BmHSP19.9 targeting P6.9 and VLF-1 to mediate the formation of defective progeny viruses in the silkworm antiviral variety 871C.

The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains.

Establishment of a Secretory Protein-Inducible CRISPR/Cas9 System for Nosema bombycis in Insect Cells.

Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.

MicroRNA bmo-miR-31-5p inhibits apoptosis and promotes BmNPV proliferation by targeting the CYP9e2 gene of Bombyx mori.

BACKGROUND: Bombyx mori nuclear polyhedrosis virus (BmNPV), as a typical baculovirus, is the primary pathogen that infects the silkworm B. mori, a lepidopteran species. Owing to the high biological safety of BmNPV in infecting insects, it is commonly utilized as a biological insecticide for pest control. Apoptosis is important in the interaction between the host and pathogenic microorganisms. MicroRNAs (miRNAs) influence immune responses and promote stability of the immune system via apoptosis. Therefore, the study of apoptosis-related miRNA in silkworms during virus infection can not only provide support for standardizing the prevention and control of diseases and insect pests, but also reduce the economic losses to sericulture caused by the misuse of biological pesticides. RESULTS: Through transcriptome sequencing, we identified a miRNA, miR-31-5p, and demonstrated that it can inhibit apoptosis in silkworm cells and promote the proliferation of BmNPV in BmE-SWU1 cells. We identified a target gene of miR-31-5p, B. mori cytochrome P450 9e2 (BmCYP9e2), and demonstrated that it can promote apoptosis in silkworm cells and inhibit the proliferation of BmNPV. Moreover, we constructed transgenic silkworm strains with miR-31-5p knockout and confirmed that they can inhibit the proliferation of BmNPV. CONCLUSION: These data indicate that miR-31-5p may exert functions of inhibiting apoptosis and promoting virus proliferation by regulating BmCYP9e2. The findings demonstrate how miRNAs influence host cell apoptosis and how they are involved in the host immune system response to viruses, providing important insights into the applications of biological insecticides for pest control. © 2024 Society of Chemical Industry.

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.

BmATAD3A mediates mitochondrial ribosomal protein expression to maintain the mitochondrial energy metabolism of the silkworm, Bombyx mori.

ATAD3A is a mitochondrial membrane protein belonging to the ATPase family that contains the AAA+ domain. It is widely involved in mitochondrial metabolism, protein transport, cell growth, development and other important life processes. It has previously been reported that the deletion of ATAD3A causes growth and development defects in humans, mice and Caenorhabditis elegans. To delve into the mechanism underlying ATAD3A defects and their impact on development, we constructed a Bombyx mori ATAD3A (BmATAD3A) defect model in silkworm larvae. We aim to offer a reference for understanding ATAD3A genetic defects and elucidating the molecular regulatory mechanisms. The results showed that knockout of the BmATAD3A gene significantly affected the weight, survival rate, ATPase production and mitochondrial metabolism of individuals after 24 h of incubation. Combined metabolomics and transcriptomics analysis further demonstrated that BmATAD3A knockout inhibits amino acid biosynthesis through the regulation of mitochondrial ribosomal protein expression. Simultaneously, our findings indicate that BmATAD3A knockout impeded mitochondrial activity and ATPase synthesis and suppressed the mitochondrial oxidative phosphorylation pathway through B. mori mitochondrial ribosomal protein L11 (BmmRpL11). These results provide novel insights into the molecular mechanisms involved in the inhibition of development caused by ATAD3A deficiency, offering a potential direction for targeted therapy in diseases associated with abnormal ATAD3A expression.

The Bombyx mori singed Gene Is Involved in the High-Temperature Resistance of Silkworms.

Temperature is an important factor in the growth, development, survival, and reproduction of organisms. The high-temperature resistance mechanism of insects may be significant for use in the prevention and control of insect pests. The silkworm, Bombyx mori, is an important Lepidoptera model species for studies on pest control in agriculture and forestry. We identified a gene in B. mori, the B. mori singed (Bmsn) gene, which is involved in the high-temperature resistance of silkworms. Sn proteins are highly conserved among species in many taxonomic groups. The overexpression of the Bmsn gene promoted the proliferation of silkworm cells, reduced oxidation, and reduced the accumulation of reactive oxygen species under stress. Interfering with the Bmsn gene had the opposite result. We constructed a transgenic B. mori strain that overexpressed the Bmsn gene. The physiological traits of the transgenic strain were significantly improved, and it had stronger high-temperature resistance. The Bmsn gene is involved in the process by which fat bodies respond to high-temperature stress. These findings provide insights into the mechanism of high-temperature resistance of insects and offer a new perspective on agricultural and forestry pest control.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Matrix metalloproteinase 2 degrades collagen I to regulate ovarian development by association with an insulin-like peptide.

