae), the red kangaroo, are notable because, despite a
relatively low BMR, its extreme fAS of 54 results in a VO2,max or
maximum metabolic rate (MMR) equivalent to the high levels
reported in a group of placental mammals, such as dogs and horses,
that were categorised as ‘athletic’ (Taylor et al., 1987; Weibel et
al., 2004). Underlying this capability in M. rufus is a large mass of
THEJOURNALOFEXPERIMENTALBIOLOGY
High aerobic capability of marsupials
locomotor muscles that have comparatively high mitochondrial and
capillary volumes (Dawson et al., 2004). Another hopping marsupial,
Bettongia penicillata, a rat-kangaroo (Family Potoroidae), though
much smaller (body mass, Mb, 1kg), also shows an elevated fAS
of 23 and a markedly high MMR (Seeherman et al., 1981; Webster
and Dawson, 2012). Again, this is associated with a large skeletal
muscle mass that has relatively high muscle mitochondrial volume
densities and both a large total capillary volume and a large total
capillary erythrocyte volume (Webster and Dawson, 2012). Overall,
the muscle and cardio-respiratory structural features of M. rufus and
B. penicillata are identical to those previously reported for ‘athletic’
placental mammals of equivalent sizes (Weibel et al., 2004).
Notably, the ratio between MMR and total muscle mitochondrial
volume (~5ml O2min–1ml–1) is, as initially proposed (Hoppeler and
Lindstedt, 1985), consistent in placentals (Weibel et al., 2004) and
macropodiform marsupials (Webster and Dawson, 2012).
Macropus rufus and B. penicillata belong to the specialised
monophyletic suborder Macropodiformes (kangaroos, wallabies and
rat-kangaroos) (Meredith et al., 2008), but do they differ aerobically
from other marsupials? Evidence for ‘athletic’ level aerobic
capacities in other marsupials is strong and comes from disparate
studies of their cardio-respiratory features (e.g. Dawson and
Needham, 1981; Hallam et al., 1989; Hallam et al., 1995; Agar et
al., 2000; Dawson et al., 2003). The generality of this assertion is
not certain because, while Hinds and colleagues (Hinds et al., 1993)
measured comparatively high fAS during locomotion for several
smaller species of marsupial, their reported MMR values did not
reach ‘athletic’ placental levels. However, the MMR value obtained
(Hinds et al., 1993) for B. penicillata was lower than those obtained
on more extensively trained animals (Seeherman et al., 1981;
Webster and Dawson, 2012). The only other marsupial for which
comparable data are available is a South American opossum,
Monodelphis domestica (Family: Didephidae) (Mb≈90g). It also has
a comparatively large fAS (Dawson and Olson, 1988; Schaeffer et
al., 2003) but its MMR does not reach an ‘athletic’ level, though
the mitochondrial volume densities of its skeletal muscles are not
lower than generally seen in similar sized placentals (Schaeffer et
al., 2003). However, there is support for relatively high aerobic
capabilities extending to smaller marsupials. This comes from
studies of field metabolic rate (FMR). Marsupials and placentals
show distinctly different patterns of variation of FMR with Mb but,
intriguingly, small marsupials (Mb<125g) have higher FMRs than
placentals of similar size (Nagy et al., 1999; Capellini et al., 2010).
These differences are particularly marked among the smallest
marsupials; at a Mb of ~15g, the FMR of S. crassicaudata is almost
double that of the predicted placental value (Nagy, 1988).
To clarify the factors underlying this disparity and to further our
understanding of the metabolic patterns and aerobic capabilities
among small marsupial species from different phylogenetic clades,
we studied S. crassicaudata (Family: Dasyuridae). This species
(Mb≈15g) is an active, quadrupedal insectivore that has often been
used as a model for Australian dasyurid marsupials and was among
the species examined by Hinds and colleagues (Hinds et al., 1993).
We focused first on gaining an accurate determination of its MMR
and then ascertained how MMR related to its muscle content and
muscle mitochondrial features such as volume density. Because of
its high MMR levels, we predicted that it would share characteristics
that match what has previously been found in the Macropodiformes,
thereby indicating a generally higher aerobic capability among
marsupials, matching that seen in placentals designated as ‘athletic’
(see Weibel et al., 2004). If so, it would point to a fundamental
structure–function relationship for oxygen delivery to muscles
1331
evolving in or before the earliest mammals. Further, such
information provides the opportunity to examine the presumed
relationships between BMR, MMR and FMR in mammals. This is
significant in view of the idea that BMR is a good predictor of energy
budgets, which is based on the notion that the allometric relationship
between Mb and BMR locks in other metabolic levels (West et al.,
1997; West et al., 1999; Brown et al., 2004).
MATERIALS AND METHODS
Animals and animal care
Fat-tailed dunnarts, S. crassicaudata (Gould 1844) of the Family
Dasyuridae (Krajewski et al., 2012) are mouse-sized insectivorous
marsupials that inhabit the surface of open habitats, usually in semiarid and arid regions of Australia. They are active nocturnal
predators, catching relatively large, invertebrate prey, such as
crickets and beetles (Morton, 1978). Our investigations were in two
parts. (1) An analysis of the mitochondrial characteristics of
locomotor muscles of S. crassicaudata, carried out at the University
of New South Wales, Sydney. The animals used in this study were
derived from colonies maintained at the University of Adelaide and
University of Wollongong. (2) A study of aerobic capacity of S.
crassicaudata during running, undertaken at the University of
Wollongong, using animals from their breeding colony that was
established 3years earlier from free-living animals collected in
western Queensland. This study was carried out under approval
given by the University of New South Wales and University of
Wollongong Animal Care and Ethics Committees (project approval
number 00-17 and 04/06, respectively).
During investigation and prior to killing, animals were kept at
an air temperature (Ta) of 23±0.4°C, with a 12h:12h light:dark cycle
(lights on at 06:00h). They were housed individually in clear plastic
containers (55×38×20cm), which were fitted with wire tops;
bedding of straw and shredded paper was provided. A mixture of
dried cat food (moistened) and canned dog food was provided ad
libitum; this was supplemented with live crickets and vitamin drops
(Penta-vite infant vitamins, Bayer, Pymble, NSW, Australia). Water
was available at all times.
Muscle sample collection and preparation
To assess the muscle mitochondrial parameters of the skeletal muscle
of the whole body, we followed a sampling procedure comparable
to that developed previously (Hoppeler et al., 1984) and used in
other studies of marsupials (Dawson et al., 2004; Webster and
Dawson, 2012). The musculature of S. crassicaudata was divided
into five functional regions, these being head/neck, foreleg, trunk,
hindleg and tail. Animals were killed by gassing (carbon dioxide)
and weighed to the nearest 0.01g on an electronic balance (Sartorius
AG, Goettingen, Germany) directly before dissection. Four animals
were dissected to estimate total skeletal muscle mass and the
proportions of muscle in the five body regions. The contributions
to Mb of skin, heart and the digestive tract were also determined.
