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Flight substrates and their regulation by a member of the

AKH/RPCH family of neuropeptides in Cerambycidae

q

Gerd Ga¨de

*

_{, Lutz Auerswald}

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

Received 2 February 2000; accepted 29 April 2000

Abstract

The pattern of metabolic changes during tethered flight with lift-generation was investigated in two South African species of long-horned beetles (family: Cerambycidae), namelyPhryneta spinatorand Ceroplesis thunbergi. Energy substrates were measured in haemolymph and flight muscles at rest, after a flight period of 1 min at an ambient temperature of 25–29°C, and 1 h thereafter. Flight diminished the levels of proline and carbohydrates in the haemolymph and proline and glycogen in the flight muscles of both species, and caused an increase in the levels of alanine in both compartments. The concentration of lipids in the haemolymph, however, was not changed upon flight in either species. The resting period of 1 h following a 1 min flight episode, was sufficient to reverse the metabolic situation in haemolymph and flight muscles to pre-flight levels in both species. Purification of an extract of the corpora cardiaca from the two beetle species on RP-HPLC, resulted in the isolation and subsequently in the identification (by mass spectrometry, Edman degradation and RP-HPLC) of an octapeptide of the AKH/RPCH family, denoted Pea-CAH-I (pGlu– Val–Asn–Phe–Ser–Pro–Asn–Trpamide), present in each species. It was demonstrated that low doses of Pea-CAH-I elicited increases in the concentration of proline, as well as carbohydrates, in the haemolymph of both species. The levels of lipids, however, remained unchanged upon injection of this peptide. It is concluded that, upon stimulation by flight, the peptide Pea-CAH-I is released from the corpus cardiacum of a cerambycid beetle and is responsible for the regulation of the major flight substrates, proline and carbo-hydrates, of these beetles. 2000 Elsevier Science Ltd. All rights reserved.

Keywords:Lift-generating flight; Carbohydrates; Lipids; Proline; AKH/RPCH peptides; Mass spectrometry;Phryneta spinator;Ceroplesis thunbergi

1. Introduction

A large number of insects are well known for their impressive flight performance which can span from short, intermittent but sometimes powerful episodes of a few minutes, to long-distance migratory flights lasting several hours. It is, therefore, not surprising that insect flight muscles belong to the most metabolically-active tissues in nature. These tissues are characterised by an entirely aerobic metabolism, and the mitochondria in the flight muscles of insects oxidise a wide variety of fuels: carbohydrates, lipids and also the amino acid proline (for reviews, see Ga¨de, 1992; Ga¨de and Auerswald, 1998). Because insects contain only small energy stores in their

q

Dedicated to Bringfried Ga¨de on the occasion of his 85th birth-day.

* Corresponding author. Tel.: +27-21-650-3615; telefax: + 27-21-650-3301.

E-mail address:ggade@botzoo.uct.ac.za (G. Ga¨de).

0022-1910/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 0 ) 0 0 0 8 2 - 2

flight muscles and haemolymph, substrates for oxidation in the working flight muscles must be replenished by the mobilisation of fuels stored in the fat body. It is gener-ally accepted, and proven for certain species, that small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH) control this process (see, for example, Ga¨de, 1996; Ga¨de, 1997a; Ga¨de et al., 1997).

Although almost 50% of all known insect species taxonomically belong to the order Coleoptera, this order was, until recently, not a favourite one for studying flight metabolism. Some years ago, we systematically started to investigate the flight metabolism of various families of the Coleoptera. Our ongoing research has elucidated flight substrates and their control in specimens of the families Cetoniinae (Zebe and Ga¨de, 1993; Lopata and Ga¨de, 1994; Auerswald et al., 1998a,b; Auerswald and Ga¨de, 1999a,b), Scarabaeinae (Ga¨de, 1997b; Auerswald and Ga¨de, 1999c), Meloidae (Auerswald and Ga¨de, 1995; Ga¨de, 1995; Ga¨de and Auerswald, 1999) and

Chrysomelidae (Weeda et al., 1979; Weeda, 1981; Ga¨de 1989, 1999; Ga¨de and Kellner, 1989).

In the present study we mainly wanted to answer the following three questions:

1. Which energy resources are used in cerambycid beetles during short episodes of flight?

2. Which neuropeptides of the AKH/RPCH family are present in the corpora cardiaca of Cerambycidae and what is their primary structure?

3. Are these neuropeptides able to elicit similar changes as observed during flight when injected in vivo into individuals of different species of cerambycids? The combined data set was then used to draw some more general conclusions of a comparative nature with an evolutionary viewpoint on flight fuels and their reg-ulating metabolic peptides in Coleoptera.

2. Materials and methods

2.1. Insects

Adult long-horned beetles of both sexes of Phryneta

spinator and Ceroplesis thunbergi were caught on fig

trees in gardens in Cape Town, South Africa, or col-lected on flowers of acacia trees in the Karoo between Laingsburg and Beaufort West, South Africa, during the austral summer. The average weight ofP. spinator(both sexes used) was 1599±233 mg (n=20) and the mean

weight of C. thunbergi (both sexes) was 746±115 mg

(n=20). Insects were kept with flowers and/or bark of

the food plant and had access to water in the laboratory for a maximum of 2 days before the start of flight experi-ments. Corpora cardiaca for peptide isolation were mainly dissected from animals 24 h after cessation of metabolic experiments (flight or bioassays).

2.2. Bioassays

Individual beetles were kept overnight at room tem-perature (22±2°C), separately, in small plastic containers (volume: 50 ml) without food but with access to water. A 1µl sample of haemolymph was taken from the neck membrane of the same individual before injection of an appropriate test solution, and again 90 min post-injec-tion. Subsequently, the concentrations of the free amino acids, proline and alanine, as well as the levels of vanil-lin-positive material (=total lipids) and anthrone-positive material (=total carbohydrates) were quantified (see below).

2.3. Flight experiments

Cerambycid beetles were flown at 25–29°C using the “pin method”, described in detail previously (Auerswald

et al., 1998a), which assures the performance of flight with lift-generation. Flight times were 1 min if not other-wise stated. SinceP. spinatorwere caught in Cape Town and were, hence, more readily available, we also investi-gated flights of 5 min duration to see if the observed effects were more pronounced. Sampling and storage of haemolymph and flight muscle tissue for the determi-nation of metabolites were as outlined previously (Auerswald et al., 1998a).

