An advantage of silkworm, Bombyx mori, is that they can undergo artificial parthenogenesis. In the current study, we investigated the hatchability associated with artificial parthenogenesis in 208 silkworm strains including native and improved strains stocked by the National Institute of Agrobiological Sciences (NIAS). The mean hatchability for all experimental strains, not including the control hybrid, was 2.51%. When 14 strains of outliers are excluded from 208 strains, the mean hatchability decreased to 1.64%. Assuming that the histogram of 194 strains fits to the Poisson distribution, the histogram partially fit to the expected Posisson distribution. We argue that the average value, 1.64%, is an important value of Bombyx mori.

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BHR4 is an ecdysteroid-induced orphan nuclear receptor whose expression appears in response to molting surges of ecdysteroid and then disappears as the ecdysteroid titer declines before each ecdysis. When the transgenic Bombyx larvae carrying an hsp70-BHR4 insert were heat-shocked during the pupal molt to missexpress BHR4, the pupal ecdysis was blocked due to a maintained ecdysteroid titer. The lack of decrease in the ecdysteroid titer was due to a stimulation of ecdysteroid biosynthesis caused by up-regulated ecdysteroid biosynthetic enzyme genes in the prothoracic glands rather than the disruption of the ecdysone cascade. Thus, the BHR4 expression during the pupal molt regulates the timing of pupal ecdysis through ecdysteroid synthesis.

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Serratia liquefaciens FK01 produces two serralysin-like metalloproteases. One of the genes, ser1, has been cloned before. Another gene, designated as ser2, was cloned in this study. The protein encoded by ser2 was a typical serralysin-like metalloprotease, since a zinc binding motif (HEXXHXUGUXH), glycine-rich repeats (GGXGXD), a Met-turn (SXMXY) and an ABC exporter motif (DXXX) were found in the deduced amino acid sequence. In order to investigate the contribution of these proteases to the virulence in an insect, protease- deficient mutants were created. The mutant strains were inoculated to the silkworm, Bombyx mori. The result of the mortality test suggested that we could not show that deficiency in ser1 and ser2 caused a virulence attenuation in the silkworm under our experimental conditions used in this study. It also suggested that other virulence factors existed in the infection in the silkworm.

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A clonal analysis system in the silkworm, Bombyx mori, was developed to achieve expression of genes of interest in portions of tissues. We established two types of transgenic silkworm strains: one expresses yeast FLP recombinase under the control of a heat shock promoter, and the other possesses the DsRed gene interrupted with the GFP gene flanked at both ends with two FLP recombinase target (FRT) sites. The F₁ eggs between the two strains were heat-shocked to induce FLP recombinase, which catalyzed intrachromosomal recombination to excise the GFP gene, resulting in the expression of DsRed in randomly formed cell clones.

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During the development of Bombyx mori, cell mitosis normally occurs in epidermal tissues of early larval instars, but not during the feeding stage of the final instar. However, in the author’s previous paper, it was shown that cell mitosis was still occurring in Knobbed mutant (K) larvae at the beginning of the molting period of the fourth instar. This result suggested that epidermal mitosis might persist through the feeding stage of the fifth larval instar. Here, cell mitosis was investigated in the epidermis of Knobbed final instar larvae. The epidermal mitosis was compared between the knobbed and the surrounding unknobbed regions of the fifth larval instar at 48hours after ecdysis. Mitotic cells were observed in the knobbed region of the second abdominal segment but not in the non-knobbed surrounding and posterior regions of the same segment. In addition, the corresponding regions of the third abdominal segment did not show mitotic cells.

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Ld652Y cells, which are derived from the gypsy moth, Lymantria dispar, undergo apoptosis upon infection with various nucleopolyhedroviruses (NPVs). To gain insight into the mechanisms underlying NPV-induced apoptosis of Ld652Y cells, we cloned and characterized an L. dispar homologue (ld-caspase-1) of Spodoptera frugiperda effector caspase sf-caspase-1. The ld-caspase-1 gene had an open reading frame of 882bp, encoding a polypeptide of 294 amino acid residues with a predicted molecular mass of 33,304Da. Multiple alignment analysis demonstrated that Ld-caspase-1 had a high degree of amino acid sequence identity (71.4-83.6%) with those of lepidopteran caspase-1 proteins sequenced previously, and consensus sequences of a catalytic site, ¹⁷⁴QACQG¹⁷⁸, and three cleavage sites, ²³DEGDA²⁷, ¹⁷⁹DKLDA¹⁸³, and ¹⁹⁰TETDG¹⁹⁴, were completely or nearly completely conserved among these homologous caspases. When expressed in Escherichia coli, Ld-caspase-1 exhibited substantial cleavage activity for the synthetic substrate Ac-DEVD-AMC, which is preferentially cleaved by human caspase-3, and negligible activity towards Ac-IETD-AMC and Ac-LEHD-AMC, which are preferentially cleaved by human caspases-8 and -9, respectively. Ld-caspase-1 transiently expressed in Ld652Y cells executed apoptosis and stimulated caspase-3-like protease activity, which were both suppressed by the pancaspase inhibitor z-VAD-fmk. Apoptosis induction and activation of caspase-3-like protease by transiently expressed Ld-caspase-1 were also observed in the lepidopteran cell lines Sf9 and BM-N, and dipteran cell line S2. Taken together, these results indicate that the Ld-caspase-1 cloned in this study functions as an effector caspase and is responsible for apoptosis execution in insect cells destined to undergo apoptosis.

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