In a further five animals the heart plus seven skeletal muscles,
including the diaphragm because of its role in ventilation, were then
dissected out and small blocks sampled for electron microscopy.
The skeletal muscles used were randomly selected, one coming from
each region except the hindleg, where two muscles were selected.
These were, m. trapezius (head/neck), m. deltoid (forelimb), m.
pectoralis minor (trunk), m. multifidi lumborum (trunk), m. gluteus
maximus and m. quadriceps (hindleg), and m. multifidi lumborum
(tail).
Two small blocks, no greater than 2mm thick, were randomly
cut from each muscle whilst being bathed in a drop of cold
THEJOURNALOFEXPERIMENTALBIOLOGY
1332 The Journal of Experimental Biology 216 (7)
glutaraldehyde fixative solution (2.5% in 0.1moll–1 sodium
cacodylate buffer, pH7.4). Sample blocks were transferred to vials
containing the buffered glutaraldehyde fixative solution for proper
immersion fixation for a minimum of 4h. Sample preparation
thereafter followed the method of previous studies (Dawson et al.,
2004; Webster and Dawson, 2012), with the blocks ultimately being
embedded in Spurr’s resin (a slow cure, low viscosity epoxy) over
a long infiltration period (3–4days) and cured at 60°C for 48h. Ultrathin sections of ~60–80nm were cut for each muscle sample using
glass knives mounted on a Reichert-Jung Ultracut microtome (Leica
Microsystems, Vienna, Austria). The sections were placed onto
copper grids (200 square mesh) and were immediately stained with
uranyl acetate in 50% ethanol for 10min.
Mitochondrial volume and inner mitochondrial membrane
surface area
Grids were viewed at 10,000× magnification with either a Hitachi
7000 (Tokyo, Japan) or JEOL 1400 (Tokyo, Japan) transmission
electron microscope (TEM). Ten grid squares were selected per
sample block using a systematic random sampling method (Howard
and Reed, 1998). Digital micrographs were taken in the top left corner
of the grid squares using an Olympus SQ (Tokyo, Japan) digital
camera and software package AnalySIS (attached to the Hitachi 7000
TEM) or a Gatan (Pleasanton, CA, USA) digital camera and software
package Gatan Digital Micrograph (attached to the JEOL 1400). For
each animal, a total of 160 micrographs were taken (10 micrographs
per block × two blocks per muscle × eight muscles).
Mitochondrial volumes were determined using the methods of
previous studies (Dawson et al., 2004; Webster and Dawson, 2012).
Briefly, mitochondria were identified and selected in digital images
by a human operator. The total percentage area covered by the
mitochondria (mitochondrial area fraction) in each micrograph was
estimated using either an image processing plug-in to Adobe
Photoshop (Adobe Systems Inc., San Jose, CA, USA) or the software
ImageJ (US National Institutes of Health, Bethesda, MD, USA).
According to the Delesse principle, the mitochondrial volume
fraction Vv(mt,f), often referred to as mitochondrial volume density,
is equivalent to the mitochondrial area fraction (Weibel, 1980;
Howard and Reed, 1998). The total mitochondrial volume V(mt,m)
for each muscle region (in ml) was calculated from Eqn1:
V(mt,m) = Mm × Vv(mt,f) × Vv(f,m) × d–1 ,
(1)
where Mm is regional muscle mass, Vv(mt,f) is the volume fraction
of mitochondria, Vv(f,m) is the volume fraction of muscle occupied
by muscle fibres, and d is the density of the muscle. For this study,
it was assumed that Vv(f,m) was equal to 1 (Hoppeler et al., 1987)
and that d was equal to 1.06gml–3 (Mendez and Keys, 1960) as the
myofibril fraction and density are considered constant in all muscles
(Mendez and Keys, 1960; Barth et al., 1992).
The surface density of the inner mitochondrial membranes was
estimated in four muscles (m. gluteus maximus, m. deltoid, heart and
diaphragm). For each animal, a total of 40 mitochondria (five
mitochondria per block × two blocks per muscle × four muscles) were
examined and micrographs taken at up to 40,000× magnification
(using the Hitachi 7000 TEM with attached Olympus digital camera).
The surface density of inner mitochondrial membranes per unit volume
of mitochondria, Sv(im,mt), was estimated using the same method
as in previous studies (Dawson et al., 2004; Webster and Dawson,
2012). An overall estimation of the total surface area of inner
membranes in each muscle is given by Eqn2:
S(im,m) = V(mt,m) × Sv(im,mt) .
(2)
Aerobic capacity
To ensure that maximum aerobic capacity (MMR) was achieved, we
followed procedures published elsewhere (Seeherman et al., 1981);
such procedures were used in comparable studies on placental
mammals (see Weibel et al., 2004). The essence of these procedures
was extensive treadmill training (running) that ensured an accurate
and reproducible MMR. Seeherman and colleagues found that at least
2–6weeks of training were needed for this to be achieved for most
of the species that they investigated (Seeherman et al., 1981). We
trained S. crassicaudata for treadmill running for 6–8weeks by
exercising them at speeds of up to 1.5ms–1, generally on alternate
days. The highest training speed at which an animal could maintain
5min of constant running, following an initial speed adjustment period
of 30s, was used during the measurement of MMR; such speeds
ranged between 1 and 1.5ms–1. The MMR obtained was the highest
2min period of instantaneous oxygen consumption when an animal
ran for at least 5min. The method for obtaining instantaneous oxygen
consumption (Bartholomew et al., 1981) involved initially determining
the washout characteristics of the chamber, at the flow rate used, by
tracking the dynamics of a sudden pulse of O2-depleted air followed
by an immediate return to room air.
For actual measurement, one S. crassicaudata was contained
within an inverted 1.2l rectangular plastic container on a stationary
treadmill belt. The treadmill speed was then adjusted to that
required, ie. the highest training speed for that individual. A
constant airflow of 2.0lmin–1 was aspirated through the container
at all treadmill speeds. Air entered through two small holes in the
front of the chamber and also through the bottom edges of the
chamber in contact with the belt. Flow rate was monitored with a
Sierra Top-Trak mass-flow meter (Sierra, Monterey, CA, USA).
Oxygen content of inlet and outlet air was measured using a Sable
Systems FC-1 oxygen analyser (Las Vegas, NV, USA), with a
detection sensitivity of 0.0005%. Water and CO2 were removed from
sampled air prior to gas analysis using Drierite and soda lime,
respectively. The O2 throughout this exercise period was determined
using appropriate corrections for the system configuration (Hill,
1972). Values were adjusted for variations in chamber air leakage
at different treadmill speeds. Air leakage was determined by
delivering a gas mix into the chamber via a mass flow controller
with a percentage O2 similar to that while a S. crassicaudata was
running. Readings were first taken while the treadmill belt was
stationary and then recorded at each belt speed used in the MMR
determinations. Corrections ranged from 7% at the lowest running
speed to 14% at the highest.
