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First published online September 19, 2006
Journal of Experimental Biology 209, 3828-3836 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02450
Lifetime performance in foraging honeybees: behaviour and physiology
1 Department of Biology, McMaster University, Hamilton, ON, L8S 4K1,
Canada
2 Department of Psychology, Neuroscience & Behaviour, McMaster
University, Hamilton, ON, L8S 4K1, Canada
3 Department of Regional Centre for Mass Spectrometry, Department of
Chemistry, McMaster University, Hamilton, ON, L8S 4K1, Canada
4 University of Waterloo, ON, N2L 3G1, Canada
* Author for correspondence (e-mail: grantm{at}mcmaster.ca)
Accepted 20 July 2006
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Thus, the purpose of this study was to assess the contribution of physiological changes to the increase in honeybee foraging performance. We investigated aspects of honeybee flight muscle biochemistry throughout the adult life, from non-foraging hive bees, through young and mature foragers, to old foragers near the end of their lifespan. Two-dimensional gel proteomic analysis on honeybee thorax muscle revealed an increase in several proteins from hive bees to mature foragers including troponin T 10a, aldolase and superoxide dismutase. By contrast, the activities (Vmax) of enzymes involved in aerobic performance, phosphofructokinase, hexokinase, pyruvate kinase and cytochrome c oxidase, did not increase in the flight muscles of hive bees, young foragers, mature foragers and old foragers. However, citrate synthase activity was found to increase with foraging experience. Hence, our results suggest plasticity in both structural and metabolic components of flight muscles with foraging experience.
Key words: insect, muscle, flight, performance, honeybee, Apis mellifera, troponin T, superoxide dismutase, metabolic enzyme, foraging
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Research on age-specific performance in several taxa including humans has
documented a strikingly similar lifetime performance curve consisting of low
initial performance rising to a much higher peak followed by decline late in
life (Clutton-Brock, 1991
;
Newton, 1988;
Stephan and Levin, 1992;
Wooler et al., 1990). In
forager honeybees (Apis mellifera), Dukas and Visscher
(Dukas and Visscher, 1994)
documented a similar pattern of more than twofold increase in the rate of food
delivery by individually marked foragers from the time they commenced foraging
until their peak several days later, followed by decreased performance of old
foragers. Whereas this increase in performance has been proposed to occur as a
result of learning, other factors such as physiological components of flight
muscle and metabolism, aerodynamic mechanisms and motivational state could
also shape this pattern. To the authors' knowledge, no research has explicitly
evaluated the contribution of any of these factors to increases in foraging
performance. In this study, we thus investigated aspects of flight muscle
biochemistry that could be associated with improved foraging performance in
honeybees.
In flying insects, ATP turnover rates increase several hundredfold from
resting rates. This dramatic increase is accompanied by matching increases in
oxygen consumption because ATP supplies involve fully aerobic metabolic
pathways (Suarez et al., 1996;
Wegener, 1996). Honeybees rely
exclusively on carbohydrates (hexoses) to power flight
(Blatt and Roces, 2001;
Rothe and Nachtigall, 1989)
and any metabolic adaptations affecting flight would act to increase the flux
capacity through the glycolytic pathway. As well, there may be increases in
oxidative stress associated with the large increase in mitochondrial oxygen
consumption during flight. To date, few studies have measured the components
(i.e. enzyme activities, Vmax) of glycolysis, Krebs cycle,
electron transport chain or antioxidant defence systems in honeybees
(Harrison, 1986;
Seehuus et al., 2006;
Suarez, 2000;
Suarez et al., 1996).
Moreover, there is currently no information available on the changes in these
components in relation to directly quantified foraging performance.
Similarly, little research has focused on differences in structural
components of flight muscles and their effects on flight performance in
insects (Domingo et al., 1998;
Marden et al., 2001;
Marden et al., 1998;
Marden et al., 1999;
Strohm and Daniels, 2003).
Specifically, the calcium sensitivity of the troponin-tropomyosin complex can
affect cross-bridge recruitment and muscle force generation necessary for
sustained flight (Marden et al.,
1999). Insect troponin T isoforms have Ca2+-binding
capabilities and generally show more developmental and tissue-specific
diversity than other isoforms of troponins I and C
(Reiser et al., 1992;
Sabry and Dhoot, 1991;
Schiaffino and Reggiani,
1996). For example, Marden et al.
(Marden et al., 1999) found
that the relative abundance of different troponin T transcripts affects muscle
calcium sensitivity and the power output in the flight muscles of dragonflies
(Libellula pulchella). Thus structural components of honeybee flight
muscles may also contribute to increased flight capacity and, in the same
manner, increased foraging performance.
