BACKGROUND
the collapse of the fukushima dai-ichi nuclear power plant  due to the great east japan earthquake on  <dig> march  <dig> resulted in the release of a massive amount of radioactive materials into the surrounding environment.
this large-scale environmental pollution has impacted the lives of organisms living in the polluted areas.
a field study revealed that the number of butterflies decreased in the polluted areas  <cit> , and morphological abnormalities have been detected in aphids from fukushima  <cit> .
however, such studies examining the biological impacts of the accident are still scarce.
our group has been studying the development and evolution of butterflies in response to environmental stress  <cit> .
one of our favourite butterflies is a small lycaenid butterfly, the pale grass blue, zizeeria maha , which is found throughout japan, except in hokkaido  <cit> .
using this butterfly as an experimental system, we previously reported various morphological abnormalities and a smaller forewing size in adults collected from highly polluted areas  <cit> .
we showed that some abnormal traits, including aberrations in the wing colour pattern, were heritable, suggesting genetic damage introduced by the accident  <cit> .
we believe that the proposed genetic damage was introduced mainly by the initial exposure of the butterflies immediately after the explosion  <cit> .
we reproduced these field-based results through an external exposure experiment in which butterfly larvae and pupae were raised near an artificial caesium radiation source as well as through an internal exposure experiment in which larvae consumed contaminated host plant leaves collected from polluted areas  <cit> .
high mortality and abnormality rates were detected  <cit> , and a small forewing size  was also observed  <cit> , similar to the outcome of the food stress in butterflies  <cit> .
in a previous study, we collected host plant leaves of oxalis corniculata  from four relatively highly polluted localities in the tohoku district , in addition to the control locality, ube   <cit> .
we further quantified caesium radioactivity in pupae that ingested contaminated leaves and determined the mathematical relationships between the ingested dose of caesium and the resultant rates of mortality and abnormalities  <cit> .
we found that the mortality and abnormality rates increased sharply under low doses of caesium ingestion, following a power function.
however, as the leaves showing the lowest concentration recorded in that study were collected from hirono , leaves from other localities with much lower contamination levels should be examined to better understand the effects of low-level radiation ingestion.figure  <dig>
collection localities for host plant leaves.
the localities examined in the present study are shown in blue letters and those examined in the previous studies  <cit>  in black letters.
the fukushima dai-ichi nuclear power plant is indicated as npp in red.
in the present study, we collected leaves from host plants of the pale grass blue butterfly from six localities showing relatively low-level contamination: motomiya, koriyama , kashiwa and musashino, , atami  and okinawa  .
these host plant leaves were given to larvae from okinawa, which were not affected by the nuclear accident to any detectable degree, under our standard rearing conditions  <cit> .
we evaluated the biological impacts of various levels of contamination on this butterfly in this manner.
furthermore, we performed the internal exposure experiment not only in the first  generation, but also in the second  generation to examine possible transgenerational effects.table  <dig>
information for the collection localities for host plant leaves
collection site
collection date
distance from the npp
ground radiation
<dig> october 2012*1
<dig> october 2012*1
*1leaves were collected only for activity measurements.
RESULTS
effects on the f <dig> generation
previous studies showed that an average larva has a body weight of  <dig>  g and consumes  <dig>  g of leaves throughout its life on average  <cit> .
we obtained the concentrations of caesium activity in leaves collected from the six indicated localities , and we calculated the amount of caesium activity ingested by each feeding group of larvae, together with the resultant mortality and abnormality rates .
the mortality rate and the abnormality rate were defined as percent dead or percent abnormal and dead , respectively.
we then examined the dose–response relationship between the ingested caesium dose and the mortality rate .
the mortality rate  increased linearly in accordance with an increase of the caesium dose  with the regression model, y =  <dig>   x +  <dig>   .
remarkably, the mortality rate of the koriyama group  was 53%.
the dose–response relationship for abnormality rates was almost identical to that for mortality rates .table  <dig>
caesium radioactivity in host plant leaves*
1
activity of
137
cs
activity of
134
cs
total for
137
cs and
134
cs
*1activity levels were calculated assuming that the larvae ate all of the leaves required for their subsequent growth at once on the first day of ingestion of the contaminated leaves and that 137cs and 134cs were released at a 1: <dig> activity ratio on  <dig> march  <dig> in a single burst from the fukushima dai-ichi npp.
ingested caesium dose and mortality and abnormality rates in the f
1
generation
the f
1
leaves from:
number of larvae
ingested caesium dose
mortality rate
total abnormality rate
*1mean values for the summer and fall results.
effects of the ingested caesium dose on the f
1
generation.
mortality rate.
relative normality rate .
because surviving adults are not necessarily healthy regarding morphological traits, we calculated relative normality values for adults in reference to the control okinawa groups as an indication of the robustness of each feeding group .
to this end, the ratio of the number of morphologically normal  adults to the original number of larvae was calculated, and the ratios in the control okinawa groups were set at 100%.
as expected, the robustness distribution  appeared to show a non-significant inverse response relationship with the caesium dose  with the regression model, y = − <dig>   x +  <dig>   .
