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.     

Phenolic acids and resistance to insect attack in Sorghum bicolor

Glucose and quinic acid esters of several hydroxybenzoic and cinnamic acids were identified in methanolic extracts of leaves of 15-day-old Sorghum bicolor together with two O-glucosides. Some of the phenolic acids were also found to occur as insoluble esters of cell wall polysaccharides. While these derivatives did not affect the feeding of Locusta migratoria on sorghum, the free acids, as a mixture, were markedly inhibitory. It was found that Sorghum contained a mixture of hydrolases which could effect the transformation of “inactive” phenolic esters and glycosides into “active” phenolic acids at a high enough concentration to significantly reduce feeding by Locusta.     

Some effects of mermithid infection on metabolic reserves and flight in Locusta

Low infections of Mermis nigrescens reduce the flight performance of adult male Locusta migratoria, and bring about changes in the levels of blood carbohydrate and protein but not lipid. The quantities of all three metabolites in the fat body are reduced, as is the amount of tissue present. Lipid content only, is markedly decreased in the flight muscle, but the water content is increased.

Blood volume and malpighian tubule activity are unaffected by mermithid infection, but the rates of lipid mobilisation in response to injections of extracts of corpora cardiaca or flight are greatly reduced compared with those in uninfected locusts. Additional carbohydrate does not correct flight performance in parasitised locusts as it does in starved animals. Poor flight performance is not due to an effect of the worm on the host endocrine system.     

The Leucophaea maderae hindgut preparation: A rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species

The isolated hindgut preparation of the cockroach, Leucophaea maderae has provided an effective bioassay tool for the isolation of certain structural types of insect myotropic peptides. Initially, the preparation was used to monitor excitatory and inhibitory activities of numerous HPLC fractions in a study that resulted in the structural characterization of 12 Leucophaea neuropeptides. Subsequently, the preparation was used as the bioassay for the isolation and structural characterization of myotropic neuropeptides of the house cricket, Acheta domesticus, and the locust, Locusta migratoria. Five novel myotropic peptides from the cricket were structurally characterized, and 32 separate myotropic compounds were isolated from nervous tissue of the locust. At present, 8 of the locust peptides have been structurally characterized. Isolation studies using this bioassay have been responsible for the discovery of 25 unique neuropeptides, 4 new peptide families, and the initial demonstration of the natural analog phenomenon in insects.     

Regulatory peptides in parasitic platyhelminths

Regulatory peptides are short chains of amino acids that regulate cell-to-cell interactions in widely divergent animal groups. Evidence is accumulating to suggest that they mediate many aspects of physiology and behaviour, serving as neurotransmitters, neuromodulators and hormones. While most data in this field derive from studies on the mammalian nervous and endocrine systems, the last decade has witnessed an upsurge of interest in invertebrate peptide biology, not least because it is likely that many regulatory peptides originated in the nervous system of invertebrates. Platyhelminths, like other invertebrate groups investigated, contain numerous neuropeptides, and here David Halton and colleagues review the evidence that these putative signalling agents serve key roles in parasite motility, reproduction and morphogenesis. The physicochemical differences between host and parasite peptides raise the possibility that selective disruption of peptidergic control systems in parasites could be an exploitable target in future chemotherapeutic strategies.     

