Allometric scaling of flight energetics in orchid bees: evolution of flux capacities and flux rates
,*
1 Department of Zoology, University of British Columbia, Vancouver, BC,
Canada V6T 1Z4
2 Smithsonian Tropical Research Institute, Balboa, Republic of
Panama
3 Department of Ecology, Evolution and Marine Biology, University of
California Santa Barbara, Santa Barbara, CA 93106-9610, USA
* Author for correspondence (e-mail: darveau{at}zoology.ubc.ca)
Accepted 6 July 2005
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Summary |
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Key words: enzyme activity, metabolic rate, evolution, allometry, insect flight, orchid bee
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Introduction |
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In analyses of the quantitative design of metabolic pathways, enzyme
maximal velocities, i.e. Vmax values
(kcatx[E], where kcat is the
catalytic efficiency and [E] is enzyme concentration), measured in
vitro, provide useful estimates of the maximum capacities for flux at
various steps (Newsholme and Crabtree,
1986; Suarez et al.,
1997
). As orthologous enzymes adapted for function at similar
temperatures display similar kcat values
(Hochachka and Somero, 2002
),
Vmax values serve as indirect measures of [E]. In
addition, Vmax values, when considered in relation to
pathway flux rates, provide insights into enzyme function in vivo.
Comparisons between flux capacities and flux rates yield valuable information
concerning the degree to which biochemical capacities are matched to
physiological loads (Suarez et al.,
1996
; Suarez,
2000
). Application of such analyses to honeybee flight muscles
revealed that their high mass-specific metabolic rates during flight require
high Vmax for glycolysis and the electron transport chain,
and that certain enzymes operate close to their maximal capacities in
vivo (Suarez et al.,
1996
; Suarez,
2000
). At near-equilibrium reactions, Vmax
values are typically much greater than net flux rates. Staples and Suarez
(1997
) found, at the
phosphoglucoisomerase step, that the Vmax value is close
to that required to catalyze the required rate of forward flux while
maintaining near-equilibrium.
In this study, we investigated the quantitative design of flight muscle
metabolic pathways in orchid bees by measuring the activities of selected
enzymes involved in fuel breakdown, glycolysis, redox balance and
mitochondrial oxidative metabolism. The relationships between body mass,
hovering flight metabolic rate and enzyme activity are analysed using the
method of phylogenetically independent contrasts
(Felsenstein, 1985;
Garland et al., 1992
).
Incorporating this study with our previous analysis of the evolution of orchid
bee flight energetics (Darveau et al.,
2005
), we analyze the correlated evolution between morphological
and biomechanical variables with metabolic and biochemical variables
associated with hovering flight.
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Materials and methods |
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Enzyme activities
Bees collected for enzyme measurements were frozen at -80°C, shipped in
dry ice to the laboratory, and stored at -80°C until measurements were
conducted. Individual thoraxes were minced with scissors and homogenized in 19
volumes of ice-cold buffer. All further manipulations were carried out in
glass or plasticware cooled in crushed ice. The homogenization buffer used on
samples for the measurement of hexokinase (HK), phosphofructokinase (PFK),
glycerol 3-phosphate dehydrogenase (GPDH), citrate synthase (CS) and
cytochrome oxidase (COX) consisted of 25 mmol l-1 Tris-potassium
phosphate pH 7.8 at 4°C, 2 mmol l-1 ethylene diamine
tetra-acetic acid (EDTA), 5 mmol l-1 dithiothreitol (DTT), 1 mmol
l-1 fructose 6-phosphate, 3.5 mmol l-1 glucose
6-phosphate and 0.5% (v/v) Triton X-100. The use of phosphate buffer and
inclusion of sugar phosphates served to stabilize PFK activity that would
otherwise have been lost (Suarez et al.,
1996; Wegener et al.,
1986
). The homogenization buffer used for samples designated for
measurement of glycogen phosphorylase (GP), trehalase (TR), and
phosphoglucoisomerase (PGI), consisted of 25 mmol l-1 Hepes, pH 7.3
at 4°C, 2 mmol l-1 EDTA, 5 mmol l-1 DTT and 0.5%
(v/v) Triton X-100. Minced thoraxes were homogenized three times for 10 s at
30 s intervals, using a Polytron homogenizer with a small tip (Brinkmann
Instruments, Rexdale, ON, Canada). Homogenates were then sonicated using a
Kontes Micro Ultrasonic Cell Disrupter (Mandel Scientific, Guelph, ON,
Canada), again three times for 10 s, at 30 s intervals. Homogenates were
centrifuged (Jouan MR 1812, Winchester, VI, USA) for 5 min at 8000
g at 4°C, and the supernatants used for assays. To ensure
that these procedures resulted in complete extraction of membrane-bound
enzymes (e.g. trehalase), preliminary studies were conducted to compare enzyme
activities in uncentrifuged homogenates and supernatant fractions. Activities
obtained were equal, showing that extraction of all enzymes was complete.
