Study on anti-BmNPV mechanism of branched-chain amino acid aminotransferases in silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

Transcriptome analysis indicates the mechanisms of BmNPV resistance in Bombyx mori midgut.

Bombyx mori nucleopolyhedrovirus (BmNPV) caused serious economic losses in sericulture. Analyzing the molecular mechanism of silkworms (B. mori) resistance to BmNPV is of great significance for the prevention and control of silkworm virus diseases and the biological control of agricultural lepidopteran pests. In order to clarify the defense mechanisms of silkworms against BmNPV, we constructed a near isogenic line BC8 with high resistance to BmNPV through the highly BmNPV-resistant strain NB and the highly BmNPV-susceptible strain 306. In this study, RNA-Seq technique was used to analyze the transcriptome level differences in the midgut of BC8 and 306 following BmNPV infection. A total of 1350 DEGs were identified. Clustering analysis showed that these genes could be divided into 8 clusters with different expression patterns. Functional annotations based on GO and KEGG analysis indicated that they were involved in various metabolism pathways. Finally, 32 BmNPV defense responsive genes were screened. They were involved in metabolism, reactive oxygen species (ROS), signal transduction and immune response, and insect hormones. The further verification shows that HSP70 should participate in resistance responses of anti-BmNPV. These findings have paved the way in further functional characterization of candidate genes and subsequently can be used in breeding of BmNPV resistance dominant silkworms.

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.

The dual roles of three MMPs and TIMP in innate immunity and metamorphosis in the silkworm, Bombyx mori.

The matrix metalloproteinases (MMPs) and their endogenous inhibitory factors, tissue inhibitors of metalloproteinases (TIMPs), are implicated in many diseases. However, the mammalian MMPs (> 20) and TIMPs (> 3) are larger in number, and so little is known about their individual roles in organisms. Hence, we have systematically studied the roles of all three MMPs and one TIMP in silkworm innate immunity and metamorphosis. We observed that MMPs and TIMP are highly expressed during the pupation stage of the silkworms, and TIMP could interact with each MMPs. High-activity MMPs and low-activity TIMP may enhance the infection of B. mori nucleopolyhedrovirus in both in vitro and in vivo. MMPs' knockout and TIMP overexpression delayed silkworm development and even caused death. Interestingly, different MMPs' knockout led to different tubular tissue dysplasia. These findings provide insights into the conserved functions of MMPs and TIMP in human organogenesis and immunoregulation.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Involvement of Epidermis Cell Proliferation in Defense Against Beauveria bassiana Infection.

Entomopathogenic fungi Beauveria bassiana can infect many species of insects and is used as a biological pesticide world-wide. Before reaching the hemocoel, B. bassiana has to penetrate the integument which is composed of a thick chitin layer and epidermal cells. Some chitinase, protease and lipase secreted by B. bassiana are probably involved in the fungal penetration of the integument. While microscopic proof is needed, it is difficult to locate the precise infection sites following the traditional method of immersion infection. Consequently, we developed a new method to inoculate conidia solution into a single fixed-site on the back of one segment. This fixed-site infection method is pathogenic but it is also dose dependent. Using the fixed-site infection protocol, it is also very convenient to track hyphae inside the cuticle layer by light and transmission electron microscopy. The fact that few hyphae were detected inside the chitin layer after fixed-site infection with mutant ΔBPS8, a protease secreted during fungi germination, indicates that this method is suitable for screening genes involved in penetrating the integument in large scale. We also found that melanization occurs before new hyphae penetrate the chitin layer. Most importantly, we discovered that fungal infection can induce epidermal cell proliferation through DNA duplication and cell division, which is essential for the host to defend against fungal infection. Taken together the fixed-site infection method may be helpful to determine the mechanism of fungal and host interaction in the integument so as to effectively exert fungal biological virulence.

Unraveling the innate immune responses of Bombyx mori hemolymph, fat body, and midgut to Bombyx mori nucleopolyhedrovirus oral infection by metabolomic analysis.