2.4. Metabolite determinations

One microlitre of haemolymph was either mixed with 100 µl of concentrated sulfuric acid for the determi-nation of (a) total vanillin-positive material (=lipids; according to the method of Zo¨llner and Kirsch, 1962) or (b) of total anthrone-positive material (=carbohydrates; according to Spik and Montreuil, 1964) or mixed with 100 µl of 80% acetonitrile for quantification of the amino acids proline and alanine (see below).

Frozen flight muscles were ground to a powder under liquid nitrogen, metabolites were extracted with per-chloric acid, glycogen was analysed by the modified anthrone method with glucose as a standard, and the amino acids, after derivatisation with dansyl chloride, were separated by HPLC and quantified (for details, see Zebe and Ga¨de, 1993).

2.5. Peptide purification and identification

Corpora cardiaca were dissected into 80% methanol, crude extracts were prepared as described previously (Ga¨de et al., 1984), redissolved in 15% acetonitrile con-taining 0.1% trifluoroacetic acid, applied to a Nucleosil C-18 column and run as outlined before (Ga¨de, 1985; see also legend to Fig. 2). One purified fraction of the extract from each beetle was used to generate spectra of mass data using a Voyager Elite (PerSeptive Biosystems, Inc., Framingham, MA, USA) matrix-assisted laser desorption/ionisation instrument in the time-of-flight configuration. Samples were prepared in α-cyano-4-hyd-roxycinnamic acid, and spectra were acquired in the positive, linear mode.

The HPLC-purified fraction from each species was enzymatically modified at the N-terminus (to remove a pyroglutamate residue; see Ga¨de et al., 1988; the enzyme used was pyroglutamate aminopeptidase isolated from

Pyrococcus furiosusand purchased from Takara, Shuzo

Co., Japan). Subsequent to HPLC chromatography (conditions as before), the deblocked material was sub-jected to Edman degradation sequencing using a model 477A sequenator connected to a model 120A on-line PTH (phenylthiohydantoin)-analyser (Applied Biosys-tems, Weiterstadt, Germany).

2.6. Synthetic peptide

The synthetic peptide Periplaneta americana car-dioacceleratory hormone-I (Pea-CAH-I; for nomencla-ture, see Raina and Ga¨de, 1988) came from Peninsula Laboratories, Inc. (Belmont, CA, USA).

3. Results

3.1. Flight experiments

The resting levels of carbohydrates, lipids and the amino acids proline and alanine in the haemolymph of the two cerambycid species were in the same order of magnitude (Fig. 1). Whereas the carbohydrate concen-trations were almost identical, lipid levels were only about 60% in P. spinator when compared to C.

thun-bergi, and the levels of alanine and proline were higher

in P. spinator. Upon 1 min of lift-generating flight, a

small but significant decrease in the concentration of carbohydrates was measured in the haemolymph of both species (Fig. 1). In contrast, no changes occurred in the levels of the total lipids. Proline, however, decreased sig-nificantly by about 20% (P. spinator) and 30% (C.

thunbergi) upon 1 min of flight with a concomitant

sub-stantial and significant increase of the alanine levels in both species. The sum of both amino acids, amounting to about 48 µmol/ml in P. spinator and about 35 µmol/ml inC. thunbergi, was not significantly different between resting and flown beetles or those which had flown for 1 min and were then rested for 1 h. In the latter case, the changes in substrate levels initiated dur-ing flight (thus, carbohydrates and amino acids) were reversed, and these levels were not significantly different from the resting values taken before the commencement of flight (Fig. 1).

Some specimens ofP. spinatorwere flown for 5 min; it is clear that changes in haemolymph carbohydrates and amino acids are significantly different from resting values and are more pronounced than after 1 min of flight. In fact, about 50% of the carbohydrates in the haemolymph were used up and about 40% of the proline (Fig. 1).

In the flight muscles, the initial (resting) concen-trations of glycogen, proline and alanine were also simi-lar in both species (Fig. 2). In both species, significant changes occurred after only 1 min of flight: the tration of glycogen was about 75% of the resting concen-tration after flight and that of proline was about 60%. Alanine was produced in significant quantities during flight in both species and the sum of the amino acids was constant. One hour of rest subsequent to a flight time of 1 min could reverse, in most cases, these changes caused by flight, however, no synthesis of glycogen to the pre-flight level was measured in P. spinator.

As with the situation in haemolymph, a longer time of flight (5 min) brought about more pronounced decreases in the concentrations of glycogen (50% decrease) and proline (66% decrease) in the flight muscles of P. spinator.

3.2. Conspecific bioassays

Injection of one gland equivalent of a crude extract of corpora cardiaca fromP. spinatorresulted in a small but significant increase in the concentration of total carbohydrates in the haemolymph of this beetle, while the level of total lipids remained unchanged (Table 1). Under similar conditions there was a substantial increase in the concentration of proline in the haemolymph and a significant decrease in the levels of alanine (Table 1). Water, which served as a control for the measure of stress induced by injection, had no significant effect on the levels of any of the measured substances in the hae-molymph.

Similarly, one gland equivalent of a crude extract of corpora cardiaca from C. thunbergi caused significant increases in carbohydrates and proline levels, but not of lipids; and alanine was significantly decreased (Table 1).

3.3. Isolation and identification of neuropeptides

A single purification step on a RP-C-18 column of four (P. spinator, Fig. 3A, C) and 17 (C. thunbergi, Fig. 3B, D) pairs of corpora cardiaca was sufficient to show a dominant UV (214 nm) absorbance peak for each spec-ies at about 8.9 min. This peak corresponded to a fluor-escent peak monitored for the characteristics of trypto-phan (276 nm excitation and 350 nm emission), which is a conserved residue at position 8 in the sequence of all known AKH peptides. An aliquot of the peak from

P. spinatorwas cleaved by pyroglutamate

aminopeptid-ase, the deblocked peptide was purified on RP-HPLC and the shortened peptide which eluted about 4 min earl-ier from the column (not shown) was sequenced by Edman degradation. The yield was: Val (52.4 pmol)– Asn (41.7)–Phe (40.9)–Ser (27.1)–Pro (21.1)–Asn (20.7)–Trp (1.5). Undeblocked material was analysed by MALDI mass spectrometry and, for both beetles, mass peaks at m/z=995 (mass plus sodium) and m/z=1011

(mass plus potassium) were detected (Fig. 4 forC.

thun-bergipeak material). The same masses were found when

synthetic Pea-CAH-I was analysed under identical con-ditions (not shown). Co-injection of peak material (retention time 8.9 min) from both beetle species and synthetic Pea-CAH-I always resulted in co-elution using different gradients and different organic modifiers such as trifluoroacetic acid and heptafluorobutyric acid (not shown).

