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Utilisation of substrates during tethered flight with and without lift generation in the African fruit beetle Pachnoda sinuata (Cetoniinae)

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Several insects are able to use a combination of carbohydrates and proline to power their flight, but the extent to which either substrate is oxidised varies widely. One end of the scale is exemplified by the tsetse fly Glossina morsitans (see review by Bursell, 1981) and different species of onitine and scarabaeine dung beetles (Gäde, 1997a,b), which have negligible stores of flight muscle glycogen and haemolymph carbohydrates and use proline almost exclusively as a

substrate. At the other end of the scale, insects such as the blowfly Phormia regina use proline only at the beginning of flight to supply the Krebs cycle with intermediates (Sacktor and Wormser-Shavit, 1966; Sacktor and Childress, 1967). Proline oxidation plays a greater role during flight in the blister beetle Decapotoma lunata, although its importance is still minor compared with that of carbohydrates (Auerswald and Gäde, 1995). In the Colorado potato beetle Leptinotarsa JEB1616

We have investigated the pattern of metabolic changes during tethered flight with and without lift generation in the African fruit beetle Pachnoda sinuata.

Two distinct metabolic phases occur during lift-generating flight. The first phase is characterised by a high rate of oxygen consumption and a rapid change in proline and alanine levels in the haemolymph and flight muscles and in glycogen level in the flight muscles. Carbohydrates are released from the fat body into the haemolymph. These carbohydrates are oxidised during the second phase. Changes in proline and alanine levels in the haemolymph and flight muscles and in glycogen level in the flight muscles are minor during the second phase and the rate of oxygen consumption is reduced.

During lift-generating flight, metabolic changes are rapid. Proline concentrations in the haemolymph and flight muscles fall dramatically during the first 30 s of flight, while alanine concentrations rise concomitantly. While haemolymph concentrations of proline and alanine remain virtually unchanged thereafter, further changes in the levels of these amino acids occur in the flight muscles during 5 min of flight. The initial levels of the two amino acids in the flight muscles are re-established over 1 h of rest following a 5 min flight, while this process takes longer in the haemolymph. The concentration of haemolymph carbohydrates increases during the first 30 s of flight and declines thereafter during 5 min of flight. The pre-flight levels are restored after 1 h of subsequent rest. The stores

of glycogen in the flight muscles are rapidly diminished during the first 10 s of flight and decrease at a lower rate during further flight lasting up to 5 min. A subsequent 1 h of rest is sufficient almost to restore pre-flight levels. Haemolymph lipid levels are slightly but significantly increased during 11 min of flight and after 1 h of subsequent rest.

During flight without lift production, the metabolic changes are considerably slower and beetles fly approximately seven times longer than during lift-generating flight.

Resting basalar (BM), ventral (DVM) and dorso-longitudinal (DLM) flight muscles show no differences in levels of proline, alanine and glycogen. After different periods of flight, during which lift and wing loading were minimised, the DVM was found to have the highest level of proline after 5 min of flight. Levels of alanine in the DVM were lower than in the DLM. There was no evidence to suggest that different flight muscles are specialised for either proline or carbohydrate utilisation.

Proline and carbohydrates participate equally in supplying energy to the flight muscles during lift-generating flight. The contribution to the energy supply by the flight muscles is 54 %, while that of the haemolymph is 46 %.

Key words: insect, flight, energy metabolism, proline, alanine, metabolite, African fruit beetle, Pachnoda sinuata.

Summary

Introduction

UTILISATION OF SUBSTRATES DURING TETHERED FLIGHT WITH AND WITHOUT

LIFT GENERATION IN THE AFRICAN FRUIT BEETLE PACHNODA SINUATA

(CETONIINAE)

LUTZ AUERSWALD, PETER SCHNEIDER* ANDGERD GÄDE†

Zoology Department, University of Cape Town, Rondebosch 7701, South Africa

*Present address: Zoologie III, Universität Heidelberg, Im Neuenheimer Feld 504, D-69120 Heidelberg, Germany †Author for correspondence (e-mail: ggade@botzoo.uct.ac.za)

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decemlineata, proline is claimed to serve as the major energy

source (Weeda et al. 1979).

