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PubMed2002

An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle.

We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages -15 to -1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N-terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.

Animals↗
PubMed2003

Copper in the silk formation process of Bombyx mori silkworm.

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.

Animals↗
PubMed1997

Eicosanoids mediate induction of immune genes in the fat body of the silkworm, Bombyx mori.

The expression of cecropin and lysozyme genes is induced in response to bacterial peptidoglycan in the fat body of the silkworm, Bombyx mori. Specific inhibitors of either phospholipase A2, cyclooxygenase or lipoxygenase significantly inhibit the induction of the immune genes both in vivo and in cultured fat body as detected by means of Northern hybridization. Arachidonic acid injected into the larvae induces the expression of the cecropin and lysozyme genes. The findings support the idea that eicosanoids mediate some process leading to the expression of immune genes in the fat body following recognition of peptidoglycan as a signal for invading bacteria.

Animals↗
PubMed1999

The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains.

We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.

Acute-Phase Proteins↗
PubMed2001

Selective transport of the mulberry leaf urease from the midgut into the larval hemolymph of the silkworm, Bombyx mori.

Just before spinning, larvae of the silkworm, Bombyx mori, absorb intact urease of the host plant (mulberry leaf) from the midgut lumen into the hemolymph. In order to investigate whether the transport of the mulberry leaf urease is selective, crude proteins extracted from the mulberry leaves were labeled with biotin and orally administered to the fifth instar larvae. The biotinylated proteins transported into the hemolymph were detected by ligand blotting using streptavidin. When the biotinylated proteins were administered to 5-day-old fifth instar larvae, a strong signal of a biotinylated protein was detected in the hemolymph 2 days after the administration. In contrast, when the biotinylated mulberry leaf proteins were administered to 3-day-old fifth instar larvae, no signal derived from the biotinylated proteins was detected in the hemolymph. The signal weakened when the biotinylated proteins had been immunoprecipitated before administering to the larvae, indicating that the signal came from the mulberry leaf urease. These results show that the transport of the mulberry leaf urease from the midgut into the hemolymph is selective and larval-stage specific. Subsequently, binding assays were carried out to test the binding ability of the mulberry leaf urease to the brush border membrane in the epithelial cells of larval midgut. The urease was not bound to the brush border membrane vesicles (BBMV) from the midgut of 3-day-old fifth instar larvae, while more than 60% of the total amount of incubated urease was bound to the BBMV from the midgut of 6-day-old fifth instar larvae. The urease binding ability of BBMV correlated with the uptake of the mulberry leaf urease. This suggests that a urease binding molecule(s) exists in the BBM of the midgut epithelium, which is involved in the uptake of the mulberry leaf urease. In addition, the uptake of the mulberry leaf urease into the hemolymph was induced by 20-hydroxyecdysone.

Journal Article↗
PubMed2002

Corazonin reduces the spinning rate in the silkworm, Bombyx mori.

The insect neuropeptide, [Arg(7)]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.

Journal Article↗
PubMed2002

Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori.

Topical application of fenoxycarb (1 &mgr;g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.

Journal Article↗
PubMed2003

In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ.

The lepidopteran hematopoietic process is poorly understood. We therefore examined the fundamental properties of hematopoiesis in the silkworm Bombyx mori using hematopoietic organ culture. In a medium containing larval plasma taken from the fourth day of the final larval stadium, over 50,000 hemocytes per hematopoietic organ were discharged within 48 h, with the number of cells comprising the hematopoietic organ simultaneously increasing from approximately 20,000 to 40,000. However, in the absence of plasma, cell numbers comprising the hematopoietic organ were unchanged and the number of discharged cells was much less. Hematopoietic organs cultured with plasma showed strong mitotic indices in a BrdU incorporation assay, but did not when cultured without plasma, indicating that plasma contains hematopoietic factor(s). The hematopoietic stimulation ability of larval plasma was observed from the last day of the penultimate larval stadium to the prepupal stage. The response of the hematopoietic organs to larval plasma was highest at the beginning of the final larval stadium and decreased with aging. Most cells discharged from the hematopoietic organ were plasmatocytes and prohemocytes, irrespective of location and developmental stage. Using this in vitro culture method, we tested the effects of 20-hydroxyecdysone (20E) and juvenile hormone-I (JH-I) on B. mori hematopoiesis. 20E showed a weak, but significant, hematopoietic activity, whereas JH-I did not, suggesting that a part of larval hematopoiesis is endocrinally regulated.