Fig. 1. Changes in metabolite concentrations in the haemolymph of (A)P. spinatorand (B)C. thunbergiduring flight and subsequent rest. Values are presented as mean±S.D. (n=3–6). Significance of changes compared with resting value *p,0.05, **p,0.005; compared with flight value †p,0.05, ††p,0.005 using pairedt-test.

3.4. Bioassays with the endogenous neuropeptide

Having established that conspecific injection of a crude extract of the corpus cardiacum of each species had hypertrehalosaemic and hyperprolinaemic effects

and that a small neuropeptide of the AKH/RPCH family of peptides could be isolated and identified from such extracts, it was of paramount importance to show that the endogenous peptide, Pea-CAH-I, was also biologically active in low doses in both species. As compiled in Table

Fig. 2. Changes in metabolite concentrations in the flight muscles of (A)P. spinatorand (B)C. thunbergi during flight and subsequent rest. Values are presented as mean±S.D. (n=3–6). Significance of changes compared with resting value *p,0.05, **p,0.005; compared with flight value †p,0.05, ††p,0.005 using pairedt-test.

1, an amount of 2 pmol of Pea-CAH-I was sufficient to increase carbohydrate and proline levels in the haemo-lymph of C. thunbergi. Five pmol had a more pro-nounced effect. With the injection of either concentration of Pea-CAH-I, alanine was elevated in the haemolymph.

Quantitatively, very similar results were achieved when Pea-CAH-I was injected into P. spinator. Here, however, significant changes in substrate concentration were only imminent at injected concentrations of 10 pmol (Table 1).

G.

Ga

¨de,

L.

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Journal

of

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Physiology

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1575–1584

Table 1

Conspecific bioassays in the cerambycid beetles,P. spinataandC. thunbergia

Treatment Lipids (mg ml-1_{) Carbohydrates (mg ml}-1_{) Proline (}µ_{mol ml}-1_{) Alanine (}µ_{mol ml}-1₎

0 min 90 min D 0 min 90 min D 0 min 90 min D 0 min 90 min D

A. Phryneta spinator

Water 4.3±2.5 3.6±2.5 −0.7±0.6 9.8±6.5 9.9±7.0 0.1±1.2 44.4±11.4 42.6±9.7 −1.8±2.7* 4.6±1.8 4.4±1.6 −0.2±1.2 Pea-CAH-I

2 pmol 7.7±3.1 8.9±2.4 1.2±0.4 45.8±5.4 47.8±5.8 2.0±0.8 4.6±1.8 3.1±1.5 −1.5±0.8 10 pmol 3.2±3.2 3.8±3.9 0.6±0.9+ _{5.2±3.9 6.4±4.4 1.2±0.6** 43.9±18.8 48.0±19.8 4.1±2.3**}++ _{4.5±0.7 3.0±0.9 −1.5±0.8*}

20 pmol 2.2±1.7 3.4±2.4 1.2±1.3+ _{6.7±3.5 8.7±3.4 2.0±0.7**}++ _{48.5±20.4 63.2±26.8 14.7±12.5*}+ _{4.4±1.8 1.3±1.3 −3.1±0.9**}+

1 pCCb _4.8±_{1.4 4.6}±_1.5 −_0.2±_{0.4 9.6}±_{1.8 11.7}±_{1.9 2.1}±_0.6**++ _{40.8±3.1 57.5±8.0 16.7±6.2**}++ _{5.0±1.9 1.9±1.6 −3.1±0.8**}++

B. Ceroplesis thunbergi

Water 7.0±1.0 7.2±1.2 0.2±0.4 10.5±1.9 10.3±2.1 −0.2±0.5 35.1±5.2 34.9±4.6 −0.2±1.0 1.8±1.4 1.6±1.4 −0.2±0.5 Pea-CAH-I

2 pmol 9.4±2.5 10.6±2.6 1.2±09*+ 34.9±4.6 38.3±2.3 3.4±3.0*+ 5.4±3.2 2.2±1.6 −3.2±3.0*+ 5 pmol 9.2±1.6 11.6±2.0 2.4±0.8**++ 35.0±4.6 42.9±3.6 7.9±1.8**++ 8.0±2.5 1.4±1.9 −6.6±3.7*+ 1 pCCb _7.3±_{2.0 7.5}±_{1.8 0.2}±_{0.7 10.0}±_{1.3 12.5}±_{1.0 2.5}±_0.9**++ _{36.9±7.0 43.6±4.6 6.7±3.0**}++ _{8.5±2.2 1.5±1.3 −7.0±2.5*}++

a _{Data are given as means}±_{S.D. (n}=_{6). Significance of change is marked by *p,0.05 and **p,0.005 using a pairedt-test or, when significantly different compared with control group, with}

+_{p,0.05 and}++_{p,0.005 using student’st-test.}

Fig. 3. Separation of a crude methanolic extract of four pairs of corpora cardiaca fromP. spinator(A, C) and 17 pairs of corpora cardiaca from

C. thunbergi(B, D) on RP-HPLC. Chromatographic conditions: a Nucleosil C-18 column (i.d. 4.6×250 mm length) plus guard column of the same material was eluted with a linear gradient of 0.11% trifluoroacetic acid (TFA; solvent A) and 0.1% TFA in 60% acetonitrile (solvent B). The gradient ran from 43% B to 53% B within 20 min at a flow rate of 1 ml min21_{. The elution was monitored with a UV detector at 214 nm (A,}

B) and a fluorescent detector at 276 nm (excitation) and 350 nm (emission; C, D). *Aliquots of peak material used for elucidation of primary structures. The arrow shows retention time of injected synthetic Pea-CAH-I.

4. Discussion

The present study had three objectives: to identify the fuels for flight and the AKH neuropeptides present in cerambycid beetles, as well as to assess whether these neuropeptides, when injected, could mimic the changes in substrate concentrations that occur during flight. Moreover, to integrate these data in a comparative, evol-utionary framework.

