Abnormalities in field-caught individuals and their F 1 offspring

We collected 144 first-voltine adults (111 males and 33 females) from 10 localities (Shiroishi, Fukushima, Motomiya, Koriyama, Hirono, Iwaki, Takahagi, Mito, Tsukuba and Tokyo) approximately 2 months after the accident on 13–18 May 2011 (Fig. 1a; Supplementary Table 1). Most of the collected adults appeared morphologically and behaviorally normal, but mild morphological abnormalities were detected in some individuals upon close inspections (Fig. 1b–e; Supplementary Table 2). The overall abnormality rate for 7 localities (excluding Shiroishi, Koriyama and Tokyo to allow comparisons with the second phase of field work in September) was 12.4% (Table 1). The male forewing size (from the base to the apical end) was different among populations (ANOVA, df = 7, F = 4.0, p = 0.00093); it was significantly reduced in the Fukushima population in comparison with the Tsukuba population (t test with pooled SD, p = 0.00091, Holm-corrected by 28 combinatorial pairs, excluding Shiroishi and Koriyama due to small sample sizes) and with the Hirono, Tokyo and Takahagi populations (t test with pooled SD, p = 0.018, 0.018 and 0.038, respectively, Holm-corrected by 28 pairs as above) (Fig. 1c). The male forewing size was negatively correlated with the ground radiation dose at the collection localities (Pearson correlation coefficient r = −0.74, df = 8, p = 0.029, Holm-corrected by 2 pairs [ground radiation dose and distance from the NPP]) (Fig. 1d).

Table 1 Overall abnormality rate (OAR) of adults Full size table

Figure 1 First-voltine collection and abnormalities. (a) Collection localities. A red dot indicates the location of the Fukushima Dai-ichi NPP. Black dots and black half dots indicate the cities from which the first-voltine adults were collected. Brown dots and brown half dots indicate cities from which the host plant leaves were collected for the internal exposure experiment. All experiments were performed in Okinawa, marked by a blue dot. Inset shows the collection localities around the NPP. (b) Representative wings with normal (leftmost) and aberrant colour patterns. Numbers 1, 2, 3 and 4 indicate the first, second, third and fourth spot arrays, respectively and “D” indicates the discal spot. Red arrows indicate loss, dislocation and weak expression of spots (left individual), weak expression and dislocation of spots (middle individual) and enlargement of spots (right individual). These samples were caught in Mito except for the leftmost aberrant specimen, which was caught in Iwaki. Scale bar, 1.0 cm. (c) Male forewing sizes from various localities. The first quartile and third quartile were indicated by horizontal bars at the bottom and top of the box, respectively. Median is indicated as the centre line inside the box. Outliers were indicated by dots. A red dot indicates the mean value and a red bar the standard deviation (SD). Holm-corrected p-values are shown, which were obtained for pairwise comparisons among 8 localities using t tests with pooled SD. Only male samples were used here because when the female samples were used to obtain eggs, broken wings resulted from the egg collection procedure. Samples from Shiroishi (n = 5) and Koriyama (n = 3) were excluded because of small sample sizes. (d) Scatter plot of the male forewing size and ground radiation dose at each collection locality. Pearson correlation coefficient r = −0.74 (Holm-corrected p = 0.029). (e) Representative morphological abnormalities. From left to right, dented eyes (Shiroishi), deformed left eye (Iwaki), deformed right palpus (Takahagi) and deformed wing shape (Fukushima). Arrowheads indicate deformation. Scale bars, 0.50 mm with the exception of the rightmost bar, which is 1.0 cm. Full size image

