Flight-related metabolism and its regulatory peptides in the spittle bug Locris arithmetica (Cicadomorpha: Cercopidae) and the stink bugs Nezara viridula (Heteroptera: Pentatomidae) and Encosternum delegorguei (Heteroptera: Tessaratomidae)

Journal of Insect Physiology 55 (2009) 1134–1144

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Flight-related metabolism and its regulatory peptides in the spittle bug Locris arithmetica (Cicadomorpha: Cercopidae) and the stink bugs Nezara viridula (Heteroptera: Pentatomidae) and Encosternum delegorguei (Heteroptera: Tessaratomidae) Gerd Ga¨de *, Heather G. Marco Zoology Department, University of Cape Town, Rondebosch 7700, South Africa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 May 2009 Received in revised form 8 August 2009 Accepted 11 August 2009

Three species of bugs (Order: Hemiptera) belonging to different suborders and different families were investigated with respect to flight-related metabolism, and the neuropeptide hormones that regulate metabolism in Encosternum delegorguei, Locris arithmetica and Nezara viridula were characterised. The concentration of two potential metabolic fuels in the haemolymph of these bugs (at rest) revealed that lipids were more abundant than carbohydrates and that lipids increased significantly when the bugs performed extensive exercise (flight) and in the resting period following the aerobic activity. Carbohydrate levels declined during flight but recovered to the pre-flight level during a 1 h resting period post-flight. Further experiments with N. viridula revealed greater lipid accumulation in the haemolymph after a 10 min flight than after a 2 min flight and significant activation of glycogen phosphorylase was recorded in the fat body immediately after flight activity. Crude extracts of corpora cardiaca (CC) from L. arithmetica and E. delegorguei were both active in mobilising carbohydrates in the cockroach Periplaneta americana. In conspecific assays, only L. arithmetica CC extract had a significant hypertrehalosaemic effect, while CC extracts from both E. delegorguei and L. arithmetica were hyperlipaemic. By a combination of liquid chromatography and mass spectrometry two octapeptides known as Peram-CAH-I and Pyrap-AKH were identified from the spittle bug, L. arithmetica, and two octapeptides known as Panbo-RPCH and Schgr-AKH-II were identified from the edible inflated stink bug, E. delegorguei. Injection of Panbo-RPCH into E. delegorguei and into the green stink bug, N. viridula had no effect on circulating carbohydrates, although glycogen phosphorylase was activated in the fat body. The circulating lipid concentration in N. viridula did not change significantly under artificially induced hypertrehalosaemia, suggesting that lipids were not being used or mobilised. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Heteroptera Flight Energy substrates AKHs Peptide sequence Mass spectrometry

1. Introduction Metabolically speaking, the flight muscles of insects are the most active tissue in nature: through entirely aerobic means, large amounts of energy are utilised during flight muscle contractions; a variety of fuels, such as lipids, carbohydrates and the amino acid proline are oxidised in the process (for reviews, see Ga¨de, 1992; Ga¨de and Auerswald, 1998). The amount of energy substrates immediately available in the flight muscles itself and in the haemolymph, however, is insufficient to fuel the contractions of flight muscles for longer than a few minutes during intense

* Corresponding author at: Zoology Department, University of Cape Town, University Avenue, J Day Building, Rondebosch ZA-7700, South Africa. Tel.: +27 216503615; fax: +27 216503301. E-mail address: [email protected] (G. Ga¨de). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.08.009

performance and, hence, substrates stored in the insect’s fat body must be mobilised to replenish the oxidised metabolites. This mobilisation is under the neuroendocrine control of peptides which are synthesised in the corpora cardiaca (CC) and which belong to the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family (for reviews, see Ga¨de, 1997, 2009). Hemiptera is the most species-rich order of exopterygote insects. The classical division of Hemiptera into two suborders, Homoptera (cicadas, leaf- and planthoppers, spittle bugs) and Heteroptera (true bugs), has since been revised to five suborders: Fulgomorpha, Cicadomorpha, Coleorrhyncha, Stenorrhyncha and Heteroptera (Gullan and Cranston, 2005). With about 80 families, Heteroptera is the largest hemipteran clade. Despite this impressive biodiversity and the resulting ecophysiological adaptations, there are only very few complete analyses on the energetic demands of flight and its endocrine regulation in the Heteroptera, viz. in the backswimmer Notonecta glauca (clade Hydrocorisae;

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Family: Notonectidae; Ga¨de et al., 2004) and the twig wilter Holopterna alata (clade Geocorisidae: Family: Coreidae; Ga¨de et al., 2006). There are some other investigations including a short report on flight in the orange winged cicada Platypleura capensis (Ga¨de and Janssens, 1994) but in these studies either flight metabolism was analysed or the regulatory peptides were identified (see review by Ga¨de, 2008). In the present study we aim to address this paucity of information by investigating aspects of flight metabolism in a member of the suborder Cicadomorpha (Family: Cercopidae), the spittle bug Locris arithmetica. This species can be found occasionally in large numbers in Kikuyu grass and is known to damage such pastures; its close relative, Locris rubens, is an endemic pest of sorghum in parts of western and central Africa (Ajayi and Oboite, 2000). Until recently, L. arithmetica was not endemic in the Western Cape Province but is more often detected there during the last years; dispersal flight is suspected in this case, thus making L. arithmetica a good study object for the current investigation. Further, the use of flight fuel is investigated in the suborder Heteroptera, in two stink bug species from different families, viz. the green stink bug Nezara viridula (Family: Pentatomidae) and the edible inflated stink bug Encosternum delegorguei (Family: Tessaratomidae). N. viridula is a well-known economically important pest insect worldwide which feeds and damages about 200 plant species including various legumes. E. delegorguei on the other hand, is a sought after source of protein for people of the Venda tribe in the northern parts of South Africa (Teffo et al., 2007). It is reported that this bug ‘‘flies in droning swarms on hot days’’ (Picker et al., 2002) and it is this behaviour that made it especially interesting to include the inflated stink bug in the current study on flight-associated metabolism. In addition to an analysis of flight fuels, we also identified the primary sequence of peptides that control fuel mobilisation in each species under investigation, except in N. viridula where the regulatory peptide is already known (Ga¨de et al., 2003). 2. Materials and methods 2.1. Insects All the insects used in this study were collected in South Africa; only adult male and female specimens were included in the investigations. Specimens of the green stink bug, N. viridula, were collected from various plants on the campus of the University of Cape Town and the senior author’s garden in Mowbray, both Western Cape Province. Specimens of the edible inflated stink bug, E. delegorguei, were collected from its host plant Dodonaea viscosa during aggregation in September in the Limpopo Province by Ms C. Dzerefos and purchased from her. Non-diapausing E. delegorguei have been observed to feed on the sap from this D. viscosa tree (Dzerefos et al., in press). Both species were collected during 2006 and 2007. N. viridula was held in the laboratory at 25  1 8C with 16 h of light and an ad lib. supply of fresh green beans and peeled dried sunflower seeds; a breeding colony was established. E. delegorguei was kept in the laboratory under the same conditions as for N. viridula for 2 days after capture before experimentation commenced; a breeding colony was not established. The body mass of the green stink bug was 156.4  14.3 mg (mean  S.D., n = 12) and of the inflated stink bug 810  158 mg (n = 11). For experimentation 2day-old N. viridula adults were selected, whereas E. delegorguei adults were of unspecified age. Specimens of the spittle bug, L. arithmetica, were collected in April 2007 in Kikuya grass next to a small lake in the vicinity of Somerset West (Western Cape Province). Adults of undetermined age and body mass of 42.1  5.2 mg (n = 12) were used in experimentation.

