Honeybee flight metabolic rate: does it depend upon air temperature?
1 Department of Biology, University of Massachusetts Boston, Boston,
Massachusetts 02125-3393, USA
2 Department of Biology, University of Vermont, Burlington, Vermont 05405,
USA
* Author for correspondence (e-mail: woody.woods{at}umb.edu)
Accepted 24 January 2005
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Summary |
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Key words: Apis mellifera, thermoregulation, flight energetics, water loss, wingbeat frequency, bee
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Introduction |
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In theory, insects could maintain thermal stability in flight by regulating
heat gain, heat loss, or both. Under shaded conditions, where short-wave
radiative heat gain is small, metabolic heat production is the primary source
of heat gain (Cooper et al.,
1985; Coelho,
1991
; Roberts and Harrison,
1999
), but its role in maintaining thermal balance over a range of
Ta values has been debated. Initial measurements of
honeybees found no association between flight metabolic rate (FMR) and
Ta (Heinrich,
1980b
), and flying honeybee heat exchange models published in the
subsequent decade accepted this as a key assumption
(Cooper et al., 1985
;
Coelho, 1991
). This was in
agreement with conclusions reached for other insect orders, where FMR was
associated with mechanical flight requirements
(Casey, 1989
;
Wolf et al., 1989
;
Heinrich, 1993
), and thermal
stability was maintained at higher Ta values through
various mechanisms for regulating heat loss (reviewed in Heinrich
1981
,
1993
;
Dudley, 2000
). More recent
studies, though, have concluded that flying honeybees, as well as other bees
and the dragonfly Anax junius, do indeed maintain thermal stability,
at least partly by varying heat production
(May, 1995
; Harrison et al.,
1996a
,b
;
Roberts et al., 1998
;
Roberts and Harrison, 1999
;
Borrell and Medeiros, 2004
;
reviewed for bees in Roberts and Harrison,
1998
; for honeybees, Harrison
and Fewell, 2002
), and other related research has supported this
mechanism, though with caution (Moffatt,
2001
). In studies reporting wingbeat frequency (WBF) as well as
FMR, both variables show generally similar relationships with
Ta, and measurements of WBF have been presented as
corroborative evidence of Ta effects on FMR
(May, 1995
;
Harrison et al., 1996b
;
Roberts et al., 1998
;
Borrell and Medeiros, 2004
).
Evidence that load and FMR in honeybees may be decoupled
(Balderrama et al., 1992
;
Moffatt, 2000
) or only weakly
associated (Feuerbacher et al.,
2003
) suggest that varying heat production during flight is a
plausible mechanism for maintaining thermal balance during flight in nature. A
recently proposed explanation (Harrison
and Fewell, 2002
) arises from the observation that force
production of honeybees in tethered flight reaches a peak value when
Tth is about 38°C and declines as
Tth increases or decreases
(Coelho, 1991
). Harrison and
Fewell (2002
) hypothesize that
FMR may follow a similar pattern, with the result that the range of
measurement Ta values chosen, along with differences in
bees' metabolic capacity between studies, may explain differing conclusions
about the role of varying heat production in maintaining thermal stability
during flight.
Another possible explanation is differences in experimental conditions.
Making precise measurements of FMR of insects requires subjecting animals to
conditions different from those encountered in nature, and the consequences of
these differences are not always obvious. While open-flow respirometry offers
the advantages of high accuracy and temporal resolution, it requires that
subjects maintain free flight within a confined space, in the absence of
natural referents, for a period of time sufficient to yield a stable signal.
Researchers have employed a variety of tactics to elicit flight under
confinement or restraint; for bees, these include visual cues
(Esch, 1976;
Esch et al., 1975
;
Wolf et al., 1989
; Jungman et
al., 1989; Nachtigall et al.,
1989
; Feller and Nachtigall,
1989
; Ellington et al.,
1990
; Hrassnig and Crailsheim,
1999
; Feuerbacher et al.,
2003
), tarsectomy, amounting to landing gear removal
(Heinrich, 1980b
), tethering
(Sotavalta, 1954
;
Esch et al., 1975
;
Esch, 1976
;
Jungmann et al.,1989
;
Feller and Nachtigall, 1989
;
Nachtigall et al., 1989
;
Coelho, 1991
;
Hrassnig and Crailsheim, 1999
)
and flight chamber motion, using either constant agitation
(Harrison and Hall, 1993
;
Harrison et al.,
1996a
,b
,
2001
;
Harrison and Fewell, 2002
) or
occasional and minimal shaking of the chamber
(Heinrich, 1980b
;
Roberts et al., 1998
;
Roberts and Harrison, 1999
).
