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Single-domain von Willebrand factor type C proteins (SVWCs), primarily found in arthropods, responds to infections caused by various pathogens. Three SVWCs have been identified in the silkworm and BmSVWC2 might play a crucial role in the immune system. However, the regulatory mechanism of BmSVWC2 remains largely unknown. This study aimed to investigate the biochemical functions of BmSVWC2 in the immune system of B. mori comprehensively. Phylogenetic analysis revealed that BmSVWC1, BmSVWC3, and BmSVWC2 were distributed in diverse groups, suggesting distinct biochemical functions. The mRNA and protein levels of BmSVWC2 increased significantly in response to bacterial infection. BmSVWC2 exhibited clear binding activity to the polysaccharide pathogen-associated molecular patterns of bacteria and fungi, enhancing bacterial clearance in vivo but not in vitro. RNA-sequencing assays of the fat body and hemocytes showed that numerous immune genes were markedly up-regulated with higher level of BmSVWC2, primarily affecting recognition, signaling, and response production of the Toll and immune deficiency (IMD) signaling pathways. This led to the production of various antimicrobial peptides and significant antibacterial activities in the hemolymph. BmSVWC2 up-regulated phagocytosis-related genes in the fat body and hemocytes, and phagocytosis assays confirmed that BmSVWC2 improved the phagocytic ability of hemocytes against bacteria. Additionally, BmSVWC2 induced the expression of nitric oxide synthetase (NOS) in the fat body, and bioassays confirmed that BmSVWC2 increased NOS activity in the fat body and hemolymph, resulting in nitric oxide accumulation. However, BmSVWC2 did not affect phenoloxidase activity, despite it caused differential expression of a few serine proteases and serine protease inhibitors. Co-immunoprecipitation and mass spectrometry assays showed that BmSVWC2 interacted with 30 K proteins, such as 30 K protein 2, 30 K pBmHPC-19, 30 K 19G1-like, 30 K protein 8, 30 K protein 7, 30 K pBmHPC-23, and low molecular mass lipoprotein 4-like. Our study provides a comprehensive characterization of BmSVWC2 and elucidates the mechanism underlying its regulation of immune responses activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Toll and immune deficiency (IMD) signaling pathways in insects are highly conserved in evolution and regulate the expression of antimicrobial peptides (AMPs) and other immune-related genes mainly through nuclear factor-kappa B (NF-κB) transcription factors. However, the differences of NF-κB transcription factors Rels and Relish in the expression regulation of AMPs and other immune-related genes in silkworm ( Bombyx mori ) have not been systematically reported. In this study, the BmRelA , BmRelB and BmRelish1 genes were cloned and their eukaryotic cell overexpression vectors were constructed. After the recombinant vectors were transfected into BmE and BmN cells, the expression of AMPs and immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of AMP genes Defensin2 and Gloverin2 was mainly regulated by Relish, the expression of Moricin was mainly regulated by RelA and RelB, and the expression of other AMP genes was jointly regulated by both. In addition, the expression levels of peptidoglycan recognition proteins (PGRPs), β-1, 3-glucan recognition proteins (βGRPs), lysozymes (Lys) and lysozyme-like proteins (LLPs), and nitric oxide synthase (NOS) were up-regulated to varying degrees in different cell lines in response to RelA, RelB and Relish1, suggesting that the expression of these immune-molecules was also regulated by Toll or IMD pathways in silkworm. Compared with the regulatory specificity of transcription factors in Drosophila Toll and IMD signaling pathways on the expression of AMPs, this study found that the regulatory patterns of Rels and Relish1 on the expression of AMPs in silkworm are more complex, which provides an experimental basis for further analysis of the effect mechanism and feedback mechanism of Toll and IMD pathways in insects.