Honeybees show age polyethism (Winston,
1987). They spend the first 1-3 weeks of their adult life
performing tasks inside the hive and then switch to foraging, which becomes
their primary task until they die, typically within 1 to 2 weeks. Some of the
physiological changes associated with the onset of foraging include a decrease
in body mass, an increase in thorax glycogen stores and an increase in
metabolic rate (Harrison,
1986). The timing of the transition from hive bee to forager is
extremely variable (Robinson,
2002). Hence physiological changes after the onset of foraging
must be related to foraging experience rather than age.
In this study, we (1) assessed honeybee foraging performance by monitoring foraging experience and rate of food delivery to the hive; (2) identified flight muscle proteins that showed changes between hive bees and peak (mature) foragers by means of a proteomic analysis; and concurrently, (3) determined the activities of flight muscle metabolic enzymes (Vmax) in bees of varying foraging experience. Besides suggesting the contribution of physiology to the increase in foraging performance, our results provide a template of potential components of flight muscles that could affect flight performance in insects.
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Materials and methods |
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), this
corresponds to an average daily increase in frame mass of 155 g. We marked
newly eclosed bees (Apis mellifera L.) with individually numbered
tags and introduced them into a two-frame observation hive containing
approximately 2000 bees. We made four introductions of 80 bees 3 days apart in
order to have bees commencing foraging over several days.
Two weeks after introducing the first bee cohort, we removed a few bees
that had already initiated foraging and began data recording. All bees
departing and entering the hive travelled through a transparent Plexiglas
tunnel. The marked bees were diverted into a side tunnel, caged and weighed on
an analytical balance with precision of 0.1 mg. The balance reported the bee
weight and time of day to the computer, and we added the bee identity, her
travel direction and whether she carried pollen. We recorded data from the
start of bee activity in the morning until 18:00 h on a total of 20 successive
days, skipping a single rainy day with no foraging activity. At the end of the
experiment, we edited the data set to include only trips longer than 5 min. We
omitted all shorter trips assuming they were orientation trips by bees about
to initiate foraging (Capaldi et al.,
2000; Dukas and Visscher,
1994; Ribbands,
1953). For each foraging trip, we calculated the trip duration in
min, the mass of forage in mg and the food delivery rate, defined as the mass
of forage over trip duration.
Concurrently, entries and exits were monitored on a separate set of marked
honeybees, which were used for proteomic and enzymatic analyses. These bees
were collected at four different life stages: hive bees (11-15 days old),
young foragers (2 days of foraging experience), mature foragers (4-11 days of
foraging experience) and old foragers (
12 days of foraging experience).
Upon collection, bees were placed on ice and dissected so that only their
thoraxes were kept. Thoraxes were immediately frozen in liquid nitrogen and
stored at -80°C for future analyses.
It was essential to compare the behaviour of the same individual bees
throughout their life to control for the possibility of a positive correlation
between foraging performance and lifespan. Hence, following published methods
(Dukas and Visscher, 1994), the
main statistical analysis involved repeated measures ANOVA on the data set of
food delivery rates over the first 7 days by the 24 bees that foraged for at
least 7 days. Only four of these 24 bees collected pollen on at least half
their foraging trips, precluding a detailed analysis of pollen foragers. To
evaluate the effect of senescence, we conducted a second analysis of the
performance of the 14 bees that foraged for at least 12 days. Sample sizes
were insufficient for analyses beyond 12 days of foraging experience.
Two-dimensional electrophoresis and proteome analysis
Similar sized thoracic sections from nine hive bees and nine mature
foragers [mass=27.0±0.6 mg and 26.3±0.1 mg (mean ±
s.e.m.), respectively] were individually homogenised in 500 µl of ice cold
two-dimensional gel lysis buffer [as detailed elsewhere
(Smith et al., 2005)] using a
motorised dounce homogeniser. The homogenate was clarified by centrifugation
(18 000 g, for 5 min at 4°C) and the supernatant desalted
using commercially available protein desalting columns (Pierce, Rockford, IL,
USA).
From each homogenate 200 µg total protein was resolved by two-dimensional (2D) gel electrophoresis. All 2D electrophoresis was carried out using the InvestigatorTM electrophoresis system (Genomic Solutions, Ann Arbor, MI, USA) according to the manufacturer's instructions and using the pre-made rehydration/solubilisation, equilibration and running buffers. Briefly, the first dimension was resolved on pHIash (pH 3-10) immobilised pH gradient (IPG) strips, using the pHaser isoelectric focussing apparatus pre-programmed ramped voltage regimen, for a total of 100 000 volt-hours, and the second dimension was resolved on trycine chemistry/10% duracryl slab gels run on the InvestigatorTM (Ann Arbor, MI, USA) 2D casting and running apparatus, again using the pre-programmed voltage regimen. After electrophoresis the gels were fixed with water, methanol and acetic acid (in accordance with the instructions provided with the gel stain) and then stained with SYPRO-ruby stain (Genomic Solutions). Imaging of the stained gels was carried out using the Perkin Elmer Pro-Express gel imaging system. The gel images were then analysed using Phoretix 2DTM analytical software version v2004 (Nonlinear Dynamics).
