Substrate usage and its regulation during flight and swimming in the backswimmer, Notonecta glauca

Abstract.  The metabolites that are generally used by insects during exercise are present in quite different concentrations in the haemolymph of the backswimmer Notonecta glauca L. Lipids are most abundant (between 10 and 20 mg/mL), whereas carbohydrates (2–3 mg/mL) and proline (approximately 1 mg/mL) are at very low concentrations. Injection of an extract of conspecific corpora cardiaca causes pronounced hyperlipaemia in the backswimmer. A neuropeptide with the same effect was isolated from the corpora cardiaca in a single high-performance liquid chromatography (HPLC) step; the primary sequence was deduced from mass spectrometric measurements (matrix-assisted laser desorption/ionization-time of flight and electrospray quadrupol time-of-flight mass spectrometry) of whole corpora cardiaca, and the mass was confirmed in the HPLC fraction that had adipokinetic activity. The biologically active octapeptide has the sequence pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, which was characterized previously from the corpora cardiaca of the Emperor dragonfly, Anax imperator , and denoted Anaim-adipokinetic hormone (AKH). The synthetic Anaim-AKH peptide causes lipid mobilization when injected at a dose of 1 pmol into N. glauca . When other synthetic AKH members that occur in Hemiptera are injected into N. glauca at the same dose, the hyperlipaemic responses are significantly lower than after injection of Anaim-AKH. Because only lipids increase upon activity, such as continuous swimming for 1 h or during a 1-h rest period after a 3-min flight episode in the laboratory, it is assumed that Anaim-AKH serves as a true adipokinetic hormone in the backswimmer during bouts of natural swimming and flight.     

Adipokinetic Hormone and Flight Fuel Related Characteristics of Density-Dependent Locust Phase Polymorphism: A Review

Recent findings on differences between the gregarious and solitary phases of locusts are reviewed in relation to flight fuel utilization, adipokinetic responses, and adipokinetic hormones. Laboratory results obtained with Locusta migratoria migratorioides show that the amount of lipid reserves, resting levels of haemolymph lipids, and hyperlipaemic responses to flight and to injection of corpus cardiacum extract or of synthetic adipokinetic hormones, are higher in crowded than in isolated locusts. No major phase-dependent differences seem to exist in flight-related carbohydrate metabolism. The adipokinetic hormone content of the corpora cardiaca is higher in younger isolated locusts than in crowded ones. Adipokinetic hormone precursor-related peptide content of the corpora cardiaca is also higher in isolated than in crowded locusts. Crowded locusts have higher lipid reserves and higher hyperlipaemic responses to flight than isolated locusts also in Schistocerca gregaria and, following injection of synthetic adipokinetic hormone, the formation of low density lipophorin is higher in crowded than in isolated locusts of this species. The laboratory results obtained with isolated and crowded locusts are extrapolated to understand the ecophysiology of the migrations of solitary and gregarious field populations of L.m. migratorioides according to available information on the differences in the migration of the two phases. It is inferred that in this species solitary locusts have a rather coarse adipokinetic strategy focused on a single prereproductive long-distance migratory flight, whereas gregarious locusts possess a fine adipokinetic balance for reiterative, sometimes unpredictably long-distance, migrations in the prereproductive, as well as reproductive, periods. The differences between the adipokinetic strategies of solitary and gregarious S. gregaria seem to be less dramatic, nevertheless, they indicate a better adaptation of the gregarious phase to prolonged flights.     

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.     

Adipokinetic hormone of the true armyworm, Pseudaletia unipuncta: immunohistochemistry, amino acid analysis, quantification and bioassay

Abstract A cluster of five to seven AKH-like immunoreactive cells lie in each lobe of the paired corpora cardiaca of the true armyworm, Pseudaletia unipuncta. These cells form a mesh work of immunoreactive processes within the corpora cardiaca, and immunoreactive tracts projecting posteriorly over the aorta.
Reversed-phase high performance liquid chromatography of extracts of the corpora cardiaca of P.unipuncta revealed a single large U.V. absorbent peak with a retention time identical to synthetic Manduca- AKH. Amino acid analysis of the contents of this peak yielded a composition identical to that of synthetic Manduca-AKH which was analysed in a parallel manner. Furthermore the material within the peak possessed adipokinetic activity when bioassayed in day 2 adult male P. unipuncta. The corpora cardiaca of similar individuals were found to contain approximately 17.6ng (17.6pmol) of Manduca-AKH equivalents per pair.
Injection of Manduca-AKH into 2-day-old adult male P.unipuncta resulted in a dose-dependent elevation in haemolymph lipid levels with a maximum level of 80–90μmg/μl obtained with 5–10 ng of Manduca-AKH. Continuous flight also elevated haemolymph lipid levels in day 4 adult males with a significant elevation evident in the first samples taken after 15 min of flight and lipid levels plateauing at approximately 100 μg/μl by about 60 min of flight.     