Short periods of flight were sufficient to clearly dem-onstrate that proline and carbohydrates are used to pro-duce the energy necessary for flight in both cerambycid species; significant decreases of these substrates occurred in the two compartments analysed, viz. haemo-lymph and flight muscles. Lipids do not play any role during flight. The production of alanine is a further clear indication that proline is partially oxidised (see also Bur-sell, 1981). It appears, thus, that a number of beetle spec-ies, or even familspec-ies, make use of the dual substrate sys-tem for flight episodes. Whereas some members of the Scarabaeinae (Ga¨de, 1997b) and Onitinae (Ga¨de, 1997c) apparently use exclusively proline as fuel for flight (as do tsetse flies; Bursell, 1981), members of the families Cetoniinae (Zebe and Ga¨de, 1993; Lopata and Ga¨de, 1994; Auerswald et al., 1998a,b; Auerswald and Ga¨de, 1999a,b), Meloidae (Auerswald and Ga¨de, 1995; Ga¨de and Auerswald, 1999) and Chrysomelidae (Weeda et al., 1979; Ga¨de, 1999) have co-opted for the carbohydrate plus proline route. There are, however, other beetle spec-ies that utilise other substrates for flight. Our own pre-liminary studies of two species of jewel beetles (Buprestidae) gave no indications for a major contri-bution of energy via proline oxidation (G. Ga¨de and L. Auerswald, unpublished). Previous work by Thompson and Bennett (1971) identified fats as the main fuel during flights of the bark beetle Dendroctonus pseudotsugae. Proline concentrations, however, were not measured in that study.

Concerning the AKH peptides occurring in beetles, it is clear that cerambycids, which are the subject of the present study, contain only one octapeptide member, denoted Pea-CAH-I. This peptide was previously found as one of two endogenous octapeptides in blattid cock-roaches (see Ga¨de, 1996), but also, together with Pea-CAH-II, in the chrysomelid Leptinotarsa decemlineata

(Ga¨de and Kellner, 1989). Pea-CAH-I was also isolated and sequenced from corpora cardiaca of the cerambycid beetle,Morimus funereus(Gerd Ga¨de and Suzana Djord-jevic, unpublished results). Chrysomelidae and Ceram-bycidae are both families in the large superfamily of Chrysomeloidea (see Crowson, 1981); gene duplication may have occurred in this case, giving rise to the second peptide in Chrysomelidae. Blister beetles (Meloidae) together with Tenebrionidae are members of the large superfamily Cucujoidea, which together with the Chry-someloidea belong to the series Cucujiformia (Crowson,

1981). Whereas tenebrionid beetles contain only the octapeptide Tem-HrTH (see Ga¨de, 1996), blister beetles contain Tem-HrTH and the decapeptide Del-CC which is an elongated Tem-HrTH (see Ga¨de, 1996). The mol-ecular phylogenetic relationship in the peptides Tem-HrTH and Pea-CAH-I is obvious: there is only a con-servative Val2 _{(Pea-CAH-I) to Leu}2 _(Tem-HrTH) exchange explained by point mutation of one of the trip-let bases in the RNA code. To date, however, we have no knowledge of whether the tenebrionid beetles use proline and carbohydrates as flight substrates.

The scarabaeid beetles belong to a completely differ-ent series to those of the Cucujiformia, namely the Scar-abaeiformia (Crowson, 1981). This is also reflected in the AKH peptides which differ substantially from those discussed above: they have either three instead of two aromatic amino acids and/or a negatively charged resi-due, aspartic acid, at position 7 (see Ga¨de et al., 1997). The physiological function of the octapeptide in the two investigated cerambycid beetles also became clear in this study. In both species, injection of the endogenous peptide in the physiological range increased the two sub-strates, proline and carbohydrates, which we have ident-ified in this study as being important fuels for flight. Up to now, such combinations of hyperprolinaemic and hyp-ertrehalosaemic effects have been unequivocally shown only in the chrysomelid potato beetle L. decemlineata

(Ga¨de, 1999), the cetoniid fruit beetle P. sinuata

(Auerswald and Ga¨de, 1999a) and the two meloid blister beetlesM. oculataandD. lunata (Ga¨de and Auerswald, 1999). The usage of a dual substrate system is remi-niscent of the situation in locusts which oxidise carbo-hydrates and lipids during flight (see Ga¨de, 1992). There are, however, some fundamental differences to note. Whereas carbohydrates are used as the initial fuel for flight in locusts, and lipid oxidation constitutes the major fuel during long-distance flight episodes, proline appears to be used in substantial quantities in the first phase of flight, and even exclusively during the pre-flight warm-up phase, as shown in the cetoniid beetle P. sinuata

(Auerswald et al., 1998a). Moreover, in locusts the mode of action of adipokinetic peptides involves the second messenger cyclic AMP for both glycogenolysis and lip-olysis (van der Horst et al., 1999). In P. sinuata we speculate that two different receptors may be present for (I) activating the breakdown of glycogen in which cyclic AMP is not involved, and (II) regulation of the synthesis of proline which is mediated by cyclic AMP (Auerswald and Ga¨de, 2000). We postulate that other beetle species, including the cerambycids investigated in this study, that power their flight muscles by the oxidation of carbo-hydrates and proline, have a similar mode of action as shown for P. sinuata. Cockroaches, on the other hand, seem to use one major substrate to provide energy for muscular activity (and flight is not the preferential mode of locomotion), namely carbohydrates. The AKH

mem-bers in various cockroach species are known and also their effect on activation of glycogen phosphorylase (see Ga¨de et al., 1997). Signal transduction, however, is again not via cyclic AMP (Orr et al., 1985; Lee and Keeley, 1994; Becker et al., 1998).

In conclusion, it seems that the following tendencies become apparent in a comparative framework:

1. insects which undertake long-distance flights, such as locusts, use the “dual carbohydrate–lipid oxidation system” with cyclic AMP as second messenger in the transduction process for mobilisation of both sub-strates;

2. insects which are good flyers but only fly middle-dis-tances and never for longer than probably 1 h, such as potato beetles, blister beetles and long-horned beetles use the “dual carbohydrate–proline oxidation system” in which cyclic AMP is only involved in the proline pathway;

3. insects undertaking mainly trivial short-distance flights, such as cockroaches, use only carbohydrates, and cyclic AMP has no role in signal transduction. It is not yet clear where flies and bees belong to in this scheme, since they are obviously not “trivial flyers” but utilise exclusively carbohydrates (see Ga¨de, 1992). This unclarity is partly due to a paucity of detailed knowledge on the regulation of glycogenolysis.

