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Environmental and genetic influences on flight metabolic rate in the honey bee, Apis mellifera
Comparative Biochemistry and Physiology Part A 133 (2002) 323–333
Review
Environmental and genetic influences on flight metabolic rate in the honey bee, Apis mellifera夞 Jon F. Harrison*, Jennifer H. Fewell Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA Received 11 June 2001; received in revised form 27 January 2002; accepted 29 January 2002
Abstract Flying honey bees demonstrate highly variable metabolic rates. The lowest reported values (approximately 0.3 W gy1) occur in tethered bees generating the minimum lift to support their body weight, free-flying 2-day old bees, winter bees, or bees flying at high air temperatures (45 8C). The highest values (approximately 0.8 W gy1) occur in foragers that are heavily loaded or flying in low-density air. In different studies, flight metabolic rate has increased, decreased, or remained constant with air temperature. Current research collectively suggests that this variation occurs because flight metabolic rates decrease at thorax temperatures above or below 38 8C. At 30 8C, approximately 30% of colonial energy is spent during typical foraging, so variation in flight metabolic rate can strongly affect colony-level energy balance. Higher air temperatures tend to increase colonial net gain rates, efficiencies and honey storage rates due to lower metabolic rates during flight and in the hive. Variation in flight metabolism has a clear genetic basis. Different genetic strains of honey bees often differ in flight metabolic rate, and these differences in flight physiology can be correlated with foraging effort, suggesting a possible pathway for selection effects on flight metabolism. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Africanized honey bee; Apis mellifera; Development; Flight; Foraging; Honey bee metabolic rate; Metabolism; Season; Temperature; Thermoregulation
1. Introduction Comparative studies can lend the impression that hovering flight metabolic rate is a defined parameter for a given species. Species-specific metabolic rates are often adopted by ecologists or evolutionary biologists for theoretical or applied 夞 This paper was presented in the session, ‘Physiology and Biochemistry of Exercise’, at the Society for Experimental Biology, April 2–6, 2001, Canterbury, UK. *Corresponding author. Tel.: q1-480-965-9459; fax: q1480-965-2519. E-mail address: [email protected] (J.F. Harrison).
purposes. However, a growing body of research suggests that intraspecific variation in flight metabolic rate can be substantial, even during what appears to the human observer as stationary hovering. In this review, we focus on a single insect species, the honey bee, Apis mellifera, to illustrate some of the causes and consequences of such variation in flight energetics. To our knowledge, there is no other species, vertebrate or invertebrate, for which flight metabolic rate has been quantified in such a wide range of environments and populations (see Harrison and Roberts, 2000 for review).
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1.1. Honey bees as a model system Honey bees have many advantages as model organisms for the study of insect flight. Their large size and endothermy facilitates the rapid metabolic measurements needed to track energetic changes associated with behavioral variation. Honey bees fly freely or on tethers in experimental chambers. They also can be individually marked, and their complex behaviors can be observed within and without the hive and compared to their flight physiology. Honey bees are also well situated for studies of the genetic basis to variation in flight physiology. Among insects, the honey bee genome is known with a precision second only to Drosophila melanogaster (Hunt et al., 1997; Page et al., 2000). Selection at the colony level produces effects in a large number of related workers that can be tested without destroying the colony as a whole (Fewell and Page, 2000). Finally, honey bee queens can be instrumentally inseminated, allowing the production of hybrids and backcrosses. 1.2. Methods for studying honey bee flight metabolism Because of their small size and high metabolic rates, honey bees are well-suited for both flowthrough and closed-system respirometry studies. Flying honey bees exclusively use aerobic carbohydrate catabolism during flight (Rothe and Nachtigall, 1989), so carbon dioxide emission and oxygen consumption provide equivalent measures of metabolic rate. Since modern carbon dioxide analyzers have greater precision than oxygen analyzers, most recent respirometry studies have measured carbon dioxide emission (Harrison and Roberts, 2000). Measurements during tethered flight have allowed quantification of the lift produced during hovering, and continuous measures of thorax temperature, concomitant with flow-through respirometry measures of metabolism (Esch, 1976; Jungmann et al., 1989; Nachtigall et al., 1989). Metabolic rates during free flight have been measured using three types of methods: hovering flight in vertical tubes or respirometry chambers, agitated flight in chambers, and forward flight in wind tunnels. In the hovering flight protocol, bees are measured (using closed- or open-system respirometry) during undisturbed, stationary hovering for
seconds to minutes in a relatively large (liters) chamber. Lights above the chamber induce flight. Bees that cling to the walls, ascend or descend are discarded. In some cases the tarsi are removed to prevent the bees from landing on the chamber walls (Heinrich, 1980a,b). Stationary hovering protocols have been used for studies of temperature (Heinrich, 1980a,b; Harrison et al., 1996a; Roberts and Harrison, 1999), air pressure (Withers, 1981), and loading (Wolf et al., 1989) effects on flight metabolic rate. Unless the tarsi are removed a majority of bees tested with a hovering protocol will cling to the vessel walls for periods of time and must be discarded. Proper lighting and chamber design can increase the number of bees that fly freely, but the proportion of bees that fly without landing remains low. To overcome this problem, we have used an agitated flight protocol combined with flowthrough respirometry, using chamber vibration to induce bees to fly in a relatively small (0.3 l) rounded chamber for 1–2 min. With this protocol, behavior of the bee in the chamber varies widely from constant to little flight. However, metabolic rates and wing beat frequencies (during flight periods) are similar to those measured for bees in continuous hovering (Harrison et al., 1996a), probably because the flight muscle can generate a similar metabolic rate during non-flight shivering as during flight. Because more than 90% of bees exhibit strong elevations in metabolic rate in response to agitation, this protocol is particularly useful for population genetic studies (Harrison and Hall, 1993; Fewell and Harrison, 2001). The agitated flight protocol is also appropriate for studies in which it is mandatory to assess the metabolic capacity of all bees including those who will not fly (e.g. studies of the effects of pathogens: Harrison et al., 2001). A re-circulating wind tunnel with optical feedback to orient the bees has been used to measure flight metabolic rate of bees carrying loads during slow forward flight (Wolf et al., 1989). Metabolic rates measured in slow forward flight were similar but not identical to those measured during undisturbed stationary hovering (Wolf et al., 1989). This sophisticated technique probably provides values most similar to natural flight. 1.3. Metabolic scope Flying honey bees with similar body temperatures have metabolic rates of 0.3–0.8 W (g
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body)y1 (see below). The thorax accounts for approximately 20–40% of body mass, and the flight muscle occupies approximately 75% of the thorax (Heinrich, 1980b). Resulting metabolic rates can reach 2–3 W gy1 of flight muscle. This is at least twofold higher than comparable estimates for flight muscle of hovering hummingbirds, and 30 times higher than human athletes exercising at VO2max (Suarez et al., 1996). The high metabolic rates of honey bee flight are partially, but not completely explained by their small body size relative to vertebrates (Harrison and Roberts, 2000). In endothermic insects like honey bees metabolic rates during flight can be 100 times higher than in resting individuals (Kammer and Heinrich, 1978), so the metabolic scopes of endothermic insects far exceed those of most vertebrates. This tremendous scope is often used to justify the assumption that essentially 100% of oxygen consumption during flight occurs in the flight muscle (Suarez et al., 1996). However, these extreme metabolic scopes depend on comparisons between cold, quiescent bees and warm, flying bees. Resting metabolic rates of honey bees vary widely, depending on the temperature and activity level of the bee (Rothe and Nachtigall, 1989; Lighton and Lovegrove, 1990). Ideally, metabolic scope would be estimated by comparing metabolic rates of flying and non-moving bees with identical body temperatures. This is a difficult comparison to make empirically, as single bees kept in a warm metabolic chamber (like most bees in a warm hive) walk and groom continuously. Walking and grooming workers, with thorax temperatures no more than 1 8C above air temperature (35 8C), have metabolic rates of 0.08 W (g body)y1 (S.E.M.s0.011, Ns15). Since the inside of a brood-rearing hive is usually near 35 8C, the metabolic rate of a bee emerging from the hive and taking flight increases four- to ten-fold, exhibiting a metabolic scope similar to typical vertebrate values. 2. Individual variation in flight metabolism 2.1. Effect of air temperature Honey bees have been reported to forage at air temperatures ranging from 5 to 45 8C (Heinrich, 1979; Cooper et al., 1985; Spangler, 1992). It should be noted that these represent ranges from
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Fig. 1. Effect of air temperature on thorax temperatures of Apis mellifera. Data in Figs. 1–4 from Heinrich (1980b), Roberts and Harrison (1999) and Harrison et al. (2001).
