Effects of Radiation From Contaminated Soil and Moss in f*ckushima on Embryogenesis and Egg Hatching of the Aphid Prociphilus oriens (2024)

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Abstract

Much attention has been directed to the extent to which radioactive substances emitted widely from the f*ckushima Daiichi Nuclear Power Plant (NPP) have affected the survival, reproduction, and abundance of wild organisms. The results of irradiation experiments conducted to date have predicted that the radioactivity measured in most areas of f*ckushima (a few to tens µSv/h) is not sufficiently high to adversely affect the development, reproduction, and survival of insects and other arthropods (United Nations Scientific Committee on the Effects of Atomic Radiation 1996). Nevertheless, an increasing number of documents have revealed a significant increase in morphological abnormalities and a rapid reduction in the abundance of wild organisms, probably because of the radioactive substances scattered from the f*ckushima Daiichi NPP (Møller et al. 2012; Hiyama et al. 2012, 2015; Akimoto 2014; Murase et al. 2015; Watanabe et al. 2015; Horiguchi et al. 2016, Otaki and Taira 2017). Studies on a lycaenid butterfly (Hiyama et al. 2012, 2015) and an eriosomatine aphid (Akimoto 2014) indicate that morphological abnormalities were frequently detected just after f*ckushima Daiichi accident, but that the proportion of abnormalities declined rapidly in just a few years. When rice plants in pots were experimentally placed under low-dose radiation (4 µSv/h) in Iitate Village, f*ckushima, for only 3 days, gene expression profiling revealed that the rice rapidly activated genes involved in DNA repair, antioxidant defense, photosynthesis, secondary metabolism, and cell death in the leaves (Hayashi et al. 2014). This report implies that plants could sense ultra-low dose of radiation and could enhance defensive abilities against radiation.

Around the f*ckushima Daiichi NPP, radioactivity in the air has been consistently decreasing since the accident (Hiyama et al. 2015); however, highly radioactive contaminated soil is still widely and commonly found in f*ckushima (Endo et al. 2012, 2014, 2015). If radioactive substances adsorbed in the soil adversely affect the diversity and abundance of soil fauna, then radioactivity could also disturb nutrient cycling within ecosystems through the reduced density of soil arthropods, which function to decompose leaf litter. However, no studies have evaluated the influence of soil contamination on soil arthropods. Mousseau et al. (2014) reported that in areas with high radioactivity around the Chernobyl NPP, as air radiation became higher, leaf litter decomposed more slowly, and the litter layer became thicker. Although they did not evaluate the abundance of soil arthropods, if accumulated radioactive substances have detrimental effects on soil arthropods, nutrient recycling could be hindered, leading to a long-term decline in forests.

In the present study, we determined whether eggs of an eriosomatine aphid species successfully hatched when experimentally placed in contaminated soil and moss sampled from f*ckushima to evaluate the effects of radiation on the embryogenesis of small-sized insects. Although aphid eggs are usually deposited on the stems or branches of the host plants and not on the soil surface, they successfully hatch if humidity on the soil surface is appropriate and if no predatory insects are present in the soil. Eggs of an eriosomatine aphid were used for the experiments primarily because morphological abnormalities were found in the eriosomatine aphid Tetraneura sorini collected in Kawamata Town, f*ckushima, just after their first sexual reproduction following the f*ckushima Daiichi accident (Akimoto 2014). The second reason is that a large number of eggs are readily available for experiments, and that information is available regarding genetic and environmental factors influencing hatching time of aphid eggs (Akimoto 2006). The hatching time distribution of aphid eggs varies substantially in response to genetic or environmental differences. For example, in a Tetraneura species, larger eggs hatch earlier under the same temperature conditions, than do smaller eggs (Akimoto 1998). In Prociphilus oriens, eggs derived from within-clone mating (selfing) hatch later with lower hatchability than do eggs derived from outbreeding (Akimoto 2006). In the galling aphid Kaltenbachiella japonica, genetic differentiation in hatching time was detected among populations on individual host trees because of local adaptation to the phenologies of their respective host trees (Komatsu and Akimoto 1995). Thus, we postulated that if radiation has harmful effects on embryogenesis, aphid eggs subjected to radiation should have lower hatchability and delayed hatching similar to the effects of hom*ozygosity of deleterious recessive genes. Provided that rapidly developing embryos are readily affected by radiation (Russell and Russell 1952; Vereecke and Pelerents 1969; Cerutti 1974), aphid eggs at diapause and hatching could be used as a bioindicator for understanding the effects of radiation.

