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Protein delivery to cells in vivo has great potential for the functional analysis of proteins in nonmodel organisms. In this study, using the butterfly wing system, we investigated a method of protein delivery to insect epithelial cells that allows for easy access, treatment, and observation in real time in vivo. Topical and systemic applications (called the sandwich and injection methods, respectively) were tested. In both methods, green/orange fluorescent proteins (GFP/OFP) were naturally incorporated into intracellular vesicles and occasionally into the cytosol from the apical surface without any delivery reagent. However, the antibodies were not delivered by the sandwich method at all, and were delivered only into vesicles by the injection method. A membrane-lytic peptide, L17E, appeared to slightly improve the delivery of GFP/OFP and antibodies. A novel peptide reagent, ProteoCarry, successfully promoted the delivery of both GFP/OFP and antibodies into the cytosol via both the sandwich and injection methods. These protein delivery results will provide opportunities for the functional molecular analysis of proteins in butterfly wing development, and may offer a new way to deliver proteins into target cells in vivo in nonmodel organisms. [ABSTRACT FROM AUTHOR]

The Fukushima nuclear accident in March 2011 caused biological impacts on the pale grass blue butterfly Zizeeria maha. At least some of the impacts are likely mediated by the host plant, resulting in "field effects". However, to obtain the whole picture of the impacts, direct exposure effects should also be evaluated. Here, we examined the distribution of experimentally ingested anthropogenic cesium-137 (137Cs) in adult butterfly bodies using imaging plate autoradiography. We showed that 137Cs ingested by larvae was incorporated into adult bodies and was biased to females, although the majority of ingested 137Cs was excreted in the pupal cuticle and excretory material during eclosion. 137Cs accumulation in adult bodies was the highest in the abdomen, followed by the thorax and other organs. These results suggest that 137Cs accumulation in reproductive organs may cause adverse transgenerational or maternal effects mediated by reactive oxygen species (ROS) on germ cells. 137Cs accumulation was detected in field individuals collected in September 2011 and September 2016 but not in May 2011, which is consistent with the abnormality dynamics known from previous studies. Taken together, these results contribute to an integrative understanding of the multifaceted biological effects of the Fukushima nuclear accident in the field. [ABSTRACT FROM AUTHOR]

Simple Summary: The larvae of many butterflies and moths show various body colors within a species. We studied the possible effect of the host plant color on the plastic larval body color of the lycaenid butterfly Zizeeria maha, which shows various larval body colors ranging from green to red, even within a sibling group. The females laid eggs normally on both green and red leaves, despite showing a green preference. The larvae grew equally by consuming either green or red leaves. The number of red larvae decreased as the larvae grew, demonstrating a stage-dependent variation. When the larvae were fed either green or red leaves across multiple generations, the red larvae were significantly more abundant in the red leaf lineage than in the green leaf lineage. Moreover, the red-fed siblings showed a significantly higher red larval frequency than the green-fed siblings in the red leaf lineage but not in the green leaf lineage. These results suggest that, in this butterfly species, the plastic larval body color for crypsis may be affected not only by the color of the leaves that the larvae consume but also by the color of the leaves that their mothers consume, in addition to a stage-dependent color variation. Many lepidopteran larvae show body color polyphenism, and their colors may be cryptic on the host plant leaves. To elucidate the effect of the host plant color on the plastic larval body color, we focused on the lycaenid butterfly Zizeeria maha, which shows various larval body colors ranging from green to red, even within a sibling group. We showed that oviposition was normally performed on both green and red leaves, despite a green preference and the fact that the larvae grew equally by consuming either green or red leaves. The number of red larvae decreased from the second instar stage to the fourth instar stage, demonstrating a stage-dependent variation. When the larvae were fed either green or red leaves across multiple generations of the lineages, the red larvae were significantly more abundant in the red leaf lineage than in the green leaf lineage. Moreover, the red-fed siblings showed a significantly higher red larval frequency than the green-fed siblings in the red-leaf lineage but not in the green-leaf lineage. These results suggest that, in this butterfly species, the plastic larval body color for crypsis may be affected not only by the color of the leaves that the larvae consume (single-generation effect) but also by the color of the leaves that their mothers consume (maternal effect), in addition to a stage-dependent color variation. [ABSTRACT FROM AUTHOR]

