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.     

Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects

When an extract of the corpora cardiaca/corpora allata from two species of wingless stick insects, Carausius morosus and Cuniculina impigra, which cause no adipokinetic or hyperglycaemic effect when injected into the donor insects themselves, is injected into adult Locusta migratoria it resulted in an increase in the haemolymph lipid concentration. The lipid elevation was time dependent, with a maximum effect about 90–180 min after injection, and was also dose-dependent. About 0.001–0.002 (C. morosus) and 0.01 (C. impigra) gland equivalents were needed to produce a significant increase; a maximal effect was reached with approx. 0.075 (C. morosus) and 0.25 (C. impigra) gland equivalents. Carausius extract was also able to elevate carbohydrate concentration in the haemolymph of Periplaneta americana. However, the effect was weak and no maximal response was reached even with a dose of 0.5 gland equivalents. Adipokinetic hormone activity was present in CC/CA extracts of larval Carausius; the activity was about 30 times lower in 1-day-old 2nd instar individuals, and approx. 5 times less at the beginning of the 6th instar than that found in adults. In both stages the hormone levels increased gradually from the beginning to the end of the instar. No age-related changes were observed during the adult stage. Further studies on the lipid-mobilising factor of C. morosus revealed that it was stored entirely in the CC and not in other nervous tissue, e.g. brain, CA, suboesophageal ganglion, thoracic and abdominal cord. The factor was heat stable for at least 1 hr at 100°C and retained its adipokinetic activity after incubation with trypsin and the exopeptidases such as carboxypeptidase A and leucine aminopeptidase. However, activity was abolished when incubated with thermolysin and α-chymotrypsin. From these experiments a close resemblance to the locust AKH, a blocked decapeptide, is suggested.     

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.     

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 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.     

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.     

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.     

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.     

Fate of glucose in haemolymph of the American cockroach, Periplaneta americana

The rate of removal of high concentrations of glucose (10 μg/μl haemolymph) from haemolymph of adult male cockroaches, Periplaneta americana, was studied in normal and ligated insects. More than 50% of the injected glucose is removed from the haemolymph of normal insects within 20 min of injection. A period of rapid trehalose synthesis occurs during the initial 10 min following injection of glucose into the haemocoele, and this is succeeded by a period of glycogen synthesis. The results are discussed in terms of earlier observations on ‘stress-induced hypertrehalosemia’ and the possible involvement of a glycogenic agent.     

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.     

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.     

Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca

Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.     

A quantitative study of adipokinetic hormone of the firebug,Pyrrhocoris apterus

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/μl.     

Comparative immunocytochemical study of release sites of insulin,glucagon and AKH-like products in Locusta migratoria,Periplaneta americana,and Carausius morosus

Summary Insulin, glucagon and adipokinetic hormone antisera were applied to the corpora cardiaca, perisympathetic organs, neurohemal areas and peripheral neurosecretory cells of three insect species, the locust Locusta migratoria, the cockroach Periplaneta americana, and the stick insect Carausius morosus. The neurohemal part of the corpora cardiaca was shown to be immunoreactive to both insulin and glucagon antisera while the glandular cells reacted to adipokinetic hormone antisera. The perisympathetic organs seem to be devoid of these three substances, but certain peripheral neurohemal areas contained AKH and glucagon immunoreactive products. The latter were found to originate in the peripheral neurosecretory cells.     

Influence of aminergic and peptidergic substances on heart beat frequency in the stick insect Carausius morosus (Insecta,Phasmatodea)

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi‐exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo‐HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo‐HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo‐HrTH‐II, thereby rendering the HrTH peptide “not active.” In ligated stick insects, 6.7 × 10^?8 M Carmo‐HrTH‐II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose‐dependent manner; inhibition was achieved with 10^?4 M of octopamine.     

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.     

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 post-prandial rest in Locusta migratoria nymphs and its hormonal regulation

Doppler radar actographs have been used to measure the fall in locomotor activity following feeding in mid-fifth instar nymphs of Locusta migratoria. Filling the crops of the insects with agar via a cannula, reduced activity in a similar manner. Haemolymph from recently-fed donors injected into insects which had been deprived of food, likewise reduced locomotor activity compared with haemolymph from deprived donors. Injections of nutrients were ineffective, but homogenates of the storage lobes of the copora cardiaca from food-deprived donors proved effective compared with similar homogenates from newly-fed donors. It is suggested that neurosecretion released from the corpora cardiaca, known to occur following crop-filling, leads to a reduction in locomotor activity.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

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.