Exfoliation of Al-Residual Multilayer MXene Using Tetramethylammonium Bases for Conductive Film Applications.

This study describes the concise exfoliation of multilayer Ti3C2T x MXene containing residual aluminum atoms. Treatment with tetramethylammonium base in a co-solvent of tetrahydrofuran and H2O produced single-layer Ti3C2T x , which was confirmed via atomic force microscopy observations, with an electrical conductivity 100+ times that of Ti3C2T x prepared under previously reported conditions. The scanning electron microscopy and X-ray diffraction measurements showed that the exfoliated single-layer Ti3C2T x MXenes were reconstructed to assembled large-domain layered films, enabling excellent macroscale electric conductivity. X-ray photoelectron spectroscopy confirmed the complete removal of residual Al atoms and the replacement of surface fluorine atoms with hydroxy groups. Using the exfoliated dispersion, a flexible transparent conductive film was formed and demonstrated in an electrical application.

Tolerance of High Oral Doses of Nonradioactive and Radioactive Caesium Chloride in the Pale Grass Blue Butterfly Zizeeria maha.

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.

Insights into eyespot color-pattern formation mechanisms from color gradients, boundary scales, and rudimentary eyespots in butterfly wings.

Butterfly eyespot color patterns are traditionally explained by the gradient model, where positional information is stably provided by a morphogen gradient from a single organizer and its output is a set of non-graded (or graded) colors based on pre-determined threshold levels. An alternative model is the induction model, in which the outer black ring and the inner black core disk of an eyespot are specified by graded signals from the primary and secondary organizers that also involve lateral induction. To examine the feasibility of these models, we analyzed eyespot color gradients, boundary scales, and rudimentary eyespots in various nymphalid butterflies. Most parts of eyespots showed color gradients with gradual or fluctuating changes with sharp boundaries in many species, but some species had eyespots that were composed of a constant color within a given part. Thus, a plausible model should be flexible enough to incorporate this diversity. Some boundary scales appeared to have two kinds of pigments, and others had "misplaced" colors, suggesting an overlapping of two signals and a difficulty in assuming sharp threshold boundaries. Rudimentary eyespots of three Junonia species revealed that the outer black ring is likely determined first and the inner yellow or red ring is laterally induced. This outside-to-inside determination together with the lateral induction may favor the induction model, in which dynamic signal interactions play a major role. The implications of these results for the ploidy hypothesis and color-pattern rules are discussed.

Developmental dynamics of butterfly wings: real-time in vivo whole-wing imaging of twelve butterfly species.

Colour pattern development of butterfly wings has been studied from several different approaches. However, developmental changes in the pupal wing tissues have rarely been documented visually. In this study, we recorded real-time developmental changes of the pupal whole wings of 9 nymphalid, 2 lycaenid, and 1 pierid species in vivo, from immediately after pupation to eclosion, using the forewing-lift method. The developmental period was roughly divided into four sequential stages. At the very early stage, the wing tissue was transparent, but at the second stage, it became semi-transparent and showed dynamic peripheral adjustment and slow low-frequency contractions. At this stage, the wing peripheral portion diminished in size, but simultaneously, the ventral epithelium expanded in size. Likely because of scale growth, the wing tissue became deeply whitish at the second and third stages, followed by pigment deposition and structural colour expression at the fourth stage. Some red or yellow (light-colour) areas that emerged early were "overpainted" by expanding black areas, suggesting the coexistence of two morphogenic signals in some scale cells. The discal spot emerged first in some nymphalid species, as though it organised the entire development of colour patterns. These results indicated the dynamic wing developmental processes common in butterflies.

Focusing on butterfly eyespot focus: uncoupling of white spots from eyespot bodies in nymphalid butterflies.

