Effect of Graded Motor Imagery Combined With Repetitive Transcranial Magnetic Stimulation on Upper Extremity Motor Function in Stroke Patients: A Randomized Controlled Trial.

OBJECTIVE: To investigate the efficacy of graded motor imagery therapy (GMI) and repetitive transcranial magnetic stimulation (rTMS) on upper extremity function alone and in combination in patients with stroke. DESIGN: This was a prospective randomized controlled trial. SETTING: A rehabilitation hospital. PARTICIPANTS: Participants (N=56) were randomized into GMI (n=19), rTMS (n=18), or combined groups (n=19). INTERVENTIONS: There were 2 interventions: (1) 20 minutes of 1 Hz rTMS. (2) 30 minutes of GMI. In addition to this, all participants receive conventional rehabilitation including 120 minutes of physical therapy and occupational therapy daily. All treatments were administered once a day, 5 days a week, for 4 weeks. The Fugl-Meyer assessment of upper extremity (FMA-UE), Action Research Arm Test (ARAT), modified Barthel Index (MBI), motor activity log (MAL), and motor evoked potential (MEP) were assessed in a blinded manner at baseline and 4 weeks after treatment, respectively. MAIN OUTCOME MEASURES: The primary endpoint was the improvement from baseline in FMA-UE for stroke patients at 4 weeks. RESULTS: After 4 weeks of treatment, the FMA-UE scores in the GMI, rTMS, and combined groups were higher than those at baseline assessment, with statistically significant differences among the 3 groups (P=.009). The efficacy of the combined group was significantly better than that of the GMI and rTMS groups (P=.015, P=.043, respectively). In the motor activity log-amount of use (MAL-AOU) score, the efficacy of the combined group was better than that of the rTMS group (P=.035). CONCLUSIONS: Both GMI and rTMS were effective in improving upper extremity function in patients with stroke, but the combination of the 2 techniques was more effective. However, GMI was better than rTMS in improving the interest of stroke patients in active training.

Subchronic Chloroform Exposure Causes Intestinal Damage and Induces Gut Microbiota Disruption and Metabolic Dysregulation in Mice.

Chloroform is a prevalent toxic environmental pollutant in urban settings, posing risks to human health through exposure via various mediums such as air and tap water. The gut microbiota plays a pivotal role in maintaining host health. However, there is a paucity of research elucidating the impact of chloroform exposure on the gut microbiota. In this investigation, 18 SPF Kunming female mice were stratified into three groups (n = 6) and subjected to oral gavage with chloroform doses equivalent to 0, 50, and 150 mg/kg of body weight over 30 days. Our findings demonstrate that subchronic chloroform exposure significantly perturbs hematological parameters in mice and induces histopathological alterations in cecal tissues, consequently engendering marked disparities in the functional composition of cecal microbiota and metabolic equilibrium of cecal contents. Ultimately, our investigation revealed a statistically robust correlation, exhibiting a high degree of significance, between the intestinal microbiome composition and the metabolites that were differentially expressed consequent to chloroform exposure.

Insecticidal potential of a Consolida ajacis extract and its major compound (ethyl linoleate) against the diamondback moth, Plutella xylostella.

The diamondback moth (Plutella xylostella) is one of the most destructive lepidopteran pests of cruciferous vegetables. However, DBM has developed resistance to current chemical and biological insecticides used for its control, indicating the necessity for finding new insecticides against it. Bio-insecticides derived from plant extracts are eco-friendly alternatives to synthetic pesticides. The aims of this study were to evaluate the insecticidal activity of Consolida ajacis seed extracts against DBM, the underlying mechanism of the control effect of promising extracts, and the identification of the main insecticidal compounds of these extracts. The results showed that ethyl acetate extract of C. ajacis seed exhibited strong contact toxicity (LC50: 5.05 mg/mL), ingestion toxicity, antifeedant, and oviposition deterrent activities against DBM, among the extracts evaluated. At 72 h, glutathiase, acetylcholinesterase, carboxylesterase, peroxidase, and superoxide dismutase activities were inhibited, but catalase activity was activated. The main compound identified from the extract was ethyl linoleate, which had the most significant insecticidal activity on the diamondback moths. This study's findings provide a better understanding of the insecticidal activity of ethyl acetate extract obtained from C. ajacis and its main component (ethyl linoleate). This will help in the development of new insecticides to control DBM.