For flying honeybees, the association between FMR and Ta
has, under different measurement conditions, variously been found negative
(Harrison et al.,
1996a
,b
;
Roberts and Harrison, 1999
),
positive (Hrassnig and Crailsheim,
1999
; Harrison et al.,
2001
), or not significant
(Heinrich, 1980b
).
Nevertheless the honeybee, with its capacity for hovering and its relative
willingness to fly in confinement, is a system of choice for addressing flight
energetics questions, and FMR has been measured more often for the honeybee
than for any other animal (Harrison and
Fewell, 2002
).
We re-examined whether, for honeybees flying in confinement, variation in FMR contributes to the maintenance of thermal stability. Our choice of an outdoor location was driven by early trials, which we report, that showed greater willingness to sustain flight in a respirometry chamber outdoors than under otherwise similar conditions indoors. We sought to explain previous differing conclusions by performing two experiments that, in combination, cover much of the methodological ground of previous work while quantifying the voluntary flight and non-flight behavior of bees flying in small chambers under largely natural outdoor lighting conditions, accounting for all bees measured. Specifically, we asked: (1) what is the relationship between FMR and Ta for honeybees in continuous, unprovoked, self-supporting flight? (2) What is the relationship between Tth and Ta? (3) What is the contribution of evaporative heat loss to body temperature stability? (4) Do wingbeat frequency (WBF) and FMR have similar relationships with Ta? (5) Does agitation of the flight chamber or incidence of non-flight behavior affect the relationship of FMT and Ta? We then examined the possibility that differences in Tth could explain differing conclusions about the relationship of FMR and Ta between studies.
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Materials and methods |
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The respirometry chamber, a 500 cc Pyrex Erlenmeyer flask, was housed in a transparent temperature control cabinet placed in a shaded outdoor location. The 0.38 m high x 0.31 m diameter temperature control cabinet consisted of a 0.64 cm Plexiglas top and bottom, secured by a wooden frame, with a sheet of 0.2 mm acetate forming a cylinder comprising the side wall; access to the chamber was through the overlapping edges of the acetate sheet. Temperature was raised by means of a warm air blower whose output was ducted by 3.2 cm PVC tubing through a fitting in the floor of the cabinet and directed away from the respirometry chamber; a 5 cm aperture in the top Plexiglas panel of the cabinet served as an exhaust. Chamber Ta values ranging from 18 to 39°C were maintained to within ±0.5°C. Measurements made at a given Ta value were distributed across different times of day so that Ta treatment was independent of both time of day (P=0.71) and the number of hours before or after solar noon (P=0.75).
If a bee ceased flying during measurement, the chamber was picked up by the lip within 0.5 min, tapped 3-4 times or shaken lightly and then set down. This procedure was repeated at approximately 0.5 min intervals if the bee did not resume flight; thus, brief chamber motion sometimes caused bees to initiate flight, but was not used to sustain flight in bees that persisted in landing.
Respirometry, body segment temperature and wingbeat frequency measurement
Carbon dioxide production and water loss were measured by differential
open-flow respirometry. Air scrubbed of water and carbon dioxide by soda
lime-Drierite-soda lime columns was flowed at 860 cc min-1 through
the respirometer chamber. Bev-A-Line tubing (Thermoplastic Processes Inc.,
Georgetown, DE, USA) was used throughout except between scrubber columns.
Ta inside the chamber was monitored to ±0.1°C
using a Physitemp BAT 12 field thermometer (Physitemp Instruments, Clifton,
NJ, USA) with its sensor inside a hypodermic needle inserted through the
chamber stopper. Data were digitized using a converter (ADC-1, Remote
Measurement Systems, Seattle, WA, USA) and recorded using a computer and
Datacan V software (Sable Systems, Henderson, NV, USA). Flight activity and
wingbeat frequency were recorded on a standard cassette recorder (Radio Shack
CTR 123) whose microphone (Realistic catalog no. 331052) was inserted through
the chamber stopper. The same recorder was used for both recording and
playback, and was powered by an external AC power supply throughout to ensure
uniform tape speed. Carbon dioxide production and water loss were measured
using a Li-Cor 6262 analyzer (Li-Cor, Lincoln, NE, USA), using 0.1 min signal
averaging, with signals recorded every 0.1 min. Corrections for dilution and
infrared band-broadening arising from the inclusion of subjects' water loss in
the excurrent air column were made by the Li-Cor unit. Each data point
presented represents the mean for the measurement period. Data from the first
minute of measurement were not used except for measurements when chamber
temperature was not elevated above ambient; 1 min of equilibration is
sufficient for honeybees to reach thermal equilibrium even at elevated
Ta (Roberts and
Harrison, 1999). The chamber was kept in the temperature control
cabinet and flushed with scrubbed air between measurements.