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For heterologous bioassays, adult male American cockroaches, Periplaneta americana, of unspecified age were used. 2.2. Bioassays Carbohydrate mobilisation in P. americana was measured as described elsewhere (Ga¨de, 1980). Conspecific biological assays on green stink bugs were performed as outlined earlier (Ga¨de et al., 2003), and assays on E. delegorguei and L. arithmetica were performed in a similar fashion: the insects were individually placed in small containers with a ball of water-soaked cotton wool and left undisturbed for 2 h before experimentation at 24  1 8C. Thereafter, 1 ml of haemolymph was taken laterally from the abdomen with a microcapillary pipette; the haemolymph was blown into 200 ml of concentrated sulphuric acid, and the insects were injected with 5 ml of distilled water as control or with the appropriate test solution diluted in distilled water. The insect was then returned to its container and a second 1 ml sample of haemolymph was taken 90 min after the injection. The haemolymph was mixed thoroughly with the sulphuric acid and 100 ml each was used for measuring the total anthrone-positive (=carbohydrates) and vanillin-positive (=lipids) material as previously described (Ga¨de and Auerswald, 2000). 2.3. Determination of glycogen phosphorylase and the concentration of stored metabolites To isolate fat body tissue, the abdomen of the bug was opened ventrally with a pair of scissors and the fat body was separated from other organs with a pair of forceps. Fat body tissue from individual bugs (E. delegorguei and N. viridula) was dissected, homogenised, processed and the activity of glycogen phosphorylase determined in the direction of glycogen breakdown as outlined previously (Ga¨de, 1981). Stored reserves of glycogen and total lipids in young adult male N. viridula were measured from resting individuals: the thorax and abdomen were quickly separated and frozen in liquid N2. The separate body parts from individual bugs were later ground into a fine powder, further homogenised by sonication in either perchloric acid for glycogen extraction, or in a mixture of chloroform and methanol for lipid extraction. Glycogen in the thorax and abdomen of green stink bugs was extracted and measured as anthrone-positive material according to Zebe and Ga¨de (1993). Total lipid content was determined gravimetrically according to Folch et al. (1957); briefly, tissues were sonicated in a chloroform:methanol solution (2:1, v/v; tissue ratio of 1:19 by weight). After phase separation, the infranatant (containing the extracted lipid) was dried to constant weight. 2.4. Flight experiments Edible stink bug: Individual bugs were subjected to liftgenerating flight (for instrumentation, see Auerswald et al., 1998a) at 30 8C created by a photographic lamp and a laminar flow of about 1.6 m s 1 created by a fan placed at one end of a rectangular metal tunnel (for details, see Auerswald et al., 1998a). After a flight of 3 min, insects were allowed to rest for 1 h. Haemolymph (1 ml) was taken from the bugs at three time points: at rest prior to flying, immediately after 3 min of flight and, finally, at rest for 1 h following flight. Spittle bugs: Only a few individuals were subjected to liftgenerating flight in a similar fashion as the edible stink bugs. All individuals flew readily for 2 min only, after which they were rested for 1 h. Green stink bugs: The first series of flight experiments were performed in a large constant temperature room at 34 8C.

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Haemolymph (0.5 ml) was taken from resting insects at the beginning of the experiment. One group (control) was kept at rest for a further 1 h after which a haemolymph sample was again withdrawn. The second group (experimental) was allowed to fly freely for a period of 1 min and 46 s after which they were confined to rest for 1 h; following this post-flight rest, a 0.5 ml haemolymph sample was again taken (see Fig. 2). All other flight experiments (tethered but lift-generating flight) were undertaken in a similar fashion as described for the edible stink bug. In another series of flight experiments, green stink bugs were injected at rest with 0.9 mg trehalose dissolved in 5 ml distilled water. In one group of animals, a haemolymph sample was taken before injection of trehalose to estimate the average carbohydrate concentration in the non-injected bug population. This value was found to be approximately 10 mg carbohydrate/ml of haemolymph. The remaining animals were kept in an inactive state and a 0.5 ml haemolymph sample was collected at a single time point from as early as 5 min after injection and up to 2 h post-injection to measure the carbohydrate concentration over time. From these experiments, an assumption was made that 5 min after injection of trehalose was an adequate period for equally distributing the trehalose in the circulatory system. Therefore, for flight experiments with trehalose-injected bugs, flight activity was only initiated after this ‘‘distribution’’ period; some bugs were engaged in an active short (about 2.5 min) or long (about 10.5 min) flight, and two haemolymph samples were removed for carbohydrate determination: before injection of trehalose, and immediately after cessation of flight. 2.5. Isolation, purification and identification of AKH peptides from the corpora cardiaca of the edible stink bug and the spittle bug Dissection of CC: Before dissection of the CC, inflated stink bugs and spittle bugs were cooled on ice for about 10 min and subsequently pinned down in a dissection dish, the head was opened dorsally with a pair of iridectomy scissors to reveal the brain and the underlying CC. The glands were dissected under a stereo microscope using fine scissors and forceps and were transferred into 80% methanol and stored at 20 8C. Extraction of peptides was performed as described previously (Ga¨de et al., 1984). The extracts were reconstituted in either distilled water for biological assays, or in 15% acetonitrile/0.01% trifluoroacetic acid (TFA) for separation by RP-HPLC. HPLC separation: For RP-HPLC, the crude extract was applied to a Nucleosil 100 C18 column (4.6 mm  250 mm, particle size 5 mm) equipped with a guard column (10 mm) of the same material and using equipment described previously (Ga¨de, 1985; see also legend to Figs. 6 and 9). Peak material was collected manually and used for bioassays and mass spectrometry. Liquid chromatography (LC)–mass spectrometry (MS): An aliquot of the dried methanolic extract was sent to Ceske Budejovice in the Czech Republic for mass spectrometric measurement. LC/MS analysis was performed on an LTQ-XL mass spectrometer (Thermo Electron, San Jose, USA) equipped with an electrospray probe operated at 4.0 kV. The dried methanolic extract from the CC was dissolved in 120 ml of aqueous 0.04% TFA and 5 ml of this solution (equivalent to less than one CC) were injected into a 100 mm  2.1 mm ID, 1.9 mm Agilent Zorbax XDBC8 column. An elution gradient from 15% to 40% B (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile) was used at a flow rate of 200 ml min 1 within 10 min. The primary sequence of the peptides was deduced from the electrospray MSN spectra obtained by the collision-induced dissociation (CID) of the detected MH+ ion and its product fragment ions.

