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PubMed2006

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori.

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.

Animals↗
PubMed2006

Induction of hemolin gene expression by bacterial cell wall components in eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding hemolin was isolated from fat body of immunized Samia cynthia ricini larvae based on subtractive suppression hybridization method. The cDNA encodes 413 amino acid residue open reading frame with an 18 residue predicted signal peptide. The expression of the gene was strongly induced in fat body and midgut by an injection of bacterial cells or peptidoglycans, but very weakly by lipopolysaccharide. The mRNA expression in the fat body was detected as early as 3 h post-injection, and reached the peak level at 12 h.

Animals↗
PubMed2005

A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin.

A cDNA clone with homology to lebocin gene was isolated from fat body of immunized Samia cynthia ricini larvae. The cDNA has an open reading frame encoding 162 amino acid residues. The deduced amino acid sequence shows significant homology to lebocin precursor proteins from Bombyx mori and Trichoplusia ni only in the "prosegment" region, but no homology to mature lebocin, a proline-rich antibacterial peptide, indicating the protein is not a precursor for lebocin antibacterial peptide. Northern analysis indicates that transcript of the lebocin-like gene is not detected in any tissues of naive larvae, but induced mainly in fat body after injection of the larvae with bacterial cells or cell wall components, such as peptidoglycan.

Amino Acid Sequence↗
PubMed2003

cDNA cloning and expression of bacteria-induced Hdd11 gene from eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding Hdd11 protein, a bacteria-induced protein of unknown function, was isolated from fat body of immunized Samia cynthia ricini larvae based on suppression subtractive hybridization. The cDNA encodes a 167 amino acid residue open reading frame with an 18 residue predicted signal peptide. The deduced amino acid sequence showed 54% and 55% identity with Hdd11 proteins from Hyphantria cunea and Manduca sexta, respectively. Expression of the gene was undetectable in naive larvae when measured by Northern blot hybridization, but strongly induced in fat body after injecting bacterial cells or peptidoglycan into the larvae. The mRNA expression in the fat body was detected as early as 3 h post injection, reached peak levels at 12 h and continued for further 60 h at significant levels. The transcript was detected at very low levels in midgut, hemocytes and malpighian tubules.

Amino Acid Sequence↗
PubMed2004

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae.

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.

Animals↗
PubMed2005

Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori.

To investigate the pathogenicity of Pseudomonas aeruginosa in insects, a gacA mutant of P. aeruginosa PA01 was constructed by site-directed mutagenesis. The mutant was designated as C1. C1 was less virulent to Bombyx mori than the parent strain. To complement the gacA gene, P. aeruginosa C1 was transformed with the broad host range plasmid pJB3Km1 carrying a 3.9-kbp gacA fragment. The expression of the gacA mRNA in C1 (pgacA) was detected. In addition, the complemented mutant restored the level and timing of pyocyanin production, indicating that functional GacA is produced in the complemented strain. However, no significant difference was observed between C1 and C1 (pgacA) with respect to the killing of B. mori larvae.

Animals↗
PubMed2005

Membrane-bound sorbitol 6-phosphatase in fat body cells controls the dynamics of sorbitol 6-phosphate, a major hemolymph sugar in the silkworm.

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 micromol/larva at its maximum and decreases to less than 1 micromol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.

Animals↗
PubMed2005

The roles of ERK and P38 MAPK signaling cascades on embryonic diapause initiation and termination of the silkworm, Bombyx mori.

To clarify the molecular mechanisms of Bombyx diapause, we focused on mitogen-activated protein kinases (MAPK), which are the major components of signal transduction cascades that regulate cell proliferation, differentiation, and stress responses. In the present study, cloning of Bombyx extracellular signal-related kinase (ERK), MAPK-ERK kinase (MEK), and p38 MAPK cDNA, revealed that their amino acid sequences have close similarity with those of other species. We analyzed the roles of the kinases in diapause initiation and termination by immuno-blotting with anti-phospho-kinase antibodies. Phospho-MEK levels remained consistently high in non-diapausing eggs, then declined after the diapausing stage in diapausing eggs, and began to increase 45 d after transfer to 5 degrees C upon diapause termination. The phospho-ERK and phospho-MEK profiles were similar, suggesting that ERK phosphorylation is regulated by MEK. The phospho-p38 MAPK levels declined 36 h after oviposition in diapausing eggs, and increased at 15-30 d at 5 degrees C in yolk cells, suggesting that p38 MAPK has a role in diapause initiation and termination. Phospho-ERK levels were maintained with diapause-interrupting treatment and declined with diapause-sustaining treatment. ERK phosphorylation is considered to have a role in diapause termination and in the resumption of development.

Amino Acid Sequence↗
PubMed2006

Differential usage of two promoters of the Broad-Complex gene in the silkworm, Bombyx mori.

The Broad-Complex gene encodes one of the key regulators of the ecdysone signal cascade. We previously isolated part of the genomic DNA and cDNAs of Broad-Complex in Bombyx mori (BmBR-C). Here, we report structures of the entire genomic DNA and 5' untranslated region (5'-UTR) of the cDNAs. BmBR-C was found to span about 158 kbp including 13 exons. In the 5'-UTR, additional alternatively spliced exons were identified. The 5' ends of the cDNAs were mapped to two different positions, the distal promoter (P(dist)) and proximal promoter (P(prox)), separated by 86 kbp. Expression from these promoters was controlled differentially. Semi-quantitative PCR using cDNAs from the carcass, silk glands and fat body revealed that expression from P(prox) was changed moderately and expression from P(dist) was weak and constant during the fourth ecdysis. At the onset of pupation, expression from P(prox) was suppressed in all tissues, but that from P(dist) was induced in the carcass and ASG. In the fat body, expression from both promoters increased in the prepupal stage. A combination of promoters differing in responsiveness to an ecdysone signal may serve to achieve a complex regulation of downstream genes in reply to a simple hormonal signal.

