A possible molecular ancestor for mollusk APGWamide,insect adipokinetic hormone,and crustacean red pigment concentrating hormone

Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar α helix–random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families. The APGWamide genes are larger than the AKH/RPCH genes. The sequence environment occupied by introns is similar in AKH/RPCH and APGWamide genes. Such similarities suggest that these peptide families might have been originated by gene rearrangements from a common ancestor having either an AKH/RPCH/APGWamide-like structure or both an AKH/RPCH-like and an APGWamide-like structures. In the former model, DNA fragments could have been gained when the ancestor evolved to mollusks and it could have lost nucleotides when the progression to mandibular arthropods took place. In the second model, AKH/RPCH-like structures could have been fused during evolution toward mandibular arthropods, whereas in mollusks they could have been lost with the possible amplification of the APGWamide-like structure. Loss of domains in exon 1 may have originated the signal peptide and the first codon of the active RPCH. In exon 2, loss of domains possibly determined the junctions of codons 2 to 5 with the loss of a APGWamide copy; exon 3 underwent fewer variations. The similarity of the mollusk APGWamide precursors is closer to that of the RPCH family than the insect AKH family, indicating an earlier evolutionary departure.     

The adipokinetic hormones of Heteroptera: a comparative study

The adipokinetic hormones (AKHs) from 15 species of heteropteran Hemiptera (encompassing eight families, six superfamilies and three infraorders) have been isolated and structurally identified using liquid chromatography coupled with mass spectrometry. None of the structures are novel and all are octapeptides. These peptide sequence data are used, together with the previously available AKH sequence data on Heteroptera, to create a larger dataset for comparative analyses. This results, in total, in AKH sequences from 30 species (spanning 13 families), which are used in a matrix confronted with the current hypotheses on the phylogeny of Heteroptera. The expanded dataset shows that all heteropterans have octapeptide AKHs; three species have two AKHs, whereas the overwhelming majority have only one AKH. From a total of 11 different AKH peptides known from Heteroptera to date, three AKHs occur frequently: Panbo‐red pigment‐concentrating hormone (RPCH) (×10), Schgr‐AKH‐II (×6) and Anaim‐AKH (×4). The heteropteran database also suggests that particular AKH variants are family‐specific. The AKHs of Heteroptera: Pentatomomorpha (all terrestrial) are not present in Nepomorpha (aquatic) and Gerromorpha: Gerridae (semiaquatic); AKHs with a Val in position 2 are absent in the Pentatomomorpha (only AKHs with Leu² are present), whereas Val² predominates in the nonterrestrial species. An unexpected diversity of AKH sequences is found in Nepomorpha, Nepoidea, Nepidae and Nepinae, whereas Panbo‐RPCH (which has been identified in all infraorders of decapod crustaceans) is present in all analysed species of Pentatomidae and also in the only species of Tessaratomidae investigated. The molecular evolution of Heteroptera with respect to other insect groups and to crustaceans is discussed     

Water scorpions (Heteroptera, Nepidae) and giant water bugs (Heteroptera, Belostomatidae): sources of new members of the adipokinetic hormone/red pigment-concentrating hormone family

Two novel octapeptide members of the AKH/RPCH family have been identified from the corpora cardiaca (CC) of two species of water bugs. The giant water bug Lethocerus indicus (family: Belostomatidae) contains a peptide code-named Letin-AKH with the sequence pGlu-Val-Asn-Phe-Ser-Pro-Tyr-Trp amide, and the water scorpion Nepa cinerea (family: Nepidae) has the peptide code-named Nepci-AKH with the sequence pGlu-Leu/Ile-Asn-Phe-Ser-Ser-Gly-Trp amide. The sequences were deduced from the multiple MS(N) electrospray mass data from crude CC extracts. Synthetic peptides were made and co-elution on reversed-phase high performance liquid chromatography (RP-HPLC) with the natural peptide from crude gland extract confirmed the accuracy of the deduced sequence for Letin-AKH and demonstrated that Nepci-AKH contains a Leu residue at position 2 and not an Ile residue. A previously characterized member of the AKH/RPCH family was identified in the stick water scorpion Ranatra linearis by mass spectrometry: Grybi-AKH (pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp amide) has the same mass (919 Da) as Nepci-AKH and differs in two positions from Nepci-AKH (residues 2 and 6). The apparent function of the peptides is to achieve lipid mobilization in the species under investigation; indications for this came from conspecific bioassays using the appropriate synthetic peptides for injecting into the insects. This function is very likely linked to dispersal flight metabolism of water bugs. Swimming activity in N. cinerea also results in an increase in lipid concentration in the hemolymph.     

