Sites of action of a peptide neurohormone that controls hindgut muscle activity in the cockroach Leucophaea maderae

A peptide neurohormone from the brain and nervous system of the Madeira cockroach Leucophaea maderae has stimulating effects on both the mechanical and electrical events of hindgut visceral muscle. The peptide initiated action potentials at silent recording sites in the circular muscles of the rectum after prior treatment with tetrodotoxin (10⁻⁶ g/ml). The neurohormone also caused an increase in the amplitude and frequency of spontaneous postsynaptic potentials. However, the isolated hindgut failed to respond to the neurohormone after depolarization in high potassium saline solutions. Both the potassium contracture and the action of the neurohormone were calcium dependent.Although some hindguts were responsive to the neurohormone in a Ca free medium, such preparations failed to respond in 0·5 mM EGTA. Moreover, 1 mM Mn blocked the action of the peptide. The sodium ion was also essential for effective hormone action. These results suggest the presence of a loosely bound source of Ca at the surface of muscle membranes that in some way interacts with the neurohormone to change muscle excitability.     

Release from the brain and acquisition by the corpus cardiacum of a neurohormone in vitro

Hindgut stimulating neurohormone (HSN) was synthesized and stored by cultured brains of the cockroach, Leucophaea maderae. The presence of either HSN or cultured corpora cardiaca in the medium caused the brains to release their accumulated HSN. The corpora cardiaca were able to sequester HSN from the medium when the concentration was above a threshold level. Thus, the corpus cardiacum may provide an homeostatic mechanism for maintaining physiological levels of neurohormone.     

The Heterodimeric Glycoprotein Hormone,GPA2/GPB5, Regulates Ion Transport across the Hindgut of the Adult Mosquito,Aedes aegypti

A family of evolutionarily old hormones is the glycoprotein cysteine knot-forming heterodimers consisting of alpha- (GPA) and beta-subunits (GPB), which assemble by noncovalent bonds. In mammals, a common glycoprotein hormone alpha-subunit (GPA1) pairs with unique beta-subunits that establish receptor specificity, forming thyroid stimulating hormone (GPA1/TSHβ) and the gonadotropins luteinizing hormone (GPA1/LHβ), follicle stimulating hormone (GPA1/FSHβ), choriogonadotropin (GPA1/CGβ). A novel glycoprotein heterodimer was identified in vertebrates by genome analysis, called thyrostimulin, composed of two novel subunits, GPA2 and GPB5, and homologs occur in arthropods, nematodes and cnidarians, implying that this neurohormone system existed prior to the emergence of bilateral metazoans. In order to discern possible physiological roles of this hormonal signaling system in mosquitoes, we have isolated the glycoprotein hormone genes producing the alpha- and beta-subunits (AedaeGPA2 and AedaeGPB5) and assessed their temporal expression profiles in the yellow and dengue-fever vector, Aedes aegypti. We have also isolated a putative receptor for this novel mosquito hormone, AedaeLGR1, which contains features conserved with other glycoprotein leucine-rich repeating containing G protein-coupled receptors. AedaeLGR1 is expressed in tissues of the alimentary canal such as the midgut, Malpighian tubules and hindgut, suggesting that this novel mosquito glycoprotein hormone may regulate ionic and osmotic balance. Focusing on the hindgut in adult stage A. aegypti, where AedaeLGR1 was highly enriched, we utilized the Scanning Ion-selective Electrode Technique (SIET) to determine if AedaeGPA2/GPB5 modulated cation transport across this epithelial tissue. Our results suggest that AedaeGPA2/GPB5 does indeed participate in ionic and osmotic balance, since it appears to inhibit natriuresis and promote kaliuresis. Taken together, our findings imply this hormone may play an important role in ionic balance when levels of Na⁺ are limited and levels of K⁺ are in excess – such as during the digestion and assimilation of erythrocytes following vertebrate blood-feeding by females.     

