Origin, structure, and composition of the spider major ampullate silk fiber revealed by genomics, proteomics, and single-cell and spatial transcriptomics.

Spiders produce nature's toughest fiber using renewable components at ambient temperatures and with water as solvent, making it highly interesting to replicate for the materials industry. Despite this, much remains to be understood about the bioprocessing and composition of spider silk fibers. Here, we identify 18 proteins that make up the spiders' strongest silk type, the major ampullate fiber. Single-cell RNA sequencing and spatial transcriptomics revealed that the secretory epithelium of the gland harbors six cell types. These cell types are confined to three distinct glandular zones that produce specific combinations of silk proteins. Image analysis of histological sections showed that the secretions from the three zones do not mix, and proteomics analysis revealed that these secretions form layers in the final fiber. Using a multi-omics approach, we provide substantial advancements in the understanding of the structure and function of the major ampullate silk gland as well as of the architecture and composition of the fiber it produces.

De Novo Long-Read Genome Assembly and Annotation of the Luna Moth (Actias luna) Fully Resolves Repeat-Rich Silk Genes.

We present the first long-read de novo assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin), a long and highly repetitive gene (>20 kb) essential in silk fiber production. There are >160,000 described species of moths and butterflies (Lepidoptera), but only within the last 5 years have we begun to recover high-quality annotated whole genomes across the order that capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mb, a contig N50 of 16.8 Mb, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A. luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

Genome-wide expression quantitative trait locus analysis reveals silk-preferential gene regulatory network in maize.

Silk of maize (Zea mays L.) contains diverse metabolites with complicated structures and functions, making it a great challenge to explore the mechanisms of metabolic regulation. Genome-wide identification of silk-preferential genes and investigation of their expression regulation provide an opportunity to reveal the regulatory networks of metabolism. Here, we applied the expression quantitative trait locus (eQTL) mapping on a maize natural population to explore the regulation of gene expression in unpollinated silk of maize. We obtained 3,985 silk-preferential genes that were specifically or preferentially expressed in silk using our population. Silk-preferential genes showed more obvious expression variations compared with broadly expressed genes that were ubiquitously expressed in most tissues. We found that trans-eQTL regulation played a more important role for silk-preferential genes compared to the broadly expressed genes. The relationship between 38 transcription factors and 85 target genes, including silk-preferential genes, were detected. Finally, we constructed a transcriptional regulatory network around the silk-preferential gene Bx10, which was proposed to be associated with response to abiotic stress and biotic stress. Taken together, this study deepened our understanding of transcriptome variation in maize silk and the expression regulation of silk-preferential genes, enhancing the investigation of regulatory networks on metabolic pathways.

A comprehensive gene expression analysis of the unique three-layered cocoon of the cecropia moth, Hyalophora cecropia.

The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.

Time-course transcriptome data of silk glands in day 0-7 last-instar larvae of Bombyx mori (w1 pnd strain).

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

Mutation in the Bombyx mori BmGMC2 gene impacts silk production and silk protein synthesis.

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers.

Ectopic expression of BmeryCA in Bombyx mori increases silk yield and mechanical properties by altering the pH of posterior silk gland.

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 α-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.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Bombyx moriSuppressor of Hairless is involved in the regulation of the silkworm cell cycle and endoreplication of the silk glands.

The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.

[Primary Structure Characterization and Biosynthesis of Spider Silk Proteins for Multifunctional Biomaterials].

Spider silk is a natural fiber known as "biosteel" with the strongest composite performance, such as high tensile strength and toughness. It is also equipped with excellent biocompatibility and shape memory ability, thus shows great potential in many fields such as biomedicine and tissue engineering. Spider silk is composed of macromolecular spidroin with rich structural diversity. The characteristics of the primary structure of natural spidroin, such as the high repeatability of amino acids in the core repetitive region, the high content of specific amino acids, the large molecular weight, and the high GC content of the spidroin gene, have brought great difficulties in heterologous expression. This review discusses focuses on the relationship between the featured motifs of the microcrystalline region in the repetitive unit of spidroin and its structure, as well as the spinning performance and the heterologous expression. The optimization design for the sequence of spidroin combined with heterologous expression strategy has greatly promoted the development of the biosynthesis of spider silk proteins. This review may facilitate the rational design and efficient synthesis of recombinant spidroin.

Integrated transcriptome and proteome analysis reveals the unique molecular features and nutritional components on the muscles in Chinese Taihe black-bone silky fowl chicken.