Proteome changes thought to be associated with the transition from hive
activity to foraging were selected according to similar criteria to those used
by Smith et al. (Smith et al.,
2005). The protein spot was present on all hive bee and all
foraging bee gels (i.e. the protein was consistently resolved) yet there was a
significant change in mean normalized spot volume, a parameter offered by the
Phoretix analytical software which combines spot area and intensity to give an
overall index of expression, between hive and foraging gels.
Selected protein spots (see Results) were cut from the gel using the Perkin Elmer Pro-Pick robotic work station and the gel plugs preserved in 2% glycerol at 4°C until they were subjected to in-gel trypsin digestion and peptide analysis.
In-gel tryptic digestion and nano electrospray quadropole time of flight mass spectroscopy analysis
The gel plugs, containing the protein spots of interest (see above), were
destained with 50 mmol l-1 ammonium bicarbonate, containing 50%
acetonitrile and air dried. The proteins were then reduced by adding 30 µl
of 10 mmol l-1 dithiotreitol (DTT) in 25 mmol l-1
ammonium bicarbonate to each gel plug and incubating for 1 h at 56°C.
After cooling to room temperature, the DTT solution was removed and the gel
plugs treated with the same volume of 100 mmol l-1 iodoacetamide in
50 mmol l-1 ammonium bicarbonate. After 60 min incubation at
ambient temperature, in the dark, the gel plugs were washed with 30 µl of
25 mmol l-1 ammonium bicarbonate for 15 min and then dehydrated
with 100% acetonitrile. After 10 min the liquid phase was removed, and the gel
plugs were completely dried in air. The proteins were then subjected to in-gel
digestion: 0.015 µg trypsin in 30 µl of 50 mmol l-1 ammonium
bicarbonate solution containing 10% acetonitrile was added to each gel plug
and these were incubated at 37°C overnight. The digested proteins were
desalted and concentrated using a Millipore C18 ZipTip prior to MS analysis
and the peptides were finally eluted in 8 µl of 50% aqueous acetonitrile
containing 0.2% formic acid. All protein digests were analyzed by a Q-TOF
Global Ultima (Micromass Waters, Manchester, UK) with a nanoES source.
Capillary voltage was typically 1.2-1.6 kV, cone voltage was 50-100 V and the
voltage was 100 V. Mass spectra in time of flight mass spectroscopy (TOF MS)
and MSMS mode were in a mass range 50-1800 m/e with a resolution of 8000 full
width at half maximum height (FWHM). Argon was used as collision gas.
Normalized spot volumes from hive bees and mature foragers 2D gels were compared by Student's t-test (Statistix analytical software).
Enzyme activities
Thoraxes from each life stage (hive bees, young foragers, mature foragers,
old foragers; mean ± s.e.m. thorax mass= 27.8±0.8 mg,
27.1±0.5 mg, 26.5±0.3 mg and 26.6±0.6 mg, respectively)
were powdered using a liquid N2-cooled mortar and pestle. Thorax
weight did not differ among life stages (ANOVA; F3,39=1.0,
P=0.41). Whole thoraxes were then homogenized on ice using a glass on
glass homogenizer for 1 min in 20 volumes of extraction buffer consisting of
75 mmol l-1 potassium phosphate (pH 7.3) and 10 mg ml-1
Lubrol® (Suarez et al.,
1996). All enzymes were measured at 37°C in a Spectromax Plus
384, 96-well microplate reader (Molecular Devices, Sunnyvale, CA, USA). Assays
were performed in triplicate and control rates without substrate were
determined for each assay.
Enzyme activity of cytochrome c oxidase (COx), phosphofructokinase
(PFK) and hexokinase (HK) were measured on fresh thorax homogenates. Enzyme
activity of pyruvate kinase (PK) and citrate synthase (CS) were measured after
having been frozen and thawed once and twice, respectively. Nine to eleven
thoraxes were used for each life stage. Assays condition were, COx: 50 mmol
l-1 potassium phosphate (pH 7.5), 50 µmol l-1
cytochrome c; PFK: 10 mmol l-1 fructose 6-phosphate (F6P) (omitted
in control), 1 mmol l-1 ATP, 0.15 mmol l-1 NADH, 2 mmol
l-1 AMP, 10 mmol l-1 MgCl2, 100 mmol
l-1 KCl, 5 mmol l-1 DTT, 1 U aldolase, 5 U triose
phosphate isomerase and 5 U 
-glycerophosphate dehydrogenase in 50 mmol
l-1 imidazole (pH 7.4); HK: 5 mmol l-1
D-glucose (omitted in 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, 50 mmol l-1 Hepes (pH 7.4); PK: 5 mmol
l-1 phosphoenol pyruvate (PEP; omitted in control), 50 mmol
l-1 imidazole (pH 7.4), 5 mmol l-1 ADP, 2.5 mmol
l-1 MgCl2, 0.15 mmol l-1 NADH, 10 mmol
l-1 fructose 1,6-phosphate and 9.25 U lactate dehydrogenase (LDH);
CS: 0.5 mmol l-1 oxaloacetate (omitted in control), 0.09 mmol
l-1 acetyl-CoA, and 0.1 mmol l-1 dithiobisnitrobenzoic
acid (DTNB) in 20 mmol l-1 Tris (pH 8.0).
