Locust Corpora Cardiaca Research Articles

Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries

Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects.

Identification of a glycogenolysis-inhibiting peptide from the corpora cardiaca of locusts.

A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide.

Several Isoforms of Locustatachykinins May Be Involved in Cyclic AMP-Mediated Release of Adipokinetic Hormones from the LocustCorpora cardiaca

Four locustatachykinins (LomTK I–IV) were identified in about equal amounts in extracts of corpora cardiaca of locusts, using reverse-phase high-performance liquid chromatography and radioimmunoassay with synthetic LomTK I–IV as standards. Brain extracts also contained the four isoforms in roughly equimolar concentrations. Retrograde tracing of the nervi corporis cardiaci II (NCC II)in vitrowith Lucifer yellow in combination with LomTK immunocytochemistry revealed that about half of the secretomotor neurons in the lateral part of the protocerebrum projecting into the glandular lobe of the corpora cardiaca (CCG) contain LomTK-immunoreactive material. Since the four LomTKs are present in the CCG, these four or five neurons in each hemisphere are likely to contain colocalized LomTK I–IV. The role of two of the LomTKs in the regulation of the release of adipokinetic hormones (AKHs) from the adipokinetic cells in the CCG in the locust was investigated. Experiments performedin vitroshowed that LomTK I and II induced release of AKH in a dose-dependent manner. These peptides also rapidly and transiently elevated the cyclic AMP-content of the CCG. The peak level of cyclic AMP occurred about 45 seconds after stimulation with LomTK. These results support the proposal that LomTKs are involved in controlling the release of the adipokinetic hormones and suggest that all LomTK isoforms may participate in this cyclic AMP-mediated event.

A possible role of SchistoFLRFamide in inhibition of adipokinetic hormone release from locust corpora cardiaca.

The distribution and actions of FMRFamide-related peptides (FaRPs) in the corpora cardiaca of the locust Locusta migratoria were studied. Antisera to FMRFamide and SchistoFLRFamide (PDVDHVFLRFamide) label neuronal processes that impinge on glandular cells in the glandular lobe of the corpora cardiaca known to produce adipokinetic hormones. Electron microscopic immunocytochemistry revealed that these FaRP-containing processes form synaptoid contacts with the glandular cells. Approximately 12% of the axon profiles present in the glandular part of the corpus cardiacum contained SchistoFLRFamide-immunoreactive material. Retrograde tracing of the axons in the nervus corporis cardiaci II with Lucifer yellow revealed 25-30 labelled neuronal cell bodies in each lateral part of the protocerebrum. About five of these in each hemisphere reacted with the SchistoFLRFamide-antiserum. Double-labelling immunocytochemistry showed that the FaRP-containing processes in the glandular lobe of the corpora cardiaca are distinct from neuronal processes, reacting with an antiserum to the neuropeptide locustatachykinin. The effect of the decapeptide SchistoFLRFamide and the tetrapeptide FMRFamide on the release of adipokinetic hormone I (AKH I) from the cells in the glandular part of the corpus cardiacum was studied in vitro. Neither the deca- nor the tetrapeptide had any effect on the spontaneous release of AKH I. Release of AKH I induced by the phosphodiesterase inhibitor IBMX, however, was reduced significantly by both peptides. These results point to an involvement of FaRPs as inhibitory modulators in the regulation of the release of adipokinetic hormone from the glandular cells.

Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca

The glandular cells of the corpus cardiacum of the locust Locusta migratoria, known to synthesize and release adipokinetic hormones (AKH), are contacted by axons immunoreactive to an antiserum raised against the locust neuropeptide locustatachykinin I (LomTK I). Electron-microscopical immunocytochemistry reveals LomTK immunoreactive axon terminals, containing granular vesicles, in close contact with the glandular cells cells. Release of AKH I from isolated corpora cardiaca of the locust has been monitored in an in vitro system where the amount of AKH I released into the incubation saline is determined by reversed phase high performance liquid chromatography with fluorometric detection. We could show that LomTK I induces release of AKH from corpora cardiaca in a dose-dependent manner when tested in a range of 10–200 μM. This is thus the first clear demonstration of a substance inducing release of AKH, correlated with the presence of the substance in fibers innervating the AKH-synthesizing glandular cells, in the insect corpora cardiaca.

