Double-stranded (ds) RNA is a useful tool for sequence-specific gene silencing, now known as RNA interference (RNAi), in a variety of organisms. Studies on pol III promoter-based expression systems have been well documented showing them to be an effective tool for inducing RNAi in mammalian cells and Drosophila Schneider 2 (S2) cells, but not in silkworm cells. In this study, we developed a Bombyx mori U6 promoter (U6p)-based short hairpin RNA (shRNA) expression system for inducing RNAi in silkworm cells. First, a transient reporter assay showed that the transfection of plasmids containing U6p-driven shRNA-expression units (U6-shRNA units) caused a decrease in the expression of the target gene in BmN cells with the efficacy dependent on the length of the hairpin stem region. Subsequently, we demonstrated the ability of the U6-shRNA unit to confer resistance to Bombyx mori nucleopolyhedrovirus (BmNPV)-resistance on BmN cells; i.e., the viral titer in the culture medium of BmN cells transfected with U6-shRNA (with the sequence of an essential viral gene)-expression plasmids was decreased to 30% of the control at 48hr post-infection. These results suggested that the U6-shRNA expression system was a useful tool for RNAi in silkworm cells.

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The basement membrane is a thin, specialized extracellular matrix surrounding most tissues in all insects, and the disintegration of the basement membrane facilitates cell movements, such as the rearrangement of cells during development. Histological analysis of the basement membrane in the wing discs, midgut, fat bodies, and silk glands of Bombyx mori provided insights into their assembly during metamorphosis. Our observations revealed that the basement membrane was not degraded in the wing discs, fat bodies, or midgut during the larval feeding stages, but was partially or completely degraded during the later spinning and early pupal stages. In contrast, the basement membrane of the silk glands was not degraded at any developmental stage, and its thickness increased during the late spinning and early pupal stages. The degradation of the basement membrane during the late spinning stages in the above tissues correlated with the expression pattern of BmADAMTS-like (Bombyx mori a disintegrin and metalloproteinase with thrombospondin-type I motif) gene expression.

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The cell line Hv-AM1 from Heliothis virescens was characterized for permissiveness and productivity, employing Helicoverpa armigera nucleopolyhedrovirus (HearNPV) and seven other NPVs, including those of Hyphantria cunea (HycuMNPV), Orgyia pseudotsugata (OpMNPV), Bombyx mori (BmNPV), Spodoptera exigua (SeMNPV), Spodoptera litura (SpltMNPV), Lymantria dispar (LdMNPV) and Autographa californica (AcMNPV). Based on budded virus (BV) yields, polyhedrin production, and polyhedra formation, the results showed for the first time that the cell line Hv-AM1 was permissive for HearNPV, HycuMNPV and OpMNPV, supporting moderate levels of BVs, polyhedrin and polyhedra production. Consistent with the previous results, the cell line Hv-AM1 was also shown to be highly permissive for AcMNPV infection, yielding high levels of BVs, polyhedrin and polyhedra. In the cell line Hv-AM1 infected with BmNPV, SpltMNPV and SeMNPV, few, if any, viral progeny were produced, regardless of the severity of the observed cytopathic effects and the significant production of viral DNA, suggesting its semipermissivity nature for these NPVs. In addition, the cell line Hv-AM1 was found to readily undergo apoptosis upon infection with BmNPV, SeMNPV and HycuMNPV, while it was refractive to LdMNPV. Thus, although the cell line Hv-AM1 is not very efficient for HearNPV production, the diverse observed responses and high permissiveness against NPVs indicate that the cell line Hv-AM1 provides a novel system for analyzing the possible mechanisms underlying the permissiveness and productivity of insect cells against NPVs.

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First, we confirmed that a trehalase specific inhibitor, validoxylamine A, injected into diapause-egg producing pupae produced significant numbers of nondiapause eggs, suggesting that biosynthesis of diapause hormone and/or its release into the hemolymph may be reduced by inhibition of trehalase activity in brain-subesophageal ganglion (SG). Next, to better understand trehalose catabolic activities in brain-SG of diapause- and nondiapause-egg producers of the silkworm, Bombyx mori, we examined the expression of trehalase-1 and -2 mRNAs during pupal-adult development. These mRNAs code for soluble and integral-membrane proteins, respectively. Remarkable differences in the mRNA amounts were not found in either the diapause- and nondiapause-egg producers, but the expression of these mRNAs increased much more rapidly prior to adult emergence, as compared with dopa decarboxylase mRNA, a key enzyme in the formation of the neurotransmitter dopamine. These results suggest involvement of trehalases within brain-SG in adult ecdysis.

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To facilitate gene functional analysis systems in non-model insects, we focused on the Tetracycline-Off (Tet-Off) system, a binary expression system using a tetracycline controlled transactivator protein (tTA) and its binding site, the tetracycline operator sequences (tetO). The Tet-Off system can control gene expression temporally by adding or removing tetracycline (Tc) or doxycycline (Dox), which inhibits the transactivational activity of tTA. In this study, we established a Tet-Off system applicable to a wide variety of insects by constructing the Drosophila heat shock protein 70 (hsp70)-tTA and tetO-Enhanced Green Fluorescent Protein (EGFP) vectors using piggyBac, the most widely used transformation vectors in insects to date. The effectiveness of these vectors was examined using transgenic flies. We show that (1) EGFP fluorescence was efficiently induced by the hsp70 promoter using the Tet-Off system, (2) Tc or Dox can effectively inactivate tTA to repress gene expression under tetO in a concentration dependent manner, and (3) removal of Tc or Dox from the diet can recover the transactivational activity of tTA, also in a concentration- and time-dependent manner. Since Drosophila hsp70 promoter and piggyBac are useful in many insects, our results indicate that the Tet-Off system used in this study provides an ideal opportunity for examining the function of genes in non-model insects as well as in Drosophila.

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Glycoconjugates consisting of silk fibroin (SF) and polycationic chitooligosaccharides (COS) were prepared by the chemical modification of SF with COS using cyanuric chloride (CY) as a coupling spacer. The growth of mouse fibroblast (L-929) cells on glass coverslips coated with the conjugate (COS-CY-SF) was investigated by a cell culture method. The number of cells grown on the COS-CY-SF conjugate-coated surfaces was the lowest compared with surfaces coated with SF and collagen during 7 days of culture, although the cells on the conjugate-coated surfaces were able to proliferate. Moreover, cell spreading on the conjugate coated-surfaces was prevented over 7 days, while the cells on the surfaces coated with SF and collagen exhibited a well-spread morphology within 5 days. These results reveal the cell-resistant properties of the COS-CY-SF conjugated-coated surfaces and the potential use of the conjugate-coatings for implant applications.

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Sericin accounts for about 25% of the total composition of silkworm cocoon shells. It has recently been found to be a natural attracting material on cosmetic fields because of its skin moisturizing effect, and inhibitory effects against lipid peroxidation and tyrosinase activity. The silkworm race “Sericin Hope” possibly provides undestroyed natural sericin, which is easily gelled and emulsified at lower concentrations. In order to improve this race, we developed “Sericin Flavo” by cross-breeding it with the green-cocoon strain “Daizo (Matsumura)”. This new race spins a high ratio of green sericin cocoons, each of which contains about 4.1mg of flavonol. These green sericin cocoons show higher antioxidant activity and a wider UV shielding effect.

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