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.

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.

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.

A Single-Wing Removal Method to Assess Correspondence Between Gene Expression and Phenotype in Butterflies: The Case of Distal-less.

It is often desirable but difficult to retrieve information on the mature phenotype of an immature tissue sample that has been subjected to gene expression analysis. This problem cannot be ignored when individual variation within a species is large. To circumvent this problem in the butterfly wing system, we developed a new surgical method for removing a single forewing from a pupa using Junonia orithya; the operated pupa was left to develop to an adult without eclosion. The removed right forewing was subjected to gene expression analysis, whereas the non-removed left forewing was examined for color patterns. As a test case, we focused on Distal-less (Dll), which likely plays an active role in inducing elemental patterns, including eyespots. The Dll expression level in forewings was paired with eyespot size data from the same individual. One third of the operated pupae survived and developed wing color patterns. Dll expression levels were significantly higher in males than in females, although male eyespots were smaller in size than female eyespots. Eyespot size data showed weak but significant correlations with the Dll expression level in females. These results demonstrate that a single-wing removal method was successfully applied to the butterfly wing system and suggest the weak and non-exclusive contribution of Dll to eyespot size determination in this butterfly. Our novel methodology for establishing correspondence between gene expression and phenotype can be applied to other candidate genes for color pattern development in butterflies. Conceptually similar methods may also be applicable in other developmental systems.

Comparative Morphological Analysis of the Immature Stages of the Grass Blue Butterflies Zizeeria and Zizina (Lepidoptera: Lycaenidae).

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

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.

Spontaneous long-range calcium waves in developing butterfly wings.

BACKGROUND: Butterfly wing color patterns emerge as the result of a regular arrangement of scales produced by epithelial scale cells at the pupal stage. These color patterns and scale arrangements are coordinated throughout the wing. However, the mechanism by which the development of scale cells is controlled across the entire wing remains elusive. In the present study, we used pupal wings of the blue pansy butterfly, Junonia orithya, which has distinct eyespots, to examine the possible involvement of Ca(2+) waves in wing development. RESULTS: Here, we demonstrate that the developing pupal wing tissue of the blue pansy butterfly displayed spontaneous low-frequency Ca(2+) waves in vivo that propagated slowly over long distances. Some waves appeared to be released from the immediate peripheries of the prospective eyespot and discal spot, though it was often difficult to identify the specific origins of these waves. Physical damage, which is known to induce ectopic eyespots, led to the radiation of Ca(2+) waves from the immediate periphery of the damaged site. Thapsigargin, which is a specific inhibitor of Ca(2+)-ATPases in the endoplasmic reticulum, induced an acute increase in cytoplasmic Ca(2+) levels and halted the spontaneous Ca(2+) waves. Additionally, thapsigargin-treated wings showed incomplete scale development as well as other scale and color pattern abnormalities. CONCLUSIONS: We identified a novel form of Ca(2+) waves, spontaneous low-frequency slow waves, which travel over exceptionally long distances. Our results suggest that spontaneous Ca(2+) waves play a critical role in the coordinated development of scale arrangements and possibly in color pattern formation in butterflies.

The Lycaenid Central Symmetry System: Color Pattern Analysis of the Pale Grass Blue Butterfly Zizeeria maha.

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

The marginal band system in nymphalid butterfly wings.

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

Ingestion of radioactively contaminated diets for two generations in the pale grass blue butterfly.

BACKGROUND: The release of radioactive materials due to the Fukushima nuclear accident has raised concern regarding the biological impacts of ingesting radioactively contaminated diets on organisms. We previously performed an internal exposure experiment in which contaminated leaves collected from polluted areas were fed to larvae of the pale grass blue butterfly, Zizeeria maha, from Okinawa, which is one of the least polluted localities in Japan. Using the same experimental system, in the present study, we further examined the effects of low-level-contaminated diets on this butterfly. Leaves were collected from two localities in Tohoku (Motomiya (161 Bq/kg) and Koriyama (117 Bq/kg)); two in Kanto (Kashiwa (47.6 Bq/kg) and Musashino (6.4 Bq/kg)); one in Tokai (Atami (2.5 Bq/kg)); and from Okinawa (0.2 Bq/kg). In addition to the effects on the first generation, we examined the possible transgenerational effects of the diets on the next generation. RESULTS: In the first generation, the Tohoku groups showed higher rates of mortality and abnormalities and a smaller forewing size than the Okinawa group. The mortality rates were largely dependent on the ingested dose of caesium. The survival rates of the Kanto-Tokai groups were greater than 80%, but the rates in the Tohoku groups were much lower. In the next generation, the survival rates in the Tohoku groups were below 20%, whereas those of the Okinawa groups were above 70%. The survival rates in the second generation were independent of the locality of the leaves ingested by the first generation, indicating that the diet in the second generation was the determinant of their survival. Moreover, a smaller forewing size was observed in the Tohoku groups in the second generation. However, the forewing size was inversely correlated with the cumulative caesium dose ingested throughout the first and second generations, indicating that the diet in the first generation also influenced the forewing size of the second generation. CONCLUSIONS: Biological effects are detectable under a low ingested dose of radioactivity from a contaminated diet. The effects are transgenerational but can be overcome by ingesting a non-contaminated diet, suggesting that at least some of the observed effects are attributable to non-genetic physiological changes.

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.

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.

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.

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.

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

BACKGROUND: Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. RESULTS: A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. CONCLUSIONS: Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems.

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.

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.

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.