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Flavanones, a class of flavonoids, are abundant in fruits, vegetables, and herbs. They are known to have several biological activities, such as anti-inflammatory and anti-cancer activities, but their effects on obesity remain unclear. Obesity is closely associated with adipocyte differentiation and lipid accumulation in adipose tissue. Therefore, in this study, we examined the effects of flavanone derivatives on adipocyte differentiation and lipid accumulation by using 3T3-L1 cells. Among the 15 flavanone derivatives studied, 4′-phenylflavanone (4PF), with a biphenyl structure, significantly inhibited adipocyte differentiation-related lipid accumulation in 3T3-L1 cells; this inhibition of lipid accumulation was dose-dependent. Gene expression analysis showed that 4PF suppressed the expression of adipogenic marker genes. Although the induction of peroxisome proliferator activator γ2 (Pparγ2), a master regulator of adipocyte differentiation, and its target genes during adipocyte differentiation was attenuated in 4PF-treated cells, 4PF did not directly regulate Pparγ2 gene expression and its activation. In contrast, 4PF suppressed mitotic clonal expansion (MCE), which is associated with changes in the expression of proliferation-related genes at the early stages of adipocyte differentiation. Taken together, these results suggest that 4PF inhibits lipid accumulation because it suppresses MCE during adipocyte differentiation. Thus, our findings may help in the development of anti-obesity drugs.

S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

A unique historical architecture was created at Shuri Castle (Shuri-jo) in the Ryukyu Islands by its "Kawara" roof tiles. After the 13th and 14th centuries, Kawara tiles were introduced to the Ryukyu Islands from several regions, including China, Korea, and mainland Japan, and evolved shapes and patterns that are unique to this island region. However, the transition of some internal features, such as the chemical components and microstructure, had not been analyzed. This study used a multi-faceted approach for such internal data and non-destructive quantitative methods to propose a new perspective on the evolution of historical Ryukyuan Kawara. We analyzed two styles of Ryukyuan Kawara from the 13th to 15th centuries and found that the material processing and firing conditions of the two styles were very similar, even though it had been suggested that they had different origins. A quantitative analysis of tiles from the 16th to 19th centuries revealed a transition in color tone to red, leading to the modern traditional Ryukyuan tiles; traces of changes in firing conditions were also found along with this transition. Finally, the study revealed that the evolution of Ryukyuan Kawara consisted of changed factors, e.g. surface color, and unchanged factors, e.g. paste density. Previous archaeological studies mainly focused on changing external characteristics, such as form and pattern; however, our analysis showed that the internal features changed, while the elemental composition and paste density remained constant from the appearance of the roof tiles until the 19th century. We propose that this is related to different responses of individual factors to external stressors, such as the social context, which may be common to other archaeological artifacts as well. Our study provides a new perspective on the evolution of Ryukyuan Kawara and presents a different discussion of and methods for the chronological study of material culture., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Aoyama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many field observations of the biological effects of the Fukushima nuclear accident have been reported in the first decade after the accident. A series of observational and experimental studies have demonstrated causal adverse effects on the pale grass blue butterfly even at the low-level radiation exposure in the "field," contrary to the dosimetric view that insects are generally tolerant of radiation exposure. However, it has been demonstrated that the pale grass blue butterfly is tolerant of high oral doses of anthropogenic radioactive cesium ( 137 Cs) under "laboratory" conditions. This field-laboratory paradox can be explained by ecological field effects; for example, radiation stress in the field causes physiological and biochemical changes in the host plant, which then trophically affects butterfly larvae. The second decade of butterfly-based Fukushima research will be devoted to demonstrating how such adverse field effects occur. Changes in the host plant's nutritional contents likely affect butterfly physiology. The host plant may also upregulate secondary metabolites that affect herbivorous insects. The plant may be affected by changes in endophytic soil microbes in radioactively contaminated areas. If demonstrated, these results will reveal that the delicate ecological balances among the butterfly, its host plant, and soil microbes have been affected by radioactive pollution in Fukushima, which has important implications for environmental policies and human health. Integr Environ Assess Manag 2022;18:1539-1550. © SETAC., (© 2022 SETAC.)

