Members of the Y-box-binding protein family regulate gene expression through both transcription and translation. Bombyx Y-box-binding protein (BYB) was found to enhance the binding activity of various transcription factors including fibroin-modulator-binding protein-1 (FMBP-1) to their target DNA sequences. We dissected BYB and analyzed the regions responsible for the enhancement. Enhancement of protein-DNA interaction by BYB was conducted independently by the conserved cold shock domain (CSD) in the N-terminal half and by the C-terminal half containing basic/aromatic (B/A) islands that are rich in arginine and aromatic residues. Expression of BYB was analyzed from the penultimate to last instar during larval development. BYB was expressed in every tissue examined, but controlled differentially. The expression pattern was comparatively dependent on the molting cycle in the carcass, fat body and anterior silk gland (ASG); the expression decreased during the fourth molt. However, in the middle silk gland (MSG) and posterior silk gland (PSG), the expression increased steadily from the fourth instar to early into the fifth instar. The results suggested that BYB exhibits tissue- and stage-dependent function (s) during larval development.

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RNA-mediated interference (RNAi) provides a rapid and potent approach for analyzing gene function in vivo. The conventional RNAi technique is performed by microinjection of double-stranded RNA (dsRNA) into syncytial blastoderm embryos (embryonic RNAi). While the embryonic RNAi method is efficient for analyses of embryogenesis, it is inappropriate for analyses of postembryonic development. To circumvent this problem, larval RNAi by the injection of dsRNA into the larval body cavity had been reported using Tribolium castaneum. In order to demonstrate the general utility of the larval RNAi method in non-model insects, we used the evolutionary conserved homeobox genes required for appendage formation, Distal-less (Dll) and aristaless (al), to affirm their function in adult development of the ladybird beetle, Harmonia axyridis. The injection of dsRNA for Harmonia Dll (Ha-Dll) and al (Ha-al) into the early stage of last instar larvae efficiently induced adult morphological defects that mimicked those of known loss-of-function phenotypes for these genes. Surprisingly, these adult defects by larval RNAi were induced with a 100% penetrance. We conclude that larval RNAi is extremely efficient for the analysis of adult development in Harmonia and that larval RNAi will become a powerful new tool for analyzing postembryonic development at a molecular level, particularly in non-model insects.

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To confirm the correspondence between sericin proteins and sericin genes of Bombyx mori, the partial amino acid sequences of cocoon sericins were determined by N-terminal sequencing of lysyl endopeptidase fragments and were then compared to the polypeptides derived from the Ser1 gene. It was determined that sericin M, which is the abundant component of sericin, was a product of the Ser1 gene, whereas sericin A, which distributes mainly in the floss and the outer layer of the cocoon, was not. In addition, sericin P was considered to be a smaller product of the Ser1 gene based on the cleavage pattern in the enzymatic digestion. It is expected that the properties of sericin A are different from those of sericins M and P because it does not have 38-amino acid repeats that constitute the major portion of the Ser1 proteins.

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New mutant larvae were discovered in a hybrid race, C21×Koishimaru, of the silkworm, Bombyx mori. Genetic analysis indicated that the mutant character was controlled by a single dominant gene belonging to the E pseudoalleles, located at position 21.1 on the 6th-linkage group. This new mutant was named ‘Extra-abdominal legs and extra crescents’, with the gene symbol E^Al. The phenotype of the E^Al mutant was as follows. E^Al heterozygous larvae showed two pairs of extra-abdominal legs on the first- and second-abdominal segments and a pair of extra-crescent markings on the first-abdominal segment. The majority of E^Al homozygous larvae developed to the adult stage. The E^Al homozygous larvae showed nearly the same characters as E^Al heterozygotes, but expression of the extra-abdominal legs and extra-crescent markings was slightly clearer than those in the heterozygous larvae. Although copulatory behavior and fertility were normal in E^Al heterozygous moths, E^Al homozygous moths were predominantly sterile in both sexes. Recombination between E^Al and E^Dn was not recognized, but those between E^Al and E^Nc were detected, of which the value was 0.08%. The strain carrying the recombinant chromosome (E^Al E^Nc/+ +) was established and maintained.

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This study was conducted to find out ecdysone responsive cells among established cell lines of Bombyx mori, and to obtain cells to clarify the regulatory mechanism of ecdysone responsive cuticle protein genes. Four cell lines were selected to examine the ecdysone-responsiveness. The aggregation of cells was observed after 20-hydroxyecdysone (20E) treatment, in cell lines of NBO1, NBO2 and NBS1228. The expression of E75A and BHR3 genes increased by the addition of 20E, and the existence of JHA accelerated the ecdysone-induced strength of the signals. The results of the real time PCR revealed that the addition of 20E strongly induced ecdysone responsive genes, E75A, BHR3 and mg-0560 EST clone. Expression of two cuticle protein genes, BmWCP2 and BmWCP10, was observed with or without 20E in the cells, but it was observed stage specifically in wing discs of Bombyx mori. We identified ecdysone responsive and cuticle-protein gene expressible cell lines of B. mori, and they will be used for the hormonal research.

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The baculoviruses Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV) have highly similar genome sequences but essentially nonoverlapping host ranges. Recent studies demonstrated the arrest of protein synthesis in BmN cells (BmNPV-permissive) infected with AcMNPV. We report here the arrest of cell proliferation in AcMNPV-infected BmN cells involving a marked decrease of ribosomal RNA (rRNA), which was not observed for BmNPV-infected BmN cells or for Sf-9 cells (AcMNPV-permissive) infected with AcMNPV or BmNPV. The arrest of protein synthesis in BmN cells using the de novo protein synthesis inhibitor, cycloheximide, didn’t cause the decrease of rRNA, suggesting a unique function of AcMNPV in its ability to decrease rRNA in BmN cells.

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