The adipokinetic hormone family in Chrysomeloidea: structural and functional considerations

The presented work is a hybrid of an overview and an original research paper on peptides belonging to the adipokinetic hormone (AKH) family that are present in the corpora cardiaca of Chrysomeloidea. First, we introduce the AKH/red pigment-concentrating hormone (RPCH) peptide family. Second, we collate the available primary sequence data on AKH peptides in Cerambycidae and Chrysomelidae, and we present new sequencing data (from previously unstudied species) obtained by liquid-chromatography coupled with ion trap electrospray ionisation mass spectrometry. Our expanded data set encompasses the primary structure of AKHs from seven species of Cerambycidae and three species of Chrysomelidae. All of these species synthesise the octapeptide code-named Peram-CAH-I (pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp amide). Whereas this is the sole AKH peptide in Cerambycidae, Chrysomelidae demonstrate a probable event of AKH gene duplication, thereby giving rise to an additional AKH. This second AKH peptide may be either Emppe-AKH (pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp amide) or Peram-CAH-II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp amide). The peptide distribution and structural data suggest that both families are closely related and that Peram-CAH-I is the ancestral peptide. We hypothesise on the molecular evolution of Emppe-AKH and Peram-CAH-II from the ancestral peptide due to nonsynonymous missense single nucleotide polymorphism in the nucleotide coding sequence of prepro-AKH. Finally, we review the biological significance of the AKH peptides as hyperprolinaemic hormones in Chrysomeloidea, i.e. they cause an increase in the circulating concentration of proline. The mobilisation of proline has been demonstrated during flight in both cerambycid and chrysomelid beetles.     

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.     

Crustacean Pigmentary-Effector Hormones: Chemistry and Functions of RPCH, PDH, and Related Peptides

Integumental color changes and eye pigment movements in crustaceansare regulated by pigmentary-effector hormones. The identifiedhormones include: an octapeptide RPCH (red pigment-concentratinghormone) and several forms of octadecapeptide PDH (pigment-dispersinghormone: -PDH, ß-PDH). RPCH-related peptides (AKHs,adipokinetic hormones) and PDH-related peptides (PDFs, pigment-dispersingfactors) occur in insects, and are recognized as members ofAKH/RPCH and PDH/PDF peptide families. The domain for maturepeptide is located between the signal peptide and precursor-relatedpeptide in AKH/RPCH precursors, and at the C-terminal end inthe PDH/PDF precursors. The precursor-related (associated) peptidesin RPCH and PDH precursors in Crustacea show little or no similarityto corresponding domains of AKH and PDF precursors in insects.Although the functions of precursor-related peptides are unknown,the mature peptides are shown to serve diverse functions. RPCH'sactions in crustaceans include: pigment concentration in oneor more types of chromatophores, dark-adaptational screeningpigment movement in distal eye pigment cells, increase of retinalsensitivity, and neuromodulation. The related AKHs largely influencemetabolism in insects, although they serve additional functions.PDHs trigger pigment dispersion in chromatophores and inducelight-adaptational screening pigment movements in extraretinulareye pigment cells. The related PDFs appear to serve as a transmitterof circadian signals in the regulation of biological rhythmsin insects. Evolutionary relationships among the PDH/PDF peptidesand directions for future research are discussed.     

Physiological actions by hypertrehalosemic hormone and adipokinetic peptides in adult Blaberus discoidalis cockroaches

Members of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family characteristically cause metabolite mobilization by the insect fat body. The present study identified several additional physiological actions in adult Blaberus discoidalis cockroaches that were influenced by synthetic Blaberus hypertrehalosemic hormone (HTH) and other AKH/RPCH family peptides. HTH elevated blood carbohydrate by 4-fold and cytochrome heme a + b synthesis of fat body mitochondria by 3-fold. Both carbohydrate and heme synthesis were dose-responsive to HTH. Carbohydrate synthesis was 10 times more sensitive to HTH than heme synthesis. Heme synthesis was also stimulated by Periplaneta cardioacceleratory hormones (CAH)-I and -II and RPCH but not by AKH-I or -II, at the doses tested. HTH showed strong cardioexcitatory activity. Long-term treatment of decapitated female B. discoidalis with juvenile hormone analog (JHA = methoprene) stimulated by 2.6-fold the rate of synthesis of secreted fat body proteins. HTH enhanced the JHA-dependent export protein synthesis by 42% above that observed with JHA alone. SDS-PAGE demonstrated that JHA determined the nature of the newly synthesized polypeptides; HTH enhanced their synthesis rate. Neither AKH-I nor HTH affected protein synthesis when added directly to isolated fat body. These results demonstrate that peptides of the AKH/RPCH family have multiple physiological actions related to fat body energy metabolism.     

