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Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(-)/sgorf76(-), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(-)/sgorf76(-). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 Society of Chemical Industry.)

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., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest to report regarding the present study., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest exists., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest exists., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 IE1 158-208 was a major nuclear localization element, and IE1 1-157 and IE1 539-559 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, IE1 1-258 , IE1 560-584 , 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.

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions. Although E2Fs have been widely studied in mammals, there have been few studies performed in insects. Here, we determined the function of E2F4 in the silkworm, Bombyx mori. We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals. Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase, whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase. These results indicate that BmE2F4 is important in cell cycle regulation. We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland. Furthermore, we identified a protein called Bm14-3-3ζ that can interact with BmE2F4 and allow it to localize in the nucleus. Overexpression of the Bm14-3-3ζ gene led to cell cycle arrest in the G1 phase, while knocking down the gene increased the proportion of cells in S phase. These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation., (© 2021 Institute of Zoology, Chinese Academy of Sciences.)

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., (© 2021 Federation of European Biochemical Societies.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cell division cycle protein 37 (Cdc37) is a molecular chaperone that actively participates in many intracellular physiological and biochemical processes as well as pathogen infection. However, the function of Cdc37 in silkworm cells under Bombyx mori nucleopolyhedrovirus (BmNPV) infection is unknown. We cloned and identified BmCdc37, a Cdc37 gene from B. mori, which is highly conserved among other species. After BmNPV infection, the expression level of the BmCdc37 gene was up-regulated and showed an expression pattern similar to the BmHsp90 gene, which relies on Cdc37 to stabilize and activate specific protein kinases. The immunofluorescence, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays all indicated that BmCdc37 interacts with BmHsp90 in silkworm cells. Both BmCdc37 and BmHsp90 promote the reproduction of BmNPV. Co-expression of BmCdc37 and BmHsp90 was better at promoting virus proliferation than overexpression alone. These findings all indicate that BmCdc37 plays an active role in the proliferation of BmNPV., (Copyright © 2021. Published by Elsevier Inc.)

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori . In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin β1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2'-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin β1.

Energy metabolism is important for the proliferation of microsporidia in infected host cells, but there is limited information on the host response. The energy metabolism response of silkworm (Bombyx mori) to microsporidia may help manage Nosema bombycis infections. We analyzed differentially expressed genes in the B.mori midgut transcriptome at two significant time points of microsporidia infection. A total of 1448 genes were up-regulated, while 315 genes were down-regulated. A high proportion of genes were involved in the phosphatidylinositol signaling system, protein processing in the endoplasmic reticulum, and glycerolipid metabolism at 48 h post infection (h p.i.), and a large number of genes were involved in the TCA cycle and protein processing at 120 h p.i. These results showed that the early stages of microsporidia infection affected the basic metabolism and biosynthesis processes of the silkworm. Knockout of Bm_nscaf2860_46 (Bombyx mori isocitrate dehydrogenase, BmIDH) and Bm_nscaf3027_062 (Bombyx mori hexokinase, BmHXK) reduced the production of ATP and inhibited microsporidia proliferation. Host fatty acid degradation, glycerol metabolism, glycolysis pathway, and TCA cycle response to microsporidia infection were also analyzed, and their importance to microsporidia proliferation was verified. These results increase our understanding of the molecular mechanisms involved in N. bombycis infection and provide new insights for research on microsporidia control. IMPORTANCE: Nosema bombycis can be vertically transmitted in silkworm eggs. The traditional prevention and control strategies for microsporidia are difficult and time-consuming, and this is a problem in silkworm culture. Research has mainly focused on host gene functions related to microsporidia infection and host immune responses after microsporidia infection. Little is known about the metabolic changes occurring in the host after infection. Understanding the metabolic changes in the silkworm host could aid in the recognition of host genes important for microsporidia infection and growth. We analyzed host metabolic changes and the main participating pathways at two time points after microsporidia infection and screened the microsporidia-dependent host energy metabolism genes BmIDH and BmHXK. The results revealed genes that are important for the proliferation of Nosema bombycis. These results illustrate how microsporidia hijack the host genome for their growth and reproduction., (Copyright © 2021. Published by Elsevier Inc.)