Statistical analysis
Comparisons between muscles were analysed using one-way
analysis of variance (ANOVA). A Student–Newman–Keuls (SNK)
multiple-range test was applied when significant differences were
indicated by the ANOVA (using Statistica for Mac, StatSoft, Tulsa,
Table1. Body mass of the fat-tailed dunnart, including contributions
of muscle and other body components
Mb (g)
Total skeletal muscle (g)
Total skeletal muscle (% Mb)
Gut + liver (% Mb)
Heart (% Mb)
Skin (% Mb)
Mb, body mass.
Values are means ± s.d., N=5.
THEJOURNALOFEXPERIMENTALBIOLOGY
15.0±1.28
4.83±0.223
32.3±1.96
14.2±1.23
0.79±0.068
17.5±0.986
High aerobic capability of marsupials
Table2. Mitochondrial volume density of muscles from different
regions of the body of the fat-tailed dunnart
Muscles sampled
Heart
Trapezius
Deltoid
Diaphragm
Pectoralis
Multifidi lumborum
Gluteus maximus
Quadriceps
Body section
Vv(mt,f) (%)
Head/neck
Foreleg
Trunk
Trunk
Trunk, tail
Hindleg
Hindleg
33.9±2.7a
12.9±1.2c
12.1±1.2c
21.1±2.9b
10.6±1.4c
14.5±2.3c
13.0±1.5c
12.9±4.0c
Vv(mt,f), volume fraction of mitochondria.
Values are means ± s.d., N=5.
Values with different superscript letters are significantly different
(Student–Newman–Keuls test, P<0.05).
OK, USA). Values are given as means ± s.d. Regression analyses
were carried out using Microsoft Excel (Microsoft, Redmond, WA,
USA).
1333
mass. The foreleg and head/neck regions equally made up most
of the residual body muscle mass, whereas the tail contained little
muscle. Although Table3 shows that there are significant
differences in Vv(mt,f) in muscle regions (F4,1=4.849, P=0.01),
the differences are relatively small and the regional mitochondrial
volumes V(mt,m) largely reflect regional muscle masses. There
is a significant difference in V(mt,m) across muscle regions
(F4,1=137.1, P=0.0001). The trunk has a significantly larger
volume (Table3), with the hindleg also having more than each
of the remaining regions. The percentage of total muscle
mitochondrial volume contained by the regions follows a similar
pattern of significant differences (F4,1=234.1, P=0.0001). The total
volume of mitochondria in the skeletal muscle, V(mt), was
0.68±0.064ml (Table3).
The mean MMR of S. crassicaudata determined from sustained
treadmill running was 4.09ml O2min–1, or 272ml O2min–1kg–1
(N=8 animals) at an average speed of 1.2ms–1 (Table4). The BMR
reported in a previous study (Dawson and Hulbert, 1970) was
0.320ml O2min–1 and thus the fAS was 12.8.
DISCUSSION
RESULTS
The mean body mass of S. crassicaudata in this investigation was
15.0g (Table1), which is similar to the mass of wild-caught
animals. The contribution of skeletal muscle to body mass in S.
crassicaudata was estimated to be 32.3±1.96% (Table1). The size
of the heart and the contributions to body mass of some other major
components, such as skin and the digestive system, are also shown
in Table1.
In S. crassicaudata, Sv(im,mt) varied little between the muscle
tissues investigated. Values were: heart, 34.0±7.8m2cm–3;
m.
gluteus
maximus,
diaphragm,
36.8±5.8m2cm–3;
2
–3
35.8±7.3m cm ; m. deltoid, 35.0±7.5m2cm–3.
The content of mitochondria in heart and a range of skeletal
muscles from S. crassicaudata is shown in Table2 as Vv(mt,f). The
heart and diaphragm contained significantly higher densities of
mitochondria than the skeletal muscles; that of the heart was
33.9±2.7%, with that of the diaphragm being 21.1±2.9% (F7,1=47.15,
P=0.0001). While values for the other muscles ranged from
14.5±2.3% for the m. multifidi lumborum of the trunk and tail to
10.6±1.4% for the m. pectoralis muscle, the differences between
them were not significant (F5,1=2.116, P=0.1).
The muscle content throughout the body showed significant
differences between regions (Table3; F4,1=51.86, P=0.0001). Both
the hindleg and trunk had significantly more muscle than other
regions and together comprised 60.6% of the total skeletal muscle
The muscle characteristics and aerobic capacity of the marsupial S.
crassicaudata (Table4) mark it as a mammal of high aerobic
capacity in relation to other studies (Weibel et al., 2004; Weibel
and Hoppeler, 2005). It compares favourably with Apodemus
sylvaticus, the European wood mouse, a similar sized placental
previously studied in detail (Hoppeler et al., 1984). Apodemus
sylvaticus is grouped with the ‘athletic’ as against the ‘sedentary or
normal’ mammals by those examining the structure–function
relationships that underpin the aerobic capabilities of placental
mammals (Weibel et al., 2004; Weibel and Hoppeler, 2005). In the
phylogenetically disparate species S. crassicaudata and A. sylvaticus,
both the MMR and the total muscle mitochondrial volume, V(mt),
are alike (Table4) but there are differences in the way the two species
achieve their high aerobic capacities (MMRs). Notably, these are
in the relative volumes of muscle and the Vv(mt,f).
The mean proportion of skeletal muscle in the body of placental
mammals, Mm/Mb (%), is 36–38% (Lindstedt and Schaeffer, 2002;
Weibel et al., 2004). Sminthopsis crassicaudata with a Mm/Mb of
32.3±1.96% and A. sylvaticus with Mm/Mb of 42.5% (Table4) fall
on either side of this mean, with A. sylvaticus having one of the
highest Mm/Mb values in the data set of Weibel and colleagues
(Weibel et al., 2004). These authors found that Mm/Mb was
independent of body mass, but was consistently higher in the
‘athletic’ group of species. The pronghorn (Antilocapra americana)
at 45% had the highest value for a placental; however, the marsupial
Table3. Distribution of muscle and muscle mitochondria in the body of the fat-tailed dunnart
Body region
Head and neck
Foreleg
Trunk
Hindleg
Tail
Muscle mass
(% total)
Vv(mt,f)
(%)
V(mt,m)
(ml)
V(mt,m)
(% total)
17.8±2.2b
19.8±1.7b
28.8±4.8a
31.8±4.7a
1.7±0.3c
Mm=4.83±0.22g
12.9±1.2b
12.1±1.2b
15.4±1.7a
13.0±2.1b
14.5±2.3a,b
V(mt)=0.68±0.064ml
0.111±0.010c
0.117±0.11c
0.237±0.027a
0.199±0.032b
0.012±0.002d
16.4±1.42c
17.3±2.17c
35.0±0.85a
29.4±2.16b
1.7±0.17d
Mm, total muscle mass.
Vv(mt,f), volume fraction of mitochondria; values were derived from the mean densities of mitochondria in the muscles sampled from these regions (Table2).
V(mt,m), mitochondrial volume of muscle regions, either as total volume of mitochondria or as a percentage of total muscle mitochondria.
V(mt), total muscle mitochondrial volume of the whole body.
Values are means ± s.d., N=5.