From the remarks above, it is evident that one of the exciting areas of future research will be to unravel whether the AKH/RPCH peptides in insects with the “dual carbohydrate–proline oxidation system” bind to completely different receptors or to similar receptors but of different subtypes to elicit the hyperprolinaemic (via cyclic AMP) or hypertrehalosaemic (without involve-ment of cyclic AMP) effect. Studies on cerambycids may shed some light in this area.

Acknowledgements

The authors would like to express their gratitude to Ms H. Marco for correcting the English text and per-forming mass spectrometric measurements and to Dr R. Kellner (Merck KGaA, Darmstadt, Germany) for sequencing. Financial support was provided by the

Volkswagen Foundation (I, 73024; Hannover,

Germany), and the University of Cape Town.

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Ga¨de, G., 1992. The hormonal integration of insect flight metabolism. Zoologische Jahrbu¨cher, Abteilung Physiologie 96, 211–225. Ga¨de, G., 1995. Isolation and identification of AKH/RPCH family

pep-tides in blister beetles (Meloidae). Physiological Entomology 20, 45–51.

Ga¨de, G., 1996. The revolution in insect neuropeptides illustrated by the adipokinetic hormone/red pigment-concentrating hormone fam-ily of peptides. Zeitschrift fu¨r Naturforschung 51c, 607–617. Ga¨de, G., 1997a. The explosion of structural information on insect

neuropeptides. In:. Herz, W., Kirby, G.W., Moore, R.E., Steglich, W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural Products, vol. 71. Springer-Verlag, Wien, pp. 1–128.

Ga¨de, G., 1997b. Distinct sequences of AKH/RPCH family members in beetle (Scarabaeus-species) corpus cardiacum contain three aro-matic amino acid residues. Biochemical and Biophysical Research Communications 230, 16–21.

Ga¨de, G., 1997c. Hyperprolinaemia caused by novel members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides isolated from corpora cardiaca of onitine beetles. Bio-chemical Journal 321, 201–206.

Ga¨de, G., 1999. Control of proline as flight substrate in long-horned and leaf beetles by AKH/RPCH neuropeptides. In: Roubos, E.W., Wendelaar Bonga, S.E., Vaudry, H., De Loof, A. (Eds.), Recent Developments in Comparative Endocrinology and Neurobiology. Shaker Publishing, Maastricht, pp. 308–310.

Ga¨de, G., Auerswald, L., 1998. Insect neuropeptides regulating sub-strate mobilisation. South African Journal of Zoology 33, 65–70. Ga¨de, G., Auerswald, L., 1999. Flight substrates in blister beetles

(Coleoptera: Meloidae) and their regulation by neuropeptides of the AKH/RPCH family. European Journal of Entomology 96, 331–335. Ga¨de, G., Goldsworthy, G.J., Kegel, G., Keller, R., 1984. Single step

purification of locust adipokinetic hormones I and II by reversed-phase high-performance liquid chromatography and the amino-acid composition of the hormone II. Hoppe Seyler’s Zeitschrift fu¨r Phy-siologische Chemie 365, 393–398.

Ga¨de, G., Hilbich, C., Beyreuther, K., Rinehart, K.L., 1988. Sequence analyses of two neuropeptides of the AKP/RPCH-family from the lubber grasshopper,Romalea microptera. Peptides 9, 681–688. Ga¨de, G., Hoffmann, K.-H., Spring, J.H., 1997. Hormonal regulation

in insects: facts, gaps and future directions. Physiological Reviews 77, 963–1032.

Ga¨de, G., Kellner, R., 1989. The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical. Peptides 10, 1287–1289.

Lee, Y.-H., Keeley, L.L., 1994. Intracellular transduction of trehalose synthesis by hypertrehalsosaemic hormone in the fat body of the tropical cockroach Blaberus discoidalis. Insect Biochemistry and Molecular Biology 24, 473–480.

Lopata, A., Ga¨de, G., 1994. Physiological action of a neuropeptide from the corpora cardiaca of the fruit beetle,Pachnoda sinuata, and its possible role in flight metabolism. Journal of Insect Physi-ology 40, 53–62.

Orr, G.L., Gole, J.W.D., Jahagirdar, A.P., Downer, R.G.H., Steele, J.E., 1985. Cyclic AMP does not mediate the action of synthetic hypertrehalosaemic peptides from the corpus cardiacum of Per-iplaneta americana. Insect Biochemistry 15, 703–709.

Raina, A.K., Ga¨de, G., 1988. Insect peptide nomenclature. Insect Bio-chemistry 18, 785–787.

Spik, G., Montreuil, J., 1964. Deux causes d’erreur dans les dosages colorimetriques des oses neutres totaux. Bulletin de la Socie´te´ Chi-mique et Biologique 46, 739–749.

Thompson, S.N., Bennett, R.B., 1971. Oxidation of fat during flight of male Douglas-fir beetles,Dentroctonus pseudotsugae. Journal of Insect Physiology 17, 1555–1563.

van der Horst, D.J., van Marrewijk, W.J.A., Vullings, H.G.B., Died-eren, J.H.B., 1999. Metabolic neurohormones: release, signal trans-duction and physiological responses of adipokinetic hormones in insects. European Journal of Entomology 96, 299–308.

Weeda, E., 1981. Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle,Leptinotarsa decemlineata. Journal of Insect Physiology 27, 411–417. Weeda, E., De Kort, C.A.D., Beenakkers, A.M.T., 1979. Fuels for

energy metabolism in the Colorado potato beetle, Leptinotarsa decemlineataSay. Journal of Insect Physiology 25, 951–955. Zebe, E., Ga¨de, G., 1993. Flight metabolism in the African fruit beetle,

Pachnoda sinuata. Journal of Comparative Physiology 163B, 107–112.

Zo¨llner, N., Kirsch, K., 1962. U¨ ber die quantitative Bestimmung von Lipoiden (Mikromethode) mittels der vielen natu¨rlichen Lipoiden (allen bekannten Plasmalipoiden) gemeinsamen Sulfophosphovan-illin Reaktion. Zeitschrift fu¨r die Gesamte Experimentelle Medizin 135, 545–561.

