The adipokinetic neuropeptide of Mantodea. Sequence elucidation and evolutionary relationships

A neuropeptide with adipokinetic activity in Locusta migratoria and the mantid Empusa pennata, and hypertrehalosaemic activity in Periplaneta americana, was isolated by reversed-phase high performance liquid chromatography from corpora cardiaca of the mantids E. pennata and Sphodromantis sp. After brief enzymatic digestion by 5-oxoprolylpeptidase the primary structure of the peptide of each species was determined by pulsed-liquid phase sequencing employing Edman degradation. The C-terminus of both peptides was blocked, as indicated by the lack of digestion with carboxypeptidase A. The peptides of both species were identical: a blocked, uncharged octapeptide with the sequence L-Glu-Val-Asn-Phe-Thr-Pro-Asn-Trp-NH2. The peptide is now called mantid adipokinetic hormone (Emp-AKH). The synthetic peptide was chromatographically indistinguishable from the natural compound and increased blood lipids in locusts and blood carbohydrates in cockroaches when administered in low doses. The structural features clearly define the peptide as a novel member of the large AKH/RPCH-family of peptides. Seven amino-acid residues are at identical positions in Emp-AKH when compared with the adipokinetic hormone of a dragonfly (Lia-AKH) and the hypertrehalosaemic hormone I from the American cockroach (Pea-CAH-I). Evolutionary relationships to other insect orders are discussed.     

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.     

In vivo studies on the release of hormones from the corpora cardiaca of locusts

Summary Sectioning of the afferent nerves (NCCl and NCCll) to the locust corpus cardiacum prevents thein vivo release of adipokinetic hormone from the glandular lobes. This failure to release the hormone during flight and the consequent lack of lipid mobilisation brings about an impairment of flight performance which can be corrected by injections of corpus cardiacum extracts. Sectioning of the NCCl and NCCll reduces markedly the activity of the corpora allata. However, the poor flight performance of allatectomised locusts is not related to an inability to mobilise lipid since injections of corpus cardiacum extract which will mobilise fat body lipid in these locusts have no effect on flight performance. The results of individual sectioning of the NCCl and NCCll suggest that a double innervation of the glandular lobes functionsin vivo to control adipokinetic hormone release but that the NCCl alone may control the release of the diuretic hormone.     

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.     

Hypertrehalosaemic activity in corpus cardiacum-corpus allatum-aorta complex and adipokinetic response of Glossina morsitans

Abstract. Extracts from the corpus cardiacum-corpus allatum-aorta (CC-CA-A) complex of Glossina morsitans morsitans Westwood contain a hypertrehalosaemic factor when assayed in Periplaneta americana L. and in G.morsitans. A slight though significant decrease, followed by an increase, in haemolymph total carbohydrate occurs when tsetse are flown for 1 h. When assayed in Locusta migratoria L., the extracts have no adipokinetic activity, but L.migratoria corpus cardiacum extract produces an adipokinetic response in the female tsetse. It is suggested that the neurosecretions contained in the tsetse CC-CA-A complex contain a hypertrehalosaemic factor whose role is to mobilize glycogen.     

Dragonfly Erythemis simplicicollis contains a novel adipokinetic neuropeptide

We have isolated a novel member of the adipokinetic hormone family of peptides from a methanolic extract of corpora cardiaca of the libellulid dragonfly Erythemis simplicicollis by using a single‐step reversed‐phase high performance liquid chromatography method and monitoring biological activity in various heterologous bioassays and a homologous one. The sequence, as determined by Edman degradation and mass spectrometry, was of an uncharged blocked octapeptide: pGlu‐Leu‐Asn‐Phe‐Thr‐Pro‐Ser‐Trp amide. The structure was confirmed by chemical synthesis. The synthetic peptide increased hemolymph lipids in the dragonfly and was active in another libellulid (Orthetrum julia‐falsum) as well, but to a lesser extent than the conspecific peptide Lia‐AKH, which is an isoform of the novel peptide differing by a Val (instead of Leu) at position 2. Since lipids are apparently used as substrate for muscle contraction during flight of Erythemis simplicicollis and the native peptide induces lipid mobilization, this novel peptide is denoted Ers‐AKH. Arch. Insect Biochem. Physiol. 40:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromatography, and amino-acid composition of factor II

