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.     

Variations de la tendance au vol soutenu du criquet Pélerin Schistocerca gregaria après implantations de corpora cardiaca

An attempt has been made to determine the influence of the corpora cardiaca on sustained flight tendency shown by Schistocerca gregaria male imagos reared in dense groups or in isolation, taking into consideration the presence or absence of the corpora allata. Corpora cardiaca either whole or in part (glandular or neurohaemal lobe) from either good or poor flyers were inserted into the abdomen of either good or poor flyers. A small amount of extra corpora cardiaca or neurohaemal lobe from good flyers favours sustained flight. A large amount from good or poor flyers inhibits flight. Allatectomized recipients react like intact animals, but locusts isolated for several generations scarcely react. Glandular lobes are without effect and so are corpora cardiaca taken from good fliers about 15 days after their connexions with the brain have been cut. Organs taken from good fliers just after a long flight are also of little effect. It is concluded that factors are elaborated by the pars intercerebralis, then stored and activated in the neurohaemal part of the corpora cardiaca: these organs alone do not control flight tendency.     

A possible site of action for adipokinetic hormone on the flight muscle of locusts

In mitochondrial preparations the oxidation of palmitate and of palmitoyl carnitine is stimulated by dilute extracts of the glandular lobes of the corpora cardiaca. Extracts of the storage lobes of the corpora cardiaca or of corpora allata are without effect. In a working single muscle preparation the entry of diglyceride into the muscle is also stimulated by tissue extracts containing adipokinetic hormone. This stimulation is, however, prevented by the addition of 2-bromostearic acid to the perfusion fluid. It is suggested that adipokinetic hormone may have a single site of action in the flight muscle and this may be in stimulating the entry of acyl groups into the mitochondria; perhaps by acting on the inner acyl carnitine transferase.     

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.     

Isolation, physiological characterization, release and sequence elucidation of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the stick insect, Sipyloidea sipylus

ABSTRACT. The corpora cardiaca of the stick insect, Sipyloidea sipylus Westwood, contain peptidic material which elevates blood lipids in migratory locusts, blood carbohydrates in American cockroaches, and activates glycogen phosphorylase in the fat body of the cockroach in a time- and dose-dependent manner. The active principle is found in appreciable amounts only in the corpora cardiaca; slight hyperlipaemia is caused by extracts made from corpora allata and abdominal ganglia, whereas brain, suboesophageal and thoracic ganglia are not active. The adipokinetic activity is already present in corpora cardiaca from second instar (first day) nymphs. The factor retains its adipokinetic activity after boiling for up to 1 h. Conspecific injections of extracts from corpora cardiaca of S.sipylus cause hypertrehalosaemia in ligated stick insects and activate glycogen phosphorylase in non-ligated S.sipylus. After incubation of corpora cardiaca in vitro in saline with high concentrations of potassium and calcium, one fraction with adipokinetic (in locusts) and hypertrehalosaemic (in stick insects) activity can be isolated from the medium by RP-HPLC. Fractionation of a methanolic extract of corpora cardiaca from S.sipylus by RP-HPLC shows that active compounds are confined to apparently three absorbance peaks. The material of the highest absorbance peak was purified to homogeneity by RP-HPLC, and its amino acid composition determined after acid hydrolysis with HCl and with methanesulfonic acid revealed the residues Asx, Thr₍₃₎, Glx, Pro, Gly, Leu, Phe and Trp. The primary structure of this hypertrehalosaemic factor is assigned as a blocked decapeptide, pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂, from its FAB spectrum and metastable scans of its FAB spectrum. The structure is confirmed by synthesis; the synthetic and natural peptide co-chromatograph, and the synthetic peptide elevates blood carbohydrates in ligated stick insects and activates fat body phosphorylase in non-ligated S.sipylus.     