Another purpose of this study was to connect the exposure dose with the developmental response of aphid eggs. In most studies that have reported morphological abnormalities in organisms in f*ckushima, the dose of radiation to which the organisms were exposed has not been reported. In the present study, we evaluated the total exposure dose during diapause and embryogenesis and elucidated aphid response to radiation.

Materials and Methods

Contaminated Soil and Moss

Approximately 100 g of soil was sampled on 19 October 2015 by one of us (K.I.) from the surface of the ground just beneath the downspout of a house in Techiro, Akaugi, Namie Town, f*ckushima Prefecture, which belonged to the “Difficult-to-Return Zone” specified by the Japanese Government in the evacuation instructions. The soil was clayey with sand, including little humus. The moss Polytrichum sp. was also collected in the vicinity and in Mizusakai-Pass on the same day. During experiments, we were not able to find any animals in the soil and moss. Permission for entering this zone for official activities was granted to K.I. by Namie Town in 2015. When the soil and moss were sampled, the radiation dose at 1 m high was 35 and 3– 6 µSV/h, respectively.

Commercially available humus (Meikoh, Ebetsu) was used as the control for the contaminated soil treatment. The humus was made in Hokkaido before the f*ckushima Daiichi accident and had been maintained in a plastic bag. The moss Polytrichum sp. was collected in Iwamizawa, Hokkaido in mid-November 2015 and was used as the control for the contaminated moss experiment. This region was not contaminated by the f*ckushima Daiichi accident (Hokkaido Government 2016). Total Cs-137 radioactivity in contaminated soil, moss, and controls was measured with Ge detectors (Canberra GX3018) at the Research Reactor Institute, Kyoto University, Kumatori, Osaka.

After all experiments were completed, all the soil and moss used for experiments were transported to f*ckushima in mid-June 2016 and returned to the exact places from which they were collected.

Collection of Aphid Eggs

Eggs of P. oriens were collected from the trunks of the host plant Fraxinus mandshurica on 7 November 2015 in Iwamizawa, Hokkaido, northern Japan, and used as a test organism for the exposure experiments. Prociphilus oriens is a common eriosomatine aphid whose winged females are abundant in autumn in Hokkaido. Winged females leave their secondary host, Abies sachalinensis, and alight on the trunks of F. mandshurica to give birth to males and sexual females. After copulation, sexual females deposit eggs in bark crevices on the trunks. To effectively collect eggs, we placed paper gauze (Haize gauze, Asahikasei^®) around branches of F. mandshurica before the appearance of winged females. Winged females aggregated on the paper gauze after landing on the tree and deposited sexual individuals. Finally, a number of eggs were deposited on the undersurface of the paper gauze, which was collected from the branches and brought to the laboratory on 7 November. All eggs used for the experiments were collected from a few F. mandshurica trees growing near each other in Iwamizawa, Hokkaido (43°11′N, 141°46′E). In the wild, eggs overwinter for approximately 5 months from mid-November (the oviposition time) to late April of the next year (the hatching time). The egg is oval, measuring 0.91 mm in the major axis and 0.36 mm in the minor axis. Because of its small size, the egg is probably affected by β-rays in addition to γ-rays.

Experimental Design

Collected eggs were preserved at 5 °C in a refrigerator and then transferred to small plastic containers (30 × 30 × 10 mm) that were lined with 2 sheets of dampened filter paper. We placed 50 eggs, using a fine writing brush, in each container, which was maintained at 5 °C until eggs were transferred onto the soil or moss. Contaminated soil sampled from f*ckushima was divided and placed into 10 cylindrical styrol containers (50 mm in the inner diameter and 30 mm high, with a screw-on lid), which were randomly allocated to 2 experiments: 4-month and 3-month experiments each with 5 replicates. Each container contained on average 9.8 g of contaminated soil. Five replicates were prepared to serve as controls for the 4-month and 3-month experiments. In the controls, uncontaminated humus of ca. 10 g from Hokkaido was placed in the container.

