Hidetoshi Iwano

Dimorphism of spores of Nosema spp. in cultured cell

Abstract A Nosema sp. from the lawn grass cutworm, Spodoptera depravata , produced two types of spores in cultured cells of Antheraea eucalypti . One was characterized by 3 to 5 coils of the polar tube, thin endospore (35 nm), early development (36 hr postinoculation), and the spontaneous discharge of a short polar tube. The other type characterized by 10 to 12 coils of the polar tube, thick endospore (91 nm), and late development (54 hr postinoculation) was the predominant spore in later culture and was similar to the purified spores harvested from diseased insects, which discharged a long polar tube. Nosema bombycis similarly produced two types of spores in cultured cell.

Effect of root exudates on growth of newly isolated nitrifying bacteria from barley rhizoplane

Abstract An ammonia-oxidizing bacterium, strain PJA1, and nitrite-oxidizing bacterium, strain PJN1, were newly isolated from the rhizoplane of barley. The cells of strain PJA1 were lobate, compartmentalized, and showed characteristics of the genus Nitrosolobus. The similarity of the 16S rRNA gene to Nitrosolobus multiformis ATCC25196 was 99.04%. The cells of strain PJN1 were rod-shaped. The similarity of the 16S rRNA gene to Nitrobacter agilis ATCC14123 was 98.57%. These newly isolated bacteria were identified as Nitrosolobus sp. PJA1 and Nitrobacter sp. PJN1, respectively. The exudates prepared from barley roots of both the pre-heading and post-heading stages promoted the growth of strain PJA1 (15–20% increase). The growth of the non-rhizoplane strains like Nitrosomonas europaea ATCC25978 was remarkably inhibited by the exudates. The growth of strain PJN1 was also remarkably promoted by the root exudates at the pre-heading stage (6 time increase), and by the root exudates at the post-heading stage (2.5 time increase). The root exudates did not effect the growth of Nitrobacter winogradskyi IFO14297, isolated from the non-rhizoplane.

Purification and expression analysis of imaginal disc growth factor in the silkworm, Bombyx mori.

In the present study, we purified and sequenced a homolog of the Drosophila imaginal disc growth factor (IDGF) from the hemolymph of Bombyx mori (BmIDGF). Antibodies against BmIDGF were produced and subsequently used in immunoblotting analyses. The immunoblotting analyses demonstrated an extremely high level of BmIDGF in the hemolymph throughout the period of rapid growth of the organs of B. mori. The results of RT-PCR showed that BmIDGF was predominantly expressed in fat bodies. Real-time RT-PCR revealed that BmIDGF transcripts in fat bodies were highly expressed during the feeding stage but significantly suppressed during the molting, wandering, and pupal stages. Starvation brought about a significant decline of BmIDGF mRNAs in the fat bodies and BmIDGF proteins in the hemolymph. After re-feeding, the BmIDGF transcripts in fat bodies and BmIDGF proteins in the hemolymph increased again. In addition, an immunocytochemical study revealed BmIDGF proteins on the surface of wing discs. The present findings suggest that the level of BmIDGF in the hemolymph was modulated by the fat body in response to nutritional conditions and that BmIDGF was transported to target organs through the hemolymph.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori.

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

Phylogenetic relationships among Bacillus thuringiensis (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.