We newly isolated seven W chromosome derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by means of six different STS primer sets, which amplify each different female specific DNA fragment. Two color fluorescence in situ hybridization (FISH) with 19L6H W-BAC probe as a standard W-chromosome painter disclosed that six out of seven new W-BAC probes strongly highlighted the whole W chromosome in B. mori. Besides the W, hybridization signals occurred in telomeric and/or subtelomeric regions of the autosomes. The remaining W-BAC, 20H9D, provided discontinuous signals, which appeared in both terminal and central compartments of the W chromosome. A representative of W-chromosome painting BACs, 19L6H, and 20H9D W-BAC were hybridized to chromosome spreads of Bombyx mandarina females. The 19L6H W-BAC bound to the whole B. mandarina W chromosome as in B. mori. In contrast, 20H9D W-BAC signals in B. mandarina W chromosome were different from those in B. mori W, indicating rearrangements of the W chromosome during karyotype evolution of the two closely related Bombyx species. Further FISH analyses using B. mori W-BAC probes in 15 lepidopteran species showed no hybridization signals in their sex chromosomes as well as autosomes. Our results suggest the absence of homologous sequences between the Bombyx W and the W chromosomes of other lepidopteran species.

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In order to obtain conclusive evidence that fibroinase, a cathepsin L-like cysteine proteinase, of silk gland digests sericin, in addition to fibroin, three sericin fractions, s-A, s-M and s-P, were prepared from cocoons by the method of Takasu et al. (2002) and subjected to enzymatic digestion by fibroinase purified from silk gland of day one B. mori pupa. Time course study showed clearly that all the sericin fractions were digested in a time dependent manner and that complete digestion was attained by 3h. Sericin fragments from each sericin fraction digested for 3h were subjected to reverse phase column chromatography. Mass of each sericin fragment was determined by MALDI TOF mass spectrometry to check the purity of each peak fraction, followed by peptide sequencing by protein sequencer. Some sequences of sericin fragments corresponded portions of the sequence of sericin protein deduced from the nucleotide sequence of sericin gene (Garel et al., 1997). By alignment of an amino acid sequence of a sericin fragment with the sericin protein sequence, estimation of cleavage sites of sericin by fibrionase of silk gland was possible. It was concluded that fibroinase of silk gland digests sericin as well as fibroin (Watanabe et al., 2006a). This is because fibroinase of silk gland is a cathepsin L-like cysteine proteinase, which is generally believed to show broad substrate specificity. Fibroinase of silk gland is highly likely the proteinase that is responsible for digestion of fibroin and sericin in the luminal contents of silk gland in the early pupa and at each molt period during larval development.

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This study demonstrates the function of BmAcer which had been isolated from the wing discs of the silkworm, Bombyx mori during metamorphosis. BmAcer was secreted from the cultured wing discs into the Grace’s medium. The existence of 20-hydroxyecdysone increased the BmAcer production. ACE activity was detected by RP-HPLC method. Incubation was operated with 100μM synthesized peptide (HHL) using as substrate for 2h at 37°C. HHL degradation was inhibited by the addition of ACE inhibitor, Captopril, in the incubation solution. High ACE activity of the hemolymph was observed in the fifth larval instar and pupal stages. Higher activity was observed in the feeding stage and the late pupal stage. These results suggest that BmAcer is secreted from the wing discs into the hemolymph and has physiological function during larval and pupal development.

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The in vitro activity of four polycationic 9-mer peptides derived from insect defensins alone and combined with clinically used antibiotics was investigated against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. These bacteria were more susceptible to the D-9-mer peptides (diastereomer) composed of only D-amino acids (MIC range: 20-100μg/ml) than to the L-9-mer peptide composed of only L-amino acids (MIC range: 400-800μg/ml). Furthermore, D-9-mer peptides exhibited synergic and additive effects in combination with antibiotics. Interestingly, an increase in bacterial resistance to antibiotics was observed whereas resistance to D-9-mer peptides was not observed, even when bacteria were cultured in medium containing these antimicrobial peptides and antibiotics for several hundred generations (MRSA: 200 generations and P. aeruginosa: 1000 generations). Moreover, one D-9-mer peptide showed continuous synergy in combination with piperacillin against P. aeruginosa during cultivation. These results suggest the potential use of these D-9-mer peptides in topical or systemic therapy for nosocomial infections.

