Beetles' choice--proline for energy output: control by AKHs

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.     

Metabolic changes in the African fruit beetle,Pachnoda sinuata,during starvation

Specimens of the fruit beetle Pachnoda sinuata were starved for up to 30 days. The weight of the beetles declined consistently throughout the starvation period. Concentrations of carbohydrates and alanine in flight muscles, fat body and haemolymph decreased rapidly after onset of starvation, while the concentration of proline remained high. Whereas the lipid concentrations in the haemolymph did not change significantly upon starvation, the lipid content in flight muscles and fat body decreased significantly.Beetles that had been starved for 14 days responded to injection of Mem-CC, the endogenous neuropeptide from its corpora cardiaca, with hyperprolinaemia and a decrease in the alanine level, but no such effect was monitored after prolonged starvation of 28 days. Regardless of the period of starvation, Mem-CC injection could not cause hypertrehalosaemia or hyperlipaemia, although carbohydrates were increased in fed beetles after injection.Flight ability of beetles that had been starved for 15 or 30 days was apparently not impaired. During such periods, beetles used proline exclusively as fuel for flight as evidenced by the increase in the level of alanine in the haemolymph and decrease of the level of proline; the concentrations of carbohydrates and lipids remained unchanged.Activities of malic enzyme and alanine aminotransferase (enzymes involved in transamination in proline metabolism), glyceraldehyde-3-phosphate dehydrogenase (enzyme of glycolysis), 3-hydroxyacyl-CoA dehydrogenase (enzyme of beta-oxidation of fatty acids) and of malate dehydrogenase (enzyme of Krebs cycle) were measured in fat body and flight muscles. In flight muscle tissue the maximum activity of NAD(+)-dependent malic enzyme increased, while that of glyceraldehyde-3-phosphate dehydrogenase decreased during starvation, and malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and alanine aminotransferase were unchanged. In fat body tissue, activities of NADP(+)-dependent malic enzyme and 3-hydroxyacyl-CoA dehydrogenase increased during food deprivation and activities of glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and alanine aminotransferase remained unchanged.     

Flight metabolism in carpenter bees and primary structure of their hypertrehalosaemic peptide

We measured the rate of oxygen consumption and carbon dioxide production as well as energy substrates in haemolymph and flight muscles of carpenter bees of the genus Xylocopa at rest and after tethered lift-generating flight. Flight of 2 min duration at an ambient temperature of 28○C elevated oxygen consumption about 70-fold above resting rate. The respiratory quotient during rest and flight was 1 indicating that carbohydrates were the exclusive substrate oxidised. This was corroborated by measurements of metabolism. Carbohydrates are in high concentrations in the haemolymph. This store was significantly diminished during a 10-min flight period. Whereas lipids did not contribute to energy provisions, the proline concentration in the haemolymph and in the flight muscles was significantly decreased upon flight, but the amount can only account for a very small contribution to overall flight metabolism. Polysaccharide reserves in flight muscles and whole abdomina are almost non-existent. However, earlier studies had identified the crop as a source of oligosaccharides (Louw and Nicolson 1983). Carbohydrate metabolism is influenced by a metabolic peptide from the corpus cardiacum. We could isolate a peptide from the corpora cardiaca of carpenter bees, which by retention time in HPLC and by its mass is very likely characterised as the octapeptide Scg-AKH-II (pGlu-Leu-Asn-Phe- Ser-Thr-Gly-Trp-NH₂) previously shown to occur in certain Orthoptera. This is the first member of the large AKH/RPCH family of peptides to be identified from a hymenopteran species. Injection of the synthetic peptide into adult carpenter bees caused carbohydrate mobilisation. We suggest that the peptide targets the high sugar stores in the crop and speculate that it may facilitate sugar passage rate through the digestive system.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Isolation and identification of AKH/RPCH family peptides in blister beetles (Meloidae)