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.     

Molecular dynamics study on an adipokinetic hormone peptide in aqueous solution

Lom-AKH-I is a member of the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family of peptides found in flying insects. A molecular dynamics simulation at room temperature (293 K) in water has been performed to survey the folding path of the Lom-AKH-I peptide in water and to establish the secondary structure of Lom-AKH-I. The obtained results indicate the presence of an undefined extended conformation.     

Invertebrate neuropeptide hormones

The development of a long-term research program on the neurosecretory hormones of arthropods is described. The purification and full characterization of the first invertebrate neurohormones, the red pigment-concentrating hormone (RPCH) and the distal retinal pigment hormone (DRPH) demonstrated that they are peptides, an octapeptide and an octadecapeptide, respectively. Physiological function studies with the pure hormones and their synthetic preparations showed that the RPCH acts as a general pigment-concentrating hormone (PCH), and that the DRPH, in addition to its light-adaptive function, also constitutes a general pigment-dispersing hormone (PDH). In the regulation of the color-adaptation of the animals, the two hormones act as antagonists. The chromatophorotropic activities are widely distributed within the arthropod neuroendocrine systems. Purification of the pigment-concentrating activities from the locust corpora cardiaca lead to the isolation and characterization of the first insect neurohormones, the adipokinetic hormones (AKH I and AKH II). These two hormones, AKH I being a decapeptide and AKH II being an octapeptide, are close structural analogs to the crustacean PCH, demonstrating a common evolution of arthropod neurohormones. The hormones of this PCH-family all cross-react, but structure-function studies of the hormones show that quite different parts of their structure are involved in their binding to the various receptors.     

The putative ancestral peptide of the adipokinetic/red-pigment-concentrating hormone family isolated and sequenced from a dragonfly

A neuropeptide with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana was purified by reversed-phase high performance liquid chromatography from the corpus cardiacum of the dragonfly, Libellula auripennis. After brief enzymatic digestion by 5-oxoprolyl-peptidase the primary structure of the peptide was determined by pulsed-liquid phase sequencing employing Edman degradation. As the peptide was not cleaved by carboxypeptidase the C-terminus was blocked, too. The peptide was assigned as a blocked uncharged octapeptide: Glu-Val-Asn-Phe-Thr-Pro-Ser-TrpNH2. The synthetic peptide was chromatographically indistinguishable from the natural compound and, upon injection in low quantities into dragonflies, elicited mainly haemolymph lipids. Therefore it is called dragonfly adipokinetic hormone (Lia-AKH). It is a new member of the large AKH/RPCH family of peptides. Because of its structural features and its origin from a very primitive insect order it is assumed to represent the putative ancestral peptide of this family. Synthesis was shown to occur in the corpus cardiacum by in vitro incorporation of tritium-labelled Trp into Lia-AKH.     

Structure-activity studies on adipokinetic hormones in Manduca sexta.

Structure-activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N-terminal was found to be important for activity of peptides; however, Manduca AcGly1AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a beta-bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.     

Preliminary characterization of enzyme activities in Malpighian tubules involved in the breakdown of adipokinetic hormones

Adipokinetic hormones (AKH) from different insect species, crustacean red pigment-concentrating hormone (RPCH), and synthetic substrates were used to characterized enzyme activities present in the Malpighian tubules (MT) of the desert locuts, Schistocerca gregaria, which are involved in the degradation of AKH. When peptides containing proline (position 6) were incubated with MT homogenate they were cleaved by a post-proline cleaving enzyme (PPCE). The presence of such an enzyme was confirmed by the breakdown of a synthetic substrate for PPCE. Peptides which do not contain proline were broken down by a post-phenylalanine cleaving enzyme (PFCE) which could be chymotrypsin or chymotryptic. This PFCE activity(ies) seem(s) to be inactive on the proline-containig peptides or their fragments or digests these at a slow rate. The C-terminal chymotrypsin fragments of the AKHs were broken down by MT homogenates with no accumulation of new intermediate products. It is not clear whether another endopeptidase, PPCE, or leucine aminopeptidase (LAP) is responsible. The MTs contain LAP activity; however, this enzyme(s) may be different from its vertebrate counterpart(s). Homogenates of MTs break down equimolar amounts of Pro-7AMC at approximately the same rate, while porcine kidney LAP (cytosol) cleaved Pro-7AMC much slower than Leu-7AMC. The demonstration of carboxypeptidase (CP) A and B activity in the MTs was not possible using conventional substrates such as hippuryl derivatives of amino acids. When CPA from porcine pancreas was added to MT homogenates hippuryl-phenylalanine was digested proving that the conditions were appropriate for CPA activity to occur. The treatment of a N-terminally blocked peptide fragment with MT homogenate led to the breakdown of the peptide giving evidence that the MT CP requires a substrate with a somewhat longer length of amino acid residues.     