Bombyx mori nucleopolyhedrovirus (BmNPV) infection causes a series of physiological and pathological changes in Bombyx mori (B. mori). Here, a metabolomic study of the innate immunity organs including hemolymph, fat body, and midgut of the silkworm strain Dazao following BmNPV challenge was conducted to reveal the metabolic variations in B. mori. Compared to the control, 4964 and 4942 features with 4077 and 4327 high-quality features were generated under positive and negative modes, respectively, from BmNPV-infected larvae. The principal component analysis and supervised learning method using partial least squares discrimination analysis demonstrated good analytical stability and experimental reproducibility of the metabolic profiles. Based on database annotations, a total of 296, 108, and 215 differential expressed metabolites (DEMs) were identified from BmNPV-infected group of hemolymph, fat body, and midgut, respectively, which were all mainly grouped into carboxylic acids and derivatives, fatty acyls, and glycerophospholipids. Kyoto Encyclopedia of Genes and Genomes Database enrichment analysis of the DEMs showed that amino acid metabolism was increased at 24 h after BmNPV infection. BmNPV induction was adopted to significantly alter a series of immune-related pathways including phospholipase D signaling pathway, FoxO signaling pathway, metabolism of xenobiotics by cytochrome P450, melanogenesis, membrane transport, carbohydrate metabolism, and lipid metabolism. The different levels of expression of several DEMs including l-glutamate, naphthalene, 3-succinoylpyridine 1-acyl-sn-glycerol 3-phosphate, and l-tyrosine which were involved in those pathways exhibited the immune responses of B. mori to BmNPV infection. Our findings are valuable for a better understanding of the antiviral mechanism of B. mori underlying the interaction between the silkworm and BmNPV.

The effects of quercetin combined with nucleopolyhedrovirus on the growth and immune response in the silkworm (Bombyx mori).

Flavonoids are secondary metabolites that help plants resist insect attack. It can resist insect attack by inhibiting insect immune defense, and pathogens can also inhibit insect immune defense. It is speculated that the combination of flavonoids and pathogens may inhibit the immune defense and have stronger toxicity to silkworm. In this study, the combined treatment of quercetin with Bombyx mori nuclear polyhedrosis virus (BmNPV) had significant negative effects on the growth and survival of silkworm compared with BmNPV group. The detoxifying enzyme activity of BmNPV group was significantly increased at 96 h, while the activity of the combined treatment group was significantly decreased with the increase of quercetin exposure time (72 or 96 h). The activity of antioxidant enzymes also showed a similar trend, that was, the activity of antioxidant enzymes in the combined treatment group also decreased significantly with the increase of quercetin exposure time, which led to the increase of reactive oxygen species content. The silkworm cells would produce lipid peroxidation, malondialdehyde content was significantly increased, so that the expression of immune-related genes (the antimicrobial peptide, Toll pathway, IMD pathway, JAK-STAT pathway, and melanin genes) were decreased, leading to the damage of the immune system of silkworm. These results indicated that quercetin combined with BmNPV could inhibit the activities of protective enzymes and lead to oxidative damage to silkworm. It can also affect the immune response of the silkworm, and thus resulting in abnormal growth. This study provides the novel conclusion that quercetin accumulation will increase the susceptibility of silkworm to pathogens.

The identification of nuclear factor Akirin with immune defense role in silkworm, Bombyx mori.

Akirins, highly conserved nuclear factors, regulate diverse physiological processes such as innate immunity. The biological functions of Akirins have extensively been studied in vertebrates and many invertebrates; however, there is no report so far on lepidopteran insects. In the present study, we identified and characterized a novel Akirin from the silkworm, Bombyx mori (designated as BmAkirin), and explored its potential roles in innate immunity. The expression analysis revealed the unequal mRNA levels of BmAkirin in all the tested tissues; however, the gene's transcription level was highest in testis, followed by ovaries and hemocytes. It also had significant expression levels at the early stages of embryonic development. Expression of BmAkirin in fat bodies and hemocytes exhibited an increase in various degrees when challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria. Immunofluorescence analysis showed BmAkirin protein was prominently localized in the nucleus. Knockdown of BmAkirin strongly reduced the expression of AMPs and decreased the survival ability of larva upon immune stimulation. Moreover, the bacterial clearance ability of larvae was also decreased following the depletion of BmAkirin. Collectively, our results demonstrate that BmAkirin plays an indispensable role in the innate immunity of Bombyx mori (B. mori) by positively modulating AMPs expression in vivo.

The hemolymph melanization response is related to defence against the AcMNPV infection in Bombyx mori.

