Wing Scales Research Articles

Vermilion and cinnabar are involved in ommochrome pigment biosynthesis in eyes but not wings of Bicyclus anynana butterflies

If the same pigment is found in different tissues in a body, it is natural to assume that the same metabolic pathways are deployed similarly in each tissue. Here we show that this is not the case for ommochromes, the red and orange pigments found in the eyes and wings of butterflies. We tested the expression and function of vermilion and cinnabar, two known fly genes in the ommochrome pathway, in the development of pigments in the eyes and in the wings of Bicyclus anynana butterflies, both traits having reddish/orange pigments. By using fluorescent in-situ hybridization (HCR3.0) we localized the expression of vermilion and cinnabar in the cytoplasm of pigment cells in the ommatidia but observed no clear expression for either gene on larval and pupal wings. We then disrupted the function of both genes, using CRISPR-Cas9, which resulted in the loss of pigment in the eyes but not in the wings. Using thin-layer chromatography and UV–vis spectroscopy we identified the presence of ommochrome and ommochrome precursors in the orange wing scales and in the hemolymph of pupae. We conclude that the wings either synthesize ommochromes locally, with yet unidentified enzymes or incorporate these pigments synthesized elsewhere from the hemolymph. Different metabolic pathways or transport mechanisms, thus, lead to the presence of ommochromes in the wings and eyes of B. anynana butterflies.

Influence of combined stress by salinity (NaCl) and heavy metals (Pb(NO3)2) on the proline, chlorophyll and lead accumulation in the tissues of the Atriplex canescens (Pursh ) Nutt.

Abstract. Salinity and heavy metals pollution are the root causes of many environmental problems. This study aims to determine the effect of NaCl-induced salinity and Pb(NO3)2 on chlorophyll, proline content, and lead accumulation in shoots and roots of Atriplex canesens. Sowing was carried out in plastic pots (16 cm in diameter and 13.8 cm high) containing a mixture of sand/compost. The irrigation of plants was performed with distilled water and Hoagland nutrient solution. After four months of growth, the plants were subjected to either 0.50 or 3% of NaCl combined with 800, 1600, and 2400 ppm of Pb(NO3)2 for a duration of one month, then we performed biochemical and chemical analyses on the plants. Results showed that the combined influence of strong salinity and lead caused a reduction of chlorophyll and increased proline and lead accumulation in plant tissues. Lead significantly accumulated in the roots compared to the shoots of the plants.

A cis-regulatory sequence of the selector gene vestigial drives the evolution of wing scaling in Drosophila species.

Scaling between specific organs and overall body size has long fascinated biologists, being a primary mechanism by which organ shapes evolve. Yet, the genetic mechanisms that underlie the evolution of scaling relationships remain elusive. Here, we compared wing and fore tibia lengths (the latter as a proxy of body size) in Drosophila melanogaster, Drosophila simulans, Drosophila ananassae and Drosophila virilis, and show that the first three of these species have roughly a similar wing-to-tibia scaling behavior. In contrast, D. virilis exhibits much smaller wings relative to their body size compared with the other species and this is reflected in the intercept of the wing-to-tibia allometry. We then asked whether the evolution of this relationship could be explained by changes in a specific cis-regulatory region or enhancer that drives expression of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and contributes to wing size. To test this hypothesis directly, we used CRISPR/Cas9 to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, we discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are significantly smaller with respect to controls, partially shifting the intercept of the wing-to-tibia scaling relationship towards that observed in D. virilis. We conclude that a single cis-regulatory element in D. virilis contributes to constraining wing size in this species, supporting the hypothesis that scaling could evolve through genetic variations in cis-regulatory elements.

ECR Spotlight – Keity Johanna Farfán Pira

ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology during our centenary year, but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Keity Johanna Farfán Pira is an author on ‘ A cis-regulatory sequence of the selector gene vestigial drives the evolution of wing scaling in Drosophila species’, published in JEB. Keity Johanna conducted the research described in this article on the selector gene vestigial and its role in wing size evolution in Drosophila species in Marcos Nahmad's lab at Cinvestav-IPN, Mexico City, Mexico. She is now a postdoctoral fellow in the lab of Erica L. Westerman at University of Arkansas, Fayetteville, AR, USA, investigating evo-devo of regulatory regions and genes in determinant processes of development and behavior in insects.

