Eiji Kanke

Isolation and expression of the ecdysteroid-inducible angiotensin- converting enzyme-related gene in wing discs of Bombyx mori

We isolated a clone encoding a putative angiotensin-converting enzyme-related gene from the wing disc cDNA library of the silkworm, Bombyx mori (refer to as BmAcer). The predicted open reading frame encoded 648 amino acids with about 50% identities with the Drosophila melanogaster angiotensin-converting enzyme Ance and Acer. Northern analysis identified a 2.2-kilobase mRNA which was abundant in wing discs two days after the beginning of wandering. An accumulation of the transcript was observed approximately 2 h after 20-hydroxyecdysone (20E) exposure in vitro and was blocked slightly by a protein synthetic inhibitor. These data suggest that the transcription of the BmAcer gene is directly 20E-inducible.

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.

Isolation and expression of an ecdysteroid-inducible neutral endopeptidase 24.11-like gene in wing discs of Bombyx mori

In the process of comparison of two cDNA libraries (W0, W2), we isolated a clone from the wing discs of Bombyx mori encoding a putative neutral endopeptidase 24.11-like gene. The predicted open reading frame encoded 772 amino acid residues, having about 53% identity with Drosophila GH07643, 36% with rat NEP, and 34% with rat ECE. This is the first NEP gene isolated in invertebrate. A 3.6-kb transcript was found to accumulate in the wing disc according to the increase of ecdysteroid titer during metamorphosis. Accumulation of the transcript was induced in wing discs with 20-hydroxyecdysone about 20h after incubation, which was inhibited by cycloheximide. This gene is ecdysone-inducible, appears to encode a functional protein, and may function during wing metamorphosis.

Fluctuation of the ploidy level in the epidermis of Bombyx mori during the penultimate and ultimate larval instars

Summary This study was carried out to clarify the fluctuation of the ploidy level in the epidermis and compare the level of polyploidy with related evidence during the penultimate and ultimate larval instar of Bombyx mori. Flow cytometry analysis revealed that the percentage of 8C and 4C cells decreased with the increase of 16C cells in the fourth larval instar, while 32C cells appeared for the first time on day 3 in the fourth larval instar. At the beginning of the fifth larval instar, most of cells were 16C or 32C, and the percentage of 16C cells was twice that of 32C cells. The percentage of 16C and 32C cells remained almost constant during the feeding stage, followed by a decrease during the spinning stage. Changes of ploidy level, DNA synthesis, and mitosis were observed when the concentration of hemolymph ecdysteroid was high in both the penultimate and ultimate larval instar. Mitosis was induced in the presence of 20E. The present results indicate that ecdysteroid affects the DNA synthesis, mitosis, and polyploidy in the epidermis in the penultimate and ultimate instar of Bombyx mori.

Ecdysteroid Titer, Responsiveness of Prothoracic Gland to Prothoracicotropic Hormone (PTTH), and PTTH Release of the Recessive Trimolter Strain of Bombyx mori in Extra-ecdysed Larvae by JHA and 20E Application

Abstract Application of 20-hydroxyecdysone (20E) or juvenile hormone analogue (JHA) to the last (fourth) larval instar of a recessive trimolter Bombyx strain induced extra larval ecdysis. The ecdysteroid titer, the PTTH-secreting activity of the brain, and the ecdysteroid-secreting activity from the PG were compared among the untreated and 20E- and JHA-treated recessive trimolter. The ecdysteroid titer in the hemolymph and ecdysteroid secretory activity of the PG decreased in the mid-fourth larval instar in untreated control larvae, while those in 20E- and JHA-treated larvae did not decrease during the same stage. An application of JHA enhanced the PTTH-secreting activity of the brain. Results indicate that the recessive trimolter strain of Bombyx mori lost the responsiveness of the PG to PTTH in the early stage of the fourth larval instar, resulting in precocious metamorphosis. Results also indicate that the application of 20E induced extra larval ecdysis by maintaining substantial levels of ecdysteroid in the hemolymph. However, JHA is suggested to induce larval ecdysis through activation of the brain and maintenance of the responsiveness of the PG to PTTH.