Biological effects of the Fukushima nuclear accident have been reported in various organisms, including the pale grass blue butterfly Zizeeria maha and its host plant Oxalis corniculata . This plant upregulates various secondary metabolites in response to low-dose radiation exposure, which may contribute to the high mortality and abnormality rates of the butterfly in Fukushima. However, this field effect hypothesis has not been experimentally tested. Here, using an artificial diet for larvae, we examined the ingestional toxicity of three radiation-dependent plant metabolites annotated in a previous metabolomic study: lauric acid (a saturated fatty acid), alfuzosin (an adrenergic receptor antagonist), and ikarugamycin (an antibiotic likely from endophytic bacteria). Ingestion of lauric acid or alfuzosin caused a significant decrease in the pupation, eclosion (survival), and normality rates, indicating toxicity of these compounds. Lauric acid made the egg-larval days significantly longer, indicating larval growth retardation. In contrast, ikarugamycin caused a significant increase in the pupation and eclosion rates, probably due to the protection of the diet from fungi and bacteria. These results suggest that at least some of the radiation-dependent plant metabolites, such as lauric acid, contribute to the deleterious effects of radioactive pollution on the butterfly in Fukushima, providing experimental evidence for the field effect hypothesis.

The biological impacts of the Fukushima nuclear accident, in 2011, on wildlife have been studied in many organisms, including the pale grass blue butterfly and its host plant, the creeping wood sorrel Oxalis corniculata . Here, we performed an LC-MS-based metabolomic analysis on leaves of this plant collected in 2018 from radioactively contaminated and control localities in Fukushima, Miyagi, and Niigata prefectures, Japan. Using 7967 peaks detected by LC-MS analysis, clustering analyses showed that nine Fukushima samples and one Miyagi sample were clustered together, irrespective of radiation dose, while two Fukushima (Iitate) and two Niigata samples were not in this cluster. However, 93 peaks were significantly different (FDR < 0.05) among the three dose-dependent groups based on background, low, and high radiation dose rates. Among them, seven upregulated and 15 downregulated peaks had single annotations, and their peak intensity values were positively and negatively correlated with ground radiation dose rates, respectively. Upregulated peaks were annotated as kudinoside D (saponin), andrachcinidine (alkaloid), pyridoxal phosphate (stress-related activated vitamin B6), and four microbe-related bioactive compounds, including antibiotics. Additionally, two peaks were singularly annotated and significantly upregulated (K 1 R 1 H 1 ; peptide) or downregulated (DHAP(10:0); decanoyl dihydroxyacetone phosphate) most at the low dose rates. Therefore, this plant likely responded to radioactive pollution in Fukushima by upregulating and downregulating key metabolites. Furthermore, plant-associated endophytic microbes may also have responded to pollution, suggesting their contributions to the stress response of the plant.

The biological consequences of the Fukushima nuclear accident have been intensively studied using the pale grass blue butterfly Zizeeria maha and its host plant, the creeping wood sorrel Oxalis corniculata . Here, we performed metabolomic analyses of Oxalis leaves from Okinawa to examine the plant metabolites that were upregulated or downregulated in response to low-dose radiation exposure from Fukushima's contaminated soil. The cumulative dose of radiation to the plants was 5.7 mGy (34 μGy/h for 7 days). The GC-MS analysis revealed a systematic tendency of downregulation among the metabolites, some of which were annotated as caproic acid, nonanoic acid, azelaic acid, and oleic acid. Others were annotated as fructose, glucose, and citric acid, involved in the carbohydrate metabolic pathways. Notably, the peak annotated as lauric acid was upregulated. In contrast, the LC-MS analysis detected many upregulated metabolites, some of which were annotated as either antioxidants or stress-related chemicals involved in defense pathways. Among them, only three metabolite peaks had a single annotation, one of which was alfuzosin, an antagonist of the α 1 -adrenergic receptor. We conclude that this Oxalis plant responded metabolically to low-dose radiation exposure from Fukushima's contaminated soil, which may mediate the ecological "field effects" of the developmental deterioration of butterflies in Fukushima.