The African fruit beetle Pachnoda sinuata was found to oxidise large amounts of proline as well as carbohydrates to power flight (Zebe and Gäde, 1993; Lopata and Gäde, 1994). This beetle occurs in large numbers in South Africa, where it feeds on a variety of flowers and fruits (Donaldson, 1979; Heinrich and McClain, 1986; Holm and Marais, 1992). The beetle’s flight apparatus consists of fibrillar muscles of which, amongst others, the dorso-longitudinal and basalar muscles perform the downstroke, during which most of the lift is generated (Schneider and Hoese, 1982). The dorso-ventral muscle is one of the upstroke muscles, which produce most of the drag.

P. sinuata is a large beetle that has already been the subject

of flight experiments (Barnett et al. 1975; Zebe and Gäde, 1993; Lopata and Gäde, 1994). All previous experiments, however, were performed using a flight mill. This reduces the requirement for lift and wing-loading and, therefore, is different from free flight. In the present study, we used a different method of tethered flight which allowed the insect to generate lift (Schneider, 1989) to investigate a more natural metabolic situation in P. sinuata during flight. We investigated the role of proline and carbohydrates in supplying energy during flight and the contribution of the different compartments (flight muscles, haemolymph, fat body) of the insect to the substrate supply. Using the flight mill method, we investigated flights of longer duration, not examined in previous studies (see above). In addition, we used both methods of tethered flight to investigate whether the muscles involved in generating lift or drag show different metabolic patterns during flight, i.e. whether they are biochemically specialised to use either proline or carbohydrate oxidation for energy provision.

Materials and methods Insects

Male fruit beetles Pachnoda sinuata flaviventris (Gory and Percheron) were caught in the vicinity of Cape Town and were kept as outlined previously (Zebe and Gäde, 1993). The body mass of the animals used averaged 1 g±23 %.

Flight experiments

The experimental conditions and the procedures for handling the animals were as outlined by Auerswald et al. (1998).

Lift-generating flight

In some beetles metabolite levels were measured after pre-flight warm-up. These experiments were performed as previously described in detail by Auerswald et al. (1998).

Flight without lift generation

Fruit beetles were fixed by the pronotum to a pipette tip using superglue; the pipette tip was then attached to the arm

(length 29.5 cm) of a flight mill. Flight velocity was recorded by means of an infrared microprocessor tachometer (RS, UK) whose light beam was directed towards a reflector on top of the flight mill.

Samples for the determination of metabolite levels in the haemolymph and flight muscles were taken and stored as described by Auerswald et al. (1998). In addition, fat body samples were taken as follows: after removal of the gut, reproductive organs and Malphigian tubules from the abdomen, the tracheae and airsacs with the surrounding fat body tissue were dissected out and stored in the same way as the flight muscles. Dissection of the two tissues took approximately 2 min. To investigate the different flight muscle types (basalar, dorso-ventral and dorso-longitudinal; see Fig. 1), whole thoraces were frozen in liquid N2. Hitting the frozen sample with a pestle in a grinder cooled by liquid N2 caused the thorax to fall apart, and the different muscles (Fig. 1) were identified and then treated separately.

Preparation of samples and measurement of metabolite levels Haemolymph

Samples (1µl) of haemolymph were either blown immediately into 100µl of concentrated H2SO4 for the measurement of levels of total lipids (Zöllner and Kirsch, 1962) or carbohydrates (Spik and Montreuil, 1964) or pipetted into 60µl of 80 % acetonitrile for amino acid analysis (see below). Tissue samples

Perchloric acid extracts from frozen tissues were made according to the method of Zebe and Gäde (1993).

Measurement of glycogen

Glycogen was extracted as previously described (Zebe and Gäde, 1993) and analysed by the modified anthrone method (Spik and Montreuil, 1964) using glucose as a standard.

DVM

DLM DLM ODM

StPM

TCxM 1

BM

[image:2.609.319.550.505.679.2]

SM

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Measurement of proline and alanine levels

Derivatisation of extracts using dansyl chloride and measurement of proline and alanine levels by HPLC were carried out as previously described (Zebe and Gäde, 1993).

Measurements of rates of oxygen consumption and respiratory quotient

The measurements were executed as outlined by Auerswald

et al. (1998).

