Ren Ishihara

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.

The life cycle of Nosema bombycis as revealed in tissue culture cells of Bombyx mori

Abstract Nosema bombycis spores inoculated into cultures of Bombyx mori cells resulted in infection of the cells. The development of the pathogen through its complete cycle to the formation of new spores was observed. A form having two nuclei, similar to those of the sporoplasm, was seen among the newly formed spores. This form leaves the host cell, migrates to a new cell, which it penetrates and infects, and the growth cycle is repeated. This “secondary infective form” is believed to be responsible for spreading the infection within the host.

Some observations on the fine structure of sporoplasm discharged from spores of a microsporidian, Nosema bombycis☆

Abstract Electron micrographs showing stages in sporoplasm discharge from spores of Nosema bombycis are presented. The released polar filament is tubular and contains metarial, possibly part of the sporoplasm, derived from the spore. The fine structure of sporoplasm discharged into preheated hemolymph of the silkworm, Bombyx mori, or injected into the silk gland of the silkworm, was observed. The sporoplasm had a smooth single membrane, usually two nuclei surrounded by a double membrane, ribosomelike particles, vesicles, a vesicular structure having coiled tubules, a concentric ring structure consisting of two or four rings and containing granules. After the sporoplasm was injected into the host tissue a membrane system was revealed. No typical mitochondria, polyribosomic cluster of ribosomes, or endoplasmic reticulum having rough surface were recognized. Polar filaments were observed piercing into both the wall and cytoplasm of the silk gland.

Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori.

The yellow color of Bombyx mori hemolymph is due to the presence of carotenoids, which are primarily associated with lipophorin particles. Carotenoids were extracted from high density lipophorin (HDLp) of B. mori and analyzed by HPLC. HDLp contained 33 micrograms of carotenoids per mg protein. Over 90% of carotenoids were lutein while alpha-carotene and beta-carotene were minor components. When larval hemolymph was subjected to density gradient ultracentrifugation, a second minor yellow band was present, which was identified as B. mori lipid transfer particle (LTP). During other life stages examined however, this second band was not visible. To determine if coloration of LTP may fluctuate during development, we determined its concentration in hemolymph and compared it to that of lipophorin. Both proteins were present during all life stages and their concentrations gradually increased. The ratio of lipophorin: LTP was 10-15:1 during the fourth and fifth instar larval stages, and 20-30:1 during the pupal and adult stages. Thus, there was no correlation between the yellow color attributed to LTP and its hemolymph concentration. It is possible that yellow coloration of the LTP fraction corresponds to developmental stages when the particle is active in carotene transport. To determine if LTP is capable of facilitating carotene transfer, we took advantage of a white hemolymph B. mori strain which, when fed artificial diet containing a low carotene content, gives rise to a lipophorin that is nearly colorless. A spectrophotometric, carotene specific, transfer assay was developed which employed wild type, carotene-rich HDLp as donor particle and colorless low density lipophorin, derived from the white hemolymph strain animals, as acceptor particle. In incubations lacking LTP carotenes remained associated with HDLp while inclusion of LTP induced a redistribution of carotenes between the donor and acceptor in a time and concentration dependent manner. Time course studies suggested the rate of LTP-mediated carotene transfer was relatively slow, requiring up to 4 h to reach equilibrium. By contrast, studies employing 3H-diacylglycerol labeled HDLp as donor particle in lipid transfer assays revealed a rapid equilibration of label between the particles. Thus, it is plausible that the slower rate of LTP-mediated carotene transfer is due to its probable sequestration in the core of HDLp.

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Abstract Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.

cDNA and deduced amino acid sequences of apolipophorin‐IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.

The spread of pebrine within a colony of the silkworm, Bombyx mori (Linnaeus).

Abstract The epizootic pattern of pebrine within a colony of the silkworm, Bombyx mori (Linnaeus), was investigated. The rate of infection was high in the first, the second, and the fifth instars, and low in the middle instars. This pattern corresponded to the change in the number of larvae excreting spores of Nosema bombycis Naegeli.