The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

A micro-RNA is the effector gene of a classic evolutionary hotspot locus.

In Lepidoptera (butterflies and moths), the genomic region around the gene cortex is a 'hotspot' locus, repeatedly used to generate intraspecific melanic wing color polymorphisms across 100-million-years of evolution. However, the identity of the effector gene regulating melanic wing color within this locus remains unknown. Here, we show that none of the four candidate protein-coding genes within this locus, including cortex, serve as major effectors. Instead, a micro-RNA (miRNA), mir-193, serves as the major effector across three deeply diverged lineages of butterflies, and its function is conserved in Drosophila. In Lepidoptera, mir-193 is derived from a gigantic long non-coding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. We show that a miRNA can drive repeated instances of adaptive evolution in animals.

Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns.

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/ß-catenin dependent (canonical) and Armadillo/ß-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

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.

Historical spatiotemporal changes in fire danger potential across biomes.

This study 1) identifies the seasons and biomes that exhibit significant (1980-2019) changes in fire danger potential, as quantified by the Canadian Fire Weather Index (FWI); 2) explores what types of fire behavior potentials may be contributing to changes in fire danger potential, as quantified by the United States Energy Release Component (ERC) and the Ignition Component (IC); 3) provides spatiotemporal insight on how fire danger potential and fire behavior potential are responding in relation to changes in seasonal precipitation totals and seasonal mean air temperature across biomes. Time series of these fire potentials, as well as seasonal mean temperature, and seasonal precipitation totals are generated using data from the 0.25° ECMWF spatial resolution Reanalysis 5th Generation (ERA5) and the Climatic Research Unit gridded Time Series (CRU TS). The Mann-Kendall test is then applied to identify significant spatiotemporal trends across each biome. Results indicate that the September-November season (SON) exhibits the greatest rate of increase in fire danger potential, followed by the June-August season (JJA), December, January-February season (DJF), and March-May season (MAM), and this is predominant over the Tropical and Subtropical Moist Broadleaf Forest Biome, as well as all vegetation types of the temperate biomes. Similarly, the temperate biomes experience the greatest rate of increase in fire intensity potential and ignition potential, but prevalent during the DJF and MAM seasons. Furthermore, there is a significant positive correlation between fire danger potential and seasonal mean air temperature during JJA in the Northern Hemisphere for the temperate biomes in North America and Europe, as well as the Tropical and Subtropical biomes in Africa. Our analysis provides quantitative insight as to how fire danger potential and fire behavior potential have been responding to changes in seasonal mean temperature and seasonal precipitation totals across different ecoregions around the world.

Laser Microdissection-Mediated Isolation of Butterfly Wing Tissue for Spatial Transcriptomics.

The assignment of specific patterns of gene expression to specific cells in a complex tissue facilitates the connection between genotype and phenotype. Single-cell sequencing of whole tissues produces single-cell transcript resolution but lacks the spatial information of the derivation of each cell, whereas techniques such as multiplex FISH localize transcripts to specific cells in a tissue but require a priori information of the target transcripts to examine. Laser dissection of tissues followed by transcriptome analysis is an efficient and cost-effective technique that provides both unbiased gene expression discovery together with spatial information. Here, we detail a laser dissection protocol for total RNA extraction from butterfly larval and pupal wing tissues, without the need of paraffin embedding or the use of a microtome, that could be useful to researchers interested in the transcriptome of specific areas of the wing during development. This protocol can bypass difficulties in extracting high quality RNA from thick fixed tissues for sequencing applications.

Antennapedia and optix regulate metallic silver wing scale development and cell shape in Bicyclus anynana butterflies.

Butterfly wing scales can develop intricate cuticular nanostructures that produce silver colors, but the underlying genetic and physical basis of such colors is mostly unexplored. Here, we characterize different types of wild-type silver scales in Bicyclus anynana butterflies and show that the varying thickness of the air layer between two cuticular laminas is most important for producing silvery broadband reflectance. We then address the function of five genes-apterous A, Ultrabithorax, doublesex, Antennapedia, and optix-in silver scale development by examining crispants with either ectopic gains or losses of silver scales. Simultaneous transformations of three parameters-loss of the upper lamina, increased lower lamina thickness, and increased pigmentation-occur when silver scales become brown and vice versa when brown scales become silver. Antennapedia and optix are high-level regulators of different silver scale types and determine cell shape in both sexes. Moreover, Antennapedia is involved in determining ridge and crossrib orientation.

