Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

BACKGROUND: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

ALKBH5-mediated CHAC1 depletion promotes malignant progression and decreases cisplatin-induced oxidative stress in gastric cancer.

The m6a demethyltransferase ALKBH5 dynamically modulates gene expression and intracellular metabolic molecules by modifying RNA m6a in cancer cells. However, ALKBH5's function in gastric cancer (GC) has remained controversial. This study demonstrates that ALKBH5 is highly expressed in GC. Silencing ALKBH5 hampers proliferation, and metastatic potential, and induces cell death in GC cells. Through a comprehensive analysis of the transcriptome and m6A sequencing, alterations in certain ALKBH5 target genes, including CHAC1, were identified. ALKBH5's demethylation effect regulates CHAC1 RNA stability, leading to reduced CHAC1 expression. Moreover, CHAC1 modulates intracellular ROS levels, influencing the chemotherapy sensitivity of gastric cancer. In summary, our study unveils the pivotal role of the ALKBH5-CHAC1-ROS axis and highlights the significance of m6A methylation in gastric cancer.

High-throughput Sequencing Analysis of Differentially Expressed microRNAs Associated With SREBP1 in Differentiated Thyroid Carcinoma.

BACKGROUND/AIM: MicroRNAs (miRNAs) interact with mRNAs and play important roles in progression and prognosis in multiple cancers. Sterol regulatory element-binding protein 1 (SREBP1) is an important lipid metabolism regulatory gene. The aim of the present study was to analyze the profiles of miRNAs that are associated with SREBP1 expression in differentiated thyroid carcinoma (DTC). MATERIALS AND METHODS: In the present study, a high-throughput small RNA sequencing (miRNA-Seq) method was used to investigate differences in miRNA profiling with versus without interference with SREBP1 expression via small interfering RNA. Real-time qPCR (qRT-PCR) was performed to confirm the results. RESULTS: A total of 1,393 conserved and 84 novel miRNAs were successfully discovered. In two separate batches, a total of 27 differentially expressed miRNAs (11 up-regulated and 16 down-regulated) were observed in BCPAP cells after SREBF1 interference with two distinct siRNA fragments, as compared to the control siRNA treatment. Hsa-miR-941, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-100-5p, and hsa-miR-21-3p were selected for validation using qRT-PCR. The qRT-PCR results were consistent with the sequencing data. Gene Ontology enrichment showed that the predicted targets of these miRNAs were mainly involved in the regulation of system development, metabolism and protein binding cellular processes, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes pathways analysis showed that the predicted target genes were involved in several signaling pathways, including the Ras, MAPK, insulin, thyroid hormone, and metabolic pathway signaling pathways. CONCLUSION: Differentially expressed miRNAs and their target genes may play an important role in the progression and prognosis of DTC that is associated with SREBP1 expression.

Spindle function and Wnt pathway inhibition by PBX1 to suppress tumor progression via downregulating DCDC2 in colorectal cancer.

PBX1 is a transcription factor that regulates a variety of genes, involved in intracellular lipid metabolism, cell proliferation, and other pathways. The promoting and inhibiting function of PBX1 in various cancer types was extensively discussed, however, there have been no studies on PBX1 proteins in colorectal cancer (CRC). This study aimed to reveal the anti-tumor function of PBX1 in CRC and the underlying molecular mechanism. Bioinformatics analysis revealed that PBX1 is downregulated in CRC, indicating that is a potential antioncogene in CRC. Overexpression of PBX1 suppresses tumor growth and metastasis in vitro and in vivo. Mechanistically, we found that PBX1 acted as a transcription factor that suppressed DCDC2 expression and inhibited spindle function. Moreover, the PBX1-DCDC2 axis controlled the Wnt pathway in CRC cells. Overexpression of DCDC2 restored CRC proliferation, metastasis abilities and Wnt pathway. In conclusion, this study suggests that PBX1 acts as a transcription factor to suppress DCDC2 expression and inhibit cell proliferation and metastasis by disrupting spindle function and the Wnt pathway in CRC.

Single-cell RNA-seq dissecting heterogeneity of tumor cells and comprehensive dynamics in tumor microenvironment during lymph nodes metastasis in gastric cancer.