At the conclusion of respirometry measurement, the chamber stopper was
removed and a plastic bag that had been kept within the temperature control
cabinet was immediately placed over the mouth of the respirometry chamber. The
bag and chamber were then removed from the cabinet and the bee was shaken into
the bag and restrained against a sheet of foam as rapidly as possible; within
10 s, thorax (Tth,), then head (Th),
then abdomen (Tab) temperatures were measured using a
hypodermic needle thermocouple probe
(Heinrich, 1993) connected to
a Physitemp BAT 12 field thermometer. Body segment temperatures are reported
only for bees that were flying for the final 30 s of respirometry measurement
and that had sustained flight for >40 s of the final min of measurement.
Body and nectar crop content mass were determined immediately after
measurements to the nearest 0.001 g using a Sartorius L4205+ pan balance
(Sartorius GMBH, Göttingen, Germany).
Analysis
Respirometry data were transformed and analyzed using Datacan V, with
washout correction performed as described in Bartholomew et al.
(1981), as implemented in
Datacan V; statistics were done in SPSS 11 for Macintosh (SPSS Inc, Chicago,
IL, USA). CO2 production values were converted to W assuming
carbohydrate metabolism (Beenakkers et al.,
1984
). Early observations that WBF of some bees declined
audibly during the measurement period made it clear that post-respirometry WBF
measurements would not be representative. However, the air pump and blower
motor contributed background noise during respirometry, making analysis of
digitized recordings made during respirometry problematical. We therefore
determined WBF for each 0.5 min of respirometry measurement from the pitch of
the flight tone (Sotavalta,
1947
). Analysis was performed by one of us (W.A.W.) who has
absolute pitch, as did Soltavalta (1947), but using a keyboard verified to be
tuned to concert pitch (A=440 Hz) as a reference. Recordings were sampled
continuously and estimated pitch noted at least twice during each 0.5 min
interval, and more frequently if pitch varied. In a double-blind verification
of this method, 9 x 30 s recording sections that did not include blower
motor noise were re-analyzed by M. Schindlinger using Cool Edit software
(Syntrillium/Adobe Systems, San Jose, CA, USA); acoustically determined values
averaged 2.0±3.5 Hz (mean ±
S.E.M.), or 1.1%, higher than digitally
determined values, a difference that was not significant (paired-sample
t-test, d.f.=8, P>0.1). Periods of flight were timed from
recordings by stopwatch, with very brief buzzing intervals of <1 s omitted
as not representing flight; these measurements were repeatable to within less
than 3%. To synchronize sound recordings with respirometry traces, a time base
correction of -12 s was applied to recordings to account for the 9
srespirometry system time delay (established by bolus injection using a 1 s
sampling interval) plus a 3 s delay arising from our 6 s signal averaging
period. Periods of self-sustaining flight with no interruption or provocation
for at least 1 min, and which did not include periods of substantial (>10%)
decline in WBF, were termed first quality flight and are reported as a subset
of our measurements.
Head and abdomen temperature excess ratios (Rh and
Rab, respectively) are the ratios of head or abdomen
temperature excess to thorax temperature excess, calculated as
Rh=(Th-Ta)/(Tth-Ta)
and
Rab=(Tab-Ta)/(Tth-Ta);
these values are predicted to be independent of Ta if heat
transfer between segments does not change, but to be associated with
Ta if thermoregulation involves changes in heat transfer
between body segments. For example, an increase in the value of
Rh or Rab at higher values of
Ta would indicate an increased shunting of heat to the
head or abdomen, suggesting use as a thermal window to dissipate thorax heat,
at least in the absence of changes in other pathways of heat exchange
(Baird, 1986;
Stavenga et al., 1993
;
Roberts and Harrison, 1999
). A
decrease to negative values in Rh or
Rab at higher Ta would indicate
evaporative cooling from the head or abdomen, respectively
(Roberts and Harrison,
1999
).
Since no pyranometer was available, we accounted for solar short-wave
radiative heat gain by making hourly measurements with a microeinstein meter
(Li-Cor), with its sensor pointed directly upwards in full sun, and both
upwards and downwards in the shade. Full sun values were indexed to direct
solar radiation in W m-2 as calculated from the solar zenith angle
for the dates and latitude of our measurements (appendix B in
Stevenson, 1985). The ratios
of the microeinstein values in shade to those in full sun were used to
establish the fraction of full sun values encountered in our shaded location.
Honeybee surface area was estimated by scaling values reported by Roberts and
Harrison (1999
) to the mean
body mass of our bees, assuming a surface area mass scaling exponent of 2/3.
Calculated short-wave radiation from overhead and from below in shade were
each assumed to strike 50% of the bee's surface area
(Kenagy and Stevenson, 1982
;
Cooper et al., 1985
).
