Systematic analysis of innate immune-related genes in the silkworm: Application to antiviral research.

The silkworm, a crucial model organism of the Lepidoptera, offers an excellent platform for investigating the molecular mechanisms underlying the innate immune response of insects toward pathogens. Over the years, researchers worldwide have identified numerous immune-related genes in silkworms. However, these identified silkworm immune genes are not well classified and not well known to the scientific community. With the availability of the latest genome data of silkworms and the extensive research on silkworm immunity, it has become imperative to systematically categorize the immune genes of silkworms with different database IDs. In this study, we present a meticulous organization of prevalent immune-related genes in the domestic silkworm, using the SilkDB 3.0 database as a reliable source for updated gene information. Furthermore, utilizing the available data, we classify the collected immune genes into distinct categories: pattern recognition receptors, classical immune pathways, effector genes and others. In-depth data analysis has enabled us to predict some potential antiviral genes. Subsequently, we performed antiviral experiments on selected genes, exploring their impact on Bombyx mori nucleopolyhedrovirus replication. The outcomes of this research furnish novel insights into the immune genes of the silkworm, consequently fostering advancements in the field of silkworm immunity research by establishing a comprehensive classification and functional understanding of immune-related genes in the silkworm. This study contributes to the broader understanding of insect immune responses and opens up new avenues for future investigations in the domain of host-pathogen interactions.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes.

INTRODUCTION: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. METHODS: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. RESULTS: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. CONCLUSION: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

BmPGPR-L4 is a negative regulator of the humoral immune response in the silkworm Bombyx mori.

Toll, immune deficiency and prophenoloxidase cascade represent vital immune signaling pathways in insects. Peptidoglycan recognition proteins (PGRPs) are innate immune receptors that activate and regulate the immune signaling pathways. Previously, we reported that BmPGPR-L4 was induced in the silkworm Bombyx mori larvae by bacteria and peptidoglycan challenges. Here, we focused on the function of BmPGRP-L4 in regulating the expression of antimicrobial peptides (AMPs). The hemolymph from BmPGRP-L4-silenced larvae exhibited an enhanced inhibitory effect on the growth of Escherichia coli, either by growth curve or inhibitory zone experiments. Coincidentally, most of the AMP genes were upregulated after RNAi of BmPGRP-L4. Oral administration of heat-inactivated E. coli and Staphylococcus aureus after RNAi of BmPGRP-L4 resulted in the increased expression of BmPGRP-L4 in different tissues of the silkworm larvae, revealing an auto-regulatory mechanism. By contrast, the expression of most AMP genes was downregulated by oral bacterial administration after RNAi of BmPGRP-L4. The above results demonstrate that BmPGRP-L4 recognizes bacterial pathogen-associated molecular patterns and negatively regulates AMP expression to achieve immunological homeostasis. As a negative regulator, BmPGPR-L4 is proposed to be involved in the feedback regulation of the immune signaling pathways of the silkworm to prevent excessive activation of the immune response.

Single domain von Willebrand factor type C "cytokines" and the regulation of the stress/immune response in insects.

The single domain von Willebrand factor type C (SVWC) appears in small secreted peptides that are arthropod-specific and are produced following environmental stress or pathogen exposure. Most research has focused on proteins with SVWC domain that are induced after virus infection and are hypothesized to function as "cytokines" to regulate the innate immune response. The expansion of SVWC genes in insect species indicates that many other functions remain to be discovered. Research in shrimp has elucidated the adaptability of Vago-like peptides in the innate immune response against bacteria, fungi and viruses after activation by Jak-STAT and/or Toll/Imd pathways in which they can act as pathogen-recognition receptors or cytokine-like signaling molecules. SVWC factors also appear in scorpion venoms and tick saliva, underlining their versatility to acquire new functions. This review discusses the discovery and function of SVWC peptides from insects to crustaceans and chelicerates and reveals the enormous gaps in knowledge that remain to be filled to understand this enigmatic group of secreted peptides.

Single-nucleus sequencing of silkworm larval midgut reveals the immune escape strategy of BmNPV in the midgut during the late stage of infection.

The midgut is an important barrier against microorganism invasion and proliferation, yet is the first tissue encountered when a baculovirus naturally invades the host. However, only limited knowledge is available how different midgut cell types contribute to the immune response and the clearance or promotion of viral infection. Here, single-nucleus RNA sequencing (snRNA seq) was employed to analyze the responses of various cell subpopulations in the silkworm larval midgut to B. mori nucleopolyhedrovirus (BmNPV) infection. We identified 22 distinct clusters representing enteroendocrine cells (EEs), enterocytes (ECs), intestinal stem cells (ISCs), Goblet cell-like and muscle cell types in the BmNPV-infected and uninfected silkworm larvae midgut at 72 h post infection. Further, our results revealed that the strategies for immune escape of BmNPV in the midgut at the late stage of infection include (1) inhibiting the response of antiviral pathways; (2) inhibiting the expression of antiviral host factors; (3) stimulating expression levels of genes promoting BmNPV replication. These findings suggest that the midgut, as the first line of defense against the invasion of the baculovirus, has dual characteristics of "resistance" and "tolerance". Our single-cell dataset reveals the diversity of silkworm larval midgut cells, and the transcriptome analysis provides insights into the interaction between host and virus infection at the single-cell level.