Fig. 2. Changes in metabolite concentrations in the flight muscles of (A)P. spinatorand (B)C. thunbergi during flight and subsequent rest. Values are presented as mean±S.D. (n=3–6). Significance of changes compared with resting value *p,0.05, **p,0.005; compared with flight value †p,0.05, ††p,0.005 using pairedt-test.

1, an amount of 2 pmol of Pea-CAH-I was sufficient to increase carbohydrate and proline levels in the haemo-lymph of C. thunbergi. Five pmol had a more pro-nounced effect. With the injection of either concentration of Pea-CAH-I, alanine was elevated in the haemolymph.

Quantitatively, very similar results were achieved when Pea-CAH-I was injected into P. spinator. Here, however, significant changes in substrate concentration were only imminent at injected concentrations of 10 pmol (Table 1).

G.

Ga

¨de,

L.

Auerswald

/

Journal

of

Insect

Physiology

46

(2000)

1575–1584

Table 1

Conspecific bioassays in the cerambycid beetles,P. spinataandC. thunbergia

Treatment Lipids (mg ml-1_{) Carbohydrates (mg ml}-1_{) Proline (}µ_{mol ml}-1_{) Alanine (}µ_{mol ml}-1₎

0 min 90 min D 0 min 90 min D 0 min 90 min D 0 min 90 min D

A. Phryneta spinator

Water 4.3±2.5 3.6±2.5 −0.7±0.6 9.8±6.5 9.9±7.0 0.1±1.2 44.4±11.4 42.6±9.7 −1.8±2.7* 4.6±1.8 4.4±1.6 −0.2±1.2 Pea-CAH-I

2 pmol 7.7±3.1 8.9±2.4 1.2±0.4 45.8±5.4 47.8±5.8 2.0±0.8 4.6±1.8 3.1±1.5 −1.5±0.8 10 pmol 3.2±3.2 3.8±3.9 0.6±0.9+ _{5.2±3.9 6.4±4.4 1.2±0.6** 43.9±18.8 48.0±19.8 4.1±2.3**}++ _{4.5±0.7 3.0±0.9 −1.5±0.8*}

20 pmol 2.2±1.7 3.4±2.4 1.2±1.3+ _{6.7±3.5 8.7±3.4 2.0±0.7**}++ _{48.5±20.4 63.2±26.8 14.7±12.5*}+ _{4.4±1.8 1.3±1.3 −3.1±0.9**}+

1 pCCb _4.8±_{1.4 4.6}±_1.5 −_0.2±_{0.4 9.6}±_{1.8 11.7}±_{1.9 2.1}±_0.6**++ _{40.8±3.1 57.5±8.0 16.7±6.2**}++ _{5.0±1.9 1.9±1.6 −3.1±0.8**}++

B. Ceroplesis thunbergi

Water 7.0±1.0 7.2±1.2 0.2±0.4 10.5±1.9 10.3±2.1 −0.2±0.5 35.1±5.2 34.9±4.6 −0.2±1.0 1.8±1.4 1.6±1.4 −0.2±0.5 Pea-CAH-I

2 pmol 9.4±2.5 10.6±2.6 1.2±09*+ 34.9±4.6 38.3±2.3 3.4±3.0*+ 5.4±3.2 2.2±1.6 −3.2±3.0*+ 5 pmol 9.2±1.6 11.6±2.0 2.4±0.8**++ 35.0±4.6 42.9±3.6 7.9±1.8**++ 8.0±2.5 1.4±1.9 −6.6±3.7*+ 1 pCCb _7.3±_{2.0 7.5}±_{1.8 0.2}±_{0.7 10.0}±_{1.3 12.5}±_{1.0 2.5}±_0.9**++ _{36.9±7.0 43.6±4.6 6.7±3.0**}++ _{8.5±2.2 1.5±1.3 −7.0±2.5*}++

a _{Data are given as means}±_{S.D. (}n=_{6). Significance of change is marked by *p,0.05 and **p,0.005 using a pairedt-test or, when significantly different compared with control group, with} +_{p,0.05 and}++_{p,0.005 using student’st-test.}

Fig. 3. Separation of a crude methanolic extract of four pairs of corpora cardiaca fromP. spinator(A, C) and 17 pairs of corpora cardiaca from C. thunbergi(B, D) on RP-HPLC. Chromatographic conditions: a Nucleosil C-18 column (i.d. 4.6×250 mm length) plus guard column of the same material was eluted with a linear gradient of 0.11% trifluoroacetic acid (TFA; solvent A) and 0.1% TFA in 60% acetonitrile (solvent B). The gradient ran from 43% B to 53% B within 20 min at a flow rate of 1 ml min21_{. The elution was monitored with a UV detector at 214 nm (A,}

B) and a fluorescent detector at 276 nm (excitation) and 350 nm (emission; C, D). *Aliquots of peak material used for elucidation of primary structures. The arrow shows retention time of injected synthetic Pea-CAH-I.

4. Discussion

The present study had three objectives: to identify the fuels for flight and the AKH neuropeptides present in cerambycid beetles, as well as to assess whether these neuropeptides, when injected, could mimic the changes in substrate concentrations that occur during flight. Moreover, to integrate these data in a comparative, evol-utionary framework.

Short periods of flight were sufficient to clearly dem-onstrate that proline and carbohydrates are used to pro-duce the energy necessary for flight in both cerambycid species; significant decreases of these substrates occurred in the two compartments analysed, viz. haemo-lymph and flight muscles. Lipids do not play any role during flight. The production of alanine is a further clear indication that proline is partially oxidised (see also Bur-sell, 1981). It appears, thus, that a number of beetle spec-ies, or even familspec-ies, make use of the dual substrate sys-tem for flight episodes. Whereas some members of the Scarabaeinae (Ga¨de, 1997b) and Onitinae (Ga¨de, 1997c) apparently use exclusively proline as fuel for flight (as do tsetse flies; Bursell, 1981), members of the families Cetoniinae (Zebe and Ga¨de, 1993; Lopata and Ga¨de, 1994; Auerswald et al., 1998a,b; Auerswald and Ga¨de, 1999a,b), Meloidae (Auerswald and Ga¨de, 1995; Ga¨de and Auerswald, 1999) and Chrysomelidae (Weeda et al., 1979; Ga¨de, 1999) have co-opted for the carbohydrate plus proline route. There are, however, other beetle spec-ies that utilise other substrates for flight. Our own pre-liminary studies of two species of jewel beetles (Buprestidae) gave no indications for a major contri-bution of energy via proline oxidation (G. Ga¨de and L. Auerswald, unpublished). Previous work by Thompson and Bennett (1971) identified fats as the main fuel during flights of the bark beetle Dendroctonus pseudotsugae. Proline concentrations, however, were not measured in that study.

