SUPPLEMENTARY INFORMATION

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

Atsuki Hiyama1†, Chiyo Nohara1†, Seira Kinjo1, Wataru Taira1,

Sinichi Gima2, Akira Tanahara2, Joji M. Otaki1＊

1 The BCPH Unit of Molecular Physiology,

Department of Chemistry, Biology and Marine Science,

Faculty of Science,

University of the Ryukyus,

Nishihara, Okinawa 903-0213,

Japan

2 Instrumental Research Center,

University of the Ryukyus,

Nishihara, Okinawa 903-0213,

Japan

† Contributed equally to this work.

* Correspondence should be addressed to J.M.O.

(E-mail: )

Supplementary Figure 1 | Rearing system of the pale grass blue butterfly Z. maha in the laboratory. (a) Cages for rearing. (b) Plants and butterflies inside a cage. Inset shows a pale grass blue butterfly feeding on a flower inside the cage.

Supplementary Figure 2 | Pupation time. (a) Pupation-time dynamics of the F1 generation of various parents’ localities. All local populations except Shiroishi differed significantly from the Tsukuba population (generalized Wilcoxon test, p < 0.00001, Holm-corrected by 28 pairs, in all population groups). For the number of individuals, see Figure 2a or Supplementary Table 3. (b) Scatter plot of half-pupation time and distances of the collection localities from the NPP. Pearson correlation coefficient r = −0.83 (df = 6, p = 0.29, Holm-corrected by 30 pairs).

Supplementary Table 1 | Localities of adult butterflies collected in May, September, and October 2011.

Collection date / City / Location / Distance from the NPP (km) / Ground radiation dose (μSv/h) / Number of collected
Total Male Female
13-18 May / Shiroishi1 / Aoki, Fukuokafukaya / 76.7 / 0.32 / 6 / 5 / 1
Fukushima2 / Omori Shiroyama Park / 62.8 / 1.13 / 4 / 3 / 1
Nitanda, Omori / 62.8 / 1.25 / 10 / 6 / 4
Motomiya2 / Keikou Park 1 / 58.8 / 3.09 / 11 / 9 / 2
Keikou Park 2* / 58.8 / 2.73 / － / － / －
Koriyama2 / Furusato-no-kawa Park / 60.9 / 2.22 / 3 / 3 / 0
Hirono2 / Futatsunuma Park / 20.7 / 1.30 / 17 / 12 / 5
Iwaki2 / Shiraiwa, Yotsukura / 33.3 / 0.46 / 5 / 3 / 2
Tairaizumizaki, Buryo / 37.6 / 0.63 / 19 / 15 / 4
Takahagi3 / Shimotetsuna / 82.3 / 0.30 / 6 / 4 / 2
Akiyama / 84.1 / 0.42 / 18 / 15 / 3
Mito3 / Kairakuen / 127.5 / 0.18 / 4 / 2 / 2
Mito Sports Park / 130.1 / 0.14 / 10 / 7 / 3
Tsukuba3 / Douhou Park 1 / 172.0 / 0.16 / 3 / 1 / 2
Douhou Park 2* / 172.0 / 0.15 / － / － / －
Sakura Sports Park 1 / 168.3 / 0.17 / 14 / 11 / 3
Sakura Sports Park 2* / 168.3 / 0.16 / － / － / －
Tokyo / Hibiya Park 1 / 225.5 / 0.15 / 14 / 14 / 0
Hibiya Park 2* / 225.5 / 0.09 / － / － / －
18-21 / Fukushima2 / Omori Shiroyama Park / 63.1 / 0.71 / 37 / 31 / 6
September / Hanamiyama / 59.2 / 2.43 / 15 / 13 / 2
Motomiya2 / Keikou Park 1 / 58.6 / 1.75 / 31 / 22 / 9
Keikou Park 2* / 58.6 / 1.49 / － / － / －
Hirono2 / Futatsunuma Park 1 / 21.1 / 0.81 / 19 / 14 / 5
Futatsunuma Park 2 / 20.8 / 0.82 / 6 / 5 / 1
Futatsunuma Park 3 / 21.3 / 0.90 / 1 / 1 / 0
Iwaki2 / Shiraiwa Yotsukura / 33.2 / 0.40 / 8 / 8 / 0
Ishimoriyama / 38.8 / 0.42 / 48 / 26 / 22
Takahagi3 / Nakagou SA Outbound / 79.8 / 0.24 / 6 / 2 / 4
Mito3 / Tano SA Outbound / 125.7 / 0.19 / 6 / 5 / 1
Tsukuba3 / Yatabe-higashi SA Outbound / 173.3 / 0.18 / 15 / 15 / 0
3-4 October / Kobe4 / Nebutani Park / 596.0 / 0.08 / 46 / 26 / 20

* Radiation measurement only. SA: service area in high way.