Abstract. Two peptides were isolated from methanolic extracts of corpora cardiaca of the blister beetle, Decupotoma lunata , by a single-step purification procedure, utilizing C-18 reversed-phase high-performance liquid chromatography (RP-HPLC) for separation, and the increase of haemolymph lipids in Locusta migratoria for bioassay. The native peptides were analysed by matrix-assisted laser-desorption ionization mass spectrometry revealing main ions at m/z 1180 and 1009 respectively which were attributed to the [M + Na]⁺ form of the respective peptides. After deblocking of the N-terminal pyroglutamate residue of each peptide, the structures of the deblocked peptides were determined by pulsed-liquid phase sequencing employing Edman chemistry. The sequences of the two peptides, (1) pGlu-Leu-Asn-Phe-Ser-Pro-Am-Trp-Gly-AsnNH₂ and (2) pGlu-Leu-Asn-Phe-Ser-Pro-Asn-TrpNH₂, characterize them as deca- and octapeptide members of the AKH/RPCH family. Whereas the decapeptide is a novel member of this family and is given the acronym Del-CC ( Decupotoma lunata corpus cardiacum peptide), the octapeptide has previously been found in tenebrionid beetles and has the acronym Tem-HrTH. The corpora cardiaca of two other species of blister beetles ( Cyaneolytta pectoralis and Mylabris coeca ) contain the same two peptides as D. lunata , as judged by RP-HPLC and biological activity. Neither a corpus cardiacum extract of Decupotoma lunata nor the synthetic peptides Del-CC and Tem-HrTH were active in mobilizing carbohydrates or lipids in the blister beetle.     

Physiological and biochemical aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle,Pachnoda sinuata

We studied several aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle Pachnoda to investigate their flight capability. The majority of adults which were forcefully removed from their pupal cocoon flew off within 5 min of exposure to bright sunlight. Most of the beetles which did not fly voluntarily were, however, capable of flight. Compared with 2-4 week old adults of the same species, cocoon-enclosed adults have higher reserves of glycogen in flight muscles and fat body, whereas the level of total carbohydrates in the haemolymph and the concentration of proline in haemolymph, flight muscles and fat body were similar.Enzymes involved in carbohydrate breakdown (MDH, GAPDH) were more active in flight muscles and fat body of cocoon-enclosed adults compared with adults, while enzymes of proline metabolism in the flight muscles (AlaT, NAD-ME) and fat body (AlaT, NADP-ME) had similar activities in cocoon-enclosed adults and adults. An enzyme of the beta-oxidation of fatty acids (HOAD) had similar activities in flight muscles and fat body of cocoon-enclosed adults and adults.Mitochondria isolated from flight muscles of adults removed prematurely from their cocoon favour the oxidation of proline and pyruvate. Pyruvate, however, is oxidized at higher rates than by mitochondria isolated from flight muscles of adults.During a short lift-generating flight, cocoon-enclosed adults proved that their flight muscles are capable of strong flight performance. During these flights, cocoon-enclosed adults consume proline and carbohydrates at a similar rate to that of adults.The endogenous AKH peptide, Mem-CC, has hyperprolinaemic and hypertrehalosaemic activity in cocoon-enclosed adults. The hypertrehalosaemic effect, however, is stronger in cocoon-enclosed adults than in adults.The content of Mem-CC in corpora cardiaca of larvae (3rd instar), cocoon-enclosed adults and 1 day-old adults is similar at 5-6 pmol per pair of corpora cardiaca, whereas it is higher in 10 day-old adults and 20 day-old adults (37 and 15 pmol per pair corpora cardiaca, respectively).From these results we conclude that cocoon-enclosed adults comply with all the prerequisites for flight performance before they leave their pupal cocoon. Furthermore, cocoon-enclosed adults have a more pronounced carbohydrate-based metabolism before they leave their cocoon compared with adults, which suggests that carbohydrate breakdown is mainly involved in such activities as leaving the cocoon and burrowing activity thereafter.     

