Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

The BTB-ZF gene Bm-mamo regulates pigmentation in silkworm caterpillars.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

Study on anti-BmNPV mechanism of branched-chain amino acid aminotransferases in silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

Lipid homeostasis is essential for oogenesis and embryogenesis in the silkworm, Bombyx mori.

Reproduction, a fundamental feature of all known life, closely correlates with energy homeostasis. The control of synthesizing and mobilizing lipids are dynamic and well-organized processes to distribute lipid resources across tissues or generations. However, how lipid homeostasis is precisely coordinated during insect reproductive development is poorly understood. Here we describe the relations between energy metabolism and reproduction in the silkworm, Bombyx mori, a lepidopteran model insect, by using CRISPR/Cas9-mediated mutation analysis and comprehensively functional investigation on two major lipid lipases of Brummer (BmBmm) and hormone-sensitive lipase (BmHsl), and the sterol regulatory element binding protein (BmSrebp). BmBmm is a crucial regulator of lipolysis to maintain female fecundity by regulating the triglyceride (TG) storage among the midgut, the fat body, and the ovary. Lipidomics analysis reveals that defective lipolysis of females influences the composition of TG and other membrane lipids in the BmBmm mutant embryos. In contrast, BmHsl mediates embryonic development by controlling sterol metabolism rather than TG metabolism. Transcriptome analysis unveils that BmBmm deficiency significantly improves the expression of lipid synthesis-related genes including BmSrebp in the fat body. Subsequently, we identify BmSrebp as a key regulator of lipid accumulation in oocytes, which promotes oogenesis and cooperates with BmBmm to support the metabolic requirements of oocyte production. In summary, lipid homeostasis plays a vital role in supporting female reproductive success in silkworms.

Harvesting Silk Fibers from Plodia interpunctella: Role of Environmental Rearing Conditions in Fiber Production and Properties.

Silk fibers are produced by a wide variety of insects. The silkworm Bombyx mori (Bombyx) was domesticated because the physical properties of its silk fibers were amenable to the production of fine textiles. Subsequently, engineers have regenerated silk fibroin to form biomaterials. The monocular focus on Bombyx silk has underutilized the expanse of diverse silk proteins produced by more than 100,000 other arthropods. This vast array of silk fibers could be utilized for biomedical engineering challenges if sufficient rearing and purification processes are developed. Herein, we show that the moth, Plodia interpunctella (Plodia), represents an alternative silk source that is easily reared in highly regulated culture environments allowing for greater consistency in the silk produced. We controlled the temperature, resource availability (larvae/gram diet), and population density (larvae/mL) with the goal of increasing silk fiber production and improving homogeneity in Plodia silk proteins. We determined that higher temperatures accelerated insect growth and reduced life cycle length. Furthermore, we established initial protocols for the production of Plodia silk with optimal silk production occurring at 24 °C, with a resource availability of 10 larvae/gram and a population density of 0.72 larvae/mL. Population density was shown to be the most prominent driving force of Plodia silk mat formation among the three parameters assessed. Future work will need to link gene expression, protein production and purification, and resulting mechanical properties as a function of environmental cues to further transition Plodia silk into regenerated silk fibroin biomaterials.

Meiosis: The silk moth and the elephant.

In most eukaryotes, balanced chromosome segregation at meiosis requires crossovers, but female Bombyx mori lack these structures. Instead, the synaptonemal complex is repurposed to compensate for this absence of crossovers, a remarkable example of exaptation.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

Bmo-miR-6498-5p suppresses Bombyx mori nucleopolyhedrovirus infection by down-regulating BmPLPP2 to modulate pyridoxal phosphate content in B. mori.

The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR-6498-5p was down-regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR-6498-5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR-6498-5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4-deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR-6498-5p has a potential anti-BmNPV role by down-regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR-6498-5p down-regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori-BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture.

Bmpali, Bmb1 and Bmcap are necessary for uric acid granule formation in Bombyx mori.

Uric acid is the end-product of nitrogen metabolism of the silkworm and other lepidopterans. The accumulation of uric acid particles in the epidermis causes the larval silkworm to appear white and opaque. However, the mechanism of uric acid granule formation is still unclear. Silkworm epidermis color is linked to the genes responsible for uric acid particle formation. We first identified two genes in the Bombyx mori genome that encode subunits of the Bloc-1 (Biogenesis of Lysosome-related Organelles Complex-1) by homology to these genes in other eukaryotes, Bmpali and Bmb1. Mutation in these genes caused a transparent phenotype in the silkworm larvae, and the loss of BmBloc-1 subunit gene Bmcap resulted in the same phenotype. These three genes are highly conserved between human and silkworm. We discovered that Bmpali, Bmcap, and Bmb1 localize in the cytoplasm of BmN cells. Yeast two-hybrid assays demonstrated that the Bmpali physically interacts with both Bmcap and Bmb1. Investigating the roles of Bmpali, Bmb1, and Bmcap is essential for uric acid granule formation understanding in Bombyx mori. These mutants present a valuable silkworm model for studying the biogenesis of lysosome-related organelles (LROs).

Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.

A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

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.

High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.

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.

Structural Insights into the Substrate Binding of Farnesyl Diphosphate Synthase FPPS1 from Silkworm, Bombyx mori.

Farnesyl diphosphate synthase (FPPS) is an important enzyme involved in the juvenile hormone (JH) biosynthesis pathway. Herein, we report the crystal structure of a type-I Lepidopteran FPPS from Bombyx mori (BmFPPS1) at 2.80 Å resolution. BmFPPS1 adopts an α-helix structure with a deep cavity at the center of the overall structure. Computational simulations combined with biochemical analysis allowed us to define the binding mode of BmFPPS1 to its substrates. Structural comparison revealed that BmFPPS1 adopts a structural pattern similar to that of type-II FPPS but exhibits a distinct substrate-binding site. These findings provide a structural basis for understanding substrate preferences and designing FPPS inhibitors. Furthermore, the expression profiles and RNA interference of BmFPPSs indicated that they play critical roles in the JH biosynthesis and larval-pupal metamorphosis. These findings enhance our understanding of the structural features of type-I Lepidopteran FPPS while providing direct evidence for the physiological role of BmFPPSs in silkworm development.