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 (137Cs) 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 KEY POINTS: A series of observational and experimental studies have demonstrated causal adverse effects of the Fukushima nuclear accident on the pale grass blue butterfly even at the low‐level radiation exposure in the field, although the butterfly is tolerant of radiation exposure in the laboratory.The 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.In response to radiation exposure, the host plant may change its own nutritional contents, may upregulate secondary metabolites that affect herbivorous insects, and may be affected by changes in endophytic soil microbes.The delicate ecological balances among the butterfly, its host plant, and soil microbes may have been affected by radioactive pollution in Fukushima, which has important implications for environmental policies and human health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 noncontaminated 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. [ABSTRACT FROM AUTHOR]

The pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae) has been used as an environmental indicator species for radioactive pollution after the Fukushima nuclear accident. Here, based on the one-individual tracking method in the field, we examined dispersal-associated and other behavioral traits of this butterfly, focusing on two subspecies, Z. maha argia in mainland Japan and Z. maha okinawana in Okinawa. The accumulated distances in the adult lifespan were 18.9 km and 38.2 km in mainland and Okinawa males, respectively, and 15.0 km and 7.8 km in mainland and Okinawa females, respectively. However, the mean distance from the starting point was only 24.2 m and 21.1 m in the mainland and Okinawa males, respectively, and 13.7 m and 7.4 m in the mainland and Okinawa females, respectively. Some quantitative differences in resting and feeding were found between subspecies and between sexes. The ARIMA (autoregressive integrated moving average) model indicated that the dispersal distance was 52.3 m (99% confidence interval value of 706.6 m) from the starting point in mainland males. These results support the idea that despite some behavioral differences, both subspecies of this butterfly are suitable as an environmental indicator because of the small dispersal ranges.

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.

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 (137Cs) 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 137Cs 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.

To systematically analyze biological changes with spatiotemporal dynamics, it is important to establish a system that is amenable for real-time in vivo imaging at various size levels. Herein, we focused on the developing pupal wing tissues in the pale grass blue butterfly, Zizeeria maha, as a system of choice for a systematic multiscale approach in vivo in real time. We showed that the entire pupal wing could be monitored throughout development using a high-resolution bright-field time-lapse imaging system under the forewing-lift configuration; we recorded detailed dynamics of the dorsal and ventral epithelia that behaved independently for peripheral adjustment. We also monitored changes in the dorsal hindwing at the compartmental level and directly observed evaginating scale buds. We also employed a confocal laser microscopy system with multiple fluorescent dyes for three-dimensional observations at the tissue and cellular levels. We discovered extensive cellular clusters that may be functionally important as a unit of cellular communication and differentiation. We also identified epithelial discal and marginal dents that may function during development. Together, this lycaenid forewing system established a foundation to study the differentiation process of epithelial cells and can be used to study biophysically challenging mechanisms such as the determination of color patterns and scale nanoarchitecture at the multiscale levels.

The pale grass blue butterfly Zizeeria maha has been used as an environmental indicator to evaluate the biological impacts of the Fukushima nuclear accident. A high morphological abnormality rate (AR) of this butterfly was detected in 2011 from radioactively contaminated areas at 37–38°N. However, the geographical AR distribution has not been documented for the entirety of northeastern Japan. Additionally, the geographical distribution of the wing color pattern modification rate (MR) of temperature-shock type remains undocumented. Here, we collected adult butterflies from many localities in northeastern Japan in 2014 and examined the local AR and MR. Both AR and MR were generally low throughout the 44 local populations surveyed. Latitudinal AR and MR distributions indicated a gap zone at approximately 39°N. The mean AR and the mean MR of the populations south of the gap zone were low (AR= 3.0%,MR= 1.1%), whereas those of the northern populations were relatively high (AR= 10.6%, MR =10.3%). Logistic regression analyses revealed that abnormalities and modifications were associated with temperature-related variables. We conclude that abnormalities and modifications are generally rare, but that their rates are higher in the northern populations than in the southern ones. These results, along with evidence from other studies, strongly suggest that the high AR detected in 2011 from contaminated areas was induced by anthropogenic radioactive mutagens. This study presents a basic dataset of the current wildlife state of Z.maha in northeastern Japan, which facilitates a future use of this butterfly species as an environmental indicator. [ABSTRACT FROM AUTHOR]