Determination of haemolymph volume

Essentially, the procedure of Clegg and Evans (1961) was used. A trace amount of [3H-G]inulin (1.315×1010Bq g−1; DuPont, USA), dissolved in 5µl of water (37 918 cts min−1), was injected ventrally into the abdomen between the last two segments using a 10µl Hamilton syringe. After 20 min, a 1µl sample of haemolymph was taken and pipetted into a scintillation vial containing 4 ml of scintillation fluid (Packard, Ultima Gold TR), and the radioactivity measured using a Tri Carb 460 scintillation counter (Packard).

Statistical analyses

For determination of significance, the Student’s t-test or paired t-test was used. For clarity, significance is depicted only when the change first became significant in comparison with the reference value. During flight mill experiments, we used the resting levels as a reference for comparison with flight values. In the experiments investigating lift-generating flight, the value after pre-flight warm-up was taken as the reference for animals that flew, except in Fig. 2B, where the 30 s value was taken. Significance levels applied were P<0.05 (†) and P<0.01 (††). For the resting period following flight, values were compared with those when flight had terminated. Significance levels are indicated as P<0.05 (*) and P<0.01 (**).

Results are presented as means ±S.D.

Results Lift-generating flight Flight duration

The majority of the beetles flew for between 1 and 5 min. Occasionally, a beetle flew for up to 20 min, but this occurred in less than 5 % of the flights (N=42).

Concentrations of metabolites in the haemolymph

Mean resting levels of proline and alanine in the haemolymph were 98 and 8µmol ml−1, respectively. The proline concentration dropped during the first 30 s of flight and levelled off at approximately 50µmol ml−1 (Fig. 2A). Mean alanine concentration increased to 42µmol ml−1during the first 30 s of flight and to 62µmol ml−1after 5 min of flight (Fig. 2A).

Proline and alanine concentrations were inversely related during rest after a flight of 5 min (Fig. 2A). Pre-flight levels had not been reached after 60 min of rest. The sum of the concentrations of the two amino acids was more or less stable

during flight and subsequent rest at approximately 110µmol ml−1, with the exception of the value at 30 s of flight, which was 90µmol ml−1.

The mean concentration of haemolymph carbohydrates at rest was approximately 7 mg ml−1. The concentration increased during the first 30 s of flight to approximately 9 mg ml−1and then dropped significantly (Fig. 2B). After 5 min of flight, a value of less than 6 mg ml−1 was reached. During 60 min of subsequent rest after 5 min of flight, the concentration rose to 7.5 mg ml−1, a return to pre-flight levels.

Mean haemolymph lipid levels showed a small but significant (P<0.001) increase of 1.5±1.1 mg ml−1 from an initial value of 10.0±3.1 to 11.5 mg ml−1in 13 beetles which stopped flying after 11.0±3.8 min. After 60 min of subsequent rest, the concentration of lipids in the haemolymph was 14.0±6.0 mg ml−1(data not shown).

*

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[image:3.609.318.565.279.601.2]

Duration of rest after 5 min of flight (min)

Fig. 2. Metabolite concentrations in the haemolymph of Pachnoda

sinuata during different durations of lift-generating flight and

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Metabolite concentrations in flight muscles

The most dramatic changes in flight muscle metabolite concentrations occurred during the first 30 s of flight (Fig. 3A,B). The mean resting proline concentration of 55µmol g−1fresh muscle mass declined to 31µmol g−1during the first 30 s of flight. In contrast to levels in the haemolymph, proline levels dropped further to reach 13µmol g−1after 5 min of flight. The resting alanine concentration of 11µmol g−1 increased to 36µmol g−1 during the first 30 s of flight (Fig. 3A). Again in contrast to the situation in the haemolymph, the level continued to increase, reaching 55µmol g−1after 5 min of flight. Pre-flight levels of proline and alanine were re-established after 1 h of rest following a 5 min flight. The sum of the concentrations of proline and alanine was within the range 64–70µmol g−1throughout the experiment.

Glycogen stores in the flight muscles amounting to 77µmol glucose equivalents g−1fresh muscle mass declined most rapidly within the first 10 s of flight to a value of 48µmol g−1 (Fig. 3B). Thereafter, glycogen levels were depleted at a less dramatic rate, reaching 12µmol g−1after 5 min of flight. After 60 min of rest, the glycogen concentration had almost returned to its pre-flight level.