The Taihe Black-Bone silky fowl chicken (BB-sfc) is a renowned dietary and medicinal chicken globally recognized for its high nutritional and medicinal value. Compared to the local Black-Bone black-feathered chicken (BB-bfc), the Taihe silky fowl chicken has higher levels of amino acids, trace elements, and unsaturated fatty acids in their muscles, which offer anti-aging, anti-cancer, and immune enhancing benefits. Despite this, the unique nutritional components, genes, and proteins in Taihe silky fowl chicken muscles are largely unknown. Therefore, we performed a comprehensive transcriptome and proteome analysis of muscle development between BB-sfc and BB-bfc chickens using RNA-Seq and TMT-based quantitative proteomics methods. RNA-Seq analysis identified 286 up-regulated genes and 190 down-regulated genes in BB-sfc chickens, with oxidoreductase activity and electron transfer activity enriched in up-regulated genes, and phospholipid homeostasis and cholesterol transporter activity enriched in down-regulated genes. Proteome analysis revealed 186 significantly increased and 287 significantly decreased proteins in Taihe BB-sfc chicken muscles, primarily affecting mitochondrial function and oxidative phosphorylation, crucial for enhancing muscle antioxidant capacity. Integrated transcriptome and proteome analysis identified 6 overlapped up-regulated genes and 8 overlapped down-regulated genes in Taihe silky fowl chicken, related to improved muscle antioxidant status. Taken together, this research provides a comprehensive database of gene expression and protein information in Taihe Black-Bone silky fowl chicken muscles, aiding in fully exploring their unique economic value in the future.

Efficient Biosynthetic Fabrication of Spidroins with High Spinning Performance.

The unique 3D structure of spider silk protein (spidroin) determines the excellent mechanical properties of spidroin fiber, but the difficulty of heterologous expression and poor spinning performance of recombinant spider silk protein limit its application. A high-yield low-molecular-weight biomimetic spidroin (Amy-6rep) is obtained by sequence modification, and its excellent spinning performance is verified by electrospinning it for use as a nanogenerator. Amy-6rep increases the highly fibrogenic microcrystalline region in the core repeat region of natural spidroin with limited sequence length and replaces the polyalanine sequence with an amyloid polypeptide through structural similarity. Due to sequence modification, the expression of Amy-6rep increased by ≈200%, and the self-assembly performance of Amy-6rep significantly increased. After electrospinning with Amy-6rep, the nanofibers exhibit good tribopower generation capacity. In this paper, a biomimetic spidroin sequence design with high yield and good spinning performance is reported, and a strategy for electrospinning to produce an artificial nanogenerator is explored.

Unveiling the Dynamic Self-Assembly of a Recombinant Dragline-Silk-Mimicking Protein.

Despite the considerable interest in the recombinant production of synthetic spider silk fibers that possess mechanical properties similar to those of native spider silks, such as the cost-effectiveness, tunability, and scalability realization, is still lacking. To address this long-standing challenge, we have constructed an artificial spider silk gene using Golden Gate assembly for the recombinant bacterial production of dragline-mimicking silk, incorporating all the essential components: the N-terminal domain, a 33-residue-long major-ampullate-spidroin-inspired segment repeated 16 times, and the C-terminal domain (N16C). This designed silk-like protein was successfully expressed in Escherichia coli, purified, and cast into films from formic acid. We produced uniformly 13C-15N-labeled N16C films and employed solid-state magic-angle spinning nuclear magnetic resonance (NMR) for characterization. Thus, we could demonstrate that our bioengineered silk-like protein self-assembles into a film where, when hydrated, the solvent-exposed layer of the rigid, ß-nanocrystalline polyalanine core undergoes a transition to an α-helical structure, gaining mobility to the extent that it fully dissolves in water and transforms into a highly dynamic random coil. This hydration-induced behavior induces chain dynamics in the glycine-rich amorphous soft segments on the microsecond time scale, contributing to the elasticity of the solid material. Our findings not only reveal the presence of structurally and dynamically distinct segments within the film's superstructure but also highlight the complexity of the self-organization responsible for the exceptional mechanical properties observed in proteins that mimic dragline silk.

Identification and function of the Pax gene Bmgsb in the silk gland of Bombyx mori.

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.

Identification of genes associated with the silk gland size using multi-omics in silkworm (Bombyx mori).