Alafrasca sticta, a new genus and species of the tribe Empoascini (Hemiptera: Cicadellidae: Typhlocybinae) with a checklist of the tribe from China.

A new monotypic genus of empoascine leafhopper, Alafrasca gen. nov. is described and illustrated for A. sticta sp. nov. from Sichuan, China as its type species. Habitus photos and illustrations of male genitalia of the new species are given. A checklist of the tribe in China is also provided.

Engineering microalgae for robust glycolate biosynthesis: Targeted knockout of hydroxypyruvate reductase 1 combined with optimized culture conditions enhance glycolate production in Chlamydomonas reinhardtii.

Microalgae-based glycolate production through the photorespiratory pathway is considered an environmentally friendly approach. However, the potential for glycolate production is limited by photoautotrophic cultivation with low cell density and existing strains. In this study, a targeted knockout approach was used to disrupt the key photorespiration enzyme, Chlamydomonas reinhardtii hydroxypyruvate reductase 1 (CrHPR1), leading to a significant increase in glycolate production of 280.1 mg/L/OD750. The highest potency yield reached 2.1 g/L under optimized mixotrophic conditions, demonstrating the possibility of synchronizing cell growth with glycolate biosynthesis in microalgae. Furthermore, the hypothesis that the cell wall-deficient mutant facilitates glycolate excretion was proposed and validated by comparing the glycolate accumulation trends of various Chlamydomonas reinhardtii strains. This study will facilitate the development of microalgae-based biotechnology and shed lights on the continuous advancement of green biomanufacturing for industrial application.

[Functions and mechanisms of testicular descent in Apodemus agrarius based on transcriptomics and metabolomics].

This study aims to explore the functions and mechanisms of testicular descent in Apodemus agrarius, and analyze the changes in genes and metabolite levels in this process. Illumina NovaSeq and liquid chromatography-mass spectrometry were used for the transcriptomic analysis and metabolomic analysis, respectively, of the normal and descending testis of A. agrarius. Gene ontology (GO) enrichment of the transcriptomic results revealed 240 differentially expressed genes (DEGs), such as Spesp1, Izumo1, Hyal5, and Fabp9. Kyoto encyclopedia of genes and genomes (KEGG) enrichment showed 52 DEGs, including Pcyt1, Pla2g4e, Gpd1l, and Lypla3. The qRT-PCR results were consistent with the transcriptomic results in terms of the expression patterns of six randomly selected genes in the normal and descending testis. The metabolomic results revealed 28 differential metabolites associated with the testicular function, including 3-dehydroquinic acid, α-linolenic acid, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate. The conjoint analysis showcased that glycerophospholipid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism may be the key metabolic pathways regulating testicular descent in A. agrarius. This study will help to understand the mechanism of testicular descent and lay a theoretical foundation for exploring the mechanisms of the population changes of A. agrarius and developing laboratory animal resources.

Biodegradable high-nitrogen iron alloy anastomotic staples: In vitro and in vivo studies.

For gastrointestinal anastomosis, metallic biodegradable staples have a broad application potential. However, both magnesium and zinc alloys have relatively low strength to withstand the repeated peristalsis of the gastrointestinal tract. In this study, we developed a novel kind of biodegradable high-nitrogen iron (HN-Fe) alloy wires (0.23 mm), which were fabricated into the staples. The tensile results showed that the ultimate tensile strength and elongation of HN-Fe wires were 1023.2 MPa and 51.0 %, respectively, which was much higher than those of other biodegradable wires. The degradation rate in vitro of HN-Fe wires was slightly higher than that of pure Fe wires. After 28 days of immersion, the tensile strength of HN-Fe wires remained not less than 240 MPa, meeting the clinical requirements. Furthermore, sixteen rabbits were enrolled to conduct a comparison experiment using HN-Fe and clinical Ti staples for gastroanastomosis. After 6 months of implantation, a homogeneous degradation product layer on HN-Fe staples was observed and no fracture occurred. The degradation rate of HN-Fe staples in vivo was significantly higher than that in vitro, and they were expected to be completely degraded in 2 years. Meanwhile, both benign cutting and closure performance of HN-Fe staples ensured that all the animals did not experience hemorrhage and anastomotic fistula during the observation. The anastomosis site healed without histopathological change, inflammatory reaction and abnormal blood routine and biochemistry, demonstrating good biocompatibility of HN-Fe staples. Thereby, the favorable performance makes the HN-Fe staples developed in this work a promising candidate for gastrointestinal anastomosis.

