A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera)

A new member of the AKH/RPCH family was isolated and identified from the corpora cardiaca of the firebug Pyrrhocoris apterus. The peptide was isolated in a single step by reversed phase HPLC and the structure deduced from the multiple MS (MS(N)) electrospray mass spectra and amino acid analysis as that of an octapeptide with the sequence pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH(2): this sequence was confirmed by synthesis. The synthetic peptide induced lipid mobilisation and stimulated locomotory activity in macropterous females. This peptide, designated as Pyrrhocoris apterus adipokinetic hormone (Pya-AKH), is the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera.     

The first decapeptide adipokinetic hormone (AKH) in Heteroptera: a novel AKH from a South African saucer bug, Laccocoris spurcus (Naucoridae, Laccocorinae)

A novel peptide of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family has been elucidated by mass spectrometry from the corpora cardiaca of an African saucer bug species, Laccocoris spurcus. It is the first decapeptide member found in the species-rich taxon Heteroptera, has the primary sequence pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Gly amide and is denoted as Lacsp-AKH. The first eight amino acids are identical to the octapeptide Anaim-AKH of the European saucer bug, Ilyocoris cimicoides. The synthetic peptide Lacsp-AKH elevates lipids upon injection into the hemolymph of L. spurcus at a low dose of 3 pmol. Swimming activity in this saucer bug also causes a significant increase in the lipid concentration in the hemolymph. Thus, both results point to an apparent function of the endogenous new decapeptide Lacsp-AKH in L. spurcus, namely, to regulate lipid mobilization. Isolation of an AKH peptide from the corpora cardiaca of the water bug Aphelocheirus aestivalis (Aphelocheiridae) resulted in the assignment of the octapeptide Anaim-AKH, supporting current phylogenies on the infraorder Nepomorpha.     

Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

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.     

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.     

Isolation and amino acid composition of the adipokinetic neuropeptide from the corpus cardiacum of the cricket, Gryllus bimaculatus

ABSTRACT. The corpora cardiaca of the cricket, Gryllus bimaculatus De Geer, contain peptidic material which elicits hyperlipaemia in migratory locusts and in crickets. Appreciable amounts of this adipokinetic factor are found only in the corpora cardiaca of the cricket, and not in other parts of the nervous system. The factor is heat-stable and retains its activity after incubation with the exopeptidases leucine aminopeptidase and carboxypeptidase A. Fractionation of a methanolic extract of corpora cardiaca from Gryllus bimaculatus by RP-HPLC shows that the adipokinetic activity is confined to a single absorbance peak. The amino acid composition of the purified adipokinetic neuropeptide was determined after acid hydrolysis with HCI and with methanesulfonic acid. The factor is an octapeptide containing the following amino acid residues in equimolar amounts: Asx, Thr, Ser, Glx, Gly, Val, Phe and Trp. This composition closely resembles the AKHI II molecule from Schistocerca species , and the Gryllus peptide can be assigned to the AKH/RPCH family of neuropeptides.     

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.     

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.     

A novel adipokinetic peptide in a water boatman (Heteroptera, Corixidae) and its bioanalogue in a saucer bug (Heteroptera, Naucoridae)

The corpora cardiaca (CC) of two water bug species, the water boatman Corixa punctata and the saucer bug Ilyocoris cimicoides, contain a substance that cause hyperlipemia in the migratory locust. The primary sequence of one octapeptide belonging to the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family was deduced from the multiple MS(N) electrospray mass data of CC material from each species. Whereas the saucer bug contains the known octapeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Anaim-AKH, the water boatman has a novel peptide identified as pGlu-Leu/Ile-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Corpu-AKH. The ambiguity about the amino acid at position 2, i.e. Leu or Ile, in Corpu-AKH was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with the synthetic peptide containing Leu at position 2. Functionally, the peptides regulate lipid mobilization, as evidenced by an adipokinetic effect after injecting synthetic Anaim-AKH and Corpu-AKH into the respective acceptor species. Swimming activity of I. cimicoides also causes hyperlipemia.     

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.     