studies across many populations, locations and seasons; our impression is that the daily range of air temperatures at which a single colony will forage is narrower. At air temperatures below 20– 25 8C honey bees do not thermoregulate. Thoracic temperatures of bees in free flight increase linearly with air temperature with a slope approaching 1 (Fig. 1). However, at higher air temperatures freeflying honey bees partially stabilize thoracic temperature, so that it increases only 0.4–0.5 8C for every 1 8C rise in air temperature (Fig. 1, Coelho, 1991a; Harrison et al., 1996a). This represents a moderate thermoregulatory ability compared to other endothermic bee species (Roberts and Harrison, 1998). Stabilization of thoracic temperature helps preserve the capacity of the flight muscle to produce maximal power when air temperature varies; however, the range of thoracic temperatures at which bees can generate sufficient force to lift their body mass is quite broad (28–47 8C, Coelho, 1991b). Thermal stability during flight is partially accomplished by variation in heat loss. At air temperatures above 33 8C evaporative water loss increases exponentially, and accounts for half of thoracic thermoregulation (Roberts and Harrison, 1999). Honeybees extrude nectar droplets from their crop onto their mandibles at high air temper-
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Fig. 2. Effect of air temperature on flight metabolic rate for three studies of honey bee flight.
atures, and this may be the primary mechanism by which evaporative water loss is regulated (Esch, 1976; Heinrich, 1980b; Cooper et al., 1985). However, there is also evidence for substantial heat loss from the abdomen at air temperatures above 40 8C, suggesting that substantial evaporation may also occur from the abdominal surface (Roberts and Harrison, 1999). The heart and aorta of the honey bee are arranged in loops within the narrow petiole between the thorax and abdomen, so that heat produced within the thorax is not transferred to the abdomen (Heinrich and Esch, 1994). As expected from this morphology, there is no evidence for effects of temperature on heat exchange between thorax and abdomen as occurs in bumblebees (Heinrich, 1980b; Roberts and Harrison, 1999). Studies of the effect of air temperature on metabolic heat production for free-flying bees have
produced mixed results (Fig. 2). Using stationary hovering bees, Heinrich (1980a) found a constant flight metabolism for bees flying at 20 and 42 8C. However, Harrison et al. (1996b) and Roberts and Harrison (1999) found that flight metabolic rate decreased by approximately half as temperatures rise from 21 to 45 8C in either stationary hovering or agitated flight protocols. Conversely, Harrison et al. (2001) found an increase in metabolic rate with temperature in winter bees tested with the agitated flight protocol at 16 and 24 8C. How can these contradictions be explained? We can not exclude the possibility that differences in experimental protocols account for this variation. However, we hypothesize that different effects of air temperature on flight metabolism occurred in these studies because: (1) honey bees differed in their metabolic capacities, leading to very different thorax temperatures at similar air temperatures; and (2) thorax temperature and flight metabolic rate are positively correlated at temperatures less than 38 8C, and negatively correlated at higher temperatures. Multiple lines of evidence suggest that individual metabolic capacities differed considerably across (and within) these studies, producing different thorax temperatures at similar air temperatures. Within the air temperature range of 20–24 8C individual flight metabolic rates vary widely (Fig. 2). At steady-state, if heat production equals heat loss MHPsŽTthyTair.CqRHLqEHL (1) where MHP equals metabolic heat production, Tth is thorax temperature, Tair is air temperature, C is whole-animal convective thermal conductance, RHL is net radiative heat loss and EHL is evaporative heat loss. In the 20–24 8C range, when bees are flying inside controlled-temperature rooms so that chamber walls have surface temperatures similar to air temperature, and solar radiation is zero, RHL and EHL together account for approximately 25% of total heat loss (Roberts and Harrison, 1999). Convective thermal conductances calculated at 20–24 8C do not differ between winter and summer-collected European bees (Harrison et al., 2001), and thus are likely to be similar across these studies. Therefore, as a first approximation, for the 20–24 8C air temperature range, Eq. (1) reduces to MHPsŽTthyTair.X
(2)
where X is a constant. This equation predicts a
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Fig. 3. Effect of air temperature on force production of tethered bees, modified from Coelho (1991b). Horizontal lines indicate the range of thorax temperatures in three studies of honey bee flight.
positive linear relationship between MHP and Tth at a given Tair. Such a linear relationship has been reported for flying and non-flying bees (Feller and Nachtigall, 1989; Jungmann et al., 1989; Harrison et al., 2001). Using the values for the three studies in the 20–24 8C air temperature range (data in Figs. 1 and 2), a linear plot of mean thorax temperature vs. mean metabolic rate has an r 2s 0.99. Thus, in the 20–24 8C temperature range, summer-collected foragers (Roberts and Harrison, 1999) had twice the MHP (Fig. 2), creating a 5 8C higher Tth than winter-collected bees (Harrison et al., 2001) with Heinrich’s bees being intermediate in both variables. The evidence in support of the hypothesis that honey bee flight metabolic rate decreases at thorax temperatures above and below 38 8C is less conclusive, but intriguing. Force production of tethered bees is maximal at a thorax temperature of 38 8C (Fig. 3, Esch, 1976; Coelho, 1991a); a reasonable but untested hypothesis is that flight metabolic rates show the same relationship with thorax temperature. In the winter bee study, thoracic temperatures varied within a range in which higher thorax temperatures are predicted to correlate with higher force production and metabolism
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(Fig. 3). In the Heinrich (1980b) study, measured thorax temperatures occurred on either side of the optimal temperature for force production, predicting a weak negative relationship between thorax temperature and metabolic rate (Fig. 3). In the Roberts and Harrison (1999) study (as well as in Harrison et al., 1996a), all measured thorax temperatures occurred in a range in which higher thorax temperatures are predicted to correlate negatively with force production and metabolism (Fig. 3). For the three studies, the direction of the correlations between thorax temperature and flight metabolic rate is consistent with predictions (Fig. 4). For the Heinrich (1980b) and the Roberts and Harrison (1999) studies, the magnitude of the slopes of the relationships are also near those predicted from Coelho’s (1991a) data. Studies of tethered flying bees also support this hypothesis. In Nachtigall’s and associate’s studies (Rothe and Nachtigall, 1989; Feller and Nachtigall, 1989; Nachtigall et al., 1989), thorax temperatures varied between 23 and 38 8C, and metabolic rates and thorax temperatures were positively correlated. Integration of these various studies suggests that the mechanisms and degree to which honey bees stabilize thoracic temperature will depend on their metabolic capacity (which varies with season and genetic factors: see below), the range of air temperatures examined, and the environmental conditions. The extent to which temperature-generated variation in metabolic rates correlates with variation
Fig. 4. Relationships between flight metabolic rate and thorax temperatures in three studies of honey bee flight.
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Fig. 5. Honey bee flight metabolic rates under various conditions. European honey bees (A. m. ligustica): non-flying, walking and grooming 35 8C bees (this study), hovering, 20 and 45 8C (Roberts and Harrison, 1999), winter bees at 20 8C (interpolated from Harrison et al., 2001), 2-day-old bees at 20 8C (this study), hovering with a 75 mg nectar load (Wolf et al., 1989), flying in 88 kPa air (Withers, 1981), Africanized honey bee (A. m. scutellata) flying at 22 8C (Harrison and Hall, 1993).