The ovary generally undergoes tissue remodeling during larval to pupal transition, which includes membrane degeneration and ovariole growth. At the same time, the hormones produced by insects significantly change during metamorphosis. However, the regulatory mechanism for ovarian development and hormones is not fully understood in insects. Herein, we found that matrix metalloproteinase 2 (MMP2) was highly expressed in the ovarian capsules and ovarioles, and the development was abnormal after knocking out MMP2 in Bombyx mori. The process of abnormal degradation of collagen I due to MMP2 deletion, which resulted in abnormal development of ovarioles and eggs, was analyzed in detail. The proteomics of ovaries in the MMP2-knock out and wild type strains showed a critically significant difference in the expression of a protein, insulin-like peptide (ILP). Additional analysis revealed significant alteration of ILP during ovarian development, and abnormal expression of ILP significantly affected ovarian development in vivo and MMP2 expression in vitro and in vivo. These results showed that MMP2 regulation of ovarian tissue remodeling is closely related to ILP expression. Our study provides new insights into the regulatory mechanism of MMP2 and ovarian development in B. mori.

A novel transcription factor, BmZFP67, regulates endomitosis switch by controlling the expression of cyclin B in silk glands.

Endomitosis is involved in developmental processes associated with an increase in metabolic cell activity, which is characterized by repeated rounds of DNA replication without cytokinesis. Endomitosis cells are widespread in protozoa, plants, animals and humans. Endomitosis cell cycle is currently viewed as a variation of the canonical cell cycle and transformed from mitotic cell cycle. However, the meaningful question about how endomitosis transformed from mitosis is still unclear. Herein, we identified a novel transcription factor in silk glands, ZFP67, which is gradually reduced in silk glands during the transition of mitosis to endomitosis. In addition, over-expressed ZFP67 in silk glands led to the transition delayed. And, knock-out of ZFP67 led to abnormal chromatin division and unsuccessful cell division. These data reveled that ZFP67 played an important role in transition of mitosis to endomitosis. Furthermore, ZFP67 can regulate the transcription of cyclin B, a key cyclin related to cell division and G2/M phase, which is demonstrated by chromatin immunoprecipitation and dual luciferase reporter system in this article. In conclusion, it can be speculated that the decreasing expression of ZFP67 in silk glands during the transition stage of mitosis-to-endomitosis resulted in the lack of cyclin B, which further led to unsuccessful cytokinesis and then promoted the transition from mitosis to endomitosis of silk gland cells.

Common strategies in silkworm disease resistance breeding research.

The silkworm, which is considered a model invertebrate organism, was the first insect used for silk production in human history and has been utilized extensively throughout its domestication. However, sericulture has been plagued by various pathogens that have  caused significant economic losses. To enhance the resistance of a host to its pathogens,numerous strategies have been developed. For instance, gene-editing techniques have been applied to a wide range of organisms, effectively solving a variety of experimental problems. This review focuses on several common silkworm pests and their pathogenic mechanisms, with a particular emphasis on breeding for disease resistance to control multiple types of silkworm diseases. The review also compares the advantages and disadvantages of transgenic technology and gene-editing systems. Finally, the paper provides a brief summary of current strategies used in breeding silkworm disease resistance, along with a discussion of the establishment of existing technologies and their future application prospects. © 2023 Society of Chemical Industry.

Apoptosis-related long non-coding RNA LINC5438 of Bombyx mori promotes the proliferation of BmNPV.