The infection caused by Nosema bombycis often known as pebrine, is a devastating sericulture disease. The infection can be transmitted to the next generation through eggs laid by infected female Bombyx mori moths (transovarial) as well as with N. bombycis contaminated food (horizontal). Most diagnoses were carried out in the advanced stages of infection until the time that infection might spread to other healthy insects. Hence, early diagnosis of pebrine is of utmost importance to quarantine infected larvae from uninfected silkworm batches and stop further spread of the infection. The findings of our study provide an insight into how the silkworm larval host defence system was activated against early N. bombycis transovarial infection. The results obtained from transcriptome analysis of infected 2 nd instar larvae revealed significant (adjusted P-value < 0.05) expression of 1888 genes of which 801 genes were found to be upregulated and 1087 genes were downregulated when compared with the control. Pathway analysis indicated activation of the immune deficiency (IMD) pathway, which shows a potential immune defence response against pebrine infection as well as suppression of the melanin synthesis pathway due to lower expression of prophenoloxidase activating enzyme (PPAE). Liquid chromatography mass spectrometry (LC-MS/MS) analysis of haemolymph from infected larvae shows the secretion of serpin binding protein of N. bombycis which might be involved in the suppression of the melanization pathway. Moreover, among the differentially expressed genes, we found that LPMC-61, yellow-y, gasp and osiris 9 can be utilised as potential markers for early diagnosis of transovarial pebrine infection in B. mori. Physiological as well as biochemical roles and functions of many of the essential genes are yet to be established, and enlightened research will be required to characterize the products of these genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

COVID-19, caused by the novel coronavirus SARS-CoV-2, has presented a significant challenge to global health, security, and the economy. Vaccination is considered a crucial measure in preventing virus transmission. The silkworm bioreactor has gained widespread usage in antigen presentation, monoclonal antibody preparation, and subunit vaccine development due to its safety, efficiency, convenience, and cost-effectiveness. In this study, we employed silkworm BmN cells and the silkworm MultiBac multigene co-expression system to successfully produce two prototype vaccines: a recombinant baculovirus vector vaccine (NPV) co-displaying the SARS-CoV-2 virus capsid protein and a capsid protein virus-like particle (VLP) vaccine. Following the purification of these vaccines, we immunized BALB/c mice to evaluate their immunogenicity. Our results demonstrated that both VLP and NPV prototype vaccines effectively elicited robust immune responses in mice. However, when equal inoculation doses between groups were compared, the recombinant NPV vaccine exhibited significantly higher serum antibody titers and increased expression of spleen cytokines and lymphocyte immune regulatory factors compared to the VLP group. These results suggested an increased immune efficacy of the recombinant NPV vaccine. Conversely, the VLP prototype vaccine displayed more pronounced effects on lymphocyte cell differentiation induction. This study successfully constructed two distinct morphological recombinant vaccine models and systematically elucidated their differences in humoral immune response and lymphocyte differentiation rate. Furthermore, it has fully harnessed the immense potential of silkworm bioreactors for vaccine research and development, providing valuable technical insights for studying mutated viruses like coronaviruses., Competing Interests: Declaration of competing interest Authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Bomb m 6 is one of the most representative allergens in silkworm pupae. The aim of the present study was to investigate the B-cell linear epitopes of Bomb m 6. Sequence propensity- and machine learning-based strategies were first used to predict 12 and 8 candidate epitopes, respectively. Then, 46 overlapping peptides [P1-P46; length, 15 amino acids (AAs); offset, 5 AAs] spanning the Bomb m 6 sequence were synthesized and tested for IgE/IgG reactivity by dot blotting. Seven IgE-binding epitopes and six IgG-binding epitopes were identified. AA61-80, AA136-150, and AA146-150 were identified as the shared dominant IgE/IgG-binding epitopes. The epitopes contain mainly α-helix and β-sheet structures and high proportions of hydrophobic AAs, and are distributed in charged regions. The major peptides of the epitopes were poorly resistant to simulated gastrointestinal fluid digestion. These findings provide a reference for the prevention of and immunotherapy for silkworm pupa allergy., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. All authors read and approved the final manuscript. All authors have seen and approved the final version of the manuscript being submitted. This article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. All authors declare that there is no conflict of interest in regards to publication of this research work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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