Based on the established rearing method24 (Supplementary Fig. 1), we obtained F 1 offspring from the female parents caught in the Fukushima area. This and all the following experiments were performed in Okinawa, located 1,750 km from the Fukushima Dai-ichi NPP (see Fig. 1a), where artificial radiation can scarcely be detected. Some of these field-caught parents had mild abnormalities (Supplementary Table 2), but those from Motomiya showed no detectable abnormalities and appeared to be morphologically and behaviorally healthy. In the F 1 generation (Supplementary Tables 3, 4), the mortality rates of larvae, prepupae and pupae and the abnormality rate of adults were high for Iwaki, Hirono, Motomiya and Fukushima and overall abnormality rate of the F 1 adults was 18.3% (Table 1), 1.5 times the overall abnormality rate of the parent generation. The eclosion-time dynamics (Fig. 2a) as well as the pupation-time dynamics (Supplementary Fig. 2a) varied among the F 1 populations from different localities. The eclosion curves of all of the local populations examined differed significantly from the eclosion curve of the Tsukuba population (generalized Wilcoxon test, p < 0.00001 in all population groups, Holm-corrected by 28 pairs). Essentially identical results were obtained in the pupation curve (Supplementary Figure 2a). The half-eclosion time was negatively correlated with the distances of the collection localities from the Fukushima Dai-ichi NPP (r = −0.91, df = 6, p = 0.045, Holm-corrected by 30 pairs [{ground radiation dose and distance from the NPP} versus {abnormality rates of four stages (called “total”), adults, wings, colour patterns, appendages and others; mortality rates of pupae, prepupae and larvae; periods of prepupae and pupae; peak days of eclosion and pupation; half days of eclosion and pupation}]) (Fig. 2b). Similarly, half-pupation time was negatively correlated, but not significant statistically (Supplementary Fig. 2b).

Figure 2 F 1 abnormalities. (a) Eclosion-time dynamics. Cumulative percentages of eclosed individuals were plotted against eclosion day. All local populations differ significantly from the Tsukuba population (generalized Wilcoxon test, Holm-corrected p < 0.00001). (b) Scatter plot of half-eclosion time and distances of the collection localities from the NPP. Half-eclosion time was derived from the eclosion-time dynamics shown in (a) as the time when 50% of the pupae eclosed. Pearson correlation coefficient r = −0.91 for half-eclosion time (Holm-corrected p = 0.045). (c) Scatter plot of abnormality rate of appendages and distances from the NPP. Pearson correlation coefficient r = −0.86 (Holm-corrected p = 0.18). (d) Representative morphological abnormalities of appendages. Miniaturized left foreleg tarsus (Fukushima F 1 , leftmost), undeveloped left middle leg tarsus (Fukushima F 1 and Hirono F 1 , second and third from the left, respectively) and undeveloped palpi (Takahagi F 1 , rightmost) were structurally abnormal, reminiscent of Drosophila Distal-less mutants. Arrowheads indicate abnormal structures. Insets show enlargements of boxed areas. Scale bar, 0.50 mm. (e) Representative morphological abnormalities of eyes. Both compound eyes were dented (Fukushima F 1 , left) and left compound eye was bar-like in shape (Hirono F 1 , right), reminiscent of Drosophila Bar mutants. Scale bar, 0.50 mm. (f) Representative wing size and shape deformation. Right hindwing was much smaller than the left hindwing of the same individual (Fukushima F 1, left), wings were folded (Takahagi F 1, middle) and wings were rumpled (Iwaki F 1 , right). Scale bar, 1.0 cm. (g) Representative wing colour-pattern modifications. The top left three individuals are F 1 individuals from an Iwaki parent and the top rightmost individual is a Hirono F 1 . The bottom samples, from left to right, are F 1 individuals from Hirono, Mito, Shiroishi, Motomiya and Motomiya. Arrows indicate modified spots. Scale bar, 1.0 cm. Full size image

We also observed a negative correlation between the F 1 abnormality rate of appendages and the distances from the NPP, although not significant statistically (r = −0.86, df = 6, p = 0.18, Holm-corrected as above) (Fig. 2c). We detected morphological malformations in various parts (Supplementary Table 4) including legs, antennae, palpi, eyes, abdomen and wings (Fig. 2d–g). In addition to dented compound eyes (Fig. 2e, left), the entire eye structure was deformed in a pattern similar to that of Drosophila Bar mutants (Fig. 2e, right). Wing aberrations, including broken or wrinkled wings, were found in many individuals (Fig. 2f). Asymmetric hindwing size reduction was observed in a few individuals (Fig. 2f, left). Colour-pattern changes were relatively frequent (Fig. 2g). In one individual, the third spot array was located closer to the second array. In another individual, spots were deleted or added, which was occasionally found only in a right or left wing. In another individual, spots were fused together. Additionally, wing-wide spot enlargement was relatively common, especially in individuals from Iwaki. This spot enlargement pattern differs from those observed at the northern range margins and from those induced by temperature shocks24,27. It also differs from those seen in sibling inbreeding24.

