Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Novel Doublesex Duplication Associated with Sexually Dimorphic Development of Dogface Butterfly Wings.

Sexually dimorphic development is responsible for some of the most remarkable phenotypic variation found in nature. Alternative splicing of the transcription factor gene doublesex (dsx) is a highly conserved developmental switch controlling the expression of sex-specific pathways. Here, we leverage sex-specific differences in butterfly wing color pattern to characterize the genetic basis of sexually dimorphic development. We use RNA-seq, immunolocalization, and motif binding site analysis to test specific predictions about the role of dsx in the development of structurally based ultraviolet (UV) wing patterns in Zerene cesonia (Southern Dogface). Unexpectedly, we discover a novel duplication of dsx that shows a sex-specific burst of expression associated with the sexually dimorphic UV coloration. The derived copy consists of a single exon that encodes a DNA binding but no protein-binding domain and has experienced rapid amino-acid divergence. We propose the novel dsx paralog may suppress UV scale differentiation in females, which is supported by an excess of Dsx-binding sites at cytoskeletal and chitin-related genes with sex-biased expression. These findings illustrate the molecular flexibility of the dsx gene in mediating the differentiation of secondary sexual characteristics.

Knockdowns of red Malphigian tubules reveal pigmentation roles in the milkweed bug.

Classical Drosophila eye color mutations have unearthed a toolkit of genes that have permitted candidate gene studies of the outstanding diversity of coloration patterns in other insects. The gene underlying the eye color phenotypes of the red Malphigian tubules (red) fly mutant was mapped to a LysM domain gene of unknown molecular function. Here, we used RNAi to test the role of a red ortholog in the pigmentation of the milkweed bug Oncopeltus fasciatus, and contrast its effect with the ommochrome biosynthetic pathway gene vermilion (ver). Pigmentation was reduced in the cuticle of embryonic legs and first instar abdomens following parental RNAi against red, but not against ver, likely reflecting an effect on pterin biogenesis. Nymphal RNAi of red and ver both resulted in adult eye depigmentation, consistent with an effect on ommochrome content. These results suggest red loss-of-function impacts biochemically distinct types of pigments, and we discuss its putative role in the biogenesis of lysosome-related organelles such as ommochromasomes and pterinosomes.

Gephebase, a database of genotype-phenotype relationships for natural and domesticated variation in Eukaryotes.

Gephebase is a manually-curated database compiling our accumulated knowledge of the genes and mutations that underlie natural, domesticated and experimental phenotypic variation in all Eukaryotes-mostly animals, plants and yeasts. Gephebase aims to compile studies where the genotype-phenotype association (based on linkage mapping, association mapping or a candidate gene approach) is relatively well supported. Human clinical traits and aberrant mutant phenotypes in laboratory organisms are not included and can be found in other databases (e.g. OMIM, OMIA, Monarch Initiative). Gephebase contains more than 1700 entries. Each entry corresponds to an allelic difference at a given gene and its associated phenotypic change(s) between two species or two individuals of the same species, and is enriched with molecular details, taxonomic information, and bibliographic information. Users can easily browse entries and perform searches at various levels using boolean operators (e.g. transposable elements, snakes, carotenoid content, Doebley). Data is exportable in spreadsheet format. This database allows to perform meta-analyses to extract global trends about the living world and the research fields. Gephebase should also help breeders, conservationists and others to identify promising target genes for crop improvement, parasite/pest control, bioconservation and genetic diagnostic. It is freely available at www.gephebase.org.

Porcupine/Wntless-dependent trafficking of the conserved WntA ligand in butterflies.

Wnt ligands are key signaling molecules in animals, but little is known about the evolutionary dynamics and mode of action of the WntA orthologs, which are not present in the vertebrates or in Drosophila. Here we show that the WntA subfamily evolved at the base of the Bilateria + Cnidaria clade, and conserved the thumb region and Ser209 acylation site present in most other Wnts, suggesting WntA requires the core Wnt secretory pathway. WntA proteins are distinguishable from other Wnts by a synapomorphic Iso/Val/Ala216 amino-acid residue that replaces the otherwise ubiquitous Thr216 position. WntA embryonic expression is conserved between beetles and butterflies, suggesting functionality, but the WntA gene was lost three times within arthropods, in podoplean copepods, in the cyclorrhaphan fly radiation, and in ensiferan crickets and katydids. Finally, CRISPR mosaic knockouts (KOs) of porcupine and wntless phenocopied the pattern-specific effects of WntA KOs in the wings of Vanessa cardui butterflies. These results highlight the molecular conservation of the WntA protein across invertebrates, and imply it functions as a typical Wnt ligand that is acylated and secreted through the Porcupine/Wntless secretory pathway.

The coding loci of evolution and domestication: current knowledge and implications for bio-inspired genome editing.

One promising application of CRISPR/Cas9 is to create targeted mutations to introduce traits of interest into domesticated organisms. However, a major current limitation for crop and livestock improvement is to identify the precise genes and genetic changes that must be engineered to obtain traits of interest. Here, we discuss the advantages of bio-inspired genome editing, i.e. the engineered introduction of natural mutations that have already been associated with traits of interest in other lineages (breeds, populations or species). To obtain a landscape view of potential targets for genome editing, we used Gephebase (www.gephebase.org), a manually curated database compiling published data about the genes responsible for evolutionary and domesticated changes across eukaryotes, and examined the >1200 mutations that have been identified in the coding regions of more than 700 genes in animals, plants and yeasts. We observe that our genetic knowledge is relatively important for certain traits, such as xenobiotic resistance, and poor for others. We also note that protein-null alleles, often owing to nonsense and frameshift mutations, represent a large fraction of the known loci of domestication (42% of identified coding mutations), compared with intraspecific (27%) and interspecific evolution (11%). Although this trend may be subject to detection, publication and curation biases, it is consistent with the idea that breeders have selected large-effect mutations underlying adaptive traits in specific settings, but that these mutations and associated phenotypes would not survive the vagaries of changing external and internal environments. Our compilation of the loci of evolution and domestication uncovers interesting options for bio-inspired and transgene-free genome editing.

Bringing immersive science to undergraduate laboratory courses using CRISPR gene knockouts in frogs and butterflies.

The use of CRISPR/Cas9 for gene editing offers new opportunities for biology students to perform genuine research exploring the gene-to-phenotype relationship. It is important to introduce the next generation of scientists, health practitioners and other members of society to the technical and ethical aspects of gene editing. Here, we share our experience leading hands-on undergraduate laboratory classes, where students formulate hypotheses regarding the roles of candidate genes involved in development, perform loss-of-function experiments using programmable nucleases and analyze the phenotypic effects of mosaic mutant animals. This is enabled by the use of the amphibian Xenopus laevis and the butterfly Vanessa cardui, two organisms that reliably yield hundreds of large and freshly fertilized eggs in a scalable manner. Frogs and butterflies also present opportunities to teach key biological concepts about gene regulation and development. To complement these practical aspects, we describe learning activities aimed at equipping students with a broad understanding of genome editing techniques, their application in fundamental and translational research, and the bioethical challenges they raise. Overall, our work supports the introduction of CRISPR technology into undergraduate classrooms and, when coupled with classroom undergraduate research experiences, enables hypothesis-driven research by undergraduates.

Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity.

Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings. In two Heliconius species, WntA specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly Agraulis, WntA removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, WntA acquired a divergent role in the patterning of interveinous patterns in the monarch, a basal nymphalid butterfly that lacks stripe-like symmetry systems. These results identify WntA as an instructive signal for the prepatterning of a biological system of exuberant diversity and illustrate how shifts in the deployment and effects of a single developmental gene underlie morphological change.

Sub-micrometer insights into the cytoskeletal dynamics and ultrastructural diversity of butterfly wing scales.

BACKGROUND: The color patterns that adorn lepidopteran wings are ideal for studying cell type diversity using a phenomics approach. Color patterns are made of chitinous scales that are each the product of a single precursor cell, offering a 2D system where phenotypic diversity can be studied cell by cell, both within and between species. Those scales reveal complex ultrastructures in the sub-micrometer range that are often connected to a photonic function, including iridescent blues and greens, highly reflective whites, or light-trapping blacks. RESULTS: We found that during scale development, Fascin immunostainings reveal punctate distributions consistent with a role in the control of actin patterning. We quantified the cytoskeleton regularity as well as its relationship to chitin deposition sites, and confirmed a role in the patterning of the ultrastructures of the adults scales. Then, in an attempt to characterize the range and variation in lepidopteran scale ultrastructures, we devised a high-throughput method to quickly derive multiple morphological measurements from fluorescence images and scanning electron micrographs. We imaged a multicolor eyespot element from the butterfly Vanessa cardui (V. cardui), taking approximately 200 000 individual measurements from 1161 scales. Principal component analyses revealed that scale structural features cluster by color type, and detected the divergence of non-reflective scales characterized by tighter cross-rib distances and increased orderedness. CONCLUSION: We developed descriptive methods that advance the potential of butterfly wing scales as a model system for studying how a single cell type can differentiate into a multifaceted spectrum of complex morphologies. Our data suggest that specific color scales undergo a tight regulation of their ultrastructures, and that this involves cytoskeletal dynamics during scale growth.

Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis.

Hox genes play crucial roles in establishing regional identity along the anterior-posterior axis in bilaterian animals, and have been implicated in generating morphological diversity throughout evolution. Here we report the identification, expression, and initial genomic characterization of the complete set of Hox genes from the amphipod crustacean Parhyale hawaiensis. Parhyale is an emerging model system that is amenable to experimental manipulations and evolutionary comparisons among the arthropods. Our analyses indicate that the Parhyale genome contains a single copy of each canonical Hox gene with the exception of fushi tarazu, and preliminary mapping suggests that at least some of these genes are clustered together in the genome. With few exceptions, Parhyale Hox genes exhibit both temporal and spatial colinearity, and expression boundaries correlate with morphological differences between segments and their associated appendages. This work represents the most comprehensive analysis of Hox gene expression in a crustacean to date, and provides a foundation for functional studies aimed at elucidating the role of Hox genes in arthropod development and evolution.

Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems.

Most butterfly wing patterns are proposed to be derived from a set of conserved pattern elements known as symmetry systems. Symmetry systems are so-named because they are often associated with parallel color stripes mirrored around linear organizing centers that run between the anterior and posterior wing margins. Even though the symmetry systems are the most prominent and diverse wing pattern elements, their study has been confounded by a lack of knowledge regarding the molecular basis of their development, as well as the difficulty of drawing pattern homologies across species with highly derived wing patterns. Here we present the first molecular characterization of symmetry system development by showing that WntA expression is consistently associated with the major basal, discal, central, and external symmetry system patterns of nymphalid butterflies. Pharmacological manipulations of signaling gradients using heparin and dextran sulfate showed that pattern organizing centers correspond precisely with WntA, wingless, Wnt6, and Wnt10 expression patterns, thus suggesting a role for Wnt signaling in color pattern induction. Importantly, this model is supported by recent genetic and population genomic work identifying WntA as the causative locus underlying wing pattern variation within several butterfly species. By comparing the expression of WntA between nymphalid butterflies representing a range of prototypical symmetry systems, slightly deviated symmetry systems, and highly derived wing patterns, we were able to infer symmetry system homologies in several challenging cases. Our work illustrates how highly divergent morphologies can be derived from modifications to a common ground plan across both micro- and macro-evolutionary time scales.

Susceptibility Pattern of Microorganisms Isolated by Percutaneous Needle Biopsy in Nonbacteremic Pyogenic Vertebral Osteomyelitis.

