Avermectin is a widely used insecticide for pest control, such as the Plutella xylostella. Despite its efficacy in pest management, concerns have been raised regarding its effect on non-target species, such as the important economic insect silkworm (Bombyx mori). We aimed to investigate the effects of avermectin application on the 3D genome architecture of silkworm midgut using high-throughput techniques such as high-throughput chromosome conformation capture (Hi-C) coupled with RNA sequencing (RNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Midgut tissue samples, during the fifth instar and three days, were collected from silkworm larvae fed mulberry leaves (control group) and leaves soaked in avermectin solution for 12 h (experimental group). Our findings revealed that avermectin treatment led to significant changes, including 386 differentially expressed genes (252 up-regulated, 134 down-regulated) and increased chromatin accessibility, particularly those involved in immune response, metabolism, and cellular stress pathways. The Hi-C data revealed more intense spatial interactions in the experimental group, leading to increased expression of detoxification proteins like ABC transporter. This study provides important insights into the molecular basis of pesticide resistance and a foundational basis for further research on the genetic and epigenetic mechanisms of insect stress responses.

The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the alpha-CA class in the silk gland, which is a Zn2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO2 to HCO3-, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers.

Silk, especially spider and insect silk, is a highly versatile biomaterial with potential applications in biomedicine, materials science, and biomimetic engineering. The primary structure of silk proteins is the basis for the mechanical properties of silk fibers. Biotechnologies such as single-molecule sequencing have facilitated an increasing number of reports on new silk genes and assembled silk proteins. Therefore, this review aims to provide a comprehensive overview of the recent advances in representative spider and insect silk proteins, focusing on identification methods, sequence characteristics, and de novo design and assembly. The review discusses three identification methods for silk genes: polymerase chain reaction (PCR)-based sequencing, PCR-free cloning and sequencing, and whole-genome sequencing. Moreover, it reveals the main spider and insect silk proteins and their sequences. Subsequent de novo assembly of artificial silk is covered and future research directions in the field of silk proteins, including new silk genes, customizable artificial silk, and the expansion of silk production and applications are discussed. This review provides a basis for the genetic aspects of silk production and the potential applications of artificial silk in material science and biomedical engineering.

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2–3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39–45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori. We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

Highly repetitive sequences play a major structural and function role in the genome. In the present study, we developed Cas9-assisted cloning and SMRT sequencing of long repetitive sequences (CACS) to sequence and manipulate highly repetitive genes from eukaryotic genomes. CACS combined Cas9-mediated cleavage of a target segment from an intact genome, Gibson assembly cloning, and PacBio SMRT sequencing. Applying CACS, we directly cloned and sequenced the complete sequences of fibroin heavy chain (FibH) genes from 17 domesticated (Bombyx mori) and 7 wild (Bombyx mandarina) silkworms. Our analysis revealed the unique fine structure organization, genetic variations, and domestication dynamics of FibH. We also demonstrated that the length of the repetitive regions determined the mechanical properties of silk fiber, which was further confirmed by Cas9 editing of FibH. CACS is a simple, robust, and efficient approach, providing affordable accessibility to highly repetitive regions of a genome. STATEMENT OF SIGNIFICANCE: Silkworm silk is the earliest and most widely used animal fiber, and its excellent performance mainly depends on the fibroin heavy chain (FibH) protein. The FibH gene is the main breakthrough in understanding the formation mechanism and improvement of silk fiber. In the study, we developed a CACS method for characterizing the fine structure and domestication landscape of 24 silkworm FibH genes. We used CRISPR/Cas9 to edit the repetitive sequence of FibH genes, revealing the relationship between FibH genes and mechanical properties of silkworm silk. Our study is helpful in modifying silk genes to manipulate other valuable highly repetitive sequences, and provides insight for silkworm breeding.

The highly repetitive and variable fibroin heavy chain (FibH) gene can be used as a silkworm identification; however, only a few complete FibH sequences are known. In this study, we extracted and examined 264 FibH gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average FibH lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively. All FibH sequences had a conserved 5' and 3' terminal non-repetitive (5' and 3' TNR, 99.74% and 99.99% identity, respectively) sequence and a variable repetitive core (RC). The RCs differed greatly, but they all shared the same motif. During domestication or breeding, the FibH gene mutated with hexanucleotide (GGTGCT) as the core unit. Numerous variations existed that were not unique to wild and domesticated silkworms. However, the transcriptional factor binding sites, such as fibroin modulator-binding protein, were highly conserved and had 100% identity in the FibH gene's intron and upstream sequences. The local and improved strains with the same FibH gene were divided into four families using this gene as a marker. Family I contained a maximum of 62 strains with the optional FibH (Opti-FibH, 15,960 bp) gene. This study provides new insights into FibH variations and silkworm breeding.