Silk gland size in silkworms (Bombyx mori) affects silk output. However, the molecular mechanisms by which genes regulate silk gland size remain unclear. In this study, silk glands from three pure silkworm strains (A798, A306 and XH) with different silk gland weight phenotypes were compared using transcriptomics and proteomics to identify differentially expressed genes (DEGs) and proteins (DEPs). When comparing A798 to A306 and A798 to XH, 830 and 469 DEGs were up-regulated, respectively. These genes were related to the gene ontology terms, metabolic process, transport activity and biosynthesis process. In addition, 372 and 302 up-regulated differentially expressed proteins were detected in A798 to A306 and A798 to XH, respectively, related to the gene ontology terms, ribosome and protein export, ribosome and polypeptide biosynthesis processes. Moreover, combined transcriptomics, proteomics and weighted correlation network analyses showed that five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711 and BGIBMGA010889) were significantly associated with the silk gland weight. Reverse Transcription-quantitative real-time Polymerase Chain Reaction (RT-qPCR) and Enzyme linked immunosorbent assay (ELISA) were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The results showed that four genes have higher expression levels in heavier silk glands. These genes are associated with glycogen metabolism, fatty acid synthesis and branched chain amino acid metabolism, thus potentially promoting growth and silk protein synthesis. These findings provide valuable insights into the molecular mechanisms underlying the relationship between silk gland weight and silk yield in silkworms.

Rapid production of chimeric silkworm/spider silk with improved mechanical properties by infection of nonpermissive Bombyx mori with recombinant AcMNPV harboring native-size of spidroin genes.

Spider silks with excellent mechanical properties attract more attention from scientists worldwide, and the dragline silk that serves as the framework of the spider's web is considered one of the strongest fibers. However, it is unfeasible for large-scale production of spider silk due to its highly territorial, cannibalistic, predatory, and solitary behavior. Herein, to alleviate some of these problems and explore aneasy way to produce spider fibers, we constructed recombinant baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) simultaneously expressing Trichonephila clavipes native ampullate spidroin 2 (MaSp-G) and spidroin 1 (MaSp-C) driven by the promoters of silkworm fibroin genes, to infect the nonpermissive Bombyx mori larvae at the fifth instar. MaSp-G and MaSp-C were co-expressed in the posterior silk glands (PSGs) of infected silkworms and successfully secreted into the lumen of the silk gland for fibroin globule assembly. The integration of MaSp-G and MaSp-C into silkworm silk fibers significantly improved the mechanical properties of these chimeric silk fibers, especially the strength and extensibility, which may be caused by the increment of ß-sheet in the chimeric silkworm/spider silk fiber. These results demonstrated that silkworms could be developed as the nonpermissive heterologous host for the mass production of chimeric silkworm/spider silk fibers via the recombinant baculovirus AcMNPV.

Physical Properties of the Second Type of Aciniform Spidroin (AcSp2) from Neoscona theisi Reveal a pH-Dependent Self-Assembly Repetitive Domain.

Orb-weaving spiders can use an array of specialized silks with diverse mechanical properties and functions for daily survival. Of all spider silk types, aciniform silk is the toughest silk fiber that combines high strength and elasticity. Although aciniform spidroins (AcSp) are the main protein in aciniform silks, their complete genes have rarely been characterized until now. Moreover, the structural and physical properties of AcSp variant proteins within the species are also unclear. Here, we present three full-length AcSp genes (named AcSp1A, AcSp1B, and AcSp2) from the orb-weaving spider Neoscona theisi and investigate the structural and mechanical features of these three AcSp repetitive domains. We demonstrate that all three AcSp proteins have mainly α-helical structural features in neutral solution and high thermal stability. Significantly, the AcSp2 repetitive domain shows a pH-dependent structural transition from α to ß conformations and can self-assemble into amyloid fibrils under acidic conditions, which is the first reported AcSp repetitive domain with pH-dependent self-assembly capacity. Compared with the other two AcSp spidroins, AcSp2 demonstrated the lowest expression level in the aciniform gland but had the highest strength for its silk fiber. Collectively, our findings provide new insight into the physical properties of each component of aciniform silk and expand the repertoire of known spidroin sequences for the synthesis of artificial silk materials.

Low concentration chlorantraniliprole-promoted Ca2+ release drives a shift from autophagy to apoptosis in the silk gland of Bombyx mori.