Moss collected from f*ckushima was divided into 10 containers of the same type (on average 2.57 g), whereas control moss was divided into 7 containers. All the containers were maintained for 4 months during winter.

In the soil experiments, 100 eggs were transferred from 2 plastic containers onto the soil surface of each container by using a fine writing brush on 27 November for the 4-month experiment and its control, whereas eggs were transferred on 28 December for the 3-month experiment and its control. Thus, 500 eggs were used for the radiation and control treatments for each of the 4-month and 3-month experiments. In the moss experiments, 50 eggs were transferred to each of the containers with contaminated and control moss. Thus, a total of 500 eggs were supplied for the contaminated moss treatment and 350 eggs for the control moss treatment.

Immediately after the eggs were placed on the surface of soil or moss, containers were transferred to a refrigerator maintained at –0.5 °C (–6 to –4 °C) and kept intact from 27 November to 29 March (for the 4-month treatment) or from 28 December to 29 March (for the 3-month treatment) at the Research Reactor Institute, Kyoto University. For the 4-month treatment, glass dosimeters (GD-302M, Chiyoda Technol), 12 mm long, were placed on the surface of the soil or moss in some containers to evaluate the accumulated exposure dose to the overwintering eggs. The glass dosimeter was enveloped in a cylindrical holder made from ABS resin. During cooling in the refrigerator, control soil and moss containers were shielded from contaminated soil and moss by lead blocks. To separately evaluate the γ- and β-ray dose radiating from the contaminated soil or moss, some glass dosimeters were wrapped with cellophane film, whereas others were wrapped with aluminum tape (0.2 mm thick) 3 times to shield β-rays.

Estimation of Accumulated Exposure Dose

The β-ray exposure dose can be estimated by subtracting the dose value of a dosimeter wrapped with aluminum tape from that of a dosimeter wrapped with cellophane film if there is no other shielding effect. However, this estimation of the β-ray dose is conservative because the plastic holder (52.5 mg/cm² ABS resin) of the glass dosimeter has an additional shielding effect on the β-rays. To correct the calculated β-ray dose, we evaluated the shielding effect of the plastic holder by determining the relationship between the thickness of a polypropylene sheet and the dose of radiation (CPM) transmitted through the polypropylene sheet using a scintillation survey meter (ALOKA TCS-352). The β-ray transit dose for a 52.5 mg/cm² polypropylene sheet and the β-ray dose without the plastic holder were estimated, and the total exposure dose to the eggs was calculated (Supplementary Figure 1).

Egg Hatching

After the 4- or 3-month cooling of the eggs on the soil and moss, all the containers were simultaneously transferred to 4 °C for acclimation on 29 March. Incubation of all eggs began on 1 April under an alternating temperature regime (19 °C for 8 h and 6 °C for 16 h) in the dark in a chamber (Sanyo, MIR-254). Preliminary experiments showed that alternating temperatures were a prerequisite for successful hatching of aphid eggs (Akimoto and Narita 2002; Akimoto 2006). Eggs were observed daily under a binocular microscope at 14:00, and hatched larvae were counted and collected into vials. After hatching ended, we recorded the proportion of eggs that hatched successfully (hatchability) from each container. However, in some moss experiments, the high humidity from the living moss caused high mortality of eggs (in container A7 and M3, one and zero larva hatched; Supplementary Table 1). Thus, hatchability was compared only in the soil experiments. Hatch date distribution was compared between the radiation treatment and the control in both the soil and moss experiments. Hatched larvae were slide-mounted, and hind femur length was measured as an index of body size (101 larvae from the 4-month contaminated soil experiment and 83 from the control were measured).