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Effect of dietary sucrose on midgut sucrase activity in the 5th instar B. mori larvae was investigated in germfree conditions from day 1, the 5th instar until day 8, the 5th instar, i.e., the day of onset of spinning. Sucrase activity per midgut was suppressed throughout the development in a dose-dependent manner, by sucrose from 1.4% up to 6.9% in wet weight basis. Sucrase specific activity showed a similar profile. The results implicate that dietary sucrose functions as an effector molecule to midgut cells in the 5th instar B. mori larvae and as an output response, produces suppressed sucrase protein. When aminopeptidase activity was measured using the same midgut preparations, there were no effects of dietary sucrose. Within 24h after 4th ecdysis, a slightly different pattern was observed; the highest activity was observed with 3% sucrose diet and it was higher than the control, indicating apparent activation. This may be due to inherent physiological conditions of midgut cells in early fifth instar larvae.

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A total of 162 clones were isolated by plaque assay with Hz-AM1 cells from uncloned field populations of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) collected from Kenya, South Africa, Zimbabwe and Thailand. Restriction endonuclease (REN) analysis showed that 25 out of the 162 clones exhibited unique REN patterns, which should be characterized as variants of HearNPV. Five clones, NNg1, NS2, NMa1, NZ3 and NT1, were randomly selected from the 25 clones and characterized in both Hz-AM1 cells and H. armigera larvae, together with the clone G4 which was isolated in China and sequenced previously. In Hz-AM1 cells, clones NNg1, NMa1, NZ3 and NS2 produced more budded virions than clones G4 and NT1, whereas a higher amount of polyhedrin was produced in clones NNg1 and G4. The dose-mortality analysis revealed that clone NNg1 expressed the highest insecticidal activity against third instar H. armigera larvae, showing a 50% lethal dose (LD₅₀) value of 10 occlusion bodies (OBs)/larva and a 50% lethal time (LT₅₀) value of 4.0 days following peroral infection with OBs from infected larvae. Clones NS2, NMa1 and NZ3 had LD₅₀ values of 32, 35 and 146 OBs/larva and LT₅₀ values of 5.1, 4.4 and 4.8 days, respectively, indicating that these three clones had insecticidal activity somewhat lower than that of clone NNg1. In contrast, clones NT1 and G4 had markedly lower insecticidal activities compared with the four other clones, recording LD₅₀ values of 826 and 3115 OBs/larva and LT₅₀ values of 5.8 and 8.3 days, respectively. These results indicate that clone NNg1 is the most promising candidate for biological control programs of H. armigera larvae among the six clones characterized in this study.

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Peptides of the ENF (Glu-Asn-Phe) family, which occur in the hemolymph of Lepidoptera, were reported to act on the plasmatocytes and heart, cause body paralysis, and inhibit body growth. In this paper we report on yet another, most surprising effect. An ENF peptide isolated from the wild silkmoth, Antheraea yamamai, and named Antya-ParP causes paralysis in the caterpillars but not in the pupae of Bombyx mori. However, adults that emerge from the treated pupae lay diapausing eggs. The embryonic diapause of B. mori is normally induced by the diapause hormone (Bommo-DH), which is produced in pupae from the suboesophageal ganglion. The effect of Antya-ParP is independent of this ganglion, showing that the Antya-ParP action is not mediated by Bommo-DH. The sequence of 23 amino acid residues of Antya-ParP shows no similarity to the Bommo-DH sequence of 24 residues. The mode of action of the two peptides seems to be identical but the threshold concentration of Bommo-DH is up to hundred times lower than that of Antya-ParP.

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A nuclease which degrades double-stranded RNA was purified from the digestive juice of fifth-instar larvae of the silkworm, Bombyx mori, using gel filtration and affinity column chromatography. The enzyme was found to have a molecular weight of 41,000 as estimated by a gel filtration method and detected as a single band with the same molecular weight on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It degraded double-stranded RNA of cytoplasmic polyhedrosis virus genome, synthetic Poly(I)/Poly(C), Poly(I) and Poly(C). It also digested copolymer Poly(AU), Poly(A) and Poly(U), but showed weak degradation of Poly(A)/Poly(U), Poly(C)/Poly(G), Poly(G) and natural DNA isolated from calf thymus. The pH range wherein the reaction occurred was greater than 7. The purified enzyme did not require Mg²⁺ to degrade CPV-dsRNA, whereas divalent cations including Mg²⁺ and Ca²⁺ were needed to degrade synthetic Poly(I)/Poly(C). The enzyme activity was suppressed by Co²⁺, Zn²⁺ and Mn²⁺.

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