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&#95;chr7&#95;8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo&#95;chr7&#95;8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo&#95;chr7&#95;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&#95;chr7&#95;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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis. Staphylococcus aureus and Cutibacterium acnes are pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized that S. aureus and C. acnes interact to increase the innate immunity-activating properties of S. aureus. In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization by S. aureus. Autoclaved-killed S. aureus (S. aureus [AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applying S. aureus [AC] treated with C. acnes culture supernatant increased the silkworm hemolymph melanization. Adding C. acnes culture supernatant to the medium decreased the pH. S. aureus [AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreated S. aureus [AC]. S. aureus [AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity of S. aureus [AC] treated with hydrochloric acid was inhibited by protease treatment of S. aureus [AC]. These results suggest that acid treatment of S. aureus induces innate immune activation in silkworms and that S. aureus proteins are involved in the induction of innate immunity in silkworms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Matsumoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown., Methods: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain., Results: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects., Conclusion: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

The 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., (© 2021 Federation of European Biochemical Societies.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021 Wiley Periodicals LLC.)

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., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier B.V.)

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 β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dong, An, Lu, Tang, Chen, Huang, Hou, Shen, Zhang, Zhang, Xia and Zhao.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Tang, Li, Zhang, Chen, Gong, Wei, Tang, Liu, Geng, Pan, Beerntsen and Ling.)

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 BmβGRP4 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 BmβGRP1 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R 2  = 0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R 2  = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

Many parasites alter the host locomotory behaviors in a way that increases their fitness and progeny transmission. Baculoviruses can manipulate host physiology and alter the locomotory behavior by inducing 'hyperactivity' (increased locomotion) or 'tree-top disease' (climbing high up to the top before dying). However, the detailed molecular mechanism underlying virus-induced this hyperactive behavior remains elusive. In the present study, we showed that BmNPV invaded into silkworm brain tissue, resulting in severe brain damage. Moreover, BmNPV infection disturbed the insect hormone balance. The content of 20-hydroxyecdysone (20E) in hemolymph was much lower during the hyperactive stage, while the dopamine (DA) titer was higher than mock infection. Exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced ELA (enhanced locomotory activity), while dopamine stimulates this behavior. More specificity, injection of dopamine or its agonist promote this hyperactive behavior in BmNPV-infected larvae. Taking together, our findings revealed the important role of hormone metabolism in BmNPV-induced ELA., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 β-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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp&#95;SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Feng, Xia, Fei, Peng, Wang, Zhou, Wang, Swevers and Sun.)