Nanoscale cuticle density variations correlate with pigmentation and color in butterfly wing scales

Nanoscale cuticle density variations correlate with pigmentation and color in butterfly wing scales

A review of the subgenus Parapisa of Apisa (Lepidoptera: Erebidae: Arctiinae) with description of a remarkable species from Cameroonian Highlands

The subgenus Parapisa of the genus Apisa is reviewed based on the examination of 104 specimens. Apisa (P.) cinereocostata and A. (P.) subargentea are redescribed and their intraspecific variation is analysed in detail. A new species A. (P.) asipa, similar in the general coloration to other Apisa taxa, but very distinctive in the male genital morphology and the shape of the wing scales, is described from Cameroon and Nigeria. Apisa (P.) cinereocostata is hypothesized to be a widespread, but highly polymorphic taxon with significant variation in body size, intensity of grey coloration, and the proportions and shape of certain morphostructures of male genitalia. Determination keys and extensive illustrations of the variation are provided to enable proper identification of specimens.

Wide-gamut structural colours on oakblue butterflies by naturally tuned photonic nanoarchitectures.

The iridescent structural colours of butterflies, generated by photonic nanoarchitectures, often function as species-specific sexual signals; therefore, they are reproduced precisely from generation to generation. The wing scales of oakblue hairstreak butterflies (genus Arhopala, Theclinae, Lycaenidae, Lepidoptera) contain multi-layer photonic nanoarchitectures, which can generate a wide range of structural colours, from violet to green. By scanning (SEM) and cross-sectional transmission electron microscopy (TEM) investigation, the colour tuning mechanism of the cover scales was explored. We revealed that the characteristic size change of structural elements in similar photonic nanoarchitectures led to different structural colours that were examined by various reflectance spectrophotometry techniques. The measured structural properties of the naturally tuned photonic nanoarchitectures were used to calculate wing reflectances, which were compared with the measurement results. We found that the simulated structural colours were systematically redshifted by 95-126 nm as compared with the measured normal-incidence reflectance results. This is attributed to the swelling of the chitinous multi-layer structures during the standard TEM sample preparation and the tilt of the cover scales, which both affect the apparent layer thicknesses in the TEM cross-sections. We proposed a simulation correction and compared the results with the layer thicknesses measured on cryogenically prepared non-embedded SEM cross-sections.

Electromagnetic response of corrugated multilayer systems inspired by the Dione vanillae butterfly scales.

Inspired by the microstructures in the wing scales of the butterfly Dione vanillae, we investigate the optical response of two multilayer structures, which include one or two corrugated interfaces. The reflectance is calculated using the C-method and is compared with that of a planar multilayer. We perform a detailed analysis of the influence of each geometric parameter and study the angular response, which is important for structures exhibiting iridescence. The results of this study aim to contribute to the design of multilayer structures with predetermined optical responses.

Temporal stability of δ2H in insect tissues: Implications for isotope-based geographic assignments

Hydrogen isotope geolocation of insects is based on the assumption that the chitin in the wings of adult migratory insects preserves the hydrogen isotope composition (δ2H) of the larval stages without influence of adult diet. Here, we test this assumption by conducting laboratory feeding experiments for monarch butterflies (Danaus plexippus) including: (1) a starvation treatment where adults were not fed and (2) an enriched treatment where adults were fed a diet isotopically enriched in deuterium (~ +78‰) compared to the larval diet. The δ2H values of adult wings were measured at different time steps along the 24-day experiment. We also investigated intra-wing differences in δ2H values caused by wing pigmentation, absence of wing scales, and presence of major wing veins. We conclude that, although the magnitude of the changes in δ2H values are small (~6‰), wing δ2H values vary based on adult diet and insect age, particularly early after eclosion (i.e., 1–4 days). We found that wing shade, wing pigmentation, and the presence of wing scales do not alter wing δ2H values. However, wing samples containing veins had systematically higher δ2H values (~9‰), suggesting that adult diet influences the hemolymph that circulates in the wing veins. We hypothesise that there is a stronger influence of adult diet on the isotope signal of wings during early adult life relative to later life because of increased metabolic and physiologic activity in young insect wings. We argue that the influence of the isotopic contribution of adult diet is generally small and is likely minimal if the wings are carefully sampled to avoid veins. However, we also demonstrated that wings are not inert tissues, and that adult feeding contributes to some of the intra-population δ2H variance. We conclude that δ2H geolocation using insect wings remains valid, but that adult feeding, butterfly age and wing vein sampling generate an inherent uncertainty limiting the precision of geolocation.

Seasonal Assessment of some Potentially Toxic Elements with Possible Animal Health Risks in Atriplex canescens (Pursh) Nutt.