Energy use by the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) for dispersal by flight

The mountain pine beetle Dendroctonus ponderosae Hopkins is a major native pest of Pinus Linnaeus (Pinaceae) in western North America. Host colonization by the mountain pine beetle is associated with an obligatory dispersal phase, during which beetles fly in search of a suitable host. Mountain pine beetles use stored energy from feeding in the natal habitat to power flight before host colonization and brood production. Lipids fuel mountain pine beetle flight, although it is not known whether other energy sources are also used during flight. In the present study, we compare the level of energy substrates, proteins, carbohydrates and lipids of individual mountain pine beetles flown on flight mills with unflown control beetles. We use a colorimetric method to measure the entire metabolite content of each individual beetle. The present study reveals that mountain pine beetles are composed of more protein and lipid than carbohydrate. Both female and male mountain pine beetles use lipids and carbohydrates as energy sources during flight. There is variation between sexes, however, in the energy substrates used for flight. Male mountain pine beetles use protein, in addition to lipids and carbohydrates, to fuel flight, whereas protein content is not different between flown and control females.     

Cyclic AMP mediates the elevation of proline by AKH peptides in the cetoniid beetle, Pachnoda sinuata

The role of cyclic nucleotides in the transduction of the hyperprolinaemic and hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata. Flight and injection of Mem-CC into the haemocoel of the beetle induce an increase of cAMP levels in the fat body of the beetle. This increase is tissue-specific and does not occur in brain and flight muscles. An elevation of cAMP levels was also found when in vitro preparations of fat body tissue were subjected to Mem-CC. Elevation of the cAMP concentration after injection of Mem-CC is time- and dose-dependent: the maximum response is measured after 1 min, and a dose of 25 pmol Mem-CC is needed. Injection of cpt-cAMP, a cAMP analogue which penetrates the cell membrane, causes a stimulation of proline synthesis but no mobilisation of carbohydrate reserves. The same is measured when IBMX, an inhibitor of phosphodiesterase, is injected. cGMP seems not to be involved in synthesis of proline nor carbohydrate release, because injection of cpt-cGMP has no influence on the levels of proline, alanine and carbohydrates in the haemolymph. Although glycogen phosphorylase of the fat body is activated by Mem-CC in a time- and dose-dependent manner, it cannot be stimulated by cpt-cAMP. The combined data suggest that cAMP is involved in regulation of proline levels by Mem-CC but not in regulation of carbohydrates. Octopamine has no effect on metabolites in the haemolymph and is not capable of activating glycogen phosphorylase, indicating that it is not involved in the regulation of substrates in this beetle. Furthermore, the requirements of the receptor of Mem-CC are different for eliciting a hypertrehalosaemic and a hyperprolinaemic effect, respectively, suggesting that differentiation in signal transduction begins at the receptor level.     

Primary structures of neuropeptides isolated from the corpora cardiaca of various cetonid beetle species determined by pulsed-liquid phase sequencing and tandem fast atom bombardment mass spectrometry.

A peptide with the same retention time on gradient reversed-phase high-performance liquid chromatography was present in the corpora cardiaca of 5 scarabaeid beetles, subfamily Cetoniinae: the three fruit beetle species Pachnoda marginata, P. sinuata and P. aemulae and the two protea beetle species Trichostetha fascularis and T. albopicta. Crude corpora cardiaca material from P. sinuata had a small hypertrehalosaemic effect in American cockroaches and a very weak hyperlipaemic activity in migratory locusts. Injections into P. sinuata caused hypertrehalosaemia when a dose of 1.0 corpora cardiaca equivalents was injected. An identical neuropeptide was isolated, by RP-HPLC, and sequenced by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal 5-oxopyrrolidine-2-carboxylic acid residue, as well as by collision-induced decomposition tandem fast atom bombardment mass spectrometry. The peptide is a blocked octapeptide: Glu-Leu-Asn-Tyr-Ser-Pro-Asp-TrpNH2, previously designated Mem-CC. The synthetic peptide is able to elicit haemolymph carbohydrates in P. sinuata upon injection of low doses. Activity studies using synthetic analogues of this peptide revealed that Tyr4 may be important for receptor recognition/binding. The peptide is synthesized in intrinsic cells of the corpus cardiacum as shown by in vitro incorporation of [3H]Trp and [14C]Tyr in Mem-CC.     