Lymph node metastasis is the common metastasis route of gastric cancer. However, until now, heterogeneities of tumor cells and tumor microenvironment in primary tumors (PT) and metastatic lymph nodes (MLN) of gastric cancer (GC) remains uncharacterized. In our study, single cell RNA sequencing was performed on tissues from PT and MLN of gastric cancer. Trajectory analysis and function enrichment analyses were conducted to decode the underlying mechanisms contributing to LN metastasis of gastric cancer. Heterogeneous composition of immune cells and distinct intercellular interactions in PT and MLN were analyzed. Based on the generated single cell transcriptome profiles, dynamics of gene expressions in cancer cells between PT and MLN were characterized. Moreover, we reconstructed the developmental trajectory of GC cells' metastasis to LN and identified two subtypes of GC cells with distinct potentials of having malignant biological behaviors. We characterized the repression of neutrophil polarization associated genes, like LCN2, which would contribute to LN metastasis, and histochemistry experiments validated our findings. Additionally, heterogeneity in neutrophils, rather than macrophages, was characterized. Immune checkpoint associated interaction of SPP1 was found active in MLN. In conclusion, we decode the dynamics of tumor cells during LN metastasis in GC and to identify a subtype of GC cells with potentials of LN metastasis. Our data indicated that the disordering the neutrophils polarization and maturation and the activation of immune checkpoint SPP1 might contribute to LN metastasis in GC, providing a novel insight on the mechanism and potential therapeutic targets of LN metastasis in GC.

m6 A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels.

BACKGROUND: Methyltransferase 3 (METTL3)-mediated N6-methyladenosine (m6 A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3-mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer. METHODS: To investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6 A-modified RNA immunoprecipitation (Me-RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre-B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3-PBX1-GCH1 axis. ELISA and liquid chromatography-mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4 ). RESULTS: Knockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3-PBX1-GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression. CONCLUSIONS: This study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3-PBX1-GCH1 axis increasing BH4 levels in GC.

Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-ß-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

Circadian regulation of night feeding and daytime detoxification in a formidable Asian pest Spodoptera litura.

Voracious feeding, trans-continental migration and insecticide resistance make Spodoptera litura among the most difficult Asian agricultural pests to control. Larvae exhibit strong circadian behavior, feeding actively at night and hiding in soil during daytime. The daily pattern of larval metabolism was reversed, with higher transcription levels of genes for digestion (amylase, protease, lipase) and detoxification (CYP450s, GSTs, COEs) in daytime than at night. To investigate the control of these processes, we annotated nine essential clock genes and analyzed their transcription patterns, followed by functional analysis of their coupling using siRNA knockdown of interlocked negative feedback system core and repressor genes (SlituClk, SlituBmal1 and SlituCwo). Based on phase relationships and overexpression in cultured cells the controlling mechanism seems to involve direct coupling of the circadian processes to E-boxes in responding promoters. Additional manipulations involving exposure to the neonicotinoid imidacloprid suggested that insecticide application must be based on chronotoxicological considerations for optimal effectiveness.

The POU Transcription Factor POU-M2 Regulates Vitellogenin Receptor Gene Expression in the Silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) play critical roles in egg formation by transporting vitellogenin (Vg) into oocytes in insects. Although the function of VgR in insects is well studied, the transcriptional regulation of this gene is still unclear. Here, we cloned the promoter of the VgR gene from Bombyx mori (BmVgR), and predicted many POU cis-response elements (CREs) in its promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that the POU transcription factor POU-M2 bound directly to the CREs of the promoter. Overexpression of POU-M2 in an ovarian cell line (BmNs) enhanced BmVgR transcription and promoter activity detected by quantitative reverse transcription PCR and luciferase reporter assays. Analyses of expression patterns indicated that POU-M2 was expressed in ovary at day two of wandering stage initially, followed by BmVgR. RNA interference of POU-M2 significantly reduced the transcription of BmVgR in ovary and egg-laying rate. Our results suggest a novel function for the POU factor in silkworm oogenesis by its involvement in BmVgR regulation and expands the understanding of POU factors in insect VgR expression.

bmo-miR-2739 and the novel microRNA miR-167 coordinately regulate the expression of the vitellogenin receptor in Bombyx mori oogenesis.