Concerning the AKH peptides occurring in beetles, it is clear that cerambycids, which are the subject of the present study, contain only one octapeptide member, denoted Pea-CAH-I. This peptide was previously found as one of two endogenous octapeptides in blattid cock-roaches (see Ga¨de, 1996), but also, together with Pea-CAH-II, in the chrysomelid Leptinotarsa decemlineata (Ga¨de and Kellner, 1989). Pea-CAH-I was also isolated and sequenced from corpora cardiaca of the cerambycid beetle,Morimus funereus(Gerd Ga¨de and Suzana Djord-jevic, unpublished results). Chrysomelidae and Ceram-bycidae are both families in the large superfamily of Chrysomeloidea (see Crowson, 1981); gene duplication may have occurred in this case, giving rise to the second peptide in Chrysomelidae. Blister beetles (Meloidae) together with Tenebrionidae are members of the large superfamily Cucujoidea, which together with the Chry-someloidea belong to the series Cucujiformia (Crowson,

1981). Whereas tenebrionid beetles contain only the octapeptide Tem-HrTH (see Ga¨de, 1996), blister beetles contain Tem-HrTH and the decapeptide Del-CC which is an elongated Tem-HrTH (see Ga¨de, 1996). The mol-ecular phylogenetic relationship in the peptides Tem-HrTH and Pea-CAH-I is obvious: there is only a con-servative Val2 _{(Pea-CAH-I) to Leu}2 _(Tem-HrTH)

exchange explained by point mutation of one of the trip-let bases in the RNA code. To date, however, we have no knowledge of whether the tenebrionid beetles use proline and carbohydrates as flight substrates.

The scarabaeid beetles belong to a completely differ-ent series to those of the Cucujiformia, namely the Scar-abaeiformia (Crowson, 1981). This is also reflected in the AKH peptides which differ substantially from those discussed above: they have either three instead of two aromatic amino acids and/or a negatively charged resi-due, aspartic acid, at position 7 (see Ga¨de et al., 1997). The physiological function of the octapeptide in the two investigated cerambycid beetles also became clear in this study. In both species, injection of the endogenous peptide in the physiological range increased the two sub-strates, proline and carbohydrates, which we have ident-ified in this study as being important fuels for flight. Up to now, such combinations of hyperprolinaemic and hyp-ertrehalosaemic effects have been unequivocally shown only in the chrysomelid potato beetle L. decemlineata (Ga¨de, 1999), the cetoniid fruit beetle P. sinuata (Auerswald and Ga¨de, 1999a) and the two meloid blister beetlesM. oculataandD. lunata (Ga¨de and Auerswald, 1999). The usage of a dual substrate system is remi-niscent of the situation in locusts which oxidise carbo-hydrates and lipids during flight (see Ga¨de, 1992). There are, however, some fundamental differences to note. Whereas carbohydrates are used as the initial fuel for flight in locusts, and lipid oxidation constitutes the major fuel during long-distance flight episodes, proline appears to be used in substantial quantities in the first phase of flight, and even exclusively during the pre-flight warm-up phase, as shown in the cetoniid beetle P. sinuata (Auerswald et al., 1998a). Moreover, in locusts the mode of action of adipokinetic peptides involves the second messenger cyclic AMP for both glycogenolysis and lip-olysis (van der Horst et al., 1999). In P. sinuata we speculate that two different receptors may be present for (I) activating the breakdown of glycogen in which cyclic AMP is not involved, and (II) regulation of the synthesis of proline which is mediated by cyclic AMP (Auerswald and Ga¨de, 2000). We postulate that other beetle species, including the cerambycids investigated in this study, that power their flight muscles by the oxidation of carbo-hydrates and proline, have a similar mode of action as shown for P. sinuata. Cockroaches, on the other hand, seem to use one major substrate to provide energy for muscular activity (and flight is not the preferential mode of locomotion), namely carbohydrates. The AKH

mem-1. insects which undertake long-distance flights, such as locusts, use the “dual carbohydrate–lipid oxidation system” with cyclic AMP as second messenger in the transduction process for mobilisation of both sub-strates;

2. insects which are good flyers but only fly middle-dis-tances and never for longer than probably 1 h, such as potato beetles, blister beetles and long-horned beetles use the “dual carbohydrate–proline oxidation system” in which cyclic AMP is only involved in the proline pathway;

3. insects undertaking mainly trivial short-distance flights, such as cockroaches, use only carbohydrates, and cyclic AMP has no role in signal transduction. It is not yet clear where flies and bees belong to in this scheme, since they are obviously not “trivial flyers” but utilise exclusively carbohydrates (see Ga¨de, 1992). This unclarity is partly due to a paucity of detailed knowledge on the regulation of glycogenolysis.

From the remarks above, it is evident that one of the exciting areas of future research will be to unravel whether the AKH/RPCH peptides in insects with the “dual carbohydrate–proline oxidation system” bind to completely different receptors or to similar receptors but of different subtypes to elicit the hyperprolinaemic (via cyclic AMP) or hypertrehalosaemic (without involve-ment of cyclic AMP) effect. Studies on cerambycids may shed some light in this area.

Acknowledgements

The authors would like to express their gratitude to Ms H. Marco for correcting the English text and per-forming mass spectrometric measurements and to Dr R. Kellner (Merck KGaA, Darmstadt, Germany) for sequencing. Financial support was provided by the Volkswagen Foundation (I, 73024; Hannover, Germany), and the University of Cape Town.

References

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Auerswald, L., Ga¨de, G., 1999a. Effects of metabolic neuropeptides

Auerswald, L., Ga¨de, G., 2000. Cyclic AMP mediates the elevation of proline by AKH peptides in the cetoniid beetle,Pachnoda sinuata. Biochimica et Biophysica Acta 1495, 78–89.