Apoptosis is a cellular defense mechanism used for the elimination of host cells infected by viruses. Viruses have evolved corresponding inhibitors of apoptosis genes to promote their replication. Anti-apoptosis-related genes, involved in baculovirus proliferation, have been proposed but it is unclear whether these genes can be manipulated in gene therapy. We constructed a transgenic silkworm, using the CRISPR/Cas9 system to knock out the BmNPV inhibitor of apoptosis 2 (iap2). The sequencing results showed that all the sequences could edit the target site of BmNPV iap2 gene. There were no differences in economic traits and growth tests between the BmNPV iap2 knockout strain transgenic silkworm lines and the control groups. However, the mortality rate was significantly reduced, the median lethal dose (LD50) was about 100 times higher than the control group, and the onset time was prolonged by 1-2 days after knocking out BmNPV iap2. In addition, the expression levels of apoptotic-related genes Bmiap2, BmICE and BmDreed were significantly affected and the activity of caspase 9 was increased after BmNPV iap2 being edited in transgenic silkworm. These results demonstrated that gene editing BmNPV iap2 could significantly inhibit BmNPV replication and proliferation. This approach provides a new strategy for antiviral research., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Rapid and convenient detection of protein-protein interactions (PPIs) is of great significance for understanding function of protein., Results: For efficiently detecting PPIs, we used the changes of proteins fluorescence localization to design a novel system, fluorescence translocation co-localization (FTCL), based on nuclear localization signal (NLS) in living cells. Depending on the original state of protein localization (both in the cytoplasm, both in the nucleus, one in the nucleus and another in the cytoplasm), two target proteins can be partitioned into the cytoplasm and nucleus by adding a NLS or mutating an existing NLS. Three independent results display that the changes of protein fluorescence co-localization were observed following co-expression of the two target proteins. At the same time, we verified the accuracy of fluorescence co-localization by co-immunoprecipitation., Conclusions: There FTCL system provided a novel detection method for PPIs, regardless of protein localization in the nucleus or cytoplasm. More importantly, this study provides a new strategy for future protein interaction studies through organelle localization (such as mitochondria, Golgi and cytomembrane, etc.).

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down-regulation of BmPP2A expression. Furthermore, up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down-regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti-BmNPV mechanism, and a possible target for the breeding of silkworm-resistant strains., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents re-replication in vertebrates and invertebrates. Previously, we identified two Geminin genes, BmGeminin1 and BmGeminn2 , in the silkworm Bombyx mori , and we found that RNA interference of BmGeminin1 led to re-replication. However, the function of BmGeminin2 remains poorly understood. In this study, we found that knockdown of BmGeminin2 can improve cell proliferation, and upregulated G2/M-associated gene- cyclinB / CDK1 expression. Then, we performed yeast two-hybrid screening to identify interacting proteins. Our results yielded 23 interacting proteins, which are involved in DNA replication, chromosome stabilization, embryonic development, energy, defense, protein processing, or structural protein. Here, we focused on BmRRS1, a chromosome congression-related protein that is closely related to cell cycle G2/M progression. The interaction between BmGeminin2 and BmRRS1 was confirmed by immunofluorescence and immunoprecipitation. Analysis of its expression profile showed that BmRRS1 was related to BmGeminin2 . In addition, BmGeminin2 overexpression downregulated the BmRRS1 transcript. Knockdown of BmGeminin2 led to upregulation of the BmRRS1 transcript. Furthermore, overexpression of BmRRS1 can upregulate G2/M-associated gene- cyclinB / CDK1 expression, and improved cell proliferation, consistent with the effects of BmGeminin2 knockout. In addition, BmRRS1 RNA interference can eliminate the impact of BmGem2 knockout on cell proliferation, the ratio of cell cycle stage and the expression of cyclinB / CDK1 . These data suggested that the cell proliferation advantage of BmGeminin2 knockout was closely related to BmRRS1 . Our findings provide insight into the functions of Geminin and the mechanisms underlying the regulation of the cell cycle in the silkworm.