when the motomiya sample was excluded as an outlier, the regression equation becomes y = − <dig>   x +  <dig>   , showing a highly significant dose–response relationship and being consistent with the results found for the mortality rates.
we measured the forewing size of the surviving adults from each feeding group, and these data were plotted against the ingested caesium dose .
the forewing size fluctuated at low doses, but it decreased at relatively high doses in both sexes.
statistically significant differences were obtained between the okinawa and koriyama groups both in males  and in females  and between the okinawa and motomiya groups both in males  and in females .
a linear regression model between the ingested caesium dose  and the forewing size  did not show statistically significant relationship in males with the equation, y = − <dig>   x +  <dig>   , but it showed a significant relationship in females with the equation, y = − <dig>   x +  <dig>   .
this sexual difference may originate from the simple fact that females have larger wings in size.figure  <dig>
relationship between the ingested caesium dose and wing size in the f
1
generation.
the relative mean wing size is shown, together with standard error bars.
the size in the okinawa group was set as  <dig> .
male forewings.
the number of individuals that were subjected to measurement is as follows: okinawa , atami , musashino , kashiwa , koriyama , and motomiya .
female forewings.
the number of individuals that were subjected to measurement is as follows: okinawa , atami , musashino , kashiwa , koriyama , and motomiya .
we generated developmental survival curves  for the six feeding groups .
these survival curves showed stage-dependent changes of the percentage of the surviving individuals.
the survival curves for the five groups examined in a previous study  <cit>  were also included in the figure for comparison.
most of the curves were largely parallel with one another, and as a whole, these survival curves were statistically different from one another .figure  <dig>
survival curves for the f
1
generation.
the six groups examined in the present study and the five groups examined in previous studies  <cit>  are plotted together.
the survival curves were roughly segregated into three clusters of the experimental groups.
the concentrations of caesium activity in the ingested host plant leaves are indicated, together with the time period of the experiments.
groups that were examined in the summer of  <dig> are indicated by brownish colours with circular symbols.
groups examined in the fall of  <dig> are indicated by purplish colours with square symbols.
groups examined in the summer of  <dig> in previous studies  <cit>  are indicated by greenish colours with triangular symbols.
we noted that the motomiya and koriyama curves showed a great deal of mortality in the larva-prepupa period.
the curves were segregated into three clusters of groups: a high survival cluster , an intermediate survival cluster , and a low survival cluster .
these three clusters were statistically different from one another .
however, the concentrations of caesium activity recorded in the leaves showed great variation within a given cluster.
this intra-cluster variation most likely occurred because these groups were implemented using three different genetic lines of this butterfly species in different time periods, suggesting intraspecific variation regarding sensitivity to a radioactively contaminated diet.
accordingly, a set of groups that were assessed within the same time period were found to be comparable to one another as they come from the same genetic line .
we discovered various morphological abnormalities in the surviving adults .
the severe and rare abnormalities shown in figure  <dig> might imply the effects of a contaminated diet.
only three individuals in the group that consumed the control okinawa leaves showed very minor morphological abnormalities, i.e., folded wings and a deformed leg, which were not comparably severe with the abnormal individuals from the other f <dig> groups.figure  <dig>
morphological abnormalities of f
1
individuals.
morphologically abnormal parts are indicated by red arrows.
malformation of the left foreleg in an atami sample.
incomplete eclosion of the antennae in an atami sample.
curled wings in a kashiwa sample.
malformed wings in a kashiwa sample.
dented right compound eye in a koriyama sample.
abnormalities of the antennae, proboscis, and wings in a motomiya sample.
effects on the f <dig> generation
the f <dig> generation was obtained from the morphologically healthy  f <dig> adults that consumed leaves from okinawa, koriyama, or motomiya.
based on the detected caesium activity in the leaves, the ingested caesium doses were calculated , together with the resultant mortality and abnormality rates , and the dose–response relationship between the ingested dose and the mortality rate was examined.
the mortality rate  appeared to be dependent on the caesium dose  ingested by the f <dig> generation with the regression model, y =  <dig>   x +  <dig>   , but not significantly by the f <dig> generation  .table  <dig>
ingested caesium dose in the f
2
generation
f
2
leaves from:
number of larvae
ingested caesium dose
2
generation*
1
f
2
leaves
okinawa
koriyama
motomiya
f
1
leaves
*1
mr, mortality rate; ar, total abnormality rate; n, number of larvae examined.
effects of the ingested caesium dose on the mortality rate in the f
2
generation.
a two-dimensional scatter plot.
the ingested caesium dose in the f <dig> generation  is provided on the x-axis.
a three-dimensional scatter plot.
the ingested caesium doses in both the f <dig> and f <dig> generations are plotted.
“f1” and “f2” indicate the ingested caesium dose in the f <dig> and f <dig> generations, respectively.
