Diuretic hormone: another peptide with widespread distribution within the insect CNS

ABSTRACT. Diuretic hormone is a peptide neurohormone of as yet unknown chemical structure. Attempts to characterize the locust hormone have concentrated on the storage lobes of the corpora cardiaca as a source of material. Bioassay data now indicates that the occurrence of diuretic activity is more widespread in the locust CNS. Dose-response curves indicate that the levels of the diuretic factor(s) in the brain, sub-oesophageal and thoracic ganglia are remarkably similar to those found in the corpora cardiaca; activity was also detected in the abdominal ganglia (1–5) and terminal abdominal ganglion. Reverse-phase HPLC has confirmed that the diuretic material found in the various parts of the CNS is chemically similar to at least one of the diuretic factors separated from the corpora cardiaca. The significance of the widespread distribution of this peptide hormone is considered.
Recent evidence of other workers has indicated the possible structural similarity between vertebrate vasopressin and the diuretic factor in the locust sub-oesophageal ganglion. Chromatographic criteria have been used to demonstrate that the diuretic hormones isolated in our work are not vasopressin-like molecules, but are chemically distinct entities.     

Diuresis or clearance: Is there a physiological role for the “diuretic hormone” of the desert beetle Onymacris?

Malpighian tubules of Namib Desert tenebrionid beetles of the genus Onymacris are strongly stimulated by homogenates of the corpora cardiaca. The corpora cardiaca of other arid-adapted tenebrionids also contain diuretic material. Biogenic amines, which could be released during the preparation of corpora cardiaca extracts, do not stimulate fluid secretion in tubules of Onymacris rugatipennis. The diuretic factor in corpora cardiaca extracts is stable to boiling and to incubation with pronase. HPLC separation of the corpora cardiaca of O. rugatipennis gives a single region with diuretic activity in both secretory and electrical bioassays. Diuretic activity can not be detected in the haemolymph of Onymacris, and injection of corpora cardiaca extracts into the beetles does not cause diuresis. Simultaneous injection of corpora cardiaca and the dye amaranth shows that the most of the dye transported by the Malpighian tubules moves anteriorly into the midgut, indicating fluid recycling by this route. The most likely function for this “diuretic hormone” is clearance of metabolic wastes from the haemolymph.     

Isolation, physiological characterization, release and sequence elucidation of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the stick insect, Sipyloidea sipylus

ABSTRACT. The corpora cardiaca of the stick insect, Sipyloidea sipylus Westwood, contain peptidic material which elevates blood lipids in migratory locusts, blood carbohydrates in American cockroaches, and activates glycogen phosphorylase in the fat body of the cockroach in a time- and dose-dependent manner. The active principle is found in appreciable amounts only in the corpora cardiaca; slight hyperlipaemia is caused by extracts made from corpora allata and abdominal ganglia, whereas brain, suboesophageal and thoracic ganglia are not active. The adipokinetic activity is already present in corpora cardiaca from second instar (first day) nymphs. The factor retains its adipokinetic activity after boiling for up to 1 h. Conspecific injections of extracts from corpora cardiaca of S.sipylus cause hypertrehalosaemia in ligated stick insects and activate glycogen phosphorylase in non-ligated S.sipylus. After incubation of corpora cardiaca in vitro in saline with high concentrations of potassium and calcium, one fraction with adipokinetic (in locusts) and hypertrehalosaemic (in stick insects) activity can be isolated from the medium by RP-HPLC. Fractionation of a methanolic extract of corpora cardiaca from S.sipylus by RP-HPLC shows that active compounds are confined to apparently three absorbance peaks. The material of the highest absorbance peak was purified to homogeneity by RP-HPLC, and its amino acid composition determined after acid hydrolysis with HCl and with methanesulfonic acid revealed the residues Asx, Thr₍₃₎, Glx, Pro, Gly, Leu, Phe and Trp. The primary structure of this hypertrehalosaemic factor is assigned as a blocked decapeptide, pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂, from its FAB spectrum and metastable scans of its FAB spectrum. The structure is confirmed by synthesis; the synthetic and natural peptide co-chromatograph, and the synthetic peptide elevates blood carbohydrates in ligated stick insects and activates fat body phosphorylase in non-ligated S.sipylus.     

Partial purification and characterization of locust allatotropin I

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse-phase high-performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2-propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC-equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one-tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.     

