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Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori. [ABSTRACT FROM AUTHOR]

Silk proteins are synthesized in the middle and posterior silk glands of silkworms, then transit into the anterior of the silk gland, where the silk fibers are produced, stored and processed. The mechanism of formation and spinning of the silk fibers has not been fully elucidated, and transcriptome analyses specific to the anterior silk gland have not been reported. In the present study, we explored gene expression profiles in five regions of silk gland samples using the RNA-Seq method. As a result, there were 959,979,570 raw reads obtained, of which 583,068,172 reads were mapped to the silkworm genome. A total of 7419 genes were found to be expressed in terms of reads per kilobase of exon model per million mapped reads ≥ 5 in at least one sample. The gene numbers and expression levels of the expressed genes differed between these regions. The differentially expressed genes were analyzed, and 282 genes were detected as up-regulated in the anterior silk gland, compared with the other parts. Functions of these genes were addressed using the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases, and seven key pathways were enriched. It suggested that the ion transportation, energy metabolism, protease inhibitors and cuticle proteins played essential roles in the process of silk formation and spinning in the anterior silk gland. In addition, 210 genes were found differently expressed between males and females, which should help to elucidate the mechanism of the quality difference in silk fibers from male and female silkworms. [ABSTRACT FROM AUTHOR]

Although the draft genome sequence of silkworm is available for a decade, its genetic variations, especially structural variations, are far from well explored. In this study, we identified 1,298,659 SNPs and 9,731 indels, of which 32 % of SNPs and 92.2 % of indels were novel compared to previous silkworm re-sequencing analysis. In addition, we applied a read depth-based approach to investigate copy number variations among 21 silkworm strains at genome-wide level. This effort resulted in 562 duplicated and 41 deleted CNV regions, and among them 442 CNV were newly identified. Functional annotation of genes affected by these genetic variations reveal that these genes include a wide spectrum of molecular functions, such as immunity and drug detoxification, which are important for the adaptive evolution of silkworms. We further validated the predicted CNV regions using q-PCR. 94.7 % (36/38) of the selected regions show divergent copy numbers compared to a single-copy gene OR2. In addition, potential presence/absence variations are also observed in our study: 11 genes are present in the reference genome, but absent in other strains. Overall, we draw an integrative map of silkworm genetic variation at genome-wide level. The identification of genetic variations in this study improves our understanding that these variants play important roles in shaping phenotypic variations between wild and domesticated silkworms. [ABSTRACT FROM AUTHOR]

The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. [ABSTRACT FROM AUTHOR]

In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation. [ABSTRACT FROM AUTHOR]

Insect pests have developed resistance to chemical insecticides, insecticidal toxins as bioinsecticides or genetic protection built into crops. Consequently, novel, orally active insecticidal toxins would be valuable biological alternatives for pest control. Here, we identified a novel insecticidal toxin, parasporal crystal toxin (PC), from Bacillus bombysepticus (Bb). PC shows oral pathogenic activity and lethality towards silkworms and Cry1Ac-resistant Helicoverpa armigera strains. In vitro assays, PC after activated by trypsin binds to BmAPN4 and BtR-175 by interacting with CR7 and CR12 fragments. Additionally, trypsin-activated PC demonstrates cytotoxicity against Sf9 cells expressing BmAPN4, revealing that BmAPN4 serves as a functional receptor that participates in Bb and PC pathogenicity. In vivo assay, knocking out BtR-175 increased the resistance of silkworms to PC. These data suggest that PC is the first protein with insecticidal activity identified in Bb that is capable of causing silkworm death via receptor interactions, representing an important advance in our understanding of the toxicity of Bb and the contributions of interactions between microbial pathogens and insects to disease pathology. Furthermore, the potency of PC as an insecticidal protein makes it a good candidate for inclusion in integrated agricultural pest management systems. [ABSTRACT FROM AUTHOR]

Bombyx mori is an economically important holometabolous lepidopteran insect. In B. mori endogenous noncoding RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs play crucial biological functions in metamorphosis and sex determination. In addition, circular RNAs (circRNAs) have been recently identified as noncoding RNAs in most common model organisms and show potential as gene regulators. However, to date, there have been few studies on the circRNAs present in the B. mori genome conducted to date. Here, we identified 3916 circRNAs by deep circular transcriptome sequencing using the silk gland of B. mori . 3155 circRNAs were found to be derived from 1727 parental genes. The circRNAs displayed tissue-specific expression between the middle silk gland (MSG) and posterior silk gland (PSG), with 2532 and 880 being upregulated circRNAs in the MSG and PSG, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parental genes from the MSG and PSG were generally annotated to similar categories and pathways. The interaction network of circRNAs and miRNAs showed that circRNAs might act as miRNA sponges or interact with miRNAs in some other way. Overall, the results revealed the complicated patterns of circRNAs in the B. mori silk gland providing a new angle from which to explore the mechanisms of complex gene regulation and efficient silk protein synthesis. [ABSTRACT FROM AUTHOR]

In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation. [ABSTRACT FROM AUTHOR]