In columns, values with different superscript letters are significantly different (P<0.05).
THEJOURNALOFEXPERIMENTALBIOLOGY
1334 The Journal of Experimental Biology 216 (7)
Table 4. Relationship between mitochondrial content of the skeletal muscle and aerobic capacity in the fat-tailed dunnart compared with that
of the ʻathleticʼ placental wood mouse and two small marsupials
Parameter
Mitochondrial content
Mb (g)
Mm/Mb (%)
Vv(mt,f) (%)
V(mt)/Mb (mlkg–1)
Aerobic capacity
VO2,max/Mb (ml O2min–1kg–1)
BMR (ml O2min–1kg–1)
BMR (ml O2min–1kg–0.72)
fAS
VO2,max/Vv(mt) (ml O2min–1ml–1)
Dunnart
Wood mouse
Rat-kangaroo
Opossum
15.0±1.28
32.3±1.96
14.0±1.33
45.0±4.26
20.3
42.5
11.0
43.5
1000
43.5
8.7
36.0
89.4
32
8.4
30.1
272±30.9
21.3±1.77
6.6
12.8±1.45
6.1±0.56
264
28.0
9.4
9.4
5.0
177
7.8
7.8
23
4.9
129
9.53
4.9
13.6
4.3
Vv(mt,f), volume fraction of mitochondria; V(mt,m)/Mb, mass-specific mitochondrial volume; VO2,max, maximum aerobic metabolic rate.
For the dunnart, values are means ± s.d.
Data sources other than the current study: fat-tailed dunnart (Sminthopsis crassicaudata), BMR from Dawson and Hulbert (1970); wood mouse (Apodemus
sylvaticus), BMR from Haim et al. (1995), other data from Hoppeler et al. (1984); rat-kangaroo (Bettongia penicillata) data from Webster and Dawson (2003,
2012); short-tailed opossum (Monodelphis domestica), BMR from Dawson and Olson (1988), other data from Schaeffer et al. (2003).
red kangaroo (M. rufus) has an Mm/Mb value of 47% (Dawson et
al., 2004). The lower Mm/Mb of S. crassicaudata compared with A.
sylvaticus, however, is offset by its relatively higher Vv(mt,f), which
is 14% versus 11% in A. sylvaticus (Table4). The very similar total
heart mitochondrial volumes in the two species reflect this balance.
This trait is a reliable predictor of the MMR of equivalent sized
species among marsupials (Dawson et al., 2003) and placentals
(Karas et al., 1987). The heart masses were 0.79% and 0.78% of
Mb, respectively, for S. crassicaudata and A. sylvaticus, while
Vv(mt,f) in the hearts of both species was approximately 34%.
The surface area of the inner mitochondrial membranes, S(im,m),
has been consistently correlated with the activity of the terminal
respiratory chain enzymes in vertebrate groups (Else and Hulbert,
1981), and appears to be functionally linked with aerobic metabolic
capacity. The surface density of inner mitochondrial membranes per
unit volume of mitochondria, Sv(im,mt), in the muscles of S.
crassicaudata is ~35m2cm–3, which is similar to that of other
marsupials (Dawson et al., 2004; Webster and Dawson, 2012) and
placentals including A. sylvaticus (Hoppeler et al., 1981; Hoppeler et
al., 1984; Schwerzmann et al., 1989). As S(im,m) equals V(mt,m)
multiplied by Sv(im,mt) (Eqn2), mitochondrial volume in skeletal
muscle can be used as a proxy for S(im,m). The high overall Vv(mt,f)
of S. crassicaudata relative to that of A. sylvaticus and those of other
placentals compiled by Weibel and colleagues (Weibel et al., 2004)
results from high mitochondrial volume densities in all muscles across
the body (Tables2, 3). The Vv(mt,f) of individual muscles, except
for the diaphragm, did not vary through the body (Table2); this would
reflect S. crassicaudata’s active quadrupedal lifestyle. The pattern
differs in the more specialised kangaroos, whereby muscle Vv(mt,f)
is markedly higher in the region of the pelvis and lower back where
the bulk of the skeletal muscle is also found (Dawson et al., 2004).
These data from S. crassicaudata considerably extend our
understanding of the overall aerobic capacities of marsupials relative
to those of placentals. Initially, an investigation of the cardiorespiratory allometry in marsupials (Dawson and Needham, 1981)
identified them as having the capability for a considerable aerobic
capacity. Dawson and Dawson further challenged the notion that
marsupials, with their low BMRs, were ‘low energy’ mammals
(Dawson and Dawson, 1982). Two small marsupials species that
they exposed to cold had generally larger fAS values, 8–9 as against
4–6 for similar-sized placental species, and aerobic capabilities
equivalent to those of the placentals. Data from Hinds and colleagues
further highlighted relatively high fAS values in a range of
marsupials (Hinds et al., 1993). In response to cold, marsupials and
placentals were able to increase aerobic metabolism above BMR
by 8.3 and 5.1 times, respectively; values during locomotion were
almost twice those observed in the cold (Hinds et al., 1993) and
fAS values were again higher in marsupials (17) than in placentals
(13.5). However, subsequent locomotor investigations indicate that
these fAS values were underestimated in marsupials (see below).
The aerobic factorial scope of M. rufus is of the order of 54
(Dawson et al., 2004). How could this be so much greater than the
value of 17 given by Hinds and colleagues (Hinds et al., 1993) for
the fAS of marsupials during locomotion? The answer comes from
investigations on placentals (see Weibel et al., 2004; Weibel and
Hoppeler, 2005). Allometric equations from these studies show that
MMR is more loosely associated with BMR than was previously
considered. When MMR is plotted against Mb for placentals, two
distinct patterns occur (Weibel et al., 2004). One group of species
has a relatively high MMR while most other species tend to have
a distinctly lower MMR; the former was designated ‘athletic’ and
the latter ‘sedentary’ (Fig.1). Furthermore, these MMR patterns vary
with Mb in a different manner from that of BMR. While the allometic
equations usually used for BMR have an exponent of 0.75, the
exponents found for MMR of ‘athletic’ and ‘sedentary’ placentals
were much steeper, 0.942 and 0.849, respectively (Fig.1). Weibel
and co-workers have shown that MMR is largely set by the energy
needs of active cells, primarily those in muscle, during maximal
work and that total skeletal muscle mitochondrial volume, V(mt),
is a superior proxy for this (Weibel et al., 2004). ‘Athletic’ species
had greater V(mt) than ‘sedentary’ species, which was due to either
greater Mm/Mb and/or higher Vv(mt,f). Overall, as initially proposed
(Hoppeler and Lindstedt, 1985), there was a strong and consistent
correlation between MMR and V(mt) (Fig.2). Our previous studies
(Dawson et al., 2004; Webster and Dawson, 2012) have shown that
both the hopping marsupials have MMRs that fall within the
‘athletic’ grouping in relation to Mb (Fig.1), and that the relationship
between MMR and V(mt) is indistinguishable from that of placentals
(Fig.2). Given the large evolutionary distance and the disparity in
body form between modern placentals and the kangaroos and ratkangaroos, we were somewhat surprised to find comparable
relationships. The volume of muscle, its total mitochondrial content
and its overall vascular supply were essentially identical in the
Macropodiformes to values seen in ‘athletic’ placental mammals.