Distal-less and spalt are distal organisers of pierid wing patterns.

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespot's central signalling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centres in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

Investigating the turbulent dynamics of small-scale surface fires.

High frequency (30 Hz) two-dimensional particle image velocimetry data recorded during a field experiment exploring fire spread from point ignition in hand-spread pine needles under calm ambient wind conditions are analysed in this study. In the initial stages, as the flame spreads approximately radially away from the ignition point in the absence of a preferred wind-forcing direction, it entrains cooler ambient air into the warmer fire core, thereby experiencing a dynamic pressure resistance. The fire-front, comprising a flame that is tilted inward, is surrounded by a region of downdraft. Coherent structures describe the initial shape of the fire-front and its response to local wind shifts while also revealing possible fire-spread mechanisms. Vortex tubes originating outside the fire spiral inward and get stretched thinner at the fire-front leading to higher vorticity there. These tubes comprise circulation structures that induce a radially outward velocity close to the fuel bed, which pushes hot gases outward, thereby causing the fire to spread. Moreover, these circulation structures confirm the presence of counter-rotating vortex pairs that are known to be a key mechanism for fire spread. The axis of the vortex tubes changes its orientation alternately towards and away from the surface of the fuel bed, causing the vortex tubes to be kinked. The strong updraft observed at the location of the fire-front could potentially advect and tilt the kinked vortex tube vertically upward leading to fire-whirl formation. As the fire evolves, its perimeter disintegrates in response to flow instabilities to form smaller fire "pockets". These pockets are confined to certain points in the flow field that remain relatively fixed for a while and resemble the behavior of a chaotic system in the vicinity of an attractor. Increased magnitudes of the turbulent fluxes of horizontal momentum, computed at certain such fixed points along the fire-front, are symptomatic of irregular fire bursts and help contextualize the fire spread. Most importantly, the time-varying transport terms of the turbulent kinetic energy budget equation computed at adjacent fixed points indicate that local fires along the fire-front primarily interact via the horizontal turbulent transport term.

Historical seasonal changes in prescribed burn windows in California.

Prescribed (Rx) burns are conducted on days when the meteorological thresholds of maximum air temperature, relative humidity, and wind speeds are all met (burn window) in order to ensure safe Rx burn practices. Limited burn windows have been consistently identified as one of the most important constraints for conducting Rx burns in California. We investigate whether burn windows across California can be extended from the typical fall season to include other opportune seasons for facilitating specific management objectives. We quantify the seasonal Rx burn efficiencies by assessing the frequency and burned areas using an aggregate of Rx datasets, and we compute the seasonal spatiotemporal trends in the number of days the set of meteorological parameters are met over thirty-five years (1984 to 2019), using the gridMET 4 km dataset. Our results indicate that while fall burns are most frequently executed (40% of the time), the spring (and to a lesser extent winter) seasons yield efficient Rx burns similar to fall because greater acres are being consumed with less burns. In addition, winter and spring seasons experience burn window opportunities (70-90% of the time) over larger areas than the other seasons, and this is predominantly over forested regions in Northern California. Our results also indicate that burn windows in the winter and spring are decreasing at a rate of one day per year over a larger spatial area than that of summer and fall. This decrease is primarily driven by changes in the number of days the relative humidity thresholds are met. Policymakers recognize the critical importance that Rx burns have on a multitude of ecosystem restoration factors, fire behavior dynamics, and firefighter safety. Therefore, there is a need to capitalize on these additional burn windows before these opportunities become less feasible in the future.

Mapping the wildland-urban interface in California using remote sensing data.