BACKGROUND: Developmental studies on butterfly wing color patterns often focus on eyespots. A typical eyespot (such as that of Bicyclus anynana) has a few concentric rings of dark and light colors and a white spot (called a focus) at the center. The prospective eyespot center during the early pupal stage is known to act as an organizing center. It has often been assumed, according to gradient models for positional information, that a white spot in adult wings corresponds to an organizing center and that the size of the white spot indicates how active that organizing center was. However, there is no supporting evidence for these assumptions. To evaluate the feasibility of these assumptions in nymphalid butterflies, we studied the unique color patterns of Calisto tasajera (Nymphalidae, Satyrinae), which have not been analyzed before in the literature. RESULTS: In the anterior forewing, one white spot was located at the center of an eyespot, but another white spot associated with either no or only a small eyespot was present in the adjacent compartment. The anterior hindwing contained two adjacent white spots not associated with eyespots, one of which showed a sparse pattern. The posterior hindwing contained two adjacent pear-shaped eyespots, and the white spots were located at the proximal side or even outside the eyespot bodies. The successive white spots within a single compartment along the midline in the posterior hindwing showed a possible trajectory of a positional determination process for the white spots. Several cases of focus-less eyespots in other nymphalid butterflies were also presented. CONCLUSIONS: These results argue for the uncoupling of white spots from eyespot bodies, suggesting that an eyespot organizing center does not necessarily differentiate into a white spot and that a prospective white spot does not necessarily signify organizing activity for an eyespot. Incorporation of these results in future models for butterfly wing color pattern formation is encouraged.

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.

Spatial patterns of correlated scale size and scale color in relation to color pattern elements in butterfly wings.

Complex butterfly wing color patterns are coordinated throughout a wing by unknown mechanisms that provide undifferentiated immature scale cells with positional information for scale color. Because there is a reasonable level of correspondence between the color pattern element and scale size at least in Junonia orithya and Junonia oenone, a single morphogenic signal may contain positional information for both color and size. However, this color-size relationship has not been demonstrated in other species of the family Nymphalidae. Here, we investigated the distribution patterns of scale size in relation to color pattern elements on the hindwings of the peacock pansy butterfly Junonia almana, together with other nymphalid butterflies, Vanessa indica and Danaus chrysippus. In these species, we observed a general decrease in scale size from the basal to the distal areas, although the size gradient was small in D. chrysippus. Scales of dark color in color pattern elements, including eyespot black rings, parafocal elements, and submarginal bands, were larger than those of their surroundings. Within an eyespot, the largest scales were found at the focal white area, although there were exceptional cases. Similarly, ectopic eyespots that were induced by physical damage on the J. almana background area had larger scales than in the surrounding area. These results are consistent with the previous finding that scale color and size coordinate to form color pattern elements. We propose a ploidy hypothesis to explain the color-size relationship in which the putative morphogenic signal induces the polyploidization (genome amplification) of immature scale cells and that the degrees of ploidy (gene dosage) determine scale color and scale size simultaneously in butterfly wings.

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.

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.

Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

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.

System-dependent regulations of colour-pattern development: a mutagenesis study of the pale grass blue butterfly.

Developmental studies on wing colour patterns have been performed in nymphalid butterflies, but efficient genetic manipulations, including mutagenesis, have not been well established. Here, we have performed mutagenesis experiments in a lycaenid butterfly, the pale grass blue Zizeeria maha, to produce colour-pattern mutants. We fed the P-generation larvae an artificial diet containing the mutagen ethyl methane sulfonate (EMS), and the F1- and F2-generation adults showed various aberrant colour patterns: dorsoventral transformation, anterioposterior background colouration gap, weak contrast, disarrangement of spots, reduction of the size of spots, loss of spots, fusion of spots, and ectopic spots. Among them, the disarrangement, reduction, and loss of spots were likely produced by the coordinated changes of many spots of a single wing around the discal spot in a system-dependent manner, demonstrating the existence of the central symmetry system. The present study revealed multiple genetic regulations for system-dependent and wing-wide colour-pattern determination in lycaenid butterflies.

Phenotypic plasticity in the range-margin population of the lycaenid butterfly Zizeeria maha.