THEJOURNALOFEXPERIMENTALBIOLOGY
High aerobic capability of marsupials
100,000
Athletic species:
MMR=199.64Mb0.934
R2=0.9959
M. rufus
1000
Sedentary species:
MMR=95.115Mb0.842
R2=0.9864
B. penicillata
100
10
1
0.001
MMR (ml O2 min–1)
10,000
9
M. rufus
8
7
6
1000
5
B. penicillata
100
4
2,3
M. domestica
1
S. crassicaudata
S. crassicaudata
0.1
10,000
10
M. domestica
0.01
11
10
V(mt)=4.88Mb1.01
1
Mb (kg)
10
100
1
0.1
1000
Fig.1. Maximum metabolic rate (MMR) as a function of body mass (Mb) in
mammals. ʻAthleticʼ mammals (filled circles and solid line) have a different
relationship between MMR and Mb from that of more ʻsedentaryʼ mammals
(open circles and dashed line). Marsupial species values may fall either on
the ʻathleticʼ line (Sminthopsis crassicaudata, Bettongia penicillata,
Macropus rufus) or on the ʻsedentaryʼ line (Monodelphis domestica).
Sminthopsis crassicaudata data are from the present study; B. penicillata,
M. rufus and M. domestica data are from previous studies (Seeherman et
al., 1981; Kram and Dawson, 1998; Schaeffer et al., 2003). Placental data
are from Weibel et al. (Weibel et al., 2004). Allometric equations shown on
the graph include all species (placentals and marsupials) but slopes and
elevations are not significantly different from previously published placentalonly equations (Weibel et al., 2004). Also shown are data for several
marsupial species (including S. crassicaudata) from previous work (Hinds
et al., 1993); the allometric equation for this data set is
MMR=131.76Mb0.882, with r2=0.9949 (triangles and dotted line). This line
falls between the ʻsedentaryʼ and ʻathleticʼ lines, and may indicate
incomplete treadmill training of the individuals used in the study; see
Discussion for details.
Thus, our data for S. crassicaudata provide a wide size range over
which marsupials have aerobic capabilities that are essentially similar
to those of ‘athletic’ placentals (Fig.1), despite the significantly lower
BMR of these marsupials (Table4). Notably, while V(mt) is high in
S. crassicaudata, the relationship between MMR and V(mt) follows
the general mammalian pattern (Fig.2). Does a pattern of ‘athleticism’
pertain for most other marsupials or do marsupials also have variable
aerobic potentials, as do placentals (Weibel et al., 2004; Weibel and
Hoppeler, 2005)? Available information is equivocal in regard to this
question. The only marsupial for which comparable information is
also available on MMR and on muscle and muscle mitochondria
volumes (Table4) is the grey short-tailed opossum (Monodelphis
domestica, Family: Didelphidae) (Schaeffer et al., 2003). The MMR
of M. domestica is relatively low (Table4, Fig.1) and falls in line
with those of the ‘sedentary’ placentals, not with ‘athletic’ small
mammals such as the placental A. sylvaticus and the marsupials, S.
crassicaudata and B. penicillata. This is somewhat surprising given
its fAS at 13.6 is relatively large, but this mostly reflects it having a
BMR that is low, even for a marsupial (Table4). The BMR of M.
domestica is approximately 70% of the value predicted for a marsupial
of its mass (including other didelphids) from allometric equations
(Dawson and Hulbert, 1970; Withers et al., 2006). However, the
relationship between MMR and V(mt) in M. domestica is similar to
that of mammals generally (Fig.2) and its Vv(mt,f) is also relatively
low (Table4).
Apart from the results for M. domestica, other data suggest that
high aerobic capacity may be a general characteristic of marsupials.
For example, marsupials tend to have larger hearts than placentals
(Dawson et al., 2003), a trait that benefits attaining high MMR.
Also, the data collected by Hinds and colleagues (Hinds et al., 1993)
1
10
100
1000
V(mt) (ml)
10,000 100,000
Fig.2. MMR as a function of total mitochondrial volume [V(mt)] in
mammals. Marsupial species data (open circles) were obtained as follows:
S. crassicaudata, present study; M. domestica, B. penicillata and M. rufus,
previous studies (Schaeffer et al., 2003; Webster and Dawson, 2012;
Dawson et al., 2004). Numbers on the right identify the placental mammal
species (filled circles) (Weibel et al., 2004): 1, wood mouse; 2, mole rat; 3,
white rat; 4, guinea pig; 5, agouti; 6, fox; 7, goat; 8, dog; 9, pronghorn; 10,
horse; 11, steer.
corroborates the greater aerobic potential of marsupials when it is
examined in detail. The MMR of marsupials during locomotion that
they report are from untrained animals, but still, with their expanded
fAS values, they mostly exceed those of trained ‘sedentary’
placentals (Fig.1). Full treadmill training presumably would increase
the MMR of many of these species to ‘athletic’ levels, as we found
for S. crassicaudata (Fig.1). A clear supporting framework for these
abilities is apparent in other species so far examined. As in ‘athletic’
placentals (Weibel et al., 2004; Weibel and Hoppeler, 2005), the
peak aerobic demands associated with maximum energy output by
muscle are met via the commensurate, matched oxygen supply
system from the lungs to the muscle mitochondria via the expanded
MMR (αMb0.93)
Metabolic rate (ml O2 min–1)
MMR (ml O2 min–1)
100,000
1335
10,000
M. rufus
1000
FMR
(αMb0.74)
(αMb0.60)
B. penicillata
100
10
BMR (αMb0.72)
S. crassicaudata
1
0.1
0.001
5
4
3
2
1
0.01
0.1
1
Mb (kg)
10
100
Fig.3. Different levels of metabolic rate (basal metabolic rate, BMR; field
metabolic rate, FMR; and MMR) as a function of Mb in mammals. Numbers
on the left: 1, marsupial BMR; 2, placental BMR; 3, placental FMR; 4,
marsupial FMR; 5, ʻathleticʼ mammal MMR. Allometric equations for the
relationships of these levels of metabolic rate to Mb are shown in Table5.
The measured values of BMR, FMR and MMR for three species of
marsupial are also shown: S. crassicaudata (circles) – BMR (Dawson and
Hulbert, 1970), FMR (Nagy et al., 1988) and MMR data (present study); B.
penicillata (squares) – BMR (Webster and Dawson, 2003), FMR (Nagy,
1994) and MMR data (Seeherman et al., 1981); and M. rufus (triangles) –
BMR (Dawson et al., 2000a), FMR (Munn et al., 2008) and MMR data
(Dawson et al., 2004).