Melanization is mediated by the prophenoloxidase (proPO) activation cascade and plays an important role in the arthropods immune system. Previously, we found that the hemolymph of the p50 strain does not perform melanization after infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, this mechanism is still unclear. In this study, the underlying mechanism of the inhibition of hemolymph melanization was investigated by analysing the AcMNPV-susceptible or -resistant silkworm strains after inoculation with AcMNPV. The results showed that the level of hemolymph melanization was higher in resistant strain C108 than in susceptible strain p50 at the late stage (72 to 120 h postinoculation). The PO activity decreased significantly at the late stage of infection (72 to 120 hpi), and the expression of BmPPO1 and BmPPO2 was downregulated in p50. However, the PO activity increased in the resistant strain C108, while the expression level of BmPPO1 and BmPPO2 displayed no significant changes. The expression of the BmPPAE gene was upregulated in two strains during viral infection. In addition, the hemolymph melanization can weaken the viral activity in vitro. Our results suggested that the silkworm hemolymph melanization response is related to defence against the AcMNPV infection.

Bombyx mori Apolipophorin-III inhibits Beauveria bassiana directly and through regulating expression of genes relevant to immune signaling pathways.

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmßGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmßGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Identification and location of sericin in silkworm with anti-sericin antibodies.

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.

Lineage-specific gene evolution of innate immunity in Bombyx mori to adapt to challenge by pathogens, especially entomopathogenic fungi.

Bombyx mori is a model species of Lepidoptera, in which 21 gene families and 220 genes have been identified as involved in immunity. However, only 45 B. mori - Drosophila melanogaster - Anopheles gambiae - Apis mellifera - Tribolium castaneum 1:1:1:1:1 orthologous genes were identified. B. mori has unique immune factors not found in D. melanogaster - A. gambiae - A. mellifera - T. castaneum. Pattern recognition receptors, signal transducers and effector genes for antifungal immune responses in B. mori have evolved through expansion and modification of existing genes. This review summarizes the current knowledge of the antifungal immune responses of B. mori and focuses on the lineage-specific gene evolution used by Lepidoptera to adapt to the challenge by pathogens, especially entomopathogenic fungi.

Bmcas-1 plays an important role in response against BmNPV infection in vitro.

Apoptosis, as one kind of innate immune system, is involved in host response against pathogens innovation. Caspases play a vital role in the execution stage of host cell apoptosis. It has been reported that Bmcaspase-1 (Bmcas-1) has a close relationship with Bombyx mori nucleopolyhedrovirus (BmNPV) infection for its differentially expressed patterns after viral infection. However, its underlying response mechanism is still unclear. The significant differential expression of Bmcas-1 in different tissues of differentially resistant strains revealed its vital role in BmNPV infection. To further validate its role in BmNPV infection, budded virus (BV)-eGFP was analyzed after knockdown and overexpression of Bmcas-1 by small interfering RNA and the pIZT-mCherry vector, respectively. The reproduction of BV-eGFP obviously increased at 72 h after knockdown of Bmcas-1, and decreased after overexpression in BmN cells. Moreover, the conserved functional domain of Cas-1 among different species and the closed evolutionary relationship of Cas-1 in Lepidoptera hinted that Bmcas-1 might be associated with apoptosis, and this was also validated by the apoptosis inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK. Therefore, Bmcas-1 plays an essential antiviral role by activating apoptosis, and this result lays a fundament for clarifying the molecular mechanism of silkworm in response against BmNPV infection and breeding of resistant strains.

A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins.

Previously, we found that nodule formation, a cellular defense response in insects, is regulated by humoral factors called C-type lectins in the hemolymph. To elucidate the factors that elicit nodule formation following the recognition of microorganisms by C-type lectins, a reproducible quantitative in vitro assay system was constructed. Then, using this system, the inhibitory activities of antisera raised against hemolymph proteases (HPs), serine protease homologues (SPHs), and pathogen-associated molecular pattern (PAMP)-recognition proteins were assessed. Among the antisera raised against HP and SPH, only that against HP8, a terminal proteinase that activates Spätzle, consistently inhibited in-vitro nodule-like aggregate formation in all three tested microorganisms, Micrococcus luteus, Escherichia coli, and Saccharomyces cerevisiae. Antisera raised against C-type lectins, BmLBP, and BmMBP also inhibited nodule-like aggregate formation, while those against ß-glucan recognition proteins and peptidoglycan recognition protein-S1 did not. Microorganisms pretreated with hemolymph, which contains HP8 and C-type lectins, also induced nodule-like aggregate formation, indicating that nodulation factors are present on microbial cells. Furthermore, antisera raised against HP8, BmLBP, and BmMBP showed inhibitory activities in the in vivo nodule formation system using Bombyx mori larvae. Thus, two humoral factors in the hemolymph of B. mori larvae, BmHP8 and C-type lectins, were found to play significant roles in eliciting the cellular defense response of nodule formation.

