cDNA cloning and transcript distribution of two different neuroparsin precursors in the desert locust, Schistocerca gregaria.

Neuroparsins were originally identified in locust corpus cardiacum extracts as folliculostatic or 'antigonadotropic' neuropeptides. This paper presents the cloning of two different neuroparsin precursor cDNAs from the brain of the desert locust, Schistocerca gregaria. The first transcript encodes the precursor (Scg-NPP1) of S. gregaria neuroparsin A and B, whereas the second codes for a novel neuroparsin-related peptide precursor (Scg-NPP2). Both precursors display significant sequence similarities with each other and with the Locusta migratoria neuroparsin (Lom-NPP) and Aedes aegypti ovary ecdysteroidogenic hormone (Aea-OEH1) precursors. Northern blot analysis revealed that these neuroparsin transcripts are present in larval and adult locust brains. Interestingly, the Scg-NPP2 mRNA content proved to be strongly regulated during the reproductive cycle in both adult males and females.

Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria.

Neurosecretory products immunologically related to either neuroparsin (NP) or ovary maturing parsin (OMP) of Locusta migratoria (Lom) were purified from the nervous corpora cardiaca of Schistocerca gregaria (Scg). The determination of both their molecular masses by mass spectrometry and their sequences by automated Edman degradation established that they are members of the NP and OMP families respectively. NP molecules of Schistocerca (Scg NPs) consisted of two major forms having about the same molecular masses as NPA and NPB of Locusta and 88% primary structure similarity. They had also the same antidiuretic activity. OMP molecules of Schistocerca (Scg OMPs) were composed in young adults of four isoforms: two long isoforms corresponding to Lom OMP, and differing by a tripeptide insertion (Pro-Ala-Ala) at position 21 and two short isoforms deprived of the 13-residue N-terminal peptide of Lom OMP and differing by the same tripeptide insertion. The PAA isoforms were observed in low amounts as compared to the other isoforms. In mature adults, only the two short isoforms were present. The complete sequence of PAA Scg OMP presents a large degree of sequence homology with Lom OMP (83%). The mixed Scg OMPs had the same biological effects as Lom OMPs. They induced precocious occurrence of both ecdysteroids and vitellogenin in the haemolymph and stimulated oöcyte growth.

Arguments for two distinct gonadotropic activities triggered by different domains of the ovary maturating parsin of Locusta migratoria.

To complete previous results concerning the role of the ovary maturating parsin of Locusta migratoria (Lom OMP), we determined, by an enzyme immunoassay, the titers of circulating ecdysteroids and analyzed circulating vitellogenin (Vg) and oöcyte growth following (1) suppression of 20 hydroxyecdysone (20E) and (2) injection of the Lom OMP, either as an entire molecule in allatectomized adults or as smaller peptides in allatectomized fifth-instar larvae females. Titers of ecdysteroids appeared unrelated to the presence of circulating Vg but increased during the first phase of vitellogenesis and injection of OMP accelerated the occurrence of circulating 20E. Nevertheless, immunoneutralization of 20E at the beginning of adult life delayed but did not prevent rapid oöcyte growth contrary to immunoneutralization of Lom OMP suggesting an additive gonadotropic effect of the neurohormone, distinct from that of 20E. Of two synthetic peptides corresponding to the C- and N-terminal gonadotropic domains of the OMP, respectively, only the C-terminal peptide was able to induce Vg in allatectomized larvae. After metamorphosis, injection of OMP did not induce Vg in adults allatectomized at the beginning of imaginal life but improved the maintenance of circulating Vg in adults allatectomized after Vg appeared in the haemolymph. This result suggests that OMP either delays the Vg mRNA decay or increases the translation of Vg mRNA. Thus, Lom OMP appears to have two distinct roles: an ecdysteroidogenic effect triggered by its C-terminal domain with the ovary as the target tissue and a protecting effect on Vg mRNA probably triggered by its other gonadotropic domain, the N-terminal, with the fat body as the target tissue.

Expression of neuroparsin cDNA in insect cells using baculovirus vectors.

The cDNA encoding neuroparsin A, a polytropic neurohormone of the locust, Locusta migratoria, was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that transcription was under control of the p10 promoter. A polypeptide having the same charge and the same apparent molecular weight as the authentic neuroparsin A and that was reactive against neuroparsin immune serum was produced in recombinant virus-infected lepidopteran cell lines but not in control virus-infected cells. The baculovirus-expressed polypeptide was purified by two steps of liquid chromatography (anion exchange and reversed phase) which were previously used to purify the natural neuroparsin. The purified baculovirus-expressed polypeptide enhanced fluid reabsorption of everted rectum preparations, as did the natural neuroparsin. Thus, this gene expression system produced a polypeptide identical to authentic neuroparsin.