It is noteworthy that we obtained relatively high abnormality rates for the F 1 populations from Motomiya, the parents of which showed no detectable abnormal phenotype. Other parents used for egg collection were also comparably normal and vigorous. The abnormal F 1 individuals obtained from healthy parents suggest that genes that are important in morphological development were damaged by radiation at the stage of germ-line cell development in the parents.

Inheritance of abnormalities by the F 2 generation

We tested the fertility of these abnormal F 1 individuals and the inheritance of their abnormal traits. We chose 10 F 1 females with abnormal traits (except one female from Shiroishi, which did not have any detectable abnormal trait) and crossed them with non-abnormal F 1 individuals from Tsukuba. Of the collection localities from which the F 1 offspring were obtained, Tsukuba was the farthest from the Fukushima Dai-ichi NPP (Supplementary Table 4) and therefore chosen as the source of the non-abnormal F 1 adults. To avoid failure due to the unexpected infertility of the males, we put 3 normal virgin males for 1 virgin female in a single cage. Although our mating system is nearly always successful and yields more than 100 offspring per female if both males and females are fertile24, 3 females out of 10 produced only a limited number of offspring, i.e., less than 2 adult offspring (Supplementary Table 5). Nonetheless, we were able to obtain a reasonable number of eggs from other females and successfully reared these offspring to the adult stage.

The F 2 generation showed a relatively high abnormality rate (Fig. 3a; Supplementary Table 6). The overall abnormality rate in the F 2 adults was 33.5% (Table 1). An important finding was that certain traits observed in the F 1 generation were inherited by the F 2 generation (Fig. 3b; Supplementary Table 6). Colour-pattern modifications were relatively frequent (Fig. 3c). Wing-wide spot enlargement was evident especially in the Iwaki F 2 generation as in the Iwaki F 1 generation discussed above. In particular, 52.4% of the Iwaki F 2 females in the strain “Iwaki1” inherited this trait; this inheritance was biased towards females (Supplementary Table 6). Abnormalities of appendages were also relatively frequent (Fig 3d). A striking antenna malformation, or a forked antenna, was observed in a F 2 individual from Takahagi (Fig 3d). This abnormality had never been seen in the F 1 and other individuals that were reared in our laboratory. These results demonstrated that the abnormal traits observed in the F 1 generation were inherited by the F 2 generation and that it is highly probable that these characteristics are caused by genetic damage introduced to the parent germ-line cells, possibly due to the Fukushima Dai-ichi NPP accident.

Figure 3 F 2 abnormalities. (a) Abnormality rate for the F 2 generation. The x-axis shows strain names that indicate the local origin of their P generation. The total number of individuals (corresponding to 100%) was indicated for each strain. See also Supplementary Table 3. (b) Identical and homologous abnormality rates. The number of individuals that show abnormal traits identical to the F 1 parents was divided by the total number of individuals obtained and expressed as a percentage. Similarly, the number of individuals that show abnormal traits in organs, such as wings and appendages, homologous with those in their F 1 parents was divided by the total number of individuals obtained and expressed as a percentage. The total number of abnormal individuals (corresponding to 100%) was indicated for each strain. (c) Representative wing colour-pattern aberrations. Arrows indicate modified spots and wing parts. The top leftmost wings are the wing-wide spot elongation type of the Iwaki F 2 , a phenotype similar to that of its F 1 parent shown in Fig. 2g. The top four samples, from left to right, are Iwaki F 2 , Takahagi F 2 , Iwaki F 2 and Fukushima F 2 individuals. All of the samples at the bottom are Fukushima F 2 individuals. The bottom middle and rightmost wings show a deformation of the hindwing shape, which were obtained from the offspring of the Fukushima F 1 parent that had the small hindwing shown in Fig 2f. Scale bar, 1.0 cm. (d) Antenna and leg malformations. The left panel shows a Takahagi F 2 individual with a malformation of left antenna, which is short and forked (arrowheads). The right panel shows a Takahagi F 2 individual with a deformation of the left hindleg femur. Insets show pictures taken from different angles. Scale bars, 0.50 mm. Full size image

