Disaggregation and fibrillation during sol-gel transition of alginate hydrogels.

The rheological and morphological characteristics of Ca-crosslinked alginate hydrogels with two different M/G ratios, α-L-guluronate (G)-rich and ß-D-mannuronate (M)-rich, each with one alginic acid concentration, were investigated. It was found that the stiffness and elasticity of alginate hydrogels are derived from the thickness and density of the fibril network structures. In aqueous alginate solution, ball-like aggregates of alginates are present. Time-resolved small-angle X-ray scattering and time-domain nuclear magnetic resonance measurements suggest that the disaggregation of alginate aggregates and loose fibrillation occur in the early stage of the sol-gel transition. After these induction stage, direct gelation is finally caused by the formation of the egg-box junction. G-rich alginate hydrogel has a higher stiffness and a thicker and denser fibril network structure than M-rich alginate hydrogel. The former also exhibits faster and more significant changes in physical properties during the sol-gel transition.

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Transcriptomic comparison between populations selected for higher and lower mobility in the red flour beetle Tribolium castaneum.

Movement is an important behavior observed in a wide range of taxa. Previous studies have examined genes controlling movement using wing polymorphic insects and genes controlling wing size. However, few studies have investigated genes controlling movement activity rather than morphological traits. In the present study, we conducted RNA sequencing using populations with higher (WL) and lower (WS) mobility established by artificial selection in the red flour beetle Tribolium castaneum and compared gene expression levels between selected populations with two replicate lines. As a result, we found significant differences between the selected populations in 677 genes expressed in one replicate line and 1198 genes expressed in another replicate line, of which 311 genes were common to the two replicate lines. Furthermore, quantitative PCR focusing on 6 of these genes revealed that neuropeptide F receptor gene (NpF) was significantly more highly expressed in the WL population than in the WS population, which was common to the two replicate lines. We discuss differences in genes controlling movement between walking activity and wing polymorphism.

Massive horizontal gene transfer and the evolution of nematomorph-driven behavioral manipulation of mantids.

To complete their life cycle, a wide range of parasites must manipulate the behavior of their hosts.1 This manipulation is a well-known example of the "extended phenotype,2" where genes in one organism have phenotypic effects on another organism. Recent studies have explored the parasite genes responsible for such manipulation of host behavior, including the potential molecular mechanisms.3,4 However, little is known about how parasites have acquired the genes involved in manipulating phylogenetically distinct hosts.4 In a fascinating example of the extended phenotype, nematomorph parasites have evolved the ability to induce their terrestrial insect hosts to enter bodies of water, where the parasite then reproduces. Here, we comprehensively analyzed nematomorphs and their mantid hosts, focusing on the transcriptomic changes associated with host manipulations and sequence similarity between host and parasite genes to test molecular mimicry. The nematomorph's transcriptome changed during host manipulation, whereas no distinct changes were found in mantids. We then discovered numerous possible host-derived genes in nematomorphs, and these genes were frequently up-regulated during host manipulation. Our findings suggest a possible general role of horizontal gene transfer (HGT) in the molecular mechanisms of host manipulation, as well as in the genome evolution of manipulative parasites. The evidence of HGT between multicellular eukaryotes remains scarce but is increasing and, therefore, elucidating its mechanisms will advance our understanding of the enduring influence of HGT on the evolution of the web of life.

Parallel evolution of opsin visual pigments in hawkmoths by tuning of spectral sensitivities during transition from a nocturnal to a diurnal ecology.

Light environments differ dramatically between day and night. The transition between diurnal and nocturnal visual ecology has happened repeatedly throughout evolution in many species. However, the molecular mechanism underlying the evolution of vision in recent diurnal-nocturnal transition is poorly understood. Here, we focus on hawkmoths (Lepidoptera: Sphingidae) to address this question by investigating five nocturnal and five diurnal species. We performed RNA-sequencing analysis and identified opsin genes corresponding to the ultraviolet (UV), short-wavelength (SW) and long-wavelength (LW)-absorbing visual pigments. We found no significant differences in the expression patterns of opsin genes between the nocturnal and diurnal species. We then constructed the phylogenetic trees of hawkmoth species and opsins. The diurnal lineages had emerged at least three times from the nocturnal ancestors. The evolutionary rates of amino acid substitutions in the three opsins differed between the nocturnal and diurnal species. We found an excess number of parallel amino acid substitutions in the opsins in three independent diurnal lineages. The numbers were significantly more than those inferred from neutral evolution, suggesting that positive selection acted on these parallel substitutions. Moreover, we predicted the visual pigment absorption spectra based on electrophysiologically determined spectral sensitivity in two nocturnal and two diurnal species belonging to different clades. In the diurnal species, the LW pigments shift 10 nm towards shorter wavelengths, and the SW pigments shift 10 nm in the opposite direction. Taken together, our results suggest that parallel evolution of opsins may have enhanced the colour discrimination properties of diurnal hawkmoths in ambient light.