Endothermic warm-up

Endothermic warm-up of P. sinuata is energy-expensive and metabolic changes occur during this process (Auerswald et al. 1998). Therefore, metabolite concentrations were determined just after the beetles had warmed up. These data were later taken into account for the calculation of rates of substrate consumption (see Discussion) to separate the energetic costs of flight from those of heat generation (see Table 1). The proline concentration after warm-up was 77.6±13.3µmol ml−1(N=5) in the haemolymph and 46.8±4.8µmol g−1muscle mass (N=5) in the flight muscles, while the respective alanine concentrations were 29.5±16.2µmol ml−1(N=5) and 21.5±4.0µmol g−1(N=5). The glycogen concentration in the flight muscles after warm-up was 71.9±6.1µmol g−1muscle mass (N=5), and no changes in the haemolypmh carbohydrate and lipid levels occurred during warm-up. These data are not depicted in the respective figures.

Rates of oxygen consumption and respiratory quotient

Because flight velocity was not directly measurable, we used the rate of oxygen consumption as an indicator of flight performance. Mean V˙O∑was highest shortly after the start of flight at 104 ml g body mass−1h−1after 2 min, but fell steadily with flight duration (Fig. 4). After 12 min of flight, V˙O∑ was 92 ml g body mass−1h−1. However, the respiratory quotient remained at approximately 0.9 throughout flight (Fig. 4).

Flight without lift generation Flight duration

All the beetles (N=48) flew for at least 45 min, and more than 10 % flew for approximately 2 h; the longest flight recorded lasted 165 min.

Concentrations of metabolites in the haemolymph

In resting beetles, the following mean concentrations of metabolites were found: proline 129µmol ml−1, alanine 14µmol ml−1, total carbohydrates 9 mg ml−1 and lipids 9 mg ml−1.

The proline concentration in the haemolymph decreased during the first 15 min of flight to 40µmol ml−1and reached a plateau of 30–40µmol ml−1after approximately 30 min of flight (Fig. 5A). The alanine concentration rose to 74µmol ml−1after 15 min of flight and then to 90–100µmol ml−1in beetles that flew for 30 min or longer (Fig. 5A). Pre-flight levels of proline were reached after 60 min of rest following a flight of 30 min, while pre-flight alanine values were reached after 15 min of rest. The sum of the concentrations of proline and alanine was more or less stable throughout the experiment (between 120 and 145µmol ml−1), with the exception of the value at 15 min of flight, which was 114µmol ml−1. During the subsequent resting period, the sum of the concentrations was lower, ranging from

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Duration of rest after 5 min of flight (min)

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101 to 129µmol ml−1, with the lowest values at 15 and 30 min after the termination of flight.

The mean level of total carbohydrates in the haemolymph dropped from the initial value of 9.4 mg ml−1 to 4.8 mg ml−1 during the first 30 min of flight (Fig. 5B). Subsequently, the concentration of carbohydrates increased to reach pre-flight levels after 90 min of flight. When beetles that had flown for 30 min rested, pre-flight values were reached after 120 min.

The mean haemolymph lipid concentration rose almost linearly during the first 60 min of flight from 9.4 to 16.3 mg ml−1 and remained at this level (Fig. 5C). In beetles that rested after 30 min of flight, no significant return towards pre-flight levels occurred.

Metabolite concentrations in flight muscles

Flight muscles contained approximately 59µmol g−1fresh muscle mass proline and 5µmol g−1alanine at rest. During the first 15 min of flight, proline concentration dropped to 22µmol g−1and the level remained at approximately 20µmol g−1after longer flight times (Fig. 6A). Concomitantly, alanine concentration increased to 38µmol g−1during a flight of 15 min and reached a plateau of more than 40µmol g−1after flights of 30 min or longer. Pre-flight levels of the two amino acids were regained after 60 min of rest following a 30 min flight. The sum of proline and alanine concentrations is very stable, at 60–65µmol g−1, throughout flight and subsequent rest. A mean glycogen concentration of approximately 65µmol g−1fresh muscle mass was measured in flight muscles of resting beetles. These stores were largely depleted during the first 15 min of flight (to 16µmol g−1; Fig. 6B). There was a negligible further decrease to approximately 10µmol g−1 during 45 min of flight. Recovery of stores after 30 min of flight took approximately 120 min.