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system is a powerful genetic engineering technique that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges, including the lack of effective host target genes and the off-target effects, limit the application of CRISPR/Cas9 in insects. To mitigate these difficulties, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes the baculovirus-inducible promoter 39K, which directs transcription of the gene encoding, the Cas9 protein, and the U6 promoter which targets the sgATAD3A site of the ATPase family AAA domain-containing protein 3 (ATAD3A) gene. The double-positive transgenic line sgATAD3A×39K-Cas9 (ATAD3A-KO) was obtained by hybridization; antiviral activity in this hybrid transgenic line is induced only after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The BmNPV-inducible system significantly reduced off-target effects and did not affect the economically important characteristics of the transgenic silkworms. Most importantly, this novel system efficiently and consistently edited target genes, inhibiting BmNPV replication after the transgenic silkworms were inoculated with occlusion bodies (OBs). The suppression of BmNPV by the virus-inducible system was comparable to that of the stably expressed CRISPR/Cas9 system. Therefore, we successfully established a highly efficient BmNPV-inducible ATAD3A-KO transgenic silkworm line, with improved gene targeting specificity and antiviral efficiency. Our study thereby provides insights into the treatment of infectious diseases and into the control of insect pests.

Lysozymes is a ubiquitous immune effector that is widely distributed in both vertebrates and invertebrates. Previous reports have shown that lysozymes significantly inhibit viral infections in vertebrates. However, the antiviral effects of lysozymes in invertebrates remain unclear. Here, we investigated the role of lysozymes in Bombyx mori (B. mori) response to viral infection by overexpressing B. mori C-lysozyme (BmC-LZM) in larvae and cells. We found that BmC-LZM was up-regulated in cells in response to viral infection. Indeed, the overexpressing of BmC-LZM significantly inhibited viral replication in cells during late-stage infection. However, this effect was reversed by BmC-LZM mRNA. BmC-LZM was successfully overexpressed in B. mori strain 871 using Baculovirus Expression Vector System (BEVS). This overexpression markedly reduced viral proliferation and increased larval survival percentage. Thus, BmC-LZM inhibited viral replication both in vivo and in vitro, indicating that BmC-LZM is involved in the insect immune response to viral infection. Our results provide a basis for further applications of lysozymes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bombyx mori nuclear polyhedrosis virus (BmNPV) is an important pathogen of silkworms. Despite extensive studies in recent decades, the interaction between BmNPV and host cells is still not clearly understood. Autophagy is an intrinsic innate immune mechanism and it controls infection autonomously in virus-infected cells. In this study, we found that BmNPV infection could trigger autophagy, as demonstrated by the formation of autophagosomes, fluorescent Autophagy-related gene 8-Green Fluorescent Protein (ATG8-GFP) punctate, and lipidated ATG8. Meanwhile, autophagic flux increased significantly when monitored by the ATG8-GFP-Red Fluorescent Protein (RFP) autophagy tandem sensor and protein degradation of p62. In addition, almost all of the identified autophagy-related genes ( Atgs ) had been up-regulated post infection in mRNA levels. Then, we screened Atgs with the greatest fold-change during virus infection. Interestingly, all of the screened Atgs positively regulated the expression of virus genes. Further studies showed that Atg7 and Atg9 could contribute to the level of autophagy caused by viral infection. Our results demonstrated that BmNPV induced host cell autophagy to benefit its infection. These results offer insight into the complex interactions between virus and host cell, and viral pathogenesis., Competing Interests: The authors declare no conflict of interest.