Mutations in a ß-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori.

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the ß-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes.

Development of 49 novel microsatellite markers from Next-generation sequencing data and a robust method for parentage tests in the emu (Dromaius novaehollandiae).

The emu is a useful and new breed of poultry, but their genetic improvement has not advanced yet due to their very recent domestication. Pedigree information is difficult to record because of their complex reproduction system (polyandry). To identify parent-offspring relationships in the emu, parentage test based on polymorphic DNA markers have to be developed. In this study, we isolated more than 25,000 microsatellite (simple sequence repeat, SSR) regions from Next-generation sequencing data via the QDD pipeline and developed 49 SSR markers with polymorphism in the Japanese farmed emu. The dinucleotide motifs, (AC)n, (AT)n and (AG)n, were the most frequently detected and were found on 10,167 (38.55%), 8,114 (30.76%) and 4,796 (18.18%) contigs, respectively. Forty-nine novel SSR markers were characterized in 20 individuals and showed NA ranged from 2 to 12, with an average of 4.2. HE/HO ranged from 0.389/0.071 to 0.702/1.000 with an average of 0.601/0.515. PIC value ranged from 0.059 to 0.886 with an average of 0.528, and 17 of 49 markers showed a higher polymorphism than 0.500. Thirty-four individuals were genotyped using 12 markers, and CERVUS simulations based on genotype showed that parents of all offspring were identified with 0.9995-1.0 probability. Thus, 49 novel SSR markers and a robust method for parentage test for the Japanese emu were developed.

Identification of an antiviral component from the venom of the scorpion Liocheles australasiae using transcriptomic and mass spectrometric analyses.

Scorpion venom contains a variety of biologically active peptides. Among them, neurotoxins are major components in the venom, but it also contains peptides that show antimicrobial activity. Previously, we identified three insecticidal peptides from the venom of the Liocheles australasiae scorpion, but activities and structures of other venom components remained unknown. In this study, we performed a transcriptome analysis of the venom gland of the scorpion L. australasiae to gain a comprehensive understanding of its venom components. The result shows that potassium channel toxin-like peptides were the most diverse, whereas only a limited number of sodium channel toxin-like peptides were observed. In addition to these neurotoxin-like peptides, many non-disulfide-bridged peptides were identified, suggesting that these components have some critical roles in the L. australasiae venom. In this study, we also isolated a component with antiviral activity against hepatitis C virus using a bioassay-guided fractionation approach. By integrating mass spectrometric and transcriptomic data, we successfully identified LaPLA2-1 as an anti-HCV component. LaPLA2-1 is a phospholipase A2 having a heterodimeric structure that is N-glycosylated at the N-terminal region. Since the antiviral activity of LaPLA2-1 was inhibited by a PLA2 inhibitor, the enzymatic activity of LaPLA2-1 is likely to be involved in its antiviral activity.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Transcriptomic comparison between beetle strains selected for short and long durations of death feigning.

The molecular basis of death feigning, an antipredator behavior that has received much attention recently, was analyzed. We compared the gene expression profiles of strains with different behaviors, i.e., different durations of death feigning, in the beetle Tribolium castaneum. Beetles artificially selected for short (S) and long (L) durations of death feigning for many generations were compared thoroughly by RNA sequencing. We identified 518 differentially expressed genes (DEGs) between the strains. The strains also showed divergence in unexpected gene expression regions. As expected from previous physiological studies, genes associated with the metabolic pathways of tyrosine, a precursor of dopamine, were differentially expressed between the S and L strains; these enzyme-encoding genes were expressed at higher levels in the L strain than in the S strain. We also found that several genes associated with insulin signaling were expressed at higher levels in the S strain than in the L strain. Quantitative real-time PCR analysis showed that the relative expression levels of Tchpd (encoding 4-hydroxyphenylpyruvate dioxygenase, Hpd) and Tcnat (encoding N-acetyltransferase, Nat) were significantly higher in the L strain than in the S strain, suggesting the influence of these enzymes on the supply of dopamine and duration of death feigning.

Interspecies Comparative Analyses Reveal Distinct Carbohydrate-Responsive Systems among Drosophila Species.

During evolution, organisms have acquired variable feeding habits. Some species are nutritional generalists that adapt to various food resources, while others are specialists, feeding on specific resources. However, much remains to be discovered about how generalists adapt to diversified diets. We find that larvae of the generalists Drosophila melanogaster and D. simulans develop on three diets with different nutrient balances, whereas specialists D. sechellia and D. elegans cannot develop on carbohydrate-rich diets. The generalist D. melanogaster downregulates the expression of diverse metabolic genes systemically by transforming growth factor ß (TGF-ß)/Activin signaling, maintains metabolic homeostasis, and successfully adapts to the diets. In contrast, the specialist D. sechellia expresses those metabolic genes at higher levels and accumulates various metabolites on the carbohydrate-rich diet, culminating in reduced adaptation. Phenotypic similarities and differences strongly suggest that the robust carbohydrate-responsive regulatory systems are evolutionarily retained through genome-environment interactions in the generalists and contribute to their nutritional adaptabilities.

Global Liver Gene Expression Analysis on a Murine Hepatic Steatosis Model Treated with Mulberry (Morus alba L.) Leaf Powder.

BACKGROUND/AIM: Mulberry (Morus alba L.) leaves (ML) contain many functional components, such as 1-deoxynojirimycin, flavonoids (rutin, quercetin, kaempferol). It is well known that 1-deoxynojirimycin functions to suppress increases in blood glucose level by α-glucosidase inhibitory activity. Thus, the molecular mechanism underlying the protective and therapeutic effects of ML supplementation was investigated on a mouse model of high-calorie diet (Western diet: WD)-induced hepatic steatosis (HS). MATERIALS AND METHODS: The C57BL/6J mouse was used for the HS model. The mice were divided into three groups: control (normal diet: ND), WD, and WD + 1% ML groups. The WD group was fed a high-calorie (high carbohydrate and high fat) diet for 12 weeks to develop HS. At week 12, all mice were sacrificed, blood was collected for biochemical tests, and the liver was obtained for histological examination and RNA sequencing (RNA-Seq). RESULTS: Liver weight, plasma triglycerides (TG), alanine aminotransferase (ALT), and alanine aminotransferase (AST) levels of both ML groups were significantly lower than those of the WD group. On histological examination of the liver, the area of fatty deposits was found to be suppressed by ML administration. In the gene expression analysis of the liver of WD- versus ML-fed mice by RNA-Seq, 722/45,706 genes exhibited a significant change in expression (corrected p-value<0.05). Gene network analysis of these genes showed that genes related to liver inflammation were inactivated and those related to regeneration of liver were activated in the ML group. CONCLUSION: ML functions to suppress HS in WD-fed mice and regulates genes related to inflammation and regeneration of liver cells.

Evolution of Gustatory Receptor Gene Family Provides Insights into Adaptation to Diverse Host Plants in Nymphalid Butterflies.

The host plant range of herbivorous insects is a major aspect of insect-plant interaction, but the genetic basis of host range expansion in insects is poorly understood. In butterflies, gustatory receptor genes (GRs) play important roles in host plant selection by ovipositing females. Since several studies have shown associations between the repertoire sizes of chemosensory gene families and the diversity of resource use, we hypothesized that the increase in the number of genes in the GR family is associated with host range expansion in butterflies. Here, we analyzed the evolutionary dynamics of GRs among related species, including the host generalist Vanessa cardui and three specialists. Although the increase of the GR repertoire itself was not observed, we found that the gene birth rate of GRs was the highest in the lineage leading to V. cardui compared with other specialist lineages. We also identified two taxon-specific subfamilies of GRs, characterized by frequent lineage-specific duplications and higher non-synonymous substitution rates. Together, our results suggest that frequent gene duplications in GRs, which might be involved in the detection of plant secondary metabolites, were associated with host range expansion in the V. cardui lineage. These evolutionary patterns imply that the capability to perceive various compounds during host selection was favored during adaptation to diverse host plants.

Elevenin regulates the body color through a G protein-coupled receptor NlA42 in the brown planthopper Nilaparvata lugens.