Distribution of allatostatin in the adult cockroach,Diploptera punctata and effects on corpora allata in vitro

Direct radiochemical measurements of juvenile hormone synthesis showed that corpora allata from adult female Diploptera punctata can be inhibited in vitro by neuropeptides extracted from several ganglia of the central nervous system of females at many stages of the reproductive cycle. Extracts of protocerebra, corpora cardiaca, suboesophageal, thoracic and ventral ganglia all elicited dose-depedent reductions in juvenile hormone synthesis. On a ‘per organ’ basis, the protocerebrum contains the most extractable material. Inhibitory activity of extracts of suboesophageal, thoracic and 6th abdominal ganglia, like that of protocerebra (Rankin et al., 1986) was trypsin sensitive.Glands of high activity were less sensitive to protocerebral extract than those of low activity. The inhibitory effect on glands of low activity was maximal within 1 h, persisted in the presence of protocerebral extract for at least 46 h, and was abolished within 1 h after corpora allata were placed in normal medium. The inhibitory effect of protocerebral extract was not altered by the addition of magnesium to the medium. The extract had a specific effect on synthetic step(s) prior to methylation and epoxidation as demonstrated by enhanced juvenile hormone synthesis in the presence of inhibitory factor and the juvenile hormone precursor, farnesoic acid.     

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.     

Le rôle du système nerveux central sur les mécanismes de l'oviposition chez Carausius morosus et Clitumnus extradentatus

Cephalic nervous connexions were found necessary for oviposition and normal egg maturation in the two species studied. Neurosecretory cells of A type from the brain, suboesophageal, thoracic, and four abdominal ganglia seem to elaborate a stimulating substance for egg laying. This neurosecretion type is not present in corpora cardiaca, corpora allata, the four last abdominal ganglia, or perisympathetic organs which have no clear-cut effect on oviposition. The circadian rhythm of egg laying appears to be entirely controlled by the liberation of this neurosecretion and by an inhibitory factor localized in the head.In Carausius, egg deposition by the valvulae of the ovipositor is regulated by these two factors whereas egg transit through the common oviduct is a distinct preliminary step under nervous control. This nervous effect from the posterior region does not exist in Clitumnus.     

Regulation of egg laying and in vitro oviductal contractions in Gryllus bimaculatus

Oviductal contractions and the control of oviposition were investigated in vivo and in vitro in Gryllus bimaculatus females. In vivo experiments showed that oviposition is controlled nervously by both the brain and the last abdominal ganglion, and that one or more neurohormones cause ovipositor movements and abdominal contractions. In vitro, the assay of nerve ganglia and corpora cardiaca extracts on the isolated oviduct showed that they markedly increase the frequency of oviductal contractions. However, the action of thoracic ganglia extracts varies according to a circadian cycle. This observation, combined with the finding that the effects of the corpora cardiaca differ from those of the brain, suggests that each of these organs contains distinct neurohormones. None of the neurotransmitters tested was as potent as brain or ganglia extracts, although octopamine, l-glutamate and proctolin do stimulate oviductal contractions at low concentrations.     

Induction of egg diapause in Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana

Induction of egg diapause in the silkworm, Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana was examined. All tissues tested such as brain, corpora cardiaca, corpora allata, suboesophageal and thoracic ganglia and nerve cords between thoracic ganglia were able to produce diapause eggs in non-diapause egg producers both by transplantation and injection of their crude homogenates. The homogenate of thoracic ganglia was effective even in pharate adults with the suboesophageal ganglion removed or in isolated abdomens of pharate adults.From these results, it was surmised that some endocrine organs, as well as the central nervous system in the cephalo-thorax of Periplaneta americana, contained the active principle responsible for egg diapause in Bombyx mori.     

Distribution of locustamyotropin-like immunoreactivity in the nervous system of Locusta migratoria.

Locustamyotropin-like immunoreactivity was visualized in the nervous system of Locusta migratoria by means of the peroxidase antiperoxidase method. Highly specific antibodies to the carboxy-terminus of the locustamyotropins were obtained by elution through an affinity column to which Lom-MT II was covalently bound. Specific cells in the nervous system of Locusta migratoria contain substances immunoreactive to anti-locustamyotropin. In total, about 100 cells immunoreactive to the Lom-MT-II antiserum were detected in the head ganglia, in the abdominal neuromeres of the metathoracic ganglion, and in the five free abdominal ganglia. In the brain, immunoreactive cell groups were situated in the inner and outer edge of the tritocerebrum. Prominent axon bundles tightly surround the tractus I to the corpora cardiaca. The corpora allata were innervated by the nervus corporis allati I coming from the corpora cardiaca and by fibers in the nervus corporis allati II originating from cell bodies in the suboesophageal ganglion. Immunoreactive cell bodies in the suboesophageal and abdominal ganglia are distributed along the anterior posterior midline axis, both dorsally and ventrally. The processes of the cell bodies in the abdominal ganglia leave the ganglia and were traced in the respective median nerves into the neurohaemal organs. Since the Lom-MT-II antiserum cross-reacts with all peptides of the locustamyotropin family that have a carboxy-terminus in common, these cells may contain one or several locustamyotropins. The Lom-MT antiserum also recognizes pheromone biosynthesis activating neurohormone, as was revealed by the intensive labeling of suboesophageal cell bodies in Bombyx mori.     