Metabolite concentrations in the fat body

The changes in the amino acid levels in the fat body during

flight were less pronounced than those in the flight muscles, and a significant change was measured only for alanine (Fig. 7A). However, during subsequent rest after 30 min of flight, the concentrations of the two amino acids increased (alanine) and decreased (proline) significantly compared with the value at the end of flight (Fig. 7A). The total concentration of proline and alanine ranged from 44 to 50µmol g−1fresh tissue mass during flight and subsequent rest. The mean level of glycogen in the fat body (155µmol g−1) was much higher than in the flight muscles. Depletion of the stores took longer than in the flight muscles. After 30 min of flight, a concentration of approximately 60µmol g−1 was reached, a value that did not change with longer flight durations (Fig. 7B). Pre-flight levels were restored after 120 min of rest following 30 min of flight.

Flight velocity

As a measure of performance during flight mill flight, we recorded flight velocity (results not depicted). The highest flight velocity (approximately 6 km h−1) was observed immediately after the initiation of flight. Velocity decreased consistently until it reached a stable level of approximately 3 km h−1after approximately 30 min of flight.

Muscle types

[image:5.609.57.567.85.279.2]

To determine whether the functional specialisation of the different muscles is accompanied by a biochemical/physiological specialisation, we measured metabolic changes separately in the dorso-longitudinal (DLM), dorso-ventral (DVM) and basalar (BM) muscles (see Fig. 1) after 30 s of lift-producing flight and after different durations of flight without lift production (Fig. 8). Resting levels of the substrates did not differ between the various muscles. No significant differences in levels between muscles were measured after 30 s of lift-generating flight (Fig. 8A). When lift was minimised, however, significant differences in levels occurred between the muscle types (Fig. 8B). These differences Table 1. Rates of oxygen consumption calculated from data on rates of substrate utilisation

Rate of Rate of O2 consumption

consumption during the (µmol g−1tissue (ml O2g−1 (ml O2g−1

first 30 s of flight* Molar ratio min−1) tissue h−1) body mass) % of total V. O∑

Flight muscle (µmol g−1 fresh tissue mass min−1)

Proline 31.6 2.5 79 106.2 8.7 17

}

53.8%

Glycogen 64.3 6 385.8 518.5 42.5 83

Proline+glycogen 51.2

Haemolymph (µmol ml−1min−1)

Proline 60.2 2.5 150.5 202.3 33.6 76.4

}

46.2%

Carbohydrates** 7.77 6 46.6 62.66 10.4 23.6

Proline+carbohydrates 44.0

Total 95.2 100%

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were largest in animals that flew for only 5 min. Proline levels were highest in the DVM in these animals and, in addition, the alanine level was significantly lower than in the DLM. Minor differences in levels occurred after both 10 and 20 min of flight without lift, but these differences were less pronounced than after a 5 min flight.

Haemolymph volume and tissue mass

To estimate the participation of proline and carbohydrates and the different parts of the body to the supply of energy during flight, respectively, we calculated the respective rates of oxygen consumption. It was necessary to know the haemolymph volume. For 12 beetles (ranging from 610–1400 mg body mass), a significant linear relationship between body mass (m, mg) and haemolymph volume (V, µl) was measured: V=0.113m+50.69, P<0.001. The mean haemolymph volume for the beetles with a mean body mass of 1020±237 mg was 165.8±29.5µl. Flight muscles were found to represent 8.2±1.9 % (N=5) of total body mass and the fat body tissue 4.1±3.8 % (N=6). Thus, a beetle with a mean fresh mass of 1020 mg contained 83.6 mg of flight muscles, 41.8 mg of fat body tissue and 166µl of haemolymph.

Discussion

In the present study, we quantify the pattern of metabolic changes in the African fruit beetle P. sinuata using a method that simulates natural flight (Schneider, 1989); our previous studies had analysed the situation only during flight without lift generation (Zebe and Gäde, 1993; Lopata and Gäde, 1994).

During lift-generating flight, we could distinguish two metabolic phases. The first phase lasted from take-off until approximately 30 s of flight. It is characterised by a steep decrease in the haemolymph and flight muscle proline levels and a mirror-image increase in the concentration of alanine concentration, the end product of partial proline degradation (Bursell, 1981; Weeda et al. 1979). The concentration of glycogen in the flight muscles is rapidly reduced, while carbohydrate levels in the haemolymph increase during this

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[image:6.609.57.441.72.438.2]

Respiratory quotient

Fig. 4. Rate of oxygen consumption and respiratory quotient of

Pachnoda sinuata measured during lift-generating flight. Data are

given as means ±S.D. (N=3–6).

