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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.

RNAi is considered a major antiviral defense mechanism in insects, but its relative importance as compared to other antiviral pathways has not been evaluated comprehensively. Here, it is attempted to give an overview of the antiviral defense mechanisms in Drosophila that involve both RNAi and non-RNAi. While RNAi is considered important in most viral infections, many other pathways can exist that confer antiviral resistance. It is noted that very few direct recognition mechanisms of virus infections have been identified in Drosophila and that the activation of immune pathways may be accomplished indirectly through cell damage incurred by viral replication. In several cases, protection against viral infection can be obtained in RNAi mutants by non-RNAi mechanisms, confirming the variability of the RNAi defense mechanism according to the type of infection and the physiological status of the host. This analysis is aimed at more systematically investigating the relative contribution of RNAi in the antiviral response and more specifically, to ask whether RNAi efficiency is affected when other defense mechanisms predominate. While Drosophila can function as a useful model, this issue may be more critical for economically important insects that are either controlled (agricultural pests and vectors of diseases) or protected from parasite infection (beneficial insects as bees) by RNAi products.

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 .

Insects rely on their innate immune system to defend against pathogens, and the Toll signaling pathway plays an important role in immune regulation. Our previous studies have shown that BmToll9-1 functions as a positive regulator in the Toll pathway. This study seeks to elucidate the role of BmToll9-1, as a sensor to bacterial challenge, in modulating larval development and downstream Toll signaling pathways. Silkworm larvae were subjected to infection with either Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus bacteria following silencing of BmToll9-1 by RNA interference (RNAi). This bacterial challenge triggered a compensatory re-induction of BmToll9-1 expression, which resulted in the recovery of larval weight and size to levels observed in untreated controls. Furthermore, upon bacterial infection of BmToll9-1-silenced larvae, there was an up-regulation in the expression of both signaling genes in the Toll pathway and downstream effector genes, with a marked preference for Gram-negative bacteria. These results highlight the involvement of BmToll9-1 in the Toll signaling pathway as a positive regulator, influencing silkworm development. Additionally, BmToll9-1 and BmToll9-2 were cross-validated to be genetically distinct genes, even though they were confirmed to be functionally analogous in the silkworm.

As obligatory intracellular parasites, viruses must rely on metabolic reprogramming of host cells to meet their replication needs. Baculovirus is an important biopesticide and a vector for the preparation of biological products. In addition, one of its representative species, Bombyx mori nucleopolyhedrovirus (BmNPV- Baculoviridae ), also causes huge losses to the insect industry. In our previous study, amino acid metabolism has been found to play a crucial role in the BmNPV infection process. However, the mechanisms by which BmNPV reprograms host amino acid metabolism remains unclear. In fact, current insights in the importance of amino acid metabolism are limited to the impact of glutamine on viral infection. Therefore, unraveling the mechanism of amino acid metabolism reprogramming induced by baculovirus would advance this field of research to a great extent. In this study, targeted metabolomics revealed that the preferred amino acids of BmNPV budded virus (BV) include arginine, lysine, proline, isoleucine, histidine and others. In addition, most of the viral amino acids were found to be increased in the hemolymph of BmNPV infected silkworms at the later stage of infection, especially arginine, valine, phenylalanine and others. Furthermore, the importance of arginine for BmNPV proliferation was validated. Next, we confirmed that the expression of the arginine transporter Slc7a6 was strongly induced by BmNPV infection and that Slc7a6 could promote arginine uptake to support BmNPV proliferation in host cells. Moreover, using Slc7a6 knockout cells which eliminate extracellular arginine uptake, we confirmed that BmNPV could induce mitochondrial autophagy, thereby supplementing intracellular arginine and providing necessary amino acids for BmNPV proliferation. Overall, these findings support a model in which baculovirus (BmNPV) enhances the uptake of exogenous amino acids by inducing the expression of amino acid transporters and activating autophagy of organelles to maintain intracellular amino acid levels, thereby facilitating virus proliferation.

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.

In contrast to mammals, insects possess a separate machinery for processing of long dsRNAs into siRNAs for the cleavage of viral RNAs. The process of RNAi is considered very efficient in all insects once the delivery in the cytoplasm occurs such as during RNA virus replication. For the application of RNAi as insecticide to succeed, efficient uptake of intact dsRNA into the cytoplasm therefore is necessary, which seems to occur by natural mechanisms in the leaf beetles for which RNAi-based insecticides already have been marketed. In most insects, relatively high amounts of dsRNA are required to trigger gene silencing which raises questions regarding potential side effects. Besides, RNAi is considered as the major antiviral defense mechanism, at least in Drosophila , but not necessarily in all other insects. Following increasing evidence from the recent literature, it has become prudent to include the sensing of dsRNA as an immune trigger to evaluate the extent of the RNAi mechanism that is triggered by dsRNA. In this review, an overview of mechanisms is presented regarding how the recognition of dsRNA as a “pathogen-associated molecular pattern”, the multiple additional functions of the canonical siRNA factors and the modulation of the function of Dicer-2 and Ago-2 by dsRNA-binding proteins may complicate the efficiency of the exo-RNAi process and aggravate its application for pest control.

Although it is reported that the protein tyrosine phosphatase gene of baculovirus (group I nucleopolyhedrovirus) can induce enhanced locomotory activity (ELA) in caterpillars, our understanding of the host factors that are involved in the regulation of the behavioral change is still limited. Previously, single-nucleus RNA sequencing (snRNA-seq) was used to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte, muscle cell types and other unannotated cells in the silkworm larvae brains. Analysis of viral transcriptomes in each brain cell subset revealed that all brain cells could be infected by Bombyx mori nucleopolyhedrovirus (BmNPV) at 96 hours post infection but infection occurred at low levels. Furthermore, we found that chemosensory protein 3 (CSP3), encoding a small secreted protein that is possibly implicated in the transport of semiochemicals, was significantly up-regulated after BmNPV infection in most of the brain cell clusters. Knockdown of BmCSP3 resulted in significantly reduced ELA in BmNPV-infected silkworm larvae. In parallel, targeted metabolomics revealed significant shifts in the abundance of specific lipids and neurotransmitters. Subsequently, structural modeling and molecular dynamics experiments indicated that CSP3 has a large hydrophobic pocket that manifests significant flexibility and likely can accommodate divergent ligand structures or mixtures of them, including known neurotransmitters of the brain and (lyso)glycerophospholipids from larval head samples. In vitro binding assays have confirmed the interaction of several neurotransmitters and an eicosanoid to purified BmCSP3 protein. Our study provides insights into the regulation of insect behavior following analysis of viral infection at the single-cell transcriptome level and reveals an unexpected function for CSP proteins in the insect brain.

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.

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.