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.

iTRAQ-based quantitative proteomic analysis of silkworm infected with Beauveria bassiana.

Beauveria bassiana is a harmful pathogen to the economically important insect silkworm, always causes serious disease to the silkworm, which results in great losses to the sericulture industry. In order to explore the silkworm (Bombyx mori) response to B. bassiana infection, differential proteomes of the silkworm responsive to B. bassiana infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) at the different stage of the 3rd instar silkworm larvae. Among the 5040 proteins identified with confidence level of ≥95 %, total 937 proteins were differentially expressed, of which 488 proteins were up-regulated and 449 proteins were down-regulated. 23, 15, 250, 649 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the B. bassiana infected larvae at 18, 24, 36, 48 h post infection (hpi) respectively. Based on GO annotations, 6, 4, 128, 316 DEPs were involved in biological processes, 12, 5, 143, 376 DEPs were involved in molecular functions, and 6, 3, 108, 256 DEPs were involved in cell components at 18, 24, 36, 48 hpi respectively. KEGG pathway analysis displayed that 18, 12, 210, 548 DEPs separately participated in 63, 35, 201, 264 signal transduction pathways at different time of infection, and moreover a higher proportion of DEPs involved in metabolic pathways. The cluster analysis on the DEPs of different infection stages distinguished a co-regulated DEP, lysozyme precursor, which was up-regulated at both the mRNA level and the protein level, indicating that the lysozyme protein kept playing an important role in defending the silkworm against B. bassiana infection. This was the first report using an iTRAQ approach to analyze proteomes of the whole silkworm against B. bassiana infection, which contributes to better understanding the defense mechanisms of silkworm to B. bassiana infection and provides important experimental data for the identification of key factors involved in the interaction between the pathogenic fungus and its host.

Insights Into the Antiviral Pathways of the Silkworm Bombyx mori.

The lepidopteran model silkworm, Bombyx mori, is an important economic insect. Viruses cause serious economic losses in sericulture; thus, the economic importance of these viruses heightens the need to understand the antiviral pathways of silkworm to develop antiviral strategies. Insect innate immunity pathways play a critical role in the outcome of infection. The RNA interference (RNAi), NF-kB-mediated, immune deficiency (Imd), and stimulator of interferon gene (STING) pathways, and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway are the major antiviral defense mechanisms, and these have been shown to play important roles in the antiviral immunity of silkworms. In contrast, viruses can modulate the prophenol oxidase (PPO), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and the extracellular signal-regulated kinase (ERK) signaling pathways of the host to elevate their proliferation in silkworms. In this review, we present an overview of the current understanding of the main immune pathways in response to viruses and the signaling pathways modulated by viruses in silkworms. Elucidation of these pathways involved in the antiviral mechanism of silkworms furnishes a theoretical basis for the enhancement of virus resistance in economic insects, such as upregulating antiviral immune pathways through transgenic overexpression, RNAi of virus genes, and targeting these virus-modulated pathways by gene editing or inhibitors.

Using silkworms to search for lactic acid bacteria that contribute to infection prevention and improvement of hyperglycemia.

Bombyx mori, the silkworm, has biological functions in common with mammals, including humans. Since the molecular design of silkworm's innate immune system is analogous to that of mammals, understanding the silkworm's innate immunity is expected to contribute to the control of infection in humans. It is also possible to use silkworms to explore foodstuffs that activate innate immunity. Lactic acid bacteria have long been used in the production of fermented foods, and in recent years, their use as supplements has been attracting attention. Using silkworms, which are laboratory animals, functional lactic acid bacteria can be explored and isolated at low cost. Fermented foods produced by this method are expected to contribute to the maintenance of human health. In addition to the immune system, humans and silkworms share a common mechanism for maintaining blood glucose homeostasis, and it is possible to construct a pathological model of diabetes and search for therapeutic substances using silkworms. Taken together, we propose that the silkworm is useful for assessing the functions of lactic acid bacterial for health purposes.