Nanoparticle-induced morphological transition of Bombyx mori nucleopolyhedrovirus: a novel method to treat silkworm grasserie disease

Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed α structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe ‘roughening’ of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09?±?6.61 % after 96 h. In this group, 61.04?±?8.03 % larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases.     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Genome-Wide Analysis of Differentially Expressed microRNA in Bombyx mori Infected with Nucleopolyhedrosis Virus

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV.     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell

The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAi-mediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV–Bme21 BEVS.     

Comparative transcriptome analysis of wing discs from Bombyx mori and Bombyx mandarina

The insect wing is developed from the wing imaginal disc which is designed from the embryonic ectoderm. To get insight into gene expression profiles in wing discs of Bombyx mori during metamorphosis, we compared the gene expression in the wing between B. mori and B. mandarina moth through RNA-seq. Out of total valid reads identified from the 5th day of 5th instar larvae of silkworm (L5), 7th day of pupae (P7), 1st day of moth (M1) and 1st day of wild silkworm moth (WM1), 20,092,004, 29,251,647, 24,654,695 and 19,753,089 reads were mapped to the mRNA reference sequences of silkworm, respectively. 9229, 7048, 9268 and 6701 differentially expressed genes (DEGs) were respectively recorded in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. Further, the peroxisome, ribosome, endocytosis and oxidative phosphorylation pathways were significantly regulated in the metamorphosis of the silkworm. Our study identified 16 orthologous genes with a positive selection from M1, which might be subjected to artificial selection in the domestication of B. mori and would play vital roles in the flight of B. mandarina.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.     

Metabolic engineering of the baculovirus-expression system via inverse "shotgun" genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

Abortive infection of the baculovirus Autographa californica nuclear polyhedrosis virus in Sf-9 cells after mutation of the putative DNA helicase gene.

Homologous recombination between the Autographa californica nuclear polyhedrosis virus (AcNPV) genome and a 0.6-kbp-long DNA fragment derived from the putative DNA helicase gene of Bombyx mori nuclear polyhedrosis virus generates eh2-AcNPV, an expanded-host-range AcNPV mutant (S. Maeda, S.G. Kamita, and A. Kondo, J. Virol. 67:6234-6238, 1993). After inoculation at a high multiplicity of infection (MOI), eh2-AcNPV replicates efficiently in both the Sf-9 (AcNPV-permissive) and BmN (non-AcNPV-permissive) cell lines. In this study, we found that after the inoculation of Sf-9 cells at a low MOI (i.e., 1 and 0.1 PFU per cell), the release of eh2-AcNPV virions was dramatically reduced (approximately 900- and 10,000-fold, respectively, at 72 h postinoculation) compared with that of wild-type AcNPV. In addition, the titer of eh2-AcNPV determined by plaque assay on Sf-9 cells was approximately 200-fold lower than that determined by plaque assay on BmN cells. Analyses of gene expression and viral DNA replication after low-MOI eh2-AcNPV inoculation of Sf-9 cells indicated that viral early genes were expressed normally. However, DNA replication and late-gene expression were significantly reduced. These findings suggested that abortive infection occurred at the stage of viral DNA replication in nearly all low-MOI eh2-AcNPV-infected Sf-9 cells. In the larvae of Spodoptera frugiperda, the organism from which Sf-9 cells are derived, the infectivity of eh2-AcNPV was lower than that of AcNPV; however, abortive infection was not found.     

Molecular cloning of <Emphasis Type="Italic">BmTUDOR-SN</Emphasis> and analysis of its role in the RNAi pathway in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> (Lepidoptera: Bombycidae)

Tudor-sn, a conserved nuclease, was first isolated from RNA-induced silencing complex (RISC) and was subsequently implicated in the RNA interference (RNAi) pathway in humans, flies and nematodes. However, in the silkworm, Bombyx mori L, the RNAi mechanism and the components of RISC were quite unclear. Here, we cloned the full-length cDNA of TUDOR-SN (BmTUDOR-SN) from the silkworm. Phylogenetic analysis revealed that BmTudor-sn had a high homology with Tudor-sn proteins in other insects. Fluorescent microscopic observation indicated that the subcellular localization of enhanced green fluorescent protein fused BmTudor-sn was mainly in the cytoplasm of silkworm BmN4 cells. Knockdown of BmTUDOR-SN did not, however, affect the RNAi efficiency in BmN4 cells.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

Identification of Enterococcus mundtii as a pathogenic agent involved in the "flacherie" disease in Bombyx mori L. larvae reared on artificial diet

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal.The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Dimerization and proper degradation of BmNPV IE2 are required for efficient virus growth in larvae of Bombyx mori (Lepidoptera: Bombycidae)

The baculovirus ie2 gene is one of the immediate early genes, and its product is known to transactivate viral promoters. However, the roles of Bombyx mori nucleopolyhedrovirus (BmNPV) ie2 in insect larvae are poorly understood. Here we investigated the functions of BmNPV IE2 in cultured cells and in insect larvae using two mutant viruses, BmIE2D and BmIE2CS. BmIE2D lacks the IE2 C-terminal coiled-coil domain that is required for IE2 dimerization. The other mutant BmIE2CS expresses an E3 ligase activity-deficient IE2 derivative, which is degraded more slowly compared with wild-type IE2. We found that ie2 mutations had little effect on BmNPV infection in cultured cells, whereas budded virus and occlusion body production was significantly reduced in the hemolymph of B. mori larvae infected with ie2 mutants. These results indicate that both dimerization and proper degradation of BmNPV IE2 are crucial steps for efficient virus growth in B. mori larvae, but not in cultured cells. Oral infection assays also revealed that the infectivity of the occluded form of ie2 mutants was normal in B. mori larvae, which is inconsistent with the results reported from ie2 mutants of Autographa californica NPV. This suggests that loss of IE2 function causes virus-specific effects in host insects.