Bacillus thuringiensis (Bt), a bacterium known for its insecticide activity, was discovered in 1901 by a Japanese scientist who was studying a silkworm disease. The ailment, known as the “sotto” disease, is very virulent as it kills the insect almost instantaneously. He observed that death occurred before the bacterium multiplied and suggested that a toxin, not bacterial infection, was involved in the early stage of the pathogenicity. Modern biochemical studies, such as isolation of the protein responsible for insecticidal activity, began in the 1950's. During the same period, the first large scale, commercial production of sprayable Bt formulations in the U. S. took place in California. The industry flourished after a high potent strain, Bt kurstaki HD1, was reported in 1970. In 1981, the first gene for the crystalliferous insecticidal protein (crystal protein) was cloned from a variant strain of HD1 and sequenced. Since then, numerous genes have been cloned and characterized. These genes, called cry and cyt, have been classified into some 30 different groups by amino acid sequences of the proteins encoded by the genes. Extensive studies in biochemistry and molecular biology of the crystal proteins and their genes have been conducted. Receptor genes for the Bt crystal protein have been cloned, and regulation of the cry gene expression in Bt has been thoroughly studied. Technical and commercial success of transgenic crop plants expressing Bt insecticidal protein genes encourages further study on this scientifically and politically interesting subject matter.

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A proton-translocating vacuolar-type ATPase (V-ATPase) was identified and characterized in the middle silk gland (MSG) of Bombyx mori. The presence of V-ATPase in a microsomal fraction was verified by immunoblots using an antiserum to the V-ATPase holoenzyme from Manduca sexta midgut and by measuring the enzyme activity as an azide- and vanadate-insensitive, bafilomycin-sensitive ATPase. The antiserum localized the V-ATPase at the apical cell surface of the posterior division of MSG, which is characterized as unusual huge vacuoles, cavities and numerous pits. The strong immunoreactions of V-ATPase were also distributed at the periphery of these structures, which appeared to be filled with the secreted sericin. These observations suggest that the plasma membrane V-ATPase in the posterior division of MSG seems to be important for the active exocytosis and secretion of sericin to establish the electrochemical proton gradients by pumping the proton out of the cell towards the glandular lumen.

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Antheraea yamamai paralytic peptide (AnyParP) is shown to induce the production of diapause eggs in the non-diapause egg producers in B. mori. In this study, we compared diapause and the body paralysis induced by a series of homologs and fragments of AnyParP. AnyParP was most effective in inducing diapause in both the polyvoltine (N₄) and bivoltine (Daizo) non-diapause egg producers. All tested members of the paralytic (ENF) peptide family exerted considerable paralytic activity in an assay of B. mori larvae. All of the AnyParP fragments were less active than the complete peptides in both diapause induction and paralytic activity. The results indicate that the entire sequence of AnyParP is essential for the two activities. Since the structure of AnyParP is very different from the diapause hormone (intrinsic egg diapause inducer), AnyParP may represent an alternative mechanism of embryonic diapause induction.

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Two unique polypeptides with apparent molecular weights (MWs) of 45, 500 (45.5K) and 44K were identified in the purified BmDNV-2 preparation from the pupal midgut of the silkworm, Bombyx mori, that had previously been infected with B. mori densovirus type 2 (BmDNV-2) at a larval stage. Immunoblot analysis and peptide mapping showed that both 45.5K and 44K polypeptides were serologically and structurally distinct from the structural polypeptides of BmDNV-2, suggesting that these two polypeptides were not related to the polypeptides for virion structure. Comparison of directly determined NH₂-terminal amino acid sequences of 45.5K and 44K polypeptides with those deduced from the nucleotide sequences of BmDNV-2 genomic DNAs implied that they were not coded by viral genome. Based on the fact that 45.5K and 44K polypeptides were related serologically and structurally but distinct in their NH₂-terminal amino acid sequences, 45.5K polypeptide seemed to contain entire or nearly entire amino acid sequence of 44K polypeptide and a unique NH₂-terminal amino acid extension. The 45.5K and 44K polypeptides were found only in the midgut of BmDNV-2-infected silkworm, and were not detected in fat body, Malpighian tubule, ovary and hemolymph of the same infected insects. In the BmDNV-2-infected midgut, conspicuous accumulation of these two polypeptides is achieved only at two specific times, one being 2-3 days postinoculation and the other at larval-pupal transformation onward. These results suggest that 45.5K and 44K polypeptides are involved in the degeneration and discharge of virus-infected midgut cells.

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To characterize Autographa californica nucleopolyhedrovirus (AcMNPV) infection of the cells derived from Bombyx mori, cytopathology, cellular viability and metabolic activity, viral DNA replication, viral polypeptide synthesis, and polyhedrin gene expression were examined. AcMNPV-infected Bombyx cells displayed cytopathology and were impaird in their ability of proliferation. Immunoblot analysis showed that viral capsid polypeptides were produced in AcMNPV-infected Bombyx cells in a large quantity that was comparable to that in conventional permissive Sf21 cells, while there was a striking decrease in the production of envelope fusion protein GP64 and viral genomic DNA in Bombyx cells as compared to that in Sf21 cells. Bombyx cells supported the production of budded virions (BVs) into culture medium, although BV yield in AcMNPV-infected Bombyx cells was lower by one to two orders of magnitude than that in AcMNPV-infected Sf21 cells. No detectable amount of polyhedrin protein was produced in AcMNPV-infected Bombyx cells, and northern blot analysis demonstrated that the defect in polyhedrin production in AcMNPV-infected Bombyx cells was due to the transcriptional restriction of polyhedrin gene expression. These results indicate that in AcMNPV-infected Bombyx cells, virus replication cycle proceeds to yield a limited number of progeny BVs into culture medium, without an optimal expression of polyhedrin gene. The present study thus demonstrates that AcMNPV-infected Bombyx cells provide a unique model system to define cellular factors responsible for host-dependent viral gene expression.

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We have developed a fully automated artificial diet feeding system for rearing silkworms. This system is capable of performing a series of tasks (mixing powdered artificial diet with hot water, transporting the food paste and delivering it to each rearing box) automatically, without an operator. Powdered artificial diet is mixed with hot water in a mixer, then transported by a delivery pump to a filling valve via a transport pipe. A 3-axis robot delivers the food to any predesignated point in the rearing box of silkworm larvae. Therefore, the development of this system makes the silkworm rearing area free from contaminants. Wet artificial diet used now needs to be cut into pieces, weighed, and stored. Using this automated system, these processes are no longer necessary. Furthermore, since the storage of wet artificial diet in a refrigerator has been eliminated, the cost of rearing facilities can be reduced.

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Using a series of leaves from mulberry seedlings, the developmental process of idioblast was surveyed by scanning electron microscopy (SEM). The 1st and 2nd leaves numbered from the shoot top had various types of idioblast protrusions on their adaxial surface; a small, round form with a sharp tip (A type) and a cone-shaped form with different sizes (B, C and D types). The surface of the 3rd leaf was rich in the E and F type protrusions which were composed of cone-shaped and basal portions. The cone-shaped portion decreased in height to form a G type protrusion showing a dome-like form with a smaller cone-shaped projection. All of the adaxial surface of the downward leaves from the 3rd leaf was occupied by the G type of idioblast protrusion. Calcium (Ca) deposition was closely associated with such sequential changes in idioblast morphology, starting Ca deposition from the D type protrusion and leading continual deposition in the egg-shaped structure of E, F and G type protrusions. Similar types of idioblasts were also present in abaxial leaf surface, which was one-fifth of adaxial idioblast density.

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