The separation of two peptides with hypertrehalosaemic activity from the corpus cardiacum of the Indian stick insect, Carausius morosus, was achieved by reversed-phase high-performance liquid chromatography using a Nucleosil C-18 column with a trifluoroacetic acid/acetonitrile gradient. The eluant was monitored at 210 nm and the hypertrehalosaemic activity was detected in ligated stick insects using a bioassay. In addition, the isolated material was potent in causing hyperlipaemia in migratory locusts and also in raising blood carbohydrates in the American cockroach. The amino-acid composition of the major peptide, hypertrehalosaemic factor II, was determined after acid hydrolysis with HCl or methanesulfonic acid. The analyses suggest that factor II is a nonapeptide which contains the following amino-acid residues: Asp, 2 Thr, Glu, Pro, Gly, Leu, Phe and Trp. This composition is almost identical to that of locust adipokinetic hormone I, lacking only one Asp residue.     

The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high performance liquid chromatography from the corpus cardiacum of the Colorado potato beetle, Leptinotarsa decemlineata. The sequences of both peptides, designated Led-CC-I and Led-CC-II, were determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. The C-terminal of both peptides were blocked and neither molecule was cleaved by carboxypeptidase. Both peptides were found to be octapeptides; Led-CC-I has the primary structure pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, and Led-CC-II has the primary sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂. These structures are identical to the two hypertrehalosaemic hormones from the American cockroach. Preliminary experiments show that the synthetic peptides are apparently involved in the control of amino acid metabolism during flight of the potato beetle.     

Structure-function studies on neurohormone D: activity of naturally-occurring hormone analogues

Summary The relative potencies of 11 naturally-occurring peptides of the adipokinetic hormone/red pigmentconcentrating hormone family (AKH/RPCH-family) have been assessed with respect to increase in heart rate in adult, female American cockroaches,Periplaneta americana, in in vitro and in vivo bioassays. In addition, analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus were also investigated. In both bioassays the N- or C-terminal-modified analogues give no or little response suggesting that blocked termini are essential for receptor-binding. In both bioassays the naturally-occurring peptide from the cockroach corpus cardiacum Pea-CAH-I (neurohormone D) is more potent than the second endogenous peptide, Pea-CAH-II. On the basis of this result and previous data it is proposed that neurohormone D is the only physiologically important true cardioactive peptide. The dose-response curves of the other peptides indicate that in octapeptides, amino acid residues at positions 2, 6, and 7 are important for receptor-recognition, and that decapeptides are not as effective as octapeptides (exception: the peptide Rom-CC-I isolated from the grasshopperRomalea microptera).     

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.     

Structure–activity relationship of adipokinetic hormone analogs in the striped hawk moth,Hippotion eson

We showed previously that the sphingid moth Hippotion eson synthesizes the highest number of adipokinetic hormones (AKHs) ever recorded, viz. five, in its corpus cardiacum: two octa-, two nona- and one decapeptide. Further, the endogenous decapeptide (Manse-AKH-II) and the other four AKHs are all active in lipid mobilization, whereas a non-lepidopteran decapeptide (Lacsp-AKH, five amino acid substitutions compared with Manse-AKH-II), was inactive in H. eson. We tested the decapeptide, Lacol-AKH, from a noctuid moth for the first time in a bioassay and it shows a maximal AKH effect in H. eson. Lacol-AKH differs from Manse-AKH-II in three places and from Lacsp-AKH in four places. We, thus, used Lacol-AKH as a lead peptide on which a series of AKH analogs are based to represent: (a) single amino acid replacements (according to the substitutions in Lacsp-AKH), (b) shorter chain lengths, (c) modified termini, and (d) a replacement of Trp in position 8. These analogs, as well as a few naturally occurring AKHs from other lepidopterans were tested in in vivo adipokinetic assays to gain insight into the ligand–receptor interaction in H. eson. Our results show that the second and third amino acids are important for biological activity in the sphingid moth. Analogs with an N-[acetylated]Glu¹ (instead of a pyroGlu), or a free C-terminus, or Ala⁸ were not active in the bioassays, while shortened Lacol-AKH analogs and the undecapeptide, non-amidated Vanca-AKH showed very reduced activity (below 25%). This information is important for the consideration of peptide mimetics to combat specific lepidopteran pest insects.     

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.     