in mechanical power output is unclear. Conceivably, variation in metabolic rate could occur without changes in power output due to processes such as substrate cycling (Surholt et al., 1990, 1991). However, during tethered flight, metabolic rate and wing-beat frequency are strongly positively correlated (Feller and Nachtigall, 1989), and hovering honey bees flying at lower temperatures (with higher metabolic rates) have higher wing beat frequencies (Spangler, 1992; Harrison et al., 1996b). However, other parameters also affect mechanical power output during flight (particularly stroke amplitude), and as yet there have been no studies to date of thermal effects on power output in free-flying honey bees. 2.2. Effects of flight speed and load carriage The primary context for flight in workers is foraging, which requires extensive changes in flight speeds and total mass transported. In the field, honey bees show tremendous flexibility in flight performance, switching rapidly between hovering and forward speeds over 10 m sy1. To date, there have been no studies of the effect of forward flight speed on metabolism in honey bees. However, in bumblebees flight metabolic rate is independent of speed up to 5 m sy1, with speed increases being accomplished primarily by variation in stroke plane angle (Casey and Ellington, 1988). Models of bumblebee flight suggest that aerodynamic power, and probably metabolic rate, must increase exponentially over higher speeds
(Dudley and Ellington, 1990), and it seems likely that a similar J-shaped pattern of metabolic rate with flight speed occurs in honey bees. Honey bee foragers can carry considerable loads during flight. Pollen loads average approximately 10 mg and can reach 30 mg (Winston, 1987). Nectar loads average approximately 10 mg, but can reach 50 mg (Winston, 1987). Forager masses vary between 60 and 100 mg, so loads range from 10 to 80% of body weight. Honey bees also carry dead bees out of the hive and fly some meters away with the corpses. Undertaker bees thus carry loads equal to their body mass during forward flight (Coelho, 1991b), though they usually cannot ascend steeply with these loads. This is a moderate load-lifting ability compared to other insects, and is consistent with the mass of the flight muscle relative to the body mass (Marden, 1987). The cost of flight increases linearly with load, increasing by approximately 40% relative to the unladen state at 30 8C to nearly 0.8 W gy1 (Fig. 5, Wolf et al., 1989). 2.3. Effect of ontogeny Metabolic rate during flight or attempted flight (in day old bees) increases approximately tenfold during ontogeny (Fig. 6). Most workers cannot fly for the first day after eclosion. Onset of the ability to fly coincides with hardening of the cuticle, a process that is probably necessary for flight. However, development of flight muscle oxidative capacity may also be important. Agitated day old
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so, this is an interesting departure from the normal situation in which elevated juvenile hormone suppresses flight muscle development in insects (reviewed by Marden, 2000). 3. Variation in flight metabolic rates across populations of honey bees
Fig. 6. Effect of ontogeny on metabolic rate during flight or attempted flight (day old bees) and flight muscle biochemical parameters. Flight metabolic rates summarized from Harrison (1986), Fewell and Harrison (2001), and data from this paper. Pyruvate kinase, citrate synthase, and glycogen data from Harrison (1986).
bees have metabolic rates of only 0.1 W gy1 (S.E.M.s0.024, Ns12), and thoracic temperatures nearly equivalent to ambient air. Two-dayold bees who can hover have flight metabolic rates of 0.3 W gy1 (S.E.M.s0.04, Ns12). The metabolic rates of bees agitated to fly increase approximately eightfold in the 2–4 days post-eclosion, coincident with tenfold increases in thoracic pyruvate kinase and citrate synthase activities, and fivefold increases in thoracic glycogen levels (Neukirch, 1982; Harrison, 1986; Moritz, 1988). Flight metabolic rates, thoracic enzyme levels and thoracic glycogen levels remain relatively constant over the 1–3 week period when the bees work within the hive. Then, at the onset of foraging, there is an approximate 15% increase in agitated flight metabolic rate, coincident with an approximate doubling of thoracic glycogen stores (Harrison, 1986; Fewell and Harrison, 2001). Cytochrome concentrations of the flight muscle also increase at least tenfold from 1 to 20 days after eclosion (Herold and Borei, 1963). It is tempting to speculate that increases in flight metabolic rate and thoracic glycogen levels might be controlled by a rise in juvenile hormone levels, which occurs in correlation with this behavioral change (Robinson, 1987; Huang et al., 1991). If
The individual variation in flight metabolic rate discussed above extends to distinct population and subspecies patterns of variation. The association of these patterns with habitat differences and genotypic variation suggest that selection plays an important role in shaping metabolic differences in this species. Perhaps the largest reported subspecies differences in metabolic rates occur between European (A. m. ligustica) and Africanized honey bees (derived from A. m. scutellata). Africanized honey bee foragers have significantly higher flight metabolic rates than European foragers (Harrison and Hall, 1993). This difference in flight metabolic rate is likely to be genetically-based; African and European foragers reared in a common natal environment show similar differences in flight metabolism (Fewell and Harrison, 2001). The higher flight metabolic rates of African bees are due to three factors: (1) smaller body size (Harrison and Hall, 1993); (2) larger thorax relative to body mass (Hepburn et al., 1999a); and (3) a higher metabolic rate per gram of thorax (Harrison and Hall, 1993). It will be important to test whether the higher flight metabolic rates of African bees correlate with greater mass-specific power output, load carriage capacity or maximal flight speeds; conceivably they could relate simply to greater behavioral activation in the agitated flight assays. Honey bee subspecies vary in wing sizes and body masses (Daly, 1991). Wing and thorax sizes also vary within a subspecies with altitude, so that high-altitude honey bees have a greater calculated mass-specific lift-generating ability (Hepburn et al., 1999a,b). The effect of such morphological variation on flight metabolism remains unstudied. Variation in flight metabolism has also been associated with variation in allele frequencies for the allozyme marker malate dehydrogenase (MDH-1). The electrophoretically fast and medium alleles of malate dehydrogenase exhibit latitudinal clines on three continents for honey bees (Nielsen et al., 1994). Bees with the fast allele have higher flight metabolic rates and wing beat frequencies (Coelho and Mitton, 1988; Harrison et
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Table 1 Energetics of ‘typical foraging’ for Apis mellifera at an air temperature of 30 8C Nectar load Nectar energetic content Energetic reward per trip Flight metabolic rate (30 8C) Trip duration Cost per trip Net gain per trip Trips per day Reward per day Cost per day during flight In-hive metabolic rate (30 8C) Daily in-hive metabolism of forager Metabolic cost per forager day Net gain rate per forager day Hive bees fed per forager % of bees which forage % of total colony energy spent foraging
30 ml 9 J mly1 (50% sugar) 270 J 2.5 J (bee min)y1 30 min 75 J 195 J, 6.5 J miny1 12 3240 J 900 J 0.16 J (bee min)y1 173 J dayy1 1073 J dayy1 2167 J dayy1 9.4 10 30
Foraging rewards and trip durations are derived from Winston (1987); within-colony metabolic rates were calculated from Kronenberg and Heller (1982), and metabolic rates during flight were taken from Roberts and Harrison (1999). Within-hive metabolic rates were considered identical for hive bees and foragers.
