Glucosidation of estradiol-17 beta in the cultured ovaries of the silkworm, Bombyx mori

Metabolic pathways of estradiol-17 beta and other vertebrate steroid hormones of cultured silkworm pupal ovaries were examined using 14C-labeled steroids. The isolated ovaries showed significant uptake and metabolic activity of the 14C-labeled estradiol-17 beta added to the medium. Analysis of the metabolized compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed extensive metabolic conversion of [14C]estradiol-17 beta and estrone; i.e., estrone was reduced to estradiol-17 beta and estradiol-17 beta was metabolized to conjugates, including estradiol-3-beta-D-glucoside and estradiol-17-alpha-D-glucoside. [14C]Testosterone was not transformed appreciably by the ovaries. Metabolic activity and physiological significance of the vertebrate steroid hormones in the silkworm ovaries are discussed.     

Identification of estradiol in the ovaries of the silkworm, Bombyx mori

Estradiol was extracted and partially purified from the ovaries of the silkworm, Bombyx mori. Identification of estradiol was done by use of radioimmunoassay (RIA) and by gas chromatography-mass spectrometry (GC-MS) after derivatization into the ethyldimethylsilyl derivative. Concentration of estradiol in the ovaries was estimated to be 176 pg/g (RIA) and 63 pg/g (GC-MS).     

Effect of the diapause hormone on trehalase activity in pupal ovaries of the silkworm, Bombyx mori L

Effect of the diapause hormone on trehalase activity was investigated in the silkworm, Bombyx mori L. Trehalase was located at different activities in various tissues of the pupae, and among the tissues tested ovary trehalase activity alone decreased after extirpation of the subesophageal ganglion (SG), the source of the diapause hormone. Removal of the SG affected neither kinetic properties of ovary trehalase nor the subcellular distribution in ovaries.     

Patterns in Genotype Composition of Indian Isolates of the Bombyx mori Nucleopolyhedrovirus and Bombyx mori Bidensovirus

The mulberry silkworm, Bombyx mori (L.), is a model organism of lepidopteran insects with high economic importance. The viral diseases of the silkworm caused by Bombyx mori nucleopolyhedrovirus (BmNPV) and Bombyx mori bidensovirus (BmBDV) inflict huge economic losses and significantly impact the sericulture industry of India and other countries. To understand the distribution of Indian isolates of the BmNPV and to investigate their genetic composition, an in-depth population structure analysis was conducted using comprehensive and newly developed genomic analysis methods. The seven new Indian BmNPV isolates from Anantapur, Dehradun, Ghumarwin, Jammu, Kashmir, Mysore and Salem grouped in the BmNPV clade, and are most closely related to Autographa californica multiple nucleopolyhedrovirus and Rachiplusia ou multiple nucleopolyhedrovirus on the basis of gene sequencing and phylogenetic analyses of the partial polh, lef-8 and lef-9 gene fragments. The whole genome sequencing of three Indian BmNPV isolates from Mysore (-My), Jammu (-Ja) and Dehradun (-De) was conducted, and intra-isolate genetic variability was analyzed on the basis of variable SNP positions and the frequencies of alternative nucleotides. The results revealed that the BmNPV-De and BmNPV-Ja isolates are highly similar in their genotypic composition, whereas the population structure of BmNPV-My appeared rather pure and homogenous, with almost no or few genetic variations. The BmNPV-De and BmNPV-Ja samples further contained a significant amount of BmBDV belonging to the Bidnaviridae family. We elucidated the genotype composition within Indian BmNPV and BmBDV isolates, and the results presented have broad implications for our understanding of the genetic diversity and evolution of BmNPV and co-occurring BmBDV isolates.     

A novel recombinant dual human SCF expressed in and purified from silkworm, Bombyx mori, possesses higher bioactivity than recombinant monomeric human SCF

A novel recombinant dual human stem cell factor (rdhSCF) gene was constructed which consisted of a full-length hSCF cDNA plus a truncated hSCF cDNA (1-145 aa), linked by a peptide (GGGGSGGGGSGG) coding region. The rdhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the polyhedrin promoter control. Silkworm larvae infected with the recombinant virus expressed rdhSCF up to 15,800 units/mL in haemolymph. The specific activity of rdhSCF purified from the haemolymph was up to 3.0 x 10(6) units/mg, about 8.6 times as high as that of monomer rhSCF from Escherichia coli, and about 9.1 times as high as that of monomer rhSCF from insect cell. The binding affinity of rdhSCF to the cell surface receptor was higher than that of monomer rhSCF.     

Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori

In insects, especially phytophagous insects, feeding behavior occurs at a regular frequency. Although a number of physiological studies have revealed various causal factors leading to feeding behavior in insects, little has been demonstrated regarding the regulatory mechanisms underlying insect feeding behavior. To confirm the presence of an endocrinological regulatory mechanism in feeding behavior, we tested the effects of several biologically active peptides on silkworm, Bombyx mori larvae feeding behaviors. To evaluate the effects of the biologically active peptides, we measured the period of latency to the first bite following sample injection into starved Bombyx larvae. Of the chemically synthesized peptides tested, myosuppressin exhibited a prolonged latency, indicating that myosuppressin is a possible inhibitory peptide in Bombyx larvae. In contrast, injections of tachykinin and short neuropeptide F, which are members of the structurally related RF-amide peptide family, had a shorter latency period, indicating that these two peptides are possible stimulatory peptides. In addition, the present study suggests that this bioassay will be advantageous for screening for peptides that regulate insect feeding behavior.     

Increases in [3H]FK-506 and [3H]L-NG-Nitro-Arginine Binding in the Rat Brain After Nigrostriatal Dopaminergic Denervation

Receptor autoradiographic technique was studied to investigate sequential changes in FK-506 binding proteins, nitric oxide synthase and dopamine uptake sites in the brain 1 week to 8 weeks after unilateral 6-hydroxydopamine injection of the medial forebrain bundle in rats. [³H]FK-506, [³H]L-N^G-nitro-arginine and [³H]mazindol were used to label FK-506 binding proteins (immunophilin), nitric oxide synthase and dopamine uptake sites, respectively. [³H]FK-506 binding showed about 13-25% increase in the ipsilateral striatum from 2 to 8 weeks after degeneration of nigrostriatal pathway. However, no significant change in [³H]FK-506 binding was observed in the ipsilateral substantia nigra during the postlesion periods. In the contralateral side, [³H]FK-506 binding also showed about 13-25% increase in the striatum from 2 to 8 weeks postlesion. The substantia nigra showed a 21% increase in [³H]FK-506 binding only 2 weeks after the lesioning. On the other hand, [³H]L-N^G-nitro-arginine binding showed about 21-31% increase in the parietal cortex and striatum 1 week or 2 weeks postlesion. In the contralateral side, a 21% increase in [³H]L-N^G-nitro-arginine binding was found in the dorsolateral striatum only 1 week postlesion. In contrast, degeneration of nigrostriatal pathway caused a conspicuous loss of [³H]mazindol binding in the ipsilateral striatum (87-96%), substantia nigra (36-73%) and ventral tegmental area (91-100%) during the postlesion periods. In the contralateral side, no significant changes in [³H]mazindol binding were observed in these areas upto 8 weeks after the postlesion. The present study demonstrates that unilateral injection of 6-hydroxydopamine into the medial forebrain bundle of rats can cause a significant increase in [3H]FK-506 and [³H]L-N^G-nitro-arginine bindings in the brains. In contrast, a marked reduction in [³H]mazindol binding is observed in the brains after the lesioning, indicating severe damage to nigrostriatal dopaminergic pathway. These results suggest that immunophilin and nitric oxide synthase may play some role in the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

Metabolism of injected [3H]ecdysone in male Sarcophaga bullata (Diptera)

Within 30 min after injection, [3H]ecdysone was rapidly partially metabolized to ecdysterone and other ecdysteroids. After 8 hr most (99%) of the tritiated material had disappeared from the hemolymph. In testes, the predominant ecdysteroid appeared to be ecdysterone but no accumulation occurred. Comparison of the dynamics of the ecdysone metabolism in abdomens and head-thorax sections showed that in the head-thorax ecdysterone was the major component, whereas in abdomens the major metabolites were highly polar products (HPP). However, the total amount of label was nearly the same in both parts. Two groups of HPP have been isolated from the abdomen fractions without testes: HPP B and HPP C. Only HPP B could be hydrolyzed by enzymes and seemed at least to contain glucuronides, beta-glucosides, and sulfate conjugates. After 4 hr most of the tritiated ecdysteroids were found in the fecal material. No male-specific metabolites have been discovered.     