Drosophila X virus-like particles as delivery carriers for improved oral insecticidal efficacy of scorpion Androctonus australis peptide against the invasive fruit fly, Drosophila suzukii.

Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects, offering a promising insecticidal component for insect pest control. However, effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph, where they can act. Here, we investigated the potential of a novel nanocarrier, Drosophila X virus-like particle (DXV-VLP), for delivering a neurotoxin from the scorpion Androctonus australis Hector (AaIT) against the invasive pest fruit fly, Drosophila suzukii. Our results show that the fusion proteins of DXV polyproteins with AaIT peptide at their C-termini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system, and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions. In addition, the AaIT peptides displayed on DXV-VLPs retained their toxicity, as demonstrated in injection bioassays that resulted in severe mortality (72%) in adults after 72 h. When fed to adults, mild mortality was observed in the group treated with DXV-AaIT (38%), while no mortality occurred in the group treated with AaIT peptide, thus indicating the significant role of DXV-VLPs in delivering AaIT peptides. Overall, this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control. Moreover, it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.

Single-Nucleus Sequencing of Fat Body Reveals Distinct Metabolic and Immune Response Landscapes in Silkworm Larvae after Bombyx mori Nucleopolyhedrovirus Infection.

The fat body plays a central role in the regulation of the life cycle of insects and acts as the major site for detoxification, nutrient storage, energy metabolism, and innate immunity. However, the diversity of cell types in the fat body, as well as how these cell subsets respond to virus infection, remains largely unknown. We used single-nucleus RNA sequencing to identify 23 distinct clusters representing adipocyte, hemocyte, epithelial cell, muscle cell, and glial cell types in the fat body of silkworm larvae. Further, by analysis of viral transcriptomes in each cell subset, we reveal that all fat body cells could be infected by Bombyx mori nucleopolyhedrovirus (BmNPV) at 72 h postinfection, and that the majority of infected cells carried at least a medium viral load, whereas most cells infected by BmNPV at 24 h postinfection had only low levels of infection. Finally, we characterize the responses occurring in the fat body cell clusters on BmNPV infection, which, on one hand, mainly reduce their metabolic functions, involving energy, carbohydrates, lipids, and amino acids, but, on the other hand, initiate a strong antiviral response. Our single-nucleus RNA sequencing analysis reveals the diversity of insect fat body cells and provides a resource of gene expression profiles for a systems-level understanding of their response to virus infection.

Plant and insect virus-like particles: emerging nanoparticles for agricultural pest management.

Virus-like particles (VLPs) represent a biodegradable, biocompatible nanomaterial made from viral coat proteins that can improve the delivery of antigens, drugs, nucleic acids, and other substances, with most applications in human and veterinary medicine. Regarding agricultural viruses, many insect and plant virus coat proteins have been shown to assemble into VLPs accurately. In addition, some plant virus-based VLPs have been used in medical studies. However, to our knowledge, the potential application of plant/insect virus-based VLPs in agriculture remains largely underexplored. This review focuses on why and how to engineer coat proteins of plant/insect viruses as functionalized VLPs, and how to exploit VLPs in agricultural pest control. The first part of the review describes four different engineering strategies for loading cargo at the inner or the outer surface of VLPs depending on the type of cargo and purpose. Second, the literature on plant and insect viruses the coat proteins of which have been confirmed to self-assemble into VLPs is reviewed. These VLPs are good candidates for developing VLP-based agricultural pest control strategies. Lastly, the concepts of plant/insect virus-based VLPs for delivering insecticidal and antiviral components (e.g., double-stranded RNA, peptides, and chemicals) are discussed, which provides future prospects of VLP application in agricultural pest control. In addition, some concerns are raised about VLP production on a large scale and the short-term resistance of hosts to VLP uptake. Overall, this review is expected to stimulate interest and research exploring plant/insect virus-based VLP applications in agricultural pest management. © 2023 Society of Chemical Industry.

Peptidoglycan Recognition Protein S5 of Bombyx mori Facilitates the Proliferation of Bombyx mori Cypovirus 1.