The neuropeptide elevenin and similar neuropeptide precursors are common in some invertebrates but their physiological function in most species has not been explored. The brown planthopper, Nilaparvata lugens (Stål) has an elevenin-like peptide and a G protein-coupled receptor (GPCR) NlA42 that is homologous to the elevenin receptor of the annelid Platynereis dumerilii. RNA interference (RNAi)-mediated knockdown of either Nl-elevenin or the NlA42 gene resulted in cuticle melanization. Ion transport peptide (ITP) also induces melanization, but unlike ITP, knockdown of NlElevenin and NlA42 did not have any effect on wing expansion or activity after eclosion. In wild condition macropterous individuals show a darker body color when compared with brachypterous individuals, but RNAi experiments suggest that insulin-signaling and Nl-elevenin signaling regulate wing morph and body color independently. NlElevenin was predominantly expressed in the brain while NlA42 was highly expressed in the abdominal integument and brain. A signal Calcium assays using aequorin indicated that NlA42 heterologously expressed in HEK293 cells exhibited responses to synthetic Nl-elevenin peptide from concentrations as low as 10-9M. These results suggest that neuropeptide Nl-elevenin is involved in the regulation of melanization through its receptor NlA42. This is the first report of a physiological function for elevenin-like peptides in insects.

Transcriptome analyses of taste organoids reveal multiple pathways involved in taste cell generation.

Taste cells undergo constant turnover throughout life; however, the molecular mechanisms governing taste cell generation are not well understood. Using RNA-Seq, we systematically surveyed the transcriptome landscape of taste organoids at different stages of growth. Our data show the staged expression of a variety of genes and identify multiple signaling pathways underlying taste cell differentiation and taste stem/progenitor cell proliferation. For example, transcripts of taste receptors appear only or predominantly in late-stage organoids. Prior to that, transcription factors and other signaling elements are upregulated. RNA-Seq identified a number of well-characterized signaling pathways in taste organoid cultures, such as those involving Wnt, bone morphogenetic proteins (BMPs), Notch, and Hedgehog (Hh). By pharmacological manipulation, we demonstrate that Wnt, BMPs, Notch, and Hh signaling pathways are necessary for taste cell proliferation, differentiation and cell fate determination. The temporal expression profiles displayed by taste organoids may also lead to the identification of currently unknown transducer elements underlying sour, salt, and other taste qualities, given the staged expression of taste receptor genes and taste transduction elements in cultured organoids.

Transcriptome Analysis of Skin from SMP30/GNL Knockout Mice Reveals the Effect of Ascorbic Acid Deficiency on Skin and Hair.

BACKGROUND/AIM: Senescence marker protein-30/gluconolactonase knockout mice (SMP-30/GNL-KO) are a very useful model for clarifying the involvement of vitamin C (VC) in aging-related diseases. In this study, the effects of VC deficiency on skin and hair growth were investigated using SMP-30/GNL-KO mice by RNA sequencing. MATERIALS AND METHODS: SMP-30/GNL-KO mice were given water containing 1.5 g/l VC until up to 8 weeks after birth to maintain a VC concentration in their organs and plasma equivalent to that in wild-type mice. The mice were then divided into two groups: a VC(+) group, where VC was administered, and a VC(-) group, where VC was not administered. Skin samples were collected at 4 and 8 weeks after the treatment. RNA was extracted from each skin sample, followed by cDNA synthesis and RNA-seq. In addition, hair growth was compared between the VC(-) and VC(+) groups after shaving. Skin samples were collected from the shaved area for histological examination by hematoxylin & eosin (HE) staining. RESULTS: RNA-seq revealed that there were 1,736 (FDR<0.001) differentially expressed genes in the VC(-) and VC(+) groups. From the functional analysis of the differentially expressed genes in the VC(-) and VC(+) groups, predicted functionalities including cell death and cytotoxicity increased in the VC(+) group. Furthermore, it was predicted that the difference in hair growth between the VC(-) and VC(+) groups was caused by the expression of genes including keratin-related genes and the Sonic hedgehog gene. It was confirmed that hair growth was significantly promoted; hair growth from hair papilla cells was also confirmed by HE staining of the shaved backs of SMP-30/GNL-KO mice in the VC(+) group. CONCLUSION: RNA-seq of the skin from VC-deficient mice showed the effects of VC deficiency on the expression of genes involved in cell growth and the hair cycle. Visual inspection suggested that changes in the expression of the genes are involved in delaying hair growth in the VC(-) group. Further research on the relationship among VC deficiency, the hair cycle, and skin cell growth may contribute to research on hair restoration and skin aging.

Expression analysis of Egr-1 ortholog in metamorphic brain of honeybee (Apis mellifera L.): Possible evolutionary conservation of roles of Egr in eye development in vertebrates and insects.