ERCC3-Related Genes May Aid in the Prognostic and Immunotherapeutic Analysis of Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has high morbidity and mortality worldwide. Excision repair cross-complement 3 (ERCC3), a key functional gene in the nucleotide excision repair (NER) pathway, is commonly mutated or overexpressed in cancers and is thought to be a key gene contributing to the development of HCC. The characteristics of immune cell infiltration in the global tumor microenvironment (TME) mediated by ERCC3 and its related key genes in HCC are still unclear. The aim of this study was to integrate the role of ERCC3-related key genes in assessing the TME cell infiltration characteristics, immunotherapy efficacy, and prognosis of HCC patients. This study provides a theoretical basis for the study of immunological mechanisms and prognosis prediction in HCC. METHODS: The HCC cohort from the TCGA database included 50 normal samples and 374 tumor samples to compare the differences in ERCC3-related gene expression and prognosis between liver tumor tissues and normal liver tissues and to analyze the extent to which different genes infiltrated TME cells by quantifying the relative abundance of 24 cells through single-sample genome enrichment analysis (ssGSEA). A risk score associated with the ERCC3 gene was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression model. RESULTS: The expression of 11 ERCC3-related genes was significantly upregulated in HCC tumor tissues compared to normal liver tissues, and high expression of these genes was significantly associated with poor prognosis in HCC patients. The key genes (11 ERCC3-related genes) were closely associated with the nucleic acid reduction signaling pathway in nucleic acid metabolism and the viral oncogenic pathway, suggesting that these key genes may play a role in tumor cell proliferation, migration, and invasion, as well as in the pathogenesis of virus-associated HCC. In addition, the infiltration characteristics of TME immune cells in normal and tumor tissues were different. Immune and mesenchymal activity was significantly lower in tumor tissues than in healthy liver tissues. This study revealed that key genes were significantly positively correlated with CTLA4 and enriched in central memory CD4 T cells, effector memory CD4 T cells, activated CD4 T cells, and type 2 T helper cells. The prognostic model constructed by regression analysis could better distinguish patients into high-risk and low-risk groups, and the survival analysis showed that the survival time of patients with high-risk score subtypes was significantly lower than that of patients with low-risk scores and that the high-risk group contained higher levels of immune-suppressive cells, which may be a mediator of immune escape. Moreover, multivariate analyses showed that the risk score profile is a reliable and unbiased biomarker for assessing the prognosis of HCC patients, and its value in predicting the outcome of immunotherapy was also confirmed. CONCLUSION: This study revealed a novel genetic signature that is significantly associated with TME cell infiltration and prognosis in HCC patients. It demonstrated that the combined action of multiple key genes associated with ERCC3 plays a crucial role in shaping the diversity and complexity of TME cell infiltrates. Evaluating the combined characteristics of multiple key genes associated with ERCC3 can help predict the outcome of immunotherapy in patients and provide new potential targets for immuno-individualized therapeutic studies on HCC.

Assessment of quality of life for the patients with cervical cancer at different clinical stages.

With improved overall survival of cervical cancer patients, the importance of the quality of life (QOL) is increasingly recognized. This study was conducted to compare the QOL of women with different stage cervical cancer before and after treatment to facilitate improved cervical cancer prevention and treatment. We used the generic Medical Outcomes Study Short Form-36 (MOS SF-36) to collect QOL information. Based on SF-36, we interviewed cervical cancer patients at West China Second Affiliated Hospital and Sichuan Cancer Hospital between May 2010 and January 2011. A total of 92 patients with precancerous lesions, 93 with early cancer, and 35 with advanced cancer responded to our survey. Average physical component summary (PCS) scores were significantly different between the three groups at every time point (P < 0.05). Average mental component summary (MCS) scores were significantly different between the three groups after treatment (P < 0.05). Average PCS and MCS scores increased gradually from the pretreatment to posttreatment period for patients with precancerous lesions. However, they reached the lowest at 1 month after treatment for patients with early and advanced cancers and rebounded between 1 and 6 months after treatment. Our results indicate that patients with precancerous lesions and early cervical cancer show better overall QOL than do those with advanced cervical cancer. Additionally, patients with early cancer recover more quickly than do those with advanced cancer in terms of both physical and mental functions. Thus, early detection and treatment initiatives may improve the QOL for patients with precancerous lesions and cervical cancer.