Scavenger receptor class B (SR-B) is an extracellular transmembrane glycoprotein that plays a vital role in innate immunity. Although SR-Bs have been widely studied in vertebrates, their functions remained to elucidate in insects. Here, we identified and characterized a scavenger receptor class B member from the silkworm, Bombyx mori (designated as BmSCRB8). BmSCRB8 is broadly expressed in various immune tissues/organs, including fat body, gut, and hemocyte. Its expression is dramatically enhanced after challenge with different types of bacteria or pathogen-associated molecular patterns (PAMPs). The recombinant BmSCRB8 protein can detect different types of bacteria by directly binding to PAMPs and significantly improve the bacterial clearance in vivo. After knockdown of BmSCRB8, the pathogenic bacterial clearance was strongly impaired, and several AMP genes were down-regulated following E. coli challenge. Moreover, pathogenic bacteria's treatment following the depletion of BmSCRB8 remarkably decreased silkworm larvae's survival rate. Taken together, these results demonstrate that BmSCRB8 acts as a pattern recognition protein and plays an essential role in silkworm innate immunity by enhancing bacterial clearance and contributing to the production of AMPs in vivo., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cathepsin L protease belongs to the papain-like cysteine proteases family, plays indispensable roles in animals' pathological and physiological processes. However, little is known about Cathepsin L in silkworm, Bombyx mori. Herein, a novel Cathepsin L-like (Cat L-like) was cloned and identified from silkworm by the rapid amplification of cDNA ends (RACE). Cat L-like contains an intact open reading frame (ORF) of 1 668 bp and encodes 556 amino acid residues, consisting of a signal peptide, typical cathepsins' inhibitor&#95;I29, and pept&#95;C1 domain. Cat L-like is specifically and highly expressed in hemocytes. The cathepsin (including Cathepsin L, B, and H) crude extract from hemocytes had typical substrate specific catalytic activities and were sensitive to pH and temperature. Cat L-like up-regulated considerably after 20-hydroxyecdysone (20-E) administration, indicating that Cat L-like may be regulated by insect hormone. The responses of Cat L-like against bacterial infection suggest it may play essential roles in silkworm immunity. Overall, our studies provide a theoretical basis and insights to further investigate the functions of Cat L-like and in insects' innate immunity mechanisms., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bombyx mori cypovirus (BmCPV) is one of the most important pathogens causing severe disease to silkworm. Emerging evidence indicates that long noncoding RNAs (lncRNAs) play importantly regulatory roles in virus infection and host immune response. To better understand the interaction between silkworm, Bombyx mori and BmCPV, we performed a comparative transcriptome analysis on lncRNAs and mRNAs between the virus-infected and noninfected silkworm larvae midgut at two time points postinoculation. A total of 16,753 genes and 1845 candidate lncRNAs were identified, among which 356 messenger RNA (mRNAs) and 41 lncRNAs were differentially expressed (DE). Target gene prediction revealed that most of DEmRNAs (123) were coexpressed with 28 DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans- regulation by BmCPV-induced lncRNAs, and a regulatory network of DElncRNAs and DEmRNAs was then constructed. According to the network, many genes involved in apoptosis, autophagy, and antiviral response, such as ATG3, PDCD6, IBP2, and MFB1, could be targeted by different DElncRNAs, implying the essential roles of these genes and lncRNAs in BmCPV infection. In all, our studies revealed for the first time the alteration of lncRNA expression in BmCPV-infected larvae and its potential influence on BmCPV replication, providing a new perspective for host-cypovirus interaction studies., (© 2021 Wiley Periodicals LLC.)

Cathepsins belongs to the cysteine protease family, which are activated by an acidic environment. They play essential biological roles in the innate immunity and development of animals. Here, we identified a 62 kDa cathepsin L-like protease from the silkworm Bombyx mori. It contained putative conserved domains, including an I29 inhibitor domain and a peptidase C1A domain. The expression analysis revealed that cathepsin L-like was highly produced in the fat body, and 20-hydroxyecdysone (20 E) induced its expression. After challenge with three different types of heat-killed pathogens (Escherichia coli, Beauveria bassiana, and Bacillus cereus), the mRNA levels of cathepsin L-like significantly increased and displayed variable expression patterns in the immune tissues, suggesting its potential role in the innate immune response. The suppression of cathepsin L-like altered the expression of immune-related genes associated with the Toll and IMD pathway. Besides, autophagy-related genes such as Atg6, Atg8, VAMP2, Vps4, and syntaxin expression were also altered, indicating that cathepsin L-like regulates innate immunity and autophagy. Fluorescence microscopic analysis exhibited that cathepsin L-like was localized in the cytoplasm, and it was activated and dispersed throughout the cytoplasm and nucleus following the induction of anti-microbial autophagy. Altogether, our data suggest that cathepsin L-like may regulate the innate immune response and anti-microbial autophagy in the silkworm, B. mori., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Microsporidia are obligate single-celled eukaryote parasites. Microsporidian infection can cause large economic losses to beneficial insects such as silkworms and honey bees. Identification of resistance biomacromolecules and breeding of transgenic lines resistant to the microsporidian Nosema bombycis are important for disease management. We previously used transcriptome analysis to identify a guanylate binding protein family BmAtlastin-n gene that was significantly upregulated after Nosema bombycis infection, and we determined that the molecule was highly expressed in resistance-related tissues such as the midgut, fat body and the epidermis. The transgenic silkworm line overexpressing BmAtlastin-n biomolecules had economic characters similar to those of non-transgenic lines. The transgenic OE-BmAtlastin-n lines had significantly improved survival after microspore infection. We used RT-PCR and H&E staining to show that the number of spores in the transgenic lines was significantly lower than in the control lines. In this study, we identified a BmAtlastin-n macromolecule with resistance to N. bombycis and developed a transgenic line. The results improved understanding of the GBP protein family and provided biomacromolecule material for the treatment and prevention of microsporidia., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang.)