“mr” indicates the mortality rate.
plots showing the same amount of f <dig> ingestion are connected by lines.
next, we produced developmental survival curves for the f <dig> generation .
as a whole, these survival curves were highly different from one another .
the curves were clearly segregated into two clusters: high and very low survival clusters.
these two groups were statistically very different from each other .
again, it was apparent that the determinant of this segregation was the f <dig> leaves and not the f <dig> leaves.
the remarkable segregation into the two clusters indicated the importance of the f <dig> diet.figure  <dig>
survival curves for the f
2
generation.
the survival curves are segregated into two clusters of the experimental groups.
for each curve, the localities of the f <dig> and f <dig> leaves are indicated, e.g., as koriyama -okinawa .
we compared the forewing sizes of the surviving adults .
the group that consumed the koriyama leaves in both the f <dig> and f <dig> generations showed very small forewings in both sexes compared to the group that consumed the okinawa leaves in both generations .
interestingly, the cumulative ingested caesium dose throughout the f <dig> and f <dig> generations showed a higher correlation with the forewing size than the ingested dose in the f <dig> generation alone in both sexes, but especially in females .
the female regression line between the cumulative ingested dose  throughout the f <dig> and f <dig> generations and the forewing size  was obtained as y = − <dig>   x +  <dig>   .figure  <dig>
forewing size variation of the f
2
generation.
male forewing size.
mean values are indicated, together with standard error bars.
the motomiya -motomiya  group showed a very small number of surviving adults, and thus, no error bars are shown for this group.
these explanations are also applicable to the female graph shown in b. the difference between the okinawa-okinawa group and the koriyama-koriyama group is statistically significant .
female forewing size.
the difference between the okinawa-okinawa group and the koriyama-koriyama group is statistically significant .
relationship between the cumulative ingested caesium dose in the f <dig> and f <dig> generations and the forewing size.
for the caesium dose, the total dose of 137cs and 134cs was used.
pearson correlation coefficient
r
and
p
-values for the f
2
generation between the ingested caesium dose and forewing size
caesium dose
r
p
male, f1 + f2
female, f1 + f2
as observed in the f <dig> generation, various morphological abnormalities were detected in the surviving f <dig> adults .
considering the very high mortality and abnormality rates recorded, it is likely that many of the recorded effects are attributable to the contaminated diets ingested by the butterflies.figure  <dig>
morphological abnormalities in f
2
individuals.
morphologically abnormal parts are indicated by red arrows.
dented left compound eye in a koriyama-koriyama sample.
eclosion failure in a koriyama-koriyama sample.
eclosion failure in a motomiya-okinawa sample.
truncated right antenna in a koriyama-okinawa sample.
eclosion failure in an okinawa-motomiya sample.
truncated right antenna in an okinawa-motomiya sample.
discussion
in the present study, we performed an internal exposure experiment using contaminated host plant leaves.
the host plant leaves were collected from the localities showing low-level contamination, including two localities in tohoku , two in kanto , and one in tokai .
a similar experiment was performed previously  <cit> , and the present study therefore serves to reproduce and validate the previous results.
furthermore, the present study extends the previous study by using the leaves that were less contaminated than those used in the previous study.
in the present study, low-dose effects were clearly detected in the butterflies in the form of high mortality and abnormality rates, low normality rates , low survival rates, and a small forewing size in the f <dig> generation of the tohoku groups.
it was somewhat surprising to find that the koriyama group, which ingested  <dig>  mbq of radioactive caesium during the larval stage from contaminated-contaminated leaves showing an activity of 117 bq/kg, displayed mortality and abnormality rates greater than 50%, although the motomiya group exhibited somewhat lower rates.
these results suggest that low-dose ingestion of approximately 100 bq/kg may be seriously toxic to certain organisms.
even among the surviving adults, a small forewing size was detected in the koriyama and motomiya groups, confirming the results concerning mortality rates, abnormality rates, normality rates, and survival rates.
for the kanto and tokai localities, which presented relatively low contamination levels ranging from  <dig>  bq/kg to  <dig>  bq/kg, both the mortality  rates and the abnormality  rates appeared to be dose dependent.
these kanto-tokai groups were tested using the same genetic lines at the same time, which were comparable to one another.
therefore, the contaminated leaves from the kanto-tokai districts may have had small but detectable effects on the fitness of the butterflies.
on the other hand, the forewing size of males in the kanto-tokai groups appeared to be larger than that of the okinawa group, despite the possible adverse effects detected for other measures .
the reasons for this size increase are not clear.
the biological responses to very low doses may simply be different from the responses to relatively high doses.
interestingly, the survival curves obtained for the different localities were segregated into three clusters: high, intermediate, and low survival clusters, not only among the groups from the previous study  <cit> , but also in the groups of the present study, despite the large difference in caesium activity levels between the previous and present studies.
in the previous study  <cit> , the half mortality rate was calculated as  <dig>  bq per larva, corresponding to constant leaf consumption of  <dig>  × 103 bq/kg throughout the larval stage.
these previous values were more than  <dig> times as high as the values we obtained for koriyama in the present study.
as a result, within a given cluster of survival curves, the levels of leaf contamination were highly variable .
however, within a given set of groups that were treated within the same time period , the survival rates were largely dependent on the caesium activity in the leaves.
these results may originate simply from a dependence of radiation sensitivity on the genetic background of butterfly lines.