Synthetic biology is an eco-friendly and sustainable approach for the production of compounds, particularly used when the production processes involve toxic reagents. In this study, we used the silk gland of silkworm to produce indigoidine, a valuable natural blue pigment that cannot be synthesized naturally in animals. We genetically engineered these silkworms by integrating the indigoidine synthetase (idgS) gene from S. lavendulae and the PPTase (Sfp) gene from B. subtilis into the silkworm genome. In the resulting Blue silkworm, indigoidine was detected at a high level in the posterior silk gland (PSG), spanning all developmental stages from larvae to adults, without affecting silkworm growth or development. This synthesized indigoidine was secreted from the silk gland and subsequently stored in the fat body, with only a small fraction being excreted by the Malpighian tubule. Metabolomic analysis revealed that Blue silkworm efficiently synthesized indigoidine by upregulating l-glutamine, the precursor of indigoidine, and succinate, which is related to energy metabolism in the PSG. This study represents the first synthesis of indigoidine in an animal and therefore opens a new avenue for the biosynthesis of natural blue pigments and other valuable small molecules.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 degrees C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding. (C) 2020 Elsevier B.V. All rights reserved.

To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the B. mori FibH gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of FibH, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of B. moriFibH, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the FibH repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.

Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNAPyl/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNAPyl. Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.

Fluoride generally exists in the natural environment, and has been reported to induce serious environmental hazard to animals, plants, and even humans via ecological cycle. Silkworm, Bombyx mori, which showed significant growth and reproductivity reduction when exposed to fluoride, has become a model to evaluate the toxicity of fluoride. However, the detailed mechanism underlying fluoride toxicity and corresponding transport proteins remain unclear. In this study, we performed RNA-seq of the larval midgut and fat body with fluoride exposure and normal treatment. Differential analysis showed that there were 4405 differentially expressed genes in fat body and 4430 DEGs in midgut with fluoride stress. By Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, we identified several key pathways involved in the fluoride exposure and poisoning. We focused on the oxidative phosphorylation and MAPK signal pathway. QRT-PCR confirmed that oxidative phosphorylation process was remarkably inhibited by fluoride exposure and resulted in the blocking of ATP synthesis. The MAPK signal pathway was stimulated via phosphorylation signal transduction. Moreover, by protein structure analysis combined with the DEGs, we screen 36 potential membrane proteins which might take part in transporting fluoride. Taken together, the results of our study expanded the underlying mechanisms of fluoride poisoning on silkworm larval growth and development, and implied potential fluoride transport proteins in silkworm.

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

The site-specific nature and ease of handling of clustered regularly interspaced palindromic repeat （CRISPR）/Cas9 has fulfilled the decades-long goal of genome engineering, and thus has pushed the biological sciences into a new era in which site-directed manipulation of DNA is no longer a short slab （Doudna and Charpentier,2014）. The incomparable power of this system also enabled unprecedented new opportunities for practical applications in agriculture,ecosystem and human health （Liu, 2017）. However, with the

Silks are natural protein biopolymers with desirable mechanical properties and play crucial roles in insect survival and reproduction. However, the mechanisms by which large amounts of silk fibroin are efficiently secreted from the protein production organs (silk glands) remain elusive. Here, we focus on a dominant silkworm mutation, naked pupa (Nd), which enables carriers to lose spinning behaviors, produce a deficiency of silk fibroin production, and result in degenerate posterior silk gland (PSG). Linkage mapping and sequencing analyses revealed a deletion of 19 bp of the fibroin heavy chain (FibH), which results in a frameshift-caused deletion of the C-terminal domain (CT) responsible for the Nd locus. Immunofluorescence and immunoblot analysis showed that the PSG cells with truncated FibH exhibit blockades in the secretion of all three fibroins (FibH, FibL, and P25) from silk gland cell to silk gland lumen (a secretion-deficiency). By comparing the hereditary characters of three naked silkworm mutations (Nd, Nd-s, and fibH-ko), we explored the relationship between dominant and recessive inheritances in naked silkworms and found that high-molecular-weight/repetitive FibH with secretion-deficiency was in positive correlation with PSG atrophy phenotype, and moreover, the repetitive region of Nd-FibH accounted for the dominant phenotypes of fibroin secretion-deficiency, PSG atrophy, and naked pupa in B. mori. Our results uncovered the molecular nature of the silkworm Nd mutation and significantly improved our understanding of fibroin synthesis and secretion in silk-spinning caterpillars.

Staphylococcus aureus is an important and common Gram-positive bacteria which causes clinical infections and food-poisoning cases. Therapeutic schedules for treatment of S. aureus infections are facing a challenge because of the emergence of multidrug resistance strains. It is urgent to find new antiinfective drugs to control S. aureus infection. S. aureus strains are capable of producing the golden carotenoid pigment: staphyloxanthin, which acts as an important virulence factor and a potential target for antivirulence drug design. This review is aimed at presenting an updated overview of this golden carotenoid pigment of S. aureus from the biosynthesis of staphyloxanthin, its function, and the genes involved in pigment production to staphyloxanthin: a novel target for antivirulence therapy.

Recent advancements in genetic engineering technology have led to the development of CRISPR interference (CRISPRi) as a precise tool for regulating gene expression. When CRISPR/dCas9 is fused with transcriptional repressors, the system can robustly silence endogenous gene expression. The CRISPR/Cas9 tool is a promising alternative in organisms (e.g., Bombyx mori) that do not respond to traditional gene suppression techniques, such as RNA interference (RNAi). However, transcriptional repressors remain poorly categorized in multiple cell types and species, leading to difficulties in optimizing performance and efficiency. Here, we tested CRISPRi usability and efficiency in Bombyx mori cells (BmE). We fused dCas9 to five transcriptional repressors including KRAB, Hairy, SID, SRDX, and ERD. All five constructs were efficient in BmE cells. In a proof-of-concept experiment, we showed that CRISPRi acting on BmSoxE (a gene involved in cell proliferation) could generate similar phenotypes as RNAi gene suppression. Moreover, CRISPRi has fewer off-target effects. Through co-transfection of BmE cells with sgRNAs, we also demonstrated that dCas9 could simultaneously repress the expression of multiple genes. Furthermore, we identified sgRNA distance from transcriptional start site (TSS) and the dCas9: sgRNA ratio as the two limiting factors of CRISPRi efficiency. Our results demonstrated that CRISPR/dCas9 is a viable and rapid alternative for functional investigations of the B. mori genome and perhaps other Lepidoptera insects.

Genome editing using standard tools (ZFN, TALEN, and CRISPR/Cas9) rely on double strand breaks to edit the genome. A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks was developed recently and quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites that can be edited by BE3 for inactivation, with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base editing in an invertebrate model.

Insect ScienceVolume 26, Issue 6 p. 1055-1058 LETTER TO THE EDITOR Targeted activation of BmCyclinE in Bombyx mori using designer TAL effectors San-Yuan Ma, Corresponding Author San-Yuan Ma masy@swu.edu.cn State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, ChinaCorrespondence: San-Yuan Ma and Qing-You Xia, State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Email: masy@swu.edu.cn, xiaqy@swu.edu.cn.Search for more papers by this authorYuan-Yuan Liu, Yuan-Yuan Liu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Department of Hematology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, ChinaSearch for more papers by this authorWei Lu, Wei Lu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorXiao-Gang Wang, Xiao-Gang Wang State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorLe Sun, Le Sun State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorKai Yu, Kai Yu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorQing-You Xia, Corresponding Author Qing-You Xia xiaqy@swu.edu.cn orcid.org/0000-0002-9263-2886 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, ChinaCorrespondence: San-Yuan Ma and Qing-You Xia, State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Email: masy@swu.edu.cn, xiaqy@swu.edu.cn.Search for more papers by this author San-Yuan Ma, Corresponding Author San-Yuan Ma masy@swu.edu.cn State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, ChinaCorrespondence: San-Yuan Ma and Qing-You Xia, State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Email: masy@swu.edu.cn, xiaqy@swu.edu.cn.Search for more papers by this authorYuan-Yuan Liu, Yuan-Yuan Liu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Department of Hematology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, ChinaSearch for more papers by this authorWei Lu, Wei Lu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorXiao-Gang Wang, Xiao-Gang Wang State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorLe Sun, Le Sun State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorKai Yu, Kai Yu State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, ChinaSearch for more papers by this authorQing-You Xia, Corresponding Author Qing-You Xia xiaqy@swu.edu.cn orcid.org/0000-0002-9263-2886 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, ChinaCorrespondence: San-Yuan Ma and Qing-You Xia, State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Email: masy@swu.edu.cn, xiaqy@swu.edu.cn.Search for more papers by this author First published: 09 June 2018 https://doi.org/10.1111/1744-7917.12613Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume26, Issue6Special Issue: Insect Genome EditingDecember 2019Pages 1055-1058 This article also appears in: Insect Genome Editing RelatedInformation