Coelenterate Neuropeptides: Structure, Action and Biosynthesis

Evolutionary "old" nervous systems such as those of coelenteratesare peptidergic: Using various radioimmunoassays we have nowisolated 13 novel neuropeptides from sea anemones and severalothers from hydrozoan polyps and medusae. These peptides areall structurally related and contain the C-terminal sequenceArg-X-NH₂ or Lys-X-NH₂, where X is Ala, Asn, Ile, Phe, Pro orTrp. Three neuropeptides have a novel N-terminal L-3-phenyllactylresidue, which protects against degradation by nonspecific aminopeptidases.The neuropeptides from sea anemones are produced by differentsets of neurones and have excitatory or inhibitory actions onisolated muscle preparations, suggesting that they are neurotransmittersor neuromodulators. We have also cloned the precursor proteinfor the sea-anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH₂).In Calliactis parasitica this precursor harbours 19 copies ofimmature Antho-RFamide (Gln-Gly-Arg-Phe-Gly) together with 7other, putative neuropeptide sequences. The precursor of Anthopleuraelegantissima contains 14 copies of Antho-RFamide and 19 other,putative neuropeptides. This shows that the biosynthetic machineryfor neuropeptides in coelenterates, the lowest animal grouphaving a nervous system, is already very efficient and similarto that of higher invertebrates, such as molluscs and insects,and vertebrates.     

Comparative peptidomics of four related hemipteran species: pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins

We performed the first comprehensive peptidomic analysis of neurohormones from hemipteran insects by analyzing the neuropeptides of two major neurohemal organs, namely the corpora cardiaca and abdominal perisympathetic organs. For the experiments we selected four related species of polyphagous stinkbugs (Pentatomidae), three of which are known to attack several important food crops. Peptide sequences were identified by MALDI-TOF mass spectrometry; tandem fragmentation of myosuppressin, sNPF, CAPA-periviscerokinins and pyrokinins revealed novel sequences not known from other insects so far. Most Leu/Ile and Glu/Lys ambiguities could be solved by either specific side-chain fragmentations or on-plate acetylation experiments. The identification of the specific sequences provides a solid basis for forthcoming pharmacological tests to study the neuroendocrine system of these pest insects. However, it should be mentioned in this context that the sequences of the peptides from different stinkbugs are likely not representative of Hemiptera in general. The forthcoming release of the genome from the reduviid Rhodnius prolixus will provide sufficient data to clear this point.     

A review of FMRFamide- and RFamide-like peptides in metazoa

Neuropeptides are a diverse class of signalling molecules that are widely employed as neurotransmitters and neuromodulators in animals, both invertebrate and vertebrate. However, despite their fundamental importance to animal physiology and behaviour, they are much less well understood than the small molecule neurotransmitters. The neuropeptides are classified into families according to similarities in their peptide sequence; and on this basis, the FMRFamide and RFamide-like peptides, first discovered in molluscs, are an example of a family that is conserved throughout the animal phyla. In this review, the literature on these neuropeptides has been consolidated with a particular emphasis on allowing a comparison between data sets in phyla as diverse as coelenterates and mammals. The intention is that this focus on the structure and functional aspects of FMRFamide and RFamide-like neuropeptides will inform understanding of conserved principles and distinct properties of signalling across the animal phyla.     

The occurrence of myotropic factors in the intestine and malpighian tubules of Locusta migratoria

1. A large number of myotropic factors, presumably of peptidic nature, were separated by means of HPLC (high-performance liquid chromatography) from extracts of foreguts, midguts, hindguts and the Malpighian tubules of Locusta migratoria.2. Some factors occur in the intestine as well as in the brain and are actually brain-gut peptides, similar to those described in vertebrates.3. Some factors occur in the whole alimentary canal, while others only occur in specific parts of the intestinal system.4. Two myotropic fractions were present in the Malpighian tubules and could not be detected in other investigated extracts.5. Some factors influenced the contraction pattern of the visceral muscle of the hindgut of Leucophea maderae as well as the oviduct of Locusta migratoria.6. Other peptidic fractions evoked a positive response on the locust oviduct, while they were inactive on the Leucophea hindgut and vice versa.7. In brief, the use of two different bioassays has enabled us to partially characterize new myotropic factors in insects. It becomes increasingly clear that the number of neuropeptides present not only in the nervous system, but also in the gastrointestinal system, of insects is much larger than has long been assumed.     

