Hanne Duve

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 or 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. Several different types of functions for the Leu-callatostatins of the blowfly are proposed in this study, but there is no evidence to suggest a role in the regulation of juvenile hormone synthesis and release.

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.

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. J. Comp. Neurol. 413:405–416, 1999.

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.

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.

The distribution of pancreatic polypeptide in the nervous system and gut of the blowfly, Calliphora vomitoria (Diptera)

SummaryThe distribution of a neuropeptide, previously shown to have the same or a very similar amino acid composition as vertebrate pancreatic polypeptide (PP), has been studied in the nervous system and gut of the blowfly, Calliphora vomitoria. Neurones immunoreactive to a bovine PP antiserum occur in the thoracic and abdominal ganglionic components of the central nervous system, in addition to the brain and suboesophageal ganglion. Pancreatic polypeptide appears to be relayed from its cells of origin to a neurohaemal organ in the dorsal sheath of the thoracic ganglion. PP immunoreactivity is also found in cells of the hypocerebral ganglion of the stomatogastric nervous system and in associated nerve fibres. The mid-gut contains PP-positive material in flask-shaped cells of its epithelial lining.

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)

Abstract.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.

Innervation of the foregut of the cockroach Leucophaea maderae and inhibition of spontaneous contractile activity by callatostatin neuropeptides

Abstract. The innervation of the gut of the cockroach Leucophaea madera (F.) has been studied by means of wholemount immunocytochemistry with antisera raised against Leu‐callatostatin, a cockroach allatostatin homologue identified from neuropeptide isolation and gene studies in the blowfly Calliphora vomitoria. Leu‐callatostatin‐imunoreactive neurones in the brain, with axon trajectories in the stomatogastric nervous system, innervate the foregut and midgut. Neurones in the last abdominal ganglion supply the hindgut and the midgut via the proctodeal nerve. In addition to a rich callatostatin‐immunoreactive nerve supply, the midgut, including the midgut caeca, contain numerous callatostatin‐immunoreactive endocrine cells. Physiological studies show that the spontaneous contractile activities of the foregut, but not the hindgut, are inhibited by callatostatin neuropeptides. Leu‐callatostatin 3 was the most potent of the range of Leu‐and Met‐callatostatins tested, with a dose‐dependent response between 10‐13 and 10‐7 M. This is similar to the results obtained with the previously identified myoinhibitory peptide of L. maderae, leucomyosuppressin. However, this peptide, with a different type of structure to the allatostatins, inhibits both foregut and hindgut motility equally. Experiments with a series of analogues of the Met‐callatostatins showed that the free acid (as opposed to the carboxyamidated peptide) and N‐terminally truncated peptides were inactive. These morphological and physiological results are thought to be representative of the, as yet unidentified, naturally occurring allatostatin homologues of L. maderae. This family of peptides should be added to the increasing list of insect gut myoinhibitory substances.

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.

Allatostatins of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea).

More than 40 peptides belonging to the -Y/FXFGL-NH(2) allatostatin superfamily have been isolated and identified from the central nervous system (CNS) of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). The peptides can be arranged in seven sub-groups according to the variable post-tyrosyl residue represented by Ala, Gly, Ser, Thr, Asn, Asp, and Glu. Two of the residues (Thr and Glu) have not been observed in this position previously in either insects or crustaceans. Also reported for the first time for allatostatins, two of the peptides are N-terminally blocked by a pyroglutamic acid residue. The yields of certain peptides with similar amino acid sequences to each other were, in some instances, very different. As an example, the yield of ANQYTFGL-NH(2) was 2pmol, compared with ASQYTFGL-NH(2), with a yield of 156 pmol. There are several possibilities to account for this. If, as in all species so far investigated, there is a single allatostatin gene in P. monodon, then it would appear that different sub-populations have contributed mutant forms of particular peptides to the extract. Another, less likely possibility is that this species has more than one allatostatin gene, producing a variable array of peptides albeit in different molar ratios. Several peptides were present apparently as a result of the loss of one or more residues at the N-terminus of a larger form, either due to N-terminal degradation or specific post-translational processing. The number of peptides identified exceeds that for any other insect or crustacean species previously investigated. None is identical to any of the 60-70 insect allatostatins so far identified, and only three are common to other crustaceans. Immunohistochemical study of the CNS of P. monodon, with the same antisera as used to monitor the purification, confirms the widespread nature and complexity of allatostatinergic neural pathways in arthropods. Thus, all neuromeres of the brain, and all except one of the ventral cord ganglia, possess allatostatin neurons and extensive areas of allatostatin-innervated neuropile. In addition to the cytological evidence that the allatostatins act as neurotransmitters, associated with tissues as varied as eyes and legs, their presence in neurohemal areas such as the sinus gland and the perineural sheath of the thoracic ganglia suggests a neuroendocrine function. As well as posing a challenge to physiologists assigning specific functions to the allatostatins, their extensive intra-species multiplicity, linked to their inter-species variability, also presents a complex problem to geneticists and evolutionists.