High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

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.

Patterning of the Drosophila L2 vein is driven by regulatory interactions between region-specific transcription factors expressed in response to Dpp signalling.

Pattern formation relies on the generation of transcriptional landscapes regulated by signalling pathways. A paradigm of epithelial patterning is the distribution of vein territories in the Drosophila wing disc. In this tissue, Decapentaplegic signalling regulates its target genes at different distances from the source of the ligand. The transformation of signalling into coherent territories of gene expression requires regulatory cross-interactions between these target genes. Here, we analyse the mechanisms generating the domain of knirps expression in the presumptive L2 vein of the wing imaginal disc. We find that knirps is regulated by four Decapentaplegic target genes encoding the transcription factors aristaless, spalt major, spalt-related and optix The expression of optix is activated by Dpp and repressed by the Spalt proteins, becoming restricted to the most anterior region of the wing blade. In turn, the expression of knirps is activated by Aristaless and repressed by Optix and the Spalt proteins. In this manner, the expression of knirps becomes restricted to those cells where Spalt levels are sufficient to repress optix, but not sufficient to repress knirps.

Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence.

The optix gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. optix knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that optix simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these optix knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that optix plays a deeply conserved role in butterfly wing pattern development.

Anteroposterior patterning of Drosophila ocelli requires an anti-repressor mechanism within the hh pathway mediated by the Six3 gene Optix.

In addition to compound eyes, most insects possess a set of three dorsal ocelli that develop at the vertices of a triangular cuticle patch, forming the ocellar complex. The wingless and hedgehog signaling pathways, together with the transcription factor encoded by orthodenticle, are known to play major roles in the specification and patterning of the ocellar complex. Specifically, hedgehog is responsible for the choice between ocellus and cuticle fates within the ocellar complex primordium. However, the interactions between signals and transcription factors known to date do not fully explain how this choice is controlled. We show that this binary choice depends on dynamic changes in the domains of hedgehog signaling. In this dynamics, the restricted expression of engrailed, a hedgehog signaling target, is key because it defines a domain within the complex where hedgehog transcription is maintained while the pathway activity is blocked. We further show that the Drosophila Six3, optix, is expressed in and required for the development of the anterior ocellus specifically, limiting the ocellar expression domain of en. This finding confirms previous genetic evidence that the spatial allocation of the primordia of anterior and posterior ocelli is differentially regulated, which may apply to the patterning of the insect head in general.

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila.

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein-protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.

CRISPR in butterflies: An undergraduate lab experience to inactivate wing patterning genes during development.

CRISPR is a technique increasingly used in the laboratory for both fundamental and applied research. We designed and implemented a lab experience for undergraduates to carry out CRISPR technology in the lab, and knockout the wing patterning genes optix and WntA in Vanessa cardui butterflies. Students obtained spectacular phenotypic mutants of butterfly wings color and patterns, awakening curiosity about how genomes encode morphology. In addition, students successfully used molecular techniques to genotype and screen wild-type caterpillar larvae and butterflies for CRISPR edits in genes. Student feedback suggests that they experienced a meaningful process of scientific inquiry by carrying out the whole CRISPR workflow process, from the design and delivery of CRISPR components through microinjection of butterfly eggs, the rearing of live animals through their complete life cycle, and molecular and phenotypic analyses of the resulting mutants. We discuss our experience using CRISP genome editing experiments in butterflies to expose students to hands-on research experiences probing gene-to-phenotype relationships in a charismatic and live organism.

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.

Refraction in Preschool Children in Kazanlak, Bulgaria.

INTRODUCTION: The refractive state of the eye changes as the eye's axial length increases and the cornea and lens flatten. In general, eyes are hyperopic at birth, become slightly more hyperopic until the age of 7, which at this point we see a myopic shift toward plano until the eyes reach their adult dimensions, usually by about the age 16. AIM: To determine the prevalence of refractive error in preschool children aged 3 - 6 in the city of Kazanlak, Bulgaria. MATERIALS AND METHODS: A cross-sectional study was conducted in a kindergarten based school within the city of Kazanlak, Bul-garia. Out of 15 kindergarten schools, 10 chose to participate in the study. The children underwent non-mydriatic refraction screening using a Plus-Optix S12с mobile camera. Myopia, hyperopia and astigmatism were defined as being a spherical equivalent (SE) ≤ -0.50 D, SE ≥ +2.00 D and cylindrical diopters ≤ -1.00 D. RESULTS: A total of 596 children were screened. Out of these 596 children; 526 were with ametropia (470 hypermetropia, 46 myopia) and 50 had astigmatism. Strabismus was found in 12 children, with a further 8 children suffering from amblyopia and finally 8 children were also found to be diagnosed with anisometropia. In 8 cases there were no results due to opacities or due to the small size of the pupil. CONCLUSION: The prevalence of refractive error in preschool children is similar to that found in other research in the field. While most cases fall into the category of emmetropia or mild hypermetropia, most of the children had never been to an ophthalmologist. A manda-tory checkup should be issued for all children below the age of seven.

Structural color in Junonia butterflies evolves by tuning scale lamina thickness.