A comparative ultrastructural and physiological study on melanophores of wild-type and periodic albino mutants of Xenopus laevis

Summary The central and visceral nervous systems of the cockroach Periplaneta americana were studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibody to bovine pancreatic polypeptide (PP). PP-like immunoreactive neuron somata are most numerous in the brain; at least 6 pairs of cell groups occur in clearly defined regions. Three pairs of cells each are also present in the suboesophageal ganglion and the thoracic ganglia, one pair of a single cell each in the first abdominal and the frontal ganglia, and 4 to 6 pairs of single cells in the terminal ganglion. No reactive cells were found in the retrocerebral complex and the second to the fifth abdominal ganglia. The axons containing PP-like immunoreactivity issue many branches that are distributed in the entire brain-retrocerebral complex, ventral cord, and visceral nervous system. PP-like immunoreactive material produced in the brain seems to be transported by three routes: protocerebrum to corpora cardiaca (-allata) through the nervi corporis cardiaci, tritocerebrum to visceral nervous system through frontal commissures, and to ventral cord through circumoesophageal connectives.A possible homology between the mammalian brain-GEP (gastro-enteropancreatic) system and the brain-midgut system of this insect is discussed.     

The hormonal content of the locust corpus cardiacum

Summary 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. This work is supported by grants from the Science Research Council and the Royal Society.     

Quantification of periviscerokinin‐1 in the nervous system of the American cockroach,Periplaneta americana: An insect neuropeptide with unusual distribution

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin‐1 (Pea‐PVK‐1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea‐PVK‐1. For this, we combined ELISA, HPLC, and MALDI‐TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea‐PVK‐1 in unseparated tissue extracts. No cross‐reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea‐PVK‐1 in its oxidized and nonoxidized form, were found in HPLC‐separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI‐TOF mass spectrometry, we were able to confirm the existence of authentic Pea‐PVK‐1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea‐PVK‐1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea‐PVK‐1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea‐PVK‐1 differs considerably from that of other known insect neuropeptides. Arch. Insect Biochem. Physiol. 40:203–211, 1999. © 1999 Wiley‐Liss, Inc.     

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

A quantitative study of adipokinetic hormone of the firebug,Pyrrhocoris apterus

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/μl.     

Stimulation of fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus

Abstract. Fluid secretion by single isolated Malpighian tubules of Acheta domesticus (L.) is stimulated by aqueous extracts of nervous and neuroendocrine tissues from the cricket. Diuretic activity, expressed as the increase in rate of secretion per microgram tissue protein (pl/mm/min/ug protein), is highest in the CA and CC.
The response to aqueous CC extracts is dose-dependent. The maximum increase in secretion rate is 300–350 pl/mm/min and the ED_max and ED₅₀ are 0.1–0.2 and 0.02–0.03 gland pairs respectively.
The diuretic activity in the CC is retained after 5min at 95C, and is freely soluble in 80% methanol. Diuretic activity is, however, greatly reduced after prolonged heating or after treatment with either pronase or chymotrypsin. It is conluded that the diuretic factor(s) is a low molecular weight peptide. A loss of activity after incubation with pyroglutamate amino peptidase suggests that some active peptides present are N-terminally blocked.
The diuretic activity of crude aqueous CC extracts is rapidly lost on standing at room temperature. This is partly prevented by precipitation of protein enzymes by either heat treatment or extraction in methanol.
Synthetic vertebrate and insect neuropeptides are generally low in activity increasing fluid secretion by no more than 60pl/mm/min. However, extracts of neuroendocrine tissues from a wide range of insect species are potent stimulants of tubule secretion. Various biogenic amines were tested and have little effect on fluid secretion; thus they cannot contribute greatly to the response of cricket tubules to aqueous tissue extracts.     

