In Silico and In Vitro Evaluation of the Molecular Mimicry of the SARS-CoV-2 Spike Protein by Common Short Constituent Sequences (cSCSs) in the Human Proteome: Toward Safer Epitope Design for Vaccine Development.

Spike protein sequences in SARS-CoV-2 have been employed for vaccine epitopes, but many short constituent sequences (SCSs) in the spike protein are present in the human proteome, suggesting that some anti-spike antibodies induced by infection or vaccination may be autoantibodies against human proteins. To evaluate this possibility of "molecular mimicry" in silico and in vitro, we exhaustively identified common SCSs (cSCSs) found both in spike and human proteins bioinformatically. The commonality of SCSs between the two systems seemed to be coincidental, and only some cSCSs were likely to be relevant to potential self-epitopes based on three-dimensional information. Among three antibodies raised against cSCS-containing spike peptides, only the antibody against EPLDVL showed high affinity for the spike protein and reacted with an EPLDVL-containing peptide from the human unc-80 homolog protein. Western blot analysis revealed that this antibody also reacted with several human proteins expressed mainly in the small intestine, ovary, and stomach. Taken together, these results showed that most cSCSs are likely incapable of inducing autoantibodies but that at least EPLDVL functions as a self-epitope, suggesting a serious possibility of infection-induced or vaccine-induced autoantibodies in humans. High-risk cSCSs, including EPLDVL, should be excluded from vaccine epitopes to prevent potential autoimmune disorders.

Spatiotemporal Characteristics Determining the Multifaceted Nature of Reactive Oxygen, Nitrogen, and Sulfur Species in Relation to Proton Homeostasis.

Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

Soil Microbes and Plant-Associated Microbes in Response to Radioactive Pollution May Indirectly Affect Plants and Insect Herbivores: Evidence for Indirect Field Effects from Chernobyl and Fukushima.

The biological impacts of the nuclear accidents in Chernobyl (1986) and Fukushima (2011) on wildlife have been studied in many organisms over decades, mainly from dosimetric perspectives based on laboratory experiments using indicator species. However, ecological perspectives are required to understand indirect field-specific effects among species, which are difficult to evaluate under dosimetric laboratory conditions. From the viewpoint that microbes play a fundamental role in ecosystem function as decomposers and symbionts for plants, we reviewed studies on microbes inhabiting soil and plants in Chernobyl and Fukushima in an attempt to find supporting evidence for indirect field-specific effects on plants and insect herbivores. Compositional changes in soil microbes associated with decreases in abundance and species diversity were reported, especially in heavily contaminated areas of both Chernobyl and Fukushima, which may accompany explosions of radioresistant species. In Chernobyl, the population size of soil microbes remained low for at least 20 years after the accident, and the abundance of plant-associated microbes, which are related to the growth and defense systems of plants, possibly decreased. These reported changes in microbes likely affect soil conditions and alter plant physiology. These microbe-mediated effects may then indirectly affect insect herbivores through food-mass-mediated, pollen-mediated, and metabolite-mediated interactions. Metabolite-mediated interactions may be a major pathway for ecological impacts at low pollution levels and could explain the decreases in insect herbivores in Fukushima. The present review highlights the importance of the indirect field effects of long-term low-dose radiation exposure under complex field circumstances.

Live Detection of Intracellular Chitin in Butterfly Wing Epithelial Cells In Vivo Using Fluorescent Brightener 28: Implications for the Development of Scales and Color Patterns.

Chitin is the major component of the extracellular cuticle and plays multiple roles in insects. In butterflies, chitin builds wing scales for structural colors. Here, we show that intracellular chitin in live cells can be detected in vivo with fluorescent brightener 28 (FB28), focusing on wing epithelial cells of the small lycaenid butterfly Zizeeria maha immediately after pupation. A relatively small number of cells at the apical surface of the epithelium were strongly FB28-positive in the cytosol and seemed to have extensive ER-Golgi networks, which may be specialized chitin-secreting cells. Some cells had FB28-positive tadpole-tail-like or rod-like structures relative to the nucleus. We detected FB28-positive hexagonal intracellular objects and their associated structures extending toward the apical end of the cell, which may be developing scale bases and shafts. We also observed FB28-positive fibrous intracellular structures extending toward the basal end. Many cells were FB28-negative in the cytosol, which contained FB28-positive dots or discs. The present data are crucial to understanding the differentiation of the butterfly wing epithelium, including scale formation and color pattern determination. The use of FB28 in probing intracellular chitin in live cells may be applicable to other insect systems.

Imaging Plate Autoradiography for Ingested Anthropogenic Cesium-137 in Butterfly Bodies: Implications for the Biological Impacts of the Fukushima Nuclear Accident.

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.

Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development.

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.

Pleiotropic Functions of Nitric Oxide Produced by Ascorbate for the Prevention and Mitigation of COVID-19: A Revaluation of Pauling's Vitamin C Therapy.