The pale grass blue butterfly Zizeeria maha has been used as an environmental indicator to evaluate the biological impacts of the Fukushima nuclear accident. A high morphological abnormality rate (AR) of this butterfly was detected in 2011 from radioactively contaminated areas at 37-38°N. However, the geographical AR distribution has not been documented for the entirety of northeastern Japan. Additionally, the geographical distribution of the wing color pattern modification rate (MR) of temperature-shock type remains undocumented. Here, we collected adult butterflies from many localities in northeastern Japan in 2014 and examined the local AR and MR. Both AR and MR were generally low throughout the 44 local populations surveyed. Latitudinal AR and MR distributions indicated a gap zone at approximately 39°N. The mean AR and the mean MR of the populations south of the gap zone were low (AR= 3.0%,MR= 1.1%), whereas those of the northern populations were relatively high (AR= 10.6%, MR =10.3%). Logistic regression analyses revealed that abnormalities and modifications were associated with temperature-related variables.We conclude that abnormalities and modifications are generally rare, but that their rates are higher in the northern populations than in the southern ones. These results, along with evidence from other studies, strongly suggest that the high AR detected in 2011 from contaminated areas was induced by anthropogenic radioactive mutagens. This study presents a basic dataset of the current wildlife state of Z.maha in northeastern Japan, which facilitates a future use of this butterfly species as an environmental indicator. [ABSTRACT FROM AUTHOR]

Larvae of the pale grass blue butterfly, Zizeeria maha (Kollar) (Lepidoptera: Lycaenidae), feed exclusively on Oxalis corniculata L. (Oxalidales: Oxalidaceae), which accumulates oxalic acid as with other Oxalidaceae species. Larvae were stimulated to feed on artificial diets containing a crude methanolic extract from host plant leaves. Fractionations and bioassays revealed that the strong feeding activity was found in the water layer, from which oxalic acid was detected as a major compound. Removal of oxalic acid as calcium oxalate precipitates by addition of calcium chloride into the water layer resulted in a significant decrease in feeding activity on the filtrate. Re-addition of oxalic acid to the filtrate recovered the feeding activity. The addition of 260 μmol oxalic acid, corresponding to 1 g fresh leaves of Oxalis, to 1 g of artificial diet significantly stimulated feeding compared with the intact artificial diet. Therefore, oxalic acid was concluded to be a major feeding stimulant for Zizeeria maha larvae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Lycaenidae is one of the larger of the world's butterfly families, based on number and diversity of species, but knowledge of roosting in this group is sparse. Zizina otis riukuensis and Zizeeria maha okinawana are two small lycaenids that are commonly found in urban settings and widely distributed across much of Asia. We conducted experiments on a university campus to determine the plant species and plant structures commonly used by these two blues when roosting. We also tested the hypothesis that gregarious roosting exists in these two blues by demonstrating the non-random distribution of roosting blues and the tight mapping of their roosts to the spatial distribution of specific plant species and/or specific plant structures, as well as by demonstrating behavioral interactions among individuals during roosting-assembly. We found that both Z. otis and Z. maha roosted primarily on flowers and fruits of Tridax procumbens and Vernonia cinerea . We also found that these blues formed conspicuous roosting aggregations with significant positive associations between the flowers and fruits of both T. procumbens and V. cinerea and the blues. Moreover, our behavioral observations showed that these blues expressed various levels of interaction during roosting gatherings. Based on these findings, we conclude that gregarious roosting exists in both Z. otis and Z. maha . To our knowledge, this paper represents one of the first demonstration of nocturnal gregarious roosting in lycaenids. This study also highlights the importance of institutional estates in providing roosting resources for butterflies in urban ecosystems.

The pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae) has been used as an environmental indicator species for radioactive pollution after the Fukushima nuclear accident. Here, based on the one-individual tracking method in the field, we examined dispersal-associated and other behavioral traits of this butterfly, focusing on two subspecies, Z. maha argia in mainland Japan and Z. maha okinawana in Okinawa. The accumulated distances in the adult lifespan were 18.9 km and 38.2 km in mainland and Okinawa males, respectively, and 15.0 km and 7.8 km in mainland and Okinawa females, respectively. However, the mean distance from the starting point was only 24.2 m and 21.1 m in the mainland and Okinawa males, respectively, and 13.7 m and 7.4 m in the mainland and Okinawa females, respectively. Some quantitative differences in resting and feeding were found between subspecies and between sexes. The ARIMA (autoregressive integrated moving average) model indicated that the dispersal distance was 52.3 m (99% confidence interval value of 706.6 m) from the starting point in mainland males. These results support the idea that despite some behavioral differences, both subspecies of this butterfly are suitable as an environmental indicator because of the small dispersal ranges. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (137Cs) 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 137Cs 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. [ABSTRACT FROM AUTHOR]