Mobilization of lipid and carbohydrate reserves in the migratory grasshopper Melanoplus sanguinipes

Abstract. The North American migratory grasshopper Melanoplus sanguinipes Fabricius (Orthoptera: Acrididae) exhibits heritable variation in predisposition to make long-duration flights, and performance of long-duration flight enhances reproductive output. As a first step in understanding the physiological basis of these phenomena, we examined the mobilization of lipid and carbohydrate reserves during flight and in response to injection of extracts of the corpora cardiaca. Extract of conspecific corpora cardiaca elevates the concentration of haemolymph lipid. Both synthetic locust adipokinetic hormone I (AKH I) and synthetic Locusta migratoria AKH II raise the concentration of lipid in the haemolymph. However, although AKH I is more active than AKH II in locusts, dose-response curves for the two peptides are similar in M.sanguinipes. Neither extract of conspecific corpora cardiaca nor locust AKH I affects haemolymph carbohydrate in this species. Haemolymph carbohydrate and total glycogen reserves are Diminished by tethered flight; in contrast, haemolymph lipid is elevated by flight. Grasshoppers identified as presumptive migrants or non-migrants do not differ significantly in body composition. Total lipid reserves did not decrease measurably after extended flight, even though total reserves of carbohydrate do not appear to be sufficient to maintain the durations of flight performed.     

Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria

Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci II (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R_F value similar to Carlsen's peptide (adipokinetic hormone II).These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta, and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in ligated locusts.It is concluded that NCC II controls the release of adipokinetic hormones during flight and that two adipokinetic hormones are released during flight. One of these hormones adipokinetic hormone II also acts as a hyperglycaemic hormone illustrating that a hyperglycaemic hormone is released, during flight.     

Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui.

A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family. The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an approximately 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0. 1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mM KCl, significant amounts of Vac-AKH were released from the glands.     

Structure and function of adipokinetic hormones of the large white butterfly Pieris brassicae

The large white butterfly Pieris brassicae L. (also called cabbage white) is very common in Europe, Asia and the northern region of Africa, and has also been found in South Africa during approximately the last 20 years. The species is considered a pest insect, with larvae attacking brassicaceous crops. The adult is a strong migratory flyer and new territory can be infested this way. As a first step to investigate methods for combating this pest species, the present study aims to determine the complement of adipokinetic peptides, here generically referred to as adipokinetic hormones (AKHs), which are required to regulate the mobilization of fuels for insect flight. Biological assays, as well as mass spectrometry, reveal information about the presence, structure and function of AKHs in P. brassicae: a methanolic extract of the corpora cardiaca has hypertrehalosaemic activity in cockroaches, does not cause hyperlipaemia in locusts, and has adipokinetic activity in P. brassicae itself. Liquid‐chromatography electrospray ion trap mass spectrometry reveals three peptides that can be associated with the AKH family: the non‐amidated undecapeptide Vanca‐AKH (pELTFTSSWGGK‐OH), the nonapeptide Manse‐AKH (pELTFTSSWG amide) and the novel octapeptide Piebr‐AKH (pELTFSSGW amide). Sequence confirmation of all three assigned structures is obtained from matching mass spectrometry spectra from synthetic and native peptides. Moreover, the synthetic peptides Manse‐AKH and Piebr‐AKH have significant hyperlipaemic (=adipokinetic) activity when injected into newly‐emerged adult cabbage white butterflies. The non‐amidated Vanca‐AKH is, apparently, incompletely processed Manse‐AKH without hormonal activity. Simulated dispersal flight is able to release AKHs, as indicated by the higher concentration of lipids in the haemolymph of adult P. brassicae after activity and rest periods.     

Adipokinetic hormone and flight metabolism in the locust

In adult male Schistocerca 20 min of tethered flight causes a halving of the haemolymph carbohydrate concentration. Injection of a proteinaceous emulsion of diglyceride 30 min before flight reduces both flight speed and carbohydrate utilisation. This effect can be overcome by the injection of trehalose immediately before the flight. If, in addition to the diglyceride, a dilute extract of the glandular lobes of the corpora cardiaca is injected immediately before flight, either with or without additional trehalose, carbohydrate utilisation is drastically reduced whereas flight speed is unaffected. It is argued that diglyceride competes with trehalose as a substrate for the flight muscles and that adipokinetic hormone from the glandular lobes of the corpora cardiaca stimulates the oxidation of diglyceride in these muscles during flight. This brings about a more complete (non-competitive) inhibition of trehalose utilisation by the flight muscles.     