More severe abnormalities 6 months after the accident

To assess the possible genetic and ecological impacts of the Fukushima nuclear accident on the populations of Z. maha, we asked if any abnormalities similar to those found in the F 1 and F 2 generations in the laboratory from the first-voltine females were observed in the field 6 months after the accident. We again collected Z. maha adults from the 7 localities (i.e., Fukushima, Motomiya, Hirono, Iwaki, Takahagi, Mito and Tsukuba; 18–21 September 2011) and from Kobe (3–4 October 2011) (see Fig. 1a). They were probably the fourth- or fifth-voltine individuals. We collected a total of 238 individuals (168 males and 70 females) from these localities (Supplementary Table 1). We observed frequent malformations of legs and antennae as well as wing colour-pattern aberrations (Fig. 4a; Supplementary Table 7). The overall abnormality rate for the 7 localities (excluding Kobe to allow comparisons with the first phase of field work in May) was 28.1%, more than double that observed in the field-collected first-voltine adults in May (Table 1). The total abnormality rate of the field-collected adults in September 2011 was correlated with the ground radiation dose at the collection localities (r = 0.84, df = 6, p = 0.13, Holm-corrected by 14 pairs [{ground radiation dose and distance from the NPP} versus {abnormality rates of adults, wings, colour patterns, appendages and others and wing sizes of males and females}]) (Fig. 4b).

Figure 4 Abnormalities in the adult samples collected in September 2011 and in their F 1 offspring. (a) Representative morphological abnormalities of the field-caught individuals. Insets are enlargement of the boxed areas. The tarsus of the left hindleg was structurally abnormal (Hirono, left), the tarsus of the right foreleg was not developed at all (Fukushima, second from left), the right antenna (an arrowhead) was elongated with abnormal structure and colouration (Motomiya, second from right) and the wing colour-patterns and wing shape were modified as indicated by arrows (Iwaki and Fukushima, right). All scale bars indicate 1.0 mm with the exception of the rightmost bar, which is 1.0 cm. (b) Scatter plot of ground radiation dose and abnormality rate of the field-caught adults. Pearson correlation coefficient r = 0.84 (Holm-corrected p = 0.13). (c) Representative abnormalities in the F 1 generation. The left three panels indicate malformations of left foreleg tarsus (an arrowhead) (Takahagi F 1 , top), tumor-like solid protuberance (arrowheads) in the ventral side of the thorax (Takahagi F 1 , middle) and dented eyes (Fukushima F 1 , bottom). Scale bars in the left three panels all indicate 1.0 mm. Wing colour-pattern modifications (arrows) of the F 1 samples were shown on the right: from left to right, Iwaki, Iwaki, Motomiya, Hirono and Takahagi (top) and Takahagi, Motomiya, Motomiya, Fukushima, Motomiya and Motomiya (bottom). Scale bar in the wing panel indicates 1.0 cm. Full size image

In the F 1 generation from the September samples, the mortality rate and the abnormality rate were relatively high (Supplementary Tables 3, 4) and abnormalities similar to those observed in the field-collected September samples were observed (Fig. 4c). The overall abnormality rate of the F 1 adults was 59.1% (Table 1). These results demonstrated that the September populations in the Fukushima area deteriorated in comparison with the May populations, possibly due to genetic damage caused by radiation from the Fukushima Dai-ichi NPP, as predicted from the results of our previous breeding experiments using the first-voltine adults.

Effects of external and internal exposures

To experimentally reproduce the abnormal phenotypes obtained from the field and in the breeding experiments, we artificially exposed larvae and pupae that were obtained from females caught in Okinawa to radiation from 137Cs, one of the major radionuclides released from the Fukushima Dai-ichi NPP, up to 55 mSv (0.20 mSv/h) or 125 mSv (0.32 mSv/h). In both exposure levels, we observed abnormal traits (Fig. 5a) and forewing size reduction in both sexes in comparison with non-irradiated controls (t test, p < 0.00001 in both sexes) (Fig. 5b). The survival curves indicated dose-dependence; the 55-mSv and 125-mSv curves differed significantly from each other (generalized Wilcoxon test, p = 0.0040, Holm-corrected by 6 pairs). We also observed significant differences between the 55-mSv curve and its control (generalized Wilcoxon test, p = 0.018, Holm-corrected as above) and between 125 mSv curve and its control (generalized Wilcoxon test, p = 0.0000026, Holm-corrected as above). The survival curves also indicated that external exposure caused frequent deaths at the prepupal stage and that the higher dose mainly affected the pre-eclosion and eclosion stages (Fig. 5c).