Activation of triacylglycerol lipase in the fat body of a beetle by adipokinetic hormone

The activation of triacylglycerol lipase and the stimulation of proline synthesis in the fat body of the fruit beetle Pachnoda sinuata by the endogenous octapeptide hormone Melme-CC (pQLNYSPDWa), which belongs to the family of insect adipokinetic hormones, were studied, and the correlation of both events investigated. At rest, the activity of triacylglycerol lipase in the fat body of the beetle was higher than in the fat body of the American cockroach, Periplaneta americana, but lower than in the migratory locust, Locusta migratoria. Triacylglycerol lipase of the beetle is activated by: (a) injection of synthetic Melme-CC and (b) the stimulus of flight. Activation of lipase by Melme-CC is time-dependent. Injection of cpt-cAMP activates triacylglycerol lipase in the fat body and causes an increase in the concentration of proline in the haemolymph at the expense of alanine. In contrast, injection of F-inositol-1,4,5-phosphate does not affect the activation state of lipase, nor the levels of amino acids in the haemolymph. High doses of octopamine do not activate lipase. Furthermore, activity of fat body lipase and proline concentration in the haemolymph both follow a circadian rhythm: both parameters are high in the morning, whereas they are low in the evening. When transfer of Melme-CC, released from the corpora cardiaca, to the thorax/abdomen is prevented by neck-ligation, the activity of lipase, as well as the circulating proline levels are low. Regression analysis revealed that activity of triacylglycerol lipase is positively correlated to proline concentration in the haemolymph, whereas there is a negative correlation of the enzyme activity and alanine level in the haemolymph. From these results we conclude that the activation of fat body triacylglycerol lipase by Melme-CC in P. sinuata stimulates proline synthesis. Proline is one of the major substrates to power flight activity in the beetle.     

Flight fuel and neuropeptidergic control of fuel mobilisation in the twig wilter, Holopterna alata (Hemiptera, Coreidae)

The corpus cardiacum of the twig wilter Holopterna alata contains a factor that elicits increases in the concentration of lipids in the haemolymph of twig wilters and migratory locusts and causes hypertrehalosaemia in American cockroaches. A hyperlipaemic neuropeptide was isolated from corpora cardiaca of H. alata in a single high-performance liquid chromatography step. The primary sequence of this peptide was assigned by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, biological assay and co-elution with the synthetic peptide. The adipokinetic peptide of H. alata is an octapeptide with the sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp amide denoted Schgr-AKH-II which was sequenced previously from the corpora cardiaca of a number of Caelifera, Ensifera and some Hymenoptera. A dose of 1pmol of synthetic Schgr-AKH-II causes a pronounced hyperlipaemic effect in the twig wilter. Physiological experiments with the twig wilter reveal that during flight periods of 3 min, the normally low carbohydrate concentration in the haemolymph is significantly diminished, whereas the lipid concentration stays constant in most cases. During a subsequent rest period of 60 min after a 3 min flight episode, however, the concentration of lipids in the haemolymph increases substantially and significantly, indicating that lipids, too, are a major fuel during flight of twig wilters. This is corroborated by the activation of the enzyme triacylglycerol (TAG) lipase in the fat body, but not in the flight muscles, by injection of 5 pmol of synthetic Schgr-AKH-II, the endogenous adipokinetic hormone that is thought to be released during flight. Moreover, in the thorax there is a significant decrease in the concentration of glycogen and lipids measured after flight plus 60 min of rest compared to non-flown twig wilters, whereas no significant changes were monitored for these substrates stored in the abdomen. When the change in lipid class composition was analysed during flight plus 60 min of rest, TAG which comprised the major class in all compartments analysed (thorax, abdomen, haemolymph) was significantly reduced in abdomen and thorax, and diacylglycerol was significantly increased in all three compartments. From all the data collected, it is concluded that lipids are the major fuel class for flight in H. alata and that the contribution of carbohydrates is minimal.     