THEJOURNALOFEXPERIMENTALBIOLOGY
1336 The Journal of Experimental Biology 216 (7)
Table5. Allometric equations relating BMR, FMR and MMR to Mb in mammals, corresponding to the lines in Fig.3
Mammalian group
a
b
Line no. in Fig.3
BMR
Marsupials
Placentals (all)
Placentals (four Orders only)2
6.68
7.57
9.04
0.72
0.72
0.72
1
n.a.
2
FMR3
Marsupials
Placentals
All ʻathleticʼ mammals
19.21
26.76
199.64
0.60
0.74
0.93
4
3
5
Metabolic rate
MMR
Source
Capellini et al., 20101
Capellini et al., 20101
Data from Capellini et al., 2010;
Heyssen and Lacy, 19852
Capellini et al., 20101
Capellini et al., 20101
Fig.1 (present study)
BMR, basal metabolic rate; FMR, field metabolic rate; MMR, maximum metabolic rate.
In each case, equations are of the form MR=aMbb, with MR in ml O2min–1 and Mb in kg.
1
Values for the intercept ʻaʼ were provided by I. Capellini (personal communication).
2
For comparison with the four placental Orders for which MMR data are available (Weibel et al., 2004), we calculated an equation relating BMR to Mb for just
these four Orders, using data for the Orders Artiodactyla, Carnivora and Rodentia from Capellini et al. (2010) and data for the horse, Equus caballus (Order
Perissodactyla), from Hayssen and Lacy (1985).
3
Conversion of FMR from units of kJday–1 (Capellini et al., 2010) to units of ml O2min–1 assumed that 1000ml of O2 provides 20.1kJ of energy.
supply of erythrocytes. This is the concept of symmorphosis
(Weibel, 2000) and it also pertains to M. rufus and B. penicillata
(Dawson et al., 2004: Webster and Dawson, 2012). Following this
concept, there are numerous other studies that lend support for a
generally high MMR in marsupials. These examined lung structure
and function of the respiratory system (Dawson and Needham, 1981;
Hallam et al., 1989; Chappell and Dawson, 1994; Dawson et al.,
2000b), heart structure and capacities (Dawson and Needham, 1981;
Dawson et al., 2003), blood oxygen affinities (Hallam et al., 1995)
and relative haematocrit levels (Agar et al., 2000).
Broadly then, clades of mammals, both placental and marsupial,
have evolved elevated aerobic capacities that can be sustained by
small species for at least several minutes and for relatively longer
periods in larger species. The evolutionary forces behind such
elevated capabilities are likely to be diverse, but the predator–prey
‘arms race’ (Vermeij, 1987) initially comes to mind. The MMR that
a mammal can attain is clearly determined by the functional
characteristics of muscle mitochondria, for which V(mt) is an
appropriate proxy (Fig.2). V(mt) results from various mixes of
Mm/Mb ratios and V(mt,m) levels of individual muscles, which can
also vary markedly. In regard to the link between MMR and BMR,
it seems to be much more loose than previously accepted. The
patterns differ considerably between marsupials and placentals, as
indicated by their differing fAS values. Although fAS shows much
plasticity (consider the value of 54 for M. rufus), there is an apparent
upper limit to MMR based at the ‘athletic’ level (see Weibel et al.,
2004; Weibel and Hoppeler, 2005), which we have shown is also
reached by marsupials. Some mammalian groups may have
evolutionarily varied their energetic profile by varying their basic
energetic structure, i.e. their BMR. For example, a relatively low
BMR in M. domestica is reflected in a low MMR (Table4), which
is seen in the converse in red-toothed shrews of the subfamily
Soricinae such as Blarina brevicauda and Sorex araneus (Dawson
and Olson, 1987; Poppitt et al., 1993). However, underlying patterns
are common to marsupials and placentals, and indicate that the basic
structure–function framework for mammalian aerobic capabilities
is ancient. It must at least predate the divergence of the therians.
The high MMR of S. crassicaudata does not completely explain
the unusual patterns in the allometry of FMR in marsupials and
placentals, whereby small marsupials have higher FMRs than
placentals (Koteja, 1991; Nagy et al., 1999; Cooper et al., 2003;
Capellini et al., 2010). The BMR of S. crassicaudata is 75% of that
predicted for a similar sized placental, yet its FMR at ~7 times BMR
(Nagy, 1988) is almost double the predicted FMR for a placental
(Fig.3). In the context of its high MMR, via a fAS of 13, S.
crassicaudata has ample aerobic capacity for such a FMR (Fig.3).
The reasons behind the high FMRs of small marsupials are
conjectural, but the fact that most small marsupials are
insectivores/carnivores could be an underlying feature. Note, that
while A. sylvaticus has a high MMR, this omnivorous rodent has a
FMR of only ~3.2 times BMR (Speakman, 1997). Nagy and coauthors highlight the plasticity of FMR in mammals and point to
numerous causes (Nagy et al., 1999; Nagy, 2005).
The fact that FMR and MMR, with its clear connection to
V(mt), may not be closely linked via BMR in mammals is
highlighted by energetic profiles displayed among marsupials. As
with placentals, these show marked impacts associated with Mb
(Fig.3, Table 5) and it is instructive to compare the overall data
for S. crassicaudata with that for the large kangaroo, M. rufus
(Fig.3). While the BMR of M. rufus is ~75% of that of a placental,
its FMR is low, 50% of that predicted for a placental (Munn et
al., 2008). That M. rufus, with a fAS of 54, has one of the highest
mammalian MMRs highlights the looseness in connections
between the energy ‘levels’ of mammals. The patterns in the levels
of energy use among mammals that we have clarified also
robustly contest the proposal that design features of the O2
transport system lock in an allometric exponent of 0.75 for the
relationship between Mb and BMR (West et al., 1997; West et
al., 1999) that extends mechanistically to MMR and FMR, as in
the ‘metabolic theory of ecology’ (Brown et al., 2004).
LIST OF SYMBOLS AND ABBREVIATIONS
d
fAS
Mb
Mm
S(im,m)
Sv(im,mt)
V(mt,m)
V(mt)
VO2,max
Vv(f,m)
Vv(mt,f)
density of muscle
factorial aerobic scope
body mass
muscle mass
total surface area of inner mitochondrial membranes
surface density of inner mitochondrial membranes per unit
volume of mitochondria
mitochondrial volume of individual muscles (or muscle regions)
total mitochondrial volume of skeletal muscle
maximal aerobic oxygen consumption
volume fraction of muscle occupied by muscle fibres
volume fraction of mitochondria
ACKNOWLEDGEMENTS
Staff of the University of New South Wales Electron Microscope Unit provided
much instruction on processing samples for electron microscopy and the use of
two models of transmission electron microscopes. Mrs Sigrid Fraser of the UNSW
Electron Microscope Unit performed sample processing not performed by the
authors.
THEJOURNALOFEXPERIMENTALBIOLOGY
High aerobic capability of marsupials
FUNDING
This work was supported by the Australian Research Council [grant nos
A199172218 to T.J.D. and DP0453021 to W.A.B.].
REFERENCES
Agar, N. S., Reinke, N. B., Godwin, I. R. and Kuchel, P. W. (2000). Comparative
biochemistry of marsupial erythrocytes: a review. Comp. Haematol. Int. 10, 148-167.