We previously identified a nuclear hormone receptor gene, BmNHR96, which promotes Bombyx mori nucleopolyhedrovirus (BmNPV) entry into silkworm cells. In an attempt to create an antiviral silkworm strain for better silk production, we used RNAi to downregulate BmNHR96 in silkworm larvae. We screened the resulting BmNHR96-RNAi silkworm strain (NHR) and also explored the antiviral mechanism in vivo. We found that the survival rate of the NHR strain was higher than that of the Dazao strain, when silkworm larvae were infected with BmNPV via oral ODV infection and BV injection. More importantly, the economic characteristics (silk yield) of the transgenic line remained unchanged. These findings reveal that RNAi of BmNHR96 could be an effective way to enhance the tolerance of B. mori to BmNPV infection., (Copyright © 2017. Published by Elsevier Inc.)

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.

DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm.

Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.

Our previous study has identified a gene, BmREEPa, which affects BmNPV invasion in silkworm cells. In this study, we interfered with BmREEPa in silkworm larvae through transgenic technology and screened BmREEPa-RNAi silkworm strains (RP). We found the mortality in RP was lower than that in Dazao, when silkworm larvae were infected with BmNPV via oral and injection routes. And the expression level of VP39 was lower in RP than in Dazao in the group infected via injection. In the oral infection group, VP39 expression level showed significant reduction at 48 h post-infection. These results revealed that the anti-BmNPV activity was enhanced in RP, and this enhancement probably presents itself during secondary infection via BVs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

B.mori nucleopolyhedrovirus (BmNPV), which produces BV and ODV two virion phenotypes in its life cycle, caused the amount of economic loss in sericulture. But the mechanism of its infection was still unclear. In this study we characterized B.mori nuclear hormone receptor 96 (BmNHR96) as a NHR96 family member, which was localized in the nucleus. We also found BmNHR96 over-expression could enhance the entry of BV as well as cellular cholesterol level. Furthermore, we validated that BmNHR96 increased membrane fusion mediated by GP64, which could probably promote BV-infection. In summary, our study suggested that BmNHR96 plays an important role in BV infection and this function probably actualized by affecting cellular cholesterol level, and our results provided insights to the mechanisms of BV-infection of B.mori., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pathogen-inducible promoters have been studied extensively and widely used in resistance breeding and gene therapy. However, few reports have been published that explore the efficacy of Bombyx mori nucleopolyhedrovirus (BmNPV)-inducible promoters in antiviral research in the Bombyx mori (Lepidoptera). Here, we screened BmNPV promoters (VP1054, P33, Bm21, Bm122, 39K, P143, and P6.9) and found that the 39K promoter had the highest BmNPV-induced transcriptional activity by dual-luciferase reporter assays system. By 5' truncation analysis, two regions of 39K promoter were critical for optimal virus-inducible activity, indicated that they could serve as a candidate to produce synthetic pathogen-induced promoters. Furthermore, we enhanced the virus-inducible activity of BmNPV 39K promoter using a hybrid enhancer comprising hr3 and polh-up (designated as HP39K). Finally, we showed that RNAi regulated by HP39K promoter could significantly inhibit the proliferation of BmNPV in silkworm cells. Taken together, our results have practical value in antiviral research of silkworm and baculovirus expression system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self-regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re-activated by re-feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20-hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E-BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

Atlastin is a member of the dynamin protein superfamily and it can mediate homotypic fusion of endoplasmic reticulum (ER) membranes, which is required for many biological processes. In this study, a new Atlastin homologous protein, BmAtlastin-n, was characterized in silkworms and was found to contain an N-terminal conserved GTPase domain and a coiled-coil middle domain. BmAtlastin-n is localized in the cytoplasm and enriched in silkworm midgut. Results also showed that overexpression of BmAtlastin-n in BmN-SWU1 cells could enhance resistance to BmNPV. To better confirm its antiviral effect, microRNA was used to knock down the expression of BmAtlastin-n in BmE-SWU1 cells with inducing the reproduction of BmNPV. A transgenic expression vector of BmAtlastin-n was constructed and introduced to silkworm embryos by microinjection. The transgenic silkworm also showed considerable antiviral capacity. In conclusion, these findings demonstrate that BmAtlastin-n plays an important role in BmNPV defense. More importantly, the current study may provide a new clue for Atlastin research.