Statistics

The proportion of eggs that hatched successfully was analyzed using the generalized linear mixed model, in which variation among containers was treated as a random effect and the radiation/control variable was treated as a fixed effect. The analysis was conducted using the glmer function in package “lme4” in R version 3.2.1 (R Core Team 2015) with a binomial error structure. Differences in hatch dates were tested with a nested Anova using JMP ver.9.0.2. (SAS Institute Inc., Cary, NC); in the statistical model, containers were treated as a random effect and nested in the radiation/control variable.

Results

Radiation Dose in Soil and Moss

The Cs-137 concentration in contaminated soil ranged from 2140 to 3300 Bq/g (for 5 containers), whereas that in contaminated moss ranged from 64 to 105 Bq/g (for 2 containers). The Cs-137 concentration in control soil was 0.01 Bq/g.

The total exposure dose to the eggs for the 4-month cooling period was estimated to range from 52.2 to 104.7 mGy when they were placed on the soil from f*ckushima, and from 3.7 to 9.9 mGy when they were placed on moss from f*ckushima (Table 1). On the control soil and moss, the total exposure dose ranged from 0.2 to 0.4 mGy. The difference in exposure dose between the dosimeter wrapped with cellophane film and that shielded by aluminum tape in R1 and R7 container indicated that the contribution of β-rays was 42.0 and 20.4 mGy, respectively (Table 1). However, these values are underestimates because the plastic holder of the glass dosimeter had a shielding effect on β-rays. Measurements of the β-ray dose transmitted through a polypropylene sheet indicated that the total estimated exposure dose to the eggs was 206 mGy (in R1) or 103 mGy (in R7) in the 4-month soil treatment, with the contribution of β-rays and γ-rays being approximately 2:1 (Supplementary Figure 1).

Response of Aphid Eggs to Radiation

The first hatching occurred on 5 April and hatching continued until 22 April for all experiments. Egg hatchability varied from 61% to 97% among the containers. In a container (CR1 in the 4-month soil experiment), probably because of fungal attack, the hatchability was reduced (61%). The generalized linear mixed model indicated no significant differences in hatchability between the radiation treatment and the control in both the 4-month soil experiment (z = −0.399, P = 0.69) and the 3-month soil experiment (z = –1.141, P = 0.25; Table 2). The among-container variation in hatchability may be due to differences in humidity and fungal growth among the containers.

No morphological abnormalities were found in any of the radiation treatments. There was no significant difference in hind femur length between larvae from the 4-month contaminated soil treatment and the control (Anova, degree of freedom [df] = 1,182, F = 1.00, P = 0.317).

Hatched larvae were collected on the day hatching occurred in the soil experiments, and larvae were found on the soil surface or the inner wall of the container. However, in the moss experiments, because the eggs were usually hidden under the moss, hatched larvae were often not detected on the hatch date, but collected when they climbed up to the top of moss stems. For this reason, there was a slight difference in hatching dates between the soil and moss experiments.

Egg hatching dates varied largely among the containers; even in controls, variation among the containers was significant (Anova, 4-month soil experiment, df = 4,397, F = 27.28, P < 0.0001; 3-month soil experiment, df = 4,417, F = 2.50, P = 0.042). When all containers were pooled, egg hatching in the radiation treatment was significantly earlier than that in the control in each of the 4-month and 3-month soil experiments, and the 4-month moss experiment (Anova; 4-month soil experiment, df = 1,799, F = 8.14, P = 0.0044, Figure 1A; 3-month soil experiment, df = 1,820, F = 14.93, P = 0.0001, Figure 1B; 4-month moss experiment, df = 1,460, F = 21.08, P < 0.0001, Figure 2). However, when variation among containers in hatching dates was considered as a random effect, the hatching-date difference between the radiation treatment and the control was not significant in the 4-month soil experiment (Nested Anova; df = 1,8.1, F = 0.41, P = 0.54), marginal in the 3-month soil experiment (df = 1,7.8, F = 5.32, P = 0.051), and significant in the 4-month moss experiment (df = 1,8.9, F = 6.89, P = 0.028). There was no significant difference in hatching dates between the controls of the 4-month and 3-month soil experiments (Nested Anova; df = 1,8.03, F = 0.0022, P = 0.96). The results of the incubation experiments are tabulated in Table 3.