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Fig. 5. Metabolite concentrations in the haemolymph of Pachnoda

sinuata during different durations of flight without lift production

[image:6.609.254.546.73.454.2]
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phase. These carbohydrates are probably released from the fat body by the action of a neuropeptide from the corpus cardiacum (Lopata and Gäde, 1994) which was identified as an octapeptide of the large AKH/RPCH family, termed Mem-CC (Gäde et al. 1992; Gäde, 1996). Flight performance in this early flight period is high, as indicated by the high rates of oxygen consumption. It is noteworthy that, at the end of this phase, minimum values of the total concentration of proline and alanine occur in the haemolymph (but not in the flight muscles). This indicates a deficit of Krebs cycle intermediates early in flight (since proline is utilised but no comparable amount of alanine arrives in the haemolymph) and that proline resynthesis in the fat body is not yet generating proline at the maximum rate. Thus, a steady state between rates of proline oxidation and resynthesis is not reached.

In the second phase, metabolite levels appear to stabilise. Proline and alanine concentrations in the haemolymph remain constant, while the concentrations in the flight muscles reach a plateau and then increase again at a slower rate. Resynthesis of proline from alanine in the fat body probably takes place at a maximum rate, which should be approximately 1.5µmol ml−1 haemolymph min−1(calculated for the first 15 min of rest after

flight; Fig. 2A). Glycogen stores in the flight muscle decrease at a much lower rate, while haemolymph carbohydrate concentration is reduced during this phase. The carbohydrates released from the fat body in the first phase now become available and is oxidised. Flight performance is also markedly lower than during the first phase. The second phase is therefore characterised by the fact that metabolism is in an equilibrium state: lower flight performance results in a lower demand for fuels and these are provided by using the carbohydrate reserves previously mobilised from the fat body. The stable respiratory quotient (RQ) during this phase suggests that proline is also contributing to the energy provision. Owing to the resynthesis of proline from alanine in the fat body (see Bursell, 1977; L. Auerswald and G. Gäde, unpublished data for P. sinuata fat body), rates of oxidation and synthesis are balanced and are responsible for the steady state observed during this phase.

These two phases observed during flight seem to contrast with the three phases described by Zebe and Gäde (1993). However, these authors studied flight on a flight mill (without lift production). They found that during the first phase, which comprised the first 2 min of flight, proline was the exclusive fuel in the flight muscles. In the light of our new results, discussed in Auerswald et al. (1998), there is a very plausible explanation: previous studies (Zebe and Gäde, 1993) did not

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Fig. 6. Metabolite concentrations in the flight muscle tissue of

Pachnoda sinuata during different durations of flight without lift

generation and subsequent rest after 30 min of flight. (A) Proline (broken line) and alanine (solid line); (B) glycogen. Values are given as means ±S.D. (N=6). For an explanation of the symbols, see Fig. 2.

Fig. 7. Metabolite concentrations in the fat body tissue of Pachnoda

sinuata during different durations of flight without lift and

[image:7.609.323.563.382.677.2]
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take the pre-flight warm-up phase into account. It is now clear from metabolic and respiratory studies that proline is the exclusive fuel during warm-up (Auerswald et al. 1998); this warm-up phase was, however, included in the metabolic changes observed during 2 min of flight in our previous study (Zebe and Gäde, 1993).

All previous flight data for P. sinuata were obtained during flight without lift, but such studies investigated only relatively short periods of flight (15–20 min; Barnett et al. 1975; Zebe and Gäde, 1993; Lopata and Gäde, 1994). Our flight mill experiments in the present study lasted up to 2 h. Using our data and those of the studies cited above, flight on a flight mill can be characterised as follows: the two phases we found during lift-generating flight are also apparent, but the metabolic changes occur much more slowly. The rapid changes discussed for the first phase of lift-generating flight (30 s) take place over 4–8 min in the flight muscles and over 8–30 min in the haemolymph during flight on the flight mill. The increase in haemolymph carbohydrate levels, which occurred during the

first phase of lift-generating flight, occurs only after more than 30 min of flight on the flight mill; at this time, fat body glycogen level reaches a low and stable value. The carbohydrates in the haemolymph are probably derived from mobilised fat body reserves. As indicated by measurements of flight velocity, flight performance reaches a low level at this stage.