al., 1996b). In heterozygotes, the difference in flight metabolic rate is approximately 3% (Harrison et al., 1996b), while homozygotes, differ by up to 20% (Coelho and Mitton, 1988). The effects of malate dehydrogenase on flight metabolic rate are similar at 21 and 38 8C, suggesting that the latitudinal cline is not related to thermal adaptations of these enzymes (Harrison et al., 1996b). The association between MDH-1 allele type and metabolic rate may also be linked to subspecies differences. Africanized bees have a much higher frequency of the fast allele than feral European populations (Hall and Smith, 1991; Loper et al., 1999). 4. Comparison of the relative magnitudes of within-individual and cross-population effects on flight metabolic A comparison of the relative magnitude of the various environmental and genetic effects on flight metabolic rate is shown in Fig. 5. Minimal metabolic rates for bees able to generate sufficient lift to support their body in tethered flight are approximately 0.3 W gy1 (Nachtigall et al., 1989). We also found values of approximately 0.3 W gy1 for bees hovering at air temperatures of 45 8C, for winter bees flying at 20 8C, and for 2-day-old bees just able to fly (Fig. 5). Increased load carriage, or flying in low-density air, increases flight metabolic rate to nearly 0.8 W gy1 (Fig. 5). A protocol
that unequivocally measures maximal metabolic rate remains to be developed for free-flying insects. However, extrapolation of the relationship between flight metabolic rate and load (Wolf et al., 1989) to the approximate maximal load carriage of 100% of body weight suggests a maximal flight metabolic rate of 0.8–0.9 W gy1, approximately 3 times the minimal metabolic rate necessary for flight. The effect of air temperature on metabolic rate during hovering spans about half of this total variation. Summer-collected, foraging-age bees flying at 20 8C have flight metabolic rates of approximately 0.6 W gy1, about double that measured at 45 8C. Effects of ontogeny and season also seem to produce variation in flight metabolic rates about half of the total variation observed (Fig. 5). Crosspopulation effects seem to be smaller than the within-individual effects. African bees have massspecific flight metabolic rates approximately 10– 20% greater than European bees at similar temperatures, with malate dehydrogenase effects of a similar or smaller magnitude (Fig. 5). 5. Colony-level energetic consequences of variation in flight metabolic rate To consider the ecological importance of variation in environmental or genetic variation in flight metabolic rates, it is necessary to relate flight energetics to the metabolism of the whole colony and to the energetic rewards of foraging. The
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energetics of ‘typical foraging’ are presented in Table 1. For a honey bee foraging at 30 8C, energy costs during foraging are estimated to account for 30% of total energy expenditure. Net energy intake should allow storage of 2110 J dayy1 of honey, or alternatively, will suffice for the energetic needs of approximately 9–10 hive bees. Because approximately 10–20% of the colony forages, this calculation suggests that energy intake should only slightly exceed energy use. Air temperature must strongly effect colonial energy balance. First, if higher air temperatures decrease metabolic rate during foraging in a manner similar to that observed in hovering summer bees (Harrison et al., 1996b; Roberts and Harrison, 1999), both net energy gain (benefit-costytime) and efficiency (benefit-costycost) during foraging will increase with air temperature. For the typical foraging flight described in Table 1, net gain rate will be 20% greater at 40 8C than at 20 8C, and efficiency nearly 50% higher. Secondly, in-hive metabolic rate decreases exponentially at higher temperatures, from approximately 450 J (bee day)y1 at 20 8C to approximately 200 J (bee day)y1 (Kronenberg and Heller, 1982). Therefore, the number of hive bees fed per forager with the nectar intake described in Table 1 increases from 4 to 12 as air temperature rises from 20 to 40 8C. These results suggest that triple the foraging effort would be required for a colony to subsist at 20 8C relative to 40 8C. Also, at the same level of foraging intake, colonial honey accumulation should triple as air temperatures rise from 20 to 40 8C. Cross-population variation in flight metabolic rates may also be important to colonial energetics and growth. African honey bees have trip frequencies approximately 20% higher than European bees in the same colony (Fewell and Harrison, 2001), similar to the difference in forager flight metabolic rate. Africanized bees also allocate a much larger proportion of their workers to pollen foraging. Because pollen contains the nutritional (and especially protein) requirements for developing brood, this shift in allocation likely translates to increased rates of worker production in Africanized colonies (Fewell and Harrison, 2001). These data suggest that variation in flight rate may contribute to ability of Africanized bees to reproduce at higher rates than European bees, and may partially explain the competitive advantage Africanized bees have relative to European bees in the neotropics.
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The variation in flight metabolic rates among subspecies and the correlation of flight metabolism with foraging work effort together suggest how natural selection may affect flight muscle metabolism in honey bees. Immediate colony growth and reproduction must be traded off against accumulation of honey stores for surviving dearth periods. In temperate climates lower metabolic rates and higher efficiencies may confer the highest long-term colony growth rates (Schmid-Hempel et al., 1985; Houston et al., 1988). However, in more tropical environments, rapid growth rates and colony reproductive rates may be advantageous (Winston, 1992). Selection at the colony level may lead to genetic changes in individuals associated with higher or lower rates of foraging behavior, which may be associated with high or low flight metabolic rates. Because it is becoming easier to identify particular genetic loci associated with such behavioral or physiological variation (Hunt et al., 1997), soon it will be possible to explicitly test hypotheses concerning the evolution of flight metabolic rate in honey bees. Acknowledgments This research was partially funded by NSF IBN 9985857 to JFH and NSF 0093410 to JHF and JFH. References Casey, T.M., Ellington, C.P., 1988. Energetics of insect flight. In: Wieser, W., Gnaiger, E. (Eds.), Energy Transformations in Cells and Organisms. Georg Thieme Verlag Stuttgart, Innsbruck, Austria, pp. 200–210. Coelho, J.R., 1991a. Heat transfer and body temperature in honey bee (Hymenoptera: Apidae) drones and workers. Environ. Entomol. 20, 1627–1635. Coelho, J.R., 1991b. The effect of thorax temperature on force production during tethered flight in honeybee (Apis mellifera) drones, workers, and queens. Physiol. Zool. 64, 823–835. Coelho, J.R., Mitton, J.B., 1988. Oxygen consumption during hovering is associated with genetic variation of enzymes in honey-bees. Funct. Ecol. 2, 141–146. Cooper, P.D., Schaffer, W.M., Buchmann, S.L., 1985. Temperature regulation of honey bees (Apis mellifera) foraging in the Sonoran desert. J. Exp. Biol. 114, 1–15. Daly, H.V., 1991. Systematics and identification of Africanized honey bees. In: Spivak, M., Fletcher, D.J.C., Breed, M.D. (Eds.), The ‘Africanized’ Honey Bee. Westview Press, Boulder, pp. 13–44. Dudley, R., Ellington, C.P., 1990. Mechanics of forward flight in bumblebees. J. Exp. Biol. 148, 53–88.