Accumulation of [3H]Sialyl-conjugates in sialidosis (sialidase-deficient) fibroblasts cultured in the presence of [3H]-N-acetylmannosamine

Skin fibroblasts from normal individuals and a patient with the infantile form of sialidosis were cultured for up to 72h in medium containing [³H]-N-acetylmannosamine. The sialidosis fibroblasts consistently accumulated more labeled compound(s) than the control cells, i.e. 37–88% more cpm per mg protein. Precipitation of sonicates of these cells with 10% trichloracetic acid, TCA, demonstrated that the excess radioactivity in the sialidosis fibroblasts was in one or more TCA soluble compounds. There was no detectable difference in the amount of label in the TCA insoluble material.The TCA soluble, labeled, material from the sialidosis and the control fibroblasts was separated, isolated and purified on AG1-X8, QAE Sephadex A-25 and Bio-Gel P-4 chromatography columns. Analysis of the isolated material showed the excess radioactivity in the sialidosis fibroblasts to be due to increased levels of [³H]sialic acid covalently bound to a variety of anionic sialyl conjugates. These compounds have been separated and partially purified.Finally, acid hydrolysis and chromatographic analysis of the TCA insoluble fractions showed that greater than 80% of the label in this material was also due to [³H]sialic acid. There was no detectable difference between the control and the sialidosis patient in the amount of label in this fraction.     

Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori

The effects of starvation and feeding on the release of bombyxin, a peptide of insulin superfamily in insects, from the larval brain of the silkworm Bombyx mori were investigated. Following starvation, the bombyxin titer in the hemolymph of larvae decreased, whereas its content in the brain increased. On the other hand, refeeding of the starved larvae resulted in an increase in the hemolymph bombyxin titer and a rapid decrease in the hormone level in the brain. These results indicate that the release of bombyxin from the brain is suppressed by starvation and stimulated by feeding. The hemolymph glucose titer also changed sharply upon starvation and refeeding, and a close relationship was observed between the changes in glucose concentrations and bombyxin titers in the hemolymph. The injection of glucose into starved larvae could mimic the effect of refeeding on the release of bombyxin, suggesting that glucose serves as the signal for the "fed" state of the animal. It is likely that glucose is a common nutritional signal for inducing the release of mammalian and insect insulins.     

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.     

The Uptake of [γ-3H] Aminobutyric Acid in the Goldfish Retina

After goldfish retinas had been incubated for 1 hr with [gamma-(3)H]aminobutyric acid, we found by autoradiography that the label was localized to a few restricted types of retinal cells. In particular, external and internal horizontal cells from light-stimulated retinas were more heavily labeled than corresponding cells from retinas kept in darkness. Some other cells and tissues in the retina also incorporated the labeled acid. Light stimulation, however, did not cause a pronounced change in the amount of label associated with these cells. Among these were some heavily labeled cells on the vitreal side of the inner nuclear layer, and scattered grains associated with the ganglion cell and optic nerve layers. Electrophoresis of retinal extracts after incubation with the labeled acid also showed that light-stimulated retinas contained about 40-100% more radioactivity than retinas kept in darkness, and that 90% of this activity remained as [gamma-(3)H]aminobutyric acid. The role of the acid in the retina is not known; it is not clear if horizontal cells normally synthesize or store it. The stimulation-dependent accumulation of the labeled acid into horizontal cells suggests that it plays a functional role in these cells.     

Basic pattern of fluctuation in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm,Bombyx mori

General features of the changes in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm Bombyx mori were analyzed by comparing the patterns of the titer changes between different races and between silkworms reared under different environmental conditions. In common to all types of the silkworms tested, we observed low PTTH titers during the phagoperiod of the final instar, a small rise in PTTH titer on the day before wandering, two middle-sized peaks of the titer at the wandering and prepupal stages, high PTTH titers during early pupal-adult development, and a gradual titer increase shortly before adult eclosion. Increases in hemolymph PTTH titer were closely correlated with increases in ecdysteroid titers and with subsequent occurrences of morphological and behavioral changes characteristic of the initiation or progression of metamorphosis. The timing of the increase in hemolymph PTTH titer on the day of wandering was photoperiodically controlled, but that timing at the later stages seemed not to be influenced by the light-dark cycle.     