BACKGROUND: Many butterfly species have been experiencing the northward range expansion and physiological adaptation, probably due to climate warming. Here, we document an extraordinary field case of a species of lycaenid butterfly, Zizeeria maha, for which plastic phenotypes of wing color-patterns were revealed at the population level in the course of range expansion. Furthermore, we examined whether this outbreak of phenotypic changes was able to be reproduced in a laboratory. RESULTS: In the recently expanded northern range margins of this species, more than 10% of the Z. maha population exhibited characteristic color-pattern modifications on the ventral wings for three years. We physiologically reproduced similar phenotypes by an artificial cold-shock treatment of a normal southern population, and furthermore, we genetically reproduced a similar phenotype after selective breeding of a normal population for ten generations, demonstrating that the cold-shock-induced phenotype was heritable and partially assimilated genetically in the breeding line. Similar genetic process might have occurred in the previous and recent range-margin populations as well. Relatively minor modifications expressed in the tenth generation of the breeding line together with other data suggest a role of founder effect in this field case. CONCLUSIONS: Our results support the notion that the outbreak of the modified phenotypes in the recent range-margin population was primed by the revelation of plastic phenotypes in response to temperature stress and by the subsequent genetic process in the previous range-margin population, followed by migration and temporal establishment of genetically unstable founders in the recent range margins. This case presents not only an evolutionary role of phenotypic plasticity in the field but also a novel evolutionary aspect of range expansion at the species level.

Inhibition of immunoglobulin G-catalyzed hydrogen peroxide generation by dexamethasone and piroxicam.

In the present study, we established a simple and physiologically acceptable in vitro assay system to measure H2O2 generated by human immunoglobulin G (IgG) and other proteins. In addition, the effects of various drugs were also tested in this method. We found that UV irradiation (280 nm) of the test solutions for 1 h at 37 degrees C produced suitable conditions to test the effects of these drugs. The test solution contained 100 microg/ml IgG in 50 mM phosphate buffer (pH 7.4), and 1% dimethylformamide (DMF), a solvent used to dissolve each drug. Phosphate anions were preferable for H2O2 generation. H2O2 concentration in the irradiated sample was determined by continuous photometric measurement of absorption (O.D.) at 340 nm for 600 sec. The decrease in O.D. was due to the oxidation of NADPH by H2O2 mediated by the glutathione redox cycle. H2O2 generation was expressed as O.D.(340 nm decrease/400 sec). IgG (100 microg/ml) generated 6-7 microM H2O2/h. With irradiation, most cytokines, proteins and enzymes failed to generate significant amounts of H2O2. The formation of H2O2 from H2O and UV light-induced singlet oxygen (1O2) was demonstrated by the inhibitory effects of 1O2 quenchers. Dexamethasone (IC50: 6 ng/ml = 1.4x10(-8) M) blocked H2O2 generation catalyzed by IgG. This action was not mediated by binding to the glucocorticoid receptor. Piroxicam (IC50: 20 ng/ml = 6.0 x 10(-6) M) and diclofenac.Na (IC50: 500 ng/ml = 1.6 x 10(-5) M), but not indomethacin, also blocked H2O2 generation. The mechanism underlying the inhibition of IgG-catalyzed H2O2 generation is not clear; however, the possibility exists that these drugs intercept, or interfere with, the approach of water molecules at the catalytic interface(s) of the IgG.

A simple method for the selection of a suitable emulsifier based on color difference.

The purpose of this study was to establish a simple and rapid method for selecting a nonionic surfactant to prepare an emulsion that can maintain a stable emulsification phase. As an index of the degree of emulsification, the white chromaticity of the prepared sample was measured using a color difference meter. When liquid paraffin was used as an oil, the color difference (dE(H)) was shown as a change in a V-shaped curve depending on changes in the hydrophile-lipophile balance (HLB) number of sorbitan ester, polyoxyethylene sorbitan ester, or polyethyleneglycol ester. Although there was a difference in the minimal value of dE(H) in these experiments, the dE(H) was similar to the required HLB of liquid paraffin (HLB=10). On the other hand, no relationship was found between the HLB number and color difference when polyglycerol ester was used. It seems that there was possibility of the effect by the lowering of apparent HLB with a collapse of the hydration. These results indicate that color difference could be utilized for the selection of surfactants, except for polyglycerol ester.