The ivory lncRNA regulates seasonal color patterns in buckeye butterflies.

Long noncoding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here, we show that a conserved lncRNA, ivory, is an important color patterning gene in the buckeye butterfly Junonia coenia. ivory overlaps with cortex, a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et al., we argue that ivory, not cortex, is the color pattern gene of interest at this locus. In J. coenia, a cluster of cis-regulatory elements (CREs) in the first intron of ivory are genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of different ivory CREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to the ivory promoter during wing pattern development, suggesting that they directly regulate ivory. This case study demonstrates how cis-regulation of a single noncoding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns.

Antibody-Mediated Protein Knockdown Reveals Distal-less Functions for Eyespots and Parafocal Elements in Butterfly Wing Color Pattern Development.

One of the important genes for eyespot development in butterfly wings is Distal-less. Its function has been evaluated via several methods, including CRISPR/Cas9 genome editing. However, functional inhibition may be performed at the right time at the right place using a different method. Here, we used a novel protein delivery method for pupal wing tissues in vivo to inactivate a target protein, Distal-less, with a polyclonal anti-Distal-less antibody using the blue pansy butterfly Junonia orithya. We first demonstrated that various antibodies including the anti-Distal-less antibody were delivered to wing epithelial cells in vivo in this species. Treatment with the anti-Distal-less antibody reduced eyespot size, confirming the positive role of Distal-less in eyespot development. The treatment eliminated or deformed a parafocal element, suggesting a positive role of Distal-less in the development of the parafocal element. This result also suggested the integrity of an eyespot and its corresponding parafocal element as the border symmetry system. Taken together, these findings demonstrate that the antibody-mediated protein knockdown method is a useful tool for functional assays of proteins, such as Distal-less, expressed in pupal wing tissues, and that Distal-less functions for eyespots and parafocal elements in butterfly wing color pattern development.

Integrating Cu2O Colloidal Mie Resonators in Structurally Colored Butterfly Wings for Bio-Nanohybrid Photonic Applications.

Colloidal Cu2O nanoparticles can exhibit both photocatalytic activity under visible light illumination and resonant Mie scattering, but, for their practical application, they have to be immobilized on a substrate. Butterfly wings, with complex hierarchical photonic nanoarchitectures, constitute a promising substrate for the immobilization of nanoparticles and for the tuning of their optical properties. The native wax layer covering the wing scales of Polyommatus icarus butterflies was removed by simple ethanol pretreatment prior to the deposition of Cu2O nanoparticles, which allowed reproducible deposition on the dorsal blue wing scale nanoarchitectures via drop casting. The samples were investigated by optical and electron microscopy, attenuated total reflectance infrared spectroscopy, UV-visible spectrophotometry, microspectrophotometry, and hyperspectral spectrophotometry. It was found that the Cu2O nanoparticles integrated well into the photonic nanoarchitecture of the P. icarus wing scales, they exhibited Mie resonance on the glass slides, and the spectral signature of this resonance was absent on Si(100). A novel bio-nanohybrid photonic nanoarchitecture was produced in which the spectral properties of the butterfly wings were tuned by the Cu2O nanoparticles and their backscattering due to the Mie resonance was suppressed despite the low refractive index of the chitinous substrate.

Socket Array Irregularities and Wing Membrane Distortions at the Eyespot Foci of Butterfly Wings Suggest Mechanical Signals for Color Pattern Determination.

Eyespot foci on butterfly wings function as organizers of eyespot color patterns during development. Despite their importance, focal structures have not been examined in detail. Here, we microscopically examined scales, sockets, and the wing membrane in the butterfly eyespot foci of both expanded and unexpanded wings using the Blue Pansy butterfly Junonia orithya. Images from a high-resolution light microscope revealed that, although not always, eyespot foci had scales with disordered planar polarity. Scanning electron microscopy (SEM) images after scale removal revealed that the sockets were irregularly positioned and that the wing membrane was physically distorted as if the focal site were mechanically squeezed from the surroundings. Focal areas without eyespots also had socket array irregularities, but less frequently and less severely. Physical damage in the background area induced ectopic patterns with socket array irregularities and wing membrane distortions, similar to natural eyespot foci. These results suggest that either the process of determining an eyespot focus or the function of an eyespot organizer may be associated with wing-wide mechanics that physically disrupt socket cells, scale cells, and the wing membrane, supporting the physical distortion hypothesis of the induction model for color pattern determination in butterfly wings.