In diverse organisms, nanostructures that coherently scatter light create structural color, but how such structures are built remains mysterious. We investigate the evolution and genetic regulation of butterfly scale laminae, which are simple photonic nanostructures. In a lineage of buckeye butterflies artificially selected for blue wing color, we found that thickened laminae caused a color shift from brown to blue. Deletion of the optix patterning gene also altered color via lamina thickening, revealing shared regulation of pigments and lamina thickness. Finally, we show how lamina thickness variation contributes to the color diversity that distinguishes sexes and species throughout the genus Junonia. Thus, quantitatively tuning one dimension of scale architecture facilitates both the microevolution and macroevolution of a broad spectrum of hues. Because the lamina is an intrinsic component of typical butterfly scales, our findings suggest that tuning lamina thickness is an available mechanism to create structural color across the Lepidoptera.

From iridescent blues to vibrant purples, many butterflies display dazzling 'structural colors' created not by pigments but by microscopic structures that interfere with light. For instance, the scales that coat their wings can contain thin films of chitin, the substance that normally makes the external skeleton of insects. In slim layers, however, chitin can also scatter light to produce color, the way that oil can create iridescence at the surface of water. The thickness of the film, which is encoded by the genes of the butterfly, determines what color will be produced. Yet, little is known about how common thin films are in butterflies, exactly how genetic information codes for them, and how their thickness and the colors they produce can evolve. To investigate, Thayer et al. used a technique called Helium Ion Microscopy and examined the wings of ten related species of butterflies, showing that thin film structures were present across this sample. However, the different species have evolved many different structural colors over the past millions of years by changing the thickness of the films. Next, Thayer et al. showed that this evolution could be reproduced at a faster pace in the laboratory using common buckeye butterflies. These insects mostly have brown wings, but they can have specks of blue created by thin film structures. Individuals with more blue on their wings were mated and over the course of a year, the thickness of the film structures increased by 74%, leading to shiny blue butterflies. Deleting a gene called optix from the insects also led to blue wings. Optix was already known to control the patterns of pigments in butterflies, but it now appears that it controls structural colors as well. From solar panels to new fabrics, microscopic structures that can scatter light are useful in a variety of industries. Understanding how these elements exist and evolve in organisms may help to better design them for human purposes.

Resolution of an exposed pars plana Baerveldt shunt in a patient with a Boston keratoprosthesis type 1 without surgery.

Patients with a keratoprosthesis often develop complications including glaucoma, requiring glaucoma drainage devices. In most of these patients, glaucoma drainage devices have been shown to be safe and effective. However, occasionally, a glaucoma drainage device in the setting of a keratoprosthesis can lead to conjunctival erosion with mechanical trauma. While repeat surgical intervention may appear necessary, we report a case of a patient who had improved conjunctival erosion and glaucoma drainage device exposure after refitting of a therapeutic contact lens. Therapeutic contact lenses can be used to maintain hydration and decrease exposure while improving cosmesis and refractive error. Complications following keratoprosthesis surgery are an understudied area, particularly regarding glaucoma drainage devices, and we seek to show that careful fitting of therapeutic contact lenses may avoid the risks of repeat surgical intervention.

Specific expression and function of the Six3 optix in Drosophila serially homologous organs.

Organ size and pattern results from the integration of two positional information systems. One global information system, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. However, the mechanisms of the Hox-signal integration in this well-studied system are incomplete. Here, we have investigated this issue by studying the expression and function of the Six3 transcription factor optix during Drosophila wing and haltere development. We find that in both organs, Dpp defines the expression domain of optix through repression, and that the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. We show that optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere. In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin; the halteres of optix mutants are also significantly reduced. In addition, in the haltere, optix is necessary for the suppression of sensory bristles.

Optical coherence tomography to evaluate the interaction of different edge designs of four different silicone hydrogel lenses with the ocular surface.

PURPOSE: To evaluate the lens edge interaction with the ocular surface with different edge designs using optical coherence tomography and to examine the effect of lens power on the lens edge interactions. METHODS: Four types of silicone hydrogel lenses with different edge designs (round-, semi-round-, chisel-, and knife-edged) at six different powers (+5.0, +3.0, +1.0, -1.0, -3.0, and -5.0 diopters) were fitted to both eyes of 20 healthy volunteers. Optical coherence tomography images were taken at the corneal center and at the limbus within 15-30 minutes after insertion. The images were evaluated with respect to two parameters: conjunctival indentation exerted by the lens edge; and the tear film gaps between the posterior surface of the lens and the ocular surface. The amount of conjunctival indentation was measured with the distortion angle of the conjunctiva at the lens edge. RESULTS: The degree of conjunctival indentation was highest with the chisel-edged design followed by the semi-round design (P<0.0001). Knife- and round-edged lenses exerted similar levels of conjunctival indentation that was significantly lower compared to chisel-edged lens (P<0.001). For each one of the tested lens edge designs, no significant difference was observed in the conjunctival indentation with respect to lens power. The chisel-edged lens produced the highest amount of conjunctival indentation for each one of the six lens powers (P<0.0001). Post-lens tear film gaps at the limbus were observed at most in the round-edge design (P=0.001). CONCLUSION: The fitting properties of contact lenses may be influenced by their edge design but not by their lens power.