The biological impacts of the Fukushima nuclear accident on the pale grass blue butterfly

Abstract

The collapse of the Fukushima Dai-ichi Nuclear Power Plant caused a massive release of radioactive materials to the environment. A prompt and reliable system for evaluating the biological impacts of this accident on animals has not been available. Here we show that the accident caused physiological and genetic damage to the pale grass blue Zizeeria maha, a common lycaenid butterfly in Japan. We collected the first-voltine adults in the Fukushima area in May 2011, some of which showed relatively mild abnormalities. The F₁ offspring from the first-voltine females showed more severe abnormalities, which were inherited by the F₂ generation. Adult butterflies collected in September 2011 showed more severe abnormalities than those collected in May. Similar abnormalities were experimentally reproduced in individuals from a non-contaminated area by external and internal low-dose exposures. We conclude that artificial radionuclides from the Fukushima Nuclear Power Plant caused physiological and genetic damage to this species.

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.

Figure 2. F₁ 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 F₁, leftmost), undeveloped left middle leg tarsus (Fukushima F₁ and Hirono F₁, second and third from the left, respectively), and undeveloped palpi (Takahagi F₁, 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 F₁, left), and left compound eye was bar-like in shape (Hirono F₁, 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 F₁, right). Scale bar, 1.0 cm. (g) Representative wing colour-pattern modifications. The top left three individuals are F₁ individuals from an Iwaki parent, and the top rightmost individual is a Hirono F₁. The bottom samples, from left to right, are F₁ individuals from Hirono, Mito, Shiroishi, Motomiya, and Motomiya. Arrows indicate modified spots. Scale bar, 1.0 cm.

Figure 3. F₂ abnormalities.

(a) Abnormality rate for the F₂ 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 F₁ 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 F₁ 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 F₂, a phenotype similar to that of its F₁ parent shown in Fig. 2g. The top four samples, from left to right, are Iwaki F₂, Takahagi F₂, Iwaki F₂, and Fukushima F₂ individuals. All of the samples at the bottom are Fukushima F₂ individuals. The bottom middle and rightmost wings show a deformation of the hindwing shape, which were obtained from the offspring of the Fukushima F₁ 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 F₂ individual with a malformation of left antenna, which is short and forked (arrowheads). The right panel shows a Takahagi F₂ individual with a deformation of the left hindleg femur. Insets show pictures taken from different angles. Scale bars, 0.50 mm.

Figure 4. Abnormalities in the adult samples collected in September 2011 and in their F₁ 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 F₁ generation. The left three panels indicate malformations of left foreleg tarsus (an arrowhead) (Takahagi F₁, top), tumor-like solid protuberance (arrowheads) in the ventral side of the thorax (Takahagi F₁, middle), and dented eyes (Fukushima F₁, bottom). Scale bars in the left three panels all indicate 1.0 mm. Wing colour-pattern modifications (arrows) of the F₁ 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.

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.