Synthesis, transport, and release of a neurohormone by cultured neuroendocrine glands from the cockroach, Leucophaea maderae

A hindgut-stimulating neurohormone synthesized in vitro by the neurosecretory cells of cultured brains of Leucophaea maderae passes through the nervi corporis cardiaci I into the corpora cardiaca and is released into the culture medium. As much as 90 per cent of the hormone breaks down in the medium during a 3-day incubation period, and the amount recovered represents only a small fraction of the amount actually released.     

Experimental evidence for a neuroendocrine control of ecdysone biosynthesis in adult females of Locusta migratoria

The considerable increase in ecdysteroid concentration which occurs in normal Locusta ovaries at the end of each cycle of oöcyte maturation is prevented if the median neurosecretory cells of the pars intercerebralis are cauterized, or if the corpora cardiaca are excised 24 hr before the onset of ecdysone synthesis in normal females. Implantation of additional brain-corpora cardiaca complexes into young vitellogenic females advances the time of ecdysone synthesis by some 12 hr. Oöcyte growth itself is not affected in these different types of experiments.It is inferred from the data of the present study that ecdysone synthesis in the follicle cell epithelium of maturing Locusta ovaries is stimulated by a neurohormone produced in the median neurosecretory cells of the pars intercerebralis and secreted into the blood via the corpora cardiaca.     

Characterization of three neurosecretory proteins from the A median neurosecretory cells of Locusta migratoria by coupled chromatographic,electrophoretic and isoelectrofocusing methods

Proteins from the nervous corpora cardiaca (N-CC) of Locusta migratoria were analysed using a combination of chromatography, electrophoresis and electrofocusing. Three major cysteine or/and cystine-rich proteins were identified as neurosecretory proteins of the median neurosecretory cells (M-NSC) by comparing the pattern of separated proteins of (1) intact N-CC and (2) depleted N-CC after intracerebral axotomy of the M-NSC.These three neurosecretory proteins are: a trimer, a dimer and a monomer. The trimer and the monomer are composed of apparent 18,000 subunits and 9000 polypeptide chains. The dimer is composed of apparent 7000 subunits composed of putative 4000 polypeptide chains. The three neurosecretory proteins are acidic (pH_i approx. 5.6 for the trimer, 5.1 for the monomer and 4.0 for the dimer). Their relationship to the neurophysins and to the neurosecretory protein isolated from brain and corpora cardiaca of the locust by Friedel et al. (1980) (Gen. comp. Endocr.41, 487–498) is discussed. The role of these three neurosecretory proteins (carrier protein, neurohormone or precursor of neurohormone) is as yet unknown.     

Effect of endocrine glands on the unfixed total haemocyte counts of the bug Halys dentata

Changes in total haemocyte counts in relation to the endocrine glands have been studied in Halys dentata. Unfixed total haemocyte counts were taken after extirpation and implantation of three endocrine structures. It is concluded that haemocyte count decreases after the extirpation of the corpora allata in the early life span of adults, while an abrupt increase was noted after the 7th day. When both the corpora allata and corpora cardiaca are extirpated, the count was significantly decreased throughout the life span of adults. However, when only the corpora cardiaca were removed there was no significant variation up to the 7th day, but later on the count gradually decreased. The implantation of brain, corpora cardiaca and allata is followed by a significant rise in the haemocyte count throughout the life span. Total haemocyte count also fluctuated after the implantation of corpora allata and brain, separately. After implantation of corpora cardiaca of one-day-old adult insects there was no variation in the total haemocyte count while the corpora cardiaca of 6-day-old adults when implanted in one-day-old adults cause a significant rise in the total haemocyte count. Therefore, it is concluded that the corpora cardiaca influences the total haemocyte count after a critical period.     

Density-gradient centrifugation isolation of hormone-containing neurosecretory granules from the cockroach,Leucophaea maderae

Neurosecretory granules (NSG) containing hindgut-stimulating neurohormone (HSN) from Leucophaea maderae were isolated by densitygradient centrifugation of cockroach brain homogenates.High concentrations of HSN were consistently found in isolates containing large numbers of NSG. HSN was measured by bioassay and the NSG were identified by electron microscopy.     

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.     

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.     