Neuroanatomical localization of sex steroid-concentrating cells in the Japanese quail (Coturnix japonica): autoradiography with [3H]-testosterone, [3H]-estradiol, and [3H]-dihydrotestosterone

Steroid autoradiography was undertaken to determine the neuroanatomical loci which might be involved in the activation of steroid-sensitive behaviors in the Japanese quail (Coturnix japonica). Male and female quail were either surgically gonadectomized or photically regressed and implanted with androgen or estrogen to restore normal sexual and courtship behavior. After gonadectomy or implant removal, each quail was injected with 250 microCi of [3H]-testosterone (3H-T), [3H]-estradiol (3H-E2), or [3H]-dihydrotestosterone (3H-DHT), sacrificed, processed for autoradiography, and the telencephalon, diencephalon, mesencephalon, and rhombencephalon were examined for labelled cells. Following 3H-T or 3H-E2 injection and autoradiography, labelled cells were found in nucleus septalis lateralis (SL), nucleus preopticus medialis (POM), nucleus paraventricularis (PVN), regio lateralis hypothalami (LHy), nucleus inferior hypothalami (IH), nucleus infundibuli (IN), nucleus intercollicularis (ICo), substantia grisea centralis (GCt), nucleus taeniae (Tn), and in the reticular formation near nucleus motorius nervi trigemini (MV). In addition, following 3H-E2 autoradiography, labelled cells were found around nucleus accumbens (Ac). Following 3H-DHT autoradiography, labelled cells were found only in SL, PVN, Tn, LHy, ICo, and CGt. No labelled cells were found in Ac, POM, IH, IN, or MV even after long exposure times. These results suggest that the nuclei labelled following 3H-E2 but not 3H-DHT administration bind exclusively the aromatized metabolites of T. Since quail show a sex difference in male-typical copulatory behavior in response to E2, labelled cells were counted in POM, LHy, IH, and Tn of male and female quail following 3H-E2 injection and autoradiography. No sex differences in the number of labelled cells were found in POM, LHy, or IH. Males were found to have more labelled cells than females in Tn. These results show that sex differences in male-typical copulatory behavior are not due to sex differences in the number of cells binding estrogens in POM. The results reported here constitute the most neuroanatomically extensive report of steroid binding cells to date for a galliform brain, the first comparison in a galliform bird of the distributions of cells labelled following injection of 3H-T, 3H-E2, and 3H-DHT and the first analysis of sex differences in numbers of estrogen-binding cells in four nuclei in the avian brain.     

Relationship between firing activity of bombyxin-producing neurosecretory cells and hemolymph bombyxin titer in the silkworm Bombyx mori

Isolated brain-retrocerebral neurohemal complex of the silkworm of Bombyx mori was stimulated electrically and the released bombyxin (an insulin-like neuropeptide) was measured using time-resolved fluoroimmunoassay. The amount of bombyxin release depended on the number of stimulus pulses delivered to the axonal tract of the bombyxin-producing (BP) neurosecretory cells, and 17 fg of bombyxin per pulse was released from a cell. The titer of bombyxin in the hemolymph of bombyxin-II injected pupae decreased exponentially, the half-life being 170 min. To relate firing activity of a population of BP cells to the hormone titer in the hemolymph, bombyxin titer and its change in the hemolymph were calculated numerically. We assumed that the amount of bombyxin release was proportional to the firing rate of BP cells and the released bombyxin was inactivated with the same time course of injected bombyxin. Our calculations suggested that the hemolymph bombyxin titer may fluctuate dynamically and the mean titer is 380 pg/ml, a level which is close to the actually determined bombyxin titer at middle stages of pupal-adult development.     