Fuels for energy metabolism in the Colorado potato beetle, Leptinotarsa decemlineata Say

During flight, the proline concentration in the flight muscles of Leptinotarsa decemlineata decreases sharply while that of alanine shows a proportional increase. In view of the known dynamics of proline turn over the observed decrease would indicate that proline plays the major role as a mobilizable energy source for flight. To a minor extent carbohydrates such as glycogen and glucose support energy metabolism during flight in the Colorado potato beetle.During a recovery period after flight the proline concentration in the fat body increases sharply within minutes, a feature which would indicate that fat body could synthesize proline maybe at the expense of alanine. Comparison of starvation and flight metabolism reveals that the metabolic changes in the two situations are different.     

Flight metabolism in the African fruit beetle,Pachnoda sinuata

Metabolite concentrations in flight muscles and in abdomen of beetles (Pachnoda sinuata) were measured after various periods of tethered flight and subsequent rest. Three distinct phases of energy metabolism are found in active flight muscles: (1) during the first minutes of flight proline is used as main substrate and concomitantly alanine accumulated as an end product; (2) the second phase is characterized by a large-scale degradation of glycogen; (3) after about 8 min of flight the metabolite levels stabilize, while flight performance appears unchanged. After the termination of flight the preflight proline concentration (70 mol·g^-1 fw) is re-established in less than 60 min, whereas restoration of resting levels of other metabolites requires longer. The pattern of maximal enzyme activities and the respiratory rates of mitochondria with different substrates confirm the significance of proline and carbohydrates as the main fuels of working flight muscles.Abbreviations CS citrate synthetase - Cytox cytochrome c oxidase - EDTA ethylenediaminetetra-acetate - fw fresh weight - GluDH glutamate dehydrogenase - GPT alanine aminotransferase - HOAD hydroxyacyl-coenzyme A dehydrogenase - HPLC high pressure liquid chromatography - ME malic enzyme - PCA perchloric acid - RQ repiratory quotient - TRA triethanolamine     

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.     

Importance of proline and other amino acids during honeybee flight

Summary. The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. ¹⁴C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing ¹⁴C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 μg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates. Received December 5, 1998, Accepted February 1, 1999     

Effects of metabolic neuropeptides from insect corpora cardiaca on proline metabolism of the African fruit beetle,Pachnoda sinuata

The effect of neuropeptides from the corpora cardiaca of the fruit beetle Pachnoda sinuata on proline metabolism has been investigated in vivo. Conspecific injections of a crude extract from corpora cardiaca cause an increase of the concentration of proline in the haemolymph by nearly 20% and a decrease of the concentration of alanine, the precursor in proline synthesis, by about 64% when compared with a water-injected group. Purification of an extract of corpora cardiaca on reversed-phase liquid chromatography revealed two distinct UV absorbance and fluorescence peaks that cause hyperprolinaemia in the fruit beetle. The major peak is the previously identified octapeptide Mem-CC; the second peak is also a peptide, but its primary sequence remains, as yet, unidentified. Synthetic Mem-CC elicited time- and dose-dependent increases/decreases of the concentrations of proline and alanine in the haemolymph respectively. Furthermore, the receptor for this peptide seems to be specific in P. sinuata: only peptides of the large family of adipokinetic hormones with an Asp, Asn or Gly residue at position 7 could elicit biological activity, whereas those with a Trp, Ser or Val residue at this position did not have any activity.     

Energy substrates for flight in the Colorado beetle,Leptinotarsa decemlineata Say

Colorado beetles could be induced to fly when suspended on a roundabout. The group which showed the greatest tendency to fly when suspended and flew for the longest period were females 10–12 days after diapause break. Flights lasted for 20–30 min and were occasionally longer. No change in haemolymph carbohydrate levels occurred during flight but significant increases in haemolymph total lipid levels and significant decreases in haemolymph proline levels were observed. ¹⁴C-proline injected into the haemolymph was utilised very rapidly in flown insects but disappeared only slowly in non-flown insects.     

