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.

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.

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.

Reduced proinsecticide activation by cytochrome P450 confers coumaphos resistance in the major bee parasite Varroa destructor.

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [14C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4 RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.

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.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the genus Cypovirus in the family Reoviridae. The results of cryo-electron microscopy showed that the BmCPV capsid consists of 60 asymmetric units, and each asymmetric unit contains one turret protein (TP), two large protrusion proteins (LPP) and two capsid shell proteins (CSP). CSP has the ability to self-assemble into virus-like particles (VLPs), and the small protrusion domain (SPD) in CSP may play an essential role in the assembly of viral capsids. In this study, three critical amino acid sites, D828, S829 and V945, in the SPD were efficiently mutated (point mutation) based on the principle of PCR circular mutagenesis. Moreover, a multi-gene expression system, Ac-MultiBac baculovirus, was used to produce eight different recombinant VLPs in vitro. Transmission electron microscopy showed that the single site and double site mutations had little effect on the efficiency and morphology of the assembly of VLPs. Still, the simultaneous mutation of the three sites had a significant impact. The experimental results demonstrate that the SPD of CSP plays an essential role in assembly of the viral capsid, which lays the foundation for further analysis of the molecular and structural mechanism of BmCPV capsid assembly.

Antiviral defense against Cypovirus 1 (Reoviridae) infection in the silkworm, Bombyx mori.

Recent years have shown a large increase in studies of infection of the silkworm (Bombyx mori) with Cypovirus 1 (previously designated as B. mori cytoplasmic polyhedrosis virus), that causes serious damage in sericulture. Cypovirus 1 has a single-layered capsid that encapsulates a segmented double-strand RNA (dsRNA) genome which are attractive features for the establishment of a biotechnological platform for the production of specialized gene silencing agents, either as recombinant viruses or as viral-like particles with nonreplicative dsRNA cargo. For both combatting viral disease and application of Cypovirus-based pest control, however, a better understanding is needed of the innate immune response caused by Cypovirus infection of the midgut of lepidopteran larvae. Studies of deep sequencing of viral small RNAs have indicated the importance of the RNA interference pathway in the control of Cypovirus infection although many functional aspects still need to be elucidated and conclusive evidence is lacking. A considerable number of transcriptome studies were carried out that revealed a complex response that hitherto remains uncharacterized because of a dearth in functional studies. Also, the uptake mechanism of Cypovirus by the midgut cells remains unclarified because of contrasting mechanisms revealed by electron microscopy and functional studies. The field will benefit from an increase in functional studies that will depend on transgenic silkworm technology and reverse genetics systems for Cypovirus 1.

Transcriptional response of immune-related genes after endogenous expression of VP1 and exogenous exposure to VP1-based VLPs and CPV virions in lepidopteran cell lines.

In insects, RNAi is considered the major antiviral immune defense pathway. DsRNAs produced during viral infection are processed by Dicer enzymes into small RNAs that function as specificity determinants to silence viral genes. By contrast, in mammals, recognition of molecules associated with viral infection, such as dsRNA, by pattern recognition receptors (PRRs) initiates a signaling cascade that culminates in the production and release of signaling proteins with antiviral function such as interferons. However, in insects, the hypothesis that components of virions can be recognized as pathogen-activated molecular patterns (PAMPs) to activate the innate immune response has not been investigated systematically. In this study, the potential of VP1, that constitutes the major capsid protein of cytoplasmic polyhedrosis virus (CPV; Reoviridae), to activate a collection of immune-related genes was examined in silkworm-derived Bm5 cells. Two different methods of VP1 administration were tested, either through endogenous expression in transformed cell lines, or through addition of purified VP1-based viral-like particles to the extracellular medium. In addition, exposure to CPV virions isolated from purified polyhedra was also performed. In general, our results do not show a robust transcriptional response of immune-related genes to VP1 or CPV virions, but two exceptions were noted. First, the expression of the antimicrobial peptide (AMP) gene Attacin was strongly induced after 24 h of exposure to VP1-based VLPs. Second, the expression levels of dcr-2, an essential gene in the RNAi pathway, were greatly increased in VP1-expressing transformed Sf21 cells but not transformed Bm5 cells, indicating the existence of species-specific effects. However, the increased expression of dcr-2 did not result in increased silencing efficiency when tested in an RNAi reporter assay. Our study indicates that the capsid protein VP1 of CPV has the potential to act as a PAMP and to induce a transcriptional response in insect cells that relate both to RNAi and protein effectors such as AMPs. The identity of the PRRs and the signaling cascade that are potentially triggered by VP1 remain to be elucidated in future experiments. While this study was performed on a small scale, it can encourage more comprehensive studies with high-throughput approaches (microarray, deep sequencing) to search more systematically whether viral capsid proteins can act as PAMPs in insects and whether their production results in the induction of immune-related genes with potential antiviral function.

Chichen type III interferon produced by silkworm bioreactor induces ISG expression and restricts ALV-J infection in vitro.

Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.

Bombyx mori nucleopolyhedrovirus lef8 gene: effects of deletion and implications for gene transduction applications.

In this study, we have deleted the lef8 gene of the baculovirus BmNPV, which encodes one of the viral RNA polymerase subunits, in order to create a knockout bacmid, Δlef8, directing cytopathology-free single-cell infections for gene transduction and recombinant protein production. However, while removal of the complete lef8 ORF produced the expected phenotype, it also affected the function of the closely linked essential gene orf40, thus hampering the mutant bacmid rescue in cultured Bombyx cells expressing recombinant LEF8. Subsequently, we determined that several diverse sequences can substitute for the orf40 5'-upstream sequences that were removed by the deletion of the lef8 gene and also showed that neither a physical linkage nor expression of the two relevant genes under native promoter control is a prerequisite for a fully functional virus. Based on these findings, we generated a rescue-competent lef8-null vector, which contained a heterologous promoter-driven orf40. This lef8-deficient vector, which produces productive infections and progeny virus lacking lef8 in deficiency-complementing cells expressing LEF8, could be used as the basis for an alternative to current silkmoth transduction systems.

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.

UNLABELLED: The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. IMPORTANCE: This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.

Chorion genes: a landscape of their evolution, structure, and regulation.

Differential regulation at the level of transcription provides a means for controlling gene expression in eukaryotes, especially during development. Insect model systems have been extensively used to decipher the molecular basis of such regulatory cascades, and one of the oldest such model systems is the regulation of chorion gene expression during ovarian follicle maturation. Recent experimental and technological advances have shed new light onto the system, allowing us to revisit it. Thus, in this review we try to summarize almost 40 years' worth of studies on chorion gene regulation while-by comparing Bombyx mori and Drosophila melanogaster models-attempting to present a comprehensive, unified model of the various regulatory aspects of choriogenesis that takes into account the evolutionary conservation and divergence of the underlying mechanisms.

The molecular and physiological impact of bisphenol A in Sesamia nonagrioides (Lepidoptera: Noctuidae).

In the present study we investigated the potential relative effects of bisphenol A (BPA) and RH-5992 (tebufenozide) on the development and metamorphosis of the corn stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). A number of morphological and molecular factors were examined in order to identify the toxic and the endocrine-relative action of these two chemicals. We observed that BPA, RH-5992 and the combination of BPA/RH-5992 caused a developmental delay by extending the transition period between larval and pupal instars. These chemicals also reduced adult emergence and caused molting malformations during development and metamorphosis. In the corn stalk borer, BPA exhibits ecdysteroid activities in a fashion similar to that of the ecdysone agonist RH-5992. These results suggest that exposure to environmentally relevant concentrations of BPA during the early stages of the corn borer's life cycle can result in various disorders that may be a consequence of endocrine disruption. The molecular mechanism by which BPA interferes with the physiological processes was also investigated. A significant induction was observed in the expression levels of the ecdysone-induced genes SnEcR and SnUSP, after injection of BPA and RH-5992. Additionally, we found that BPA acts as a very weak agonist of ecdysteroids in Bombyx mori derived Bm5 cell lines. From these cellular and molecular assays, our results brought evidence that BPA, like RH-5992, interferes with the ecdysteroidal pathways of the lepidopteran insect species.

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.

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.

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.

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis.

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3µM) and Bm5 cells (EC50=5.3µM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71µM and 0.00089µM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039µM; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18µM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.

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.

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.

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.