Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy.

A comparative study of the co-localisation of three different families of neuropeptides, viz. allatostatins of the Y/FXFGL-NH(2) type, Manduca sexta allatostatin (Mas-AS) and allatotropin, in the frontal ganglion of lepidopteran larvae has been carried out by means of immunocytochemistry and confocal laser scanning microscopy. The simultaneous application of three types of fluorochrome-conjugated antibodies reveals triple co-localisation in an anterodorsal pair of neurones in the frontal ganglion of the noctuids Heliothis virescens and Lacanobia oleracea. There is no evidence of differential axonal transport, since all parts of these neurones show complete co-localisation of all three peptides. Prominent axons of the ganglionic neurones project in the recurrent nerve to the foregut and stomodeal valve. Over the crop, lateral and sub-lateral branches follow the course of circular muscle fibres and terminate in varicosities. All three neuropeptides have previously been shown to be myoregulatory on the foregut; the Y/FXFGL-NH(2) allatostatins and Mas-AS are inhibitory, whereas allatotropin is excitatory. The morphological evidence of co-localisation of physiologically antagonistic peptides within the same terminals suggests that an extremely complex mechanism controls the contractile activities of the foregut. A posterodorsal pair of neurones in the frontal ganglion have prominent axons projecting via the frontal connectives to the brain and in the recurrent nerve to the stomodeal valve where extensive branching suggests control over the valve movements. Studies of another noctuid, Spodoptera frugiperda, and the sphingid, M. sexta, show interesting variations in the co-localisation phenomenon.

Morphological and physiological comparisons of two types of allatostatin in the brain and retrocerebral complex of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae).

The cellular localisation of two types of allatostatin in the brain and retrocerebral complex has been studied in larvae of Lacanobia oleracea (Noctuidae) using antisera against Manduca sexta allatostatin (Mas-AS) and two members of the Y/FXFGL-NH(2) allatostatin family. The axons of two groups of Mas-AS-immunoreactive neurosecretory cells in the pars lateralis form part of the nervi corporis cardiaci (NCC 1). They exit the brain as the combined NCC 1 and NCC 2 and pass through the corpora cardiaca (CC), where they divide to innervate the corpora allata (CA) and the mandibular (salivary) gland. The presence of Mas-AS immunoreactivity in the CA is consistent with the inhibitory action of this peptide on juvenile hormone (JH) biosynthesis in L. oleracea. Immunoreactivity in the mandibular gland nerve suggests an additional, as yet unidentified role for this peptide. Cells of the pars intermedialis, the main contributors to NCC 2, do not show Mas-AS immunoreactivity. The distribution of Y/FXFGL-NH(2) immunoreactivity is different from that of Mas-AS. Although there are fewer cells in the pars lateralis, immunoreactivity is observed in certain neurones of the pars intermedialis and the tritocerebrum. Axons of these latter neurones contribute to NCC 2 and NCC 3, respectively, and, combined with those from NCC 1, result in the prominent occurrence of Y/FXFGL-NH(2) immunoreactivity in the CC, particularly in the storage lobe. The CA has far less Y/FXFGL-NH(2) immunoreactivity compared with Mas-AS. In bioassays, the Y/FXFGL-NH(2) allatostatins did not inhibit JH synthesis by CA of L. oleracea.

Triple co-localisation of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut.

The triple co-localisation of peptidergic material immunoreactive to antisera raised against allatostatins of the Y/FXFGL-NH2 type, Manduca sexta allatostatin (Mas-AS), and allatotropin has been demonstrated in a single pair of anterodorsal neurones in the frontal ganglion of the tomato moth, Lacanobia oleracea (Noctuidae). Another pair of posterior neurones contain only Y/FXFGL-NH2-type allatostatin immunoreactivity. The neurites of all four cells trifurcate, and axons project to the brain in the frontal connectives and to the foregut in the recurrent nerve. Axons from the anterior neurones, within the recurrent nerve, have prominent lateral branches supplying muscles of the crop, and axons from both anterior and posterior cells show profuse branching and terminal arborisations in the region of the stomodeal valve. The brain contributes Y/FXFGL-NH2-immunoreactive material, but not allatotropin or Mas-AS, to the recurrent nerve via NCC 1+2 and NCC 3. All three peptides have a reversible effect on the spontaneous (peristaltic) contractions of the foregut (crop) in vitro. Thus, both types of allatostatin are inhibitory at 10(-12) to 10(-7) M, whereas allatotropin is strongly myostimulatory at 10(-14) M. This is the first demonstration of the gut myoinhibitory effects of Mas-AS and, taken together with the effects of Y/FXFGL-NH2-type allatostatins and allatotropin, reveals a different functional aspect to that normally attributed to these three peptides, i.e. control of juvenile hormone synthesis by the corpus allatum.