Psychological needs satisfaction and smartphone addiction among Chinese adolescents: The mediating roles of social anxiety and loneliness.

Objectives: Although previous studies have initially noted that psychological needs satisfaction (PNS) might be a significant risk factor for technology addiction (e.g. online gaming addiction and Internet addiction), specific mechanisms involved in the association between PNS and adolescent smartphone addiction are largely unknown. Based on self-determination theory, this cross-sectional study constructed a multiple mediation model to examine whether PNS will influence adolescent smartphone addiction through the mediating roles of social anxiety and loneliness. Methods: Eight hundred and ninety-nine Chinese adolescents answered the questionnaire including measures of PNS, social anxiety, loneliness, and smartphone addiction. SPSS 24.0 was used for common method bias test, reliability test, and correlation analysis, and Mplus 7.4 was used to examine the mediating roles of social anxiety and loneliness in the multiple mediation model. Results: This study found that (1) PNS was negatively associated with adolescent smartphone addiction; (2) loneliness significantly mediated the association between PNS and smartphone addiction while the mediating role of social anxiety in this association was nonsignificant; and (3) social anxiety and loneliness also sequentially mediated this association. Conclusion: This study further enriched potential mechanisms linking PNS and smartphone addiction among adolescents, which may contribute to intervention and prevention programs for adolescent smartphone addiction from the perspective of improving both PNS and negative emotions including social anxiety and loneliness.

Parent-adolescent relationships, peer relationships, and adolescent mobile phone addiction: The mediating role of psychological needs satisfaction.

INTRODUCTION: Prior studies have shown that parent-adolescent relationships and peer relationships may be important factors associated with adolescent mobile phone addiction (MPA). The present study aims to further explore the direct effects of parent-adolescent and peer relationships on adolescent MPA as well as the indirect effects through the mediating roles of autonomy, competence, and relatedness needs satisfaction. METHODS: Our sample consisted of 1766 Chinese adolescents (53.10% male; Mage = 13.33, SD = 1.94, range from 10 to 18 years) who completed questionnaires regarding parent-adolescent relationships, peer relationships, psychological needs satisfaction, and MPA. SPSS 24.0 was used to analyze correlations among variables and Mplus 7.4 was used to test the structural equation model in this study. RESULTS: (1) positive parent-adolescent relationships were negatively associated with adolescent MPA, while peer relationships did not show a significant association with MPA; (2) autonomy and competence needs satisfaction significantly mediated the effects of parent-adolescent and peer relationships on MPA, while the mediating role of relatedness need satisfaction between parent-adolescent and peer relationships and MPA was not significant; (3) the mediating effect of competence need satisfaction between peer relationships and MPA was significantly stronger than that between parent-adolescent relationships and MPA. CONCLUSIONS: This study explored the different mechanisms by which parent-adolescent and peer relationships influence adolescent MPA. These discoveries may contribute to intervention and prevention programs for adolescent MPA.

Identification and Functional Analysis of Glutathione S-Transferases from Sitophilus zeamais in Olfactory Organ.

Odorant-degrading enzymes (ODEs) play an important role in rapidly degrading and inactivating odorant molecules that have completed information transmission, as well as in maintaining the stability and sensitivity of insect olfactory sensing systems. Glutathione S-transferases (GSTs), as a group of ODEs, supposedly bear the ability to catalyze the conjugation of glutathione (GSH) and xenobiotic odorant molecules in the degrading process. However, there are few reports regarding the role of the GST genes of Sitophilus zeamais in the degrading process. Thus, we characterized 13 full-length genes encoding GST sequences from S. zeamais, of which only SzeaGSTd1 contained a high abundance in the antennae. Ligand-binding assays implied that SzeaGSTd1 was able to catalyze the conjugation of GSH with 2, 4-dinitrochlorobenzene (CDNB). We investigated whether recombinant SzeaGSTd1 bears the ability to degrade the volatile molecules of the host; among the host volatiles, and found capryl alcohol to be a suitable substrate for SzeaGSTd1. These results strongly suggest that SzeaGSTd1 probably plays a role in auxiliary host location by degrading the host volatiles of capryl alcohol and exhibits a potential biological function in the olfactory sensing system of S. zeamais. Knowledge of the potential functions of SzeaGSTd1 will provide new ideas for biological control strategies for S. zeamais.

