Transgenic overexpression of bmo-miR-6498-5p increases resistance to Nosema bombycis in the silkworm, Bombyx mori.

Microsporidia are unfriendly microorganisms, and their infections cause considerable damage to economically or environmentally important insects like silkworms and honeybees. Thus, the identification of measures to improve host resistance to microsporidia infections is critically needed. Here, an overexpressed miR-6498-5p transgenic silkworm line was constructed. Importantly, the survival rates and median lethal doses of the transgenic line were clearly higher after infection with Nosema bombycis. H&E staining and RT-qPCR analyses revealed an inhibitory effect on the proliferation of N. bombycis in the transgenic larvae. Metabolomics analysis further revealed the presence of 56 differential metabolites between the two lines. KEGG analysis of these 56 metabolites found that they were involved in various amino acid and vitamin metabolism pathways. Notably, VB6 metabolism was enriched among the metabolites, and the pathway was well known for its involvement in the synthesis, interconversion, and degradation of amino acids. These suggest that miR-6498-5p modifies parasitic environments to inhibit the proliferation of N. bombycis by affecting the host amino acid metabolism. These results demonstrate the potential of microRNAs as biomolecules that can promote resistance to microsporidia and provide new insights and a new approach to generate microsporidia-resistant biological materials.IMPORTANCEMicrosporidia have an extremely wide host range and are capable of infecting a wide variety of insects and vertebrates, including humans, and their lethality to multiple species often poses significant environmental management challenge. Here, we successfully constructed a microsporidium-resistant line in the silkworm, based on the overexpression of miR-6498-5p. Our results strongly support the hypothesis that miR-6498-5p efficiently suppresses the proliferation of Nosema bombycis by regulating the host VB6 metabolism, a key pathway for enzymes involved in amino acid transport and protein metabolism. Our study provides new insights for understanding host anti-pathogen defenses toward microsporidia.

Bombyx mori nucleopolyhedrovirus LEF-2 disrupts the cell cycle in the G2/M phase by triggering a host cell DNA damage response.

It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.

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.

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.

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.

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.

Transcriptome analysis reveals changes in silkworm energy metabolism during Nosema bombycis infection.

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.

Bombyx mori cell division cycle protein 37 promotes the proliferation of BmNPV.

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.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

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 ß1 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 ß1.

A novel system to rapidly detect protein-protein interactions (PPIs) based on fluorescence co-localization.

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.).

Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis.

The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line. The double-positive HSP70-Cas9(+)×sgATAD3A(+) lines were obtained by hybridization and activation of the CRISPR/Cas9 system under the condition of microsporidia infection, although it is silenced in uninfected individuals. Genome editing analysis showed that the system could efficiently edit the BmATAD3A gene and induce large deletions. It is notable that the HSP70-induced system could effectively improve the survival rate of transgenic silkworm after microsporidia infection and inhibit the expression of key microsporidia genes. Moreover, no significant developmental differences between the transgenic silkworms infected with microsporidia and normal individuals were observed. In this study, we effectively inhibited microsporidia proliferation in transgenic individuals through disruptive techniques, thereby providing a method for microsporidia treatment and prevention, paving the way for economically advantageous insect breeding.

BmAtg13 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus.

Autophagy is a cell adaptive response that involves the process of microbial infections. Our previous study has indicated that Bombyx mori nucleopolyhedrovirus (BmNPV) infection triggers the complete autophagic process in BmN-SWU1 cells, which is beneficial to the viral infection. Autophagy-related (ATG) protein ATG13, as part of the ULK complex (a serine-threonine kinase complex composed of ULK1, ULK2, ATG13, ATG101, and FIP200), is the most upstream component of the autophagy pathway, and how it affects virus infections will improve our understanding of the interaction between the virus and the host. This study has determined that the overexpression of the BmAtg13 gene promotes the expression of viral genes and increases viral production in BmN-SWU1 cells, whereas knocking down the BmAtg13 gene suppresses BmNPV replication. Moreover, the BmAtg13 overexpression transgenic line contributed to viral replication and increased mortality rate of BmNPV infection. In contrast, the BmAtg13 knockout transgenic line reduced viral replication 96 h post-infection. Furthermore, BmATG13 directly interacted with viral protein BRO-B, forming a protein complex. Taken together, the findings of this study suggest that BmATG13 may be utilized by the BRO-B protein to promote BmNPV replication and proliferation, which, in turn, provides important insights into the mechanism that autophagy influences viral infection.

Resistant silkworm strain block viral infection independent of melanization.

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.

Performance of chemical chelating agent stabilization and cement solidification on heavy metals in MSWI fly ash: A comparative study.

Heavy metal stabilization by chemical chelating agent and solidification by cement are technologies currently used to reduce the leaching of heavy metals in municipal solid waste incineration (MSWI) fly ash. This paper studied the physico-chemical properties of the fly ash, heavy metals content in the fly ash, and the leaching concentration of the heavy metals from fly ash. The effect of four chelating agents namely dithiocarbamate (1#), dithiocarbamic acid dipotassium salt (2#), amino dithiocarbamate chelating resin (3#) and thiourea (4#) on the stabilization and leaching of heavy metals were investigated. Different addition ratios (1%, 2% and 3% w/w) of the chelating agents, various curing time (7, 14, 28 days), and different amounts of the cement (10%, 15% and 20% w/w) were used in order to find the agent with the optimum stabilization and leaching of heavy metals as well as find the effect of combining the agent and cement. The results showed that the dithiocarbamate (1#) chelating agent had the best stabilizing performance due to the three-dimensional structure after their reaction. The addition of cement to the fly ash led to the immobilization of heavy metals due to the C-S-H gel formation. The technology of cement solidification and chelating agent stabilization was optimal from the point of economic cost and the complexity aspect.

Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms.

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.

Bombyx mori protein BmREEPa and BmPtchd could form a complex with BmNPV envelope protein GP64.

Our previous studies have indicated that Bombyx mori receptor expression enhancing protein a (BmREEPa) could participate in BV invasion in vivo and in vitro, however, the mechanism is still unclear. In this study, we screened BmREEPa interacting protein through co-immunoprecipitation and finally identified a membrane protein, Bombyx mori patched domain containing protein (BmPtchd, KR338939), which contains receptor activity. Further studies showed that BmPtchd, BmREEPa and Glycoprotein 64 could form a protein complex and the expression level of BmREEPa and BmPtchd could be affected by cellular cholesterol level. These findings may provide an important basis for explaining the invasion mechanism of Bombyx mori Nucleopolyhedrovirus budded virus.

BmNHR96 participate BV entry of BmN-SWU1 cells via affecting the cellular cholesterol level.

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.

Transgenic RNAi of BmREEPa in silkworms can enhance the resistance of silkworm to Bombyxmori Nucleopolyhedrovirus.

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.

In vivo RNA interference of BmNHR96 enhances the resistance of transgenic silkworm to BmNPV.

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.