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.     

Secretion of hyperglycaemic hormone from the corpus cardiacum of flying blowflies, Calliphora erythrocephala

In flies with an intact brain-corpus cardiacum system, the haemolymph trehalose concentration is kept near the resting level (～23 g/l.) during continuous flight for at least 45 min. Upon cardiacectomy or corpus cardiacum denervation haemolymph trehalose decreases during the first 15 min of flight to one-third of the resting level, and complete flight exhaustion occurs within 45 min. In such flight-exhausted flies a slow, apparently hormone-independent, increase of haemolymph trehalose occurs during subsequent rest. Squeezing of denervated (i.e. non-secreting) corpora cardiaca in situ in flight-exhausted flies leads temporarily to a steep rise of haemolymph trehalose level and restoration of flight performance.     

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.     

Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata

In the adult males of the Colorado potato beetle, Leptinotarsa decemlineata, corpora cardiaca extract injected in vivo gives rise to an increase in glucose and a decrease in alanine concentration of the haemolymph. The regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle was investigated further in vitro. Proline regulates its own synthesis by a feedback inhibition. Moreover, a factor present in extracts from the corpora cardiaca stimulates synthesis in the fat body in vitro. This effect was demonstrated with corpora cardiaca extracts from beetles, locusts and cokroaches. Also, synthetic adipokinetic hormone stimulates proline synthesis in the fat body of the Colorado beetle. In addition, a release of glucose from the fat body of the beetle could be evoked by the addition of locust and beetle corpora cardiaca extracts or synthetic adipokinetic hormone. The physiological significance of both effects is discussed.     

Flight substrates and their regulation by a member of the AKH/RPCH family of neuropeptides in Cerambycidae

The pattern of metabolic changes during tethered flight with lift-generation was investigated in two South African species of long-horned beetles (family: Cerambycidae), namely Phryneta spinator and 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 degrees 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 carbohydrates, of these beetles.     

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.     

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.     

The role of the glandular lobes of the corpora cardiaca during flight in Locusta

ABSTRACT. Flight performance in Locusta is reduced following severance of the major afferent nerves to the corpora cardiaca or removal of the glandular lobes of the corpora cardiaca. These operations prevent the release of adipokinetic hormone and the consequent mobilization of stored lipid. However, locusts deprived of about 90% of their glandular lobe tissue, while flying poorly, did mobilize lipid. It is suggested that the remaining glandular parenchyma cells are capable of secreting enough hormone to stimulate lipid mobilization, but that the concentration may be inadequate to encourage lipid utilization. After removal of all the glandular lobe parenchyma, the blood carbohydrate concentration was temporarily depressed. Nevertheless flight performance was equally poor, both when haemolymph carbohydrate levels were low and when they had returned to normal. After the injection of trehalose into operated control locusts and locusts deprived of their glandular lobes, flight was still markedly poorer in the operated insects, even though the injection of trehalose prevented adipokinetic hormone release in the intact locust. It seems that the poor flight performance of locusts deprived of their glandular lobes cannot be fully explained by the simple absence of adipokinetic hormone.     

Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

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.     

Identification of the adipokinetic hormone of the large milkweed bug Oncopeltus fasciatus

Abstract The adipokinetic hormone (AKH) of the large milkweed bug Oncopeltus fasciatus is isolated from an acidified methanolic extract of 200 corpora cardiaca, purified by single step reversed phase high‐performance liquid chromatography (HPLC) and N‐terminally deblocked using pyroglutamate aminopeptidase. The sequence is identified by Edman degradation and matrix assisted laser desorption/ionization‐time of flight mass spectroscopy as pGlu‐Leu‐Asn‐Phe‐Ser‐Pro‐Asn‐Trp amide. This structure is confirmed by chemical synthesis and coelution of native and synthetic peptide on HPLC. The AKH of O. fasciatus is identical to Tenmo‐HrTH, a member of the adipokinetic/red pigment‐concentrating hormone peptide family that had been isolated earlier from several tenebrionid beetles. Tenmo‐HrTH causes a significant rise in the concentration of haemolymph lipids when injected into adult male and female O. fasciatus, but displays no hyperglycaemic activity. There is no indication of the presence of other AKHs in O. fasciatus. The large milkweed bug represents the first member of the seed bugs (Lygaeidae) for which the endogenous AKH has been identified.