Effects of precocene I and II on flight behaviour in Oncopeltus fasciatus, the migratory milkweed bug

Treatment of newly emerged adult Oncopeltus fasciatus with the corpus allatum inhibitors. 7-methoxy-2,2-dimethyl chromene or 6,7-dimethoxy-2,2-dimethyl chromene (preocene I and II) results in an inhibition of long-term flight (presumed migratory) behaviour in both males and females and inhibition of oögenesis in females. Treatment of reproductive females with precocene briefly stimulates flight behaviour (which ceases in such females as oviposition begins) and then subsequently inhibits it. Oviposition also ceases in such females and oöcyte resorption occurs. Topical application or injections of JH III to precocenetreated animals results in immediate restoration of the tendency to make long tethered flights while corpora cardiaca extract injections, corpora cardiaca implantation, sham implantation, sham injections and sham topical applications were ineffective in restoring prolonged flight behaviour to precocene-treated animals.Restoration of flight by JH III injection was observed within 1 hr after treatment with the hormone. These results indicate that JH is necessary for prolonged flight and presumably migratory behaviour in this species and its stimulatory effect on flight behaviour is immediate. Possible mechanisms of action of the hormone on flight behaviour are discussed.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.     

Restricted specificity of a hyperglycaemic factor from the corpus cardiacum of the stick insect Carausius morosus

Extracts of the corpora cardiaca of the stick insect Carausius morosus elevate the haemolymph carbohydrate concentration in adult and 6th-instar larvae which are ligated behind the first pair of legs, but not in non-ligated (intact) insects. The increase in haemolymph sugars is due to trehalose elevation, is time dependent (with a maximal effect about 90–120 min after injection), and is dose dependent (needing 0.005 gland equivalents for a significant effect and a tenfold higher dose for a maximal response). The hyperglycaemic factor is localised entirely in the corpora cardiaca and appears to be specific to stick insects; corpora cardiaca extracts of two lepidopteran species (Acherontia atropos and Aglais urticae) and of Locusta migratoria have no effect, whereas corpora cardiaca extracts of the stick insects Cuniculina impigra and Sipyloidea sipylus have similar activity to those from C. morosus. This specificity is also shown when S. sipylus is used as the recipient. Synthetic adipokinetic hormone and red pigment concentrating hormone possess no hyperglycaemic activity in the stick-insect system. Two peaks of hyperglycaemic activity were obtained after column chromatography of corpora cardiaca extract of C. morosus on Sephadex G-25 and Sephadex LH-20. The factor seems to act via activation of fat-body glycogen phosphorylase, which, although 60% active in the control insects, is significantly increased to approx. 85% upon corpora cardiaca injection. However, the activation is demonstrated in ligated and intact insects. No significant decrease in the glycogen level of the fat body is observed after corpora cardiaca injection.     

Adipokinetic hormone controls lipid metabolism in adults and carbohydrate metabolism in larvae of Manduca sexta

The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpora cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH.     

Re-evaluation of the role of corpora cardiaca in calling and oviposition behaviour of giant silk moths

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating.Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.     

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.     

Adipokinetic response of a flightless grasshopper (Barytettix psolus): Functional components,defective response

The mature flightless grasshopper Barytettix psolus shows a very small adipokinetic response when injected with extracts of its own corpora cardiaca, although the fat body contains enough lipid for a strong response. When these extracts were injected into Melanoplus differentialis, a grasshopper capable of flight, or the moth Manduca sexta, much stronger adipokinetic responses were observed. Upon analysis of B. psolus extracts by HPLC, two components with adipokinetic activity were obtained. The major component appears to be identical to locust adipokinetic hormone (AKH) I. Extracts of B. psolus corpora cardiaca also activated fat body glycogen phosphorylase in B. psolus. This activation, however, did not result in an increase in hemolymph sugar, probably because of low levels of glycogen in the fat body. B. psolus hemolymph contains a high-density lipophorin (HDLp) consisting of the apolipophorins (apoLp) I and II and lipid. Both apoproteins are glycosylated. The hemolymph also contains apoLp-III, although this apoprotein apparently does not associate with HDLp to form a low-density lipophorin (LDLp) following AKH or corpora cardiaca extract injections. When B. psolus lipophorin and AKH were injected into Schistocerca americana, lipophorin took up lipids and combined with apoLp-III, forming LDLp. ApoLp-III from B. psolus injected into S. americana can also form LDLp, demonstrating that the components are functional. A lipid transfer particle isolated from M. sexta and injected into B. psolus does not improve the adipokinetic response. Thus, it appears that the adipokinetic response of B. psolus is not deficient because of the lack of AKH or functional lipophorins, but may be caused by the lack of a full response to AKH by fat body or the deficiency in hemolymph of some as yet unknown factor.     