Bombyx mori cypovirus 1 (BmCPV1), a primary pathogen of the silkworm, is a typical dsRNA virus belonging to the Reoviridae family. In this study, a total of 2520 differentially expressed genes (DEGs) were identified by RNA-seq analysis of the silkworm midgut after BmCPV1 infection and Gene Ontology (GO) functional annotation showed that the DEGs predominantly functioned in binding (molecular function), cell (cellular component), and cellular processes (biological process). Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation revealed that the DEGs were mainly distributed in global and overview metabolism maps, translation, and signal transduction. Among the identified DEGs, BmPGRP-S5 belongs to the peptidoglycan recognition protein (PGRP) family. Previous studies have revealed that PGRPs were involved in the interactions between silkworm and BmCPV1. Here, we explored the effect of BmPGRP-S5 on BmCPV1 replication and demonstrated that BmPGRP-S5 promotes the proliferation of BmCPV1 in BmN cells through overexpression or knockdown experiments. Knocking down of BmPGRP-S5 in silkworm larvae similarly promoted the proliferation of BmCPV1. Through experimental validation, we therefore determined that BmPGRP-S5 acts as a proviral host factor for BmCPV1 infection. This study clarifies the proliferation mechanism of BmCPV1 and provides new insights into the functional role of BmPGRP-S5.

What Are the Functional Roles of Piwi Proteins and piRNAs in Insects?

Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered.

Silencing of the immune gene BmPGRP-L4 in the midgut affects the growth of silkworm (Bombyx mori) larvae.

Peptidoglycan recognition proteins (PGRPs) are one of the receptors in insects' immune pathways, essential for insects to recognize the exogenous pathogens in order to activate the Toll and immune deficiency (IMD) pathway. In the silkworm Bombyx mori, previous studies focused on the short PGRPs and less is known about the long PGRPs. In this study, a long PGRP in silkworm BmPGRP-L4 was cloned and its expression and function were analysed. The results showed that BmPGRP-L4 contains a transmembrane region, a conserved PGRP domain, and an amidase-2 domain. The expression profile demonstrated that BmPGRP-L4 existed in diverse tissues including epidermis, fat body, midgut, and silk glands, with remarkably high expression in the midgut in the 5th instar. Oral infection with Escherichia coli and Staphylococcus aureus significantly induced BmPGRP-L4 in the midgut and epidermis, as well as in the fat body and silk glands. Peptidoglycan also induced the expression of BmPGRP-L4 in midgut tissue ex vivo and BmN4 cells in vitro. RNAi of BmPGRP-L4 was effective in the midgut and epidermis, while the efficiency in the fat body was transient. RNAi-mediated knock-down of BmPGRP-L4 reduced the weight and growth of the silkworm, possibly due to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

The piRNA pathway is required for nucleopolyhedrovirus replication in Lepidoptera.

The Piwi-interacting RNA (piRNA) pathway has been shown to be involved in the antiviral defense against RNA viruses, especially in mosquitoes, but its universality has been questioned. Here, we used the Bombyx mori nucleopolyhedrovirus (BmNPV) -infected silkworm as a model to explore the effects of the key factors of piRNA pathway, BmAgo3 and Siwi, on replication of a large DNA virus (belonging to the family of Baculoviridae). We demonstrated that BmAgo3 and Siwi could promote the replication of BmNPV through both overexpression and knockdown experiments in BmN cell lines and silkworm larvae. In addition, we also studied the effect of PIWI-class genes on Autographa californica nucleopolyhedrovirus (AcMNPV) replication in the Spodoptera frugiperda cell line Sf9. By knocking down the expression of PIWI-class genes in Sf9, we found that Piwi-like-1 and Piwi-like-2-3 could inhibit AcMNPV replication, while Piwi-like-4-5 promoted virus replication. Our study provides compelling evidence that the piRNA pathway affects host infection by exogenous viruses in Lepidoptera. Also, our results reflect the diversity of the roles of PIWI-class genes in virus infection of the host across species. This study is the first to explore the interaction of PIWI-class proteins with DNA viruses, providing new insights into the functional roles of the piRNA pathway.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B. mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B. mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

Functional characterization of putative ecdysone transporters in lepidopteran pests.

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone enters cells through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone importer (EcI) from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that EcI likely appeared only at the root of the arthropod lineage. Partial disruption of EcI in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of EcI in the lepidoptera cell line RP-HzGUT-AW1(MG) demonstrated this protein's ability to control ecdysone mediated signaling in gene regulation, its role in ecdysone mediated cell death, and its sensitivity to rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

Sirt5 Inhibits BmNPV Replication by Promoting a Relish-Mediated Antiviral Pathway in Bombyx mori.