The complete mitochondrial genome sequence and phylogenetic position of Xylota coquilletti (Soo-Hyun Jeong, Ho-Yeon Han 2019) (Diptera: Syrphidae: Eristalinae: Xylotini).

The complete mitochondrial genome sequence of the Xylota coquilletti (Diptera: Syrphidae: Eristalinae: Xylotini) was sequenced and reported for the first time. The whole genome was 15,920 bp in length with the 37 classical eukaryotic mitochondrial genes and a control region. The nucleotide composition was included by 40.5% A, 39.6% T, 11.7% C, and 8.2% G, meaning that A + T (80.1%) was much greater than C + G (19.9%). It consisted of 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and a control region (CR). Phylogenetic analyses were performed using 13 PCGs and it was found that Xylota coquilletti was sister to Ferdinadea cupera. All this information could complement the mitochondrial data for a new tribe of Eristalinae.

Ecology and genetic structure of the invasive spotted lanternfly Lycorma delicatula in Japan where its distribution is slowly expanding.

Lycorma delicatula has expanded its distribution from China to Japan, Korea, and the USA, causing significant economic damage to vineyards in the latter two countries. However, in Japan, L. delicatula has long been limited to the Hokuriku region, central Japan, and no significant damage to crops has been reported since it was first reported there in 2009. Manipulation experiments and field observations in the Hokuriku region, where winter precipitation is extremely high, revealed that egg numbers and hatchability were significantly reduced in exposed places, especially when wax was excluded from the egg mass. Phylogenetic analysis showed that the population in Japan could be divided into at least two groups. Most L. delicatula samples from Hokuriku formed a clade with those from northwestern China. Samples from Okayama, where the distribution of L. delicatula was recently confirmed, had the same haplotype as those from central China, Korea, and the USA. These results suggest that environmental factors and genetic characteristics of L. delicatula are involved in the relatively slow expansion of its distribution in Hokuriku. Conversely, in Okayama, where precipitation is relatively low, the rapidly increasing haplotype in Korea and the USA was detected, leading to concerns that its distribution will expand further.

One-step fabrication of Cu-based metal organic framework multilayer core-shell microspheres for efficiently catalyzing the oxygen reduction reaction.

Micro/nanomaterials with multilayer core-shell structures are receiving widespread attention due to their potential in energy storage and conversion systems. However, simple fabrication of multilayered core-shell structured micro/nanomaterials with a consistent composition still faces a great challenge. Herein, a simple one-step solvothermal method is used to fabricate Cu-based metal organic framework multilayer core-shell microspheres (Cu-MOF-MCSMSs) as efficient oxygen reduction reaction (ORR) catalysts. The systematic structural evolution of Cu-MOF-MCSMSs is from microspheres to core-shell microspheres and then to multilayer core-shell microspheres. Additionally, different transition metal cations and anions can also influence the structures, compositions and thus ORR activities of the synthesized MOFs. The representative Cu-MOF-MCSMSs exhibit high ORR activity and cycling stability. The simple method can provide a good guide to fabricate other micro/nanomaterials with multilayer core-shell structures and desirable properties.

High-quality reference genomes of swallowtail butterflies provide insights into their coloration evolution.

Swallowtail butterflies (Papilionidae) are a historically significant butterfly group due to their colorful wing patterns, extensive morphological diversity, and phylogenetically important position as a sister group to all other butterflies and have been widely studied regarding ecological adaption, phylogeny, genetics, and evolution. Notably, they contain a unique class of pigments, i.e., papiliochromes, which contribute to their color diversity and various biological functions such as predator avoidance and mate preference. To date, however, the genomic and genetic basis of their color diversity and papiliochrome origin in a phylogenetic and evolutionary context remain largely unknown. Here, we obtained high-quality reference genomes of 11 swallowtail butterfly species covering all tribes of Papilioninae and Parnassiinae using long-read sequencing technology. Combined with previously published butterfly genomes, we obtained robust phylogenetic relationships among tribes, overcoming the challenges of incomplete lineage sorting (ILS) and gene flow. Comprehensive genomic analyses indicated that the evolution of Papilionidae-specific conserved non-exonic elements (PSCNEs) and transcription factor binding sites (TFBSs) of patterning and transporter/cofactor genes, together with the rapid evolution of transporters/cofactors, likely promoted the origin and evolution of papiliochromes. These findings not only provide novel insights into the genomic basis of color diversity, especially papiliochrome origin in swallowtail butterflies, but also provide important data resources for exploring the evolution, ecology, and conservation of butterflies.