In the present investigation Br (Bromine), Cr (Chromium) and Pb (Lead) concentrations were assessed employing X-ray florescence spectrometry to evaluate seasonal variation of these elements in the xero-halophyte Atriplex canescens (Pursh) Nutt., a shrub with high pastoral value. The results showed that A. canescens and its surrounding soil have similar accumulation patterns for Br and Pb across seasons, but Cr concentrations in shrubs are higher in spring than other seasons. The seasonal mean contents of trace elements in A. canescens descend in the following order: Cr (8.33µg g- 1) > Br (5.34µg g- 1) > Pb (0.24µg g- 1). The maximum element transfer factor (ETF) of Cr and Pb was recorded in summer and autumn. However, no significant difference between seasons was found for Br. Principle component analysis (PCA) showed that Br, Cr and Pb were associated negatively with soil during all seasons. In contrast, A. canescens was only associated to Cr contents in the spring. Results provide evidence also that the levels of Br, Cr and Pb were within the safety-limits recommended by the National Research Council (NRC) guidelines for animal nutrition.

Transcriptomic Analysis Provides Insight into the ROS Scavenging System and Regulatory Mechanisms in Atriplex canescens Response to Salinity

Atriplex canescens is a representative halophyte with excellent tolerance to salt. Previous studies have revealed certain physiological mechanisms and detected functional genes associated with salt tolerance. However, knowledge on the ROS scavenging system and regulatory mechanisms in this species when adapting to salinity is limited. Therefore, this study further analyzed the transcriptional changes in genes related to the ROS scavenging system and important regulatory mechanisms in A. canescens under saline conditions using our previous RNA sequencing data. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation revealed that the differentially expressed genes (DEGs) were highly enriched in signal transduction- and reactive oxygen species-related biological processes, including "response to oxidative stress", "oxidoreductase activity", "protein kinase activity", "transcription factor activity", and "plant hormone signal transduction". Further analyses suggested that the transcription abundance of many genes involved in SOD, the AsA-GSH cycle, the GPX pathway, PrxR/Trx, and the flavonoid biosynthesis pathway were obviously enhanced. These pathways are favorable for scavenging excessive ROS induced by salt and maintaining the integrity of the cell membrane. Meanwhile, many vital transcription factor genes (WRKY, MYB, ZF, HSF, DREB, and NAC) exhibited increased transcripts, which is conducive to dealing with saline conditions by regulating downstream salt-responsive genes. Furthermore, a larger number of genes encoding protein kinases (RLK, CDPK, MAPK, and CTR1) were significantly induced by saline conditions, which is beneficial to the reception/transduction of salt-related signals. This study describes the abundant genetic resources for enhancing the salt tolerance in salt-sensitive plants, especially in forages and crops.

Characterization of a bioderived imaging sensor based on a Morpho butterfly’s wing

The interest in imaging sensors with broad spectral ranges has increased, driven by both the industry as well as the scientific community. Thus, the need to improve their performance and lower their market cost seems to be higher than ever. Nature provides an unexpected solution to this requirement, as the wing scales of a Morpho butterfly exhibit radiation induced displacement which can be measured using digital holography. The scales on a wing thus act as pixels on an area detector. However, no detailed characterization of the imaging performance of such a detector was reported. We address this issue and perform spatial and temporal characterization of a Morpho wing as an imaging sensor using digital holographic interferometry and thermal imaging. We believe that this is the first image acquisition using the wing of a butterfly, showing that the sensor has linear response at 405, 450, 660 and 980nm wavelengths, with sensitivity that depends linearly on the absorption of the specimen. The temporal response is demonstrated to be reproducible, with the 55ms time constant, while the spatial resolution of 1,46lp/mm at 20% contrast was achieved. The obtained results confirm the imaging capabilities of a Morpho wing and open the path toward novel bioinspired imaging techniques.

High and lowland dependent wing phenotypic variation of the dark blue tiger butterfly, Tirumala septentrionis (Butler, 1874) (Lepidoptera: Nymphalidae) with FE-SEM wing scales nanomorphology

Butterflies display remarkable variation in wing shape and size, which is associated with tremendous ecological diversity. In butterflies, wing color patterns have been extensively studied, but wing shape and size diversity is less understood. In the present study, we investigated the altitude-specific wing asymmetry (right and left wings), sexual asymmetry, and wing covariation (proximal-distal and anterior-posterior wing organization) of Tirumala septentrionis (Butler, 1874). Using the geometric morphometric (GMM) method, we discovered that broad and expanded wings were found in lowland areas, whereas long and more rounded slender wings were found in highland areas. Furthermore, the ANOVA analysis, found significant (p < 0.05) altitude-specific shape and size differences in forewings and hindwings of both sexes. Thus, compared to size variations, the shape can be utilized as a valuable tool to distinguish sex (male and female), wing asymmetry (right and left) as well as altitudes (high and lowlands) specific variations of T. septentrionis. Furthermore, we noticed that lowland butterfly population had more asymmetric wings than the highland in the wing asymmetry analysis. We suspected that the unique wing morphology observed in the highland T. septentrionis population favored long-distance flight (indication of a migratory population). In contrast, the lowland population wing morphology indicated a newly colonized butterfly population. In addition to GMM analysis, we documented the FE-SEM wing scale morphology of T. septentrionis collected from two different altitudes. Interestingly, we observed that there is no difference in scale morphology related to altitude variations. Finally, we concluded that altitude-specific variations were observed mainly on wing shape and size, rather than the wing scales. These morphological variations were primarily associated with an adaptive response like migration, host searching, flight energy management and so on.

Phytoremediation Potential and Physiological Mechanisms Underlying Metallic Extraction of Suaeda glauca, Artemisia desertorum, and Atriplex canescens

Mining activities have led to serious environmental (soil erosion, degradation of vegetation, and groundwater contamination) and human health (musculoskeletal problems, diarrheal conditions, and chronic diseases) issues at desert mining areas in northwest China. Native plant species grown naturally in desert regions show a unique tolerance to arid and semiarid conditions and are potential candidates for soil phytoremediation. Here, an ex situ experiment involving pot planting of seedlings of three native plant species (Suaeda glauca, Artemisia desertorum, and Atriplex canescens) was designed to explore their phytoremediation potential and the underlying physiological mechanism. For Zn and Cu, the three plants were all with a biological accumulation coefficient (BAC) greater than 1. For Cd, Ni, and Pb, Atriplex canescens had the highest bioaccumulation concentrations (521.52, 862.23, and 1734.59 mg/kg), with BAC values (1.06, 1.30, 1.25) greater than 1, which indicates that Atriplex canescens could be a broad-spectrum metal extraction plant. Physiological analysis (antioxidation, extracellular secretions, photosynthesis, and hydraulics) showed that the three desert plants exploited their unique strategy to protect against the stress of complex metals in soils. Moreover, the second growing period was the main heavy metal accumulation and extraction stage concomitant with highest water use efficiency (iWUE). Taken together, the three desert plants exhibited the potent heavy metal extraction ability and physiological and ecological adaptability to a harsh polluted environment in arid desert areas, providing potential resources for the bioremediation of metal-contaminated soils in an arid and semiarid desert environment.

Cover Caption

As a species of butterflies, the blue tiger (Tirumala limniace) folds its wings to utilize the cooling power of wing scales when overheated by sunshine. However, it remained unclear what the cooling system looks like. It is found that light scales, dark scales, and scales on wing veins and margins are distinctly different in the nanostructure. The scale hierarchy of T. limniace diversifies through a single wing at the micro to nano‐level on both surface and section, corresponding to the different cooling efficiency (see pages 1761‐1772). Photo provided by Yu‐Jie Wang.

AcHKT1;2 is a candidate transporter mediating the influx of Na+ into the salt bladder of Atriplex canescens

AcHKT1;2 is a candidate transporter mediating the influx of Na+ into the salt bladder of Atriplex canescens

Growth and nutrient uptake of haloxylon and atriplex as affected by mycorrhizal symbiosis under combined drought and salinity stresses

This study was conducted to determine the effect of arbuscular mycorrhizal fungi (AMF) symbiosis on plant growth and nutrient uptake under combined drought and salinity stresses. A pot experiment was carried out with a factorial arrangement of treatments in a completely randomized design with three replications. Experimental treatments included two plant types ( Atriplex canescens and Haloxylon ammodendron) with three levels of inoculation of fungal species ( Funneliformis geosporus, Funneliformis mosseae, and control), two levels of soil salinity stress (7 and 14 dS m–1), and two levels of drought stress (50% and 80% of management allowable depletion). Vegetative parameters, as well as root N, P, and K concentrations and uptakes, mycorrhizal growth response, mycorrhizal nitrogen response, mycorrhizal phosphorus response, mycorrhizal potassium response, and root colonization were measured. The results showed that the application of AMF increased the plant growth variables such as stem diameter, root length, shoot dry weights, and shoot to root ratio as well as nitrogen and phosphorus uptakes. The application of both AMF types was effective as compared to the control. However, F. mosseae indicated better performance especially, in terms of the effect on plant growth variables. Also, F. mosseae was more effective to relieve stress (i.e., salinity and drought) than F. geosporus. There was a significant difference between plant types and H. ammodendron had better efficiency than A. canescens under drought and salinity stresses. Based on the results, planting of H. ammodendron inoculated with F. mosseae might be recommended for soil conservation in the arid environments.