Due to the mixed distribution of buildings and vegetation, wildland-urban interface (WUI) areas are characterized by complex fuel distributions and geographical environments. The behavior of wildfires occurring in the WUI often leads to severe hazards and significant damage to man-made structures. Therefore, WUI areas warrant more attention during the wildfire season. Due to the ever-changing dynamic nature of California's population and housing, the update frequency and resolution of WUI maps that are currently used can no longer meet the needs and challenges of wildfire management and resource allocation for suppression and mitigation efforts. Recent developments in remote sensing technology and data analysis algorithms pose new opportunities for improving WUI mapping methods. WUI areas in California were directly mapped using building footprints extracted from remote sensing data by Microsoft along with the fuel vegetation cover from the LANDFIRE dataset in this study. To accommodate the new type of datasets, we developed a threshold criteria for mapping WUI based on statistical analysis, as opposed to using more ad-hoc criteria as used in previous mapping approaches. This method removes the reliance on census data in WUI mapping, and does not require the calculation of housing density. Moreover, this approach designates the adjacent areas of each building with large and dense parcels of vegetation as WUI, which can not only refine the scope and resolution of the WUI areas to individual buildings, but also avoids zoning issues and uncertainties in housing density calculation. Besides, the new method has the capability of updating the WUI map in real-time according to the operational needs. Therefore, this method is suitable for local governments to map local WUI areas, as well as formulating detailed wildfire emergency plans, evacuation routes, and management measures.

Reimagine fire science for the anthropocene.

Fire is an integral component of ecosystems globally and a tool that humans have harnessed for millennia. Altered fire regimes are a fundamental cause and consequence of global change, impacting people and the biophysical systems on which they depend. As part of the newly emerging Anthropocene, marked by human-caused climate change and radical changes to ecosystems, fire danger is increasing, and fires are having increasingly devastating impacts on human health, infrastructure, and ecosystem services. Increasing fire danger is a vexing problem that requires deep transdisciplinary, trans-sector, and inclusive partnerships to address. Here, we outline barriers and opportunities in the next generation of fire science and provide guidance for investment in future research. We synthesize insights needed to better address the long-standing challenges of innovation across disciplines to (i) promote coordinated research efforts; (ii) embrace different ways of knowing and knowledge generation; (iii) promote exploration of fundamental science; (iv) capitalize on the "firehose" of data for societal benefit; and (v) integrate human and natural systems into models across multiple scales. Fire science is thus at a critical transitional moment. We need to shift from observation and modeled representations of varying components of climate, people, vegetation, and fire to more integrative and predictive approaches that support pathways toward mitigating and adapting to our increasingly flammable world, including the utilization of fire for human safety and benefit. Only through overcoming institutional silos and accessing knowledge across diverse communities can we effectively undertake research that improves outcomes in our more fiery future.

Spatial and temporal pattern of wildfires in California from 2000 to 2019.

The environmental pollution, property losses and casualties caused by wildfires in California are getting worse by the year. To minimize the interference of wildfires on economic and social development, and formulate targeted mitigation strategies, it is imperative to understand the scale and extent of previous wildfire occurrences. In this study, we first investigated the temporal distributions of past wildfires in California divided by size and causes and analyzed the changes observed in the past two decades against the last century. The trend of wildfires in different time scales (yearly and monthly), as well as the distribution of wildfires across different spatial scales (administrative units, climate divisions in California from 2000 to 2019) were also studied. Furthermore, to extract the significant variables on the risk of wildfire occurrence, multivariate analyses of environmental and human-related variables with wildfire densities were carried out. The results show that the wildfire density distribution of the burned area in California conforms to the characteristics of the Pareto distribution. Over the past two decades, the frequency of small (< 500 acres), human-caused wildfires has increased most rapidly, and they are widely distributed in central and western California. The wildfire season has lengthened and the peak months have been advanced from August to July. In terms of the variables related to the risk of wildfire occurrence, the temperature, vapor pressure deficit, grass cover, and the distance to roads are crucial. This study reveals the relationship between environmental and social background conditions and the spatial-temporal distribution of wildfires, which can provide a reference for wildfire management, the formulation of future targeted wildfire emergency plans, and the planning of future land use in California.

Overcoming challenges in implementing measures across multiple centers of a chain of hospitals to combat COVID-19 pandemic.