Sulfakinin neuropeptides in a crustacean. Isolation, identification andtissue localization in the tiger prawn Penaeus monodon.

The sulfakinin (SK) family of neuropeptides are characterized by a C-terminal octapeptide sequence that begins with two acidic residues (most commonly DD), and ends with YGHMRF-NH2, usually with the tyrosyl residue sulfated. So far, sulfakinins have only been identified in insects and the present study was initiated to investigate if the family is more widely distributed within the arthropods. Purification of an extract of the central nervous system of the giant tiger prawn Penaeus monodon has revealed three novel members of the sulfakinin peptide family. One of the peptides, Pem SKI, has the sequence

Characterisation of the helicostatin peptide precursor gene from Helicoverpa armigera (Lepidoptera: Noctuidae).

The gene encoding the helicostatin peptide family was isolated from a Helicoverpa armigera genomic DNA library. The deduced precursor sequence allowed unambiguous identification of all helicostatin peptides and verified the sequences of eight peptides previously isolated. The gene consists of at least three exons and encodes a precursor of 225 amino acids that contains three blocks of tandemly arranged helicostatin peptides including seven copies of the C-terminal sequence -YXFGL followed by a single Gly residue for carboxylamidation. Complete endoproteolytic processing at all possible dibasic cleavage sites would generate the seven helicostatin octapeptides previously purified from larval extracts. If processing was not complete at the third pair of basic amino acids the octadecapeptide (helicostatin IIa) would also be released. Two novel putative helicostatin peptide sequences were identified; YSKFNFGL and ERDMHRFSFGL, both of which had the C-terminal pentapeptide -FXFGL in place of the more usual -YXFGL sequence. Comparison of the helicostatin precursor with that of the cockroaches, locust and flies revealed variation in size, sequence and organisation of the 'allatostatin' precursors across different insect orders. In situ hybridisation histochemistry established that helicostatins are expressed in neurones of the central nervous system and endocrine cells of the midgut, indicating that the helicostatins are true brain-gut peptides. Northern blot analysis identified a single transcript of 1.6 kb in mRNA from whole larvae, isolated central nervous system and gut tissue.

Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion.

The frontal ganglion and associated neuronal pathways in larvae of the noctuid moth Helicoverpa armigera have been studied immunocytochemically with antisera against the endogenous neuropeptides, the allatostatins (helicostatins), and allatotropin. Two pairs of large ganglionic neurones contain allatostatin immunoreactivity, with the anteriormost of these pairs showing colocalisation with allatotropin. Allatostatin and allatotropin axons exit the frontal ganglion in the recurrent nerve and traverse the surface of the crop to give terminal arborisations around the stomodeal valve. There is a greater degree of lateral branching of allatotropin axons compared with allatostatin axons over the crop musculature. In vitro experiments show that the two types of peptides have antagonistic effects on the spontaneous myoactivity of the crop musculature. Allatotropin is myostimulatory at concentrations as low as 10(-16) M, enhancing both frequency and amplitude of peristaltic waves of contraction. All members of the helicostatin family inhibit peristalsis completely at concentrations of 10(-7)-10(-6) M and, to varying degrees, at 10(-10)-10(-8) M. On the basis of this evidence, it is suggested that peptidergic neurones of the frontal ganglion play a major part in regulating foregut motility through the antagonistic actions of the allatostatins and allatotropin.

Localization of allatostatin-immunoreactive material in the central nervous system, stomatogastric nervous system, and gut of the cockroach Blattella germanica.

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH2 C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed.

Identification, tissue localisation and physiological effect in vitro of a neuroendocrine peptide identical to a dipteran Leu-callatostatin in the codling moth Cydia pomonella (Tortricidae: Lepidoptera).