Insects are the largest group of animals. Nearly all organisms, including insects, have viral pathogens. An important domesticated economic insect is the silkworm moth Bombyx mori . B. mori nucleopolyhedrovirus (BmNPV) is a typical baculovirus and a primary silkworm pathogen. It causes major economic losses in sericulture. Baculoviruses are used in biological pest control and as a bioreactor. Silkworm and baculovirus comprise a well-established model of insect-virus interactions. Several recent studies have focused on this model and provided novel insights into viral infections and host defense. Here, we focus on baculovirus invasion, silkworm immune response, baculovirus evasion of host immunity, and enhancement of antiviral efficacy. We also discuss major issues remaining and future directions of research on silkworm antiviral immunity. Elucidation of the interaction between silkworm and baculovirus furnishes a theoretical basis for targeted pest control, enhanced pathogen resistance in economically important insects, and bioreactor improvement., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, Goldsmith and Xia.)

Nosema bombycis is a unicellular spore-forming obligate parasite, related to fungi, and causes infections in economically important animals and are opportunistic human pathogens. However, the mechanisms of host response to N. bombycis remain unclear. STING (stimulator of interferon genes) is an adapter protein involved in the innate immune response to pathogens. In this study, a transgenic gRNA vector containing BmSTING was constructed and microinjected to generate the transgenic line BmSTING Δ6bp/WT and BmSTING Δ5bp/WT in silkworms. The expression of BmSTING was significantly reduced in BmSTING Δ5bp/WT compared to non-transgenic silkworm. The mortality and LC3 (microtubule-associated protein 1 light chain 3) level in BmSTING Δ6bp/WT and BmSTING Δ5bp/WT was significantly decreased in the early infection stage of N. bombycis, but the transgenic silkworms died rapidly in the later stage. Furthermore, both BmSTING and LC3 were increased in BmE cell lines after infection with N. bombycis. This study highlights the role of STING-dependent pathways response to microsporidia in silkworm, Bombyx mori., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ&#95;0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ&#95;0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) are the most important pattern recognition receptors (PRRs) in insects. PGRPs can recognize pathogenic microorganism peptidoglycans (PGs) and play an important role in innate immunity. Twelve PGRPs have been identified in silkworms. However, the specific roles played by these PGPRs in the silkworm innate immune system have not been elucidated to date. In this study, we systematically investigated the biological functions of BmPGRP-S1 in silkworms. We observed that BmPGRP-S1 was highly expressed in silkworm immune-related organs and was upregulated in response to bacterial challenges. Furthermore, we determined that BmPGRP-S1 can bind to bacteria or PGs and activate antimicrobial peptide (AMP) expression. Inhibition of the expression of BmPGRP-S1 by siRNA reduced AMP gene expression in silkworms. Further experiments demonstrated that BmPGRP-S1 is involved in IMD pathway activation to induce AMP expression. Taken together, these results demonstrate that BmPGRP-S1 serves as a receptor to activate AMP gene expression through the IMD pathway to address bacterial challenges., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antifungal innate immunity is an important defence used by insects against entomogenous fungi. However, the downstream target antifungal peptides of different immune signalling pathways are unknown. We found that the Toll, Janus kinase/signal transducer and activator of transcription (Jak/STAT) and Immunodeficiency (IMD) signalling pathways in the silkworm, Bombyx mori, can be activated by Beauveria bassiana. Inhibition of the Toll, IMD and Jak/STAT signalling pathways reduced the antifungal activities of silkworm haemolymph. We verified the target antifungal peptides of different immune signalling pathways. The expression patterns of five anti-fungal peptide genes in silkworm larvae and BmN cells were detected after blocking or over-expressing the immune signalling pathways. The Toll signalling pathways mediated the expression of Bmcecropin A, Bmattacin 1 and Bmgloverin 2; IMD signalling pathways mediated Bmenbocin 1, Bmgloverin 2 and Bmattacin 1; Jak/STAT signalling pathways mediated Bmstorage protein 30K-19G1 (Bmsp 1), Bmattacin 1 and Bmcecropin A. These data indicated that anti-microbial peptide genes in B. mori evolved through expansion and selection of existing genes to adapt to the challenge of invasive microorganisms such as fungi. This information provides insight into the antifungal immune responses in B. mori and aids understanding of insect immune regulation mechanisms., (© 2020 The Royal Entomological Society.)