Morphological investigations on the wing scales of four species of common Indian butterflies

Wing scales of butterflies exhibit extreme diversity in shape, size, colour, and number of spines. They are sub microscopic with a length of 300 to 600µm and a breadth of 150 to 400µm. A typical scale possessed a flat body with basal pedicel and apical crown which is provided with a varying number of pointed edges called spines. Investigations were carried outon the morphology of wing scales in four species of common butterflies viz., Pachilopta hector (Linnaeus 1758), Troides minos (Cramer 1779), Jamides celeno (Cramer1775) and Eurema andersonii (Linnaeus 1758). Wings of P. hector possessed nine types of scales, the crown of all are with pointed spines of varying numbers, ranging from one to five. The southern birdwing T. minos, possessed nine types of scales. A major portion of wings with black colour is due to black coloured scales but white bands of the forewings are due to transparent and colourless scales. The prominent yellow colour on the hind wings of this butterfly is due to the presence of a single type of scale with a round crown that is devoid of spines and is fully packed with yellow pigment. Jamides celeno possessed twenty different types of scales, most of them are devoid of spines and the ridges within the scales are not clear. Scales on the upper surface of the wing with ashy blue colour and scales of white bands on the lower surface of the wings are identical and are transparent and colourless. The common grass yellow E. andersonii possessed twentyfive different types of scales, of which thirteen are on the black margins of wings and twelve are in the yellow portions of wings. Almost half of the total number of scales in the yellow portions of the wing is transparent and colourless and in coloured scales distribution of pigment is not uniform.This is the first report on the different types of wings scales in the selected butterflies.

Back to the Future: Quantifying Wing Wear as a Method to Measure Mosquito Age.

Vector biologists have long sought the ability to accurately quantify the age of wild mosquito populations, a metric used to measure vector control efficiency. This has proven difficult due to the difficulties of working in the field and the biological complexities of wild mosquitoes. Ideal age grading techniques must overcome both challenges while also providing epidemiologically relevant age measurements. Given these requirements, the Detinova parity technique, which estimates age from the mosquito ovary and tracheole skein morphology, has been most often used for mosquito age grading despite significant limitations, including being based solely on the physiology of ovarian development. Here, we have developed a modernized version of the original mosquito aging method that evaluated wing wear, expanding it to estimate mosquito chronological age from wing scale loss. We conducted laboratory experiments using adult Anopheles gambiae held in insectary cages or mesocosms, the latter of which also featured ivermectin bloodmeal treatments to change the population age structure. Mosquitoes were age graded by parity assessments and both human- and computational-based wing evaluations. Although the Detinova technique was not able to detect differences in age population structure between treated and control mesocosms, significant differences were apparent using the wing scale technique. Analysis of wing images using averaged left- and right-wing pixel intensity scores predicted mosquito age at high accuracy (overall test accuracy: 83.4%, average training accuracy: 89.7%). This suggests that this technique could be an accurate and practical tool for mosquito age grading though further evaluation in wild mosquito populations is required.

Early evolution of wing scales prior to the rise of moths and butterflies

Although scales are a defining and conspicuous feature of moths and butterflies (Lepidoptera),1-3 their earliest evolution predates the group but is shrouded by a dearth of fossil evidence. Herein, we report two new species in mid-Cretaceous Kachin amber, representing lineages closely related to Lepidoptera: one represents the extinct Tarachoptera, with dense scales on the fore- and hindwings, while the other is an early lineage of caddisflies, with a hindwing covered by a single layer of angustifoliate scales. A novel phylogenetic analysis of 174 morphological characters and 73 extant and fossil representatives of Mecopterida demonstrates a monophyletic origin of scales in the common ancestor of Tarachoptera, Trichoptera, and Lepidoptera; that Tarachoptera are monophyletic but their scale morphology is plesiomorphic for the whole group; and that scales were lost early in caddisfly evolution before reappearing multiple times within the clade. Collectively, these fossils provide clarity into the origin and early evolution of scales before their diversification among the moths and butterflies.