Modulatory Effects of Biogenic Amines on Adipokinetic Hormone Secretion from Locust Corpora Cardiaca in Vitro

The control of the release of adipokinetic hormones from neurosecretory cells within the glandular lobes of the corpus cardiacum involving axons running through the paired nervus corporis cardiaci II is unclear. Cyclic-AMP (cAMP) is clearly a second messenger. The effects of four biogenic amines (octopamine, dopamine, tyramine, and serotonin) on the release of adipokinetic hormone-I were investigated in vitro. None had an effect on its own; they all potentiated the hormone release induced by cAMP-activating agents. Dopamine and serotonin were only present in the neurohemal part of the corpus cardiacum. Octopamine and tyramine were not detectable in the corpus cardiacum.

Actions of Ion-Transport Peptide From Locust Corpus Cardiacum on Several Hindgut Transport Processes

ABSTRACT Schistocerca gregaria ion-transport peptide (Scg-ITP), a neuropeptide isolated from locust corpora cardiaca, stimulates ileal Cl − transport (Isc) in a dose-dependent manner and causes increases in Na+, K+ (IK) and fluid reabsorption (Jv) as previously observed with crude extracts of corpus cardiacum and with cyclic AMP. Unlike cyclic AMP, Scg-ITP does not stimulate ileal NH4+ secretion. H+ secretion (JH) in the ileum, which is not affected by cyclic AMP, is almost completely abolished by Scg-ITP. Although ITP may act via cyclic AMP as second messenger to stimulate NaCl, KC1 and fluid reabsorption, it apparently acts through a different intracellular pathway to influence xJH. Scg-ITP is unlikely to be the chloride transport stimulating hormone previously reported to act on the rectum, because it did not produce a maximum rectal Isc response and had no effect on either rectal Jv (which is Cl− -depcndent) or IK.

Functional cross-reactivities and transduction systems at the rectal level by antidiuretic peptides from cockroach and locust corpora cardiaca

Antidiuretic methanol-soluble substances were extracted from the glandular lobes of corpora cardiaca of the cockroaches Blaberus craniifer and Leucophaea maderae. These substances interact with a similar factor from Locusta migratoria corpora cardiaca glandular lobes to stimulate the rectal fluid reabsorption of the different species in a dose-dependent manner. The cockroach methanol-soluble substances were peptides stable at room temperature but not when submitted to high temperature. The molecular weight of Leucophaea antidiuretic peptide is > 6000 Da. These substances are structurally different from the antidiuretic neuroproteins previously isolated, neuroparsins and neuroparsin-like proteins, since treatment with immune serum against locust neuroparsins does not prevent their antidiuretic activity. Intracellular transduction of the antidiuretic effect of the peptides is mediated at the rectal level in both groups of insects (cockroaches and locust) by a similar second messenger: cyclic adenosine monophosphate. The results are consistent with the existence of several related antidiuretic peptides from the glandular lobes of the corpora cardiaca of cockroaches and locust. Because of the observed cross-reactivities, it is likely that the receptors able to recognize the different peptides are also related to each other.

A new function for the locust neuroparsins: Stimulation of water reabsorption

Antidiuretic activity of 21 anion exchange HPLC fractions from locust corpora cardiaca was evaluated. The first two fractions, which were the only ones immunoreactive with neuroparsin immune serum, enhance the fluid reabsorption with dose-response curves very similar to those of crude extracts of the nervous corpora cardiaca (NCC). Incubation of both the immunoreactive fractions and NCC crude extracts with neuroparsin immune serum results in a disappearance of the antidiuretic activity. Thus, the antidiuretic factor of the NCC having been partially characterized is very likely to be the neuroparsins.