Identification of the adipokinetic hormone of the large milkweed bug Oncopeltus fasciatus

Abstract The adipokinetic hormone (AKH) of the large milkweed bug Oncopeltus fasciatus is isolated from an acidified methanolic extract of 200 corpora cardiaca, purified by single step reversed phase high‐performance liquid chromatography (HPLC) and N‐terminally deblocked using pyroglutamate aminopeptidase. The sequence is identified by Edman degradation and matrix assisted laser desorption/ionization‐time of flight mass spectroscopy as pGlu‐Leu‐Asn‐Phe‐Ser‐Pro‐Asn‐Trp amide. This structure is confirmed by chemical synthesis and coelution of native and synthetic peptide on HPLC. The AKH of O. fasciatus is identical to Tenmo‐HrTH, a member of the adipokinetic/red pigment‐concentrating hormone peptide family that had been isolated earlier from several tenebrionid beetles. Tenmo‐HrTH causes a significant rise in the concentration of haemolymph lipids when injected into adult male and female O. fasciatus, but displays no hyperglycaemic activity. There is no indication of the presence of other AKHs in O. fasciatus. The large milkweed bug represents the first member of the seed bugs (Lygaeidae) for which the endogenous AKH has been identified.     

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.     

The first identified neuropeptide in the insect order Megaloptera: a novel member of the adipokinetic hormone family in the alderfly Sialis lutaria

This is the first report on the structural identity of a neuropeptide of the insect order Megaloptera. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca glands of the alderfly, Sialis lutaria. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Thr-Phe-Thr-Pro-Ser-Trp amide. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by comparing retention time on reversed-phase HPLC and establishing co-elution with the synthetic Leu(2)-form which also had exactly the same MS(2) mass spectra as the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which has already more than 40 members. Interestingly, the primary structure is identical to that predicted from genome information for the adipokinetic hormone of the yellow fever mosquito, Aedes aegypti. Since alderflies are not known for their active flight metabolism but produce a rather high number of eggs, it is anticipated that the alderfly is a good study object to establish a possible role of the novel peptide to regulate fat mobilization from the fat body and transport into the egg, thereby playing a role in the control of reproductive processes.     

Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera).

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.     

Hypertrehalosaemic peptides in the honeybee (Apis mellifera): purification,identification and function

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation.     

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 content of the corpora cardiaca in gregarious and solitary migratory locusts

Abstract. The adipokinetic hormone (AKH-I and AKH-II) content of the corpora cardiaca from adult males of crowded (gregarious) and isolated (solitary) Locusta migratoria migratorioides (Reiche & Fairmaire) was quantified by reverse-phase high-performance liquid chromatography.Significantly less total hormone was found in the corpora cardiaca of crowded locusts than in those glands of isolated locusts at the age of 12–19 days after fledging.The ratio of AKH-I/AKH-II was higher in crowded than in isolated locusts at this age.From the age of 12–19 days to that of 25–30 days, AKH content increased significantly in the corpora cardiaca of crowded locusts, but no such increase was found in the glands of isolated locusts, and at 25–30 days there were no significant differences in the AKH content of the glands from crowded and isolated locusts.     

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.     

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.     

Isolation and identification of the AKH III precursor-related peptide from Locusta migratoria

We have isolated an 8770Da peptide from extracts of corpora cardiaca of adult male and female Locusta migratoria. The N-terminal amino-acid sequence as partially established by Edman degradation is Ala-Leu-Gly-Ala-Pro-Ala-Ala-Gly-Asp. These nine amino acids correspond to the first nine N-terminal amino acids of the adipokinetic hormone precursor-related peptide gamma-chain (APRP-gamma), a peptide that is predicted from the gene encoding the adipokinetic hormone III precursor. The APRP-gamma chain has a monoisotopic mass of 4387Da and contains two cysteine residues. It is known that both AKH I and AKH II precursors occur as dimers. After processing they give rise to the active hormones and three dimeric (two homodimers and one heterodimer) adipokinetic hormone precursor related peptides (APRPs). Based on the mass of 8770Da and the established N-terminal sequence tag, we conclude that the isolated peptide is a homodimer consisting of two APRP-gamma units, covalently linked to each other by two disulphide bounds. In analogy with the previous identified APRPs (APRP-1, APRP-2, and APRP-3), this APRP will be designated as APRP-4.