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Esch, H., 1976. Body temperature and flight performance of honey bees in a servo-mechanically controlled wind tunnel. J. Comp. Physiol. 109, 65–277. Feller, P., Nachtigall, W., 1989. Flight of the honey bee II. Inner- and surface thorax temperatures and energetic criteria, correlated to flight parameters. J. Comp. Physiol. B 158, 719–727. Fewell, J.H., Page, R.E., 2000. Colony level selection effects on individual and colony foraging task performance in honey bees, Apis mellifera. Behav. Ecol. Sociobiol. 48, 173–181. Fewell, J.H., Harrison, J.F., 2001. Variation in worker behavior of African and European honey bees. In: Page, R.E., Erickson, E. (Eds.), Proceedings of the Second International Congress on Africanized Bees and Bee Mites. Hall, H.G., Smith, D.R., 1991. Distinguishing African and European honeybee matrilines using amplified mitochondrial DNA. Proc. Nat. Acad. Sci. 88, 4548–4552. Harrison, J., 1986. Caste-specific changes in honey bee flight capacity. Physiol. Zool. 59, 175–187. Harrison, J.F., Camazine, S., Marden, J.H., Kirkton, S.D., Rozo, A., Yang, X., 2001. Mite not make it home: tracheal mites reduce the safety margin of oxygen delivery of flying honeybees. J. Exp. Biol. 204, 805–814. Harrison, J.F., Hall, H.G., 1993. African–European honeybee hybrids have low nonintermediate metabolic capacities. Nature 363, 258–260. Harrison, J.F., Fewell, J.H., Roberts, S.P., Hall, H.G., 1996a. Achievement of thermal stability by varying metabolic heat production in flying honeybees. Science 274, 88–90. Harrison, J.F., Nielsen, D.I., Page, R.E.J., 1996b. Malate dehydrogenase phenotype, temperature and colony effects on flight metabolic rate in the honey-bee, Apis mellifera. Funct. Ecol. 10, 81–88. Harrison, J.F., Roberts, S.P., 2000. Flight respiration and energetics. Ann. Rev. Physiol. 62, 179–205. Heinrich, B., 1979. Thermoregulation of African and European honeybees during foraging, attack, and hive exits and returns. J. Exp. Biol. 80, 217–229. Heinrich, B., 1980a. Mechanisms of body temperature regulation in honeybees, Apis mellifera I. Regulation of head temperature. J. Exp. Biol. 85, 61–72. Heinrich, B., 1980b. Mechanisms of body temperature regulation in honeybees, Apis mellifera II. Regulation of thoracic temperature at high air temperatures. J. Exp. Biol. 85, 73–87. Heinrich, B., Esch, H., 1994. Thermoregulation in bees. Am. Sci. 82, 164–170. Hepburn, H.R., Radloff, S.E., Fuchs, S., 1999a. Flight machinery dimensions of honeybees, Apis mellifera. J. Comp. Physiol. B 169, 107–112. Hepburn, H.R., Youthed, C., Illgner, P., Radloff, S.E., Brown, R.E., 1999b. Production of aerodynamic power in mountain honeybees (Apis mellifera). Naturwissenschaften 85, 389–390. Herold, R.C., Borei, H., 1963. Cytochrome changes during honey bee flight muscle development. Devel. Biol. 8, 67–79. Houston, A.P., Schmid-Hempel, P., Kacelnik, A., 1988. Foraging strategy, worker mortality, and the growth of the colony in social insects. Am. Nat. 131, 107–114. Huang, Z.-Y., Robinson, G.E., Tobe, S.S., et al., 1991. Hormonal regulation of behavioural development in the honey
bee is based on changes in the rate of juvenile hormone biosynthesis. J. Insect Physiol. 37, 733–741. Hunt, G.J., Guzman-Novoa, E., Fondrk, M.K., Page, R.E., 1997. Quantitative trait loci for honey bee stinging behavior and body size. Genetics 148, 1203–1213. Jungmann, R., Rothe, U., Nachtigall, W., 1989. Flight of the honey bee I. Thorax surface temperature and thermoregulation during flight. J. Comp. Physiol. B 158, 711–718. Kammer, A.E., Heinrich, B., 1978. Insect flight metabolism. Adv. Ins. Phys. 13, 133–228. Kronenberg, F., Heller, H.C., 1982. Colonial thermoregulation in honey bees (Apis mellifera). J. Comp. Physiol. B 148, 65–76. Lighton, J.R.B., Lovegrove, B.G., 1990. A temperatureinduced switch from diffusive to conective ventilation in the honeybee. J. Exp. Biol. 154, 509–516. Loper, G.M., Fewell, J., Smith, D.R., Sheppard, W.S., Schiff, N., 1999. Honey bee foraging task organization. In: Hoopingarner, R., Connor, L. (Eds.), Apiculture for the 21st Century. Wicwas Press, Cheshire, Connecticut, pp. 47–51. Marden, J.H., 1987. Maximum lift production during takeoff in flying animals. J. Exp. Biol. 130, 235–258. Marden, J.H., 2000. Variation in the size, composition, and function of insect flight muscles. Ann. Rev. Physiol. 62, 157–178. Moritz, R.F.A., 1988. Biochemical changes during honeybee flight muscle development. In: Nachtigall, W. (Ed.), The Flying Honeybee; Aspects of Energetics. Gustav Fischer, Stuttgart, New York, pp. 51–65. Nachtigall, W., Rothe, U., Feller, P., Jungmann, R., 1989. Flight of the honey bee. III. Flight metabolic power calculated from gas analysis, thermoregulation and fuel consumption. J. Comp. Physiol. B 158, 729–737. Neukirch, A., 1982. Dependence of the life-span of the honeybee (Apis mellifera) upon flight performance and energy consumption. J. Comp. Physiol. B 146, 35–40. Nielsen, D.I., Page, R.E., Crosland, M.W.J., 1994. Clinal variation and selection of malate dehydrogenase allozymes in honey bee populations. Experientia 50, 867–871. Page, R.E., Fondryk, M.K., Hunt, G.J., et al., 2000. Genetic dissection of honeybee (Apis melifera L.) foraging behavior. J. Heredity 91, 474–479. Roberts, S.P., Harrison, J.F., 1998. Mechanisms of thermoregulation in flying bees. Am. Zool. 38, 492–502. Roberts, S.P., Harrison, J.F., 1999. Mechanisms of thermal stability during flight in the honeybee Apis mellifera. J. Exp. Biol. 202, 1523–1533. Robinson, G.E., 1987. Regulation of honey bee age polyethism by juvenile hormone. Behav. Ecol. Sociobiol.. Rothe, U., Nachtigall, W., 1989. Flight of the honey bee IV. Respiratory quotients and metabolic rates during sitting, walking and flying. J. Comp. Physiol. B 158, 739–749. Schmid-Hempel, P., Kacelnik, A., Houston, A.I., 1985. Honeybees maximize efficiency by not filling their crop. Behav. Ecol. Sociobiol. 17, 61–66. Spangler, H.G., 1992. The influence of temperature on the wingbeat frequencies of free-flying honey bees, Apis mellifera L. (Hymenoptera: Apidae). Bee Sci. 2, 181–186. Suarez, R.K., Lighton, J.R.B., Joos, B., Roberts, S.P., Harrison, J.F., 1996. Energy metabolism, enzymatic flux capacities, and metabolic flux rates in flying honeybees. Proc. Nat. Acad. Sci., USA 93, 12616–12620.
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Review
Environmental and genetic influences on flight metabolic rate in the honey bee, Apis mellifera夞 Jon F. Harrison*, Jennifer H. Fewell Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA Received 11 June 2001; received in revised form 27 January 2002; accepted 29 January 2002
Abstract Flying honey bees demonstrate highly variable metabolic rates. The lowest reported values (approximately 0.3 W gy1) occur in tethered bees generating the minimum lift to support their body weight, free-flying 2-day old bees, winter bees, or bees flying at high air temperatures (45 8C). The highest values (approximately 0.8 W gy1) occur in foragers that are heavily loaded or flying in low-density air. In different studies, flight metabolic rate has increased, decreased, or remained constant with air temperature. Current research collectively suggests that this variation occurs because flight metabolic rates decrease at thorax temperatures above or below 38 8C. At 30 8C, approximately 30% of colonial energy is spent during typical foraging, so variation in flight metabolic rate can strongly affect colony-level energy balance. Higher air temperatures tend to increase colonial net gain rates, efficiencies and honey storage rates due to lower metabolic rates during flight and in the hive. Variation in flight metabolism has a clear genetic basis. Different genetic strains of honey bees often differ in flight metabolic rate, and these differences in flight physiology can be correlated with foraging effort, suggesting a possible pathway for selection effects on flight metabolism. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Africanized honey bee; Apis mellifera; Development; Flight; Foraging; Honey bee metabolic rate; Metabolism; Season; Temperature; Thermoregulation
1. Introduction Comparative studies can lend the impression that hovering flight metabolic rate is a defined parameter for a given species. Species-specific metabolic rates are often adopted by ecologists or evolutionary biologists for theoretical or applied 夞 This paper was presented in the session, ‘Physiology and Biochemistry of Exercise’, at the Society for Experimental Biology, April 2–6, 2001, Canterbury, UK. *Corresponding author. Tel.: q1-480-965-9459; fax: q1480-965-2519. E-mail address: [email protected] (J.F. Harrison).
purposes. However, a growing body of research suggests that intraspecific variation in flight metabolic rate can be substantial, even during what appears to the human observer as stationary hovering. In this review, we focus on a single insect species, the honey bee, Apis mellifera, to illustrate some of the causes and consequences of such variation in flight energetics. To our knowledge, there is no other species, vertebrate or invertebrate, for which flight metabolic rate has been quantified in such a wide range of environments and populations (see Harrison and Roberts, 2000 for review).