Linus Pauling, who was awarded the Nobel Prize in Chemistry, suggested that a high dose of vitamin C (l-ascorbic acid) might work as a prevention or treatment for the common cold. Vitamin C therapy was tested in clinical trials, but clear evidence was not found at that time. Although Pauling's proposal has been strongly criticized for a long time, vitamin C therapy has continued to be tested as a treatment for a variety of diseases, including coronavirus infectious disease 2019 (COVID-19). The pathogen of COVID-19, SARS-CoV-2, belongs to the ß-coronavirus lineage, which includes human coronavirus, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). This review intends to shed new light on vitamin C antiviral activity that may prevent SARS-CoV-2 infection through the chemical production of nitric oxide (NO). NO is a gaseous free radical that is largely produced by the enzyme NO synthase (NOS) in cells. NO produced by upper epidermal cells contributes to the inactivation of viruses and bacteria contained in air or aerosols. In addition to enzymatic production, NO can be generated by the chemical reduction of inorganic nitrite (NO2-), an alternative mechanism for NO production in living organisms. Dietary vitamin C, largely contained in fruits and vegetables, can reduce the nitrite in saliva to produce NO in the oral cavity when chewing foods. In the stomach, salivary nitrite can also be reduced to NO by vitamin C secreted from the epidermal cells of the stomach. The strong acidic pH of gastric juice facilitates the chemical reduction of salivary nitrite to produce NO. Vitamin C contributes in multiple ways to the host innate immune system as a first-line defense mechanism against pathogens. Highlighting chemical NO production by vitamin C, we suggest that controversies on the therapeutic effects of vitamin C in previous clinical trials may partly be due to less appreciation of the pleiotropic functions of vitamin C as a universal bioreductant.

The Plastic Larval Body Color of the Pale Grass Blue Butterfly Zizeeria maha (Lepidoptera: Lycaenidae) in Response to the Host Plant Color: The Maternal Effect on Crypsis.

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.

Butterfly Wing Color Pattern Modification Inducers May Act on Chitin in the Apical Extracellular Site: Implications in Morphogenic Signals for Color Pattern Determination.

Butterfly wing color patterns are modified by various treatments, such as temperature shock, injection of chemical inducers, and covering materials on pupal wing tissue. Their mechanisms of action have been enigmatic. Here, we investigated the mechanisms of color pattern modifications usingthe blue pansy butterfly Junoniaorithya. We hypothesized that these modification-inducing treatments act on the pupal cuticle or extracellular matrix (ECM). Mechanical load tests revealed that pupae treated with cold shock or chemical inducers were significantly less rigid, suggesting that these treatments made cuticle formation less efficient. A known chitin inhibitor, FB28 (fluorescent brightener 28), was discovered to efficiently induce modifications. Taking advantage of its fluorescent character, fluorescent signals from FB28 were observed in live pupae in vivo from the apical extracellular side and were concentrated at the pupal cuticle focal spots immediately above the eyespot organizing centers. It was shown that chemical modification inducers and covering materials worked additively. Taken together, various modification-inducing treatments likely act extracellularly on chitin or other polysaccharides to inhibit pupal cuticle formation or ECM function, which probably causes retardation of morphogenic signals. It is likely that an interactive ECM is required for morphogenic signals for color pattern determination to travel long distances.

Phenotypic Plasticity of the Mimetic Swallowtail Butterfly Papilio polytes: Color Pattern Modifications and Their Implications in Mimicry Evolution.

Butterfly wing color patterns are sensitive to environmental stress, such as temperature shock, and this phenotypic plasticity plays an important role in color pattern evolution. However, the potential contributions of phenotypic plasticity to mimicry evolution have not been evaluated. Here, we focused on the swallowtail butterfly Papilio polytes, which has nonmimetic and mimetic forms in females, to examine its plastic phenotypes. In the nonmimetic form, medial white spots and submarginal reddish spots in the ventral hindwings were enlarged by cold shock but were mostly reduced in size by heat shock. These temperature-shock-induced color pattern modifications were partly similar to mimetic color patterns, and nonmimetic females were more sensitive than males and mimetic females. Unexpectedly, injection of tungstate, a known modification inducer in nymphalid and lycaenid butterflies, did not induce any modification, but fluorescent brightener 28, another inducer discovered recently, induced unique modifications. These results suggest that phenotypic plasticity in nonmimetic females might have provided a basis of natural selection for mimetic color patterns during evolution.

Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima.

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 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.

Metabolomic Profiles of the Creeping Wood Sorrel Oxalis corniculata in Radioactively Contaminated Fields in Fukushima: Dose-Dependent Changes in Key Metabolites.

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 (K1R1H1; 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.

Metabolomic Response of the Creeping Wood Sorrel Oxalis corniculata to Low-Dose Radiation Exposure from Fukushima's Contaminated Soil.

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 α1-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.

Nutrient Imbalance of the Host Plant for Larvae of the Pale Grass Blue Butterfly May Mediate the Field Effect of Low-Dose Radiation Exposure in Fukushima: Dose-Dependent Changes in the Sodium Content.