Cost-benefit analysis of screening for esophageal and gastric cardiac cancer.

In 2005, a program named "Early Detection and Early Treatment of Esophageal and Cardiac Cancer" (EDETEC) was initiated in China. A total of 8279 residents aged 40-69 years old were recruited into the EDETEC program in Linzhou of Henan Province between 2005 and 2008. Howerer, the cost-benefit of the EDETEC program is not very clear yet. We conducted herein a cost-benefit analysis of screening for esophageal and cardiac cancer. The assessed costs of the EDETEC program included screening costs for each subject, as well as direct and indirect treatment costs for esophageal and cardiac severe dysplasia and cancer detected by screening. The assessed benefits of this program included the saved treatment costs, both direct and indirect, on esophageal and cardiac cancer, as well as the value of prolonged life due to screening, as determined by the human capital approach. The results showed the screening cost of finding esophageal and cardiac severe dysplasia or cancer ranged from RMB 2707 to RMB 4512, and the total cost on screening and treatment was RMB 13 115-14 920. The cost benefit was RMB 58 944-155 110 (the saved treatment cost, RMB 17 730, plus the value of prolonged life, RMB 41 214-137 380). The ratio of benefit-to-cost (BCR) was 3.95-11.83. Our results suggest that EDETEC has a high benefit-to-cost ratio in China and could be instituted into high risk areas of China.

Facile construction of S-containing Co-based metal organic framework core-shell microspheres as an efficient bifunctional oxygen electrocatalyst.

A cost-effective non-noble metal bifunctional electrocatalyst towards the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER) is very important for energy-related applications. Micro/nanomaterials with core-shell structures have emerged as potential non-noble metal catalyst candidates. Herein, an efficient bifunctional oxygen electrocatalyst, S-containing Co-based metal organic framework core-shell microspheres (Co-MOF-CSMSs), has been designed and constructed by using 2,2':5',2''-terthiophene-5,5''-dicarboxylic acid as a novel ligand through a facile one-step hydrothermal method. Due to the integrated favorable structural characteristics of the core-shell structure and MOFs for electrocatalysis, Co-MOF-CSMSs are revealed as a good bifunctional electrocatalyst for the ORR and OER, including an onset potential of 0.93 V vs. RHE (reversible hydrogen electrode), a half-wave potential of 0.78 V vs. RHE and an overpotential of 0.35 V at 10 mA cm-2. This work provides a low-cost and facile method to design and construct advanced micro/nanomaterials with core-shell structures to targetedly develop high-performance bifunctional oxygen electrocatalysts.

Chromosome-level reference genome assembly and gene editing of the dead-leaf butterfly Kallima inachus.

The leaf resemblance of Kallima (Nymphalidae) butterflies is an important ecological adaptive mechanism that increases their survival. However, the genetic mechanism underlying ecological adaptation remains unclear owing to a dearth of genomic information. Here, we determined the karyotype (n = 31) of the dead-leaf butterfly Kallima inachus, and generated a high-quality, chromosome-level assembly (568.92 Mb; contig N50: 19.20 Mb). We also identified candidate Z and W chromosomes. To our knowledge, this is the first study to report on these aspects of this species. In the assembled genome, 15,309 protein-coding genes and 49.86% repeat elements were annotated. Phylogenetic analysis showed that K. inachus diverged from Melitaea cinxia (no leaf resemblance), both of which are in Nymphalinae, around 40 million years ago. Demographic analysis indicated that the effective population size of K. inachus decreased during the last interglacial period in the Pleistocene. The wings of adults with the pigmentary gene ebony knocked out using CRISPR/Cas9 showed phenotypes in which the orange dorsal region and entire ventral surface darkened, suggesting its vital role in the ecological adaption of dead-leaf butterflies. Our results provide important genome resources for investigating the genetic mechanism underlying protective resemblance in dead-leaf butterflies and insights into the molecular basis of protective coloration.