5' Untranslated Regions↗
PubMed2006

Distinct effects of different temperatures on diapause termination, yolk morphology and MAPK phosphorylation in the silkworm, Bombyx mori.

Previously, we showed that chilling of diapausing Bombyx eggs activated ERK/MAPK in yolk cells coincidentally with acquisition of developmental competence, and that ERK regulates diapause termination via activating transcription of key enzyme genes for ecdysteroid and sorbitol metabolism. To further elucidate the molecular mechanisms of temperature signaling and diapause termination, effects of different temperatures on diapause termination, yolk morphology, and ERK and p38 MAPK phosphorylation were analyzed. We confirmed the coincidence of diapause termination and changes of phosphorylated levels of ERK and p38 MAPK at various temperatures. Our study also shows that different temperatures have distinct effects on diapause termination and yolk morphology, however, these two effects did not always coincide. Our analyses suggest the possibility that the effects on diapause termination and yolk morphology are independently regulated by ERK. We observed that a temperature shift alters diapause intensity reversibly, and coincidental changes of phosphorylated ERK levels suggest that ERK may have a role in regulating the diapause intensity. The present study suggests that ERK and p38 MAPK have roles for transmitting the temperature signals that are important for diapause termination.

Animals↗
PubMed2004

Molecular cloning and expression pattern of an armadillo homologue from the mulberry silkworm Bombyx mori.

Armadillo/beta-catenin, encoded by the segment polarity gene armadillo functions as a downstream effector of canonical Wnt signals. The expression patterns of Bm wnt-1, -Ci and -engrailed, suggested the presence of Wnt-1 network in the middle silkglands of Bombyx mori. To test this, a homologue of armadillo from B. mori (Bm arm) was cloned by PCR using degenerate primers, designed based on the conserved regions of the protein, and characterized. The cloned region harboured one complete and two incomplete Armadillo (Arm) repeat motifs and was highly conserved across species. Genomic Southern analysis suggested Bm arm to be a multi-copy gene. The expression of Bm arm RNA was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The RNA was expressed uniformly in the embryos, whereas the Arm protein was localized in a segmentally reiterated striped pattern, in conformity with its predicted segment polarity nature. Bm arm was also expressed in the entire silkgland and the transition from third to fourth and fifth larval intermoults was accompanied by an increase in the transcript levels. However, the Arm protein was predominantly localized to the middle silkglands, especially the middle and posterior sub-compartments. The silkglands represented a novel expression domain for arm in Bombyx, and the results were consistent with the existence of a canonical Wnt network in the middle silkglands.

Amino Acid Sequence↗
PubMed1999

Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori.

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.

Amino Acid Sequence↗
PubMed1997

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm, Bombyx mori.

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain-corpora cardiaca-corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.

Journal Article↗
PubMed1999

Disturbance of adult eclosion by fenoxycarb in the silkworm, Bombyx mori.

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.

Journal Article↗
PubMed1999

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori.

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

Journal Article↗
PubMed1999

Oxygen consumption in relation to sorbitol utilization at the termination of diapause in eggs of the silkworm, Bombyx mori.

Rates of oxygen consumption were followed throughout the entire period of diapause in eggs of Bombyx mori. In non-diapause eggs at 25 degrees C, O(2) uptake was divisible into three phases, corresponding to morphogenetic processes. In diapause eggs at 25 degrees C, O(2) uptake showed a peak (100 &mgr;l/g eggs/h) at 1 day and then suddenly dropped to reach a level of 8-10 &mgr;l/g eggs/h at 10 days and thereafter. To break diapause, eggs were exposed to 5 degrees C for varying periods. When O(2) uptake was measured at 5 degrees C, it remained at 6 &mgr;l/g eggs/h. When eggs were chilled for increasing periods and O(2) uptake was measured immediately after warming to 25 degrees C, the rates increased after a lag phase. In HCl-treated eggs, O(2) uptake increased immediately after acid-treatment. In all cases, highly increasing O(2) uptake at 25 degrees C coincided with termination of diapause. These results were discussed in relation to sorbitol utilization at the termination of diapause.

Journal Article↗
PubMed1999

Induction of dauer larvae by application of fenoxycarb early in the 5th instar of the silkworm, Bombyx mori.

Fenoxycarb application from 0 h until 60 h of the 5th instar of Bombyx mori induced 100% dauer larvae. Between the 60 and 78 h, the ratio of fenoxycarb-induced dauer larvae decreased, and the ratio of supernumerary instar moulting larvae increased. After application of fenoxycarb at the 48 h of the 5th instar, the haemolymph prothoracicotropic hormone (PTTH) titer was higher in fenoxycarb-treated larvae than in control larvae. Furthermore, brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes from fenoxycarb-treated larvae released higher amounts of PTTH in vitro than the Br-CC-CA complexes of control larvae. Prothoracic glands (PGs) of fenoxycarb-treated larvae at the 48 h of the 5th instar exhibited basal and PTTH-stimulated secretory activities similar to that of control PGs until the 72 h of the 5th instar. After that time point, both basal and PTTH-stimulated secretory activity of PGs from fenoxycarb-treated larvae significantly decreased and remained low for the rest of the investigated period. The combined results suggest that the application of fenoxycarb affects the ability of the PGs to be stimulated by PTTH and the induction of dauer larvae in Bombyx mori is not due to inhibition of PTTH release from Br-CC-CA complexes.

Journal Article↗