Egg hatching time may have been affected by environmental harshness, which could be represented by egg hatchability. A statistical model including radiation (absent/present), period (4-month/3-month), hatchability, and its interactions indicated that in the soil experiments, hatching dates were significantly affected by the interaction between radiation and hatchability (Table 4). This result suggests that environmental conditions in the containers affected hatching dates differentially between the radiation and control treatments. When the containers with a hatchability of more than 80% were used (6 containers from each of the radiation treatment and the control), hatching time in the radiation treatment was significantly earlier than in the control (Nested Anova, radiation, df = 1,8.98, F = 7.49, P = 0.023), and the difference between the 4-month and 3-month experiments was not significant (Nested Anova, period, df = 1,8.97, F = 1.13, P = 0.315). In contrast, when the containers with a hatchability of less than 80% were used, no significant difference was detected between the radiation treatment and the control (radiation, df = 1,5.02, F = 0.55, P = 0.493), or between the 4-month and 3-month experiments (period, df = 1,5.02, F = 0.01, P = 0.922).

Discussion

A survey conducted in 2012 indicated that morphological abnormalities occurred frequently (13.4%) in first instars of the eriosomatine aphid T. sorini at a contaminated area in f*ckushima (Akimoto 2014). Morphological abnormalities were postulated to occur because eggs were irradiated directly from radioactive particles that were accumulated on the surface of the host trees. The present study primarily attempted to reproduce morphological abnormalities using radiation from contaminated soil or moss. Secondly, we attempted to evaluate the relationship between the total exposure dose of test insects and their developmental responses because information regarding exposure dose to wild organisms in f*ckushima is lacking.

In the present study, the eggs were subjected to low-dose radiation on the contaminated soil or moss for most of diapause and embryogenesis. On contaminated soil, the cumulative exposure dose was estimated to be ca. 100–200 mGy, of which the contribution of β-rays was approximately twice that of γ-rays. Endo et al. (2014) estimated that the ratio of β-ray to γ-ray dose rate was 2:1 on the surface of contaminated soil in f*ckushima more than 200 days after the f*ckushima Daiichi accident. Thus, the estimations of β- and γ-ray doses to the eggs in our study closely agreed with the results of Endo et al. (2014) for contaminated soil in f*ckushima. In contrast, for the eggs on the contaminated moss, there were only slight differences between the dose values of 2 types of glass dosimeter (–1.1 to 0.9 mGy), suggesting that the contribution of β-rays was negligible. Therefore, we estimated that the eggs on the contaminated moss were exposed to a cumulative γ-ray dose of 4–10 mGy. The effects of these radiation doses were too small to lead to a reduction in hatchability or an increase in morphological abnormalities. Nevertheless, we detected slight differences in hatching time between the radiation treatments and the controls.

Although P. oriens eggs were collected from a few trees at a locality and well randomized when transferred to containers, we detected significantly large variation in hatching dates among the containers in the control. This result implies that the among-container differences in hatching dates resulted from differences in ambient conditions among the containers, not from genetic differences among eggs. In contrast, when 20 P. oriens eggs were transferred to 22 containers lined with dampened filter paper, we did not detect significant among-container differences in hatching dates (Akimoto unpublished data). Thus, uncontrolled effects of the soil surfaces, differences in humidity and fungal or bacterial growth, may have affected hatching dates even in the controls. Therefore, to detect the effects of radiation, it is needed to remove the among-container differences in the background.