The pale grass blue butterfly Zizeeria maha is sensitive to low-dose radioactive pollution from the Fukushima nuclear accident in the field but is also highly tolerant to radioactive cesium ( 137 Cs) in an artificial diet in laboratory experiments. To resolve this field-laboratory paradox, we hypothesize that the butterfly shows vulnerability in the field through biochemical changes in the larval host plant, the creeping wood sorrel Oxalis corniculata , in response to radiation stress. To test this field-effect hypothesis, we examined nutrient contents in the host plant leaves from Tohoku (mostly polluted areas including Fukushima), Niigata, and Kyushu, Japan. Leaves from Tohoku showed significantly lower sodium and lipid contents than those from Niigata. In the Tohoku samples, the sodium content (but not the lipid content) was significantly negatively correlated with the radioactivity concentration of cesium ( 137 Cs) in leaves and with the ground radiation dose. The sodium content was also correlated with other nutrient factors. These results suggest that the sodium imbalance of the plant may be caused by radiation stress and that this nutrient imbalance may be one of the reasons that this monophagous butterfly showed high mortality and morphological abnormalities in the field shortly after the accident in Fukushima.

The Fukushima nuclear accident in March 2011 caused the massive release of anthropogenic radioactive materials from the Fukushima Dai-ichi Nuclear Power Plant to its surrounding environment. Its biological effects have been studied using the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae), but the overwintering states of this butterfly remain elusive. Here, we conducted a series of field surveys in March 2018, March 2019, and April 2019 in Fukushima and its vicinity to clarify the overwintering states of this butterfly at the time of the Fukushima nuclear accident. We discovered overwintering individuals in situ associated with the host plant Oxalis corniculata under natural straw mulch as first-instar to fourth-instar larvae in March 2018 and 2019. No other developmental stages were found. The body length and width were reasonably correlated with the accumulated temperature. On the basis of a linear regression equation between body size and accumulated temperature, together with other data, we deduced that the pale grass blue butterfly occurred as fourth-instar larvae in Fukushima and its vicinity at the time of the accident. This study paves the way for subsequent dosimetric analyses that determine the radiation doses absorbed by the butterfly after the accident., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The biological effects of the Fukushima nuclear accident have been examined in the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae). In previous internal exposure experiments, larvae were given field-collected contaminated host plant leaves that contained up to 43.5 kBq/kg (leaf) of radioactive caesium. Larvae ingested up to 480 kBq/kg (larva), resulting in high mortality and abnormality rates. However, these results need to be compared with the toxicological data of caesium. Here, we examined the toxicity of both nonradioactive and radioactive caesium chloride on the pale grass blue butterfly. Larvae were fed a caesium-containing artificial diet, ingesting up to 149 MBq/kg (larva) of radioactive caesium ( 137 Cs) or a much higher amount of nonradioactive caesium. We examined the pupation rate, eclosion rate, survival rate up to the adult stage, and the forewing size. In contrast to previous internal exposure experiments using field-collected contaminated leaves, we could not detect any effect. We conclude that the butterfly is tolerant to ionising radiation from 137 Cs in the range tested but is vulnerable to radioactive contamination in the field. These results suggest that the biological effects in the field may be mediated through ecological systems and cannot be estimated solely based on radiation doses.

High morphological abnormality and mortality rates have been reported in the pale grass blue butterfly, Zizeeria maha, since the Fukushima nuclear accident. However, it remains uncertain if these effects are restricted to this butterfly. Here, we evaluated the effects of ingesting cabbage leaves grown with contaminated soils from Fukushima on the development and hemocytes of the cabbage white butterfly, Pieris rapae. Contaminated cabbage leaves containing various low levels of anthropogenic 134 Cs and 137 Cs radioactivity (less than natural 40 K radioactivity) were fed to larvae from Okinawa, the least contaminated locality in Japan. Negative developmental and morphological effects were detected in the experimental groups. The cesium (but not potassium) radioactivity concentration was negatively correlated with the granulocyte percentage in hemolymph, and the granulocyte percentage was positively correlated with the pupal eclosion rate, the adult achievement rate, and the total normality rate. These results demonstrated that ingesting low-level radiocesium contaminants in Fukushima (but not natural radiopotassium) imposed biologically negative effects on the cabbage white butterfly, as in the pale grass blue butterfly, at both cellular and organismal levels.

Adverse biological impacts of the Fukushima nuclear accident have been revealed using the pale grass blue butterfly, Zizeeria maha, since 2012, which were often considered incompatible with the conventional understanding of radiation biology. This discrepancy likely originates from different system conditions and methodologies. In this article, we first respond to comments from the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) regarding our study; "technical errors" in unit usage and mathematical models noted by UNSCEAR are not errors but reflect our research philosophy not to introduce theoretical assumptions associated with unit conversion and mathematical fit. Second, we review our recent studies to support the original 2012 conclusions. Because the high morphological abnormality rate and small body size detected in Fukushima in 2011 have already ceased, likely through adaptive evolution, their present geographical distributions were investigated throughout Japan. Local populations showing relatively high abnormality rates and small body sizes were rare and basically restricted to Miyagi and its northern populations excluding the Fukushima populations, supporting the causal involvement of the accident. Lastly, we stress the importance of understanding the whole picture of the biological impacts of the Fukushima accident. In addition to the direct radiation impacts, indirect impacts through unknown radiation-associated mechanisms, such as immunological responses to insoluble particulate matter and nutritional deficiencies in plants and animals, would be in effect. Further environmental studies beyond conventional radiation biology and physics are necessary to understand the complex responses of organisms, including humans, to the Fukushima nuclear accident., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The pale grass blue butterfly, Zizeeria maha, has been used to evaluate biological impacts of the Fukushima nuclear accident in March 2011. Here, we examined the possibility that butterflies have adapted to be robust in the contaminated environment. Larvae (n = 2432) were obtained from adult butterflies (n = 20) collected from 7 localities with various contamination levels in May 2012, corresponding to the 7th generation after the accident. When the larvae were reared on non-contaminated host plant leaves from Okinawa, the normality rates of natural exposure without artificial irradiation (as an indication of robustness) were high not only in the least contaminated locality but also in the most contaminated localities. The normality rates were similarly obtained when the larvae were reared on non-contaminated leaves with external irradiation or on contaminated leaves from Fukushima to deliver internal irradiation. The normality rate of natural exposure and that of external or internal exposure were correlated, suggesting that radiation resistance (or susceptibility) likely reflects general state of health. The normality rate of external or internal exposure was divided by the relative normality rate of natural exposure, being defined as the resistance value. The resistance value was the highest in the populations of heavily contaminated localities and was inversely correlated with the distance from the Fukushima Dai-ichi nuclear power plant. These results suggest that the butterfly population might have adapted to the contaminated environment within approximately 1 year after the accident. The present study may partly explain the decrease in mortality and abnormality rates later observed in the contaminated areas., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The pale grass blue butterfly has been used to assess the biological effects of the Fukushima nuclear accident. Zizeeria and Zizina are two closely related genera of grass blue butterflies that are widely distributed in tropical to temperate Asia, Australia, and Africa, making them suitable environmental indicators for these areas. However, the morphological features of the immature stages have been examined only in fragmentary fashion. Here, we reared Zizeeria maha argia, Zizeeria maha okinawana, Zizeeria karsandra karsandra, Zizina emelina emelina, Zizina otis labradus, and Zizina otis riukuensis using a standard rearing method that was developed for Zizeeria maha, and comparatively identified morphological traits to effectively classify the immature stages of species or subspecies. Morphological information on these and other subspecies including Zizeeria knysna knysna and Zizina otis antanossa from Africa was also collected from literature. The subspecies were all reared successfully. The subspecies all had dorsal nectary and tentacle organs with similar morphology. For the subspecies of Zizeeria maha, only minor morphological differences were noted. Similarly, the subspecies of Zizina otis shared many traits. Most importantly, Zizeeria and Zizina differed in the shape of the sensory hairs that accompany the dorsal nectary organ; Zizeeriahad pointed hairs, and Zizina had blunt or rounded hairs. However, Zizina emelina exhibited several intermediate features between these two genera. Overall, the morphological traits did not completely reflect the conventional systematic relationships. This comparative study describes the efficient rearing of the grass blue butterflies and provides a morphological basis for the use of these species as environmental indicators.