1: Miyagi Prefectore. 2: Fukushima Prefecture. 3: Ibaraki Prefecture. 4: Hyogo Prefecture.

Supplementary Table 2 | Abnormality rate of adult butterflies collected in May 2011.

Abnormality rate (%)
Total / Wings / Color patterns / Appendages / Others
City / Total / Male / Female / Total / Male / Female / Total / Male / Female / Total / Male / Female / Total / Male / Female
Shiroishi / 33.3 / 20.0 / 100 / 0 / 0 / 0 / 0 / 0 / 0 / 16.7 / 0 / 100 / 33.3 / 20.0 / 100
Fukushima / 21.4 / 11.1 / 40.0 / 7.1 / 0 / 20.0 / 0 / 0 / 0 / 14.3 / 11.1 / 20.0 / 0 / 0 / 0
Motomiya / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
Koriyama / 33.3 / 33.3 / ― / 0 / 0 / ― / 0 / 0 / ― / 33.3 / 33.3 / ― / 0 / 0 / ―
Hirono / 11.8 / 8.3 / 20.0 / 0 / 0 / 0 / 0 / 0 / 0 / 5.9 / 8.3 / 0 / 5.9 / 0 / 20.0
Iwaki / 8.3 / 11.1 / 0 / 0 / 0 / 0 / 4.2 / 5.6 / 0 / 0 / 0 / 0 / 4.2 / 5.6 / 0
Takahagi / 8.3 / 10.5 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 8.3 / 10.5 / 0 / 0 / 0 / 0
Mito / 35.7 / 33.3 / 40.0 / 0 / 0 / 0 / 14.3 / 0 / 40.0 / 14.3 / 22.2 / 20.0 / 7.1 / 11.1 / 0
Tsukuba / 5.9 / 8.3 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 5.9 / 8.3 / 0
Tokyo / 7.1 / 7.1 / ― / 0 / 0 / ― / 0 / 0 / ― / 7.1 / 7.1 / ― / 0 / 0 / ―

―: Not applicable.

Supplementary Table 3 | Number of individuals and number of dead or abnormal individuals of the F1 generation from the adults collected in May and in September-October 2011.

Larva / Pupa / Adult / Dead individual / Abnormal / Total (dead
City / Total / Male / Female / Larva / Prepupa / Pupa / (Adult) / + abnormal)
May / Shiroishi / 158 / 154 / 151 / 72 / 79 / 2 / 2 / 3 / 31 / 38
Fukushima / 346 / 272 / 239 / 135 / 104 / 38 / 36 / 33 / 46 / 153
Motomiya / 232 / 161 / 142 / 75 / 67 / 49 / 22 / 18 / 26 / 115
Hirono / 470 / 288 / 261 / 138 / 123 / 156 / 26 / 27 / 60 / 269
Iwaki / 499 / 271 / 230 / 123 / 107 / 153 / 75 / 41 / 57 / 326
Takahagi / 229 / 182 / 166 / 78 / 88 / 17 / 30 / 16 / 18 / 81
Mito / 339 / 256 / 240 / 123 / 117 / 67 / 16 / 16 / 28 / 127
Tsukuba / 243 / 194 / 175 / 86 / 89 / 19 / 30 / 19 / 28 / 96
Sep / Fukushima / 322 / 274 / 255 / 125 / 130 / 47 / 1 / 19 / 149 / 216
and / Motomiya / 401 / 323 / 271 / 134 / 137 / 74 / 4 / 52 / 174 / 304
Oct / Hirono / 149 / 73 / 11 / 7 / 4 / 66 / 10 / 62 / 10 / 148
Iwaki / 135 / 113 / 109 / 51 / 58 / 22 / 0 / 4 / 69 / 95
Takahagi / 290 / 256 / 240 / 112 / 128 / 34 / 0 / 16 / 131 / 181
Kobe / 266 / 236 / 223 / 108 / 110 / 27 / 3 / 13 / 43 / 86

Supplementary Table 4 | Mortality rate and abnormality rate of the F1 generation from the adults collected in May and in September-October 2011.