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)

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.     

Colonisation ability of the threatened tenebrionid beetle Oplocephala haemorrhoidalis and its common relative Bolitophagus reticulatus

Abstract. 1. Life-history traits associated with colonisation ability were compared in the threatened tenebrionid beetle Oplocephala haemorrhoidalis and its common relative Bolitophagus reticulatus . Both species feed and breed exclusively in fruiting bodies of the wood-decaying fungus Fomes fomentarius .
2. The presence and status of flight wings, flight muscles, and mature eggs were determined by dissection. Flight willingness was studied in a field experiment, and flight duration in a flight-mill experiment.
3. Females of O. haemorrhoidalis had fewer but larger eggs in their abdomen than B. reticulatus females.
4. All beetles of both species had fully developed flight wings but a larger proportion of B. reticulatus than O. haemorrhoidalis had developed flight muscles.
5. Bolitophagus reticulatus was more willing to take off than O. haemorrhoidalis , however both species, especially O. haemorrhoidalis , were powerful fliers, with many individuals being able to fly several kilometres. Oplocephala haemorrhoidalis tended to make few flights of long duration whereas B. reticulatus made several, but mostly shorter, flights.
6. The results indicate that B. reticulatus has a suite of life-history traits that makes it better adapted than O. haemorrhoidalis to exploit the scattered trees with fruiting bodies present in managed forests. This may explain why O. haemorrhoidalis is restricted primarily to sites with a high density of suitable substrates that have been available continuously for a long time.     

Specificity and localisation of lipoprotein lipase in the flight muscles of Locusta migratoria

Using natural lipoproteins as substrates, lipase activity has been measured in leg muscle, fat body, midgut and flight muscles of Locusta migratoria. The enzymic activity in the flight muscles is higher than in those other tissues tested, confirming the potential of the flight muscles to utilise lipids at high rates. In addition, a membrane-bound lipoprotein lipase can be extracted from flight muscle. The flight muscle enzyme activity shows a marked substrate specificity; at lipoprotein concentrations equivalent to those found normally in flown or resting locusts respectively, the enzyme hydrolyses diacylglycerols associated with lipoprotein A+ (present in the haemolymph of flown or adipokinetic hormone-injected locusts) at about 4 times the rate of those associated with lipoprotein Ayellow (which is the major lipoprotein in resting locusts). In addition, the hydrolysis of lipids carried by lipoprotein Ayellow is dramatically reduced in the presence of lipoprotein A+. These observations indicate that the enzyme plays a specific role in the uptake of lipids at the flight muscles to ensure a smooth transition from carbohydrate to lipid based metabolism during flight.     

The primary structure of the hypertrehalosemic neuropeptide from tenebrionid beetles: a novel member of the AKH/RPCH family

A hypertrehalosemic neuropeptide from the corpora cardiac of the two tenebrionid beetle species, Tenebrio molitor and Zophobas rugipes, was purified by high performance liquid chromatography, and its sequence determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. Additionally, the C-terminus of the peptide was blocked as shown by the lack of breakdown using carboxypeptidase. In both species an identical octapeptide, designated Tem-HrTH, with the following amino acid sequence, was found: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2. This primary sequence has an 88% homology with the hypertrehalosemic hormone I (Pea-CAH-I) from the American cockroach as well as with the red pigment-concentrating hormone (RPCH) of prawns. Injection of the synthetic peptide into larvae or young adults of T. molitor or adult Z. rugipes increases the hemolymph carbohydrate levels in a dose-dependent manner. Thin layer chromatography identified the elevated sugar component of the hemolymph as the disaccharide trehalose. Carbohydrate release from larval fat body in vitro was also shown upon administration of a low concentration of synthetic Tem-HrTH.