Localization of Lom-AG-myotropin I-like substances in the male reproductive and nervous tissue of the locust,Locusta migratoria

Summary Lom-AG myotropin I (Lom-AG-MTI) was the first peptide to be isolated from the male accessory reproductive glands of the locust, Locust migratoria. It shows no sequence similarity to any of the peptides identified from vertebrate or invertebrate tissues. A polyclonal antiserum was used to localize Lom-AG-MTI-like material in the male reproductive system and nervous system of the locust. Immunoreactivity was found in two of the hyaline gland tubules. In the brain, cell bodies were detected in the proto- and deuterocerebrum as well as the frontal ganglion. Nerve fibers were stained in the neuropils of the brain and throughout the labial nerves into the recurrent nerve. Thoracic and last abdominal ganglia contained neurons which could be stained with Lom-AG-MTI antiserum. The pronounced reactivity in the central nervous system suggests a possible neuroregulatory function of the peptide.     

Pyrokinin/PBAN-like peptides in the central nervous system of mosquitoes

The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides is characterized by a common C-terminal pentapeptide, FXPRLamide, which is required for diverse physiological functions in various insects. Polyclonal antisera against the C-terminus was utilized to determine the location of cell bodies and axons in the central nervous systems of larval and adult mosquitoes. Immunoreactive material was detected in three groups of neurons in the subesophageal ganglion of larvae and adults. The corpora cardiaca of both larvae and adults contained immunoreactivity indicating potential release into circulation. The adult and larval brains had at least one pair of immunoreactive neurons in the protocerebrum with the adult brain having additional immunoreactive neurons in the dorsal medial part of the protocerebrum. The ventral ganglia of both larvae and adults each contained one pair of neurons that sent their axons to a perisympathetic organ associated with each abdominal ganglion. These results indicate that the mosquito nervous system contains pyrokinin/PBAN-like peptides and that these peptides could be released into the hemolymph. The peptides in insects and mosquitoes are produced by two genes, capa and pk/pban. Utilizing PCR protocols, we demonstrate that products of the capa gene could be produced in the abdominal ventral ganglia and the products of the pk/pban gene could be produced in the subesophageal ganglion. Two receptors for pyrokinin peptides were differentially localized to various tissues.     

Distribution of SchistoFLRFamide-like immunoreactivity in the adult ventral nervous system of the locust,Schistocerca gregaria

SchistoFLRFamide (PDVDHVFLRF-NH₂) is one of the major endogenous neuropeptides of the FMRF-amide family found in the nervous system of the locust,Schistocerca gregaria. To gain insights into the potential physiological roles of this neuropeptide we have examined the distribution of SchistoFLRFamide-like immunoreactivity in the ventral nervous system of adult locusts by use of a newly developed N-terminally specific antibody. SchistoFLRFamide-like immunoreactivity in the ventral nerve cord is found in a subgroup of the neurones that are immunoreactive to an antiserum raised against bovine pancreatic polypeptide (BPP). In the suboesophageal ganglion three groups of cells stain, including one pair of large posterior ventral cells. These cells are the same size, in the same location in the ganglion and have the same branching pattern as a pair of BPP immunoreactive cells known to innervate the heart and retrocerebral glandular complex of the locust. In the thoracic and abdominal ganglia two and three sets of cells, respectively, stain with both the SchistoFLRFamide and BPP antisera. In the abdominal ganglia the immunoreactive cells project via the median nerves to the intensely immunoreactive neurohaemal organs.     

PBAN/pyrokinin peptides in the central nervous system of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species. This research was supported in part by a US-Israel Binational Science Foundation Grant (no. 2003367).     

Occurrence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect,Locusta migratoria

Summary Using a radioimmunoassay developed for the determination of crustacean cardioactive peptide (CCAP), immunoreactive material was detected in extracts of locust nervous tissue. Serial dilutions of a brain extract gave a displacement curve parallel to the CCAP standard curve. One locust nervous system was calculated to contain approximately 1.4 pmol CCAP-like material.In order to investigate whether the immunoreactive substance was similar or identical to the crustacean neuropeptide, isolation and complete characterization was carried out using 800 locust nervous systems. The isolation procedure consisted of pre-purification of the crude extract on a Sep-Pak cartridge, affinity chromatography on a column which was prepared by coupling of anti-CCAP antibody to CNBr-activated Sepharose, and reversed phase high performance liquid chromatography (HPLC). In the HPLC-profile immunoreactivity was confined to a single peak which co-chromatographed with authentic CCAP. The peptide was carboxymethylated and analyzed in an automated gas-phase sequencer. Its amino acid sequence, is identical to that of CCAP fromCarcinus maenas.Synthetic CCAP was tested on the isolated locust hindgut in vitro. The peptide proved to be a potent enhancer of gut contractions, with a significant effect being observable at concentrations of 10^–10 M. It is concluded that in the locust CCAP may function as a myotropic peptide.     

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.     

An FMRFamide antiserum differentiates between populations of antigens in the ventral nervous system of the locust,Schistocerca gregaria

Summary The distribution of FMRFamide immunoreactive neurones in the ventral nerve cord of the locust, Schistocerca gregaria, is described. These neurones are found only in the suboesophagael and thoracic ganglia, although immunoreactive processes are found in the neuropils of the abdominal ganglia. Many of these neurones also react with an antiserum raised against bovine pancreatic polypeptide (BPP), but this antiserum also reveals another population of cells in the abdominal ganglia. The staining obtained with the BPP antiserum is blocked by preabsorption of the antiserum with FMRFamide; the converse is not true: FMRFamide-like immunoreactivity is not suppressed by preincubation with BPP. These results suggest that there are at least two endogenous peptide antigens in the locust nerve cord: one is found in cells of the suboesophageal and thoracic ganglia, and the other is found in cells of the abdominal ganglia.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Mass spectrometric analysis of the perisympathetic organs in locusts: identification of novel periviscerokinins

A mass spectrometric analysis carried out to determine the peptidome of the abdominal perisympathetic organs in the locust species Locusta migratoria and Schistocerca gregaria yielded a number of predominant ion peaks, among which are Lom-PVK (AAGLFQFPRVamide) and Scg-MT-2 (TSSLFPHPRLamide). In addition, three novel peptides were identified: Lom-PVK-2 (identical in Schistocerca): GLLAFPRVamide, Lom-PVK-3: DGGEPAAPLWFGPRVamide, and Scg-PVK-3: DGAETPGAAASLWFGPRVamide. An extensive mass spectrometric study of the central nervous system showed that the periviscerokinins (-PRVamides) and Scg-MT-2 (-FXXPRLamide) are restricted to the abdominal ganglia and their perisympathetic organs, while the pyrokinins (-FXPRLamides) are present only in the brain-retrocerebral complex. Sequence comparison with the Drosophila genes supports a conserved gene structure whereby a capability-like gene encodes the periviscerokinins that are expressed in the abdominal ganglia and stored in the perisympathetic organs, while a hugin-like gene encodes the pyrokinins that are expressed in the head ganglia and stored in the retrocerebral complex.     

Molecular cloning of two distinct precursor genes of NdWFamide,a d-tryptophan-containing neuropeptide of the sea hare, Aplysia kurodai

NdWFamide (NdWFa) is a d-tryptophan-containing cardioexcitatory neuropeptide in gastropod mollusks, such as Aplysia kurodai and Lymanea stagnalis. In this study, we have cloned two cDNA encoding distinct precursors for NdWFa from the abdominal ganglion of A. kurodai. One of the predicted precursor proteins consisted of 90 amino acids (NWF90), and the other consisted of 87 amino acids (NWF87). Both of the predicted precursor proteins have one NWFGKR sequence preceded by the N-terminal signal peptide. Sequential double staining by in situ hybridization (ISH) and immunostaining with anti-NdWFa antibody suggested that NdWFa-precursor and NdWFa peptide co-exist in neurons located in the right-upper quadrant region of the abdominal ganglion. In ISH, NWF90-specific signal and NWF87-specific one were found in different subsets of neurons in the abdominal ganglia of Aplysia. The expression level of NWF90 gene estimated by RT-PCR is much higher than that of NWF87 gene. These results suggest that NWF90 precursor is the major source of NdWFa in Aplysia ganglia.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC₅₀ = 18 nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.     

Gastrin-cholecystokinin-like immunoreactivity in the central nervous system of the milkweed bug,Spilostethus pandurus. Ultrastructural aspects of the reactive A cells of the brain

Summary— All the ganglia belonging to the central nervous system of adults of the milkweed bug Spilostethus pandurus (Hemiptera) were screened immunohistochemically for vertebrate gastrin-cholecystokinin (CCK-8(s))-like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin-positive, ie they are A-type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A-type cells reactive to CCK antiserum were identified, at the ultrastructural level, by combining paraldehyde fuschin staining of semithin sections with a post-embedding immunogold technique carried out on adjacent ultrathin sections. The neurosecretory cells contain numerous vesicles of elevated electron density. These data suggest that members of the CCK peptide family are present in the central nervous system of Spilostethus pandurus.     

Developmental and regional-specific expression of FLRFamide peptides in the tobacco hornworm,Manduca sexta,suggests functions at ecdysis

The developmental profile of a family of three FLRFamide (Phe-Leu-Arg-Phe-NH₂) peptides in the tobacco hornworm, Manduca sexta, revealed regional-specific expression patterns within the segmental ganglia. Levels of the three peptides—F7G (GNSFLRFamide), F7D (DPSFLRFamide), and F10 (pEDVVHSFLRFamide)—were always higher in the thoracic than abdominal ganglia. The predominant peptide also differed regionally, with F7G being highest in the thoracic ganglia and F7G and F10 being equivalent in the abdominal ganglia. Furthermore, we found regional-specific transient declines in ganglion peptide levels temporally correlated to ecdysis. Thoracic ganglion peptide levels declined at each molt, while abdominal ganglion levels declined over a period of 2 days after ecdysis. The decline in central levels was accompanied by an increase in levels in peripheral neurohemal sites, the transverse nerves (TNs). These observations suggest peptides were released from neurosecretory cells (NSCs) at ecdysis. Distinct sets of thoracic and abdominal NSCs and their processes in peripheral neurohemal sites were immunoreactive, supporting the biochemical data. These results also suggest the regional differences may arise from cellular-specific expression patterns for this family of peptides. In addition, fine immunoreactive processes were observed traveling between TNs and skeletal muscles, suggestive of myotropic actions. We propose that the release of different M. sexta FLRFamides from regionally distinct NSCs leads to a coordinated modulation of skeletal and visceral muscles that facilitate ecdysis. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 469–485, 1998     

Isolation,identification and synthesis of locustamyotropin II,an additional neuropeptide of Locusta migratoria: Member of the cephalomyotropic peptide family

An eight residue neuropeptide (Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-NH₂) has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata-suboesophageal ganglion complexes of Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The peptide designated as locustamyotropin II, or Lom-MT II according to Raina and Gäde (Insect Biochem.18, 785–787, 1988), has a Phe-X-Pro-Arg-Leu-NH₂ carboxyl-terminal in common with the previously identified locustamyotropin I. Locustamyotropin II is also related to leucopyrokinin (Lem-PK), a blocked myotropic neuropeptide isolated from cockroach heads. Both peptides have identical carboxyterminal pentapeptide sequences. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. Lom-MT II differs from Lem-PK in the first three aminoterminal residues. In contrast to Lem-PK and like Lom-MT I, the novel locust peptide is not N-terminally blocked. Lom-MT II has a stimulatory effect on the motility of the oviduct of Locusta but not on the hindgut.     

Discovery of multiple neuropeptide families in the phylum Platyhelminthes

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era.     

Peptide B induction of bag-cell activity in Aplysia: localization of sites of action to the cerebral and pleural ganglia

The bag cells of the marine mollusc Aplysia are model neuroendocrine cells involved in the initiation of egg laying and its associated behaviors, but the natural stimulus triggering bag-cell activity is not known. The atrial gland of A. californica, an exocrine organ in the reproductive tract, contains two structurally related peptides (A and B) which can induce an afterdischarge in vitro, and these peptides can be used to probe the central nervous system for sites where extrinsic excitatory input onto the bag-cell system might occur. These sites were identified in a series of lesion and ablation experiments. The entire central nervous system was removed from an animal and placed in a chamber with two compartments which could be independently perfused, allowing peptides (atrial gland extract or purified peptide B) to be selectively applied to specific regions of the nervous system while bag-cell activity was monitored using extracellular suction electrodes. Afterdischarges were consistently and reliably induced when peptides were applied to the cerebral ganglion, the pleural ganglia, the cerebropleural connectives, or the rostral 10-15% of the pleurovisceral connectives, provided that an intact neuronal pathway connected the site of peptide application with the bag cells. In contrast, afterdischarges were never observed when peptides were selectively applied to the buccal or pedal ganglia and only rarely observed when applied to the abdominal ganglion and caudal pleurovisceral connectives. These results support the hypothesis that bag-cell processes and/or neuron(s) presynaptically excitatory to the bag cells are located in the pleural and cerebral ganglia and narrow the region of the central nervous system where the critical initiator element(s) can be identified.