Mortality rate (%) / Abnormality rate (%) / Total
City / Larva / Pre- pupa / Pupa / Adult / Wings / Color patterns / Appendages / Others / Abnormality rate (%)
May / Shiroishi / 1.3 / 1.3 / 2.0 / 20.5 / 13.3 / 6.6 / 1.3 / 0.7 / 24.1
Fukushima / 11.0 / 11.7 / 12.1 / 19.3 / 11.7 / 2.1 / 3.8 / 4.2 / 44.2
Motomiya / 21.1 / 12.0 / 11.2 / 18.3 / 9.9 / 8.5 / 4.2 / 0.7 / 49.6
Hirono / 33.2 / 8.3 / 9.4 / 23.0 / 13.0 / 5.8 / 6.9 / 1.9 / 57.2
Iwaki / 30.7 / 21.7 / 15.1 / 24.8 / 8.7 / 13.0 / 6.5 / 1.3 / 65.3
Takahagi / 7.4 / 14.2 / 8.8 / 10.8 / 7.8 / 1.8 / 3.6 / 1.2 / 35.4
Mito / 19.8 / 5.9 / 6.3 / 11.7 / 8.3 / 3.3 / 0.8 / 1.7 / 37.5
Tsukuba / 7.8 / 13.4 / 9.8 / 16.0 / 9.7 / 2.3 / 1.1 / 3.4 / 39.5
Sep / Fukushima / 14.60 / 0.36 / 6.93 / 58.43 / 43.53 / 20.48 / 10.98 / 0.39 / 67.08
and / Motomiya / 18.45 / 1.22 / 16.10 / 64.21 / 49.82 / 19.23 / 18.45 / 5.90 / 75.81
Oct / Hirono / 44.30 / 12.05 / 84.93 / 90.91 / 72.73 / 36.36 / 45.45 / 0 / 99.33
Iwaki / 16.30 / 0 / 3.54 / 63.30 / 44.04 / 37.04 / 15.60 / 0 / 70.37
Takahagi / 11.72 / 0 / 6.25 / 54.58 / 40.83 / 14.16 / 14.58 / 0.83 / 62.41
Kobe / 10.15 / 1.26 / 5.51 / 19.28 / 15.25 / 2.26 / 3.14 / 0.45 / 32.33

Supplementary Table 5 | Number of eggs, larva, and adults of the

F2 generation from the adults collected in May 2011.

Abnormality / Egg / Larva / Adult
Strain / of F1 / Total / Male / Female
Takahagi 1 / Foreleg / － / 115 / 62 / 28 / 34
Iwaki 1 / Color pattern / － / 49 / 51 / 30 / 21
Iwaki 2 / Pulpus / 88 / － / 22 / 13 / 9
Iwaki 3 / Pulpus / 54 / － / 2 / 0 / 2
Hirono 1 / Eye / 23 / 0 / 0 / 0 / 0
Hirono 2 / Color pattern / － / 153 / 83 / 35 / 48
Motomiya 1 / Color pattern / 11 / － / 1 / 0 / 1
Fukushima 1 / Asymmetrical hindwing size / － / 138 / 102 / 51 / 51
Fukushima 2 / Middle leg / － / － / 36 / 12 / 24
Shiroishi 1 / Normal (non-abnormal) / － / 86 / 76 / 42 / 34

－: Not recorded.

Supplementary Table 6 | Abnormality rate of inheritance of the F2 generation from the adults collected in May 2011.