2.6. Synthetic peptides The following synthetic peptides were either previously purchased from Peninsula Laboratories (now: Bachem-Peninsula Laboratories, Belmont, CA, USA) or previously custom-synthesised by Dr. R. Kellner (Merck KGaA, Darmstadt, Germany) and K.D. Clark (Department of Entomology, University of Georgia, USA): adipokinetic hormone I of the migratory locust (code-name: LocmiAKH-I), adipokinetic hormone II of the desert locust (Schgr-AKHII), adipokinetic hormone of the fire bug (Pyrap-AKH), hypertrehalosaemic or cardioacceleratory hormone I of the American cockroach (Peram-CAH-I) and red pigment-concentrating hormone of the boreal shrimp (Panbo-RPCH). 2.7. Statistical analyses Comparisons between experimental groups were done as outlined in the legends of figures and tables. 3. Results 3.1. Haemolymph metabolite levels and flight experiments Lipids and carbohydrates are considered as putative fuel for flight in bugs. To survey the amount of readily available fuel, the concentration of lipids and carbohydrates in the haemolymph was determined in specimens 1 day after they were caught in the wild (inflated stink bug and spittle bug) or 1–2 days after the imaginal moult (green stink bug). The highest lipid concentration in the haemolymph (58.1  24.9 mg ml 1; mean  S.D., n = 6) was measured in E. delegorguei with a 10-fold lower concentration of carbohydrates measured in its haemolymph (5.5  1.7 mg ml 1). The other stink bug, N. viridula, also had a high concentration of lipids (20.6  4.7 mg ml 1; n = 6) but the carbohydrate concentration (11.9  3.6 mg ml 1) was only 2-fold lower. This relationship was also true for lipids (5.4  2.7 mg ml 1; n = 5) and carbohydrates (2.7  0.4 mg ml 1) in L. arithmetica, which clearly had the lowest concentration of both metabolites. Although high individual variation in metabolite concentration was common in those insects that were just collected in the field, the measured circulating lipid level was always higher than the carbohydrate level. The high individual variation in metabolite concentration was not so apparent in bugs that were kept in captivity for 4 days and more with constant access to food and water; hence, flight experiments with E. delegorguei and L. arithmetica specimens were carried out a few days after capture. Ten specimens of E. delegorguei were subjected to liftgenerating, tethered flight: two did not fly at all and two others flew only for a few seconds; these animals were excluded from further experimentation. The remaining six insects were subjected to a period of 3 min of flight and, thereafter, were rested for 1 h. As depicted in Fig. 1A, the high level of lipids in the haemolymph prior to flight muscle activity was slightly and significantly increased after 3 min of flight with a further small but significant increase measured after the 1 h post-flight resting period. The concentration of carbohydrates in the haemolymph was significantly decreased after flight and significantly increased after the postflight resting period of 1 h and even overshot the initial resting level (Fig. 1A). All of the L. arithmetica specimens flew readily for about 2 min. During this short flight period the concentration of lipids in the haemolymph did not change significantly, whereas the carbohydrate concentration was diminished 2-fold (Fig. 1B). A resting period of 1 h after the 2 min of flight resulted in a 2-fold increase in the lipids, while the carbohydrates were almost back to resting level (Fig. 1B).

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Fig. 2. Concentration of lipids in the haemolymph of two groups of Nezara viridula showing the adipokinetic effect of flight. Haemolymph was sampled from bugs that were kept at rest for 60 min (n = 16); one group of bugs (n = 10) rested for a further 60 min and then a second haemolymph sample was collected, while the remaining bugs (n = 6) were forced to engage in free flight (mean time of 1 min 46  24 s) followed by 60 min rest before a second haemolymph sample was collected. Values are presented as mean  S.D. *Significantly different from pre-flight resting values (P = 0.005; paired t-test).

Fig. 1. Changes in the concentration of metabolites in the haemolymph during flight and subsequent rest of (A) Encosternum delegorguei and (B) Locris arithmetica. E. delegorguei specimens flew for 3 min and L. arithmetica flew for 2 min; post-flight rest of 1 h in both species. Values are presented as mean  S.D. (n = 6 for E. delegorguei; n = 5 for L. arithmetica). *Significantly different (P = 0.001–0.05) from preflight resting values (calculated by paired t-test). **Significantly different (P = 0.001– 0.002) from flight value as calculated by paired t-test.