For each enzyme, we tested whether enzyme activity increased from hive bees to young foragers, mature foragers and old foragers by performing an analysis of variance linear contrast (ANOVA linear contrast; SPSS version 12.0, SPSS Inc.). Post hoc analysis was performed using the Dunn-Sidak test.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Overall, we recorded the behaviour of 38 bees. These bees commenced foraging at an average age of 14±0.3 days. The average foraging life span of the 27 bees that died before the end of the experiment was 9.7±0.9 days, and the median foraging span was 8 days.
Two-dimensional electrophoresis, proteome analysis and protein identification
The minimum and maximum number of proteins resolved from hive and foraging
bees were 194 and 259, and 197 and 276, respectively.
Fig. 2 depicts a typical 2D gel
from a foraging bee and also indicates the five spots that fulfilled the
selection criteria given above (see Materials and methods, 2D electrophoresis
and proteome analysis) and were selected for identification
(Table 1). There were no
completely consistent inductions or deletions in the proteome; i.e. there were
no examples of protein spots that were absent from all hive bees but induced
in all foragers, nor any which were present in all hive bees and deleted from
all foragers. Quantitative proteome changes were selected according to the
criteria used by Smith et al. (Smith et
al., 2005). Specifically, the protein spot was present on all hive
bee and all foraging bee gels (i.e. the protein was consistently resolved) yet
there was a significant change in mean normalized spot volume, a parameter
offered by the Phoretix analytical software (see Materials and methods) which
combines spot area and intensity to give an overall index of protein
expression.
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Fig. 2 also indicates four further proteins that were identified (using the same method as previously described) but did not show differential expression between hive and foraging bees: tropomyosin, tropomyosin 2, actin and enolase (Table 2). The mean ± s.e.m. normalized spot volumes of these proteins in hive and foraging bees are; 8.82±1.45 and 8.38±1.41, 3.29±0.66 and 2.88±0.15, 2.62±0.62 and 2.28±0.33, and 2.93±0.58 and 3.71±0.35, respectively. These proteins were selected for identification primarily because, being some of the more abundant, they could be used to improve the digestion and analysis procedure of the proteins that did exhibit expression changes (refer to Table 1). As a result we were able to optimise the amount of trypsin to ensure adequate digestion but avoid interference from an excessive mass spectrometry trypsin signature. Overall, this proved invaluable in obtaining accurate identification; particularly of the lower abundance proteins listed in Table 1 and Fig. 3.
Although these proteins did not show significant changes in expression they are included: (a) to increase the knowledge of the bee proteome and (b), in the case of tropomyosin and tropomyosin 2, to reinforce the very precise nature of the change in the major structural proteins.
Flight muscle enzyme activity
Of the five metabolic enzymes measured, only CS activity significantly
increased with foraging experience (Fig.
4; ANOVA linear contrast; F1,36=6.2,
P=0.018). Activities of glycolytic enzymes PK, PFK and HK, as well as
electron transport chain enzyme COx, did not increase with foraging
experience. In fact, contrary to our prediction, there was a decreasing trend
in PFK and COx activities, while PK and HK activities did not show any pattern
with foraging experience. Pairwise comparisons also showed no significant
differences between any life stages (Dunn-Sidak post hoc test;
P0.16).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Compared to hive bees, foragers at peak foraging
performance (mature foragers) showed an increase in an isoform of an important
regulatory muscle structural component, Troponin T 10A. As well, we expected
a priori that foragers would show increases in the activities of
several metabolic enzymes important in aerobic capacity. Results from our
in vitro analysis of enzymatic flux capacity followed our prediction
for CS, but not for COx or any of the glycolytic enzymes measured, which are
classically defined as regulatory for overall glycolytic flux. In contrast,
proteomic analysis showed that the glycolytic enzyme aldolase increased in
mature foragers compared to hive bees. Thus, although the contribution of
glycolytic enzymes for flight performance is currently unclear, our results
suggest plasticity in both structural and metabolic components of flight
muscles with foraging experience.