Bombyx mori latent virus (BmLV) is a positive, single-stranded insect RNA virus closely related to plant maculaviruses. BmLV was first isolated from Bombyx mori ovary-derived cell line BmN-4, and this virus has already infected most B. mori-derived cultured cell lines. We previously reported that small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways function cooperatively to maintain the amount of BmLV RNA for normal BmN-4 cell growth. On the other hand, BmLV does not propagate in B. mori larvae. Here we conducted BmLV injection into the larval body cavities of B. mori, and examined BmLV accumulation in larval ovaries where siRNA and piRNA pathways are both active, to investigate whether this in vivo resistance is governed by small RNA pathways. Expression levels of RNA-dependent RNA polymerase, coat protein, and p15 genes in BmLV-injected larval ovaries were extremely low compared with those in B. mori cultured cells, indicating that B. mori larval ovaries are more resistant to BmLV than B. mori cultured cells. We also sequenced small RNAs prepared from BmLV-injected larval ovaries and mapped them onto the BmLV genome. Although their amounts were very small, we were able to detect BmLV-derived small RNAs in the ovaries. According to their length distribution and nucleotide bias, they were likely to be siRNAs and piRNAs. These results suggest that B. mori ovaries can potentially produce small RNAs against BmLV, but the resistance of larval ovaries against BmLV is not dependent on RNA silencing pathways., (© 2020 Wiley Periodicals LLC.)