Effect of agitation
In a separate set of measurements, we sought to determine whether the
degree of flight chamber agitation required to maintain flight affected the
association between FMR and Ta. Bees were captured and
handled as described previously, but during a different year and from a
different colony maintained by B. H. in Hinesburg, VT, USA, between 08:30 h
and 17:30 h during late July and early August. Respirometry was done as
previously described except that we used Tygon tubing (Saint-Gobain
Performance Plastics, Bridgewater, NJ, USA), since its elasticity assured
secure seals at fittings when the chamber was being vigorously shaken. Because
of Tygon tubing's hygroscopic properties (J. R. B. Lighton, personal
communication), temporal response for water loss is much slower and the values
less trustworthy; we therefore do not report water loss data for this
experiment. No temperature control cabinet was used; instead, chamber
Ta was allowed to track ambient temperature in shade. WBF
was not recorded. Bees were kept airborne by administering chamber agitation
only when they attempted to land. Behavior, as recorded in field notes and by
markers in the Datacan V files, was divided into three categories. In the
first, which we term `non-agitated flight', bees flew with few or no landing
attempts (not extending their legs or changing body angle in apparent
preparation for landing). In the second, `agitated flight', bees repeatedly
attempted to land and were kept in the air by frequent or constant chamber
agitation. In the third, `intermediate flight', behavior was varied or
intermediate, with no portion of the measurement clearly representing either
specific behavior. Respirometry data analysis was as in the primary
experiment.
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Results |
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For bees in first-quality flight, FMR was not associated with Ta (Fig. 2A). FMR was also independent of time of day (P=0.92) and of the number of hours before or after solar noon (P=0.21). There was, however, a positive relationship between WBF and Ta (Fig. 2B, r2=0.34, P=0.01), though this rested upon measurements at Ta values between 19 and 31°C; for Ta values between 25 and 37°C, there was no association (N=14, r2<0.01, P=0.89). We did not find an association between WBF and FMR during periods of first-quality flight (r2=0.11, P=0.15). However, we did find a negative association between metabolic energy expenditure per wingbeat and Ta (Fig. 3), amounting to a reduction of 14.4% between 20 and 37°C, though significance was marginal (N=19, r2=0.21, P=0.046).
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Body temperature
Body segment temperatures are reported for the 36 of 78 honeybees that were
flying at the end of respirometry and had flown for more than 2/3 of the final
1 min and for the final 30 s immediately prior to body temperature
measurements (Fig. 5). 13 of
the 19 bees displaying 1 min or more of first-quality flight (Figs
2,
3,
4) met these criteria and are
included in Fig. 5. Data for
four bees whose WBF had declined by between 18 and 25% during measurement and
that had empty honeycrops, suggesting energy reserve depletion, but that flew
during the final minute as described, are shown but are excluded from the
displayed regressions. Although Tth was significantly
affected by Ta, the slope of the least-squares regression
is relatively shallow at 0.181; bees maintained relatively stable thorax
temperatures (Tth) of 38.5±2.1°C (mean ±
S.D., N=31) over Ta values
ranging from 18 to 39°C. Th and
Tab were likewise significantly affected by
Ta (Fig.
5), though the relationships were distinctly nonlinear, with both
Th and Tab falling significantly below
Ta at Ta values >34°C
(one-tailed paired-sample t-test, d.f.=9; for Th,
P<0.01; for Tab, P<0.0001).
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At Ta values <28°C, the slopes of both the head and abdomen temperature excess ratios (Rh and Rab) vs Ta were not significantly different from zero (Fig. 6A; P for each slope<0.01), while above 34°C the slopes were nearly vertical (Fig. 6A). The abrupt change in these ratios corresponded to a sharp increase in evaporative heat loss in the same Ta range (Fig. 6B).
|
Calculated direct solar radiation at our 44.47°N latitude at solar
zenith on August 29 was 882 W m-2. At solar noon under clear
conditions, microeinstein values with the sensor oriented upward in the shade
were 4.4% of values with the sensor similarly oriented in direct sunlight;
with the sensor oriented downward in shade, values were 2.3% of those in full
sunlight. Corresponding values 1 h before sunset, the approximate time of each
day's final measurements, were 2.8 and 1.9% of full noon sun values. Mean
calculated body surface area, based on mean body mass of 0.090±0.001 g
(± S.E.M.) was 265 mm-2;
calculated short-wave heat gain by individual bees therefore ranged from 58 to
84 mW g-1. Since Ta treatment was independent
of both time of day and mean hours before or after solar noon, bees
experienced a mean heat gain of about 71 mW g-1 across
Ta treatments. Occasional broken low cloud cover (present
during a minor portion of our measurements) would be expected to reduce these
values by 15-30% (Monteith,
1973).