Apoptosis, as an important part of the immune response, is one of the core events in the host-virus interaction. Studies have shown that long non-coding RNAs (lncRNAs) play important roles in the process of cell apoptosis and pathophysiology. To investigate the apoptosis-related lncRNAs involved in Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworms, transcriptome sequencing was conducted based on silkworm cells infected with BmNPV before and after B. mori inhibitor of apoptosis (Bmiap) gene knockout. A total of 23 differentially expressed lncRNAs were identified as being associated with the mitochondrial apoptosis pathway. Moreover, we demonstrated that B. mori LINC5438 has the function of inhibiting apoptosis in silkworm cells. Overexpression of LINC5438 promoted the proliferation of BmNPV, while interference with LINC5438 inhibited its proliferation, indicating that LINC5438 plays an important role in BmNPV infection. Our results also showed that LINC5438 can regulate the expression of Bmiap, BmDronc, BmICE, and its predicted target gene BmAIF, suggesting that LINC5438 may function through the mitochondrial pathway. These findings provide important insights into the mechanisms of virus-host interaction and the applications of baculoviruses as biological insecticides.

Transcriptome analysis reveals that knocking out BmNPV iap2 induces apoptosis by inhibiting the oxidative phosphorylation pathway.

Apoptosis is essential for the normal growth, development, and immunity defense of living organisms, and its function and mechanisms have been intensively studied. When viral infection occurs, apoptosis is triggered, causing programmed death of the infected cells. Meanwhile, viruses have also evolved countermeasures to inhibit apoptosis in host cells. We previously constructed a transgenic silkworm line with significantly improved resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) by knocking out the BmNPV inhibitor of apoptosis 2 (iap2) gene. However, the mechanism of how IAP2 induces apoptosis still needs to be further investigated. Here, the transcriptomes of Cas9(-)/sgiap2 (-) and Cas9(+)/sgiap2(+) strains were analyzed at 48 h after BmNPV infection, and a total of 709 differential genes were obtained. A KEGG analysis revealed that the differentially expressed genes were enriched in the oxidative phosphorylation, proteasome, and ribosome pathways. In the oxidative phosphorylation pathway, 41 differentially expressed genes were downregulated, and 12 of these genes were verified by qRT-PCR. More importantly, the knockout of BmNPV iap2 led to the inhibition of the oxidative phosphorylation pathway, followed by activated oxidative stress triggered apoptosis, thereby inhibiting the replication of BmNPV in vitro and vivo. The results provide a basis for the analysis of the initiation of apoptosis that can inhibit virus proliferation, and the study presents new ideas for the subsequent creation of resistant material.

Baculovirus LEF-11 interacts with BmIMPI to induce cell cycle arrest in the G2/M phase for viral replication.

Viruses arrest the host cell cycle and using multiple functions of host cells is an important approach for their replication. Baculovirus arrests infected insect cells at both the late S and G2/M phase, but the strategy employed by baculovirus is not clearly understood. Our research suggests that the Bombyx mori nucleopolyhedrovirus (BmNPV) could arrest the cell cycle in the G2/M phase to promote virus replication, and also that the viral protein LEF-11 could inhibit host cell proliferation and arrest the cell cycle by inhibiting the cell cycle checkpoint proteins BmCyclinB and BmCDK1. Furthermore, we found that LEF-11 interacts with BmIMPI to regulate cell proliferation, but not by direct interaction with BmCyclinB or BmCDK1. In addition, our findings showed that BmIMPI was important and necessary for LEF-11 induced cell cycle arrest in the G2/M phase. Moreover, BmIMPI was found to interact with BmCyclinB and BmCDK1, and down-regulate the expression of BmCyclinB and BmCDK1 to compromise the cell cycle and cell proliferation. Taken together, the data presented demonstrated that baculovirus LEF-11 regulates BmIMPI to inhibit host cell proliferation and provide a new insight into the molecular mechanisms employed by viruses to induce cell cycle arrest.

Identification of the Key Functional Domains of Bombyx mori Nucleopolyhedrovirus IE1 Protein.