Beetles flew for longer when lift was minimised. While the maximum flight duration of lift-generating flight was 20 min, some individuals flew for almost 3 h on the flight mill. This indicates that the energy expenditure of flight mill flight is much lower than that of lift-generating flight. This is in accordance with the observations of Heinrich (1971), who measured V˙O∑values of 45–50 ml O2g body mass−1h−1during true free flight, but only 21 ml g body mass−1h−1 during tethered flight without lift generation in the sphinx moth

Manduca sexta (see also review by Casey, 1989).

The qualitative changes seen during both types of flight experiments are comparable. This means that, although flight mill experiments do not resemble natural conditions, investigations using this method may have merits. However, the quantitative differences caused by minimising lift production must be taken into account.

Interestingly, haemolymph lipid concentration increases significantly during both types of flight, but no restoration of the pre-flight level occurs during subsequent rest. Similar data are reported for the Colorado potato beetle Leptinotarsa

decemlineata during tethered flight (Mordue and de Kort, 1978;

Weeda et al. 1979). However, the maximum increases of 1.5 mg ml−1after 11 min of lift-generating flight and 9.1 mg ml−1after 2 h of flight on the flight mill were small in P. sinuata compared with that of approximately 20 mg ml−1after 20 min of tethered flight in L. migratoria (Goldsworthy et al. 1972), which uses fat as the predominant fuel for flight. These results suggest that lipids are not used as a flight substrate in P. sinuata. This idea is supported by the fact that haemolymph lipid levels do not increase during starvation in P. sinuata (L. Auerswald and G. Gäde, unpublished results) as they do in L. migratoria (Jutsum

et al. 1975). In addition, in contrast to the locust, where AKH

peptides initiate the release of lipids for flight (see Goldsworthy and Mordue, 1989; Gäde, 1992), haemolymph lipid levels in P.

sinuata were not influenced by the fruit beetle’s own AKH

peptide, Mem-CC (Lopata and Gäde, 1994). During flight, the RQ did not fall, as would be expected if fatty acids were oxidised during prolonged flight. Thus, taken together, these data suggest that lipids are not oxidised by the flight muscles, but that they might be used for other purposes.

We assumed that, during flight where lift is minimised, the muscles involved in generating lift should show a slower depletion of substrates compared with lift-producing flight. We therefore measured metabolic changes separately in three different muscles of the flight apparatus. As expected, metabolic changes during generating flight in the lift-generating downstroke muscles (BM and DLM) and in the DVM, one of the upstroke muscles responsible for producing drag, did not differ, because all the muscles are used. However, during flight where lift is minimised, we again found no large

90

0 30 60

Amino acid (

µ

mol

g

1fresh

muscle mass) and glycogen concentrations

(

µ

mol

glucose

equivalents

g

1fresh

muscle

mass)

75

0 25 50

Pro

A

B

Ala Glyc Pro Ala Glyc

Rest

5 min ‡‡ ‡‡

10 min 20 min

Flight with lift production lasting 30 s

BM DVM DLM

[image:8.609.46.292.74.387.2]

Pro Ala Glyc Pro Ala Glyc Pro Ala Glyc Flight without lift generation lasting

Fig. 8. Metabolite concentrations in different flight muscles of

Pachnoda sinuata. (A) At rest and after lift-generating flight (N=5;

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differences. Unexpectedly, the DVM was found to have used less proline than the DLM and BM and to have generated less alanine than the DLM. This was an unexpected result, suggesting a major participation of the downstroke muscles (BM and DLM) in generating both lift and drag. This pattern of substrate use, in addition to the similar resting values of metabolites in the various muscles, excludes a biochemical specialisation of certain muscles to oxidise one or other fuel.