that is, direct comparison between different butterfly lines may be difficult, even if the experimental set-ups are virtually identical with the exception of their genetic makeup.
therefore, our results suggest the existence of intraspecific individual variation of sensitivity to low-level radiation, which is consistent with the existing literature  <cit> .
the biological effects of ingesting the contaminated diets were more severe in the f <dig> generation, despite the normal morphological appearance of their f <dig> parents.
the differences can be illustrated as follows: the mortality rates of the koriyama f <dig> group and the koriyama f <dig> group obtained from the koriyama f <dig> adults were  <dig> % and 79%, respectively, while the mortality rates of the motomiya f <dig> group and the motomiya f <dig> group obtained from the motomiya f <dig> adults were  <dig> % and 99%, respectively.
as the mortality rates of the okinawa f <dig> group and the okinawa f <dig> group obtained from the okinawa f <dig> adults were 8% and 17%, respectively, the increase in mortality rates observed in the f <dig> generation cannot be readily attributed to the transgenerational accumulation of radiation effects.
however, effects of the f <dig> generation on the f <dig> generation were discernable in the form of a small forewing size and its high correlation with the cumulative caesium dose, especially in females.
it is important to note that a small forewing size can be induced not only through feeding of contaminated leaves, but also through long-term low-dose external irradiation in this species  <cit> .
furthermore, a small forewing size has been detected in field-caught butterflies from the polluted areas  <cit> .
therefore, the small forewing size observed in the present study implies that internal radiation exposure may have contributed to the high mortality rates recorded.
nevertheless, the f <dig> effect was small, and it could largely be overcome by the consumption of okinawa leaves or amplified by the consumption of koriyama or motomiya leaves in the f <dig> generation.
that is, the effects were transgenerational, but largely non-genetic physiological effects that could be overcome by dietary changes.
mechanistically, these outcomes may be explained by maternal effects, epigenetic effects, genomic instability, or similar phenomena.
these results potentially conflict with our previous findings showing that morphological abnormalities, including wing colour pattern aberrations, found in the f <dig> generation were heritable by the f <dig> generation  <cit> .
in this previous experiment, we used p-generation adults collected from the field in the spring of  <dig>  these p-generation individuals were directly exposed to all of the radioactive materials from the explosion of the npp when they were larvae, which could have resulted in genetic damage.
we then employed morphologically abnormal f <dig> adults to obtain the f <dig> generation using non-contaminated leaves.
in the case of the possible genetic damage indicated in  <dig>  the larvae may have consumed radioactive materials adsorbed on the surface of leaves.
the contributions of β-ray-emitting nuclides in the earlier experiment cannot be ignored  <cit> .
in contrast, the present study was performed in  <dig>  when the major radioactive species present was caesium, and the surface adsorption of radioactive materials was likely much less extreme.
therefore, the probability of genetic damage resulting from the ingestion of a contaminated diet was rather low in the present study.
both experimental set-ups are reasonable for examining heritable effects that are likely based on either genetic or physiological  changes.
in the examination of forewing size, we noted a sex difference.
females showed a higher correlation with the ingested dose of radioactivity in both the f <dig> and f <dig> generations than males.
the reasons for this sex difference are not clear.
however, higher sensitivity of females to cold shock, a type of environmental stress, has been reported in this species of butterfly  <cit> .
hence, it is likely that females are generally more sensitive to stress than males of this species.
the internal exposure of organisms living in a polluted area through a contaminated diet is unavoidable.
indeed, the possible biological effects of contaminated diets on humans after the chernobyl accident have been documented  <cit> .
however, to the best of our knowledge, controlled experimental evaluations of internal exposure via a consumed diet are scarce in any organism in the field of radiation biology, with a few possible exceptions  <cit> , although there may be some anecdotal or non-published studies that were performed following the chernobyl accident.
thus, our experimental results constitute an important contribution to the research field.
accumulation of such scientific evidence is required to establish the low-dose biological effects of radiation  <cit> .
CONCLUSIONS
our experimental system for detecting the biological effects of internal exposure to radiation via contaminated diets revealed that low-dose ingestion likely causes death , disease , and growth retardation , at least in some individuals of the pale grass blue butterfly.
the effects of ingesting a contaminated diet on survival rates and forewing size were shown to be transgenerational.
however, the effects of ingesting a contaminated diet in the f <dig> generation could be largely overcome by ingesting a non-contaminated diet in the f <dig> generation.
it is likely that at least some of the biological effects of internal exposure to low doses of radiation are attributed to non-genetic physiological  changes.