Effect of behavior
The mean fraction of the measurement period spent in flight (FTF) for the
full sample of 78 honeybees was 0.52; FTF was not affected by time of day
(P=0.48) or by the number of hours before or after solar noon
(P=0.73). For all 78 bees, FTF showed a weak decline as
Ta increased, though it was not significant
(N=78, r2=0.04, P=0.08). However, for
the 19 bees that displayed 1 or more min of first-quality flight
(Fig. 4), this relationship was
strongly significant (r2=0.63, P<0.0001).
FTF had a pronounced effect on the relationship between metabolic rate and Ta (Fig. 8). For bees that flew for 80% of more of the time, the relationship was not significant (N=14, r2=0.02, P=0.66). However, for bees flying between 40 and 79% of the time, the slope of metabolic rate on Ta was steeper and strongly significant (N=36, r2=0.31, P<0.0001), and became still steeper and more strongly significant for bees that flew for less than 40% of the measurement period (N=28, r2=0.60, P<0.00001).
For the full sample of 78 bees regardless of FTF, mean WBF over the measurement period was independent of Ta for bees whose WBF declined by <5% (Fig. 7, N=54, r2<0.01, P=0.69). Among bees whose WBF declined by >5%, several at low Ta yielded the lowest WBF values we recorded; however, WBF was still independent of Ta (Fig. 7, regression not shown; N=24, r2=0.06, P=0.25). FTF had little effect on this relationship, with none of the three categories showing a significant association (for 0-39% flight, N=28, r2=0.07, P=0.17; for 40-79% flight, N=36, r2<0.01, P=0.66; for 80-100% flight, N=14, r2=0.02, P=0.63).
Effect of agitation
The response of metabolic rate to Ta differed between
honeybees in agitated and non-agitated flight. For bees in non-agitated
flight, metabolic rate was independent of air temperature
(Fig. 2A). In contrast, for
honeybees in agitated flight, metabolic rate decreased as chamber air
temperature increased, with values at 38°C less than two thirds of those
at 22°C (Fig. 2B). Metabolic rate values at lower temperatures were similar for agitated and
non-agitated fliers, while differences in values at higher temperatures
accounted for the difference between the two categories
(Fig. 2). After discarding data
that included pronounced declines in metabolic rate, possibly indicating
depletion of energy reserves, there was no relationship between mean time from
the beginning of measurement to the midpoint of the trace section averaged and
the rate of CO2 production (N=59,
r2=0.06, P=0.19).
Measurements of the first eight honeybees in this data set were completed
indoors. Since only one of these bees met our criteria for non-agitated flight
(Fig. 9), we moved the
apparatus outdoors into a shaded location, where non-agitated flight behavior
was much more frequent (30 of 51 bees), and there completed all further
measurements. While collecting the data in our primary data set (Figs
1,
2,
3,
4,
5,
6,
7,
8), we made 20 additional
indoor measurements; eight of these bees displayed non-agitated flight. In
all, only 32% (9) of the 28 bees measured indoors displayed non-agitated
flight, compared with 59% (37) of the 51 bees measured outdoors. Because
metabolic rate values in our primary data set were overall about 15% lower,
raising the possibility of seasonal or colony effects (reviewed in
Harrison and Fewell, 2002),
these supplementary measurements are not included in the regressions in
Fig. 9. However, their
inclusion did not alter the conclusions for either flight quality category
(for non-agitated flight, MR=-0.903Ta+676.3,
N=39, r2<0.01, P=0.78; for agitated
flight, MR=-19.47Ta+1122, N=28,
r2=0.52, P<0.0001).
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Discussion |
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What could account for these differing outcomes? Harrison and Fewell
(2002) have hypothesized that
a combination of two factors may provide an explanation. First, honeybees in
the different studies may have had different metabolic capacities. Such
variation has been attributed to differences between colonies
(Harrison et al., 1996b
),
genotypes (Harrison and Hall,
1993
; Harrison et al.,
1996b
), seasons (Harrison et
al., 2001
) and foraging task
(Feuerbacher et al., 2003
).
Second, FMR and Tth may be positively correlated below a
particular Tth and negatively correlated above it;
Harrison and Fewell (2002
)
propose that a Tth of 38°C, at which Coelho
(1991
) found maximum flight
force production for tethered honeybees
(Fig. 11A), might correspond
to a maximum value for FMR, with FMR decreasing as Tth
either increased or decreased.
|
The relationship between flight force production (FFP) and FMR in
free-flying honeybees has not been directly tested. However, for our and other
studies of honeybees, FMR during self-supporting flight and FFP during
tethered flight show similar patterns of response to Tth
(Fig. 11). As
Ta increases to 47°C, both FMR and FFP fall to the
minimum values associated with flight
(Coelho, 1991;
Harrison and Fewell, 2002
),
and the ratio of minimum values for flight to maximum values attained is
similar for FMR and FFP (Fig.