Two-Photon Polymerization of Butterfly Wing Scale Inspired Surfaces with Anisotropic Wettability.

Wings of Morph aega butterflies are natural surfaces that exhibit anisotropic liquid wettability. The direction-dependent arrangement of the wing scales creates orientation-turnable microstructures with two distinct contact modes for liquid droplets. Enabled by recent developments in additive manufacturing, such natural surface designs coupled with hydrophobicity play a crucial role in applications such as self-cleaning, anti-icing, and fluidic manipulation. However, the interplay among resolution, architecture, and performance of bioinspired structures is barely achieved. Herein, inspired by the wing scales of the Morpho aega butterfly, full-scale synthetic surfaces with anisotropic wettability fabricated by two-photon polymerization are reported. The quality of the artificial butterfly scale is improved by optimizing the laser scanning strategy and the objective lens movement path. The corresponding contact angles of water on the fabricated architecture with various design parameters are measured, and the anisotropic fluidic wettability is investigated. Results demonstrate that tuning the geometrical parameters and spatial arrangement of the artificial wing scales enables anisotropic behaviors of the droplet's motion. The measured results also indicate a reverse phenomenon of the fabricated surfaces in contrast to their natural counterparts, possibly attributed to the significant difference in equilibrium wettability between the fabricated microstructures and the natural Morpho aega surface. These findings are utilized to design next-generation fluid-controllable interfaces for manipulating liquid mobility on synthetic surfaces.

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.

Investigating the Effect of Reflectance Tuning on Photocatalytic Dye Degradation with Biotemplated ZnO Photonic Nanoarchitectures Based on Morpho Butterfly Wings.

Photonic nanoarchitectures of butterfly wings can serve as biotemplates to prepare semiconductor thin films of ZnO by atomic layer deposition. The resulting biotemplated ZnO nanoarchitecture preserves the structural and optical properties of the natural system, while it will also have the features of the functional material. The ZnO-coated wings can be used directly in heterogeneous photocatalysis to decompose pollutants dissolved in water upon visible light illumination. We used the photonic nanoarchitectures of different Morpho butterflies with different structural colors as biotemplates and examined the dependence of decomposition rates of methyl orange and rhodamine B dyes on the structural color of the biotemplates and the thickness of the ZnO coating. Using methyl orange, we measured a ten-fold increase in photodegradation rate when the 20 nm ZnO-coated wings were compared to similarly coated glass substrates. Using rhodamine B, a saturating relationship was found between the degradation rate and the thickness of the deposited ZnO on butterfly wings. We concluded that the enhancement of the catalytic efficiency can be attributed to the slow light effect due to a spectral overlap between the ZnO-coated Morpho butterfly wings reflectance with the absorption band of dyes, thus the photocatalytic performance could be changed by the tuning of the structural color of the butterfly biotemplates. The photodegradation mechanism of the dyes was investigated by liquid chromatography-mass spectroscopy.

Tensile mechanical properties and finite element simulation of the wings of the butterfly Tirumala limniace.

This study examined the morphological characteristics and mechanical properties of the wings of Tirumala limniace. The wings of this butterfly, including the forewings and hindwings, are composed mainly of a flexible wing membrane and supporting wing veins. Scanning electron microscopy was employed to observe specific positions of the wing membrane and veins and reveal the morphological characteristics. Tensile experiments were conducted to evaluate the mechanical properties of the wings and proved that the multifiber layer structures have a significantly fixed orientation of fiber alignment. A butterfly wing model reconstructed in reverse based on the finite element method was used to analyze the static characteristics of the wing structure in detail. Evaluation of stress and strain after applying uniform loading, perpendicular loading, and torsion revealed that minor wing deformation occurred and was concentrated near the main wing vein, which verifies the steadiness of the butterfly wing structure. Additionally, the flapping of butterfly wings was simulated using computational fluid dynamics to study the flow field near the butterfly wings and the distribution of pressure gradient on the wings. The results confirmed the effect of wing veins on maintaining the flight performance.