Auerswald, L., Schneider, P., Ga¨de, G., 1998a. Proline powers the pre-flight warm-up in the African fruit beetle, Pachnoda sinuata (Cetoniinae). Journal of Experimental Biology 201, 1651–1657. Auerswald, L., Schneider, P., Ga¨de, G., 1998b. Utilisation of substrates

during tethered flight with and without lift in the African fruit beetle, Pachnoda sinuata (Cetoniinae). Journal of Experimental Biology 201, 2333–2342.

Becker, A., Liewald, J.F., Wegener, G., 1998. Signal transduction in isolated fat body from the cockroach Blaptica dubiaexposed to hypertrehalosaemic neuropeptide. Journal of Comparative Physi-ology 168B, 159–167.

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Ga¨de, G., 1985. Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromato-graphy, and amino acid composition of factor II. Biological Chem-istry Hoppe-Seyler 366, 195–199.

Ga¨de, G., 1989. Characterisation of neuropeptides of the AKH/RPCH-family from corpora cardiaca of Coleoptera. Journal of Compara-tive Physiology 159B, 589–596.

Ga¨de, G., 1992. The hormonal integration of insect flight metabolism. Zoologische Jahrbu¨cher, Abteilung Physiologie 96, 211–225. Ga¨de, G., 1995. Isolation and identification of AKH/RPCH family

pep-tides in blister beetles (Meloidae). Physiological Entomology 20, 45–51.

Ga¨de, G., 1996. The revolution in insect neuropeptides illustrated by the adipokinetic hormone/red pigment-concentrating hormone fam-ily of peptides. Zeitschrift fu¨r Naturforschung 51c, 607–617. Ga¨de, G., 1997a. The explosion of structural information on insect

neuropeptides. In:. Herz, W., Kirby, G.W., Moore, R.E., Steglich, W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural Products, vol. 71. Springer-Verlag, Wien, pp. 1–128.

Ga¨de, G., 1997b. Distinct sequences of AKH/RPCH family members in beetle (Scarabaeus-species) corpus cardiacum contain three aro-matic amino acid residues. Biochemical and Biophysical Research Communications 230, 16–21.

Ga¨de, G., 1997c. Hyperprolinaemia caused by novel members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides isolated from corpora cardiaca of onitine beetles. Bio-chemical Journal 321, 201–206.

Ga¨de, G., 1999. Control of proline as flight substrate in long-horned and leaf beetles by AKH/RPCH neuropeptides. In: Roubos, E.W., Wendelaar Bonga, S.E., Vaudry, H., De Loof, A. (Eds.), Recent Developments in Comparative Endocrinology and Neurobiology. Shaker Publishing, Maastricht, pp. 308–310.

Ga¨de, G., Auerswald, L., 1998. Insect neuropeptides regulating sub-strate mobilisation. South African Journal of Zoology 33, 65–70. Ga¨de, G., Auerswald, L., 1999. Flight substrates in blister beetles

(Coleoptera: Meloidae) and their regulation by neuropeptides of the AKH/RPCH family. European Journal of Entomology 96, 331–335. Ga¨de, G., Goldsworthy, G.J., Kegel, G., Keller, R., 1984. Single step

purification of locust adipokinetic hormones I and II by reversed-phase high-performance liquid chromatography and the amino-acid composition of the hormone II. Hoppe Seyler’s Zeitschrift fu¨r Phy-siologische Chemie 365, 393–398.

Ga¨de, G., Hilbich, C., Beyreuther, K., Rinehart, K.L., 1988. Sequence analyses of two neuropeptides of the AKP/RPCH-family from the lubber grasshopper,Romalea microptera. Peptides 9, 681–688. Ga¨de, G., Hoffmann, K.-H., Spring, J.H., 1997. Hormonal regulation

in insects: facts, gaps and future directions. Physiological Reviews 77, 963–1032.

Ga¨de, G., Kellner, R., 1989. The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical. Peptides 10, 1287–1289.

Lee, Y.-H., Keeley, L.L., 1994. Intracellular transduction of trehalose synthesis by hypertrehalsosaemic hormone in the fat body of the tropical cockroach Blaberus discoidalis. Insect Biochemistry and Molecular Biology 24, 473–480.

Lopata, A., Ga¨de, G., 1994. Physiological action of a neuropeptide from the corpora cardiaca of the fruit beetle,Pachnoda sinuata, and its possible role in flight metabolism. Journal of Insect Physi-ology 40, 53–62.

Orr, G.L., Gole, J.W.D., Jahagirdar, A.P., Downer, R.G.H., Steele, J.E., 1985. Cyclic AMP does not mediate the action of synthetic hypertrehalosaemic peptides from the corpus cardiacum of Per-iplaneta americana. Insect Biochemistry 15, 703–709.

Raina, A.K., Ga¨de, G., 1988. Insect peptide nomenclature. Insect Bio-chemistry 18, 785–787.

Spik, G., Montreuil, J., 1964. Deux causes d’erreur dans les dosages colorimetriques des oses neutres totaux. Bulletin de la Socie´te´ Chi-mique et Biologique 46, 739–749.

Thompson, S.N., Bennett, R.B., 1971. Oxidation of fat during flight of male Douglas-fir beetles,Dentroctonus pseudotsugae. Journal of Insect Physiology 17, 1555–1563.

van der Horst, D.J., van Marrewijk, W.J.A., Vullings, H.G.B., Died-eren, J.H.B., 1999. Metabolic neurohormones: release, signal trans-duction and physiological responses of adipokinetic hormones in insects. European Journal of Entomology 96, 299–308.

Weeda, E., 1981. Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle,Leptinotarsa decemlineata. Journal of Insect Physiology 27, 411–417. Weeda, E., De Kort, C.A.D., Beenakkers, A.M.T., 1979. Fuels for

energy metabolism in the Colorado potato beetle, Leptinotarsa decemlineataSay. Journal of Insect Physiology 25, 951–955. Zebe, E., Ga¨de, G., 1993. Flight metabolism in the African fruit beetle,

Pachnoda sinuata. Journal of Comparative Physiology 163B, 107–112.

Zo¨llner, N., Kirsch, K., 1962. U¨ ber die quantitative Bestimmung von Lipoiden (Mikromethode) mittels der vielen natu¨rlichen Lipoiden (allen bekannten Plasmalipoiden) gemeinsamen Sulfophosphovan-illin Reaktion. Zeitschrift fu¨r die Gesamte Experimentelle Medizin 135, 545–561.