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PubMed2006

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.

Animals↗
PubMed2004

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori.

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Animals↗
PubMed2004

Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L.

Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.

Animals↗
PubMed2005

Parasitization of fifth instar tasar silkworm, Antheraea mylitta, by the uzi fly, Blepharipa zebina; a host-parasitoid interaction and its effect on host's nutritional parameters and parasitoid development.

The uzi fly, Blepharipa zebina, is a well-known larval endoparasitoid of the tropical tasar silkworm, Antheraea mylitta. The present study dealt with the effect of the number of maggots developing per host on host nutritional parameters, parasitoid development and reproduction. Nutritional indices for ingestion, digestion, approximate digestibility, relative consumption rate, relative growth rate, and gain in body weight declined significantly with the increase in parasitoid burden, but the efficiency of conversion of digested food recorded a significant increase. The efficiency of conversion of ingested food remained little affected. The developmental period was significantly extended in larvae parasitized with 5 and 10 maggots per larva (mpl). Cocoon shell weight decreased by 27-63.5% in parasitized groups (1, 2, and 5 mpl) while larvae parasitized with 10 mpl could not spin cocoons. The maggot development period, recovery percentage, and fecundity of the uzi fly declined significantly with the increase in number of maggots developing per host.

Animals↗
PubMed2004

Silkworm-pheophorbide alpha mediated photodynamic therapy against B16F10 pigmented melanoma.

In order to apply photodynamic therapy (PDT) to pigmented melanoma, the efficacy of PDT mediated by pheophorbide alpha from silkworm excreta (SPbalpha) and commercial Photofrin against B16F10 melanoma was comparatively studied from the in vivo assay using C57BL/6J mice. From in vitro PDT assay, the proliferation of B16F10 cells treated with SPbalpha (more than 0.5 microg/ml) and light illumination (1.2 J/cm2) were significantly inhibited with the necrotic response. This indicated that the photocytotoxicity of SPbalpha (665 nm) was not influenced by melanin from melanoma. From the assessment of the in vivo photosensitizing activity, the tumor growth was further delayed in groups treated with SPbalpha/PDT compared to that treated with Photofrin /PDT. The survival rate of tumor bearing mice treated with SPbalpha/PDT was closely associated with its photosensitizing effect. In addition, the photosensitizing effect of SPbalpha/PDT showed a dose dependent tendency in light illumination. These results demonstrated that B16F10 melanoma cells were significantly photosensitized by SPbalpha/PDT, regardless of the influence of melanin from melanoma, and SPbalpha/PDT at very low drug dose (1 mg/kg) and light dose (1.2 J/cm2) showed the photosensitizing efficacy surpassing Photofrin against B16F10 melanoma in mice system.

Animals↗
PubMed2006

Estradiol-17beta: tracing its metabolic significance in female fatbody of fifth instar larvae of silkworm, Bombyx mori L (race: Nistari).

In recent years, various vertebrate peptide and steroid hormones have been identified in invertebrates, estradiol-17beta (E2) being a major one. We have specifically shown NADP-malate dehydrogenase (NADP-MDH) activity in fifth instar larval fatbody of female silkworm, Bombyx mori, as an E2 responsive parameter. Interestingly enough, estradiol-induced increase in the enzyme activity could be counteracted by simultaneous application of specific E2-inhibitor, ICI-182780. Further, a nice correlation was obtained among the E2 titre, specific *E2 binding and expression of NADP-MDH activity in fatbody during different days of normal fifth instar larval development. Though the nature of the binding sites is quite similar to known steroid receptors of vertebrate, the reported absence of estrogen receptor gene in some insects poses a question. A recent finding regarding the presence of an estrogen-related receptor ortholog in fruit fly may provide some answers. The specific effects elicited by estradiol in the female fatbody of this insect support its possibility of having an important metabolic function. This role played by E2, whether hormonal or not, is yet to be identified.

Adipose Tissue↗
PubMed2005

Synthesis of prolyl 4-hydroxylase alpha subunit and type IV collagen in hemocytic granular cells of silkworm, Bombyx mori: Involvement of type IV collagen in self-defense reaction and metamorphosis.

The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.

Amino Acid Sequence↗
PubMed2005

Comparative analysis of the development of the mandibular salivary glands and the labial silk glands in the mulberry silkworm, Bombyx mori.

The mulberry silkworm, Bombyx mori has a pair of salivary glands arising from the mandibular segment, in addition to the labial silk glands which are generally considered as modified salivary glands. Here we report the characterization of salivary glands and the comparative gene expression profiling of the silk and salivary glands. The two independent salivary glands made up by 330 cells, grow about 1000 fold during larval development. These individual glands extend up to the T(1) thoracic segment unlike silk glands with fused anterior ends and extending up to the caudal region. The salivary glands also undergo endomitosis resembling the silk glands. The B. mori homologue of the homeotic gene Deformed (BmDfd) was expressed in the mandibular and maxillary segments in stage 17 embryo and got localized to the centre of the mandibular segment at stage 18 to form the salivary gland placodes. The expression was also seen in the distal ends of the leg appendages after blastokinesis (stage 22). Only low variations in BmDfd expression ranging from 1.6 to 2.1 fold were apparent during embryonic development. BmDfd expression was observed in the salivary glands all through the larval instars but not in the silk glands. The transcription factor, Forkhead and the segment polarity gene, Wingless were expressed throughout the salivary glands, the latter confirming the absence of physiological compartmentation within these glands unlike the silk glands. The expression of Amylase and Fibrohexamerin was restricted to the salivary and silk glands, respectively and therefore, served as molecular markers for these tissues.

