Spontaneous synthesis of cecropin B, an antibacterial peptide, was observed in the molting stage in two lepidopteran insects, Bombyx mori and Antheraea pernyi, by immunohistochemical staining. The natural induction of ceropin B gene expression was also observed in artificially induced molting of B. mori larvae, suggesting that the natural induction is tightly linked to ecdysis. As insect molting is strictly controlled by ecdysteroids, a possibility that the hormone induces antibacterial peptide gene expression was tested by using a fat body primary culture of B. mori. The results showed that 20-hydroxyecdysone failed to induce gene expression of three antibacterial peptides, cecropin B, attacin, and lebocin, suggesting that unknown endogenous trigger(s) is involved in the natural induction. The results suggest that insects contain a novel ecdysis-linked immune system, which is strikingly different from that of vertebrates.

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α-Amylases are major digestive enzymes in bruchids that infest seeds of starchy grain legumes. To cope with these insect pests, some species of the genus Phaseolus have developed seed protection systems involved in various defense proteins. Proteinaceous α-amylase inhibitor (αAl) is one of these defense proteins that has been verified through genetic engineering to be a useful tool for protection of legume seeds from bruchids. In the present study, a cDNA (CCA) encoding α-amylase was isolated from the larvae of the azuki bean weevil, Callosobruchus chinensis. The CCA possessed an open reading frame encoding a polypeptide of 490 amino acid residues including the predicted signal sequence of 16 amino acid residues, that had an estimated molecular weight of 52, 223 after cleavage of predicted signal sequence. Deduced amino acid sequence showed that three amino acid residues (Glu²³⁷, Asp²⁰¹, Asp³⁰³) are involved in catalysis and three histidine residues (His¹¹⁵, His²⁰⁵, His³⁰¹) responsible for substrate binding conserved in this α-amylase. In baculovirus expression system, CCA expressed C. chinensis α-amylase that was active in a broad pH range from 4.5 to 6.5. Consistent with native α-amylase from C. chinensis larvae, the activity of recombinant C. chinensis α-amylase expressed in the baculovirus expression system was inhibited by αAl-1 and αAl-Pa1 from the cultivated common bean, Phaseolus vulgaris, and the tepary bean, Phaseolus acutifolius, respectively, but not by αAl-2 from certain wild common bean accessions. These results indicate that CCA and its expressed recombinant C. chinensis α-amylase obtained in this study are useful to elucidate the molecular basis for the specific interactions between bruchid α-amylases and bean αAls.

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We have successfully developed an efficient gene transfer system using the recombinant baculovirus (AcNPV) in the silkworm, Bombyx mori. A driver-reporter cassette with an EGFP reporter was inserted into AcNPV bacmid using the Bac-to-bac baculovirus expression system. The gene transfer was accomplished by injection of the recombinant AcNPV into larvae or pupae. A cassette driven by the Bombyx actin A3 promoter clearly gave EGFP fluorescence in the whole body, including the central nervous system (CNS). To apply this system for analysis of neurohormone gene expression mechanisms, cassettes were constructed with the promoters of three insect neuropeptide hormone genes: bombyxin, a prothoracicotropic hormone (PTTH), and diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN). The bombyxin promoter directed the EGFP expression to the median 8 neurosecretory cells of the brain, PTTH to the lateral 4 neurosecretory cells of the brain and DH-PBAN to 14 cells of the suboesophageal ganglion, which have been identified to be the neuropeptide hormone-producing cells. This assay is time saving, convenient, and highly reproducible, so that the present system provides a new tool for analysis of the neuropeptide hormone gene expression in the CNS.

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Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was applied to sericin fiber spun by Sericin-hope silkworms, developed to produce silk protein sericin in large amounts, and to a native sericin solution before spinning. Polarized ATR-FTIR measurement showed little orientation of sericin molecules in sericin fiber. Secondary structures of sericin fiber and native sericin were analyzed by Fourier self-deconvolution and curve fitting. The drying process of the native sericin solution was also followed to determine the effect of dehydration on sericin conformation. These analyses showed that β-sheets in native sericin increased with drying and that the secondary structure of air-dried sericin was similar to that of sericin fiber. These observations suggest that drying is a significant factor in the structural transition of sericin during fiber formation and that sericin undergoes only modest structural changes by spinning compared with fibroin.