 the collapse of the fukushima dai-ichi nuclear power plant due to the great east japan earthquake on march resulted in the release of a massive amount of radioactive materials into the surrounding environment. this large-scale environmental pollution has impacted the lives of organisms living in the polluted areas. a field study revealed that the number of butterflies decreased in the polluted areas, and morphological abnormalities have been detected in aphids from fukushima. however, such studies examining the biological impacts of the accident are still scarce. our group has been studying the development and evolution of butterflies in response to environmental stress. one of our favourite butterflies is a small lycaenid butterfly, the pale grass blue, zizeeria maha, which is found throughout japan, except in hokkaido. using this butterfly as an experimental system, we previously reported various morphological abnormalities and a smaller forewing size in adults collected from highly polluted areas. we showed that some abnormal traits, including aberrations in the wing colour pattern, were heritable, suggesting genetic damage introduced by the accident. we believe that the proposed genetic damage was introduced mainly by the initial exposure of the butterflies immediately after the explosion. we reproduced these field-based results through an external exposure experiment in which butterfly larvae and pupae were raised near an artificial caesium radiation source as well as through an internal exposure experiment in which larvae consumed contaminated host plant leaves collected from polluted areas. high mortality and abnormality rates were detected, and a small forewing size was also observed, similar to the outcome of the food stress in butterflies. in a previous study, we collected host plant leaves of oxalis corniculata from four relatively highly polluted localities in the tohoku district, in addition to the control locality, ube. we further quantified caesium radioactivity in pupae that ingested contaminated leaves and determined the mathematical relationships between the ingested dose of caesium and the resultant rates of mortality and abnormalities. we found that the mortality and abnormality rates increased sharply under low doses of caesium ingestion, following a power function. however, as the leaves showing the lowest concentration recorded in that study were collected from hirono, leaves from other localities with much lower contamination levels should be examined to better understand the effects of low-level radiation ingestion.figure collection localities for host plant leaves. the localities examined in the present study are shown in blue letters and those examined in the previous studies in black letters. the fukushima dai-ichi nuclear power plant is indicated as npp in red. in the present study, we collected leaves from host plants of the pale grass blue butterfly from six localities showing relatively low-level contamination: motomiya, koriyama, kashiwa and musashino,, atami and okinawa. these host plant leaves were given to larvae from okinawa, which were not affected by the nuclear accident to any detectable degree, under our standard rearing conditions. we evaluated the biological impacts of various levels of contamination on this butterfly in this manner. furthermore, we performed the internal exposure experiment not only in the first generation, but also in the second generation to examine possible transgenerational effects.table information for the collection localities for host plant leaves collection site collection date distance from the npp ground radiation october 2012*1 october 2012*1 *1leaves were collected only for activity measurements.  effects on the f generation previous studies showed that an average larva has a body weight of  g and consumes  g of leaves throughout its life on average. we obtained the concentrations of caesium activity in leaves collected from the six indicated localities, and we calculated the amount of caesium activity ingested by each feeding group of larvae, together with the resultant mortality and abnormality rates. the mortality rate and the abnormality rate were defined as percent dead or percent abnormal and dead, respectively. we then examined the dose–response relationship between the ingested caesium dose and the mortality rate. the mortality rate increased linearly in accordance with an increase of the caesium dose with the regression model, y =  x + . remarkably, the mortality rate of the koriyama group was 53%. the dose–response relationship for abnormality rates was almost identical to that for mortality rates.table caesium radioactivity in host plant leaves* 1 activity of 137 cs activity of 134 cs total for 137 cs and 134 cs *1activity levels were calculated assuming that the larvae ate all of the leaves required for their subsequent growth at once on the first day of ingestion of the contaminated leaves and that 137cs and 134cs were released at a 1: activity ratio on march in a single burst from the fukushima dai-ichi npp. ingested caesium dose and mortality and abnormality rates in the f 1 generation the f 1 leaves from: number of larvae ingested caesium dose mortality rate total abnormality rate *1mean values for the summer and fall results. effects of the ingested caesium dose on the f 1 generation. mortality rate. relative normality rate. because surviving adults are not necessarily healthy regarding morphological traits, we calculated relative normality values for adults in reference to the control okinawa groups as an indication of the robustness of each feeding group. to this end, the ratio of the number of morphologically normal adults to the original number of larvae was calculated, and the ratios in the control okinawa groups were set at 100%. as expected, the robustness distribution appeared to show a non-significant inverse response relationship with the caesium dose with the regression model, y = − x + . when the motomiya sample was excluded as an outlier, the regression equation becomes y = − x + , showing a highly significant dose–response relationship and being consistent with the results found for the mortality rates. we measured the forewing size of the surviving adults from each feeding group, and these data were plotted against the ingested caesium dose. the forewing size fluctuated at low doses, but it decreased at relatively high doses in both sexes. statistically significant differences were obtained between the okinawa and koriyama groups both in males and in females and between the okinawa and motomiya groups both in males and in females. a linear regression model between the ingested caesium dose and the forewing size did not show statistically significant relationship in males with the equation, y = − x + , but it showed a significant relationship in females with the equation, y = − x + . this sexual difference may originate from the simple fact that females