Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670]     

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-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.     

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

Topical application of fenoxycarb (1 μg 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.     

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.     

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.     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

in silico identification of protein-protein interactions in Silkworm,Bombyx mori

The Domesticated silkworm, Bombyx mori, an economically important insect has been used as a lepidopteran molecular model next only to Drosophila. Compared to the genomic information in silkworm, the protein-protein interaction data are limited. Therefore experimentally identified PPI maps from five model organisms such as E.coli, C.elegans, D.melanogaster, H. sapiens, S. cerevisiae were used to infer the PPI network of silkworm using the well-recognized Interlog based method. Among the 14623 silkworm proteins, 7736 protein-protein interaction pairs were predicted which include 2700 unique proteins of the silkworms. Using the iPfam interaction domains and the gene expression data, these predictions were validated. In that 625 PPI pairs of predicted network were associated with the iPfam domain-domain interactions and the random network has average of 9. In the gene expression method, the average PCC value of the predicted network and random network was 0.29 and 0.23100±0.00042 respectively. It reveals that the predicted PPI networks of silkworm are highly significant and reliable. This is the first PPI network for the silkworm which will provide a framework for deciphering the cellular processes governing key metabolic pathways in the silkworm, Bombyx mori and available at SilkPPI (http://210.212.197.30/SilkPPI/).     

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth''s olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.     

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.     

Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori

Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under diet-deprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced meal-starts were dependent on the time elapsed since the larvae's previous meals. Provided that more than 1h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae.     

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.     

Effect of tyramine and stress on sex-pheromone production in the pre- and post-mating silkworm moth, Bombyx mori

Tyramine (TA) increased significantly after mating, whereas there were no significant differences in octopamine (OA) and dopamine (DA) levels in the brain-suboesophageal ganglion (SOG) complexes between virgin and mated females. The effects of various biogenic amines were tested on pheromone production of virgin and mated females of the silkworm moth, Bombyx mori. After 8h a significant reduction by TA (46%) was observed. Meanwhile, when OA or DA was injected, a significant increase of pheromone titer was observed in both virgin and mated females. This study also presents evidence for an increase in levels of OA and DA in the brain-SOG complexes in response to mechanical stress in B. mori female. TA suppressed pheromone production in an in vitro pheromone gland (PG) homogenate preparation, thus suggesting that the target of TA is the PG. TA inhibited pheromone production in vitro in a dose-dependent manner and DA had a lower inhibitory activity than TA, whereas OA had no effect, suggesting that TA is a candidate for regulating pheromone production in the PG, although other factors could be responsible for the pheromonostatic function.     

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.     

C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori

Two flavonoids containing the l-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.