Since a colony of N. viridula was established for this study, more insects were available for a variety of experiments. First, lipid concentrations in the haemolymph of N. viridula were determined in a group of inactive bugs and in bugs that had rested for 1 h following a short period of free flight (Fig. 2). The high concentration of lipids was unchanged in the inactive group after 1 h but had increased significantly in insects that were resting for 1 h subsequent to flight activity (Fig. 2). During these free flight experimentations, the stink bugs could not be stimulated to fly for more extensive periods, hence, tethered flight with lift-generation was introduced for N. viridula. In this set-up, bugs could be subjected to short (about 2 min) or long (about 10 min) flights as well as imposing 1 h of rest after a short flight; both circulating metabolites were measured. Fig. 3A shows significant changes in the lipid concentration during flight: the pre-flight resting concentration of nearly 19 mg ml 1 was increased to 22– 23 mg ml 1 during short flight and short flight plus rest, respectively; immediately after the long flight period the lipid concentration increased to almost 26 mg ml 1 (Fig. 3A). Carbohydrates were clearly used during long flights, decreasing from almost 10 mg ml 1 to 2 mg ml 1 and were used to a lesser extent during short flights (Fig. 3B); during the resting post-flight period no significant change to the carbohydrate concentration in the haemolymph was measured (Fig. 3B). The activation state of glycogen phosphorylase in the fat body of the same individuals was also measured in this experiment: at rest the enzyme is about

Fig. 3. Changes in the concentration of circulating lipids (A) and carbohydrates (B), as well as in the activation of the enzyme glycogen phosphorylase in the fat body (C) of Nezara viridula at rest, just after flights of 2 min (short flight) and 10 min (long flight) and 60 min after a short flight. Values are presented as mean  S.D.; n = 7 for pre-flight rest and short flight; n = 6 for long flight and n = 8 rest following the short flight. *Significantly different from pre-flight resting value (P = 0.0004; paired t-test). Statistical comparisons between groups were performed by ANOVA followed by Tukey test: the same letter indicates no significant difference between those groups.

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15% activated (Fig. 3C). When the phosphorylase was measured 15 min after the bugs had performed short flights or immediately after long flights, glycogen phosphorylase was significantly activated to 46% and 40%, respectively (Fig. 3C). A resting period of 1 h after the short flight was sufficient to bring the activation state of the phosphorylase back to 23%; this was not significantly different from the initial resting value (Fig. 3C). The concentration of stored metabolites, i.e. energy reserves in the form of glycogen and total lipids in thorax and abdomen of resting N. viridula was determined: glycogen in the thorax amounted to 1.3  0.1 mg glucose equivalents per gram fresh weight and in the abdomen glycogen content was measured as 1.1  0.1 mg glucose equivalents per gram fresh weight (n = 3); lipid in the thorax and abdomen was determined as 148.9  38.7 mg lipid per gram fresh weight and 163.6  0.8 mg lipid per gram fresh weight, respectively (n = 10). The endogenous AKH of the green stink bug, Panbo-RPCH, does not elicit hypertrehalosaemia when injected into N. viridula (Ga¨de et al., 2003). Additionally, in some insects hypertrehalosaemia inhibits the release of AKH from the CC in vitro (Passier et al., 1997; Flanigan and Ga¨de, 1999). To examine the effect of elevated carbohydrates in the green stink bug on the concentrations of lipids and carbohydrates during flight, trehalose was injected into resting N. viridula. First, the concentration of total carbohydrates in the haemolymph was measured over time while the bugs were kept at rest. As depicted in Fig. 4 the level of total carbohydrates rose from about 10 mg ml 1 before injection to 60 mg ml 1 5 min after the injection (this time period was deemed adequate for the injected trehalose to be distributed equally in the haemolymph). In the next 5 min the carbohydrate concentration did not change but from 15 min after injection onwards there was a decrease to about 30 mg ml 1 measured 2 h after the injection (Fig. 4). In the next series of experiments, a haemolymph sample was collected from individual stink bugs before they were injected with trehalose; after 5 min at rest for equal distribution of the injected trehalose in the circulatory system, the bugs were then subjected to a period of short (about 2 min and 30 s) or long (about 10 min and 30 s) liftgenerating tethered flight and immediately after flight a second sample of haemolymph was withdrawn to measure the concentration of lipids and carbohydrates in circulation. During all treatments there was no significant change in the concentration of lipids in the haemolymph (Fig. 5B). The carbohydrate level remained significantly higher during both flight times than during

Fig. 4. Concentration of circulating carbohydrates in Nezara viridula before and after injection of 0.9 mg trehalose (in 5 ml distilled water). Bugs were kept at rest and 0.5 ml haemolymph was sampled before injection, or at a different time point after injection up to 120 min. Values are presented as mean  S.D.; n = 5 for each time point. The concentration of trehalose is significantly higher in all of the injected groups when compared with the non-injected value (ANOVA with Dunnett test).

Fig. 5. Changes in the concentration of carbohydrates (A) and lipids (B) in the haemolymph of Nezara viridula in response to a long flight (10 min 28  1 min 20 s) or short flight (2 min 32  53 s) under artificially high trehalosaemic conditions. Before injecting 0.9 mg trehalose into resting bugs, 0.5 ml haemolymph was sampled. Before subjecting bugs to tethered, lift-generating flight a 5 min period of rest was upheld for the injected trehalose to distribute in the haemolymph. Following flight, a haemolymph sample was withdrawn. Values are presented as mean  S.D.; n = 6– 10. Statistical analyses performed by ANOVA with Tukey test.

rest before trehalose was injected (Fig. 5A) but the values are lower than those measured in trehalose-injected stink bugs that were kept at rest for the same period of time (see Fig. 4; note: 7.5 min corresponds to 5 min distribution of injectate plus 2.5 min flight, and 15.5 min corresponds to 5 min distribution plus 10.5 min flight). 3.2. Biological activity of spittle bug and inflated stink bug corpus cardiacum extract In a first series of experiments it was tested whether crude extracts of retrocerebral glands of the spittle bug and the inflated stink bug had carbohydrate-mobilising activity in a well-known test system, i.e. the American cockroach. The equivalent of 0.5 pairs of CC from L. arithmetica and E. delegorguei, respectively, proved positive in mobilising carbohydrates (Table 1). Compared with the maximal possible response for carbohydrate release in cockroaches, which was achieved by injecting 10 pmol of the endogenous cockroach peptide Peram-CAH-I, the increase in carbohydrates in cockroaches after injection of the spittle bug extract amounted to about 80%; in contrast, although active, the extract from the inflated stink bug had only about 20% activity. The second series of experiments tested whether the crude gland extract of the spittle bug and the inflated stink bug had metabolic effects in the respective donor species itself. As depicted in Table 1, injection of own CC extract (0.5 gland equivalents) resulted in a significant hyperlipaemic and hypertrehalosaemic effect in L. arithmetica, whereas 0.5 gland equivalents of E. delegorguei only caused significant hyperlipaemia in the inflated stink bug while the carbohydrate levels in the haemolymph were unaffected (Table 1). Thus, both heterologous and conspecific