11). The effect of Ta on FMR for hovering or
slow forward flight has been measured only for air temperature values that
yielded relationships that were nominally linear and were so reported;
accordingly, the range of air temperatures measured in each study, together
with between-study differences in the bees' capacity for heat production and
loss at a given Ta, can determine slope of FMR on
Ta by shifting the endpoints to the left or right along
what may be an overall nonlinear function
(Fig. 11). Examining FMR and
Ta for discrete Ta ranges in each
study suggests an even closer relationship between FMR and FFP than do the
reported linear relationships of FMR and Ta
(Fig. 11). In our study,
independence of FMR and Ta may therefore be explained by
the relatively narrow Tth range, the bees defended, all
falling within the 36-41.5°C range, for which Coelho
(1991
) found that FFP did not
fall below 95% of the maximum value reached at 38°C. Similarly, the
inverse association between FMR and Ta in the Roberts and
Harrison (1999
) study may be
attributable to the overall higher and broader range of
Tth values encountered, with the lowest value at 38°C
(Fig. 11). In the Heinrich
(1980b
) study, mean
Tth fell either above or below the 36 to 41.5°C
range.
Metabolic rates in our midsummer experiment (agitation effects) are similar
to those for honeybees measured in midsummer in Arizona (Harrison et al.,
1996a,b
;
fig. 9 in Roberts and Harrison,
1999
), while values for our late-season measurements (primary
dataset) are intermediate between those collected in midsummer and in
temperate midwinter (Harrison et al.,
2001
; Harrison and Fewell,
2002
). The 15% difference between our datasets is several times
greater than has been associated with colony or genotype effects
(Harrison et al., 1996a
;
Harrison and Fewell, 2002
),
but leaves open the possibilities of effects of season
(Harrison et al., 2001
) or
foraging task (Feuerbacher et al.,
2003
).
Dependence of body temperature on air temperature
The slope of Tth on Ta between 18
and 38°C was 0.18, or shallower by at least half those found for honeybees
in untethered flight; previous values for indoor studies range from 0.39 to
0.52 (Heinrich, 1979,
1980b
; Harrison et al., 1996;
Roberts and Harrison, 1999
),
and for outdoor studies from 0.37 to 0.44
(Cooper et al., 1985
;
Coelho, 1991
;
Feuerbacher et al., 2003
).
What could explain the twofold difference in the slope of
Tth on Ta between our study and
others? A direct approach to this question would be to calculate heat budgets
for different Ta values. We do not follow that approach
because of the lack of temporal synchrony of the body temperature measurements
with FMR and evaporative heat loss (EHL) data, critical for animals that
exhibit rapid heating and cooling rates, and because we lack data for surface
temperature and convective heat loss. However, it is useful to compare our
results with previous research in terms of what we do know about all possible
paths of heat exchange. Sources of heat gain in our case include metabolic
heat production and short-wave radiation. These gains must be balanced by heat
losses that include net long-wave radiation, convection and evaporation. In
the only other study to measure FMR, EHL and body segment temperatures of
honeybees in voluntary flight (Roberts and
Harrison, 1999
), mean Tth at
Ta=21°C was only 0.6°C higher than in our study
despite mean FMR values that were about 100 mW g-1 (16%) higher,
EHL that was somewhat (about 30 mW g-1) lower, and long-wave heat
flux that was presumably similar. This roughly 70 mW g-1, or net
remaining difference, may be attributable to the calculated short-wave heat
gain value range of 58-84 mW g-1 in our study. At higher
Ta values (34-39°C), bees in our study defended lower
Tth values; at Ta=38°C, predicted
Tth was 40.2°C in our study and 44.4°C in Roberts
and Harrison (1999
). This
difference in Tth corresponds to differences between the
two studies in the responses of temperature excess ratios and EHL to
Ta. In our case, Rh and
Rab became lower and more variable at
Ta values above 34°C
(Fig. 6), while in the Roberts
and Harrison (1999
) study this
change did not occur until Ta exceeded 39°C; EHL
reached 100-200 mW g-1 at Ta values of 35 to
36°C in our case but only at 30-40°C in Roberts and Harrison
(1999
). Curiously, EHL also
increased as Ta values fell below 21°C; it is possible
that this was due to excretion of excess water resulting from a positive water
balance (see Nicolson and Louw,
1982
; Bertsch,
1984
) and contributed little to EHL.
The head serves as a thermal window for excess thoracic heat through
evaporaton of regurgitated nectar (Esch,
1976; Heinrich,
1980a
,b
).