Vitellogenin receptors (VgRs) play crucial roles in oogenesis by mediating endocytosis of vitellogenin and other nutrients in ovipara. We conducted small RNA sequencing and screening with a luciferase reporter system, and found that bmo-miR-2739 and a novel miRNA (novel-miR-167) coordinately regulate the expression of VgR in Bombyx mori (BmVgR). Further analyses suggested that these two miRNAs direct target repression by binding directly to the BmVgR 3' untranslated region. Forced expression of either miRNA using the piggyBac system blocked vitellogenin (Vg) transport and retarded ovariole development. Antagomir silencing of bmo-miR-2739 or novel-miR-167 resulted in increased amounts of BmVgR protein in the ovaries and BmVgR mRNA in the fat body. This evidence, combined with spatiotemporal expression profiles, revealed that these two miRNAs function together to fine-tune the amount of BmVgR protein for ovarian development. Additionally, novel-miR-167 was mainly responsible for the post-transcriptional repression of BmVgR in non-ovarian tissues. The results of this study contribute to our understanding of the function of miRNAs during ovarian development of a lepidopteran and suggest a new strategy for controlling insect reproduction.

SREBP1 as a potential biomarker predicts levothyroxine efficacy of differentiated thyroid cancer.

BACKGROUND: SREBP1 is a well-known transcript factor regulating lipogenesis. It has been reported to play an important role in tumor progress in recent years. However, the roles of SREBP1 in differentiated thyroid cancer (DTC) are uncertain. Based on this, we aimed to investigate the expression of SREBP1 and the influence of SREBP1 on DTC patients. METHODS: qRT-PCR and immunohistochemistry were used to detect the expression of SREBPs in DTC tissues and the adjacent normal tissues. The following methods, including the MTS, colony-forming assay, flow cytometry and Hoechst staining were used to detect the biological function of thyroid cancer cells based on SREBP1 interference or not. RESULTS: the expression of SREBP1 was significantly different among DTCs, thyroid nodules and the adjacent normal tissues. Briefly, SREBP1 was upregulated follow with the malignancy, but there was no significant difference of SREBP2 between thyroid nodules and the adjacent normal tissues. Further, the ROC curve showed that SREBP1 has higher diagnostic value than SREBP2. SREBP1 expression was significantly related to the tumor size and lymph node metastasis in DTCs. In vitro, the proliferation of thyroid cancer cells was suppressed obviously after interfered with SREBP1, and the apoptotic cells was increased. Further, SREBP1 expression was also associated with the short-term efficacy of levothyroxine in DTC patients. CONCLUSION: this is the first time to report that SREBP1 is an oncogene and a pro-proliferation factor in thyroid cancer, indicating that SREBP1 may serve as a potential biomarker and therapeutic target in thyroid cancer.

Down-Regulation of APTR and it's Diagnostic Value in Papillary and Anaplastic Thyroid Cancer.

APTR has been employed as a potential biomarker attributing to it was involved in carcinogenesis and malignancy's progression. However, the roles of APTR in papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) are unclear. In the present study, we aimed to explore the relative expression of APTR in PTC and ATC tissues and the relation between APTR expression and PTC clinicopathological features. We analyzed APTR expression in PTC and ATC by investigating data obtained from the Gene Expression Omnibus (GEO) database. Then, we tested 76-pair PTC and adjacent normal samples by qRT-PCR, and the result was in accordance with the analysis in GEO datasets. Chi-square (χ2) analysis was employed to evaluate the association between APTR and PTC clinical features. These results showed that APTR was negatively related to TNM stages, distant metastasis. In addition, we further evaluated the feasibility of using APTR to detect PTC and ATC patients by the receiver operating characteristic (ROC) and the area under curve (AUC). These findings implied that down-regulation of APTR is correlated with tumorigenesis, also indicated that the potential diagnostic value of APTR for detecting PTC and ATC patients.

LncRNAs SNHG12 and LINC00152 were associated with progression of patients with papillary thyroid carcinoma.

Aim: To investigate the clinical roles of LINC00152 and SNHG12 in papillary thyroid carcinoma (PTC). Methods: LINC00152 and SNHG12 expression was sought and analysis in gene expression omnibus, The Cancer Genome Atlas and GEPIA datasets. Tumor and adjacent normal tissues were collected from 97 PTC and 44 benign thyroid nodules patients. The expression was evaluated by quantitative real-time polymerase chain reaction. The association between the expression level and clinicopathologic characteristics was analyzed by χ2 test. Receiver operating characteristic curves were plotted to evaluate the diagnostic value. Results: The expression of SNHG12 and LINC00152 were significantly higher in PTC tissues than in adjacent normal tissues not only in gene expression omnibus database but the validated samples. More interesting, LINC00152 expression level was also significantly higher in PTC tissues than that in benign thyroid nodules. The upregulation of LINC00152 and SNHG12 was associated with the malignant progression of PTC. Receiver operating characteristic curve analysis also demonstrated that there was a good trend, which indicates that they may have certain diagnostic value. Conclusion: LINC00152 and SNHG12 might serve as serve as potential related molecules of PTC.

