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.     

Some problems of homeostasis of haemolymph metabolites in the locust

Sectioning of the afferent nerves (NCCI and II) to the locust corpora cardiaca, glandular lobe removal, cardiacectomy, or removal of the median neurosecretory cells of the brain, have no long-term effect on blood lipid concentration. After removal of the glandular lobe, haemolymph carbohydrate concentration is lowered and remains significantly so from the second to the sixth day after the operation but returns to normal within 10 to 15 days. Severance of the afferent nerves to the corpora cardiaca does not, however, affect blood carbohydrate concentration. The injection of a concentrated extract of glandular lobes into locusts deprived of their glandular lobes does not elicit a hyperglycaemic effect even when blood carbohydrate levels are low. Cauterization of the median neurosecretory cells of the brain, sectioning of the NCCI and II, or removal of the glandular lobes of the corpora cardiaca have no effect on haemolymph protein.After dilution of haemolymph constituents by the injection of water, carbohydrate and protein concentrations are not rapidly restored to their initial values. The lipid concentration, however, rapidly returns to its pre-injection level due to the mobilization of 16:16, 16:18, and 18:18 diglycerides. This occurs even in glandular lobe deprived, median neurosecretory cell cauterized, or headless locusts. These diglycerides are mobilized following the injection of solutions containing lipid, carbohydrate, and/or protein, and are the same diglycerides that are released from the fat body in response to adipokinetic hormone. It is concluded that the injection of large volumes of fluid causes lipid mobilization but adipokinetic hormone does not apear to be involved, and the mechanism of blood lipid homeostasis in the resting locust is not clear.     

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.     

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.     

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.     

Diel rhythms of adipokinetic hormone, fat body response, and haemolymph lipid and sugar levels in the house cricket

Abstract. The adipokinetic hormone (Grb-AKH) content in the corpora car-diaca of the house cricket, Acheta domesticus , varied during a day with two peaks in the scotophase and one peak in the photophase. There were two distinct peaks of total lipid concentration in the blood, one early in the photophase and the other early in the scotophase. Fat body sensitivity to adipokinetic hormone also varied in close synchrony with the lipid rhythm. It was not possible to attribute the rhythm of blood lipid titre unequivocally to either the rhythm of adipokinetic hormone content in the corpora cardiaca or to the rhythm of sensitivity of the fat body to the hormone. In adult crickets the blood carbohydrate titre had two peaks in adult females, one towards the end of scotophase and another in the late photophase (c. 12h apart), but a single peak at the end of scotophase was apparent in last instar larvae. The blood carbohydrate rhythm persists in DD and is therefore endogenous. Carbohydrates were not mobilized by the Grb-AKH, therefore could not be involved in the blood carbohydrate rhythm. Exposure to various day lengths caused shifts in the patterns of the carbohydrate rhythms, but imposition of a cyclic temperature regime had no effect on the rhythm.     

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.     

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.     

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.     

The effect of Metarhizium anisopliae var acridum on haemolymph energy reserves and flight capability in the desert locust, Schistocerca gregaria

Adult desert locusts, Schistocerca gregaria , 3 days after inoculation with the entomopathogenic fungus Metarhizium anisopliae var acridum , had significantly less carbohydrate and lipid in the haemolymph than controls. This was not due to reduced food intake as 3 days of complete starvation had no effect on haemolymph titres of energy reserves in controls. Furthermore injection of an extract of the corpora cardiaca (the source of adipokinetic hormone, AKH) caused a large significant increase in haemolymph lipid in mycosed locusts, indicating the availability of significant quantities of lipid in the fat body, the target for AKH. Haemolymph carbohydrate declined significantly during tethered flight of control locusts but not in mycosed individuals. An injected supplement of trehalose significantly boosted flight performance of mycosed insects but not controls. The results are discussed in the light of the hypothesis that the poor flight capability of mycosed locusts is due in part to a fungus-induced reduction in mobile energy reserves.     

Age-related changes in the response to adipokinetic hormone in Locusta migratoria

ABSTRACT. In fifth instar nymphs of Locusta there is only a feeble adipokinetic response to extracts of corpora cardiaca. In fledglings, this poor response persists for a few days but then increases dramatically to reach a plateau by day 8. The response declines to almost zero as the locusts age beyond 35 days of adult life. This pattern of change in response is similar in both males and females but there are some differences in magnitude depending upon whether the response is measured as changes in haemolymph total lipid (vanillin-positive material) or total diglyceride (gas liquid chromatography). The poor response to adipokinetic hormone in nymphs and newly fledged locusts is not a result of shortage of stored lipid in the fat body and cannot be improved by injection of extra hormone.     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.     

A comparative study on the isolation of adipokinetic and hypertrehalosaemic factors from insect corpora cardiaca

ABSTRACT. Extracts of corpora cardiaca from two cockroaches, Nauphoeta cinerea Olivier and Leucophaea maderae F., from a cricket, Gryllus bimaculatus De Geer, from the Colorado potato beetle, Leptinotarsa decemlineata Say, and from the sphinx moth, Sphinx ligustri L. were assayed for adipokinetic and hypertrehalosaemic activity, in acceptor locusts ( Locusta migratoria L.) and cockroaches ( Periplaneta americana L.) respectively. Both bioassays give positive results with all corpus cardiacum material tested except that from the sphinx moth; in this insect haemolymph lipid concentrations (but not those of the total carbohydrate) are, however, increased after injection of an extract of corpora cardiaca from the same species. A similar result is obtained when specimens of G. bimaculatus are injected with an extract of corpora cardiaca from G. bimaculatus. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high-performance liquid chromatography. Gland extracts from the two cockroach species each show a single absorbance peak which has hypertrehalosaemic activity, but with a (common) retention time distinct from all previously described arthropod neuropeptides. The corpora cardiaca of G. bimaculatus contain also a novel adipokinetic factor with a retention time distinct from previously characterized arthropod hormones, as well as from the new cockroach factor described in this study. The two hypertrehalosaemic factors from the corpora cardiaca of the potato beetle coelute with the hypertrehalosaemic hormones I and II of the American cockroach. The active (adipokinetic) compound from glands of S. ligustri appears to coelute with locust adipokinetic hormone I.     

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.     

The hormonal content of the locust corpus cardiacum

Summary The amounts of adipokinetic and diuretic hormone in the separate storage and glandular lobes of the locust corpora cardiaca during the imaginal moult and up to the onset of sexual maturation have been measured. The levels of the hormones are high prior to the imaginal moult, fall at emergence and increase during the somatic growth period. The effects of surgical interference with the neuroendocrine system upon the hormonal content of the corpora cardiaca have been investigated. Cautery of the brain neurosecretory cells or allatectomy in mature locusts has no effect on the content of adipokinetic hormone. Diuretic hormone is absent from the storage lobes of locusts deprived of their cerebral neurosecretory cells but normal levels are present in the corpora cardiaca of allatectomised animals. Severance of the nervus corporis cardiacum I and II reduces the level of diuretic hormone in the storage lobes of the corpora cardiaca but is without effect on the levels of adipokinetic hormone in the glandular lobes. This work is supported by grants from the Science Research Council and the Royal Society.     

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.     

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.     

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.     

The hormonal control of haemolymph lipid during flight in Locusta migratoria

Fractionation of methanolic extracts of haemolymph on thin layer chromatography, followed by bioassay, has been used to measure the titres of adipokinetic hormones I and II in the haemolymph of flown locusts. These titres have been correlated with the elevation in haemolymph lipid. Haemolymph lipid elevates in a biphasic manner during locust flight. A rise in lipid occurs during the first 10 min of flight. Lipid levels then plateau between 10 and 20 min. A second, more pronounced elevation begins at 20 min and continues for up to 60 min. The titre of adipokinetic hormone I elevates 10–15 min after flight commences while that of hormone II elevates between 15–30 min. Adipokinetic hormone I contributes 80% of the activity at 30 min but only 45% at 60 min. It is suggested that the elevation in haemolymph lipid during the first 10 min of flight may not be induced by adipokinetic hormone I or II. The role of octopamine in this initial elevation is proposed and discussed.     

Substrate utilization and flight speed during tethered flight in the locust

Mature laboratory locusts normally exhibit a characteristic pattern of change in flight speed with time. They fly at high speed for the first few minutes, during which carbohydrate forms the major fuel, but then slow to a cruising speed when lipid is used almost exclusively. Locusts flown for 30 min, rested for 2hr, and then reflown, exhibit an identical pattern of flight, even though they oxidise only half the amount of carbohydrate used in the first flight. The injection of adipokinetic hormone before the first flight elicits a low initial flight speed for 10 to 15 min but then the locusts accelerate to a constant higher speed. The injection of hormone before the second flight, when blood lipid levels are already high, reduces the utilization of carbohydrate by the flight muscles dramatically but results in constant high-speed flight.