Determination of locust AKH-I by radioimmunoassay and the identification of an AKH-like factor in the locust brain

The N-terminal glutamic acid analog of the decapeptide locust adipokinetic hormone-I, [Glu 1]AKH-I, was prepared by solid-phase synthesis and derivatized to [4-hydroxyphenyl propionyl-Glu 1]AKH-I. This derivative was radioiodinated on the 4-hydroxyphenyl function ([ 125I]AKH analog). [Glu 1]AKH-I was coupled to bovine serum albumin to produce rabbit antiserum. This, together with the [ 125I]AKH analog was utilized to develop a highly selective radioimmunoassay procedure (RIA) for detecting AKH-I in locusts. Methanol extracts of locust brain contain two factors (BI and BII) that inhibit protein synthesis in excised fat body tissue of vitellogenic female locusts. BI also elicits lipid mobilization when injected into adult male locusts. The HPLC retention time of BI on an RP-18 column is identical to that of locust adipokinetic hormone (AKH-I), both synthetic and derived from adult corpora cardiaca (CC). The second brain factor (BII) does not exhibit lipid mobilizing activity. One tissue-equivalent of BI contains 33 ng of AKH-I as determined by RIA, compared to 450 ng native HPLC-separated AKH-I in locust CC.

Anti-juvenile effect of neuroparsin A, a neuroprotein isolated from locust corpora cardiaca

Neuroparsin A, a sulfur-containing protein synthesized by the medial part of the brain of Locusta and transported to the corpora cardiaca (CC) via the nervi corporis cardiaci I (Girardie et al., 1987), was satisfactorily isolated using electro-elution. A specific immune serum against electro-eluted neuroparsin A was generated. On serial histological sections of the brain treated with the immune serum, only the median neurosecretory cells [stained in blue following the double staining Victoria blue-paraldehyde fuchsin (Al type)] were revealed using peroxidase-antiperoxidase technique. Inactivation of neuroparsin A by antigen-antibody complex formation following injections of immune serum induced green pigmentation, intermediary forms and precocious sexual maturation. These symptoms also follow juvenile hormone (JH) injections. Injections of immune serum antineuroparsin A or the electro-eluted neuroparsin A produced opposite effects on oocyte growth but had no effect on the rate of JH biosynthesis evaluated by radiochemical assay. The neurohormone neuroparsin A could be the median humoral inhibiting factor of the JH system which was previously demonstrated (Girardie, 1966, 1967) in the central area of the pars intercerebralis.

An active principle from the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust, inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly. From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts.

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.

Hormonal activation of protein kinase and lipid mobilization in the locust fat body in vitro

Cyclic nucleotide-activated protein kinase in locust fat body is elevated by extracts containing adipokinetic hormone (AKH) from locust corpora cardiaca or by synthetic redpigment-concentrating hormone (RPCH) which also exhibits lipid-mobilizing activity. Maximal activation is first attained in vitro 90 sec after RPCH is added to the incubation medium and 3.5 min after addition of AKH extract. This activation reflects the increase in endogenous cyclic nucleotides. Incubation of fat body in the presence of exogenous cyclic nucleotides activates lipase, and RPCH stimulation ultimately elevates the level of transported diacylglycerol 2.3-fold. RPCH stimulates fat body lipase in vivo.