Pyogenic vertebral osteomyelitis (VO) is diagnosed according to several lines of evidence: clinical, biological, radiological, and histological. Definitive diagnosis requires the isolation of a causative pathogen or histological confirmation. The aim of our study was to describe the microorganisms isolated by percutaneous needle biopsy (PNB) and to analyze their susceptibility patterns, in order to assess the possibility of empirical combination therapy for the treatment of nonbacteremic patients without resorting to PNB. Based on a French prospective multicenter study of 351 patients with VO, we compiled clinical, biological, and radiological findings for 101 patients with microbiologically confirmed VO. Based on antibiotic susceptibility testing of PNB isolated pathogens, the suitabilities of four antibiotic combinations were analyzed: ofloxacin plus rifampin, levofloxacin plus rifampin, ciprofloxacin plus clindamycin, and ciprofloxacin plus amoxicillin-clavulanate. The main causative pathogens identified were coagulase-negative Staphylococcus spp. (26% of isolates), followed by Staphylococcus aureus (21%), Streptoccocus spp. (13%), and enterobacteria (21%). Empirical antibiotic combination therapy was effective in nearly 75% of cases, and the different combinations gave similar results, except for ofloxacin-rifampin, which was effective in only 58% of cases. A "perfect" empirical antibiotic therapy does not exist. If PNB is not possible, a combination of a fluoroquinolone with clindamycin or rifampin can be used, but the high risk of microbiological failure does not allow the exclusion of PNB. (This study has been registered with EudraCT, number 2006-000951-18, and ClinicalTrials.gov, number NCT00764114.).

Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand.

Although animals display a rich variety of shapes and patterns, the genetic changes that explain how complex forms arise are still unclear. Here we take advantage of the extensive diversity of Heliconius butterflies to identify a gene that causes adaptive variation of black wing patterns within and between species. Linkage mapping in two species groups, gene-expression analysis in seven species, and pharmacological treatments all indicate that cis-regulatory evolution of the WntA ligand underpins discrete changes in color pattern features across the Heliconius genus. These results illustrate how the direct modulation of morphogen sources can generate a wide array of unique morphologies, thus providing a link between natural genetic variation, pattern formation, and adaptation.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Analysis of the genetic loci of pigment pattern evolution in vertebrates.

Vertebrate pigmentation patterns are amongst the best characterised model systems for studying the genetic basis of adaptive evolution. The wealth of available data on the genetic basis for pigmentation evolution allows for analysis of trends and quantitative testing of evolutionary hypotheses. We employed Gephebase, a database of genetic variants associated with natural and domesticated trait variation, to examine trends in how cis-regulatory and coding mutations contribute to vertebrate pigmentation phenotypes, as well as factors that favour one mutation type over the other. We found that studies with lower ascertainment bias identified higher proportions of cis-regulatory mutations, and that cis-regulatory mutations were more common amongst animals harbouring a higher number of pigment cell classes. We classified pigmentation traits firstly according to their physiological basis and secondly according to whether they affect colour or pattern, and identified that carotenoid-based pigmentation and variation in pattern boundaries are preferentially associated with cis-regulatory change. We also classified genes according to their developmental, cellular, and molecular functions. We found a greater proportion of cis-regulatory mutations in genes implicated in upstream developmental processes compared to those involved in downstream cellular functions, and that ligands were associated with a higher proportion of cis-regulatory mutations than their respective receptors. Based on these trends, we discuss future directions for research in vertebrate pigmentation evolution.

Representation of Imprecision in Deep Neural Networks for Image Classification.

Quantification and reduction of uncertainty in deep-learning techniques have received much attention but ignored how to characterize the imprecision caused by such uncertainty. In some tasks, we prefer to obtain an imprecise result rather than being willing or unable to bear the cost of an error. For this purpose, we investigate the representation of imprecision in deep-learning (RIDL) techniques based on the theory of belief functions (TBF). First, the labels of some training images are reconstructed using the learning mechanism of neural networks to characterize the imprecision in the training set. In the process, a label assignment rule is proposed to reassign one or more labels to each training image. Once an image is assigned with multiple labels, it indicates that the image may be in an overlapping region of different categories from the feature perspective or the original label is wrong. Second, those images with multiple labels are rechecked. As a result, the imprecision (multiple labels) caused by the original labeling errors will be corrected, while the imprecision caused by insufficient knowledge is retained. Images with multiple labels are called imprecise ones, and they are considered to belong to meta-categories, the union of some specific categories. Third, the deep network model is retrained based on the reconstructed training set, and the test images are then classified. Finally, some test images that specific categories cannot distinguish will be assigned to meta-categories to characterize the imprecision in the results. Experiments based on some remarkable networks have shown that RIDL can improve accuracy (AC) and reasonably represent imprecision both in the training and testing sets.

Early origin and diverse phenotypic implementation of iridescent UV patterns for sexual signaling in pierid butterflies.

Iridescent ultraviolet (IUV) patterns on pierid butterfly wings are phenotypic adaptations commonly used as sexual signals, generated by scales with ultrastructural modifications. Pierid IUV patterns are sexually dichromatic, with reduced size in females, where conspicuous sexual signaling balances courtship against ecological predation. There have been no phylogenetic reconstructions of IUV within Pieridae and little morphological characterization of phenotypic diversity. Our genus-wide characterization of IUV revealed the uniform similarity of stacked lamellar ridges on the dorsal surface of cover scales. We tested a hypothesis of single versus multiple origins by reconstructing a phylogeny of 534 species (~43.2% described species), with all genera represented, and a trait matrix of 734 species (~59.4%) screened for IUV. A single, early dimorphic origin of IUV followed by several losses and gains received strong support, concluding that IUV patterns and structural coloration are old traits. Collectively, these results support the homology of IUV scales and patterns that diversified within several lineages, suggesting an interplay between female-mediated sexual selection and ecological predatory selection.

Genetics of yellow-orange color variation in a pair of sympatric sulphur butterflies.

Continuous color polymorphisms can serve as a tractable model for the genetic and developmental architecture of traits. Here we investigated continuous color variation in Colias eurytheme and Colias philodice, two species of sulphur butterflies that hybridize in sympatry. Using quantitative trait locus (QTL) analysis and high-throughput color quantification, we found two interacting large-effect loci affecting orange-to-yellow chromaticity. Knockouts of red Malpighian tubules (red), likely involved in endosomal maturation, result in depigmented wing scales. Additionally, the transcription factor bric-a-brac can act as a modulator of orange pigmentation. We also describe the QTL architecture of other continuously varying traits, together supporting a large-X effect model where the genetic control of species-defining traits is enriched on sex chromosomes. This study sheds light on the range of possible genetic architectures that can underpin a continuously varying trait and illustrates the power of using automated measurement to score phenotypes that are not always conspicuous to the human eye.

Evidence for a single, ancient origin of a genus-wide alternative life history strategy.

Understanding the evolutionary origins and factors maintaining alternative life history strategies (ALHS) within species is a major goal of evolutionary research. While alternative alleles causing discrete ALHS are expected to purge or fix over time, one-third of the ~90 species of Colias butterflies are polymorphic for a female-limited ALHS called Alba. Whether Alba arose once, evolved in parallel, or has been exchanged among taxa is currently unknown. Using comparative genome-wide association study (GWAS) and population genomic analyses, we placed the genetic basis of Alba in time-calibrated phylogenomic framework, revealing that Alba evolved once near the base of the genus and has been subsequently maintained via introgression and balancing selection. CRISPR-Cas9 mutagenesis was then used to verify a putative cis-regulatory region of Alba, which we identified using phylogenetic foot printing. We hypothesize that this cis-regulatory region acts as a modular enhancer for the induction of the Alba ALHS, which has likely facilitated its long evolutionary persistence.