Background: The border ocellus, or eyespot, is a conspicuous color pattern element in butterfly wings. For two decades, it has been hypothesized that transcription factors such as Distal-less (Dll) are responsible for eyespot pattern development in butterfly wings, based on their expression in the prospective eyespots. In particular, it has been suggested that Dll is a determinant for eyespot size. However, functional evidence for this hypothesis has remained incomplete, due to technical difficulties., Results: Here, we show that ectopically expressed Dll induces ectopic elemental color patterns in the adult wings of the blue pansy butterfly, Junonia orithya (Lepidoptera, Nymphalidae). Using baculovirus-mediated gene transfer, we misexpressed Dll protein fused with green fluorescent protein (GFP) in pupal wings, resulting in ectopic color patterns, but not the formation of intact eyespots. Induced changes included clusters of black and orange scales (a basic feature of eyespot patterns), black and gray scales, and inhibition of cover scale development. In contrast, ectopic expression of GFP alone did not induce any color pattern changes using the same baculovirus-mediated gene transfer system., Conclusions: These results suggest that Dll plays an instructive role in the development of color pattern elements in butterfly wings, although Dll alone may not be sufficient to induce a complete eyespot. This study thus experimentally supports the hypothesis of Dll function in eyespot development.

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.

One important public concern in Japan is the potential health effects on animals and humans that live in the Tohoku-Kanto districts associated with the ingestion of foods contaminated with artificial radionuclides from the collapsed Fukushima Dai-ichi Nuclear Power Plant. Additionally, transgenerational or heritable effects of radiation exposure are also important public concerns because these effects could cause long-term changes in animal and human populations. Here, we concisely review our findings and implications related to the ingestional and transgenerational effects of radiation exposure on the pale grass blue butterfly, Zizeeria maha, which coexists with humans. The butterfly larval ingestion of contaminated leaves found in areas of human habitation, even at low doses, resulted in morphological abnormalities and death for some individuals, whereas other individuals were not affected, at least morphologically. This variable sensitivity serves as a basis for the adaptive evolution of radiation resistance. The distribution of abnormality and mortality rates from low to high doses fits well with a Weibull function model or a power function model. The offspring generated by morphologically normal individuals that consumed contaminated leaves exhibited high mortality rates when fed contaminated leaves; importantly, low mortality rates were restored when they were fed non-contaminated leaves. Our field monitoring over 3 years (2011-2013) indicated that abnormality and mortality rates peaked primarily in the fall of 2011 and decreased afterwards to normal levels. These findings indicate high impacts of early exposure and transgenerationally accumulated radiation effects over a specific period; however, the population regained normality relatively quickly after ∼15 generations within 3 years., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

The body size of the pale grass blue butterfly, Zizeeria maha, has been used as an environmental indicator of radioactive pollution caused by the Fukushima nuclear accident. However, geographical and temporal size distributions in Japan and temperature effects on size have not been established in this species. Here, we examined the geographical, temporal, and temperature-dependent changes of the forewing size of Z. maha argia in Japan. Butterflies collected in 2012 and 2013 from multiple prefectures throughout Japan demonstrated an inverse relationship of latitude and forewing size, which is the reverse of Bergmann's cline. The Fukushima population was significantly larger than the Aomori and Miyagi populations and exhibited no difference from most of the other prefectural populations. When monitored at a single geographic locality every other month, forewing sizes were the largest in April and the smallest in August. Rearing larvae at a constant temperature demonstrated that forewing size followed the temperature-size rule. Therefore, the converse Bergmann's rule and the temperature-size rule coexist in this multivoltine species. Our study establishes this species as a useful environmental indicator and supports the idea that the size reduction observed only in Fukushima Prefecture in 2011 was caused by the environmental stress of radioactive pollution.