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We have developed an insect rearing facility and automated rearing machine, to establish a fully automated large-scale insect rearing system to produce a new materials or a substances using useful functions of insects. In this study, we assembled a three-dimensional automated warehouse type silkworm rearing machine, to enable auto mechanization of rearing work. Six shelves in 4 rows type rearing machine has been constructed and control program was coded on Visual BASIC control language. After evaluated an operation and control function of this system, we found a possibility of controlling the automatic operation of the rearing system. Also, some physical properties of rearing system were examined. It was ascertained that the vibration and noise of this system does not affect the development of silkworm. Result of rearing test, this system can be used as a fully automated rearing of the silkworm of fifth instar.

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We fitted a probability distribution to the directions of a silkworm body and proposed a time series model for variations of the silkworm’s position during cocoon construction. The analysis of real data showed that the directions of the body approximately followed a mixture distribution of two Fisher densities on a unit sphere except at an early stage of spinning. In addition, it revealed that the autoregressive model we proposed was appropriate to represent periodicity for the variation of positions fixed by the silkworm.

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The in planta transformation method was used for mulberry plants [Morus alba L., F₁ generation obtained by natural pollination of a Chinese variety, Canzhuan No.4 (female)]. For transformation, an avirulent mutant of Agrobacterium tumefaciens (M-21), isolated by us, was used; the mutant is capable of integrating its T-DNA into chromosomes of host plants but produces no galls since a gene (tryptophan monooxygenase gene, iaaM) involved in auxin (indole acetic acid, IAA) biosynthesis in the T-DNA region is destructed by transposon5 (Tn5) insertion. All other genes including the ipt gene for cytokinin biosynthesis in the T-DNA region are intact. Young mulberry trees in pots were decapitated and meristems of axillary buds were inoculated by water suspension of A. tumefaciens after being pricked with a needle. The inoculated four plants were allowed to grow in pots. All of the four transformants in the T₀ generation showed characteristic phenotypes different from those of non-transformed plants, which seemed to be due to overproduction of cytokinin. The transgene (Tn5-inserted T-DNA) was detected in all of four transformants by nested PCR. The offspring of transformants (T₀) produced by stem cuttage also showed remarkable phenotypes different from those of offspring of non-transformed plants. The transgene was transmitted in all of the offspring of transformants. Thus, the transgenic mulberry plants can be produded simply and efficiently by the in planta transformation method as detailed in this paper.

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Considering the automatic raw silk reeling machine as a “black box” in which a large number of cooked cocoons are being reeled or prepared for the reeling, and using the queues model with infinitely many servers to describe the dynamic process of the number of cooked cocoons staying in the reeling machine, we gave a method to estimate the distribution of the number of cooked cocoons in the “black box” according to the time series information of the numbers of the input and the output cooked cocoons. By computer simulation, we discussed on the errors of the estimated distribution and its applications to the control of reeling process.

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An amplification-mediated method for the analysis of the methylation status was designed and applied to Bombyx mori DNA. The current method, named PAM, utilized the differential methylation sensitivities of four-base cutters, HapII and MspI. In order to reduce background signals, the first HapII digestion step was followed by the ligation to the HapII adaptor, while the second digestion by the same enzyme was followed by the Klenow-filling of the 5′ protruding ends. Then comes the step of digestion with either MspI or HapII, and further both digests were subjected to ligation with the MspI adaptor and to PCR with primers annealing to the MspI adaptor. The PCR products found in the MspI digest, not in the HapII digest, are expected to represent specifically the DNA sequences flanked by the two methylated CpGs in the context of CCGG. In fact, these types of specific signals were given by more than twenty fragments derived from DNA of a moth. By adding another primer that anneals to the HapII adaptor at the PCR step allowed the detection of DNA fragments neighbored by a methylated site at either of the sides. The PAM method using genomic DNA of five larval tissues exhibited differences in the methylation pattern among them. These findings indicate that the PAM method is a powerful tool for screening genome-wide methylation status in the B. mori cells.

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Last instar larvae of the silkworm, Bombyx mori, were fed an artificial diet containing plant-derived 20-hydroxyecdysone (20E) solution to establish simple recipes for molt manipulation applicable to physiological, biochemical, and molecular biological studies. 20E diet from day 0 or day 1 induced an extra larval molt, while 20E diet from day 2 or thereafter induced precocious pupation, depending on the hemolymph JH titer. We advocate three treatments for stable molt manipulation: (1) rapid induction of an extra larval molt within 2.5 days after feeding 400 ppm of 20E from day 0, (2) late induction of an extra larval molt after feeding 20 ppm from day 0 for 1 week, and (3) 2 days’ acceleration of pupation after feeding 20 ppm from day 4. In the first two treatments, the generated 6th instar larvae eventually died without pupation, while the precocious pupae generated in the third treatment metamorphosed normally into adults. We also measured the hemolymph ecdysteroid titer to clarify the endocrinological environment of 20E-fed larvae.

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