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Table 1 Biological activity of a crude methanolic extract of corpora cardiaca from Locris arithmetica and Encosternum delegorguei, and of the respective synthetic peptides in CCs of L. arithmetica and E. delegorguei. Hypertrehalosaemic activity of the crude CC extracts was also assayed in a heterologous test using Periplaneta americana. Treatment

Haemolymph lipids (mg ml n

0 min

1

Haemolymph carbohydrates (mg ml

1

P

n

Difference

NS 0.001 0.01 0.01

Acceptor insect: L. arithmetica 6 0.6  0.4 1.2  0.9 11 1.8  1.3 3.2  2.0 6 1.0  0.4 1.5  0.2 6 1.0  0.3 1.7  0.4

NS 0.002 0.0005 0.001

Acceptor insect: E. delegorguei 5 14.5  4.2 11.6  1.7 5 8.8  3.8 10.7  4.4 5 11.4  3.8 12.8  3.8 Not determined

)

90 min

Control (5 ml distilled water) L. arithmetica extract (0.5 gland pair equiv.) Peram-CAH-I (5 pmol) Pyrap-AKH (5 pmol)

Acceptor insect: L. arithmetica 6 6.4  2.1 5.8  2.0 9.5  4.2 11 5.8  1.6 6 3.9  0.9 8.2  1.9 6 4.8  1.2 10.3  3.4

Control (10 ml distilled water) E. delegorguei extract (0.5 gland pair equiv.) Panbo-RPCH (10 pmol) Schgr-AKH-II (10 pmol)

Acceptor insect: E. 5 55.6  12.5 5 56.9  7.8 5 55.2  8.9 5 53.6  9.9

delegorguei 56.7  12.3 64.1  8.2 65.4  10.9 64.6  7.4

Difference 0.6  0.6 3.7  2.8 4.3  1.8 5.5  2.9

1.1  1.1 7.2  2.2 10.2  2.2 11.0  3.0

a

Acceptor insect: P. americana Control (10 ml distilled water) E. delegorguei extract (0.5 gland pair equiv.) L. arithmetica extract (0.5 gland pair equiv.) Peram-CAH-I (10 pmol)

0 min

90 min

Acceptor insect: P. americana 5 14.1  2.1 14.0  1.2 6 14.3  2.0 18.9  2.9 6 13.8  1.9 32.7  6.3 5 13.5  1.4 37.8  6.9

Not determined

) Pa

0.6  0.7 1.4  0.9 0.5  0.4 0.7  0.4

NS 0.001 0.05 0.01

2.9  4.4 1.9  6.1 1.4  3.7

NS NS NS

0.1  1.5 4.6  1.7 18.9  5.8 24.3  7.5

NS 0.001 0.002 0.002

Data are presented as mean  S.D. a Paired t-test was used to calculate the significance between pre- and post-injection. NS, not significant.

bioassays indicated the presence of peptides of the AKH/RPCH family in the CC of the two species. 3.3. Isolation and structural analyses of AKH peptides from the CC of the spittle bug and the inflated stink bug A similar strategy was followed to isolate and structurally characterise the AKH from the two bug species. For the sake of brevity, however, details for the spittle bug will be presented here, whereas the results from the inflated stink bug will be mentioned briefly only. A typical chromatogram of 10 pair equivalents of CC from L. arithmetica is presented in Fig. 6. The trace shown represents the fluorescence, which is characteristic of the presence of the amino acid tryptophan (at position 8 in all members of the AKH/RPCH family). Material from peaks numbered I–V were collected and

Fig. 6. Fluorescence profile of reverse phase high-performance chromatography (RP-HPLC) separation: methanolic crude extract of 10 corpora cardiaca pair equivalents from Locris arithmetica. Separation was attained with a Nucleosil 100 C18 column (4.6 mm  250 mm) at 30 8C and a linear gradient of 20–80% solvent B in 30 min at a flow rate of 1 ml min 1 (solvents: A = 0.11% trifluoracetic acid [TFA] in water; B = 0.1% TFA in 60% acetonitrile in water). Eluant passed through a fluorescence detector (276 nm excitation, 350 nm emission). Peak material (I–V) was collected by hand, evaporated in a vacuum concentrator and reconstituted in distilled water for bioassays: a dot below a peak shows it had biological activity.

tested in the cockroach bioassay at a concentration of one gland equivalent. Only material that eluted at 19.3 min and 22.3 min (peak IV and V), respectively, was active in causing hypertrehalosaemia in cockroaches (data not shown). Analysis of a crude methanolic extract from the spittle bug (about one gland equivalent) on liquid chromatography coupled to mass spectrometry revealed large peaks at 6.62 min and 7.88 min (Fig. 7) with m/z 973.4 and 1001.4, respectively for [M+H]+ (Fig. 7B and C); furthermore, for both masses there is a peak that is 22 mass units larger, i.e. m/z 995.3 and 1023.3, respectively, indicating a peptide

Fig. 7. LC/MS analysis of the corpora cardiaca material from Locris arithmetica. (A) The total ion chromatogram; (B and C) electrospray ionisation (ESI) mass spectra of the peaks shown in (A) with retention time of 6.62 min and 7.88 min, respectively. For LC/MS conditions, see Section 2.5.