Immunochemical analysis of the distribution of the new ovary maturating neurohormone during development of the African locust,Locusta migratoria

Summary Following our prior identification of a gonadotropic neurohormone isolated from the neurosecretory lobe of the corpora cardiaca of the African locust, we have raised a polyclonal antiserum against this new molecule. In the present paper, we characterize this antiserum using enzyme-linked immunosorbent assay and Western blotting. The latter procedure reveals that the immune serum specifically recognizes the neurohormone, which we have termed ovary maturating parsin. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to analyze the distribution of this gonadotropic neurohormone throughout the central nervous system during development. It is produced only by the type-B neurosecretory cells of the pars intercerebralis-corpora cardiaca system and is present both in males and females throughout life from embryo to adult. This permanent expression suggests that the neurohormone may have functions other than its primary direct gonadotropic role in females.     

Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata

In the adult males of the Colorado potato beetle, Leptinotarsa decemlineata, corpora cardiaca extract injected in vivo gives rise to an increase in glucose and a decrease in alanine concentration of the haemolymph. The regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle was investigated further in vitro. Proline regulates its own synthesis by a feedback inhibition. Moreover, a factor present in extracts from the corpora cardiaca stimulates synthesis in the fat body in vitro. This effect was demonstrated with corpora cardiaca extracts from beetles, locusts and cokroaches. Also, synthetic adipokinetic hormone stimulates proline synthesis in the fat body of the Colorado beetle. In addition, a release of glucose from the fat body of the beetle could be evoked by the addition of locust and beetle corpora cardiaca extracts or synthetic adipokinetic hormone. The physiological significance of both effects is discussed.     

Restricted specificity of a hyperglycaemic factor from the corpus cardiacum of the stick insect Carausius morosus

Extracts of the corpora cardiaca of the stick insect Carausius morosus elevate the haemolymph carbohydrate concentration in adult and 6th-instar larvae which are ligated behind the first pair of legs, but not in non-ligated (intact) insects. The increase in haemolymph sugars is due to trehalose elevation, is time dependent (with a maximal effect about 90–120 min after injection), and is dose dependent (needing 0.005 gland equivalents for a significant effect and a tenfold higher dose for a maximal response). The hyperglycaemic factor is localised entirely in the corpora cardiaca and appears to be specific to stick insects; corpora cardiaca extracts of two lepidopteran species (Acherontia atropos and Aglais urticae) and of Locusta migratoria have no effect, whereas corpora cardiaca extracts of the stick insects Cuniculina impigra and Sipyloidea sipylus have similar activity to those from C. morosus. This specificity is also shown when S. sipylus is used as the recipient. Synthetic adipokinetic hormone and red pigment concentrating hormone possess no hyperglycaemic activity in the stick-insect system. Two peaks of hyperglycaemic activity were obtained after column chromatography of corpora cardiaca extract of C. morosus on Sephadex G-25 and Sephadex LH-20. The factor seems to act via activation of fat-body glycogen phosphorylase, which, although 60% active in the control insects, is significantly increased to approx. 85% upon corpora cardiaca injection. However, the activation is demonstrated in ligated and intact insects. No significant decrease in the glycogen level of the fat body is observed after corpora cardiaca injection.     

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.     

Specific action of activation factor I on RNA synthesist in prothoracic glands in insects

The activation factor I, one of the two prothoracotropical factors obtained by gel filtration techniques from extracts of corpora cardiaca of cockroaches, induces a significant stimulation of uridine incorporation into the RNA of cultured larval prothoracic glands of Periplaneta americana and Galleria mellonella. This effect shows a clear dose-response relationship. Results obtained with actinomycin D permit the conclusion that increase of the uridine incorporation reflects a true stimulation of RNA synthesis of these glands. α-Amanitin does not block the stimulation of the total RNA induced by the activation factor I.The activation factor I has no influence on the uridine incorporation in other tested larval tissues and organs of cockroaches of the same age (brains, corpora cardiaca-corpora allata complexes, salivary glands, muscle tissue, midgut, fat body tissue).A close relationship may be suggested from this specific action of activation factor I on the RNA synthesis of prothoracic glands with α-ecdysone synthesis specifically proved in these organs.     

In vivo and in vitro photoperiodic induction of diapause using isolated brain-suboesophageal ganglion complexes of the silkworm,Bombyx mori

