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. © 1990.