Animals↗
PubMed1997

Neuroendocrine control of diapause hormone secretion in the silkworm, Bombyx mori.

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.

Journal Article↗
PubMed1998

Phe-X-Pro-Arg-Leu-NH(2) peptide producing cells in the central nervous system of the silkworm, Bombyx mori.

Members of the neuropeptide family having Phe-X-Pro-Arg-Leu-NH(2) (FXPRLamide; X=Ser, Thr, Val, or Gly) at the C-terminus serve as regulators of oviduct and visceral muscle contraction, sex pheromone production, and diapause induction. Antibody raised against Bombyx mori diapause hormone recognized a variety of FXPRLamide peptides. Using this antibody, the antigen was immunocytochemically localized in the central nervous system (CNS) of the silkworm, Bombyx mori. Immunoreactive somata were observed in all ganglia of the CNS including the brain. Twelve somata localized at the midline of the suboesophageal ganglion (SG) were most intensely stained, and their neurite projections reached the retrocerebral complex. Thus, these cells in the SG exhibited typical features of neuroendocrine neurons. Marked reduction in immunoreactivity was observed in a pair of neurosecretory cells in the labial neuromere in SG of diapause type pupae, which indicates an active release of FXPRLamide peptides from these cells. No clear connection to neurohemal sites were observed in immunoreactive cells in the brain, thoracic or abdominal ganglia, suggesting that the immunoreactive peptides in these organs are likely to serve as neurotransmitters or neuromodulators.

Journal Article↗
PubMed1998

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head critical period.

The endocrine regulation of larval-pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3-4 days before the gut purge ([Sakurai, 1984]). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval-pupal metamorphosis.

Journal Article↗
PubMed1998

Reduction of glycogen in eggs of the silkworm, Bombyx mori, by use of a trehalase inhibitor, trehazolin, and diapause induction in glycogen-reduced eggs.

A new trehalase inhibitor, trehazolin, caused a potent inhibition of ovary trehalase in the silkworm, Bombyx mori. A single injection of trehazolin into pupae (40&mgr;g/animal) did not interfere with the accumulation of proteins and lipids, but markedly reduced glycogen content in eggs accompanied by a remarkable increase in hemolymph trehalose levels. The most potent effect of trehazolin was expressed in eggs that developed at the mid-stage of pupal-adult development. In these eggs glycogen content was reduced to a trace level, less than 3% of that of the control. The reduced glycogen content was almost restored to the control level by injection of glucose but not by trehalose. Trehazolin treatment influenced oviposition and larval hatching, whereas embryogenesis went on normally in glycogen-reduced eggs. Injection of synthetic diapause hormone into non-diapause type hosts induced an incidence of 45% diapause in the eggs and increased their glycogen content. Surprisingly, injection of trehazolin never affected diapause induction by the hormone, despite considerably reduced glycogen content in these eggs. Thus, our findings provide a new method for production of eggs containing various amounts of glycogen, and a novel system for analyzing diapause-associated metabolism besides the well-known glycogen-sorbitol metabolism.

Journal Article↗
PubMed1999

Artificial insemination using trypsin-treated sperm in the silkworm, Bombyx mori.

A new method was developed for artificial insemination of the silkworm. Virgin females artificially inseminated by this method oviposited fertilized eggs at a rate almost similar to that of females mated with males. Using inactive sperm collected from the seminal vesicles, we confirmed the previous finding by Omura, S., 1936a, Artificial insemination of Bombyx mori, Journal of Faculty of Agriculture, Hokkaido Imperial University 38, 135-150, and Omura, S., 1938, Studies of the reproductive system of the male of Bombyx mori II, Post-testicular organs and post-testicular behaviour of the spermatozoa, Journal of Faculty of Agriculture, Hokkaido Imperial University 40, 129-170, that the sperm of B. mori requires the secretion of the glandula prostatica for activation. Sperm also could be activated by trypsin. At an optimal concentration, 0.3 &mgr;g trypsin/ml in 50% semen solution, the fertilization rate as well as the number of eggs oviposited was almost equivalent to that obtained in normally mated moths. These results may contribute not only to basic studies on fertilization and reproduction in Lepidoptera but also to the development of long-term preservation of genetic resources by using cryopreserved sperm of B. mori and other Lepidoptera insects.