By use of a bivoltine silkworm race which shows a long-day photoperiodic response after induction during the last (5th) instar, we tried to programme photoperiodic induction in the isolated brain-suboesophageal ganglion complex in vivo and in vitro. A pair of the complexes from a newly ecdysed 5th-instar female was transplanted into the abdomen of a late 5th-instar larva and exposed to long-day (20 h light: 4 h dark) or short-day (8 h light: 16 h dark) conditions for 3 cycles. The short-day-exposed complexes elicited the production of diapause eggs in the recipient silkworms destined to become non-diapause egg producers, whereas the long-day-exposed brain complexes produced non-diapause eggs. Transplant experiments of the brain-suboesophageal ganglion complex using isolated abdomens showed a similar result. The brain complexes from newly ecdysed females of the 5th-instar were cultured in Grace's insect medium under 20 h light: 4 h dark or 8 h light: 16 h dark for 4 cycles, respectively. After in vitro culture, a pair of complexes was implanted into the abdomen of a late 5th-instar larva destined to become a non-diapause egg producer, and the diapause incidence in the resultant moths was examined. The brain complexes which received the short-day cycles induced a large portion of diapause eggs, whereas those which received the long-day conditions induced non-diapause eggs. The connection of corpora cardiaca and corpora allata with the brain complex had no influence on the result. Suboesophageal ganglia which had been cultured in vitro and implanted elicited a remarkable production of diapause eggs, but cultured brains were ineffective in producing diapause eggs, regardless of the photoperiod experienced. These results demonstrate that photoperiodic induction of the silkworm can be programmed in in vivo and in vitro culture systems, and that components of the photoperiodic clock (photoreceptor, clock, and counter system) are located in the brain-suboesophageal ganglion complex, possibly in the brain itself.     

Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans

Aqueous extracts of brain, thoracic ganglion or corpora cardiaca of female Glossina morsitans were shown to contain a substance which inhibited the synthesis of lipid from l[U-¹⁴C] leucine by fat cells incubated in vitro. The highest concentration of this substance was found in the corpora cardiaca; approximately 1 × 10^?6 gland pairs μl^?1 were required for maximum inhibition. At concentrations greater than 1 × 10^?4 gland pairs μl^?1 the lipid synthesis inhibiting factor (hereafter referred to as the LSIF) was inactivated by the presence of a substance which could be removed by gel filtration. The concentration of LSIF in the corpora cardiaca and midbrain varied throughout the reproductive cycle of the female. Net release of LSIF from the midbrain occurred between the 2nd and 7th day of the 9-day reproductive cycle. Net release from the corpora cardiaca began on day 5 and continued until the end of the interlarval period on day 9. Results are consistent with the hypothesis that LSIF is synthesised mainly in the medial neurosecretory cells of the midbrain whereas the corpora cardiaca are the site of storage and release into the haemolymph. LSIF was present in midbrain and corpora cardiaca extracts from male G. morsitans but at lower concentrations than in females. No variation in LSIF concentration could be correlated with the feeding cycle. LSIF activity was not detected in fresh haemolymph but was found at high concentration in boiled haemolymph, suggesting the presence of an inhibitor which was inactivated at high temperature. Preliminary investigations into the nature of LSIF have shown it to be inactivated by proteolytic enzymes and to be recoverable in a single peak from a Sephadex G15 column.Results support the view that LSIF is a peptide hormone which, in conjunction with an inhibitor, controls the lipid synthetic ability of the fat cells of the adult female tsetse fly throughout the reproductive cycle.     

Re-evaluation of the role of corpora cardiaca in calling and oviposition behaviour of giant silk moths

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating.Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.     

Synthesis and storage of a neurohormone in insect brains in vitro

The build-up of neurosecretory material in the median neurosecretory cells and fibre tracts of cultured cockroach brains was demonstrated by staining and bioassay. Examination of the cultured brains by electron microscopy showed active production of neurosecretory granules after 3 days in vitro. The close correlation of the results obtained by these different methods of assay leaves little doubt that a neurohormone is being synthesized and stored.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.     

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.     

Experimentelle Untersuchungen zum Freisetzungsmechanismus von Neurohormonen nach elektrischer Reizung der Corpora cardiaca von Periplaneta americana in vitro

Releasing of neurohormones by in vitro stimulation of the corpora cardiaca of Periplaneta americana by means of suction electrodes and by simultaneous application of sympathicomimetics and -lytics as well as parasympathicomimetics and -lytics was investigated. The release of neurohormone D by stimulation of N.c.c. I in the presence of atropine in bath-fluid is inhibited. Contrary to this, the presence of eserine stimulates release. Application of reserpine, as well as of the sympathicolytics tolazolin, retinin, and guanethidin, does not influence release of this hormone in connexion with the stimulation of N.c.c. I. These results indicate that the release of neurohormone D by stimulation of N.c.c. I is regulated by cholinergic components. On the other hand, release of the hyperlgykaemic factor by electrical stimulation of N.c.c. II is controlled by adrenergic components. This conclusion results from the increase of the release rate caused by reserpine. Sympathicolytics decreased the release rate. Atropine and eserine do not influence hormone release in connexion with the stimulation of N.c.c. II.