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Fig. 8. The CID MS2-ESI spectrum of (A) the ion with m/z 973.4, and (B) m/z 1001.4 from corpora cardiaca of Locris arithmetica (see Fig. 7). The insets show the sequence of the assigned peptides code-named Peram-CAH-I (A) and Pyrap-AKH (B), together with the theoretical calculated masses for b- and y-type fragment ions, which are observed in the respective MS2 mass spectrum.

in its Na+ form [M+Na]+ (Fig. 7B and C). The complete primary sequence of these two compounds were elucidated by CID of the [M+H]+ ions at m/z 973.4 and 1001.4 (Fig. 8A and B). Characteristic y- and b-type product ions, together with diagnostic y-NH3 and bH2O ions, were detected and made the assignment as members of the AKH/RPCH family easy (see insets, Fig. 8). Peptide identification as Peram-CAH-I and Pyrap-AKH, respectively, was further substantiated in two ways: 1. A crude extract of 30 gland equivalents of L. arithmetica CC was prepared as described in Section 2.5; an aliquot (corresponding

to 15 CC) was run on RP-HPLC as before but with a shallow gradient between 43% and 53% of solvent B: there were two distinctive fluorescence peaks with peak I at 9.2 min being a poorly resolved doublet (Fig. 9A). The remainder of the extract was divided in two portions of 7.5 gland equivalents and each of these was ‘‘spiked’’ with either 20 pmol of synthetic PeramCAH-I or Pyrap-AKH and then chromatographed (Fig. 9B and C). In each run either the shoulder of peak I (Fig. 9B) or peak II (Fig. 9C) increased in height, thus, indicating that synthetic Peram-CAH-I co-elutes with the shoulder peak of peak I and synthetic Pyrap-AKH co-elutes with peak II, thereby confirming

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Fig. 10. Activation of glycogen phosphorylase (as percent of total activity) in the fat body of Encosternum delegorguei (A) and Nezara viridula (B) after the injection of distilled water (control) and the endogenous AKH peptide, Panbo-RPCH (5 pmol). Fat body tissue was dissected 15 min after injection. Values are presented as mean  S.D. (n = 5–6). *Significantly different to control values (P = 0.0002–0.005; Student’s t-test).

identified as the Gly-extended non-amidated forms of Panbo-RPCH and Schgr-AKH-II, respectively (data not shown). 3.4. Biological action of synthetic peptides

Fig. 9. Fluorescence profiles of RP-HPLC separations. (A) Methanolic crude extracts of 15 CC equivalents from Locris arithmetica. A co-injection of an aliquot of the CC extract (7.5 CC equivalents) and 20 pmol Peram-CAH-I (B), or co-injection with 20 pmol Pyrap-AKH (C). The same HPLC set-up was used as for Fig. 6 but a shallow gradient of 43–53% solvent B in 20 min was employed here.

the assigned primary sequences, including the assigned Leu at position two of the peak II material, since an Ile2 compound would have a different retention time under the chosen HPLC conditions (see Ga¨de et al., 2007a). 2. Synthetic Peram-CAH-I and Pyrap-AKH were analysed with the LC/MS system and the peptides had the same retention time, mass data and CID spectra as peak I and peak II, respectively, of the crude extract (data not shown). The LC/MS analysis for the CC material from the inflated stink bug revealed two peaks that were very close together, at 7.86 min and 7.90 min, with m/z 930.5 and 934.4, respectively (data not shown). In addition, there were minor peaks at 7.91 min (m/z 988.5) and 7.96 min (m/z 992.5). The CID spectra of all these peaks (data not shown) were easy to interpret and gave assignments to AKH family peptides with the primary sequences of pELNFSPGW amide, known as Panbo-RPCH, and pELNFSTGW amide, known as Schgr-AKH-II. The ambiguity of Leu versus Ile at position 2 in each peptide was resolved by running the respective synthetic peptides under the same chromatographic conditions: retention time, as well as CID spectrum was identical to that of the corresponding native peptide. The two larger masses, 988.5 and 992.5, were

Having identified the endogenous AKHs of the bugs, it was of interest to show in some experiments that these peptides (in the form of chemically synthesised material) were, indeed, responsible for the metabolic changes noted earlier when using crude gland extract and/or flight as stimulus. Both endogenous peptides of the spittle bug, Peram-CAH-I and Pyrap-AKH, had significant effects in elevating the circulating concentrations of lipids and carbohydrates (Table 1). In the inflated stink bug, Panbo-RPCH and Schgr-AKH-II had a hyperlipaemic effect (Table 1) and Panbo-RPCH was able to activate glycogen phosphorylase in the fat body from 22% (control injection of distilled water) to 46% after peptide injection (Fig. 10A). The effect of Panbo-RPCH on haemolymph levels of metabolites in N. viridula had been reported earlier (Ga¨de et al., 2003); in the present study, a significant activation of glycogen phosphorylase is measured in the fat body of N. viridula from 25% (water-injected) to 55% (PanboRPCH-injected; Fig. 10B). 4. Discussion It is a known fact that the flight muscle tissue of insects is the most active tissue when engaged in aerobic metabolism. It is further well-known that energy for this high metabolic demand is recruited from stored metabolites (usually in the fat body) via the haemolymph, and that a family of neuropeptide hormones, generically known as the adipokinetic hormone (AKH) family, regulates this mobilisation of energy substrates (Ga¨de, 2009). Insects that display dispersal flight are, hence, good models for studying flight-related metabolism. The current study attempts to start filling in the knowledge gap with respect to flight metabolism in the diverse group of the species-rich insect order, Hemiptera, by looking at three bug species (from two suborders, Cicadomorpha and Heteroptera) that engage in active flight and have relevance to human populations either as agricultural pests or as a source of protein-rich food.

