The silk gland of the domesticated silkworm Bombyx mori, is a remarkable organ that produces vast amounts of silk with exceptional properties. Little is known about which silk gland cells execute silk protein synthesis and its precise spatiotemporal control. Here, we use single-cell RNA sequencing to build a comprehensive cell atlas of the silkworm silk gland, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotate all 10 cell types and determine their distributions in each region of the silk gland. Additionally, we decode the developmental trajectory and gene expression status of silk gland cells. Finally, we discover marker genes involved in the regulation of silk gland development and silk protein synthesis. Altogether, this work reveals the heterogeneity of silkworm silk gland cells and their gene expression dynamics, affording a deeper understanding of silk-producing organs at the single-cell level.

There is a recent emerging theory that suggests a cross-link between pathogens and cancer. In this context, we examined the association between theMycobacterium tuberculosis(MTB) with its L-forms (MTB-L) and lung cancer. In the present study, we have optimized and applied a highly sensitive assay to detect the presence of MTB and MTB-L in 187 lung cancer samples and 39 samples of other cancer origins. By carefully controlling confounding factors, we have found that 62% of the lung cancer samples are MTB-L positive, while only 5.1% of the other cancer samples are MTB-L positive. Through generalized linear models and random forest models, we have further identified a set of clinical end-points that are strongly associated with MTB-L presence. Our finding provides the basis for future studies to investigate the underlying mechanism linking MTB-L infection to lung cancer development.

The steroid hormone 20-hydroxyecdysone (20E), which is known to regulate insect molting and metamorphosis, is crucial for the normal development of silk glands (SGs) in the silkworm Bombyx mori. However, how the 20E signaling pathway and its core members function in the SG remains largely unclear. Here, we report that the orphan nuclear receptor BmHR3, a 20E-response factor, plays an essential role in regulating SG development and silk protein synthesis. First, we showed that tissue-specific BmHR3 overexpression and knockout result in severe developmental defects in posterior silk glands (PSGs). Second, we revealed that BmHR3 dysfunction in PSGs dramatically represses the transcription of silk fibroin protein-coding genes, thereby inhibiting fibroin protein synthesis. Finally, we confirmed that BmHR3 can regulate fibroin protein-coding gene expression via direct and indirect mechanisms. This study elucidates the vital function of BmHR3 in B. mori SG and provides valuable information for thoroughly understanding the regulatory roles of 20E signaling in specialized insect organs.

Silk production is a classic example of a domestication trait, yet the cell-type-specific driver of its enhancement in the silkworm Bombyx mori remains unresolved. To address this, we integrated extensive bulk RNA-seq data with a single-nucleus RNA-seq atlas of silk glands (SGs) from domestic B. mori and wild B. mandarina for deconvolution analysis. This identified phenotype-associated cell subpopulations (Scissor+ and Scissor− cells) that enrich in B. mori and B. mandarina, respectively. Transcriptomic characterization revealed that B. mori SG cells exhibit a pervasive “pro-synthesis” transcriptional state, with concerted upregulation of silk protein genes and metabolic pathways. Conversely, B. mandarina cells maintained a “protective–adaptive” state, enriched for stress response and xenobiotic metabolism genes. Pseudotime analysis further delineated the cell state transitions, pinpointing key dynamic gene expression linked to high silk yield. Our findings demonstrate that domestication reshaped the silk gland cellular landscape, promoting a systemic shift toward a synthesis-optimized cell state. This study offers a new framework at the cellular level to elucidate the evolution of complex traits under selection.

The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely used to analyze gene function in many organisms, including the silkworm ( Bombyx mori ), a model lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein affects normal endogenous gene expression have rarely been studied. Here, we analyzed the transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and control groups, respectively. These DEGs were further annotated by gene function classification and pathway assessment using public databases. In addition, 506 DEGs were shared (common) between both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly downregulated; many of them were annotated to be involved in metabolic processes such as “fat digestion and absorption”, “glycine, serine and threonine metabolism” and “glutathione metabolism” and in signal transduction pathways such as the “Rap1 signaling pathway”, “MAPK signaling pathway” and “Hippo signaling pathway”. Overall, this work enhances understanding of the effects of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study gene function.

The silk glands (SGs) of silkworms specifically synthesize silk proteins, thus strongly influencing the yield and quality of silk. Here, we present a protocol for isolating SG nuclei from silkworms and obtaining high-quality tissue slices for spatial transcriptomics. We describe steps for rearing, dissecting, and nucleus isolation. We then detail procedures for embedding, frozen section, and RNA capturing and sequencing. This protocol enables the exploration of the spatial distribution of SG cells at single-cell resolution. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.