Figure 5 External and internal exposures. (a) Representative abnormalities obtained by external exposure. Left hindleg tibia and tarsus, antennae, palpi and an eye showed abnormal structures (All exposed at 125 mSv with the exception of the left bottom individual, which was exposed at 55 mSv. All scale bars, 1.0 mm). Aberrant wing colour patterns are indicated by arrows and boxes (Left wings exposed at 55 mSv and right wings at 125 mSv. Scale bar, 1.0 cm). Inset shows the enlarged boxed area. (b) Forewing size reduction in the externally exposed individuals at 55 mSv (t test). (c) Survival curves of individuals that were exposed externally. Differences between the exposed at 55 mSv and its control (Holm-corrected p = 0.018), between the exposed at 125 mSv and its control (Holm-corrected p = 0.0000026) and between the exposed at 55 mSv and at 125 mSv (Holm-corrected p = 0.0040) were statistically significant (generalized Wilcoxon test). (d) Survival curves of individuals that ingested contaminated leaves from different localities. The host plant collection localities are shown. All curves differed from the non-contaminated Ube curve (generalized Wilcoxon test, Holm-corrected p < 0.000001). The Hirono curve was different from the Fukushima curve (Holm-corrected p = 0.0017) and from the Iitate flatland curve (Holm-corrected p = 0.00035) (generalized Wilcoxon test). (e) Scatter plot of the 137Cs activity of the host plant and pupal mortality rate (r = 0.91) and colour-pattern abnormality rate (r = 0.96). (f) Forewing size reduction in the internally exposed individuals (t test). (g) Representative abnormalities of individuals that ingested contaminated leaves. From the top left to the right bottom, the panels show right antenna malformation (Iitate montane region), right palpus abnormality (Fukushima), dented left compound eye (Iitate flatland), eclosion failure (Fukushima), bent wings (Fukushima), additional bent wings (Hirono), aberrant wing colour patterns (Fukushima) and an ectopic black spot beside the discal spot (Iitate flatland; enlargement in the inset). Arrowheads indicate abnormal parts and arrows indicate deformed wing spots. Scale bars for the top four panels indicate 1.0 mm and those for the bottom four panels indicate 5.0 mm. Full size image

To evaluate the effects of internal exposure caused by ingested food, we fed host plant leaves collected from the Fukushima and other areas (i.e., Fukushima, Iitate montane region, Iitate flatland, Hirono and Ube; see Fig. 1; Supplementary Table 8) to Okinawa larvae, which had never been exposed to artificial radionuclides. We confirmed that these leaves indeed contained high activities of 134Cs and 137Cs (Supplementary Table 8). Almost all individuals that consumed leaves from the non-contaminated locality (i.e., Ube) survived, whereas many individuals that consumed leaves from the contaminated localities could not survive well (Fig. 5d). Survival curves in the 4 groups fed leaves from the contaminated localities differed significantly from that in the Ube group (generalized Wilcoxon test, p < 0.000001 in all groups, Holm-corrected by 10 pairs). A dose-dependent trend was indicated by the significant differences between the Hirono and Fukushima curves (p = 0.0017, Holm-corrected as above) and between the Hirono and Iitate flatland curves (p = 0.00035, Holm-corrected as above). The pupal mortality rate (r = 0.91, df = 3, p = 1.0, Holm-corrected by 32 pairs [{ground radiation dose, ground β-ray dose, activity of 137Cs in host plant and activity of 134Cs in host plant} versus {abnormality rates of four stages (total), adults, wings, colour patterns, appendages and others and mortality rates of pupae and larvae}]) and colour-pattern abnormality rate (r = 0.96, df = 3, p = 0.34, Holm-corrected as above) showed high r values with the 137Cs activity of the collected leaves, although not significant statistically (Fig. 5e). Forewing size difference was observed among males (ANOVA, df = 4, F = 25, p < 0.0000001) and among females (ANOVA, df = 4, F = 8.0, p = 0.0000073) (Fig. 5f). In comparison with the Ube samples, forewing size reduction in males was detected in the Fukushima population (Student t test, p < 0.000001, Holm-corrected by 10 pairs), in the Iitate flatland population (Welch t test, p = 0.00015, Holm-corrected as above) and in the Iitate montane region males (Welch t-test, p = 0.00015, Holm-corrected as above) (Fig. 5f). Similarly, forewing size reduction in females was observed between the Ube and Iitate flatland samples (Student t test, p = 0.000041, Holm-corrected as above). Morphological abnormalities, including colour-pattern aberrations (Fig. 5g), were detected in the adults that ingested contaminated leaves during the larval period.