Spectral Engineering of Hybrid Biotemplated Photonic/Photocatalytic Nanoarchitectures.

Solar radiation is a cheap and abundant energy for water remediation, hydrogen generation by water splitting, and CO2 reduction. Supported photocatalysts have to be tuned to the pollutants to be eliminated. Spectral engineering may be a handy tool to increase the efficiency or the selectivity of these. Photonic nanoarchitectures of biological origin with hierarchical organization from nanometers to centimeters are candidates for such applications. We used the blue wing surface of laboratory-reared male Polyommatus icarus butterflies in combination with atomic layer deposition (ALD) of conformal ZnO coating and octahedral Cu2O nanoparticles (NP) to explore the possibilities of engineering the optical and catalytic properties of hybrid photonic nanoarchitectures. The samples were characterized by UV-Vis spectroscopy and optical and scanning electron microscopy. Their photocatalytic performance was benchmarked by comparing the initial decomposition rates of rhodamine B. Cu2O NPs alone or on the butterfly wings, covered by a 5 nm thick layer of ZnO, showed poor performance. Butterfly wings, or ZnO coated butterfly wings with 15 nm ALD layer showed a 3 to 3.5 times enhancement as compared to bare glass. The best performance of almost 4.3 times increase was obtained for the wings conformally coated with 15 nm ZnO, deposited with Cu2O NPs, followed by conformal coating with an additional 5 nm of ZnO by ALD. This enhanced efficiency is associated with slow light effects on the red edge of the reflectance maximum of the photonic nanoarchitectures and with enhanced carrier separation through the n-type ZnO and the p-type Cu2O heterojunction. Properly chosen biologic photonic nanoarchitectures in combination with carefully selected photocatalyst(s) can significantly increase the photodegradation of pollutants in water under visible light illumination.

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.

Spectral tuning of biotemplated ZnO photonic nanoarchitectures for photocatalytic applications.

The photocatalytic activity of a flat surface can be increased by micro- and nanostructuring the interface to increase the area of the contact surface between the photocatalyst and the solute, and moreover, to optimize charge carrier transfer. Further enhancement can be achieved by using photonic nanostructures, which exhibit photonic band gap (PBG). Structurally coloured butterfly wings offer a rich 'library' of PBGs in the visible spectral range which can be used as naturally tuned sample sets for biotemplating. We used conformal atomic layer deposition of ZnO on the wings of various butterfly species (Arhopala asopia, Hypochrysops polycletus, Morpho sulkowskyi, Polyommatus icarus) possessing structural colour extending from the near UV to the blue wavelength range, to test the effects arising from the nanostructured surfaces and from the presence of different types of PBGs. Aqueous solutions of rhodamine B were used to test the enhancement of photocatalytic activity that was found for all ZnO-coated butterfly wings. The best reaction rate of decomposing rhodamine B when illuminated with visible light was found in 15 nm ZnO coated M. sulkowskyi wing, the reflectance of which had the highest overlap with the absorption band of the dye and had the highest reflectance intensity.

Stability-Enhanced Emission Based on Biophotonic Crystals in Liquid Crystal Random Lasers.

A new design of a bio-random laser based on a butterfly wing structure and ITO glass is proposed in this article. Firstly, the butterfly wing structure was integrated in a liquid crystal cell made of ITO glass. The integrated liquid crystal cell was injected with liquid crystal and dye to obtain a bio-random laser. A non-biological random laser was obtained with a capillary glass tube, liquid crystal and dye. The excitation spectra and thresholds were recorded to evaluate the performance of the biological and non-biological random lasers. The results show that the excitation performance stability of the bio-random laser is improved and the number of spikes in the spectra is reduced compared with the non-biological random laser. Finally, the equivalent cavity length of the biological and non-biological random lasers was compared and the optical field distribution inside the butterfly wing structure was analyzed. The data show that the improvement of the excitation performance stability of the bio-random laser is related to the localization of the optical field induced by the photonic crystal structure in the butterfly wing.

