Identification of the cockroach neuropeptide Pea-CAH-II as a second adipokinetic hormone in the firebug Pyrrhocoris apterus

A new member of the AKH/RPCH family was isolated from the corpora cardiaca of the firebug Pyrrhocoris apterus. It is the second adipokinetic peptide identified in this species. The peptide was characterized and its structure was deduced from the multiple MS(N) electrospray mass spectra as that of an octapeptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH(2.) The peptide differs from the original P. apterus AKH (Pya-AKH) by one amino acid in position 3. Topical application and/or injection of the peptide induced lipid mobilization, but was inactive in mobilization of carbohydrates.     

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.     

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.     

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.     

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 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.     

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 comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Isolation and structure elucidation of a novel adipokinetic hormone (Lom-AKH-III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria

A new adipokinetic hormone (named Lom-AKH-III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (less than Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be [less than Glu]-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is less than Glu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I (less than Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species. Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.     

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.