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4.1. Fuels for flight 4.1.1. Cicadomorpha To date, only one other study has addressed the issue of flight and energy substrates in non-heteropteran Hemiptera: an 8-fold higher concentration of carbohydrates than total lipids was measured in haemolymph of the cicada, P. capensis, and it was shown that carbohydrates are apparently exclusively oxidised during the erratic natural flight of this cicada (Ga¨de and Janssens, 1994). The current study reveals a very different situation in the spittle bug, L. arithmetica: in most individuals circulating lipids are 2-fold higher than carbohydrates, although 4–10-fold higher levels were recorded in some insects (see Table 1) and in others there was almost no difference in metabolite concentrations (see Fig. 1B). Interestingly, the absolute concentration of lipids was always around 5 mg ml 1 haemolymph, while the carbohydrate concentration varied substantially from 4.8 mg ml 1 to 0.6 mg ml 1. These data may hint at a strong correlation between feeding and the circulating concentration of carbohydrates in the spittle bug: high concentration just after feeding and much lower values some hours thereafter. Because insects were collected from wild populations, physiological variations were expected in the spittle bugs due to variable age and life histories. Regardless of the starting concentration of carbohydrates in L. arithmetica specimens, flight, injection of a crude CC extract or injection of endogenous synthetic peptides always affected the carbohydrate concentration in the haemolymph. These results caution against only using the actual/absolute carbohydrate level to deduce which metabolite is oxidised as fuel for flight metabolism. It is noteworthy to emphasise here, that the method of lift-generating flight employed in this study, results in maximal contraction of the insect’s flight muscles, hence, even a short period of flight equals a very energy-demanding performance which allows one to make accurate deductions about metabolic fuel utilised by the insect (see also discussion in Auerswald et al., 1998b). A flight of about 2 min resulted in a measurable (albeit small on the basis of the actual amount oxidised) decrease in the concentration of carbohydrates in the haemolymph, whereas no change was noted in the lipid levels (Fig. 1B). During the period of inactivity following flight, carbohydrates are replenished in the haemolymph of the spittle bug presumably by the same mechanism as in stink bugs (see later): glycogen phosphorylase is activated by the endogenous AKH peptides, glycogen in the fat body is converted and mobilised to a readily available form of carbohydrate (trehalose) in the haemolymph and so the pool of available substrate increases to the pre-flight resting level. The fact that the circulating lipid concentration did not decrease during aerobic activity of L. arithmetica may be explained by two possibilities, viz. the relatively short flight time (and the relative abundance of lipids already in the haemolymph), and the mobilisation of lipid from storage proceeding at approximately the same rate as its utilisation by the active flight muscles. After cessation of muscle contraction, however, when lipid mobilisation is still fully activated, lipids continue to be released from fat body stores but are not oxidised by the non-contracting flight muscles, and this results in a 100% increase of lipids in the haemolymph (Fig. 1B). This phenomenon with respect to lipids also occurs during flight of heteropteran Hemiptera (current study—see below; Ga¨de et al., 2004, 2006). From present data with L. arithmetica it appears that flight may initially be fuelled by carbohydrates but that there may be a strong reliance also on a lipid-based flight metabolism. Conspecific biological assays support this notion: the injection of crude CC extract or injection of the endogenous synthetic AKH peptides into resting spittle bugs caused mobilisation of carbohydrates and lipids (Table 1). This interpretation is also in keeping with previous data on flight

metabolism in various locusts and grasshoppers (Goldsworthy, 1983; Ga¨de and Marco, 2009). 4.1.2. Heteropteran stink bugs In both stink bug species, E. delegorguei and N. viridula, haemolymph lipid concentrations were quite high with about 50 mg ml 1 (inflated stink bug) and 25 mg ml 1 (green stink bug) and carbohydrates amounted to much lower concentrations around 10 mg ml 1 in both species. This, combined with the low concentration of glycogen found in thorax and abdomen of N. viridula were strong indications that lipid metabolism is very likely more important in both species during extensive activity than the breakdown of carbohydrates. Unfortunately we did not have sufficient specimens of E. delegorguei to measure metabolites in the thorax and abdomen, but the low concentration of glycogen found in N. viridula is in keeping with data found previously in a number of other heteropteran species (Ward et al., 1982; Canavoso et al., 2003; Ga¨de et al., 2004, 2006) and, thus, we expect to find low concentrations also in the inflated stink bug. On the other hand, visual inspection of adult E. delegorguei has revealed that they have a substantial fat body during September when our experiments were executed (Dzerefos et al., in press). Data of the current study show that glycogen levels of N. viridula are about 50% lower than what was measured in another plant sap feeder, the twig wilter H. alata (Ga¨de et al., 2006). Hence, we did not attempt to monitor changes of stored metabolites (especially glycogen) in N. viridula during flight because it was surmised that this low concentration of glycogen would not make a significant contribution towards energy supply during muscle contraction and that lipids were the primary source. That lipid oxidation in the flight muscles is the main source of energy production during high intensity work such as flight, is supported by the following data in both stink bug species in the current study: high concentrations of lipids in the haemolymph (both species), thorax and abdomen (N. viridula); increase of lipids in the haemolymph after short flight periods (3 min E. delegorguei; 2 min N. viridula) and especially after longer flight (10 min N. viridula) or during the resting period following a bout of flight (both species) when muscle contraction has ceased but lipids are continued to be released; and a hyperlipaemic response when CC extract or low concentration of endogenous AKH are injected (both species; Ga¨de et al., 2003 for N. viridula). Thus, it appears that a lipid-based metabolism is adopted by all feeding types of heteropteran bugs, i.e. obligatory haematophagous triatomine species (Ward et al., 1982; Canavoso et al., 2003), predatory water bugs (Ga¨de et al., 2004, 2007a,b) and plant sap feeders (Ga¨de et al., 2003, 2006; present study). In the current study we wanted to go a step beyond that of previous work done on Heteroptera to initiate investigations into details of the carbohydrate pathway and also its possible interaction with lipid oxidation. For example, it is known from work on migratory locusts that carbohydrates are also oxidised to a low extent continuously during extended flights (Van der Horst et al., 1978). This process would very likely need to be ‘‘fuelled’’ by breakdown of stored glycogen from the fat body, which, in turn, would involve an activated glycogen phosphorylase. Such an activation of glycogen phosphorylase was shown to occur in the fat body of L. migratoria (Ga¨de, 1981; Vroemen et al., 1995) even though an overt hypertrehalosaemic effect could not be measured. In the current study, the same situation seems to prevail in both stink bugs, E. delegorguei and N. viridula: injection of CC extract or the appropriate endogenous AKH in its synthetic form does not elevate the total carbohydrate concentration in the haemolymph but the active form of glycogen phosphorylase in the fat body is increased (both species) and the enzyme is also activated by the stimulus of flight (N. viridula). These data suggest that AKH is released during flight, activates the enzyme and mobilises