Two stages of juvenile hormone-mediated growth of secondary follicles in Culex pipiens

To investigate the effect of juvenile hormone deprivation on the growth of secondary follicles during the second gonotrophic cycle, female mosquitoes, Culex pipiens L., were allatectomized daily after the first blood meal. Allatectomy on days 1–3 suppressed growth of secondary follicles indicating that juvenile hormone was required for a second gonotrophic cycle. When allatectomy was performed 4 days or more after the first blood meal, secondary follicles grew, indicating the presence of juvenile hormone. However, if mosquitoes were allatectomized before oviposition, only 25% developed a second batch of eggs after a second blood meal.Allatectomies performed 1 and 24 h after oviposition indicated that additional juvenile hormone was released after deposition of the first batch of eggs, and that this second release was needed for secondary follicles to complete previtellogenic growth. Thus, in C. pipiens, secondary follicles undergo two periods of juvenile hormone-mediated growth-one before and one after oviposition.     

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.     

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.     

Argas (Persicargas) arboreus: juvenile hormone analog termination of diapause and oviposition control

The juvenile hormone analog acetaldehyde, 2-(2-ethoxyethoxy) ethyl p-(methylthio) phenyl acetal, topically applied in a concentration of 10 μg/5 μl acetone/female, terminated diapause and induced vitellogenesis and oviposition in fed female Argas (Persicargas) arboreus Kaiser, Hoogstraal, and Kohls (Argasidae). This is the first reported hormonal termination of diapause in argasid ticks. A full bloodmeal and mating were critical factors controlling oviposition in nondiapausing females, and may enhance juvenile hormone activity. Experiments suggest that tick bodies possess sites of hormonal activities similar to those in other arthropods.     

The effects of mating,exogenous juvenile hormone and a juvenile hormone analogue on pheromone titre,calling and oviposition in the omnivorous leafroller moth (Platynota stultana)

Mating in Platynota stultana resulted in the termination of calling, the gradual reduction of pheromone in the pheromone glands to non-detectable levels (<0.1 ng/♀) within 14 h, and oviposition of the first batch of eggs 20–24 h after copulation. Decapitation of virgin females resulted in a similar decline in pheromone titre, and also eliminated oviposition and calling. Pheromone production appears to be controlled via the head. Mating probably terminates neural or hormonal input required for pheromone production and/or removes neural or hormonal inhibition of pheromone degradation. A juvenile hormone analogue (ZR-512) and juvenile hormones I, II and III applied exogenously to virgin females elicited oviposition comparable to mated females and terminated calling within 48 h. The juvenile hormone analogue also appeared to block pheromone production in virgin females. These results suggest that juvenile hormone may be involved in the switch from virgin to mated behaviour in this species.     

Male accessory gland substance of Melanoplus sanguinipes: An oviposition stimulant under the control of the corpus allatum

The accessory glands of male Melanoplus sanguinipes contain an oviposition stimulant. Injection of gland extracts from mature males induces oviposition in 75 per cent of capable virgin females within 24 hr. Injection of gland extracts from allatectomized males produces no stimulatory effect. Gland extracts from mature males contain two antigens which cannot be detected in gland extracts from allatectomized males. However, both antigens can be detected in gland extracts from allatectomized males 3 days after treatment with juvenile hormone.Anion-exchange chromatography of mature gland extracts yielded two fractions which, when injected into virgin females, induced oviposition in 71 per cent of capable insects within 24 hr. These two fractions are immunologically identical to the two antigens which are absent from gland extracts of allatectomized males. We suggest that synthesis of the oviposition stimulant in Melanoplus is controlled by the corpus allatum.Injection of brain extract also induces oviposition in 100 per cent of capable virgin females within 24 hr. A possible rôle for the brain in the oviposition process is discussed.     