Foraging performance
Honeybee foragers increased their rate of forage uptake as they gained
foraging experience. Dukas and Visscher, who performed a similar experiment
(Dukas and Visscher, 1994),
found a comparable, but more gradual increase in the rate of forage uptake. We
believe that because our study site offered abundant food sources, honeybee
foragers had an easier task finding and collecting food, which resulted in a
steep increase in the rate of forage uptake with experience. Besides changes
affecting flight metabolism and perhaps muscle efficiency associated with
aerodynamic mechanisms (Feuerbacher et
al., 2003), learning could also play an important role in the
observed increase in foraging performance with experience. Bees are known to
learn tasks such as flower handling and navigational skills, which could
strongly affect the rate of forage uptake
(Dukas and Visscher, 1994).
Research in progress is focussing on the relative contribution of learning and
decision to the observed increase in foraging performance (R.D., unpublished
data).
Flight muscle structural components
Insect indirect flight muscle contraction is controlled by a supramolecular
complex. As in vertebrates, troponin is a major regulatory protein of this
structure. Moreover, insect muscle troponin T is a calcium-binding regulatory
protein with many alternatively spliced isoforms
(Domingo et al., 1998;
Herranz et al., 2005). In
honeybees, there was a significant increase in the troponin T 10A isoform
(mutually excluded exon 10A) in mature foragers compared to hive bees whereas
other components of this supramolecular complex, tropomyosin and tropomyosin 2
did not change between these two casts. Muscle activation results from
Ca2+ binding to troponin C, which leads to a conformational change
in troponin I and tropomyosin to allow cross-bridge formations
(Tobacman, 1996). Troponin T
joins troponin C and I to tropomyosin, and is thought to affect calcium
sensitivity by forming interactions among neighbouring troponin complexes
(Marden et al., 1998). In
dragonflies, an age-related change in the troponin T isoform was found to
affect calcium sensitivity of muscle activation
(Fitzhugh and Marden, 1997),
and the relative abundance of troponin transcripts explains much of the
variation in aerodynamic power output during free flight
(Marden et al., 1999).
Although dragonfly flight muscle is synchronous and honeybee flight muscle is
asynchronous, it is possible that increases in troponin T 10A in honeybees
affect calcium sensitivity in a way similar to dragonflies. In addition,
because the troponin T 10A isoform is associated with the acquisition of
flight in honeybees (Domingo et al.,
1998), it most likely affects flight performance in the same
manner as that seen in dragonflies. So, although this troponin isoform first
appears as part of a developmental-specific program early in life [observed in
5-day old hive bees (Domingo et al.,
1998)], we show that the level of expression of this contractile
protein further increases as bees become foragers. Thus, our results suggest
that increases in troponin T 10A could ultimately affect foraging performance.
Further research on the functional role of this protein is warranted.
Flight muscle metabolic enzymes
Because foraging flight in honeybees is fuelled entirely by the oxidation
of hexose sugars, we examined key enzymes in glycolysis (HK, PFK, PK), a Krebs
cycle enzyme (CS) and an electron transport chain component (COx). Of these,
only CS showed increases in activity as bees gained foraging experience.
Enzyme activities, or, more specifically, maximal enzymatic flux capacities
(Vmax), represent the upper limits of flux at a particular
step of a biochemical pathway in vitro, but can provide insights on
flux in vivo (Newsholme and
Crabtree, 1986; Suarez et al.,
1996; Suarez et al.,
1997). Vmax is a function of enzyme
concentration and catalytic efficiency (kcat). Since
kcat is constant
(Suarez et al., 2000), the
differences in Vmax are necessarily due to differences in
enzyme concentrations.
HK, PFK, and PK activities in flight muscles did not change with increasing
foraging experience. Since these three enzymes are thought to be major
regulatory steps of glycolysis (Hochachka
and Sommero, 2002), we assume that they exert most of the control
of the overall glycolytic flux. Based on our enzyme activity measurements, the
overall flux capacity of glycolysis does not seem to be upregulated with
foraging experience. It seems that most of the metabolic capacity of the
flight muscles is already present relatively early in the bee life, before the
onset of foraging. However, proteomic analysis revealed an increase in
aldolase abundance in flight muscles of mature foragers compared to hive bees.
The aldolase step in glycolysis is generally thought to be a near-equilibrium
reaction and thus its contribution in determining overall flux through
pathways is somewhat controversial (Brooks,
1996; Fell, 1998;
Pierce and Crawford, 1997).
However, there is some evidence for near-equilibrium steps of pathways
affecting flight metabolism (Coelho and
Mitton, 1988; Harrison et al.,
1996). On the other hand, since equilibrium constants differ from
in vitro values (Connett,
1985), it is not clear how aldolase functions in vivo.
Moreover, changes in protein abundance may not reflect in vivo
changes of enzyme flux capacity. For example, recent research on
Drosophila flight muscle suggests that co-localization of some
glycolytic enzymes, including aldolase, along the sarcomeres is required to
sustain flight (Sullivan et al.,
2003; Wojtas et al.,
1997). Hence, not only enzymes need to be present but
co-localization is also required for proper muscle function. One more aspect
of near-equilibrium enzymes is that they must maintain higher forward flux
capacities to maintain a given pathway flux rate relative to enzymes that are
far from equilibrium (Staples and Suarez,
1997). Thus, changes in Vmax of
near-equilibrium enzymes must be greater than changes in
Vmax of allosteric enzymes. So it is possible that small
changes in Vmax of HK, PFK and PK were not detected in
this study because these changes were smaller than the error associated with
enzyme measurements. Possibly honeybees have already reached an upper limit in
the amount of enzymes that can be packed within a muscle cell without
affecting contractile components, and, therefore, the enzymes operate at a
greater fractional velocity v/Vmax in
vivo instead of increasing Vmax itself
(Suarez et al., 1996). If this
is the case, the overall flux of glycolysis could be upregulated even though
no changes in enzyme activity (Vmax) were observed.
Moreover, Suarez et al. (Suarez et al.,
2005) have proposed for body size variation in metabolism in
orchid bees (Apidea: Euglossini), that variation in flux at any step in
metabolism may be under hierarchical control (changes in enzyme concentration
[E]) or metabolic control (variation in product, substrate or modulator
concentration) or both. So, even though we did not see any changes in
Vmax in vitro, in vivo small changes in
[product]/[substrate] or [modulators] may increase total flux rate through
glycolysis in forager versus hive honeybees.
We measured CS and COx, mitochondrial enzymes involved in the Krebs cycle
and in the electron transport chain, respectively, because they are important
determinants of aerobic capacity. No increases in COx activities were observed
but CS showed a 14% increase in activity as bees gained foraging experience.
Comparative studies on insects and mammals have shown that mitochondrial
enzyme densities are constant per unit cristae membrane surface area
(Suarez et al., 2000). Hence,
the modest increase in CS activity observed suggests that honeybees slightly
augment flight muscle mitochondrial density as they gain foraging experience,
which could enhance aerobic capacity and flight muscle ATP turnover rates.
Enhanced antioxidant defence in foragers
Our proteomic analysis revealed that Cu/Zn superoxide dismutase (Cu/ZnSOD,
cytosolic) was increased in mature foragers compared to hive bees. Cu/ZnSOD is
involved in antioxidant defence in cells by catalyzing the reaction that
converts superoxide radicals into hydrogen peroxide
(Fridovich, 1995). Cu/ZnSOD
activity is increased in highly oxidative muscles
(Powers et al., 1999) offering
an enhanced antioxidant defence. It is thought that prolonged muscular
exercise increases Cu/ZnSOD activity in mammalian muscle cells to cope with
increases in reactive oxygen species
(Powers et al., 1999).
Recently, the over expression of the Cu/ZnSOD gene has been linked to
decreased oxidative damage and increased longevity in Drosophila
(Parkes et al., 1998;
Spencer et al., 2003). It is
thus possible that the higher levels of Cu/ZnSOD in mature foragers compared
to hive bees allow them to cope with the increased production of free radicals
associated with the high aerobic demands of flight.
Conclusion
Our results suggest a physiological basis for increased foraging ability in
mature foragers compared to hive bees. Increases in troponin T 10A isoform in
mature foragers and increases in CS activity with foraging experience may
contribute to enhanced flight performance, but causation between these
proteins and foraging success needs to be established. In vitro
glycolytic enzyme flux capacities (Vmax) could not explain
the observed increase in foraging performance but proteomic analysis revealed
that aldolase protein content was increased in mature foragers. Thus,
physiological flux rates through glycolysis need to be assessed in relation to
foraging experience to further elucidate the physiological determinants of
lifetime performance in honeybees. In addition, further experiments are
required to assess the functional role of troponin T 10A and its contribution
to the foraging performance of honeybees.
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Acknowledgments |
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References |
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Blatt, J. and Roces, F. (2001). Haemolymph
sugar levels in foraging honeybees (Apis mellifera carnica):
dependence on metabolic rate and in vivo measurement of maximal rates
of trehalose synthesis. J. Exp. Biol.
204,2709
-2716.
Brooks, S. P. (1996). Equilibrium enzymes in metabolic pathways. Biochem. Cell Biol. 74,411 -416.[Medline]
Capaldi, E. A., Smith, A. D., Osborne, J. L., Fahrbach, S. E., Farris, S. M., Reynolds, D. R., Edwards, A. S., Martin, A., Robinson, G. E., Poppy, G. M. et al. (2000). Ontogeny of orientation flight in the honeybee revealed by harmonic radar. Nature 403,537 -540.[CrossRef][Medline]
Clutton-Brock, T. H. (1991). The Evolution of Parental Care. Princeton, NJ: Princeton University Press.
Coelho, J. R. and Mitton, J. B. (1988). Oxygen consumption during hovering is associated with genetic variation of enzymes in honey-bees. Funct. Ecol. 2, 141-146.
Connett, R. J. (1985). In vivo glycolytic
equilibria in dog gracilis muscle. J. Biol. Chem.
260,3314
-3320.
Domingo, A., Gonzalez-Jurado, J., Maroto, M., Diaz, C., Vinos, J., Carrasco, C., Cervera, M. and Marco, R. (1998). Troponin-T is a calcium-binding protein in insect muscle: in vivo phosphorylation, muscle-specific isoforms and developmental profile in Drosophila melanogaster. J. Muscle Res. Cell Motil. 19,393 -403.[CrossRef][Medline]
Dukas, R. and Visscher, P. K. (1994). Lifetime learning by foraging honey-bees. Anim. Behav. 48,1007 -1012.[CrossRef]
Fell, D. A. (1998). Increasing the flux in metabolic pathways: a metabolic control analysis perspective. Biotechnol. Bioeng. 58,121 -124.[CrossRef][Medline]
Feuerbacher, E., Fewell, J. H., Roberts, S. P., Smith, E. F. and
Harrison, J. F. (2003). Effects of load type (pollen or
nectar) and load mass on hovering metabolic rate and mechanical power output
in the honey bee Apis mellifera. J. Exp.
Biol. 206,1855
-1865.
Fitzhugh, G. and Marden, J. (1997). Maturational changes in troponin T expression, Ca2+-sensitivity and twitch contraction kinetics in dragonfly flight muscle. J. Exp. Biol. 200,1473 -1482.[Abstract]
Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64, 97-112.[CrossRef][Medline]
Harrison, J. F., Nielsen, D. I. and Page, R. E. (1996). Malate dehydrogenase phenotype, temperature and colony effects on flight metabolic rate in the honey-bee, Apis mellifera.Funct. Ecol. 10,81 -88.[CrossRef]
Harrison, J. M. (1986). Caste-specific changes in honeybee flight capacity. Physiol. Zool. 59,175 -187.
Herranz, R., Mateos, J., Mas, J. A., Garcia-Zaragoza, E.,
Cervera, M. and Marco, R. (2005). The coevolution of insect
muscle TpnT and TpnI gene isoforms. Mol. Biol. Evol.
22,2231
-2242.
Hochachka, P. W. and Somero, G. N. (2002). Biochemical Adaptation: Mechanism and Process in Physiological Evolution. New York: Oxford University Press.
Marden, J. H., Fitzhugh, G. H. and Wolf, M. R. (1998). From molecules to mating success: integrative biology of muscle maturation in a dragonfly. Am. Zool. 38,528 -544.
Marden, J. H., Fitzhugh, G. H., Wolf, M. R., Arnold, K. D. and
Rowan, B. (1999). Alternative splicing, muscle calcium
sensitivity, and the modulation of dragonfly flight performance.
Proc. Natl. Acad. Sci. USA
96,15304
-15309.
Marden, J. H., Fitzhugh, G. H., Girgenrath, M., Wolf, M. R. and Girgenrath, S. (2001). Alternative splicing, muscle contraction and intraspecific variation: associations between troponin T transcripts, Ca2+ sensitivity and the force and power output of dragonfly flight muscles during oscillatory contraction. J. Exp. Biol. 204,3457 -3470.
Newsholme, E. A. and Crabtree, B. (1986). Maximum catalytic activity of some key enzymes in provision of physiologically useful information about metabolic fluxes. J. Exp. Zool. 239,159 -167.[CrossRef][Medline]
Newton, I. (1988). Age and reproduction in the sparrowhawk. In Reproductive Success (ed. T. H. Clutton-Brock), pp. 201-219. Chicago: University of Chicago Press.
Pappin, D. J. C., Hojrup, P. and Bleasby, A. J. (1993). Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3, 327-332.[CrossRef][Medline]
Parkes, T. L., Elia, A. J., Dickinson, D., Hilliker, A. J., Phillips, J. P. and Boulianne, G. L. (1998). Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19,171 -174.[CrossRef][Medline]
Pierce, V. A. and Crawford, D. L. (1997).
Phylogenetic analysis of glycolytic enzyme expression.
Science 276,256
-259.
Powers, S. K., Ji, L. L. and Leeuwenburgh, C. (1999). Exercise training-induced alterations in skeletal muscle antioxidant capacity: a brief review. Med. Sci. Sports Exerc. 31,987 -997.[Medline]
Reiser, P. J., Greaser, M. L. and Moss, R. L.
(1992). Developmental-changes in Troponin-T isoform expression
and tension production in chicken single skeletal-muscle fibers. J.
Physiol. Lond. 449,573
-588.
Ribbands, C. R. (1953). The Behavior and Social Life of Honeybees. London: Bee Research Association.