Our results allow us to calculate the contributions of the different fuels (carbohydrates and proline) as well as the contribution of the major compartments (flight muscles, haemolymph) to the overall energy consumption for a ‘model beetle’ (as outlined in the Results) during the first 30 s of lift-generating flight by using the rate of substrate utilisation and the equation for the partial oxidation of proline (see Auerswald and Gäde, 1995). The data shown in Table 1 demonstrate clearly that proline and carbohydrates contribute to a similar extent (44.4

versus 55.6 %, respectively) to the energy required for flight. A

major part of flight substrates in P. sinuata is supplied by the flight muscle tissue itself (54 %; Table 1). The haemolymph also supplies a large amount of energy (46 %), but this is a relatively smaller amount than we found in the blister beetle Decapotoma

lunata (75 %; Auerswald and Gäde, 1995). This difference is

mainly caused by the fact that P. sinuata has a much lower relative haemolymph volume (163.7µl g−1body mass) than D.

lunata (347.2µl g−1body mass). The sum of the two compartments amounts to a body-mass-specific rate of oxygen consumption of 95.2 ml g−1h−1. Because we did not measure metabolic changes in the fat body during lift-generating flight, we assumed from the data obtained for flight mill flight that the rate of substrate depletion in the fat body is similar to that in flight muscle tissue. Despite the low percentage of body mass due to the fat body, V˙O∑ is then well above 100 ml g−1body mass h−1, taking the rate of resynthesis of proline into account (see Figs 2A, 3A for resting values after flight). This value is in good agreement with our results obtained from measuring V˙O∑ in vivo (approximately 104 ml O2g−1body mass h−1, Fig. 4).

In conclusion, we have shown that proline and carbohydrates are both major substrates for energy supply during flight in P.

sinuata. In addition, we have demonstrated differences in

substrate use between the two flight methods as well as the high energy demand required of lift-generating flight.

We are grateful to Dr Sue Nicolson for checking the English and commenting on the manuscript. This study was financially supported by a grant (to G.G.) and a PhD bursary (to L.A.) from the Foundation for Research Development (Pretoria, RSA) and funds from the University of Cape Town (to P.S. and G.G.).

References

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pre-flight warm-up in the African fruit beetle Pachnoda sinuata (Cetoniinae). J. exp. Biol. 201, 1651–1657.

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GÄDE, G. (1997b). Distinct sequences of AKH/RPCH family members in beetle (Scarabaeus-species) corpus cardiacum contain three aromatic amino acid residues. Biochem. biophys. Res.

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GÄDE, G., LOPATA, A. KELLNER, R. AND RINEHART, K. L. (1992). Primary structures of neuropeptides isolated from the corpora cardiaca of various cetonid beetle species determined by pulsed-liquid phase sequencing and tandem fast atom bombardment mass spectrometry. Biol. Chem. Hoppe-Seyler 373, 133–142.

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of the locust. J. comp. Physiol. 79, 85–96.

GOLDSWORTHY, G. J. AND MORDUE, W. (1989). Adipokinetic hormones: functions and structures. Biol. Bull. mar. biol. Lab.,

Woods Hole 177, 218–224.

HEINRICH, B. (1971). Temperature regulation of the sphinx moth,

Manduca sexta. I. Flight energetics and body temperatures during

free and tethered flight. J. exp. Biol. 54, 141–152.

HEINRICH, B. ANDMCCLAIN, E. (1986). ‘Laziness’ and hypothermia as a foraging strategy in flowering scarabs (Coleoptera: Scarabaeidae). Physiol. Zool. 59, 273–282.

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JUTSUM, A. R., AGARWAL, H. C. AND GOLDSWORTHY, G. J. (1975). Starvation and haemolymph lipids in Locusta migratoria

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SACKTOR, B. ANDCHILDRESS, C. C. (1967). Metabolism of proline in insect flight muscle and its significance in stimulating the oxidation of pyruvate. Archs Biochem. Biophys. 120, 583–588.

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ZEBE, E. ANDGÄDE, G. (1993). Flight metabolism in the African fruit beetle, Pachnoda sinuata. J. comp. Physiol. B 163, 107–112. ZÖLLNER, N. AND KIRSCH, K. (1962). Über die quantitative

Figure

Fig. 1. Flight muscles of Pachnoda sinuata seen from the front of themetathorax: BM, basalar muscle; DVM, dorso-ventral muscle; DLM,dorso-longitudinal muscle; SM, subalar muscle; ODM, obliquedorsal muscle; StPM, sternopleural muscle; TCxM 1, tergocoxalmuscle 1.
Fig. 2. Metabolite concentrations in the haemolymph of Pachnodasimplicity, symbols are given only at the first time point at which afor animals that flew
Fig. 3. Metabolite concentrations in the flight muscle tissue of thecomplete flight muscles of durations of lift-generating flight and subsequent rest after 5 min offlight
Table 1. Rates of oxygen consumption calculated from data on rates of substrate utilisation
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References

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