The second decade of the blue butterfly in Fukushima: Untangling the ecological field effects after the Fukushima nuclear accident.

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.

A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Cryptochrome 1 mediates light-dependent inclination magnetosensing in monarch butterflies.

Many animals use the Earth's geomagnetic field for orientation and navigation. Yet, the molecular and cellular underpinnings of the magnetic sense remain largely unknown. A biophysical model proposed that magnetoreception can be achieved through quantum effects of magnetically-sensitive radical pairs formed by the photoexcitation of cryptochrome (CRY) proteins. Studies in Drosophila are the only ones to date to have provided compelling evidence for the ultraviolet (UV)-A/blue light-sensitive type 1 CRY (CRY1) involvement in animal magnetoreception, and surprisingly extended this discovery to the light-insensitive mammalian-like type 2 CRYs (CRY2s) of both monarchs and humans. Here, we show that monarchs respond to a reversal of the inclination of the Earth's magnetic field in an UV-A/blue light and CRY1, but not CRY2, dependent manner. We further demonstrate that both antennae and eyes, which express CRY1, are magnetosensory organs. Our work argues that only light-sensitive CRYs function in animal light-dependent inclination-based magnetic sensing.

Light environment drives evolution of color vision genes in butterflies and moths.

Opsins, combined with a chromophore, are the primary light-sensing molecules in animals and are crucial for color vision. Throughout animal evolution, duplications and losses of opsin proteins are common, but it is unclear what is driving these gains and losses. Light availability is implicated, and dim environments are often associated with low opsin diversity and loss. Correlations between high opsin diversity and bright environments, however, are tenuous. To test if increased light availability is associated with opsin diversification, we examined diel niche and identified opsins using transcriptomes and genomes of 175 butterflies and moths (Lepidoptera). We found 14 independent opsin duplications associated with bright environments. Estimating their rates of evolution revealed that opsins from diurnal taxa evolve faster-at least 13 amino acids were identified with higher dN/dS rates, with a subset close enough to the chromophore to tune the opsin. These results demonstrate that high light availability increases opsin diversity and evolution rate in Lepidoptera.

Evolution of Phototransduction Genes in Lepidoptera.

Vision is underpinned by phototransduction, a signaling cascade that converts light energy into an electrical signal. Among insects, phototransduction is best understood in Drosophila melanogaster. Comparison of D. melanogaster against three insect species found several phototransduction gene gains and losses, however, lepidopterans were not examined. Diurnal butterflies and nocturnal moths occupy different light environments and have distinct eye morphologies, which might impact the expression of their phototransduction genes. Here we investigated: 1) how phototransduction genes vary in gene gain or loss between D. melanogaster and Lepidoptera, and 2) variations in phototransduction genes between moths and butterflies. To test our prediction of phototransduction differences due to distinct visual ecologies, we used insect reference genomes, phylogenetics, and moth and butterfly head RNA-Seq and transcriptome data. As expected, most phototransduction genes were conserved between D. melanogaster and Lepidoptera, with some exceptions. Notably, we found two lepidopteran opsins lacking a D. melanogaster ortholog. Using antibodies we found that one of these opsins, a candidate retinochrome, which we refer to as unclassified opsin (UnRh), is expressed in the crystalline cone cells and the pigment cells of the butterfly, Heliconius melpomene. Our results also show that butterflies express similar amounts of trp and trpl channel mRNAs, whereas moths express ∼50× less trp, a potential adaptation to darkness. Our findings suggest that while many single-copy D. melanogaster phototransduction genes are conserved in lepidopterans, phototransduction gene expression differences exist between moths and butterflies that may be linked to their visual light environment.

Developmental and hemocytological effects of ingesting Fukushima's radiocesium on the cabbage white butterfly Pieris rapae.

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 134Cs and 137Cs radioactivity (less than natural 40K 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.

Transgenerational effects of historic radiation dose in pale grass blue butterflies around Fukushima following the Fukushima Dai-ichi Nuclear Power Plant meltdown accident.