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carbohydrates which are used continuously during flight at a low rate, and, as clearly seen in E. delegorguei (Fig. 1A), carbohydrate levels are replenished in the haemolymph during rest after flight— presumably from the (small) stores of glycogen in the fat body. Another hypothesis we wanted to address in N. viridula was whether high concentrations of trehalose in the haemolymph inhibit the release of AKH. Houben (1976) had made two important observations in the migratory locust: first, the total amount of injected trehalose could not be accounted for in the haemolymph— the actual concentration was always lower than the theoretical calculated value. Secondly, the release of AKH from the corpus cardiacum of L. migratoria can be prevented when locusts are injected with 5 mg trehalose just before a not too-intensive flight, and intermittently during flight. In addition, in vitro experiments with locusts revealed that 80 mM trehalose in the incubation medium (this concentration is also measured in resting locusts), inhibited the release of AKH, whereas 40 mM trehalose (which is typically measured in locusts after a short period of flight), did not (Passier et al., 1997; Flanigan and Ga¨de, 1999). Both phenomena have been detected in the current study with the green stink bug: (a) the time curve of a single injection of trehalose (Fig. 4) showed a clear decrease of the carbohydrate concentration in the haemolymph of resting stink bugs, suggesting that the trehalose is taken up from the haemolymph, possibly by the action of insulin-like peptides (ILPs) and stored in fat cells as glycogen. ILPs have been shown to occur in numerous insects and have been structurally identified in some species (Wu and Brown, 2006). (b) The trehalose-injected N. viridula show a decreased level of carbohydrate after flight (this is a bigger decrease than what was observed in resting, hypertrehalosaemic bugs; see Fig. 4), while the lipid concentration remains in a steady state (Fig. 5); this suggests that the injected trehalose (and not lipid) was used as fuel for muscle contractions and, that possibly ILPs were released upon the high carbohydrate concentration (see Wu and Brown, 2006) and, thereby, the release of AKH was prevented. In conclusion then, it appears that similar mechanisms of AKH-release or inhibition are operative in Orthoptera and Heteroptera. 4.2. Characterisation of the peptides of the AKH/RPCH family There are at least five major peaks that have characteristic Trp fluorescence discernible when a CC extract of the spittle bug is purified on HPLC; however, only material from two of these peaks had biological activity in in vivo hypertrehalosaemic assays. Identification by LC/MS methodology, co-elution with appropriate synthetic peptides and biological activity characterised the material unequivocally as peptides of the AKH/RPCH family previously elucidated and code-named Peram-CAH-I and Pyrap-AKH. These octapeptides differ at two positions, 2 and 5; both substitutions, Val2 to Leu2 and Ser5 to Thr5, are conservative exchanges achieved by a single mutation in one base of the codon triplet. Both octapeptides are very different in primary structure to the decapeptides of the only other known non-heteropteran AKH of Hemiptera, peptides denoted Placa-HrTH from various species of cicada (Ga¨de, 2009). Hence, it does not appear that AKH peptides in the Cicadomorpha are all identical or unique for this taxon. Peram-CAH-I is the characteristic AKH of blattid cockroaches and has also been found in a number of beetle species from the families Chrysomelidae and Cerambycidae (Ga¨de, 2009). Pyrap-AKH, on the other hand, is known from a few heteropteran species, a grasshopper and the red flour beetle Tribolium castaneum (Ga¨de, 2009). In the present study, we also characterised two members of the AKH/RPCH family in the edible stink bug. This is quite remarkable because, to date, only two species of the hemipteran family Pyrrocoridae, Pyrrhocoris apterus and Dysdercus intermedius, are known to have two AKHs: Pyrap-AKH and Peram-CAH-II which differ from each other by a single amino acid at position three (Asn

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versus Thr); all other hemipteran species investigated, and that is by far the majority of species studied, contain only one AKH (Ga¨de, 2009). In E. delegorguei we find the two peptides Schgr-AKH-II and Panbo-RPCH; again these octapeptides differ from each other by one amino acid at position 6 (Thr versus Pro) and it is thought that during, or after a gene duplication event, a single point mutation has occurred. Schgr-AKH-II is a peptide that is, likely, ancestral in ensiferan and caeliferan Orthoptera but also occurs in many Hymenoptera (Ga¨de, 2009). In heteropteran Hemiptera it is present in members of the family Coreidae. Panbo-RPCH, on the other hand, is the ‘‘mainstay’’ member of the peptide family in decapod crustaceans and, in insects, it has only been found, to date, in species of the heteropteran family Pentatomidae, i.e. N. viridula and Coenomorpha spp. (Ga¨de, 2009). The subset of AKH peptides found here for the edible stink bug, Schgr-AKH-II and Panbo-RPCH, links the family of Tessaratomidae closely to the Pentatomidae and Coreidae. Further ideas of possible evolutionary trends in the AKH peptides in the Heteroptera will be presented in a forthcoming contribution (D. Kodrik, H.G. Marco, P. Sˇimek, R. Socha, P. Stys and G. Ga¨de, in preparation). It is worth noting that Gly-extended nonamidated forms of Panbo-RPCH and Schgr-AKH-II were also identified in minor quantities in the inflated stink bug in the present study. Such C-terminal extended AKHs have been sequenced before in various insects (Ko¨llisch et al., 2000, 2003; Baggerman et al., 2002; Audsley and Weaver, 2003; Predel et al., 2004; Ga¨de et al., 2008). For most of these peptides it is not clear whether they are biologically active and none of them have been shown to be released in vitro or in vivo hence, nothing is known about their titer and half-life in the haemolymph relative to those of the mature amidated peptides. Future experiments addressing these issues should help to distinguish which of these forms may be incompletely or alternatively processed precursors; only the latter would result in a functional true hormone. From a functional point of view it is important to reiterate here that the peptides identified for the different species in this study all result in hyperlipaemia as shown by the increase of lipids in the haemolymph after injection of a low dose of synthetic peptide. This, then, affirms earlier reports on a number of water bugs (Ga¨de et al., 2004, 2007a,b) and on the twig wilter (Ga¨de et al., 2006) where we had also shown that these insects use a lipid-based metabolism for high activity purposes, and that the control is achieved via AKHs. Activation of phosphorylase is necessary to support metabolism via a continuous low turnover of carbohydrates, as shown to occur in locusts during flight activity (Van der Horst et al., 1978) and, although an overt effect on an increase of carbohydrates in the haemolymph after injection of AKH is not measurable, the higher activity of glycogen phosphorylase in the fat body is a clear result that we have established in this study in response to AKH. Acknowledgements Thanks are due to the National Research Foundation (Pretoria, Republic of South Africa; grants: FA 20071300002 and IFR2008071500048 to GG) and the University of Cape Town (University Research Council blockgrants to GG and HGM) for partial financial support, to Dr P. Sˇimek (Academy of Sciences of the ˇ eske´ Bude˘jovice, Czech Republic) for mass Czech Republic, C spectrometric measurements and to Dr L. Auerswald (Marine and Coastal Management, Cape Town, RSA) for help with some conspecific bioassays. References Ajayi, O., Oboite, F.A., 2000. Importance of spittle bugs, Locris rubens (Erichson) and Poophilus costalis (Walker) on sorghum in West and Central Africa, with emphasis on Nigeria. Annals of Applied Biology 136, 9–14.

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