[Effect of olive leaves (Olea europea) feeding on the in vitro JH III biosynthesis by the corpora allata in Schistocerca gregaria during vitellogenesis]. / Action d'une alimentation à base de feuilles d'olivier (Olea europea) sur la biosynthèse in vitro de la JH III par les corpora allata chez Schistocerca gregaria au cours de la vitellogenèse.

Per os administration of olive leaves (Olea europea) to females of Schistocerca gregaria results in stopping vitellogenesis. These vitellogenins are not synthesised by the fat body in the heamolymph. The vitellogenin inhibition is induced by the stopping of juvenile hormone JH III by the corpora allata. These corpora allata (Medicago sp.) Synthesise 10 times less JH III than those of alfalfa fed females.

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone.

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM-resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the anti-native neurohormone serum and the same biological activity as the native neurohormone.

Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain-corpora cardiaca complex of various insect species.

Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.

Immunogold detection of two neurohormones: the locust ovary maturing parsin and neuroparsin.

The cellular localization of two neurohormones of the locust pars intercerebralis-corpora cardiaca system: the ovary maturing parsin and neuroparsin, was investigated using electron microscopic immunocytochemistry (post-embedding immunogold labelling). The ovary maturing parsin and neuroparsin containing cells were first identified in semithin sections treated by combined histochemical- and immunostaining. The neuroparsin cells were paraldehyde fuchsin positive (A-type cells) and the ovary maturing parsin cells were paraldehyde fuchsin negative when semithin sections were stained with paraldehyde fuchsin and immunostained with anti-ovary maturing parsin serum. The ovary maturing parsin and neuroparsin producing cells were identified on immunogold labelled ultrathin sections adjacent to double stained semithin sections. Ovary maturing parsin cells have larger more numerous vesicles of greater electron density than neuroparsin cells. The neuroparsin cells contained more lysosomal structures than the ovary maturing parsin cells suggesting different neurosecretory dynamics. Thus, immunogold labelling with antisera specific for each neurohormone demonstrates the individual nature of these two neurosecretory cells in the pars intercerebralis of the Locust.

[Immunological similarities between two cerebral substances from Schistocerca gregaria and two neurohormones from Locusta migratoria]. / Similitudes immunologiques entre les substances cérébrales de Schistocerca gregaria et deux neurohormones de Locusta migratoria.

Two neurohormones produced by two distinctive neurosecretory median cells (A1, B) of the protocerebum have been characterized in Locusta migratoria; on with a multiple functions one called neuroparsin and one stimulating the ovarian maturation called lom OMP. Using the specific immunoserum of these two neurohormones in Schistocerca gregaria, we could demonstrate the occurring of molecules related to the neuroparsin and lom OMP of Locusta. Through histology studies of the brain and corpora cardiaca complex, the immunoserum revealed the presence of the two types of these neurosecretory cells suggesting the occurring in Locusta as well as Schistocerca of the same cells releasing molecules immunologically apparented to neuroparsin and lom OMP. The results were confirmed by electrophoretic separation of corpora cardiaca extract under unreduced conditions followed by a transfer on immobilon membrane.

Effects of two neuronal antidiuretic molecules, neuroparsin and 5-hydroxytryptamine, on cytosolic free calcium monitored with indo-1 in epithelial and muscular cells of the African locust rectum.

Using the probe indo-1 in a microspectrofluorimetric study, it was demonstrated that the locust antidiuretic neurohormone, neuroparsin, enhanced cytosolic free Ca2+ concentrations, measured as percentages of the ratio F405/F480 (R), in epithelial cells of the African locust rectum. 5-hydroxytryptamine, whose antidiuretic effect was previously established, enhanced R in longitudinal muscular cells, and was able to increase R slightly in epithelial cells. The possibility of reciprocal Ca2+ movements between muscular and epithelial cells is discussed. Both of the neuronal molecules, which act via distinct transduction pathways (phosphoinositide turnover for neuroparsin, and Ca(2+)-dependent adenylate cyclase for 5-hydroxytryptamine), stimulated an increase in R by causing Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. Cyclic nucleotides (cAMP and cGMP) lowered R in epithelial cells, the cGMP effect being interpreted as a feedback control on phosphoinositide turnover and resulting in the ability to re-establish cAMP production to levels incompatible with high PLC activity. In longitudinal muscular cells, the increase in R due to cAMP suggests the involvement of 5-hydroxytryptamine in stimulation of adenylate cyclase activity. Furthermore, results enabled the localization in epithelial cells of the transduction pathways mediating the actions of another antidiuretic factor extracted from the glandular lobes of the locust corpora cardiaca.