Immunohistochemical localization of gastrin-cholecystokinin-like material in the central nervous system of the migratory locust

Brain, corpora cardiaca (CC)-corpora allata (CA) complex, suboesophageal ganglion, thoracic and abdominal ganglia of adults, larvae and embryos of Locusta migratoria have been immunohistochemically screened for gastrin cholecystokinin (CCK-8(s]-like material. In adult, numerous immunoreactive neurons and nerve fibres are located, with a marked symmetry, in various parts of the brain and throughout the ventral nerve cord. In the median part of the brain, cell bodies belonging neither to cellular type A1 nor A2 (following Victoria blue-paraldehyde fuchsin staining) are immunopositive; their processes terminate in the upper protocerebral neuropile. In lateral parts of the brain, external cell bodies send axons into CC and some up to CA, other internal have processes which terminate in the neuropile of the brain. Two of these latter cells react also with methionine-enkephalin antiserum. In the ventral nerve cord, in addition to numerous perikarya, immunoreactive arborizations terminate in the neuropile or in close association with the sheath, at the dorsal part of all ganglia. This CCK-8(s) distribution pattern is observed only at the two last larval instars, but is precociously detected in the abdominal nerve cord of embryos, one day before hatching.     

Immunohistochemical localization of gastrin-cholecystokinin-like material in the central nervous system of the migratory locust

Summary Brain, corpora cardiaca (CC)-corpora allata (CA) complex, suboesophageal ganglion, thoracic and abdominal ganglia of adults, larvae and embryos of Locusta migratoria have been immunohistochemically screened for gastrin cholecystokinin (CCK-8(s))-like material. In adult, numerous immunoreactive neurons and nerve fibres are located, with a marked symmetry, in various parts of the brain and throughout the ventral nerve cord. In the median part of the brain, cell bodies belonging neither to cellular type A1 nor A2 (following Victoria blue-paraldehyde fuchsin staining) are immunopositive; their processes terminate in the upper protocerebral neuropile. In lateral parts of the brain, external cell bodies send axons into CC and some up to CA, other internal have processes which terminate in the neuropile of the brain. Two of these latter cells react also with methionine-enkephalin antiserum. In the ventral nerve cord, in addition to numerous perikarya, immunore-active arborizations terminate in the neuropile or in close association with the sheath, at the dorsal part of all ganglia.This CCK-8(s) distribution pattern is observed only at the two last larval instars, but is precociously detected in the abdominal nerve cord of embryos, one day before hatching.     

Diuresis in a desert beetle? hormonal control of the malpighian tubules ofOnymacris plana (Coleoptera: Tenebrionidae)

Summary Malpighian tubules of a desert tenebrionid beetle,Onymacris plana, have been studied as isolated preparations. Under control conditions tubules of female beetles secreted fluid at an average rate of 3.3 nl/min, but this rate was increased 20–25 times by a diuretic hormone (DH).Homogenates of the brain, corpora cardiaca (CC) and prothoracic ganglion induced striking increases in tubule secretion rates, which sometimes exceeded 100 nl/min. The increased rates were sustained for 3 h without renewal of the medium. Diuretic activity was also present in the other thoracic ganglia. High K treatment caused release of DH from the CC only.Exogenous cyclic AMP (1 mM) stimulated the isolated tubules ofO. plana, but to a lesser extent than the DH. The cationic composition of the secreted fluid resembled that of most other insect tubules, with high K and low Na concentrations. Stimulation with DH doubled the Na concentration.The DH was not inactivated by the tubules themselves, but was destroyed by contact with the haemolymph. An inactivation mechanism is vital in the apparently contradictory situation of a desert beetle possessing a diuretic hormone. The role of the cryptonephric system during diuresis is unknown.Abbreviations DH diuretic hormone - cAMP adenosine 3:5-cyclic monophosphoric acid - CC corpora cardiaca     

The inhibition of lipid synthesis in vitro in the locust, Schistocerca gregaria, by factors from the corpora cardiaca

ABSTRACT. The rate of lipid synthesis from [¹⁴C]acetate in fat body from Schistocerca americana gregaria has been studied in vitro. Maximum incorporation is found on days 6–10 in adults and day 4 of the fifth stadium. The label appeared in the fatty acid components of triacyl-glycerol, diacylglycerol and phospholipid.
Lipid synthesis in vitro was inhibited by extracts of corpora cardiaca, and such inhibition was most marked (up to 85%) in fat bodies from insects at stages where fatty acid synthesis was greatest. HPLC separation of corpora cardiaca extracts gave several active fractions of which the most active was adipokinetic hormone 1 (AKH-1).