Journal Article↗
PubMed1999

Effect of 1-(4-phenoxyphenoxypropyl)imidazole (KS-175) on larval growth in the silkworm Bombyx mori.

1-(4-Phenoxyphenoxypropyl)imidazole (KS-175), which has two types of characteristic moieties of insect growth regulators (IGRs), the phenoxyphenoxyalkyl group of juvenile hormone analogs (JHAs) and imidazole of 1,5-disubstituted imidazole such as KK-42, was tested for its biological activity on the silkworm, Bombyx mori. Penultimate (4th) instar larvae topically treated with KS-175 did not molt for more than 20 days. This activity was different from that reported for any IGRs. After the treatment, ecdysteroid levels in the hemolymph did not increase and the cells of the prothoracic gland had shrunk. When the treated penultimate larvae were fed an artificial diet supplemented with 20 ppm of 20-hydroxyecdysone, the larvae molted to the ultimate (5th) instar with a timing similar to that of control larvae fed a diet with or without 20-hydroxyecdysone. These results suggest that topical application of KS-175 irreversibly damages ecdysone biosynthesis in the prothoracic glands.

Journal Article↗
PubMed2000

Artificial insemination using cryopreserved sperm in the silkworm, Bombyx mori.

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.

Journal Article↗
PubMed2001

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori.

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.

Amino Acid Sequence↗
PubMedDate not supplied

Immune proteins and their gene expression in the silkworm, Bombyx mori.

Several self-defense proteins have been isolated from the silkworm, Bombyx mori and their amino acid sequences determined. These proteins include novel antibacterial proteins designated lebocin and moricin, and a novel lectin designated hemocytin, an insect homologue of mammalian von Willebrand factor. Antibacterial mechanisms of lebocin and moricin have been analyzed and their ability to form ion channels in bacterial membranes play an important role in defense against bacterial infection. cDNAs and genes encoding these proteins have been cloned to examine their induction mechanisms upon bacterial infection. Regulatory motifs such as the kappaB-like and GATA sequence have been identified in the B. mori antibacterial proteins. On the other hand, hemocytin gene expression was confirmed to occur upon bacterial infection and before pupation under naive conditions, suggesting that hemocytin plays an important role in both immunity and metamorphosis. Moreover, this review also describes the releasing mechanisms of a bacterial cell wall component, lipopolysaccharide (LPS), from intact bacteria, clearance of LPS from B. mori hemolymph and a possible signal transduction pathway for antibacterial protein gene expression.

Amino Acid Sequence↗
PubMed2002

Flufenoxuron, an insect growth regulator, promotes peroral infection by nucleopolyhedrovirus (BmNPV) budded particles in the silkworm, Bombyx mori L.

A novel method was developed to infect perorally the silkworm Bombyx mori L. with budded particles of nucleopolyhedrovirus (BmNPV) using flufenoxuron, an insect growth regulator. NPV vectors are used to obtain proteins that occur naturally in minute amounts. NPV vectors are constructed conventionally by replacing the polyhedrin gene with the foreign gene of interest. These vectors thus do not produce polyhedra. The budded virus particle suspension of these vectors is produced in a cell culture and used as the stock inoculum. Budded NPV particles do not infect their host perorally. The inoculum is injected manually into the individual host using a syringe. It was found that B. mori L. fed on the insect growth regulator flufenoxuron were sensitive to BmNPV budded particles given perorally. Over 90% of B. mori L. ingesting BmNPV budded particles (1.3 x 10(6) TCID(50) units per larva) after consuming an artificial diet for 24 h, containing 0.1% (w/w) flufenoxuron died of the viral infection. The peroral inoculation of BmNPV budded particles by flufenoxuron may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.

Animals↗