PURPOSE: Coronavirus outbreak was first reported towards the end of 2019 and has now been declared a pandemic by the World Health Organization. Post declaration, India announced lockdown and various guidelines were issued by Health authorities worldwide for the organizations to function in this emergency aeon. Different organizations strategized differently to respond to COVID 19 pandemic. The aim was to implement COVID 19 guidelines across all the centres of a chain of Eye hospitals in India in short span of time overcoming the challenges. METHODS: A SMART strategy was confabulated within the team. There were numerous challenges like lack of time, rapidly changing guidelines, no travelling scenarios, training and educating the staff in huge numbers, rapidly evolving situation, change management, need of increased vigilance, and language barrier at certain centres, sustaining quality assurance and motivating the staff in stressful times. The challenges were surpassed using a power packed strategy, dedicated efforts and team work grasping the available tools and technology from a single location. This template can serve us and other hospitals to respond in a better way for similar future challenges. RESULTS: The COVID 19 protocols were implemented across all the centres successfully. The entire Quality assurance system drifted to online platform and education and awareness methods took the digital thread. Teamwork synergized with the aforementioned things and we came out with flying colours achieving the goals in short span of time overcoming all the barriers. CONCLUSION: Difficult times bring challenges and challenges teach us the innovative ways to convert them into opportunities. A well planned strategy, best utilization of available resources, team work and continuous monitoring can bring the best outcomes forward.

Molecular mechanisms underlying simplification of venation patterns in holometabolous insects.

How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. Here, we examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia We also test the function of some of these genes in B. anynana We identify both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered and rhomboid gene expression in butterflies, and propose how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, via silencing of vein-inducing programs at Spalt-expression boundaries, and via changes in expression of vein maintenance genes.

Effects of canopy midstory management and fuel moisture on wildfire behavior.

Increasing trends in wildfire severity can partly be attributed to fire exclusion in the past century which led to higher fuel accumulation. Mechanical thinning and prescribed burns are effective techniques to manage fuel loads and to establish a higher degree of control over future fire risk, while restoring fire prone landscapes to their natural states of succession. However, given the complexity of interactions between fine scale fuel heterogeneity and wind, it is difficult to assess the success of thinning operations and prescribed burns. The present work addresses this issue systematically by simulating a simple fire line and propagating through a vegetative environment where the midstory has been cleared in different degrees, leading to a canopy with almost no midstory, another with a sparse midstory and another with a dense midstory. The simulations are conducted for these three canopies under two different conditions, where the fuel moisture is high and where it is low. These six sets of simulations show widely different fire behavior, in terms of fire intensity, spread rate and consumption. To understand the physical mechanisms that lead to these differences, detailed analyses are conducted to look at wind patterns, mean flow and turbulent fluxes of momentum and energy. The analyses also lead to improved understanding of processes leading to high intensity crowning behavior in presence of a dense midstory. Moreover, this work highlights the importance of considering fine scale fuel heterogeneity, seasonality, wind effects and the associated fire-canopy-atmosphere interactions while considering prescribed burns and forest management operations.

Dissection of Larval and Pupal Wings of Bicyclus anynana Butterflies.

The colorful wings of butterflies are emerging as model systems for evolutionary and developmental studies. Some of these studies focus on localizing gene transcripts and proteins in wings at the larval and pupal stages using techniques such as immunostaining and in situ hybridization. Other studies quantify mRNA expression levels or identify regions of open chromatin that are bound by proteins at different stages of wing development. All these techniques require dissection of the wings from the animal but a detailed video protocol describing this procedure has not been available until now. Here, we present a written and accompanying video protocol where we describe the tools and the method we use to remove the larval and pupal wings of the African Squinting Bush Brown butterfly Bicyclus anynana. This protocol should be easy to adapt to other species.

Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process.

Eyespots on the wings of nymphalid butterflies represent colorful examples of pattern formation, yet the developmental origins and mechanisms underlying eyespot center differentiation are still poorly understood. Using CRISPR-Cas9 we re-examine the function of Distal-less (Dll) as an activator or repressor of eyespots, a topic that remains controversial. We show that the phenotypic outcome of CRISPR mutations depends upon which specific exon is targeted. In Bicyclus anynana, exon 2 mutations are associated with both missing and ectopic eyespots, and also exon skipping. Exon 3 mutations, which do not lead to exon skipping, produce only null phenotypes, including missing eyespots, lighter wing coloration and loss of scales. Reaction-diffusion modeling of Dll function, using Wnt and Dpp as candidate morphogens, accurately replicates these complex crispant phenotypes. These results provide new insight into the function of Dll as a potential activator of eyespot development, scale growth and melanization, and suggest that the tuning of Dll expression levels can generate a diversity of eyespot phenotypes, including their appearance on the wing.This article has an associated 'The people behind the papers' interview.

CRISPR-Cas9 Mediated Genome Editing in Bicyclus anynana Butterflies.

CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation.