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Using heavy-ion microbeam, we report target irradiation of selected compartments within the diapause-terminated egg and its mutational consequences in the silkworm, Bombyx mori. On one hand, carbon-ion exposure of embryo to 0.5-6 Gy increased the somatic mutation frequency, suggesting targeted radiation effects. On the other, such increases were not observed when yolk was targeted, suggesting a lack of nontargeted bystander effect.

To carry out the radio-microsurgery study using silkworm, Bombyx mori, we have already developed the specific irradiation systems for eggs and third to fifth instar larvae. In this study, a modified application consisting of the first instar silkworm larvae was further developed using heavy-ion microbeams. This system includes aluminum plates with holes specially designed to fix the first instar silkworm larvae during irradiation, and Mylar films were used to adjust energy deposited for planning radiation doses at certain depth. Using this system, the suppression of abnormal proliferation of epidermal cells in the knob mutant was examined. Following target irradiation of the knob-forming region at the first instar stage with 180-mum-diameter microbeam of 220 MeV carbon (12C) ions, larvae were reared to evaluate the effects of irradiation. The results indicated that the knob formation at the irradiated segment was specially suppressed in 5.9, 56.4, 66.7 and 73.6% of larvae irradiated with 120, 250, 400 and 600 Gy, respectively, but the other knob formations at the non-irradiated segments were not suppressed in either irradiation. Although some larva did not survive undesired non-targeted exposure, our present results indicate that this method would be useful to investigate the irradiation effect on a long developmental period of time. Moreover, our system could also be applied to other species by targeting tissues, or organs during development and metamorphosis in insect and animals.

In order to investigate whether eggs of the black-striped strain (P(S)) of the silkworm, Bombyx mori, represent an appropriate model for estimating the biological effect of cosmic radiation, radiosensitivity of the eggs against X-rays and heavy ion particles was examined as ground-based experiments. The exposure of diapause eggs to X-rays or heavy ion particles resulted in somatic mutations appearing as a white spot on the black integument during larval stage. Irradiation of non-diapause eggs with X-rays demonstrated a significant difference in frequency of the mutation between fractionated and single administration doses, but no difference was observed in diapause eggs. Incidence of the mutation as induced by carbon ion beams for 15-day old eggs was higher for eggs that had been kept at 15 degrees C than those kept at 25 degrees C. Neon beam irradiation of diapause eggs displayed dose- and linear energy transfer (LET)-dependent effects, causing a maximal rate of the mutation at 150 keV/microm. These results confirm that B. mori eggs represent valid models for estimating the biological effects of cosmic radiation.

To study the effects of heavy-ion radiosurgery on the hemopoietic function of a silkworm, hemopoietic organs of larvae were locally irradiated with carbon-ion beams, and the changes in the hemocyte density and in the hemocyte function were investigated. When the larvae were irradiated by 50 Gy to 300 Gy carbon ions on the 3rd day of the 4th instar, the hemocyte densities did not change for a while, though they gradually increased at a later stage, but were finally still significantly lower than those of unirradiated controls. The hemocyte densities of the larvae irradiated at different developmental stages showed suppressed increments, and carbon-ion irradiation given to larvae at early stages compared to the later stages had a significant suppressive effect on the hemocyte densities. On unilateral irradiated larvae a hemocyte intermediate increment between those of bilateral irradiated larvae and unirradiated controls was observed. The percentage of dead hemocytes was obviously higher for irradiated larvae than unirradiated controls during the later 5th instar. Thus, it is evident that carbon-ion radiosurgery on hemopoietic organs of silkworm induced not only a quantitative change, but also a qualitative change in the hemocytes.