Amylases↗
PubMed2004

Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm.

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3mg purified (His)(6)-tagged CI-b1 from 200ml of culture medium. Studies on (His)(6)-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against alpha-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.

Animals↗
PubMedDate not supplied

An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin.

The insect baculovirus expression system is one of the most effective eukaryotic expression systems known, and has been widely used to produce numerous recombinant proteins. The current traditional inoculation method consists of injecting recombinant baculovirus directly into insects, thus causing potential contamination to the environment due to virus diffusion during the inoculation process. In the present experiment, we directly introduced baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus. This new method produced the same infection results as the traditional method. A new safe infection technique without direct use of the virus was thus developed.

Animals↗
PubMed2005

Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori.

We have developed a modified method to detect phenoloxidase activity on hemocytes by using freshly prepared l-DOPA (1 mg/ml in 35% ethanol) to fix and incubate larval hemocytes. This method is more sensitive than the common method, in which hemocytes were fixed in 4% formaldehyde and then incubated with 2 mg/ml l-DOPA in water separately. Phenoloxidase assayed using this modified method can be inhibited by phenyltiourea (phenoloxidase inhibitor). After incubation with l-DOPA solution in ethanol, most prohemocytes, all plasmatocytes and young granulocytes are stained brown due to oxidation of l-DOPA into pigments, indicating that they have phenoloxidase. Oenocytoids are dimly stained because many of their cell inclusions have been released during the treatment. Large propidium-iodide-negative prohemocytes have strong phenoloxidase activity and are easily misunderstood as propidium-iodide-positive oenocytoids if the fluorescent method is not used for identification. Thus, in addition to oenocytoids and plasmatocytes, some prohemocytes and granulocytes in the silkworm also have phenoloxidase.

Animals↗
PubMed2004

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.

Animals↗
PubMed2006

Structures, regulatory regions, and inductive expression patterns of antimicrobial peptide genes in the silkworm Bombyx mori.

Antimicrobial peptides (AMPs) are a group of immune proteins that protect the host from infection. In Drosophila, seven groups of inducible AMPs have been identified, with activities against fungi and gram-positive and gram-negative bacteria. On the basis of the silkworm genome sequence and expressed sequence tags, we identified 35 AMP genes, mostly belonging to the cecropin, moricin, and gloverin gene families. We predicted the core promoters required for gene transcription and the cis-regulatory elements for NF-kappaB/Rel and GATA transcription factors. The expression profiles of these genes after an immune challenge with lipopolysaccharide were examined by reverse transcription PCR. Members of the cecropin B and gloverin A subfamilies were intensely expressed in the fat body after induction. In contrast, those of the moricin B subfamily were not expressed under the same conditions. Such results suggest that these regulatory elements and their positions in the upstream regions play an important role in regulating the transcription of these defense genes.

Amino Acid Sequence↗
PubMed2002

Behavior of silkworm yolk protein on phospholipid membranes.

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of -70 mV for yolk and -198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.

Animals↗
PubMed2002

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast.

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.

Animals↗
PubMed1997

Prothoracicotropic hormone-producing neurosecretory cells in the silkworm, Bombyx mori, express a muscarinic acetylcholine receptor.

Using an anti-muscarinic acetylcholine receptor (mAChR) antibody and an anti-prothoracicotropic hormone (PTTH) antibody, double immunofluorescence staining was performed on brain sections of the silkworm, Bombyx mori. Four pairs of dorsolateral neurosecretory cells, along with some intercerebral neurosecretory cells, were immunoreactive to anti-mAChR antibody. Among these immunoreactive cells, two pairs of dorsolateral neurosecretory cells were identified to be PTTH-producing neurosecretory cells. Nerve fibers in the median and paramedian protocerebral areas, and nerve terminals in the corpus allatum also showed immunoreactivity to the anti-mAChR antibody. Some of these nerve terminals expressing mAChRs were overlapped by immunostaining with the anti-PTTH antibody. These results indicated that PTTH-producing neurosecretory cells of Bombyx mori expressed an mAChR, and that muscarinic, cholinergic transmission might directly regulate PTTH release from neurosecretory cells.

Animals↗
PubMed2003

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm, Bombyx mori.

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.

Animals↗
PubMed2002

Solution structure of moricin, an antibacterial peptide, isolated from the silkworm Bombyx mori.

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.

Amino Acid Sequence↗