Abnormality rate
Adult of F2 / Identical to F1 / Homologous with F1
Strain name / Total / Male / Female / Total / Male / Female / Total / Male / Female
Takahagi 1 / 43.6 / 53.6 / 35.3 / 3.2 / 0 / 5.9 / 19.4 / 17.9 / 20.6
Iwaki 1 / 56.9 / 33.3 / 90.5 / 23.5 / 3.3 / 52.4 / 43.1 / 20.0 / 76.2
Iwaki 2 / 36.4 / 30.8 / 44.4 / 9.1 / 7.7 / 11.1 / 13.6 / 7.7 / 22.2
Iwaki 3 / 50.0 / 0 / 50.0 / 0 / － / 0 / 0 / － / 0
Hirono 2 / 35.0 / 28.6 / 39.6 / 3.6 / 2.9 / 4.2 / 19.3 / 5.7 / 29.2
Motomiya 1 / 100 / 0 / 100 / 0 / － / 0 / 100 / － / 100
Fukushima 1 / 28.4 / 27.5 / 29.4 / 8.8 / 5.9 / 11.8 / 18.6 / 19.6 / 17.7
Fukushima 2 / 25.0 / 8.3 / 33.3 / 0 / 0 / 0 / 11.1 / 0 / 16.7
Shiroishi 1 / 22.4 / 23.8 / 20.6 / 0 / 0 / 0 / 0 / 0 / 0

－: Not applicable.

Supplementary Table 7 | Abnormality rate of butterflies collected in September-October 2011.

Abnormality rate (%)
Total / Wings / Color patterns / Appendages / Others
City / Total / Male / Female / Total / Male / Female / Total / Male / Female / Total / Male / Female / Total / Male / Female
Fukushima / 38.5 / 43.2 / 12.5 / 5.8 / 6.8 / 0 / 17.3 / 18.2 / 12.5 / 26.9 / 31.8 / 0 / 1.9 / 2.3 / 0
Motomiya / 35.5 / 31.8 / 44.4 / 16.1 / 18.2 / 11.1 / 12.9 / 13.6 / 11.1 / 16.1 / 13.6 / 22.2 / 0 / 0 / 0
Hirono / 19.2 / 10.0 / 50.0 / 0 / 0 / 0 / 0 / 0 / 0 / 19.2 / 10.0 / 50.0 / 0 / 0 / 0
Iwaki / 28.6 / 29.4 / 27.3 / 3.6 / 0 / 9.1 / 1.8 / 0 / 4.6 / 19.6 / 23.5 / 13.6 / 1.8 / 2.9 / 0
Takahagi / 16.7 / 0 / 25.0 / 0 / 0 / 0 / 16.7 / 0 / 25.0 / 16.7 / 0 / 25.0 / 0 / 0 / 0
Mito / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
Tsukuba / 6.7 / 6.7 / ― / 0 / 0 / ― / 0 / 0 / ― / 6.7 / 6.7 / ― / 0 / 0 / ―
Kobe / 13.0 / 15.4 / 10.0 / 2.2 / 3.9 / 0 / 2.2 / 0 / 5.0 / 8.7 / 11.5 / 5.0 / 0 / 0 / 0

―: Not applicable.

Supplementary Table 8 | Localities and radioactivities of collected host plant leaves for internal radiation exposure experiment.

Collection
date / City / Location / Distance from the NPP
（km） / Ground radiation dose
（μSv/h） / β-ray dose of host plant (cpm/kg) / Activity of 137Cs in host plant (Bq/kg) / Activity of 134Cs in host plant (Bq/kg)
15, 17, 28 July / Ube*,1 / Higashisue Oaza / 966.0 / 0.18 / 6.33×102 / 5.3 (±0.2)
×10-1 / 3.9 (±0.1)
×10-1
Kounan-kita
17-19 July and 2 August / Hirono2 / Futatsunuma Park / 20.7 / 1.06 / 4.35×103 / 7.71 (±0.08)
×102 / 1.83  (±0.06)
×102
17-19 July and 2 August / Fukushima*,2 / Takahata Okabe / 61.1 / 3.57 / 2.13×104 / 4.17 (±0.04)
×103 / 3.16 (±0.03)
×103
Hanamiyama
17-19 July and 2 August / Iitate flatland**,2 / Ainosawa Campsite / 40.0 / 6.22 / 2.73×104 / 5.42 (±0.05)
×103 / 4.07 (±0.04)
×103
Omori Fukaya
Onishi Iitoi
17-19 July and 2 August / Iitate montane region2 / Nagadoro / 33.7 / 18.86 / 7.61×104 / 2.31 (±0.01)
×104 / 1.75 (±0.01)
×104

* Collected at two different sites. ** Collected at three different sites. 1: Yamaguchi Prefecture. 2: Fukushima Prefecture.

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