Seiji Tsuzuki

Ecdysteroid-inducible genes in the programmed cell death during insect metamorphosis

The anterior silk gland of the silkworm, Bombyx mori, undergoes programmed cell death (PCD) during pupal metamorphosis and PCD is triggered by 20-hydroxyecdysone (20E) in vitro. In order to identify the genes responsible for the PCD, we subtracted cDNAs prepared from the anterior silk glands incubated in the presence or absence of 20E in vitro. After a series of screenings by dot blot hybridization, DNA sequencing and reverse transcription polymerase chain reaction (RT-PCR), we obtained seven novel genes that were activated by 20E in vitro. Nucleotide sequence analysis indicated that two cDNAs (EN78 and EC08) did not have any obvious region to encode proteins, while five genes, designated EC74, EN86, EN03, EN10 and EN16, encoded proteins that are similar to inorganic phosphate cotransporter, TIA-1-like protein, chitinase-related protein, translation-initiation-factor subunit and annexin, respectively. Expression profiles of the genes after 20E stimulation indicated that four genes could be classified as early genes, while two are delayed early genes. The genes identified may provide insight into the PCD induced by a steroid hormone.

Larval Growth and Diapause in a Tropical Moth, Omphisa fuscidentalis Hampson

Abstract The bamboo borer, Omphisa fuscidentalis, is a moth found in northern Thailand, Lao and Myanmar and its larvae feed on the inner pulp of bamboo shoots. In a tropical highland (about 500 m sea level) forest at 19°N near Chiang Mai, Thailand, the larvae feed on at least 5 bamboo species. Nucleotide sequence analysis of the region of mitochondrial cytochrome C oxidase subunit 1 gene amplified by the polymerase chain reaction (PCR) verified that larvae collected from different bamboos belong to the same species. Adults appeared in early August and laid clusters of eggs on the newly grown bamboo shoot. The newly hatched larvae bore a hole in the shoot, enter an internode of the shoot and feed on the inner pulp. After maturation in September, the larvae remain in an internodal cavity of bamboo for up to 9 months, from September to the following June. Number of larval instars was estimated by measuring the width of head capsules remained in internodes of bamboo shoots. The growth curve of the width fitted to Dyars law and the mature larvae were estimated to be 5th instar. Mature larvae were collected in the field each month and their body weight, head capsule width, protein and fat contents and hemolymph ecdysteroid titer were measured. Body weight continuously decreased during the 9 months whereas head capsule width remained constant. Fat content fluctuated during this period while protein level remained at a similar level until March, after which it significantly increased. During this period, hemolymph ecdysteroid concentrations remained low. Current results show that the bamboo borer larvae enter diapause at the end of feeding period of the fifth (last) larval instar and the larval diapause lasts until June.

Bombyxin F1 Gene : Structure and Expression of a New Bombyxin Family Gene That Forms a Pair with Bombyxin B10 Gene

Abstract Bombyxin F1 gene, a new bombyxin family gene, has been identified. The F1 gene forms a pair with bombyxin B10 gene with an opposite transcriptional orientation and the gene pair F1/B10 is located between bombyxin gene pairs B9/C1 and A7/B7 in a bombyxin gene cluster. The nucleotide sequence of the F1 gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family-A, family-b, family-C, family-D, and family-E bombyxin genes; the bombyxin F1 gene and preprobombyxin F1 share no more than 62% and 53% sequence identities with other bombyxin members, respectively. Harr-plot analysis indicated that the spacer of the F1/B10 gene pair has low sequence similarity with that of other bombyxin gene pairs characterized. The bombyxin F1 mRNA in Bombyx mori brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs. Genomic Southern hybridization indicated that the Bombyx haploid genome contains a single copy of the family-F bombyxin gene.

Structure and expression of bombyxin E1 gene: a novel family gene that encodes bombyxin-IV, an insect insulin-related neurosecretory peptide.

A bombyxin gene encoding precursor molecule for bombyxin-IV, one of the insulin-related neurosecretory peptide of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family A, B, C and D bombyxin genes. The gene encoding the bombyxin-IV precursor was therefore defined into a novel family E and designated as gene E1. The bombyxin E1 transcript in Bombyx brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs, and the amount of the E1 transcript did not change markedly during the fifth larval instar. Genomic Southern hybridization indicated that the Bombyx haploid genome contained a single copy of the bombyxin family E gene.