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.     

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.     

Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans

Aqueous extracts of brain, thoracic ganglion or corpora cardiaca of female Glossina morsitans were shown to contain a substance which inhibited the synthesis of lipid from l[U-¹⁴C] leucine by fat cells incubated in vitro. The highest concentration of this substance was found in the corpora cardiaca; approximately 1 × 10^?6 gland pairs μl^?1 were required for maximum inhibition. At concentrations greater than 1 × 10^?4 gland pairs μl^?1 the lipid synthesis inhibiting factor (hereafter referred to as the LSIF) was inactivated by the presence of a substance which could be removed by gel filtration. The concentration of LSIF in the corpora cardiaca and midbrain varied throughout the reproductive cycle of the female. Net release of LSIF from the midbrain occurred between the 2nd and 7th day of the 9-day reproductive cycle. Net release from the corpora cardiaca began on day 5 and continued until the end of the interlarval period on day 9. Results are consistent with the hypothesis that LSIF is synthesised mainly in the medial neurosecretory cells of the midbrain whereas the corpora cardiaca are the site of storage and release into the haemolymph. LSIF was present in midbrain and corpora cardiaca extracts from male G. morsitans but at lower concentrations than in females. No variation in LSIF concentration could be correlated with the feeding cycle. LSIF activity was not detected in fresh haemolymph but was found at high concentration in boiled haemolymph, suggesting the presence of an inhibitor which was inactivated at high temperature. Preliminary investigations into the nature of LSIF have shown it to be inactivated by proteolytic enzymes and to be recoverable in a single peak from a Sephadex G15 column.Results support the view that LSIF is a peptide hormone which, in conjunction with an inhibitor, controls the lipid synthetic ability of the fat cells of the adult female tsetse fly throughout the reproductive cycle.     

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.     

Hormone stimulated lipolysis and proline synthesis in the fat body of the adult tsetse fly, Glossina morsitans

G. morsitans fat cells incubated in vitro with l-[U-¹⁴C]-leucine incorporated the radiolabel, mainly into triglycerides. Aqueous extracts of corpora cardiaca, midbrain, or thoracic ganglion stimulated the release of radiolabelled material from prelabelled fat cells in vitro. Corpora cardiaca extracts were the most active, approx. 1 × 10^?3 gland pairs/μl elicited the maximal response. At concentrations above 1 × 10^?3 gland pairs/μl the activity of corpora cardiaca extracts was inhibited by a substance which could be removed by gel filtration. The stimulatory factor in nervous-tissue extracts was destroyed by proteolytic enzymes and was recoverable in a single peak by Sephadex G15 gel filtration. Results suggest that it is a peptide hormone produced mainly by the median neurosecretory cells of the midbrain with the corpora cardiaca being the site of storage and release. No hormone was detectable in fresh haemolymph, but it was found at high concentration in boiled haemolymph, implying the presence of a heat labile inhibitor.Under the in vitro conditions used the hormone stimulated the synthesis of proline from alanine and the hydrolysis of triglycerides to free fatty acids. The probable functions of the hormone are to stimulate proline synthesis in response to demand for flight and/or to mobilise lipid for larval nutrition. The relative importance of these apparent functions in vivo could not be determined.     

Immunocytochemical localization of prolactin-like antigenic determinatns in the neuroendocrine system of the honeybee (Apis mellifica)

Summary In the brain of the adult worker bee (Apis mellifica) prolactin-like (PRL) immunoreactive cells were localized in the lateral neurosecretory cell region and the subesophageal ganglion by means of the PAP procedure. These cells emit nerve fibres which pass through the neuropile of the brain to the corpora cardiaca where a great number of immunoreactive axon terminals is present. Tests with antisera against rat pituitary prolactin and human luteinizing hormone were negative. These results indicate that hPRL material is produced in neurosecretory cells of the bee brain and transferred via axons to the corpora cardiaca for storage and subsequent release into haemolymph.This work is part of the Ph. D. thesis of K.P.S.     

Immunocytochemical localization of prolactin-like antigenic determinants in the neuroendocrine system of the honeybee (Apis mellifica)

In the brain of the adult worker bee (Apis mellifica) prolactin-like (PRL) immunoreactive cells were localized in the lateral neurosecretory cell region and the subesophageal ganglion by means of the PAP procedure. These cells emit nerve fibers which pass through the neuropile of the brain to the corpora cardiaca where a great number of immunoreactive axon terminals is present. Test with antisera against rat pituitary prolactin and human luteinizing hormone were negative. These results indicate that hPRL material is produced in neurosecretory cells of the bee brain and transferred via axons to the corpora cardiaca for storage and subsequent release into haemolymph.     

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.     

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.     

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.     

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.     

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.     

Flight fuel related differences between solitary and gregarious locusts (Locusta migratoria migratorioides)

Abstract. Flight fuel relations of crowded and isolated Locusta migratoria migratorioides were investigated in younger (12–16 days after fledging) and older (27–30 or 27–32 days after fledging) adult males.No phase polymorphism dependent differences were found in resting haemolymph carbohydrate levels of the younger locusts.In the older age group, resting haemolymph carbohydrate levels were slightly though significantly higher in the isolated than in the crowded locusts.Injection of various doses of synthetic adipokinetic hormones (AKHs) did not induce marked changes in haemolymph carbohydrate levels and no differences were found between crowded and isolated locusts.A 30 min flight led to the same decrease in haemolymph carbohydrate levels of isolated and crowded locusts, 43.3% and 44.6% of the resting levels, respectively.We concluded, therefore, that the results do not seem to indicate that isolated locusts rely more heavily on carbohydrates as flight fuel than crowded locusts.Hyperlipaemic responses to flight were less intense in isolated than in crowded locusts, but phase polymorphism dependent differences in flight-induced increase of haemolymph lipid levels were not parallel in 12–16-day-old and 27–32-day-old males.In the younger age group the difference was mainly in the duration of flight needed to induce full response which appeared already after 20 min of flight in the crowded locusts, but only after 45 or 60 min of flight in the isolated ones.In contrast, the older isolated locusts showed markedly lower haemolymph lipid elevations than the crowded locusts even after 30, 45 or 60 min of flight.The hypothesis is forwarded that isolated locusts have a rather coarse adipokinetic strategy focused on a single long-distance migratory flight, whereas gregarious locusts possess a fine adipokinetic balance for reiterative migratory flights and saving fuel reserves for unpredictable long-distance migrations.     

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.     

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.     

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.     

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.     

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.     

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.     

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.