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 (137Cs) 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 (137Cs) 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 Fractal Geometry of the Nymphalid Groundplan: Self-Similar Configuration of Color Pattern Symmetry Systems in Butterfly Wings.

The nymphalid groundplan is an archetypical color pattern of nymphalid butterflies involving three major symmetry systems and a discal symmetry system, which share the basic morphogenesis unit. Here, the morphological and spatial relationships among these symmetry systems were studied based on cross-species comparisons of nymphalid hindwings. Based on findings in Neope and Symbrenthia, all three major symmetry systems can be expressed as bands, spots, or eyespot-like structures, suggesting equivalence (homology) of these systems in developmental potential. The discal symmetry system can also be expressed as various structures. The discal symmetry system is circularly surrounded by the central symmetry system, which may then be surrounded by the border and basal symmetry systems, based mainly on findings in Agrias, indicating a unified supersymmetry system covering the entire wing. The border symmetry system can occupy the central part of the wing when the central symmetry system is compromised, as seen in Callicore. These results suggest that butterfly color patterns are hierarchically constructed in a self-similar fashion, as the fractal geometry of the nymphalid groundplan. This self-similarity is likely mediated by the serial induction of organizers, and symmetry breaking of the system morphology may be generated by the collision of opposing signals during development.

Morphological and Spatial Diversity of the Discal Spot on the Hindwings of Nymphalid Butterflies: Revision of the Nymphalid Groundplan.

Diverse butterfly wing color patterns are understood through the nymphalid groundplan, which mainly consists of central, border, and basal symmetry systems and a discal spot. However, the status of the discal spot remains unexplored. Here, the morphological and spatial diversity of the discal spot was studied in nymphalid hindwings. The discal spot is expressed as a small or narrow spot, a pair of parallel bands, a diamond or oval structure, a large dark spot, a few fragmented spots, or a white structure. In some cases, the discal spot is morphologically similar to and integrated with the central symmetry system (CSS). The discal spot is always located in a distal portion of the discal cell defined by the wing veins, which is sandwiched by the distal and proximal bands of the CSS (dBC and pBC) and is rarely occupied by border ocelli. The CSS occasionally has the central band (cBC), which differs from the discal spot. These results suggest that the discal spot is an independent and diverse miniature symmetry system nested within the CSS and that the locations of the discal spot and the CSS are determined by the wing veins at the early stage of wing development.

Butterfly eyespot color pattern formation requires physical contact of the pupal wing epithelium with extracellular materials for morphogenic signal propagation.

BACKGROUND: Eyespot color pattern formation on butterfly wings is sensitive to physical damage and physical distortion as well as physical contact with materials on the surface of wing epithelial tissue at the pupal stage. Contact-mediated eyespot color pattern changes may imply a developmental role of the extracellular matrix in morphogenic signal propagation. Here, we examined eyespot responses to various contact materials, focusing on the hindwing posterior eyespots of the blue pansy butterfly, Junonia orithya. RESULTS: Contact with various materials, including both nonbiological and biological materials, induced eyespot enlargement, reduction, or no change in eyespot size, and each material was characterized by a unique response profile. For example, silicone glassine paper almost always induced a considerable reduction, while glass plates most frequently induced enlargement, and plastic plates generally produced no change. The biological materials tested here (fibronectin, polylysine, collagen type I, and gelatin) resulted in various responses, but polylysine induced more cases of enlargement, similar to glass plates. The response profile of the materials was not readily predictable from the chemical composition of the materials but was significantly correlated with the water contact angle (water repellency) of the material surface, suggesting that the surface physical chemistry of materials is a determinant of eyespot size. When the proximal side of a prospective eyespot was covered with a size-reducing material (silicone glassine paper) and the distal side and the organizer were covered with a material that rarely induced size reduction (plastic film), the proximal side of the eyespot was reduced in size in comparison with the distal side, suggesting that signal propagation but not organizer activity was inhibited by silicone glassine paper. CONCLUSIONS: These results suggest that physical contact with an appropriate hydrophobic surface is required for morphogenic signals from organizers to propagate normally. The binding of the apical surface of the epithelium with an opposing surface may provide mechanical support for signal propagation. In addition to conventional molecular morphogens, there is a possibility that mechanical distortion of the epithelium that is propagated mechanically serves as a nonmolecular morphogen to induce subsequent molecular changes, in accordance with the distortion hypothesis for butterfly wing color pattern formation.

Dispersibility of the Pale Grass Blue Butterfly Zizeeria maha (Lepidoptera: Lycaenidae) Revealed by One-Individual Tracking in the Field: Quantitative Comparisons between Subspecies and between Sexes.

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.

Overwintering States of the Pale Grass Blue Butterfly Zizeeria maha (Lepidoptera: Lycaenidae) at the Time of the Fukushima Nuclear Accident in March 2011.

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.