The novel pesticide chlorantraniliprole (CAP) is widely used for pest control in agriculture, and the safety for non-target organisms of trace residues in the environment has received widespread attention. In the present study, exposure to low concentrations of CAP resulted in abnormal silk gland development in the B. mori, and induced the release of intracellular Ca2+ in addition to the triggering of Ca2+-dependent gene transcription. Moreover, the CAP treatment group exhibited down-regulation of oxidative phosphorylation and antioxidant enzyme-related genes in the silk gland, resulting in peroxide accumulation. Furthermore, transcript levels of autophagy-related genes were significantly up-regulated and protein levels of LC3-I and LC3-II were up-regulated, indicating an increase in autophagy. The protein levels of ATG5 and NtATG5 were also significantly up-regulated. While the protein levels of caspase3 and active caspase3 were significantly up-regulated consistent with the transcript levels of key genes in the apoptotic signaling pathway, ultimately affecting silk protein synthesis. Overall, these findings indicate that low concentration CAP induced abnormal development in the silk gland of B. mori by causing intracellular Ca2+ overload, which inhibits oxidative phosphorylation pathway and the removal of reactive oxygen species, leading to a driving a shift from autophagy to apoptosis. The findings herein provided a basis for evaluating the safety of CAP environmental residues on non-target organisms.

The promoting effects of pyriproxyfen on autophagy and apoptosis in silk glands of non-target insect silkworm, Bombyx mori.

Pyriproxyfen is a juvenile hormone analogue. The physiological effects of its low-concentration drift during the process of controlling agricultural and forestry pests on non-target organisms in the ecological environment are unpredictable, especially the effects on organs that play a key role in biological function are worthy of attention. The silk gland is an important organ for silk-secreting insects. Herein, we studied the effects of trace pyriproxyfen on autophagy and apoptosis of the silk gland in the lepidopteran model insect, Bombyx mori (silkworm). After treating fifth instar silkworm larvae with pyriproxyfen for 24 h, we found significant shrinkage, vacuolization, and fragmentation in the posterior silk gland (PSG). In addition, the results of autophagy-related genes of ATG8 and TUNEL assay also demonstrated that autophagy and apoptosis in the PSG of the silkworm was induced by pyriproxyfen. RNA-Seq results showed that pyriproxyfen treatment resulted in the activation of juvenile hormone signaling pathway genes and inhibition of 20-hydroxyecdysone (20E) signaling pathway genes. Among the 1808 significantly differentially expressed genes, 796 were upregulated and 1012 were downregulated. Among them, 30 genes were identified for autophagy-related signaling pathways, such as NOD-like receptor signaling pathway and mTOR signaling pathway, and 30 genes were identified for apoptosis-related signaling pathways, such as P53 signaling pathway and TNF signaling pathway. Further qRT-PCR and in vitro gland culture studies showed that the autophagy-related genes Atg5, Atg6, Atg12, Atg16 and the apoptosis-related genes Aif, Dronc, Dredd, and Caspase1 were responsive to the treatment of pyriproxyfen, with transcription levels up-regulated from 24 to 72 h. In addition, ATG5, ATG6, and Dronc genes had a more direct response to pyriproxyfen treatment. These results suggested that pyriproxyfen treatment could disrupt the hormone regulation in silkworms, promoting autophagy and apoptosis in the PSG. This study provides more evidence for the research on the damage of juvenile hormone analogues to non-target organisms or organs in the environment, and provides reference information for the scientific and rational use of juvenile hormone pesticides.

Juvenile hormone regulates silk gene expression by m6A RNA methylation.

Juvenile hormone (JH) is an indispensable insect hormone that is critical in regulating insect development and physiology. N6-methyladenosine (m6A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. However, the relationship between m6A and JH remains largely unknown. Here, we found that the application of a Juvenile hormone analog (JHA) extended the larval period of Bombyx mori and increased the weight and thickness of the cocoon. Interestingly, global transcriptional patterns revealed that m6A-related genes are specifically regulated by JHA in the posterior silk gland (PSG) that synthesizes the major component of cocoon silk. By transcriptome and m6A sequencing data conjointly, we discovered that JHA significantly regulated the m6A modification in the PSG of B. mori and many m6A-containing genes are related to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. Notably, 547 genes were significantly regulated by JHA at both the m6A modification and expression levels, especially 16 silk-associated genes, including sericin2, seroin1, Serine protease inhibitors 4 (BmSPI4), Serine protease inhibitors 5 (BmSPI5), and LIM domain-binding protein 2 (Ldb). Among them, 11 silk associated genes were significantly affected by METTL3 knockdown, validating that these genes are targets of m6A modification. Furthermore, we confirm that JHA directly regulates the expression of BmSPI4 and BmSPI5 through m6A modification of CDS regions. These results demonstrate the essential role of m6A methylation regulated by JH in PSG, and elucidate a novel mechanism by which JH affects silk gland development via m6A methylation. This study uncovers that m6A modification is a critical factor mediating the effect of JH in insects.