At air temperatures below 30°C, mean Th excess was
about 7°C. However, at Ta values from 35 to 38°C,
mean Th was more than 2°C lower than
Ta. This crossover occurs at a Ta
value >6°C lower than in indoor measurements of untethered honeybees
(Heinrich, 1980b
;
Roberts and Harrison,
1999
).
Tab closely tracked Ta when the
latter was <30°C, but showed a surprising decline to about 4°C
below Ta when the latter was between 35 and 38°C.
There is no previous evidence that the abdomen plays more than a minor role as
a heat exchanger in honeybees (Heinrich,
1980b). Honeybees maintain impressively high
Tth despite small body size and lack of insulation, at
least partly by preventing the loss of heat to the abdomen, which is joined to
the thorax by a narrow petiole that has a countercurrent heat exchanger (see
Heinrich, 1980b
); there is no
evidence that this heat exchange is circumvented at higher air temperatures in
order to take advantage of the abdomen as a thermal window, as occurs in
bumblebees (Heinrich, 1976
,
1980b
). Nevertheless,
Tab did drop below Ta when the latter
was >42°C in previous indoor measurements
(Heinrich, 1980b
;
Roberts and Harrison, 1999
),
and is associated with higher total EHL, for which several possible mechanisms
have been proposed (Roberts and Harrison,
1999
).
Regulation of lower thoracic temperatures
Why did honeybees in our study regulate lower and more stable
Tth at higher air temperatures, apparently through higher
EHL? Honeybees flying at air temperatures where they could maintain
Tth at or near the 38°C level, associated with maximum
metabolic rate and force production, do not always do so. Attacking bees
regulate Tth at about 38°C, whereas the mean
Tth of outgoing foragers from the same hive is about
3°C lower (Heinrich,
1979), and bees imbibing 40-60% sucrose maintain
Tth at 36°C, while bees imbibing 10-30% sucrose
maintain Tth of only 33°C
(Waddington, 1990
). Those
studies were made at lower air temperatures, where allowing
Tth to fall results in a lower rate of energy use. At
higher air temperatures, however, similar energy savings could be realized by
limiting active evaporative heat loss and allowing Tth to
rise above 38°C, yielding dividends of improved water balance and greater
flight range (see Cooper et al.,
1985
). Direct experimental evidence for such a strategy is
lacking, but the observation that Tth decreased as wing
loading increased for honeybees foraging at Ta values
between 35 and 40°C (Cooper et al.,
1985
) is suggestive. Since flying honeybees can have FMR values as
high as 800 mW g-1, but can maintain flight with FMR as low as
about 300 mW g-1 (Coelho,
1991
; Harrison and Fewell,
2002
), there appears to be a considerable margin within which a
trade-off between energy and water conservation on the one hand and mechanical
power production on the other could be varied through the active mechanism of
evaporative heat loss at high Ta.
Dependence of wingbeat frequency on air temperature
In studies that have measured WBF and FMR during hovering, agitated or
tethered flight, the two variables have similar associations with
Ta in honeybees (Esch,
1976; Feller and Nachtigall,
1989
; Harrison et al., 1996;
Roberts and Harrison, 1999
)
and in other insects (May,
1995
; Roberts et al.,
1998
; Borrel and Medeiros, 2004). However, as with FMR, the slope
of the relationship between WBF and Ta for honeybees is
variously found to be strongly positive
(Feller and Nachtigall, 1989
;
Esch, 1976
), slightly positive
(this study), zero (Spangler,
1992
) or negative (Spangler,
1992
; Harrison et al., 1996;
Roberts and Harrison, 1999
).
These differences appear to be explained by differences in
Tth range between studies. Where positive slopes of WBF on
Ta are reported, Tth values all fell
below 36°C (Esch, 1976
;
Feller and Nachtigall, 1989
),
and where negative slopes are reported, Tth values were
mostly above 37°C; in our study, where the slope was only slightly
positive, Tth fell between 36 and 40°C. Thus, the
relationship of WBF to Tth appears to follow the same
general pattern as the relationships of FFP and FMR to
Tth.
Dependence of energy expenditure per wingbeat on air temperature
This is the first report of a temperature effect on energy expenditure per
wingbeat for honeybees in voluntary flight; as air temperature increased from
20 to 37°C for first-quality flight, energy expenditure per wingbeat
diminished by 14.4%. An examination of data from other studies, however,
suggests that this phenomenon may not be uncommon among flying insects
(Table 1). In the case of
honeybees, the finding may explain an apparent discrepancy noted between
tethered flight experiments (Heinrich,
1993): although the oxygen consumption per action potential is
independent of Ta in both honeybees
(Bastian and Esch, 1970
) and
bumblebees (Kammer and Heinrich,
1974
), mechanical power output at a given action-potential
frequency increases with temperature (Esch
et al., 1975
). This is consistent with the positive relationship
of Ta and muscle efficiency in hovering orchid bees
(Borrell and Medeiros, 2004
).
