Takeshi Kawarabata

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.

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Abstract Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 108 spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.

Highly infectious free virions in the hemolymph of the silkworm (Bombyx mori) infected with a nuclear polyhedrosis virus

Abstract Density gradient centrifugation and electron microscope studies on the free virions of a nuclear polyhedrosis virus in the infected hemolymph of the silkworm showed that the free virus particle was a virion without an envelope. Only the nucleocapsid was observed in the highly infectious fraction of the infected hemolymph. The unenveloped virion in the hemolymph was nearly a hundred times more infectious than the enveloped virion, liberated from the polyhedra, when hemocoelic inoculation was employed. It was estimated that more than 80% of the infectivitiy in the infected hemolymph was due to the unenveloped virion.

Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori.

Abstract Comparative infectivity and virus neutralization studies on occluded and nonoccluded viruses of Bombyx mori nuclear polyhedrosis revealed that the infectious unit causing peroral infection differed from that causing hemocoelic infection. There were functional differences between the occluded (mainly virons with envelopes) and the nonoccluded virus (mainly virions without envelopes) preparations. The peroral infection was largely due to the virion with an envelope (peroral infectious unit), and the hemocoelic infection was due largely to the virion without an envelope (hemocoelic infectious unit). The apparent change of the virions with envelope to those without envelopes was detected as a slight increase in hemocoelic infectivity when the occluded virus was diluted and incubated at 4°C for more than 6 days.

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua

SummaryHemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14±0.45 μm and 2.84±0.14 μm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10±0.21 μm ×2.00±0.11 μm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.

Coinfection of Spodoptera exigua and Spodoptera frugiperda cell lines with the nuclear polyhedrosis viruses of Autographa californica and Spodoptera exigua

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a broad host range among Lepidoptera. In contrast, the Spodoptera exigua MNPV (SeMNPV) can replicate efficiently only in S. exigua larvae or S. exigua-derived cell lines. In this study, we examined the coinfection of S. exigua Se301 and Spodoptera frugiperda IPLB-SF21AEII (Sf21) cell lines with SeMNPV and AcMNPV recombinant (Ac360-501β-gal) which was constructed for expression of β-galactosidase under control of the polyhedrin promoter. Coinfection led to the restriction as the level of late gene expression, nonoccluded virus production, and DNA replication of Ac360-501β-gal in both Se301 and Sf21 cell lines. In contrast, Ac360-501β-gal supported the SeMNPV replication in Sf21 cells. Occurrence of recombinants, between Ac360-501β-gal and SeMNPV, with expanded host range was not observed in coinfected Sf21 cells. This suggests that Ac360-501β-gal supports the SeMNPV replication through trans-activation.

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Abstract Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.

Spherical Parasporal Inclusions of the Lepidoptera-Specific and Coleoptera-Specific Bacillus thuringiensis Strains: A Comparative Electron Microscopic Study

Abstract. Four Lepidoptera-specific Bacillus thuringiensis strains that belong to the four H serogroups (serovars sumiyoshiensis, fukuokaensis, darmstadiensis, and japonensis) and a Coleoptera (Scarabaeidae)-specific strain belonging to serovar japonensis were examined for comparative ultrastructure of spherical parasporal inclusions. The prominent feature of the inclusions of the Lepidoptera-specific strains was the existence of thick, highly electron-dense envelopes surrounding a homogeneous protein matrix. The envelopes were 15.0–66.7 nm thick and consisted of 5–12 layers of membrane. This is also the case with inclusions of a Coleoptera-specific strain. The ultrastructure of inclusions from the five strains was in marked contrast to that of the bipyramidal parasporal inclusions produced by a Lepidoptera-specific serovar sotto strain.

Identification of Insertion and Deletion Genes in Autographa californica Nucleopolyhedrovirus Variants Isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-Nigrum

The genomic DNA of four Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum was analyzed in comparison with the AcMNPV E2 strain. Restriction endonuclease analysis revealed a deletion and an insertion in collinear regions of the four variants. Polymerase chain reaction analysis indicated that, in the four variants, the deletion occurred in the region corresponding to AcMNPV C6 ORF86 (pnk/pnl). Also the insertion, with a length of approximately 1.1 kb, was commonly identified in the fragments corresponding to the PstI-J fragment (18.5 m.u.–21.2 m.u.) of AcMNPV E2. Sequencing analysis of the variant from S. litura showed that the insertion contains an additional open reading frame encoding 322 amino acids between homologues of AcMNPV ORF30 and ORF31 (the superoxide dismutase gene). This ORF has 82.8% amino acid identity to Bombyx mori NPV T3 ORF 22 (bro-a, one of the baculovirus repeated ORFs) and thus, it was named Splt-bro-a. Southern blot hybridization study indicated that the other three variants also contain Splt-bro-a homologue. In addition, the labeled Splt-bro-a gene weakly hybridized to the PstI-D fragment (99.0 m.u.–8.0 m.u.) of AcMNPV E2. This fragment contains AcMNPV ORF2, a member of bro family. The signal was also observed on the corresponding fragment of the four variants. This result suggested that two bro genes are present in the four variants, although AcMNPV E2 and C6 are known to contain a single bro gene.

Cloning of a microsporidian, Nosema bombycis (Microsporida ; Nosematidae) in insect cell cultures by a limiting dilution method

Several clones of Nosema bombycis NS 001 were established using a limiting dilution of Antheraea eucalypti cell cultures inoculated with sporoplasms emerged from N. bombycis spores. The infected A. eucalypti cell cultures were diluted and transferred to 96-well plastic microplates to isolate a single infected cell in the well. Certain numbers of wells containing the single A. eucalypti cell were randomly selected, because infection of the individual cells isolated in each well was not possible to determine by phase-contrast microscopy at this time. These wells were provided with either uninfected Bombyx mori S. P. C. Bm36 or A. eucalypti cells to produce clones of N. bombycis NIS 001 from presumably a single sporoplasm parasitized in the single cell. Spores of morphological diversity were observed among the clones of N. bombycis NIS 001 isolated by this method. Generally, the characteristics of spore morphology were maintained consistently after several passages of the clones in vitro. Results of the latex adhesion test indicated that the spore surface antigens of seven clones were homologous.