Genome-wide annotation and comparative analysis of cuticular protein genes in the noctuid pest Spodoptera litura.

Insect cuticle is considered an adaptable and versatile building material with roles in the construction and function of exoskeleton. Its physical properties are varied, as the biological requirements differ among diverse structures and change during the life cycle of the insect. Although the bulk of cuticle consists basically of cuticular proteins (CPs) associated with chitin, the degree of cuticular sclerotization is an important factor in determining its physical properties. Spodoptera litura, the tobacco cutworm, is an important agricultural pest in Asia. Compared to the domestic silkworm, Bombyx mori, another lepidopteran whose CP genes have been well annotated, S. litura has a shorter life cycle, hides in soil during daytime beginning in the 5th instar and is exposed to soil in the pupal stage without the protection of a cocoon. In order to understand how the CP genes may have been adapted to support the characteristic life style of S. litura, we searched its genome and found 287 putative cuticular proteins that can be classified into 9 CP families (CPR with three groups (RR-1, RR-2, RR-3), CPAP1, CPAP3, CPF, CPFL, CPT, CPG, CPCFC and CPLCA), and a collection of unclassified CPs named CPH. There were also 112 cuticular proteins enriched in Histidine residues with content varying from 6% to 30%, comprising many more His-rich cuticular proteins than B. mori. A phylogenetic analysis between S. litura, M. sexta and B. mori uncovered large expansions of RR-1 and RR-2 CPs, forming large gene clusters in different regions of S. litura chromosome 9. We used RNA-seq analysis to document the expression profiles of CPs in different developmental stages and tissues of S. litura. The comparative genomic analysis of CPs between S. litura and B. mori integrated with the unique behavior and life cycle of the two species offers new insights into their contrasting ecological adaptations.

Down-regulation of DANCR acts as a potential biomarker for papillary thyroid cancer diagnosis.

Long non-coding RNAs (lncRNAs) have been reported to be dysregulated and play a crucial role in the progression of cancer. LncRNA DANCR has recently been revealed to be involved in tumorigenesis of numerous types of cancer, including osteosarcoma, gastric cancer, breast cancer, hepatocellular carcinoma, and colorectal cancer. However, the expression profiles and biological relevance of DANCR in papillary thyroid cancer (PTC) have not yet been reported. In the present study, the expression level of DANCR in PTC tissues and adjacent normal tissues was detected by reverse transcription-quantitative PCR in PTC patients, and then we analyzed the association with clinical pathological characteristics of patients and DANCR expressions. These results demonstrated that the expression of DANCR was notably decreased in tumor tissues in comparison with adjacent normal tissues (P<0.001). Furthermore, the present study found that DANCR expression level was correlated to T grade (P<0.01) and TNM stage (P=0.017). The present study demonstrated that DANCR was associated with PTC aggressive clinical features and may serve as a diagnostic biomarker for detecting PTC patients.

Identification and validation of potential target genes in papillary thyroid cancer.

Thyroid cancer (TC) is one of the most common endocrine malignancies, and the incidence of TC has almost tripled over the past three decades. This increase may partially own to overdiagnosis and approximately 15-30% of cytological indeterminate thyroid nodules cannot be evaluated by means of fine-needle aspiration. The present study aimed to identify potential crucial genes of PTC and provide new sights into improving the diagnosis of thyroid lesions for future study. We adopted an integrated analysis of Gene expression profiles of PTC patients and adjacent normal controls and data from The Cancer Genome Atlas databases (TCGA). The differentially expressed genes (DEGs) were screened using the Limma package in R software. Connectivity Map (CMap) was used to predict potential drugs for PTC. STRING and Cytoscape software were employed to perform GO, KEGG pathway enrichment analysis and module analysis for DEGs. RT-qPCR was used to validate hub genes screened using module analysis. A total of 218 DEGs were screened, including 55 down-regulated and 163 up-regulated DEGs. GO analysis showed that these DEGs were primary enriched in cell adhesion, extracellular region and glycosaminoglycan binding. KEGG pathway analysis revealed that DEGs primarily participated in ECM-receptor interaction. PPI network and module analysis identified seven-hub genes, including FN1, SERPINA1, ECM1, MMRN1, PROS1, CFD, TIMP1. RT-qPCR results validated that the expression levels of seven-hub genes were consistent with the bioinformatics analysis. These findings have identified seven-hub genes which may helpful for the development of gene panel for thyroid nodules diagnosis.

