When an excised section of larval integument is implanted into a larva, the growth rate of the epidermal cells of the implanted tissue increases to form a cyst in vivo. In order to clarify the processes associated with in vivo cyst formation in the sweet potato hornworm, Agrius convolvuli, we investigated the progression thereof using light- and electron microscopy. Immunohistochemical techniques using eCBP (epidermal carotenoid binding protein) as a specific marker for detecting larval epidermal cells, and BrdU-incorporation for examining DNA synthesis in the epidermal cells were also undertaken. The results indicated that in vivo cyst formation proceeded through the following five phases: (1) hemocytes aggregate around the periphery of the transplanted integument, (2) forming a thick mass of aggregated cells. This is followed by (3) DNA synthesis, not only in the cells near the periphery, but also in the entire epidermal cell layer. Then, (4) the epidermal cells at the leading edge penetrate the cell mass until continuity of the integument is complete, (5) finally they secrete new cuticle inside the cyst. Given the simplicity, high reproducibility and ease with which these experimental procedures can be conducted, the system of in vivo cyst formation in insect is a useful model for studying the more detailed mechanisms associated with wound recovery, such as epidermal elongation at the site of integument damage.

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The effect of heavy-ion irradiation on the differentiation of epidermal cells was examined in the silkworm, Bombyx mori. Local irradiation of fifth instar day 1 larvae with carbon-ions (400Gy) resulted in no marked morphological changes in the instar. The detection of few TUNEL-positive cells indicated that apoptosis of the epidermal cells did not occur in response to irradiation. However, the resultant moths were observed to loose their scales in the area irradiated in a dose dependent manner. Similar results were also obtained when newly hatched larvae were irradiated. Light- and scanning electron microscopy revealed that the pupal epidermal cells secreted adult cuticle, but that differentiation of scale and socket cells was completely inhibited at the site of irradiation. On the other hand, when effects of ion-beam irradiation were examined in an epidermal mutant strain (Knobbed: K), it was found that the paired protuberances (knobs) at the sites of larval markings disappeared in subsequent larval, pupal and adult stages when newly hatched larvae were locally irradiated. These findings suggest that knobs formed by the excessive proliferation of epidermal cells did not develop because cell division in these areas was suppressed by heavy-ion irradiation. It therefore appears that epidermal cells are considerably resistant to heavy-ion beams and that epidermal differentiation, such as that associated with scale and knob differentiation, are suppressed by the inhibition of cell division after irradiation.

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Fibroinase activity of silk gland of B. mori was determined during whole larval development and in early pupa. For this purpose we synthesized highly sensitive peptide substrates, fluorescent quenched peptides, 7-methoxycoumarin-4-acetic acid (MCA)-Ala-Gly-Tyr-Gly-Ala-Gly-Lys (2,4-dinitrophenyl group (Dnp))-Gly (hereafter referred to as MCA-Tyr-Dnp) and MCA-Ala-Gly-Phe-Gly-Ala-Gly-Lys (Dnp)-Gly (MCA-Phe-Dnp) based on the cleavage specificity of fibroinase of silk gland to preferentially split the peptide bond between Gly and Ala in the fibroin peptide (Watanabe et al., 2004a) and on the tetrapeptide produced by hydrolysis of B. mori liquid fibroin by fibroinase of silk gland for 12h as a building block of substrate, which was identified in this study. The kinetic parameters of these two substrates were determined using fibroinase of silk gland from different developmental periods, i.e., from the fourth molt period and from day 1 pupa. MCA-Tyr-Dnp gave similar k_cat/K_m values, while MCA-Phe-Dnp gave different values. To obtain comparable data on the fibroinase activity of silk gland during B. mori development was the objective of this study, MCA-Tyr-Dnp was adopted as a substrate. Assay was conducted both at pH4.0 and at pH5.0. Similar activity levels were obtained at both pH’s. Fibroinase activity of silk gland increased directly proportional to a log scale from day 1, the first instar larva until the middle period of the fifth instar larva by 10,000-fold. Considerable activity was observed in the feeding period in each larval instar including the fifth larval instar, the latter result of which confirmed the previous results using liquid fibroin as a substrate. Activity was also observed at each molt period and a peak of activity was observed clearly at the second, third, and fourth molt periods. These observations in the earlier period of larval development of B. mori were interpreted as a further support of a dual role of fibroinase of B. mori silk gland depending on the developmental period, i.e., at the feeding period for digestion of obsolete protein synthesis machinery and cellular organells in lysosomes, and at the molt period for digestion of fibroin and sericin accumulated in the lumen contents of silk gland, respectively.

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We cloned and sequenced cDNA of Bombyx mandarina vitellogenin (BmaVg) inhabiting Japan. The complete sequence of BmaVg cDNA without a poly A tail is 5733 bp long and encodes 1780 amino acid residues among which are typical highly conserved sequences and common features found in most insects. Multiple alignment of vitellogenin sequences of Japanese Bombyx mandarina, the Chinese one, and Bombyx mori showed that the nucleotide sequence of Japanese BmaVg cDNA shared 99.5% identity with Chinese BmaVg and 98.2% identity with Bombyx mori vitellogenin (BmoVg), and that the amino acid sequence of Japanese BmaVg cDNA shared 99.6% homology with Chinese BmaVg and 97.5% homology with BmoVg. These findings indicate that BmaVg (JPN) is more similar to BmaVg (CHN) than to BmoVg, which was also confirmed through a comparison of the mutation frequencies of both nucleotide and amino acid sequences. The phylogenetic tree of Vgs demonstrates that BmaVgs of individuals collected from eighteen prefectures in Japan primarily cluster in a group with BmaVg (CHN) and without BmoVg, suggesting that the sequence variation between the two BmaVgs arose after divergence of B. mori from B. mandarina.

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