A Review on Pharmacological Properties of Christia vespertilionis

@#Commonly referred to as ‘daun rerama’, Christia vespertilionis has increased in popularity in traditional and modern medicine. This review aims to report the relevance of this plant in terms of its traditional uses, pharmacological actions, phytoconstituents, extractions methods, and identify the research gaps and future potentials. The review is conducted as per PRISMA guidelines; a database search was conducted in Web of Science, Science Direct, Scopus and Google Scholar from 1996 to 2021. Results show that to date, phytochemicals such as alkaloids, flavonoids, quinones, and others have been identified, corresponding to its range of pharmacological activities that include anti-cancer, anti-malaria, and antioxidant. There have also been claims of antidiabetic activity but not supported by enough scientific evidence. Study on molecular and gene expression was still lacking. There is a good future in the research of this plant with many potential aspects to be investigated.

Biotemplated g-C3N4/Au Periodic Hierarchical Structures for the Enhancement of Photocatalytic CO2 Reduction with Localized Surface Plasmon Resonance.

Graphitic carbon nitride (g-C3N4) is a promising photocatalyst for CO2 reduction to alleviate the greenhouse effect. However, the low light absorption, small specific surface area, and rapid charge recombination limit the photocatalytic efficiency of g-C3N4. Herein, we demonstrate a bioinspired nanoarchitecturing strategy to significantly improve the light harvesting and charge separation of the g-C3N4/Au composite, as proven by the remarkable photocatalytic CO2 reduction. Specifically, a biotemplating approach is employed to transfer the sophisticated hierarchical structures and the related light-harvesting functionality of Troides helena butterfly wings to the g-C3N4/Au composite. The resulting g-C3N4/Au composite shows high photocatalytic efficiency under UV-visible excitation with triethanolamine as the sacrificial agent. The yields of CO and CH4 are 331.57 and 39.71 µmol/g/h, respectively, which are ∼36 times and ∼88 times that of pure g-C3N4 under the same conditions. Detailed experiments and the finite-difference time-domain method suggest that the superb photocatalytic activity should be ascribed to the unique periodic hierarchical structure which assists the light absorption and the localized surface plasmon resonance for promoted charge separation in addition to the more effective CO2 diffusion and larger specific surface area. Our work provides a new path for the design and optimization of photocatalysts based on biological structures that are usually unattainable artificially.

Butterfly wing architectures inspire sensor and energy applications.

Natural biological systems are constantly developing efficient mechanisms to counter adverse effects of increasing human population and depleting energy resources. Their intelligent mechanisms are characterized by the ability to detect changes in the environment, store and evaluate information, and respond to external stimuli. Bio-inspired replication into man-made functional materials guarantees enhancement of characteristics and performance. Specifically, butterfly architectures have inspired the fabrication of sensor and energy materials by replicating their unique micro/nanostructures, light-trapping mechanisms and selective responses to external stimuli. These bio-inspired sensor and energy materials have shown improved performance in harnessing renewable energy, environmental remediation and health monitoring. Therefore, this review highlights recent progress reported on the classification of butterfly wing scale architectures and explores several bio-inspired sensor and energy applications.

High-Quality Genome Assembly and Comprehensive Transcriptome of the Painted Lady Butterfly Vanessa cardui.