Contribution à l'étude infrastructurale du système neurosécréteur rétrocérébral chez Locusta migratoria migratorioides (R. et F.)

Résumé L'étude infrastructurale des corpora cardiaca de Locusta révèle l'existence de 3 types de fibres neurosécrétrices (portion neurohémale) et d'un seul type de cellules glandulaires (portion endocrine intrinsèque). Elle permet également d'envisager les modalités du rejet des sécrétions exogènes et endogènes.

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Ultrastructural study of the retrocerebral neurosecretory system in Locusta migratoria migratorioides (R. et F.)I. The corpora cardiaca

Summary An ultrastructural study of the corpora cardiaca of Locusta migratoria migratorioides shows three neurosecretory fiber types in the neurohaemal part of these organs; only one cellular type constitutes the intrinsic glandular part of the corpora cardiaca. This study also shows sites of release of stored extrinsic neurosecretory material and of products elaborated by intrinsic glandular cells of the corpora cardiaca.

     

Peptidomics of the pars intercerebralis-corpus cardiacum complex of the migratory locust, Locusta migratoria.

The pars intercerebralis-corpora cardiaca system (PI-CC) of insects is the endocrinological equivalent of the hypothalamus-pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.     

Relationship of adipokinetic hormone I and II to migratory propensity in the grasshopper, Melanoplus sanguinipes

This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species.     

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.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.     

Diuresis or clearance: Is there a physiological role for the “diuretic hormone” of the desert beetle Onymacris?

Malpighian tubules of Namib Desert tenebrionid beetles of the genus Onymacris are strongly stimulated by homogenates of the corpora cardiaca. The corpora cardiaca of other arid-adapted tenebrionids also contain diuretic material. Biogenic amines, which could be released during the preparation of corpora cardiaca extracts, do not stimulate fluid secretion in tubules of Onymacris rugatipennis. The diuretic factor in corpora cardiaca extracts is stable to boiling and to incubation with pronase. HPLC separation of the corpora cardiaca of O. rugatipennis gives a single region with diuretic activity in both secretory and electrical bioassays. Diuretic activity can not be detected in the haemolymph of Onymacris, and injection of corpora cardiaca extracts into the beetles does not cause diuresis. Simultaneous injection of corpora cardiaca and the dye amaranth shows that the most of the dye transported by the Malpighian tubules moves anteriorly into the midgut, indicating fluid recycling by this route. The most likely function for this “diuretic hormone” is clearance of metabolic wastes from the haemolymph.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

Fuels for flight in Locusta

In Locusta migratoria, carbohydrate is the predominant energy source for the first 20 to 30 min of flight. During this time carbohydrate is utilized at the rate of 120 μg/min, but thereafter it is used at only 8 to 9 per cent of the initial rate. Initially carbohydrate is not mobilized to any extent from body stores. Lipid, however, is mobilized (from stores in the fat body) in response to adipokinetic hormone released from the glandular lobes of the corpora cardiaca. The mobilized lipid is diglyceride, and 16 : 18 and 18 : 18 diglycerides predominate. The rate of diglyceride mobilization is rapid, both during flight and in response to extracts of corpora cardiaca. The release of adipokinetic hormone during flight has also been estimated and is found to be more rapid than previously suggested. It is calculated that the rate of lipid utilization increases during flight from virtually zero during the first few minutes to 35 μg/min (15 to 30 min) and finally reaches about 85 μg/min during the second 30 min of flight.