Since honeybees are myogenic fliers, with stretch-activated wing muscles, this
effect is probably not due to greater overlap of muscle contractions at lower
temperatures, leaving open the possibility that muscle viscosity or internal
friction decreases at higher temperatures, or that elastic energy storage is
affected by temperature (Heinrich,
1993
; Borrell and Medeiros,
2004
).
|
Could agitation be a confounding factor?
For the full sample of our primary data set, honeybees spent an average of
52% of the measurement period in flight, with this value declining at higher
Ta values (Fig.
8), at which foragers will often cease flight in order to cool
(Cooper et al., 1985). As the
fraction of time spent in flight decreased, the slope of FMR on
Ta became steeper (Fig.
8). For bees flying less that 40% of the time, the slope fell
between that for workers that were not flying at all
(Cahill and Lustick, 1976
) and
that for bees that spent more than 80% of the time flying
(Fig. 8). Workers that are not
flying nevertheless maintain high thorax temperatures by shivering
their wing muscles (Cahill and Lustick,
1976
); at lower air temperatures, this behavior has a metabolic
cost similar to that of flight (Figs
8,
9), but as air temperature
increases, shivering diminishes and non-flight metabolic rate declines to a
small fraction of its value at lower Ta
(Fig. 8;
Cahill and Lustick, 1976
).
Thus, to the extent that non-flight behavior is included in respirometry
measurements of honeybee workers, we might expect a more negative slope of
metabolic rate on Ta, and that this effect would be
distinct from that of Tth on FMR
(Fig. 11). This may explain
why, for bees with Tth in the 36-41.5°C range over
which FMR and FFP are relatively unchanged, there is an inverse relationship
between FMR and Ta for bees under constant agitation (for
Ta=20-27°C, W
g-1thorax=2.53-0.048Ta,
N=69, r2=0.23, P<0.0001; data from
Harrison et al., 1996) but not for bees in voluntary flight [for
Ta=20-29°C, mW
g-1=674.1-3.19Ta, N=10,
r2=0.03, P=0.64; data from Roberts and Harrison
(1999
); see
Fig. 2A, all measurements, in
the present study]. This is supported by the results of our separate agitation
effects experiment, in which bees were agitated only as much as necessary to
keep them in the air, with bees' willingness to fly without agitation as our
primary independent variable. The slope of FMR on Ta for
bees in our study that required constant or frequent agitation to remain
airborne is similar to that for bees that were constantly agitated regardless
of willingness to fly (Harrison et al., 1996), suggesting that metabolic
measurements made under constant agitation, while valuable where a large
sample size is required under constant conditions, may not be representative
of flight where Ta is an independent variable.
Indoor vs outdoor flight
In outdoor measurements, the incidence of voluntary flight was about twice
that for indoor measurements. Foraging honeybees are known to navigate using
familiar landmarks and polarized light
(Dyer, 1996;
Esch and Burns, 1996
; Wehner
et al., 1997; Srinivasan et al.,
1996
,
1997
), cues that were present
in our study but not in other respirometry measurements of honeybees.
Honeybees also have a high flicker fusion frequency
(Miall, 1978
), and show higher
WBF and slower, more interrupted flight under fluorescent lighting of 100 Hz,
similar to that in many laboratories, than under fluorescent lighting of 300
Hz (van Praagh, 1972
). In
addition, honeybees judge distance to objects during flight by motion parallax
(Srinivasan et al., 1991
,
1996
), and in our outdoor
measurements bees had a largely unobstructed view of natural objects at
natural distances. When, during the agitation effects experiment, we briefly
placed visual barriers close to the side of the chamber the bee was facing,
the animal nearly always re-oriented itself to face in a direction with an
unobstructed view. It was probable, though, that bees could also perceive the
chamber wall, possibly because of the ripples in the glass surface, since most
sustained circling, bobbing or hovering flight toward the center of the
chamber rather than persistently flying against the glass.
The salient difference in methods between our study and others of honeybees in voluntary flight under confinement was the presence of more nearly natural visual stimuli, and the principal difference in results was the bees' defense of a narrower Tth range which, in turn, appears to explain our finding of independence of FMR and Ta. We cannot conclude that any of our findings other than the frequency of voluntary flight were affected by our outdoor location. However, providing a more natural visual environment may be more important than previously recognized for obtaining representative flight under containment, and partitioning the effects of different components of the full suite of natural visual stimuli, may help explain remaining differences in results between respirometry studies of honeybees, as well as other insects, in flight.
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References |
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