We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors.

Class B scavenger receptors (SR-Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR-Bs; it is necessary to study these SR-Bs for revealing their function. In this study, we cloned the full-length coding sequence of BmSCRBQ4, a SR-B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue., (© 2014 Institute of Zoology, Chinese Academy of Sciences.)

Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 μM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 μM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

We previously established and characterized two insect cell lines (BmN-SWU1 and BmN-SWU2) from Bombyx mori ovaries. Here, we examined their differential susceptibilities to Bombyx mori nucleopolyhedrovirus (BmNPV) despite having originated from the same tissue source. BmN-SWU1 cells were susceptible and supported high titers of BmNPV replication, while BmN-SWU2 cells were resistant to BmNPV infection. Subcellular localization analysis demonstrated that very few BmNPV particles could be imported into BmN-SWU2 cells. However, initiation of BmNPV DNA replication but not amplification was detected in BmN-SWU2 cells after transfection with vA4prm-VP39-EGFP bacmid DNA. BmNPV transcription assays showed that late and very late but not early viral genes apparently were blocked in BmNSWU2 cells by unknown mechanisms. Further syncytium formation assays demonstrated that the BmNPV envelope fusion protein GP64 could not mediate BmN-SWU2 host cell-cell membrane fusion. Taken together, these results indicate that these two cell lines represent optimal tools for investigating host-virus interactions and insect antiviral mechanisms.

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What's more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein.

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Pumilio protein is the founding member of the PUF family of RNA-binding proteins, which contains 8 repeat Puf domains and plays important roles during embryogenesis and post-embryogenesis by binding the Nanos response element (NRE) of specific target genes in eukaryotes. In addition, many other proteins containing the Puf domain were identified but with different functions from the Pumilio protein in various species. Taking advantage of the newly assembled genome sequences, in this study we performed a genome-wide analysis of PUF genes in silkworm and other 27 species. In the silkworm, three PUF genes were identified, named Bmpumilio, Bmpenguin and Bmnop by homology analysis. In fungi and animals, four evolutionarily conservational PUF gene families were identified, Group-A, -B, -C and -D. While Group-A, -C, and -D are present in all fungi and animals, Group-B was only identified in fungi. Interestingly, the number and features of the Puf domains are distinct in each group, suggesting different roles for these proteins in every group. The EST and microarray data showed that the mRNA of the three PUF genes can be widely detected in all tissues of the silkworm. Our results provide some new insights into the functions and evolutionary characteristics of PUF proteins.

The flap endonuclease-1 (FEN-1) gene is involved in DNA replication and repair, and it maintains genomic stability as well as the accuracy of DNA replication under normal growth conditions. However, FEN-1 also plays an important role in apoptosis and cancer development. We cloned the BmFEN-1 gene from Bombyx mori, which was 1343 bp in length and possessed an 1143 bp ORF (123-1266). It consists of seven introns and eight exons that encode a protein with 380 amino acids that has the typical XPG domain. The N-terminal motif is located at amino acids 95-105, and the proliferating cell nuclear antigen interaction motif is located at amino acids 337-344. RNA interference-mediated reduction of BmFEN-1 expression induced cell cycle arrest in S phase in BmE-SWU1 cells. These results suggest that BmFEN-1 can inhibit apoptosis and promote cell proliferation.

Background: Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes., Results: From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis., Conclusions: Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori.

A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2n=56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID(50)) at a cell density of 10(5) cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Mapping of silkworm Bmrbp1 gene in silkworm chromosome may lay the basis to investigate function of silkworm Bmrbp1 gene and to establish silkworm physical genetic map. In this study, FISH (fluorescence in situ hybridization) was carried out on meiotic chromosome using one sex regulation gene rbp1 of Bombyx mori as a probe. The experimental results demonstrated that: this gene was a single-copy gene of autosome, located at the site with relative length of 38.806 +/- 1.080% in the 5th chromosome of silkworm meiosis.

Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace's medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n=56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained.

DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines.