Search

Double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is a tool in functional gene study and pest control. However, RNAi efficiency in Lepidoptera is low compared to the RNAi sensitive Coleoptera. Previous studies on RNAi in the silkworm Bombyx mori, the lepidopteran model insect, were performed by injection only. Successful oral RNAi in the silkworm has never been reported yet. This study aims to develop a successful oral dsRNA delivery method to the silkworm larvae. Chitosan is an economical and biodegradable polymer. Chitosan/dsRNA nanoparticles were prepared by self-assembly. These nanoparticles were found to be stable when incubated in the midgut juice of the silkworm larvae, whereas naked dsRNA underwent complete degradation. Chitosan/dsRNA nanoparticles targeting various immune genes in oral administration to the silkworm larvae mediated significant knockdown of gene transcript. This silencing effect resulted in smaller larvae and cocoons when the silkworms were fed with Chitosan/dsRNA nanoparticles targeting BmToll9-2 gene, indicating that immune genes might be used as targets in pest control. Optimization of the chitosan/dsRNA nanoparticles maintained an RNAi effect from 3-5 days. The efficient RNAi was due to the persistence of nanoparticle-shielded dsRNA in the larvae. The above findings contribute to the first oral RNAi report in the silkworm, which facilitates the application of RNAi in insects and oral RNAi in pest control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

The discovery that infections of viruses are pervasive among insects has considerable potential for future applications, such as new strategies for pest control through the manipulation of virus-host interactions. However, few studies can be found that aim to minimize (for beneficial insects) or maximize (for pests) virus impact or virulence. Viruses generally employ molecular mechanisms that deviate from the cells' to increase their replication efficiency and to avoid the immune response. In this research, a screening system is presented for the detection of molecules that interfere with the internal ribosomal entry site (IRES) of Cricket paralysis virus (Dicistroviridae) which has been well characterized in previous research. Over-expression and RNAi experiments identified the importance of eIF4A, a component of the cap-dependent translation initiation complex, to modify the activity of IRES-mediated translation. Application of Rocaglamide A (RocA), a natural product from Aglaia plants and inhibitor of eIF4A, resulted in strong stimulation of IRES-mediated translation in reporter assays as well as increased CrPV genome replication and virion production in lepidopteran Hi5 cells. At 100 nM of RocA, dsRNA molecules accumulated in infected cells, corresponding to full-length genome (9.5 kb) and a smaller fragment (0.8 kb) with unknown function. Treatment of silkworm larvae with RocA by injection or topically was highly toxic while no strong stimulation of CrPV infection could be observed. The prospect of the use of rocaglamates as insecticides and enhancers of CrPV infection is discussed together with its potential impact on mammalian cells., (© 2025 Wiley Periodicals LLC.)

Toll receptors play important roles in the development and innate immunity of insects. Previously, we reported the immunological function of BmToll9-2 in silkworm, Bombyx mori , larvae. In this study, we focused on the role of BmToll9-2 as a regulator in the Toll signaling pathway. The expressions of most signaling genes in the Toll pathway, as well as immune effectors, were reduced after the RNAi of BmToll9-2 . Coincidentally, hemolymph from BmToll9-2 -silenced larvae exhibited decreased antibacterial activity in the growth of Escherichia coli , demonstrated either by growth curve or inhibitory zone experiments. The oral administration of heat-inactivated E. coli and Staphylococcus aureus following the RNAi of BmToll9-2 up-regulated the expression of most signaling genes in the Toll pathway and downstream immune effectors. The above results indicate that BmToll9-2 is positively involved in the Toll signaling pathway. As a positive regulator, BmToll9-2 is shown to be activated preferentially against E. coli and, in turn, positively modulates the humoral immune response in antibacterial activity., Competing Interests: The authors declare no conflicts of interest.

Toll receptors are important regulators of insects' innate immune system which, upon binding of pathogen molecules, activate a conserved signal transduction cascade known as the Toll pathway. RNA interference (RNAi) is a powerful tool to study the function of genes via reverse genetics. However, due to the reported refractory of RNAi efficiency in lepidopteran insects, successful reports of silencing of Toll receptors in the silkworm Bombyx mori have not been reported yet. In this study, a Toll receptor of the silkworm Bombyx Toll9-2 (BmToll9-2) was cloned and its expression and function were analyzed. The results showed that BmToll9-2 contains an ectodomain (ECD) with a signal peptide and nine leucine-rich repeats, a transmembrane helix, and a cytoplasmic region with a Toll/interleukin-1 domain. Phylogenetic analysis indicates that BmToll9-2 clusters with other insect Toll9 receptors and mammalian Toll-like receptor 4. Oral infection of exogenous pathogens showed that the Gram-negative bacterium Escherichia coli and its main cell wall component lipopolysaccharide (LPS), as well as the Gram-positive bacterium Staphylococcus aureus and its main cell wall component peptidoglycan, significantly induce BmToll9-2 expression in vivo. LPS also induced the expression of BmToll9-2 in BmN4 cells in vitro. These observations indicate its role as a sensor in the innate immunity to exogenous pathogens and as a pathogen-associated receptor that is responsive to LPS. RNAi of BmToll9-2 was effective in the midgut and epidermis. RNAi-mediated knock-down of BmToll9-2 reduced the weight and growth of the silkworm. Bacterial challenge following RNAi upregulated the expression of BmToll9-2 and rescued the weight differences of the silkworm, which may be related to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae., (© 2024 Wiley Periodicals LLC.)

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

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., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

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., (© 2024 Wiley Periodicals LLC.)

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., (© 2024 The Authors. Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC.)

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., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of financial interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

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., (© 2023 Society of Chemical Industry.)

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., (© 2023 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

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., (Copyright © 2023 by The American Association of Immunologists, Inc.)

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.