Adverse environmental conditions cause serious economic losses in sericulture; Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary biotic stress and high temperature is the major abiotic stress in this industry. B. mori heat shock protein 19.9 (Bmhsp19.9) overexpression was previously demonstrated to protect transgenic silkworm H19.9 against extreme temperature. This study analyzed the role of Bmhsp19.9 in H19.9A and H19.9B silkworm lines and BmE cells infected with BmNPV at regular and high temperatures. qPCR results showed that Bmhsp19.9 expression was upregulated in BmE cells and silkworm after BmNPV challenge. Bmhsp19.9 overexpression significantly inhibited BmNPV proliferation in BmE cells. The viral DNA content was significantly decreased in transgenic H19.9 silkworm compared to the control. These results suggested that Bmhsp19.9 was involved in antiviral immunity against BmNPV. Furthermore, Bmhsp19.9 overexpression protected BmE cells against BmNPV under high temperature shock. This indicates that Bmhsp19.9 is a promising candidate for improving silkworm resistance to biotic and abiotic stresses, thereby reducing sericulture losses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Integrins are transmembrane glycoproteins that are broadly distributed in living organisms. As a heterodimer, they contain an α and a β subunit, which are reported to be associated with various physiological and pathological processes. In the present study, a 2502 bp full-length cDNA sequence of Bmintegrin β1 was obtained from the silkworm, Bombyx mori. Bmintegrin β1 belongs to the β subunit of the integrin family and contains several typical structures of integrins. Gene expression profile analysis demonstrated that Bmintegrin β1 was ubiquitously expressed in all tested tissues and organs, with the maximum expression levels in fat body and hemocytes. The immunofluorescence results showed that Bmintegrin β1 was located in the cell membrane and widely distributed in fat bodies and different types of hemocytes. Bmintegrin β1 expression was remarkably increased after challenging with different kinds of bacteria and pathogen-associated molecular patterns (PAMPs). Further investigation revealed that Bmintegrin β1 could participate in the agglutination of pathogenic bacteria possibly through direct binding with the relative bacteria and PAMPs. Altogether, this study provides a novel insight into the immune functional features of Bmintegrin β1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction and Objectives: Moths are a significant source of indoor and outdoor aeroallergens. High prevalence of IgE-mediated sensitization was demonstrated in a group of patients with allergic respiratory diseases. There are no studies on adult stage of these moth species allergens involved in allergic respiratory reactions - the aim of this study., Material and Methods: 36 participants were included in an experimental study, submitted to skin prick test with Bombyx mori wing extract and six other common allergens, as well as Western blot analysis with incubated nitrocellulose membrane impregnated with silkworm moth extract and human IgE-antibody. The participants were divided into 3 groups: 1) 21 allergic patients whose skin prick test was positive to Bombyx mori wing extract, 2) eight allergic patients whose skin prick test was positive to mite and negative to Bombyx mori extract 3) seven negative non-allergic subjects., Results: Among the 21 participants from group 1, 19 serum samples reacted to Bombyx mori extract by Western blot. All of them reacted to a protein at 80 kDa and five other proteins (66, 50, 45, 37 and 30 kDa) were identified in more than 50% of the individuals tested, considered as major allergenic proteins. Sera from seven out of eight patients sensitized to house dust mite demonstrated IgE-reactivity to Bombyx mori extract by Western blot analysis. Serum samples from healthy participants did not react at all., Conclusion: Six major reactive proteins by immunoblot analysis from moth's wings sensitized patients can be potential allergens. The one at 80 kDa is the major protein, seen in all IgE-reactive patients from group 1 and in none from group 2, yet to be identified. Future studies should be conducted to better characterize these proteins., (Copyright © 2020 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

The disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) has always been difficult to control, resulting in tremendous economic losses in the sericulture industry. Although much has been learned about the impact of noncoding RNAs on pathogenesis, the role of circular RNA (circRNA) in insect immunity remains unclear. To explore circRNA regulation involved in BmNPV infection, we used transcriptome analysis of BmN cells with or without BmNPV infection to generate circRNA data set. A total of 444 novel circRNAs were identified in BmN cells, with 198 pervasively distributed both in the control group and BmNPV-infection group. The host genes were enriched inMAPK signaling pathway, dorso-ventral axis formation, and ECM-receptor interaction, which were required for the normal larval growth. A total of 75 circRNAs were differentially expressed (DE) on BmNPV infection. Six downregulated circRNAs were validated by Sanger sequencing and qRT-PCR. DEcircRNA-miRNA-DEmRNA network was constructed based on the six validated circRNAs. Pathway analysis indicated that the predicted target genes were mainly enriched in the metabolic pathway and immune-related signaling pathway. Our results may provide a basis for further studies on circRNA function in BmN cells challenged by BmNPV infection and offer an insight into the molecular mechanism on silkworm-virus interaction., (© 2020 Wiley Periodicals LLC.)

