| Abstract |
1. An egg-mass of tortricids at the black-headed stage was sterilized by
dipping in 70% ethanol for 1 minute and subsequently in a 0.1% aqueous
solution of mercury chloride for 20 minutes, and then rinsed with sterile
distilled water and wiped with filter paper under aseptic conditions.
Thereafter, the egg-mass was put into a screw-capped test tube containing
0.5ml of medium. The tube was inclined with light reaching the bottom part.
After 2 or 3 days incubation at 25 Celsius, the hatched larvae were
transferred together with the medium into a watch glass, and cut under
aseptic conditions. The explants thus obtained were transferred into
plastic culture flasks (Falcon 3013) together with 4ml of IPL41S medium
and incubated.
2. Most of the explants underwent degenerative changes with melanization
and collapsed, but the remains survived for several months. From these
materials, free cells and/or hollow vesicles were obtained after several
changes of the medium at 10 to 20 day intervals using one-half of fresh
medium until the cells entered the proliferative stage and almost all the
bottom surface of the culture flask was covered by cells. After a long
initial period of adaptation which ranged from 161 to 346 days depending
on the tortricid species used, 8 cell lines were eventually established.
3. The cell lines were maintained by the addition of one-half of fresh
medium until the 5th to 7th subcultures. Thereafter, the ratio of cell
suspension and fresh medium was shifted to 1:4 to 1:9, as the number of
cells increased in each cell line, at weekly intervals.
4. All of the serum used were evaluated based on the growth curves using
the continuous cell line designated as IPLB-Sf21AE11.
5. The two cell lines derived from Adoxophyes orana fasciata were
designated as FTRS-AoL1 and FTRS-AoL2. The cell lines designated as
FTRS-AfL, FTRS-PhL, FTRS-AbL81 and FTRS-HmL45 were derived from Adoxophyes
sp. (smaller tea tortrix), Pandemis heparana, Archippus breviplicanus and
Homona magnanima, respectively. Both the FTRS-H1L1 and FTRS-H1L2 lines
were derived from Hoshinoa longicellana.
6. In FTRS-AoL1, FTRS-AfL and FTRS-AbL81, spindle-shaped and spherical
cells were predominant, while in FTRS-AoL2, FTRS-PhL and FTRS-HmL45 the
cells were mainly spherical. The diameter of a single cell of these lines
was about 25 mum. In FTRS-H1L1 and FTRS-H1L2, the cells assumed an
epithelium like morphology.
7. Results of semi-quantitative analyses carried out by using an API ZYM
kit, showed that the activity of the enzymes concerned was high in all the
cell lines, except for the activity of beta-glucuronidase and beta-
glucosaminidase. The pattern of activity of beta-glucuronidase in the
FTRS-HmL45 cells was different from that of the other cell lines. The
activity of beta-glucosaminidase, in the cell lines FTRS-AfL, FTRS-PhL,
FTRS-AbL81 and FTRS-H1L2 was distinctly weaker than that of the other lines.
8. Inoculation tests of strains of 2 nuclear polyhedrosis viruses (NPVs)
and an entomopox virus (EPV), namely Plutella NPV (PxNPV), Adoxophyes
NPV (AoNPV) and Adoxophyes EPV (AoEPV) to the cell lines were performed.
In each of the cell lines FTRS-AoL1, FTRS-AoL2, FTRS-AfL, FTRS-PhL and
FTRS-HmL45, only a few cells were infected with PxNPV. About one third of
the cells in FTRS-AoL1 and about 10% of the cells in FTRS-AoL2 were
infected with AoNPV. In FTRS-AfL, only a small percentage of the cells was
infected with this virus. About half of the cells of FTRS-HmL45 were
infected with AoEPV, while the percentages of infected cells were very low
in FTRS-AoL1, FTRS-AoL2, FTRS-AfL, FTRS-PhL and FTRS-AbL81.
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