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

To ensure survival under unfavorable environmental conditions and synchronize populations, most insect species enter diapause, which is a seasonal adaptation that evolved as a specific subtype of dormancy (1, 2). Diapause is not a passive response to changing conditions but rather an actively induced state that precedes adverse natural situations. Therefore, this diapause phenotype is accompanied by changes in energy metabolism or storage to improve cold/stress tolerance in later life stages, or progeny via reproductive switch (3). Although it has been generally suggested that brain/neuroendocrine systems are associated with this seasonal reproductive plasticity in both vertebrates and invertebrates (3, 4), the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration into morphological, physiological, behavioral, and reproductive responses, known as the diapause syndrome, remains unclear (3).The silkworm Bombyx mori is a typical insect that arrests normal development during early embryogenesis, which is accompanied by metabolic changes in diapause (5, 6). The development of diapause-destined embryos is arrested during the G2 cell cycle stage immediately after the formation of the cephalic lobe and telson and sequential segmentation of the mesoderm (7). The bivoltine strain of B. mori has two generations per year, and progeny diapause is transgenerationally induced as a maternal effect and is determined by the environmental temperature, photoperiod, and nutrient conditions during embryonic and larval development of the mother (5, 6). The temperature signal during the mother’s embryonic development predominantly affects diapause determination, even if silkworms of the bivoltine Kosetsu strain are exposed to all cases of photoperiods during embryonic and larval development. In the Kosetsu strain, when eggs are incubated at 25 °C under continuous darkness, the resultant female moths (25DD) lay diapause eggs in almost all cases. In contrast, incubation of eggs at 15 °C in dark condition results in moths (15DD) that lay nondiapause eggs in almost all cases (6).Embryonic diapause is induced by the diapause hormone (DH) signaling pathway, which consists of highly sensitive and specific interactions between a neuropeptide, DH, and DH receptor (DHR) (6, 8). DH is exclusively synthesized in seven pairs of neurosecretory cells (DH-PBAN–producing neurosecretory cells [DHPCs]) located within the subesophageal ganglion (SG) in the mother’s generation (6). DH is released into the hemolymph during pupal–adult development and acts on the DHR, which belongs to the G protein-coupled receptors (GPCRs) (9). DH levels in the hemolymph are higher in the 25DD than 15DD pupae in the middle of pupal–adult development when the developing ovaries are sensitive to DH (6). Furthermore, the embryonic Bombyx TRPA1 ortholog (BmTRPA1) acts as a thermosensitive channel that is activated at temperatures above ∼21 °C and affects diapause induction through DH release (10). However, there remain questions about the thermal information that is received by BmTRPA1 and linked to DH signaling to induce diapause.From the 1950s, it has been suggested that the DH release was controlled by signals derived from certain region(s) in the brain based on surgical experiments, such as midsagittal bisection or transection (11–13). Especially, the operation in nondiapause producers changed them to diapause producers while transection of the protocerebrum had no effect on the diapause producers. These surgical results suggested the involvement of the protocerebrum in the inhibitory control of DH secretion (12, 14). Furthermore, the accumulation of the ovarian 3-hydroxykynurenine (3-OHK) pigment that accompanies the diapause syndrome was affected by injection with γ-aminobutyric acid (GABA) and the plant alkaloid picrotoxin (PTX), which is a widely used ionotropic GABA and glycine receptor antagonist (15, 16), and the selective ionotropic GABA receptor (GABAR) antagonist bicuculline. This suggests that a GABAergic neurotransmission via ionotropic GABAR is involved in DH secretion, which may be active in nondiapause producers but inactive in diapause producers throughout the pupal–adult development (14, 17). In general, ionotropic GABAR is composed of homo- or hetero-pentameric subunits. All known GABAR subunits display a similar structural scheme, with a large N-terminal extracellular domain involved in the formation of a ligand-binding pocket and a pore domain made of four transmembrane alpha-helices (TM1–TM4) (16, 18). Four homologous sequences of the ionotropic GABAR subunit genes were identified as RDL, LCCH3, GRD, and a GRD-like sequence named 8916 in various insects (19). However, the in vivo physiological roles of both signals derived from the brain and the GABAergic pathway in diapause induction have not been previously investigated.Corazonin (Crz) is an undecapeptide neurohormone sharing a highly conserved amino acid (a.a.) sequence across insect lineages and is involved in different physiological functions, such as heart contraction (20), stress response (21, 22), various metabolic activities (23–25), female fecundity (26), melanization of locust cuticles (27), regulation of ecdysis (28, 29), and control of caste identity (30). Moreover, Crz belongs to the gonadotropin-releasing hormone (GnRH) superfamily alongside adipokinetic hormone (AKH) and AKH/Crz-related peptide (ACP). Duplicates of an ancestral GnRH/Crz signaling system occurred in a common ancestor of protostomes and deuterostomes through coevolution of the ligand receptor (31, 32).Herein, we demonstrated that the hierarchical pathway consists of a GABAergic and Crz signaling system modulating progeny diapause induction by acting on DH release. We propose that the PTX-sensitive GABAergic signal may act to chronically suppress Crz release in dorsolateral Crz neurons (under nondiapause conditions) and that diapause conditions (or PTX injection) inhibits GABAergic signaling, resulting in accelerated Crz release, which in turn induces DH release. GABA signaling may be finely tuned by the temperature-dependent expression of the plasma membrane GABA transporter (GAT), which differs between the 25DD and 15DD conditions. Furthermore, this signaling pathway possesses similar features to the GnRH signaling system with respect to seasonal reproductive plasticity in vertebrates.