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.     

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.     

Adipokinetic and hyperglycaemic factors of different insect species: Separation with high performance liquid chromatography

Corpus cardiacum extracts from the phasmids, Carausius morosus, Cuniculina impigra, Sipyloidea sipylus, Acrophylla wuelfingi, Eurycantha goliath, Bacillus rossius and Extatosoma tiaratum, from the Orthopterans, Locusta migratoria and Gryllus bimaculatus, from the Dictyopterans, Periplaneta americana, Gromphadorrhina coquereliana and Blaberus craniifer, from the Coleopterans Tenebrio molitor and Pachnoda sp., synthetic adipokinetic hormone and synthetic crustacean red pigment-concentrating hormone (RPCH) were injected into locusts, cockroaches and ligated stick insects as bioassay systems for adipokinetic and hyperglycaemic substances, respectively. The locust and cockroach bioassay gave positive results with all corpus cardiacum material tested (however the lipid response in locusts upon injection of T. molitor corpus cardiacum extract was very poor). The stick insect bioassay was quite specific for stick insect corpus cardiacum material; only corpus cardiacum extracts from a few other species (G. bimaculatus, P. americana, G. coquereliana and Pachnoda sp.) showed weak activity. All other extracts, including synthetic adipokinetic hormone and RPCH, failed to induce a response.Separations of corpus cardiacum extracts from L. migratoria, P. americana, T. molitor, C. morosus and S. sipylus were achieved on reversed-phase high-performance liquid chromatography (RP-HPLC). Locust corpus cardiacum extract showed two absorbance peaks with adipokinetic activity, adipokinetic hormones I and II. The peaks with hyperglycaemic activity from P. americana corpus cardiacum extracts had different retention times to those of locust adipokinetic hormones I and II. Stick insect corpus cardiacum extracts revealed also 2 absorbance peaks with adipokinetic activity, the major one co-eluting with RPCH. The active compound from corpus cardiacum extracts of T. molitor appeared to elute close to locust adipokinetic hormone I.     

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.     

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.     

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.     

Neuroendocrine control of blood lipids in the locust, Locusta migratoria

In order to determine the best conditions, the influence of various parameters on the haemolymph lipid concentration were studied. These parameters are the age, the sex and the feeding of the animals, the time and the number of the haemolymph sample-taking and the temperature of the locust culture. A large in vivo increase in haemolymph lipid concentration was obtained in locusts which received extracts of the whole CC and of their glandular or neurohemal lobes. Reversely, a decrease in this concentration was obtained in locusts operated 7 days before (cardiacectomy or glandular lobe removal). Moreover the pars intercerebralis extracts increased the level of haemolymph lipids. We conclude that adipokinetic factors are present, both in the glandular lobes of the CC and in their neurohemal lobes. It is likely that the latter partly originate from the pars intercerebralis. Results of allatectomy and injections of corpora allata extracts led to the conclusion that corpora allata contain an adipokinetic factor, the juvenile hormone. and factors that inhibit the haemolymph lipid concentration. Finally, from different injections of neurotransmitters and drugs it is argued that it is mainly octopamine which is involved in the mechanism governing the increase of the level of haemolymph lipids.     

Cerebral neurosecretory cells and flight in the locust

The effect on flight performance of various superficial lesions of the pars intercerebralis in and around the area of the MNSC (median groups of cerebral neurosecretory cells) have been studied 18 hr after surgery. Only lesions involving areas immediately lateral to the MNSC produce an impairment of flight performance. The release of adipokinetic hormone during flight was studied in these locusts by measuring the changes in haemolymph lipid during flight. It has not been possible to identify any of the areas tested as being concerned with the control of the release of adipokinetic hormone since lipid mobilization was not prevented by any of the operations studied.The poor flight performance in locusts in which the MNSC were destroyed by cautery on day 1 of adult life can be prevented by regular topical application of a synthetic juvenile hormone analogue. It is argued that the effects of removal of the MNSC on the development of flight performance are most likely a consequence of reduced activity of the corpora allata.     

The effects of corpora cardiaca on tethered flight in the Locust

Summary Removal of the cerebral neurosecretory cells tas no effect on the tethered flight pattern of adult maleLocusta. However, the flight pattern is markedly influenced by removal of the glandular lobes of the corpora cardiaca or by injection of 0.01 pair of corpora cardiaca. Changes in flight speed, brought about by such injections, may reflect differences in substrate utilisation in response to hormonal factor(s) from the corpora cardiaca. It is suggested that one effect of corpus cardiacum factor(s) may be the suppression of carbohydrate utilisation by the flight muscles.     

Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h(-1) per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.     

Allatectomy and flight performance in maleLocusta migratoria

Summary Flight performance in locusts is a function of age, with its peak value at 18 days after emergence. While allatectomy retards the normal development of flight capability, it also has the effect of slowing down the decline in flight performance characteristic of operated control locusts as they age. Periodic topical application of synthetic juvenile hormone remedies the initial effect of allatectomy but its effectiveness wears off with age. The period of optimum flight performance is prolonged in locusts allatectomised when mature.A characteristic features of the flight pattern of immature-allatectomised and matureallatectomised locusts when flown about one week after the operation is a rapid decline in flight speed during the first 20 minutes of flight. Eventually, as the allatectomised locusts age, they assume the flight pattern of normal locusts and subsequent differences in flight performance between operated and normal locusts are confined to differences in flight intensity.Allatectomy has no marked effect on the preflight haemolymph total lipid and carbohydrate levels, the mobilisation of lipid and the amount of carbohydrate depleted. The quantity of lipid mobilised is, however, related to flight performance in both allatectomised and operated control locusts. Locusts which fly faster mobilise more lipid. The lipids mobilised by the adipokinetic hormone are 1618; 1818 and 1616 diglycerides in order of abundance. Allatectomy has no effect on the nature of these diglycerides released during flight.     

A possible role of Schisto FLRFamide in inhibition of adipokinetic hormone release from locust corpora cardiaca

The distribution and actions of FMRFamide-related peptides (FaRPs) in the corpora cardiaca of the locust Locusta migratoria were studied. Antisera to FMRFamide and SchistoFLRFamide (PDVDHVFLRFamide) label neuronal processes that impinge on glandular cells in the glandular lobe of the corpora cardiaca known to produce adipokinetic hormones. Electron microscopic immunocytochemistry revealed that these FaRP-containing processes form synaptoid contacts with the glandular cells. Approximately 12% of the axon profiles present in the glandular part of the corpus cardiacum contained SchistoFLRFamide-immunoreactive material. Retrograde tracing of the axons in the nervus corporis cardiaci II with Lucifer yellow revealed 25–30 labelled neuronal cell bodies in each lateral part of the protocerebrum. About five of these in each hemisphere reacted with the SchistoFLRFamide-antiserum. Double-labelling immunocytochemistry showed that the FaRP-containing processes in the glandular lobe of the corpora cardiaca are distinct from neuronal processes, reacting with an antiserum to the neuropeptide locustatachykinin. The effect of the decapeptide SchistoFLRFamide and the tetrapeptide FMRFamide on the release of adipokinetic hormone I (AKH I) from the cells in the glandular part of the corpus cardiacum was studied in vitro. Neither the deca- nor the tetrapeptide had any effect on the spontaneous release of AKH I. Release of AKH I induced by the phosphodiesterase inhibitor IBMX, however, was reduced significantly by both peptides. These results point to an involvement of FaRPs as inhibitory modulators in the regulation of the release of adipokinetic hormone from the glandular cells.     

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.     

Is octopamine a transmitter mediating hormone release in insects?

The release of hyperlipemic hormone from the glandular cells of the corpus cardiacum (CC) of Locusta migratoria is under the synaptic control of axons in nervus corpus cardiacum II (NCC II). The effects of aminergic agonists and antagonists on the release of the hyperlipemic hormone induced by electrical stimulation of NCC II have been examined. CC isolated from reserpine-injected locusts did not release hormone when subjected to electrical stimulation of NCC II but continued to release hormone in response to high-potassium saline. The electrically stimulated release of hormone from isolated CC was abolished by the alpha-adrenergic blocking agent, phenoxybenzamine, but potentiated by the beta-adrenergic blocking agent, propranolol. Phenoxybenzamine did not interfere with release induced by high-potassium saline. It is suggested that the postsynaptic receptors on the glandular cells are similar to the alpha-adrenergic receptors of vertebrates. Octopamine was found to be present in the glandular lobe of the CC at concentrations of 0.62 pmole per gland pair. Reserpine depleted the content to 0.3 pmole per pair. Bathing the CC in 10(-7) M octopamine resulted in the release of hyperlipemic hormone, and this release was blocked by phenoxybenzamine. It is concluded that the neurotransmitter involved in the synapse between axons of NCC II and the cells releasing hyperlipemic hormone is aminergic, possibly octopaminergic. Octopamine may well be a transmitter mediating hormone release in insects.     