Genetic polymorphism contributes to 131I radiotherapy-induced toxicities in patients with differentiated thyroid cancer.

AIM: To investigate the association between SNPs in DNA damage response pathways and toxicities following 131I radiotherapy of differentiated thyroid cancer (DTC). Materials & methods: We identified 22 functional SNPs of genes in DNA damage response pathways. MassArray was used to sequence SNP genotypes in 203 DTC patients. Hardy-Weinberg equilibrium and the associations between the two alleles of each SNP and toxicity reactions were evaluated using χ2 analysis. RESULTS: Ataxia-telangiectasia mutated (ATM) rs620815 T-allele carriers were at increased risk of 131I radiation-induced gastrointestinal reaction compared with C allele carriers. TNFα rs1800629 GA genotype may increase the incidence of neck pain compared with GG genotype. Furthermore, TNFα rs1800629, ATM rs11212570, NF-κß rs230493, and TGF-ß rs1800469, rs2241716 were associated with throat pain following 131I radiotherapy. CONCLUSION: The identified SNPs might serve as novel biomarkers for DTC treated with 131I radiotherapy.

Upregulation of long noncoding RNA MALAT1 in papillary thyroid cancer and its diagnostic value.

AIM: To investigate the expression level of lncRNA MALAT1 in papillary thyroid cancer (PTC) and evaluate its clinical diagnostic value as a biomarker in PTC. METHODS: MALAT1 lncRNA expression in tissues was detected by qRT-PCR. The diagnostic value of MALAT1 as a biomarker in PTC was evaluated with receiver operating characteristics. RESULTS: MALAT1 expression was upregulated in PTC tissues compared with paired corresponding noncancerous tissues. We also found that upregulated MALAT1 expression was correlated with tumor size, lymph node metastases (p = 0.011) and WHO disease stage. The area under the curve was 0.6320, 0.7192, 0.7089 and 0.7000 for PTC, lymph node metastasis, extrathyroidal extension and WHO disease stage prediction, respectively. CONCLUSION: Our finding suggests that MALAT1 may exert oncogenic function in PTC and may be a potential diagnostic marker for this cancer.

REST, not REST4, is a risk factor associated with radiotherapy plus chemotherapy efficacy in glioma.

BACKGROUND/AIM: Repressor element silencing transcription factor (REST) is a transcription repressor, expressed in several malignancies. This study aims to evaluate the prognostic values of REST and its splicing variant REST4 in glioma, and investigate the potential correlation between REST and REST4. METHODS: REST and REST4 expression values were evaluated by qRT-PCR in 89 patients with gliomas and 10 with normal brain tissues. RESULTS: Upregulation of REST was related to higher World Health Organization (WHO) grade, larger tumor size, higher ki67, and higher p53 positive rate. After radiotherapy+temozolomide (RT+TMZ) treatment, low REST expression patients could get better therapeutic efficacy (P=0.031). The positive rate of REST4 expression was only 13.5% in glioma tissues, and REST4 expression was not associated with clinical characteristics and REST expression in this study. CONCLUSIONS: REST was a prognostic factor in glioma, while REST4 was not. REST expression can be a predictor in evaluating the survival outcome of gliomas patients treated with RT+TMZ after surgery.

A new approach for comprehensively describing heterogametic sex chromosomes.

Notwithstanding the rapid developments in sequencing techniques, Y and W sex chromosomes have still been mostly excluded from whole genome sequencing projects due to their high repetitive DNA content. Therefore, Y and W chromosomes are poorly described in most species despite their biological importance. Several methods were developed for identifying Y or W-linked sequences among unmapped scaffolds. However, it is not enough to discover functional regions from short unmapped scaffolds. Here, we provide a new and simple strategy based on k-mer comparison for comprehensive analysis of the W chromosome in Bombyx mori. Using this novel method, we effectively assembled de novo 1281 W-derived genome contigs (totaling 1.9 Mbp), and identified 156 W-linked transcript RNAs and 345 W-linked small RNAs. This method will help in the elucidation of mechanisms of sexual development and exploration of W chromosome biological functions, and provide insights into the evolution of sex chromosomes. Moreover, we showed this method can be employed in identifying heterogametic sex chromosomes (W and Y chromosomes) in many other species where genomic information is still scarce.