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 1 6 3 - 0
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1.1. Honey bees as a model system Honey bees have many advantages as model organisms for the study of insect flight. Their large size and endothermy facilitates the rapid metabolic measurements needed to track energetic changes associated with behavioral variation. Honey bees fly freely or on tethers in experimental chambers. They also can be individually marked, and their complex behaviors can be observed within and without the hive and compared to their flight physiology. Honey bees are also well situated for studies of the genetic basis to variation in flight physiology. Among insects, the honey bee genome is known with a precision second only to Drosophila melanogaster (Hunt et al., 1997; Page et al., 2000). Selection at the colony level produces effects in a large number of related workers that can be tested without destroying the colony as a whole (Fewell and Page, 2000). Finally, honey bee queens can be instrumentally inseminated, allowing the production of hybrids and backcrosses. 1.2. Methods for studying honey bee flight metabolism Because of their small size and high metabolic rates, honey bees are well-suited for both flowthrough and closed-system respirometry studies. Flying honey bees exclusively use aerobic carbohydrate catabolism during flight (Rothe and Nachtigall, 1989), so carbon dioxide emission and oxygen consumption provide equivalent measures of metabolic rate. Since modern carbon dioxide analyzers have greater precision than oxygen analyzers, most recent respirometry studies have measured carbon dioxide emission (Harrison and Roberts, 2000). Measurements during tethered flight have allowed quantification of the lift produced during hovering, and continuous measures of thorax temperature, concomitant with flow-through respirometry measures of metabolism (Esch, 1976; Jungmann et al., 1989; Nachtigall et al., 1989). Metabolic rates during free flight have been measured using three types of methods: hovering flight in vertical tubes or respirometry chambers, agitated flight in chambers, and forward flight in wind tunnels. In the hovering flight protocol, bees are measured (using closed- or open-system respirometry) during undisturbed, stationary hovering for
seconds to minutes in a relatively large (liters) chamber. Lights above the chamber induce flight. Bees that cling to the walls, ascend or descend are discarded. In some cases the tarsi are removed to prevent the bees from landing on the chamber walls (Heinrich, 1980a,b). Stationary hovering protocols have been used for studies of temperature (Heinrich, 1980a,b; Harrison et al., 1996a; Roberts and Harrison, 1999), air pressure (Withers, 1981), and loading (Wolf et al., 1989) effects on flight metabolic rate. Unless the tarsi are removed a majority of bees tested with a hovering protocol will cling to the vessel walls for periods of time and must be discarded. Proper lighting and chamber design can increase the number of bees that fly freely, but the proportion of bees that fly without landing remains low. To overcome this problem, we have used an agitated flight protocol combined with flowthrough respirometry, using chamber vibration to induce bees to fly in a relatively small (0.3 l) rounded chamber for 1–2 min. With this protocol, behavior of the bee in the chamber varies widely from constant to little flight. However, metabolic rates and wing beat frequencies (during flight periods) are similar to those measured for bees in continuous hovering (Harrison et al., 1996a), probably because the flight muscle can generate a similar metabolic rate during non-flight shivering as during flight. Because more than 90% of bees exhibit strong elevations in metabolic rate in response to agitation, this protocol is particularly useful for population genetic studies (Harrison and Hall, 1993; Fewell and Harrison, 2001). The agitated flight protocol is also appropriate for studies in which it is mandatory to assess the metabolic capacity of all bees including those who will not fly (e.g. studies of the effects of pathogens: Harrison et al., 2001). A re-circulating wind tunnel with optical feedback to orient the bees has been used to measure flight metabolic rate of bees carrying loads during slow forward flight (Wolf et al., 1989). Metabolic rates measured in slow forward flight were similar but not identical to those measured during undisturbed stationary hovering (Wolf et al., 1989). This sophisticated technique probably provides values most similar to natural flight. 1.3. Metabolic scope Flying honey bees with similar body temperatures have metabolic rates of 0.3–0.8 W (g
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body)y1 (see below). The thorax accounts for approximately 20–40% of body mass, and the flight muscle occupies approximately 75% of the thorax (Heinrich, 1980b). Resulting metabolic rates can reach 2–3 W gy1 of flight muscle. This is at least twofold higher than comparable estimates for flight muscle of hovering hummingbirds, and 30 times higher than human athletes exercising at VO2max (Suarez et al., 1996). The high metabolic rates of honey bee flight are partially, but not completely explained by their small body size relative to vertebrates (Harrison and Roberts, 2000). In endothermic insects like honey bees metabolic rates during flight can be 100 times higher than in resting individuals (Kammer and Heinrich, 1978), so the metabolic scopes of endothermic insects far exceed those of most vertebrates. This tremendous scope is often used to justify the assumption that essentially 100% of oxygen consumption during flight occurs in the flight muscle (Suarez et al., 1996). However, these extreme metabolic scopes depend on comparisons between cold, quiescent bees and warm, flying bees. Resting metabolic rates of honey bees vary widely, depending on the temperature and activity level of the bee (Rothe and Nachtigall, 1989; Lighton and Lovegrove, 1990). Ideally, metabolic scope would be estimated by comparing metabolic rates of flying and non-moving bees with identical body temperatures. This is a difficult comparison to make empirically, as single bees kept in a warm metabolic chamber (like most bees in a warm hive) walk and groom continuously. Walking and grooming workers, with thorax temperatures no more than 1 8C above air temperature (35 8C), have metabolic rates of 0.08 W (g body)y1 (S.E.M.s0.011, Ns15). Since the inside of a brood-rearing hive is usually near 35 8C, the metabolic rate of a bee emerging from the hive and taking flight increases four- to ten-fold, exhibiting a metabolic scope similar to typical vertebrate values. 2. Individual variation in flight metabolism 2.1. Effect of air temperature Honey bees have been reported to forage at air temperatures ranging from 5 to 45 8C (Heinrich, 1979; Cooper et al., 1985; Spangler, 1992). It should be noted that these represent ranges from
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Fig. 1. Effect of air temperature on thorax temperatures of Apis mellifera. Data in Figs. 1–4 from Heinrich (1980b), Roberts and Harrison (1999) and Harrison et al. (2001).
studies across many populations, locations and seasons; our impression is that the daily range of air temperatures at which a single colony will forage is narrower. At air temperatures below 20– 25 8C honey bees do not thermoregulate. Thoracic temperatures of bees in free flight increase linearly with air temperature with a slope approaching 1 (Fig. 1). However, at higher air temperatures freeflying honey bees partially stabilize thoracic temperature, so that it increases only 0.4–0.5 8C for every 1 8C rise in air temperature (Fig. 1, Coelho, 1991a; Harrison et al., 1996a). This represents a moderate thermoregulatory ability compared to other endothermic bee species (Roberts and Harrison, 1998). Stabilization of thoracic temperature helps preserve the capacity of the flight muscle to produce maximal power when air temperature varies; however, the range of thoracic temperatures at which bees can generate sufficient force to lift their body mass is quite broad (28–47 8C, Coelho, 1991b). Thermal stability during flight is partially accomplished by variation in heat loss. At air temperatures above 33 8C evaporative water loss increases exponentially, and accounts for half of thoracic thermoregulation (Roberts and Harrison, 1999). Honeybees extrude nectar droplets from their crop onto their mandibles at high air temper-
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Fig. 2. Effect of air temperature on flight metabolic rate for three studies of honey bee flight.
atures, and this may be the primary mechanism by which evaporative water loss is regulated (Esch, 1976; Heinrich, 1980b; Cooper et al., 1985). However, there is also evidence for substantial heat loss from the abdomen at air temperatures above 40 8C, suggesting that substantial evaporation may also occur from the abdominal surface (Roberts and Harrison, 1999). The heart and aorta of the honey bee are arranged in loops within the narrow petiole between the thorax and abdomen, so that heat produced within the thorax is not transferred to the abdomen (Heinrich and Esch, 1994). As expected from this morphology, there is no evidence for effects of temperature on heat exchange between thorax and abdomen as occurs in bumblebees (Heinrich, 1980b; Roberts and Harrison, 1999). Studies of the effect of air temperature on metabolic heat production for free-flying bees have
produced mixed results (Fig. 2). Using stationary hovering bees, Heinrich (1980a) found a constant flight metabolism for bees flying at 20 and 42 8C. However, Harrison et al. (1996b) and Roberts and Harrison (1999) found that flight metabolic rate decreased by approximately half as temperatures rise from 21 to 45 8C in either stationary hovering or agitated flight protocols. Conversely, Harrison et al. (2001) found an increase in metabolic rate with temperature in winter bees tested with the agitated flight protocol at 16 and 24 8C. How can these contradictions be explained? We can not exclude the possibility that differences in experimental protocols account for this variation. However, we hypothesize that different effects of air temperature on flight metabolism occurred in these studies because: (1) honey bees differed in their metabolic capacities, leading to very different thorax temperatures at similar air temperatures; and (2) thorax temperature and flight metabolic rate are positively correlated at temperatures less than 38 8C, and negatively correlated at higher temperatures. Multiple lines of evidence suggest that individual metabolic capacities differed considerably across (and within) these studies, producing different thorax temperatures at similar air temperatures. Within the air temperature range of 20–24 8C individual flight metabolic rates vary widely (Fig. 2). At steady-state, if heat production equals heat loss MHPsŽTthyTair.CqRHLqEHL (1) where MHP equals metabolic heat production, Tth is thorax temperature, Tair is air temperature, C is whole-animal convective thermal conductance, RHL is net radiative heat loss and EHL is evaporative heat loss. In the 20–24 8C range, when bees are flying inside controlled-temperature rooms so that chamber walls have surface temperatures similar to air temperature, and solar radiation is zero, RHL and EHL together account for approximately 25% of total heat loss (Roberts and Harrison, 1999). Convective thermal conductances calculated at 20–24 8C do not differ between winter and summer-collected European bees (Harrison et al., 2001), and thus are likely to be similar across these studies. Therefore, as a first approximation, for the 20–24 8C air temperature range, Eq. (1) reduces to MHPsŽTthyTair.X
(2)
where X is a constant. This equation predicts a
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Fig. 3. Effect of air temperature on force production of tethered bees, modified from Coelho (1991b). Horizontal lines indicate the range of thorax temperatures in three studies of honey bee flight.