Enzyme activities were measured in duplicate using a Perkin-Elmer Lambda 2
UV-Visible spectrophotometer (Norwalk, CT, USA) equipped with a Lauda
circulating water bath (Brinkman Instruments) adjusted to maintain cuvette
temperatures (monitored using a temperature probe) at 37°C. HK, PFK, GPDH,
PGI, TR, GP reactions were monitored by following the rate of appearance or
disappearance of reduced nicotinamide adenine dinucleotide (NADH) or
nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm using a
millimolar extinction coefficient () of 6.22. The CS reaction was
monitored 5,5' dithiobis-2-nitrobenzoic acid (DTNB) at 412 nm using
=13.6. The COX reaction was measured by monitoring oxidized cytochrome
c at 550 nm using
=29.5. Control (background) rates, obtained
without one substrate (indicated below), were measured and subtracted from
rates obtained with all substrates present.
Assay conditions and substrate concentrations required to elicit Vmax were as follows: HK: 50 mmol l-1 Hepes, pH 7.0, 5 mmol l-1 D-glucose (omitted from control), 4 mmol l-1 ATP, 10 mmol l-1 MgCl2, 100 mmol l-1 KCl, 0.5 mmol l-1 NADP+, 5 mmol l-1 DTT, 1 U glucose 6-phosphate dehydrogenase. PFK: 50 mmol l-1 Tris-HCl, pH 8.0, 5 mmol l-1 fructose 6-phosphate (omitted from control), 10 mmol l-1 MgCl2, 100 mmol l-1 KCl, 2 mmol l-1 ATP, 0.15 mmol l-1 NADH, 0.01 mmol l-1 fructose 2,6-bisphosphate, 5 mmol l-1 DTT, 1 U aldolase, 5 U triosephosphate isomerase, 5 U glyceraldehyde 3-phosphate dehydrogenase. GPDH: 50 mmol l-1 imidazol pH 7.0, 1 mmol l-1 dihydroxyacetonephosphate (omitted from control), 0.15 mmol l-1 NADH. CS: 50 mmol l-1 Tris-HCl, pH 8.0, 0.5 mmol l-1 oxaloacetate (omitted from control), 0.3 mmol l-1 acetyl-CoA, 0.1 mmol l-1 DTNB. COX: 50 mmol l-1 potassium phosphate, pH 7.5, 0.05 mmol l-1 reduced cytochrome c. TR: 50 mmol l-1 potassium phosphate pH 6.6, 1.1 mmol l-1 MgCl2, 0.5 mmol l-1 NADP+, 1.1 mmol l-1 ATP, 10 mmol l-1 trehalose (omitted from control), 2.5 U of hexokinase and glucose 6-phosphate dehydrogenase. GP: 100 mmol l-1 potassium phosphate pH 7.4, 2 mg ml-1 glycogen, 0.5 mmol l-1 NADP+, 4 µmol l-1 glucose 1,6-biphosphate, 2 mmol l-1 AMP, 10 mmol l-1 MgCl2, 10 U phosphoglucomutase and 2.5 U glucose 6-phosphate dehydrogenase. PGI: 50 mmol l-1 Tris-HCl, pH 8.0, 0.5 mmol l-1 fructose 6-phosphate, 0.5 mmol l-1 NADP+, 2.5 U glucose 6-phosphate dehydrogenase. All chemicals were from Sigma Chemical Company (Oakville, ON, Canada).
Respiration rate measurements in vitro
As individual bees do not possess sufficient flight muscle mitochondria for
isolation, we used crude homogenates of individual thoraxes to measure rates
of substrate oxidation in vitro. Bees were captured in the field and
placed in a refrigerator at 4°C until used for measurements. Before
preparing the thoraxes for homogenization, individual bees had to be warmed up
until leg movements were noticeable. For reasons that remain unknown, this
warm-up step was required prior to dissection and thoracic homogenization for
O2 consumption to occur. Preparation of homogenates from cold
thoraxes resulted in no detectable respiration. After each thorax was
dissected from the insect, further manipulations were performed on ice.