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.     

Pyrgomorphid grasshoppers of the genus Phymateus contain species-specific decapeptides of the AKH/RPCH family regulating lipid-mobilization during flight

Abstract. Using heterologous and conspecific bioassays, two peptides have been isolated from methanolic extracts of corpora cardiaca from the pyrgomorphid grasshopper Phymateus morbillosus L.The structures of both peptides were elucidated by a combination of Edman degradation, after deblocking the N-terminal pyroglutamic acid residue, and mass spectrometric techniques.One peptide is an octapeptide (pGlu-Leu-Asn-Phe-Ser-Thr-Gly-TrpNH₂) which also occurs in other insects and is code-named Scg-AKH-II.The second peptide is a novel decapeptide member of the AKH/RPCH family (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-SerNH₂ code-named here Phm-AKH.It is the first example of a different peptide in the same genus.The analysis of changes of metabolites in the haemolymph, fat body and flight muscles of male P.morbillosus during a 30 min flight and rest after flight reveal an overall picture of flight metabolism similar to that of Locusta migratoria. Carbohydrate-fuelled metabolism is pronounced during the first 15 min of flight, whereas lipid-based metabolism is mainly used thereafter.By analogy with work on L.migratoria , it is concluded that the endogenous peptides of P.morbillosus regulate these metabolic events.     

Vanessa cardui adipokinetic hormone (Vanca-AKH) in butterflies and a moth

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.     

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata

Summary A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the LomAKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.     

Chemical Properties and Physiological Actions of Crustacean Chromatophorotropins

The crustacean pigment-translocating hormones, the red pigment-concentratinghormone (RPCH), an octapeptide, and the light-adapting distalretinal pigment hormone (DRPH), an octadecapeptide, are thefirst invertebrate neurohormones to be fully characterized.Studies with both purified and synthetic hormones show that,in certain decapods, RPCH is a general pigment-concentratinghormone (PCH), affecting the pigments of all kinds of chromatophores(erythrophores, xanthophores, leucophores and melanophores);the DRPH seems to serve not only light-adapting function, butalso act as a general chromatophore pigment-dispersing hormone(PDH). The two hormones thus function as antagonists when regulatingthe color-adaptation of the decapod crustaceans. PCH activityis widely distributed within the arthropod endocrine systems.The first characterized insect neurohormones, the locust adipokinetichormones (AKH I and AKH II), show close structural similaritiesto the crustacean hormone, indicating a common evolution ofsome of the arthropod neurohormones. Physiological studies ofthe three hormones (RPCH, AKH I, and AKH II) and their syntheticanalogs show that they crossreact, i.e., they all exhibit pigment-concentratingactivity when tested on decapod crustaceans, adipokinetic activitywhen tested on locusts, and hyperglycemic activity when testedon cockroaches, although each of the hormones is more potentin its own system. Structure-function studies show, however,that quite different binding-site requirements exist for thehormones in activating their receptors on the various targettissues. The physiological specificity in their action thereforeseems to depend on a differential evolution of the hormone receptors.     

The potential of adipokinetic hormone to teach us about neuropeptides

Genetic manipulation and purification techniques are facilitating research into the biology of arthropod neuropeptides. The red pigment concentrating hormone (RPCH)/ adipokinetic hormone (AKH) family are a conserved group of peptides which were first recognized for their hormonal activities. Biosynthesis of AKH in the grasshopper seems to proceed via a large protein precursor (12 kDa) in the cell body of the neuron, which implies precise coordination of synthesis and neural activity. Beginning with a 10 amino acid stretch of known precursor protein sequence, an oligonucleotide primer approach is being used to clone a recombinant AKH gene. Once achieved, this will allow fundamental questions on the biochemistry and physiology of neuropeptides to be addressed. Such work in an arthropod system raises the intriguing possibility of studying neuropeptide level and synthetic activity in single, defined neurons.     

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.