The painted lady butterfly, Vanessa cardui, has the longest migration routes, the widest hostplant diversity, and one of the most complex wing patterns of any insect. Due to minimal culturing requirements, easily characterized wing pattern elements, and technical feasibility of CRISPR/Cas9 genome editing, V. cardui is emerging as a functional genomics model for diverse research programs. Here, we report a high-quality, annotated genome assembly of the V. cardui genome, generated using 84× coverage of PacBio long-read data, which we assembled into 205 contigs with a total length of 425.4 Mb (N50 = 10.3 Mb). The genome was very complete (single-copy complete Benchmarking Universal Single-Copy Orthologs [BUSCO] 97%), with contigs assembled into presumptive chromosomes using synteny analyses. Our annotation used embryonic, larval, and pupal transcriptomes, and 20 transcriptomes across five different wing developmental stages. Gene annotations showed a high level of accuracy and completeness, with 14,437 predicted protein-coding genes. This annotated genome assembly constitutes an important resource for diverse functional genomic studies ranging from the developmental genetic basis of butterfly color pattern, to coevolution with diverse hostplants.

ASIA syndrome symptoms induced by gluteal biopolymer injections: Case-series and narrative review.

BACKGROUND: The number of plastic surgery procedures have been rising in the last few years. The morbi-mortality due to illegal use of biopolymers is a public health problem. One of the clinical consequences, foreign body modelling reaction, may be a precursor of ASIA (Autoimmune/Inflammatory disease induced by adjuvants) syndrome.The objective of this article is to present a case-series study of patients who developed ASIA syndrome following gluteal injection with biopolymers and emphasize the importance of toxic exposure in triggering autoimmune responses. A surgical technique used on some of the patients in the study is described. METHODS: A group of thirteen patients, diagnosed with foreign body modelling reaction, who developed ASIA syndrome confirmed by approved criteria was followed between May 2016 and May 2018. The "Butterfly Wings Technique," a new surgical procedure for patients who have medium to severe compromise, was used on five of them.A narrative literature review was done to look for subjects with ASIA syndrome and gluteal biopolymer infiltration. RESULTS: All the patients in the present case-series with foreign body modelling reaction developed ASIA syndrome. Some of them had a background of familial autoimmunity. Five of the patients were surgically treated and saw a clinical improvement after the extraction of the biopolymer with the proposed technique.The narrative literature review identified 7 articles related to the disease through the database search. CONCLUSIONS: We suggest that foreign body modelling reaction should be considered a precursor to ASIA syndrome. New research projects will be needed in the future to evaluate the factors that determine when ASIA syndrome is triggered in a patient with this reaction.

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.

Tissue-specific developmental regulation and isoform usage underlie the role of doublesex in sex differentiation and mimicry in Papilio swallowtails.

Adaptive phenotypes often arise by rewiring existing developmental networks. Co-option of transcription factors in novel contexts has facilitated the evolution of ecologically important adaptations. doublesex (dsx) governs fundamental sex differentiation during embryonic stages and has been co-opted to regulate diverse secondary sexual dimorphisms during pupal development of holometabolous insects. In Papilio polytes, dsx regulates female-limited mimetic polymorphism, resulting in mimetic and non-mimetic forms. To understand how a critical gene such as dsx regulates novel wing patterns while maintaining its basic function in sex differentiation, we traced its expression through metamorphosis in P. polytes using developmental transcriptome data. We found three key dsx expression peaks: (i) eggs in pre- and post-ovisposition stages; (ii) developing wing discs and body in final larval instar; and (iii) 3-day pupae. We identified potential dsx targets using co-expression and differential expression analysis, and found distinct, non-overlapping sets of genes-containing putative dsx-binding sites-in developing wings versus abdominal tissue and in mimetic versus non-mimetic individuals. This suggests that dsx regulates distinct downstream targets in different tissues and wing colour morphs and has perhaps acquired new, previously unknown targets, for regulating mimetic polymorphism. Additionally, we observed that the three female isoforms of dsx were differentially expressed across stages (from eggs to adults) and tissues and differed in their protein structure. This may promote differential protein-protein interactions for each isoform and facilitate sub-functionalization of dsx activity across its isoforms. Our findings suggest that dsx employs tissue-specific downstream effectors and partitions its functions across multiple isoforms to regulate primary and secondary sexual dimorphism through insect 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.