Isolation and amino terminal sequence of melanization and reddish coloration hormone (MRCH) from the silkworm,Bombyx mori

Cuticular melanization associated with the gregarious phase of the common armyworm larvae, Leucania separata, is caused by a neurohormone, melanization and reddish coloration hormone (MRCH). Two molecular species of MRCH were isolated from 211,000 heads of adult Bombyx mori with conventional column chromatography and reversed-phase high performance liquid chromatography. As little as 6 ng of purified MRCH elicited marked melanization in the cuticle of an L. separata larva. Automated Edman degradation confirmed 16 residues of N-terminal amino acid sequences of the purified MRCHs; these showed homology with each other.     

Juvenile hormone regulation of the second biting cycle in Culex pipiens

Juvenile hormone regulation of the second biting cycle was studied in Culex pipiens by allatectomizing mosquitoes at daily intervals after the first blood meal. Mosquitoes oviposited and were tested for biting 2 days later. Allatectomy on days 1 through 4 prevented biting in a high percentage of females, indicating that juvenile hormone was required for a second biting cycle. When allatectomy was delayed 6 or more days after the first blood meal, the mosquitoes took a second blood meal after oviposition even though eggs were retained at the time of allatectomy. Thus, egg retention did not prevent mosquitoes from releasing juvenile hormone for a second biting cycle. Unoperated mosquitoes also bit when they were forced to retain eggs after the first blood meal. However, these mosquitoes only fed on blood if they were confined on or near the host; they would not feed in cages where host seeking was required to locate the host. Based on these findings, it appeared that oviposition was necessary to initiate host-seeking behaviour, whereas juvenile hormone release initiated biting. These results, in conjunction with earlier work demonstrating juvenile hormone induction of the first biting cycle, indicated that alternating periods of juvenile hormone production and oviposition regulate the cyclic patterns of biting and host-seeking behaviour throughout the life of the mosquito.     

Identification of the juvenile hormone from the cricket, Teleogryllus commodus, and juvenile hormone titre changes

In the cricket, Teleogryllus commodus, eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.     

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.     

Experimental and ultrastructural study of the control of ovulation and parturition in the tsetse fly Glossina fuscipes (Diptera)

Injections of haemolymph, organ extracts and various other substances, as well as in vitro experiments, show that ovary and oviduct extracts on the one hand, and dibutyryl cyclic AMP on the other, enhance ovulation, whereas parturition is stimulated by extracts of the brain, thoracic ganglionic mass, nerve XVII, different parts of the genital apparatus and perhaps proctolin. On the contrary, the proximal neurohaemal organs (corpus cardiacum and perisympathetic organs) appear to contain a substance inhibiting parturition. Lastly, normal intact flies cannot respond to extracts facilitating parturition in decapitated flies. Neuromuscular junctions probably containing peptides and neurotransmitters are described in the muscles of the ovaries, oviducts and the vaginal aperture. Other junctions containing neurotransmitters only are present in uterine and intersegmental muscles. The neurohaemal areas of nerve XVII contain 3 types of peptidergic terminals. From our overall results it is concluded that ovulation is regulated by neurosecretory products released at neuromuscular junctions in the ovaries and oviducts. Parturition control is more complicated. A first neurohormone (parturition-stimulating hormone) appears to be produced in the nerve centres and released in neurohaemal areas located on nerve XVII in the vicinity of the uterus; it enhances the contraction of this organ. A second neurohormone, the parturition-inhibiting hormone may be released in the corpora cardiaca and the median perisympathetic organ. A cephalic nervous factor might exert inhibitory action.     

The influence of light and sea water temperature on the reproductive cycle of Lineus ruber (Heteronemertea)

The cerebral ganglia of Lineus ruber secretes a neurohormone named gonad-inhibiting hormone (GIH) controlling reproduction. As L. ruber undergoes repeated annual cycles of reproduction during its life, the way the neuroendocrine activity of the worm is seasonally regulated is an important ecophysiological question. In other words, what environmental ‘key factors’ influences the beginning of spawning season in L. ruber? In the present work we show that light is not a timer which controls the annual reproductive cycle progress of L. ruber. On the other hand our results suggest that thermoperiod is a good candidate for this function. Further work is still necessary to clarify the transduction mechanism of thermal stimuli in GIH discharge.     

Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig

Corpora allata of adult female Euborellia annulipes, incubated in medium containing ³H-methionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 ± 1.6% of the total juvenile hormone III synthesized was released and 96.5% ± 0.3 in medium supplemented with 60 μM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 ± 0.004 mm), hormone production was only 0.59 ± 0.13 fmol hr⁻/corpus allatum; production increased to 1.52 ± 0.25 fmol hr⁻¹/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 ± 0.13 fmol hr⁻¹/corpus allatum. The addition of 60 μM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of l-methionine in the medium also enhanced hormone biosynthesis. Maximal hormone production was 32.8 ± 10.9 fmol hr⁻¹/corpus allatum, at an l-methionine concentration of 51 μM.     

Ecdysone,juvenile hormone and oögenesis in Rhodnius prolixus

Oviposition and oögenesis can be inhibited in female Rhodnius prolixus by ecdysone given by the digestive tract. The inhibition is dose-dependent, and doses higher than 4.0 ng ecdysone/mg body weight drastically reduce the size and shape of the whole ovaries. In ecdysone-treated insects, normal oviposition and oögenesis can be re-established by a subsequent blood meal without ecdysone, or by the application of a juvenile hormone analogue.These results suggest that ecdysone inhibits juvenile hormone production.     

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Control of oocyte maturation in sexually mature Drosophila females

In many sexually mature insects egg production and oviposition are tightly coupled to copulation. Sex-Peptide is a 36-amino-acid peptide synthesized in the accessory glands of Drosophila melanogaster males and transferred to the female during copulation. Sex-Peptide stimulates vitellogenic oocyte progression through a putative control point at about stage 9 of oogenesis. Here we show that application of the juvenile hormone analogue methoprene mimics the Sex-Peptide-mediated stimulation of vitellogenic oocyte progression in sexually mature virgin females. Apoptosis is induced by 20-hydroxyecdysone in nurse cells of stage 9 egg chambers at physiological concentrations (10(-7) M). 20-Hydroxyecdysone thus acts as an antagonist of early vitellogenic oocyte development. Simultaneous application of juvenile hormone analogue, however, protects early vitellogenic oocytes from 20-hydroxyecdysone-induced resorption. These results suggest that the balance of these hormones in the hemolymph regulates whether oocytes will progress through the control point at stage 9 or undergo apoptosis. These data are further supported by a molecular analysis of the regulation of yolk protein synthesis and uptake into the ovary by the two hormones. We conclude that juvenile hormone is a downstream component in the Sex-Peptide response cascade and acts by stimulating vitellogenic oocyte progression and inhibiting apoptosis. Since juvenile hormone analogue does not elicit increased oviposition and reduced receptivity, Sex-Peptide must have an additional, separate effect on these two postmating responses.     

Seminal fluid reduces female longevity and stimulates egg production and sperm trigger oviposition in a moth

Previous studies suggest that a number of factors in relation to mating may reduce female longevity and stimulate egg production and oviposition. However, it is still not clear whether these factors act on these parameters independently or in a collective way. Here we carried out a series of experiments including mating trials and seminal fluid injection to determine the factors responsible for reducing female longevity and stimulating egg production and oviposition in relation to mating in the moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Results show that seminal fluid and sperm work collectively to allow females to achieve maximum realized fecundity (number of eggs laid) in E. kuehniella but these factors play different roles in the process and their actions are independent. Seminal fluid signals females to allocate resources to ova, resulting in shorter longevity and greater egg production while eupyrene (not apyrene) sperm in the spermatheca trigger females to lay maximum number of eggs. We suggest that the receptors for seminal fluid signal may be located in the female reproductive tract and haemolymph, and those for sperm signal may be in the spermatheca. Hypotheses that females prolong their longevity by oosorption, physical injuries by males reduce female longevity, and mechanical stimulation by males triggers oviposition, are not substantiated in the present study.