have larger wings in size.figure relationship between the ingested caesium dose and wing size in the f 1 generation. the relative mean wing size is shown, together with standard error bars. the size in the okinawa group was set as. male forewings. the number of individuals that were subjected to measurement is as follows: okinawa, atami, musashino, kashiwa, koriyama, and motomiya. female forewings. the number of individuals that were subjected to measurement is as follows: okinawa, atami, musashino, kashiwa, koriyama, and motomiya. we generated developmental survival curves for the six feeding groups. these survival curves showed stage-dependent changes of the percentage of the surviving individuals. the survival curves for the five groups examined in a previous study were also included in the figure for comparison. most of the curves were largely parallel with one another, and as a whole, these survival curves were statistically different from one another.figure survival curves for the f 1 generation. the six groups examined in the present study and the five groups examined in previous studies are plotted together. the survival curves were roughly segregated into three clusters of the experimental groups. the concentrations of caesium activity in the ingested host plant leaves are indicated, together with the time period of the experiments. groups that were examined in the summer of are indicated by brownish colours with circular symbols. groups examined in the fall of are indicated by purplish colours with square symbols. groups examined in the summer of in previous studies are indicated by greenish colours with triangular symbols. we noted that the motomiya and koriyama curves showed a great deal of mortality in the larva-prepupa period. the curves were segregated into three clusters of groups: a high survival cluster, an intermediate survival cluster, and a low survival cluster. these three clusters were statistically different from one another. however, the concentrations of caesium activity recorded in the leaves showed great variation within a given cluster. this intra-cluster variation most likely occurred because these groups were implemented using three different genetic lines of this butterfly species in different time periods, suggesting intraspecific variation regarding sensitivity to a radioactively contaminated diet. accordingly, a set of groups that were assessed within the same time period were found to be comparable to one another as they come from the same genetic line. we discovered various morphological abnormalities in the surviving adults. the severe and rare abnormalities shown in figure  might imply the effects of a contaminated diet. only three individuals in the group that consumed the control okinawa leaves showed very minor morphological abnormalities, i.e., folded wings and a deformed leg, which were not comparably severe with the abnormal individuals from the other f groups.figure morphological abnormalities of f 1 individuals. morphologically abnormal parts are indicated by red arrows. malformation of the left foreleg in an atami sample. incomplete eclosion of the antennae in an atami sample. curled wings in a kashiwa sample. malformed wings in a kashiwa sample. dented right compound eye in a koriyama sample. abnormalities of the antennae, proboscis, and wings in a motomiya sample. effects on the f generation the f generation was obtained from the morphologically healthy f adults that consumed leaves from okinawa, koriyama, or motomiya. based on the detected caesium activity in the leaves, the ingested caesium doses were calculated, together with the resultant mortality and abnormality rates, and the dose–response relationship between the ingested dose and the mortality rate was examined. the mortality rate appeared to be dependent on the caesium dose ingested by the f generation with the regression model, y =  x + , but not significantly by the f generation.table ingested caesium dose in the f 2 generation f 2 leaves from: number of larvae ingested caesium dose 2 generation* 1 f 2 leaves okinawa koriyama motomiya f 1 leaves *1 mr, mortality rate; ar, total abnormality rate; n, number of larvae examined. effects of the ingested caesium dose on the mortality rate in the f 2 generation. a two-dimensional scatter plot. the ingested caesium dose in the f generation is provided on the x-axis. a three-dimensional scatter plot. the ingested caesium doses in both the f and f generations are plotted. “f1” and “f2” indicate the ingested caesium dose in the f and f generations, respectively. “mr” indicates the mortality rate. plots showing the same amount of f ingestion are connected by lines. next, we produced developmental survival curves for the f generation. as a whole, these survival curves were highly different from one another. the curves were clearly segregated into two clusters: high and very low survival clusters. these two groups were statistically very different from each other. again, it was apparent that the determinant of this segregation was the f leaves and not the f leaves. the remarkable segregation into the two clusters indicated the importance of the f diet.figure survival curves for the f 2 generation. the survival curves are segregated into two clusters of the experimental groups. for each curve, the localities of the f and f leaves are indicated, e.g., as koriyama -okinawa. we compared the forewing sizes of the surviving adults. the group that consumed the koriyama leaves in both the f and f generations showed very small forewings in both sexes compared to the group that consumed the okinawa leaves in both generations. interestingly, the cumulative ingested caesium dose throughout the f and f generations showed a higher correlation with the forewing size than the ingested dose in the f generation alone in both sexes, but especially in females. the female regression line between the cumulative ingested dose throughout the f and f generations and the forewing size was obtained as y = − x + .figure forewing size variation of the f 2 generation. male forewing size. mean values are indicated, together with standard error bars. the motomiya -motomiya group showed a very small number of surviving adults, and thus, no error bars are shown for this group. these explanations are also applicable to the female graph shown in b. the difference between the okinawa-okinawa group and the koriyama-koriyama group is statistically significant. female forewing size. the difference between the okinawa-okinawa group and the koriyama-koriyama group is statistically significant. relationship between the cumulative ingested caesium dose in the f and f generations and the forewing size. for the caesium dose, the total dose of 137cs and 134cs was used. pearson correlation coefficient r and p -values for the f 2 generation between the ingested caesium dose and forewing size caesium dose r p male, f1 + f2 female, f1 + f2 as observed in the