Sequence analyses of two neuropeptides of the AKH/RPCH-family from the lubber grasshopper, Romalea microptera

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high-performance liquid chromatography from the corpus cardiacum of the lubber grasshopper, Romalea microptera. The sequences of both peptides, designated Ro I and Ro II, were determined by gas-phase sequencing employing Edman degradation after the N-terminal pyroglutamate residue was enzymatically deblocked, as well as by fast atom bombardment mass spectrometry. Ro I was found to be a decapeptide with the primary structure: pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, whereas Ro II is an octapeptide with the structure: pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2. Ro II is identical with AKH-G isolated from the cricket Gryllus bimaculatus. Synthetic materials having the assigned structures were found to be chromatographically, mass spectrometrically, and biologically indistinguishable from the natural peptides, confirming the sequences and establishing the Romalea peptides as members of the AKH/RPCH-family of peptides.     

20-Hydroxy-ecdysone as a modulator of electrical activity in neurosecretory cells of Rhodnius prolixus

Ovariectomy of the adult female Rhodnius results in a reduction of the electrical activity of the corpus cardiacum. Injection of 20-hydroxy-ecdysone into ovariectomized animals increases the activity to control values. Incubation of heads isolated from ovariectomized females in 1.0 × 10^?7 M 20-hydroxy-ecdysone elicits a recruitment of large-amplitude spikes and the appearance of bursting activity in the corpus cardiacum of mated females which is characteristic of neurohormone release. Simultaneous recordings from the median neurosecretory cells and the corpus cardiacum demonstrate that the action neurosecretory cells.     

Isolation and identification of AKH/RPCH family peptides in blister beetles (Meloidae)

Abstract. Two peptides were isolated from methanolic extracts of corpora cardiaca of the blister beetle, Decupotoma lunata , by a single-step purification procedure, utilizing C-18 reversed-phase high-performance liquid chromatography (RP-HPLC) for separation, and the increase of haemolymph lipids in Locusta migratoria for bioassay. The native peptides were analysed by matrix-assisted laser-desorption ionization mass spectrometry revealing main ions at m/z 1180 and 1009 respectively which were attributed to the [M + Na]⁺ form of the respective peptides. After deblocking of the N-terminal pyroglutamate residue of each peptide, the structures of the deblocked peptides were determined by pulsed-liquid phase sequencing employing Edman chemistry. The sequences of the two peptides, (1) pGlu-Leu-Asn-Phe-Ser-Pro-Am-Trp-Gly-AsnNH₂ and (2) pGlu-Leu-Asn-Phe-Ser-Pro-Asn-TrpNH₂, characterize them as deca- and octapeptide members of the AKH/RPCH family. Whereas the decapeptide is a novel member of this family and is given the acronym Del-CC ( Decupotoma lunata corpus cardiacum peptide), the octapeptide has previously been found in tenebrionid beetles and has the acronym Tem-HrTH. The corpora cardiaca of two other species of blister beetles ( Cyaneolytta pectoralis and Mylabris coeca ) contain the same two peptides as D. lunata , as judged by RP-HPLC and biological activity. Neither a corpus cardiacum extract of Decupotoma lunata nor the synthetic peptides Del-CC and Tem-HrTH were active in mobilizing carbohydrates or lipids in the blister beetle.     

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 effect of constant darkness on the content of adipokinetic hormone, adipokinetic response and walking activity in macropterous females of Pyrrhocoris apterus (L.)

Abstract.  Changes in the content of adipokinetic hormone (AKH), the adipokinetic response and the walking activity of 10-day-old adult macropterous females of the firebug, Pyrrhocoris apterus (L.), reared under long-day (LD) photoperiod (LD 18 : 6 h) are compared with those exposed for 3 days to constant darkness (DD). Diel changes of all the parameters studied in LD females persist in females kept in constant dark. A positive correlation exists between diel changes of AKH content in the central nervous system (CNS) in the LD and DD females, and a negative correlation in the AKH level in haemolymph and walking activity. In addition, there is a positive correlation between diel changes of AKH level in haemolymph and walking activity in macropterous females reared under LD conditions, as well as in those transferred to constant darkness. The data suggest that there is some feedback between the release of AKH from CNS into the haemolymph and walking activity in macropterous females. Preliminary studies on the simultaneous expression of mRNA for the period gene and a positive reaction to an antibody against AKH in the same corpus cardiacum cells suggest that the period gene may be involved in regulating the AKH content in this gland.     

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.