In vitro growth of locust embryonic pars intercerebralis neurosecretory cells.

Neurosecretory brain cells from embryonic locusts cultured in serum-free medium failed to show any visible signs of growth. In contrast, the same neurons co-cultured with CNS explants (brain-retrocerebral complexes and thoracic ganglia) show excellent axonal growth: sprouting occurs after one day of co-culture and increases within the first week. These results indicate the production of an active neurite outgrowth stimulating factor by co-cultured CNS explants. The similarity of the stimulating effects by the two explants on neurite outgrowth rule out brain neurohormones as probable candidates for the stimulating factor. In addition, neither insulin nor neuroparsin added to the culture medium to test their trophic effect improves the growth of the cells. Conditioned medium derived from cultures of brain-retrocerebral complexes produced no neurite outgrowth, suggesting that the active factor released in the medium by brain explants does not remain free in solution but binds to the substratum. Finally, neurons co-cultured with CNS explants attached to the bottom of the culture dish develop neurites only when in close proximity to the explants. This observation strongly suggests the binding of an active neurite outgrowth stimulating factor to the substratum in the vicinity of the explants. As a control for CNS explants, the action of non-nervous tissue was tested: a similar, but less extensive neurotrophic effect, was observed with esophagus segments co-cultured with neurosecretory brain cells. These results demonstrate that locust neurosecretory neurons isolated in cell culture require combined explants for elaborating processes and suggest that the neurite promoting effect is mediated by a substrate-associated molecule(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides.

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi = 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.

Gastrin-cholecystokinin-like and neuroparsin-like immunoreactivities in the brain and retrocerebral neuroendocrine complex of the cockroach Blattella germanica.

Using single and double labelling techniques respectively, brain-corpora cardiaca-corpora allata complexes of the cockroach Blattella germanica have been immunohistochemically investigated with antisera raised against either the vertebrate peptide gastrin-cholecystokinin (CCK-8(s] and/or the locust neurohormone neuroparsin (NPA). Single immunolabelling with anti-CCK-8(s) reveals immunoreactive perikarya and processes in median and lateral parts of protocerebrum, optic lobes, deutocerebrum and tritocerebrum. Some fibres originating in median and lateral protocerebrum are intrinsic to the brain, whereas others terminate in the nervous areas of the corpora cardiaca. Single immunolabelling with anti-NPA reveals immunoreactive cell bodies in the median part of the protocerebrum and their processes terminate both in the nervous area of the corpora cardiaca and between the intrinsic secretory cells of this neurohaemal organ. Double immunolabelling with anti-CCK-8(s) and anti-NPA enables a description of the anatomical relations between the processes and the endings of these two neurosecretory systems.

Tryptophanyl-tRNA synthetase-like immunoreactivity in the central nervous system and midgut of the migratory locust. Comparisons with gastrin-cholecystokinin-like and octopamine-like immunoreactivity.

A tryptophanyl-tRNA synthetase (TrpRS)-immunoreactivity is localized in various neurosecretory cells of all ganglia of the central nervous system of the Orthoptera Locusta migratoria, except in deutocerebrum, and in endocrine cells of the midgut. It has been observed that TrpRS-like material never co-localizes either with CCK-like or octopamine-like material. TrpRS immunoreactive perikarya and processes that ramify extensively throughout the neuropiles have been detected in the protocerebrum, optic lobes, tritocerebrum, suboesophageal, thoracic and abdominal ganglia. In the lateral protocerebrum, a particular TrpRS pathway different from the lateral gastrin cholecystokinin (CCK-8(s] pathway is revealed, certain of these processes terminating in the glandular part of the corpora cardiaca. In the metathoracic ganglion, have been observed numerous immunoreactive cell bodies and processes in the neuropiles. Some of them constitute a major pathway and which are distinct from octopamine (OA) cells but in close vicinity with the latter. In the midgut immunopositive TrpRS-like cells are dispersed among the regenerative and digestive cells of the epithelium; they are different from gastrin-cholecystokinin positive cells. The various TrpRS-like immunoreactivities identified in Locusta indicate that TrpRS-like material may occur in different tissues of organisms other than Vertebrates. These results suggest also that TrpRS-like enzyme could be involved in functions other than aminoacylation, as in Vertebrates.

