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.

Applications and Potentials of a Silk Fibroin Nanoparticle Delivery System in Animal Husbandry.

Silk fibroin (SF), a unique natural polymeric fibrous protein extracted from Bombyx mori cocoons, accounts for approximately 75% of the total mass of silk. It has great application prospects due to its outstanding biocompatibility, biodegradability, low immunogenicity, and mechanical stability. Additionally, it is non-toxic and environmentally friendly. Nanoparticle delivery systems constructed with SF can improve the bioavailability of the carriers, increase the loading rates, control the release behavior of the deliverables, and enhance their action efficiencies. Animal husbandry is an integral part of agriculture and plays a vital role in the development of the rural economy. However, the pillar industry experiences a lot of difficulties, like drug abuse while treating major animal diseases, and serious environmental pollution, restricting sustainable development. Interestingly, the limited use cases of silk fibroin nanoparticle (SF NP) delivery systems in animal husbandry, such as veterinary vaccines and feed additives, have shown great promise. This paper first reviews the SF NP delivery system with regard to its advantages, disadvantages, and applications. Moreover, we describe the application status and developmental prospects of SF NP delivery systems to provide theoretical references for further development in livestock production and promote the high-quality and healthy development of animal husbandry.

Insights into midgut cell types and their crucial role in antiviral immunity in the lepidopteran model Bombyx mori.

The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect's physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect's innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect's immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms.

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.

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.

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.

Apolipoprotein D facilitate the proliferation of BmNPV.

The silkworm, Bombyx mori, a model Lepidopteran specie, is an important economic insect. It is specifically infected by Bombyx mori nucleopolyhedrovirus (BmNPV), causing huge losses to the sericulture industry. Therefore, the understandings of the interaction mechanism between BmNPV and the host will help to provide the theoretical basis for the sericulture industry to control BmNPV. Apolipoprotein D (ApoD) is a member of lipid transport family and capable of binding to a variety of lipophilic ligands. ApoD is mainly used in neurodegenerative disease research in mammals, and there is little research on ApoD against viruses. Here, we explored the effects of Bombyx mori Apolipoprotein D (BmApoD) on BmNPV replication. We knocked out and overexpressed BmApoD in BmN cells and infected them with Bombyx mori nucleopolyhedrovirus (BmNPV). The results showed that BmApoD promote the replication of BmNPV in BmN cells. It was also confirmed that BmApoD promote the replication of BmNPV after knocking down BmApoD in silkworm larvae. This study is the first to explore the role of ApoD in insect-virus interactions, providing new insights into the functional role of ApoD.

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.

Global Metabolic Profiling of Baculovirus Infection in Silkworm Hemolymph Shows the Importance of Amino-Acid Metabolism.

Viruses rely on host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Infection of the silkworm, Bombyx mori, by Bombyx mori nucleopolyhedrovirus (BmNPV), has been studied extensively in the past to unravel interactions between baculoviruses and their lepidopteran hosts. To understand the interaction between the host metabolic responses and BmNPV infection, we analyzed global metabolic changes associated with BmNPV infection in silkworm hemolymph. Our metabolic profiling data suggests that amino acid metabolism is strikingly altered during a time course of BmNPV infection. Amino acid consumption is increased during BmNPV infection at 24 h post infection (hpi), but their abundance recovered at 72 hpi. Central carbon metabolism, on the other hand, particularly glycolysis and glutaminolysis, did not show obvious changes during BmNPV infection. Pharmacologically inhibiting the glycolytic pathway and glutaminolysis also failed to reduce BmNPV replication, revealing that glycolysis and glutaminolysis are not essential during BmNPV infection. This study reveals a unique amino acid utilization process that is implemented during BmNPV infection. Our metabolomic analysis of BmNPV-infected silkworm provides insights as to how baculoviruses induce alterations in host metabolism during systemic infection.

Identification of Silkworm Hemocyte Subsets and Analysis of Their Response to Baculovirus Infection Based on Single-Cell RNA Sequencing.