Barth, E., Stämmler, G., Speiser, B. and Schaper, J. (1992). Ultrastructural
quantitation of mitochondria and myofilaments in cardiac muscle from 10 different
animal species including man. J. Mol. Cell. Cardiol. 24, 669-681.
Bartholomew, G. A. D., Vleck, D. and Vleck, C. M. (1981). Instantaneous
measurements of oxygen consumption during pre-flight warm-up and post-flight
cooling in sphingid and saturniid moths. J. Exp. Biol. 90, 17-32.
Bininda-Emonds, O. R. P., Cardillo, M., Jones, K. E., MacPhee, R. D. E., Beck, R.
M. D., Grenyer, R., Price, S. A., Vos, R. A., Gittleman, J. L. and Purvis, A.
(2007). The delayed rise of present-day mammals. Nature 446, 507-512.
Brown, J. H., Gillooly, J. F., Allen, A. P., Savage, A. M. and West, G. B. (2004).
Toward a metabolic theory of ecology. Ecology 85, 1771-1789.
Capellini, I., Venditti, C. and Barton, R. A. (2010). Phylogeny and metabolic scaling
in mammals. Ecology 91, 2783-2793.
Chappell, M. A. and Dawson, T. J. (1994). Ventilatory accommodation of changing
oxygen consumption in dasyurid marsupials. Physiol. Zool. 67, 418-437.
Cooper, C. E., Withers, P. C. and Bradshaw, S. D. (2003). Field metabolic rate and
water turnover of the numbat (Myrmecobius fasciatus). J. Comp. Physiol. B 173,
687-693.
Dawson, T. J. (1973). ʻPrimitive mammalsʼ. In Comparative Physiology of
Thermoregulation, Vol. 3, Special Aspects of Thermoregulation (ed. G. C. Whittow),
pp. 1-46. New York and London: Academic Press.
Dawson, T. J. (1989). Responses to cold of monotremes and marsupials. In Advances
in Comparative and Environmental Physiology, Vol. 4, Animal Adaptation to Cold
(ed. L. C. H. Wang), pp. 255-288. Berlin: Springer-Verlag.
Dawson, T. J. and Dawson, W. R. (1982). Metabolic scope and conductance in
response to cold of some dasyurid marsupials and Australian rodents. Comp.
Biochem. Physiol. 71A, 59-64.
Dawson, T. J. and Hulbert, A. J. (1970). Standard metabolism, body temperature,
and surface areas of Australian marsupials. Am. J. Physiol. 218, 1233-1238.
Dawson, T. J. and Needham, A. D. (1981). Cardiovascular characteristics of two
resting marsupials: an insight into the cardio-respiratory allometry of marsupials. J.
Comp. Physiol. 145B, 95-100.
Dawson, T. J. and Olson, J. M. (1987). The summit metabolism of the short-tailed
shrew Blarina brevicauda: a high summit is further elevated by cold acclimation.
Physiol. Zool. 60, 631-639.
Dawson, T. J. and Olson, J. M. (1988). Thermogenic capabilities of the opossum
Monodelphis domestica when warm and cold acclimated: similarities between
American and Australian marsupials. Comp. Biochem. Physiol. 89A, 85-91.
Dawson, T. J., Blaney, C. E., Munn, A. J., Krockenberger, A. and Maloney, S. K.
(2000a). Thermoregulation by kangaroos from mesic and arid habitats: influence of
temperature on routes of heat loss in eastern grey kangaroos (Macropus giganteus)
and red kangaroos (Macropus rufus). Physiol. Biochem. Zool. 73, 374-381.
Dawson, T. J., Munn, A. J., Blaney, C. E., Krockenberger, A. and Maloney, S. K.
(2000b). Ventilatory accommodation of oxygen demand and respiratory water loss in
kangaroos from mesic and arid environments, the eastern grey kangaroo (Macropus
giganteus) and the red kangaroo (M. rufus). Physiol. Biochem. Zool. 73, 382-388.
Dawson, T. J., Webster, K. N., Mifsud, B., Raad, E., Lee, E. and Needham, A. D.
(2003). Functional capacities of marsupial hearts: size and mitochondrial parameters
indicate higher aerobic capabilities than generally seen in placental mammals. J.
Comp. Physiol. B 173, 583-590.
Dawson, T. J., Mifsud, B., Raad, M. C. and Webster, K. N. (2004). Aerobic
characteristics of red kangaroo skeletal muscles: is a high aerobic capacity matched
by muscle mitochondrial and capillary morphology as in placental mammals? J. Exp.
Biol. 207, 2811-2821.
Else, P. L. and Hulbert, A. J. (1981). Comparison of the ʻmammal machineʼ and the
ʻreptile machineʼ: energy production. Am. J. Physiol. 240, R3-R9.
Haim, A., McDevitt, R. M. and Speakman, J. R. (1995). Thermoregulatory responses
to manipulations of photoperiod in wood mice Apodemus sylvaticus from high
latitudes (57°N). J. Therm. Biol. 20, 437-443.
Hallam, J. F., Dawson, T. J. and Holland, R. A. B. (1989). Gas exchange in the lung
of a dasyurid marsupial: morphometric estimation of diffusion capacity and blood
oxygen uptake kinetics. Respir. Physiol. 77, 309-322.
Hallam, J. F., Holland, R. A. B. and Dawson, T. J. (1995). The blood of carnivorous
marsupials: low hemoglobin oxygen affinity. Physiol. Zool. 68, 342-354.
Hayssen, V. and Lacy, R. C. (1985). Basal metabolic rates in mammals: taxonomic
differences in the allometry of BMR and body mass. Comp. Biochem. Physiol. 81A,
741-754.
Hill, R. W. (1972). Determination of oxygen consumption by use of the paramagnetic
oxygen analyzer. J. Appl. Physiol. 33, 261-263.
Hinds, D. S., Baudinette, R. V., MacMillen, R. E. and Halpern, E. A. (1993).
Maximum metabolism and the aerobic factorial scope of endotherms. J. Exp. Biol.
182, 41-56.
Hoppeler, H. and Lindstedt, S. L. (1985). Malleability of skeletal muscle in
overcoming limitations: structural elements. J. Exp. Biol. 115, 355-364.
Hoppeler, H., Mathieu, O., Krauer, R., Claassen, H., Armstrong, R. B. and Weibel,
E. R. (1981). Design of the mammalian respiratory system. VI Distribution of
mitochondria and capillaries in various muscles. Respir. Physiol. 44, 87-111.
Hoppeler, H., Lindstedt, S. L., Uhlmann, E., Niesel, A., Cruz-Orive, L. M. and
Weibel, E. R. (1984). Oxygen consumption and the composition of skeletal muscle
1337
tissue after training and inactivation in the European woodmouse (Apodemus
sylvaticus). J. Comp. Physiol. B 155, 51-61.
Hoppeler, H., Kayar, S. R., Claasen, H., Uhlmann, E. and Karas, R. H. (1987).