Purification and characteristics of the chloride transport stimulating factor from locust corpora cardiaca: a new peptide

Corpora cardiaca (CC), cAMP, and hemolymph all increase short-circuit current (Isc) and electropotential difference (PD) across locust rectum by stimulating electrogenic transport of Cl− from the lumen. Using ΔIsc as a bioassay, we have purified the water-soluble stimulant (CTSH) from CC using gel filtration chromatography, DEAE-Sephadex anion exchange, cellulose acetate electrophoresis, and thin-layer chromatography. A single peak of CTSH activity was observed after all these procedures, although small amounts of CTSH activity occasionally remained in the high molecular weight (MW) protein precipitate. CTSH was purified more than 100-fold on Bio-Gel P-30 columns. It has a MW of 8 000 – 12 000, is destroyed by trypsin digestion, and has a net negative charge over the pH range (5–10) at which it is most stable. Various properties (i.e., stability at 20 °C, localization in CC, MW, Rf values) and reciprocal bioassay s indicate that CTSH is different from diuretic, antidiuretic, and adipokinetic hormones from locust CC. No difference in the properties of CTSH from glandular (GL) and storage lobes (SL) of CC were noted, although 80% of activity was in the SL. The concentration of purified CTSH required to cause maximal stimulation of rectal Isc is less than 7 nM.

Isolation of granules containing adipokinetic hormone from locust corpora cardiaca by differential centrifugation

Using differential centrifugation a fraction rich in AKH has been isolated from the glandular lobes of the corpus cardiacum of Schistocerca. This fraction, obtained by centrifugation at 30,000 g in 0.44 M sucrose, when examined under the electron microscope, was seen to consist of electron dense granules of size range 100–600 nm. These granules are identical with the neurosecretory granules seen in the intact glandular lobe cells.

Characterization of a second peptide with adipokinetic and red pigment-concentrating activity from the locust corpora cardiaca

A new peptide with both adipokinetic activity in the locust and red pigment-concentrating activity in the shrimp, can be readily separated from the adipokinetic hormone in extracts of corpora cardiaca (CC) from Schistocerca americana gregaria by the use of gel chromatography and the Leander adspersus bioassay. The new peptide accounts for 20% of the total biological activity in locust CC, and it is located principally in the CC glandular lobe. The amino acid composition of the pure peptide is: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. This composition is similar to, but not identical with the red pigment-concentrating hormone of the shrimp.

Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight.

Adipokinetic hormone, isolated from locust corpora cardiaca, has been identified as a blocked peptide: PCA-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2. The detailed structure is based on mass spectrometric data, substantiated in part by dansyl-Edman and carboxypeptidase data on thermolytic fragments. This is the first peptide hormone from an insect neuroendocrine organ to be fully characterised.

The hormonal content of the locust corpus cardiacum

The amounts of adipokinetic and diuretic hormone in the separate storage and glandular lobes of the locust corpora cardiaca during the imaginal moult and up to the onset of sexual maturation have been measured. The levels of the hormones are high prior to the imaginal moult, fall at emergence and increase during the somatic growth period. The effects of surgical interference with the neuroendocrine system upon the hormonal content of the corpora cardiaca have been investigated. Cautery of the brain neurosecretory cells or allatectomy in mature locusts has no effect on the content of adipokinetic hormone. Diuretic hormone is absent from the storage lobes of locusts deprived of their cerebral neurosecretory cells but normal levels are present in the corpora cardiaca of allatectomised animals. Severance of the nervus corporis cardiacum I and II reduces the level of diuretic hormone in the storage lobes of the corpora cardiaca but is without effect on the levels of adipokinetic hormone in the glandular lobes.

In vivo studies on the release of hormones from the corpora cardiaca of locusts

Sectioning of the afferent nerves (NCCl and NCCll) to the locust corpus cardiacum prevents thein vivo release of adipokinetic hormone from the glandular lobes. This failure to release the hormone during flight and the consequent lack of lipid mobilisation brings about an impairment of flight performance which can be corrected by injections of corpus cardiacum extracts. Sectioning of the NCCl and NCCll reduces markedly the activity of the corpora allata. However, the poor flight performance of allatectomised locusts is not related to an inability to mobilise lipid since injections of corpus cardiacum extract which will mobilise fat body lipid in these locusts have no effect on flight performance. The results of individual sectioning of the NCCl and NCCll suggest that a double innervation of the glandular lobes functionsin vivo to control adipokinetic hormone release but that the NCCl alone may control the release of the diuretic hormone.