Individual thoraxes were minced with scissors and homogenized in 19 volumes of
ice-cold 10 mmol l-1 Tris, pH 7.4, 1 mmol l-1 EGTA, 250
mmol l-1 sucrose, by a single, 10 s, low speed homogenization using
a Polytron homogenizer (Brinkmann Instruments).
Rates of mitochondrial respiration in the crude thoracic homogenates were
measured at 37°C in a 1.6 ml water-jacketed Gilson glass chamber, equipped
with a Clark-type O2 electrode (YSI, Yellow Springs, OH, USA). The
assay buffer, consisting of 10 mmol l-1 Tris, pH 7.4, 1 mmol
l-1 EGTA, 25 mmol l-1 KH2PO4, 154
mmol l-1 KCl, was equilibrated with room air to an oxygen content
of 406 nmol O ml-1 (Reynafarje
et al., 1985) before measurements. After the addition of 50 µl
of homogenate, 10 µl of 1 mol l-1 pyruvate and 10 µl 1 mol
l-1 proline were added and respiration was initiated by adding 20
µl of 40 mmol l-1 ADP.
Data analysis
All data are presented as species mean values ±
S.D. (standard deviation), but all analyses presented
below were also performed using individual data points. Species samples were
randomized to reduce the effect of assay date on mean estimates of species
enzyme activity, and assay date was also included in statistical analyses. The
effect of body mass Mb on the different variables was
tested using log-transformed data to linearise the relationship, expressed as
Y=aXb.
Analysis of enzyme fractional velocity was conducted by calculating the
in vivo pathway flux rate, divided by the maximal enzyme activity
(µmol min-1 g-1 thorax) x100 to express it as a
percentage (Suarez et al.,
1996). Carbon dioxide production rates were converted to µmol
min-1 g-1 of glycolytic flux rate, Krebs cycle rate and
electron transport chain rate.
Data were also analyzed using phylogenetically independent contrasts (PIC;
Felsenstein, 1985) using the
PDAP module (Midford et al.,
2003
) included in Mesquite
(Maddison and Maddison, 2004
).
We used the hypothesized phylogeny based on cytochrome b (cyt
b) sequence from our previous work
(Darveau et al., 2005
) and
applied two models of character evolution, using raw cyt b genetic
distance for the gradual model, and branch lengths set to 1 for the
speciational model. We ensured the contrasts were adequately standardized by
plotting the absolute value of standardized independent contrasts against
their S.D. (Garland
et al., 1992
). We also tested for branch length and topology
uncertainty by performing simulations using 10 000 trees obtained from a
Bayesian analysis and reported the frequency distribution of independent
contrast correlation coefficient, using Mesquite.
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Results |
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Among mitochondrial oxidative enzymes, the Krebs cycle enzyme CS is positively related to body mass (Fig. 3A). Two clusters of data points are apparent, represented by Euglossa species as one group and Exaerete, Eulaema and Eufriesea as another. The activity of the electron transport chain enzyme, COX, is independent of species body mass (Fig. 3B). Mitochondrial respiration rates were measured in eight species of orchid bees using crude thoracic homogenates. No significant relationship between body mass and mass-specific mitochondrial respiration rate was detectable (Fig. 3C).
|
Flight energetics and metabolic design
We correlated the enzyme Vmax with mass-specific
metabolic rates (Darveau et al.,
2005) in 14 species for which both values are available. The
activities of HK, COX and GP are positively correlated with mass-specific
metabolic rates (Fig. 4A-C),
yielding exponents of 0.97, 0.47 and 0.36, respectively.
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The correlation between hovering flight mass-specific metabolic rates and enzyme activities were also analyzed using both our proposed cyt b phylogeny and 10 000 trees obtained from Bayesian analysis. For HK, the relationship was significant using all phylogenies (Fig. 9A). For COX and GP activity, most relationships were non-significant (Fig. 9B,C).
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Discussion |
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Given the effect of body mass on metabolic rate during hovering flight in
orchid bees, we hypothesized that body mass might also influence the
quantitative design of flight muscle bioenergetic pathways. A direct
relationship between flight muscle metabolic rate and enzyme activity would
yield an approximately threefold lower activity for large orchid bee species.
It is somewhat surprising that no decreases in CS or COX activities were
found, given the apparent decline in mitochondrial volume densities reported
by Casey et al. (1992) in
orchid bees, as well as the finding of allometry in mitochondrial enzyme
activities in mammalian (Emmett and
Hochachka, 1981
) and fish
(Somero and Childress, 1980
)
skeletal muscles. In the study of Casey et al.
(1992
), mitochondrial volume
ranged from about 43% in small species to 30% in large species. However, large
variation was found within a given body mass, e.g. the mitochondrial volume
densities of 150 mg species ranged from 35% to 43%. Such large variation in
mitochondrial content may partly explain the patterns in CS and COX
Vmax values and mitochondrial respiration rates
(Fig. 3A-C) reported here.
Thus, unlike mass-specific metabolic rates during hovering, mitochondrial
oxidative capacities in the flight muscles do not scale allometrically.
In contrast with CS and COX, Vmax values at the HK step
decline in parallel with wingbeat frequencies and mass-specific metabolic
rates (Fig. 5). The similarity
in scaling relationships between the Vmax for HK and
metabolic rate suggests a functional connection between this enzymatic step
and the overall rate of pathway flux. This result is particularly striking
when considered in relation to previous reports of positive scaling of
glycolytic enzyme Vmax values in studies of mammals
(Emmett and Hochachka, 1981),
fishes (Somero and Childress,
1980
), reptiles (Baldwin et
al., 1995
), amphibians (Miller
et al., 1993
) and crustaceans
(Baldwin et al., 1999
). Such
patterns, however, occur in animals in which glycolysis is involved primarily
in anaerobic ATP production. For example, in fishes, the energetic cost of
`anaerobic', burst locomotion increases with increasing body mass and positive
scaling of glycolytic capacity is thought to be required to provide the energy
for such activity (Somero and Childress,
1980
). In contrast, glycolysis operates as part of an obligately
aerobic system for ATP synthesis in insect flight muscles
(Hochachka and Somero, 1973
;
Suarez, 2000
). It seems likely
that the differences between these results and ours may be partly be due to
the evolution of an aerobic role by what was once an anaerobic pathway.
Flux capacities in relation to physiological flux rates
The relationships between physiological flux rates and enzyme
Vmax values have not been evaluated within an evolutionary
framework to date. In considering such data from intraspecific studies, Fell
(2000) argued that the concept
of rate-limiting step should be replaced with the concept of multi-site
modulation of biochemical pathways. This is, of course, consistent with
metabolic control theory (Fell,
1997
), but it leads to the prediction that
Vmax values at multiple steps (potentially, all steps)
should be co-adjusted to account for pathway flux variation
(Fell, 2000
). Our results show
that the Vmax values of most enzymes are independent of
the variation in flux rates. Thus, among orchid bees, evolutionary changes in
physiological pathway flux rates do not require the proportional changes in
activity of all enzymes. Hochachka et al.
(1998
) considered the
glycolytic pathway of red and white muscles in fish and grouped enzymatic
reactions in two different categories, i.e. low and high activity enzymes,
corresponding to those that catalyze reactions held far from or
near-equilibrium, respectively. Hochachka et al.
(1998
) proposed that the
largest differences in Vmax values are found at steps
catalyzed by enzymes catalyzing near-equilibrium reactions. Contrary to this,
our results show that the activity of PGI does not correlate with flux rate.
An alternative hypothesis proposed by Fell
(2000
) is that the low
activity, regulatory enzymes should change the most in relation to pathway
flux. This is supported by the HK results, but not by those obtained for PFK.
It appears, therefore, that no simple rule allows the prediction of how
Vmax values should relate to pathway flux rates across
species.
Although metabolic control theory and its application as metabolic control
analysis have led to widespread abandonment of the concept of single
rate-limiting steps in metabolism, examples do exist of reactions that have
high flux control coefficients. Studies of cardiac muscle
(Kashiwaya et al., 1994),
skeletal muscle (Puigjaner et al.,
1997
; Fueger et al.,
2004
) and myotubes (Whitesell
et al., 2003
) fueled strictly on glucose show that most of the
control of glycolysis is shared between the HK and glucose transport. HK is
known to be a regulator of glycolysis in many tissue types
(Kashiwaya et al., 1994
;
Cardenas et al., 1998
),
including insect flight muscle (Saktor, 1975;
Storey, 1980
). Its role as a
regulator of glycolytic flux in a tissue highly dependent upon exogenous
glucose might explain the parallel evolution of HK Vmax,
mass-specific metabolic rate and wingbeat frequency, as well as the
conservation of HK fractional velocity among orchid bees. The latter may allow
the maintenance of the enzyme's regulatory role over the range of flux rates
observed across species. It is tempting to speculate that the conservation of
fractional velocity in this reaction allows HK to maintain its flux control
coefficient, i.e. its degree of control over glycolytic flux, across
species.