A wide range of hemocyte types exist in insects but a full definition of the different subclasses is not yet established. The current knowledge of the classification of silkworm hemocytes mainly comes from morphology rather than specific markers, so our understanding of the detailed classification, hemocyte lineage and functions of silkworm hemocytes is very incomplete. Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of the baculoviruses and a major pathogen that specifically infects silkworms (Bombyx mori) and causes serious losses in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of hemocytes in BmNPV and mock-infected larvae to comprehensively identify silkworm hemocyte subsets and determined specific molecular and cellular characteristics in each hemocyte subset before and after viral infectmadion. A total of 20 cell clusters and their potential marker genes were identified in silkworm hemocytes. All of the hemocyte clusters were infected by BmNPV at 3 days after inoculation. Interestingly, BmNPV infection can cause great changes in the distribution of hemocyte types. The cells appearing in the infection group mainly belong to prohemocytes (PR), while plasmatocytes (PL) and granulocytes (GR) are very much reduced. Furthermore, we found that BmNPV infection suppresses the RNA interference (RNAi) and immune response in the major hemocyte types. In summary, our results revealed the diversity of silkworm hemocytes and provided a rich resource of gene expression profiles for a systems-level understanding of their functions in the uninfected condition and as a response to BmNPV.

Heat Shock Protein 75 (TRAP1) facilitate the proliferation of the Bombyx mori nucleopolyhedrovirus.

The viruses utilize multiple cellular proteins to facilitate their proliferation. The Heat Shock Protein (HSP), the highly conserved protein in eukaryotes and prokaryotes, plays a critical role in facilitating viral proliferation. However, less is known about the role of the HSPs in the life cycles of the Baculoviruses. We constructed recombinant Bombyx mori nucleopolyhedrovirus and discovered the Heat Shock Protein 75 (TRAP1) in the B. mori ovary (BmN) cells by the co-immunoprecipitation experiment using the GP64 (glycoprotein 64) as the bait protein. Tissue expression profile analysis of B. mori indicated that the TRAP1 gene has higher expression levels in the ovary, midgut, and hemolymph. Down-regulation of TRAP1 via RNA interference (RNAi) and geldanamycin (GA, a TRAP1 inhibitor) treatment can reduce the expression level of the major capsid protein VP39 (viral protein 39) of BmNPV. In contrast, the up-regulation of TRAP1 via overexpression can increase the expression level of the VP39. These results indicated that the TRAP1 of B. mori could facilitate the proliferation of the BmNPV. This study provided new insights into the function of TRAP1, and the basic mechanisms of the baculoviruses life cycle for disease prevention.

The piRNA response to BmNPV infection in the silkworm fat body and midgut.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that causes huge losses to the silkworm industry but the piRNA responses during BmNPV infection in the silkworm remain uninvestigated. Here, silkworm piRNA profiles of uninfected and BmNPV-infected fat body and midgut were determined by high-through sequencing in the early stages of BmNPV infection. A total of 2675 and 3396 genome-derived piRNAs were identified from fat body and midgut, respectively. These genome-derived piRNAs mainly originated from unannotated instead of transposon regions in the silkworm genome. In total, 572 piRNAs were associated with 280 putative target genes in fat body and 805 piRNAs with 380 target genes in midgut. Compared to uninfected tissues, 322 and 129 piRNAs were significantly upregulated in BmNPV-infected fat body and midgut, respectively. In addition, 276 and 117 piRNAs were significantly downregulated. Moreover, differentially expressed (DE) piRNAs during BmNPV infection differed significantly between fat body and midgut. Putative DE piRNA-targeted genes were associated with "response to stimulus" and "environmental information processing" in fat body after infection with BmNPV, which may indicate an active piRNA response to BmNPV infection in fat body. This study may lay the foundation for future research of the potential roles of the piRNA pathway and specific piRNAs in BmNPV pathogenesis.

Antimicrobial Peptides as Potential Antiviral Factors in Insect Antiviral Immune Response.

Antimicrobial peptides (AMPs) with antiviral activity (antiviral peptides: AVPs) have become a research hotspot and already show immense potential to become pharmaceutically available antiviral drugs. AVPs have exhibited huge potential in inhibiting viruses by targeting various stages of their life cycle. Insects are the most speciose group of animals that inhabit almost all ecosystems and habitats on the land and are a rich source of natural AMPs. However, insect AVP mining, functional research, and drug development are still in their infancy. This review aims to summarize the currently validated insect AVPs, explore potential new insect AVPs and to discuss their possible mechanism of synthesis and action, with a view to providing clues to unravel the mechanisms of insect antiviral immunity and to develop insect AVP-derived antiviral drugs.