A neuroendocrine peptide of the Leu-callatostatin family, LPVYNFGL-NH2, has been isolated from tissue extracts of 5th instar larvae of the codling moth, Cydia pomonella (Lepidoptera). It is identical to a peptide previously isolated from the blowfly, Calliphora vomitoria (Diptera). The distribution of this peptide within the tissues of C. pomonella has been mapped by immunocytochemistry using antisera raised against LPVYNFGL-NH2. Midgut endocrine cells contain Leu-callatostatin immunoreactivity, as do several paired Leu-callatostatin neurones in the brain and ventral nerve cord. Within the visceral nervous system, the frontal ganglion contains four Leu-callatostatin neurones. Axons from these cells combine with others originating from neurones in the brain and project within the nervi cardiostomatogastrici to innervate the tissues of the foregut. In particular, the oesophageal valve has a prominent ring of Leu-callatostatin-immunoreactive fibres. The synthetic peptide, LPVYNFGL-NH2, has a potent reversible inhibitory effect in vitro on all visible forms of spontaneous contractile activity of the foregut, including closure of the oesophageal valve. Complete myoinhibition is observed at peptide concentrations from 10(-10 )to 10(-16) M. These results, in conjunction with the results of similar studies on cockroaches, crickets and flies, suggest that the Leu-callatostatins are a ubiquitous family of insect neuroendocrine peptides with an important role in the control of gut motility.

Lepidopteran peptides of the allatostatin superfamily.

Peptides of the allatostatin superfamily with the C-terminal amino acid sequence -YXFGL-NH2 have been isolated and identified from the lepidopterans, the codling moth, Cydia pomonella (Tortricidae) and the bollworm, Helicoverpa armigera (Noctuidae). The peptides, designated cydiastatins and helicostatins respectively, were monitored during purification with radioimmunoassays based on the callatostatins of the blowfly Calliphora vomitoria. The eight peptides from each of the two species appear to form an homologous series with four identical and three that differ by a single amino acid. This study demonstrates the ubiquitous nature of this family of peptides in insects.

Isolation and identification of multiple neuropeptides of the allatostatin superfamily in the shore crab Carcinus maenas.

20 neuropeptides belonging to the allatostatin superfamily were isolated from extracts of cerebral and thoracic ganglia of the shore crab Carcinus maenas. They were purified by HPLC, monitored by radioimmunoassay and identified by mass spectrometry and amino acid sequencing. The allatostatins are characterised by a common C-terminal pentapeptide sequence -YXFGL-NH2. Previously such peptides have only been reported from insects. In insects the variable post-tyrosyl residue is restricted to Ala, Asn, Asp, Gly or Ser. In C. maenas, however, there are only two types; thirteen of the peptides having a post-tyrosyl Ala and the other seven, a post-tyrosyl Ser. The crab peptides include the shortest allatostatins so far identified (YAFGL-NH2 and YSFGL-NH2) as well as the longest, a 27-residue peptide. The total of 20 peptides exceeds the highest number of allatostatins found in any of the insects investigated so far (14 in Periplaneta americana). It is of interest that, despite their clear homology, none of the peptides of C. maenas is identical to any of the more than 50 known insect allatostatins. The crab allatostatins show evidence of gene duplication and mutation that has resulted in several sub-groups with close structural similarities. For example, there are four heptapeptides with the common C-terminus -PYAFGL-NH2 that differ only at the N-terminal residue, which is either Glu, Asp, Asn or Ser. Other motifs, variously extended at the N-terminus, include -GPY(A/S)FGL-NH2 (three peptides), -DMY(A/S)FGL-NH2 (three peptides), and -GQY(A/S)FGL-NH2 (two peptides). Unique among the allatostatin superfamily, one of the crab peptides has a Tyr for Phe substitution at position three from the C-terminus (GGPYSYGL-NH2). Immunocytochemistry has provided clues to the functions of the allatostatins in crustaceans by showing their widespread presence in the central and stomatogastric nervous systems.

Identification of the dipteran Leu-callatostatin peptide family: characterisation of the prohormone gene from Calliphora vomitoria and Lucilia cuprina.

The prohormone gene encoding the Leu-callatostatin peptides has been isolated from a Calliphora vomitoria genomic DNA library and its homologue was cloned from genomic and cDNA libraries of another blowfly species, Lucilia cuprina. Gene and prohormone structure and organisation are essentially identical in the two species. The Leu-callatostatin gene consists of at least 3 exons. The prohormone is encoded on exons two and three and the two blocks of putative Leu-callatostatin peptides are carried on separate exons. It is 180 amino-acids long, begins with a short signal peptide and contains two blocks of tandemly arranged Leu-callatostatin peptides separated by an acidic spacer region. The prohormone contains 5 copies of the C-terminal sequence -YX FGL characteristic of the Leu-callatostatin family. Complete endoproteolytic processing at all possible pairs of basic amino acids would generate 5 different Leu-callatostatin octapeptides. Two larger Leu-callatostatins could be released if processing was not complete at two of the sites. None of the 3 peptides encoded in the first block was identified in previous purification studies of the callatostatin peptides. The second block, located at the carboxyl end of the prohormone, contains two peptide sequences identical to the previously isolated Leu-callatostatins 1 and 4. The absence of independent copies of Leu-callatostatins 2 and 3 on the prohormone establishes that endoproteolytic cleavage of the precursor does not invariably proceed to completion and that Leu-callatostatin 2 must be derived by N-terminal processing of the parent peptide Leu-callatostatin 1. Reverse transcriptase PCR analysis of mRNA from brain and midgut, the two major sites of Leu-callatostatin expression, shows that the prohormone sequence at these two sites is identical, ruling out the possibility that different populations of peptides are expressed in these two tissues as a result of alternative RNA splicing.