Silent information regulators (Sirtuins) belong to the family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases (HDACs) that have diverse functions in cells. Mammalian Sirtuins have seven isoforms (Sirt1-7) which have been found to play a role in viral replication. However, Sirtuin members of insects are very different from mammals, and the function of insect Sirtuins in regulating virus replication is unclear. The silkworm, Bombyx mori, as a model species of Lepidoptera, is also an important economical insect. B. mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects silkworms and causes serious losses in the sericulture industry. Here, we used the infection of the silkworm by BmNPV as a model to explore the effect of Sirtuins on virus replication. We initially knocked down all silkworm Sirtuins, and then infected with BmNPV to analyze its replication. Sirt2 and Sirt5 were found to have potential antiviral functions in the silkworm. We further confirmed the antiviral function of silkworm Sirt5 through its effects on viral titers during both knockdown and overexpression experiments. Additionally, Suramin, a Sirt5 inhibitor, was found to promote BmNPV replication. In terms of molecular mechanism, it was found that silkworm Sirt5 might promote the immune pathway mediated by Relish, thereby enhancing the host antiviral response. This study is the first to explore the role of Sirtuins in insect-virus interactions, providing new insights into the functional role of members of the insect Sirtuin family.

Characterization of virus-like particles assembled by co-expression of BmCPV capsid shell protein and large protrusion protein.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the Reoviridae family of the Cypovirus genus. Previous studies have shown that the BmCPV major capsid shell protein (CSP) has the ability to self-assemble into virus-like particles (VLPs), and cryo-electron microscopy of the BmCPV virions has revealed a tight mutual binding region between CSP and another capsid protein known as the Large Protrusion Protein (LPP), which further stabilizes the capsid shell. In this study, the multi-gene baculovirus expression system, Ac-MultiBac, was used to produce both solely CSP-based and CSP-LPP co-assembled VLPs. Transmission electron microscopy (TEM) results showed that addition of LPP did not affect the assembly of VLPs resulting in almost identical structure in both cases. However, ex vivo administration of VLPs to silkworm midgut tissue showed that CSP-based VLPs did not induce a significant transcriptional response in the innate immunity and RNAi gene cascades, compared to the co-assembled CSP-LPP based VLPs and the natural BmCPV virions isolated from polyhedra. The experimental results indicate that CSP and LPP attach tightly ("Plug and Display" model with CSP acting as "catcher" and LPP as "tag") to form VLPs that have a structure similar to that of the native CPV virions. Moreover, our results showed that the formation of VLPs with the two BmCPV capsid proteins is feasible, which can form the basis for the production of BmCPV-based VLPs as a new type of biological material to display exogenous proteins.

Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles.

Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.

Identification of Helicoverpa armigera promoters for biotechnological applications.

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

Analysis of luciferase dsRNA production during baculovirus infection of Hi5 cells: RNA hairpins expressed by very late promoters do not trigger gene silencing.

The baculovirus expression vector system (BEVS) has become an important platform for the expression of recombinant proteins and is especially useful for the production of large protein complexes such as virus-like particles (VLPs). An important application for VLPs is their use as vehicles for targeted delivery of drugs or toxins which requires the development of methods for efficient loading with the intended cargo. Our research intends to employ the BEVS for the production of VLPs for the delivery of insecticidal dsRNA molecules to targeted insect pests (as "dsRNA-VLPs"). A convenient strategy would be the co-expression of long dsRNAs with viral capsid proteins and their simultaneous encapsulation during VLP assembly but the capacity of the BEVS for the production of long dsRNA has not been assessed so far. In this study, the efficiency of production of long RNA hairpins targeting the luciferase gene ("dsLuc") by the polyhedrin promoter during baculovirus infection was evaluated. However, RNAi reporter assays could not detect significant amounts of dsLuc in Hi5 cells infected with recombinant baculovirus, even in the presence of co-expressed dsRNA-binding protein B2-GFP or the employment of the MS2-MCP system. Nevertheless, dot blot analyses using anti-dsRNA antibody revealed that baculovirus-mediated expression of B2-GFP resulted in significant increases in dsRNA levels in infected cells that may correspond to hybridized complementary viral transcripts. Using B2-GFP as a genetically encoded sensor, dsRNA foci were detected in the nuclei that partially co-localized with DAPI staining, consistent with their localization at the virogenic stroma. Co-localization experiments with the baculovirus proteins vp39, Ac93, ODV-E25 and gp64 indicated limited overlap between B2-GFP and the ring zone compartment where assembly of nucleocapsids and virions occurs. Stability experiments showed that exogenous dsRNA is resistant to degradation in extracts of non-infected and infected Hi5 cells and it is proposed that strong unwinding activity at the virogenic stroma in the infected nuclei may neutralize the annealing of complementary RNA strands and block the production of long dsRNAs. Because the strong stability of exogenous dsRNA, transfection can be explored as an alternative method for delivery of cargo for dsRNA-VLPs during their assembly in baculovirus-infected Hi5 cells.