The hyperglycaemic activity of locust adipokinetic hormone

ABSTRACT. Pure adipokinetic hormone (AKH) extracted from the glandular lobes of locusts induces hyperglycaemia in cockroaches. Larger doses (20–200 pmol) of AKH are required to induce hyperglycaemia in the cockroach than are required to produce hyperlipaemia in locusts (1–20 pmol).     

Hypolipemia,hypoglycemia, and inactivation of glycogen phosphorylase in Locusta migratoria

Feeding starved adult migratory locusts, Locusta migratoria, caused decreases of hemolymph lipid concentrations and of the percentage of active fat body glycogen phosphorylase which suggested that a molecule(s) from the neurosecretory system or the midgut may have been released to regulate metabolism. Fat body phosphorylase was also inactivated after insects were transferred from 0 to 25 ° C. In adults with elevated hemolymph lipid levels after the injection of small doses of corpus cardiacum extract (CC), feeding did not induce a decrease in hemolymph lipid concentrations. It appears that the processes initiated by feeding could not override the effects of the continued presence of adipokinetic hormone(s) (AKHs) in the hemolymph or their long-term effects. Aqueous, methanolic, or ethanolic extracts of brains or storage lobes (SL) of fed locust CC did not lead to decreases of hemolymph lipid concentrations. Bovine insulin was equally inactive when tested at doses which were previously reported to reduce lipid levels. Fractions of ethanolic brain extracts from 3-day-starved males collected after high-performance size-exclusion chromatography, however, produced hypoglycemic effects in fed males. Two biologically active fractions were found, one with high (≥ 10 kDa) and one with low molecular weight (approximately 1 kDa). © 1995 Wiley-Liss, Inc.     

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.     

Morphology of neurones in the storage part of the corpus cardiacum of Locusta migratoria: no evidence for their involvement in the regulation of adipokinetic cell activity

The storage part of the corpus cardiacum of Locusta migratoria consists of two compartments: a neural part on the haemocoelic side containing neuronal cell bodies that are protected by a blood-brain barrier, and a neurohaemal part adjacent to the aorta. Intracellular filling of the neurones in the neural part with Lucifer yellow followed by confocal laser scanning microscopy has revealed that these neurones can be divided into several classes. None of the neurones has processes extending into the glandular part of the corpus cardiacum. They are, therefore, not directly involved in the regulation of adipokinetic cell activity.     

Activation of fat body glycogen phosphorylase in Locusta migratoria by corpus cardiacum extract and synthetic adipokinetic hormone

Between 10 and 20 per cent of the total glycogen phosphorylase in the fat body of mature Locusta migratoria of both sexes is in the active form. Injection of an aqueous corpus cardiacum (CC) extract results in a rapid activation: within 2 min the level of active phosphorylase is significantly increased and full activation is reached within 10 to 20 min. As little as 0.002 CC gland equivalents stimulate fat body glycogen phosphorylase significantly and maximum activation is obtained with 0.05 CC gland equivalents. From experiments with known quantities of injected synthetic adipokinetic hormone (SAKH), it appears that this hormone cannot account for all the activation. This is supported by results obtained when extracts of carefully isolated storage lobes are injected; at the dose used here these have no adipokinetic activity, but activate fat body phosphorylase. Furthermore, when locusts are ‘stressed’ by rotation, although no adipokinetic hormone is released, an activation of phosphorylase occurs. Starvation causes also an increase in the active form of the enzyme. The fat body receptor sites of the locust recognise also the crustacean red pigment concentrating hormone (RPCH), whose structure closely resembles that of the locust adipokinetic hormone, leading to activation of the phosphorylase. However, RPCH is about 2.5–5 times less potent than SAKH. Crude CC extracts of a stick insect (Carausius morosus), a cockroach (Periplaneta americana) and the tobacco hornworm (Manduca sexta) activate locust fat body phosphorylase, although this last extract has no effect on lipid elevation. On the other hand, CC extracts of the death's head hawk moth (Acherontia atropos) and purified crustacean hyperglycaemic hormone from a crayfish (Orconectes limosus) have no effect.     

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.