positive linear relationship between MHP and Tth at a given Tair. Such a linear relationship has been reported for flying and non-flying bees (Feller and Nachtigall, 1989; Jungmann et al., 1989; Harrison et al., 2001). Using the values for the three studies in the 20–24 8C air temperature range (data in Figs. 1 and 2), a linear plot of mean thorax temperature vs. mean metabolic rate has an r 2s 0.99. Thus, in the 20–24 8C temperature range, summer-collected foragers (Roberts and Harrison, 1999) had twice the MHP (Fig. 2), creating a 5 8C higher Tth than winter-collected bees (Harrison et al., 2001) with Heinrich’s bees being intermediate in both variables. The evidence in support of the hypothesis that honey bee flight metabolic rate decreases at thorax temperatures above and below 38 8C is less conclusive, but intriguing. Force production of tethered bees is maximal at a thorax temperature of 38 8C (Fig. 3, Esch, 1976; Coelho, 1991a); a reasonable but untested hypothesis is that flight metabolic rates show the same relationship with thorax temperature. In the winter bee study, thoracic temperatures varied within a range in which higher thorax temperatures are predicted to correlate with higher force production and metabolism
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(Fig. 3). In the Heinrich (1980b) study, measured thorax temperatures occurred on either side of the optimal temperature for force production, predicting a weak negative relationship between thorax temperature and metabolic rate (Fig. 3). In the Roberts and Harrison (1999) study (as well as in Harrison et al., 1996a), all measured thorax temperatures occurred in a range in which higher thorax temperatures are predicted to correlate negatively with force production and metabolism (Fig. 3). For the three studies, the direction of the correlations between thorax temperature and flight metabolic rate is consistent with predictions (Fig. 4). For the Heinrich (1980b) and the Roberts and Harrison (1999) studies, the magnitude of the slopes of the relationships are also near those predicted from Coelho’s (1991a) data. Studies of tethered flying bees also support this hypothesis. In Nachtigall’s and associate’s studies (Rothe and Nachtigall, 1989; Feller and Nachtigall, 1989; Nachtigall et al., 1989), thorax temperatures varied between 23 and 38 8C, and metabolic rates and thorax temperatures were positively correlated. Integration of these various studies suggests that the mechanisms and degree to which honey bees stabilize thoracic temperature will depend on their metabolic capacity (which varies with season and genetic factors: see below), the range of air temperatures examined, and the environmental conditions. The extent to which temperature-generated variation in metabolic rates correlates with variation
Fig. 4. Relationships between flight metabolic rate and thorax temperatures in three studies of honey bee flight.
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Fig. 5. Honey bee flight metabolic rates under various conditions. European honey bees (A. m. ligustica): non-flying, walking and grooming 35 8C bees (this study), hovering, 20 and 45 8C (Roberts and Harrison, 1999), winter bees at 20 8C (interpolated from Harrison et al., 2001), 2-day-old bees at 20 8C (this study), hovering with a 75 mg nectar load (Wolf et al., 1989), flying in 88 kPa air (Withers, 1981), Africanized honey bee (A. m. scutellata) flying at 22 8C (Harrison and Hall, 1993).
in mechanical power output is unclear. Conceivably, variation in metabolic rate could occur without changes in power output due to processes such as substrate cycling (Surholt et al., 1990, 1991). However, during tethered flight, metabolic rate and wing-beat frequency are strongly positively correlated (Feller and Nachtigall, 1989), and hovering honey bees flying at lower temperatures (with higher metabolic rates) have higher wing beat frequencies (Spangler, 1992; Harrison et al., 1996b). However, other parameters also affect mechanical power output during flight (particularly stroke amplitude), and as yet there have been no studies to date of thermal effects on power output in free-flying honey bees. 2.2. Effects of flight speed and load carriage The primary context for flight in workers is foraging, which requires extensive changes in flight speeds and total mass transported. In the field, honey bees show tremendous flexibility in flight performance, switching rapidly between hovering and forward speeds over 10 m sy1. To date, there have been no studies of the effect of forward flight speed on metabolism in honey bees. However, in bumblebees flight metabolic rate is independent of speed up to 5 m sy1, with speed increases being accomplished primarily by variation in stroke plane angle (Casey and Ellington, 1988). Models of bumblebee flight suggest that aerodynamic power, and probably metabolic rate, must increase exponentially over higher speeds
(Dudley and Ellington, 1990), and it seems likely that a similar J-shaped pattern of metabolic rate with flight speed occurs in honey bees. Honey bee foragers can carry considerable loads during flight. Pollen loads average approximately 10 mg and can reach 30 mg (Winston, 1987). Nectar loads average approximately 10 mg, but can reach 50 mg (Winston, 1987). Forager masses vary between 60 and 100 mg, so loads range from 10 to 80% of body weight. Honey bees also carry dead bees out of the hive and fly some meters away with the corpses. Undertaker bees thus carry loads equal to their body mass during forward flight (Coelho, 1991b), though they usually cannot ascend steeply with these loads. This is a moderate load-lifting ability compared to other insects, and is consistent with the mass of the flight muscle relative to the body mass (Marden, 1987). The cost of flight increases linearly with load, increasing by approximately 40% relative to the unladen state at 30 8C to nearly 0.8 W gy1 (Fig. 5, Wolf et al., 1989). 2.3. Effect of ontogeny Metabolic rate during flight or attempted flight (in day old bees) increases approximately tenfold during ontogeny (Fig. 6). Most workers cannot fly for the first day after eclosion. Onset of the ability to fly coincides with hardening of the cuticle, a process that is probably necessary for flight. However, development of flight muscle oxidative capacity may also be important. Agitated day old
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so, this is an interesting departure from the normal situation in which elevated juvenile hormone suppresses flight muscle development in insects (reviewed by Marden, 2000). 3. Variation in flight metabolic rates across populations of honey bees
Fig. 6. Effect of ontogeny on metabolic rate during flight or attempted flight (day old bees) and flight muscle biochemical parameters. Flight metabolic rates summarized from Harrison (1986), Fewell and Harrison (2001), and data from this paper. Pyruvate kinase, citrate synthase, and glycogen data from Harrison (1986).
bees have metabolic rates of only 0.1 W gy1 (S.E.M.s0.024, Ns12), and thoracic temperatures nearly equivalent to ambient air. Two-dayold bees who can hover have flight metabolic rates of 0.3 W gy1 (S.E.M.s0.04, Ns12). The metabolic rates of bees agitated to fly increase approximately eightfold in the 2–4 days post-eclosion, coincident with tenfold increases in thoracic pyruvate kinase and citrate synthase activities, and fivefold increases in thoracic glycogen levels (Neukirch, 1982; Harrison, 1986; Moritz, 1988). Flight metabolic rates, thoracic enzyme levels and thoracic glycogen levels remain relatively constant over the 1–3 week period when the bees work within the hive. Then, at the onset of foraging, there is an approximate 15% increase in agitated flight metabolic rate, coincident with an approximate doubling of thoracic glycogen stores (Harrison, 1986; Fewell and Harrison, 2001). Cytochrome concentrations of the flight muscle also increase at least tenfold from 1 to 20 days after eclosion (Herold and Borei, 1963). It is tempting to speculate that increases in flight metabolic rate and thoracic glycogen levels might be controlled by a rise in juvenile hormone levels, which occurs in correlation with this behavioral change (Robinson, 1987; Huang et al., 1991). If
The individual variation in flight metabolic rate discussed above extends to distinct population and subspecies patterns of variation. The association of these patterns with habitat differences and genotypic variation suggest that selection plays an important role in shaping metabolic differences in this species. Perhaps the largest reported subspecies differences in metabolic rates occur between European (A. m. ligustica) and Africanized honey bees (derived from A. m. scutellata). Africanized honey bee foragers have significantly higher flight metabolic rates than European foragers (Harrison and Hall, 1993). This difference in flight metabolic rate is likely to be genetically-based; African and European foragers reared in a common natal environment show similar differences in flight metabolism (Fewell and Harrison, 2001). The higher flight metabolic rates of African bees are due to three factors: (1) smaller body size (Harrison and Hall, 1993); (2) larger thorax relative to body mass (Hepburn et al., 1999a); and (3) a higher metabolic rate per gram of thorax (Harrison and Hall, 1993). It will be important to test whether the higher flight metabolic rates of African bees correlate with greater mass-specific power output, load carriage capacity or maximal flight speeds; conceivably they could relate simply to greater behavioral activation in the agitated flight assays. Honey bee subspecies vary in wing sizes and body masses (Daly, 1991). Wing and thorax sizes also vary within a subspecies with altitude, so that high-altitude honey bees have a greater calculated mass-specific lift-generating ability (Hepburn et al., 1999a,b). The effect of such morphological variation on flight metabolism remains unstudied. Variation in flight metabolism has also been associated with variation in allele frequencies for the allozyme marker malate dehydrogenase (MDH-1). The electrophoretically fast and medium alleles of malate dehydrogenase exhibit latitudinal clines on three continents for honey bees (Nielsen et al., 1994). Bees with the fast allele have higher flight metabolic rates and wing beat frequencies (Coelho and Mitton, 1988; Harrison et
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Table 1 Energetics of ‘typical foraging’ for Apis mellifera at an air temperature of 30 8C Nectar load Nectar energetic content Energetic reward per trip Flight metabolic rate (30 8C) Trip duration Cost per trip Net gain per trip Trips per day Reward per day Cost per day during flight In-hive metabolic rate (30 8C) Daily in-hive metabolism of forager Metabolic cost per forager day Net gain rate per forager day Hive bees fed per forager % of bees which forage % of total colony energy spent foraging
30 ml 9 J mly1 (50% sugar) 270 J 2.5 J (bee min)y1 30 min 75 J 195 J, 6.5 J miny1 12 3240 J 900 J 0.16 J (bee min)y1 173 J dayy1 1073 J dayy1 2167 J dayy1 9.4 10 30
Foraging rewards and trip durations are derived from Winston (1987); within-colony metabolic rates were calculated from Kronenberg and Heller (1982), and metabolic rates during flight were taken from Roberts and Harrison (1999). Within-hive metabolic rates were considered identical for hive bees and foragers.