In a previous comparison of distantly related species
(Suarez et al., 1996), it was
reported that fractional velocities of the PFK reaction increase with flux
rate to a maximum of about 50%. Consistent with this finding and, in contrast
with the results obtained with HK, the present study shows that
%Vmax at the PFK step increases from 4% to 22%, as
mass-specific metabolic rate increases and body mass declines across species.
Such a decline in `excess capacity' at this and other nonequilibrium reactions
with increasing flux across species
(Suarez et al., 1996
) is also
observed at other steps in the present interspecific study of orchid bees.
Because some enzyme Vmax values scale isometrically (PGI,
PFK, GPDH, COX), or allometrically, but with exponents less than pathway flux
rates (TR and GP), fractional velocities at these steps decline with
increasing mass among orchid bees. Thus, species that sustain higher metabolic
rates tend to have higher fractional velocities at these reactions and less
excess capacity. These results beg the question of the functional significance
of excess capacity at the biochemical level, and the role of evolution in
selecting for or against excess enzyme protein expression. At least a partial
explanation is provided by Staples and Suarez
(1997
) who argue that, in the
case of near-equilibrium reactions, Vmax values higher
than net flux rates do not necessarily represent excess capacities. In the
case of PGI in honeybee flight muscles, the high Vmax
values empirically measured are actually required for the enzyme to maintain
near-equilibrium, as well as the net forward flux required for flight. In a
companion paper (Suarez et al.,
2005
), some of the functional consequences of isometry in PGI
Vmax values in orchid bees are explored.
Broader implications
In our previous analysis of the evolution of hovering flight metabolic
rate, we established a link between form and function of the flight apparatus.
Here, we extend this analysis by examining the biochemical correlates of
mass-related variation in form and function. By considering the results
obtained from both studies, it is possible to establish the connection between
the structural design of the flight apparatus and the molecular design of
energy producing pathways. Over the range of body mass found in this group of
bees, wing size and wing loading influence the scaling of wingbeat
frequencies. Wingbeat frequencies, in turn, determine muscle power output
(Pennycuick and Rezende, 1984)
and, therefore, metabolic rate (Darveau et
al., 2005
). Authors of recent models explaining the effect of body
mass on metabolic rate suggest that there is no reason to believe that the
metabolic machinery (such as enzyme activity) should scale with species body
mass (e.g. Banavar et al.,
2002
). Instead, it is proposed that metabolic rate is a
consequence of limitations in the rates of material supply to cells
(Banavar et al., 1999
;
West et al., 1999
). Such a
limitation has not been observed in bees at the level of the tracheal system
(Harrison et al., 2001
;
Joos et al., 1997
). Rather, it
appears that the rate of energy expenditure by the contractile apparatus sets
metabolic rates and the manner in which they scale. Such a conceptual model is
supported by data demonstrating the correlated evolution of body mass, wing
morphology, flight metabolic rate and enzymatic flux capacity. That
biomechanics can adequately explain flight metabolic rate scaling in this
group of bees is an alternative view to currently popular theories based on
the assumption of supply limitations. Of course, the mechanisms and processes
that account for the high metabolic rates during flight differ from those that
contribute to resting metabolic rates (the metabolic state most often referred
to in metabolic scaling studies). Nonetheless, the connection between these
two metabolic states remains a subject of great interest, as studies performed
on mammals (Ricklefs et al.,
1996
; Krosniunas and Gerstner,
2003
), birds (Rezende et al.,
2002
) and insects (Reinhold,
1999
) show a positive correlation (or, in some studies, correlated
evolution) between resting and maximal metabolic rates. Our results support
the suggestion (Chown et al.,
2004
) that, ideally, integrative analyses of body mass effects on
energetics should combine mechanistic and evolutionary perspectives, as well
as micro- and macrophysiological approaches.
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Acknowledgments |
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Footnotes |
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