Specific genes quickly transcribed after extracellular stimuli without de novo protein synthesis are known as immediate early genes (IEGs) and are thought to contribute to learning and memory processes in the mature nervous system of vertebrates. A recent study revealed that the homolog of Early growth response protein-1 (Egr-1), which is one of the best-characterized vertebrate IEGs, shared similar properties as a neural activity-dependent gene in the adult brain of insects. With regard to the roles of vertebrate Egr-1 in neural development, the contribution to the development and growth of visual systems has been reported. However, in insects, the expression dynamics of the Egr-1 homologous gene during neural development remains poorly understood. Our expression analysis demonstrated that AmEgr, a honeybee homolog of Egr-1, was transiently upregulated in the developing brain during the early to mid pupal stages. In situ hybridization and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry revealed that AmEgr was mainly expressed in post-mitotic cells in optic lobes, the primary visual center of the insect brain. These findings suggest the evolutionarily conserved role of Egr homologs in the development of visual systems in vertebrates and insects.

Global Liver Gene Expression Analysis on a Murine Metabolic Syndrome Model Treated by Low-molecular-weight Lychee Fruit Polyphenol (Oligonol®).

BACKGROUND/AIM: Oligonol® (OLG) is a low-molecular-weight lychee fruit polyphenol mainly containing catechin-type monomers and oligomers of proanthocyanidins. Dietary OLG supplementation reportedly improves lipid metabolism disorder and lowers the visceral fat level in animal and human studies. Thus, we investigated the mechanism behind the protective and beneficial effects of OLG on a Western diet (WD)-induced metabolic syndrome (MetS) of a murine model. MATERIALS AND METHODS: Using the C57BL/6J mouse for the MetS model, mice were divided into three groups: control (normal diet: ND), Western diet (WD) and WD + 0.5% OLG (OLG) groups. The WD group was fed a high-calorie (high fructose plus high fat) diet for 12 weeks to develop MetS. At week 12, all mice were sacrificed and the blood and liver were obtained for histological and biological examinations and RNA sequencing (RNA-Seq). RESULTS: Body weight, liver weight, plasma triglycerides (TG), total cholesterol (T-Cho) and alanine aminotransferase (ATS) levels of both OLG groups were significantly lower than those of the WD group. On histological examination of the liver, the area of fatty deposits was shown to be suppressed by OLG administration. Expression gene analysis in the liver of WD- versus OLG-fed mice by RNA-Seq showed that 464/45,706 genes exhibited a significant change of expression (corrected p-value <0.05, absolute value of fold change (FC) ≥2). Gene network analysis showed that genes related to hepatic steatosis, liver inflammation and tumor invasion were inactivated in the OLG group. In particular, the lipid metabolism-related genes Lpin1, Adig and Cidea were regulated by OLG administration. CONCLUSION: OLG may function to suppress MetS and the progression of geriatric diseases in WD-fed mice by regulating the expression of lipid metabolism, inflammation and tumor-related genes in the liver.

The complete mitochondrial genome of the Japanese honeybee, Apis cerana japonica (Insecta: Hymenoptera: Apidae).

In this study, we analyzed the complete mitochondrial genome of the Japanese honeybee Apis cerana japonica. The mitochondrial genome of A. c. japonica is a circular molecule of 15 917 bp and is similar to that of A. c. cerana. It contains 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one A + T-rich control region. All protein-coding genes are initiated by ATT and ATG codons and are terminated by the typical stop codon TAA or TAG, except for the start codon of ATP8 which ends with C. All tRNA genes typically form a cloverleaf secondary structure, except for tRNA-Ser (AGN).

Rough eyes of the Northeast-Asian wood white, Leptidea amurensis.

The northeast-Asian wood white, Leptidea amurensis (Lepidoptera, Pieridae), belongs to the Dismorphiinae, a subfamily of the family Pieridae. We studied the structure of the compound eye in this species through a combination of anatomy, molecular biology and intracellular electrophysiology, with a particular focus on the evolution of butterfly eyes. We found that their eyes consist of three types of ommatidia, with a basic set of one short-, one middle- and one long-wavelength-absorbing visual pigment. The spectral sensitivities of the photoreceptors are rather simple, and peak in the ultraviolet, blue and green wavelength regions. The ommatidia have neither perirhabdomal nor fluorescent pigments, which modulate photoreceptor spectral sensitivities in a number of other butterfly species. These features are primitive, but the eyes of Leptidea exhibit another unique feature: the rough appearance of the ventral two-thirds of the eye. The roughness is due to the irregular distribution of facets of two distinct sizes. As this phenomenon exists only in males, it may represent a newly evolved sex-related feature.