Adaptive variation in the mammalian respiratory system in relation to energetic
demand: III. Skeletal muscles: setting the demand for oxygen. Respir. Physiol. 69,
27-46.
Howard, C. V. and Reed, M. G. (1998). Unbiased Stereology: Three-Dimensional
Measurement in Microscopy. Oxford: BIOS Scientific Publishers Ltd.
Karas, R. H., Taylor, C. R., Rosler, K. and Hoppeler, H. (1987). Adaptive variation in
the mammalian respiratory system in relation to energetic demand: V. Limits to
oxygen transport by the circulation. Respir. Physiol. 69, 65-79.
Koteja, P. (1991). On the relation between basal and field metabolic rates in birds and
mammals. Funct. Ecol. 5, 56-64.
Krajewski, C., Anderson, F. E., Woolley, P. A. and Westerman, M. (2012).
Molecular evidence for a deep clade of dunnarts (Marsupialia: Dasyuridae:
Sminthopsis). J. Mammal. Evol. 19, 265-276.
Kram, R. and Dawson, T. J. (1998). Energetics and biomechanics of locomotion by
red kangaroos (Macropus rufus). Comp. Biochem. Physiol. 120B, 41-49.
Lindstedt, S. L. and Schaeffer, P. J. (2002). Use of allometry in predicting anatomical
and physiological parameters of mammals. Lab. Anim. 36, 1-19.
Martin, C. J. (1903). Thermal adjustment and respiratory exchange in monotremes
and marsupials. Philos. Trans. R. Soc. Lond. B Biol. Sci. 195, 1-37.
McNab, B. K. (1980). Food habits, energetics, and the population biology of mammals.
Am. Nat. 116, 106-124.
McNab, B. K. (2005). Uniformity in the basal metabolic rate of marsupials: its causes
and consequences. Rev. Chil. Hist. Nat. 78, 183-198.
Mendez, J. and Keys, A. (1960). Density and composition of mammalian muscle.
Metabolism 9, 184-188.
Meredith, R. W., Westerman, M. and Springer, M. S. (2009). A phylogeny and time
scale for living kangaroos and kin (Macropodiformes: Marsupialia) based on nuclear
DNA sequences. Aust. J. Zool. 56, 395-410.
Morton, S. R. (1978). An ecological study of Sminthopsis crassicaudata (Marsupialia:
Dasyuridae), parts 1, 2 and 3. Aust. Wildl. Res. 5, 151-211.
Munn, A., Dawson, T. J., McLeod, S. R., Croft, D. B., Thompson, M. B. and
Dickson, C. R. (2009). Field metabolic rate and water turnover of red kangaroos
and sheep in an arid rangeland: an empirically derived dry-sheep-equivalent for
kangaroos. Aust. J. Zool. 57, 23-28.
Nagy, K. A. (1994). Field bioenergetics of mammals: what determines field metabolic
rates? Aust. J. Zool. 42, 43-53.
Nagy, K. A. (2005). Field metabolic rate and body size. J. Exp. Biol. 208, 1621-1625.
Nagy, K. A., Lee, A. K., Martin, R. W. and Fleming. M. R. (1988). Field metabolic
rate and food requirement of a small dasyurid marsupial, Sminthopsis crassicaudata.
Aust. J. Zool. 36, 293-299.
Nagy, K. A., Girard, I. A. and Brown, T. K. (1999). Energetics of free-ranging
mammals, reptiles, and birds. Annu. Rev. Nutr. 19, 247-277.
Poppitt, S. D., Speakman, J. R. and Racey, P. A. (1993). The energetics of
reproduction in the common shrew (Sorex araneus): a comparison of indirect
calorimetry and the doubly labelled water method. Physiol. Zool. 66, 964-982.
Schaeffer, P. J., Villarin, J. J. and Lindstedt, S. L. (2003). Chronic cold exposure
increases skeletal muscle oxidative structure and function in Monodelphis domestica,
a marsupial lacking brown adipose tissue. Physiol. Biochem. Zool. 76, 877-887.
Schwerzmann, K., Hoppeler, H., Kayar, S. R. and Weibel, E. R. (1989). Oxidative
capacity of muscle and mitochondria: correlation of physiological, biochemical, and
morphometric characteristics. Proc. Natl. Acad. Sci. USA 86, 1583-1587.
Seeherman, H. J., Taylor, C. R., Maloiy, G. M. O. and Armstrong, R. B. (1981).
Design of the mammalian respiratory system. II. Measuring maximum aerobic
capacity. Respir. Physiol. 44, 11-23.
Speakman, J. (1997). Factors influencing the daily energy expenditure of small
mammals. Proc. Nutr. Soc. 56, 1119-1136.
Taylor, C. R., Maloiy, G. M. O., Weibel, E. R., Langman, V. A., Kamau, J. M. Z.,
Seeherman, H. J. and Heglund, N. C. (1981). Design of the mammalian respiratory
system. III Scaling maximum aerobic capacity to body mass: wild and domestic
mammals. Respir. Physiol. 44, 25-37.
Taylor, C. R., Karas, R. H., Weibel, E. R. and Hoppeler, H. (1987). Adaptive
variation in the mammalian respiratory system in relation to to energetic demand: II.
Reaching the limits to oxygen flow. Respir. Physiol. 69, 7-26.
Vermeij, G. J. (1987). Evolution and Escalation: an Ecological History of Life.
Princeton, NJ: Princeton University Press.
Webster, K. N. and Dawson, T. J. (2003). Locomotion energetics and gait
characteristics of a rat-kangaroo, Bettongia penicillata, have some kangaroo-like
features. J. Comp. Physiol. B 173, 549-557.
Webster, K. N. and Dawson, T. J. (2012). The high aerobic capacity of a small,
marsupial rat-kangaroo (Bettongia penicillata) is matched by the mitochondrial and
capillary morphology of its skeletal muscles. J. Exp. Biol. 215, 3223-3230.
Weibel, E. R. (1980). Stereological Methods. London: Academic Press.
Weibel, E. R. (2000). Symmorphosis: on Form and Function in Shaping Life.
Cambridge, MA: Harvard University Press.
Weibel, E. R. and Hoppeler, H. (2005). Exercise-induced maximal metabolic rate
scales with muscle aerobic capacity. J. Exp. Biol. 208, 1635-1644.
Weibel, E. R., Bacigalupe, L. D., Schmitt, B. and Hoppeler, H. (2004). Allometric
scaling of maximal metabolic rate in mammals: muscle aerobic capacity as
determinant factor. Respir. Physiol. Neurobiol. 140, 115-132.
West, G. B., Brown, J. H. and Enquist, B. J. (1997). A general model for the origin of
allometric scaling laws in biology. Science 276, 122-126.
West, G. B., Brown, J. H. and Enquist, B. J. (1999). The fourth dimension of life:
fractal geometry and allometric scaling of organisms. Science 284, 1677-1679.
Withers, P. C., Cooper, C. E. and Larcombe, A. N. (2006). Environmental correlates
of physiological variables in marsupials. Physiol. Biochem. Zool. 79, 437-453.
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