f generation, various morphological abnormalities were detected in the surviving f adults. considering the very high mortality and abnormality rates recorded, it is likely that many of the recorded effects are attributable to the contaminated diets ingested by the butterflies.figure morphological abnormalities in f 2 individuals. morphologically abnormal parts are indicated by red arrows. dented left compound eye in a koriyama-koriyama sample. eclosion failure in a koriyama-koriyama sample. eclosion failure in a motomiya-okinawa sample. truncated right antenna in a koriyama-okinawa sample. eclosion failure in an okinawa-motomiya sample. truncated right antenna in an okinawa-motomiya sample. discussion in the present study, we performed an internal exposure experiment using contaminated host plant leaves. the host plant leaves were collected from the localities showing low-level contamination, including two localities in tohoku, two in kanto, and one in tokai. a similar experiment was performed previously, and the present study therefore serves to reproduce and validate the previous results. furthermore, the present study extends the previous study by using the leaves that were less contaminated than those used in the previous study. in the present study, low-dose effects were clearly detected in the butterflies in the form of high mortality and abnormality rates, low normality rates, low survival rates, and a small forewing size in the f generation of the tohoku groups. it was somewhat surprising to find that the koriyama group, which ingested mbq of radioactive caesium during the larval stage from contaminated-contaminated leaves showing an activity of 117 bq/kg, displayed mortality and abnormality rates greater than 50%, although the motomiya group exhibited somewhat lower rates. these results suggest that low-dose ingestion of approximately 100 bq/kg may be seriously toxic to certain organisms. even among the surviving adults, a small forewing size was detected in the koriyama and motomiya groups, confirming the results concerning mortality rates, abnormality rates, normality rates, and survival rates. for the kanto and tokai localities, which presented relatively low contamination levels ranging from  bq/kg to  bq/kg, both the mortality rates and the abnormality rates appeared to be dose dependent. these kanto-tokai groups were tested using the same genetic lines at the same time, which were comparable to one another. therefore, the contaminated leaves from the kanto-tokai districts may have had small but detectable effects on the fitness of the butterflies. on the other hand, the forewing size of males in the kanto-tokai groups appeared to be larger than that of the okinawa group, despite the possible adverse effects detected for other measures. the reasons for this size increase are not clear. the biological responses to very low doses may simply be different from the responses to relatively high doses. interestingly, the survival curves obtained for the different localities were segregated into three clusters: high, intermediate, and low survival clusters, not only among the groups from the previous study, but also in the groups of the present study, despite the large difference in caesium activity levels between the previous and present studies. in the previous study, the half mortality rate was calculated as  bq per larva, corresponding to constant leaf consumption of  × 103 bq/kg throughout the larval stage. these previous values were more than times as high as the values we obtained for koriyama in the present study. as a result, within a given cluster of survival curves, the levels of leaf contamination were highly variable. however, within a given set of groups that were treated within the same time period, the survival rates were largely dependent on the caesium activity in the leaves. these results may originate simply from a dependence of radiation sensitivity on the genetic background of butterfly lines. that is, direct comparison between different butterfly lines may be difficult, even if the experimental set-ups are virtually identical with the exception of their genetic makeup. therefore, our results suggest the existence of intraspecific individual variation of sensitivity to low-level radiation, which is consistent with the existing literature. the biological effects of ingesting the contaminated diets were more severe in the f generation, despite the normal morphological appearance of their f parents. the differences can be illustrated as follows: the mortality rates of the koriyama f group and the koriyama f group obtained from the koriyama f adults were % and 79%, respectively, while the mortality rates of the motomiya f group and the motomiya f group obtained from the motomiya f adults were % and 99%, respectively. as the mortality rates of the okinawa f group and the okinawa f group obtained from the okinawa f adults were 8% and 17%, respectively, the increase in mortality rates observed in the f generation cannot be readily attributed to the transgenerational accumulation of radiation effects. however, effects of the f generation on the f generation were discernable in the form of a small forewing size and its high correlation with the cumulative caesium dose, especially in females. it is important to note that a small forewing size can be induced not only through feeding of contaminated leaves, but also through long-term low-dose external irradiation in this species. furthermore, a small forewing size has been detected in field-caught butterflies from the polluted areas. therefore, the small forewing size observed in the present study implies that internal radiation exposure may have contributed to the high mortality rates recorded. nevertheless, the f effect was small, and it could largely be overcome by the consumption of okinawa leaves or amplified by the consumption of koriyama or motomiya leaves in the f generation. that is, the effects were transgenerational, but largely non-genetic physiological effects that could be overcome by dietary changes. mechanistically, these outcomes may be explained by maternal effects, epigenetic effects, genomic instability, or similar phenomena. these results potentially conflict with our previous findings showing that morphological abnormalities, including wing colour pattern aberrations, found in the f generation were heritable by the f generation. in this previous experiment, we used p-generation adults collected from the field in the spring of these p-generation individuals were directly exposed to all of the radioactive materials from the explosion of the npp when they were larvae, which could have resulted in genetic damage. we then employed morphologically abnormal f adults to obtain the f generation using non-contaminated leaves. in the case of the possible genetic damage indicated in the larvae may have consumed radioactive materials adsorbed on the surface of leaves. the contributions