al., 1996b). In heterozygotes, the difference in flight metabolic rate is approximately 3% (Harrison et al., 1996b), while homozygotes, differ by up to 20% (Coelho and Mitton, 1988). The effects of malate dehydrogenase on flight metabolic rate are similar at 21 and 38 8C, suggesting that the latitudinal cline is not related to thermal adaptations of these enzymes (Harrison et al., 1996b). The association between MDH-1 allele type and metabolic rate may also be linked to subspecies differences. Africanized bees have a much higher frequency of the fast allele than feral European populations (Hall and Smith, 1991; Loper et al., 1999). 4. Comparison of the relative magnitudes of within-individual and cross-population effects on flight metabolic A comparison of the relative magnitude of the various environmental and genetic effects on flight metabolic rate is shown in Fig. 5. Minimal metabolic rates for bees able to generate sufficient lift to support their body in tethered flight are approximately 0.3 W gy1 (Nachtigall et al., 1989). We also found values of approximately 0.3 W gy1 for bees hovering at air temperatures of 45 8C, for winter bees flying at 20 8C, and for 2-day-old bees just able to fly (Fig. 5). Increased load carriage, or flying in low-density air, increases flight metabolic rate to nearly 0.8 W gy1 (Fig. 5). A protocol
that unequivocally measures maximal metabolic rate remains to be developed for free-flying insects. However, extrapolation of the relationship between flight metabolic rate and load (Wolf et al., 1989) to the approximate maximal load carriage of 100% of body weight suggests a maximal flight metabolic rate of 0.8–0.9 W gy1, approximately 3 times the minimal metabolic rate necessary for flight. The effect of air temperature on metabolic rate during hovering spans about half of this total variation. Summer-collected, foraging-age bees flying at 20 8C have flight metabolic rates of approximately 0.6 W gy1, about double that measured at 45 8C. Effects of ontogeny and season also seem to produce variation in flight metabolic rates about half of the total variation observed (Fig. 5). Crosspopulation effects seem to be smaller than the within-individual effects. African bees have massspecific flight metabolic rates approximately 10– 20% greater than European bees at similar temperatures, with malate dehydrogenase effects of a similar or smaller magnitude (Fig. 5). 5. Colony-level energetic consequences of variation in flight metabolic rate To consider the ecological importance of variation in environmental or genetic variation in flight metabolic rates, it is necessary to relate flight energetics to the metabolism of the whole colony and to the energetic rewards of foraging. The
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energetics of ‘typical foraging’ are presented in Table 1. For a honey bee foraging at 30 8C, energy costs during foraging are estimated to account for 30% of total energy expenditure. Net energy intake should allow storage of 2110 J dayy1 of honey, or alternatively, will suffice for the energetic needs of approximately 9–10 hive bees. Because approximately 10–20% of the colony forages, this calculation suggests that energy intake should only slightly exceed energy use. Air temperature must strongly effect colonial energy balance. First, if higher air temperatures decrease metabolic rate during foraging in a manner similar to that observed in hovering summer bees (Harrison et al., 1996b; Roberts and Harrison, 1999), both net energy gain (benefit-costytime) and efficiency (benefit-costycost) during foraging will increase with air temperature. For the typical foraging flight described in Table 1, net gain rate will be 20% greater at 40 8C than at 20 8C, and efficiency nearly 50% higher. Secondly, in-hive metabolic rate decreases exponentially at higher temperatures, from approximately 450 J (bee day)y1 at 20 8C to approximately 200 J (bee day)y1 (Kronenberg and Heller, 1982). Therefore, the number of hive bees fed per forager with the nectar intake described in Table 1 increases from 4 to 12 as air temperature rises from 20 to 40 8C. These results suggest that triple the foraging effort would be required for a colony to subsist at 20 8C relative to 40 8C. Also, at the same level of foraging intake, colonial honey accumulation should triple as air temperatures rise from 20 to 40 8C. Cross-population variation in flight metabolic rates may also be important to colonial energetics and growth. African honey bees have trip frequencies approximately 20% higher than European bees in the same colony (Fewell and Harrison, 2001), similar to the difference in forager flight metabolic rate. Africanized bees also allocate a much larger proportion of their workers to pollen foraging. Because pollen contains the nutritional (and especially protein) requirements for developing brood, this shift in allocation likely translates to increased rates of worker production in Africanized colonies (Fewell and Harrison, 2001). These data suggest that variation in flight rate may contribute to ability of Africanized bees to reproduce at higher rates than European bees, and may partially explain the competitive advantage Africanized bees have relative to European bees in the neotropics.
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The variation in flight metabolic rates among subspecies and the correlation of flight metabolism with foraging work effort together suggest how natural selection may affect flight muscle metabolism in honey bees. Immediate colony growth and reproduction must be traded off against accumulation of honey stores for surviving dearth periods. In temperate climates lower metabolic rates and higher efficiencies may confer the highest long-term colony growth rates (Schmid-Hempel et al., 1985; Houston et al., 1988). However, in more tropical environments, rapid growth rates and colony reproductive rates may be advantageous (Winston, 1992). Selection at the colony level may lead to genetic changes in individuals associated with higher or lower rates of foraging behavior, which may be associated with high or low flight metabolic rates. Because it is becoming easier to identify particular genetic loci associated with such behavioral or physiological variation (Hunt et al., 1997), soon it will be possible to explicitly test hypotheses concerning the evolution of flight metabolic rate in honey bees. Acknowledgments This research was partially funded by NSF IBN 9985857 to JFH and NSF 0093410 to JHF and JFH. References Casey, T.M., Ellington, C.P., 1988. Energetics of insect flight. In: Wieser, W., Gnaiger, E. (Eds.), Energy Transformations in Cells and Organisms. Georg Thieme Verlag Stuttgart, Innsbruck, Austria, pp. 200–210. Coelho, J.R., 1991a. Heat transfer and body temperature in honey bee (Hymenoptera: Apidae) drones and workers. Environ. Entomol. 20, 1627–1635. Coelho, J.R., 1991b. The effect of thorax temperature on force production during tethered flight in honeybee (Apis mellifera) drones, workers, and queens. Physiol. Zool. 64, 823–835. Coelho, J.R., Mitton, J.B., 1988. Oxygen consumption during hovering is associated with genetic variation of enzymes in honey-bees. Funct. Ecol. 2, 141–146. Cooper, P.D., Schaffer, W.M., Buchmann, S.L., 1985. Temperature regulation of honey bees (Apis mellifera) foraging in the Sonoran desert. J. Exp. Biol. 114, 1–15. Daly, H.V., 1991. Systematics and identification of Africanized honey bees. In: Spivak, M., Fletcher, D.J.C., Breed, M.D. (Eds.), The ‘Africanized’ Honey Bee. Westview Press, Boulder, pp. 13–44. Dudley, R., Ellington, C.P., 1990. Mechanics of forward flight in bumblebees. J. Exp. Biol. 148, 53–88.
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