In P. oriens, deleterious recessive genes function to delay egg hatching (Akimoto 2006). Therefore, we predicted delayed hatching and reduced hatchability for P. oriens eggs subjected to radiation. However, this prediction was negated, and the eggs in the radiation treatments tended to hatch earlier than those in the controls. In the soil experiments, the effects of radiation were small enough to be hidden in the effects of environmental harshness in the containers. Thus, we were able to detect the effect of radiation on egg hatching by focusing only on the containers where high hatchability was recorded. In containers with high hatchability, radiation led to earlier hatching than in the control. Mortality of eggs during incubation may have induced a bias in hatching dates. If fungal attacks increase with time during incubation, eggs that have the trait of late hatching are more likely to be killed by fungi. Thus, in containers with high mortality, only eggs hatching early may have survived. This bias may have resulted in difficulty in detecting the effect of radiation. Because of limited information on the organisms’ response to low-dose radiation, it is difficult to explain this result in terms of a physiological or adaptive aspect. Nevertheless, it should be emphasized that aphid eggs can respond to such a low dose as a few mGy by advancing embryogenesis.

To date, a γ-ray dose ranging from dozens to hundreds of Gy has been experimentally irradiated on insects to examine their physiological and reproductive responses. These experiments have demonstrated that insects are highly tolerant to radiation (Cole et al. 1959; Elbadry 1965; Elvin et al. 1966; Burgess and Bennett 1971; Burditt et al. 1989). The LD-50 of γ-radiation ranged from 1.3 Gy for house fly eggs to 1900 Gy for body louse nymphs and Pharaoh ant queens (Cole et al. 1959). Therefore, our results are not contradictory to the results of previous studies; we were not able to detect morphological abnormalities or increased mortality with a dose less than 200 mGy. This observation poses a question of why high rates of morphological abnormalities and mortality were observed for T. sorini in 2012. One reason may be that the initial exposure dose was tremendously high, with the effects of short-lived radionuclides, I-131 and Te-132, which have much attenuated. Another reason may be differences between species in radiation resistivity. Prociphilus oriens eggs are larger than T. sorini eggs, so that the difference in egg size may be related to differences in radiation resistivity.

There is limited information on the effect of low-dose radiation on insects. However, an irradiation experiment demonstrated that low-dose radiation on Drosophila melanogaster led to a “hormetic” effect on longevity and developmental rates (Shameer et al. 2015). When male D. melanogaster was irradiated with various doses of γ-radiation, the male and the F1 offspring had deleterious effects if exposure dose was high (from 40 to 50 Gy); that is, the lifespan of the male was shortened, and the developmental time of the offspring was increased. In contrast, if males were irradiated by a dose of 1 or 2 Gy, their lifespan was significantly increased, and the “egg to adult” developmental time of the F1 offspring was shortened. At medium radiation doses ranging from 4 to 10 Gy, no significant difference from the control was detected in male lifespan and offspring developmental time. Whether these “hormetic” effects lead to fitness benefits for males could not be determined until the effects of low-dose radiation on other fitness components are fully evaluated. However, this experimental result is similar to our results in that low-dose radiation led to a shortening of hatching time in aphid eggs. Irradiation experiments using ultra-low-dose γ-rays have not been attempted in animals, although the physiological responses of rice to an air dose rate of 4 µSv/h were evaluated using gene expression analysis (Hayashi et al. 2014). Even under this ultra-low-dose radiation, genes governing DNA repair and antioxidant defense were reported to be intensely activated as a defense against radiation.

Because the effects of low-dose radiation on small animals have not been evaluated, our results may be valuable for predicting the effects of contaminated soil in f*ckushima on soil-dwelling insects and other arthropods. The results of the present study suggest that the current level of soil contamination (3 million Bq/kg) may not have a serious effect on small animals in the soil. However, the present study evaluated radiation effects on embryogenesis for 3 and 4 months only. Therefore, examination of the effects of radiation on animals reproducing over several generations in contaminated soil is required.

Supplementary Material

Supplementary data are available at Journal of Heredity online.

Funding

This work was supported by Grants-in-Aid (grant numbers 17370028, 23370037) for Scientific Research from the Japan Society for the Promotion of Science to S.A.; Grants-in-Aid (grant number 15H01850 to T.I.).

Acknowledgments

We thank Namie Town for formally permitting us to enter “Difficult-to-Return Zone” for official activities.

Data Availability

We have deposited the primary data used for the analysis of egg hatch as follows: Egg hatch date in 3 treatments: Dryad.

References

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