of β-ray-emitting nuclides in the earlier experiment cannot be ignored. in contrast, the present study was performed in when the major radioactive species present was caesium, and the surface adsorption of radioactive materials was likely much less extreme. therefore, the probability of genetic damage resulting from the ingestion of a contaminated diet was rather low in the present study. both experimental set-ups are reasonable for examining heritable effects that are likely based on either genetic or physiological changes. in the examination of forewing size, we noted a sex difference. females showed a higher correlation with the ingested dose of radioactivity in both the f and f generations than males. the reasons for this sex difference are not clear. however, higher sensitivity of females to cold shock, a type of environmental stress, has been reported in this species of butterfly. hence, it is likely that females are generally more sensitive to stress than males of this species. the internal exposure of organisms living in a polluted area through a contaminated diet is unavoidable. indeed, the possible biological effects of contaminated diets on humans after the chernobyl accident have been documented. however, to the best of our knowledge, controlled experimental evaluations of internal exposure via a consumed diet are scarce in any organism in the field of radiation biology, with a few possible exceptions, although there may be some anecdotal or non-published studies that were performed following the chernobyl accident. thus, our experimental results constitute an important contribution to the research field. accumulation of such scientific evidence is required to establish the low-dose biological effects of radiation.  our experimental system for detecting the biological effects of internal exposure to radiation via contaminated diets revealed that low-dose ingestion likely causes death, disease, and growth retardation, at least in some individuals of the pale grass blue butterfly. the effects of ingesting a contaminated diet on survival rates and forewing size were shown to be transgenerational. however, the effects of ingesting a contaminated diet in the f generation could be largely overcome by ingesting a non-contaminated diet in the f generation. it is likely that at least some of the biological effects of internal exposure to low doses of radiation are attributed to non-genetic physiological changes. 
the release of radioactive materials due to the fukushima nuclear accident has raised concern regarding the biological impacts of ingesting radioactively contaminated diets on organisms. in addition to the effects on the first generation, we examined the possible transgenerational effects of the diets on the next generation. using the same experimental system, in the present study, we further examined the effects of low-level-contaminated diets on this butterfly. the effects are transgenerational but can be overcome by ingesting a non-contaminated diet, suggesting that at least some of the observed effects are attributable to non-genetic physiological changes. however, the forewing size was inversely correlated with the cumulative caesium dose ingested throughout the first and second generations, indicating that the diet in the first generation also influenced the forewing size of the second generation. the mortality rates were largely dependent on the ingested dose of caesium. fukushima nuclear accidentingestioninternal exposurelow-dose exposurepale grass blue butterflyradioactive contaminationtransgenerational effectissue-copyright-statement© the author 2014 in the first generation, the tohoku groups showed higher rates of mortality and abnormalities and a smaller forewing size than the okinawa group. in the next generation, the survival rates in the tohoku groups were below 20%, whereas those of the okinawa groups were above 70%. the survival rates of the kanto-tokai groups were greater than 80%, but the rates in the tohoku groups were much lower. moreover, a smaller forewing size was observed in the tohoku groups in the second generation. we previously performed an internal exposure experiment in which contaminated leaves collected from polluted areas were fed to larvae of the pale grass blue butterfly, zizeeria maha, from okinawa, which is one of the least polluted localities in japan. the survival rates in the second generation were independent of the locality of the leaves ingested by the first generation, indicating that the diet in the second generation was the determinant of their survival.
 the release of radioactive materials due to the fukushima nuclear accident has raised concern regarding the biological impacts of ingesting radioactively contaminated diets on organisms. we previously performed an internal exposure experiment in which contaminated leaves collected from polluted areas were fed to larvae of the pale grass blue butterfly, zizeeria maha, from okinawa, which is one of the least polluted localities in japan. using the same experimental system, in the present study, we further examined the effects of low-level-contaminated diets on this butterfly. leaves were collected from two localities in tohoku and koriyama ); two in kanto and musashino ); one in tokai ); and from okinawa. in addition to the effects on the first generation, we examined the possible transgenerational effects of the diets on the next generation.  in the first generation, the tohoku groups showed higher rates of mortality and abnormalities and a smaller forewing size than the okinawa group. the mortality rates were largely dependent on the ingested dose of caesium. the survival rates of the kanto-tokai groups were greater than 80%, but the rates in the tohoku groups were much lower. in the next generation, the survival rates in the tohoku groups were below 20%, whereas those of the okinawa groups were above 70%. the survival rates in the second generation were independent of the locality of the leaves ingested by the first generation, indicating that the diet in the second generation was the determinant of their survival. moreover, a smaller forewing size was observed in the tohoku groups in the second generation. however, the forewing size was inversely correlated with the cumulative caesium dose ingested throughout the first and second generations, indicating that the diet in the first generation also influenced the forewing size of the second generation.  biological effects are detectable under a low ingested dose of radioactivity from a contaminated diet. the effects are transgenerational but can be overcome by ingesting a non-contaminated diet, suggesting that at least some of the observed effects are attributable to non-genetic physiological changes. keywords fukushima nuclear accidentingestioninternal exposurelow-dose exposurepale grass blue butterflyradioactive contaminationtransgenerational effectissue-copyright-statement© the author 2014 