Bt protein, produced by Bacillus thuringiensis, can bind receptors to destroy the physiological functions of the insect midgut. It is unknown whether Bt can also target the hindgut and influence its defense against fecal bacteria. Here we show that Crystal protein 1Ab (Cry1Ab), a Bt protein, was detected in the larval hindgut contents of Bombyx mori after ingestion of this toxin protein. The number of fecal bacteria that can be inhibited by the hindgut prophenoloxidase-induced melanization was significantly enhanced after oral ingestion of Cry1Ab. Although the hindgut contents became brown, the activity of hindgut phenoloxidase was decreased. LC-MS/MS analysis of the hindgut lumen contents revealed that many new proteins including several proteases were newly secreted. The enhanced secretion of proteases cleaved prophenoloxidase to decrease its activity, including the corresponding activity to inhibit the fecal bacteria. In addition, after ingestion of Cry1Ab, the pylorus (between the midgut and hindgut) could not autonomously contract due to the physical detachment of the acellular cuticle-like membrane from the epidermal cells, which prevented the movement of food from the midgut to the hindgut. Some cells in the cryptonephry of the hindgut became swollen and degraded, possibly due to the presence of Cry1Ab in the hindgut. These findings demonstrate that the inhibition of feces bacteria by the hindgut prophenoloxidase-induced melanization is out of control after Cry1Ab ingestion., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The silkworm, Bombyx mori, is utilized as a research model in many aspects of biological studies, including genetics, development and immunology. Previous biochemical and genomic studies have elucidated the silkworm immunity in response to infections elicited by bacteria, fungi, microsporidia, and viruses. The intestine serves as the front line in the battle between insects and ingested harmful microorganisms. In this study, we performed RNA sequencing (RNA-seq) of the larval silkworm midgut after oral infection with the Gram-positive bacterium Bacillus bombysepticus and the Gram-negative bacterium Yersinia pseudotuberculosis. This enables us to get a comprehensive understanding of the midgut responses to bacterial infection. We found that B. bombysepticus induced much stronger immune responses than Y. pseudotuberculosis did. Bacterial infection resulted in more energy consumption including carbohydrates and fatty acids. The midgut immune system was characterized by the generation of reactive oxygen species and antimicrobial peptides. The former played a critical role in eliminating invading bacteria during early stage, while the latter executed during late stage. Our results provide an integrated insight into the midgut systematic responses to bacterial infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a serious disease harmful to silk industry, which is one of the major sources of financial support for farmers in many developing countries. So far, there is still no good way to prevent or treat this disease. In this study, titanium dioxide nanoparticles (TiO 2 NPs) were used to pretreat silkworm larvae, and good results were achieved in improving silkworm immunity and alleviating the damage of cytoplasmic polyhedrosis virus. The results showed that nano-titanium dioxide pretreatment could inhibit the proliferation of BmCPV in the midgut of silkworm, activate JAK/STAT and PI3K-AKT immune signaling pathways, and upregulate the expression of key immune genes, so as to improve the immunity of silkworm and enhance the resistance of silkworm to BmCPV.

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., (© 2019 Wiley Periodicals, Inc.)

Programmed cell death 2 (PDCD2) is a highly conserved eukaryotic protein indispensable for various physiological processes such as cell proliferation, development, and apoptosis. In the present study, we identified a Zinc finger protein RP-8 from the silkworm, Bombyx mori (BmZfrp8), the ortholog of PDCD2 protein. The quantitative real-time PCR analysis revealed the ubiquitous distribution of BmZfrp8 in the different tissues; however, the gene's transcription level was highest in those of the silk gland, testis, and ovary. Additionally, the expression levels of BmZfrp8 were unequal on different days of embryonic development, and it reached the highest level on the 5th day of early development. The challenge with pathogens influenced the expression level of BmZfrp8 in both hemocyte and fat body when compared with the control. Administration of 20-hydroxyecdysone significantly enhanced the BmZfrp8 expression in hemocyte. The knock-down of BmZfrp8 by double-stranded RNA suppressed the expression of developmental pathway associated genes as well as cell cycle-associated genes. Furthermore, the RNAi treated cells also showed cell cycle arrest compared to the control group. Taken together, BmZfrp8 may have a critical biological role in of B. mori, since it regulates the expression of the developmental pathway and cell cycle-associated genes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)