The nymphalid groundplan has been proposed to explain diverse butterfly wing color patterns. In this model, each symmetry system is composed of a core element and a pair of paracore elements. The development of this elemental configuration has been explained by the induction model for positional information. However, the diversity of color patterns in other butterfly families in relation to the nymphalid groundplan has not been thoroughly examined. Here, we examined aberrant color pattern phenotypes of a lycaenid butterfly, Zizeeria maha, from mutagenesis and plasticity studies as well as from field surveys. In several mutants, the third and fourth spot arrays were coordinately positioned much closer to the discal spot in comparison to the normal phenotype. In temperature-shock types, the third and fourth array spots were elongated inwardly or outwardly from their normal positions. In field-caught spontaneous mutants, small black spots were located adjacent to normal black spots. Analysis of these aberrant phenotypes indicated that the spots belonging to the third and fourth arrays are synchronously changeable in position and shape around the discal spot. Thus, these arrays constitute paracore elements of the central symmetry system of the lycaenid butterflies, and the discal spot comprises the core element. These aberrant phenotypes can be explained by the black-inducing signals that propagate from the prospective discal spot, as predicted by the induction model. These results suggest the existence of long-range developmental signals that cover a large area of a wing not only in nymphalid butterflies, but also in lycaenid butterflies.

Background: Long-term monitoring of the biological impacts of the radioactive pollution caused by the Fukushima nuclear accident in March 2011 is required to understand what has occurred in organisms living in the polluted areas. Here, we investigated spatial and temporal changes of the abnormality rate (AR) in both field-caught adult populations and laboratory-reared offspring populations of the pale grass blue butterfly, Zizeeria maha, which has generation time of approximately one month. We monitored 7 localities (Fukushima, Motomiya, Hirono, Iwaki, Takahagi, Mito, and Tsukuba) every spring and fall over 3 years (2011-2013)., Results: The adult ARs of these localities quickly increased and peaked in the fall of 2011, which was not observed in non-contaminated localities. In the offspring generation, the total ARs, which include deaths at the larval, prepupal, and pupal stages and morphological abnormalities at the adult stage, peaked either in the fall of 2011 or in the spring of 2012, with much higher levels than those of the parent field populations, suggesting that high incidence of deaths and abnormalities might have occurred in the field populations. Importantly, the elevated ARs of the field and offspring populations settled back to a normal level by the fall of 2012 and by the spring of 2013, respectively. Similar results were obtained not only in the spatiotemporal dynamics of the number of individuals caught per minute but also in the temporal dynamics of the correlation coefficient between the adult abnormality rate and the ground radiation dose or the distance from the Power Plant., Conclusions: These results demonstrated an occurrence and an accumulation of adverse physiological and genetic effects in early generations, followed by their decrease and leveling off at a normal level, providing the most comprehensive record of biological dynamics after a nuclear accident available today. This study also indicates the importance of considering generation time and adaptive evolution in evaluating the biological impacts of artificial pollution in wild organisms.

Butterfly wing color patterns are highly complex and diverse, but they are believed to be derived from the nymphalid groundplan, which is composed of several color pattern systems. Among these pattern systems, the marginal band system, including marginal and submarginal bands, has rarely been studied. Here, we examined the color pattern diversity of the marginal band system among nymphalid butterflies. Marginal and submarginal bands are usually expressed as a pair of linear bands aligned with the wing margin. However, a submarginal band can be expressed as a broken band, an elongated oval, or a single dot. The marginal focus, usually a white dot at the middle of a wing compartment along the wing edge, corresponds to the pupal edge spot, one of the pupal cuticle spots that signify the locations of color pattern organizing centers. A marginal band can be expressed as a semicircle, an elongated oval, or a pair of eyespot-like structures, which suggest the organizing activity of the marginal focus. Physical damage at the pupal edge spot leads to distal dislocation of the submarginal band in Junonia almana and in Vanessa indica, suggesting that the marginal focus functions as an organizing center for the marginal band system. Taken together, we conclude that the marginal band system is developmentally equivalent to other symmetry systems. Additionally, the marginal band is likely a core element and the submarginal band a paracore element of the marginal band system, and both bands are primarily specified by the marginal focus organizing center.

Background: 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 (Motomiya (161 Bq/kg) and Koriyama (117 Bq/kg)); two in Kanto (Kashiwa (47.6 Bq/kg) and Musashino (6.4 Bq/kg)); one in Tokai (Atami (2.5 Bq/kg)); and from Okinawa (0.2 Bq/kg). In addition to the effects on the first generation, we examined the possible transgenerational effects of the diets on the next generation., Results: 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., Conclusions: 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.

A massive amount of radioactive materials has been released into the environment by the Fukushima Dai-ichi Nuclear Power Plant accident, but its biological impacts have rarely been examined. Here, we have quantitatively evaluated the relationship between the dose of ingested radioactive cesium and mortality and abnormality rates using the pale grass blue butterfly, Zizeeria maha. When larvae from Okinawa, which is likely the least polluted locality in Japan, were fed leaves collected from polluted localities, mortality and abnormality rates increased sharply at low doses in response to the ingested cesium dose. This dose-response relationship was best fitted by power function models, which indicated that the half lethal and abnormal doses were 1.9 and 0.76 Bq per larva, corresponding to 54,000 and 22,000 Bq per kilogram body weight, respectively. Both the retention of radioactive cesium in a pupa relative to the ingested dose throughout the larval stage and the accumulation of radioactive cesium in a pupa relative to the activity concentration in a diet were highest at the lowest level of cesium ingested. We conclude that the risk of ingesting a polluted diet is realistic, at least for this butterfly, and likely for certain other organisms living in the polluted area.

Background: On August 9th 2012, we published an original research article in Scientific Reports, concluding that artificial radionuclides released from the Fukushima Dai-ichi Nuclear Power Plant exerted genetically and physiologically adverse effects on the pale grass blue butterfly Zizeeria maha in the Fukushima area. Immediately following publication, many questions and comments were generated from all over the world. Here, we have clarified points made in the original paper and answered questions posed by the readers., Results: The following points were clarified. (1) There are many advantages to using the pale grass blue butterfly as an indicator species. (2) The forewings of the individuals collected in Fukushima were significantly smaller than in the northern and southern localities. (3) We observed growth retardation in the butterflies from the Fukushima area. (4) The aberrant colour patterns in the butterflies obtained in the Fukushima area were different from the colour patterns induced by temperature and sibling crosses but similar to those induced by external and internal exposures to the artificial radionuclides and by a chemical mutagen, suggesting that genetic mutations caused the aberrations. (5) This species of butterfly has been plentiful in Fukushima area for at least half a century. We here present specimens collected from Fukushima Prefecture before the accident. (6) Mutation accumulation was detected by the increase in the abnormality rates from May 2011 to September 2011. (7) The abnormal traits were heritable. (8) Our sampling localities were not affected by the tsunami. (9) We used a high enough number of samples to obtain statistically significant results. (10) The standard rearing method was followed, producing normal adults in the control groups. (11) The exposure experiments successfully reproduced the results of the field work. This species of butterfly is vulnerable to long-term low-dose internal and external exposures; however, insect cells are known to be resistant to short-term high-dose irradiation. This discrepancy is reconcilable based on the differences in the experimental conditions., Conclusions: We are just beginning to understand the biological effects of long-term low-dose exposures in animals. Further research is necessary to accurately assess the possible biological effects of the accident.

It is generally accepted that butterfly wing color-patterns have ecological and behavioral functions that evolved through natural selection. However, particular wing color-patterns may be produced physiologically in response to environmental stress, and they may lack significant function. These patterns would represent an extreme expression of phenotypic plasticity and can eventually be fixed genetically in a population. Here, three such cases in butterflies are concisely reviewed, and their possible mechanisms of genetic assimilation are discussed. First, a certain modified color-pattern of Vanessa indica induced by temperature treatments resembles the natural color-patterns of its closely related species of the genus Vanessa (sensu stricto). Second, a different type of color-pattern modification can be induced in Vanessa cardui as a result of a general stress response. This modified pattern is very similar to the natural color-pattern of its sister species Vanessa kershawi. Third, a field observation was reported, together with experimental support, to show that the color-pattern diversity of a regional population of Zizeeria maha increased at the northern range margin of this species in response to temperature stress. In these three cases, modified color-patterns are unlikely to have significant functions, and these cases suggest that phenotypic plasticity plays an important role in butterfly wing color-pattern evolution. A neutral or non-functional trait can be assimilated genetically if it is linked, like a parasitic trait, with another functional trait. In addition, it is possible that environmental stress causes epigenetic modifications of genes related to color-patterns and that their transgenerational inheritance facilitates the process of genetic assimilation of a neutral or non-functional trait.

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.