Apolipophorin III: a lipid-triggered molecular switch

Apolipophorin III (apoLp-III) is a low molecular weight exchangeable apolipoprotein that plays an important role in the enhanced neutral lipid transport during insect flight. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is the active form of the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. ApoLp-III is well characterized in two evolutionally divergent species: Locusta migratoria and Manduca sexta. The two apolipoproteins interact in a similar manner with model phospholipid vesicles, and transform them into discoidal particles. Their low intrinsic stability in the lipid-free state likely facilitates interaction with lipid surfaces. Low solution pH also favors lipid binding interaction through increased exposure of hydrophobic surfaces on apoLp-III. While secondary structure is maintained under acidic conditions, apoLp-III tertiary structure is altered, adopting molten globule-like characteristics. In studies of apoLp-III interaction with natural lipoproteins, we found that apoLp-III is readily displaced from the surface of L. migratoria low-density lipophorin by recombinant apoLp-III proteins from either L. migratoria or M. sexta. Thus, despite important differences between these two apoLp-IIIs (amino acid sequence, presence of carbohydrate), their functional similarity is striking. This similarity is also illustrated by the recently published NMR solution structure of M. sexta apoLp-III wherein its molecular architecture closely parallels that of L. migratoria apoLp-III.     

New physiological activities of myosuppressin,sulfakinin and NVP-like peptide in <Emphasis Type="Italic">Zophobas atratus</Emphasis> beetle

Three neuropeptides Zopat-MS-2 (pEDVDHVFLRFa), Zopat-SK-1 (pETSDDYGHLRFa) and Zopat-NVPL-4trunc. (GRWGGFA), recently isolated from the neuroendocrine system of the Zophobas atratus beetle, were tested for their myotropic and hyperglycaemic activities in this species. These peptides exerted differentiated dose-dependent and tissue specific physiological effects. Zopat-MS-2 inhibited contractions of the isolated heart, ejaculatory duct, oviduct and hindgut of adult beetles and induced bimodal effects in the heart contractile activity of pupae in vivo. It also increased the haemolymph free sugar level in larvae of this species, apart from myotropic activity. Zopat-SK-1 showed myostimulatory action on the isolated hindgut of the adult beetles, but it decreased contractions of the heart, ejaculatory duct and oviduct. Injections of this peptide at a dose of 2 μg also caused delayed cardioinhibitory effects on the heartbeat of the pupae. Together with the ability to increase free sugar level in the haemolymph of larvae these were new physiological activities of sulfakinins in insects. Zopat-NVPL-4trunc. inhibited the muscle contractions of the two organs: hindgut and ejaculatory duct but it was inactive on the oviduct and the heart of the adult beetles. This peptide also increased free sugar level concentration in the haemolymph of Z. atratus larvae. These physiological actions are the first biological activities discovered for this group of the insect peptides. The present work showed pleiotropic activity of three neuropeptides and indicates that the visceral muscle contractions and the haemolymph sugar homeostasis in Z. atratus are regulated by complex mechanisms.     

Prolactin-releasing peptides

Physiologic control of prolactin (PRL) secretion is largely dependent upon levels of dopamine accessing the adenohypophysis via the hypophysial portal vessels. However, it is clear that other factors of hypothalamic origin can modulate hormone secretion in the absence or presence of dopamine. Several neuropeptides have been identified as PRL releasing factors (PRFs) but none of these peptides appears to be a major determinant of PRL secretion in vivo. There remain uncharacterized activities in hypothalamic extracts that can alter secretion and production of the hormone. In addition, there exist a wide variety of substances (neurotransmitters, neuromodulators, neuropeptides) that can act within the hypothalamus to modify the neuroendocrine regulation of PRL secretion. These factors may not be considered true PRFs because their actions are not exerted directly at the level of the lactotroph; however, they can act in brain to stimulate PRL release in vivo and therefore might be considered PRL releasing peptides (PRPs).     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Two major proteins from locust plasma are involved in coagulation and are specifically precipitated by laminarin, a ß-1,3-glucan

Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260 000 (P260) and 85 000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260–P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260–P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260–P85 complex was added to a fully activated proPO system.     