Robinson, G. E. (2002). Genomics and integrative analyses of division of labor in honeybee colonies. Am. Nat. 160,S160 -S172.[CrossRef]
Rothe, U. and Nachtigall, W. (1989). Flight of the honey bee. 4. Respiratory quotients and metabolic rates during sitting, walking and flying. J. Comp. Physiol. B 158,739 -749.
Sabry, M. A. and Dhoot, G. K. (1991). Identification and pattern of transitions of some developmental and adult isoforms of fast Troponin-T in some human and rat skeletal-muscles. J. Muscle Res. Cell Motil. 12,447 -454.[CrossRef][Medline]
Sammataro, D. (1998). The Beekeepers Handbook. Ithaca: Cornell University.
Schiaffino, S. and Reggiani, C. (1996).
Molecular diversity of myofibrillar proteins: gene regulation and functional
significance. Physiol. Rev.
76,371
-423.
Seehuus, S.-C., Norberg, K., Gimsa, U., Krekling, T. and Amdam,
G. V. (2006). Reproductive protein protects functionally
sterile honey bee workers from oxidative stress. Proc. Natl. Acad.
Sci. USA 103,962
-967.
Smith, R. W., Wood, C. M., Cash, P., Diao, L. and Part, P. (2005). Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics. Biochim. Biophys. Acta 1749,81 -93.[Medline]
Spencer, C. C., Howell, C. E., Wright, A. R. and Promislow, D. E. L. (2003). Testing an `aging gene' in long-lived Drosophila strains: increased longevity depends on sex and genetic background. Aging Cell 2, 123-130.[CrossRef][Medline]
Staples, J. F. and Suarez, R. K. (1997). Honeybee flight muscle phosphoglucose isomerase: matching enzyme capacities to flux requirements at a near-equilibrium reaction. J. Exp. Biol. 200,1247 -1254.[Abstract]
Stephan, P. E. and Levin, S. G. (1992). Striking the Mother Lode in Science: The Importance of Age, Place, and Time. New York: Oxford University Press.
Strohm, E. and Daniels, W. (2003). Ultrastructure meets reproductive success: performance of a sphecid wasp is correlated with the fine structure of the flight-muscle mitochondria. Proc. R. Soc. Lond. B Biol. Sci. 270,749 -754.[Medline]
Suarez, R. K. (2000). Energy metabolism during insect flight: biochemical design and physiological performance. Physiol. Biochem. Zool. 73,765 -771.[CrossRef][Medline]
Suarez, R. K., Lighton, J. R. B., Joos, B., Roberts, S. P. and
Harrison, J. F. (1996). Energy metabolism, enzymatic flux
capacities, and metabolic flux rates in flying honeybees. Proc.
Natl. Acad. Sci. USA 93,12616
-12620.
Suarez, R. K., Staples, J. F., Lighton, J. R. B. and West, T.
G. (1997). Relationships between enzymatic flux capacities
and metabolic flux rates: nonequilibrium reactions in muscle glycolysis.
Proc. Natl. Acad. Sci. USA
94,7065
-7069.
Suarez, R. K., Staples, J. F., Lighton, J. R. B. and Mathieu-Costello, O. (2000). Mitochondrial function in flying honeybees (Apis mellifera): respiratory chain enzymes and electron flow from complex III to oxygen. J. Exp. Biol. 203,905 -911.[Abstract]
Suarez, R. K., Darveau, C. A. and Hochachka, P. W.
(2005). Roles of hierarchical and metabolic regulation in the
allometric scaling of metabolism in Panamanian orchid bees. J. Exp.
Biol. 208,3603
-3607.
Sullivan, D. T., MacIntyre, R., Fuda, N., Fiori, J., Barrilla,
J. and Ramizel, L. (2003). Analysis of glycolytic enzyme
co-localization in Drosophila flight muscle. J. Exp.
Biol. 206,2031
-2038.
Tobacman, L. S. (1996). Thin filament-mediated regulation of cardiac contraction. Annu. Rev. Physiol. 58,447 -481.[CrossRef][Medline]
Wegener, G. (1996). Flying insects: model systems in exercise physiology. Experientia 52, 404.[CrossRef][Medline]
Winston, M. L. (1987). The Biology of the Honey Bee. Cambridge: Harvard University Press.
Wojtas, K., Slepecky, N., vonKalm, L. and Sullivan, D. (1997). Flight muscle function in Drosophila requires colocalization of glycolytic enzymes. Mol. Biol. Cell 8,1665 -1675.[Abstract]
Wooler, R. D., Bradley, J. S., Skira, I. J. and Serventy, D. L. (1990). Reproductive success of short-tailed shearwater Puffinus tenuirostris in relation to their age and breeding experience. J. Anim. Ecol. 59,161 -170.[CrossRef]
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