Identification of the dipteran Leu-callatostatin peptide family: the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria.

Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence -YXFGL-NH2. Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH2 and ARPYSFGL-NH2, whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH2. Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH2 and LPVYNFGL-NH2. The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH2 (monophasic dose-response curve with an IC50 = 10 fM) and for LPVYNFGL-NH2 (biphasic dose-response curve with IC50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

Conformational preferences of the calliFMRFamides and their free-acid analogues.

A molecular dynamics study was undertaken to determine the conformational basis for the differing activities of the insect neuropeptide hormones calliFMRFamide 3 (SPSQDFMRF-NH2). calliFMRFamide 5 (APGQDFMRF-NH2) and their corresponding free-acid analogues (SPSQDFM-RF-OH and APGQDFMRF-OH) in two insect bioassays. A simulated annealing protocol was used to determine the range of conformers available to the linear peptides. Analysis of the conformers obtained indicated that all the peptides exhibited distinct secondary structure preferences. These, when correlated with their biological activities, enabled the formulation of putative conformation-activity relationships for the peptides.

The sulfakinins of the blowfly Calliphora vomitoria. Peptide isolation, gene cloning and expression studies.

The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide. Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His- Met-Arg-Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)- Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide. His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp-Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurons of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous.

Isolation, identification and functional significance of [Hyp2]Met-callatostatin and des Gly-Pro Met-callatostatin, two further post-translational modifications of the blowfly neuropeptide Met-callatostatin.

Two post-translationally modified neuropeptides of the Met-callatostatin (Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2) family have been identified from head extracts of the blowfly Calliphora vomitoria. They are the octapeptide, [Hyp2]Met-callatostatin, (Gly-Hyp-Pro-Tyr-Asp-Phe-Gly-Met-NH2) and the truncated hexapeptide, des Gly-Pro Met-callatostatin (Pro-Tyr-Asp-Phe-Gly-Met-NH2). The existence of the [Hyp2]Met-callatostatin variant, in addition to the previously identified [Hyp3]Met-callatostatin peptide, suggests that the motif for prolyl hydroxylation in C. vomitoria is more variable than those known from mammalian and other invertebrate studies where, in those regulatory peptides containing a pair of adjacent prolyl residues so far studied, e.g., bradykinin, and the mosquito peptide Aea HP-I, only one of the pair (the second) is known to undergo hydroxylation. The truncated hexapeptide, des Gly-Pro Met-callatostatin could be produced as a result of the action of a dipeptidyl peptidase II type of enzyme which is known from mammalian studies to be unique in its ability to cleave between the two prolyl residues of an Xaa-Pro-Pro- sequence, where Xaa is any unprotected NH2-terminal amino acid. This enzyme is, however, considered unlikely to be able to cleave the Gly-Hyp-Pro-sequence, which would suggest a functional significance for such a post-translational modification. For this reason, it is of interest that [Hyp2]Met-callatostatin (and earlier, [Hyp3]Met-callatostatin) have been shown to be potent inhibitors of the spontaneous contractions of the hindgut of C. vomitoria (biphasic dose-response curve with IC50 values of 10(-14) M and 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry.

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.

Allatostatic neuropeptides from the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Identification, immunolocalization and activity.

Four allatostatic neuropeptides were isolated from extracts of the brain of the cockroach Blattella germanica. The primary structures of these peptides were assigned as Leu-Tyr-Asp-Phe-Gly-Leu-NH2 (BLAST-1), Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-2), Ala-Gly-Ser-Asp-Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-3) and Ala-Pro-Ser-Ser-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (BLAST-4). Each of the peptides showed C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The four peptides inhibited in vitro juvenile hormone production by corpora allata from virgin females of B. germanica. Immunoreactivity against allatostatins was seen in the lateral neurosecretory neurons and in the axonal pathway leading to the corpora allata.

[Hyp3]Met-callatostatin. Identification and biological properties of a novel neuropeptide from the blowfly Calliphora vomitoria.

A novel, hydroxyproline-containing neuropeptide, Gly-Pro-Hyp-Tyr-Asp-Phe-Gly-Met-NH2, designated [HYP3]Met-callatostatin, has been identified from extracts of heads of the blowfly Calliphora vomitoria. The peptide is a naturally occurring hydroxylate analogue of Met-callatostatin, a previously identified allatostatin-like peptide, and is present to the extent of 20% of the nonhydroxylated form. In bioassays, both forms of the peptide show allatostatic activity by inhibiting juvenile hormone synthesis and release in the cockroaches Periplaneta americana, Diploptera punctata, and Blattella germanica (IC50 = 100 pM-10 nM). They do not, however, influence juvenile hormone bisepoxide synthesis and release in the blowfly. In flies, [Hyp3]Met-callatostatin inhibits the peristaltic movements of the hindgut, showing a biphasic response (IC50 = 0.5 pM and 0.5 microM) compared with the monophasic response of Met-callatostatin (IC50 = 100 nM). Immunocytochemical studies with Met-callatostatin antisera provide the cytological basis for a myoinhibitory role in the gut since the axons of immunoreactive neurons in the abdominal ganglion project to the ileum. There are also endocrine cells in the midgut that, by releasing the peptides into the hemolymph, would allow the Met-callatostatins to fulfill a neurohormonal role on muscles of the gut and heart. In contrast, there are no Met-callatostatin neural pathways from the brain to the corpus allatum, the gland that produces juvenile hormone. NH2-terminal degradation of Met-callatostatins incubated with the hemolymph of P. americana results in cleavage of the Pro-Tyr bond giving the pentapeptide Tyr-Asp-Phe-Gly-Met-NH2 as a degradation product. In contrast, the Hyp-Tyr bond resists cleavage. With hemolymph from C. vomitoria, no immunoassayable degradation product has been observed with either peptide.

Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria.

The Leu-callatostatins are a series of four neuropeptides isolated from nervous tissues of the blowfly Calliphora vomitoria that show C-terminal sequence homology to the allatostatins of cockroaches. The allatostatins have an important role in the reproductive processes of insects as inhibitors of the synthesis and release of juvenile hormone from the corpus allatum. In this study, the distribution of the Leu-callatostatin-immunoreactive neurones and endocrine cells has been mapped in C. vomitoria and, in contrast to the cockroach allatostatins, it has been shown that there is no cytological basis to suggest that the dipteran peptides act as regulators of juvenile hormone. Although occurring in various neurones in the brain and thoracico-abdominal ganglion, there is no evidence of Leu-callatostatin-immunoreactive pathways linking the brain to the corpus allatum, or of immunoreactive terminals in this gland. Three different types of functions for the Leu-callatostatins are suggested by the occurrence of immunoreactive material in cells and by the pathways that have been identified. (1) A role in neurotransmission or neuromodulation appears evident from immunoreactive neurones in the medulla of the optic lobes, and from immunoreactive material in the central body and in descending interneurones in the suboesophageal ganglion that project to the neuropile of the thoracico-abdominal ganglion. (2) Leu-callatostatin neurones directly innervate muscles of the hindgut and the heart. Immunoreactive fibres from neurones of the abdominal ganglion pass by way of the median abdominal nerve to ramify extensively over several areas of the hindgut. Physiological experiments with synthetic peptides show that the Leu-callatostatins are potent inhibitors of peristaltic movements of the ileum. Leu-callatostatin 3 is active at 10(-16) to 10(-13) M. This form of regulatory control over gut motility appears to be highly specific since the patterns of contraction in other regions are unaffected by these peptides. (3) Evidence that the Leu-callatostatins act as neurohormones comes from the presence of varicosities in axons passing through the corpus cardiacum (but not the corpus allatum) and also from material in extraganglionic neurosecretory cells in the thorax. Fibres from these peripheral neurones are especially prominent over the large nerve bundles supplying the legs. There are also a considerable number of Leu-callatostatin-immunoreactive endocrine cells in a specific region of the midgut. The conclusion from this study is that although conservation of the structure of the allatostatin-type of peptides is evident through a long period of evolution it cannot be assumed that all of their functions have also been conserved.(ABSTRACT TRUNCATED AT 400 WORDS)

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria.

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH2 and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu3-Glu4- in place of -Asp3-Asp4-). Only four pairs of sulfakinin-immunoreactive neurons have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.