Neurotransmitters and neuropeptides in the developing human central nervous system. A review

This is a review on the ontogenesis of major neurotransmitters and neuropeptides in the developing human central nervous system. In general, the molecules under study appeared early in development, usually in the first trimester. Cholinergic neurons were found to be present around the time of neuropeptide formation. The newly formed neuropeptidergic fibers extended towards the cholinergic centers where both might interact. In the major centers of the central nervous system, neuropeptides were also noted to colocalize with various neurotransmitters. For example, in the facial nucleus, enkepahlin and substance P fibers coexisted with cholinergic and catecholaminergic neurons, suggesting complex interactions. In the interpeduncular nucleus, peptidergic neurons acting as interneurons clearly modulated the afferent input to this nucleus. In the hippocampus and in sensory organs such as the retina, there were indications that neuropeptides and gamma-amino butyric acid coexisted. We hypothesize that interactions of neurotransmitters and peptides in neurons and fibers early in development play an indispensable role in the morphogenesis of the human central nervous system.     

Phe-X-Pro-Arg-Leu-NH(2) peptide producing cells in the central nervous system of the silkworm,Bombyx mori

Members of the neuropeptide family having Phe-X-Pro-Arg-Leu-NH(2) (FXPRLamide; X=Ser, Thr, Val, or Gly) at the C-terminus serve as regulators of oviduct and visceral muscle contraction, sex pheromone production, and diapause induction. Antibody raised against Bombyx mori diapause hormone recognized a variety of FXPRLamide peptides. Using this antibody, the antigen was immunocytochemically localized in the central nervous system (CNS) of the silkworm, Bombyx mori. Immunoreactive somata were observed in all ganglia of the CNS including the brain. Twelve somata localized at the midline of the suboesophageal ganglion (SG) were most intensely stained, and their neurite projections reached the retrocerebral complex. Thus, these cells in the SG exhibited typical features of neuroendocrine neurons. Marked reduction in immunoreactivity was observed in a pair of neurosecretory cells in the labial neuromere in SG of diapause type pupae, which indicates an active release of FXPRLamide peptides from these cells. No clear connection to neurohemal sites were observed in immunoreactive cells in the brain, thoracic or abdominal ganglia, suggesting that the immunoreactive peptides in these organs are likely to serve as neurotransmitters or neuromodulators.     

Newly discovered functions for some myotropic neuropeptides in locusts

The field of neuropeptide research in insects during the past twenty years can be characterized by the enormous number of peptides that have been identified. In the locusts, Locusta migratoria and Schistocerca gregaria only, structural information is now available for more than 60 peptides. Quite a number of these peptides were isolated on the basis of their effect on visceral muscle contraction in vitro. A very limited number of reports describe the 'in vivo' function of a myotropic neuropeptide. Moreover, for most of the brain neuropeptides, we ignore whether they have a hormonal function. In this paper, we describe the recently discovered in vivo effects of some of the myotropic peptides, identified in locusts in the past decade. Schistocerca-neuropeptide F accelerates egg development; locustasulfakinin inhibits food intake and [His(7)]-corazonin induces body color pigmentation.     

Opioid mechanisms in insects,with special attention toLeucophaea maderae

1. This review article provides information on the evolutionary history of neuroendocrine and related regulatory mechanisms. It focuses on the presence, diverse roles, and modes of operation of one class of neuropeptides, the endogenous opioids, in insects. 2. Opioid peptides, closely resembling those of vertebrates, have been identified in the brain and related neuroendocrine structures by means of immunocytochemistry and high-pressure liquid chromatography. 3. The demonstration of naloxone-sensitive, high-affinity binding sites for Met-enkephalin-like neuropeptides in the brain and digestive tract of Leucophaea deserves special attention because it provides new insights into the functional significance of opiate receptors paralleling those known in vertebrates. 4. Possible roles of receptor-mediated opioid systems in the insects discussed are regulation of the cyclicity of the female reproductive system, maintenance of normal midgut function mediated by the recurrent nerve, and locomotor activity.     

Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.