The immediate early protein 1 (IE1) acts as a transcriptional activator and is essential for viral gene transcription and viral DNA replication. However, the key regulatory domains of IE1 remain poorly understood. Here, we analyzed the sequence characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 and identified the key functional domains of BmNPV IE1 by stepwise truncation. Our results showed that BmNPV IE1 was highly similar to Autographa californica nucleopolyhedrovirus (AcMNPV) IE1, but was less conserved with IE1 of other baculoviruses, the C-terminus of IE1 was more conserved than the N-terminus, and BmNPV IE1 was also necessary for BmNPV proliferation. Moreover, we found that IE1158-208 was a major nuclear localization element, and IE11-157 and IE1539-559 were minor nuclear localization elements, but the combination of these two minor elements was equally sufficient to fully mediate the nuclear entry of IE1. Meanwhile, IE11-258, IE1560-584, and the association of amino acids 258 and 259 were indispensable for the transactivation activity of BmNPV IE1. These results systematically resolve the functional domains of BmNPV IE1, which contribute to the understanding of the mechanism of baculovirus infection and provide a possibility to synthesize a small molecule IE1-truncated mutant as an agonist or antagonist.

BmCDK5 Affects Cell Proliferation and Cytoskeleton Morphology by Interacting with BmCNN in Bombyx mori.

The ordered cell cycle is important to the proliferation and differentiation of living organisms. Cyclin-dependent kinases (CDKs) perform regulatory functions in different phases of the cell cycle process to ensure order. We identified a homologous gene of the Cyclin-dependent kinase family, BmCDK5, in Bombyx mori. BmCDK5 contains the STKc_CDK5 domain. The BmCDK5 gene was highly expressed in S phase. Overexpression of the BmCDK5 gene accelerates the process of the cell cycle's mitotic period (M) and promotes cell proliferation; knocking out the BmCDK5 gene inhibited cell proliferation. Furthermore, we identified a protein, BmCNN, which can interact with BmCDK5 and represents the same express patterns as the BmCDK5 gene in the cell cycle phase and the spatial-temporal expression of B. mori. This study revealed that BmCDK5 and BmCNN play roles in promoting cell proliferation and regulating cytoskeleton morphology, but do not induce expression changes in microtubule protein. Therefore, our findings provide a new insight; the BmCDK5 gene has a regulatory effect on the cell cycle and proliferation of B. mori, which is presumably due to the interaction between BmCDK5 and BmCNN regulating changes in the cytoskeleton.

Analysis of Silver Nanoparticles for the Treatment and Prevention of Nucleopolyhedrovirus Affecting Bombyx mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) causes major economic losses in sericulture. A number of agents have been employed to treat viral diseases. Silver nanoparticles (AgNPs) have wide applications in biomedical fields due to their unique properties. The anti-BmNPV effect of AgNPs has been evaluated, however, there are insufficient studies concerning its toxicity to other organisms and the environment. We chemically synthesized biocompatible BSA-AgNPs with a diameter range of 2-4 nm and characterized their physical properties. The toxicity of AgNPs towards cells and larvae with different concentrations was examined; the results indicated a biofriendly effect on cells and larvae within specific concentration ranges. The SEM observation of the surface of BmNPV after treatment with AgNPs suggested that AgNPs could destroy the polyhedral structure, and the same result was obtained by Coomassie blue staining. Further assays confirmed the weakened virulence of AgNPs-treated BmNPV toward cells and larvae. AgNPs also could effectively inhibit the replication of BmNPV in infected cells and larvae. In summary, our research provides valuable data for the further development of AgNPs as an antiviral drug for sericulture.

Stable transformation of fluorescent proteins into Nosema bombycis by electroporation.

BACKGROUND: Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. METHODS: In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. RESULTS: We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. CONCLUSIONS: We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

The dual roles of three MMPs and TIMP in innate immunity and metamorphosis in the silkworm, Bombyx mori.

The matrix metalloproteinases (MMPs) and their endogenous inhibitory factors, tissue inhibitors of metalloproteinases (TIMPs), are implicated in many diseases. However, the mammalian MMPs (> 20) and TIMPs (> 3) are larger in number, and so little is known about their individual roles in organisms. Hence, we have systematically studied the roles of all three MMPs and one TIMP in silkworm innate immunity and metamorphosis. We observed that MMPs and TIMP are highly expressed during the pupation stage of the silkworms, and TIMP could interact with each MMPs. High-activity MMPs and low-activity TIMP may enhance the infection of B. mori nucleopolyhedrovirus in both in vitro and in vivo. MMPs' knockout and TIMP overexpression delayed silkworm development and even caused death. Interestingly, different MMPs' knockout led to different tubular tissue dysplasia. These findings provide insights into the conserved functions of MMPs and TIMP in human organogenesis and immunoregulation.