Amino acid sequence of locust neuroparsins.

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.

Immunohistochemical investigation of locust neuroparsin-like substances in several insects, in some other invertebrates, and vertebrates.

Anti-neuroparsin serum was immunohistochemically tested on brain and/or neurohemal organ from 40 insect species belonging to 13 orders, and from 8 non-insect invertebrate and 5 vertebrate representatives using the peroxidase-antiperoxidase procedure. In insects, immunostaining of only the A1 type of the protocerebral median neurosecretory cells was revealed in all species tested of Odonata, Dictyoptera, Isoptera and Orthoptera and in 2 species from the 9 other orders out of 13 orders tested. No immunostaining was detected in vertebrate and non-insect invertebrate species except in 2 annelid species out of 4 tested. The distribution of neuroparsin-like products in Coelomata appears to be restricted mainly to 4 phylogenetically close insect orders.

Production and characterization of antibodies to neuroparsins A and B isolated from the corpora cardiaca of the locust.

Antisera were raised in rabbits against neuroparsins A and B which were purified to near homogeneity using electroelution from 7.5% polyacrylamide electrophoresis gels. They were characterized using immunohistochemical, enzyme-linked immunosorbent assay (ELISA), and protein-blotting methods. The antineuroparsin A and the antineuroparsin B sera have different titers and sensitivities (higher titer for antineuroparsin A, higher sensitivity for antineuroparsin B). They exhibit very good specificity. The immunohistochemical study of the entire central nervous system using either antineuroparsin A or antineuroparsin B sera shows that only the A1 type of the protocerebral median neurosecretory cells are immunostained. Moreover, among the numerous proteins of the median region of the brain and of the corpora cardiaca, each immune serum recognized only neuroparsin A or neuroparsin B. Displacement curves obtained for each immune serum by competition between either neuroparsin A or neuroparsin B demonstrate that the neuroparsin A is recognized as well as neuroparsin B, with both antisera supporting the concept that these two proteins are chemically related. The nonspecific binding of neuroparsins to an antisomatostatin immune serum used at 1/100 dilution indicates that any cross-reactivities of invertebrate molecules obtained with very low dilutions of antisera to vertebrate molecules must be considered carefully before concluding any immunological relation between invertebrate and vertebrate products.

Immunohistochemical localization of dopamine in the brain of the insect Locusta migratoria migratorioides in comparison with the catecholamine distribution determined by the histofluorescence technique.

As part of a follow-up study to our previous investigation of the catecholaminergic neurosecretory cells in the brain of adult female locusts (Locusta migratoria migratorioides) by means of the formaldehyde-induced fluorescence method, we have attempted to specify the identity of the amines present in these cells by an immunohistological technique. Using a recently developed anti-dopamine serum, we have demonstrated that the majority of the catecholaminergic median neurosecretory cells contain dopamine. Moreover, dopamine is present in some cell bodies of other zones of the brain, i.e. the median subocellar neurosecretory cells, perikarya in external areas of the protocerebrum, below the calyces, around the pedunculus, in the optic lobes (between the lobula and the medulla, between the medulla and the lamina), and in external zones of the tritocerebrum. Among the structured neuropils, which were particularly fluorescent in the formaldehyde-induced fluorescence method, only the pedunculus, the posterior part of the central body, the external zones of the alpha- and beta lobes and the proximal part of the lamina contain little dopamine.

Undifferentiated cells present in the pars intercerebralis of larval and adult locusts are glial precursors. Autoradiographic and ultrastructural study in vivo and in vitro.

The intercerebral part of the protocerebrum from embryos, larvae and imagos of Locusta migratoria was investigated in vivo and after culture of the brain in vitro using light and electron microscopy. The results showed the presence in embryo and persistence in larva and adult of 2 clusters of mitotically active embryonic cells in the inner part of each half of the pars intercerebralis. The fate of these undifferentiated cells was investigated during postembryonic life by in vitro and in vivo labeling with tritiated thymidine combined with counts of nervous cells of the pars intercerebralis. Autoradiographic results confirmed the mitotic activity of the undifferentiated cells and established the pattern of this activity which declines from the third larval instar to adult stage. Mitoses were never seen in neurons and glial cells. Neurons were unlabeled and their number was constant. Glial cells were labeled and their number increases throughout postembryonic life with a pattern of proliferation similar to the pattern of mitotic activity of the undifferentiated cells. These observations indicate that the undifferentiated cells of the pars intercerebralis of the locust represent a source of glial cells and could be called glioblasts.