Low dose radiation effects have been investigated in Chernobyl for many years but there is uncertainty about initial doses received by many animal species. However, the Fukushima Dai-ichi Nuclear Power Plant accident opens an opportunity to study the effects of the initial low historic dose on directly exposed species and their progeny during a time where the contaminating radionuclides are decaying. In this paper, it is proposed that historic acute exposure and its resulting non-targeted effects (NTEs) may be partially involved in the high mortality/abnormality rates seen across generations of pale grass blue butterflies (Zizeeria maha) around Fukushima. Data from Hiyama et al. (2012) on the morphological abnormality frequencies in Z. maha collected around Fukushima and their progeny were used in this paper. Two dose reconstruction methods based on the Gaussian plume model were used to determine the external absorbed dose to the first exposed generation from both ground shine and plume shine. One method involved the use of the dose rate recorded at the time of collection and only took Cs-137 into account. The other involved using release rates and atmospheric conditions to determine the doses and considered Cs-137 and Cs-134. The reconstructed doses were plotted against the mortality rates and abnormality frequencies across generations. The mortality rates of the progeny from irradiated progenitors increased linearly with the increasing historic radiation doses reconstructed using both Cs-137 and Cs-134 sources. Additionally, a higher level of morphological abnormalities was observed in progeny than in the progenitors. The mean abnormality frequencies also increased throughout generations. As these results are a sign of NTEs being involved, it can be suggested that increasing mutation levels across generations may result, in part, from NTEs induced by the initial low dose received by the first exposed generation. However, continual accumulation of mutations over generations in their natural contaminated habitats remains a likely contributor into the observed outcome.

Gene expression profiling across ontogenetic stages in the wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation.

In temperate latitudes, many insects enter diapause (dormancy) during the cold season, a period during which developmental processes come to a standstill. The wood white (Leptidea sinapis) is a butterfly species distributed across western Eurasia that shows photoperiod-induced diapause with variation in critical day-length across populations at different latitudes. We assembled transcriptomes and estimated gene expression levels at different developmental stages in experimentally induced directly developing and diapausing cohorts of a single Swedish population of L. sinapis to investigate the regulatory mechanisms underpinning diapause initiation. Different day lengths resulted in expression changes of developmental genes and affected the rate of accumulation of signal molecules, suggesting that diapause induction might be controlled by increased activity of monoamine neurotransmitters in larvae reared under short-day light conditions. Expression differences between light treatment groups of two monoamine regulator genes (DDC and ST) were observed already in instar III larvae. Once developmental pathways were irreversibly set at instar V, a handful of genes related to dopamine production were differentially expressed leading to a significant decrease in expression of global metabolic genes and increase in expression of genes related to fatty acid synthesis and sequestration. This is in line with a time-dependent (hour-glass) model of diapause regulation where a gradual shift in the concentration of monoamine neurotransmitters and their metabolites during development of larvae under short-day conditions leads to increased storage of fat, decreased energy expenditures, and ultimately developmental stasis at the pupal stage.

Current Status of the Blue Butterfly in Fukushima Research.

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.

Robustness and Radiation Resistance of the Pale Grass Blue Butterfly from Radioactively Contaminated Areas: A Possible Case of Adaptive Evolution.

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.

Changes in structural and pigmentary colours in response to cold stress in Polyommatus icarus butterflies.

While numerous papers have investigated the effects of thermal stress on the pigmentary colours of butterfly wings, such studies regarding structural colours are mostly lacking, despite the important role they play in sexual communication. To gain insight into the possible differences between the responses of the two kinds of colouration, we investigated the effects of prolonged cold stress (cooling at 5 °C for up to 62 days) on the pupae of Polyommatus icarus butterflies. The wing surfaces coloured by photonic crystal-type nanoarchitectures (dorsal) and by pigments (ventral) showed markedly different behaviours. The ventral wing surfaces exhibited stress responses proportional in magnitude to the duration of cooling and showed the same trend for all individuals, irrespective of their sex. On the dorsal wing surface of the males, with blue structural colouration, a smaller magnitude response was found with much more pronounced individual variations, possibly revealing hidden genetic variations. Despite the typical, pigmented brown colour of the dorsal wing surface of the females, all cooled females exhibited a certain degree of blue colouration. UV-VIS spectroscopy, optical microscopy, and scanning and transmission electron microscopy were used to evaluate the magnitude and character of the changes induced by the prolonged cold stress.

Ingestional and transgenerational effects of the Fukushima nuclear accident on the pale grass blue butterfly.

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.

Spatiotemporal abnormality dynamics of the pale grass blue butterfly: three years of monitoring (2011-2013) after the Fukushima nuclear accident.

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.

Genetic and ecological studies of animals in Chernobyl and Fukushima.

Recent advances in genetic and ecological studies of wild animal populations in Chernobyl and Fukushima have demonstrated significant genetic, physiological, developmental, and fitness effects stemming from exposure to radioactive contaminants. The few genetic studies that have been conducted in Chernobyl generally show elevated rates of genetic damage and mutation rates. All major taxonomic groups investigated (i.e., birds, bees, butterflies, grasshoppers, dragonflies, spiders, mammals) displayed reduced population sizes in highly radioactive parts of the Chernobyl Exclusion Zone. In Fukushima, population censuses of birds, butterflies, and cicadas suggested that abundances were negatively impacted by exposure to radioactive contaminants, while other groups (e.g., dragonflies, grasshoppers, bees, spiders) showed no significant declines, at least during the first summer following the disaster. Insufficient information exists for groups other than insects and birds to assess effects on life history at this time. The differences observed between Fukushima and Chernobyl may reflect the different times of exposure and the significance of multigenerational mutation accumulation in Chernobyl compared to Fukushima. There was considerable variation among taxa in their apparent sensitivity to radiation and this reflects in part life history, physiology, behavior, and evolutionary history. Interestingly, for birds, population declines in Chernobyl can be predicted by historical mitochondrial DNA base-pair substitution rates that may reflect intrinsic DNA repair ability.

Fukushima's biological impacts: the case of the pale grass blue butterfly.

To evaluate the effects of the Fukushima nuclear accident on the surrounding area, we studied the pale grass blue butterfly Zizeeria maha, the most common butterfly in Japan. We here review our important findings and their implications. We found forewing size reduction, growth retardation, high mortality rates, and high abnormality rates in the field and reared samples. The abnormality rates observed in September 2011 were higher than those observed in May 2011 in almost all localities, implying transgenerational accumulation of genetic damage. Some of the abnormal traits in the F1 generation were inherited by the F2 generation. In a particular cross, the F2 abnormality rate scored 57%. The forewing size reduction and high mortality and abnormality rates were reproduced in external and internal exposure experiments conducted in our laboratory using Okinawa larvae. We observed the possible real-time evolution of radiation resistance in the Fukushima butterflies, which, in retrospect, indicates that field sampling attempts at the very early stages of such accidents are required to understand the ecodynamics of polluted regions. We propose, as the postulates of pollutant-induced biological impacts, that the collection of phenotypic data from the field and their relevant reproduction in the laboratory should be the basis of experimental design to demonstrate the biological effects of environmental pollutants and to investigate the molecular mechanisms responsible for these effects.

The biological impacts of ingested radioactive materials on the pale grass blue butterfly.

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.

Behavioural thermoregulation and the relative roles of convection and radiation in a basking butterfly.

Poikilothermic animals are often reliant on behavioural thermoregulation to elevate core-body temperature above the temperature of their surroundings. Butterflies are able to do this by altering body posture and location while basking, however the specific mechanisms that achieve such regulation vary among species. The role of the wings has been particularly difficult to describe, with uncertainty surrounding whether they are positioned to reduce convective heat loss or to maximise heat gained through radiation. Characterisation of the extent to which these processes affect core-body temperature will provide insights into the way in which a species׳ thermal sensitivity and morphological traits have evolved. We conducted field and laboratory measurements to assess how basking posture affects the core-body temperature of an Australian butterfly, the common brown (Heteronympha merope). We show that, with wings held open, heat lost through convection is reduced while heat gained through radiation is simultaneously maximised. These responses have been incorporated into a biophysical model that accurately predicts the core-body temperature of basking specimens in the field, providing a powerful tool to explore how climate constrains the distribution and abundance of basking butterflies.

Low-dose radiation, scientific scrutiny, and requirements for demonstrating effects.

Recent nuclear accidents have prompted renewed interest in the fitness consequences of low-dose radiation. Hiyama et al. provided information on such effects in the Japanese pale grass blue butterfly in a paper that has been viewed more than 300,000 times, prompting a barrage of criticism. These exchanges highlight the role of scrutiny in studies with potential effects on humans, but also raise questions about minimum requirements for demonstrating biological effects.

The Fukushima nuclear accident and the pale grass blue butterfly: evaluating biological effects of long-term low-dose exposures.

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.